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approximately , one - third of patients with symptomatic vte manifests pe , whereas two - thirds manifest dvt alone . both dvt and pe can be clinically silent ( asymptomatic ) and hence not suspected . if undiagnosed , asymptomatic vte can lead to chronic venous disease or recurrent vte and long - term debilitating sequelae such as postthrombotic syndrome and chronic thromboembolic pulmonary hypertension . vte is not only disabling but also prolongs hospital stay and increases the cost of treatment . along with myocardial infarction and arrhythmia ( due to electrolyte imbalance ) , pe is one of the commonest causes of sudden unexplained deaths in hospitalized patients . it is estimated that 20 million cases of lower extremity dvt occur in the usa alone . the prevailing notion that the incidence of vte in asians is less than that in the western population has been disproved by recent studies . the incidence of postoperative dvt in indian patients undergoing major lower limb surgery is as high ( 43.2% and 60% patients in the groups with and without prophylaxis , respectively ) as seen in the western world . given the growing burden of vte in india and lack of substantial indian data on characteristics of vte patients , use of diagnostics tools , prophylaxis , treatment options , and clinical outcomes in vte , there was a need to systematically collect such data . data on patient characteristics , clinical outcomes , predictors of mortality in acute dvt , management strategies and temporal trends in vte . the intent was to collect and provide data that would reflect actual day - to - day clinical practice , rather than results of highly controlled clinical trials with restricted study populations and imposed experimental intervention . consecutive medical records of inpatients and outpatients between january 2006 and december 2010 , meeting eligibility criteria ( confirmed diagnosis of acute or acute - on - chronic dvt by doppler ultrasound scan and/or pe by chest computed tomography scan , pulmonary angiography or v / q scan ) were identified and collected from the general medical records and/or radiology departments at each of the three participating hospitals . hospital data were used to obtain the total number of patients who were annually registered at the hospital from 2006 to 2010 . data were processed and analyzed using sas ( version 9.1 , statistical analysis system ) . for the purpose of analysis , acute - on - chronic descriptive statistics were used to present patient characteristics , management strategies , and clinical outcomes of patients . annual incidence rates ( 95% ci ) of vte per 100,000 hospital registrations over a period of 5 years were reported for each site . fisher 's exact test was used to determine differences in the incidence of acute dvt ( pe ) over the years 20062010 . armitage trend test was used to examine the direction ( positive or negative ) of the trend . as primary analyses the remaining 41% ( 393/949 ) medical records were not included because they did not satisfy the inclusion criteria . data from seven patients were excluded as there was no radiologically confirmed diagnosis of pe . a total of 64% ( 352/549 ) patients had acute dvt without pe , 23% ( 124/549 ) had acute dvt with pe , and 13% ( 73/549 ) had pe . eighty - seven percent ( 476/549 ) of patients had acute dvt ( pe ) , and 36% ( 197/549 ) had pe ( acute dvt ) [ figure 1 ] . overall distribution of venous thromboembolism patients ( n = 549 ) a total of 21% ( 115/549 ) of patients visited the hospitals directly without being referred by a physician . venous thromboembolism patients referred from different medical specialties ( n=434 ) the demographic characteristics of the vte patients are mentioned in table 2 . demographic characteristics of venous thromboembolism patients ( n=549 ) a total of 182 patients had evidence of one risk factor , 126 had evidence of two risk factors , 70 had evidence of three risk factors and 31 had four or more risk factors recorded . patients undergoing orthopedic surgery constituted 22% ( 33/152 ) of all surgical patients [ table 3 ] . risk factors for venous thromboembolism based on a review of the available records , 157 patients had a single co - morbidity , 81 had two co - morbidities , 23 had three co - morbidities , and 16 had four or more co - morbidities . ( myocardial infarction , heart failure , chronic obstructive pulmonary disease , ventilator dependency , sepsis , or pneumonia ) [ table 4 ] . co - morbidities in venous thromboembolism patients of the 476 patients with dvt , 2% ( 9 ) had upper extremity dvt , 97% ( 462 ) had lower extremity dvt and the site of dvt was not known in 5 patients . a total of 31% ( 143/462 ) patients had dvt in the right limb , 54% ( 249/462 ) in the left limb and 9% ( 41/462 ) in both limbs ( site not known in 29 patients ) . of the 462 patients with lower extremity dvt , 61% had proximal dvt , 13% had distal dvt , and 7% had proximal and distal dvt . a total of 39% ( 215/549 ) patients were diagnosed with vte during their hospital stay , 54% ( 296/549 ) were admitted to hospital with a diagnosis of vte , and 7% ( 38/549 ) were diagnosed and continued to be managed in the outpatient department [ figure 2 ] . place of detection of venous thromboembolism ( n = 549 ) duration of hospitalization after diagnosis of venous thromboembolism a smaller proportion of patients ( 15% ; 81/549 ) was diagnosed with vte during the postoperative period . figure 3 shows the proportion of patients with vte at different time points during the postoperative period . of those diagnosed beyond 6 weeks , 21% ( 3/14 ) had orthopedic surgery ( hip fracture surgery ) . diagnosis of venous thromboembolism during the postoperative period ( n = 81 ) the most common ( 73% ) symptom was swelling of the limb among patients with vte [ table 6 ] . symptoms in venous thromboembolism patients in merely 4% of all the patients , dvt was also confirmed by venography . pe was confirmed by pulmonary angiography in 27% of all the patients [ table 7 ] . . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . five percent ( 25/515 ) of patients were recommended lmwh alone , and 76% ( 393/515 ) were recommended either warfarin or acenocoumarol alone for long - term anticoagulation . the median duration of initial anticoagulation was 5 days while that of long - term anticoagulation was 180 days ( 6 months ) . anticoagulants were needed to be stopped because of bleeding in only 2% ( 9/515 ) patients . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . however , a formal site - wise statistical analysis could not be performed to analyse trends in the incidence rates in acute dvt ( pe ) and pe alone as there were zero observations in some instances . incidence of acute deep vein thrombosis ( with or without pulmonary embolism ) over a 5 years period ( 20062010 ) at three sites demographic characteristics of venous thromboembolism patients ( n=549 ) a total of 182 patients had evidence of one risk factor , 126 had evidence of two risk factors , 70 had evidence of three risk factors and 31 had four or more risk factors recorded . patients undergoing orthopedic surgery constituted 22% ( 33/152 ) of all surgical patients [ table 3 ] . risk factors for venous thromboembolism based on a review of the available records , 157 patients had a single co - morbidity , 81 had two co - morbidities , 23 had three co - morbidities , and 16 had four or more co - morbidities . ( myocardial infarction , heart failure , chronic obstructive pulmonary disease , ventilator dependency , sepsis , or pneumonia ) [ table 4 ] . 2% ( 9 ) had upper extremity dvt , 97% ( 462 ) had lower extremity dvt and the site of dvt was not known in 5 patients . a total of 31% ( 143/462 ) patients had dvt in the right limb , 54% ( 249/462 ) in the left limb and 9% ( 41/462 ) in both limbs ( site not known in 29 patients ) . of the 462 patients with lower extremity dvt , 61% had proximal dvt , 13% had distal dvt , and 7% had proximal and distal dvt . a total of 39% ( 215/549 ) patients were diagnosed with vte during their hospital stay , 54% ( 296/549 ) were admitted to hospital with a diagnosis of vte , and 7% ( 38/549 ) were diagnosed and continued to be managed in the outpatient department [ figure 2 ] . place of detection of venous thromboembolism ( n = 549 ) duration of hospitalization after diagnosis of venous thromboembolism a smaller proportion of patients ( 15% ; 81/549 ) was diagnosed with vte during the postoperative period . figure 3 shows the proportion of patients with vte at different time points during the postoperative period . of those diagnosed beyond 6 weeks diagnosis of venous thromboembolism during the postoperative period ( n = 81 ) the most common ( 73% ) symptom was swelling of the limb among patients with vte [ table 6 ] . a total of 182 patients had evidence of one risk factor , 126 had evidence of two risk factors , 70 had evidence of three risk factors and 31 had four or more risk factors recorded . patients undergoing orthopedic surgery constituted 22% ( 33/152 ) of all surgical patients [ table 3 ] . based on a review of the available records , 157 patients had a single co - morbidity , 81 had two co - morbidities , 23 had three co - morbidities , and 16 had four or more co - morbidities . ( myocardial infarction , heart failure , chronic obstructive pulmonary disease , ventilator dependency , sepsis , or pneumonia ) [ table 4 ] . of the 476 patients with dvt , 2% ( 9 ) had upper extremity dvt , 97% ( 462 ) had lower extremity dvt and the site of dvt was not known in 5 patients . a total of 31% ( 143/462 ) patients had dvt in the right limb , 54% ( 249/462 ) in the left limb and 9% ( 41/462 ) in both limbs ( site not known in 29 patients ) . of the 462 patients with lower extremity dvt , 61% had proximal dvt , 13% had distal dvt , and 7% had proximal and distal dvt . a total of 39% ( 215/549 ) patients were diagnosed with vte during their hospital stay , 54% ( 296/549 ) were admitted to hospital with a diagnosis of vte , and 7% ( 38/549 ) were diagnosed and continued to be managed in the outpatient department [ figure 2 ] . place of detection of venous thromboembolism ( n = 549 ) duration of hospitalization after diagnosis of venous thromboembolism a smaller proportion of patients ( 15% ; 81/549 ) was diagnosed with vte during the postoperative period . figure 3 shows the proportion of patients with vte at different time points during the postoperative period . of those diagnosed beyond 6 weeks diagnosis of venous thromboembolism during the postoperative period ( n = 81 ) the most common ( 73% ) symptom was swelling of the limb among patients with vte [ table 6 ] pe was confirmed by pulmonary angiography in 27% of all the patients [ table 7 ] . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . five percent ( 25/515 ) of patients were recommended lmwh alone , and 76% ( 393/515 ) were recommended either warfarin or acenocoumarol alone for long - term anticoagulation . the median duration of initial anticoagulation was 5 days while that of long - term anticoagulation was 180 days ( 6 months ) . anticoagulants were needed to be stopped because of bleeding in only 2% ( 9/515 ) patients . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . however , a formal site - wise statistical analysis could not be performed to analyse trends in the incidence rates in acute dvt ( pe ) and pe alone as there were zero observations in some instances . incidence of acute deep vein thrombosis ( with or without pulmonary embolism ) over a 5 years period ( 20062010 ) at three sites pe was confirmed by pulmonary angiography in 27% of all the patients [ table 7 ] . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . five percent ( 25/515 ) of patients were recommended lmwh alone , and 76% ( 393/515 ) were recommended either warfarin or acenocoumarol alone for long - term anticoagulation . the median duration of initial anticoagulation was 5 days while that of long - term anticoagulation was 180 days ( 6 months ) . anticoagulants were needed to be stopped because of bleeding in only 2% ( 9/515 ) patients . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . however , a formal site - wise statistical analysis could not be performed to analyse trends in the incidence rates in acute dvt ( pe ) and pe alone as there were zero observations in some instances . incidence of acute deep vein thrombosis ( with or without pulmonary embolism ) over a 5 years period ( 20062010 ) at three sites to our knowledge , this is the first multicenter , retrospective registry in india involving patients with vte that reflect real - world clinical practice . in contrast with the western data in which vte is predominantly a disease of older age , 44% patients in our study were between 40 and 59 years of age while 34% were below 40 years , particularly those with pe . in a study from north india , men constituted 70% of our registry , more than those reported from vellore registry ( 48% ) , but similar to those reported in the endorse ( epidemiologic international day for the evaluation of patients at risk for vte in the acute hospital care setting ) study ( 69% ) . one of the reasons for this could be significantly high levels of homocysteine ( thrombophilia marker ) in males as compared to females as reported in an indian study . fewer indian women use oral contraceptives and postmenopausal hormone replacement therapy , which are known to be risk factors for thrombosis . this is supported by the fact that only 1% of women in this registry reported the use of oral contraceptives , and none reported use of hormonal replacement therapy . a total of 28% of the overall referrals were from cardiologists . the majority ( 82% ) of the referrals were from medical rather than surgical ( 15% ) specialties as against a referral rate of 93% from surgeons at vellore . our finding complements that from the endorse study in which 55% of the medical patients at risk of vte had cardiovascular disease . majority ( 53% ) of patients in our study had co - morbid cardiovascular disease including diabetes mellitus ; it is possible that these patients visited a cardiologist for their cardiovascular ailment ( s ) and were then referred by the cardiologist to vascular disease specialist ( investigator ) . most ( 89% ) of these patients had swelling of the ( lower ) limb . it is possible that these patients may not have felt the need to visit a specialist for a symptom like swelling of limb , instead visited their family physician . it is very encouraging to know that family physicians suspected dvt in these situations and referred the patient to a specialist . patients with a history of vte are about 8 times more likely to develop a new episode during a subsequent high - risk period compared with patients without a history of dvt or pe . prior history of dvt was the most ( 34% ) common risk factor in patients who had only dvt , whereas past history of pe , trauma , and immobilization for more than 3 days were the most common risk factors in patients who had only pe . our results ( major lower limb surgery as a risk factor in 3% patients ) appear to be consistent with those reported in the endorse study , which reported dvt in 4.4% patients undergoing major lower limb surgery . other studies from india have reported a dvt incidence rate ranging from 8% to 20% in major lower limb surgery . however , in our study , only 7% of patients had malignancy as a predisposing factor . among the malignancies , genitourinary cancer had the highest incidence ( 45% ) . hypertension ( 25% ) was the most common co - morbidity followed by diabetes mellitus ( 19% ) in this patient population . in addition , obesity ( 11% ) was a common risk factor in dvt complicated by pe . our findings support an asian ( korean ) study that demonstrated prevalence of the metabolic syndrome in 48% patients with vte . co - morbid neurological disease ( other than stroke ) and ventilator dependency were also commonly found in patients with dvt ( 10% ) and pe ( 11% ) respectively . both these conditions immobilize patients for prolonged periods of time , predisposing them to vte . venography and pulmonary angiography are the gold standard for diagnosis of dvt and pe respectively . in our study , venography was used in just 4% patients and pulmonary angiography in less than one - third of the patients . perhaps the relatively high cost of these tests and limited availability of such procedures may be the limiting factors . overall , most ( 93% ) patients were managed as inpatients ( 39% diagnosed with vte during hospital stay and 54% admitted to hospital with a diagnosis of vte ) . a mean duration of hospitalization of 79 days after diagnosis of vte is supported by published data . in selected low - risk patients , outpatient treatment of dvt and pe may be considered . this approach was observed in a small proportion ( 7% ) of patients who were managed on an outpatient basis , nearly all ( 97% ) of whom had only dvt . the reported prevalence of postsurgical vte in our study ( 15% ) was half of that ( 30% ) reported in vellore registry . this could be explained by higher referral rate from surgeons at vellore compared to that of our sites . most ( 40% ; 32/81 ) dvt cases were diagnosed between 2 and 6 postoperative weeks , but pe in most cases ( 70% ; 7/10 ) was diagnosed during the first postoperative week . we notice that acute dvt complicated by pe was less ( 6% ; 7/124 ) frequently diagnosed during the postoperative period as against 18% ( 64/352 ) and 14% ( 10/73 ) of acute dvt alone and pe alone , respectively . the use and duration of anticoagulants in our registry appears to be consistent with the american college of chest physicians treatment guidelines , which recommend at least 5 days of initial anticoagulation with parenteral anticoagulation ( lmwh , fondaparinux , intravenous ufh , or subcutaneous ufh ) and at least 3 months of long - term anticoagulation treatment with vitamin k antagonist . bleeding is the most serious complication of anticoagulation treatment and is a major concern for clinicians particularly as the patient 's age advances . in this registry , anticoagulant treatment was needed to be stopped because of bleeding in only 2% of the study population . the prospective reite registry has reported a rate of 3% for major / fatal bleeds . thus , the fear of bleeding complications , which decreases the use of anticoagulant treatment , appears to be minimal . dvt complicated by pe ( 60% ) and pe alone ( 75% ) were more frequently shifted to intensive care unit than those who had dvt alone ( 25% ) . similar to published data in which hospital readmission rate for vte was 5% for primary and 14% for secondary diagnosis , we report a hospital readmission rate of 6% ; however we do not know the cause for readmission . the death rate was 7% among those diagnosed with vte during hospital stay as against a rate of 1% among those who were hospitalized with a diagnosis of vte . over 90% of patients treated on an outpatient basis obtained symptomatic relief with treatment . in our study , the hospital discharge rate ( 97% ) was more than triple and death rate was a quarter of that reported by pandey et al . ( hospital discharge rate 31% and death rate 16% ) at a university hospital in delhi . our data show a significant increase in acute dvt ( pe ) from 2006 to 2010 . this can be explained by the increased awareness of vte in india as well as the advent of better diagnostic modalities , such as duplex ultrasonography becoming more readily available and accepted . although there was no significant change in the number of pe cases from 2006 to 2010 , the burden of pe is almost double ( 13% of all vte ) of 7% , rate reported at christian medical college , vellore during a 10-year period from 1996 to 2005 . our finding is consistent with a study from north india that reported a 16% incidence of pe in adult medical autopsies . this study has the expected limitations of any retrospective review including the availability of complete records for all patients , although a robust review of the data on medical charts was conducted . controlling for bias and confounders is difficult as there is no randomization and no blinding . follow - up data of patients after hospital discharge were not available . in cases of death , further , the clinic charts reviewed in this study included a mix of those from vascular surgery and hematology departments , limiting the generalizability of the study results . despite these limitations , this study provides large amount of useful information in a short span of time on patient characteristics , clinical outcomes , management strategies , and temporal trends in vte , based on real world data that reflect actual day - to - day clinical practice over a period of 5 years across three sites in india . we believe that this information will serve as a guide in the optimal implementation of vte prophylaxis and treatment , to improve patient outcomes and to decrease the occurrence of vte in india . real world data reflecting actual day - to - day clinical practice in vte over a period of 5 years across three sites in india showed that vte is not uncommon in indian patients and that acute dvt was responsible for the substantial burden of vte . we believe that this information will serve as a guide in the optimal implementation of vte prophylaxis and treatment , to improve patient outcomes and to decrease the occurrence of vte in india . liesel c. dsilva is and dr . sadhna j. joglekar was full - time employee of glaxosmithkline pharmaceuticals limited .

background and aim : there is lack of substantial indian data on venous thromboembolism ( vte ) . the aim of this study was to provide real - world information on patient characteristics , management strategies , clinical outcomes , and temporal trends in vte.subjects and methods : multicentre retrospective registry involving 549 medical records of patients with confirmed diagnosis of vte ( deep vein thrombosis [ dvt ] confirmed by doppler ultrasonography ; pulmonary embolism [ pe ] by computed tomography , pulmonary angiography and/or v / q scan ) from 2006 to 2010 at three indian tertiary care hospitals.results:acute dvt without pe , acute dvt with pe , and pe alone were reported in 64% ( 352/549 ) , 23% ( 124/549 ) , and 13% ( 73/549 ) patients , respectively . mean age was 47 ( 16 ) years , and 70% were males . h / o dvt ( 34% ) , surgery including orthopedic surgery ( 28% ) , trauma ( 16% ) , and immobilization > 3 days ( 14% ) were the most common risk factors for vte . hypertension ( 25% ) , diabetes ( 19% ) , and neurological disease ( other than stroke ) ( 8% ) were the most common co - morbidities . most ( 94% ) were treated with heparin alone ( 82% ) or fondaparinux ( 2% ) for initial anticoagulation ; low molecular weight heparin alone ( 5% ) or warfarin / acenocoumarol ( 76% ) for long - term anticoagulation . anticoagulant treatment was stopped because of bleeding in 2% ( 9/515 ) patients . mortality was 7% among patients diagnosed with vte during hospital stay versus 1% in those hospitalized with diagnosed vte . the annual incidence of dvt ( pe ) increased from 2006 to 2010.conclusion:acute dvt alone was responsible for the substantial burden of vte in indian patients . bleeding was not the limiting factor for anticoagulant treatment in most patients .

Introduction Subjects and Methods Results Demographics and characteristics of venous thromboembolism patients Risk factors for venous thromboembolism patients Co-morbidities in venous thromboembolism patients Clinical presentation of venous thromboembolism Management strategies for venous thromboembolism patients Diagnostic tools for venous thromboembolism Anticoagulant treatment in venous thromboembolism Annual incidence of acute deep venous thrombosis including the trend over a period of 5 years Discussion Conclusion None Financial support and sponsorship Conflicts of interest

approximately , one - third of patients with symptomatic vte manifests pe , whereas two - thirds manifest dvt alone . if undiagnosed , asymptomatic vte can lead to chronic venous disease or recurrent vte and long - term debilitating sequelae such as postthrombotic syndrome and chronic thromboembolic pulmonary hypertension . the prevailing notion that the incidence of vte in asians is less than that in the western population has been disproved by recent studies . the incidence of postoperative dvt in indian patients undergoing major lower limb surgery is as high ( 43.2% and 60% patients in the groups with and without prophylaxis , respectively ) as seen in the western world . given the growing burden of vte in india and lack of substantial indian data on characteristics of vte patients , use of diagnostics tools , prophylaxis , treatment options , and clinical outcomes in vte , there was a need to systematically collect such data . data on patient characteristics , clinical outcomes , predictors of mortality in acute dvt , management strategies and temporal trends in vte . consecutive medical records of inpatients and outpatients between january 2006 and december 2010 , meeting eligibility criteria ( confirmed diagnosis of acute or acute - on - chronic dvt by doppler ultrasound scan and/or pe by chest computed tomography scan , pulmonary angiography or v / q scan ) were identified and collected from the general medical records and/or radiology departments at each of the three participating hospitals . hospital data were used to obtain the total number of patients who were annually registered at the hospital from 2006 to 2010 . for the purpose of analysis , acute - on - chronic descriptive statistics were used to present patient characteristics , management strategies , and clinical outcomes of patients . annual incidence rates ( 95% ci ) of vte per 100,000 hospital registrations over a period of 5 years were reported for each site . fisher 's exact test was used to determine differences in the incidence of acute dvt ( pe ) over the years 20062010 . data from seven patients were excluded as there was no radiologically confirmed diagnosis of pe . a total of 64% ( 352/549 ) patients had acute dvt without pe , 23% ( 124/549 ) had acute dvt with pe , and 13% ( 73/549 ) had pe . eighty - seven percent ( 476/549 ) of patients had acute dvt ( pe ) , and 36% ( 197/549 ) had pe ( acute dvt ) [ figure 1 ] . overall distribution of venous thromboembolism patients ( n = 549 ) a total of 21% ( 115/549 ) of patients visited the hospitals directly without being referred by a physician . venous thromboembolism patients referred from different medical specialties ( n=434 ) the demographic characteristics of the vte patients are mentioned in table 2 . demographic characteristics of venous thromboembolism patients ( n=549 ) a total of 182 patients had evidence of one risk factor , 126 had evidence of two risk factors , 70 had evidence of three risk factors and 31 had four or more risk factors recorded . risk factors for venous thromboembolism based on a review of the available records , 157 patients had a single co - morbidity , 81 had two co - morbidities , 23 had three co - morbidities , and 16 had four or more co - morbidities . co - morbidities in venous thromboembolism patients of the 476 patients with dvt , 2% ( 9 ) had upper extremity dvt , 97% ( 462 ) had lower extremity dvt and the site of dvt was not known in 5 patients . of the 462 patients with lower extremity dvt , 61% had proximal dvt , 13% had distal dvt , and 7% had proximal and distal dvt . a total of 39% ( 215/549 ) patients were diagnosed with vte during their hospital stay , 54% ( 296/549 ) were admitted to hospital with a diagnosis of vte , and 7% ( 38/549 ) were diagnosed and continued to be managed in the outpatient department [ figure 2 ] . place of detection of venous thromboembolism ( n = 549 ) duration of hospitalization after diagnosis of venous thromboembolism a smaller proportion of patients ( 15% ; 81/549 ) was diagnosed with vte during the postoperative period . figure 3 shows the proportion of patients with vte at different time points during the postoperative period . of those diagnosed beyond 6 weeks , 21% ( 3/14 ) had orthopedic surgery ( hip fracture surgery ) . diagnosis of venous thromboembolism during the postoperative period ( n = 81 ) the most common ( 73% ) symptom was swelling of the limb among patients with vte [ table 6 ] . symptoms in venous thromboembolism patients in merely 4% of all the patients , dvt was also confirmed by venography . pe was confirmed by pulmonary angiography in 27% of all the patients [ table 7 ] . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . five percent ( 25/515 ) of patients were recommended lmwh alone , and 76% ( 393/515 ) were recommended either warfarin or acenocoumarol alone for long - term anticoagulation . the median duration of initial anticoagulation was 5 days while that of long - term anticoagulation was 180 days ( 6 months ) . anticoagulants were needed to be stopped because of bleeding in only 2% ( 9/515 ) patients . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . however , a formal site - wise statistical analysis could not be performed to analyse trends in the incidence rates in acute dvt ( pe ) and pe alone as there were zero observations in some instances . incidence of acute deep vein thrombosis ( with or without pulmonary embolism ) over a 5 years period ( 20062010 ) at three sites demographic characteristics of venous thromboembolism patients ( n=549 ) a total of 182 patients had evidence of one risk factor , 126 had evidence of two risk factors , 70 had evidence of three risk factors and 31 had four or more risk factors recorded . risk factors for venous thromboembolism based on a review of the available records , 157 patients had a single co - morbidity , 81 had two co - morbidities , 23 had three co - morbidities , and 16 had four or more co - morbidities . 2% ( 9 ) had upper extremity dvt , 97% ( 462 ) had lower extremity dvt and the site of dvt was not known in 5 patients . of the 462 patients with lower extremity dvt , 61% had proximal dvt , 13% had distal dvt , and 7% had proximal and distal dvt . a total of 39% ( 215/549 ) patients were diagnosed with vte during their hospital stay , 54% ( 296/549 ) were admitted to hospital with a diagnosis of vte , and 7% ( 38/549 ) were diagnosed and continued to be managed in the outpatient department [ figure 2 ] . place of detection of venous thromboembolism ( n = 549 ) duration of hospitalization after diagnosis of venous thromboembolism a smaller proportion of patients ( 15% ; 81/549 ) was diagnosed with vte during the postoperative period . figure 3 shows the proportion of patients with vte at different time points during the postoperative period . of those diagnosed beyond 6 weeks diagnosis of venous thromboembolism during the postoperative period ( n = 81 ) the most common ( 73% ) symptom was swelling of the limb among patients with vte [ table 6 ] . based on a review of the available records , 157 patients had a single co - morbidity , 81 had two co - morbidities , 23 had three co - morbidities , and 16 had four or more co - morbidities . of the 476 patients with dvt , 2% ( 9 ) had upper extremity dvt , 97% ( 462 ) had lower extremity dvt and the site of dvt was not known in 5 patients . of the 462 patients with lower extremity dvt , 61% had proximal dvt , 13% had distal dvt , and 7% had proximal and distal dvt . a total of 39% ( 215/549 ) patients were diagnosed with vte during their hospital stay , 54% ( 296/549 ) were admitted to hospital with a diagnosis of vte , and 7% ( 38/549 ) were diagnosed and continued to be managed in the outpatient department [ figure 2 ] . place of detection of venous thromboembolism ( n = 549 ) duration of hospitalization after diagnosis of venous thromboembolism a smaller proportion of patients ( 15% ; 81/549 ) was diagnosed with vte during the postoperative period . figure 3 shows the proportion of patients with vte at different time points during the postoperative period . of those diagnosed beyond 6 weeks diagnosis of venous thromboembolism during the postoperative period ( n = 81 ) the most common ( 73% ) symptom was swelling of the limb among patients with vte [ table 6 ] pe was confirmed by pulmonary angiography in 27% of all the patients [ table 7 ] . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . five percent ( 25/515 ) of patients were recommended lmwh alone , and 76% ( 393/515 ) were recommended either warfarin or acenocoumarol alone for long - term anticoagulation . the median duration of initial anticoagulation was 5 days while that of long - term anticoagulation was 180 days ( 6 months ) . anticoagulants were needed to be stopped because of bleeding in only 2% ( 9/515 ) patients . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . however , a formal site - wise statistical analysis could not be performed to analyse trends in the incidence rates in acute dvt ( pe ) and pe alone as there were zero observations in some instances . incidence of acute deep vein thrombosis ( with or without pulmonary embolism ) over a 5 years period ( 20062010 ) at three sites pe was confirmed by pulmonary angiography in 27% of all the patients [ table 7 ] . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . five percent ( 25/515 ) of patients were recommended lmwh alone , and 76% ( 393/515 ) were recommended either warfarin or acenocoumarol alone for long - term anticoagulation . the median duration of initial anticoagulation was 5 days while that of long - term anticoagulation was 180 days ( 6 months ) . anticoagulants were needed to be stopped because of bleeding in only 2% ( 9/515 ) patients . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . however , a formal site - wise statistical analysis could not be performed to analyse trends in the incidence rates in acute dvt ( pe ) and pe alone as there were zero observations in some instances . incidence of acute deep vein thrombosis ( with or without pulmonary embolism ) over a 5 years period ( 20062010 ) at three sites to our knowledge , this is the first multicenter , retrospective registry in india involving patients with vte that reflect real - world clinical practice . in contrast with the western data in which vte is predominantly a disease of older age , 44% patients in our study were between 40 and 59 years of age while 34% were below 40 years , particularly those with pe . in a study from north india , men constituted 70% of our registry , more than those reported from vellore registry ( 48% ) , but similar to those reported in the endorse ( epidemiologic international day for the evaluation of patients at risk for vte in the acute hospital care setting ) study ( 69% ) . fewer indian women use oral contraceptives and postmenopausal hormone replacement therapy , which are known to be risk factors for thrombosis . the majority ( 82% ) of the referrals were from medical rather than surgical ( 15% ) specialties as against a referral rate of 93% from surgeons at vellore . majority ( 53% ) of patients in our study had co - morbid cardiovascular disease including diabetes mellitus ; it is possible that these patients visited a cardiologist for their cardiovascular ailment ( s ) and were then referred by the cardiologist to vascular disease specialist ( investigator ) . patients with a history of vte are about 8 times more likely to develop a new episode during a subsequent high - risk period compared with patients without a history of dvt or pe . prior history of dvt was the most ( 34% ) common risk factor in patients who had only dvt , whereas past history of pe , trauma , and immobilization for more than 3 days were the most common risk factors in patients who had only pe . hypertension ( 25% ) was the most common co - morbidity followed by diabetes mellitus ( 19% ) in this patient population . our findings support an asian ( korean ) study that demonstrated prevalence of the metabolic syndrome in 48% patients with vte . co - morbid neurological disease ( other than stroke ) and ventilator dependency were also commonly found in patients with dvt ( 10% ) and pe ( 11% ) respectively . venography and pulmonary angiography are the gold standard for diagnosis of dvt and pe respectively . perhaps the relatively high cost of these tests and limited availability of such procedures may be the limiting factors . overall , most ( 93% ) patients were managed as inpatients ( 39% diagnosed with vte during hospital stay and 54% admitted to hospital with a diagnosis of vte ) . a mean duration of hospitalization of 79 days after diagnosis of vte is supported by published data . in selected low - risk patients , outpatient treatment of dvt and pe may be considered . the reported prevalence of postsurgical vte in our study ( 15% ) was half of that ( 30% ) reported in vellore registry . most ( 40% ; 32/81 ) dvt cases were diagnosed between 2 and 6 postoperative weeks , but pe in most cases ( 70% ; 7/10 ) was diagnosed during the first postoperative week . we notice that acute dvt complicated by pe was less ( 6% ; 7/124 ) frequently diagnosed during the postoperative period as against 18% ( 64/352 ) and 14% ( 10/73 ) of acute dvt alone and pe alone , respectively . the use and duration of anticoagulants in our registry appears to be consistent with the american college of chest physicians treatment guidelines , which recommend at least 5 days of initial anticoagulation with parenteral anticoagulation ( lmwh , fondaparinux , intravenous ufh , or subcutaneous ufh ) and at least 3 months of long - term anticoagulation treatment with vitamin k antagonist . in this registry , anticoagulant treatment was needed to be stopped because of bleeding in only 2% of the study population . thus , the fear of bleeding complications , which decreases the use of anticoagulant treatment , appears to be minimal . dvt complicated by pe ( 60% ) and pe alone ( 75% ) were more frequently shifted to intensive care unit than those who had dvt alone ( 25% ) . the death rate was 7% among those diagnosed with vte during hospital stay as against a rate of 1% among those who were hospitalized with a diagnosis of vte . our data show a significant increase in acute dvt ( pe ) from 2006 to 2010 . this can be explained by the increased awareness of vte in india as well as the advent of better diagnostic modalities , such as duplex ultrasonography becoming more readily available and accepted . although there was no significant change in the number of pe cases from 2006 to 2010 , the burden of pe is almost double ( 13% of all vte ) of 7% , rate reported at christian medical college , vellore during a 10-year period from 1996 to 2005 . this study has the expected limitations of any retrospective review including the availability of complete records for all patients , although a robust review of the data on medical charts was conducted . despite these limitations , this study provides large amount of useful information in a short span of time on patient characteristics , clinical outcomes , management strategies , and temporal trends in vte , based on real world data that reflect actual day - to - day clinical practice over a period of 5 years across three sites in india . we believe that this information will serve as a guide in the optimal implementation of vte prophylaxis and treatment , to improve patient outcomes and to decrease the occurrence of vte in india . real world data reflecting actual day - to - day clinical practice in vte over a period of 5 years across three sites in india showed that vte is not uncommon in indian patients and that acute dvt was responsible for the substantial burden of vte . we believe that this information will serve as a guide in the optimal implementation of vte prophylaxis and treatment , to improve patient outcomes and to decrease the occurrence of vte in india .

if undiagnosed , asymptomatic vte can lead to chronic venous disease or recurrent vte and long - term debilitating sequelae such as postthrombotic syndrome and chronic thromboembolic pulmonary hypertension . along with myocardial infarction and arrhythmia ( due to electrolyte imbalance ) , pe is one of the commonest causes of sudden unexplained deaths in hospitalized patients . the prevailing notion that the incidence of vte in asians is less than that in the western population has been disproved by recent studies . the incidence of postoperative dvt in indian patients undergoing major lower limb surgery is as high ( 43.2% and 60% patients in the groups with and without prophylaxis , respectively ) as seen in the western world . given the growing burden of vte in india and lack of substantial indian data on characteristics of vte patients , use of diagnostics tools , prophylaxis , treatment options , and clinical outcomes in vte , there was a need to systematically collect such data . data on patient characteristics , clinical outcomes , predictors of mortality in acute dvt , management strategies and temporal trends in vte . the intent was to collect and provide data that would reflect actual day - to - day clinical practice , rather than results of highly controlled clinical trials with restricted study populations and imposed experimental intervention . consecutive medical records of inpatients and outpatients between january 2006 and december 2010 , meeting eligibility criteria ( confirmed diagnosis of acute or acute - on - chronic dvt by doppler ultrasound scan and/or pe by chest computed tomography scan , pulmonary angiography or v / q scan ) were identified and collected from the general medical records and/or radiology departments at each of the three participating hospitals . hospital data were used to obtain the total number of patients who were annually registered at the hospital from 2006 to 2010 . for the purpose of analysis , acute - on - chronic descriptive statistics were used to present patient characteristics , management strategies , and clinical outcomes of patients . annual incidence rates ( 95% ci ) of vte per 100,000 hospital registrations over a period of 5 years were reported for each site . fisher 's exact test was used to determine differences in the incidence of acute dvt ( pe ) over the years 20062010 . armitage trend test was used to examine the direction ( positive or negative ) of the trend . a total of 64% ( 352/549 ) patients had acute dvt without pe , 23% ( 124/549 ) had acute dvt with pe , and 13% ( 73/549 ) had pe . eighty - seven percent ( 476/549 ) of patients had acute dvt ( pe ) , and 36% ( 197/549 ) had pe ( acute dvt ) [ figure 1 ] . overall distribution of venous thromboembolism patients ( n = 549 ) a total of 21% ( 115/549 ) of patients visited the hospitals directly without being referred by a physician . venous thromboembolism patients referred from different medical specialties ( n=434 ) the demographic characteristics of the vte patients are mentioned in table 2 . demographic characteristics of venous thromboembolism patients ( n=549 ) a total of 182 patients had evidence of one risk factor , 126 had evidence of two risk factors , 70 had evidence of three risk factors and 31 had four or more risk factors recorded . risk factors for venous thromboembolism based on a review of the available records , 157 patients had a single co - morbidity , 81 had two co - morbidities , 23 had three co - morbidities , and 16 had four or more co - morbidities . ( myocardial infarction , heart failure , chronic obstructive pulmonary disease , ventilator dependency , sepsis , or pneumonia ) [ table 4 ] . co - morbidities in venous thromboembolism patients of the 476 patients with dvt , 2% ( 9 ) had upper extremity dvt , 97% ( 462 ) had lower extremity dvt and the site of dvt was not known in 5 patients . a total of 31% ( 143/462 ) patients had dvt in the right limb , 54% ( 249/462 ) in the left limb and 9% ( 41/462 ) in both limbs ( site not known in 29 patients ) . of the 462 patients with lower extremity dvt , 61% had proximal dvt , 13% had distal dvt , and 7% had proximal and distal dvt . a total of 39% ( 215/549 ) patients were diagnosed with vte during their hospital stay , 54% ( 296/549 ) were admitted to hospital with a diagnosis of vte , and 7% ( 38/549 ) were diagnosed and continued to be managed in the outpatient department [ figure 2 ] . place of detection of venous thromboembolism ( n = 549 ) duration of hospitalization after diagnosis of venous thromboembolism a smaller proportion of patients ( 15% ; 81/549 ) was diagnosed with vte during the postoperative period . figure 3 shows the proportion of patients with vte at different time points during the postoperative period . diagnosis of venous thromboembolism during the postoperative period ( n = 81 ) the most common ( 73% ) symptom was swelling of the limb among patients with vte [ table 6 ] . symptoms in venous thromboembolism patients in merely 4% of all the patients , dvt was also confirmed by venography . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . five percent ( 25/515 ) of patients were recommended lmwh alone , and 76% ( 393/515 ) were recommended either warfarin or acenocoumarol alone for long - term anticoagulation . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . however , a formal site - wise statistical analysis could not be performed to analyse trends in the incidence rates in acute dvt ( pe ) and pe alone as there were zero observations in some instances . incidence of acute deep vein thrombosis ( with or without pulmonary embolism ) over a 5 years period ( 20062010 ) at three sites demographic characteristics of venous thromboembolism patients ( n=549 ) a total of 182 patients had evidence of one risk factor , 126 had evidence of two risk factors , 70 had evidence of three risk factors and 31 had four or more risk factors recorded . risk factors for venous thromboembolism based on a review of the available records , 157 patients had a single co - morbidity , 81 had two co - morbidities , 23 had three co - morbidities , and 16 had four or more co - morbidities . ( myocardial infarction , heart failure , chronic obstructive pulmonary disease , ventilator dependency , sepsis , or pneumonia ) [ table 4 ] . a total of 31% ( 143/462 ) patients had dvt in the right limb , 54% ( 249/462 ) in the left limb and 9% ( 41/462 ) in both limbs ( site not known in 29 patients ) . of the 462 patients with lower extremity dvt , 61% had proximal dvt , 13% had distal dvt , and 7% had proximal and distal dvt . a total of 39% ( 215/549 ) patients were diagnosed with vte during their hospital stay , 54% ( 296/549 ) were admitted to hospital with a diagnosis of vte , and 7% ( 38/549 ) were diagnosed and continued to be managed in the outpatient department [ figure 2 ] . place of detection of venous thromboembolism ( n = 549 ) duration of hospitalization after diagnosis of venous thromboembolism a smaller proportion of patients ( 15% ; 81/549 ) was diagnosed with vte during the postoperative period . figure 3 shows the proportion of patients with vte at different time points during the postoperative period . of those diagnosed beyond 6 weeks diagnosis of venous thromboembolism during the postoperative period ( n = 81 ) the most common ( 73% ) symptom was swelling of the limb among patients with vte [ table 6 ] . a total of 182 patients had evidence of one risk factor , 126 had evidence of two risk factors , 70 had evidence of three risk factors and 31 had four or more risk factors recorded . based on a review of the available records , 157 patients had a single co - morbidity , 81 had two co - morbidities , 23 had three co - morbidities , and 16 had four or more co - morbidities . of the 476 patients with dvt , 2% ( 9 ) had upper extremity dvt , 97% ( 462 ) had lower extremity dvt and the site of dvt was not known in 5 patients . a total of 31% ( 143/462 ) patients had dvt in the right limb , 54% ( 249/462 ) in the left limb and 9% ( 41/462 ) in both limbs ( site not known in 29 patients ) . of the 462 patients with lower extremity dvt , 61% had proximal dvt , 13% had distal dvt , and 7% had proximal and distal dvt . a total of 39% ( 215/549 ) patients were diagnosed with vte during their hospital stay , 54% ( 296/549 ) were admitted to hospital with a diagnosis of vte , and 7% ( 38/549 ) were diagnosed and continued to be managed in the outpatient department [ figure 2 ] . place of detection of venous thromboembolism ( n = 549 ) duration of hospitalization after diagnosis of venous thromboembolism a smaller proportion of patients ( 15% ; 81/549 ) was diagnosed with vte during the postoperative period . figure 3 shows the proportion of patients with vte at different time points during the postoperative period . of those diagnosed beyond 6 weeks diagnosis of venous thromboembolism during the postoperative period ( n = 81 ) the most common ( 73% ) symptom was swelling of the limb among patients with vte [ table 6 ] pe was confirmed by pulmonary angiography in 27% of all the patients [ table 7 ] . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . five percent ( 25/515 ) of patients were recommended lmwh alone , and 76% ( 393/515 ) were recommended either warfarin or acenocoumarol alone for long - term anticoagulation . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . however , a formal site - wise statistical analysis could not be performed to analyse trends in the incidence rates in acute dvt ( pe ) and pe alone as there were zero observations in some instances . incidence of acute deep vein thrombosis ( with or without pulmonary embolism ) over a 5 years period ( 20062010 ) at three sites pe was confirmed by pulmonary angiography in 27% of all the patients [ table 7 ] . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . five percent ( 25/515 ) of patients were recommended lmwh alone , and 76% ( 393/515 ) were recommended either warfarin or acenocoumarol alone for long - term anticoagulation . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . however , a formal site - wise statistical analysis could not be performed to analyse trends in the incidence rates in acute dvt ( pe ) and pe alone as there were zero observations in some instances . incidence of acute deep vein thrombosis ( with or without pulmonary embolism ) over a 5 years period ( 20062010 ) at three sites to our knowledge , this is the first multicenter , retrospective registry in india involving patients with vte that reflect real - world clinical practice . in contrast with the western data in which vte is predominantly a disease of older age , 44% patients in our study were between 40 and 59 years of age while 34% were below 40 years , particularly those with pe . in a study from north india , men constituted 70% of our registry , more than those reported from vellore registry ( 48% ) , but similar to those reported in the endorse ( epidemiologic international day for the evaluation of patients at risk for vte in the acute hospital care setting ) study ( 69% ) . one of the reasons for this could be significantly high levels of homocysteine ( thrombophilia marker ) in males as compared to females as reported in an indian study . fewer indian women use oral contraceptives and postmenopausal hormone replacement therapy , which are known to be risk factors for thrombosis . this is supported by the fact that only 1% of women in this registry reported the use of oral contraceptives , and none reported use of hormonal replacement therapy . our finding complements that from the endorse study in which 55% of the medical patients at risk of vte had cardiovascular disease . majority ( 53% ) of patients in our study had co - morbid cardiovascular disease including diabetes mellitus ; it is possible that these patients visited a cardiologist for their cardiovascular ailment ( s ) and were then referred by the cardiologist to vascular disease specialist ( investigator ) . patients with a history of vte are about 8 times more likely to develop a new episode during a subsequent high - risk period compared with patients without a history of dvt or pe . prior history of dvt was the most ( 34% ) common risk factor in patients who had only dvt , whereas past history of pe , trauma , and immobilization for more than 3 days were the most common risk factors in patients who had only pe . our results ( major lower limb surgery as a risk factor in 3% patients ) appear to be consistent with those reported in the endorse study , which reported dvt in 4.4% patients undergoing major lower limb surgery . among the malignancies , genitourinary cancer had the highest incidence ( 45% ) . our findings support an asian ( korean ) study that demonstrated prevalence of the metabolic syndrome in 48% patients with vte . co - morbid neurological disease ( other than stroke ) and ventilator dependency were also commonly found in patients with dvt ( 10% ) and pe ( 11% ) respectively . in our study , venography was used in just 4% patients and pulmonary angiography in less than one - third of the patients . overall , most ( 93% ) patients were managed as inpatients ( 39% diagnosed with vte during hospital stay and 54% admitted to hospital with a diagnosis of vte ) . we notice that acute dvt complicated by pe was less ( 6% ; 7/124 ) frequently diagnosed during the postoperative period as against 18% ( 64/352 ) and 14% ( 10/73 ) of acute dvt alone and pe alone , respectively . the use and duration of anticoagulants in our registry appears to be consistent with the american college of chest physicians treatment guidelines , which recommend at least 5 days of initial anticoagulation with parenteral anticoagulation ( lmwh , fondaparinux , intravenous ufh , or subcutaneous ufh ) and at least 3 months of long - term anticoagulation treatment with vitamin k antagonist . bleeding is the most serious complication of anticoagulation treatment and is a major concern for clinicians particularly as the patient 's age advances . thus , the fear of bleeding complications , which decreases the use of anticoagulant treatment , appears to be minimal . similar to published data in which hospital readmission rate for vte was 5% for primary and 14% for secondary diagnosis , we report a hospital readmission rate of 6% ; however we do not know the cause for readmission . this can be explained by the increased awareness of vte in india as well as the advent of better diagnostic modalities , such as duplex ultrasonography becoming more readily available and accepted . although there was no significant change in the number of pe cases from 2006 to 2010 , the burden of pe is almost double ( 13% of all vte ) of 7% , rate reported at christian medical college , vellore during a 10-year period from 1996 to 2005 . our finding is consistent with a study from north india that reported a 16% incidence of pe in adult medical autopsies . in cases of death , further , the clinic charts reviewed in this study included a mix of those from vascular surgery and hematology departments , limiting the generalizability of the study results . despite these limitations , this study provides large amount of useful information in a short span of time on patient characteristics , clinical outcomes , management strategies , and temporal trends in vte , based on real world data that reflect actual day - to - day clinical practice over a period of 5 years across three sites in india . we believe that this information will serve as a guide in the optimal implementation of vte prophylaxis and treatment , to improve patient outcomes and to decrease the occurrence of vte in india . real world data reflecting actual day - to - day clinical practice in vte over a period of 5 years across three sites in india showed that vte is not uncommon in indian patients and that acute dvt was responsible for the substantial burden of vte . we believe that this information will serve as a guide in the optimal implementation of vte prophylaxis and treatment , to improve patient outcomes and to decrease the occurrence of vte in india .

the need for magnetic resonance imaging ( mri ) in patients with an implanted pacemaker or implantable cardioverter - defibrillator ( icd ) is a growing clinical issue . it is estimated that as many as 75% of active cardiac device recipients will become indicated for mri . currently , the vast majority of such devices are contraindicated for use with an mri . in european heart rhythm association survey , published recently for non - mri - certified icds ( 0.51.5 t field strength ) , the totally subcutaneous icd ( s - icd ) system , an implantable defibrillator with no leads that touch the heart , has recently been demonstrated to be a safe and effective defibrillator option for patients at risk for sudden cardiac death . it provides shock therapy and post - shock pacing therapy , but no long - term bradycardia pacing . although it has been shown as an alternative to the standard transvenous icd , its compatibility with mri remains unclear . various types of clinical mri systems currently use a superconductive magnet that creates a static magnetic field strength , typically 1.5 or 3 t. the use of mri with most pacemakers and icds is considered a contraindication due to potential hazards , including heating of the electrode that resides in or on the heart , damage to myocardium , elevation of pacing thresholds , unintended induction of ventricular tachycardia ( vt ) or ventricular fibrillation ( vf ) , pacing inhibition , permanent device malfunction , and distortion of the mri scan . recently , mr - conditional. mr - conditional indicates a lack of known hazards in a specified mri environment with specified conditions of use . due to the variety of mri scanners and scanning protocols , it is not practical to test even a single device under all conditions . hence , mr - conditional labelling dictates that the device is safe for use under certain scanning conditions , as well as how the cardiac device should be programmed before an exposure to the magnetic field in a mri scanner . the literature , although limited , provides some guidance for imaging patients with implanted pacemakers or icds that do not have mr - conditional labelling . this single - centre prospective non - controlled study describes the first use of mri in patients with an implanted s - icd . patients with implanted s - icd systems ( boston scientific sqrx model 1010 and q - trak model 3010 ) were enrolled for mri testing over a period of 18 months . the s - icd system implanted in this patient cohort was composed of a can implanted in a left mid - lateral pocket and a para - sternal subcutaneous electrode . the s - icd is currently not certified for use with an mri ; therefore , the ethics committee of homolka hospital , prague , czech republic approved our clinical study . patients with newly implanted s - icd systems ( < 6 weeks ) were excluded , and none of the patients had any intravascular leads . the patients were randomized for either a cardiac , brain , cervical , or lumbar spinal scan . one of the subjects underwent an additional knee examination , due to reported chronic pain . a total of 15 patients were enrolled into this study ( 12 males and three females , aged 2283 years , mean 53 years . subjects in our cohort ( table 1 ) underwent a total of 22 mri scans between 6 june 2012 and 24 december 2013 . in total , five brain scans , three cardiac scans , 12 lumbar scans , one knee , and one cervical spine scan were conducted ( table 2 ) . however , in one patient a minor disc protrusion was found , in other mri revealed stenosis of intervertebral foramen which was causing radicular pain of the nerve root l4 and based on this examination the patient was referred to ct - navigated periradicular therapy . table 1summary of patient anatomical data and scan locations , along with noted clinical eventsidagesexbmidgef , % indication for s - icdheating0164f20.5hcmp / vfs85secondary preventionnone0283m30.0post - mi / smvts post - catheter ablation/35secondary prevention ( post - transvenous icd extraction)none0331m25.3arvc / d / smvts68secondary preventionin - tolerable re - scanned0458m23.6post - mi / post - cabg30primary preventionnone0577m25.5post - mi30primary preventionnone0663m27.0post - mi30primary preventionnone0768m23.7post - mi / vfs / vts60secondary prevention post - transvenous icd extraction / svc occlusiontolerable0822m29.4brugada sy / vfs68secondary preventionin - tolerable re - scanned0959m27.1dcmp / vfs / post - mitral valve surgery/60secondary prev./post - transvenous icd extractionnone1041f24.6arvc / d70primary preventionnone1123f21.5lqts / vf60secondary preventionnone1266m36.9post - mi / vf / post - cabg50secondary prevention / post - repeat transvenous icd extractiontolerable1348m22.9dcmp(non - compaction)/vfs35secondary preventionnone1470m29systolic dysfunction of lv35primary preventionnone1526m33brugada sy65primary preventionnonehcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . table 2parmeters of s - icd and patient sensation during individual mri scansscan # idbody partheating sensationsshock zone ( b.p.m.)condit . shock zone ( b.p.m.)bat % episode num.101brainnone2302101001202brainnone240220861303l spinein - tolerable240220831403brainnone240220831504brainnone220190691605l spinenone220210541706l spinenone240220681807l spinetolerable240220582908l spinein - tolerablenananana1008brainnonenananana1108l spinenone2302108411209heartnone2402208911310l spinenone2301807911410heartnonenananana1511heartnone2301909711612l spinetolerable2001709721712l spinenone2001709421813c spinenone23019010041913l spinenone23019010042014l spinenone2301908612115kneenone25021010012215l spinenone2502101001s - icd parameters acquired prior- and post - mri were without any change , therefore only one value is presented.indices : na , not available ; l spine , lumbar spine ; c spine , cervical spine . summary of patient anatomical data and scan locations , along with noted clinical events hcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . parmeters of s - icd and patient sensation during individual mri scans s - icd parameters acquired prior- and post - mri were without any change , therefore only one value is presented . indices : na , not available ; l spine , lumbar spine ; c spine , cervical spine . studies were performed using a siemens avanto 1.5 t mri scanner ( vb17 software , quantum gradient coils ) . all scans were run in normal operating mode , which is limited to 2 w / kg whole body averaged specific absorption rate ( sar ) . clinically relevant mri sequences were used for evaluation ( see table 3 ) . table 3types of pulse sequences typically used for imaging of respective anatomical areasscan locationscan sequencesflairdwiflashfsehastesestirtruefispbrainxxxxheartxxxxcervical spinexxkneexxxxlumbar spinexxflair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession.fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . types of pulse sequences typically used for imaging of respective anatomical areas flair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession . fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . patients were asked to report immediately any pain , torqueing movement , or heating sensation in the area of the pocket or the electrode by pressing an emergency bulb . furthermore , all patients were questioned immediately following the mri procedure to ascertain any discomfort in the vicinity of the can or electrode . pulse oximetry and standard lead electrocardiogram ( ecg ) if discomfort occurred , the patient was asked if the scan could be repeated at a later time using a revised scan sequence or the subject was again randomized for another anatomical area . since none of the components of the s - icd system are on or in the heart , heating near or around however , heating near the electrode or can with the s - icd system may still cause serious patient discomfort . therefore , along with education of subjects , each patient was instrumented by taping an oesophageal temperature probe ( beta - therm model g22k7mcd8 ) on the skin over the mid - lateral implant site to record any temperature excursions that might be correlated to patient symptoms of heating / discomfort near the pocket . to minimize the risk of inappropriate therapy , the s - icd system was programmed to therapy each s - icd system was evaluated prior to and immediately after the scan to verify proper functioning , including interrogation , sensing , and battery voltage . after the completion of the mri , long - term regular clinical follow - up and checking of the device were performed . patients with implanted s - icd systems ( boston scientific sqrx model 1010 and q - trak model 3010 ) were enrolled for mri testing over a period of 18 months . the s - icd system implanted in this patient cohort was composed of a can implanted in a left mid - lateral pocket and a para - sternal subcutaneous electrode . the s - icd is currently not certified for use with an mri ; therefore , the ethics committee of homolka hospital , prague , czech republic approved our clinical study . patients with newly implanted s - icd systems ( < 6 weeks ) were excluded , and none of the patients had any intravascular leads . the patients were randomized for either a cardiac , brain , cervical , or lumbar spinal scan . one of the subjects underwent an additional knee examination , due to reported chronic pain . a total of 15 patients were enrolled into this study ( 12 males and three females , aged 2283 years , mean 53 years . subjects in our cohort ( table 1 ) underwent a total of 22 mri scans between 6 june 2012 and 24 december 2013 . in total , five brain scans , three cardiac scans , 12 lumbar scans , one knee , and one cervical spine scan were conducted ( table 2 ) . however , in one patient a minor disc protrusion was found , in other mri revealed stenosis of intervertebral foramen which was causing radicular pain of the nerve root l4 and based on this examination the patient was referred to ct - navigated periradicular therapy . table 1summary of patient anatomical data and scan locations , along with noted clinical eventsidagesexbmidgef , % indication for s - icdheating0164f20.5hcmp / vfs85secondary preventionnone0283m30.0post - mi / smvts post - catheter ablation/35secondary prevention ( post - transvenous icd extraction)none0331m25.3arvc / d / smvts68secondary preventionin - tolerable re - scanned0458m23.6post - mi / post - cabg30primary preventionnone0577m25.5post - mi30primary preventionnone0663m27.0post - mi30primary preventionnone0768m23.7post - mi / vfs / vts60secondary prevention post - transvenous icd extraction / svc occlusiontolerable0822m29.4brugada sy / vfs68secondary preventionin - tolerable re - scanned0959m27.1dcmp / vfs / post - mitral valve surgery/60secondary prev./post - transvenous icd extractionnone1041f24.6arvc / d70primary preventionnone1123f21.5lqts / vf60secondary preventionnone1266m36.9post - mi / vf / post - cabg50secondary prevention / post - repeat transvenous icd extractiontolerable1348m22.9dcmp(non - compaction)/vfs35secondary preventionnone1470m29systolic dysfunction of lv35primary preventionnone1526m33brugada sy65primary preventionnonehcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . table 2parmeters of s - icd and patient sensation during individual mri scansscan # idbody partheating sensationsshock zone ( b.p.m.)condit . shock zone ( b.p.m.)bat % episode num.101brainnone2302101001202brainnone240220861303l spinein - tolerable240220831403brainnone240220831504brainnone220190691605l spinenone220210541706l spinenone240220681807l spinetolerable240220582908l spinein - tolerablenananana1008brainnonenananana1108l spinenone2302108411209heartnone2402208911310l spinenone2301807911410heartnonenananana1511heartnone2301909711612l spinetolerable2001709721712l spinenone2001709421813c spinenone23019010041913l spinenone23019010042014l spinenone2301908612115kneenone25021010012215l spinenone2502101001s - icd parameters acquired prior- and post - mri were without any change , therefore only one value is presented.indices : na , not available ; l spine , lumbar spine ; c spine , cervical spine . summary of patient anatomical data and scan locations , along with noted clinical events hcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . parmeters of s - icd and patient sensation during individual mri scans s - icd parameters acquired prior- and post - mri were without any change , therefore only one value is presented . indices : na , not available ; l spine , lumbar spine ; c spine , cervical spine . studies were performed using a siemens avanto 1.5 t mri scanner ( vb17 software , quantum gradient coils ) . all scans were run in normal operating mode , which is limited to 2 w / kg whole body averaged specific absorption rate ( sar ) . clinically relevant mri sequences were used for evaluation ( see table 3 ) . table 3types of pulse sequences typically used for imaging of respective anatomical areasscan locationscan sequencesflairdwiflashfsehastesestirtruefispbrainxxxxheartxxxxcervical spinexxkneexxxxlumbar spinexxflair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession.fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . types of pulse sequences typically used for imaging of respective anatomical areas flair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession . fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . patients were asked to report immediately any pain , torqueing movement , or heating sensation in the area of the pocket or the electrode by pressing an emergency bulb . furthermore , all patients were questioned immediately following the mri procedure to ascertain any discomfort in the vicinity of the can or electrode . pulse oximetry and standard lead electrocardiogram ( ecg ) if discomfort occurred , the patient was asked if the scan could be repeated at a later time using a revised scan sequence or the subject was again randomized for another anatomical area . since none of the components of the s - icd system are on or in the heart , heating near or around the electrode can not harm the myocardium . however , heating near the electrode or can with the s - icd system may still cause serious patient discomfort . therefore , along with education of subjects , each patient was instrumented by taping an oesophageal temperature probe ( beta - therm model g22k7mcd8 ) on the skin over the mid - lateral implant site to record any temperature excursions that might be correlated to patient symptoms of heating / discomfort near the pocket . to minimize the risk of inappropriate therapy , the s - icd system was programmed to therapy each s - icd system was evaluated prior to and immediately after the scan to verify proper functioning , including interrogation , sensing , and battery voltage . after the completion of the mri , the s - icd system was reprogrammed to original settings . long - term regular clinical follow - up and checking of the device were performed . no anomalies were noted via pulse oximetry or ecg during the scans for any of the patients . eleven of 15 patients reported no sensation or pain from heating of the can , two of 15 patients reported feeling some heating , and two patients reported intolerable heating ( see table 2 ) . in patients with intolerable heating , the scan was halted within seconds and changed to a scan of the brain , which proceeded without incident . patient reports of heating in the vicinity of the can occurred only during lumbar scans with a thermistor probe ; no such reports occurred during scans of the brain , cardiac area , cervical spine , or without the probe . in two cases where heating in the vicinity of the can was reported by the patient , the scan sequence was altered to reduce the intensity of radiofrequency ( rf ) field exposure by reducing the turbo factor ( e.g. from 21 to 7 ) , increasing the repetition time ( e.g. to > 4000 ms ) , and reducing the flip angle ( e.g. from 170 to 120 ) . the target values were chosen arbitrarily to maintain image contrast ( flip angle ) and keep scan time at reasonable limits ( turbo factor and repetition time ) . less heating was noted by patients after these modifications to the scan parameters were made . 03 ) was observed to have a skin lesion , appearing to be a circular rash or ulcer on the surface of the skin over the can , approximately 35 mm in diameter . the cause of this skin anomaly is not known ; it was later noted to have fully healed at a follow - up 10 days after the scan . to ascertain the effect of heating due to the instrumented thermistor catheter , the two patients who experienced the heating ( examinations 9 and 16 , see table 2 ) were rescanned several weeks later without the thermistor catheter in place ( examinations 11 and 17 ) . first , modified sequence ( with even lower amount of energy deposited in the tissue ) was used , which caused no heating . as no sensation was reported by the subjects , they were asked to report even a minimal discomfort , and the lumbar scans were performed using the same settings that resulted in heating with the thermistor catheter in place in the first imaging session . the results of the rescans revealed that no heating was felt by the patients when the thermistor catheter was absent . there were no noted changes to battery voltage , ability to detect the qrs signal or stored diagnostic data . pacing thresholds can not be assessed by the s - icd system , so this was not evaluated . none of the patients reported any pulling or twisting of the can or pain from heating of the s - icd electrode . for scans of the brain , lumbar spine , knee , and cervical spine , no effect from image artefact was noted in the anatomical area of interest . however , for scans of the cardiac area , image artefact was noted to interfere with the ability to see parts of the left ventricle , though the right ventricle of the heart was unaffected and could be imaged usefully . this was due to the can and not the electrode ( see figure 1 ) , modifications to the protocol for the lumbar spine resulted in a lower signal - to - noise ratio ; however , the images remain in diagnostic quality ( see figure 2 ) . figure 1kinetic study in four - chamber view : the systolic ( a and c ) and diastolic ( b and d ) images of cine sequences , four - chamber view . the steady - state free precession ( ssfp ) sequence ( a and b ) shows more artefacts . in ssfp kinetic study , an inflow of dark blood from the left pulmonary veins was seen . it could be caused by s - icd but also by metallic ring in mitral annulus . the spoiled gradient echo ( gre ) sequence ( c and d ) is better , but an artefact at the lateral wall is obvious . figure 2lumbar spine imaging with icd : low sar t2 fse sequence ( upper image ) compared with normal t2 fse in the same subject ( lower image , for the scanning parameters see the discussion section ) . kinetic study in four - chamber view : the systolic ( a and c ) and diastolic ( b and d ) images of cine sequences , four - chamber view . the steady - state free precession ( ssfp ) sequence ( a and b ) shows more artefacts . in ssfp kinetic study , an inflow of dark blood from the left pulmonary veins was seen . it could be caused by s - icd but also by metallic ring in mitral annulus . the spoiled gradient echo ( gre ) sequence ( c and d ) is better , but an artefact at the lateral wall is obvious . lumbar spine imaging with icd : low sar t2 fse sequence ( upper image ) compared with normal t2 fse in the same subject ( lower image , for the scanning parameters see the discussion section ) . there were no noted changes to battery voltage , ability to detect the qrs signal or stored diagnostic data . pacing thresholds can not be assessed by the s - icd system , so this was not evaluated . none of the patients reported any pulling or twisting of the can or pain from heating of the s - icd electrode . for scans of the brain , lumbar spine , knee , and cervical spine , no effect from image artefact was noted in the anatomical area of interest . however , for scans of the cardiac area , image artefact was noted to interfere with the ability to see parts of the left ventricle , though the right ventricle of the heart was unaffected and could be imaged usefully . this was due to the can and not the electrode ( see figure 1 ) , modifications to the protocol for the lumbar spine resulted in a lower signal - to - noise ratio ; however , the images remain in diagnostic quality ( see figure 2 ) . figure 1kinetic study in four - chamber view : the systolic ( a and c ) and diastolic ( b and d ) images of cine sequences , four - chamber view . the steady - state free precession ( ssfp ) sequence ( a and b ) shows more artefacts . in ssfp kinetic study , an inflow of dark blood from the left pulmonary veins was seen . it could be caused by s - icd but also by metallic ring in mitral annulus . the spoiled gradient echo ( gre ) sequence ( c and d ) is better , but an artefact at the lateral wall is obvious . figure 2lumbar spine imaging with icd : low sar t2 fse sequence ( upper image ) compared with normal t2 fse in the same subject ( lower image , for the scanning parameters see the discussion section ) . kinetic study in four - chamber view : the systolic ( a and c ) and diastolic ( b and d ) images of cine sequences , four - chamber view . the steady - state free precession ( ssfp ) sequence ( a and b ) shows more artefacts . in ssfp kinetic study , an inflow of dark blood from the left pulmonary veins was seen . it could be caused by s - icd but also by metallic ring in mitral annulus . the spoiled gradient echo ( gre ) sequence ( c and d ) is better , but an artefact at the lateral wall is obvious . lumbar spine imaging with icd : low sar t2 fse sequence ( upper image ) compared with normal t2 fse in the same subject ( lower image , there are several reports in the current literature about mr - conditional pacemakers from several companies , but very limited reports about mr - conditional icds . biotronik announced in late 2011 release of their first mr - compatible icd device and defibrillator leads pro mri , but in the conditions of use excluded scanning of the torso and focused more on the extremities examination . in european heart rhythm association survey , 60% of centres did not implant any mri - certified icds , 34.3% implanted < 10 icd devices , and only 5.6% implanted 10 and more icds ; one - fifth of responders stated that mri - certified icds should be implanted in all patients but lack of reimbursement was indicated as a possible obstacle to implant more mri - certified pacemakers / icds by 47.1% of responding centres . none of the components of the s - icd system are on or in the heart . the s - icd depends less upon being in direct contact with the myocardium to function and instead uses far - field sensing and stimulation to provide the shock and post - shock pacing therapy . as a consequence , unlike transvenous systems heating near or around the electrode can not harm the myocardium , which could present with possible safety consequences such as an elevation in pacing thresholds or scarring of the myocardium , but it may still cause serious patient discomfort . because the s - icd is larger than modern transvenous icd 's , there may be more potential for the can to experience heating due to the magnetic gradient or rf field . we report results from what we believe is the first experience of mri scanning in patients with an implanted s - icd and in various anatomical areas . overall , mri was performed safely in all patients , which is in contrast to the current literature with mri imaging in patients with electrical - active devices which are not mri - conditional . in our study , the primary clinically significant event attributable to the mri scan was the occurrence of heating in the area of the pocket in the four patients that underwent lumbar scans . it was not known if this was due to the s - icd can itself or an artefact of the thermistor catheter used to measure skin temperature over the pocket . this required a revision of our protocol , which was to re - scan two of the patients who complained of heating . re - scanning of these patients without the thermistor probe resulted in no complaints of heating , so it is assumed that the thermistor catheter itself heated during the lumbar scans and caused the discomfort . as further evidence , all the heating complaints occurred during rf - intensive scan sequences ( namely fast spin echo ) with the temperature probe located axially near the centre of the bore , where rf fields are the highest . the thermistor catheter is constructed of insulated conductive cables connected to electrodes and should couple to the rf fields efficiently , causing heating at the electrodes and pain or damage on the surface of the skin where the probe was placed over the s - icd can . if the heating was due to the s - icd can itself , it would more likely occur during gradient - intensive scan sequences ( which can generate eddy currents on can surfaces and internal components ) and at locations in the bore where there are high gradient fields , such as near the bore edges . however , when the patient was scanned with gradient - intensive scan sequences ( e.g. flair dwi ) and with the s - icd system in high gradient field locations in the bore ( e.g. such as during a brain scan ) , patients did not detect any heating or discomfort . in addition , the subcutaneous lead , which was not instrumented with a thermistor catheter , never resulted in any heating sensation noted by the patient , even when exactly the same sequence that resulted in heating in the first session was used . the use of mri - compatible temperature monitors such as fibre optic temperature probes would have provided better confirmation of possible skin temperature elevation and would not have been affected by the rf fields . for cardiac imaging , the main problem to solve is metallic artefact , especially on the gradient - echo sequences . like in research performed by nazarian et al . , several scan protocols were used to see if any yielded different effects or reduced the qualitative extent of artefact . gradient mode was changed from normal to whisper , resulting in slower ramping of the field and therefore diminishing the changes of the magnetic field in time . artefacts when present were limited to blurring of the left ventricle during cardiac scans and most yielded clinically useful information . standard interrogation of the s - icd revealed no adverse effects upon the functioning of the system . while no adverse effects upon the post - scan s - icd device function were noted , not all possible scanning protocols were tested . it should be noted that , four of the s - icd 's were exposed to repeat mri scans without adverse effects to device function . in addition , because the s - icd does not provide long - term bradycardia pacing , it is assumed that pacemaker - dependent patients would not be implanted with this system . the inhibition of the pacemaker function during the scanning sequence and possible pacing threshold changes are a unique concern in patients implanted with transvenous icds . this study included only 15 patients and 22 scans done on the same 1.5 t mri scanner . thus , even these preliminary results should only be applied to 1.5 t mri scanners ( similarly as reported in the present literature for other implantable devices ) . device functionality was tested immediately after the scan but not for long - term effects . in addition , not all device functions were tested although the s - icd system does have a beeper / interrogation warning if battery levels or memory irregularities occur . however , patients were scheduled for regular check - up and no defect of the device was observed in following 725 months ( mean observation time 18 months ) . delayed enhancement mri for determining cardiac scarring was also not tested . also , there are other anatomical areas that were not evaluated , such as shoulder and knees . this study included only 15 patients and 22 scans done on the same 1.5 t mri scanner . thus , even these preliminary results should only be applied to 1.5 t mri scanners ( similarly as reported in the present literature for other implantable devices ) . device functionality was tested immediately after the scan but not for long - term effects . in addition , not all device functions were tested although the s - icd system does have a beeper / interrogation warning if battery levels or memory irregularities occur . however , patients were scheduled for regular check - up and no defect of the device was observed in following 725 months ( mean observation time 18 months ) . delayed enhancement mri for determining cardiac scarring was also not tested . also , there are other anatomical areas that were not evaluated , such as shoulder and knees . while more data are required to support a claim of mri - conditional , this study is the study to demonstrate the feasibility of exposing s - icd patients to mri using the scanning and monitor protocol described , with some precautionary measures including : ( i ) programming the device therapy off ; ( ii ) limiting the sar to 2.0 w / kg ; ( iii ) continuous monitoring of the patients pulse oximetry and ecg by qualified personnel and especially for any feelings of heating ; ( iv ) evaluate device function post scan ; ( v ) availability of full resuscitation facilities at the mri site . given the variables of different mri scanners , the decision to perform mri on patients with an implanted s - icd system should be balanced against the potential risks . in our study , the only heating was very likely introduced by not fully mri - compatible thermometer probe ; subjects rescanned without the probe did not report any abnormalities during the scan of any body area listed ( brain , cervical and lumbar spine , heart , and knee ) . this study was supported by iga mz r nt12094/2011 , research project charles university in prague , prvouk p34 and unce 204010/2012 . funding to pay the open access publication charges for this article was provided by iga mz r nt12094/2011 .

aimsour aim was to evaluate the potential for safely imaging patients with a new type of implantable cardioverter - defibrillator called the subcutaneous implantable cardioverter - defibrillator ( s - icd ) in a 1.5 t magnetic resonance imaging ( mri ) scanner . with the increasing number of patients with cardiac implantable devices who are indicated for mri , there is a growing need for establishing mri compatibility of cardiac implantable devices.methods and resultspatients with implanted s - icd systems underwent one or more types of anatomical mri scans . the s - icd was programmed off and patients were monitored throughout the imaging procedure . device function was evaluated pre- and post - scan . patients were asked to report immediately any pain , torqueing movement , or heating sensation in the area of the pocket or electrode . fifteen patients underwent a total of 22 examinations at 1.5 t. scans included brain , spine , knee , and heart . two patients were re - scanned due to complaints of heating over the can during lumbar scans , which was caused by a thermistor probe placed on the skin to measure skin temperature . all the remaining scans occurred without incident . no evidence of device malfunction was observed.conclusionthis study is the first to domonstrate the feasibility of exposing s - icd patients to mri using the scanning and monitoring protocol described . more data are required to support s - icd as a mri conditional device .

Introduction Methods Patient selection Magnetic resonance imaging Device assessment Results Subcutaneous implantable cardioverter-defibrillator status Image artefacts Discussion Limitations Conclusions Funding

the need for magnetic resonance imaging ( mri ) in patients with an implanted pacemaker or implantable cardioverter - defibrillator ( icd ) is a growing clinical issue . in european heart rhythm association survey , published recently for non - mri - certified icds ( 0.51.5 t field strength ) , the totally subcutaneous icd ( s - icd ) system , an implantable defibrillator with no leads that touch the heart , has recently been demonstrated to be a safe and effective defibrillator option for patients at risk for sudden cardiac death . various types of clinical mri systems currently use a superconductive magnet that creates a static magnetic field strength , typically 1.5 or 3 t. the use of mri with most pacemakers and icds is considered a contraindication due to potential hazards , including heating of the electrode that resides in or on the heart , damage to myocardium , elevation of pacing thresholds , unintended induction of ventricular tachycardia ( vt ) or ventricular fibrillation ( vf ) , pacing inhibition , permanent device malfunction , and distortion of the mri scan . this single - centre prospective non - controlled study describes the first use of mri in patients with an implanted s - icd . patients with implanted s - icd systems ( boston scientific sqrx model 1010 and q - trak model 3010 ) were enrolled for mri testing over a period of 18 months . the s - icd system implanted in this patient cohort was composed of a can implanted in a left mid - lateral pocket and a para - sternal subcutaneous electrode . patients with newly implanted s - icd systems ( < 6 weeks ) were excluded , and none of the patients had any intravascular leads . a total of 15 patients were enrolled into this study ( 12 males and three females , aged 2283 years , mean 53 years . subjects in our cohort ( table 1 ) underwent a total of 22 mri scans between 6 june 2012 and 24 december 2013 . in total , five brain scans , three cardiac scans , 12 lumbar scans , one knee , and one cervical spine scan were conducted ( table 2 ) . table 1summary of patient anatomical data and scan locations , along with noted clinical eventsidagesexbmidgef , % indication for s - icdheating0164f20.5hcmp / vfs85secondary preventionnone0283m30.0post - mi / smvts post - catheter ablation/35secondary prevention ( post - transvenous icd extraction)none0331m25.3arvc / d / smvts68secondary preventionin - tolerable re - scanned0458m23.6post - mi / post - cabg30primary preventionnone0577m25.5post - mi30primary preventionnone0663m27.0post - mi30primary preventionnone0768m23.7post - mi / vfs / vts60secondary prevention post - transvenous icd extraction / svc occlusiontolerable0822m29.4brugada sy / vfs68secondary preventionin - tolerable re - scanned0959m27.1dcmp / vfs / post - mitral valve surgery/60secondary prev./post - transvenous icd extractionnone1041f24.6arvc / d70primary preventionnone1123f21.5lqts / vf60secondary preventionnone1266m36.9post - mi / vf / post - cabg50secondary prevention / post - repeat transvenous icd extractiontolerable1348m22.9dcmp(non - compaction)/vfs35secondary preventionnone1470m29systolic dysfunction of lv35primary preventionnone1526m33brugada sy65primary preventionnonehcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . )bat % episode num.101brainnone2302101001202brainnone240220861303l spinein - tolerable240220831403brainnone240220831504brainnone220190691605l spinenone220210541706l spinenone240220681807l spinetolerable240220582908l spinein - tolerablenananana1008brainnonenananana1108l spinenone2302108411209heartnone2402208911310l spinenone2301807911410heartnonenananana1511heartnone2301909711612l spinetolerable2001709721712l spinenone2001709421813c spinenone23019010041913l spinenone23019010042014l spinenone2301908612115kneenone25021010012215l spinenone2502101001s - icd parameters acquired prior- and post - mri were without any change , therefore only one value is presented.indices : na , not available ; l spine , lumbar spine ; c spine , cervical spine . parmeters of s - icd and patient sensation during individual mri scans s - icd parameters acquired prior- and post - mri were without any change , therefore only one value is presented . table 3types of pulse sequences typically used for imaging of respective anatomical areasscan locationscan sequencesflairdwiflashfsehastesestirtruefispbrainxxxxheartxxxxcervical spinexxkneexxxxlumbar spinexxflair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession.fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . patients were asked to report immediately any pain , torqueing movement , or heating sensation in the area of the pocket or the electrode by pressing an emergency bulb . furthermore , all patients were questioned immediately following the mri procedure to ascertain any discomfort in the vicinity of the can or electrode . since none of the components of the s - icd system are on or in the heart , heating near or around however , heating near the electrode or can with the s - icd system may still cause serious patient discomfort . therefore , along with education of subjects , each patient was instrumented by taping an oesophageal temperature probe ( beta - therm model g22k7mcd8 ) on the skin over the mid - lateral implant site to record any temperature excursions that might be correlated to patient symptoms of heating / discomfort near the pocket . to minimize the risk of inappropriate therapy , the s - icd system was programmed to therapy each s - icd system was evaluated prior to and immediately after the scan to verify proper functioning , including interrogation , sensing , and battery voltage . patients with implanted s - icd systems ( boston scientific sqrx model 1010 and q - trak model 3010 ) were enrolled for mri testing over a period of 18 months . the s - icd system implanted in this patient cohort was composed of a can implanted in a left mid - lateral pocket and a para - sternal subcutaneous electrode . patients with newly implanted s - icd systems ( < 6 weeks ) were excluded , and none of the patients had any intravascular leads . the patients were randomized for either a cardiac , brain , cervical , or lumbar spinal scan . subjects in our cohort ( table 1 ) underwent a total of 22 mri scans between 6 june 2012 and 24 december 2013 . in total , five brain scans , three cardiac scans , 12 lumbar scans , one knee , and one cervical spine scan were conducted ( table 2 ) . table 1summary of patient anatomical data and scan locations , along with noted clinical eventsidagesexbmidgef , % indication for s - icdheating0164f20.5hcmp / vfs85secondary preventionnone0283m30.0post - mi / smvts post - catheter ablation/35secondary prevention ( post - transvenous icd extraction)none0331m25.3arvc / d / smvts68secondary preventionin - tolerable re - scanned0458m23.6post - mi / post - cabg30primary preventionnone0577m25.5post - mi30primary preventionnone0663m27.0post - mi30primary preventionnone0768m23.7post - mi / vfs / vts60secondary prevention post - transvenous icd extraction / svc occlusiontolerable0822m29.4brugada sy / vfs68secondary preventionin - tolerable re - scanned0959m27.1dcmp / vfs / post - mitral valve surgery/60secondary prev./post - transvenous icd extractionnone1041f24.6arvc / d70primary preventionnone1123f21.5lqts / vf60secondary preventionnone1266m36.9post - mi / vf / post - cabg50secondary prevention / post - repeat transvenous icd extractiontolerable1348m22.9dcmp(non - compaction)/vfs35secondary preventionnone1470m29systolic dysfunction of lv35primary preventionnone1526m33brugada sy65primary preventionnonehcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . )bat % episode num.101brainnone2302101001202brainnone240220861303l spinein - tolerable240220831403brainnone240220831504brainnone220190691605l spinenone220210541706l spinenone240220681807l spinetolerable240220582908l spinein - tolerablenananana1008brainnonenananana1108l spinenone2302108411209heartnone2402208911310l spinenone2301807911410heartnonenananana1511heartnone2301909711612l spinetolerable2001709721712l spinenone2001709421813c spinenone23019010041913l spinenone23019010042014l spinenone2301908612115kneenone25021010012215l spinenone2502101001s - icd parameters acquired prior- and post - mri were without any change , therefore only one value is presented.indices : na , not available ; l spine , lumbar spine ; c spine , cervical spine . parmeters of s - icd and patient sensation during individual mri scans s - icd parameters acquired prior- and post - mri were without any change , therefore only one value is presented . table 3types of pulse sequences typically used for imaging of respective anatomical areasscan locationscan sequencesflairdwiflashfsehastesestirtruefispbrainxxxxheartxxxxcervical spinexxkneexxxxlumbar spinexxflair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession.fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . patients were asked to report immediately any pain , torqueing movement , or heating sensation in the area of the pocket or the electrode by pressing an emergency bulb . furthermore , all patients were questioned immediately following the mri procedure to ascertain any discomfort in the vicinity of the can or electrode . since none of the components of the s - icd system are on or in the heart , heating near or around the electrode can not harm the myocardium . however , heating near the electrode or can with the s - icd system may still cause serious patient discomfort . therefore , along with education of subjects , each patient was instrumented by taping an oesophageal temperature probe ( beta - therm model g22k7mcd8 ) on the skin over the mid - lateral implant site to record any temperature excursions that might be correlated to patient symptoms of heating / discomfort near the pocket . to minimize the risk of inappropriate therapy , the s - icd system was programmed to therapy each s - icd system was evaluated prior to and immediately after the scan to verify proper functioning , including interrogation , sensing , and battery voltage . after the completion of the mri , the s - icd system was reprogrammed to original settings . eleven of 15 patients reported no sensation or pain from heating of the can , two of 15 patients reported feeling some heating , and two patients reported intolerable heating ( see table 2 ) . in patients with intolerable heating , the scan was halted within seconds and changed to a scan of the brain , which proceeded without incident . patient reports of heating in the vicinity of the can occurred only during lumbar scans with a thermistor probe ; no such reports occurred during scans of the brain , cardiac area , cervical spine , or without the probe . 03 ) was observed to have a skin lesion , appearing to be a circular rash or ulcer on the surface of the skin over the can , approximately 35 mm in diameter . as no sensation was reported by the subjects , they were asked to report even a minimal discomfort , and the lumbar scans were performed using the same settings that resulted in heating with the thermistor catheter in place in the first imaging session . pacing thresholds can not be assessed by the s - icd system , so this was not evaluated . none of the patients reported any pulling or twisting of the can or pain from heating of the s - icd electrode . for scans of the brain , lumbar spine , knee , and cervical spine , no effect from image artefact was noted in the anatomical area of interest . none of the patients reported any pulling or twisting of the can or pain from heating of the s - icd electrode . for scans of the brain , lumbar spine , knee , and cervical spine , no effect from image artefact was noted in the anatomical area of interest . this was due to the can and not the electrode ( see figure 1 ) , modifications to the protocol for the lumbar spine resulted in a lower signal - to - noise ratio ; however , the images remain in diagnostic quality ( see figure 2 ) . biotronik announced in late 2011 release of their first mr - compatible icd device and defibrillator leads pro mri , but in the conditions of use excluded scanning of the torso and focused more on the extremities examination . none of the components of the s - icd system are on or in the heart . the s - icd depends less upon being in direct contact with the myocardium to function and instead uses far - field sensing and stimulation to provide the shock and post - shock pacing therapy . because the s - icd is larger than modern transvenous icd 's , there may be more potential for the can to experience heating due to the magnetic gradient or rf field . we report results from what we believe is the first experience of mri scanning in patients with an implanted s - icd and in various anatomical areas . in our study , the primary clinically significant event attributable to the mri scan was the occurrence of heating in the area of the pocket in the four patients that underwent lumbar scans . it was not known if this was due to the s - icd can itself or an artefact of the thermistor catheter used to measure skin temperature over the pocket . this required a revision of our protocol , which was to re - scan two of the patients who complained of heating . re - scanning of these patients without the thermistor probe resulted in no complaints of heating , so it is assumed that the thermistor catheter itself heated during the lumbar scans and caused the discomfort . the thermistor catheter is constructed of insulated conductive cables connected to electrodes and should couple to the rf fields efficiently , causing heating at the electrodes and pain or damage on the surface of the skin where the probe was placed over the s - icd can . if the heating was due to the s - icd can itself , it would more likely occur during gradient - intensive scan sequences ( which can generate eddy currents on can surfaces and internal components ) and at locations in the bore where there are high gradient fields , such as near the bore edges . flair dwi ) and with the s - icd system in high gradient field locations in the bore ( e.g. in addition , the subcutaneous lead , which was not instrumented with a thermistor catheter , never resulted in any heating sensation noted by the patient , even when exactly the same sequence that resulted in heating in the first session was used . standard interrogation of the s - icd revealed no adverse effects upon the functioning of the system . while no adverse effects upon the post - scan s - icd device function were noted , not all possible scanning protocols were tested . it should be noted that , four of the s - icd 's were exposed to repeat mri scans without adverse effects to device function . this study included only 15 patients and 22 scans done on the same 1.5 t mri scanner . thus , even these preliminary results should only be applied to 1.5 t mri scanners ( similarly as reported in the present literature for other implantable devices ) . thus , even these preliminary results should only be applied to 1.5 t mri scanners ( similarly as reported in the present literature for other implantable devices ) . while more data are required to support a claim of mri - conditional , this study is the study to demonstrate the feasibility of exposing s - icd patients to mri using the scanning and monitor protocol described , with some precautionary measures including : ( i ) programming the device therapy off ; ( ii ) limiting the sar to 2.0 w / kg ; ( iii ) continuous monitoring of the patients pulse oximetry and ecg by qualified personnel and especially for any feelings of heating ; ( iv ) evaluate device function post scan ; ( v ) availability of full resuscitation facilities at the mri site . given the variables of different mri scanners , the decision to perform mri on patients with an implanted s - icd system should be balanced against the potential risks .

in european heart rhythm association survey , published recently for non - mri - certified icds ( 0.51.5 t field strength ) , the totally subcutaneous icd ( s - icd ) system , an implantable defibrillator with no leads that touch the heart , has recently been demonstrated to be a safe and effective defibrillator option for patients at risk for sudden cardiac death . various types of clinical mri systems currently use a superconductive magnet that creates a static magnetic field strength , typically 1.5 or 3 t. the use of mri with most pacemakers and icds is considered a contraindication due to potential hazards , including heating of the electrode that resides in or on the heart , damage to myocardium , elevation of pacing thresholds , unintended induction of ventricular tachycardia ( vt ) or ventricular fibrillation ( vf ) , pacing inhibition , permanent device malfunction , and distortion of the mri scan . hence , mr - conditional labelling dictates that the device is safe for use under certain scanning conditions , as well as how the cardiac device should be programmed before an exposure to the magnetic field in a mri scanner . this single - centre prospective non - controlled study describes the first use of mri in patients with an implanted s - icd . the s - icd system implanted in this patient cohort was composed of a can implanted in a left mid - lateral pocket and a para - sternal subcutaneous electrode . patients with newly implanted s - icd systems ( < 6 weeks ) were excluded , and none of the patients had any intravascular leads . however , in one patient a minor disc protrusion was found , in other mri revealed stenosis of intervertebral foramen which was causing radicular pain of the nerve root l4 and based on this examination the patient was referred to ct - navigated periradicular therapy . table 1summary of patient anatomical data and scan locations , along with noted clinical eventsidagesexbmidgef , % indication for s - icdheating0164f20.5hcmp / vfs85secondary preventionnone0283m30.0post - mi / smvts post - catheter ablation/35secondary prevention ( post - transvenous icd extraction)none0331m25.3arvc / d / smvts68secondary preventionin - tolerable re - scanned0458m23.6post - mi / post - cabg30primary preventionnone0577m25.5post - mi30primary preventionnone0663m27.0post - mi30primary preventionnone0768m23.7post - mi / vfs / vts60secondary prevention post - transvenous icd extraction / svc occlusiontolerable0822m29.4brugada sy / vfs68secondary preventionin - tolerable re - scanned0959m27.1dcmp / vfs / post - mitral valve surgery/60secondary prev./post - transvenous icd extractionnone1041f24.6arvc / d70primary preventionnone1123f21.5lqts / vf60secondary preventionnone1266m36.9post - mi / vf / post - cabg50secondary prevention / post - repeat transvenous icd extractiontolerable1348m22.9dcmp(non - compaction)/vfs35secondary preventionnone1470m29systolic dysfunction of lv35primary preventionnone1526m33brugada sy65primary preventionnonehcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . summary of patient anatomical data and scan locations , along with noted clinical events hcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . table 3types of pulse sequences typically used for imaging of respective anatomical areasscan locationscan sequencesflairdwiflashfsehastesestirtruefispbrainxxxxheartxxxxcervical spinexxkneexxxxlumbar spinexxflair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession.fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . types of pulse sequences typically used for imaging of respective anatomical areas flair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession . since none of the components of the s - icd system are on or in the heart , heating near or around however , heating near the electrode or can with the s - icd system may still cause serious patient discomfort . therefore , along with education of subjects , each patient was instrumented by taping an oesophageal temperature probe ( beta - therm model g22k7mcd8 ) on the skin over the mid - lateral implant site to record any temperature excursions that might be correlated to patient symptoms of heating / discomfort near the pocket . to minimize the risk of inappropriate therapy , the s - icd system was programmed to therapy each s - icd system was evaluated prior to and immediately after the scan to verify proper functioning , including interrogation , sensing , and battery voltage . after the completion of the mri , long - term regular clinical follow - up and checking of the device were performed . the s - icd system implanted in this patient cohort was composed of a can implanted in a left mid - lateral pocket and a para - sternal subcutaneous electrode . patients with newly implanted s - icd systems ( < 6 weeks ) were excluded , and none of the patients had any intravascular leads . however , in one patient a minor disc protrusion was found , in other mri revealed stenosis of intervertebral foramen which was causing radicular pain of the nerve root l4 and based on this examination the patient was referred to ct - navigated periradicular therapy . table 1summary of patient anatomical data and scan locations , along with noted clinical eventsidagesexbmidgef , % indication for s - icdheating0164f20.5hcmp / vfs85secondary preventionnone0283m30.0post - mi / smvts post - catheter ablation/35secondary prevention ( post - transvenous icd extraction)none0331m25.3arvc / d / smvts68secondary preventionin - tolerable re - scanned0458m23.6post - mi / post - cabg30primary preventionnone0577m25.5post - mi30primary preventionnone0663m27.0post - mi30primary preventionnone0768m23.7post - mi / vfs / vts60secondary prevention post - transvenous icd extraction / svc occlusiontolerable0822m29.4brugada sy / vfs68secondary preventionin - tolerable re - scanned0959m27.1dcmp / vfs / post - mitral valve surgery/60secondary prev./post - transvenous icd extractionnone1041f24.6arvc / d70primary preventionnone1123f21.5lqts / vf60secondary preventionnone1266m36.9post - mi / vf / post - cabg50secondary prevention / post - repeat transvenous icd extractiontolerable1348m22.9dcmp(non - compaction)/vfs35secondary preventionnone1470m29systolic dysfunction of lv35primary preventionnone1526m33brugada sy65primary preventionnonehcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . )bat % episode num.101brainnone2302101001202brainnone240220861303l spinein - tolerable240220831403brainnone240220831504brainnone220190691605l spinenone220210541706l spinenone240220681807l spinetolerable240220582908l spinein - tolerablenananana1008brainnonenananana1108l spinenone2302108411209heartnone2402208911310l spinenone2301807911410heartnonenananana1511heartnone2301909711612l spinetolerable2001709721712l spinenone2001709421813c spinenone23019010041913l spinenone23019010042014l spinenone2301908612115kneenone25021010012215l spinenone2502101001s - icd parameters acquired prior- and post - mri were without any change , therefore only one value is presented.indices : na , not available ; l spine , lumbar spine ; c spine , cervical spine . summary of patient anatomical data and scan locations , along with noted clinical events hcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . table 3types of pulse sequences typically used for imaging of respective anatomical areasscan locationscan sequencesflairdwiflashfsehastesestirtruefispbrainxxxxheartxxxxcervical spinexxkneexxxxlumbar spinexxflair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession.fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . types of pulse sequences typically used for imaging of respective anatomical areas flair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession . since none of the components of the s - icd system are on or in the heart , heating near or around the electrode can not harm the myocardium . therefore , along with education of subjects , each patient was instrumented by taping an oesophageal temperature probe ( beta - therm model g22k7mcd8 ) on the skin over the mid - lateral implant site to record any temperature excursions that might be correlated to patient symptoms of heating / discomfort near the pocket . to minimize the risk of inappropriate therapy , the s - icd system was programmed to therapy each s - icd system was evaluated prior to and immediately after the scan to verify proper functioning , including interrogation , sensing , and battery voltage . after the completion of the mri , the s - icd system was reprogrammed to original settings . patient reports of heating in the vicinity of the can occurred only during lumbar scans with a thermistor probe ; no such reports occurred during scans of the brain , cardiac area , cervical spine , or without the probe . in two cases where heating in the vicinity of the can was reported by the patient , the scan sequence was altered to reduce the intensity of radiofrequency ( rf ) field exposure by reducing the turbo factor ( e.g. 03 ) was observed to have a skin lesion , appearing to be a circular rash or ulcer on the surface of the skin over the can , approximately 35 mm in diameter . to ascertain the effect of heating due to the instrumented thermistor catheter , the two patients who experienced the heating ( examinations 9 and 16 , see table 2 ) were rescanned several weeks later without the thermistor catheter in place ( examinations 11 and 17 ) . as no sensation was reported by the subjects , they were asked to report even a minimal discomfort , and the lumbar scans were performed using the same settings that resulted in heating with the thermistor catheter in place in the first imaging session . for scans of the brain , lumbar spine , knee , and cervical spine , no effect from image artefact was noted in the anatomical area of interest . however , for scans of the cardiac area , image artefact was noted to interfere with the ability to see parts of the left ventricle , though the right ventricle of the heart was unaffected and could be imaged usefully . this was due to the can and not the electrode ( see figure 1 ) , modifications to the protocol for the lumbar spine resulted in a lower signal - to - noise ratio ; however , the images remain in diagnostic quality ( see figure 2 ) . figure 1kinetic study in four - chamber view : the systolic ( a and c ) and diastolic ( b and d ) images of cine sequences , four - chamber view . figure 2lumbar spine imaging with icd : low sar t2 fse sequence ( upper image ) compared with normal t2 fse in the same subject ( lower image , for the scanning parameters see the discussion section ) . kinetic study in four - chamber view : the systolic ( a and c ) and diastolic ( b and d ) images of cine sequences , four - chamber view . lumbar spine imaging with icd : low sar t2 fse sequence ( upper image ) compared with normal t2 fse in the same subject ( lower image , for the scanning parameters see the discussion section ) . for scans of the brain , lumbar spine , knee , and cervical spine , no effect from image artefact was noted in the anatomical area of interest . however , for scans of the cardiac area , image artefact was noted to interfere with the ability to see parts of the left ventricle , though the right ventricle of the heart was unaffected and could be imaged usefully . this was due to the can and not the electrode ( see figure 1 ) , modifications to the protocol for the lumbar spine resulted in a lower signal - to - noise ratio ; however , the images remain in diagnostic quality ( see figure 2 ) . figure 1kinetic study in four - chamber view : the systolic ( a and c ) and diastolic ( b and d ) images of cine sequences , four - chamber view . figure 2lumbar spine imaging with icd : low sar t2 fse sequence ( upper image ) compared with normal t2 fse in the same subject ( lower image , for the scanning parameters see the discussion section ) . kinetic study in four - chamber view : the systolic ( a and c ) and diastolic ( b and d ) images of cine sequences , four - chamber view . lumbar spine imaging with icd : low sar t2 fse sequence ( upper image ) compared with normal t2 fse in the same subject ( lower image , there are several reports in the current literature about mr - conditional pacemakers from several companies , but very limited reports about mr - conditional icds . biotronik announced in late 2011 release of their first mr - compatible icd device and defibrillator leads pro mri , but in the conditions of use excluded scanning of the torso and focused more on the extremities examination . in european heart rhythm association survey , 60% of centres did not implant any mri - certified icds , 34.3% implanted < 10 icd devices , and only 5.6% implanted 10 and more icds ; one - fifth of responders stated that mri - certified icds should be implanted in all patients but lack of reimbursement was indicated as a possible obstacle to implant more mri - certified pacemakers / icds by 47.1% of responding centres . none of the components of the s - icd system are on or in the heart . the s - icd depends less upon being in direct contact with the myocardium to function and instead uses far - field sensing and stimulation to provide the shock and post - shock pacing therapy . we report results from what we believe is the first experience of mri scanning in patients with an implanted s - icd and in various anatomical areas . overall , mri was performed safely in all patients , which is in contrast to the current literature with mri imaging in patients with electrical - active devices which are not mri - conditional . in our study , the primary clinically significant event attributable to the mri scan was the occurrence of heating in the area of the pocket in the four patients that underwent lumbar scans . re - scanning of these patients without the thermistor probe resulted in no complaints of heating , so it is assumed that the thermistor catheter itself heated during the lumbar scans and caused the discomfort . the thermistor catheter is constructed of insulated conductive cables connected to electrodes and should couple to the rf fields efficiently , causing heating at the electrodes and pain or damage on the surface of the skin where the probe was placed over the s - icd can . if the heating was due to the s - icd can itself , it would more likely occur during gradient - intensive scan sequences ( which can generate eddy currents on can surfaces and internal components ) and at locations in the bore where there are high gradient fields , such as near the bore edges . however , patients were scheduled for regular check - up and no defect of the device was observed in following 725 months ( mean observation time 18 months ) . however , patients were scheduled for regular check - up and no defect of the device was observed in following 725 months ( mean observation time 18 months ) . while more data are required to support a claim of mri - conditional , this study is the study to demonstrate the feasibility of exposing s - icd patients to mri using the scanning and monitor protocol described , with some precautionary measures including : ( i ) programming the device therapy off ; ( ii ) limiting the sar to 2.0 w / kg ; ( iii ) continuous monitoring of the patients pulse oximetry and ecg by qualified personnel and especially for any feelings of heating ; ( iv ) evaluate device function post scan ; ( v ) availability of full resuscitation facilities at the mri site . in our study , the only heating was very likely introduced by not fully mri - compatible thermometer probe ; subjects rescanned without the probe did not report any abnormalities during the scan of any body area listed ( brain , cervical and lumbar spine , heart , and knee ) .

most adults with autoimmune diabetes non - insulin - requiring at diagnosis become so within 36 years ( 1 , 2 ) . the optimal treatment for this second largest group of patients with diabetes is still unknown ( 37 ) . adult patients with autoimmune diabetes usually have larger remaining -cell mass at diagnosis and many develop -cell destruction more slowly . latent autoimmune diabetes in adults ( lada ) is therefore a suitable group for evaluating new therapies in autoimmune diabetes and may also serve as a model for intervention in classical type 1 diabetes ( 3 , 4 , 69 ) . the incidence of autoimmune diabetes is about equal in almost all age groups ( 10 , 11 ) . abrupt onset , often with ketoacidosis , is most frequent during childhood , a more modest onset is more frequent in adolescents and younger adults , and among adults and elders a slowly progressive onset , termed lada , is frequent ( 3 , 4 , 11 , 12 ) . classical type 1 diabetes and lada patients often have normal c - peptide levels at diagnosis , but further progressive decline occurs after onset , and insulin dependency occurs almost inevitably ( 3 , 4 , 8 , 9 , 13 ) . most trials in early type 1 diabetes have been performed in children , whose remaining -cell mass is limited , and short - term evaluation of intervention may be difficult also due to not infrequently occurring remission periods ( 14 , 15 ) . no therapy has yet been demonstrated to promote long - term insulin independency ( 3 , 5 , 7 , 16 ) . rodent studies have demonstrated potential positive effects of insulin treatment ( 17 , 18 ) . a pilot study of small doses of insulin versus sulphonylurea ( su ) to ten ica - positive patients with slowly progressive -cell failure favoured insulin for the preservation of c - peptide ( 19 ) . c - peptide is the outcome measure of choice of -cell function in trials of autoimmune diabetes ( 20 ) . even modest preservation of -cell function has been demonstrated to have positive effects on the frequency of hypoglycaemic events , and on the prevalence of retinopathy ( 21 ) . to investigate the effect of early insulin treatment in lada patients , for 3 years , on residual -cell function and metabolic control , compared with a group initially treated with diet and/or oral hypoglycaemic agents ( oha ) . adults , aged 30 years , diagnosed with diabetes in lund and kronoberg counties in southern sweden , non - insulin - requiring at diagnosis and positive to at least one of gadabs and/or icas were eligible for participation . two thirds had to be excluded due to mental conditions or severe physical illness , but also unwillingness to risk the early start of insulin injections . the majority of the patients were randomised into two groups , in blocks of eight , by pre - prepared closed envelopes kept at the two hospital policlinics . however complete strict randomisation was not possible , as some patients refused randomisation to possible insulin treatment before it was unavoidable . there were 20 patients in the intervention group ( i ) , treated with insulin from baseline , starting with 26 units intermediate - acting insulin at night ; and 17 patients in the control group ( c ) who received regular treatment with dietoha , mostly metformin , and some su ( 5/17 ; 30% ) . for both the groups , goals for glucose levels were in accordance with general guidelines ( fasting plasma glucose ( fpg ) 4.57 , preprandial pg 57 and postprandial decisions to increase treatment in doses , number of doses and addition of ohas or insulin were at the discretion of the treating physician . if two doses / day of intermediate - acting insulin or mix - insulin were not satisfactory , doses of direct or rapid - acting insulin before meals were added , resulting in 14 doses / day . glucagon - stimulation tests were performed at baseline and after 12 , 24 and 36 months during annual policlinic visits at the two research clinics . after an overnight fast , c - peptide was determined before and 6 min after i.v . c - peptide was analysed by commercial ria ( md315 , euro - diagnostica ab , malm , sweden ) , total variation ( sum of intra - and inter - assay variations ) 7% , reference range 0.251.0 c - peptide level , to reflect a preserved normal -cell function , was arbitrarily set at 0.5 nmol / l . icas were analysed with immunofluorescence assay , with detection limit 9 jdf - u , sensitivity 100% and specificity 88% . gadabs were analysed with radioimmuno - precipitation with lower reference limit at an index of 0.08 , corresponding to 21 who - u / ml , sensitivity 70% and specificity 100% . all analyses were standardised according to the diabetes antibody standardization program ( 25 , 26 ) . metabolic control was assessed by hbalc ( mono - s ) , and values were converted to dcct standard ( 27 ) . regarding the metabolic syndrome , complete information was available regarding only body mass index ( bmi ) and prevalence of hypertension , defined as blood pressure > 140/80 mmhg at the baseline visit , or taking antihypertensive medication . all subjects provided informed consent . analyses were carried out according to intention - to - treat . for comparisons between the groups , student 's t - test , or fisher 's exact test and the mann c - peptide and hbalc at baseline and after 12 , 24 , and 36 months were analysed with repeated measures anova using time as covariate ; for three - group analyses , anova with bonferroni correction was performed where appropriate . risk factors and relations were analysed in several models with simple , multiple , linear and logistic regressions ( forward stepwise , wald ) . spss software , version 17.0 ( chicago , il , usa ) , was used . analyses were carried out according to intention - to - treat . for comparisons between the groups , student 's t - test , or fisher 's exact test and the mann c - peptide and hbalc at baseline and after 12 , 24 , and 36 months were analysed with repeated measures anova using time as covariate ; for three - group analyses , anova with bonferroni correction was performed where appropriate . risk factors and relations were analysed in several models with simple , multiple , linear and logistic regressions ( forward stepwise , wald ) . spss software , version 17.0 ( chicago , il , usa ) , was used . for the baseline characteristics of the groups , see table 1 . there were no significant gender differences in the whole study , except that in the control group there were only men with hypertension ( p=0.03 ) . most subjects were overweight since both the i and c groups had mean bmi27 kg / m . median duration of diabetes at inclusion in the study was 5.0 ( quartiles 3.09.0 ) months . in both the groups , 90% of the patients , 18/20 in i and 15/17 in c , completed 36 months of follow - up . the conventionally treated ( c ) , 30% ( 5/17 ) started insulin treatment due to clinical necessity within 6612182430 months . for the levels of glucagon - stimulated c - peptide , see table 2 and fig . c - peptide levels were unchanged for four patients , increased by mean 0.73 ( 0.5 ) nmol / l for six and declined in all others after 36 months . mean glucagon - stimulated c - peptide decreased significantly in both the groups during the 36 months ( p<0.0001 ) . there was a significant time trend for the decrease in c - peptide of 0.17 nmol / l per year ( p=0.03 ) over 36 months without any significant difference between the groups . in repeated measures , anova with time as covariate , that analyses the changes in c - peptide over time with the levels at baseline taken into account , no differences could be found regarding mean - stimulated c - peptide at any time point , although with the mann whitney u test the difference in c - peptide at baseline ( cp0 ) was significant , p=0.03 . there were large variations in c - peptide levels between different individuals , at all time points , within both the groups ( p<0.0001 ) , explaining all the variation between them ( fig . cp0 explained 43% of level of c - peptide at 36 months ( cp36 ) , r 0.43 ( p<0.0001 ) . furthermore , age was the only other factor that had a weak and non - significant influence on cp36 , explaining about 5% of cp36 , r 0.049 ( p=0.2 ) . gender , baseline or end - of - study values of bmi , titres of gadab or ica , hbalc or diabetes duration before the study start did not influence c - peptide at end - of - study . it was 86% ( 13/15 ) in c , and 61% ( 11/18 ) in i ( p=0.13 ) . the odds ratio ( or ) for having a cp36 0.5 nmol / l was 2.4 for every increase in cp0 with 0.10 nmol / l ( p=0.02 ) , and 1.06 for each increase in baseline age by 1 year ( p=0.03 ) . if corrected for cp0 and age , the two factors found to have any influence on cp36 , the insulin treated had a non - significant or of 2.5 ( 0.04184 ) of having a cp36 of 0.5 the results of levels of fasting c - peptide ( fcp ) , when comparing the two treatment groups ( data not shown ) , were in accordance with the described results of stimulated c - peptide . among the controls , the level of hbalc had increased significantly at 36 months from 7.0 ( 1.3)% to 7.5 ( 1.5)% ( p=0.006 ) ( fig . , there was no significant difference between hbalc at baseline , 7.3 ( 1.3)% , and after 36 months , 7.2 ( 0.7)% ( p=0.6 ) ( fig . 2 ) . for the levels of hbalc at baseline and during follow - up , see table 2 and fig . the differences between the groups in absolute levels of hbalc were not significant either at baseline or after 12 , 24 or 36 months . the levels of hbalc were not influenced by age , gender , bmi , antibody prevalences or titres or c - peptide levels . at baseline , prevalences of gadab were 94% , ica 67% , 72% had both antibodies , 22% gadab only and 6% only ica , with no significant differences between the two treatment groups . of the 32 patients whose ia-2a status was known , 7 ( 22% ) were positive , all of them were also positive to gadab and all but one was positive to ica , meaning that 75% of all were positive to at least two antibodies . at baseline , mean indexes of gadab were 0.78 ( 0.39 ) in i , 0.78 ( 0.48 ) in c ( ns ) and mean ica titres were 29.4 ( 40 ) in i , 72.4 ( 119 ) jdf - u in c ( ns ) . at baseline , there were no significant differences in the prevalences of any of the three antibodies between the two treatment groups or between genders , different ages , duration of diabetes before study start or levels of bmi or hbalc . neither were there any significant differences between the groups in titres of gadab or ica after 12 , 24 or 36 months . the titres of gadab or ica were not related to patient age , gender , bmi , diabetes duration , treatment or hbalc at baseline or during the follow - up . c - peptide at baseline or 36 months was not influenced by baseline titre of gadab , ica or ia-2a . no episodes of major hypoglycaemia were reported for any of the patients , and only a few minor ones . mean weight at baseline was 77.4 ( 14.5 , range 57.8110 ) kg in group i , and 83.0 ( 17.8 , range 50.8117 ) kg in group c ( ns ) ; at end - of - study 79.3 ( 12.4 , 57.7101 ) kg in i , 82.3 ( 14.8 , range 50.4115 ) kg in c ( ns ) . mean weight change during the study was 2.5 ( 4.8 , range 8.8 to + 9.3 ) kg in i ; 1.0 ( 10.5 , range 27.3 to + 16.4 ) kg in c ( ns ) . for the three groups , those treated with insulin from baseline , those never treated with insulin and those who were originally treated with dietoha , but had to start insulin treatment during the study , the influences of age , bmi , hbalc , diabetes duration before study start or antibody titres were analysed , with no significant findings except for the influence of cp0 on cp36 . the tests for all the relevant parameters were also carried out with the 37 patients divided into groups of ever- versus never - insulin treated ( during the study ) , again with no significant results ( p=0.120.87 ) , apart from the significant influence of cp0 on cp36 ( p<0.0001 ) . for the levels of glucagon - stimulated c - peptide , see table 2 and fig . c - peptide levels were unchanged for four patients , increased by mean 0.73 ( 0.5 ) nmol / l for six and declined in all others after 36 months . mean glucagon - stimulated c - peptide decreased significantly in both the groups during the 36 months ( p<0.0001 ) . there was a significant time trend for the decrease in c - peptide of 0.17 nmol / l per year ( p=0.03 ) over 36 months without any significant difference between the groups . in repeated measures , anova with time as covariate , that analyses the changes in c - peptide over time with the levels at baseline taken into account , no differences could be found regarding mean - stimulated c - peptide at any time point , although with the mann whitney u test the difference in c - peptide at baseline ( cp0 ) was significant , p=0.03 . there were large variations in c - peptide levels between different individuals , at all time points , within both the groups ( p<0.0001 ) , explaining all the variation between them ( fig . cp0 explained 43% of level of c - peptide at 36 months ( cp36 ) , r 0.43 ( p<0.0001 ) . furthermore , age was the only other factor that had a weak and non - significant influence on cp36 , explaining about 5% of cp36 , r 0.049 ( p=0.2 ) . gender , baseline or end - of - study values of bmi , titres of gadab or ica , hbalc or diabetes duration before the study start did not influence c - peptide at end - of - study . it was 86% ( 13/15 ) in c , and 61% ( 11/18 ) in i ( p=0.13 ) . the odds ratio ( or ) for having a cp36 0.5 nmol / l was 2.4 for every increase in cp0 with 0.10 nmol / l ( p=0.02 ) , and 1.06 for each increase in baseline age by 1 year ( p=0.03 ) . if corrected for cp0 and age , the two factors found to have any influence on cp36 , the insulin treated had a non - significant or of 2.5 ( 0.04184 ) of having a cp36 of 0.5 the results of levels of fasting c - peptide ( fcp ) , when comparing the two treatment groups ( data not shown ) , were in accordance with the described results of stimulated c - peptide . among the controls , the level of hbalc had increased significantly at 36 months from 7.0 ( 1.3)% to 7.5 ( 1.5)% ( p=0.006 ) ( fig . , there was no significant difference between hbalc at baseline , 7.3 ( 1.3)% , and after 36 months , 7.2 ( 0.7)% ( p=0.6 ) ( fig . 2 ) . for the levels of hbalc at baseline and during follow - up , see table 2 and fig . 2 . the differences between the groups in absolute levels of hbalc were not significant either at baseline or after 12 , 24 or 36 months . the levels of hbalc were not influenced by age , gender , bmi , antibody prevalences or titres or c - peptide levels . at baseline , prevalences of gadab were 94% , ica 67% , 72% had both antibodies , 22% gadab only and 6% only ica , with no significant differences between the two treatment groups . of the 32 patients whose ia-2a status was known , 7 ( 22% ) were positive , all of them were also positive to gadab and all but one was positive to ica , meaning that 75% of all were positive to at least two antibodies . at baseline , mean indexes of gadab were 0.78 ( 0.39 ) in i , 0.78 ( 0.48 ) in c ( ns ) and mean ica titres were 29.4 ( 40 ) in i , 72.4 ( 119 ) jdf - u in c ( ns ) . at baseline , there were no significant differences in the prevalences of any of the three antibodies between the two treatment groups or between genders , different ages , duration of diabetes before study start or levels of bmi or hbalc . neither were there any significant differences between the groups in titres of gadab or ica after 12 , 24 or 36 months . the titres of gadab or ica were not related to patient age , gender , bmi , diabetes duration , treatment or hbalc at baseline or during the follow - up . c - peptide at baseline or 36 months was not influenced by baseline titre of gadab , ica or ia-2a . no episodes of major hypoglycaemia were reported for any of the patients , and only a few minor ones . mean weight at baseline was 77.4 ( 14.5 , range 57.8110 ) kg in group i , and 83.0 ( 17.8 , range 50.8117 ) kg in group c ( ns ) ; at end - of - study 79.3 ( 12.4 , 57.7101 ) kg in i , 82.3 ( 14.8 , range 50.4115 ) kg in c ( ns ) . mean weight change during the study was 2.5 ( 4.8 , range 8.8 to + 9.3 ) kg in i ; 1.0 ( 10.5 , range 27.3 to + 16.4 ) kg in c ( ns ) . for the three groups , those treated with insulin from baseline , those never treated with insulin and those who were originally treated with dietoha , but had to start insulin treatment during the study , the influences of age , bmi , hbalc , diabetes duration before study start or antibody titres were analysed , with no significant findings except for the influence of cp0 on cp36 . the tests for all the relevant parameters were also carried out with the 37 patients divided into groups of ever- versus never - insulin treated ( during the study ) , again with no significant results ( p=0.120.87 ) , apart from the significant influence of cp0 on cp36 ( p<0.0001 ) . few prospective intervention studies have been conducted in lada patients and there is still no general agreement on the best treatment aimed to preserve -cell function ( 57 , 16 ) . there has not been any general consensus definition of lada , which complicates comparisons and pooling of results . the most common denominators are adult age , positivity to at least one pancreatic autoantibody , and non - insulin dependency at diagnosis ( 4 ) . age , bmi , duration of diabetes and of insulin independency , which of the antibodies that were analysed , gadab titres and the expression of the essential outcome variable c - peptide vary ( 2 , 4 , 5 , 2830 ) . a cochrane review also noted the heterogeneity between studies , and the conclusion about early insulin treatment was uncertain ( 5 ) . our study included patients aged 30 years , non - insulin dependent at the times of diagnosis and inclusion and positive to at least one pancreatic autoantibody , for 75% two antibodies , thereby fulfilling the main criteria for lada ( 4 ) . the results of the study indicated that none of the baseline parameters , except initial c - peptide level , significantly influenced the outcome , eliminating the importance of several criteria in comparisons with other studies . in other studies , patients aged > 65 years have often been excluded , but lada exists also in these older age groups ( 11 ) . the decline in residual -cell function was progressive for the majority of our lada patients , as is usual in autoimmune diabetes ( 4 , 8 , 13 , 16 ) . we observed great variation in the rates and magnitudes of -cell loss between patients and between different time periods during the study , with no consistent patterns . mechanisms such as more step - wise losses due to , for instance , partial remissions might explain this ( 15 ) . the decline in c - peptide was irrespective of age , gender , bmi , antibody titres , hbalc or treatment modality . the lack of influence of bmi , age , diabetes duration and baseline hbalc on disease progression was also seen in a non - interventional observation study of lada that followed 13 ab - positive patients by stimulated c - peptide for 2 years ( 16 ) . similar to ukpds , we found no association between gadab levels and disease progress ( 31 ) , in contrast to observational studies that described this ( 30 , 32 ) . we could not , in a number of regression analyses , define any other factor besides cp0 that significantly influenced the level of cp36 . the significance of initial c - peptide level was also demonstrated in a large swedish study of new - onset 1534 year olds and in the tokyo intervention study ( 28 , 33 ) . the length of our study may explain that some patients with initially higher levels of c - peptide , overrepresented in the control group , by 36 months had not yet lost enough -cell function to be clinically insulin dependent . some antibody - positive patients were described to take up to 12 years to become insulin dependent , but practically all eventually did ( 34 ) . a significant beneficial effect of early insulin treatment on the preservation of -cell function could not be demonstrated , but level of hbalc after 36 months was better preserved in the insulin treated , in keeping with observations by chaillous et al . incidentally , the shape of the curve of the development of hbalc levels over time for the conventionally treated group in our study had a likeness to that observed in the ukpds ( 35 ) . our study , as most prospective intervention studies of lada , was not large . the tokyo study , with 60 patients , found a preference for insulin treatment versus su , possibly due to the differences in treatment , but longer duration of diabetes , up to 5 years without insulin before inclusion , rendering a selection of patients with better endogenous insulin production from the start , would have excluded those who progressed earlier to insulin dependence , so the trial population differed from ours ( 28 ) . baseline level of c - peptide was an important independent predictor of the ability to preserve a sufficient amount of c - peptide over time , just as in our study . many reports end with a general recommendation of insulin treatment in lada , but the evidence has not been compelling , as concluded by the 2007 cochrane review , which scrutinised seven insulin intervention studies in lada , two insulin versus su , the rest insulin alone versus different combinations of insulin+oha ( 5 ) . ukpds recorded hbalc , weight and treatment , randomised to insulin versus su , and found that 60% of the su treated were insulin dependent after 2 years ( 36 ) . one conclusion was that su might promote insulin dependency and apart from not recommending su the cochrane review found no preference for any special type of treatment for the lada group ( 5 ) . in our study after 3 years , 65% of the conventionally treated patients were not yet treated with insulin . in contrast to both ukpds and the tokyo study , only 30% of our control patients were treated with su , which could be of importance for -cell function . in another study , of 54 patients in four groups , the insulin treated , all with low fcp , 0.3 nmol , received either insulin alone or combined with rosiglitazone ( rgz ) , and the oha treated , all with fcp > 0.3 to our knowledge , this study is the first prospective controlled european intervention study of treatment in lada . differences in defining the lada population regarding age , diabetes duration before start of intervention , antibody prevalences and gadab titres , bmi and , not least , baseline levels of c - peptide , if available , have contributed to difficulties in interpreting and comparing the results of the few existing lada intervention studies ( 4 , 5 , 28 , 29 , 37 ) . in this study , none of these factors , except baseline level of c - peptide , significantly affected the outcome . the similar findings also seen in trials of classical type 1 diabetes and of prevention of autoimmune diabetes in high - risk individuals substantiate the observation of the influence of initial level of c - peptide on outcome level ( 38 , 39 ) . we also saw a non - significant or favouring insulin treatment for preservation of -cell function , indicating the possibility that a larger study population and/or a longer period of follow - up might demonstrate significant preference for early insulin treatment also regarding -cell function . this study indicated that early insulin treatment in lada patients lead to better preservation of level of metabolic control , and that it was safe and well tolerated . the decline in c - peptide was progressive irrespective of age , gender , bmi , hbalc levels and antibody titres . only baseline level of c - peptide significantly influenced c - peptide level after 3 years . m landin - olsson has received a part - time professorship sponsored by novo - nordisk scandinavia . the other authors have no dualities of interest in relation to this article . the work was financed by the healthcare regions of skane and kronoberg , southern sweden ; lund university funding of clinical research ( alf ) ; and the swedish council of medical research .

objectivesthe optimal treatment of latent autoimmune diabetes in adults ( lada ) is not established . we explored whether early insulin treatment , which has shown beneficial effects in rodents and in human pilot studies , would result in better preservation of -cell function or metabolic control , compared with conventional treatment.subjects and methodsglucagon - stimulated c - peptide and hbalc were evaluated at baseline and after 12 , 24 and 36 months in 37 patients recently diagnosed with diabetes , aged 30 years , non - insulin - requiring and gadab and/or ica positive . twenty patients received early insulin and 17 received conventional treatment ( dietoral hypoglycaemic agents ( oha ) , metformin , some and/or sulfonylurea ) and insulin when necessary.resultslevel of metabolic control , hbalc , was preserved in the early insulin treated , while it significantly deteriorated in the conventionally treated . there was no significant difference between the groups in c - peptide after 12 , 24 or 36 months , or in the decline of c - peptide . only baseline c - peptide predicted a c - peptide of 0.5 nmol / l at 36 months . gender , body mass index , antibody titres or hbalc did not influence the levels of c - peptide or hbalc at baseline or end - of - study , or the decline in c - peptide . among the dietoha - treated , 5/17 ( 30% ) developed insulin dependency during the follow - up . no major hypoglycaemic events occurred.conclusionsearly insulin treatment in lada leads to better preservation of metabolic control and was safe . superior preservation of c - peptide could not be significantly demonstrated . only baseline level of c - peptide significantly influenced c - peptide level after 3 years . further studies exploring the best treatment in lada are warranted .

Introduction Objective Subjects and methods Statistical analyses Results -Cell function Metabolic control Autoimmunity Adverse events Three-group analysis and ever- versus never-insulin treated Discussion Further considerations Conclusions Declaration of interest Funding

most adults with autoimmune diabetes non - insulin - requiring at diagnosis become so within 36 years ( 1 , 2 ) . latent autoimmune diabetes in adults ( lada ) is therefore a suitable group for evaluating new therapies in autoimmune diabetes and may also serve as a model for intervention in classical type 1 diabetes ( 3 , 4 , 69 ) . classical type 1 diabetes and lada patients often have normal c - peptide levels at diagnosis , but further progressive decline occurs after onset , and insulin dependency occurs almost inevitably ( 3 , 4 , 8 , 9 , 13 ) . a pilot study of small doses of insulin versus sulphonylurea ( su ) to ten ica - positive patients with slowly progressive -cell failure favoured insulin for the preservation of c - peptide ( 19 ) . c - peptide is the outcome measure of choice of -cell function in trials of autoimmune diabetes ( 20 ) . even modest preservation of -cell function has been demonstrated to have positive effects on the frequency of hypoglycaemic events , and on the prevalence of retinopathy ( 21 ) . to investigate the effect of early insulin treatment in lada patients , for 3 years , on residual -cell function and metabolic control , compared with a group initially treated with diet and/or oral hypoglycaemic agents ( oha ) . adults , aged 30 years , diagnosed with diabetes in lund and kronoberg counties in southern sweden , non - insulin - requiring at diagnosis and positive to at least one of gadabs and/or icas were eligible for participation . there were 20 patients in the intervention group ( i ) , treated with insulin from baseline , starting with 26 units intermediate - acting insulin at night ; and 17 patients in the control group ( c ) who received regular treatment with dietoha , mostly metformin , and some su ( 5/17 ; 30% ) . glucagon - stimulation tests were performed at baseline and after 12 , 24 and 36 months during annual policlinic visits at the two research clinics . c - peptide was analysed by commercial ria ( md315 , euro - diagnostica ab , malm , sweden ) , total variation ( sum of intra - and inter - assay variations ) 7% , reference range 0.251.0 c - peptide level , to reflect a preserved normal -cell function , was arbitrarily set at 0.5 nmol / l . regarding the metabolic syndrome , complete information was available regarding only body mass index ( bmi ) and prevalence of hypertension , defined as blood pressure > 140/80 mmhg at the baseline visit , or taking antihypertensive medication . for comparisons between the groups , student 's t - test , or fisher 's exact test and the mann c - peptide and hbalc at baseline and after 12 , 24 , and 36 months were analysed with repeated measures anova using time as covariate ; for three - group analyses , anova with bonferroni correction was performed where appropriate . for comparisons between the groups , student 's t - test , or fisher 's exact test and the mann c - peptide and hbalc at baseline and after 12 , 24 , and 36 months were analysed with repeated measures anova using time as covariate ; for three - group analyses , anova with bonferroni correction was performed where appropriate . there were no significant gender differences in the whole study , except that in the control group there were only men with hypertension ( p=0.03 ) . in both the groups , 90% of the patients , 18/20 in i and 15/17 in c , completed 36 months of follow - up . the conventionally treated ( c ) , 30% ( 5/17 ) started insulin treatment due to clinical necessity within 6612182430 months . for the levels of glucagon - stimulated c - peptide , see table 2 and fig . c - peptide levels were unchanged for four patients , increased by mean 0.73 ( 0.5 ) nmol / l for six and declined in all others after 36 months . mean glucagon - stimulated c - peptide decreased significantly in both the groups during the 36 months ( p<0.0001 ) . there was a significant time trend for the decrease in c - peptide of 0.17 nmol / l per year ( p=0.03 ) over 36 months without any significant difference between the groups . in repeated measures , anova with time as covariate , that analyses the changes in c - peptide over time with the levels at baseline taken into account , no differences could be found regarding mean - stimulated c - peptide at any time point , although with the mann whitney u test the difference in c - peptide at baseline ( cp0 ) was significant , p=0.03 . there were large variations in c - peptide levels between different individuals , at all time points , within both the groups ( p<0.0001 ) , explaining all the variation between them ( fig . cp0 explained 43% of level of c - peptide at 36 months ( cp36 ) , r 0.43 ( p<0.0001 ) . gender , baseline or end - of - study values of bmi , titres of gadab or ica , hbalc or diabetes duration before the study start did not influence c - peptide at end - of - study . the odds ratio ( or ) for having a cp36 0.5 nmol / l was 2.4 for every increase in cp0 with 0.10 nmol / l ( p=0.02 ) , and 1.06 for each increase in baseline age by 1 year ( p=0.03 ) . if corrected for cp0 and age , the two factors found to have any influence on cp36 , the insulin treated had a non - significant or of 2.5 ( 0.04184 ) of having a cp36 of 0.5 the results of levels of fasting c - peptide ( fcp ) , when comparing the two treatment groups ( data not shown ) , were in accordance with the described results of stimulated c - peptide . among the controls , the level of hbalc had increased significantly at 36 months from 7.0 ( 1.3)% to 7.5 ( 1.5)% ( p=0.006 ) ( fig . , there was no significant difference between hbalc at baseline , 7.3 ( 1.3)% , and after 36 months , 7.2 ( 0.7)% ( p=0.6 ) ( fig . for the levels of hbalc at baseline and during follow - up , see table 2 and fig . the differences between the groups in absolute levels of hbalc were not significant either at baseline or after 12 , 24 or 36 months . the levels of hbalc were not influenced by age , gender , bmi , antibody prevalences or titres or c - peptide levels . at baseline , prevalences of gadab were 94% , ica 67% , 72% had both antibodies , 22% gadab only and 6% only ica , with no significant differences between the two treatment groups . at baseline , there were no significant differences in the prevalences of any of the three antibodies between the two treatment groups or between genders , different ages , duration of diabetes before study start or levels of bmi or hbalc . neither were there any significant differences between the groups in titres of gadab or ica after 12 , 24 or 36 months . the titres of gadab or ica were not related to patient age , gender , bmi , diabetes duration , treatment or hbalc at baseline or during the follow - up . c - peptide at baseline or 36 months was not influenced by baseline titre of gadab , ica or ia-2a . mean weight at baseline was 77.4 ( 14.5 , range 57.8110 ) kg in group i , and 83.0 ( 17.8 , range 50.8117 ) kg in group c ( ns ) ; at end - of - study 79.3 ( 12.4 , 57.7101 ) kg in i , 82.3 ( 14.8 , range 50.4115 ) kg in c ( ns ) . for the three groups , those treated with insulin from baseline , those never treated with insulin and those who were originally treated with dietoha , but had to start insulin treatment during the study , the influences of age , bmi , hbalc , diabetes duration before study start or antibody titres were analysed , with no significant findings except for the influence of cp0 on cp36 . the tests for all the relevant parameters were also carried out with the 37 patients divided into groups of ever- versus never - insulin treated ( during the study ) , again with no significant results ( p=0.120.87 ) , apart from the significant influence of cp0 on cp36 ( p<0.0001 ) . for the levels of glucagon - stimulated c - peptide , see table 2 and fig . c - peptide levels were unchanged for four patients , increased by mean 0.73 ( 0.5 ) nmol / l for six and declined in all others after 36 months . mean glucagon - stimulated c - peptide decreased significantly in both the groups during the 36 months ( p<0.0001 ) . there was a significant time trend for the decrease in c - peptide of 0.17 nmol / l per year ( p=0.03 ) over 36 months without any significant difference between the groups . in repeated measures , anova with time as covariate , that analyses the changes in c - peptide over time with the levels at baseline taken into account , no differences could be found regarding mean - stimulated c - peptide at any time point , although with the mann whitney u test the difference in c - peptide at baseline ( cp0 ) was significant , p=0.03 . there were large variations in c - peptide levels between different individuals , at all time points , within both the groups ( p<0.0001 ) , explaining all the variation between them ( fig . cp0 explained 43% of level of c - peptide at 36 months ( cp36 ) , r 0.43 ( p<0.0001 ) . gender , baseline or end - of - study values of bmi , titres of gadab or ica , hbalc or diabetes duration before the study start did not influence c - peptide at end - of - study . the odds ratio ( or ) for having a cp36 0.5 nmol / l was 2.4 for every increase in cp0 with 0.10 nmol / l ( p=0.02 ) , and 1.06 for each increase in baseline age by 1 year ( p=0.03 ) . if corrected for cp0 and age , the two factors found to have any influence on cp36 , the insulin treated had a non - significant or of 2.5 ( 0.04184 ) of having a cp36 of 0.5 the results of levels of fasting c - peptide ( fcp ) , when comparing the two treatment groups ( data not shown ) , were in accordance with the described results of stimulated c - peptide . among the controls , the level of hbalc had increased significantly at 36 months from 7.0 ( 1.3)% to 7.5 ( 1.5)% ( p=0.006 ) ( fig . , there was no significant difference between hbalc at baseline , 7.3 ( 1.3)% , and after 36 months , 7.2 ( 0.7)% ( p=0.6 ) ( fig . for the levels of hbalc at baseline and during follow - up , see table 2 and fig . the differences between the groups in absolute levels of hbalc were not significant either at baseline or after 12 , 24 or 36 months . the levels of hbalc were not influenced by age , gender , bmi , antibody prevalences or titres or c - peptide levels . at baseline , prevalences of gadab were 94% , ica 67% , 72% had both antibodies , 22% gadab only and 6% only ica , with no significant differences between the two treatment groups . at baseline , mean indexes of gadab were 0.78 ( 0.39 ) in i , 0.78 ( 0.48 ) in c ( ns ) and mean ica titres were 29.4 ( 40 ) in i , 72.4 ( 119 ) jdf - u in c ( ns ) . at baseline , there were no significant differences in the prevalences of any of the three antibodies between the two treatment groups or between genders , different ages , duration of diabetes before study start or levels of bmi or hbalc . neither were there any significant differences between the groups in titres of gadab or ica after 12 , 24 or 36 months . the titres of gadab or ica were not related to patient age , gender , bmi , diabetes duration , treatment or hbalc at baseline or during the follow - up . c - peptide at baseline or 36 months was not influenced by baseline titre of gadab , ica or ia-2a . mean weight at baseline was 77.4 ( 14.5 , range 57.8110 ) kg in group i , and 83.0 ( 17.8 , range 50.8117 ) kg in group c ( ns ) ; at end - of - study 79.3 ( 12.4 , 57.7101 ) kg in i , 82.3 ( 14.8 , range 50.4115 ) kg in c ( ns ) . for the three groups , those treated with insulin from baseline , those never treated with insulin and those who were originally treated with dietoha , but had to start insulin treatment during the study , the influences of age , bmi , hbalc , diabetes duration before study start or antibody titres were analysed , with no significant findings except for the influence of cp0 on cp36 . the tests for all the relevant parameters were also carried out with the 37 patients divided into groups of ever- versus never - insulin treated ( during the study ) , again with no significant results ( p=0.120.87 ) , apart from the significant influence of cp0 on cp36 ( p<0.0001 ) . few prospective intervention studies have been conducted in lada patients and there is still no general agreement on the best treatment aimed to preserve -cell function ( 57 , 16 ) . the most common denominators are adult age , positivity to at least one pancreatic autoantibody , and non - insulin dependency at diagnosis ( 4 ) . age , bmi , duration of diabetes and of insulin independency , which of the antibodies that were analysed , gadab titres and the expression of the essential outcome variable c - peptide vary ( 2 , 4 , 5 , 2830 ) . our study included patients aged 30 years , non - insulin dependent at the times of diagnosis and inclusion and positive to at least one pancreatic autoantibody , for 75% two antibodies , thereby fulfilling the main criteria for lada ( 4 ) . the results of the study indicated that none of the baseline parameters , except initial c - peptide level , significantly influenced the outcome , eliminating the importance of several criteria in comparisons with other studies . the decline in residual -cell function was progressive for the majority of our lada patients , as is usual in autoimmune diabetes ( 4 , 8 , 13 , 16 ) . we observed great variation in the rates and magnitudes of -cell loss between patients and between different time periods during the study , with no consistent patterns . the decline in c - peptide was irrespective of age , gender , bmi , antibody titres , hbalc or treatment modality . the lack of influence of bmi , age , diabetes duration and baseline hbalc on disease progression was also seen in a non - interventional observation study of lada that followed 13 ab - positive patients by stimulated c - peptide for 2 years ( 16 ) . the significance of initial c - peptide level was also demonstrated in a large swedish study of new - onset 1534 year olds and in the tokyo intervention study ( 28 , 33 ) . the length of our study may explain that some patients with initially higher levels of c - peptide , overrepresented in the control group , by 36 months had not yet lost enough -cell function to be clinically insulin dependent . a significant beneficial effect of early insulin treatment on the preservation of -cell function could not be demonstrated , but level of hbalc after 36 months was better preserved in the insulin treated , in keeping with observations by chaillous et al . incidentally , the shape of the curve of the development of hbalc levels over time for the conventionally treated group in our study had a likeness to that observed in the ukpds ( 35 ) . the tokyo study , with 60 patients , found a preference for insulin treatment versus su , possibly due to the differences in treatment , but longer duration of diabetes , up to 5 years without insulin before inclusion , rendering a selection of patients with better endogenous insulin production from the start , would have excluded those who progressed earlier to insulin dependence , so the trial population differed from ours ( 28 ) . baseline level of c - peptide was an important independent predictor of the ability to preserve a sufficient amount of c - peptide over time , just as in our study . many reports end with a general recommendation of insulin treatment in lada , but the evidence has not been compelling , as concluded by the 2007 cochrane review , which scrutinised seven insulin intervention studies in lada , two insulin versus su , the rest insulin alone versus different combinations of insulin+oha ( 5 ) . in our study after 3 years , 65% of the conventionally treated patients were not yet treated with insulin . in contrast to both ukpds and the tokyo study , only 30% of our control patients were treated with su , which could be of importance for -cell function . in another study , of 54 patients in four groups , the insulin treated , all with low fcp , 0.3 nmol , received either insulin alone or combined with rosiglitazone ( rgz ) , and the oha treated , all with fcp > 0.3 to our knowledge , this study is the first prospective controlled european intervention study of treatment in lada . differences in defining the lada population regarding age , diabetes duration before start of intervention , antibody prevalences and gadab titres , bmi and , not least , baseline levels of c - peptide , if available , have contributed to difficulties in interpreting and comparing the results of the few existing lada intervention studies ( 4 , 5 , 28 , 29 , 37 ) . in this study , none of these factors , except baseline level of c - peptide , significantly affected the outcome . the similar findings also seen in trials of classical type 1 diabetes and of prevention of autoimmune diabetes in high - risk individuals substantiate the observation of the influence of initial level of c - peptide on outcome level ( 38 , 39 ) . we also saw a non - significant or favouring insulin treatment for preservation of -cell function , indicating the possibility that a larger study population and/or a longer period of follow - up might demonstrate significant preference for early insulin treatment also regarding -cell function . this study indicated that early insulin treatment in lada patients lead to better preservation of level of metabolic control , and that it was safe and well tolerated . the decline in c - peptide was progressive irrespective of age , gender , bmi , hbalc levels and antibody titres . only baseline level of c - peptide significantly influenced c - peptide level after 3 years .

latent autoimmune diabetes in adults ( lada ) is therefore a suitable group for evaluating new therapies in autoimmune diabetes and may also serve as a model for intervention in classical type 1 diabetes ( 3 , 4 , 69 ) . abrupt onset , often with ketoacidosis , is most frequent during childhood , a more modest onset is more frequent in adolescents and younger adults , and among adults and elders a slowly progressive onset , termed lada , is frequent ( 3 , 4 , 11 , 12 ) . classical type 1 diabetes and lada patients often have normal c - peptide levels at diagnosis , but further progressive decline occurs after onset , and insulin dependency occurs almost inevitably ( 3 , 4 , 8 , 9 , 13 ) . most trials in early type 1 diabetes have been performed in children , whose remaining -cell mass is limited , and short - term evaluation of intervention may be difficult also due to not infrequently occurring remission periods ( 14 , 15 ) . a pilot study of small doses of insulin versus sulphonylurea ( su ) to ten ica - positive patients with slowly progressive -cell failure favoured insulin for the preservation of c - peptide ( 19 ) . c - peptide is the outcome measure of choice of -cell function in trials of autoimmune diabetes ( 20 ) . even modest preservation of -cell function has been demonstrated to have positive effects on the frequency of hypoglycaemic events , and on the prevalence of retinopathy ( 21 ) . to investigate the effect of early insulin treatment in lada patients , for 3 years , on residual -cell function and metabolic control , compared with a group initially treated with diet and/or oral hypoglycaemic agents ( oha ) . adults , aged 30 years , diagnosed with diabetes in lund and kronoberg counties in southern sweden , non - insulin - requiring at diagnosis and positive to at least one of gadabs and/or icas were eligible for participation . the majority of the patients were randomised into two groups , in blocks of eight , by pre - prepared closed envelopes kept at the two hospital policlinics . there were 20 patients in the intervention group ( i ) , treated with insulin from baseline , starting with 26 units intermediate - acting insulin at night ; and 17 patients in the control group ( c ) who received regular treatment with dietoha , mostly metformin , and some su ( 5/17 ; 30% ) . for both the groups , goals for glucose levels were in accordance with general guidelines ( fasting plasma glucose ( fpg ) 4.57 , preprandial pg 57 and postprandial decisions to increase treatment in doses , number of doses and addition of ohas or insulin were at the discretion of the treating physician . if two doses / day of intermediate - acting insulin or mix - insulin were not satisfactory , doses of direct or rapid - acting insulin before meals were added , resulting in 14 doses / day . c - peptide was analysed by commercial ria ( md315 , euro - diagnostica ab , malm , sweden ) , total variation ( sum of intra - and inter - assay variations ) 7% , reference range 0.251.0 c - peptide level , to reflect a preserved normal -cell function , was arbitrarily set at 0.5 nmol / l . regarding the metabolic syndrome , complete information was available regarding only body mass index ( bmi ) and prevalence of hypertension , defined as blood pressure > 140/80 mmhg at the baseline visit , or taking antihypertensive medication . for comparisons between the groups , student 's t - test , or fisher 's exact test and the mann c - peptide and hbalc at baseline and after 12 , 24 , and 36 months were analysed with repeated measures anova using time as covariate ; for three - group analyses , anova with bonferroni correction was performed where appropriate . for comparisons between the groups , student 's t - test , or fisher 's exact test and the mann c - peptide and hbalc at baseline and after 12 , 24 , and 36 months were analysed with repeated measures anova using time as covariate ; for three - group analyses , anova with bonferroni correction was performed where appropriate . risk factors and relations were analysed in several models with simple , multiple , linear and logistic regressions ( forward stepwise , wald ) . there were no significant gender differences in the whole study , except that in the control group there were only men with hypertension ( p=0.03 ) . median duration of diabetes at inclusion in the study was 5.0 ( quartiles 3.09.0 ) months . in both the groups , 90% of the patients , 18/20 in i and 15/17 in c , completed 36 months of follow - up . c - peptide levels were unchanged for four patients , increased by mean 0.73 ( 0.5 ) nmol / l for six and declined in all others after 36 months . mean glucagon - stimulated c - peptide decreased significantly in both the groups during the 36 months ( p<0.0001 ) . there was a significant time trend for the decrease in c - peptide of 0.17 nmol / l per year ( p=0.03 ) over 36 months without any significant difference between the groups . in repeated measures , anova with time as covariate , that analyses the changes in c - peptide over time with the levels at baseline taken into account , no differences could be found regarding mean - stimulated c - peptide at any time point , although with the mann whitney u test the difference in c - peptide at baseline ( cp0 ) was significant , p=0.03 . there were large variations in c - peptide levels between different individuals , at all time points , within both the groups ( p<0.0001 ) , explaining all the variation between them ( fig . cp0 explained 43% of level of c - peptide at 36 months ( cp36 ) , r 0.43 ( p<0.0001 ) . furthermore , age was the only other factor that had a weak and non - significant influence on cp36 , explaining about 5% of cp36 , r 0.049 ( p=0.2 ) . gender , baseline or end - of - study values of bmi , titres of gadab or ica , hbalc or diabetes duration before the study start did not influence c - peptide at end - of - study . the odds ratio ( or ) for having a cp36 0.5 nmol / l was 2.4 for every increase in cp0 with 0.10 nmol / l ( p=0.02 ) , and 1.06 for each increase in baseline age by 1 year ( p=0.03 ) . if corrected for cp0 and age , the two factors found to have any influence on cp36 , the insulin treated had a non - significant or of 2.5 ( 0.04184 ) of having a cp36 of 0.5 the results of levels of fasting c - peptide ( fcp ) , when comparing the two treatment groups ( data not shown ) , were in accordance with the described results of stimulated c - peptide . among the controls , the level of hbalc had increased significantly at 36 months from 7.0 ( 1.3)% to 7.5 ( 1.5)% ( p=0.006 ) ( fig . , there was no significant difference between hbalc at baseline , 7.3 ( 1.3)% , and after 36 months , 7.2 ( 0.7)% ( p=0.6 ) ( fig . for the levels of hbalc at baseline and during follow - up , see table 2 and fig . of the 32 patients whose ia-2a status was known , 7 ( 22% ) were positive , all of them were also positive to gadab and all but one was positive to ica , meaning that 75% of all were positive to at least two antibodies . at baseline , mean indexes of gadab were 0.78 ( 0.39 ) in i , 0.78 ( 0.48 ) in c ( ns ) and mean ica titres were 29.4 ( 40 ) in i , 72.4 ( 119 ) jdf - u in c ( ns ) . at baseline , there were no significant differences in the prevalences of any of the three antibodies between the two treatment groups or between genders , different ages , duration of diabetes before study start or levels of bmi or hbalc . the titres of gadab or ica were not related to patient age , gender , bmi , diabetes duration , treatment or hbalc at baseline or during the follow - up . mean weight at baseline was 77.4 ( 14.5 , range 57.8110 ) kg in group i , and 83.0 ( 17.8 , range 50.8117 ) kg in group c ( ns ) ; at end - of - study 79.3 ( 12.4 , 57.7101 ) kg in i , 82.3 ( 14.8 , range 50.4115 ) kg in c ( ns ) . mean weight change during the study was 2.5 ( 4.8 , range 8.8 to + 9.3 ) kg in i ; 1.0 ( 10.5 , range 27.3 to + 16.4 ) kg in c ( ns ) . for the three groups , those treated with insulin from baseline , those never treated with insulin and those who were originally treated with dietoha , but had to start insulin treatment during the study , the influences of age , bmi , hbalc , diabetes duration before study start or antibody titres were analysed , with no significant findings except for the influence of cp0 on cp36 . the tests for all the relevant parameters were also carried out with the 37 patients divided into groups of ever- versus never - insulin treated ( during the study ) , again with no significant results ( p=0.120.87 ) , apart from the significant influence of cp0 on cp36 ( p<0.0001 ) . for the levels of glucagon - stimulated c - peptide , see table 2 and fig . c - peptide levels were unchanged for four patients , increased by mean 0.73 ( 0.5 ) nmol / l for six and declined in all others after 36 months . mean glucagon - stimulated c - peptide decreased significantly in both the groups during the 36 months ( p<0.0001 ) . there was a significant time trend for the decrease in c - peptide of 0.17 nmol / l per year ( p=0.03 ) over 36 months without any significant difference between the groups . in repeated measures , anova with time as covariate , that analyses the changes in c - peptide over time with the levels at baseline taken into account , no differences could be found regarding mean - stimulated c - peptide at any time point , although with the mann whitney u test the difference in c - peptide at baseline ( cp0 ) was significant , p=0.03 . there were large variations in c - peptide levels between different individuals , at all time points , within both the groups ( p<0.0001 ) , explaining all the variation between them ( fig . cp0 explained 43% of level of c - peptide at 36 months ( cp36 ) , r 0.43 ( p<0.0001 ) . furthermore , age was the only other factor that had a weak and non - significant influence on cp36 , explaining about 5% of cp36 , r 0.049 ( p=0.2 ) . gender , baseline or end - of - study values of bmi , titres of gadab or ica , hbalc or diabetes duration before the study start did not influence c - peptide at end - of - study . the odds ratio ( or ) for having a cp36 0.5 nmol / l was 2.4 for every increase in cp0 with 0.10 nmol / l ( p=0.02 ) , and 1.06 for each increase in baseline age by 1 year ( p=0.03 ) . if corrected for cp0 and age , the two factors found to have any influence on cp36 , the insulin treated had a non - significant or of 2.5 ( 0.04184 ) of having a cp36 of 0.5 the results of levels of fasting c - peptide ( fcp ) , when comparing the two treatment groups ( data not shown ) , were in accordance with the described results of stimulated c - peptide . among the controls , the level of hbalc had increased significantly at 36 months from 7.0 ( 1.3)% to 7.5 ( 1.5)% ( p=0.006 ) ( fig . , there was no significant difference between hbalc at baseline , 7.3 ( 1.3)% , and after 36 months , 7.2 ( 0.7)% ( p=0.6 ) ( fig . for the levels of hbalc at baseline and during follow - up , see table 2 and fig . of the 32 patients whose ia-2a status was known , 7 ( 22% ) were positive , all of them were also positive to gadab and all but one was positive to ica , meaning that 75% of all were positive to at least two antibodies . at baseline , mean indexes of gadab were 0.78 ( 0.39 ) in i , 0.78 ( 0.48 ) in c ( ns ) and mean ica titres were 29.4 ( 40 ) in i , 72.4 ( 119 ) jdf - u in c ( ns ) . at baseline , there were no significant differences in the prevalences of any of the three antibodies between the two treatment groups or between genders , different ages , duration of diabetes before study start or levels of bmi or hbalc . mean weight at baseline was 77.4 ( 14.5 , range 57.8110 ) kg in group i , and 83.0 ( 17.8 , range 50.8117 ) kg in group c ( ns ) ; at end - of - study 79.3 ( 12.4 , 57.7101 ) kg in i , 82.3 ( 14.8 , range 50.4115 ) kg in c ( ns ) . mean weight change during the study was 2.5 ( 4.8 , range 8.8 to + 9.3 ) kg in i ; 1.0 ( 10.5 , range 27.3 to + 16.4 ) kg in c ( ns ) . for the three groups , those treated with insulin from baseline , those never treated with insulin and those who were originally treated with dietoha , but had to start insulin treatment during the study , the influences of age , bmi , hbalc , diabetes duration before study start or antibody titres were analysed , with no significant findings except for the influence of cp0 on cp36 . the tests for all the relevant parameters were also carried out with the 37 patients divided into groups of ever- versus never - insulin treated ( during the study ) , again with no significant results ( p=0.120.87 ) , apart from the significant influence of cp0 on cp36 ( p<0.0001 ) . few prospective intervention studies have been conducted in lada patients and there is still no general agreement on the best treatment aimed to preserve -cell function ( 57 , 16 ) . age , bmi , duration of diabetes and of insulin independency , which of the antibodies that were analysed , gadab titres and the expression of the essential outcome variable c - peptide vary ( 2 , 4 , 5 , 2830 ) . our study included patients aged 30 years , non - insulin dependent at the times of diagnosis and inclusion and positive to at least one pancreatic autoantibody , for 75% two antibodies , thereby fulfilling the main criteria for lada ( 4 ) . the results of the study indicated that none of the baseline parameters , except initial c - peptide level , significantly influenced the outcome , eliminating the importance of several criteria in comparisons with other studies . the decline in residual -cell function was progressive for the majority of our lada patients , as is usual in autoimmune diabetes ( 4 , 8 , 13 , 16 ) . we observed great variation in the rates and magnitudes of -cell loss between patients and between different time periods during the study , with no consistent patterns . the lack of influence of bmi , age , diabetes duration and baseline hbalc on disease progression was also seen in a non - interventional observation study of lada that followed 13 ab - positive patients by stimulated c - peptide for 2 years ( 16 ) . the significance of initial c - peptide level was also demonstrated in a large swedish study of new - onset 1534 year olds and in the tokyo intervention study ( 28 , 33 ) . the length of our study may explain that some patients with initially higher levels of c - peptide , overrepresented in the control group , by 36 months had not yet lost enough -cell function to be clinically insulin dependent . a significant beneficial effect of early insulin treatment on the preservation of -cell function could not be demonstrated , but level of hbalc after 36 months was better preserved in the insulin treated , in keeping with observations by chaillous et al . incidentally , the shape of the curve of the development of hbalc levels over time for the conventionally treated group in our study had a likeness to that observed in the ukpds ( 35 ) . the tokyo study , with 60 patients , found a preference for insulin treatment versus su , possibly due to the differences in treatment , but longer duration of diabetes , up to 5 years without insulin before inclusion , rendering a selection of patients with better endogenous insulin production from the start , would have excluded those who progressed earlier to insulin dependence , so the trial population differed from ours ( 28 ) . baseline level of c - peptide was an important independent predictor of the ability to preserve a sufficient amount of c - peptide over time , just as in our study . many reports end with a general recommendation of insulin treatment in lada , but the evidence has not been compelling , as concluded by the 2007 cochrane review , which scrutinised seven insulin intervention studies in lada , two insulin versus su , the rest insulin alone versus different combinations of insulin+oha ( 5 ) . in another study , of 54 patients in four groups , the insulin treated , all with low fcp , 0.3 nmol , received either insulin alone or combined with rosiglitazone ( rgz ) , and the oha treated , all with fcp > 0.3 to our knowledge , this study is the first prospective controlled european intervention study of treatment in lada . differences in defining the lada population regarding age , diabetes duration before start of intervention , antibody prevalences and gadab titres , bmi and , not least , baseline levels of c - peptide , if available , have contributed to difficulties in interpreting and comparing the results of the few existing lada intervention studies ( 4 , 5 , 28 , 29 , 37 ) . the similar findings also seen in trials of classical type 1 diabetes and of prevention of autoimmune diabetes in high - risk individuals substantiate the observation of the influence of initial level of c - peptide on outcome level ( 38 , 39 ) . we also saw a non - significant or favouring insulin treatment for preservation of -cell function , indicating the possibility that a larger study population and/or a longer period of follow - up might demonstrate significant preference for early insulin treatment also regarding -cell function .

following a clinically predictable progression pattern , advanced prostate cancer ( pca ) metastasizes to distant organs , with a striking predisposition for bone . despite a decreasing overall mortality rate for pca patients , the development of effective treatments has been hindered , in part , by the lack of cell lines and/or xenograft models that accurately recapitulate the complex metastatic microenvironment . without appropriate models to reflect the disease , mechanistic studies to accurately elucidate the players involved in pca progression in bone have been difficult to implement , impeding the development of clinically effective therapeutics targeted to bone metastases . some of the more commonly used pca cell lines ( e.g. , pc-3 and du-145 ) offer convenience to investigators because they adhere well to tissue culture plastic and therefore are amenable to high throughput screening of drug libraries . however , these same cell lines , when inoculated in bone , generate a largely osteolytic response , in contrast to the predominantly osteoblastic nature of the native human disease . to more accurately model the disease , we have employed the lncap cell line progression series ( lncap , c4 , c4 - 2 , and c4 - 2b ) , with c4 - 2b cells forming osseous lesions in bone . however , we and other investigators continue to search for pca cell models with greater fidelity to the disease that will foster the translation of preclinical findings into the clinic , particularly to satisfy the need to identify new treatments that will eradicate pca metastases growing in bone . to address the need for the highest - possible fidelity in pca cell sources , patient - derived xenograft ( pdx ) models have been established for the preclinical investigation of various aspects of pca biology including angiogenesis , identification of castrate - resistant stem - like cells , and effect of anti - androgen therapies . pdx models are generated when tumor tissue from the patient is surgically resected and engrafted directly into immunocompromised mice . tumors are subsequently maintained solely in vivo via mouse - to - mouse passage , requiring both careful monitoring of the tumor burden and labor intensive animal transfers . through serial passaging in mice and the absence of any in vitro manipulation , pdx tumors remain biologically stable , preserving much of the molecular , genetic , and histological features as well as heterogeneity of the original tumor . however , given the high costs of animal maintenance , lengthy latency period following engraftment , variable engraftment rates , and rare access to patient tissue specimens , pdx in vivo models are generally not yet widely employed in cancer research . most notably , given the poor viability exhibited when grown in vitro under standard tissue culture conditions , it is extremely challenging to culture pdx bone metastatic pca cells for any brief ex vivo manipulation needed to conduct controlled mechanistic studies in vivo . mirroring the in vitro behavior of pdx prostate tumors , pca cell lines belonging to the lncap series also adhere poorly to two - dimensional ( 2d ) tissue culture surfaces , precluding them from use in standard drug screening platforms . to circumvent this problem , our laboratory recently demonstrated the feasibility of using three - dimensional ( 3d ) hyaluronan ( ha)-based hydrogel systems to support the growth and viability of these pca cell lines for mechanistic studies and drug testing . while the commonly employed human tumor spheroid model is an alternative to culturing these poorly adherent cells , hydrogel encapsulation provides the means to fully recapitulate the tumor microenvironment with precise , tunable control over architectural and mechanical cues and/or critical cell specifically , as a ubiquitous component of the bone marrow where bone metastatic pca cells reside , ha plays an active role in regulating several biological processes , including tumorigenesis , strongly justifying its use as an extracellular matrix analogue for culturing bone metastatic tumor cells in vitro . on the basis of the suitability of ha - based hydrogel systems for the culture of pca cell lines , we hypothesized that these systems would similarly support the viability of pdx pca cells in vitro . hence , in the present study , we developed a novel protocol to encapsulate pdx pca cells within 3d ha - based hydrogels and examined tumor cell morphology , viability , proliferative capacity and phenotype . we also tested the potential of this 3d pdx pca model as a diagnostic platform for evaluating rapid patient - specific drug response , a significant advance toward achieving a more personalized therapeutic regimen . phenol red - free dulbecco s modified eagle medium / nutrient mixture f-12 ( dmem / f-12 ) , t - medium , penicillin / streptomycin , trypsin - edta , l - glutamine , live / dead viability / cytotoxicity kit , and quant - it picogreen dsdna assay kit were obtained from life technologies ( grand island , ny ) . fetal bovine serum ( fbs ) phosphate buffered saline ( pbs ) was obtained from lonza ( walkersville , md ) . thiol - modified ha ( ha - sh , glycosil , average mw = 240 kda , degree of thiolation = 1 mol / mg ha - sh ) and poly(ethylene glycol)-diacrylate ( peg - da , extralink , average mw = 3350 da ) were obtained from biotime inc . this ha - sh / peg - da hydrogel system previously has been characterized extensively . dmso , l - cysteine , and papain papaya latex were obtained from sigma - aldrich ( st . louis , mo ) . primary antibodies used were human - specific anti - nuclei antibody from millipore ( mab1281 , billerica , ma ) , anti - androgen receptor from santa cruz biotechnology ( sc-816 , dallas , tx ) , anti - cleaved caspase-3 from cell signaling technology ( nb11089717 , danver , ma ) , and anti - ki-67 from novus biologicals ( # 9664s , littleton , co ) . sylgard 184 poly(dimethylsiloxane ) ( pdms ) elastomer kit was from dow corning ( midland , mi ) . mda pca 183 and mda pca 118b cells were routinely maintained as subcutaneous tumors in cb-17 scid mice ( charles river ) . all experiments for the propagation of pdx tumors in mice were conducted under iacuc approval from the university of texas md anderson cancer center . the mda pca 183 xenograft was derived from androgen - dependent prostate carcinoma , whereas the mda pca 118b was derived from androgen receptor - negative castrate - resistant prostate carcinoma . for this study , mda pca 183 and mda pca 118b pdxs of passage 12 and 8 respectively , were used . on the day of harvest , tumor specimens were removed immediately , rinsed six times with pbs , minced with a scalpel blade , and digested with accumax enzymatic solution for 15 min at 37 c . the enzyme solution was inactivated with fbs , the resultant tumor slurry was filtered through a 70 m cell strainer , and the filtrate was centrifuged . the supernatant was removed , the resulting cell pellet was resuspended , and a cell count was performed . custom - made pdms molds were made by mixing sylgard 184 elastomer and cross - linker 10:1 ( v / v ) according to manufacturer s instructions . the liquid silicone solution was centrifuged at 4 c/3000 rpm/5 min to remove bubbles and poured into a square aluminum frame with a 1.5 mm - thick spacer . an x-660 automated co2 laser cutter ( universal laser systems , scottsdale , az ) was used at 2.5% speed and 60% power to cut the slabs into 24 60 mm rectangles with multiple 6 mm diameter cylindrical cavities . the resultant molds were steam - autoclaved before each use and were sealed onto sterile glass slides before being employed as molds for hydrogel fabrication . each cylindrical cavity could hold approximately 50 l of hydrogel . prior to pdx tumor culture , ha - sh and peg - da were solubilized at 10 and 20 mg / ml , respectively , in degassed water per the manufacturer s instructions . the solutions were mixed at 4:1 ( v / v ) ratio , 35 l of the combined solution was placed in each cavity of the silicone molds , and the mixture was allowed to cross - link for 1 h at 37 c . the bottom layer of acellular hydrogel served as a cushion layer to prevent cell clusters from settling out of the cell hydrogel construct during culture . following tumor harvest , were plated on 6-well plates at densities determined to yield hydrogel constructs with 150,000 and 300,000 encapsulated mda pca 183 and mda pca 118b cells respectively , per construct . mda pca 183 and mda pca 118b cells were cultured in dmem / f-12 supplemented with 10% and 30% ( v / v ) fbs , respectively , in the presence of 100 u / ml of penicillin and 100 g / ml of streptomycin . tumor cells then were incubated at 37 c with 5% co2 for 2 days , after which tumor cell aggregates that formed in suspension from all wells were collected into sterile 15 ml tubes and gently centrifuged . supernatant was removed from each tube , and the remaining cell pellets were resuspended in complete dmem / f-12 medium , combined , and split into microcentrifuge tubes and centrifuged again . notably , as these cells form clusters in suspension , we could not perform cell counts immediately prior to encapsulation . instead , cell counting for the preparation of hydrogel constructs was performed at the step of 2d culture in 6-well plates ( immediately after the tumors were dissociated ) . the resulting cell pellets then were resuspended in solutions of ha - sh and peg - da ( 4:1 v / v ) as described above , and 25 l of the cell - hydrogel suspension was immediately pipetted into each mold cavity , over the 35 l cushion layer of cross - linked ha - sh / peg - da . seeded hydrogels were returned to the incubator for 45 min , then immersed in cell culture medium and incubated overnight . the following day , each mold cavity was scored with a 26-gauge needle , cell - hydrogel constructs were transferred into 24-well plates and submerged in culture medium . the c4 - 2b bone metastatic pca cell line was maintained in t - medium containing 5% fbs ( v / v ) and 2 mm l - glutamine in the presence of 100 u / ml of penicillin and 100 g / ml of streptomycin . for the docetaxel drug study , 50,000 cells were encapsulated within ha - sh / peg - da as described above for the pdx tumors . a lower cell density was used for the cell line because high - density culture was associated with poor cell viability ( data not shown ) . cell - hydrogel constructs ( n = 3 ) were maintained for 2 days before treatment with docetaxel for 3 days . docetaxel was diluted in dimethyl sulfoxide ( dmso ) such that the final concentration of dmso was 1% ( v / v ) in complete medium across all drug concentrations . morphology of the cells encapsulated within the hydrogel was monitored by differential interference contrast microscopy at days 1 , 3 , 5 , and 7 postencapsulation using a nikon eclipse te300 inverted microscope and nis elements software ( nikon instruments , melville , ny ) . fluorescently labeled samples were imaged using a nikon a1-rsi confocal microscope and images processed using the nikon nis - elements ar software ( nikon instruments , melville , ny ) . cell viability was assessed using the live / dead viability / cytotoxicity kit as per the manufacturer s instructions . briefly , cell - hydrogel constructs at the designated time - points were incubated in 2 m calcein - am and 4 m ethidium homodimer-1 in pbs for 30 min at 37 c before confocal imaging . cell - hydrogel constructs ( n = 3 or 4 ) were collected into individual microcentrifuge tubes at the designated time - points , flash - frozen using liquid nitrogen , and stored at 80 c . frozen samples then were lyophilized overnight and digested in pbe buffer ( 0.10 m na2hpo4 and 0.010 m na2edta in demineralized water at ph 6.5 ) containing 125 g / ml papain in the presence of 14.5 mm l - cysteine at 65 c overnight . the digested samples then were sonicated using a probe sonicator , and the liquid supernatant was assayed using the quant - it picogreen dsdna quantification assay as per the manufacturer s instructions . excitation and emission wavelengths of 485 and 528 nm , respectively , were used to measure the fluorescence ( flx800 fluorescence microplate reader ; biotek instruments ) . cell - hydrogel constructs were washed with pbs and fixed with 4% ( v / v ) paraformaldehyde for 10 min at room temperature . after fixation , constructs were washed with pbs and stored at 4 c until staining . constructs were immersed in 0.2% ( v / v ) triton x-100 for 5 min at room temperature to permeabilize cells , then blocked with 500 l of 3% ( w / v ) bsa and 0.2% triton x-100 in pbs at 4 c overnight . all antibodies were diluted at 1:200 in 3% bsa and 0.2% triton - x-100 in pbs . antibody staining was performed using 200 l of the mixed solution to each sample , which were placed on a rocking platform shaker at 4 c overnight . samples were washed with pbs before adding fluorophore - labeled secondary antibodies directed against the appropriate host . secondary antibodies were diluted 1:500 in 3% bsa and 0.2% triton - x-100 in pbs , and 200 l of that solution was added to each sample . dapi ( 5 g / ml ) was added to each sample at room temperature for 5 min . when phalloidin was used , it was diluted 1:20 in pbs , and 100 l of that mixture was added to each sample for 15 min . in initial experiments , following tumor digestion , we encapsulated the entire pdx cell population directly into hydrogels . when we did so , a large number of dead cells was transferred to the hydrogels as observed after 1 day in 3d culture ( data not shown ) . these dead cells likely were generated during the tumor harvest and digestion , and also contain mouse - derived cells that die immediately within the hydrogels . as the presence of high numbers of dead cells would complicate any biochemical assays that were envisioned , we sought to develop an alternative procedure designed to encapsulate cells with high viability ( figure 1a ) . ( a ) mda pca 183 and mda pca 118b pdx tumors ( indicated by red arrows ) that were grown subcutaneously in scid mice were harvested , then mechanically and enzymatically digested . the tumor slurry was subsequently plated onto 6-well plates and cultured for 2 days during which the majority of mouse - derived cells ( red ) attached . the resulting tumor aggregates that formed ( blue ) then were encapsulated within ha - sh / peg - da hydrogels and cultured for up to 14 days . ( b ) dissociated tumor cells plated on tissue culture plastic for 2 days formed multicellular clusters in suspension with other cell types adhering to the plastic surface . ( c ) mixture of dapi - stained ( blue ) cells within tumoroids and adherent cells after 2 days in culture . scale bars = 100 m . in optimizing the culture conditions for the pdx pca cells , we observed that most of the mda pca 183 and mda pca 118b pca cells that had undergone mechanical and enzymatic digestion and been plated onto 6-well plates formed multicellular aggregates in suspension after 2 days in culture , presumably reflecting their characteristic poor adherence onto tissue culture plastic ( figure 1b ) . additionally , during this period , we noted that a population of cells in the tumor slurry ( shown in red in figure 1a ) adhered to the tissue culture plastic surface . leveraging this phenomenon , we collected the aggregates in suspension ( leaving behind the adherent cells ) and found that the process of gentle centrifugation resulted in the removal of dead cells . given that the mda pca 183 and mda pca 118b tumors were grown as subcutaneous tumors in mice , they carry along with them a subpopulation of mouse - derived cells in addition to the human pca cells . indeed , we found that among the poorly adherent pca cells that formed multicellular aggregates in suspension after 2 days on tissue culture plastic , the cells that had adhered stained positive for vimentin ( figure 1c ) . henceforth , we employed this pre - encapsulation 2d culture method , which not only removes dead cells , but also enriches the pdx tumor population via the depletion of mesenchymal cells . a summary of the process used to form 3d pdx tumoroids within the ha - sh / peg - da hydrogel is illustrated in figure 1a . in the first week after encapsulation , mda pca 183 cells were maintained as large tumoroids with most having diameters between 60100 m . mda pca 118b cells were maintained as smaller tumoroids , typically smaller than 60 m in diameter ( figure 2a and supplementary figure 1 ) . cells in tumoroids were in close contact in multicellular clusters , observed via staining with phalloidin for f - actin ( figure 2b ) . to demonstrate that human pca cells comprise the 3d pdx tumoroids , we stained the encapsulated cells for epithelial cell adhesion molecule ( epcam ) , an established marker for epithelial cells . as expected , both the mda pca 183 and mda pca 118b 3d pdx tumoroids stained positive for epcam ( figure 2c ) , indicating that the tumoroids form from self - sorting epithelial cells despite the presence of mesenchymal cells in the original xenograft tumors . additionally , we also confirmed this by probing for the human - specific anti - nuclei antibody and found that the majority of cells within the tumoroids stained positive ( data not shown ) , indicating that the 3d pdx tumoroids form from largely human cells . next , we asked if the pca cells in tumoroids retained their androgen receptor activation status . probing specifically for the androgen receptor , we found that while the androgen receptor was mainly localized in the nucleus of cells in the mda pca 183 tumoroids , nuclear localization of the receptor was not observed in the mda pca 118b cells as would be expected in vivo . generation of pdx tumoroids encapsulated within 3d ha - sh / peg - da hydrogels . ( a ) mda pca 183 and mda pca 118b cells remained as multicellular tumoroids post - encapsulation , over 1 week in culture . cells were stained with dapi ( blue ) or phalloidin ( green ) ; a merged image is shown on the right panel . ( c ) hydrogel - encapsulated mda pca 183 and mda pca 118b cells were stained with dapi ( blue ) , and with antibodies against epcam ( green ) and the androgen receptor ( red ) . while the mda pca 183 cells exhibited nuclear localization of the androgen receptor ( indicated by yellow arrows ) , the mda pca 118b cells stained negative for nuclear localization of the androgen receptor . encapsulation of the nonadherent tumoroids into ha - sh / peg - da hydrogels maintained cell cell contacts for at least 2 weeks in culture . given that these pdx pca tumors exhibit poor viability in vitro on 2d , we next investigated whether the encapsulated pdx tumoroids remained viable within the hydrogels over time using the live / dead viability / cytotoxicity assay . as shown in figure 3 , cells in both the mda pca 183 and mda pca 118b tumoroids were predominantly viable at days 1 , 5 , and 14 . remarkably , pdx cells had the highest survival when they had formed large clusters with other cells . dead or dying cells in each hydrogel were observed to be one of two types : either single cells that had not aggregated with other cells to form multicellular clusters or cells on the periphery of each cluster ( particularly prominent in figure 3b , day 14 ) . beyond viability , to establish if the 3d pdx tumoroids demonstrate tumor growth characteristics necessary for the model to serve as a drug - testing platform , particularly for drug candidates that target actively dividing cells , we monitored their growth over 1 week in culture , using dna content as a surrogate measure of cellularity . notably , the overall cellularity of the mda pca 118b constructs was lower than that of the mda pca 183 because the mda pca 118b cells formed smaller clusters , therefore the retrieval of cells for hydrogel encapsulation was less efficient than from the mda pca 183 cultures . this is apparent from the finding that even though a higher theoretical seeding density ( 150,000 and 300,000 cells per construct for mda pca 183 and 118b , respectively ) was employed for the mda pca 118b constructs , the average initial dna content of the mda pca 118b constructs still was lower than that of the mda pca 183 constructs ( day 1 , figure 4a ) . in analyzing the cellularity of the 3d pdx constructs over time , while dna content remained constant with no significant difference over time for the mda pca 183 tumoroids , there was a significant decrease in cellularity for the mda pca 118b from day 1 to 5 , after which an increase in cellularity was observed ( figure 4a ) . probing the 3d pdx tumoroids for ki-67 and cleaved caspase-3 , markers of proliferation and apoptosis , respectively , we found that differences in the proportion of proliferative and apoptotic cells at day 5 in culture was not apparent for either mda pca 183 or 118b ( figure 4b , c ) . panels in ( a ) and ( b ) show mda pca 183 and 118b after 1 , 5 , and 14 days in culture , respectively . cells were stained with calcein - am ( green , left panel ) or ethidium homodimer-1 ( red , middle panel ) . ( a ) average cellularity of the 3d mda pca 183 and 118b constructs over 1 week in culture . upper panel of ( b ) and ( c ) shows the mda pca 183 and mda 118b tumoroids , respectively , stained with dapi ( left panel , blue ) and antibodies against ki-67 ( middle panel , red ) . lower panel of ( b ) and ( c ) shows the mda pca 183 and mda pca 118b tumoroids , respectively , stained with dapi ( left panel , blue ) and antibodies against cleaved caspase-3 ( middle panel , red ) the right panel shows a merged image where green indicates f - actin . having demonstrated that the encapsulated 3d pdx tumoroids are ( 1 ) made up of viable pca cells , ( 2 ) maintain in vivo - like androgen receptor distribution , and ( 3 ) are proliferative in the hydrogels , we next determined the suitability of this in vitro platform for drug testing of primary pca cells . given the inherent differences in origin between the mda pca 118b and mda pca 183 pdx models , we hypothesized that they would demonstrate a differential sensitivity to chemotherapeutic drugs . to test this hypothesis , we exposed the 3d mda pca 183 and mda pca 118b tumoroids to docetaxel , currently part of the first line regimen to treat patients with castrate - resistant metastatic pca . surprisingly , we found that not only was there no overall difference in docetaxel sensitivity between the 3d mda pca 183 and mda pca 118b tumoroids , no significant reduction in cell number was detected within the range of docetaxel concentrations tested for either 3d tumoroid systems ( figure 5a ) . to confirm these findings , we performed the live / dead viability / cytotoxicity assay and found that cells were largely viable even at the highest docetaxel concentration ( figure 5b ) . additionally , there were no apparent differences in the proportion of apoptotic cells as indicated by cleaved caspase-3 staining at the various docetaxel concentrations tested ( supplementary figure 2 ) . to demonstrate that the lack of cell - kill was cell source - dependent , we similarly exposed hydrogel - encapsulated cells from a bone metastatic prostate cancer cell line , c4 - 2b , to docetaxel . interestingly , a significant decrease in cellularity was already apparent between 1 and 10 nm ( figure 5a , b ) . this finding was corroborated by an observed increase in the proportion of ethidium homodimer-1-stained cells with increasing concentration of docetaxel ( figure 5b ) . notably , given that viable c4 - 2b cells were present at 10,000 nm as observed by calcein - am staining , it is likely that the decrease in the proportion of calcein - am - stained c4 - 2b cells due to drug cytotoxicity could not be clearly discerned when compared to controls . we also showed that the pdx cells were readily killed with 1 m sodium azide ; thus , it was likely not an issue of drug accessing the pca cells ( data not shown ) . ( a ) average cellularity of 3d mda pca 183 and 118b constructs after exposure to increasing docetaxel concentrations , compared to the c4 - 2b cell line , also hydrogel - encapsulated . c4 - 2b cells exhibited significantly lower resistance to docetaxel as compared to both pdxs ( p < 0.05 ) . ( b ) viability of cells after exposure to 0 , 10 , or 10,000 nm docetaxel , assessed by calcein - am staining ( green , left panels ) and ethidium homodimer-1 ( red , middle panels ) . in this study , we assessed a 3d ha - based hydrogel system for the culture of primary pdx bone metastatic pca tumors , well - known to exhibit poor viability when cultured on tissue culture plastic in vitro . given the ubiquity of ha in the bone marrow extracellular matrix and our previous studies that demonstrated the feasibility of using 3d ha - based hydrogels to culture poorly adherent bone metastatic pca cell lines , we hypothesized that these biologically active hydrogels would serve as excellent matrices to support the long - term culture of primary pdx pca tumor tissue in vitro . the inability of bone metastatic pca cells , whether derived from cell lines or primary pdx tumor tissue , to grow in 2d culture is an established phenomenon that indicates the lack of critical components from the bone microenvironment upon which these highly adapted cells depend . in optimizing the culture conditions for the 3d pdx pca cells , we found that the cancer cells failed to adhere to tissue culture plastic , as was expected . instead , these cells aggregated to form multicellular aggregates in suspension , where the formation of cell cell contact is a likely mechanism employed by the pca cells to adapt to the loss of native cell - matrix interactions . indeed , as proposed by shen and kramer , cancer cells are capable of undergoing synoikis , a term that was used to describe the avoidance of apoptosis by relying on intercellular adhesions in the absence of cell matrix interactions . focusing specifically on squamous cell carcinoma cells , the authors demonstrated that e - cadherin - mediated cell cell contact resulted in the generation of compensatory survival signaling via the epidermal growth factor receptor . interestingly , a similar observation was made by a landmark report by kondo et al . cell interactions were necessary for the survival of primary patient - derived colorectal cancer cells in suspension culture . in optimizing the culture method for the primary pdx pca cells , we leveraged the ability of the cancer cells to spontaneously aggregate to form multicellular clusters in suspension as a means to reduce the presence of contaminant cells such as dead cells , including those that were originally from the tumor tissue itself ( in regions of necrosis ) and those that were generated during the process of tumor digestion . we found that the application of gentle centrifugation to retrieve the pca aggregates in suspension after 2 days on tissue culture plastic was an effective method to separate the pca cells from dead cells in suspension prior to encapsulation within the hydrogel . given that the pdx pca cells were maintained in mice prior to harvest , the cancer cells were also likely contaminated with mouse - derived cells ( such as blood cells , endothelial cells , or fibroblasts ) , which consequently introduces a source of heterogeneity and complicates downstream biochemical studies . furthermore , considering the importance of the tumor microenvironment in influencing tumor behavior , the presence of mouse - derived stroma may confound studies evaluating species - specific stroma - targeting drug candidates , hence making it a major hindrance in current pdx tumor models . indeed , while the pca cells formed aggregates in suspension , a population of cells was consistently observed to adhere to the tissue culture plastic surface . for example , in mda pca 183 cultures , we found that the adherent cells expressed vimentin and originated from mouse tissue ( figure 1c ) . while further characterization is necessary to determine the actual yield of the cancer cells , these results , in conjunction with the observation that epcam , an epithelial cell marker , was near - ubiquitously expressed in all of the hydrogel - encapsulated pdx clusters ( figure 2c ) , highly suggest that the pre - encapsulation 2d culture serves as a desirable selective method to generate viable and enriched pdx pca cells in 3d . in this study , we optimized the culture of pdx pca cells in vitro , which to our knowledge is unprecedented . while robust growth was limited by a balance between proliferation and apoptosis , viability of the hydrogel - encapsulated pca cells was notably well maintained even up to 14 days in culture ( figure 3 ) . ten to 20% of cells within the 3d pdx tumoroids stained positive for ki-67 at day 5 in culture ( figure 4b , c ) , indicating that a subset of cells was still progressing through the cell cycle . comparing the proportion of proliferative cells within the 3d mda pca 118b pdx tumoroids to their in vivo counterparts , we found that our estimate of ki-67-positive cells is well within the range of that in vivo . we are currently modifying the ha - based hydrogel to study the effects of ecm moieties and other cell types on the growth behavior of the pdx pca tumoroids in 3d culture . with the shift in focus from traditional chemotherapy to biologically targeted approaches based on a mechanistic understanding of pca biology , preservation of salient features of the original tumor is a critical criterion in evaluating the success of this culture system as a drug - testing platform . to this end the androgen receptor is an important modulator for prostate growth and currently the primary therapeutic target in pca . additionally , this nuclear receptor regulates psa expression , which is used clinically as a biochemical marker to track the progression of pca and response to therapy . predominantly cytoplasmic in the absence of ligands , ligand binding to the androgen receptor results in conformational changes , nuclear translocation of the ligand - bound receptor , and transcriptional activity . mirroring the in vivo tumor , detection of nuclear androgen receptor in the 3d mda pca 183 tumoroids indicates that the ha - sh / peg - da hydrogel system is capable of maintaining critical androgen receptor signaling in these androgen - dependent cells . similarly , nuclear androgen receptor was not observed in the 3d mda pca 118b tumoroids as would be expected in in vivo , indicating that these 3d pdx models may have utility as drug - testing platforms evaluating androgen receptor - targeting agents currently under intense clinical investigation . drug discovery and screening has historically relied on cancer cell lines , which have generated a wealth of knowledge in cancer biology . however , the dichotomy in drug efficacy between preclinical and clinical testing is increasingly apparent , primarily attributed to the genetic and epigenetic changes that accrue when cells acclimatize to the in vitro culture environment in extended periods of cell culture . indeed , gillet et al . evaluated the multidrug resistance transcriptome of six cancer types and found no correlation between clinical samples and established cell lines , underscoring the need for new in vitro cancer models and primary tumor models . hypothesizing that inherent differences exist between traditional pca cell lines and pdx pca tumors , we evaluated the response of the 3d pdx pca tumoroids ( mda pca 183 and 118b ) to docetaxel and compared their response to the c4 - 2b bone metastatic prostate cancer cell line . docetaxel is a microtubule stabilizer and is currently part of the first - line standard treatment for metastatic castrate - resistant pca . we found that despite the differences between the mda pca 183 and 118b cells in patient origin , their overall drug sensitivity to docetaxel was similar . given that both pdx - derived cultures showed a low number of proliferating cells and that docetaxel targets actively dividing cells , it thus was unsurprising that both demonstrated similar resistance to the drug in 3d culture . however , interestingly , the 3d pdx pca cells exhibited a significantly higher resistance to the drug as compared to the c4 - 2b cells . it is unlikely that the increased resistance is due to impaired diffusion of the drug into the hydrogel since it demonstrated clear efficacy against the hydrogel - encapsulated c4 - 2b cells . while results from this drug study are promising , studies are ongoing to establish clinical relevance and understand the mechanisms underlying this difference . with the increasing awareness that irreversible biological changes occur when conventional cell lines are established in vitro , beyond prostate cancer , there is general shift toward the use of pdx tumor models for cancer research . however , their utility depends on the ability to manipulate the tumor cells ex vivo prior to implantation . given the alterations in biological properties associated with 2d culture , a few groups have taken steps to optimize methods to reliably grow primary tumor cells in 3d . one such example is in colorectal cancer , where it has been shown that primary colorectal cancer cells can be cultured as 3d spheroids or colospheres in vitro , enabling the manipulation of the cancer cells before engraftment in vivo for controlled studies . in our current study , we demonstrate for the first time that primary bone metastatic pca cells can similarly be cultured in vitro with the use of a 3d ha - based hydrogel system . lastly , this 3d pdx model may ultimately be adapted to a rapid and high throughput platform for assessing drug efficacy , rational drug combinations , and development of predictive biomarkers for novel targeted therapies , while reducing the need for low throughput animal hosts . as an example , we showed here that 3d pdx pca cells exhibited an increased resistance to docetaxel as compared to a standard cell line commonly used in pca research . ultimately , it is envisioned that the use of 3d pdx models may greatly accelerate the advancement of novel drug candidates together with predictive biomarkers that enable patient selection when translated into early phase clinical trials . on the basis of previous success culturing bone metastatic pca cell lines using 3d ha - based hydrogels , we demonstrate for the first time that never in 2d pdx pca tumors can be cultured in vitro and maintained for at least 2 weeks . the resulting hydrogel - encapsulated 3d pdx tumoroids retained viability , proliferative capacity , and the androgen receptor expression , indicating that this novel 3d pdx model may serve as a valuable platform for drug development . while it has yet to be tested , this system promises the possibility of culturing tumor tissue directly from the patient for rapid drug screening , thereby eliminating the middle mouse and its associated problems , a major leap toward personalized medicine .

the lack of effective therapies for bone metastatic prostate cancer ( pca ) underscores the need for accurate models of the disease to enable the discovery of new therapeutic targets and to test drug sensitivities of individual tumors . to this end , the patient - derived xenograft ( pdx ) pca model using immunocompromised mice was established to model the disease with greater fidelity than is possible with currently employed cell lines grown on tissue culture plastic . however , poorly adherent pdx tumor cells exhibit low viability in standard culture , making it difficult to manipulate these cells for subsequent controlled mechanistic studies . to overcome this challenge , we encapsulated pdx tumor cells within a three - dimensional hyaluronan - based hydrogel and demonstrated that the hydrogel maintains pdx cell viability with continued native androgen receptor expression . furthermore , a differential sensitivity to docetaxel , a chemotherapeutic drug , was observed as compared to a traditional pca cell line . these findings underscore the potential impact of this novel 3d pdx pca model as a diagnostic platform for rapid drug evaluation and ultimately push personalized medicine toward clinical reality .

Introduction Materials and Methods Results Discussion Conclusions

following a clinically predictable progression pattern , advanced prostate cancer ( pca ) metastasizes to distant organs , with a striking predisposition for bone . despite a decreasing overall mortality rate for pca patients , the development of effective treatments has been hindered , in part , by the lack of cell lines and/or xenograft models that accurately recapitulate the complex metastatic microenvironment . without appropriate models to reflect the disease , mechanistic studies to accurately elucidate the players involved in pca progression in bone have been difficult to implement , impeding the development of clinically effective therapeutics targeted to bone metastases . some of the more commonly used pca cell lines ( e.g. however , these same cell lines , when inoculated in bone , generate a largely osteolytic response , in contrast to the predominantly osteoblastic nature of the native human disease . to more accurately model the disease , we have employed the lncap cell line progression series ( lncap , c4 , c4 - 2 , and c4 - 2b ) , with c4 - 2b cells forming osseous lesions in bone . however , we and other investigators continue to search for pca cell models with greater fidelity to the disease that will foster the translation of preclinical findings into the clinic , particularly to satisfy the need to identify new treatments that will eradicate pca metastases growing in bone . to address the need for the highest - possible fidelity in pca cell sources , patient - derived xenograft ( pdx ) models have been established for the preclinical investigation of various aspects of pca biology including angiogenesis , identification of castrate - resistant stem - like cells , and effect of anti - androgen therapies . pdx models are generated when tumor tissue from the patient is surgically resected and engrafted directly into immunocompromised mice . most notably , given the poor viability exhibited when grown in vitro under standard tissue culture conditions , it is extremely challenging to culture pdx bone metastatic pca cells for any brief ex vivo manipulation needed to conduct controlled mechanistic studies in vivo . mirroring the in vitro behavior of pdx prostate tumors , pca cell lines belonging to the lncap series also adhere poorly to two - dimensional ( 2d ) tissue culture surfaces , precluding them from use in standard drug screening platforms . to circumvent this problem , our laboratory recently demonstrated the feasibility of using three - dimensional ( 3d ) hyaluronan ( ha)-based hydrogel systems to support the growth and viability of these pca cell lines for mechanistic studies and drug testing . while the commonly employed human tumor spheroid model is an alternative to culturing these poorly adherent cells , hydrogel encapsulation provides the means to fully recapitulate the tumor microenvironment with precise , tunable control over architectural and mechanical cues and/or critical cell specifically , as a ubiquitous component of the bone marrow where bone metastatic pca cells reside , ha plays an active role in regulating several biological processes , including tumorigenesis , strongly justifying its use as an extracellular matrix analogue for culturing bone metastatic tumor cells in vitro . on the basis of the suitability of ha - based hydrogel systems for the culture of pca cell lines , we hypothesized that these systems would similarly support the viability of pdx pca cells in vitro . hence , in the present study , we developed a novel protocol to encapsulate pdx pca cells within 3d ha - based hydrogels and examined tumor cell morphology , viability , proliferative capacity and phenotype . we also tested the potential of this 3d pdx pca model as a diagnostic platform for evaluating rapid patient - specific drug response , a significant advance toward achieving a more personalized therapeutic regimen . notably , as these cells form clusters in suspension , we could not perform cell counts immediately prior to encapsulation . the c4 - 2b bone metastatic pca cell line was maintained in t - medium containing 5% fbs ( v / v ) and 2 mm l - glutamine in the presence of 100 u / ml of penicillin and 100 g / ml of streptomycin . morphology of the cells encapsulated within the hydrogel was monitored by differential interference contrast microscopy at days 1 , 3 , 5 , and 7 postencapsulation using a nikon eclipse te300 inverted microscope and nis elements software ( nikon instruments , melville , ny ) . in initial experiments , following tumor digestion , we encapsulated the entire pdx cell population directly into hydrogels . ( b ) dissociated tumor cells plated on tissue culture plastic for 2 days formed multicellular clusters in suspension with other cell types adhering to the plastic surface . in optimizing the culture conditions for the pdx pca cells , we observed that most of the mda pca 183 and mda pca 118b pca cells that had undergone mechanical and enzymatic digestion and been plated onto 6-well plates formed multicellular aggregates in suspension after 2 days in culture , presumably reflecting their characteristic poor adherence onto tissue culture plastic ( figure 1b ) . additionally , during this period , we noted that a population of cells in the tumor slurry ( shown in red in figure 1a ) adhered to the tissue culture plastic surface . indeed , we found that among the poorly adherent pca cells that formed multicellular aggregates in suspension after 2 days on tissue culture plastic , the cells that had adhered stained positive for vimentin ( figure 1c ) . henceforth , we employed this pre - encapsulation 2d culture method , which not only removes dead cells , but also enriches the pdx tumor population via the depletion of mesenchymal cells . a summary of the process used to form 3d pdx tumoroids within the ha - sh / peg - da hydrogel is illustrated in figure 1a . to demonstrate that human pca cells comprise the 3d pdx tumoroids , we stained the encapsulated cells for epithelial cell adhesion molecule ( epcam ) , an established marker for epithelial cells . additionally , we also confirmed this by probing for the human - specific anti - nuclei antibody and found that the majority of cells within the tumoroids stained positive ( data not shown ) , indicating that the 3d pdx tumoroids form from largely human cells . next , we asked if the pca cells in tumoroids retained their androgen receptor activation status . probing specifically for the androgen receptor , we found that while the androgen receptor was mainly localized in the nucleus of cells in the mda pca 183 tumoroids , nuclear localization of the receptor was not observed in the mda pca 118b cells as would be expected in vivo . while the mda pca 183 cells exhibited nuclear localization of the androgen receptor ( indicated by yellow arrows ) , the mda pca 118b cells stained negative for nuclear localization of the androgen receptor . given that these pdx pca tumors exhibit poor viability in vitro on 2d , we next investigated whether the encapsulated pdx tumoroids remained viable within the hydrogels over time using the live / dead viability / cytotoxicity assay . beyond viability , to establish if the 3d pdx tumoroids demonstrate tumor growth characteristics necessary for the model to serve as a drug - testing platform , particularly for drug candidates that target actively dividing cells , we monitored their growth over 1 week in culture , using dna content as a surrogate measure of cellularity . notably , the overall cellularity of the mda pca 118b constructs was lower than that of the mda pca 183 because the mda pca 118b cells formed smaller clusters , therefore the retrieval of cells for hydrogel encapsulation was less efficient than from the mda pca 183 cultures . this is apparent from the finding that even though a higher theoretical seeding density ( 150,000 and 300,000 cells per construct for mda pca 183 and 118b , respectively ) was employed for the mda pca 118b constructs , the average initial dna content of the mda pca 118b constructs still was lower than that of the mda pca 183 constructs ( day 1 , figure 4a ) . in analyzing the cellularity of the 3d pdx constructs over time , while dna content remained constant with no significant difference over time for the mda pca 183 tumoroids , there was a significant decrease in cellularity for the mda pca 118b from day 1 to 5 , after which an increase in cellularity was observed ( figure 4a ) . having demonstrated that the encapsulated 3d pdx tumoroids are ( 1 ) made up of viable pca cells , ( 2 ) maintain in vivo - like androgen receptor distribution , and ( 3 ) are proliferative in the hydrogels , we next determined the suitability of this in vitro platform for drug testing of primary pca cells . given the inherent differences in origin between the mda pca 118b and mda pca 183 pdx models , we hypothesized that they would demonstrate a differential sensitivity to chemotherapeutic drugs . to test this hypothesis , we exposed the 3d mda pca 183 and mda pca 118b tumoroids to docetaxel , currently part of the first line regimen to treat patients with castrate - resistant metastatic pca . to demonstrate that the lack of cell - kill was cell source - dependent , we similarly exposed hydrogel - encapsulated cells from a bone metastatic prostate cancer cell line , c4 - 2b , to docetaxel . ( a ) average cellularity of 3d mda pca 183 and 118b constructs after exposure to increasing docetaxel concentrations , compared to the c4 - 2b cell line , also hydrogel - encapsulated . c4 - 2b cells exhibited significantly lower resistance to docetaxel as compared to both pdxs ( p < 0.05 ) . in this study , we assessed a 3d ha - based hydrogel system for the culture of primary pdx bone metastatic pca tumors , well - known to exhibit poor viability when cultured on tissue culture plastic in vitro . given the ubiquity of ha in the bone marrow extracellular matrix and our previous studies that demonstrated the feasibility of using 3d ha - based hydrogels to culture poorly adherent bone metastatic pca cell lines , we hypothesized that these biologically active hydrogels would serve as excellent matrices to support the long - term culture of primary pdx pca tumor tissue in vitro . the inability of bone metastatic pca cells , whether derived from cell lines or primary pdx tumor tissue , to grow in 2d culture is an established phenomenon that indicates the lack of critical components from the bone microenvironment upon which these highly adapted cells depend . in optimizing the culture conditions for the 3d pdx pca cells , we found that the cancer cells failed to adhere to tissue culture plastic , as was expected . focusing specifically on squamous cell carcinoma cells , the authors demonstrated that e - cadherin - mediated cell cell contact resulted in the generation of compensatory survival signaling via the epidermal growth factor receptor . cell interactions were necessary for the survival of primary patient - derived colorectal cancer cells in suspension culture . in optimizing the culture method for the primary pdx pca cells , we leveraged the ability of the cancer cells to spontaneously aggregate to form multicellular clusters in suspension as a means to reduce the presence of contaminant cells such as dead cells , including those that were originally from the tumor tissue itself ( in regions of necrosis ) and those that were generated during the process of tumor digestion . we found that the application of gentle centrifugation to retrieve the pca aggregates in suspension after 2 days on tissue culture plastic was an effective method to separate the pca cells from dead cells in suspension prior to encapsulation within the hydrogel . given that the pdx pca cells were maintained in mice prior to harvest , the cancer cells were also likely contaminated with mouse - derived cells ( such as blood cells , endothelial cells , or fibroblasts ) , which consequently introduces a source of heterogeneity and complicates downstream biochemical studies . furthermore , considering the importance of the tumor microenvironment in influencing tumor behavior , the presence of mouse - derived stroma may confound studies evaluating species - specific stroma - targeting drug candidates , hence making it a major hindrance in current pdx tumor models . indeed , while the pca cells formed aggregates in suspension , a population of cells was consistently observed to adhere to the tissue culture plastic surface . for example , in mda pca 183 cultures , we found that the adherent cells expressed vimentin and originated from mouse tissue ( figure 1c ) . while further characterization is necessary to determine the actual yield of the cancer cells , these results , in conjunction with the observation that epcam , an epithelial cell marker , was near - ubiquitously expressed in all of the hydrogel - encapsulated pdx clusters ( figure 2c ) , highly suggest that the pre - encapsulation 2d culture serves as a desirable selective method to generate viable and enriched pdx pca cells in 3d . while robust growth was limited by a balance between proliferation and apoptosis , viability of the hydrogel - encapsulated pca cells was notably well maintained even up to 14 days in culture ( figure 3 ) . comparing the proportion of proliferative cells within the 3d mda pca 118b pdx tumoroids to their in vivo counterparts , we found that our estimate of ki-67-positive cells is well within the range of that in vivo . we are currently modifying the ha - based hydrogel to study the effects of ecm moieties and other cell types on the growth behavior of the pdx pca tumoroids in 3d culture . with the shift in focus from traditional chemotherapy to biologically targeted approaches based on a mechanistic understanding of pca biology , preservation of salient features of the original tumor is a critical criterion in evaluating the success of this culture system as a drug - testing platform . to this end the androgen receptor is an important modulator for prostate growth and currently the primary therapeutic target in pca . mirroring the in vivo tumor , detection of nuclear androgen receptor in the 3d mda pca 183 tumoroids indicates that the ha - sh / peg - da hydrogel system is capable of maintaining critical androgen receptor signaling in these androgen - dependent cells . similarly , nuclear androgen receptor was not observed in the 3d mda pca 118b tumoroids as would be expected in in vivo , indicating that these 3d pdx models may have utility as drug - testing platforms evaluating androgen receptor - targeting agents currently under intense clinical investigation . evaluated the multidrug resistance transcriptome of six cancer types and found no correlation between clinical samples and established cell lines , underscoring the need for new in vitro cancer models and primary tumor models . hypothesizing that inherent differences exist between traditional pca cell lines and pdx pca tumors , we evaluated the response of the 3d pdx pca tumoroids ( mda pca 183 and 118b ) to docetaxel and compared their response to the c4 - 2b bone metastatic prostate cancer cell line . however , interestingly , the 3d pdx pca cells exhibited a significantly higher resistance to the drug as compared to the c4 - 2b cells . it is unlikely that the increased resistance is due to impaired diffusion of the drug into the hydrogel since it demonstrated clear efficacy against the hydrogel - encapsulated c4 - 2b cells . with the increasing awareness that irreversible biological changes occur when conventional cell lines are established in vitro , beyond prostate cancer , there is general shift toward the use of pdx tumor models for cancer research . however , their utility depends on the ability to manipulate the tumor cells ex vivo prior to implantation . given the alterations in biological properties associated with 2d culture , a few groups have taken steps to optimize methods to reliably grow primary tumor cells in 3d . in our current study , we demonstrate for the first time that primary bone metastatic pca cells can similarly be cultured in vitro with the use of a 3d ha - based hydrogel system . lastly , this 3d pdx model may ultimately be adapted to a rapid and high throughput platform for assessing drug efficacy , rational drug combinations , and development of predictive biomarkers for novel targeted therapies , while reducing the need for low throughput animal hosts . as an example , we showed here that 3d pdx pca cells exhibited an increased resistance to docetaxel as compared to a standard cell line commonly used in pca research . on the basis of previous success culturing bone metastatic pca cell lines using 3d ha - based hydrogels , we demonstrate for the first time that never in 2d pdx pca tumors can be cultured in vitro and maintained for at least 2 weeks . the resulting hydrogel - encapsulated 3d pdx tumoroids retained viability , proliferative capacity , and the androgen receptor expression , indicating that this novel 3d pdx model may serve as a valuable platform for drug development . while it has yet to be tested , this system promises the possibility of culturing tumor tissue directly from the patient for rapid drug screening , thereby eliminating the middle mouse and its associated problems , a major leap toward personalized medicine .

despite a decreasing overall mortality rate for pca patients , the development of effective treatments has been hindered , in part , by the lack of cell lines and/or xenograft models that accurately recapitulate the complex metastatic microenvironment . without appropriate models to reflect the disease , mechanistic studies to accurately elucidate the players involved in pca progression in bone have been difficult to implement , impeding the development of clinically effective therapeutics targeted to bone metastases . to more accurately model the disease , we have employed the lncap cell line progression series ( lncap , c4 , c4 - 2 , and c4 - 2b ) , with c4 - 2b cells forming osseous lesions in bone . however , we and other investigators continue to search for pca cell models with greater fidelity to the disease that will foster the translation of preclinical findings into the clinic , particularly to satisfy the need to identify new treatments that will eradicate pca metastases growing in bone . to address the need for the highest - possible fidelity in pca cell sources , patient - derived xenograft ( pdx ) models have been established for the preclinical investigation of various aspects of pca biology including angiogenesis , identification of castrate - resistant stem - like cells , and effect of anti - androgen therapies . mirroring the in vitro behavior of pdx prostate tumors , pca cell lines belonging to the lncap series also adhere poorly to two - dimensional ( 2d ) tissue culture surfaces , precluding them from use in standard drug screening platforms . to circumvent this problem , our laboratory recently demonstrated the feasibility of using three - dimensional ( 3d ) hyaluronan ( ha)-based hydrogel systems to support the growth and viability of these pca cell lines for mechanistic studies and drug testing . while the commonly employed human tumor spheroid model is an alternative to culturing these poorly adherent cells , hydrogel encapsulation provides the means to fully recapitulate the tumor microenvironment with precise , tunable control over architectural and mechanical cues and/or critical cell specifically , as a ubiquitous component of the bone marrow where bone metastatic pca cells reside , ha plays an active role in regulating several biological processes , including tumorigenesis , strongly justifying its use as an extracellular matrix analogue for culturing bone metastatic tumor cells in vitro . on the basis of the suitability of ha - based hydrogel systems for the culture of pca cell lines , we hypothesized that these systems would similarly support the viability of pdx pca cells in vitro . hence , in the present study , we developed a novel protocol to encapsulate pdx pca cells within 3d ha - based hydrogels and examined tumor cell morphology , viability , proliferative capacity and phenotype . phenol red - free dulbecco s modified eagle medium / nutrient mixture f-12 ( dmem / f-12 ) , t - medium , penicillin / streptomycin , trypsin - edta , l - glutamine , live / dead viability / cytotoxicity kit , and quant - it picogreen dsdna assay kit were obtained from life technologies ( grand island , ny ) . thiol - modified ha ( ha - sh , glycosil , average mw = 240 kda , degree of thiolation = 1 mol / mg ha - sh ) and poly(ethylene glycol)-diacrylate ( peg - da , extralink , average mw = 3350 da ) were obtained from biotime inc . primary antibodies used were human - specific anti - nuclei antibody from millipore ( mab1281 , billerica , ma ) , anti - androgen receptor from santa cruz biotechnology ( sc-816 , dallas , tx ) , anti - cleaved caspase-3 from cell signaling technology ( nb11089717 , danver , ma ) , and anti - ki-67 from novus biologicals ( # 9664s , littleton , co ) . the resulting cell pellets then were resuspended in solutions of ha - sh and peg - da ( 4:1 v / v ) as described above , and 25 l of the cell - hydrogel suspension was immediately pipetted into each mold cavity , over the 35 l cushion layer of cross - linked ha - sh / peg - da . the c4 - 2b bone metastatic pca cell line was maintained in t - medium containing 5% fbs ( v / v ) and 2 mm l - glutamine in the presence of 100 u / ml of penicillin and 100 g / ml of streptomycin . morphology of the cells encapsulated within the hydrogel was monitored by differential interference contrast microscopy at days 1 , 3 , 5 , and 7 postencapsulation using a nikon eclipse te300 inverted microscope and nis elements software ( nikon instruments , melville , ny ) . in optimizing the culture conditions for the pdx pca cells , we observed that most of the mda pca 183 and mda pca 118b pca cells that had undergone mechanical and enzymatic digestion and been plated onto 6-well plates formed multicellular aggregates in suspension after 2 days in culture , presumably reflecting their characteristic poor adherence onto tissue culture plastic ( figure 1b ) . indeed , we found that among the poorly adherent pca cells that formed multicellular aggregates in suspension after 2 days on tissue culture plastic , the cells that had adhered stained positive for vimentin ( figure 1c ) . to demonstrate that human pca cells comprise the 3d pdx tumoroids , we stained the encapsulated cells for epithelial cell adhesion molecule ( epcam ) , an established marker for epithelial cells . as expected , both the mda pca 183 and mda pca 118b 3d pdx tumoroids stained positive for epcam ( figure 2c ) , indicating that the tumoroids form from self - sorting epithelial cells despite the presence of mesenchymal cells in the original xenograft tumors . additionally , we also confirmed this by probing for the human - specific anti - nuclei antibody and found that the majority of cells within the tumoroids stained positive ( data not shown ) , indicating that the 3d pdx tumoroids form from largely human cells . probing specifically for the androgen receptor , we found that while the androgen receptor was mainly localized in the nucleus of cells in the mda pca 183 tumoroids , nuclear localization of the receptor was not observed in the mda pca 118b cells as would be expected in vivo . given that these pdx pca tumors exhibit poor viability in vitro on 2d , we next investigated whether the encapsulated pdx tumoroids remained viable within the hydrogels over time using the live / dead viability / cytotoxicity assay . dead or dying cells in each hydrogel were observed to be one of two types : either single cells that had not aggregated with other cells to form multicellular clusters or cells on the periphery of each cluster ( particularly prominent in figure 3b , day 14 ) . beyond viability , to establish if the 3d pdx tumoroids demonstrate tumor growth characteristics necessary for the model to serve as a drug - testing platform , particularly for drug candidates that target actively dividing cells , we monitored their growth over 1 week in culture , using dna content as a surrogate measure of cellularity . notably , the overall cellularity of the mda pca 118b constructs was lower than that of the mda pca 183 because the mda pca 118b cells formed smaller clusters , therefore the retrieval of cells for hydrogel encapsulation was less efficient than from the mda pca 183 cultures . this is apparent from the finding that even though a higher theoretical seeding density ( 150,000 and 300,000 cells per construct for mda pca 183 and 118b , respectively ) was employed for the mda pca 118b constructs , the average initial dna content of the mda pca 118b constructs still was lower than that of the mda pca 183 constructs ( day 1 , figure 4a ) . in analyzing the cellularity of the 3d pdx constructs over time , while dna content remained constant with no significant difference over time for the mda pca 183 tumoroids , there was a significant decrease in cellularity for the mda pca 118b from day 1 to 5 , after which an increase in cellularity was observed ( figure 4a ) . probing the 3d pdx tumoroids for ki-67 and cleaved caspase-3 , markers of proliferation and apoptosis , respectively , we found that differences in the proportion of proliferative and apoptotic cells at day 5 in culture was not apparent for either mda pca 183 or 118b ( figure 4b , c ) . lower panel of ( b ) and ( c ) shows the mda pca 183 and mda pca 118b tumoroids , respectively , stained with dapi ( left panel , blue ) and antibodies against cleaved caspase-3 ( middle panel , red ) the right panel shows a merged image where green indicates f - actin . having demonstrated that the encapsulated 3d pdx tumoroids are ( 1 ) made up of viable pca cells , ( 2 ) maintain in vivo - like androgen receptor distribution , and ( 3 ) are proliferative in the hydrogels , we next determined the suitability of this in vitro platform for drug testing of primary pca cells . to test this hypothesis , we exposed the 3d mda pca 183 and mda pca 118b tumoroids to docetaxel , currently part of the first line regimen to treat patients with castrate - resistant metastatic pca . surprisingly , we found that not only was there no overall difference in docetaxel sensitivity between the 3d mda pca 183 and mda pca 118b tumoroids , no significant reduction in cell number was detected within the range of docetaxel concentrations tested for either 3d tumoroid systems ( figure 5a ) . to demonstrate that the lack of cell - kill was cell source - dependent , we similarly exposed hydrogel - encapsulated cells from a bone metastatic prostate cancer cell line , c4 - 2b , to docetaxel . notably , given that viable c4 - 2b cells were present at 10,000 nm as observed by calcein - am staining , it is likely that the decrease in the proportion of calcein - am - stained c4 - 2b cells due to drug cytotoxicity could not be clearly discerned when compared to controls . in this study , we assessed a 3d ha - based hydrogel system for the culture of primary pdx bone metastatic pca tumors , well - known to exhibit poor viability when cultured on tissue culture plastic in vitro . given the ubiquity of ha in the bone marrow extracellular matrix and our previous studies that demonstrated the feasibility of using 3d ha - based hydrogels to culture poorly adherent bone metastatic pca cell lines , we hypothesized that these biologically active hydrogels would serve as excellent matrices to support the long - term culture of primary pdx pca tumor tissue in vitro . the inability of bone metastatic pca cells , whether derived from cell lines or primary pdx tumor tissue , to grow in 2d culture is an established phenomenon that indicates the lack of critical components from the bone microenvironment upon which these highly adapted cells depend . instead , these cells aggregated to form multicellular aggregates in suspension , where the formation of cell cell contact is a likely mechanism employed by the pca cells to adapt to the loss of native cell - matrix interactions . indeed , as proposed by shen and kramer , cancer cells are capable of undergoing synoikis , a term that was used to describe the avoidance of apoptosis by relying on intercellular adhesions in the absence of cell matrix interactions . focusing specifically on squamous cell carcinoma cells , the authors demonstrated that e - cadherin - mediated cell cell contact resulted in the generation of compensatory survival signaling via the epidermal growth factor receptor . in optimizing the culture method for the primary pdx pca cells , we leveraged the ability of the cancer cells to spontaneously aggregate to form multicellular clusters in suspension as a means to reduce the presence of contaminant cells such as dead cells , including those that were originally from the tumor tissue itself ( in regions of necrosis ) and those that were generated during the process of tumor digestion . we found that the application of gentle centrifugation to retrieve the pca aggregates in suspension after 2 days on tissue culture plastic was an effective method to separate the pca cells from dead cells in suspension prior to encapsulation within the hydrogel . given that the pdx pca cells were maintained in mice prior to harvest , the cancer cells were also likely contaminated with mouse - derived cells ( such as blood cells , endothelial cells , or fibroblasts ) , which consequently introduces a source of heterogeneity and complicates downstream biochemical studies . furthermore , considering the importance of the tumor microenvironment in influencing tumor behavior , the presence of mouse - derived stroma may confound studies evaluating species - specific stroma - targeting drug candidates , hence making it a major hindrance in current pdx tumor models . while further characterization is necessary to determine the actual yield of the cancer cells , these results , in conjunction with the observation that epcam , an epithelial cell marker , was near - ubiquitously expressed in all of the hydrogel - encapsulated pdx clusters ( figure 2c ) , highly suggest that the pre - encapsulation 2d culture serves as a desirable selective method to generate viable and enriched pdx pca cells in 3d . comparing the proportion of proliferative cells within the 3d mda pca 118b pdx tumoroids to their in vivo counterparts , we found that our estimate of ki-67-positive cells is well within the range of that in vivo . we are currently modifying the ha - based hydrogel to study the effects of ecm moieties and other cell types on the growth behavior of the pdx pca tumoroids in 3d culture . with the shift in focus from traditional chemotherapy to biologically targeted approaches based on a mechanistic understanding of pca biology , preservation of salient features of the original tumor is a critical criterion in evaluating the success of this culture system as a drug - testing platform . predominantly cytoplasmic in the absence of ligands , ligand binding to the androgen receptor results in conformational changes , nuclear translocation of the ligand - bound receptor , and transcriptional activity . mirroring the in vivo tumor , detection of nuclear androgen receptor in the 3d mda pca 183 tumoroids indicates that the ha - sh / peg - da hydrogel system is capable of maintaining critical androgen receptor signaling in these androgen - dependent cells . similarly , nuclear androgen receptor was not observed in the 3d mda pca 118b tumoroids as would be expected in in vivo , indicating that these 3d pdx models may have utility as drug - testing platforms evaluating androgen receptor - targeting agents currently under intense clinical investigation . however , the dichotomy in drug efficacy between preclinical and clinical testing is increasingly apparent , primarily attributed to the genetic and epigenetic changes that accrue when cells acclimatize to the in vitro culture environment in extended periods of cell culture . evaluated the multidrug resistance transcriptome of six cancer types and found no correlation between clinical samples and established cell lines , underscoring the need for new in vitro cancer models and primary tumor models . hypothesizing that inherent differences exist between traditional pca cell lines and pdx pca tumors , we evaluated the response of the 3d pdx pca tumoroids ( mda pca 183 and 118b ) to docetaxel and compared their response to the c4 - 2b bone metastatic prostate cancer cell line . one such example is in colorectal cancer , where it has been shown that primary colorectal cancer cells can be cultured as 3d spheroids or colospheres in vitro , enabling the manipulation of the cancer cells before engraftment in vivo for controlled studies . in our current study , we demonstrate for the first time that primary bone metastatic pca cells can similarly be cultured in vitro with the use of a 3d ha - based hydrogel system . lastly , this 3d pdx model may ultimately be adapted to a rapid and high throughput platform for assessing drug efficacy , rational drug combinations , and development of predictive biomarkers for novel targeted therapies , while reducing the need for low throughput animal hosts . ultimately , it is envisioned that the use of 3d pdx models may greatly accelerate the advancement of novel drug candidates together with predictive biomarkers that enable patient selection when translated into early phase clinical trials . on the basis of previous success culturing bone metastatic pca cell lines using 3d ha - based hydrogels , we demonstrate for the first time that never in 2d pdx pca tumors can be cultured in vitro and maintained for at least 2 weeks . while it has yet to be tested , this system promises the possibility of culturing tumor tissue directly from the patient for rapid drug screening , thereby eliminating the middle mouse and its associated problems , a major leap toward personalized medicine .

organisms often employ more than one mechanism to accomplish a task . for instance , animals typically navigate with multiple input channels. the classic example is homing by the rock dove columba livia , for which magnetic fields , the sun , landmarks and geophysical processes have all been shown to be used ( wiltschko and wiltschko 2003 ) . the range of behaviours or mechanisms organisms employ may be puzzling . at times , an apparent simplicity is observed . either have chemical defences to ward off herbivores or have symbiotic relationships with protective ants ( rehr et al . 1973 ) . hosts parasitised by eurasian cuckoos cuculus canorus famously reject cuckoo eggs , but never reject cuckoo chicks ( davies 2000 ) . this simplicity may be the result of evolutionary lag but , more interestingly , may also be caused by one strategy making another maladaptive by reducing predator abundance ( planqu et al . different mechanisms may complement one another , and true redundancy is often hard to show ( able and bingman 1987 ) . indeed , the existence of a suite of mechanisms against a broad ensemble of predators is readily understandable . another striking example of a system in which different mechanisms augment and complement one another , but now at a collective level , is house hunting in social insects . this has become one of the model systems to study distributed decision making in animals . when the nest is destroyed , the colony has to decide collectively where to settle next during a time of crisis ( franks et al . individual ants or bees have been shown to combine sophisticated assessments of potential nest sites ( seeley 1977 ; seeley and morse 1978 ; mallon and franks 2000 ; franks et al . 2003b ) with various recruitment mechanisms to collate information , and thus make collective decisions ( mallon et al . 2001 ; pratt et al . 2002 ; pratt 2005 ; seeley and visscher 2003 , 2004 ; visscher 2007 ) . when the old nest is destroyed , a fraction of ants goes out scouting to find a new home . upon finding a nest , the nest is assessed ( mallon and franks 2000 ) and ants start recruiting other ants to it with a latency that is inversely proportional to the perceived nest quality ( mallon et al . 2001 ) , using a process called forward tandem running ( mglich et al . 1974 ) . during a forward tandem run , a knowledgeable ant teams up with a nave ant . the leader slowly progresses towards the new nest , each time waiting for the follower to catch up , thereby teaching her the way ( franks and richardson 2006 ) . through this slow recruitment process , once a nest population reaches a certain quorum threshold , the recruiters switch from slow tandem running to much faster social carrying , and transport the remaining passive ants and brood to the new nests ( pratt et al . this description has been the basis of several models ( pratt et al . 2002 ; pratt et al . . however , a behaviour commonly employed by these ants is usually not included ( but see pratt et al . recruiter ants are not only engaged in social carrying , but also regularly perform tandem runs from the new back to the old nest . these so - called reverse tandem runs ( mglich 1978 ) are often more common than forward tandem runs ( mallon et al . 2002 ) , but their function is much less well understood . to maximise fitness , the colony should emigrate as quickly as possible to avoid predation and other hazards . therefore , during house hunting , a fast build up of recruiters is essential . why then do ants mix fast carrying with slow reverse tandem running , when they already have forward tandem running at their disposal ? in particular , through the use of mathematical models , we explore under what circumstances reverse tandem running can have a positive influence on emigration speed . we present two mathematical models to investigate the possible role of reverse tandem runs in ant colony emigrations . reverse tandem running does not contribute to the decision - making process of which new nest to choose ( pratt et al . 2002 ; franks et al . only a small fraction of the ants in a colony are actively involved during emigrations ( pratt et al . 2002 ; langridge 2006 ) . in this paper , we thus divide the colony s n ants into fractions fn of active ants a , and ( 1 f)n passive ants p. this assumption to divide ants into active and passive is a crucial one , without which the models collapse . the rates at which active ants leave the nest and become scouts , and scouts recruiters , are given by and k , respectively . forward tandem running occurs at a rate until the quorum q is met , after which recruiters carry passive ants and brood at a rate . to incorporate reverse tandem runs , we need to model which ants follow these tandem runs . available data from nest - choice experiments ( mallon et al . 2001 ) suggest two possibilities : either the reverse tandem runs are followed by ants that have not found the new nest yet , or by ants that have been carried to the new nest . these two options are not necessarily mutually exclusive : the carried ants could have been scouts . model 1 assumes that reverse tandem runs are followed by uncommitted scouts in the arena , and model 2 assumes they are followed by passive ants that were carried to the new nest . 1diagrams of the two different models for which reverse tandem runs are hypothesised to increase speed . the parameters are explained in table 1 diagrams of the two different models for which reverse tandem runs are hypothesised to increase speed . the parameters are explained in table 1 to capture in detail the influence of reverse tandem runs on emigration dynamics , we need to consider the following points : both tandem running and social carrying involve a pair of ants from two different classes . hence , recruitment can only occur if ants of both participating classes are available;once the quorum has been met , recruiters can not carry and perform reverse tandem runs simultaneously ( we also assume recruiters are not involved in other activities than these two ) . both tandem running and social carrying involve a pair of ants from two different classes . hence , recruitment can only occur if ants of both participating classes are available ; once the quorum has been met , recruiters can not carry and perform reverse tandem runs simultaneously ( we also assume recruiters are not involved in other activities than these two ) . we assume that ants of both classes are well mixed in the part of the arena ( or nest ) where they meet . with populations of ants of size x and y meeting , the number of ants that on average meet is then proportional to xy/(x + y ) . importantly , the smallest class limits the interaction rate , as is to be expected . we thus also have to specify how much post - quorum time recruiters spend on carrying or reverse tandem running ( they are assumed not to spend any time on other behaviours ) . before the quorum is met , the rate at which active ants at the old nest , a , become recruiters , r , through tandem running is given by ra/(a + r ) . now let f be the fraction of post - quorum time spent on reverse tandem runs , and the remainder 1 f spent on social carrying . then the mean number of scouts becoming recruiters through reverse tandem runs is 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f\lambda rs/(s+r).$$\end{document}for carried ants becoming recruiters through reverse tandem running , we have by analogy 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f\lambda rc/(c+r).$$\end{document}similarly , the mean number of passive ants p that become carried ants is given by ( 1 f ) rp/(p + r ) . recruiter ants should not perform reverse tandem runs when there are no scouts or carried ants left . therefore , we replace f by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar f$\end{document } in eqs . 1 and 2 , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar f = \min\{s f , f\}$\end{document } and min { cf , f } , respectively . the min operation is for computational reasons only and ensures that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar f$\end{document } decreases continuously but rapidly to zero as s or c decreases , respectively . we drop the bar on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar f$\end{document } in the rest of the paper . forward tandem running only occurs before the quorum is met and carrying and reverse tandem running only after . these are modelled with functions l , c and r , respectively , as follows . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { l{\left ( { \lambda , r , q , a } \right ) } = } { \left\ { { \begin{array}{*{20}l } { { \lambda \frac{{ra}}{{r + a } } } \hfill } & { { { \text{if}}\,r < q , } \hfill } \\ { 0 \hfill } & { { { \text{otherwise } } , } \hfill } \\ \end{array } } \right . } \\ { c{\left ( { \phi , r , q , p } \right ) } = } { \left\ { { \begin{array}{*{20}l } { { \phi \frac{{rp}}{{r + p } } } \hfill } & { { { \text{if}}\,r \geqslant q , } \hfill } \\ { 0 \hfill } & { { { \text{otherwise } } , } \hfill } \\ \end{array } } \right . } \\ \end{array}$$\end{document}and , setting b to a for model 1 , and to p for model 2 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r(\lambda , r , q , b ) = \left\ { \begin{array}{ll } \lambda \frac{rb}{r+b}&\quad\text{if } r \ge q,\\ 0 & \quad\text{otherwise}. \end{array } \right.$$\end{document}to aid the reader , we state the full equations for both models . the equations for model 1 are given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\ { \begin{array}{ll } \dot a & = -\mu a - l(\lambda , r , q , a),\\ \dot s & = \mu a - ks - f r(\lambda , r , q , s)\\ \dot r & = ks + l(\lambda , r , q , a ) + f r(\lambda , r , q , s)\\ \dot p & = -(1-f ) c(\phi , r , q , p),\\ \dot c & = ( 1-f ) c(\phi , r , q , p ) , \end{array } \right.$$\end{document}with initial conditions ( a , s , r , p , c)(0 ) = ( fn 2 , ,,(1 f)n,0 ) . model 2 is specified by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\ { \begin{array}{ll } \dot a & = -\mu a -l(\lambda , r , q , a),\\ \dot s & = \mu a - ks,\\ \dot r & = ks + l(\lambda , r , q , a ) + f r(\lambda , r , q , c),\\ \dot p & = -(1-f ) c(\phi , r , q , p),\\ \dot c & = ( 1-f ) c(\phi , r , q , p ) - f r(\lambda , r , q , c ) , \end{array } \right.$$\end{document}with the same initial conditions as model 1 . models 1 and 2 only differ in the placement of the term fr(,r , q , c ) . in both models , the in the initial conditions is necessary to avoid singularities in the denominators of the interaction functions l , r and c. we have used = 0.01 throughout . the main hypothesis we tested on both these models was : reverse tandem runs speed up the emigration if recruiter numbers failed to increase early in the emigration ; this occurs through a combination of the new nest being hard to find and forward tandem running being prohibited . we tested this hypothesis by finding the fastest emigration strategy for given parameter settings and determining whether reverse tandem runs formed part of this optimal strategy . we first fixed the scouting parameter , , the fraction of active ants at the old nest , f , and k , the rate at which scouts become recruiters . then quorum size q and fraction of post - quorum reverse tandem running time f were varied to optimise emigration speed . emigrations were termed completed when all passive ants and active ants at the old nest had disappeared . in particular , we set the threshold for emigration completeness at p + a = 0.01 . optimal strategies were found using the nelder mead simplex method ( nelder and mead 1965 ) . parameters and f were varied on an equidistant 2020 grid spanning [ 0.01,0.2 ] [0.05,0.5 ] . the ranges of these parameters are inspired by experimental estimates in ( pratt et al . table 1values or ranges , where applicable , for the parameters used in models 1 and 2 depicted in fig . 1parametersdescriptionvalue / rangencolony size250ffraction of active ants[0.05,0.5]qquorum thresholdn.a.ffraction of post - quorum reverse tandem running timen.a.rate at which active ants at old nest become scouts ( ant min)[0.01,0.2]rate at which ants following tandem runs become recruiters ( ant min)0.1rate at which passive ants are carried to new nest ( ant min)0.2krate at which scouts independently become recruiters ( ant min){0.0001,0.001}parameter choices for , and n were taken from the ranges in pratt et al . values or ranges , where applicable , for the parameters used in models 1 and 2 depicted in fig . 1 parameter choices for , and n were taken from the ranges in pratt et al . for both models , the optimal emigration strategy included reverse tandem runs for a wide range of parameters , together with low quorum thresholds ( fig . 2 ) . fixing k whilst varying and f , the optimal strategy often contained more reverse than forward tandem runs for a large part of the parameter range ( fig . 2 ) . the fraction of time spent on reverse tandem running f and the quorum threshold q were negatively correlated . when either the fraction of active ants f decreased or the scouting parameter increased , fraction f increased , and the quorum q decreased . choosing a higher recruitment latency by decreasing k gave more reverse tandem running and lower quorum thresholds ( fig . 2 ) . 2optimal fractions of post - quorum time spent on reverse tandem runs ( top figures ) , and optimal quorum thresholds ( bottom figures ) for models 1 ( left two columns ) and 2 ( right two columns ) for varying fractions of active ants f and scouting probabilities and for two values of recruitment latencies k. other parameter values used are given in table 1 . reverse tandem runs are part of the optimal emigration strategy when scouting probability is high or when fraction of active ants is low . lowering k enhances the use of reverse tandem running optimal fractions of post - quorum time spent on reverse tandem runs ( top figures ) , and optimal quorum thresholds ( bottom figures ) for models 1 ( left two columns ) and 2 ( right two columns ) for varying fractions of active ants f and scouting probabilities and for two values of recruitment latencies k. other parameter values used are given in table 1 . reverse tandem runs are part of the optimal emigration strategy when scouting probability is high or when fraction of active ants is low . lowering k enhances the use of reverse tandem running note that , although models 1 and 2 broadly give similar predictions , they differ in the amount of post - quorum time spent on reverse tandem runs . in model 1 , this reaches a full 100% in model 1 , but never so in model 2 . overall , the models predict that reverse tandem running should be used more than forward tandem running , and the quorum threshold lowered , if the recruitment latency increases by decreasing k ( scouting ants wait longer before starting their first recruitment act ) , in combination with either a decreasing fraction of active ants f , or an increasing scouting parameter . for all but very large f , the optimal quorum threshold corresponded to the time when all active ants have left the old nest to go scouting . in the absence of multiple new nests , the decision when to switch from forward tandem running to social carrying is thus best made at the old nest . recruiters should thus apply the following rule : continue forward tandem running until there are no ants left to perform them with and then switch to social carrying ; if few forward tandem runs have been performed ( by the recruiters ) , combine carrying with reverse tandem runs ; otherwise , do not . the numbers of forward tandem runs , reverse tandem runs , the numbers of carried ants in the new nest and total emigration time were computed for each of the optimal strategies of models 1 and 2 ( fig . first , for a large parameter range , there are more reverse tandem runs than forward tandem runs . this is broadly consistent with the experimental data from nest choice experiments in mallon et al . numbers there range between 3 and 17 forward tandems , and between 9 and 25 reverse tandems , and reverse tandems were always performed more often than forward tandems . second , as a validation of our optimisation method , note that the optimal emigration time varies smoothly under parameter changes , as is to be expected for this type of model . third , for model 1 , despite a clear drop in the post - quorum time spent on reverse tandem runs with increasing f ( see fig . 2 , top left ) , the number of reverse tandem runs in fact varies smoothly . fourth , the number of carried ants that remained in the nest at the end of the emigration is clearly different between models 1 and 2 . in model 1 , this number is just ( 1 f)n , the number of passive ants in the colony . in model 2 , however , over half of the colony may end up being recruiters by drawing recruits from the carried class using reverse tandem runs . 3numbers of forward and reverse tandem runs , number of carried ants and emigration time , computed for each of the optimal strategies for models 1 ( top row ) and 2 ( bottom row ) , illustrated in fig . 2 . here we have only illustrated k = 0.001 numbers of forward and reverse tandem runs , number of carried ants and emigration time , computed for each of the optimal strategies for models 1 ( top row ) and 2 ( bottom row ) , illustrated in fig . to maximise their fitness , ants should try to achieve the fastest emigrations to minimise vulnerability ( franks et al . therefore , the active ants either have to become scouts , discover a new site and then become recruiters or wait at the old nest until a recruiter leads them to the new nest . both of these processes may be hampered : when all the active ants go out scouting , recruiter numbers slowly increase if the new nest is hard to find or if those few can not find any active ant back at the old nest to tandem run with . in terms of the models , this could occur if scouts slowly become recruiters ( low value for k ) , in combination with either a small class of active ants at the old nest ( f is small ) or all active ants having gone scouting ( high value for scouting rate ) . under either or both of these circumstances , the model predicts that ants should not waste time trying to recruit by forward tandem runs but should do the next best thing and use a low quorum threshold to quickly switch to carrying . the recruiters should then invest a fraction of their time to recruit scouts or carried ants using reverse tandem runs , thus boosting the recruiter population and speeding up the emigration . conversely , the model also predicts that reverse tandem runs should not be used if either the new nest is easy to find ( recruiter numbers then build quickly anyway ) , or when there are many ants to follow a forward tandem run . ants have been shown to leave their intact old nest if the new nest is sufficiently better , but have lower standards when their nest is destroyed ( dornhaus et al . reverse tandem runs were mainly observed when the old nest was destroyed , combined with few forward tandem runs . the model offers a simple explanation for this : the greater panic might have caused fewer scouts to remain at the old nest , thereby obstructing early recruitment . whilst investigating speed accuracy trade - offs , franks et al . , this difference in reverse tandem running activity between mild and harsher conditions was found to be non - significant ( franks et al . although evidence for which ants follow reverse tandem runs is scarce , both models give the same qualitative predictions . note , however , that the dynamics of the different ant classes during a simulated emigration in model 2 poorly match observed experimental dynamics ( see , e.g. , planqu et al . the final number of ants active in an emigration is much greater than the original number of active ants . these recruiter numbers far exceed observed numbers of active ants ( available in table 3 in pratt et al . indeed , simulated emigrations often end with half the colony being recruiters and less than half carried into the new nest ( fig . 3 , bottom row , third from left ) . 4examples of temporal dynamics for models 1 and 2 . at = 0.05 , f = 0.1447 , we have taken parameters optimal for models 1 and 2 , respectively . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics examples of temporal dynamics for models 1 and 2 . at = 0.05 , f = 0.1447 , we have taken parameters optimal for models 1 and 2 , respectively . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics figure 2 ( top left ) shows that recruiters in model 1 should use a sequential strategy if f is small ( and is large ) : when the quorum is met , they first spend all of their time on reverse tandem runs until all scouts have become recruiters , and then switch to carrying . in contrast , the total number of recruiters is bounded by fn , the number of active ants in the colony . as f decreases , the remaining recruiters take longer to carry all the passive ants . hence , the time costs for not having the recruiters increases and the time to recruit the remaining scouts decreases ( as there are fewer scouts too ) . hence , in this situation , recruiters should devote their post - quorum time , first , all on reverse tandem running before starting carrying . one does not have to make many hands if the work was light to start with . note that this behaviour for f 0 is different for model 2 . in this paper top right ) , as there is no end to building recruiter numbers but by completing the entire emigration . this argument also explains another difference between these models : the number of reverse tandem runs during an emigration . in model 1 , there is a clear maximum for intermediate f , whereas in model 2 the number of reverse tandems strictly decreases with f ( fig . 3 , second from left , top and bottom ) . contrary to the models in this paper , two previous models of house hunting by t. albipennis ants ( pratt et al . the predictions of the current models proved to be strongly dependent on the assumption of non - linearity of these terms . the corresponding linear models predicted that reverse tandem running should not be used for practically any parameter choices in f and or k. another ingredient in this model shared with the previous ( pratt et al . 2006 ) , models of this collective decision - making system is the division between active and passive ants . all we know at present is that a limited fraction of ants is actively engaged during an emigration . we have thus also explored models in which this division was absent , using both linear and non - linear interaction terms such as those in models 1 and 2 presented in this paper . in none of these models the division between active and passive ants is thus a crucial ingredient for reverse tandem running to have a positive impact on emigration speed , which should be experimentally validated . several hypotheses on the potential role of reverse tandem running have been put forward ( pratt et al . 2002 ) . first , the ants might have a home nest , which changes during the emigration , thereby reversing the direction of any recruitment events from home to another nest ( pratt et al . if true , this would predict a change of direction when about half of the colony had been displaced . moreover , this hypothesis does not offer a suggestion why tandem running often occurs early in the emigration . in other words , it might explain the direction , but not the occurrence itself . second , it has been suggested that reverse tandem running may re - allocate recruitment ( pratt et al . reverse tandem runs were nearly always observed between the best nest and the old nest . the models in this paper do not incorporate choice between nest sites , but we conjecture that early flexible commitment ( planqu et al . 2006 ) will be more efficient in redirecting ants to better nests than late recruitment . other experimental results also corroborate that reverse tandem running does not influence the decision - making process ( franks et al . the first models in which reverse tandem runs have been explicitly incorporated to analyse their role have yielded clear predictions : under a range of conditions , we expect a negative correlation between levels of early and late recruitment . this finding lends itself well to simple experiments , and we aim to present those in the near future . the build up of recruiter numbers serves two purposes : to decide on a nest and to increase the number of ants actively involved in transport . this in itself is a side - effect of the distributed nature of this system . this suggests that such additional backup behaviours could be a common feature of decentralised collective decision - making systems . although evidence for which ants follow reverse tandem runs is scarce , both models give the same qualitative predictions . note , however , that the dynamics of the different ant classes during a simulated emigration in model 2 poorly match observed experimental dynamics ( see , e.g. , planqu et al . the final number of ants active in an emigration is much greater than the original number of active ants . these recruiter numbers far exceed observed numbers of active ants ( available in table 3 in pratt et al . indeed , simulated emigrations often end with half the colony being recruiters and less than half carried into the new nest ( fig . 3 , bottom row , third from left ) . 4examples of temporal dynamics for models 1 and 2 . at = 0.05 , f = 0.1447 , we have taken parameters optimal for models 1 and 2 , respectively . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics examples of temporal dynamics for models 1 and 2 . at = 0.05 , f = 0.1447 , we have taken parameters optimal for models 1 and 2 , respectively . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics figure 2 ( top left ) shows that recruiters in model 1 should use a sequential strategy if f is small ( and is large ) : when the quorum is met , they first spend all of their time on reverse tandem runs until all scouts have become recruiters , and then switch to carrying . the total number of recruiters is bounded by fn , the number of active ants in the colony . as f decreases , the remaining recruiters take longer to carry all the passive ants . hence , the time costs for not having the recruiters increases and the time to recruit the remaining scouts decreases ( as there are fewer scouts too ) . hence , in this situation , recruiters should devote their post - quorum time , first , all on reverse tandem running before starting carrying . one does not have to make many hands if the work was light to start with . note that this behaviour for f 0 is different for model 2 . in this paper top right ) , as there is no end to building recruiter numbers but by completing the entire emigration . this argument also explains another difference between these models : the number of reverse tandem runs during an emigration . in model 1 , there is a clear maximum for intermediate f , whereas in model 2 the number of reverse tandems strictly decreases with f ( fig . 3 , second from left , top and bottom ) . contrary to the models in this paper , two previous models of house hunting by t. albipennis ants ( pratt et al . the predictions of the current models proved to be strongly dependent on the assumption of non - linearity of these terms . the corresponding linear models predicted that reverse tandem running should not be used for practically any parameter choices in f and or k. another ingredient in this model shared with the previous ( pratt et al . 2006 ) , models of this collective decision - making system is the division between active and passive ants . all we know at present is that a limited fraction of ants is actively engaged during an emigration . we have thus also explored models in which this division was absent , using both linear and non - linear interaction terms such as those in models 1 and 2 presented in this paper . in none of these models the division between active and passive ants is thus a crucial ingredient for reverse tandem running to have a positive impact on emigration speed , which should be experimentally validated . several hypotheses on the potential role of reverse tandem running have been put forward ( pratt et al . 2002 ) . first , the ants might have a home nest , which changes during the emigration , thereby reversing the direction of any recruitment events from home to another nest ( pratt et al . if true , this would predict a change of direction when about half of the colony had been displaced . moreover , this hypothesis does not offer a suggestion why tandem running often occurs early in the emigration . in other words , it might explain the direction , but not the occurrence itself . second , it has been suggested that reverse tandem running may re - allocate recruitment ( pratt et al . reverse tandem runs were nearly always observed between the best nest and the old nest . the models in this paper do not incorporate choice between nest sites , but we conjecture that early flexible commitment ( planqu et al . 2006 ) will be more efficient in redirecting ants to better nests than late recruitment . other experimental results also corroborate that reverse tandem running does not influence the decision - making process ( franks et al . the first models in which reverse tandem runs have been explicitly incorporated to analyse their role have yielded clear predictions : under a range of conditions , we expect a negative correlation between levels of early and late recruitment . this finding lends itself well to simple experiments , and we aim to present those in the near future . the build up of recruiter numbers serves two purposes : to decide on a nest and to increase the number of ants actively involved in transport . the decision - making process and the implementation of this decision are thus conflated . this in itself is a side - effect of the distributed nature of this system . this suggests that such additional backup behaviours could be a common feature of decentralised collective decision - making systems .

to perform tasks , organisms often use multiple procedures . explaining the breadth of such behavioural repertoires is not always straightforward . during house hunting , colonies of temnothorax albipennis ants use a range of behaviours to organise their emigrations . in particular , the ants use tandem running to recruit nave ants to potential nest sites . initially , they use forward tandem runs ( ftrs ) in which one leader takes a single follower along the route from the old nest to the new one . later , they use reverse tandem runs ( rtrs ) in the opposite direction . tandem runs are used to teach active ants the route between the nests , so that they can be involved quickly in nest evaluation and subsequent recruitment . when a quorum of decision - makers at the new nest is reached , they switch to carrying nestmates . this is three times faster than tandem running . as a rule , having more ftrs early should thus mean faster emigrations , thereby reducing the colony s vulnerability . so why do ants use rtrs , which are both slow and late ? it would seem quicker and simpler for the ants to use more ftrs ( and higher quorums ) to have enough knowledgeable ants to do all the carrying . in this study , we present the first testable theoretical explanation for the role of rtrs . we set out to find the theoretically fastest emigration strategy for a set of emigration conditions . we conclude that rtrs can have a positive effect on emigration speed if ftrs are limited . in these cases , low quorums together with lots of reverse tandem running give the fastest emigration .

Introduction Materials and methods Results Discussion Critique on model 2 Reverse tandem activity Nonlinearities in the models and divisions between active and passive ants Hypothesised explanations

the range of behaviours or mechanisms organisms employ may be puzzling . another striking example of a system in which different mechanisms augment and complement one another , but now at a collective level , is house hunting in social insects . when the nest is destroyed , the colony has to decide collectively where to settle next during a time of crisis ( franks et al . individual ants or bees have been shown to combine sophisticated assessments of potential nest sites ( seeley 1977 ; seeley and morse 1978 ; mallon and franks 2000 ; franks et al . when the old nest is destroyed , a fraction of ants goes out scouting to find a new home . upon finding a nest , the nest is assessed ( mallon and franks 2000 ) and ants start recruiting other ants to it with a latency that is inversely proportional to the perceived nest quality ( mallon et al . 2001 ) , using a process called forward tandem running ( mglich et al . the leader slowly progresses towards the new nest , each time waiting for the follower to catch up , thereby teaching her the way ( franks and richardson 2006 ) . through this slow recruitment process , once a nest population reaches a certain quorum threshold , the recruiters switch from slow tandem running to much faster social carrying , and transport the remaining passive ants and brood to the new nests ( pratt et al . recruiter ants are not only engaged in social carrying , but also regularly perform tandem runs from the new back to the old nest . these so - called reverse tandem runs ( mglich 1978 ) are often more common than forward tandem runs ( mallon et al . to maximise fitness , the colony should emigrate as quickly as possible to avoid predation and other hazards . therefore , during house hunting , a fast build up of recruiters is essential . why then do ants mix fast carrying with slow reverse tandem running , when they already have forward tandem running at their disposal ? in particular , through the use of mathematical models , we explore under what circumstances reverse tandem running can have a positive influence on emigration speed . we present two mathematical models to investigate the possible role of reverse tandem runs in ant colony emigrations . reverse tandem running does not contribute to the decision - making process of which new nest to choose ( pratt et al . in this paper , we thus divide the colony s n ants into fractions fn of active ants a , and ( 1 f)n passive ants p. this assumption to divide ants into active and passive is a crucial one , without which the models collapse . to incorporate reverse tandem runs , we need to model which ants follow these tandem runs . 2001 ) suggest two possibilities : either the reverse tandem runs are followed by ants that have not found the new nest yet , or by ants that have been carried to the new nest . model 1 assumes that reverse tandem runs are followed by uncommitted scouts in the arena , and model 2 assumes they are followed by passive ants that were carried to the new nest . 1diagrams of the two different models for which reverse tandem runs are hypothesised to increase speed . the parameters are explained in table 1 diagrams of the two different models for which reverse tandem runs are hypothesised to increase speed . the parameters are explained in table 1 to capture in detail the influence of reverse tandem runs on emigration dynamics , we need to consider the following points : both tandem running and social carrying involve a pair of ants from two different classes . hence , recruitment can only occur if ants of both participating classes are available;once the quorum has been met , recruiters can not carry and perform reverse tandem runs simultaneously ( we also assume recruiters are not involved in other activities than these two ) . hence , recruitment can only occur if ants of both participating classes are available ; once the quorum has been met , recruiters can not carry and perform reverse tandem runs simultaneously ( we also assume recruiters are not involved in other activities than these two ) . we thus also have to specify how much post - quorum time recruiters spend on carrying or reverse tandem running ( they are assumed not to spend any time on other behaviours ) . before the quorum is met , the rate at which active ants at the old nest , a , become recruiters , r , through tandem running is given by ra/(a + r ) . now let f be the fraction of post - quorum time spent on reverse tandem runs , and the remainder 1 f spent on social carrying . then the mean number of scouts becoming recruiters through reverse tandem runs is 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f\lambda rs/(s+r).$$\end{document}for carried ants becoming recruiters through reverse tandem running , we have by analogy 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f\lambda rc/(c+r).$$\end{document}similarly , the mean number of passive ants p that become carried ants is given by ( 1 f ) rp/(p + r ) . forward tandem running only occurs before the quorum is met and carrying and reverse tandem running only after . in both models , the in the initial conditions is necessary to avoid singularities in the denominators of the interaction functions l , r and c. we have used = 0.01 throughout . the main hypothesis we tested on both these models was : reverse tandem runs speed up the emigration if recruiter numbers failed to increase early in the emigration ; this occurs through a combination of the new nest being hard to find and forward tandem running being prohibited . we tested this hypothesis by finding the fastest emigration strategy for given parameter settings and determining whether reverse tandem runs formed part of this optimal strategy . we first fixed the scouting parameter , , the fraction of active ants at the old nest , f , and k , the rate at which scouts become recruiters . then quorum size q and fraction of post - quorum reverse tandem running time f were varied to optimise emigration speed . emigrations were termed completed when all passive ants and active ants at the old nest had disappeared . in particular , we set the threshold for emigration completeness at p + a = 0.01 . 1parametersdescriptionvalue / rangencolony size250ffraction of active ants[0.05,0.5]qquorum thresholdn.a.ffraction of post - quorum reverse tandem running timen.a.rate at which active ants at old nest become scouts ( ant min)[0.01,0.2]rate at which ants following tandem runs become recruiters ( ant min)0.1rate at which passive ants are carried to new nest ( ant min)0.2krate at which scouts independently become recruiters ( ant min){0.0001,0.001}parameter choices for , and n were taken from the ranges in pratt et al . for both models , the optimal emigration strategy included reverse tandem runs for a wide range of parameters , together with low quorum thresholds ( fig . fixing k whilst varying and f , the optimal strategy often contained more reverse than forward tandem runs for a large part of the parameter range ( fig . 2optimal fractions of post - quorum time spent on reverse tandem runs ( top figures ) , and optimal quorum thresholds ( bottom figures ) for models 1 ( left two columns ) and 2 ( right two columns ) for varying fractions of active ants f and scouting probabilities and for two values of recruitment latencies k. other parameter values used are given in table 1 . reverse tandem runs are part of the optimal emigration strategy when scouting probability is high or when fraction of active ants is low . lowering k enhances the use of reverse tandem running optimal fractions of post - quorum time spent on reverse tandem runs ( top figures ) , and optimal quorum thresholds ( bottom figures ) for models 1 ( left two columns ) and 2 ( right two columns ) for varying fractions of active ants f and scouting probabilities and for two values of recruitment latencies k. other parameter values used are given in table 1 . reverse tandem runs are part of the optimal emigration strategy when scouting probability is high or when fraction of active ants is low . lowering k enhances the use of reverse tandem running note that , although models 1 and 2 broadly give similar predictions , they differ in the amount of post - quorum time spent on reverse tandem runs . overall , the models predict that reverse tandem running should be used more than forward tandem running , and the quorum threshold lowered , if the recruitment latency increases by decreasing k ( scouting ants wait longer before starting their first recruitment act ) , in combination with either a decreasing fraction of active ants f , or an increasing scouting parameter . for all but very large f , the optimal quorum threshold corresponded to the time when all active ants have left the old nest to go scouting . in the absence of multiple new nests , the decision when to switch from forward tandem running to social carrying is thus best made at the old nest . recruiters should thus apply the following rule : continue forward tandem running until there are no ants left to perform them with and then switch to social carrying ; if few forward tandem runs have been performed ( by the recruiters ) , combine carrying with reverse tandem runs ; otherwise , do not . the numbers of forward tandem runs , reverse tandem runs , the numbers of carried ants in the new nest and total emigration time were computed for each of the optimal strategies of models 1 and 2 ( fig . first , for a large parameter range , there are more reverse tandem runs than forward tandem runs . third , for model 1 , despite a clear drop in the post - quorum time spent on reverse tandem runs with increasing f ( see fig . 2 , top left ) , the number of reverse tandem runs in fact varies smoothly . fourth , the number of carried ants that remained in the nest at the end of the emigration is clearly different between models 1 and 2 . in model 1 , this number is just ( 1 f)n , the number of passive ants in the colony . in model 2 , however , over half of the colony may end up being recruiters by drawing recruits from the carried class using reverse tandem runs . here we have only illustrated k = 0.001 numbers of forward and reverse tandem runs , number of carried ants and emigration time , computed for each of the optimal strategies for models 1 ( top row ) and 2 ( bottom row ) , illustrated in fig . therefore , the active ants either have to become scouts , discover a new site and then become recruiters or wait at the old nest until a recruiter leads them to the new nest . both of these processes may be hampered : when all the active ants go out scouting , recruiter numbers slowly increase if the new nest is hard to find or if those few can not find any active ant back at the old nest to tandem run with . in terms of the models , this could occur if scouts slowly become recruiters ( low value for k ) , in combination with either a small class of active ants at the old nest ( f is small ) or all active ants having gone scouting ( high value for scouting rate ) . under either or both of these circumstances , the model predicts that ants should not waste time trying to recruit by forward tandem runs but should do the next best thing and use a low quorum threshold to quickly switch to carrying . the recruiters should then invest a fraction of their time to recruit scouts or carried ants using reverse tandem runs , thus boosting the recruiter population and speeding up the emigration . conversely , the model also predicts that reverse tandem runs should not be used if either the new nest is easy to find ( recruiter numbers then build quickly anyway ) , or when there are many ants to follow a forward tandem run . ants have been shown to leave their intact old nest if the new nest is sufficiently better , but have lower standards when their nest is destroyed ( dornhaus et al . reverse tandem runs were mainly observed when the old nest was destroyed , combined with few forward tandem runs . the model offers a simple explanation for this : the greater panic might have caused fewer scouts to remain at the old nest , thereby obstructing early recruitment . , this difference in reverse tandem running activity between mild and harsher conditions was found to be non - significant ( franks et al . although evidence for which ants follow reverse tandem runs is scarce , both models give the same qualitative predictions . indeed , simulated emigrations often end with half the colony being recruiters and less than half carried into the new nest ( fig . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics examples of temporal dynamics for models 1 and 2 . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics figure 2 ( top left ) shows that recruiters in model 1 should use a sequential strategy if f is small ( and is large ) : when the quorum is met , they first spend all of their time on reverse tandem runs until all scouts have become recruiters , and then switch to carrying . in contrast , the total number of recruiters is bounded by fn , the number of active ants in the colony . hence , the time costs for not having the recruiters increases and the time to recruit the remaining scouts decreases ( as there are fewer scouts too ) . hence , in this situation , recruiters should devote their post - quorum time , first , all on reverse tandem running before starting carrying . this argument also explains another difference between these models : the number of reverse tandem runs during an emigration . contrary to the models in this paper , two previous models of house hunting by t. albipennis ants ( pratt et al . the corresponding linear models predicted that reverse tandem running should not be used for practically any parameter choices in f and or k. another ingredient in this model shared with the previous ( pratt et al . we have thus also explored models in which this division was absent , using both linear and non - linear interaction terms such as those in models 1 and 2 presented in this paper . in none of these models the division between active and passive ants is thus a crucial ingredient for reverse tandem running to have a positive impact on emigration speed , which should be experimentally validated . several hypotheses on the potential role of reverse tandem running have been put forward ( pratt et al . first , the ants might have a home nest , which changes during the emigration , thereby reversing the direction of any recruitment events from home to another nest ( pratt et al . moreover , this hypothesis does not offer a suggestion why tandem running often occurs early in the emigration . second , it has been suggested that reverse tandem running may re - allocate recruitment ( pratt et al . reverse tandem runs were nearly always observed between the best nest and the old nest . the models in this paper do not incorporate choice between nest sites , but we conjecture that early flexible commitment ( planqu et al . other experimental results also corroborate that reverse tandem running does not influence the decision - making process ( franks et al . the first models in which reverse tandem runs have been explicitly incorporated to analyse their role have yielded clear predictions : under a range of conditions , we expect a negative correlation between levels of early and late recruitment . although evidence for which ants follow reverse tandem runs is scarce , both models give the same qualitative predictions . indeed , simulated emigrations often end with half the colony being recruiters and less than half carried into the new nest ( fig . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics examples of temporal dynamics for models 1 and 2 . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics figure 2 ( top left ) shows that recruiters in model 1 should use a sequential strategy if f is small ( and is large ) : when the quorum is met , they first spend all of their time on reverse tandem runs until all scouts have become recruiters , and then switch to carrying . the total number of recruiters is bounded by fn , the number of active ants in the colony . hence , in this situation , recruiters should devote their post - quorum time , first , all on reverse tandem running before starting carrying . this argument also explains another difference between these models : the number of reverse tandem runs during an emigration . contrary to the models in this paper , two previous models of house hunting by t. albipennis ants ( pratt et al . the corresponding linear models predicted that reverse tandem running should not be used for practically any parameter choices in f and or k. another ingredient in this model shared with the previous ( pratt et al . we have thus also explored models in which this division was absent , using both linear and non - linear interaction terms such as those in models 1 and 2 presented in this paper . in none of these models the division between active and passive ants is thus a crucial ingredient for reverse tandem running to have a positive impact on emigration speed , which should be experimentally validated . several hypotheses on the potential role of reverse tandem running have been put forward ( pratt et al . first , the ants might have a home nest , which changes during the emigration , thereby reversing the direction of any recruitment events from home to another nest ( pratt et al . moreover , this hypothesis does not offer a suggestion why tandem running often occurs early in the emigration . second , it has been suggested that reverse tandem running may re - allocate recruitment ( pratt et al . reverse tandem runs were nearly always observed between the best nest and the old nest . the models in this paper do not incorporate choice between nest sites , but we conjecture that early flexible commitment ( planqu et al . other experimental results also corroborate that reverse tandem running does not influence the decision - making process ( franks et al . the first models in which reverse tandem runs have been explicitly incorporated to analyse their role have yielded clear predictions : under a range of conditions , we expect a negative correlation between levels of early and late recruitment .

the classic example is homing by the rock dove columba livia , for which magnetic fields , the sun , landmarks and geophysical processes have all been shown to be used ( wiltschko and wiltschko 2003 ) . this simplicity may be the result of evolutionary lag but , more interestingly , may also be caused by one strategy making another maladaptive by reducing predator abundance ( planqu et al . indeed , the existence of a suite of mechanisms against a broad ensemble of predators is readily understandable . another striking example of a system in which different mechanisms augment and complement one another , but now at a collective level , is house hunting in social insects . when the nest is destroyed , the colony has to decide collectively where to settle next during a time of crisis ( franks et al . when the old nest is destroyed , a fraction of ants goes out scouting to find a new home . upon finding a nest , the nest is assessed ( mallon and franks 2000 ) and ants start recruiting other ants to it with a latency that is inversely proportional to the perceived nest quality ( mallon et al . the leader slowly progresses towards the new nest , each time waiting for the follower to catch up , thereby teaching her the way ( franks and richardson 2006 ) . through this slow recruitment process , once a nest population reaches a certain quorum threshold , the recruiters switch from slow tandem running to much faster social carrying , and transport the remaining passive ants and brood to the new nests ( pratt et al . recruiter ants are not only engaged in social carrying , but also regularly perform tandem runs from the new back to the old nest . to maximise fitness , the colony should emigrate as quickly as possible to avoid predation and other hazards . in particular , through the use of mathematical models , we explore under what circumstances reverse tandem running can have a positive influence on emigration speed . we present two mathematical models to investigate the possible role of reverse tandem runs in ant colony emigrations . reverse tandem running does not contribute to the decision - making process of which new nest to choose ( pratt et al . only a small fraction of the ants in a colony are actively involved during emigrations ( pratt et al . in this paper , we thus divide the colony s n ants into fractions fn of active ants a , and ( 1 f)n passive ants p. this assumption to divide ants into active and passive is a crucial one , without which the models collapse . the rates at which active ants leave the nest and become scouts , and scouts recruiters , are given by and k , respectively . forward tandem running occurs at a rate until the quorum q is met , after which recruiters carry passive ants and brood at a rate . 2001 ) suggest two possibilities : either the reverse tandem runs are followed by ants that have not found the new nest yet , or by ants that have been carried to the new nest . model 1 assumes that reverse tandem runs are followed by uncommitted scouts in the arena , and model 2 assumes they are followed by passive ants that were carried to the new nest . 1diagrams of the two different models for which reverse tandem runs are hypothesised to increase speed . the parameters are explained in table 1 diagrams of the two different models for which reverse tandem runs are hypothesised to increase speed . the parameters are explained in table 1 to capture in detail the influence of reverse tandem runs on emigration dynamics , we need to consider the following points : both tandem running and social carrying involve a pair of ants from two different classes . hence , recruitment can only occur if ants of both participating classes are available;once the quorum has been met , recruiters can not carry and perform reverse tandem runs simultaneously ( we also assume recruiters are not involved in other activities than these two ) . hence , recruitment can only occur if ants of both participating classes are available ; once the quorum has been met , recruiters can not carry and perform reverse tandem runs simultaneously ( we also assume recruiters are not involved in other activities than these two ) . we assume that ants of both classes are well mixed in the part of the arena ( or nest ) where they meet . with populations of ants of size x and y meeting , the number of ants that on average meet is then proportional to xy/(x + y ) . before the quorum is met , the rate at which active ants at the old nest , a , become recruiters , r , through tandem running is given by ra/(a + r ) . now let f be the fraction of post - quorum time spent on reverse tandem runs , and the remainder 1 f spent on social carrying . then the mean number of scouts becoming recruiters through reverse tandem runs is 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f\lambda rs/(s+r).$$\end{document}for carried ants becoming recruiters through reverse tandem running , we have by analogy 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f\lambda rc/(c+r).$$\end{document}similarly , the mean number of passive ants p that become carried ants is given by ( 1 f ) rp/(p + r ) . the equations for model 1 are given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\ { \begin{array}{ll } \dot a & = -\mu a - l(\lambda , r , q , a),\\ \dot s & = \mu a - ks - f r(\lambda , r , q , s)\\ \dot r & = ks + l(\lambda , r , q , a ) + f r(\lambda , r , q , s)\\ \dot p & = -(1-f ) c(\phi , r , q , p),\\ \dot c & = ( 1-f ) c(\phi , r , q , p ) , \end{array } \right.$$\end{document}with initial conditions ( a , s , r , p , c)(0 ) = ( fn 2 , ,,(1 f)n,0 ) . model 2 is specified by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\ { \begin{array}{ll } \dot a & = -\mu a -l(\lambda , r , q , a),\\ \dot s & = \mu a - ks,\\ \dot r & = ks + l(\lambda , r , q , a ) + f r(\lambda , r , q , c),\\ \dot p & = -(1-f ) c(\phi , r , q , p),\\ \dot c & = ( 1-f ) c(\phi , r , q , p ) - f r(\lambda , r , q , c ) , \end{array } \right.$$\end{document}with the same initial conditions as model 1 . in both models , the in the initial conditions is necessary to avoid singularities in the denominators of the interaction functions l , r and c. we have used = 0.01 throughout . the main hypothesis we tested on both these models was : reverse tandem runs speed up the emigration if recruiter numbers failed to increase early in the emigration ; this occurs through a combination of the new nest being hard to find and forward tandem running being prohibited . we first fixed the scouting parameter , , the fraction of active ants at the old nest , f , and k , the rate at which scouts become recruiters . 1parametersdescriptionvalue / rangencolony size250ffraction of active ants[0.05,0.5]qquorum thresholdn.a.ffraction of post - quorum reverse tandem running timen.a.rate at which active ants at old nest become scouts ( ant min)[0.01,0.2]rate at which ants following tandem runs become recruiters ( ant min)0.1rate at which passive ants are carried to new nest ( ant min)0.2krate at which scouts independently become recruiters ( ant min){0.0001,0.001}parameter choices for , and n were taken from the ranges in pratt et al . for both models , the optimal emigration strategy included reverse tandem runs for a wide range of parameters , together with low quorum thresholds ( fig . fixing k whilst varying and f , the optimal strategy often contained more reverse than forward tandem runs for a large part of the parameter range ( fig . when either the fraction of active ants f decreased or the scouting parameter increased , fraction f increased , and the quorum q decreased . 2optimal fractions of post - quorum time spent on reverse tandem runs ( top figures ) , and optimal quorum thresholds ( bottom figures ) for models 1 ( left two columns ) and 2 ( right two columns ) for varying fractions of active ants f and scouting probabilities and for two values of recruitment latencies k. other parameter values used are given in table 1 . lowering k enhances the use of reverse tandem running optimal fractions of post - quorum time spent on reverse tandem runs ( top figures ) , and optimal quorum thresholds ( bottom figures ) for models 1 ( left two columns ) and 2 ( right two columns ) for varying fractions of active ants f and scouting probabilities and for two values of recruitment latencies k. other parameter values used are given in table 1 . lowering k enhances the use of reverse tandem running note that , although models 1 and 2 broadly give similar predictions , they differ in the amount of post - quorum time spent on reverse tandem runs . overall , the models predict that reverse tandem running should be used more than forward tandem running , and the quorum threshold lowered , if the recruitment latency increases by decreasing k ( scouting ants wait longer before starting their first recruitment act ) , in combination with either a decreasing fraction of active ants f , or an increasing scouting parameter . in the absence of multiple new nests , the decision when to switch from forward tandem running to social carrying is thus best made at the old nest . the numbers of forward tandem runs , reverse tandem runs , the numbers of carried ants in the new nest and total emigration time were computed for each of the optimal strategies of models 1 and 2 ( fig . second , as a validation of our optimisation method , note that the optimal emigration time varies smoothly under parameter changes , as is to be expected for this type of model . third , for model 1 , despite a clear drop in the post - quorum time spent on reverse tandem runs with increasing f ( see fig . fourth , the number of carried ants that remained in the nest at the end of the emigration is clearly different between models 1 and 2 . in model 1 , this number is just ( 1 f)n , the number of passive ants in the colony . 3numbers of forward and reverse tandem runs , number of carried ants and emigration time , computed for each of the optimal strategies for models 1 ( top row ) and 2 ( bottom row ) , illustrated in fig . here we have only illustrated k = 0.001 numbers of forward and reverse tandem runs , number of carried ants and emigration time , computed for each of the optimal strategies for models 1 ( top row ) and 2 ( bottom row ) , illustrated in fig . therefore , the active ants either have to become scouts , discover a new site and then become recruiters or wait at the old nest until a recruiter leads them to the new nest . both of these processes may be hampered : when all the active ants go out scouting , recruiter numbers slowly increase if the new nest is hard to find or if those few can not find any active ant back at the old nest to tandem run with . in terms of the models , this could occur if scouts slowly become recruiters ( low value for k ) , in combination with either a small class of active ants at the old nest ( f is small ) or all active ants having gone scouting ( high value for scouting rate ) . under either or both of these circumstances , the model predicts that ants should not waste time trying to recruit by forward tandem runs but should do the next best thing and use a low quorum threshold to quickly switch to carrying . note , however , that the dynamics of the different ant classes during a simulated emigration in model 2 poorly match observed experimental dynamics ( see , e.g. indeed , simulated emigrations often end with half the colony being recruiters and less than half carried into the new nest ( fig . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics examples of temporal dynamics for models 1 and 2 . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics figure 2 ( top left ) shows that recruiters in model 1 should use a sequential strategy if f is small ( and is large ) : when the quorum is met , they first spend all of their time on reverse tandem runs until all scouts have become recruiters , and then switch to carrying . in contrast , the total number of recruiters is bounded by fn , the number of active ants in the colony . hence , the time costs for not having the recruiters increases and the time to recruit the remaining scouts decreases ( as there are fewer scouts too ) . in this paper top right ) , as there is no end to building recruiter numbers but by completing the entire emigration . in model 1 , there is a clear maximum for intermediate f , whereas in model 2 the number of reverse tandems strictly decreases with f ( fig . contrary to the models in this paper , two previous models of house hunting by t. albipennis ants ( pratt et al . the predictions of the current models proved to be strongly dependent on the assumption of non - linearity of these terms . 2006 ) , models of this collective decision - making system is the division between active and passive ants . we have thus also explored models in which this division was absent , using both linear and non - linear interaction terms such as those in models 1 and 2 presented in this paper . in none of these models the division between active and passive ants is thus a crucial ingredient for reverse tandem running to have a positive impact on emigration speed , which should be experimentally validated . first , the ants might have a home nest , which changes during the emigration , thereby reversing the direction of any recruitment events from home to another nest ( pratt et al . the models in this paper do not incorporate choice between nest sites , but we conjecture that early flexible commitment ( planqu et al . the first models in which reverse tandem runs have been explicitly incorporated to analyse their role have yielded clear predictions : under a range of conditions , we expect a negative correlation between levels of early and late recruitment . this finding lends itself well to simple experiments , and we aim to present those in the near future . note , however , that the dynamics of the different ant classes during a simulated emigration in model 2 poorly match observed experimental dynamics ( see , e.g. notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics examples of temporal dynamics for models 1 and 2 . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics figure 2 ( top left ) shows that recruiters in model 1 should use a sequential strategy if f is small ( and is large ) : when the quorum is met , they first spend all of their time on reverse tandem runs until all scouts have become recruiters , and then switch to carrying . the total number of recruiters is bounded by fn , the number of active ants in the colony . hence , the time costs for not having the recruiters increases and the time to recruit the remaining scouts decreases ( as there are fewer scouts too ) . in model 1 , there is a clear maximum for intermediate f , whereas in model 2 the number of reverse tandems strictly decreases with f ( fig . contrary to the models in this paper , two previous models of house hunting by t. albipennis ants ( pratt et al . the predictions of the current models proved to be strongly dependent on the assumption of non - linearity of these terms . 2006 ) , models of this collective decision - making system is the division between active and passive ants . we have thus also explored models in which this division was absent , using both linear and non - linear interaction terms such as those in models 1 and 2 presented in this paper . in none of these models the division between active and passive ants is thus a crucial ingredient for reverse tandem running to have a positive impact on emigration speed , which should be experimentally validated . first , the ants might have a home nest , which changes during the emigration , thereby reversing the direction of any recruitment events from home to another nest ( pratt et al . the first models in which reverse tandem runs have been explicitly incorporated to analyse their role have yielded clear predictions : under a range of conditions , we expect a negative correlation between levels of early and late recruitment .

alterations of the immunologic milieu within the peritoneal cavity create a hostile environment in endometriosis that may impair gamete interaction and early embryo development . endometriomas are a common form of endometriosis that may be present in 20% to 40% of women with endometriosis who undergo in vitro fertilization ( ivf ) treatment . both endometriomas and endometrioma resection surgery can have detrimental effects on the outcome of ivf due to the possible reduction in the number of developing follicles and subsequently on the retrieved number of follicles . various protocols , which vary in duration , are used to achieve controlled ovarian hyperstimulation ( coh ) during ivf treatment . long gonadotropin releasing hormone agonist ( gnrh - a ) and gnrh antagonist ( gnrh - ant ) protocols are the most commonly used protocols aiming to suppress the premature luteinizing hormone ( lh ) surge and to optimize ivf treatment outcomes . in the literature , mostly long and ultralong gnrh - a protocols have been evaluated in endometriosis patients , and ultralong gnrh - a protocol was associated with a better ivf outcome . to our knowledge , there is no study in the current literature that compares the effectiveness of long gnrh - a and gnrh - ant protocols in the management of ivf cycles in endometriosis patients who have undergone laparoscopic endometrioma resection surgery . the aim of this study was to compare the ivf outcome of long gnrh - a and gnrh - ant protocols in endometriosis patients who have undergone laparoscopic endometrioma resection surgery . a total of 86 patients who were undergoing ivf / intracytoplasmic sperm injection ( icsi ) cycles , and who had undergone laparoscopic resection surgery for endometrioma in the infertility clinic of istanbul university school of medicine ( istanbul , turkey ) between january 1 , 2002 and january 1 , 2012 , were included in this retrospective study . all the patients underwent ivf / icsi cycles within 6 months following the resection surgery . the study protocol was approved by the ethics committee of istanbul university school of medicine , and informed consent was waived due to the retrospective nature of this study . inclusion criteria were age between 18 and 39 years , a larger unilateral or bilateral endometrioma ( > 4 cm ) detected with vaginal ultrasonography , moderate to severe endometriosis as classified during surgery ( stages iii iv ) , total antral follicle count ( afc ) 5 prior to surgery , day-3 serum follicle - stimulating hormone ( fsh ) value of < 10 iu / mc prior to surgery , normal hormone panel and regular menstrual cycles , couples undergoing the first ivf / icsi cycle after the surgery , normal uterine documented by hysterosalpingography or hysteroscopy , no previous ivf attempts , and complete cyst excision . exclusion criteria were minimal to mild endometriosis ( stages i ii ) , infertility due to male factor or a history of poor response , afc of 5 , day-3 serum fsh value of 10 iu / mc , thaw cycles , previous endometrioma resection surgery , endometrioma recurrence , detection of hydrosalpinx , no previous hormonal therapy in the last 3 months , and drainage and/or aspiration of a cyst . the patients were divided into 2 groups according to the gnrh analogue that they received . forty - four patients received the long gnrh - a protocol , whereas the remaining 42 patients received the gnrh - ant protocol . , the ovary covering the endometrioma was separated from the pelvic sidewall and adhesions were lysed . after the cyst capsule was identified , 2 grasping forceps were used to gently strip the capsule from the ovary . the correct dissection plane was carefully maintained to avoid bleeding and potential damage to the primordial follicles . the ovarian cortex was left open and the pelvis was liberally irrigated at the end of the procedure . endometrioma was detected by vaginal ultrasonography and confirmed by the pathologic examination of the cyst wall extracted during laparoscopy . the stage of endometriosis was confirmed during laparoscopy for endometrioma resection according to the revised american fertility society classification . vaginal ultrasonography was performed on all patients on the third day of the ivf cycle to evaluate follicular activity and the afc . coh was started in patients if their ultrasonogram findings did not reveal a follicular cyst over 20 mm . patients received gnrh - a or gnrh- , either puregon ( schering - plough , merck & co , kenilworth , new jersey ) or gonal - f ( emd serono , rockland , massachusetts ) , in line with the preference of the clinician . the initial dose was determined according to patient 's age , ovarian reserve , afc , body mass index , and response to prior stimulation regime ( if applicable ) . it was then adjusted according to the response of ovarian follicles , which were followed - up via vaginal ultrasonography . coh treatment was started on the second or third day of menstrual bleeding with 225 to 300 iu of recombinant fsh ( gonal - f or puregon ) . in the long gnrh - a protocol , pituitary desensitization was achieved in the luteal phase by the administration of 0.5 mg leuprolide acetate / day or 0.1 mg triptorelin acetate / day on day 21 of the previous cycle . ovarian suppression criteria were an e2 concentration < 50 pg / ml in the serum and follicle size of 10 mm in the ovary . after starting coh treatment , the dose of agonist administered was decreased by half and continued until the human chorionic gonadotropin ( hcg ) injection day . in the gnrh - ant protocol , multiple doses of the cetrorelix ( cetrotide , emd serono ; 0.25 mg , subcutaneous ) was injected daily , when the leading follicle was 12 to 13 mm in diameter and continued until the hcg injection day . follicles 17 mm in size were observed , 5000 to 10 000 iu hcg was injected to achieve follicular maturation . a 17-guage needle was utilized for oocyte retrieval , which was done under general anesthesia . after denudation and 2-hour incubation of the oocyte - corona complexes , icsi was performed . on the day of the embryo transfer , the embryos with the best morphologic appearance were chosen . the selection of embryos was based on the number of blastomeres , absence of fragmentation , and the most advanced stage of development . grade 1 embryos had to contain 6 to 8 blastomeres with no multinucleation and fragmentations . the embryo transfer protocol was based on the age of the patient , the number and quality of embryos , and history of prior assisted conception attempts . two or 3 high - quality embryos were transferred to each patient . in the present study , the transfer took place on either day 2 or 3 . the first morning after oocyte retrieval on the fourth day after embryo transfer , the -hcg level in the blood was measured and recorded . if the -hcg level was > 5 miu / ml in either measurement , it was considered positive -hcg , and patients with such levels were regarded as biochemically pregnant . at the sixth week of gestation , outcomes of the cycles with coh protocols including the antagonist and gnrh agonist were evaluated . parameters included : day-3 fsh levels , day-3 and hcg injection day e2 levels , number of antral follicles , number of follicles on hcg injection day , duration of coh , number of metaphase ii ( mii ) oocytes , fertilization rate , number of grade 1 embryos , number of embryos transferred , and rates of biochemical ( positive -hcg ) and ongoing pregnancies . all statistical calculations were performed using the statistical package for social sciences ( version 20.0 , spss inc . , chicago , illinois ) . the student t test or mann - whitney u test was used to compare the mean values between the stimulation protocols . differences in outcome rates were analyzed using a chi - square test or fisher exact test . in all statistical analyses , a total of 86 patients who were undergoing ivf / intracytoplasmic sperm injection ( icsi ) cycles , and who had undergone laparoscopic resection surgery for endometrioma in the infertility clinic of istanbul university school of medicine ( istanbul , turkey ) between january 1 , 2002 and january 1 , 2012 , were included in this retrospective study . all the patients underwent ivf / icsi cycles within 6 months following the resection surgery . the study protocol was approved by the ethics committee of istanbul university school of medicine , and informed consent was waived due to the retrospective nature of this study . inclusion criteria were age between 18 and 39 years , a larger unilateral or bilateral endometrioma ( > 4 cm ) detected with vaginal ultrasonography , moderate to severe endometriosis as classified during surgery ( stages iii iv ) , total antral follicle count ( afc ) 5 prior to surgery , day-3 serum follicle - stimulating hormone ( fsh ) value of < 10 iu / mc prior to surgery , normal hormone panel and regular menstrual cycles , couples undergoing the first ivf / icsi cycle after the surgery , normal uterine documented by hysterosalpingography or hysteroscopy , no previous ivf attempts , and complete cyst excision . exclusion criteria were minimal to mild endometriosis ( stages i ii ) , infertility due to male factor or a history of poor response , afc of 5 , day-3 serum fsh value of 10 iu / mc , thaw cycles , previous endometrioma resection surgery , endometrioma recurrence , detection of hydrosalpinx , no previous hormonal therapy in the last 3 months , and drainage and/or aspiration of a cyst . the patients were divided into 2 groups according to the gnrh analogue that they received . forty - four patients received the long gnrh - a protocol , whereas the remaining 42 patients received the gnrh - ant protocol . the ovary covering the endometrioma was separated from the pelvic sidewall and adhesions were lysed . after the cyst capsule was identified , 2 grasping forceps were used to gently strip the capsule from the ovary . the correct dissection plane was carefully maintained to avoid bleeding and potential damage to the primordial follicles . the ovarian cortex was left open and the pelvis was liberally irrigated at the end of the procedure . endometrioma was detected by vaginal ultrasonography and confirmed by the pathologic examination of the cyst wall extracted during laparoscopy . the stage of endometriosis was confirmed during laparoscopy for endometrioma resection according to the revised american fertility society classification . vaginal ultrasonography was performed on all patients on the third day of the ivf cycle to evaluate follicular activity and the afc . coh was started in patients if their ultrasonogram findings did not reveal a follicular cyst over 20 mm . patients received gnrh - a or gnrh- , either puregon ( schering - plough , merck & co , kenilworth , new jersey ) or gonal - f ( emd serono , rockland , massachusetts ) , in line with the preference of the clinician . the initial dose was determined according to patient 's age , ovarian reserve , afc , body mass index , and response to prior stimulation regime ( if applicable ) . it was then adjusted according to the response of ovarian follicles , which were followed - up via vaginal ultrasonography . coh treatment was started on the second or third day of menstrual bleeding with 225 to 300 iu of recombinant fsh ( gonal - f or puregon ) . in the long gnrh - a protocol , pituitary desensitization was achieved in the luteal phase by the administration of 0.5 mg leuprolide acetate / day or 0.1 mg triptorelin acetate / day on day 21 of the previous cycle . ovarian suppression criteria were an e2 concentration < 50 pg / ml in the serum and follicle size of 10 mm in the ovary . after starting coh treatment , the dose of agonist administered was decreased by half and continued until the human chorionic gonadotropin ( hcg ) injection day . in the gnrh - ant protocol , multiple doses of the cetrorelix ( cetrotide , emd serono ; 0.25 mg , subcutaneous ) was injected daily , when the leading follicle was 12 to 13 mm in diameter and continued until the hcg injection day . ovarian follicular development was observed via vaginal ultrasound at a 1- to 3-day frequency . when 3 follicles 17 mm in size were observed , 5000 to 10 000 iu hcg was injected to achieve follicular maturation . a 17-guage needle was utilized for oocyte retrieval , which was done under general anesthesia . after denudation and 2-hour incubation of the oocyte - corona complexes , icsi was performed . on the day of the embryo transfer , the embryos with the best morphologic appearance were chosen . the selection of embryos was based on the number of blastomeres , absence of fragmentation , and the most advanced stage of development . grade 1 embryos had to contain 6 to 8 blastomeres with no multinucleation and fragmentations . the embryo transfer protocol was based on the age of the patient , the number and quality of embryos , and history of prior assisted conception attempts . two or 3 high - quality embryos were transferred to each patient . in the present study , the transfer took place on either day 2 or 3 . the first morning after oocyte retrieval , on the fourth day after embryo transfer , the -hcg level in the blood was measured and recorded . if the -hcg level was > 5 miu / ml in either measurement , it was considered positive -hcg , and patients with such levels were regarded as biochemically pregnant . at the sixth week of gestation , outcomes of the cycles with coh protocols including the antagonist and gnrh agonist were evaluated . parameters included : day-3 fsh levels , day-3 and hcg injection day e2 levels , number of antral follicles , number of follicles on hcg injection day , duration of coh , number of metaphase ii ( mii ) oocytes , fertilization rate , number of grade 1 embryos , number of embryos transferred , and rates of biochemical ( positive -hcg ) and ongoing pregnancies . all statistical calculations were performed using the statistical package for social sciences ( version 20.0 , spss inc . , chicago , illinois ) . the student t test or mann - whitney u test was used to compare the mean values between the stimulation protocols . differences in outcome rates were analyzed using a chi - square test or fisher exact test . in all statistical analyses , the characteristics of 86 patients who underwent surgery for endometrioma and stage iii to iv endometriosis are presented in table 1 . patients in the 2 groups ( long gnrh - a and gnrh - ant ) were matched in terms of age , afc , basal fsh and estradiol levels , primary infertility ratio , body mass index , and period of infertility ( table 1 ) . in addition , the number of unilateral and bilateral endometriomas was matched both within and between the groups ( table 1 ) . characteristics of the patients abbreviations : bmi , body mass index ; fsh , follicle - stimulating hormone ; gnrh - a , gonadotropin - releasing hormone agonist ; gnrh - ant , gonadotropin - releasing hormone antagonist ; ns , not significant . p < .05 indicates statistically significant differences between long gnrh agonist and gnrh antagonist groups . the comparison of ivf / icsi cycle outcomes between the 2 different protocols used after endometrioma resection surgery is presented in table 2 . the total gonadotropin dose ( 3167.0 1124.4 vs 3261.1 1653.9 ; p = .712 ) , duration of hyperstimulation 11.00 2.13 vs 10.16 1.98 ; 0.825 ) , e2 levels on hcg day ( 1645.2 1156 vs 1779.3 1241 ; p = .654 ) , fertilization rate ( 75.75% 32.98 vs 71.32% 32.94 ; p = .210 ) , the number of transferred embryos ( 2.24 1.11 vs 1.98 1.00 ; p = .113 ) were similar in long gnrh - a and gnrh - ant protocols . comparison of the long gnrh agonist and gnrh antagonist protocols abbreviations : hcg , human chorionic gonadotropin ; hmg , human menopausal gonadotropin ; other abbreviations as in table 1 . p < .05 indicates statistically significant differences between long gnrh agonist and gnrh antagonist groups . the number of follicles on hcg day ( 11.68 7.09 vs 8.44 6.09 ; p < .001 ) , number of retrieved mii oocytes ( 7.93 5.43 vs 5.25 5.51 ; p < .001 ) , total number of grade 1/2 embryos ( 5.82 3.00 vs 4.65 2.14 ; there were no significant differences in positive -hcg pregnancy rates ( 11 of 44 [ 25% ] vs 9 of 42 [ 21.4% ] ; p = .269 ) and ongoing pregnancy rates ( 9 of 44 [ 20.5% ] vs 8 of 42 [ 19.1% ] ; p = .302 ) between long gnrh - a and gnrh - ant protocols . in ivf cycles , along with the adverse effects of endometriosis on growing follicles , oocytes , embryos , and endometrium , ovarian endometrioma itself and its surgical resection may have an additional negative impact on the outcome . choosing the appropriate ovarian stimulation protocol may prevent these adverse effects . in the present study , we tried to evaluate the efficacy of long gnrh - a and gnrh - ant protocols on patients that were specifically facing these detrimental factors due to prior laparoscopic endometrioma resection surgery . to our knowledge , there are no studies in the literature that specifically compare long gnrh - a and gnrh - ant protocols in patients with advanced endometriosis who have undergone laparoscopic endometrioma resection surgery . in the present study , the number of retrieved mii oocytes , grade 1 embryos , and aspirated follicles were higher in patients who received the gnrh - ant protocol , yet our findings did not reveal a statistically significant difference in pregnancy rates between the 2 protocols in which a similar number of embryos were transferred . furthermore , a similar number of embryo transfers revealed comparable fertilization and pregnancy rates between the 2 protocols . hence , the actual impact of gnrh analogues on ivf treatment may be on the ovarian response rather than on oocyte and embryo quality . coagulation with bipolar cautery is usually considered a safe and well - accepted approach in laparoscopic endometrioma resection surgery , which was also the technique used in the present study . however , some studies in the literature indicated that this technique was associated with decreased ovarian reserve postoperatively . recently , some studies investigated a vasopressin injection technique that may help clearly define the boundary between the cyst and the ovarian stroma . there are multiple studies in the literature that evaluated the effects of endometriosis on ivf outcomes . a meta - analysis about stage iii to iv endometriosis , which involved 22 studies , concluded that the fertilization , implantation and pregnancy rates , and the number of retrieved oocytes decreased in patients with endometriosis compared with the control subjects with tubal factor infertility . it has been argued that women with severe endometriosis were 36% less likely to achieve pregnancy than those with mild endometriosis were . the same researchers presented that women with stage iii to iv endometriosis had fewer numbers of retrieved oocytes , lower e2 levels , and lower fertilization and implantation rates in comparison with women with stage i to ii endometriosis and women with infertility due to other causes . documented that patients with stage i to ii endometriosis had a lower fertilization rate , and patients with stage iii to iv endometriosis had fewer oocytes retrieved . furthermore , when the researchers split the stage iii and iv patients with and without endometriomas , it was reported that the endometrioma group had significantly lower pregnancy , live birth , and ongoing pregnancy rates . coccia et al showed the detrimental effects of severe endometriosis on ivf outcome via significantly reduced pregnancy rates ( clinical pregnancy rate per embryo transfer : 9.7% for stage iii to iv , 25% for stage i to ii , and 26.1% for tubal factor ) . in a very recent systematic review and meta - analysis , harb et iv ) was associated with poor implantation and clinical pregnancy rates in women undergoing ivf treatment . in addition , kitajima et al argued that presence of endometrioma is associated with diminished ovarian reserve . although there is no consensus on how to manage patients with advanced endometriosis and endometriomas , gnrh analogues have been used for the treatment of endometriosis for several years . the use of gnrh - a during ivf treatment in patients with advanced endometriosis has led to increased pregnancy rates in some studies . gnrh - a may be suppressing endometriosis lesions in such patients . in support of this argument , patients who faced long - term suppression before the initiation of hyperstimulation with gonadotropins during ivf treatment had higher pregnancy rates . the most efficient ivf / icsi stimulation protocol in endometriosis patients is argued to be the ultralong ( long - term pituitary down - regulation with a gnrh - a ) protocol , studied by many researchers , including surrey et al . a cochrane systematic review and meta - analysis performed by sallam et al established the advantages of this protocol with an odds ratio of 4.28 ( in favor of long - term pituitary down - regulation with a gnrh - a ) . alternatively , advantages of the antagonist ( eg , easier and faster prevention of premature lh surge , flexibility of use , increased patient comfort due to shorter usage ) make it an attractive choice for any ivf program . in a recent study by rodriguez - purata et al , pregnancy rate after coh with either gnrh - a or gnrh - ant was found to be equally effective in patients with endometriosis ( stage i to iv ) . the number of retrieved oocytes might have an indirect effect on pregnancy rates , because higher - quality embryos can lead to more good - quality oocytes . in the literature , the results are varied when the protocols are compared with the number of retrieved oocytes . there was no statistical difference in some studies , whereas a meta - analysis by ludwig et al suggested fewer retrieved oocytes in patients who received a gnrh - ant protocol . albano et al carried out a multicenter study and also documented that patients who received a gnrh - ant protocol revealed significantly fewer numbers of retrieved oocytes . in this study , the number of retrieved mii oocytes and follicles were higher in patients who received a gnrh - a protocol . it can be argued that higher gonadotropin doses may lead to more aspirated follicles and retrieved oocytes . however , we used similar total doses of gonadotropin in the present study in both groups , yet significantly fewer numbers of aspirated follicles and retrieved oocytes were recorded in patients who received the gnrh - ant protocol . when the protocols are compared for the pregnancy rates , there are contradicting results . several studies and various meta - analyses have been carried out to compare pregnancy rates of gnrh - a versus gnrh - ant protocols . a meta - analysis of 14 randomized studies found no significant difference between the 2 gnrh analogues in the pregnancy rate . similarly , ludwig et al did not report a difference at the pregnancy rate between gnrh - a versus gnrh - ant protocols . in addition pu et al reported no statistical difference to the number of oocytes retrieved , the number of mature oocytes retrieved , the cycle cancellation and clinical pregnancy rates between gnrh - a versus gnrh - ant protocols in poor ovarian responders . on the other hand , a cochrane meta - analysis by al - inany and aboulghar , which took into account 5 randomized studies , suggested that the usage of gnrh - ant protocol compared with gnrh - a provided lower pregnancy rates . however , the conclusions of this cochrane meta - analysis can not be generalized for the endometriosis patients . merviel et al also reported lower pregnancy rates in patients who used gnrh - ant protocols . because of the risks involved with multiple pregnancies , which is a common occurrence in ivf treatments , the standard practice in many countries became the transfer of 1 ( elective single embryo transfer ) or 2 embryos instead of 3 . in turkey , the turkish ministry of health established a mandatory standard of single embryo transfer in march 2010 for all women under the age of 35 years , in their first 2 cycles . hence , cryopreserved embryos became an appealing alternative for patients who were unable get pregnant during their ivf treatment or who want to have another pregnancy later in their lives . with cryopreserved embryos , patients have the chance of a frozen - thawed embryo transfer , which can alleviate the emotional , financial , and physical burdens of an additional ivf treatment . in the present study , the gnrh - a protocol revealed increased numbers of oocytes and embryos , so the patients who received this protocol had an opportunity to cryopreserve and transfer embryos later . additionally , cryopreserved embryo transfer might have an advantage for women with endometriosis , because ovarian hyperstimulation is not required in frozen - thawed embryo transfer , which may potentially activate the endometriosis . limitations of the present study were the involvement of > 1 clinician and their preferences for different protocols ( long gnrh - a versus gnrh - ant ) and its retrospective design . in addition , differences in the skills of the surgeons , although in the present study all surgeons were experienced , might be considered as another limitation , as the ability to preserve normal ovarian tissue after endometrioma resection surgery varies according to the skill of the surgeon and the technique used . in our clinic , patients with endometrioma who have a diminished ovarian reserve ( afc < 5 , basal fsh 10 iu ) do not undergo surgery solely because of infertility . hence , another limitation was the fact that the subset of patients with poor response was omitted from the study . gnrh antagonists have been available commercially since the early 2000s , whereas gnrh agonists have been available for much longer . in our clinic , although the learning curve was not steep , it could still be regarded a limitation of this study . long gnrh - a and gnrh - ant protocols both present similar ivf outcomes in patients with endometriosis who have undergone laparoscopic endometrioma resection surgery . long gnrh - a protocol may lead to a higher number of embryos , which can be cryopreserved , providing the possibility of additional embryo transfers without having to go through the process of ovarian stimulation again . however , further prospective research on larger sample sizes , in which live birth rates especially are evaluated , have to be carried out to compare the efficiency and success rates of the 2 protocols .

background and objectives : the aim of this study was to compare the in vitro fertilization ( ivf ) outcomes of long gonadotropin - releasing hormone agonist ( gnrh - a ) and gnrh - antagonist ( gnrh - ant ) protocols in endometriosis patients who have undergone laparoscopic endometrioma resection surgery . to our knowledge , there is no study in the current literature that compares the effectiveness of long gnrh - a and gnrh - ant protocols in management of ivf cycles in endometriosis patients who underwent laparoscopic endometrioma resection surgery.methods:eighty-six patients with stage iii to iv endometriosis who had undergone laparoscopic resection surgery for endometrioma were divided into 2 groups : those who had ovarian stimulation with a long gnrh - a protocol ( n = 44 ) , and those who had ovarian stimulation with a gnrh - ant protocol ( n = 42).results : the number of follicles on human chorionic gonadotropin injection day , duration of hyperstimulation , number of retrieved metaphase ii oocytes , and total number of grade 1 embryos were statically significantly higher in the long gnrh - a protocol . there were no significant differences in positive -human chorionic gonadotropin pregnancy rates ( 25% vs 21.4% ; p = .269 ) and ongoing pregnancy rates per patient ( 20.5% vs 19.1% ; p = .302 ) between the 2 protocols.conclusions:long gnrh - a and gnrh - ant protocols both present similar ivf outcomes in patients with endometriosis who have undergone laparoscopic endometrioma resection surgery . a long gnrh - a protocol may lead to a higher number of embryos that can be cryopreserved , providing the possibility of additional embryo transfers without having to go through the process of ovarian stimulation again .

INTRODUCTION MATERIALS AND METHODS Subjects Laparoscopic Endometrioma Resection Surgery Controlled Ovarian Hyperstimulation Long GnRH-a Protocol GnRH-ant Protocol Ovarian Follicular Development and Oocyte Retrieval Embryo Transfer and Luteal Phase Support Evaluation of Assisted Reproductive Technique Results Statistical Analysis RESULTS DISCUSSION CONCLUSIONS

endometriomas are a common form of endometriosis that may be present in 20% to 40% of women with endometriosis who undergo in vitro fertilization ( ivf ) treatment . both endometriomas and endometrioma resection surgery can have detrimental effects on the outcome of ivf due to the possible reduction in the number of developing follicles and subsequently on the retrieved number of follicles . long gonadotropin releasing hormone agonist ( gnrh - a ) and gnrh antagonist ( gnrh - ant ) protocols are the most commonly used protocols aiming to suppress the premature luteinizing hormone ( lh ) surge and to optimize ivf treatment outcomes . in the literature , mostly long and ultralong gnrh - a protocols have been evaluated in endometriosis patients , and ultralong gnrh - a protocol was associated with a better ivf outcome . to our knowledge , there is no study in the current literature that compares the effectiveness of long gnrh - a and gnrh - ant protocols in the management of ivf cycles in endometriosis patients who have undergone laparoscopic endometrioma resection surgery . the aim of this study was to compare the ivf outcome of long gnrh - a and gnrh - ant protocols in endometriosis patients who have undergone laparoscopic endometrioma resection surgery . a total of 86 patients who were undergoing ivf / intracytoplasmic sperm injection ( icsi ) cycles , and who had undergone laparoscopic resection surgery for endometrioma in the infertility clinic of istanbul university school of medicine ( istanbul , turkey ) between january 1 , 2002 and january 1 , 2012 , were included in this retrospective study . the study protocol was approved by the ethics committee of istanbul university school of medicine , and informed consent was waived due to the retrospective nature of this study . exclusion criteria were minimal to mild endometriosis ( stages i ii ) , infertility due to male factor or a history of poor response , afc of 5 , day-3 serum fsh value of 10 iu / mc , thaw cycles , previous endometrioma resection surgery , endometrioma recurrence , detection of hydrosalpinx , no previous hormonal therapy in the last 3 months , and drainage and/or aspiration of a cyst . the patients were divided into 2 groups according to the gnrh analogue that they received . forty - four patients received the long gnrh - a protocol , whereas the remaining 42 patients received the gnrh - ant protocol . patients received gnrh - a or gnrh- , either puregon ( schering - plough , merck & co , kenilworth , new jersey ) or gonal - f ( emd serono , rockland , massachusetts ) , in line with the preference of the clinician . in the long gnrh - a protocol , pituitary desensitization was achieved in the luteal phase by the administration of 0.5 mg leuprolide acetate / day or 0.1 mg triptorelin acetate / day on day 21 of the previous cycle . after starting coh treatment , the dose of agonist administered was decreased by half and continued until the human chorionic gonadotropin ( hcg ) injection day . in the gnrh - ant protocol , multiple doses of the cetrorelix ( cetrotide , emd serono ; 0.25 mg , subcutaneous ) was injected daily , when the leading follicle was 12 to 13 mm in diameter and continued until the hcg injection day . the selection of embryos was based on the number of blastomeres , absence of fragmentation , and the most advanced stage of development . grade 1 embryos had to contain 6 to 8 blastomeres with no multinucleation and fragmentations . the embryo transfer protocol was based on the age of the patient , the number and quality of embryos , and history of prior assisted conception attempts . if the -hcg level was > 5 miu / ml in either measurement , it was considered positive -hcg , and patients with such levels were regarded as biochemically pregnant . parameters included : day-3 fsh levels , day-3 and hcg injection day e2 levels , number of antral follicles , number of follicles on hcg injection day , duration of coh , number of metaphase ii ( mii ) oocytes , fertilization rate , number of grade 1 embryos , number of embryos transferred , and rates of biochemical ( positive -hcg ) and ongoing pregnancies . the student t test or mann - whitney u test was used to compare the mean values between the stimulation protocols . in all statistical analyses , a total of 86 patients who were undergoing ivf / intracytoplasmic sperm injection ( icsi ) cycles , and who had undergone laparoscopic resection surgery for endometrioma in the infertility clinic of istanbul university school of medicine ( istanbul , turkey ) between january 1 , 2002 and january 1 , 2012 , were included in this retrospective study . the study protocol was approved by the ethics committee of istanbul university school of medicine , and informed consent was waived due to the retrospective nature of this study . inclusion criteria were age between 18 and 39 years , a larger unilateral or bilateral endometrioma ( > 4 cm ) detected with vaginal ultrasonography , moderate to severe endometriosis as classified during surgery ( stages iii iv ) , total antral follicle count ( afc ) 5 prior to surgery , day-3 serum follicle - stimulating hormone ( fsh ) value of < 10 iu / mc prior to surgery , normal hormone panel and regular menstrual cycles , couples undergoing the first ivf / icsi cycle after the surgery , normal uterine documented by hysterosalpingography or hysteroscopy , no previous ivf attempts , and complete cyst excision . exclusion criteria were minimal to mild endometriosis ( stages i ii ) , infertility due to male factor or a history of poor response , afc of 5 , day-3 serum fsh value of 10 iu / mc , thaw cycles , previous endometrioma resection surgery , endometrioma recurrence , detection of hydrosalpinx , no previous hormonal therapy in the last 3 months , and drainage and/or aspiration of a cyst . the patients were divided into 2 groups according to the gnrh analogue that they received . forty - four patients received the long gnrh - a protocol , whereas the remaining 42 patients received the gnrh - ant protocol . the stage of endometriosis was confirmed during laparoscopy for endometrioma resection according to the revised american fertility society classification . patients received gnrh - a or gnrh- , either puregon ( schering - plough , merck & co , kenilworth , new jersey ) or gonal - f ( emd serono , rockland , massachusetts ) , in line with the preference of the clinician . in the long gnrh - a protocol , pituitary desensitization was achieved in the luteal phase by the administration of 0.5 mg leuprolide acetate / day or 0.1 mg triptorelin acetate / day on day 21 of the previous cycle . after starting coh treatment , the dose of agonist administered was decreased by half and continued until the human chorionic gonadotropin ( hcg ) injection day . in the gnrh - ant protocol , multiple doses of the cetrorelix ( cetrotide , emd serono ; 0.25 mg , subcutaneous ) was injected daily , when the leading follicle was 12 to 13 mm in diameter and continued until the hcg injection day . the selection of embryos was based on the number of blastomeres , absence of fragmentation , and the most advanced stage of development . grade 1 embryos had to contain 6 to 8 blastomeres with no multinucleation and fragmentations . the embryo transfer protocol was based on the age of the patient , the number and quality of embryos , and history of prior assisted conception attempts . if the -hcg level was > 5 miu / ml in either measurement , it was considered positive -hcg , and patients with such levels were regarded as biochemically pregnant . at the sixth week of gestation , outcomes of the cycles with coh protocols including the antagonist and gnrh agonist were evaluated . parameters included : day-3 fsh levels , day-3 and hcg injection day e2 levels , number of antral follicles , number of follicles on hcg injection day , duration of coh , number of metaphase ii ( mii ) oocytes , fertilization rate , number of grade 1 embryos , number of embryos transferred , and rates of biochemical ( positive -hcg ) and ongoing pregnancies . the student t test or mann - whitney u test was used to compare the mean values between the stimulation protocols . in all statistical analyses , the characteristics of 86 patients who underwent surgery for endometrioma and stage iii to iv endometriosis are presented in table 1 . patients in the 2 groups ( long gnrh - a and gnrh - ant ) were matched in terms of age , afc , basal fsh and estradiol levels , primary infertility ratio , body mass index , and period of infertility ( table 1 ) . in addition , the number of unilateral and bilateral endometriomas was matched both within and between the groups ( table 1 ) . characteristics of the patients abbreviations : bmi , body mass index ; fsh , follicle - stimulating hormone ; gnrh - a , gonadotropin - releasing hormone agonist ; gnrh - ant , gonadotropin - releasing hormone antagonist ; ns , not significant . p < .05 indicates statistically significant differences between long gnrh agonist and gnrh antagonist groups . the comparison of ivf / icsi cycle outcomes between the 2 different protocols used after endometrioma resection surgery is presented in table 2 . the total gonadotropin dose ( 3167.0 1124.4 vs 3261.1 1653.9 ; p = .712 ) , duration of hyperstimulation 11.00 2.13 vs 10.16 1.98 ; 0.825 ) , e2 levels on hcg day ( 1645.2 1156 vs 1779.3 1241 ; p = .654 ) , fertilization rate ( 75.75% 32.98 vs 71.32% 32.94 ; p = .210 ) , the number of transferred embryos ( 2.24 1.11 vs 1.98 1.00 ; p = .113 ) were similar in long gnrh - a and gnrh - ant protocols . comparison of the long gnrh agonist and gnrh antagonist protocols abbreviations : hcg , human chorionic gonadotropin ; hmg , human menopausal gonadotropin ; other abbreviations as in table 1 . p < .05 indicates statistically significant differences between long gnrh agonist and gnrh antagonist groups . the number of follicles on hcg day ( 11.68 7.09 vs 8.44 6.09 ; p < .001 ) , number of retrieved mii oocytes ( 7.93 5.43 vs 5.25 5.51 ; p < .001 ) , total number of grade 1/2 embryos ( 5.82 3.00 vs 4.65 2.14 ; there were no significant differences in positive -hcg pregnancy rates ( 11 of 44 [ 25% ] vs 9 of 42 [ 21.4% ] ; p = .269 ) and ongoing pregnancy rates ( 9 of 44 [ 20.5% ] vs 8 of 42 [ 19.1% ] ; p = .302 ) between long gnrh - a and gnrh - ant protocols . in ivf cycles , along with the adverse effects of endometriosis on growing follicles , oocytes , embryos , and endometrium , ovarian endometrioma itself and its surgical resection may have an additional negative impact on the outcome . choosing the appropriate ovarian stimulation protocol may prevent these adverse effects . in the present study , we tried to evaluate the efficacy of long gnrh - a and gnrh - ant protocols on patients that were specifically facing these detrimental factors due to prior laparoscopic endometrioma resection surgery . to our knowledge , there are no studies in the literature that specifically compare long gnrh - a and gnrh - ant protocols in patients with advanced endometriosis who have undergone laparoscopic endometrioma resection surgery . in the present study , the number of retrieved mii oocytes , grade 1 embryos , and aspirated follicles were higher in patients who received the gnrh - ant protocol , yet our findings did not reveal a statistically significant difference in pregnancy rates between the 2 protocols in which a similar number of embryos were transferred . furthermore , a similar number of embryo transfers revealed comparable fertilization and pregnancy rates between the 2 protocols . coagulation with bipolar cautery is usually considered a safe and well - accepted approach in laparoscopic endometrioma resection surgery , which was also the technique used in the present study . there are multiple studies in the literature that evaluated the effects of endometriosis on ivf outcomes . a meta - analysis about stage iii to iv endometriosis , which involved 22 studies , concluded that the fertilization , implantation and pregnancy rates , and the number of retrieved oocytes decreased in patients with endometriosis compared with the control subjects with tubal factor infertility . the same researchers presented that women with stage iii to iv endometriosis had fewer numbers of retrieved oocytes , lower e2 levels , and lower fertilization and implantation rates in comparison with women with stage i to ii endometriosis and women with infertility due to other causes . documented that patients with stage i to ii endometriosis had a lower fertilization rate , and patients with stage iii to iv endometriosis had fewer oocytes retrieved . furthermore , when the researchers split the stage iii and iv patients with and without endometriomas , it was reported that the endometrioma group had significantly lower pregnancy , live birth , and ongoing pregnancy rates . coccia et al showed the detrimental effects of severe endometriosis on ivf outcome via significantly reduced pregnancy rates ( clinical pregnancy rate per embryo transfer : 9.7% for stage iii to iv , 25% for stage i to ii , and 26.1% for tubal factor ) . although there is no consensus on how to manage patients with advanced endometriosis and endometriomas , gnrh analogues have been used for the treatment of endometriosis for several years . the use of gnrh - a during ivf treatment in patients with advanced endometriosis has led to increased pregnancy rates in some studies . gnrh - a may be suppressing endometriosis lesions in such patients . in support of this argument , patients who faced long - term suppression before the initiation of hyperstimulation with gonadotropins during ivf treatment had higher pregnancy rates . the most efficient ivf / icsi stimulation protocol in endometriosis patients is argued to be the ultralong ( long - term pituitary down - regulation with a gnrh - a ) protocol , studied by many researchers , including surrey et al . a cochrane systematic review and meta - analysis performed by sallam et al established the advantages of this protocol with an odds ratio of 4.28 ( in favor of long - term pituitary down - regulation with a gnrh - a ) . in a recent study by rodriguez - purata et al , pregnancy rate after coh with either gnrh - a or gnrh - ant was found to be equally effective in patients with endometriosis ( stage i to iv ) . the number of retrieved oocytes might have an indirect effect on pregnancy rates , because higher - quality embryos can lead to more good - quality oocytes . in the literature , the results are varied when the protocols are compared with the number of retrieved oocytes . there was no statistical difference in some studies , whereas a meta - analysis by ludwig et al suggested fewer retrieved oocytes in patients who received a gnrh - ant protocol . albano et al carried out a multicenter study and also documented that patients who received a gnrh - ant protocol revealed significantly fewer numbers of retrieved oocytes . in this study , the number of retrieved mii oocytes and follicles were higher in patients who received a gnrh - a protocol . it can be argued that higher gonadotropin doses may lead to more aspirated follicles and retrieved oocytes . however , we used similar total doses of gonadotropin in the present study in both groups , yet significantly fewer numbers of aspirated follicles and retrieved oocytes were recorded in patients who received the gnrh - ant protocol . when the protocols are compared for the pregnancy rates , there are contradicting results . several studies and various meta - analyses have been carried out to compare pregnancy rates of gnrh - a versus gnrh - ant protocols . a meta - analysis of 14 randomized studies found no significant difference between the 2 gnrh analogues in the pregnancy rate . similarly , ludwig et al did not report a difference at the pregnancy rate between gnrh - a versus gnrh - ant protocols . in addition pu et al reported no statistical difference to the number of oocytes retrieved , the number of mature oocytes retrieved , the cycle cancellation and clinical pregnancy rates between gnrh - a versus gnrh - ant protocols in poor ovarian responders . on the other hand , a cochrane meta - analysis by al - inany and aboulghar , which took into account 5 randomized studies , suggested that the usage of gnrh - ant protocol compared with gnrh - a provided lower pregnancy rates . merviel et al also reported lower pregnancy rates in patients who used gnrh - ant protocols . in the present study , the gnrh - a protocol revealed increased numbers of oocytes and embryos , so the patients who received this protocol had an opportunity to cryopreserve and transfer embryos later . limitations of the present study were the involvement of > 1 clinician and their preferences for different protocols ( long gnrh - a versus gnrh - ant ) and its retrospective design . in addition , differences in the skills of the surgeons , although in the present study all surgeons were experienced , might be considered as another limitation , as the ability to preserve normal ovarian tissue after endometrioma resection surgery varies according to the skill of the surgeon and the technique used . in our clinic , patients with endometrioma who have a diminished ovarian reserve ( afc < 5 , basal fsh 10 iu ) do not undergo surgery solely because of infertility . in our clinic , although the learning curve was not steep , it could still be regarded a limitation of this study . long gnrh - a and gnrh - ant protocols both present similar ivf outcomes in patients with endometriosis who have undergone laparoscopic endometrioma resection surgery . long gnrh - a protocol may lead to a higher number of embryos , which can be cryopreserved , providing the possibility of additional embryo transfers without having to go through the process of ovarian stimulation again . however , further prospective research on larger sample sizes , in which live birth rates especially are evaluated , have to be carried out to compare the efficiency and success rates of the 2 protocols .

endometriomas are a common form of endometriosis that may be present in 20% to 40% of women with endometriosis who undergo in vitro fertilization ( ivf ) treatment . both endometriomas and endometrioma resection surgery can have detrimental effects on the outcome of ivf due to the possible reduction in the number of developing follicles and subsequently on the retrieved number of follicles . long gonadotropin releasing hormone agonist ( gnrh - a ) and gnrh antagonist ( gnrh - ant ) protocols are the most commonly used protocols aiming to suppress the premature luteinizing hormone ( lh ) surge and to optimize ivf treatment outcomes . in the literature , mostly long and ultralong gnrh - a protocols have been evaluated in endometriosis patients , and ultralong gnrh - a protocol was associated with a better ivf outcome . to our knowledge , there is no study in the current literature that compares the effectiveness of long gnrh - a and gnrh - ant protocols in the management of ivf cycles in endometriosis patients who have undergone laparoscopic endometrioma resection surgery . the aim of this study was to compare the ivf outcome of long gnrh - a and gnrh - ant protocols in endometriosis patients who have undergone laparoscopic endometrioma resection surgery . a total of 86 patients who were undergoing ivf / intracytoplasmic sperm injection ( icsi ) cycles , and who had undergone laparoscopic resection surgery for endometrioma in the infertility clinic of istanbul university school of medicine ( istanbul , turkey ) between january 1 , 2002 and january 1 , 2012 , were included in this retrospective study . all the patients underwent ivf / icsi cycles within 6 months following the resection surgery . inclusion criteria were age between 18 and 39 years , a larger unilateral or bilateral endometrioma ( > 4 cm ) detected with vaginal ultrasonography , moderate to severe endometriosis as classified during surgery ( stages iii iv ) , total antral follicle count ( afc ) 5 prior to surgery , day-3 serum follicle - stimulating hormone ( fsh ) value of < 10 iu / mc prior to surgery , normal hormone panel and regular menstrual cycles , couples undergoing the first ivf / icsi cycle after the surgery , normal uterine documented by hysterosalpingography or hysteroscopy , no previous ivf attempts , and complete cyst excision . exclusion criteria were minimal to mild endometriosis ( stages i ii ) , infertility due to male factor or a history of poor response , afc of 5 , day-3 serum fsh value of 10 iu / mc , thaw cycles , previous endometrioma resection surgery , endometrioma recurrence , detection of hydrosalpinx , no previous hormonal therapy in the last 3 months , and drainage and/or aspiration of a cyst . forty - four patients received the long gnrh - a protocol , whereas the remaining 42 patients received the gnrh - ant protocol . after the cyst capsule was identified , 2 grasping forceps were used to gently strip the capsule from the ovary . vaginal ultrasonography was performed on all patients on the third day of the ivf cycle to evaluate follicular activity and the afc . patients received gnrh - a or gnrh- , either puregon ( schering - plough , merck & co , kenilworth , new jersey ) or gonal - f ( emd serono , rockland , massachusetts ) , in line with the preference of the clinician . the initial dose was determined according to patient 's age , ovarian reserve , afc , body mass index , and response to prior stimulation regime ( if applicable ) . in the long gnrh - a protocol , pituitary desensitization was achieved in the luteal phase by the administration of 0.5 mg leuprolide acetate / day or 0.1 mg triptorelin acetate / day on day 21 of the previous cycle . ovarian suppression criteria were an e2 concentration < 50 pg / ml in the serum and follicle size of 10 mm in the ovary . after starting coh treatment , the dose of agonist administered was decreased by half and continued until the human chorionic gonadotropin ( hcg ) injection day . in the gnrh - ant protocol , multiple doses of the cetrorelix ( cetrotide , emd serono ; 0.25 mg , subcutaneous ) was injected daily , when the leading follicle was 12 to 13 mm in diameter and continued until the hcg injection day . on the day of the embryo transfer , the embryos with the best morphologic appearance were chosen . the selection of embryos was based on the number of blastomeres , absence of fragmentation , and the most advanced stage of development . the embryo transfer protocol was based on the age of the patient , the number and quality of embryos , and history of prior assisted conception attempts . the first morning after oocyte retrieval on the fourth day after embryo transfer , the -hcg level in the blood was measured and recorded . if the -hcg level was > 5 miu / ml in either measurement , it was considered positive -hcg , and patients with such levels were regarded as biochemically pregnant . at the sixth week of gestation , outcomes of the cycles with coh protocols including the antagonist and gnrh agonist were evaluated . parameters included : day-3 fsh levels , day-3 and hcg injection day e2 levels , number of antral follicles , number of follicles on hcg injection day , duration of coh , number of metaphase ii ( mii ) oocytes , fertilization rate , number of grade 1 embryos , number of embryos transferred , and rates of biochemical ( positive -hcg ) and ongoing pregnancies . in all statistical analyses , a total of 86 patients who were undergoing ivf / intracytoplasmic sperm injection ( icsi ) cycles , and who had undergone laparoscopic resection surgery for endometrioma in the infertility clinic of istanbul university school of medicine ( istanbul , turkey ) between january 1 , 2002 and january 1 , 2012 , were included in this retrospective study . inclusion criteria were age between 18 and 39 years , a larger unilateral or bilateral endometrioma ( > 4 cm ) detected with vaginal ultrasonography , moderate to severe endometriosis as classified during surgery ( stages iii iv ) , total antral follicle count ( afc ) 5 prior to surgery , day-3 serum follicle - stimulating hormone ( fsh ) value of < 10 iu / mc prior to surgery , normal hormone panel and regular menstrual cycles , couples undergoing the first ivf / icsi cycle after the surgery , normal uterine documented by hysterosalpingography or hysteroscopy , no previous ivf attempts , and complete cyst excision . exclusion criteria were minimal to mild endometriosis ( stages i ii ) , infertility due to male factor or a history of poor response , afc of 5 , day-3 serum fsh value of 10 iu / mc , thaw cycles , previous endometrioma resection surgery , endometrioma recurrence , detection of hydrosalpinx , no previous hormonal therapy in the last 3 months , and drainage and/or aspiration of a cyst . forty - four patients received the long gnrh - a protocol , whereas the remaining 42 patients received the gnrh - ant protocol . vaginal ultrasonography was performed on all patients on the third day of the ivf cycle to evaluate follicular activity and the afc . patients received gnrh - a or gnrh- , either puregon ( schering - plough , merck & co , kenilworth , new jersey ) or gonal - f ( emd serono , rockland , massachusetts ) , in line with the preference of the clinician . the initial dose was determined according to patient 's age , ovarian reserve , afc , body mass index , and response to prior stimulation regime ( if applicable ) . in the long gnrh - a protocol , pituitary desensitization was achieved in the luteal phase by the administration of 0.5 mg leuprolide acetate / day or 0.1 mg triptorelin acetate / day on day 21 of the previous cycle . ovarian suppression criteria were an e2 concentration < 50 pg / ml in the serum and follicle size of 10 mm in the ovary . after starting coh treatment , the dose of agonist administered was decreased by half and continued until the human chorionic gonadotropin ( hcg ) injection day . in the gnrh - ant protocol , multiple doses of the cetrorelix ( cetrotide , emd serono ; 0.25 mg , subcutaneous ) was injected daily , when the leading follicle was 12 to 13 mm in diameter and continued until the hcg injection day . on the day of the embryo transfer , the embryos with the best morphologic appearance were chosen . the selection of embryos was based on the number of blastomeres , absence of fragmentation , and the most advanced stage of development . the embryo transfer protocol was based on the age of the patient , the number and quality of embryos , and history of prior assisted conception attempts . the first morning after oocyte retrieval , on the fourth day after embryo transfer , the -hcg level in the blood was measured and recorded . at the sixth week of gestation , outcomes of the cycles with coh protocols including the antagonist and gnrh agonist were evaluated . parameters included : day-3 fsh levels , day-3 and hcg injection day e2 levels , number of antral follicles , number of follicles on hcg injection day , duration of coh , number of metaphase ii ( mii ) oocytes , fertilization rate , number of grade 1 embryos , number of embryos transferred , and rates of biochemical ( positive -hcg ) and ongoing pregnancies . in all statistical analyses , the characteristics of 86 patients who underwent surgery for endometrioma and stage iii to iv endometriosis are presented in table 1 . patients in the 2 groups ( long gnrh - a and gnrh - ant ) were matched in terms of age , afc , basal fsh and estradiol levels , primary infertility ratio , body mass index , and period of infertility ( table 1 ) . in addition , the number of unilateral and bilateral endometriomas was matched both within and between the groups ( table 1 ) . characteristics of the patients abbreviations : bmi , body mass index ; fsh , follicle - stimulating hormone ; gnrh - a , gonadotropin - releasing hormone agonist ; gnrh - ant , gonadotropin - releasing hormone antagonist ; ns , not significant . the comparison of ivf / icsi cycle outcomes between the 2 different protocols used after endometrioma resection surgery is presented in table 2 . the total gonadotropin dose ( 3167.0 1124.4 vs 3261.1 1653.9 ; p = .712 ) , duration of hyperstimulation 11.00 2.13 vs 10.16 1.98 ; 0.825 ) , e2 levels on hcg day ( 1645.2 1156 vs 1779.3 1241 ; p = .654 ) , fertilization rate ( 75.75% 32.98 vs 71.32% 32.94 ; p = .210 ) , the number of transferred embryos ( 2.24 1.11 vs 1.98 1.00 ; p = .113 ) were similar in long gnrh - a and gnrh - ant protocols . comparison of the long gnrh agonist and gnrh antagonist protocols abbreviations : hcg , human chorionic gonadotropin ; hmg , human menopausal gonadotropin ; other abbreviations as in table 1 . the number of follicles on hcg day ( 11.68 7.09 vs 8.44 6.09 ; p < .001 ) , number of retrieved mii oocytes ( 7.93 5.43 vs 5.25 5.51 ; p < .001 ) , total number of grade 1/2 embryos ( 5.82 3.00 vs 4.65 2.14 ; there were no significant differences in positive -hcg pregnancy rates ( 11 of 44 [ 25% ] vs 9 of 42 [ 21.4% ] ; p = .269 ) and ongoing pregnancy rates ( 9 of 44 [ 20.5% ] vs 8 of 42 [ 19.1% ] ; p = .302 ) between long gnrh - a and gnrh - ant protocols . in ivf cycles , along with the adverse effects of endometriosis on growing follicles , oocytes , embryos , and endometrium , ovarian endometrioma itself and its surgical resection may have an additional negative impact on the outcome . in the present study , we tried to evaluate the efficacy of long gnrh - a and gnrh - ant protocols on patients that were specifically facing these detrimental factors due to prior laparoscopic endometrioma resection surgery . to our knowledge , there are no studies in the literature that specifically compare long gnrh - a and gnrh - ant protocols in patients with advanced endometriosis who have undergone laparoscopic endometrioma resection surgery . in the present study , the number of retrieved mii oocytes , grade 1 embryos , and aspirated follicles were higher in patients who received the gnrh - ant protocol , yet our findings did not reveal a statistically significant difference in pregnancy rates between the 2 protocols in which a similar number of embryos were transferred . hence , the actual impact of gnrh analogues on ivf treatment may be on the ovarian response rather than on oocyte and embryo quality . coagulation with bipolar cautery is usually considered a safe and well - accepted approach in laparoscopic endometrioma resection surgery , which was also the technique used in the present study . there are multiple studies in the literature that evaluated the effects of endometriosis on ivf outcomes . a meta - analysis about stage iii to iv endometriosis , which involved 22 studies , concluded that the fertilization , implantation and pregnancy rates , and the number of retrieved oocytes decreased in patients with endometriosis compared with the control subjects with tubal factor infertility . the same researchers presented that women with stage iii to iv endometriosis had fewer numbers of retrieved oocytes , lower e2 levels , and lower fertilization and implantation rates in comparison with women with stage i to ii endometriosis and women with infertility due to other causes . documented that patients with stage i to ii endometriosis had a lower fertilization rate , and patients with stage iii to iv endometriosis had fewer oocytes retrieved . furthermore , when the researchers split the stage iii and iv patients with and without endometriomas , it was reported that the endometrioma group had significantly lower pregnancy , live birth , and ongoing pregnancy rates . coccia et al showed the detrimental effects of severe endometriosis on ivf outcome via significantly reduced pregnancy rates ( clinical pregnancy rate per embryo transfer : 9.7% for stage iii to iv , 25% for stage i to ii , and 26.1% for tubal factor ) . in a very recent systematic review and meta - analysis , harb et iv ) was associated with poor implantation and clinical pregnancy rates in women undergoing ivf treatment . although there is no consensus on how to manage patients with advanced endometriosis and endometriomas , gnrh analogues have been used for the treatment of endometriosis for several years . the use of gnrh - a during ivf treatment in patients with advanced endometriosis has led to increased pregnancy rates in some studies . in support of this argument , patients who faced long - term suppression before the initiation of hyperstimulation with gonadotropins during ivf treatment had higher pregnancy rates . the most efficient ivf / icsi stimulation protocol in endometriosis patients is argued to be the ultralong ( long - term pituitary down - regulation with a gnrh - a ) protocol , studied by many researchers , including surrey et al . a cochrane systematic review and meta - analysis performed by sallam et al established the advantages of this protocol with an odds ratio of 4.28 ( in favor of long - term pituitary down - regulation with a gnrh - a ) . alternatively , advantages of the antagonist ( eg , easier and faster prevention of premature lh surge , flexibility of use , increased patient comfort due to shorter usage ) make it an attractive choice for any ivf program . in a recent study by rodriguez - purata et al , pregnancy rate after coh with either gnrh - a or gnrh - ant was found to be equally effective in patients with endometriosis ( stage i to iv ) . in this study , the number of retrieved mii oocytes and follicles were higher in patients who received a gnrh - a protocol . however , we used similar total doses of gonadotropin in the present study in both groups , yet significantly fewer numbers of aspirated follicles and retrieved oocytes were recorded in patients who received the gnrh - ant protocol . several studies and various meta - analyses have been carried out to compare pregnancy rates of gnrh - a versus gnrh - ant protocols . a meta - analysis of 14 randomized studies found no significant difference between the 2 gnrh analogues in the pregnancy rate . in addition pu et al reported no statistical difference to the number of oocytes retrieved , the number of mature oocytes retrieved , the cycle cancellation and clinical pregnancy rates between gnrh - a versus gnrh - ant protocols in poor ovarian responders . on the other hand , a cochrane meta - analysis by al - inany and aboulghar , which took into account 5 randomized studies , suggested that the usage of gnrh - ant protocol compared with gnrh - a provided lower pregnancy rates . however , the conclusions of this cochrane meta - analysis can not be generalized for the endometriosis patients . because of the risks involved with multiple pregnancies , which is a common occurrence in ivf treatments , the standard practice in many countries became the transfer of 1 ( elective single embryo transfer ) or 2 embryos instead of 3 . in turkey , the turkish ministry of health established a mandatory standard of single embryo transfer in march 2010 for all women under the age of 35 years , in their first 2 cycles . in the present study , the gnrh - a protocol revealed increased numbers of oocytes and embryos , so the patients who received this protocol had an opportunity to cryopreserve and transfer embryos later . limitations of the present study were the involvement of > 1 clinician and their preferences for different protocols ( long gnrh - a versus gnrh - ant ) and its retrospective design . in addition , differences in the skills of the surgeons , although in the present study all surgeons were experienced , might be considered as another limitation , as the ability to preserve normal ovarian tissue after endometrioma resection surgery varies according to the skill of the surgeon and the technique used . long gnrh - a and gnrh - ant protocols both present similar ivf outcomes in patients with endometriosis who have undergone laparoscopic endometrioma resection surgery .

mycotic keratitis is a severe problem in most of the developing countries whereas specific antifungal agents are expensive and commercially unavailable as ready compounds for topical ocular use . it is endemic and this is favored by certain ecological factors that already exist in those communities . other problems like difficulties in its clinical diagnosis , laboratory results , and treatment are also incriminated . mycotic keratitis is an important cause of corneal morbidity , scarring , and blindness caused by fungal invasion through corneal epithelium . the disease was found to be more common among agricultural communities than others . according to predisposing factors , clinical features , and response to treatment , fungal corneal pathogens were divided into filamentous fungi , yeast , and yeast like and dimorphic fungi . the most common filamentous fungi causing keratomycosis are aspergillus , fusarium , curvularia , alternaria , and cladosporium , while candida albicans is the most common form of yeasts [ 2 , 3 ] . mycotic keratitis probably arises as a result of an interaction between different agent factors as invasive ability and toxicity of fungal strains and host factors due to local or systemic causes . the invasiveness of fungal strains is aided by certain properties such as the capacity of fungus to adhere to the cells and to produce enzymes and toxins that destroy anatomical defenses [ 4 , 5 ] . fungi especially filamentous species rarely infect healthy cornea spontaneously and are usually predisposed by trauma with vegetable or organic matter . on the other hand candida albicans is a common infection in compromised or immune - suppressed cornea [ 6 , 7 ] . identification of pathogenic corneal fungi is performed either by microscopic stained corneal scrapes or by their cultural features . other methods for fungus identification depend on the types of enzymes produced , immune diffusion , electrophoresis , and elisa . the three major groups of antifungal agents are as follows : polyenes , as amphotericin b and nystatin , azoles , as itraconazole and fluconazole , pyrimidines , as flucytosine [ 11 , 12 ] . polyenes , as amphotericin b and nystatin , azoles , as itraconazole and fluconazole , pyrimidines , as flucytosine [ 11 , 12 ] . the study was conducted from the first of january 2011 to the end of december 2013 at the outpatient clinic of ophthalmology department in tanta university hospital . a total number of 66303 patients with different ophthalmological complaints were examined during that period . our target included those cases diagnosed clinically as fungal keratitis ; they were 361 cases during that period . clinical diagnosis of mycotic keratitis was dependent on some clinical criteria including the following : thick area of keratitis , thick hypopyon coagulum with or without level , immune rings , satellite lesions , stromal infiltrate with feathery edge , healing usually with dense leucoma with large solitary blood vessel , area of epithelial defect usually smaller than stromal infiltration . thick area of keratitis , thick hypopyon coagulum with or without level , stromal infiltrate with feathery edge , healing usually with dense leucoma with large solitary blood vessel , area of epithelial defect usually smaller than stromal infiltration . the other group included all cases of nonmycotic keratitis who attended the clinic during the same period . all cases of mycotic and nonmycotic keratitis were submitted to a complete ophthalmological examination by the slit lamp using direct oblique , scleral scatter , and retroillumination techniques . a questionnaire sheet was designed and filled by the researchers for cases and control including the following . ( 1 ) biological and sociodemographic data : age , sex , occupation , family size , crowding index ( family members / number of rooms ) , residence , source of water whether indoor or outdoor , and sewage disposal whether pipes system or conservancy system . ( 2 ) history taking for the following : some risk factors such as recent drug intake ( prolonged antibiotics and local or systemic corticosteroids ) , corneal trauma whether organic or nonorganic , systemic diseases ( diabetes mellitus , tuberculosis , and cancer ) , ocular surgery , local eye diseases such as glaucoma , and the time of onset of complaints till examination . ( 3 ) laboratory investigations : all cases diagnosed clinically as fungal keratitis were subjected to the following : sampling : under surface anesthesia , the active edge and the bed of ulcer were scraped using platinum spatula or sterile surgical blade ( number 15 ) with the aid of slit lamp biomicroscopy or surgical microscope to ensure that adequate corneal material was obtained and to avoid corneal perforation . so disposable calcium alginate cotton tipped sterile applicators were used for those cases.isolation media : sabouraud dextrose agar was prepared with 0.05% chloramphenicol , autoclaved , and spread on sterile petri plates . plates were incubated at room temperature ; fungal growth was identified and then sensitivity tests were carried out in vitro by using different antifungal agents . according to the results of sensitivity tests , the appropriate antifungal agent was used as topical drops , subconjunctival injection , and intracameral wash and even systemically if needed depending on the severity of the case . sampling : under surface anesthesia , the active edge and the bed of ulcer were scraped using platinum spatula or sterile surgical blade ( number 15 ) with the aid of slit lamp biomicroscopy or surgical microscope to ensure that adequate corneal material was obtained and to avoid corneal perforation . isolation media : sabouraud dextrose agar was prepared with 0.05% chloramphenicol , autoclaved , and spread on sterile petri plates . plates were incubated at room temperature ; fungal growth was identified and then sensitivity tests were carried out in vitro by using different antifungal agents . according to the results of sensitivity tests , the appropriate antifungal agent was used as topical drops , subconjunctival injection , and intracameral wash and even systemically if needed depending on the severity of the case . the treatment regimen used for all cases was as follows : amphotericin b ( 2 mg / ml ) ( fungizone ; bristol - myers squibb ) was used topically till the results of culture appeared;when the culture results appeared , the appropriate antifungal agent was used;the antifungal drugs prescribed included amphotericin b 0.15% ( fungizone ; bristol - myers squibb ) prepared as fortified drops , fluconazole 0.2% ( diflucan ; pfizer ) taken directly in eye dropper from the vial , natamycin 5% ( natamet ; sun pharmaceutical industries ) , and itraconazole 1% ( itral ; jawa pharmaceuticals ) . amphotericin b ( 2 mg / ml ) ( fungizone ; bristol - myers squibb ) was used topically till the results of culture appeared ; when the culture results appeared , the appropriate antifungal agent was used ; the antifungal drugs prescribed included amphotericin b 0.15% ( fungizone ; bristol - myers squibb ) prepared as fortified drops , fluconazole 0.2% ( diflucan ; pfizer ) taken directly in eye dropper from the vial , natamycin 5% ( natamet ; sun pharmaceutical industries ) , and itraconazole 1% ( itral ; jawa pharmaceuticals ) . adjunctive therapy atropine sulphate ( isopto atropine ; alcon ) eye drops , 3 times / day.gatifloxacin ( zimmer ; allergan ) eye drops , 5 times / day , weak corticosteroid drops of fluorometholone ( fml ; allergan ) twice daily added if there is immune ring or associated uveitis . atropine sulphate ( isopto atropine ; alcon ) eye drops , 3 times / day . gatifloxacin ( zimmer ; allergan ) eye drops , 5 times / day , weak corticosteroid drops of fluorometholone ( fml ; allergan ) twice daily added if there is immune ring or associated uveitis . weak corticosteroid drops of fluorometholone ( fml ; allergan ) twice daily added if there is immune ring or associated uveitis . for cases not responding to treatment within the first week , a debridement of superficial corneal layers was done to enhance good penetration of antifungal therapy . statistical presentation and analysis of the present study were conducted , using the mean , standard deviation , chi square , and t - test by spss v.20 . a total number of 66303 patients attended the outpatient clinic of ophthalmology department in tanta university during the period in which the study was conducted . our target was 361 patients with mycotic keratitis representing 0.54% of the total attendants , while those with nonmycotic keratitis accounted for 473 cases representing 0.71% of the total attendants of the clinic during that period . the mean age for cases of mycotic keratitis was 49.18 2.28 years , while mean age for the nonmycotic group was 50.88 2.60 years . table 1 shows that males in patients of mycotic keratitis were affected about twice more than females . the same was found with the nonmycotic keratitis group ; they were 66.6% males and 33.4% females . however the difference was statistically insignificant between mycotic and nonmycotic groups as regards the sex ( = 6.4 , p = 0.739).52.4% of cases of mycotic keratitis were at the age group of 4060 years while it was recorded that 51.4% of cases of nonmycotic keratitis were at the same age group . the difference between mycotic and nonmycotic keratitis was statistically not significant regarding the different age groups ( = 6.21 , p = 0.258 ) . the mean age for cases of mycotic keratitis was 49.18 2.28 years , while mean age for the nonmycotic group was 50.88 2.60 years . table 1 shows that males in patients of mycotic keratitis were affected about twice more than females . the same was found with the nonmycotic keratitis group ; they were 66.6% males and 33.4% females . however the difference was statistically insignificant between mycotic and nonmycotic groups as regards the sex ( = 6.4 , p = 0.739 ) . 52.4% of cases of mycotic keratitis were at the age group of 4060 years while it was recorded that 51.4% of cases of nonmycotic keratitis were at the same age group . the difference between mycotic and nonmycotic keratitis was statistically not significant regarding the different age groups ( = 6.21 , p = 0.258 ) . as shown in table 2 farmers ( unskilled workers ) represented the majority of cases ( 68.4% ) in mycotic keratitis group while they represented only 50.3% in the nonmycotic keratitis group . professionals and semiprofessionals represented only 2.2% of the mycotic group compared to 19.9% among the nonmycotic group . housewives were more among the mycotic cases than the nonmycotic cases ; they were 20% and 13.3% , respectively . the difference was statistically significant between cases of mycotic and nonmycotic keratitis as regards their occupations ( = 21.41 , p = 0.001 ) . as shown in table 3 , more than one - third of cases of mycotic keratitis were of large family size ( > 6 persons ) compared to the nonmycotic group ( 38.2% and 20.3% ) , respectively , while the percentage of cases with small family size ( < 4 persons ) was less than the nonmycotic group ; they were 20% and 36.6% , respectively . the difference was statistically significant ( = 10.67 , p = 0.004 ) . table 3 showed that 67.3% of cases with mycotic keratitis lived in overcrowded houses with crowding index > 2 . there was no significant statistical difference between mycotic and nonmycotic keratitis regarding the crowding index ( = 3.03 , p = 0.081 ) . table 4 showed that in cases of mycotic keratitis , 64.3% were rural residents compared to 56.2% among the nonmycotic group . the difference was statistically not significant between mycotic and nonmycotic groups ( = 1.55 , p = 0.212 ) . as shown in table 4 , outdoor water supply represented about three - quarters ( 72.9% ) of the houses of mycotic cases and more than half of those of cases with nonmycotic keratitis ( 58.1% ) . the difference was statistically significant ( = 4.95 , p = 0.025 ) . table 4 showed that in cases of mycotic keratitis only 22.7% of patients had pipes system of sewage disposal in their houses compared to 45% in the houses of nonmycotic group with a significant statistical difference ( = 10.78 , p = 0.001 ) . from table 5 , it was found that among the mycotic group 61.5% of cases came within the first week from the onset of complaints while about a quarter ( 22.4% ) of cases came for ocular examination from 14 days up to 30 days . in nonmycotic corneal ulcers the majority ( 85% ) came within the first week of complaints and no one came after 14 days . the difference between the two groups was statistically significant as regards the different periods of time ( = 25.63 , p = 0.3 ) . table 6 and figure 1 revealed that aspergillus species constituted the majority of cases with positive cultures : aspergillus flavus in 29.1% of cases and aspergillus niger in 16.1% , and the least frequent species was candida albicans ( 3% ) . negative cultures occurred in 15.5% of cases in spite of their improvement with antifungal therapy and their typical clinical findings for diagnosis . table 7 showed that trauma either organic or nonorganic was the most frequent risk factor for both of mycotic ( 58.4% ) and nonmycotic ( 75% ) groups . organic ocular trauma was the most frequent risk factor in mycotic keratitis ( 38.2% ) while nonorganic trauma was the most risky for the nonmycotic group ( 45% ) . other risk factors included ocular surgery which represented 32.7% of mycotic cases compared to 0% among nonmycotic group . prolonged local antibiotics therapy was risky for 6.1% of mycotic keratitis compared to 0% among nonmycotic group . systemic diseases represented the least frequent risk for mycotic keratitis ( 2.2% of the cases ) while it was 10.4% in nonmycotic keratitis . all cases of mycotic keratitis that had a history of ocular surgery had a mild course of corticosteroid therapy except in 22 cases that had a prolonged heavy course . among the mycotic keratitis cases , 127 cases ( 35.2% ) showed healing with faint nebulae , 193 cases ( 53.4% ) with dense vascularized leucoma , 23 cases with descemetocele ( 6.4% ) , and 10 cases ( 2.8% ) with perforation , and 8 cases ( 2.2% ) ended by endophthalmitis as shown in figure 2 . corneal infections including fungal types are responsible for about 50% of corneal scarring in the developing world which is a significant cause of blindness . mycotic keratitis is a growing health problem in the developing countries representing from one - third up to 56% of total ocular infections . world health organization reported that about 10 million all over the world were blind due to corneal infections in its program for prevention of blindness ( global initiative vision by the year 2020 ) [ 13 , 14 ] . in the present study , about half the cases of mycotic keratitis were at the age group from 40 to 60 years , and males were about two times more affected than females . these findings were close to that of baradkar and others in 2008 in their study in india whereas they concluded that fungal keratitis affected males three times more than females and were more frequent in the age group from 20 to 50 years . males are more affected because of their outdoor activity and movement in the surrounding environment which expose them to different risks such as corneal trauma . there were no significant statistical differences between cases of mycotic keratitis and those of nonmycotic keratitis as regards the age groups ( = 6.21 , p = 0.258 ) and sex ( = 6.4 , p = 0.739 ) . ( i ) in this study , mycotic keratitis affected farmers ( unskilled workers ) more than other occupations in 68.4% of the cases followed by housewives in 20% . these findings were consistent with moharram and his colleagues in 1999 in their study in assiut where 47% of cases of fungal keratitis were farmers and 20% were housewives . farming was the main job for most of cases of fungal keratitis in other studies [ 11 , 1517 ] . most of fungal species such as aspergillus , penicillium , mucorrhacimosis species , and cladosporium are soil fungi , so they contaminate vegetables and fruits . farmers are usually exposed to trauma by organic matter ( such as dried rice stems or maize ) which facilitates invasion of the cornea by the fungi . there was a significant statistical difference between cases of mycotic keratitis and the nonmycotic keratitis regarding their occupations ( = 21.41 , p = 0.001 ) . farmers were more recorded among cases while professionals and semiprofessionals were more recorded among the control . professionals might be exposed to some risk factors such as the nonorganic trauma but rarely exposed to organic trauma which is associated mostly with fungal contamination . in this study organic trauma was the most risky factor for fungal keratitis while nonorganic trauma was the most risky for the control group and this might explain the former finding . ( ii ) this study reported that cases of mycotic keratitis belonged to large families ( 46 persons in 41.8% and more than 6 persons in 38.2% ) , with a high crowding index ( 67.3% of cases with crowding index > 2 ) , and about two - thirds ( in 64.3% ) of them were rural . there was a statistically significant difference between mycotic and nonmycotic cases as regards their family size ( larger families among mycotic keratitis cases ) ( = 10.67 , p = 0.004 ) . there were insignificant statistical differences between both groups regarding crowding index ( = 3.03 , p = 0.081 ) and rural residence ( = 1.55 , p = 0.212 ) . however in both of mycotic and nonmycotic keratitis the percentage of the disease was higher in large families , with a high crowding index and rural residence . these were explained by the unsound health behavior and lack of health awareness especially in those living in overcrowded houses with bad sanitation . these findings were in agreement with other studies [ 5 , 18 , 19 ] which reported that fungal and microbial keratitis associated with low socioeconomic status and rural residence . ( iii ) as regards water sources , the outdoor water supply accounted for 72.9% of houses of mycotic cases compared to 58.1% among the nonmycotic cases with significant statistical difference ( = 4.95 , p = 0.025 ) . concerning sewage disposal , more than three - quarters of houses of mycotic keratitis ( 77.3% ) were by conservancy system compared to 55% of the houses in the nonmycotic cases with a significant statistical difference ( = 10.78 , p = 0.001 ) . ( iv ) in conclusion , it was revealed that cases of mycotic keratitis lived in more deteriorated environment than the nonmycotic group . this was consistent with gopinathan and his colleagues who reported in 2009 that cases of mycotic keratitis lived in poor insanitary environment . rautaraya in 2011 reported that eye washing with contaminated water could cause mycotic and nonmycotic keratitis . outdoor water and insanitary sewage disposal systems could affect personal hygiene and self - care with poor sanitation of houses together with spread of flies and insects that transmit organisms easily to healthy or unhealthy corneas . in this study , 61.5% of cases of mycotic keratitis came within the first week of their onset of complaints to seek medical care compared to the majority ( 85% ) in the nonmycotic group . about a quarter of cases of mycotic keratitis ( 22.4% ) came to seek medical care after two weeks from onset of complaints compared to no one in the nonmycotic group . the difference in mean time between both groups was statistically significant ( = 25.63 , p = 0.3 ) . first , some patients developed mycotic keratitis after ocular surgery and thought that their complaints were a normal sequence of the surgery , so they delayed seeking medical care . second , some other patients were posttraumatic and thought that the symptoms were a normal sequence of trauma . lastly , some patients were treated by mistake at first as nonmycotic keratitis till they were managed as fungal keratitis . ( i ) it was found that 84.5% of patients were positive , while 15.5% were negative in spite of their typical clinical findings and their improvement with antifungal therapy . these may be explained by some fungi present in deep stroma and so could not be obtained if superficial scraping was done or if scraping was obtained only from one area . these findings were in consistence with rautaraya who reported that 25.4% of their patients showed negative fungal growth in spite of their typical clinical findings for microbial keratitis . ( ii ) for the type of fungusisolated , it was found that aspergillus species were the most common ( 45.2% ) , either flavus ( 29.1% ) or niger ( 16.1% ) . these results were in agreement with rautaraya and his colleagues in india who demonstrated that aspergillus species constituted the majority ( 27.9% ) of fungal growth in their study . these findings may be explained by the more spread of aspergillus species in the environment specially spores which can survive hot and dry weather for long time . aspergillus species existed in the soil and animal skin , so they were more frequent among the farmers in this study . ( iii ) among aspergillus species it was found that aspergillus flavus constituted 29.1% and aspergillus niger 16.1% . this was consistent with the findings of rautaraya study that demonstrated 15.8% for aspergillus flavus and 12.3% for other aspergillus species . ( iv ) candida species were the least frequent type ( only in 3% ) ; that is in agreement with what was reported by rautaraya and others that candida species were the least common in their study ( 0.9% ) . our results revealed that , among a total number of 66303 cases examined during the time of the study , mycotic keratitis occurred among 0.54% of cases and the nonmycotic keratitis in 0.71% . a study that was done from february 1991 to june 2001 by gopinathan and others in india demonstrated 5897 cases of suspected infectious keratitis ; 3563 ( 60.4% ) were culture proven ( bacterial : 1849 , 51.9% ; fungal : 1360 , 38.2% ; acanthamoeba : 86 , 2.4% ; mixed : 268 , 7.5% ) . among fungal cases aspergillus species were responsible for 28.9% of cases , fusarium in 35.6% , dematiaceous fungi in 19.3% , other hyaline fungi in 15.4% , and candida only in 0.8% . between september 1985 and august 1987 , 405 patients with corneal ulceration were examined at tribhuvan university teaching hospital in kathmandu , nepal . males and females the most common predisposing cause of ulceration was corneal trauma , usually with organic agricultural materials . pure bacterial cultures were obtained from 256 ( 63.2% ) of the patients , whereas pure fungal cultures were obtained from 27 ( 6.7% ) of the patients . in 41 patients ( 10.1% ) , of 68 positive fungal isolates obtained , 32 ( 47.0% ) were identified as aspergillus species . a 13-year study that was done in paraguay from 1988 to 2001 and included 660 patients with infectious keratitis revealed that 79% of cases were culture positive of which 49% was fungal growth . in a study done in ghana in 1995 , one or more organisms were cultured from 114 of 199 patients ( 57.3% ) , with the most common being fusarium species , pseudomonas aeruginosa , and staphylococcus epidermidis . fungi , alone or in combination , were isolated from 56% of the patients who had fungal growth , in total 122 patients ( 61.3% ) . this work reported that , among cases of mycotic keratitis , organic trauma was the most prevalent risk factor ( 38.2% ) , followed by ocular surgery in 32.7% of cases then the nonorganic trauma in 20.2% followed by the use of topical antibiotics in 6.1% , and systemic disease ( such as diabetes ) was the least frequent in only 2.2% . on the other hand , the nonorganic trauma was the most frequent risk factor for nonmycotic keratitis ( 45% ) followed by the organic trauma ( 35% ) . farmers were more exposed to organic trauma ( with rice or maize stems or others ) during farming so they were more exposed to mycotic keratitis . as regards the risk of ocular surgery among the cases , fungal contamination might occur preoperatively in those cases with negative cultures or postoperatively due to lowered body immunity ( by the stressful surgery ) and/or the use of a course of local and systemic corticosteroids with the surgery . in consistence with our findings , ocular trauma was the most frequent risk for either mycotic or nonmycotic keratitis in other studies [ 5 , 1522 ] . community awareness of the risk factors.restriction of the abuse of topical corticosteroids or antibiotics.mycotic keratitis should be suspected in every patient with a corneal lesion , specially the resistant one , and should be ruled out before commencing steroids and antibiotics.for a successful treatment , we need the following : proper early clinical diagnosis , proper laboratory diagnosis , initiating a broad spectrum antifungal drug till laboratory findings prove the specific drug , proper frequency of antifungal drugs , proper timing for termination of therapy . community awareness of the risk factors . mycotic keratitis should be suspected in every patient with a corneal lesion , specially the resistant one , and should be ruled out before commencing steroids and antibiotics . for a successful treatment , we need the following : proper early clinical diagnosis , proper laboratory diagnosis , initiating a broad spectrum antifungal drug till laboratory findings prove the specific drug , proper frequency of antifungal drugs , proper timing for termination of therapy . proper early clinical diagnosis , proper laboratory diagnosis , initiating a broad spectrum antifungal drug till laboratory findings prove the specific drug , proper frequency of antifungal drugs , proper timing for termination of therapy .

purpose . this work aims to study the problems encountered with and the different epidemiological features of patients with fungal keratitis . patients and methods . all cases with keratitis attending the outpatient clinic of ophthalmology department at tanta university hospital during three years from the first of january 2011 to the end of december 2013 were selected and carefully examined and cases with mycotic keratitis were further examined and investigated . results . from 66303 attendants during this period with different complaints , there were 361 cases ( 0.54% ) with mycotic keratitis and 473 cases ( 0.71% ) of nonmycotic origin . mycotic keratitis is common between 40 and 60 years , more in farmers ( 64% ) , families with large number and large crowding index , rural than urban residence , and patients with outdoor water sources and insanitary sewage disposal . positive fungal cultures were obtained in 84.5% and were negative in 15.5% of cases in spite of their typical clinical findings for diagnosis and their improvement with antifungal therapy . conclusion . mycotic keratitis is more frequent in farmers , rural areas , outdoor water supply , insanitary sewage disposal , and patients preceded with organic trauma . atypical clinical findings were found in some cases and not all cases improved with specific antifungal therapy .

1. Introduction 2. Patients and Methods 3. Results 4. Discussion 5. Recommendation

mycotic keratitis is a severe problem in most of the developing countries whereas specific antifungal agents are expensive and commercially unavailable as ready compounds for topical ocular use . other problems like difficulties in its clinical diagnosis , laboratory results , and treatment are also incriminated . mycotic keratitis is an important cause of corneal morbidity , scarring , and blindness caused by fungal invasion through corneal epithelium . according to predisposing factors , clinical features , and response to treatment , fungal corneal pathogens were divided into filamentous fungi , yeast , and yeast like and dimorphic fungi . the most common filamentous fungi causing keratomycosis are aspergillus , fusarium , curvularia , alternaria , and cladosporium , while candida albicans is the most common form of yeasts [ 2 , 3 ] . the invasiveness of fungal strains is aided by certain properties such as the capacity of fungus to adhere to the cells and to produce enzymes and toxins that destroy anatomical defenses [ 4 , 5 ] . other methods for fungus identification depend on the types of enzymes produced , immune diffusion , electrophoresis , and elisa . the study was conducted from the first of january 2011 to the end of december 2013 at the outpatient clinic of ophthalmology department in tanta university hospital . a total number of 66303 patients with different ophthalmological complaints were examined during that period . our target included those cases diagnosed clinically as fungal keratitis ; they were 361 cases during that period . clinical diagnosis of mycotic keratitis was dependent on some clinical criteria including the following : thick area of keratitis , thick hypopyon coagulum with or without level , immune rings , satellite lesions , stromal infiltrate with feathery edge , healing usually with dense leucoma with large solitary blood vessel , area of epithelial defect usually smaller than stromal infiltration . thick area of keratitis , thick hypopyon coagulum with or without level , stromal infiltrate with feathery edge , healing usually with dense leucoma with large solitary blood vessel , area of epithelial defect usually smaller than stromal infiltration . the other group included all cases of nonmycotic keratitis who attended the clinic during the same period . all cases of mycotic and nonmycotic keratitis were submitted to a complete ophthalmological examination by the slit lamp using direct oblique , scleral scatter , and retroillumination techniques . ( 1 ) biological and sociodemographic data : age , sex , occupation , family size , crowding index ( family members / number of rooms ) , residence , source of water whether indoor or outdoor , and sewage disposal whether pipes system or conservancy system . ( 2 ) history taking for the following : some risk factors such as recent drug intake ( prolonged antibiotics and local or systemic corticosteroids ) , corneal trauma whether organic or nonorganic , systemic diseases ( diabetes mellitus , tuberculosis , and cancer ) , ocular surgery , local eye diseases such as glaucoma , and the time of onset of complaints till examination . ( 3 ) laboratory investigations : all cases diagnosed clinically as fungal keratitis were subjected to the following : sampling : under surface anesthesia , the active edge and the bed of ulcer were scraped using platinum spatula or sterile surgical blade ( number 15 ) with the aid of slit lamp biomicroscopy or surgical microscope to ensure that adequate corneal material was obtained and to avoid corneal perforation . according to the results of sensitivity tests , the appropriate antifungal agent was used as topical drops , subconjunctival injection , and intracameral wash and even systemically if needed depending on the severity of the case . sampling : under surface anesthesia , the active edge and the bed of ulcer were scraped using platinum spatula or sterile surgical blade ( number 15 ) with the aid of slit lamp biomicroscopy or surgical microscope to ensure that adequate corneal material was obtained and to avoid corneal perforation . according to the results of sensitivity tests , the appropriate antifungal agent was used as topical drops , subconjunctival injection , and intracameral wash and even systemically if needed depending on the severity of the case . the treatment regimen used for all cases was as follows : amphotericin b ( 2 mg / ml ) ( fungizone ; bristol - myers squibb ) was used topically till the results of culture appeared;when the culture results appeared , the appropriate antifungal agent was used;the antifungal drugs prescribed included amphotericin b 0.15% ( fungizone ; bristol - myers squibb ) prepared as fortified drops , fluconazole 0.2% ( diflucan ; pfizer ) taken directly in eye dropper from the vial , natamycin 5% ( natamet ; sun pharmaceutical industries ) , and itraconazole 1% ( itral ; jawa pharmaceuticals ) . amphotericin b ( 2 mg / ml ) ( fungizone ; bristol - myers squibb ) was used topically till the results of culture appeared ; when the culture results appeared , the appropriate antifungal agent was used ; the antifungal drugs prescribed included amphotericin b 0.15% ( fungizone ; bristol - myers squibb ) prepared as fortified drops , fluconazole 0.2% ( diflucan ; pfizer ) taken directly in eye dropper from the vial , natamycin 5% ( natamet ; sun pharmaceutical industries ) , and itraconazole 1% ( itral ; jawa pharmaceuticals ) . for cases not responding to treatment within the first week , a debridement of superficial corneal layers was done to enhance good penetration of antifungal therapy . statistical presentation and analysis of the present study were conducted , using the mean , standard deviation , chi square , and t - test by spss v.20 . a total number of 66303 patients attended the outpatient clinic of ophthalmology department in tanta university during the period in which the study was conducted . our target was 361 patients with mycotic keratitis representing 0.54% of the total attendants , while those with nonmycotic keratitis accounted for 473 cases representing 0.71% of the total attendants of the clinic during that period . the mean age for cases of mycotic keratitis was 49.18 2.28 years , while mean age for the nonmycotic group was 50.88 2.60 years . table 1 shows that males in patients of mycotic keratitis were affected about twice more than females . however the difference was statistically insignificant between mycotic and nonmycotic groups as regards the sex ( = 6.4 , p = 0.739).52.4% of cases of mycotic keratitis were at the age group of 4060 years while it was recorded that 51.4% of cases of nonmycotic keratitis were at the same age group . the difference between mycotic and nonmycotic keratitis was statistically not significant regarding the different age groups ( = 6.21 , p = 0.258 ) . the mean age for cases of mycotic keratitis was 49.18 2.28 years , while mean age for the nonmycotic group was 50.88 2.60 years . table 1 shows that males in patients of mycotic keratitis were affected about twice more than females . 52.4% of cases of mycotic keratitis were at the age group of 4060 years while it was recorded that 51.4% of cases of nonmycotic keratitis were at the same age group . as shown in table 2 farmers ( unskilled workers ) represented the majority of cases ( 68.4% ) in mycotic keratitis group while they represented only 50.3% in the nonmycotic keratitis group . as shown in table 3 , more than one - third of cases of mycotic keratitis were of large family size ( > 6 persons ) compared to the nonmycotic group ( 38.2% and 20.3% ) , respectively , while the percentage of cases with small family size ( < 4 persons ) was less than the nonmycotic group ; they were 20% and 36.6% , respectively . table 3 showed that 67.3% of cases with mycotic keratitis lived in overcrowded houses with crowding index > 2 . there was no significant statistical difference between mycotic and nonmycotic keratitis regarding the crowding index ( = 3.03 , p = 0.081 ) . as shown in table 4 , outdoor water supply represented about three - quarters ( 72.9% ) of the houses of mycotic cases and more than half of those of cases with nonmycotic keratitis ( 58.1% ) . table 4 showed that in cases of mycotic keratitis only 22.7% of patients had pipes system of sewage disposal in their houses compared to 45% in the houses of nonmycotic group with a significant statistical difference ( = 10.78 , p = 0.001 ) . from table 5 , it was found that among the mycotic group 61.5% of cases came within the first week from the onset of complaints while about a quarter ( 22.4% ) of cases came for ocular examination from 14 days up to 30 days . in nonmycotic corneal ulcers the majority ( 85% ) came within the first week of complaints and no one came after 14 days . table 6 and figure 1 revealed that aspergillus species constituted the majority of cases with positive cultures : aspergillus flavus in 29.1% of cases and aspergillus niger in 16.1% , and the least frequent species was candida albicans ( 3% ) . negative cultures occurred in 15.5% of cases in spite of their improvement with antifungal therapy and their typical clinical findings for diagnosis . organic ocular trauma was the most frequent risk factor in mycotic keratitis ( 38.2% ) while nonorganic trauma was the most risky for the nonmycotic group ( 45% ) . prolonged local antibiotics therapy was risky for 6.1% of mycotic keratitis compared to 0% among nonmycotic group . all cases of mycotic keratitis that had a history of ocular surgery had a mild course of corticosteroid therapy except in 22 cases that had a prolonged heavy course . among the mycotic keratitis cases , 127 cases ( 35.2% ) showed healing with faint nebulae , 193 cases ( 53.4% ) with dense vascularized leucoma , 23 cases with descemetocele ( 6.4% ) , and 10 cases ( 2.8% ) with perforation , and 8 cases ( 2.2% ) ended by endophthalmitis as shown in figure 2 . mycotic keratitis is a growing health problem in the developing countries representing from one - third up to 56% of total ocular infections . in the present study , about half the cases of mycotic keratitis were at the age group from 40 to 60 years , and males were about two times more affected than females . these findings were close to that of baradkar and others in 2008 in their study in india whereas they concluded that fungal keratitis affected males three times more than females and were more frequent in the age group from 20 to 50 years . males are more affected because of their outdoor activity and movement in the surrounding environment which expose them to different risks such as corneal trauma . there were no significant statistical differences between cases of mycotic keratitis and those of nonmycotic keratitis as regards the age groups ( = 6.21 , p = 0.258 ) and sex ( = 6.4 , p = 0.739 ) . ( i ) in this study , mycotic keratitis affected farmers ( unskilled workers ) more than other occupations in 68.4% of the cases followed by housewives in 20% . these findings were consistent with moharram and his colleagues in 1999 in their study in assiut where 47% of cases of fungal keratitis were farmers and 20% were housewives . farming was the main job for most of cases of fungal keratitis in other studies [ 11 , 1517 ] . most of fungal species such as aspergillus , penicillium , mucorrhacimosis species , and cladosporium are soil fungi , so they contaminate vegetables and fruits . there was a significant statistical difference between cases of mycotic keratitis and the nonmycotic keratitis regarding their occupations ( = 21.41 , p = 0.001 ) . professionals might be exposed to some risk factors such as the nonorganic trauma but rarely exposed to organic trauma which is associated mostly with fungal contamination . in this study organic trauma was the most risky factor for fungal keratitis while nonorganic trauma was the most risky for the control group and this might explain the former finding . ( ii ) this study reported that cases of mycotic keratitis belonged to large families ( 46 persons in 41.8% and more than 6 persons in 38.2% ) , with a high crowding index ( 67.3% of cases with crowding index > 2 ) , and about two - thirds ( in 64.3% ) of them were rural . there was a statistically significant difference between mycotic and nonmycotic cases as regards their family size ( larger families among mycotic keratitis cases ) ( = 10.67 , p = 0.004 ) . there were insignificant statistical differences between both groups regarding crowding index ( = 3.03 , p = 0.081 ) and rural residence ( = 1.55 , p = 0.212 ) . however in both of mycotic and nonmycotic keratitis the percentage of the disease was higher in large families , with a high crowding index and rural residence . ( iii ) as regards water sources , the outdoor water supply accounted for 72.9% of houses of mycotic cases compared to 58.1% among the nonmycotic cases with significant statistical difference ( = 4.95 , p = 0.025 ) . concerning sewage disposal , more than three - quarters of houses of mycotic keratitis ( 77.3% ) were by conservancy system compared to 55% of the houses in the nonmycotic cases with a significant statistical difference ( = 10.78 , p = 0.001 ) . this was consistent with gopinathan and his colleagues who reported in 2009 that cases of mycotic keratitis lived in poor insanitary environment . outdoor water and insanitary sewage disposal systems could affect personal hygiene and self - care with poor sanitation of houses together with spread of flies and insects that transmit organisms easily to healthy or unhealthy corneas . in this study , 61.5% of cases of mycotic keratitis came within the first week of their onset of complaints to seek medical care compared to the majority ( 85% ) in the nonmycotic group . about a quarter of cases of mycotic keratitis ( 22.4% ) came to seek medical care after two weeks from onset of complaints compared to no one in the nonmycotic group . lastly , some patients were treated by mistake at first as nonmycotic keratitis till they were managed as fungal keratitis . ( i ) it was found that 84.5% of patients were positive , while 15.5% were negative in spite of their typical clinical findings and their improvement with antifungal therapy . these findings were in consistence with rautaraya who reported that 25.4% of their patients showed negative fungal growth in spite of their typical clinical findings for microbial keratitis . these results were in agreement with rautaraya and his colleagues in india who demonstrated that aspergillus species constituted the majority ( 27.9% ) of fungal growth in their study . our results revealed that , among a total number of 66303 cases examined during the time of the study , mycotic keratitis occurred among 0.54% of cases and the nonmycotic keratitis in 0.71% . among fungal cases aspergillus species were responsible for 28.9% of cases , fusarium in 35.6% , dematiaceous fungi in 19.3% , other hyaline fungi in 15.4% , and candida only in 0.8% . males and females the most common predisposing cause of ulceration was corneal trauma , usually with organic agricultural materials . pure bacterial cultures were obtained from 256 ( 63.2% ) of the patients , whereas pure fungal cultures were obtained from 27 ( 6.7% ) of the patients . in 41 patients ( 10.1% ) , of 68 positive fungal isolates obtained , 32 ( 47.0% ) were identified as aspergillus species . a 13-year study that was done in paraguay from 1988 to 2001 and included 660 patients with infectious keratitis revealed that 79% of cases were culture positive of which 49% was fungal growth . in a study done in ghana in 1995 , one or more organisms were cultured from 114 of 199 patients ( 57.3% ) , with the most common being fusarium species , pseudomonas aeruginosa , and staphylococcus epidermidis . this work reported that , among cases of mycotic keratitis , organic trauma was the most prevalent risk factor ( 38.2% ) , followed by ocular surgery in 32.7% of cases then the nonorganic trauma in 20.2% followed by the use of topical antibiotics in 6.1% , and systemic disease ( such as diabetes ) was the least frequent in only 2.2% . farmers were more exposed to organic trauma ( with rice or maize stems or others ) during farming so they were more exposed to mycotic keratitis . as regards the risk of ocular surgery among the cases , fungal contamination might occur preoperatively in those cases with negative cultures or postoperatively due to lowered body immunity ( by the stressful surgery ) and/or the use of a course of local and systemic corticosteroids with the surgery . community awareness of the risk factors.restriction of the abuse of topical corticosteroids or antibiotics.mycotic keratitis should be suspected in every patient with a corneal lesion , specially the resistant one , and should be ruled out before commencing steroids and antibiotics.for a successful treatment , we need the following : proper early clinical diagnosis , proper laboratory diagnosis , initiating a broad spectrum antifungal drug till laboratory findings prove the specific drug , proper frequency of antifungal drugs , proper timing for termination of therapy . mycotic keratitis should be suspected in every patient with a corneal lesion , specially the resistant one , and should be ruled out before commencing steroids and antibiotics .

mycotic keratitis is a severe problem in most of the developing countries whereas specific antifungal agents are expensive and commercially unavailable as ready compounds for topical ocular use . according to predisposing factors , clinical features , and response to treatment , fungal corneal pathogens were divided into filamentous fungi , yeast , and yeast like and dimorphic fungi . the most common filamentous fungi causing keratomycosis are aspergillus , fusarium , curvularia , alternaria , and cladosporium , while candida albicans is the most common form of yeasts [ 2 , 3 ] . mycotic keratitis probably arises as a result of an interaction between different agent factors as invasive ability and toxicity of fungal strains and host factors due to local or systemic causes . the invasiveness of fungal strains is aided by certain properties such as the capacity of fungus to adhere to the cells and to produce enzymes and toxins that destroy anatomical defenses [ 4 , 5 ] . other methods for fungus identification depend on the types of enzymes produced , immune diffusion , electrophoresis , and elisa . the three major groups of antifungal agents are as follows : polyenes , as amphotericin b and nystatin , azoles , as itraconazole and fluconazole , pyrimidines , as flucytosine [ 11 , 12 ] . polyenes , as amphotericin b and nystatin , azoles , as itraconazole and fluconazole , pyrimidines , as flucytosine [ 11 , 12 ] . the study was conducted from the first of january 2011 to the end of december 2013 at the outpatient clinic of ophthalmology department in tanta university hospital . clinical diagnosis of mycotic keratitis was dependent on some clinical criteria including the following : thick area of keratitis , thick hypopyon coagulum with or without level , immune rings , satellite lesions , stromal infiltrate with feathery edge , healing usually with dense leucoma with large solitary blood vessel , area of epithelial defect usually smaller than stromal infiltration . thick area of keratitis , thick hypopyon coagulum with or without level , stromal infiltrate with feathery edge , healing usually with dense leucoma with large solitary blood vessel , area of epithelial defect usually smaller than stromal infiltration . ( 1 ) biological and sociodemographic data : age , sex , occupation , family size , crowding index ( family members / number of rooms ) , residence , source of water whether indoor or outdoor , and sewage disposal whether pipes system or conservancy system . ( 2 ) history taking for the following : some risk factors such as recent drug intake ( prolonged antibiotics and local or systemic corticosteroids ) , corneal trauma whether organic or nonorganic , systemic diseases ( diabetes mellitus , tuberculosis , and cancer ) , ocular surgery , local eye diseases such as glaucoma , and the time of onset of complaints till examination . ( 3 ) laboratory investigations : all cases diagnosed clinically as fungal keratitis were subjected to the following : sampling : under surface anesthesia , the active edge and the bed of ulcer were scraped using platinum spatula or sterile surgical blade ( number 15 ) with the aid of slit lamp biomicroscopy or surgical microscope to ensure that adequate corneal material was obtained and to avoid corneal perforation . according to the results of sensitivity tests , the appropriate antifungal agent was used as topical drops , subconjunctival injection , and intracameral wash and even systemically if needed depending on the severity of the case . sampling : under surface anesthesia , the active edge and the bed of ulcer were scraped using platinum spatula or sterile surgical blade ( number 15 ) with the aid of slit lamp biomicroscopy or surgical microscope to ensure that adequate corneal material was obtained and to avoid corneal perforation . according to the results of sensitivity tests , the appropriate antifungal agent was used as topical drops , subconjunctival injection , and intracameral wash and even systemically if needed depending on the severity of the case . the treatment regimen used for all cases was as follows : amphotericin b ( 2 mg / ml ) ( fungizone ; bristol - myers squibb ) was used topically till the results of culture appeared;when the culture results appeared , the appropriate antifungal agent was used;the antifungal drugs prescribed included amphotericin b 0.15% ( fungizone ; bristol - myers squibb ) prepared as fortified drops , fluconazole 0.2% ( diflucan ; pfizer ) taken directly in eye dropper from the vial , natamycin 5% ( natamet ; sun pharmaceutical industries ) , and itraconazole 1% ( itral ; jawa pharmaceuticals ) . amphotericin b ( 2 mg / ml ) ( fungizone ; bristol - myers squibb ) was used topically till the results of culture appeared ; when the culture results appeared , the appropriate antifungal agent was used ; the antifungal drugs prescribed included amphotericin b 0.15% ( fungizone ; bristol - myers squibb ) prepared as fortified drops , fluconazole 0.2% ( diflucan ; pfizer ) taken directly in eye dropper from the vial , natamycin 5% ( natamet ; sun pharmaceutical industries ) , and itraconazole 1% ( itral ; jawa pharmaceuticals ) . adjunctive therapy atropine sulphate ( isopto atropine ; alcon ) eye drops , 3 times / day.gatifloxacin ( zimmer ; allergan ) eye drops , 5 times / day , weak corticosteroid drops of fluorometholone ( fml ; allergan ) twice daily added if there is immune ring or associated uveitis . for cases not responding to treatment within the first week , a debridement of superficial corneal layers was done to enhance good penetration of antifungal therapy . our target was 361 patients with mycotic keratitis representing 0.54% of the total attendants , while those with nonmycotic keratitis accounted for 473 cases representing 0.71% of the total attendants of the clinic during that period . the mean age for cases of mycotic keratitis was 49.18 2.28 years , while mean age for the nonmycotic group was 50.88 2.60 years . however the difference was statistically insignificant between mycotic and nonmycotic groups as regards the sex ( = 6.4 , p = 0.739).52.4% of cases of mycotic keratitis were at the age group of 4060 years while it was recorded that 51.4% of cases of nonmycotic keratitis were at the same age group . the difference between mycotic and nonmycotic keratitis was statistically not significant regarding the different age groups ( = 6.21 , p = 0.258 ) . the mean age for cases of mycotic keratitis was 49.18 2.28 years , while mean age for the nonmycotic group was 50.88 2.60 years . table 1 shows that males in patients of mycotic keratitis were affected about twice more than females . however the difference was statistically insignificant between mycotic and nonmycotic groups as regards the sex ( = 6.4 , p = 0.739 ) . 52.4% of cases of mycotic keratitis were at the age group of 4060 years while it was recorded that 51.4% of cases of nonmycotic keratitis were at the same age group . the difference between mycotic and nonmycotic keratitis was statistically not significant regarding the different age groups ( = 6.21 , p = 0.258 ) . as shown in table 2 farmers ( unskilled workers ) represented the majority of cases ( 68.4% ) in mycotic keratitis group while they represented only 50.3% in the nonmycotic keratitis group . professionals and semiprofessionals represented only 2.2% of the mycotic group compared to 19.9% among the nonmycotic group . the difference was statistically significant between cases of mycotic and nonmycotic keratitis as regards their occupations ( = 21.41 , p = 0.001 ) . as shown in table 3 , more than one - third of cases of mycotic keratitis were of large family size ( > 6 persons ) compared to the nonmycotic group ( 38.2% and 20.3% ) , respectively , while the percentage of cases with small family size ( < 4 persons ) was less than the nonmycotic group ; they were 20% and 36.6% , respectively . table 4 showed that in cases of mycotic keratitis , 64.3% were rural residents compared to 56.2% among the nonmycotic group . as shown in table 4 , outdoor water supply represented about three - quarters ( 72.9% ) of the houses of mycotic cases and more than half of those of cases with nonmycotic keratitis ( 58.1% ) . table 4 showed that in cases of mycotic keratitis only 22.7% of patients had pipes system of sewage disposal in their houses compared to 45% in the houses of nonmycotic group with a significant statistical difference ( = 10.78 , p = 0.001 ) . from table 5 , it was found that among the mycotic group 61.5% of cases came within the first week from the onset of complaints while about a quarter ( 22.4% ) of cases came for ocular examination from 14 days up to 30 days . in nonmycotic corneal ulcers the majority ( 85% ) came within the first week of complaints and no one came after 14 days . the difference between the two groups was statistically significant as regards the different periods of time ( = 25.63 , p = 0.3 ) . table 6 and figure 1 revealed that aspergillus species constituted the majority of cases with positive cultures : aspergillus flavus in 29.1% of cases and aspergillus niger in 16.1% , and the least frequent species was candida albicans ( 3% ) . negative cultures occurred in 15.5% of cases in spite of their improvement with antifungal therapy and their typical clinical findings for diagnosis . table 7 showed that trauma either organic or nonorganic was the most frequent risk factor for both of mycotic ( 58.4% ) and nonmycotic ( 75% ) groups . organic ocular trauma was the most frequent risk factor in mycotic keratitis ( 38.2% ) while nonorganic trauma was the most risky for the nonmycotic group ( 45% ) . systemic diseases represented the least frequent risk for mycotic keratitis ( 2.2% of the cases ) while it was 10.4% in nonmycotic keratitis . among the mycotic keratitis cases , 127 cases ( 35.2% ) showed healing with faint nebulae , 193 cases ( 53.4% ) with dense vascularized leucoma , 23 cases with descemetocele ( 6.4% ) , and 10 cases ( 2.8% ) with perforation , and 8 cases ( 2.2% ) ended by endophthalmitis as shown in figure 2 . mycotic keratitis is a growing health problem in the developing countries representing from one - third up to 56% of total ocular infections . in the present study , about half the cases of mycotic keratitis were at the age group from 40 to 60 years , and males were about two times more affected than females . these findings were close to that of baradkar and others in 2008 in their study in india whereas they concluded that fungal keratitis affected males three times more than females and were more frequent in the age group from 20 to 50 years . there were no significant statistical differences between cases of mycotic keratitis and those of nonmycotic keratitis as regards the age groups ( = 6.21 , p = 0.258 ) and sex ( = 6.4 , p = 0.739 ) . ( i ) in this study , mycotic keratitis affected farmers ( unskilled workers ) more than other occupations in 68.4% of the cases followed by housewives in 20% . farming was the main job for most of cases of fungal keratitis in other studies [ 11 , 1517 ] . most of fungal species such as aspergillus , penicillium , mucorrhacimosis species , and cladosporium are soil fungi , so they contaminate vegetables and fruits . there was a significant statistical difference between cases of mycotic keratitis and the nonmycotic keratitis regarding their occupations ( = 21.41 , p = 0.001 ) . in this study organic trauma was the most risky factor for fungal keratitis while nonorganic trauma was the most risky for the control group and this might explain the former finding . ( ii ) this study reported that cases of mycotic keratitis belonged to large families ( 46 persons in 41.8% and more than 6 persons in 38.2% ) , with a high crowding index ( 67.3% of cases with crowding index > 2 ) , and about two - thirds ( in 64.3% ) of them were rural . there was a statistically significant difference between mycotic and nonmycotic cases as regards their family size ( larger families among mycotic keratitis cases ) ( = 10.67 , p = 0.004 ) . however in both of mycotic and nonmycotic keratitis the percentage of the disease was higher in large families , with a high crowding index and rural residence . ( iii ) as regards water sources , the outdoor water supply accounted for 72.9% of houses of mycotic cases compared to 58.1% among the nonmycotic cases with significant statistical difference ( = 4.95 , p = 0.025 ) . concerning sewage disposal , more than three - quarters of houses of mycotic keratitis ( 77.3% ) were by conservancy system compared to 55% of the houses in the nonmycotic cases with a significant statistical difference ( = 10.78 , p = 0.001 ) . ( iv ) in conclusion , it was revealed that cases of mycotic keratitis lived in more deteriorated environment than the nonmycotic group . outdoor water and insanitary sewage disposal systems could affect personal hygiene and self - care with poor sanitation of houses together with spread of flies and insects that transmit organisms easily to healthy or unhealthy corneas . in this study , 61.5% of cases of mycotic keratitis came within the first week of their onset of complaints to seek medical care compared to the majority ( 85% ) in the nonmycotic group . about a quarter of cases of mycotic keratitis ( 22.4% ) came to seek medical care after two weeks from onset of complaints compared to no one in the nonmycotic group . these findings were in consistence with rautaraya who reported that 25.4% of their patients showed negative fungal growth in spite of their typical clinical findings for microbial keratitis . ( ii ) for the type of fungusisolated , it was found that aspergillus species were the most common ( 45.2% ) , either flavus ( 29.1% ) or niger ( 16.1% ) . these results were in agreement with rautaraya and his colleagues in india who demonstrated that aspergillus species constituted the majority ( 27.9% ) of fungal growth in their study . ( iv ) candida species were the least frequent type ( only in 3% ) ; that is in agreement with what was reported by rautaraya and others that candida species were the least common in their study ( 0.9% ) . our results revealed that , among a total number of 66303 cases examined during the time of the study , mycotic keratitis occurred among 0.54% of cases and the nonmycotic keratitis in 0.71% . a study that was done from february 1991 to june 2001 by gopinathan and others in india demonstrated 5897 cases of suspected infectious keratitis ; 3563 ( 60.4% ) were culture proven ( bacterial : 1849 , 51.9% ; fungal : 1360 , 38.2% ; acanthamoeba : 86 , 2.4% ; mixed : 268 , 7.5% ) . among fungal cases aspergillus species were responsible for 28.9% of cases , fusarium in 35.6% , dematiaceous fungi in 19.3% , other hyaline fungi in 15.4% , and candida only in 0.8% . in a study done in ghana in 1995 , one or more organisms were cultured from 114 of 199 patients ( 57.3% ) , with the most common being fusarium species , pseudomonas aeruginosa , and staphylococcus epidermidis . fungi , alone or in combination , were isolated from 56% of the patients who had fungal growth , in total 122 patients ( 61.3% ) . this work reported that , among cases of mycotic keratitis , organic trauma was the most prevalent risk factor ( 38.2% ) , followed by ocular surgery in 32.7% of cases then the nonorganic trauma in 20.2% followed by the use of topical antibiotics in 6.1% , and systemic disease ( such as diabetes ) was the least frequent in only 2.2% . on the other hand , the nonorganic trauma was the most frequent risk factor for nonmycotic keratitis ( 45% ) followed by the organic trauma ( 35% ) . as regards the risk of ocular surgery among the cases , fungal contamination might occur preoperatively in those cases with negative cultures or postoperatively due to lowered body immunity ( by the stressful surgery ) and/or the use of a course of local and systemic corticosteroids with the surgery . community awareness of the risk factors.restriction of the abuse of topical corticosteroids or antibiotics.mycotic keratitis should be suspected in every patient with a corneal lesion , specially the resistant one , and should be ruled out before commencing steroids and antibiotics.for a successful treatment , we need the following : proper early clinical diagnosis , proper laboratory diagnosis , initiating a broad spectrum antifungal drug till laboratory findings prove the specific drug , proper frequency of antifungal drugs , proper timing for termination of therapy . for a successful treatment , we need the following : proper early clinical diagnosis , proper laboratory diagnosis , initiating a broad spectrum antifungal drug till laboratory findings prove the specific drug , proper frequency of antifungal drugs , proper timing for termination of therapy .

every year , more than 9 million babies die in utero or in the first few weeks of life , and the majority of these deaths occur in developing countries , where teratogens play a major role.1 in fact , the term teratogen is used to designate products with teratogenic potential at clinical doses used in humans.2 it indicates an exposure to a physical or chemical agent that can interfere with normal development of cells and tissues and results in abnormalities and malformations in newborns.3 currently , reproductive toxicologists consider the four major manifestations of abnormal fetal development to be growth alteration , functional deficit , structural malformation , and death.4 teratogenicity can be triggered by many materials , such as medications and licit substances like alcohol , caffeine , and tobacco.3 despite the lack of information on the safety of drug use during pregnancy , most pregnant women will likely be exposed to drugs . indeed , fetal exposure can occur either before or after a woman knows she is pregnant . many epidemiological studies have demonstrated that using drugs is associated with an increased risk of birth defects,2 depending on types , timing , frequency , intensity , and duration of exposure.2 on the other hand , exposure to smoke , coffee , and alcohol during pregnancy can contribute to perinatal complications and poor neonatal outcomes , consequences that have been well identified in epidemiological studies . effectively , maternal smoking during pregnancy is an established risk factor for miscarriage , perinatal mortality , low birth weight , premature births , and small babies.59 despite the risks , many women still smoke during pregnancy : 17% in england and wales,10 and 14% in the us.11 in lebanon , a study conducted by chaaya and collaborators showed that pregnant women were partially knowledgeable about the health risks of cigarette and water - pipe smoking , and they had permissive attitudes towards all forms of smoking . moreover , almost one - quarter ( 23% ) of participants reported smoking during pregnancy , with 17% smoking only cigarettes , 4% smoking only water pipe , and 1.5% smoking both.12 smoking is responsible for 15% of all preterm births and increases the risk of perinatal mortality threefold.13 although caffeine has been demonstrated to produce birth defects and fetal mortality in animal models , its effects in humans at normal levels of consumption are much less certain . a high level of coffee consumption is however associated with an increased risk of fetal death ; it was linked to various adverse pregnancy outcomes , including fetal loss , birth defects , and fetal growth retardation.14 finally , drinking alcohol during pregnancy can cause miscarriage , premature birth , stillbirth , low birth weight,15 and a range of lifelong disorders , known as fetal alcohol spectrum disorders.16 the most known , fetal alcohol syndrome , is one of the leading known preventable causes of mental retardation and birth defects.16 to our knowledge , no data exist about exposure of pregnant women to these substances in lebanon . while the harmful effects of different medications and licit substance use during pregnancy may potentially constitute a major public health concern worldwide , few epidemiological studies have examined the actual prevalence of risky substance exposure during pregnancy and its relation to perinatal problems in lebanon . these data are important when making recommendations to health - care providers on screening substance use during pregnancy and for helping pregnant women to avoid these substances . therefore , this study aimed to assess risky exposure of pregnant lebanese women to drugs , tobacco , caffeine , and alcohol ; a secondary objective was to assess their effect on postnatal outcomes . consecutive delivering women at term were addressed after delivery in five university tertiary care hospitals of beirut and mount lebanon between january and june 2012 ; one of the hospitals was public , while the four others were private . after oral informed consent , women were interviewed face - to - face by trained interviewers ( interviewers were sixth - year pharmacy interns ) . we note that preterm delivering women , complicated pregnancies , and women with non - singleton fetuses were excluded from the study . one day after delivery , women were told about the objective of the study , which was to describe their exposures to and consumption of different substances during pregnancy . anonymity and confidentiality of the results were ensured , but no incentive was given for participants . a standardized questionnaire was administered to them , inquiring about the following characteristics and exposures during pregnancy : sociodemographic characteristics , parity , medical problems before and during pregnancy , drugs and supplements taken during pregnancy , exposure to water - pipe and cigarette smoking ( active and passive smoking ) , and consumption of alcohol , caffeine , and caffeinated beverages . moreover , medical files of both mothers and their respective children were checked to confirm information given by mothers , and to assess the health outcome of the babies until hospital discharge : weight , height , cranial perimeter , appearance , pulse , grimace , activity , and respiration score ( apgar score ) at 5 and 10 minutes , and the presence of a medical problem requiring transfer to the neonatal care unit ( ncu ) . for apgar score , each of the five criteria is scored from 0 to 2 : 2 is reserved for strictly normal states , 0 for major anomalies , and 1 for all intermediate states . the normal newborn has an apgar score greater than 7 at 5 and 10 minutes of life.17 exposure to drugs was firstly assessed by drug name , and further classification was performed according to fda classification of drugs during pregnancy:18 category a ( adequate and well - controlled studies have failed to demonstrate a risk to the fetus in the first trimester of pregnancy , and there is no evidence of risk in later trimesters ) , category b ( animal reproduction studies have failed to demonstrate a risk to the fetus , and there are no adequate and well - controlled studies in pregnant women ) , category c ( animal reproduction studies have shown an adverse effect on the fetus , and there are no adequate and well - controlled studies in humans , but potential benefits may warrant use of the drug in pregnant women despite potential risks ) , category d ( there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans , but potential benefits may warrant use of the drug in pregnant women despite potential risks ) , and category x ( studies in animals or humans have demonstrated fetal abnormalities and/or there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience , and the risks involved in use of the drug in pregnant women clearly outweigh potential benefits).18 drugs with mixed categories were classified according to the higher level of risk . smoking , caffeine , and alcohol exposure were assessed by a dichotomous variable of yes / no exposure ; in the case of a positive answer , mean frequency of exposure was assessed . for neonates , weight at birth , apgar score at 5 and 10 minutes after birth,19 and health problems that required transfer to the ncu were evaluated ( fever , infection , respiratory distress , and severe hyperbilirubinemia ) . babies were considered underweight if their birth weight was lower than 2750 g ; this broader definition than the usual one was adopted because of the low number of babies with birth weight < 2500 g. data entry and analysis were performed using spss version 18.0 ( ibm , armonk , ny , usa ) . for apgar score at 5 and 10 minutes , a transformation into logarithmic function to allow for a normal distribution of these variables was performed . percentages were shown for qualitative variables , while means and standard deviation were given for quantitative variables . the chi - squared test was used to compare between - group percentages , while student s t - test or analysis of variance were used to compare means between two or more groups , respectively . missing data were not replaced . furthermore , multivariate analyses were performed : a multiple linear regression when the dependent variable was quantitative ( apgar scores for 5 and 10 minutes , and a logistic regression when the dependent variable was dichotomous [ underweight baby , medical problem that required transfer to the ncu ] ) . backward stepwise models were retained , after ensuring models adequacy using appropriate methods ( normality of residuals for multiple regression and hosmer lemeshow test for logistic regression ) . probabilities were used for stepwise removal ( p = 0.10 ) and for stepwise entry ( p = 0.05 ) . independent variables were age of the mother , geographical origin , body mass index before pregnancy , education level , occupation , parity , delivery type , medical problem types during pregnancy , weight gain during pregnancy , number of live children , delivery term , and exposure to medications and licit substances . the absence of collinearity between factors was checked by verifying the correlation coefficient of items and the variance inflation factor of retained items , which remained lower than 10 . consecutive delivering women at term were addressed after delivery in five university tertiary care hospitals of beirut and mount lebanon between january and june 2012 ; one of the hospitals was public , while the four others were private . after oral informed consent , women were interviewed face - to - face by trained interviewers ( interviewers were sixth - year pharmacy interns ) . we note that preterm delivering women , complicated pregnancies , and women with non - singleton fetuses were excluded from the study . one day after delivery , women were told about the objective of the study , which was to describe their exposures to and consumption of different substances during pregnancy . anonymity and confidentiality of the results were ensured , but no incentive was given for participants . a standardized questionnaire was administered to them , inquiring about the following characteristics and exposures during pregnancy : sociodemographic characteristics , parity , medical problems before and during pregnancy , drugs and supplements taken during pregnancy , exposure to water - pipe and cigarette smoking ( active and passive smoking ) , and consumption of alcohol , caffeine , and caffeinated beverages . moreover , medical files of both mothers and their respective children were checked to confirm information given by mothers , and to assess the health outcome of the babies until hospital discharge : weight , height , cranial perimeter , appearance , pulse , grimace , activity , and respiration score ( apgar score ) at 5 and 10 minutes , and the presence of a medical problem requiring transfer to the neonatal care unit ( ncu ) . for apgar score , each of the five criteria is scored from 0 to 2 : 2 is reserved for strictly normal states , 0 for major anomalies , and 1 for all intermediate states . the normal newborn has an apgar score greater than 7 at 5 and 10 minutes of life.17 exposure to drugs was firstly assessed by drug name , and further classification was performed according to fda classification of drugs during pregnancy:18 category a ( adequate and well - controlled studies have failed to demonstrate a risk to the fetus in the first trimester of pregnancy , and there is no evidence of risk in later trimesters ) , category b ( animal reproduction studies have failed to demonstrate a risk to the fetus , and there are no adequate and well - controlled studies in pregnant women ) , category c ( animal reproduction studies have shown an adverse effect on the fetus , and there are no adequate and well - controlled studies in humans , but potential benefits may warrant use of the drug in pregnant women despite potential risks ) , category d ( there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans , but potential benefits may warrant use of the drug in pregnant women despite potential risks ) , and category x ( studies in animals or humans have demonstrated fetal abnormalities and/or there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience , and the risks involved in use of the drug in pregnant women clearly outweigh potential benefits).18 drugs with mixed categories were classified according to the higher level of risk . smoking , caffeine , and alcohol exposure were assessed by a dichotomous variable of yes / no exposure ; in the case of a positive answer , mean frequency of exposure was assessed . for neonates , weight at birth , apgar score at 5 and 10 minutes after birth,19 and health problems that required transfer to the ncu were evaluated ( fever , infection , respiratory distress , and severe hyperbilirubinemia ) . babies were considered underweight if their birth weight was lower than 2750 g ; this broader definition than the usual one was adopted because of the low number of babies with birth weight < 2500 g. data entry and analysis were performed using spss version 18.0 ( ibm , armonk , ny , usa ) . for apgar score at 5 and 10 minutes , a transformation into logarithmic function to allow for a normal distribution of these variables was performed . percentages were shown for qualitative variables , while means and standard deviation were given for quantitative variables . the chi - squared test was used to compare between - group percentages , while student s t - test or analysis of variance were used to compare means between two or more groups , respectively . missing data were not replaced . furthermore , multivariate analyses were performed : a multiple linear regression when the dependent variable was quantitative ( apgar scores for 5 and 10 minutes , and a logistic regression when the dependent variable was dichotomous [ underweight baby , medical problem that required transfer to the ncu ] ) . backward stepwise models were retained , after ensuring models adequacy using appropriate methods ( normality of residuals for multiple regression and hosmer lemeshow test for logistic regression ) . probabilities were used for stepwise removal ( p = 0.10 ) and for stepwise entry ( p = 0.05 ) . independent variables were age of the mother , geographical origin , body mass index before pregnancy , education level , occupation , parity , delivery type , medical problem types during pregnancy , weight gain during pregnancy , number of live children , delivery term , and exposure to medications and licit substances . the absence of collinearity between factors was checked by verifying the correlation coefficient of items and the variance inflation factor of retained items , which remained lower than 10 . among 367 consecutive delivering women who were approached , 350 ( 95.4% ) agreed to participate ; nonresponders expressed that they were not interested in participating . table 1 describes the general characteristics of the 350 women recruited from beirut and mount lebanon . most of them ( 69.4% ) had a normal mean of bmi and a university degree ; 21.7% of participants reported medical problems , mainly anemia . congenital malformations were detected in two neonates , and 16 neonates were transferred to the ncu due to medical problems . the means of the apgar score done 5 and 10 minutes after delivery were 8.49 and 9.66 , respectively . most of the women in our study were taking such supplements as iron ( 76.90% ) , folic acid ( 66.90% ) , multiple vitamins ( 66% ) , and calcium ( 48.9% ) . a total of 259 ( 74% ) women reported drinking caffeinated beverages during pregnancy , whereas only 1.1% of them drank alcohol . however , 47.5% declared being exposed to smoking : 32% were exposed to passive smoking , 6.3% were active cigarette smokers , and 8.3% were water - pipe smokers . among drugs , category b and c drugs were the most taken , with 72.9% and 34.9% , respectively , while category d represented 10.6% and there was a very low percentage for category x drugs ( 0.3% ) . in bivariate analysis , education level was not associated with substance and drug consumption , except for active smoking , which was significantly less common in university - educated women versus others ( 9.5% versus 25% , p < 0.001 ) . women aged 33 years and older consumed significantly more alcohol and drugs of categories b and d than younger women ( p < 0.05 ) ; however , no significant differences were observed between geographical regions ( p > 0.05 ) ( results not shown ) . in order to test the effect of different sociodemographic and clinical characteristics as well as different behaviors during pregnancy on neonates medical problems , development and on the risk of having underweight babies , we found that active tobacco smoking was significantly associated with an elevated risk of medical problems ( p < 0.047 ) ; babies born from tobacco - smoking mothers had approximately three times more risk of having medical problems requiring ncu transfer . coffee consumption may also be strongly associated with neonatal medical problems ( adjusted odds ratio [ aor ] = 4.37 ) . as expected , we also noticed that having a late delivery is highly associated with a decrease in the risk of neonates developing medical problems , or having underweight babies ( p < 0.001 ) . we observed that consuming category c drugs ( aor = 2.01 , confidence interval 1.064.02 ) or being a passive smoker ( aor = 2.2 , confidence interval = 1.134.31 ) were significantly associated with increased risk of having underweight babies . active tobacco smoking was associated with a higher risk of underweight babies ( aor > 1 ) , but the results did not reach statistical significance ( p = 0.151 ) . concerning the relationship between the apgar score and drug and other substance use during pregnancy , we found that higher frequencies of weekly water pipe and of daily cigarette pack consumption were associated with a lower apgar score at 5 or 10 minutes . the exposure to category d drugs during pregnancy was also significantly related to a smaller apgar 10 . in addition , we found that having gestational diabetes was negatively associated with apgar score . finally , exposure to category x drugs may negatively affect the apgar 5 ( p = 0.07 ) ( table 4 ) . among 367 consecutive delivering women who were approached , 350 ( 95.4% ) agreed to participate ; nonresponders expressed that they were not interested in participating . table 1 describes the general characteristics of the 350 women recruited from beirut and mount lebanon . most of them ( 69.4% ) had a normal mean of bmi and a university degree ; 21.7% of participants reported medical problems , mainly anemia . congenital malformations were detected in two neonates , and 16 neonates were transferred to the ncu due to medical problems . the means of the apgar score done 5 and 10 minutes after delivery were 8.49 and 9.66 , respectively . most of the women in our study were taking such supplements as iron ( 76.90% ) , folic acid ( 66.90% ) , multiple vitamins ( 66% ) , and calcium ( 48.9% ) . a total of 259 ( 74% ) women reported drinking caffeinated beverages during pregnancy , whereas only 1.1% of them drank alcohol . however , 47.5% declared being exposed to smoking : 32% were exposed to passive smoking , 6.3% were active cigarette smokers , and 8.3% were water - pipe smokers . among drugs , category b and c drugs were the most taken , with 72.9% and 34.9% , respectively , while category d represented 10.6% and there was a very low percentage for category x drugs ( 0.3% ) . in bivariate analysis , education level was not associated with substance and drug consumption , except for active smoking , which was significantly less common in university - educated women versus others ( 9.5% versus 25% , p < 0.001 ) . women aged 33 years and older consumed significantly more alcohol and drugs of categories b and d than younger women ( p < 0.05 ) ; however , no significant differences were observed between geographical regions ( p > 0.05 ) ( results not shown ) . in order to test the effect of different sociodemographic and clinical characteristics as well as different behaviors during pregnancy on neonates medical problems , development and on the risk of having underweight babies , logistic regression models were used ( table 3 ) . we found that active tobacco smoking was significantly associated with an elevated risk of medical problems ( p < 0.047 ) ; babies born from tobacco - smoking mothers had approximately three times more risk of having medical problems requiring ncu transfer . coffee consumption may also be strongly associated with neonatal medical problems ( adjusted odds ratio [ aor ] = 4.37 ) . as expected , we also noticed that having a late delivery is highly associated with a decrease in the risk of neonates developing medical problems , or having underweight babies ( p < 0.001 ) . we observed that consuming category c drugs ( aor = 2.01 , confidence interval 1.064.02 ) or being a passive smoker ( aor = 2.2 , confidence interval = 1.134.31 ) were significantly associated with increased risk of having underweight babies . active tobacco smoking was associated with a higher risk of underweight babies ( aor > 1 ) , but the results did not reach statistical significance ( p = 0.151 ) . concerning the relationship between the apgar score and drug and other substance use during pregnancy , we found that higher frequencies of weekly water pipe and of daily cigarette pack consumption were associated with a lower apgar score at 5 or 10 minutes . the exposure to category d drugs during pregnancy was also significantly related to a smaller apgar 10 . in addition , we found that having gestational diabetes was negatively associated with apgar score . finally , exposure to category x drugs may negatively affect the apgar 5 ( p = 0.07 ) ( table 4 ) . our study evaluated the exposure of pregnant lebanese women to risky substances and their influence on birth outcomes . we noticed that the participants were exposed to multiple factors that may affect the neonates health . indeed , most of them drank caffeinated beverages ( 75% ) , while substantial proportions were exposed to smoking ( active and passive , cigarette and water pipe ) , alcohol , and drugs . our results are more or less similar to those of others for coffee,20,21 smoking,22,23 alcohol,24 and drugs.25,26 for active smoking , they are lower than those of chaaya and collaborators,12 probably due to the higher education level of our population . the high consumption of caffeinated beverages is not surprising , given that the use of caffeinated substances is not forbidden by doctors during pregnancy , since reports about its harm on the fetus are scant and inconclusive,27 as opposed to alcohol or tobacco consumption . it is troubling , however , given that coffee consumption was associated with higher risk of neonates medical problems with a tendency to significance ( p = 0.064 ) ; in fact , the occurrence of congenital malformations , fetal growth retardation , small - for - date babies , miscarriages ( spontaneous abortions ) , behavioral effects , and maternal fertility problems that presumably resulted from caffeine consumption have all been reported.28 in a danish study of 7346 pregnant women , authors found that consumption of coffee during pregnancy was associated with a higher risk of fetal death , especially losses occurring after 20 completed weeks of gestation.29 the increased risk was linked to various adverse pregnancy outcomes , including fetal loss , birth defects , fetal growth retardation , and increased number of uterine contraction peaks . thus , coffee consumption during pregnancy has been subject to preventive action in some countries,30 and was recommended to be restricted by several researchers.31,32 on the other hand , the trend we found may be due to the fact that caffeine is present at different concentrations in many beverages , including coffee , tea , and colas , as well as chocolate , and in our study we did not differentiate and divide our analysis into the types of caffeinated beverages , but took all in at once . several other associations of substance use during pregnancy emerged from this study : our results showed a significant association between mothers tobacco smoking and elevated risk of medical problems . indeed , babies born from tobacco - smoking mothers had approximately three times more risk of having medical problems requiring ncu transfer . paralleling our results , numerous studies have shown strong associations between maternal cigarette smoking and neonatal morbidity and mortality.3335 in other studies , smoking was recognized as a major risk factor for poor birth outcomes that needed transfer to the ncu.36 furthermore , some researchers found that maternal smoking was associated with an increased risk of sudden infant death syndrome . krl and collaborators confirmed that active smoking during pregnancy had a negative effect on the cerebral mass of the neonate ; the deficiency in cerebral mass increased with greater smoking intensity.37 the risk to the developing fetus seems primarily due to the nicotine : the biological mechanisms of how nicotine affects fetal development have been examined in extensive human and laboratory studies , which showed that nicotine crosses the placenta and targets specific neurotransmitter receptors in the fetal brain , eliciting abnormal cell proliferation and increasing susceptibility to hypoxia - induced brain damage.3,38 in addition , we demonstrated , consistently with other research,30 that babies born to women who smoked a water pipe during pregnancy had a higher proportion of immediate need for extra medical or emergency care . for active cigarette smoking , although we found a relationship between active smoking and underweight , the relationship we found did not reach statistical significance , probably due to the low prevalence of active smokers or the low smoking dose . similarly , we found a doubling of the risk of underweight in babies of women who were exposed to passive smoking ; this is consistent with other studies in which high environmental tobacco - smoke exposure was associated with low birth weight , preterm birth , and most strongly preterm birth.39 on average , birth weight was reduced by 120 g per pack of cigarettes ( or cigar / pipe equivalent ) smoked daily by the father.40 alcohol use was much less reported during pregnancy than other substances ( 1.1% ) in this study . this is due to the low number of pregnant women that were reported drinking alcohol during their pregnancy . our participants seemed to be consuming medications belonging to categories b ( 72.9% ) and c ( 34.9% ) , whereas category d ( 10.6% ) and x ( 0.3% ) drugs were much less taken . this indicates that pregnant lebanese women may have been more likely to stay away from the most dangerous drug categories ( d and x ) , as might have been recommended by their doctor and/or pharmacist . other explanations would be that they may have less access to or need of these drugs , in addition to social desirability in responding , or , they may be underreporting these drugs for fear of legal consequences of disclosing this information . however , we think that the latter explanations are less likely , and we doubt that lebanese women are aware of the classification of drugs during pregnancy . nevertheless , confirmation of drug use with urine samples would have given more credibility to prevalence rates ; further studies are suggested to take into account this last point . our results are however similar to those of other countries : for instance , about two - thirds of women in the us take one or more prescription medications during pregnancy . nevertheless , it was found that more that 90% of the medications approved by the fda from 1980 to 2000 had insufficient data to determine safety in pregnancy.41 in addition , in a national study done in brazil on 610 pregnant women , results were similar to ours , and category d and x drugs were taken during pregnancy in low percentages.42 furthermore , in the multivariate analysis , the use of these medications during pregnancy was associated with the newborn s status in several ways : category c medication intake was associated with underweight birth , while the use of d- and x - category drugs were associated with increased risk of immediate need for intensive care . first , a selection bias is possible , since we worked with women who delivered in university tertiary care hospitals of the capital and surroundings ; they may not represent the childbearing lebanese population , particularly those of lower socioeconomic status from remote areas . we expect our results to be underestimating the true prevalence of exposure to toxic substances during pregnancy . moreover , the data are cross - sectional , and therefore no causal relationships can be inferred ; analytical results are presented indicatively . second , the risk of developing birth defects depends on exposure , timing , dosing , and route of administration;2 this information was missing from our data , and while reporting substance use for analysis , there was no specification of timing of exposure , dose , or route of administration for these substances . in fact , we did not collect information about these factors because of probable nondifferential memory bias ; this may lead to crucial differences in results between using substances in the first trimester and afterwards . the information bias could also be differential , women with children who had health problems being more able to remember exposures than women with healthy children ; this bias is expected to induce false - positive associations . third , a medical problem that occurred in newborns was not detailed because of the low number of babies that had such a problem , which precluded detailed analysis , and we could not perform any analysis on congenital malformations since only two cases were found . finally , a confounding bias is possible , since we may not have taken all potential confounders into account or the sample size may have precluded showing some associations . for example , although anemia would be expected to affect birth weight , it was removed from the final model in our study ; this may be due to an underpowering of our sample size or to a low severity of anemia , given particularly that the majority of women were taking iron supplements . although larger - scale studies may be necessary to adjust or confirm our analytical results , we do not expect that drastic changes in essential results would occur , especially concerning substantial levels of exposure to some substances and medications . our study demonstrated that lebanese women were exposed during pregnancy to medications and licit substances that affected neonates health . some risk factors associated with neonatal medical problems were identified , including active tobacco smoking and coffee consumption . consuming category c drugs or being a passive smoker was associated with increased risk of having underweight babies . higher weekly water - pipe exposure , number of daily cigarette packs , or exposure to category d drugs during pregnancy were associated with a lower apgar score . our findings have implications for clinical obstetric practice and prevention and intervention programs : clinicians should screen all pregnant women for smoking , exposure to caffeine , and other substance use and give them appropriate advice and explain all risks that may develop while using these substances .

backgroundthe harmful effects of medication and licit substance use during pregnancy may potentially constitute a major public health concern . our study aims to assess risky exposure of lebanese pregnant women to drugs , tobacco , caffeine , and alcohol , and to determine their effect on postnatal outcomes.methodswomen at term were addressed after delivery in five university hospitals of beirut and mount lebanon between february and june 2012 . a standardized questionnaire was administered to them . moreover , medical files of both mothers and their respective newborns were checked to confirm information given by mothers , and to assess the health outcome of the babies.resultsamong the interviewed 350 women , active and passive smoking of tobacco ( cigarette or water pipe ) , and consumption of category c , d , and x drugs were common during pregnancy in lebanon ; they were shown to negatively affect the neonatal outcome in multivariate analyses : they significantly decreased apgar scores and increased the risk of underweight and medical complications of babies ( p < 0.05).conclusionour study demonstrated that lebanese women were exposed during pregnancy to multiple medications and licit substances that affected the neonates health . our findings have implications for clinical obstetric practice and prevention programs in lebanon . efforts should be made to decrease exposure to harmful substances during pregnancy .

Introduction Materials and methods Population and data collection Definitions and classification Statistical analysis Results Description of the sample Characteristics and outcome of the neonates Exposure to drugs and supplements, and behavior during pregnancy Multivariate analyses Discussion Conclusion

every year , more than 9 million babies die in utero or in the first few weeks of life , and the majority of these deaths occur in developing countries , where teratogens play a major role.1 in fact , the term teratogen is used to designate products with teratogenic potential at clinical doses used in humans.2 it indicates an exposure to a physical or chemical agent that can interfere with normal development of cells and tissues and results in abnormalities and malformations in newborns.3 currently , reproductive toxicologists consider the four major manifestations of abnormal fetal development to be growth alteration , functional deficit , structural malformation , and death.4 teratogenicity can be triggered by many materials , such as medications and licit substances like alcohol , caffeine , and tobacco.3 despite the lack of information on the safety of drug use during pregnancy , most pregnant women will likely be exposed to drugs . many epidemiological studies have demonstrated that using drugs is associated with an increased risk of birth defects,2 depending on types , timing , frequency , intensity , and duration of exposure.2 on the other hand , exposure to smoke , coffee , and alcohol during pregnancy can contribute to perinatal complications and poor neonatal outcomes , consequences that have been well identified in epidemiological studies . effectively , maternal smoking during pregnancy is an established risk factor for miscarriage , perinatal mortality , low birth weight , premature births , and small babies.59 despite the risks , many women still smoke during pregnancy : 17% in england and wales,10 and 14% in the us.11 in lebanon , a study conducted by chaaya and collaborators showed that pregnant women were partially knowledgeable about the health risks of cigarette and water - pipe smoking , and they had permissive attitudes towards all forms of smoking . moreover , almost one - quarter ( 23% ) of participants reported smoking during pregnancy , with 17% smoking only cigarettes , 4% smoking only water pipe , and 1.5% smoking both.12 smoking is responsible for 15% of all preterm births and increases the risk of perinatal mortality threefold.13 although caffeine has been demonstrated to produce birth defects and fetal mortality in animal models , its effects in humans at normal levels of consumption are much less certain . a high level of coffee consumption is however associated with an increased risk of fetal death ; it was linked to various adverse pregnancy outcomes , including fetal loss , birth defects , and fetal growth retardation.14 finally , drinking alcohol during pregnancy can cause miscarriage , premature birth , stillbirth , low birth weight,15 and a range of lifelong disorders , known as fetal alcohol spectrum disorders.16 the most known , fetal alcohol syndrome , is one of the leading known preventable causes of mental retardation and birth defects.16 to our knowledge , no data exist about exposure of pregnant women to these substances in lebanon . while the harmful effects of different medications and licit substance use during pregnancy may potentially constitute a major public health concern worldwide , few epidemiological studies have examined the actual prevalence of risky substance exposure during pregnancy and its relation to perinatal problems in lebanon . these data are important when making recommendations to health - care providers on screening substance use during pregnancy and for helping pregnant women to avoid these substances . therefore , this study aimed to assess risky exposure of pregnant lebanese women to drugs , tobacco , caffeine , and alcohol ; a secondary objective was to assess their effect on postnatal outcomes . consecutive delivering women at term were addressed after delivery in five university tertiary care hospitals of beirut and mount lebanon between january and june 2012 ; one of the hospitals was public , while the four others were private . one day after delivery , women were told about the objective of the study , which was to describe their exposures to and consumption of different substances during pregnancy . a standardized questionnaire was administered to them , inquiring about the following characteristics and exposures during pregnancy : sociodemographic characteristics , parity , medical problems before and during pregnancy , drugs and supplements taken during pregnancy , exposure to water - pipe and cigarette smoking ( active and passive smoking ) , and consumption of alcohol , caffeine , and caffeinated beverages . moreover , medical files of both mothers and their respective children were checked to confirm information given by mothers , and to assess the health outcome of the babies until hospital discharge : weight , height , cranial perimeter , appearance , pulse , grimace , activity , and respiration score ( apgar score ) at 5 and 10 minutes , and the presence of a medical problem requiring transfer to the neonatal care unit ( ncu ) . the normal newborn has an apgar score greater than 7 at 5 and 10 minutes of life.17 exposure to drugs was firstly assessed by drug name , and further classification was performed according to fda classification of drugs during pregnancy:18 category a ( adequate and well - controlled studies have failed to demonstrate a risk to the fetus in the first trimester of pregnancy , and there is no evidence of risk in later trimesters ) , category b ( animal reproduction studies have failed to demonstrate a risk to the fetus , and there are no adequate and well - controlled studies in pregnant women ) , category c ( animal reproduction studies have shown an adverse effect on the fetus , and there are no adequate and well - controlled studies in humans , but potential benefits may warrant use of the drug in pregnant women despite potential risks ) , category d ( there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans , but potential benefits may warrant use of the drug in pregnant women despite potential risks ) , and category x ( studies in animals or humans have demonstrated fetal abnormalities and/or there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience , and the risks involved in use of the drug in pregnant women clearly outweigh potential benefits).18 drugs with mixed categories were classified according to the higher level of risk . smoking , caffeine , and alcohol exposure were assessed by a dichotomous variable of yes / no exposure ; in the case of a positive answer , mean frequency of exposure was assessed . furthermore , multivariate analyses were performed : a multiple linear regression when the dependent variable was quantitative ( apgar scores for 5 and 10 minutes , and a logistic regression when the dependent variable was dichotomous [ underweight baby , medical problem that required transfer to the ncu ] ) . independent variables were age of the mother , geographical origin , body mass index before pregnancy , education level , occupation , parity , delivery type , medical problem types during pregnancy , weight gain during pregnancy , number of live children , delivery term , and exposure to medications and licit substances . consecutive delivering women at term were addressed after delivery in five university tertiary care hospitals of beirut and mount lebanon between january and june 2012 ; one of the hospitals was public , while the four others were private . one day after delivery , women were told about the objective of the study , which was to describe their exposures to and consumption of different substances during pregnancy . a standardized questionnaire was administered to them , inquiring about the following characteristics and exposures during pregnancy : sociodemographic characteristics , parity , medical problems before and during pregnancy , drugs and supplements taken during pregnancy , exposure to water - pipe and cigarette smoking ( active and passive smoking ) , and consumption of alcohol , caffeine , and caffeinated beverages . moreover , medical files of both mothers and their respective children were checked to confirm information given by mothers , and to assess the health outcome of the babies until hospital discharge : weight , height , cranial perimeter , appearance , pulse , grimace , activity , and respiration score ( apgar score ) at 5 and 10 minutes , and the presence of a medical problem requiring transfer to the neonatal care unit ( ncu ) . the normal newborn has an apgar score greater than 7 at 5 and 10 minutes of life.17 exposure to drugs was firstly assessed by drug name , and further classification was performed according to fda classification of drugs during pregnancy:18 category a ( adequate and well - controlled studies have failed to demonstrate a risk to the fetus in the first trimester of pregnancy , and there is no evidence of risk in later trimesters ) , category b ( animal reproduction studies have failed to demonstrate a risk to the fetus , and there are no adequate and well - controlled studies in pregnant women ) , category c ( animal reproduction studies have shown an adverse effect on the fetus , and there are no adequate and well - controlled studies in humans , but potential benefits may warrant use of the drug in pregnant women despite potential risks ) , category d ( there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans , but potential benefits may warrant use of the drug in pregnant women despite potential risks ) , and category x ( studies in animals or humans have demonstrated fetal abnormalities and/or there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience , and the risks involved in use of the drug in pregnant women clearly outweigh potential benefits).18 drugs with mixed categories were classified according to the higher level of risk . smoking , caffeine , and alcohol exposure were assessed by a dichotomous variable of yes / no exposure ; in the case of a positive answer , mean frequency of exposure was assessed . furthermore , multivariate analyses were performed : a multiple linear regression when the dependent variable was quantitative ( apgar scores for 5 and 10 minutes , and a logistic regression when the dependent variable was dichotomous [ underweight baby , medical problem that required transfer to the ncu ] ) . independent variables were age of the mother , geographical origin , body mass index before pregnancy , education level , occupation , parity , delivery type , medical problem types during pregnancy , weight gain during pregnancy , number of live children , delivery term , and exposure to medications and licit substances . table 1 describes the general characteristics of the 350 women recruited from beirut and mount lebanon . most of the women in our study were taking such supplements as iron ( 76.90% ) , folic acid ( 66.90% ) , multiple vitamins ( 66% ) , and calcium ( 48.9% ) . however , 47.5% declared being exposed to smoking : 32% were exposed to passive smoking , 6.3% were active cigarette smokers , and 8.3% were water - pipe smokers . among drugs , category b and c drugs were the most taken , with 72.9% and 34.9% , respectively , while category d represented 10.6% and there was a very low percentage for category x drugs ( 0.3% ) . women aged 33 years and older consumed significantly more alcohol and drugs of categories b and d than younger women ( p < 0.05 ) ; however , no significant differences were observed between geographical regions ( p > 0.05 ) ( results not shown ) . in order to test the effect of different sociodemographic and clinical characteristics as well as different behaviors during pregnancy on neonates medical problems , development and on the risk of having underweight babies , we found that active tobacco smoking was significantly associated with an elevated risk of medical problems ( p < 0.047 ) ; babies born from tobacco - smoking mothers had approximately three times more risk of having medical problems requiring ncu transfer . as expected , we also noticed that having a late delivery is highly associated with a decrease in the risk of neonates developing medical problems , or having underweight babies ( p < 0.001 ) . we observed that consuming category c drugs ( aor = 2.01 , confidence interval 1.064.02 ) or being a passive smoker ( aor = 2.2 , confidence interval = 1.134.31 ) were significantly associated with increased risk of having underweight babies . active tobacco smoking was associated with a higher risk of underweight babies ( aor > 1 ) , but the results did not reach statistical significance ( p = 0.151 ) . concerning the relationship between the apgar score and drug and other substance use during pregnancy , we found that higher frequencies of weekly water pipe and of daily cigarette pack consumption were associated with a lower apgar score at 5 or 10 minutes . finally , exposure to category x drugs may negatively affect the apgar 5 ( p = 0.07 ) ( table 4 ) . table 1 describes the general characteristics of the 350 women recruited from beirut and mount lebanon . most of the women in our study were taking such supplements as iron ( 76.90% ) , folic acid ( 66.90% ) , multiple vitamins ( 66% ) , and calcium ( 48.9% ) . however , 47.5% declared being exposed to smoking : 32% were exposed to passive smoking , 6.3% were active cigarette smokers , and 8.3% were water - pipe smokers . among drugs , category b and c drugs were the most taken , with 72.9% and 34.9% , respectively , while category d represented 10.6% and there was a very low percentage for category x drugs ( 0.3% ) . women aged 33 years and older consumed significantly more alcohol and drugs of categories b and d than younger women ( p < 0.05 ) ; however , no significant differences were observed between geographical regions ( p > 0.05 ) ( results not shown ) . in order to test the effect of different sociodemographic and clinical characteristics as well as different behaviors during pregnancy on neonates medical problems , development and on the risk of having underweight babies , logistic regression models were used ( table 3 ) . we found that active tobacco smoking was significantly associated with an elevated risk of medical problems ( p < 0.047 ) ; babies born from tobacco - smoking mothers had approximately three times more risk of having medical problems requiring ncu transfer . as expected , we also noticed that having a late delivery is highly associated with a decrease in the risk of neonates developing medical problems , or having underweight babies ( p < 0.001 ) . active tobacco smoking was associated with a higher risk of underweight babies ( aor > 1 ) , but the results did not reach statistical significance ( p = 0.151 ) . concerning the relationship between the apgar score and drug and other substance use during pregnancy , we found that higher frequencies of weekly water pipe and of daily cigarette pack consumption were associated with a lower apgar score at 5 or 10 minutes . finally , exposure to category x drugs may negatively affect the apgar 5 ( p = 0.07 ) ( table 4 ) . our study evaluated the exposure of pregnant lebanese women to risky substances and their influence on birth outcomes . we noticed that the participants were exposed to multiple factors that may affect the neonates health . indeed , most of them drank caffeinated beverages ( 75% ) , while substantial proportions were exposed to smoking ( active and passive , cigarette and water pipe ) , alcohol , and drugs . it is troubling , however , given that coffee consumption was associated with higher risk of neonates medical problems with a tendency to significance ( p = 0.064 ) ; in fact , the occurrence of congenital malformations , fetal growth retardation , small - for - date babies , miscarriages ( spontaneous abortions ) , behavioral effects , and maternal fertility problems that presumably resulted from caffeine consumption have all been reported.28 in a danish study of 7346 pregnant women , authors found that consumption of coffee during pregnancy was associated with a higher risk of fetal death , especially losses occurring after 20 completed weeks of gestation.29 the increased risk was linked to various adverse pregnancy outcomes , including fetal loss , birth defects , fetal growth retardation , and increased number of uterine contraction peaks . thus , coffee consumption during pregnancy has been subject to preventive action in some countries,30 and was recommended to be restricted by several researchers.31,32 on the other hand , the trend we found may be due to the fact that caffeine is present at different concentrations in many beverages , including coffee , tea , and colas , as well as chocolate , and in our study we did not differentiate and divide our analysis into the types of caffeinated beverages , but took all in at once . several other associations of substance use during pregnancy emerged from this study : our results showed a significant association between mothers tobacco smoking and elevated risk of medical problems . krl and collaborators confirmed that active smoking during pregnancy had a negative effect on the cerebral mass of the neonate ; the deficiency in cerebral mass increased with greater smoking intensity.37 the risk to the developing fetus seems primarily due to the nicotine : the biological mechanisms of how nicotine affects fetal development have been examined in extensive human and laboratory studies , which showed that nicotine crosses the placenta and targets specific neurotransmitter receptors in the fetal brain , eliciting abnormal cell proliferation and increasing susceptibility to hypoxia - induced brain damage.3,38 in addition , we demonstrated , consistently with other research,30 that babies born to women who smoked a water pipe during pregnancy had a higher proportion of immediate need for extra medical or emergency care . similarly , we found a doubling of the risk of underweight in babies of women who were exposed to passive smoking ; this is consistent with other studies in which high environmental tobacco - smoke exposure was associated with low birth weight , preterm birth , and most strongly preterm birth.39 on average , birth weight was reduced by 120 g per pack of cigarettes ( or cigar / pipe equivalent ) smoked daily by the father.40 alcohol use was much less reported during pregnancy than other substances ( 1.1% ) in this study . our participants seemed to be consuming medications belonging to categories b ( 72.9% ) and c ( 34.9% ) , whereas category d ( 10.6% ) and x ( 0.3% ) drugs were much less taken . this indicates that pregnant lebanese women may have been more likely to stay away from the most dangerous drug categories ( d and x ) , as might have been recommended by their doctor and/or pharmacist . however , we think that the latter explanations are less likely , and we doubt that lebanese women are aware of the classification of drugs during pregnancy . nevertheless , it was found that more that 90% of the medications approved by the fda from 1980 to 2000 had insufficient data to determine safety in pregnancy.41 in addition , in a national study done in brazil on 610 pregnant women , results were similar to ours , and category d and x drugs were taken during pregnancy in low percentages.42 furthermore , in the multivariate analysis , the use of these medications during pregnancy was associated with the newborn s status in several ways : category c medication intake was associated with underweight birth , while the use of d- and x - category drugs were associated with increased risk of immediate need for intensive care . first , a selection bias is possible , since we worked with women who delivered in university tertiary care hospitals of the capital and surroundings ; they may not represent the childbearing lebanese population , particularly those of lower socioeconomic status from remote areas . we expect our results to be underestimating the true prevalence of exposure to toxic substances during pregnancy . second , the risk of developing birth defects depends on exposure , timing , dosing , and route of administration;2 this information was missing from our data , and while reporting substance use for analysis , there was no specification of timing of exposure , dose , or route of administration for these substances . third , a medical problem that occurred in newborns was not detailed because of the low number of babies that had such a problem , which precluded detailed analysis , and we could not perform any analysis on congenital malformations since only two cases were found . our study demonstrated that lebanese women were exposed during pregnancy to medications and licit substances that affected neonates health . our findings have implications for clinical obstetric practice and prevention and intervention programs : clinicians should screen all pregnant women for smoking , exposure to caffeine , and other substance use and give them appropriate advice and explain all risks that may develop while using these substances .

every year , more than 9 million babies die in utero or in the first few weeks of life , and the majority of these deaths occur in developing countries , where teratogens play a major role.1 in fact , the term teratogen is used to designate products with teratogenic potential at clinical doses used in humans.2 it indicates an exposure to a physical or chemical agent that can interfere with normal development of cells and tissues and results in abnormalities and malformations in newborns.3 currently , reproductive toxicologists consider the four major manifestations of abnormal fetal development to be growth alteration , functional deficit , structural malformation , and death.4 teratogenicity can be triggered by many materials , such as medications and licit substances like alcohol , caffeine , and tobacco.3 despite the lack of information on the safety of drug use during pregnancy , most pregnant women will likely be exposed to drugs . many epidemiological studies have demonstrated that using drugs is associated with an increased risk of birth defects,2 depending on types , timing , frequency , intensity , and duration of exposure.2 on the other hand , exposure to smoke , coffee , and alcohol during pregnancy can contribute to perinatal complications and poor neonatal outcomes , consequences that have been well identified in epidemiological studies . effectively , maternal smoking during pregnancy is an established risk factor for miscarriage , perinatal mortality , low birth weight , premature births , and small babies.59 despite the risks , many women still smoke during pregnancy : 17% in england and wales,10 and 14% in the us.11 in lebanon , a study conducted by chaaya and collaborators showed that pregnant women were partially knowledgeable about the health risks of cigarette and water - pipe smoking , and they had permissive attitudes towards all forms of smoking . moreover , almost one - quarter ( 23% ) of participants reported smoking during pregnancy , with 17% smoking only cigarettes , 4% smoking only water pipe , and 1.5% smoking both.12 smoking is responsible for 15% of all preterm births and increases the risk of perinatal mortality threefold.13 although caffeine has been demonstrated to produce birth defects and fetal mortality in animal models , its effects in humans at normal levels of consumption are much less certain . a high level of coffee consumption is however associated with an increased risk of fetal death ; it was linked to various adverse pregnancy outcomes , including fetal loss , birth defects , and fetal growth retardation.14 finally , drinking alcohol during pregnancy can cause miscarriage , premature birth , stillbirth , low birth weight,15 and a range of lifelong disorders , known as fetal alcohol spectrum disorders.16 the most known , fetal alcohol syndrome , is one of the leading known preventable causes of mental retardation and birth defects.16 to our knowledge , no data exist about exposure of pregnant women to these substances in lebanon . while the harmful effects of different medications and licit substance use during pregnancy may potentially constitute a major public health concern worldwide , few epidemiological studies have examined the actual prevalence of risky substance exposure during pregnancy and its relation to perinatal problems in lebanon . therefore , this study aimed to assess risky exposure of pregnant lebanese women to drugs , tobacco , caffeine , and alcohol ; a secondary objective was to assess their effect on postnatal outcomes . consecutive delivering women at term were addressed after delivery in five university tertiary care hospitals of beirut and mount lebanon between january and june 2012 ; one of the hospitals was public , while the four others were private . one day after delivery , women were told about the objective of the study , which was to describe their exposures to and consumption of different substances during pregnancy . a standardized questionnaire was administered to them , inquiring about the following characteristics and exposures during pregnancy : sociodemographic characteristics , parity , medical problems before and during pregnancy , drugs and supplements taken during pregnancy , exposure to water - pipe and cigarette smoking ( active and passive smoking ) , and consumption of alcohol , caffeine , and caffeinated beverages . moreover , medical files of both mothers and their respective children were checked to confirm information given by mothers , and to assess the health outcome of the babies until hospital discharge : weight , height , cranial perimeter , appearance , pulse , grimace , activity , and respiration score ( apgar score ) at 5 and 10 minutes , and the presence of a medical problem requiring transfer to the neonatal care unit ( ncu ) . the normal newborn has an apgar score greater than 7 at 5 and 10 minutes of life.17 exposure to drugs was firstly assessed by drug name , and further classification was performed according to fda classification of drugs during pregnancy:18 category a ( adequate and well - controlled studies have failed to demonstrate a risk to the fetus in the first trimester of pregnancy , and there is no evidence of risk in later trimesters ) , category b ( animal reproduction studies have failed to demonstrate a risk to the fetus , and there are no adequate and well - controlled studies in pregnant women ) , category c ( animal reproduction studies have shown an adverse effect on the fetus , and there are no adequate and well - controlled studies in humans , but potential benefits may warrant use of the drug in pregnant women despite potential risks ) , category d ( there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans , but potential benefits may warrant use of the drug in pregnant women despite potential risks ) , and category x ( studies in animals or humans have demonstrated fetal abnormalities and/or there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience , and the risks involved in use of the drug in pregnant women clearly outweigh potential benefits).18 drugs with mixed categories were classified according to the higher level of risk . smoking , caffeine , and alcohol exposure were assessed by a dichotomous variable of yes / no exposure ; in the case of a positive answer , mean frequency of exposure was assessed . for neonates , weight at birth , apgar score at 5 and 10 minutes after birth,19 and health problems that required transfer to the ncu were evaluated ( fever , infection , respiratory distress , and severe hyperbilirubinemia ) . babies were considered underweight if their birth weight was lower than 2750 g ; this broader definition than the usual one was adopted because of the low number of babies with birth weight < 2500 g. data entry and analysis were performed using spss version 18.0 ( ibm , armonk , ny , usa ) . furthermore , multivariate analyses were performed : a multiple linear regression when the dependent variable was quantitative ( apgar scores for 5 and 10 minutes , and a logistic regression when the dependent variable was dichotomous [ underweight baby , medical problem that required transfer to the ncu ] ) . independent variables were age of the mother , geographical origin , body mass index before pregnancy , education level , occupation , parity , delivery type , medical problem types during pregnancy , weight gain during pregnancy , number of live children , delivery term , and exposure to medications and licit substances . the absence of collinearity between factors was checked by verifying the correlation coefficient of items and the variance inflation factor of retained items , which remained lower than 10 . consecutive delivering women at term were addressed after delivery in five university tertiary care hospitals of beirut and mount lebanon between january and june 2012 ; one of the hospitals was public , while the four others were private . we note that preterm delivering women , complicated pregnancies , and women with non - singleton fetuses were excluded from the study . one day after delivery , women were told about the objective of the study , which was to describe their exposures to and consumption of different substances during pregnancy . a standardized questionnaire was administered to them , inquiring about the following characteristics and exposures during pregnancy : sociodemographic characteristics , parity , medical problems before and during pregnancy , drugs and supplements taken during pregnancy , exposure to water - pipe and cigarette smoking ( active and passive smoking ) , and consumption of alcohol , caffeine , and caffeinated beverages . moreover , medical files of both mothers and their respective children were checked to confirm information given by mothers , and to assess the health outcome of the babies until hospital discharge : weight , height , cranial perimeter , appearance , pulse , grimace , activity , and respiration score ( apgar score ) at 5 and 10 minutes , and the presence of a medical problem requiring transfer to the neonatal care unit ( ncu ) . the normal newborn has an apgar score greater than 7 at 5 and 10 minutes of life.17 exposure to drugs was firstly assessed by drug name , and further classification was performed according to fda classification of drugs during pregnancy:18 category a ( adequate and well - controlled studies have failed to demonstrate a risk to the fetus in the first trimester of pregnancy , and there is no evidence of risk in later trimesters ) , category b ( animal reproduction studies have failed to demonstrate a risk to the fetus , and there are no adequate and well - controlled studies in pregnant women ) , category c ( animal reproduction studies have shown an adverse effect on the fetus , and there are no adequate and well - controlled studies in humans , but potential benefits may warrant use of the drug in pregnant women despite potential risks ) , category d ( there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans , but potential benefits may warrant use of the drug in pregnant women despite potential risks ) , and category x ( studies in animals or humans have demonstrated fetal abnormalities and/or there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience , and the risks involved in use of the drug in pregnant women clearly outweigh potential benefits).18 drugs with mixed categories were classified according to the higher level of risk . smoking , caffeine , and alcohol exposure were assessed by a dichotomous variable of yes / no exposure ; in the case of a positive answer , mean frequency of exposure was assessed . for neonates , weight at birth , apgar score at 5 and 10 minutes after birth,19 and health problems that required transfer to the ncu were evaluated ( fever , infection , respiratory distress , and severe hyperbilirubinemia ) . babies were considered underweight if their birth weight was lower than 2750 g ; this broader definition than the usual one was adopted because of the low number of babies with birth weight < 2500 g. data entry and analysis were performed using spss version 18.0 ( ibm , armonk , ny , usa ) . furthermore , multivariate analyses were performed : a multiple linear regression when the dependent variable was quantitative ( apgar scores for 5 and 10 minutes , and a logistic regression when the dependent variable was dichotomous [ underweight baby , medical problem that required transfer to the ncu ] ) . independent variables were age of the mother , geographical origin , body mass index before pregnancy , education level , occupation , parity , delivery type , medical problem types during pregnancy , weight gain during pregnancy , number of live children , delivery term , and exposure to medications and licit substances . most of the women in our study were taking such supplements as iron ( 76.90% ) , folic acid ( 66.90% ) , multiple vitamins ( 66% ) , and calcium ( 48.9% ) . however , 47.5% declared being exposed to smoking : 32% were exposed to passive smoking , 6.3% were active cigarette smokers , and 8.3% were water - pipe smokers . among drugs , category b and c drugs were the most taken , with 72.9% and 34.9% , respectively , while category d represented 10.6% and there was a very low percentage for category x drugs ( 0.3% ) . in bivariate analysis , education level was not associated with substance and drug consumption , except for active smoking , which was significantly less common in university - educated women versus others ( 9.5% versus 25% , p < 0.001 ) . women aged 33 years and older consumed significantly more alcohol and drugs of categories b and d than younger women ( p < 0.05 ) ; however , no significant differences were observed between geographical regions ( p > 0.05 ) ( results not shown ) . in order to test the effect of different sociodemographic and clinical characteristics as well as different behaviors during pregnancy on neonates medical problems , development and on the risk of having underweight babies , we found that active tobacco smoking was significantly associated with an elevated risk of medical problems ( p < 0.047 ) ; babies born from tobacco - smoking mothers had approximately three times more risk of having medical problems requiring ncu transfer . as expected , we also noticed that having a late delivery is highly associated with a decrease in the risk of neonates developing medical problems , or having underweight babies ( p < 0.001 ) . we observed that consuming category c drugs ( aor = 2.01 , confidence interval 1.064.02 ) or being a passive smoker ( aor = 2.2 , confidence interval = 1.134.31 ) were significantly associated with increased risk of having underweight babies . concerning the relationship between the apgar score and drug and other substance use during pregnancy , we found that higher frequencies of weekly water pipe and of daily cigarette pack consumption were associated with a lower apgar score at 5 or 10 minutes . most of the women in our study were taking such supplements as iron ( 76.90% ) , folic acid ( 66.90% ) , multiple vitamins ( 66% ) , and calcium ( 48.9% ) . however , 47.5% declared being exposed to smoking : 32% were exposed to passive smoking , 6.3% were active cigarette smokers , and 8.3% were water - pipe smokers . in bivariate analysis , education level was not associated with substance and drug consumption , except for active smoking , which was significantly less common in university - educated women versus others ( 9.5% versus 25% , p < 0.001 ) . women aged 33 years and older consumed significantly more alcohol and drugs of categories b and d than younger women ( p < 0.05 ) ; however , no significant differences were observed between geographical regions ( p > 0.05 ) ( results not shown ) . in order to test the effect of different sociodemographic and clinical characteristics as well as different behaviors during pregnancy on neonates medical problems , development and on the risk of having underweight babies , logistic regression models were used ( table 3 ) . we found that active tobacco smoking was significantly associated with an elevated risk of medical problems ( p < 0.047 ) ; babies born from tobacco - smoking mothers had approximately three times more risk of having medical problems requiring ncu transfer . as expected , we also noticed that having a late delivery is highly associated with a decrease in the risk of neonates developing medical problems , or having underweight babies ( p < 0.001 ) . we observed that consuming category c drugs ( aor = 2.01 , confidence interval 1.064.02 ) or being a passive smoker ( aor = 2.2 , confidence interval = 1.134.31 ) were significantly associated with increased risk of having underweight babies . concerning the relationship between the apgar score and drug and other substance use during pregnancy , we found that higher frequencies of weekly water pipe and of daily cigarette pack consumption were associated with a lower apgar score at 5 or 10 minutes . the high consumption of caffeinated beverages is not surprising , given that the use of caffeinated substances is not forbidden by doctors during pregnancy , since reports about its harm on the fetus are scant and inconclusive,27 as opposed to alcohol or tobacco consumption . it is troubling , however , given that coffee consumption was associated with higher risk of neonates medical problems with a tendency to significance ( p = 0.064 ) ; in fact , the occurrence of congenital malformations , fetal growth retardation , small - for - date babies , miscarriages ( spontaneous abortions ) , behavioral effects , and maternal fertility problems that presumably resulted from caffeine consumption have all been reported.28 in a danish study of 7346 pregnant women , authors found that consumption of coffee during pregnancy was associated with a higher risk of fetal death , especially losses occurring after 20 completed weeks of gestation.29 the increased risk was linked to various adverse pregnancy outcomes , including fetal loss , birth defects , fetal growth retardation , and increased number of uterine contraction peaks . thus , coffee consumption during pregnancy has been subject to preventive action in some countries,30 and was recommended to be restricted by several researchers.31,32 on the other hand , the trend we found may be due to the fact that caffeine is present at different concentrations in many beverages , including coffee , tea , and colas , as well as chocolate , and in our study we did not differentiate and divide our analysis into the types of caffeinated beverages , but took all in at once . several other associations of substance use during pregnancy emerged from this study : our results showed a significant association between mothers tobacco smoking and elevated risk of medical problems . paralleling our results , numerous studies have shown strong associations between maternal cigarette smoking and neonatal morbidity and mortality.3335 in other studies , smoking was recognized as a major risk factor for poor birth outcomes that needed transfer to the ncu.36 furthermore , some researchers found that maternal smoking was associated with an increased risk of sudden infant death syndrome . krl and collaborators confirmed that active smoking during pregnancy had a negative effect on the cerebral mass of the neonate ; the deficiency in cerebral mass increased with greater smoking intensity.37 the risk to the developing fetus seems primarily due to the nicotine : the biological mechanisms of how nicotine affects fetal development have been examined in extensive human and laboratory studies , which showed that nicotine crosses the placenta and targets specific neurotransmitter receptors in the fetal brain , eliciting abnormal cell proliferation and increasing susceptibility to hypoxia - induced brain damage.3,38 in addition , we demonstrated , consistently with other research,30 that babies born to women who smoked a water pipe during pregnancy had a higher proportion of immediate need for extra medical or emergency care . for active cigarette smoking , although we found a relationship between active smoking and underweight , the relationship we found did not reach statistical significance , probably due to the low prevalence of active smokers or the low smoking dose . similarly , we found a doubling of the risk of underweight in babies of women who were exposed to passive smoking ; this is consistent with other studies in which high environmental tobacco - smoke exposure was associated with low birth weight , preterm birth , and most strongly preterm birth.39 on average , birth weight was reduced by 120 g per pack of cigarettes ( or cigar / pipe equivalent ) smoked daily by the father.40 alcohol use was much less reported during pregnancy than other substances ( 1.1% ) in this study . nevertheless , it was found that more that 90% of the medications approved by the fda from 1980 to 2000 had insufficient data to determine safety in pregnancy.41 in addition , in a national study done in brazil on 610 pregnant women , results were similar to ours , and category d and x drugs were taken during pregnancy in low percentages.42 furthermore , in the multivariate analysis , the use of these medications during pregnancy was associated with the newborn s status in several ways : category c medication intake was associated with underweight birth , while the use of d- and x - category drugs were associated with increased risk of immediate need for intensive care . second , the risk of developing birth defects depends on exposure , timing , dosing , and route of administration;2 this information was missing from our data , and while reporting substance use for analysis , there was no specification of timing of exposure , dose , or route of administration for these substances . our findings have implications for clinical obstetric practice and prevention and intervention programs : clinicians should screen all pregnant women for smoking , exposure to caffeine , and other substance use and give them appropriate advice and explain all risks that may develop while using these substances .

female mice ( c57bl/6j strain ) 6 to 8 weeks old were obtained from jackson laboratory ( bar harbor , me , usa ) . female tlr4ko mice used in the study came from a colony established in our vivarium that was started with ( c.c3-tlr4/j strain ) mice purchased from jackson laboratory . animal studies were approved by the institutional animal care and use committee at the baylor college of medicine . all studies adhered to the association for research in vision and ophthalmology statement for use of animals in ophthalmic and visual research . of the total number of animals used in each group , 5 to 6 were used for pcr , 4 for immunobead assay , 3 for immunofluorescence ( interleukin-12a [ il-12a ] ) , and 3 for immunohistochemistry ( il-1 ) . the groups in the immunostaining experiments were untreated plus lps ( ut+lps ) , ut+water , desiccating stress day 5 ( ds5)+lps , and ds5+water . additionally , 6 ut and 2 ds5 controls without topical treatment were used in the immunohistochemistry experiment . three ut and 2 ds5 controls without topical treatment were used for immunofluorescent staining . in the cell culture studies , 5 tlr4ko mice and 7 c57bl/6j mice mice were exposed to desiccating stress ( ds ) to create a murine model of dry eye , similar to methods previously described . ds was induced by housing the mice in an environmentally controlled room with relative humidity 30% . mice were placed in customized cages with two sides constructed of wire to allow exposure to air drafts created by fans . tear secretion was pharmacologically inhibited by subcutaneous injection of scopolamine hydrobromide ( 0.5 mg/0.2 ml ; sigma - aldrich corp . , st . louis , mo , usa ) 4 times daily ( 8:30 am , 11:00 am , 1:00 pm , and 4:30 pm ) . nonstressed ( ns ) control mice were housed in a vivarium with 50% to 75% relative humidity , no exposure to air drafts , and no scopolamine injections . to determine whether tlr4 could be activated on the ocular surface of untreated mice , ns mice were treated with ultrapure lps from salmonella enterica serovar minnesota mutant r595 ( invivogen , san diego , ca , usa ) . mice were treated topically ( 5 l / eye ) with lps dissolved in endotoxin - free water ( sigma - aldrich corp . ) at a dose of 1 g/l or 10 g/l . the higher dose of lps ( 10 g/l ) was used only in gene expression experiments , not in experiments for protein analysis . five microliters of endotoxin - free water per eye was used as a vehicle control . an additional experiment for pcr analysis was conducted using this method and endotoxin - free saline ( nacl 0.9% ; enzo life sciences , farmingdale , ny , usa ) as the vehicle . for protein analysis using an immunobead assay , conjunctiva was extracted after 24 hours . to determine whether de led to increased expression of inflammatory cytokines after tlr4 activation , ds5 mice were treated with 5 l / eye lps ( 1 g/l ) or water on day 5 of desiccating stress . after 4 hours , mice were euthanized to extract corneal epithelium and conjunctiva for rna analysis . corneal epithelium and conjunctiva were extracted to measure gene expression of inflammatory mediators via pcr . tissue samples from cornea and conjunctiva were pooled separately from both eyes to give one cornea and one conjunctiva sample per mouse . rna was extracted using the rneasy plus micro kit ( qiagen , valencia , ca , usa ) according to the manufacturer 's protocol . concentration of isolated rna was measured using a nanodrop 2000 spectrophotometer ( thermo fisher scientific , waltham , ma , usa ) . after rna isolation , cdna was synthesized using ready - to - go you - prime first - strand beads ( ge healthcare bio - sciences , pittsburgh , pa , usa ) as previously reported . after cdna synthesis , pcr was run on a steponeplus real - time pcr system ( applied biosystems , grand island , ny , usa ) . ct values for each gene were normalized to the ct values of the housekeeping gene for each sample . the housekeeping gene used for these experiments was hypoxanthine guanine phosphoribosyl transferase ( hprt ) . fold differences in expression were calculated after comparing values for each gene to the those in the untreated group . each experiment in this study was completed with its own specific group of untreated mice from the same batch of mice used in that particular experiment . primers ( life technologies , grand island , ny , usa ) used in this study included interferon- ( ifn- ; abi assay i d mm00801778_m1 ) , il-1 ( abi assay i d mm00434228_m1 ) , il-6 ( abi assay i d mm00446190_m1 ) , il-12a ( abi assay i d mm00434165_m1 ) , cxcl10 ( abi assay i d mm00445235_m1 ) , tumor necrosis factor- ( tnf- ; abi assay i d mm00443260_g1 ) , and hprt ( abi assay i d mm00446968_m1 ) . tissue was extracted and placed in 100 l of radioimmunoprecipitation assay buffer ( sigma - aldrich corp . ) treated with a complete , edta - free protease inhibitor cocktail tablet ( roche , basel , switzerland ) . excised tissue was chopped with surgical scissors , sonicated , and kept on ice for 30 minutes . samples were normalized for total protein concentration by using pierce bca protein assay kit ( life technologies ) . then , concentrations of cxcl10 were analyzed using an immunobead assay ( emd millipore , billerica , ma , usa ) according to the manufacturer 's instructions . the assay was run using a luminex 100 system ( luminex , austin , tx , usa ) . immunofluorescent staining was used to visualize il-12a protein in the conjunctiva after administration of lps . tissue sections ( one slide with 2 samples per animal ) were fixed in acetone at 20c for 10 minutes . then , nonspecific sites on the samples were blocked for 60 minutes with 20% goat serum ( sigma - aldrich corp . ) in phosphate - buffered saline ( pbs ) . the primary antibody ( il-12a ab203031 ; abcam , cambridge , ma , usa ) , diluted 1:100 in 5% goat serum in pbs , was then added for 1 hour . after being washed with pbs , sections were incubated with alexa fluor 488 affinipure goat anti - rabbit immunoglobulin g ( igg ; h+l ; product 111545144 ; jackson immunoresearch laboratories inc . , west grove , pa , usa ) diluted in pbs for 1 hour in the dark . pictures were taken at 40 magnification using an eclipse e400 microscope equipped with a digital camera ( ds - qi1mc ; nikon instruments inc , melville , ny , usa ) . immunohistochemistry was used to analyze protein expression of il-1. briefly , tissue sections were fixed in acetone at 20c for 10 minutes . after washing in pbs , samples were incubated with 0.3% hydrogen peroxide in pbs to quench endogenous peroxidases and washed again in pbs . sections were then treated with an avidin / biotin blocking kit ( catalog sp-2001 ; vector laboratories , burlingame , ca , usa ) . after being rinsed in pbs , the samples were blocked for 1 hour with 20% goat serum ( sigma - aldrich corp . ) in pbs . the primary antibody ( rabbit anti - mouse il-1 ; d4t2d ; cell signaling technology , danvers , ma , usa ) was then diluted 1:50 in 5% goat serum in pbs and applied to the samples for 1 hour . after being washed , the secondary antibody , biotinylated goat anti - rabbit igg ( product 550338 ; bd pharmingen , franklin lakes , nj , usa ) was applied to the samples for 30 minutes . sections were washed and treated with a vectastain elite abc kit ( catalog no . pk-6100 ; vector laboratories ) for 30 minutes . then , the sections were washed again and incubated with solution from a vector novared peroxidase substrate kit ( catalog no . . then the sections were counterstained for 2 minutes with mayer hematoxylin ( using lillie modification ) ( scytek laboratories , logan , ut , usa ) . finally , permount ( fisher scientific , pittsburgh , pa , usa ) was applied , and a coverslip was attached . slides were examined and photographed at 40 magnification , using an eclipse e400 microscope equipped with a digital camera ( model ds - fi1 ; nikon ) . a second set of images were recorded at increased magnification , using an a1p mp upright microscope with a motorized stage equipped with a color camera ( model ds - u3 ; nikon ) and a 100/1.45 oil ( plan apochromat ofn25 dic ; nikon ) objective . cell culture studies were performed to identify which cell types on the ocular surface responded to lps stimulation . lps was added directly to either epithelial or dendritic cell ( dc ) cultures in vitro . for epithelial cell cultures , cornea or conjunctiva explants were extracted from female c57bl/6j or tlr4ko mice . conjunctiva explants were excised from the forniceal conjunctiva of 6- to 8-week - old female c57bl/6 mice or tlr4ko mice and left for 15 minutes at 37c in keratinocyte serum - free medium ( ksfm ) ( thermo fisher scientific ) supplemented with 3% fetal bovine serum , 1.25 g / ml amphotericin b ( product 15290 - 018 ; thermo fisher scientific ) , 0.5 l / ml gentamicin ( product 15750 - 060 ; thermo fisher scientific ) , and 5 g / ml dispase ii ( product 04942078001 ; roche ) . for cornea cultures , explants were left for 30 minutes in supplemented hormone epithelium medium ( shem ) containing dulbecco modified eagle medium / f12 medium ( product d8437 ; sigma - aldrich corp . ) supplemented with 5 ng / ml epidermal growth factor ( egf ) , 5 g / ml insulin , 5 g / ml transferrin , 5 ng / ml sodium selenite , 0.5 g / ml hydrocortisone , 30 ng / ml cholera toxin a , 0.5% dimethyl sulfoxide , 50 g / ml gentamicin , 1.25 g / ml amphotericin b , 5% fetal bovine serum , and 5 g / ml dispase ii ( roche ) . corneal and conjunctival explants were then plated at one explant per well in 24-well plates . conjunctiva cultures received 500 l / well of ksfm with 80 ng / ml mouse egf ( bd biosciences , san jose , ca , usa ) and cornea cultures received 500 l / well shem . after 3 days , cornea cultures received 500 l of fresh shem / well for a total of 1 ml medium / well ; medium was changed every 3 days . for conjunctiva cultures , wells received another 500 l of ksfm on day 7 for a total of 1 ml of medium / well . on day 11 , wells were left untreated or treated with 1 l of 1 g/l lps dissolved in endotoxin - free water . briefly , dcs were isolated from c57bl/6j mouse bone marrow and cultured for 10 days using rpmi 1640 medium ( thermo fisher scientific ) . on day 10 , cells were left untreated or treated with 1 l of 1 g/l lps dissolved in endotoxin - free water . all statistical analyses were performed using prism version 6.01 software ( graphpad , inc , la jolla , ca , usa ) . within each experiment for pcr and immunobead assay analysis , 1-way anova was used to determine overall statistical significance , and the holm - sidak multiple comparison test was performed to find differences between groups . two - way anovas were used to analyze statistical significance between experiments , with sidak multiple comparisons test used to analyze the significance between groups of different experiments . for the pcr experiments where saline was used as the vehicle and for pcr analysis of the cell culture studies , an unpaired student t test with welch 's correction was used to determine statistical significance among groups . for c57bl/6j mouse cell culture experiments , the number of samples per group were 4 samples per dc group , 4 samples per untreated conjunctiva cell group , and 5 samples per lps - treated conjunctiva cell group . for the cornea , 2 untreated and 3 lps - treated samples were used to test for il-1. six untreated and 4 lps - treated cornea culture samples were used to test for tnf-. ct values were set at 40 for untreated dc samples that did not contain enough il-1 to quantify . for the tlr4ko mouse cell culture experiments , 5 samples were used in each cornea and conjunctiva group . female mice ( c57bl/6j strain ) 6 to 8 weeks old were obtained from jackson laboratory ( bar harbor , me , usa ) . female tlr4ko mice used in the study came from a colony established in our vivarium that was started with ( c.c3-tlr4/j strain ) mice purchased from jackson laboratory . animal studies were approved by the institutional animal care and use committee at the baylor college of medicine . all studies adhered to the association for research in vision and ophthalmology statement for use of animals in ophthalmic and visual research . of the total number of animals used in each group , 5 to 6 were used for pcr , 4 for immunobead assay , 3 for immunofluorescence ( interleukin-12a [ il-12a ] ) , and 3 for immunohistochemistry ( il-1 ) . the groups in the immunostaining experiments were untreated plus lps ( ut+lps ) , ut+water , desiccating stress day 5 ( ds5)+lps , and ds5+water . additionally , 6 ut and 2 ds5 controls without topical treatment were used in the immunohistochemistry experiment . three ut and 2 ds5 controls without topical treatment were used for immunofluorescent staining . in the cell culture studies , 5 tlr4ko mice and 7 c57bl/6j mice mice were exposed to desiccating stress ( ds ) to create a murine model of dry eye , similar to methods previously described . ds was induced by housing the mice in an environmentally controlled room with relative humidity 30% . mice were placed in customized cages with two sides constructed of wire to allow exposure to air drafts created by fans . tear secretion was pharmacologically inhibited by subcutaneous injection of scopolamine hydrobromide ( 0.5 mg/0.2 ml ; sigma - aldrich corp . , st . louis , mo , usa ) 4 times daily ( 8:30 am , 11:00 am , 1:00 pm , and 4:30 pm ) . nonstressed ( ns ) control mice were housed in a vivarium with 50% to 75% relative humidity , no exposure to air drafts , and no scopolamine injections . to determine whether tlr4 could be activated on the ocular surface of untreated mice , ns mice were treated with ultrapure lps from salmonella enterica serovar minnesota mutant r595 ( invivogen , san diego , ca , usa ) . mice were treated topically ( 5 l / eye ) with lps dissolved in endotoxin - free water ( sigma - aldrich corp . ) at a dose of 1 g/l or 10 g/l . the higher dose of lps ( 10 g/l ) was used only in gene expression experiments , not in experiments for protein analysis . five microliters of endotoxin - free water per eye was used as a vehicle control . an additional experiment for pcr analysis was conducted using this method and endotoxin - free saline ( nacl 0.9% ; enzo life sciences , farmingdale , ny , usa ) as the vehicle . for protein analysis using an immunobead assay , conjunctiva was extracted after 24 hours . to determine whether de led to increased expression of inflammatory cytokines after tlr4 activation , ds5 mice were treated with 5 l / eye lps ( 1 g/l ) or water on day 5 of desiccating stress . after 4 hours , mice were euthanized to extract corneal epithelium and conjunctiva for rna analysis . corneal epithelium and conjunctiva were extracted to measure gene expression of inflammatory mediators via pcr . tissue samples from cornea and conjunctiva were pooled separately from both eyes to give one cornea and one conjunctiva sample per mouse . rna was extracted using the rneasy plus micro kit ( qiagen , valencia , ca , usa ) according to the manufacturer 's protocol . concentration of isolated rna was measured using a nanodrop 2000 spectrophotometer ( thermo fisher scientific , waltham , ma , usa ) . after rna isolation , cdna was synthesized using ready - to - go you - prime first - strand beads ( ge healthcare bio - sciences , pittsburgh , pa , usa ) as previously reported . after cdna synthesis , pcr was run on a steponeplus real - time pcr system ( applied biosystems , grand island , ny , usa ) . ct values for each gene were normalized to the ct values of the housekeeping gene for each sample . the housekeeping gene used for these experiments was hypoxanthine guanine phosphoribosyl transferase ( hprt ) . fold differences in expression were calculated after comparing values for each gene to the those in the untreated group . each experiment in this study was completed with its own specific group of untreated mice from the same batch of mice used in that particular experiment . primers ( life technologies , grand island , ny , usa ) used in this study included interferon- ( ifn- ; abi assay i d mm00801778_m1 ) , il-1 ( abi assay i d mm00434228_m1 ) , il-6 ( abi assay i d mm00446190_m1 ) , il-12a ( abi assay i d mm00434165_m1 ) , cxcl10 ( abi assay i d mm00445235_m1 ) , tumor necrosis factor- ( tnf- ; abi assay i d mm00443260_g1 ) , and hprt ( abi assay i d mm00446968_m1 ) . tissue was extracted and placed in 100 l of radioimmunoprecipitation assay buffer ( sigma - aldrich corp . ) treated with a complete , edta - free protease inhibitor cocktail tablet ( roche , basel , switzerland ) . excised tissue was chopped with surgical scissors , sonicated , and kept on ice for 30 minutes . samples were normalized for total protein concentration by using pierce bca protein assay kit ( life technologies ) . then , concentrations of cxcl10 were analyzed using an immunobead assay ( emd millipore , billerica , ma , usa ) according to the manufacturer 's instructions . the assay was run using a luminex 100 system ( luminex , austin , tx , usa ) . immunofluorescent staining was used to visualize il-12a protein in the conjunctiva after administration of lps . tissue sections ( one slide with 2 samples per animal ) were fixed in acetone at 20c for 10 minutes . then , nonspecific sites on the samples were blocked for 60 minutes with 20% goat serum ( sigma - aldrich corp . ) in phosphate - buffered saline ( pbs ) . the primary antibody ( il-12a ab203031 ; abcam , cambridge , ma , usa ) , diluted 1:100 in 5% goat serum in pbs , was then added for 1 hour . after being washed with pbs , sections were incubated with alexa fluor 488 affinipure goat anti - rabbit immunoglobulin g ( igg ; h+l ; product 111545144 ; jackson immunoresearch laboratories inc . , west grove , pa , usa ) diluted in pbs for 1 hour in the dark . pictures were taken at 40 magnification using an eclipse e400 microscope equipped with a digital camera ( ds - qi1mc ; nikon instruments inc , melville , ny , usa ) . immunohistochemistry was used to analyze protein expression of il-1. briefly , tissue sections were fixed in acetone at 20c for 10 minutes . after washing in pbs , samples were incubated with 0.3% hydrogen peroxide in pbs to quench endogenous peroxidases and washed again in pbs . sections were then treated with an avidin / biotin blocking kit ( catalog sp-2001 ; vector laboratories , burlingame , ca , usa ) . after being rinsed in pbs , the samples were blocked for 1 hour with 20% goat serum ( sigma - aldrich corp . ) in pbs . the primary antibody ( rabbit anti - mouse il-1 ; d4t2d ; cell signaling technology , danvers , ma , usa ) was then diluted 1:50 in 5% goat serum in pbs and applied to the samples for 1 hour . after being washed , the secondary antibody , biotinylated goat anti - rabbit igg ( product 550338 ; bd pharmingen , franklin lakes , nj , usa ) sections were washed and treated with a vectastain elite abc kit ( catalog no . pk-6100 ; vector laboratories ) for 30 minutes . then , the sections were washed again and incubated with solution from a vector novared peroxidase substrate kit ( catalog no . sk-4800 ; vector laboratories ) for 15 minutes . . then the sections were counterstained for 2 minutes with mayer hematoxylin ( using lillie modification ) ( scytek laboratories , logan , ut , usa ) . after this , finally , permount ( fisher scientific , pittsburgh , pa , usa ) was applied , and a coverslip was attached . slides were examined and photographed at 40 magnification , using an eclipse e400 microscope equipped with a digital camera ( model ds - fi1 ; nikon ) . a second set of images were recorded at increased magnification , using an a1p mp upright microscope with a motorized stage equipped with a color camera ( model ds - u3 ; nikon ) and a 100/1.45 oil ( plan apochromat ofn25 dic ; nikon ) objective . cell culture studies were performed to identify which cell types on the ocular surface responded to lps stimulation . lps was added directly to either epithelial or dendritic cell ( dc ) cultures in vitro . for epithelial cell cultures , cornea or conjunctiva explants were extracted from female c57bl/6j or tlr4ko mice . conjunctiva explants were excised from the forniceal conjunctiva of 6- to 8-week - old female c57bl/6 mice or tlr4ko mice and left for 15 minutes at 37c in keratinocyte serum - free medium ( ksfm ) ( thermo fisher scientific ) supplemented with 3% fetal bovine serum , 1.25 g / ml amphotericin b ( product 15290 - 018 ; thermo fisher scientific ) , 0.5 l / ml gentamicin ( product 15750 - 060 ; thermo fisher scientific ) , and 5 g / ml dispase ii ( product 04942078001 ; roche ) . for cornea cultures , explants were left for 30 minutes in supplemented hormone epithelium medium ( shem ) containing dulbecco modified eagle medium / f12 medium ( product d8437 ; sigma - aldrich corp . ) supplemented with 5 ng / ml epidermal growth factor ( egf ) , 5 g / ml insulin , 5 g / ml transferrin , 5 ng / ml sodium selenite , 0.5 g / ml hydrocortisone , 30 ng / ml cholera toxin a , 0.5% dimethyl sulfoxide , 50 g / ml gentamicin , 1.25 g / ml amphotericin b , 5% fetal bovine serum , and 5 g / ml dispase ii ( roche ) . corneal and conjunctival explants were then plated at one explant per well in 24-well plates . conjunctiva cultures received 500 l / well of ksfm with 80 ng / ml mouse egf ( bd biosciences , san jose , ca , usa ) and cornea cultures received 500 l / well shem . after 3 days , cornea cultures received 500 l of fresh shem / well for a total of 1 ml medium / well ; medium was changed every 3 days . for conjunctiva cultures , wells received another 500 l of ksfm on day 7 for a total of 1 ml of medium / well . on day 11 , wells were left untreated or treated with 1 l of 1 g/l lps dissolved in endotoxin - free water . after 4 hours , two to three wells were collected for each conjunctiva sample . direct stimulation of dcs by lps was also tested in vitro . briefly , dcs were isolated from c57bl/6j mouse bone marrow and cultured for 10 days using rpmi 1640 medium ( thermo fisher scientific ) . on day 10 , cells were left untreated or treated with 1 l of 1 g/l lps dissolved in endotoxin - free water . all statistical analyses were performed using prism version 6.01 software ( graphpad , inc , la jolla , ca , usa ) . within each experiment for pcr and immunobead assay analysis , 1-way anova was used to determine overall statistical significance , and the holm - sidak multiple comparison test was performed to find differences between groups . two - way anovas were used to analyze statistical significance between experiments , with sidak multiple comparisons test used to analyze the significance between groups of different experiments . for the pcr experiments where saline was used as the vehicle and for pcr analysis of the cell culture studies , an unpaired student t test with welch 's correction was used to determine statistical significance among groups . for c57bl/6j mouse cell culture experiments , the number of samples per group were 4 samples per dc group , 4 samples per untreated conjunctiva cell group , and 5 samples per lps - treated conjunctiva cell group . for the cornea , 2 untreated and 3 lps - treated samples were used to test for il-1. six untreated and 4 lps - treated cornea culture samples were used to test for tnf-. ct values were set at 40 for untreated dc samples that did not contain enough il-1 to quantify . for the tlr4ko mouse cell culture experiments , 5 samples were used in each cornea and conjunctiva group . in the first set of experiments , lps eye drops were administered to nonstressed mice to determine whether tlr4 activation on the ocular surface could lead to the release of proinflammatory mediators . gene expression was measured via rt - qpcr 4 hours after application of lps . in general , lps administration led to a dose - dependent increase in inflammatory mediator expression on the ocular surface . concentrations of 0.1 g/l and 0.01 g/l were also tested ( results not shown ) . lps concentrations of 10 g/l and 1 g/l led to enhanced expression of certain genes ( figs . 1 , 2 ) . in most cases , only small differences in expression were observed between the 1 g/l and 10 g/l concentrations , indicating that maximal tlr4 activation was reached at the 1 g/l concentration ; however , higher concentrations were not tested . in the cornea , application of 1 g/l lps significantly increased expression of il-1 ( 2.17 0.31-fold ) and cxcl10 ( 36.93 7.75-fold ) ( fig . in addition , the conjunctiva displayed more dramatic increases in gene expression after lps administration . for instance , 1 g/l lps enhanced il-1 ( 21.27 9.61-fold ) and cxcl10 ( 185 41.28-fold ) . ifn- ( 13.45 4.49-fold ) , and il-12a ( 3.99 0.27-fold ) were also significantly increased in the conjunctiva after application of 1 g/l lps . tnf- ( 7.02 3.37-fold ) and il-6 ( 20.12 10.48-fold ) were also increased after 1 g/l lps , but the results did not reach statistical significance . increases in il-6 expression ( 38.97 6.44-fold ) in the conjunctiva after 10 g/l lps did reach statistical significance ( fig . ns mice were treated with lps or endotoxin - free water and euthanized after 4 hours to analyze cytokine / chemokine expression . il-1 and cxc10 expression in the cornea significantly increased after lps administration . average of two experiments is shown ( n = 3 per group in each experiment ) . error bars are sds . ( a ) endotoxin - free saline was used as an alternate vehicle for lps . in this segment of the graph , bars depict one experiment ( n = 5 per group ) and error bars are shown as sem . lps administration led to a significant increase in il-1 when dissolved in either water or saline . * * * p 0.001 ; * * p 0.01 . ns mice were treated with lps or vehicle and euthanized after 4 hours for gene expression analysis . average of two experiments is shown ( n 3 per group in each experiment ) . error bars are sds . ( a ) endotoxin - free saline was used as an alternate vehicle for lps . in this segment of the graph , bars depict one experiment ( n = 5 per group ) , and error bars are sem . * * * * p 0.0001 ; * * * p 0.001 ; * * p 0.01 ; * p 0.05 . because water may affect the ocular surface as a hypotonic solution , endotoxin - free saline was evaluated as an alternative vehicle to lps . the choice of vehicle may affect gene expression ; however , the increase in il-1 remained significant when saline was used as the diluent . figures 1a and 2a show that 1 g/l lps dissolved in saline led to a significant increase in il-1 in both the cornea and the conjunctiva . tlr4 activation by lps was verified by repeating identical experiments in tlr4ko mice ( results not shown ) . non - stressed tlr4ko mice were treated with 1 g/l lps and euthanized 4 hours later to measure expression of inflammatory mediators by pcr . increases in gene expression by lps were abolished or greatly diminished in tlr4ko mice . a slight and nonsignificant increase in il-1 was observed in the conjunctiva of lps - treated tlr4ko mice , which may suggest noncanonical inflammasome activation by lps . because no significant increases in gene expression were observed , these findings indicate the stimulatory effects of lps are mediated by tlr4-dependent pathways on the ocular surface . in order to verify that increased gene expression of inflammatory mediators also resulted in an increase in protein expression , concentrations of cxcl10 were measured by an immunobead assay in conjunctival tissue extracts obtained 24 hours after application of lps . a significant increase ( p 0.05 ) in cxcl10 protein was found in the conjunctiva of ns mice treated with 1 g/l lps compared to ns mice treated with vehicle ( endotoxin - free water ) ( fig . conjunctiva was extracted after 24 hours and analyzed for cxcl10 by using an immunobead assay . nonstressed mice were given lps ( 1 g/l ) or vehicle and euthanized after 4 hours . immunohistochemical staining demonstrated that il-1 increased in the cornea of ns mice treated with lps compared to ns mice treated with water ( fig . 4a ) . in figure 4b , higher magnification ( 100 ) was used to display increased il-1 specifically in the corneal epithelium of ns+lps mice . figure 4c shows an increase in il-12a - positive cells in ns mice treated with lps . ( a ) immunohistochemical staining shows increased il-1 expression ( red ) in the central cornea ( stroma ) after administration of lps ( 1 g/l ) to nonstressed mice . expression of il-1 observed in ds5+lps mice is enhanced compared to that in ns+lps mice . ( b ) higher magnification ( 100 ) was used to examine enhanced expression of il-1 in the corneal epithelium of ns+lps and ds5+lps mice . ( c ) immunofluorescent staining demonstrates that application of lps ( 1 g/l ) increases expression of il-12a in the conjunctiva of nonstressed and ds5 mice . the white boxes indicate il-12a - positive cells ( green ) , which are shown enlarged in the upper corners . lps was also tested in vitro to determine which cell types may be responsible for the expression of inflammatory mediators observed in the in vivo studies . figure 5 shows the effect of lps when it is added to explant cell cultures from non - stressed mice . addition of lps to cell culture medium led to an increase in il-1 and tnf- in cornea ( fig . no increase in expression was observed when lipopolysaccharide was added to cornea and conjunctiva cultures from tlr4ko mice ( results not shown ) . increased expression levels of il-1 and tnf- are demonstrated after addition of 1 g of lps to cornea ( a ) , conjunctiva ( b ) , and dendritic ( c ) cell cultures . * * * p 0.0001 ; * * p 0.01 ; * p 0.05 . after tlr4 activation was demonstrated on the ocular surface in nonstressed mice , the next set of experiments was designed to determine whether tlr4 activation is enhanced during dry eye disease . mice were exposed to ds for 5 days , then an lps drop was administered to the eye . corneal epithelium and conjunctiva were extracted after 4 hours for analysis of gene expression by rt - qpcr . compared to control ns eyes , lps increased expression of il-1 ( 3-fold ) in the cornea and il-12a ( 2-fold ) in the conjunctiva of dry eyes . lps administration after 5 days of desiccating stress led to increased expression of il-1 ( cornea ) and il-12a ( conjunctiva ) . expression levels are higher in mice exposed to desiccating stress than in those in nonstressed mice . each series represents an average sd of two experiments ( n 3 per group in each experiment ) . separate sets of ns untreated mice were used to calculate fold increase for each series ( ns control represents two separate sets of mice for each series ) . * * * * p 0.0001 ; * * * p 0.001 ; * * p 0.01 . in a separate experiment using the same methods , figures 4a and 4b further support the hypothesis that lps administration leads to enhanced expression of inflammatory mediators during dry eye disease . enhanced protein expression of il-1 was observed in ds5 mice treated with lps compared to ns mice treated with lps ( figs . figure 4a shows enhanced expression of il-1 in the corneal stroma of ds5+lps mice , and figure 4b demonstrates the same increase in the corneal epithelium . in addition , an increase in il-12a positive cells was noted in ds5 mice treated with lps compared to ds controls ( fig . in the first set of experiments , lps eye drops were administered to nonstressed mice to determine whether tlr4 activation on the ocular surface could lead to the release of proinflammatory mediators . gene expression was measured via rt - qpcr 4 hours after application of lps . in general , lps administration led to a dose - dependent increase in inflammatory mediator expression on the ocular surface . concentrations of 0.1 g/l and 0.01 g/l were also tested ( results not shown ) . lps concentrations of 10 g/l and 1 g/l led to enhanced expression of certain genes ( figs . 1 , 2 ) . in most cases , only small differences in expression were observed between the 1 g/l and 10 g/l concentrations , indicating that maximal tlr4 activation was reached at the 1 g/l concentration ; however , higher concentrations were not tested . in the cornea , application of 1 g/l lps significantly increased expression of il-1 ( 2.17 0.31-fold ) and cxcl10 ( 36.93 7.75-fold ) ( fig . in addition , the conjunctiva displayed more dramatic increases in gene expression after lps administration . for instance , 1 g/l lps enhanced il-1 ( 21.27 9.61-fold ) and cxcl10 ( 185 41.28-fold ) . ifn- ( 13.45 4.49-fold ) , and il-12a ( 3.99 0.27-fold ) were also significantly increased in the conjunctiva after application of 1 g/l lps . tnf- ( 7.02 3.37-fold ) and il-6 ( 20.12 10.48-fold ) were also increased after 1 g/l lps , but the results did not reach statistical significance . increases in il-6 expression ( 38.97 6.44-fold ) in the conjunctiva after 10 g/l lps did reach statistical significance ( fig . ns mice were treated with lps or endotoxin - free water and euthanized after 4 hours to analyze cytokine / chemokine expression . il-1 and cxc10 expression in the cornea significantly increased after lps administration . average of two experiments is shown ( n = 3 per group in each experiment ) . error bars are sds . ( a ) endotoxin - free saline was used as an alternate vehicle for lps . in this segment of the graph , bars depict one experiment ( n = 5 per group ) and error bars are shown as sem . lps administration led to a significant increase in il-1 when dissolved in either water or saline . * * * p 0.001 ; * * p 0.01 . ns mice were treated with lps or vehicle and euthanized after 4 hours for gene expression analysis . average of two experiments is shown ( n 3 per group in each experiment ) . error bars are sds . ( a ) endotoxin - free saline was used as an alternate vehicle for lps . in this segment of the graph , bars depict one experiment ( n = 5 per group ) , and error bars are sem . * * * * p 0.0001 ; * * * p 0.001 ; * * p 0.01 ; * p 0.05 . because water may affect the ocular surface as a hypotonic solution , endotoxin - free saline was evaluated as an alternative vehicle to lps . the choice of vehicle may affect gene expression ; however , the increase in il-1 remained significant when saline was used as the diluent . figures 1a and 2a show that 1 g/l lps dissolved in saline led to a significant increase in il-1 in both the cornea and the conjunctiva . tlr4 activation by lps was verified by repeating identical experiments in tlr4ko mice ( results not shown ) . non - stressed tlr4ko mice were treated with 1 g/l lps and euthanized 4 hours later to measure expression of inflammatory mediators by pcr . increases in gene expression by lps were abolished or greatly diminished in tlr4ko mice . a slight and nonsignificant increase in il-1 was observed in the conjunctiva of lps - treated tlr4ko mice , which may suggest noncanonical inflammasome activation by lps . because no significant increases in gene expression were observed , these findings indicate the stimulatory effects of lps are mediated by tlr4-dependent pathways on the ocular surface . in order to verify that increased gene expression of inflammatory mediators also resulted in an increase in protein expression , concentrations of cxcl10 were measured by an immunobead assay in conjunctival tissue extracts obtained 24 hours after application of lps . a significant increase ( p 0.05 ) in cxcl10 protein was found in the conjunctiva of ns mice treated with 1 g/l lps compared to ns mice treated with vehicle ( endotoxin - free water ) ( fig . conjunctiva was extracted after 24 hours and analyzed for cxcl10 by using an immunobead assay . nonstressed mice were given lps ( 1 g/l ) or vehicle and euthanized after 4 hours . immunohistochemical staining demonstrated that il-1 increased in the cornea of ns mice treated with lps compared to ns mice treated with water ( fig . 4a ) . in figure 4b , higher magnification ( 100 ) was used to display increased il-1 specifically in the corneal epithelium of ns+lps mice . figure 4c shows an increase in il-12a - positive cells in ns mice treated with lps . ( a ) immunohistochemical staining shows increased il-1 expression ( red ) in the central cornea ( stroma ) after administration of lps ( 1 g/l ) to nonstressed mice . expression of il-1 observed in ds5+lps mice is enhanced compared to that in ns+lps mice . ( b ) higher magnification ( 100 ) was used to examine enhanced expression of il-1 in the corneal epithelium of ns+lps and ds5+lps mice . ( c ) immunofluorescent staining demonstrates that application of lps ( 1 g/l ) increases expression of il-12a in the conjunctiva of nonstressed and ds5 mice . the white boxes indicate il-12a - positive cells ( green ) , which are shown enlarged in the upper corners . lps was also tested in vitro to determine which cell types may be responsible for the expression of inflammatory mediators observed in the in vivo studies . figure 5 shows the effect of lps when it is added to explant cell cultures from non - stressed mice . addition of lps to cell culture medium led to an increase in il-1 and tnf- in cornea ( fig . no increase in expression was observed when lipopolysaccharide was added to cornea and conjunctiva cultures from tlr4ko mice ( results not shown ) . increased expression levels of il-1 and tnf- are demonstrated after addition of 1 g of lps to cornea ( a ) , conjunctiva ( b ) , and dendritic ( c ) cell cultures . * * * p 0.0001 ; * * p 0.01 ; * p 0.05 . after tlr4 activation was demonstrated on the ocular surface in nonstressed mice , the next set of experiments was designed to determine whether tlr4 activation is enhanced during dry eye disease . mice were exposed to ds for 5 days , then an lps drop was administered to the eye . corneal epithelium and conjunctiva were extracted after 4 hours for analysis of gene expression by rt - qpcr . compared to control ns eyes , lps increased expression of il-1 ( 3-fold ) in the cornea and il-12a ( 2-fold ) in the conjunctiva of dry eyes . lps administration after 5 days of desiccating stress led to increased expression of il-1 ( cornea ) and il-12a ( conjunctiva ) . expression levels are higher in mice exposed to desiccating stress than in those in nonstressed mice . each series represents an average sd of two experiments ( n 3 per group in each experiment ) . separate sets of ns untreated mice were used to calculate fold increase for each series ( ns control represents two separate sets of mice for each series ) . * * * * p 0.0001 ; * * * p 0.001 ; * * p 0.01 . in a separate experiment using the same methods , protein expression was analyzed in ds mice by immunostaining . figures 4a and 4b further support the hypothesis that lps administration leads to enhanced expression of inflammatory mediators during dry eye disease . enhanced protein expression of il-1 was observed in ds5 mice treated with lps compared to ns mice treated with lps ( figs . figure 4a shows enhanced expression of il-1 in the corneal stroma of ds5+lps mice , and figure 4b demonstrates the same increase in the corneal epithelium . in addition , an increase in il-12a positive cells was noted in ds5 mice treated with lps compared to ds controls ( fig . dry eye is a costly disease and is highly prevalent in the elderly . with an aging population and limited treatment options available for dry eye , it is important to define all the factors that can contribute to inflammation on the ocular surface . toll - like receptors are known to promote inflammation , but their role in dry eye has not been fully explored . tlr4 expression was found to increase in the conjunctiva in our mouse model of dry eye . moreover , inhibition of tlr4 decreases expression of some inflammatory mediators during experimental de . however , additional research is needed to understand inflammatory mediators associated with tlr4 activation on the ocular surface . theoretically , commensal or pathogenic lps - producing bacteria , as well as endogenous tlr4 ligands , could make a significant contribution to the inflammation observed in dry eye . importantly , gram - negative bacterial sequences have been found in the normal ocular microbiome . endogenous tlr4 ligands are thought to include extracellular matrix breakdown components , fibrinogen cleavage products , and heat shock proteins . heat shock protein 70 has been linked to de , but the presence of other endogenous tlr4 ligands has not yet been fully investigated . potentially , disruption of ocular surface homeostasis in dry eye could lead to generation of endogenous tlr4 ligands . despite this possibility , the ability of tlr4 to produce a proinflammatory response in the eye during normal and diseased states has not been fully explored . this study indicates that tlr4 is present on the eye and that tlr4 activation can lead to increased production of proinflammatory mediators . in fact , lps application to the ocular surface can lead to the release of several inflammatory mediators linked to dry eye . the greatest variety of inflammatory mediators and most dramatic rises in gene expression were noted in the conjunctiva ( fig . this was expected , as the greater permeability of the conjunctival epithelium may make tlr4-positive cells more accessible to lps than in the cornea . il-6 is one proinflammatory cytokine that was expressed in the conjunctiva after lps treatment ( fig . additionally , tnf- appeared to increase in a dose - dependent manner , though it did not reach statistical significance . tnf- is also important in th17 differentiation and has been shown to stimulate mmp-9 production in vitro , which can lead to further corneal epithelial disease . both il-6 and tnf- have been found to be increased in mouse models of dry eye and in humans with the disease . in addition to generating th17 pathway cytokines , lps administration also led to mediators associated with the th1 response . ifn- is the defining cytokine of the th1 response , which contributes to ocular surface disease and tear dysfunction . upregulation of ifn- by lps is important as ifn- expression is increased in mice and humans with de . furthermore , ifn- has been linked to goblet cell loss and is capable of inducing apoptosis in the conjunctival epithelium . increased production of ifn- after treatment with lps is also significant , in part , because of the ability of ifn- to amplify the immune response . ifn- increases production of th1 chemokines ( cxcl9 , cxcl10 , and cxcl11 ) , which , in turn , attract greater numbers of ifn-producing cells . also , ifn- increases expression of cd80 and cd86 on dendritic cells , which is required for t cell activation . finally , ifn- leads to increased il-12 receptor expression . interestingly , il-12a , the main cytokine responsible for th1 differentiation , was also significantly upregulated in the conjunctiva after lps stimulation ( fig . 2 ) . upregulation of il-12a , in conjunction with ifn- , suggests lps stimulation can promote the th1 response that is critical to dry eye pathology . it is particularly interesting that application of lps to non - stressed mice leads to production of inflammatory mediators in the cornea ( fig . there are some conflicting findings in the literature as to whether or not human corneal epithelial cells can respond to lps in vitro . figure 5 demonstrates cultured mouse cornea epithelial cells do respond to lps in vitro . in vivo , this study suggests that normal , unwounded mouse corneas can also respond to tlr4 activation in vivo . in the cornea ( and conjunctiva ) , lps administration significantly increased expression of cxcl10 and il-1 in ns mice ( fig . 1 ) . both the cxcl10 protein and mrna are known to increase in a mouse model of dry eye . increased cxcl10 expression has also been found in human dry eye . similarly , il-1 , a proinflammatory cytokine , is increased in mice with experimental dry eye and in humans with the disease . blockade of il-1 pathways during dry eye has been shown to be efficacious in murine models of de . in the end , lps was found to enhance expression of cytokines and chemokines on a nonstressed ocular surface , which demonstrates that tlr4 activation could contribute to homeostatic immune surveillance . the cell culture studies demonstrate that mouse cornea and conjunctiva epithelial cells , as well as dendritic cells , can respond to lps on the ocular surface . the production of inflammatory mediators observed in the in vivo studies may be from a combination of direct and indirect stimulation . figure 5 suggests that direct activation of epithelial and dendritic cells by lps is possible . indirect stimulation of epithelial cells by lps - stimulated dcs ( or other immune cells ) may also contribute to production of proinflammatory mediators . figure 5 demonstrates that il-1 and tnf- , both of which may stimulate additional mediator release from epithelial cells , were upregulated in lps - stimulated dc culture . indeed , il-1 has been shown to increase production of il-1 and il-6 in limbal epithelial stem cells , and tnf- has been shown to increase cxcl10 production in human corneal epithelial cells . although lps stimulation can lead to inflammation in a normal eye , this study suggests that tlr4 activation is enhanced during dry eye disease . figures 4 and 6 display increased expression of il-1 in the cornea after lps administration when mice are exposed to ds . this finding supports our hypothesis that disruption of the corneal epithelial barrier during de leads to greater exposure of tlr4 located in the wing and basal layers of the corneal epithelium . tlr4 is then more accessible in the cornea , leading to greater activation by lps . disruption of the corneal epithelial barrier has been established in our mouse model . however , other mechanisms are still a possibility , including increased cell surface expression of tlr4 in the corneal epithelium during de . nevertheless , enhanced expression of il-1 in the cornea during de after lps stimulation may impact clinical severity . for instance , il-1 has been shown to upregulate mmp-9 , which can lead to disruption of the corneal epithelium . in turn , this could lead to even greater exposure of tlr4 and contribute to a damaging inflammatory cycle . surprisingly , figure 6 also shows that lps administration during de increases expression of il-12a in the conjunctiva . more research needs to be conducted to assess the biological activity of il-35 on the ocular surface . upregulation of il-12 by lps may indicate that tlr4 activation could be important in the early stages of dry eye disease . microbial stress may lead to enhanced release of il-12 at the initiation of de , amplifying the intensity of the disease . tlr4 stimulation could potentiate the induction of the de immune response by changing the cytokine milieu and activating th1-polarizing dendritic cells . additional research is needed to determine the mechanism behind the increase in il-12a . in theory considering this , increased sensitivity of those cell types to lps during ds is a possibility that needs further investigation . in conclusion , this study demonstrates that not only is tlr4 capable of producing a proinflammatory response on the ocular surface but that response is enhanced during de disease . tlr4 activation during de appears to be important , especially in the initial stages of de . in the cornea , disruption of in the conjunctiva , enhanced expression of il-12a after tlr4 activation may promote th1 differentiation , leading to more severe disease . the role of commensal bacteria and endogenous tlr4 ligands in activation of tlr4 during de also needs further exploration . despite this , the proinflammatory reactions produced by tlr4 on the ocular surface illustrate that tlr4 is a potential pharmaceutical target for dry eye inflammation .

purposetoll - like receptor 4 ( tlr4 ) alerts cells to the presence of bacteria by initiating an inflammatory response . we hypothesize that disruption of the ocular surface barrier in dry eye enhances tlr4 signaling . this study determined whether dry eye enhances expression of inflammatory mediators in response to topically applied tlr4 ligand.methodsa single dose of lipopolysaccharide ( lps ) or vehicle ( endotoxin - free water ) was applied to the cornea of nonstressed ( ns ) mice or mice subjected to 5 days of desiccating stress ( ds ) . after 4 hours , corneal epithelium and conjunctiva were extracted to analyze expression of inflammatory mediators via pcr . protein expression was confirmed by immunobead assay and immunostaining.resultstopically applied lps increased expression of inflammatory mediators il-1 , cxcl10 , il-12a , and ifn- in the conjunctiva , and il-1 and cxcl10 in the cornea of ns mice compared to that in untreated controls . lps in ds mice produced 3-fold increased expression of il-1 in cornea and 2-fold increased expression in il-12a in conjunctiva compared to that in lps - treated control mice.conclusionslps increased expression of inflammatory cytokines on the ocular surface . this expression was further increased in dry eye , which suggests that epithelial barrier disruption enhances exposure of lps to tlr4 + cells and that the inflammatory response to endotoxin - producing commensal or pathogenic bacteria may be more severe in dry eye disease .

Materials and Methods Mice Murine Model of Dry Eye LPS Treatment RNA Isolation PCR Protein Isolation and Analysis Immunofluorescent Staining Immunohistochemistry Cell Culture Statistical Analysis Results TLR4 Activation in Nonstressed Mice TLR4 Activation in Experimental Dry Eye Discussion

in the cell culture studies , 5 tlr4ko mice and 7 c57bl/6j mice mice were exposed to desiccating stress ( ds ) to create a murine model of dry eye , similar to methods previously described . nonstressed ( ns ) control mice were housed in a vivarium with 50% to 75% relative humidity , no exposure to air drafts , and no scopolamine injections . to determine whether tlr4 could be activated on the ocular surface of untreated mice , ns mice were treated with ultrapure lps from salmonella enterica serovar minnesota mutant r595 ( invivogen , san diego , ca , usa ) . five microliters of endotoxin - free water per eye was used as a vehicle control . to determine whether de led to increased expression of inflammatory cytokines after tlr4 activation , ds5 mice were treated with 5 l / eye lps ( 1 g/l ) or water on day 5 of desiccating stress . after 4 hours , mice were euthanized to extract corneal epithelium and conjunctiva for rna analysis . corneal epithelium and conjunctiva were extracted to measure gene expression of inflammatory mediators via pcr . primers ( life technologies , grand island , ny , usa ) used in this study included interferon- ( ifn- ; abi assay i d mm00801778_m1 ) , il-1 ( abi assay i d mm00434228_m1 ) , il-6 ( abi assay i d mm00446190_m1 ) , il-12a ( abi assay i d mm00434165_m1 ) , cxcl10 ( abi assay i d mm00445235_m1 ) , tumor necrosis factor- ( tnf- ; abi assay i d mm00443260_g1 ) , and hprt ( abi assay i d mm00446968_m1 ) . immunohistochemistry was used to analyze protein expression of il-1. after being washed , the secondary antibody , biotinylated goat anti - rabbit igg ( product 550338 ; bd pharmingen , franklin lakes , nj , usa ) was applied to the samples for 30 minutes . for c57bl/6j mouse cell culture experiments , the number of samples per group were 4 samples per dc group , 4 samples per untreated conjunctiva cell group , and 5 samples per lps - treated conjunctiva cell group . of the total number of animals used in each group , 5 to 6 were used for pcr , 4 for immunobead assay , 3 for immunofluorescence ( interleukin-12a [ il-12a ] ) , and 3 for immunohistochemistry ( il-1 ) . in the cell culture studies , 5 tlr4ko mice and 7 c57bl/6j mice mice were exposed to desiccating stress ( ds ) to create a murine model of dry eye , similar to methods previously described . nonstressed ( ns ) control mice were housed in a vivarium with 50% to 75% relative humidity , no exposure to air drafts , and no scopolamine injections . to determine whether tlr4 could be activated on the ocular surface of untreated mice , ns mice were treated with ultrapure lps from salmonella enterica serovar minnesota mutant r595 ( invivogen , san diego , ca , usa ) . mice were treated topically ( 5 l / eye ) with lps dissolved in endotoxin - free water ( sigma - aldrich corp . ) the higher dose of lps ( 10 g/l ) was used only in gene expression experiments , not in experiments for protein analysis . to determine whether de led to increased expression of inflammatory cytokines after tlr4 activation , ds5 mice were treated with 5 l / eye lps ( 1 g/l ) or water on day 5 of desiccating stress . after 4 hours , mice were euthanized to extract corneal epithelium and conjunctiva for rna analysis . corneal epithelium and conjunctiva were extracted to measure gene expression of inflammatory mediators via pcr . primers ( life technologies , grand island , ny , usa ) used in this study included interferon- ( ifn- ; abi assay i d mm00801778_m1 ) , il-1 ( abi assay i d mm00434228_m1 ) , il-6 ( abi assay i d mm00446190_m1 ) , il-12a ( abi assay i d mm00434165_m1 ) , cxcl10 ( abi assay i d mm00445235_m1 ) , tumor necrosis factor- ( tnf- ; abi assay i d mm00443260_g1 ) , and hprt ( abi assay i d mm00446968_m1 ) . immunofluorescent staining was used to visualize il-12a protein in the conjunctiva after administration of lps . immunohistochemistry was used to analyze protein expression of il-1. the primary antibody ( rabbit anti - mouse il-1 ; d4t2d ; cell signaling technology , danvers , ma , usa ) was then diluted 1:50 in 5% goat serum in pbs and applied to the samples for 1 hour . after 4 hours , two to three wells were collected for each conjunctiva sample . on day 10 , cells were left untreated or treated with 1 l of 1 g/l lps dissolved in endotoxin - free water . in the first set of experiments , lps eye drops were administered to nonstressed mice to determine whether tlr4 activation on the ocular surface could lead to the release of proinflammatory mediators . gene expression was measured via rt - qpcr 4 hours after application of lps . in the cornea , application of 1 g/l lps significantly increased expression of il-1 ( 2.17 0.31-fold ) and cxcl10 ( 36.93 7.75-fold ) ( fig . ifn- ( 13.45 4.49-fold ) , and il-12a ( 3.99 0.27-fold ) were also significantly increased in the conjunctiva after application of 1 g/l lps . ns mice were treated with lps or endotoxin - free water and euthanized after 4 hours to analyze cytokine / chemokine expression . il-1 and cxc10 expression in the cornea significantly increased after lps administration . ns mice were treated with lps or vehicle and euthanized after 4 hours for gene expression analysis . because water may affect the ocular surface as a hypotonic solution , endotoxin - free saline was evaluated as an alternative vehicle to lps . figures 1a and 2a show that 1 g/l lps dissolved in saline led to a significant increase in il-1 in both the cornea and the conjunctiva . non - stressed tlr4ko mice were treated with 1 g/l lps and euthanized 4 hours later to measure expression of inflammatory mediators by pcr . a slight and nonsignificant increase in il-1 was observed in the conjunctiva of lps - treated tlr4ko mice , which may suggest noncanonical inflammasome activation by lps . because no significant increases in gene expression were observed , these findings indicate the stimulatory effects of lps are mediated by tlr4-dependent pathways on the ocular surface . in order to verify that increased gene expression of inflammatory mediators also resulted in an increase in protein expression , concentrations of cxcl10 were measured by an immunobead assay in conjunctival tissue extracts obtained 24 hours after application of lps . a significant increase ( p 0.05 ) in cxcl10 protein was found in the conjunctiva of ns mice treated with 1 g/l lps compared to ns mice treated with vehicle ( endotoxin - free water ) ( fig . nonstressed mice were given lps ( 1 g/l ) or vehicle and euthanized after 4 hours . immunohistochemical staining demonstrated that il-1 increased in the cornea of ns mice treated with lps compared to ns mice treated with water ( fig . ( a ) immunohistochemical staining shows increased il-1 expression ( red ) in the central cornea ( stroma ) after administration of lps ( 1 g/l ) to nonstressed mice . expression of il-1 observed in ds5+lps mice is enhanced compared to that in ns+lps mice . ( b ) higher magnification ( 100 ) was used to examine enhanced expression of il-1 in the corneal epithelium of ns+lps and ds5+lps mice . ( c ) immunofluorescent staining demonstrates that application of lps ( 1 g/l ) increases expression of il-12a in the conjunctiva of nonstressed and ds5 mice . lps was also tested in vitro to determine which cell types may be responsible for the expression of inflammatory mediators observed in the in vivo studies . addition of lps to cell culture medium led to an increase in il-1 and tnf- in cornea ( fig . increased expression levels of il-1 and tnf- are demonstrated after addition of 1 g of lps to cornea ( a ) , conjunctiva ( b ) , and dendritic ( c ) cell cultures . after tlr4 activation was demonstrated on the ocular surface in nonstressed mice , the next set of experiments was designed to determine whether tlr4 activation is enhanced during dry eye disease . corneal epithelium and conjunctiva were extracted after 4 hours for analysis of gene expression by rt - qpcr . compared to control ns eyes , lps increased expression of il-1 ( 3-fold ) in the cornea and il-12a ( 2-fold ) in the conjunctiva of dry eyes . lps administration after 5 days of desiccating stress led to increased expression of il-1 ( cornea ) and il-12a ( conjunctiva ) . in a separate experiment using the same methods , figures 4a and 4b further support the hypothesis that lps administration leads to enhanced expression of inflammatory mediators during dry eye disease . enhanced protein expression of il-1 was observed in ds5 mice treated with lps compared to ns mice treated with lps ( figs . figure 4a shows enhanced expression of il-1 in the corneal stroma of ds5+lps mice , and figure 4b demonstrates the same increase in the corneal epithelium . in the first set of experiments , lps eye drops were administered to nonstressed mice to determine whether tlr4 activation on the ocular surface could lead to the release of proinflammatory mediators . in the cornea , application of 1 g/l lps significantly increased expression of il-1 ( 2.17 0.31-fold ) and cxcl10 ( 36.93 7.75-fold ) ( fig . ifn- ( 13.45 4.49-fold ) , and il-12a ( 3.99 0.27-fold ) were also significantly increased in the conjunctiva after application of 1 g/l lps . ns mice were treated with lps or endotoxin - free water and euthanized after 4 hours to analyze cytokine / chemokine expression . il-1 and cxc10 expression in the cornea significantly increased after lps administration . ns mice were treated with lps or vehicle and euthanized after 4 hours for gene expression analysis . because water may affect the ocular surface as a hypotonic solution , endotoxin - free saline was evaluated as an alternative vehicle to lps . figures 1a and 2a show that 1 g/l lps dissolved in saline led to a significant increase in il-1 in both the cornea and the conjunctiva . non - stressed tlr4ko mice were treated with 1 g/l lps and euthanized 4 hours later to measure expression of inflammatory mediators by pcr . a slight and nonsignificant increase in il-1 was observed in the conjunctiva of lps - treated tlr4ko mice , which may suggest noncanonical inflammasome activation by lps . because no significant increases in gene expression were observed , these findings indicate the stimulatory effects of lps are mediated by tlr4-dependent pathways on the ocular surface . in order to verify that increased gene expression of inflammatory mediators also resulted in an increase in protein expression , concentrations of cxcl10 were measured by an immunobead assay in conjunctival tissue extracts obtained 24 hours after application of lps . a significant increase ( p 0.05 ) in cxcl10 protein was found in the conjunctiva of ns mice treated with 1 g/l lps compared to ns mice treated with vehicle ( endotoxin - free water ) ( fig . nonstressed mice were given lps ( 1 g/l ) or vehicle and euthanized after 4 hours . immunohistochemical staining demonstrated that il-1 increased in the cornea of ns mice treated with lps compared to ns mice treated with water ( fig . expression of il-1 observed in ds5+lps mice is enhanced compared to that in ns+lps mice . ( b ) higher magnification ( 100 ) was used to examine enhanced expression of il-1 in the corneal epithelium of ns+lps and ds5+lps mice . ( c ) immunofluorescent staining demonstrates that application of lps ( 1 g/l ) increases expression of il-12a in the conjunctiva of nonstressed and ds5 mice . lps was also tested in vitro to determine which cell types may be responsible for the expression of inflammatory mediators observed in the in vivo studies . addition of lps to cell culture medium led to an increase in il-1 and tnf- in cornea ( fig . increased expression levels of il-1 and tnf- are demonstrated after addition of 1 g of lps to cornea ( a ) , conjunctiva ( b ) , and dendritic ( c ) cell cultures . after tlr4 activation was demonstrated on the ocular surface in nonstressed mice , the next set of experiments was designed to determine whether tlr4 activation is enhanced during dry eye disease . corneal epithelium and conjunctiva were extracted after 4 hours for analysis of gene expression by rt - qpcr . compared to control ns eyes , lps increased expression of il-1 ( 3-fold ) in the cornea and il-12a ( 2-fold ) in the conjunctiva of dry eyes . lps administration after 5 days of desiccating stress led to increased expression of il-1 ( cornea ) and il-12a ( conjunctiva ) . in a separate experiment using the same methods , protein expression was analyzed in ds mice by immunostaining . figures 4a and 4b further support the hypothesis that lps administration leads to enhanced expression of inflammatory mediators during dry eye disease . enhanced protein expression of il-1 was observed in ds5 mice treated with lps compared to ns mice treated with lps ( figs . figure 4a shows enhanced expression of il-1 in the corneal stroma of ds5+lps mice , and figure 4b demonstrates the same increase in the corneal epithelium . with an aging population and limited treatment options available for dry eye , it is important to define all the factors that can contribute to inflammation on the ocular surface . tlr4 expression was found to increase in the conjunctiva in our mouse model of dry eye . however , additional research is needed to understand inflammatory mediators associated with tlr4 activation on the ocular surface . theoretically , commensal or pathogenic lps - producing bacteria , as well as endogenous tlr4 ligands , could make a significant contribution to the inflammation observed in dry eye . potentially , disruption of ocular surface homeostasis in dry eye could lead to generation of endogenous tlr4 ligands . in fact , lps application to the ocular surface can lead to the release of several inflammatory mediators linked to dry eye . the greatest variety of inflammatory mediators and most dramatic rises in gene expression were noted in the conjunctiva ( fig . ifn- increases production of th1 chemokines ( cxcl9 , cxcl10 , and cxcl11 ) , which , in turn , attract greater numbers of ifn-producing cells . interestingly , il-12a , the main cytokine responsible for th1 differentiation , was also significantly upregulated in the conjunctiva after lps stimulation ( fig . it is particularly interesting that application of lps to non - stressed mice leads to production of inflammatory mediators in the cornea ( fig . in vivo , this study suggests that normal , unwounded mouse corneas can also respond to tlr4 activation in vivo . in the cornea ( and conjunctiva ) , lps administration significantly increased expression of cxcl10 and il-1 in ns mice ( fig . similarly , il-1 , a proinflammatory cytokine , is increased in mice with experimental dry eye and in humans with the disease . in the end , lps was found to enhance expression of cytokines and chemokines on a nonstressed ocular surface , which demonstrates that tlr4 activation could contribute to homeostatic immune surveillance . the cell culture studies demonstrate that mouse cornea and conjunctiva epithelial cells , as well as dendritic cells , can respond to lps on the ocular surface . the production of inflammatory mediators observed in the in vivo studies may be from a combination of direct and indirect stimulation . although lps stimulation can lead to inflammation in a normal eye , this study suggests that tlr4 activation is enhanced during dry eye disease . figures 4 and 6 display increased expression of il-1 in the cornea after lps administration when mice are exposed to ds . this finding supports our hypothesis that disruption of the corneal epithelial barrier during de leads to greater exposure of tlr4 located in the wing and basal layers of the corneal epithelium . nevertheless , enhanced expression of il-1 in the cornea during de after lps stimulation may impact clinical severity . for instance , il-1 has been shown to upregulate mmp-9 , which can lead to disruption of the corneal epithelium . surprisingly , figure 6 also shows that lps administration during de increases expression of il-12a in the conjunctiva . more research needs to be conducted to assess the biological activity of il-35 on the ocular surface . in conclusion , this study demonstrates that not only is tlr4 capable of producing a proinflammatory response on the ocular surface but that response is enhanced during de disease . in the cornea , disruption of in the conjunctiva , enhanced expression of il-12a after tlr4 activation may promote th1 differentiation , leading to more severe disease . despite this , the proinflammatory reactions produced by tlr4 on the ocular surface illustrate that tlr4 is a potential pharmaceutical target for dry eye inflammation .

all studies adhered to the association for research in vision and ophthalmology statement for use of animals in ophthalmic and visual research . of the total number of animals used in each group , 5 to 6 were used for pcr , 4 for immunobead assay , 3 for immunofluorescence ( interleukin-12a [ il-12a ] ) , and 3 for immunohistochemistry ( il-1 ) . in the cell culture studies , 5 tlr4ko mice and 7 c57bl/6j mice mice were exposed to desiccating stress ( ds ) to create a murine model of dry eye , similar to methods previously described . to determine whether tlr4 could be activated on the ocular surface of untreated mice , ns mice were treated with ultrapure lps from salmonella enterica serovar minnesota mutant r595 ( invivogen , san diego , ca , usa ) . mice were treated topically ( 5 l / eye ) with lps dissolved in endotoxin - free water ( sigma - aldrich corp . ) the higher dose of lps ( 10 g/l ) was used only in gene expression experiments , not in experiments for protein analysis . to determine whether de led to increased expression of inflammatory cytokines after tlr4 activation , ds5 mice were treated with 5 l / eye lps ( 1 g/l ) or water on day 5 of desiccating stress . primers ( life technologies , grand island , ny , usa ) used in this study included interferon- ( ifn- ; abi assay i d mm00801778_m1 ) , il-1 ( abi assay i d mm00434228_m1 ) , il-6 ( abi assay i d mm00446190_m1 ) , il-12a ( abi assay i d mm00434165_m1 ) , cxcl10 ( abi assay i d mm00445235_m1 ) , tumor necrosis factor- ( tnf- ; abi assay i d mm00443260_g1 ) , and hprt ( abi assay i d mm00446968_m1 ) . conjunctiva explants were excised from the forniceal conjunctiva of 6- to 8-week - old female c57bl/6 mice or tlr4ko mice and left for 15 minutes at 37c in keratinocyte serum - free medium ( ksfm ) ( thermo fisher scientific ) supplemented with 3% fetal bovine serum , 1.25 g / ml amphotericin b ( product 15290 - 018 ; thermo fisher scientific ) , 0.5 l / ml gentamicin ( product 15750 - 060 ; thermo fisher scientific ) , and 5 g / ml dispase ii ( product 04942078001 ; roche ) . supplemented with 5 ng / ml epidermal growth factor ( egf ) , 5 g / ml insulin , 5 g / ml transferrin , 5 ng / ml sodium selenite , 0.5 g / ml hydrocortisone , 30 ng / ml cholera toxin a , 0.5% dimethyl sulfoxide , 50 g / ml gentamicin , 1.25 g / ml amphotericin b , 5% fetal bovine serum , and 5 g / ml dispase ii ( roche ) . for c57bl/6j mouse cell culture experiments , the number of samples per group were 4 samples per dc group , 4 samples per untreated conjunctiva cell group , and 5 samples per lps - treated conjunctiva cell group . all studies adhered to the association for research in vision and ophthalmology statement for use of animals in ophthalmic and visual research . of the total number of animals used in each group , 5 to 6 were used for pcr , 4 for immunobead assay , 3 for immunofluorescence ( interleukin-12a [ il-12a ] ) , and 3 for immunohistochemistry ( il-1 ) . in the cell culture studies , 5 tlr4ko mice and 7 c57bl/6j mice mice were exposed to desiccating stress ( ds ) to create a murine model of dry eye , similar to methods previously described . to determine whether tlr4 could be activated on the ocular surface of untreated mice , ns mice were treated with ultrapure lps from salmonella enterica serovar minnesota mutant r595 ( invivogen , san diego , ca , usa ) . the higher dose of lps ( 10 g/l ) was used only in gene expression experiments , not in experiments for protein analysis . to determine whether de led to increased expression of inflammatory cytokines after tlr4 activation , ds5 mice were treated with 5 l / eye lps ( 1 g/l ) or water on day 5 of desiccating stress . primers ( life technologies , grand island , ny , usa ) used in this study included interferon- ( ifn- ; abi assay i d mm00801778_m1 ) , il-1 ( abi assay i d mm00434228_m1 ) , il-6 ( abi assay i d mm00446190_m1 ) , il-12a ( abi assay i d mm00434165_m1 ) , cxcl10 ( abi assay i d mm00445235_m1 ) , tumor necrosis factor- ( tnf- ; abi assay i d mm00443260_g1 ) , and hprt ( abi assay i d mm00446968_m1 ) . conjunctiva explants were excised from the forniceal conjunctiva of 6- to 8-week - old female c57bl/6 mice or tlr4ko mice and left for 15 minutes at 37c in keratinocyte serum - free medium ( ksfm ) ( thermo fisher scientific ) supplemented with 3% fetal bovine serum , 1.25 g / ml amphotericin b ( product 15290 - 018 ; thermo fisher scientific ) , 0.5 l / ml gentamicin ( product 15750 - 060 ; thermo fisher scientific ) , and 5 g / ml dispase ii ( product 04942078001 ; roche ) . supplemented with 5 ng / ml epidermal growth factor ( egf ) , 5 g / ml insulin , 5 g / ml transferrin , 5 ng / ml sodium selenite , 0.5 g / ml hydrocortisone , 30 ng / ml cholera toxin a , 0.5% dimethyl sulfoxide , 50 g / ml gentamicin , 1.25 g / ml amphotericin b , 5% fetal bovine serum , and 5 g / ml dispase ii ( roche ) . for c57bl/6j mouse cell culture experiments , the number of samples per group were 4 samples per dc group , 4 samples per untreated conjunctiva cell group , and 5 samples per lps - treated conjunctiva cell group . in the first set of experiments , lps eye drops were administered to nonstressed mice to determine whether tlr4 activation on the ocular surface could lead to the release of proinflammatory mediators . in general , lps administration led to a dose - dependent increase in inflammatory mediator expression on the ocular surface . in most cases , only small differences in expression were observed between the 1 g/l and 10 g/l concentrations , indicating that maximal tlr4 activation was reached at the 1 g/l concentration ; however , higher concentrations were not tested . in the cornea , application of 1 g/l lps significantly increased expression of il-1 ( 2.17 0.31-fold ) and cxcl10 ( 36.93 7.75-fold ) ( fig . ifn- ( 13.45 4.49-fold ) , and il-12a ( 3.99 0.27-fold ) were also significantly increased in the conjunctiva after application of 1 g/l lps . the choice of vehicle may affect gene expression ; however , the increase in il-1 remained significant when saline was used as the diluent . figures 1a and 2a show that 1 g/l lps dissolved in saline led to a significant increase in il-1 in both the cornea and the conjunctiva . a slight and nonsignificant increase in il-1 was observed in the conjunctiva of lps - treated tlr4ko mice , which may suggest noncanonical inflammasome activation by lps . because no significant increases in gene expression were observed , these findings indicate the stimulatory effects of lps are mediated by tlr4-dependent pathways on the ocular surface . in order to verify that increased gene expression of inflammatory mediators also resulted in an increase in protein expression , concentrations of cxcl10 were measured by an immunobead assay in conjunctival tissue extracts obtained 24 hours after application of lps . a significant increase ( p 0.05 ) in cxcl10 protein was found in the conjunctiva of ns mice treated with 1 g/l lps compared to ns mice treated with vehicle ( endotoxin - free water ) ( fig . immunohistochemical staining demonstrated that il-1 increased in the cornea of ns mice treated with lps compared to ns mice treated with water ( fig . ( a ) immunohistochemical staining shows increased il-1 expression ( red ) in the central cornea ( stroma ) after administration of lps ( 1 g/l ) to nonstressed mice . ( b ) higher magnification ( 100 ) was used to examine enhanced expression of il-1 in the corneal epithelium of ns+lps and ds5+lps mice . ( c ) immunofluorescent staining demonstrates that application of lps ( 1 g/l ) increases expression of il-12a in the conjunctiva of nonstressed and ds5 mice . lps was also tested in vitro to determine which cell types may be responsible for the expression of inflammatory mediators observed in the in vivo studies . increased expression levels of il-1 and tnf- are demonstrated after addition of 1 g of lps to cornea ( a ) , conjunctiva ( b ) , and dendritic ( c ) cell cultures . after tlr4 activation was demonstrated on the ocular surface in nonstressed mice , the next set of experiments was designed to determine whether tlr4 activation is enhanced during dry eye disease . compared to control ns eyes , lps increased expression of il-1 ( 3-fold ) in the cornea and il-12a ( 2-fold ) in the conjunctiva of dry eyes . in a separate experiment using the same methods , figures 4a and 4b further support the hypothesis that lps administration leads to enhanced expression of inflammatory mediators during dry eye disease . enhanced protein expression of il-1 was observed in ds5 mice treated with lps compared to ns mice treated with lps ( figs . figure 4a shows enhanced expression of il-1 in the corneal stroma of ds5+lps mice , and figure 4b demonstrates the same increase in the corneal epithelium . in addition , an increase in il-12a positive cells was noted in ds5 mice treated with lps compared to ds controls ( fig . in the first set of experiments , lps eye drops were administered to nonstressed mice to determine whether tlr4 activation on the ocular surface could lead to the release of proinflammatory mediators . in general , lps administration led to a dose - dependent increase in inflammatory mediator expression on the ocular surface . in most cases , only small differences in expression were observed between the 1 g/l and 10 g/l concentrations , indicating that maximal tlr4 activation was reached at the 1 g/l concentration ; however , higher concentrations were not tested . in the cornea , application of 1 g/l lps significantly increased expression of il-1 ( 2.17 0.31-fold ) and cxcl10 ( 36.93 7.75-fold ) ( fig . ifn- ( 13.45 4.49-fold ) , and il-12a ( 3.99 0.27-fold ) were also significantly increased in the conjunctiva after application of 1 g/l lps . the choice of vehicle may affect gene expression ; however , the increase in il-1 remained significant when saline was used as the diluent . figures 1a and 2a show that 1 g/l lps dissolved in saline led to a significant increase in il-1 in both the cornea and the conjunctiva . a slight and nonsignificant increase in il-1 was observed in the conjunctiva of lps - treated tlr4ko mice , which may suggest noncanonical inflammasome activation by lps . because no significant increases in gene expression were observed , these findings indicate the stimulatory effects of lps are mediated by tlr4-dependent pathways on the ocular surface . in order to verify that increased gene expression of inflammatory mediators also resulted in an increase in protein expression , concentrations of cxcl10 were measured by an immunobead assay in conjunctival tissue extracts obtained 24 hours after application of lps . a significant increase ( p 0.05 ) in cxcl10 protein was found in the conjunctiva of ns mice treated with 1 g/l lps compared to ns mice treated with vehicle ( endotoxin - free water ) ( fig . immunohistochemical staining demonstrated that il-1 increased in the cornea of ns mice treated with lps compared to ns mice treated with water ( fig . ( a ) immunohistochemical staining shows increased il-1 expression ( red ) in the central cornea ( stroma ) after administration of lps ( 1 g/l ) to nonstressed mice . ( b ) higher magnification ( 100 ) was used to examine enhanced expression of il-1 in the corneal epithelium of ns+lps and ds5+lps mice . ( c ) immunofluorescent staining demonstrates that application of lps ( 1 g/l ) increases expression of il-12a in the conjunctiva of nonstressed and ds5 mice . lps was also tested in vitro to determine which cell types may be responsible for the expression of inflammatory mediators observed in the in vivo studies . increased expression levels of il-1 and tnf- are demonstrated after addition of 1 g of lps to cornea ( a ) , conjunctiva ( b ) , and dendritic ( c ) cell cultures . after tlr4 activation was demonstrated on the ocular surface in nonstressed mice , the next set of experiments was designed to determine whether tlr4 activation is enhanced during dry eye disease . compared to control ns eyes , lps increased expression of il-1 ( 3-fold ) in the cornea and il-12a ( 2-fold ) in the conjunctiva of dry eyes . enhanced protein expression of il-1 was observed in ds5 mice treated with lps compared to ns mice treated with lps ( figs . figure 4a shows enhanced expression of il-1 in the corneal stroma of ds5+lps mice , and figure 4b demonstrates the same increase in the corneal epithelium . in addition , an increase in il-12a positive cells was noted in ds5 mice treated with lps compared to ds controls ( fig . with an aging population and limited treatment options available for dry eye , it is important to define all the factors that can contribute to inflammation on the ocular surface . despite this possibility , the ability of tlr4 to produce a proinflammatory response in the eye during normal and diseased states has not been fully explored . this study indicates that tlr4 is present on the eye and that tlr4 activation can lead to increased production of proinflammatory mediators . in fact , lps application to the ocular surface can lead to the release of several inflammatory mediators linked to dry eye . the greatest variety of inflammatory mediators and most dramatic rises in gene expression were noted in the conjunctiva ( fig . both il-6 and tnf- have been found to be increased in mouse models of dry eye and in humans with the disease . ifn- is the defining cytokine of the th1 response , which contributes to ocular surface disease and tear dysfunction . increased production of ifn- after treatment with lps is also significant , in part , because of the ability of ifn- to amplify the immune response . ifn- increases production of th1 chemokines ( cxcl9 , cxcl10 , and cxcl11 ) , which , in turn , attract greater numbers of ifn-producing cells . interestingly , il-12a , the main cytokine responsible for th1 differentiation , was also significantly upregulated in the conjunctiva after lps stimulation ( fig . upregulation of il-12a , in conjunction with ifn- , suggests lps stimulation can promote the th1 response that is critical to dry eye pathology . it is particularly interesting that application of lps to non - stressed mice leads to production of inflammatory mediators in the cornea ( fig . in the cornea ( and conjunctiva ) , lps administration significantly increased expression of cxcl10 and il-1 in ns mice ( fig . in the end , lps was found to enhance expression of cytokines and chemokines on a nonstressed ocular surface , which demonstrates that tlr4 activation could contribute to homeostatic immune surveillance . the cell culture studies demonstrate that mouse cornea and conjunctiva epithelial cells , as well as dendritic cells , can respond to lps on the ocular surface . the production of inflammatory mediators observed in the in vivo studies may be from a combination of direct and indirect stimulation . figure 5 demonstrates that il-1 and tnf- , both of which may stimulate additional mediator release from epithelial cells , were upregulated in lps - stimulated dc culture . indeed , il-1 has been shown to increase production of il-1 and il-6 in limbal epithelial stem cells , and tnf- has been shown to increase cxcl10 production in human corneal epithelial cells . this finding supports our hypothesis that disruption of the corneal epithelial barrier during de leads to greater exposure of tlr4 located in the wing and basal layers of the corneal epithelium . however , other mechanisms are still a possibility , including increased cell surface expression of tlr4 in the corneal epithelium during de . tlr4 stimulation could potentiate the induction of the de immune response by changing the cytokine milieu and activating th1-polarizing dendritic cells . in conclusion , this study demonstrates that not only is tlr4 capable of producing a proinflammatory response on the ocular surface but that response is enhanced during de disease . in the cornea , disruption of in the conjunctiva , enhanced expression of il-12a after tlr4 activation may promote th1 differentiation , leading to more severe disease . despite this , the proinflammatory reactions produced by tlr4 on the ocular surface illustrate that tlr4 is a potential pharmaceutical target for dry eye inflammation .

diabetes mellitus is a growing public health concern , presently affecting 25.8 million or 8.3% of the american population and nearly 387 million people worldwide . while the availability of novel drugs , techniques , and surgical intervention has improved the survival rate of individuals with diabetes , the prevalence of diabetes is still rising in the united states , with the number of people with diabetes projected to double by 2025 . t2d is a result of chronic insulin resistance and loss of -cell mass and function . both in experimental animals and people , obesity is a leading pathogenic factor for developing insulin resistance , which is always associated with the impairment in energy metabolism , causing increased intracellular fat content in skeletal muscle , liver , fat , and pancreatic islets . constant insulin resistance will progress to t2d when -cells are unable to secrete adequate amount of insulin to compensate for decreased insulin sensitivity , which is largely due to insulin secretory dysfunction and significant loss of functional -cells [ 48 ] . indeed , those individuals with t2d always manifest increased -cell apoptosis and reduced -cell mass [ 6 , 7 , 9 ] . as such , the search for novel agents that simultaneously promote insulin sensitivity and -cell survival may provide a more effective strategy to prevent the onset of diabetes . recently , naturally occurring polyphenolic compounds have been the focal point of medicinal research interest due to their pharmacological implications associated with human disease with considerable attention devoted to managing diabetes [ 11 , 12 ] . polyphenols exist naturally as secondary plant metabolites and are the largest source of human dietary antioxidants , with a typical daily intake of roughly 1 g / day . kaempferol ( 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4h-1-benzopyran-4-one ) is a flavonol , of low molecular weight ( 286.2 g / mol ) , that has been identified in many plants used in traditional medicine , including equisetum spp . , sophora japonica , and ginkgo biloba , and edible plants , including beans , broccoli , cabbage , gooseberries , grapes , kale , strawberries , tea , and tomatoes [ 15 , 16 ] . dietary intake of kaempferol containing foods has been epidemiologically associated with a reduced risk of certain cancers and cardiovascular diseases . it has been reported that kaempferol has antioxidative , antimicrobial , anti - inflammatory , lipolytic , and anticancer [ 20 , 21 ] effects . however , to date , the studies regarding its effect on the pathogenesis of diabetes are very limited . we recently demonstrated that kaempferol treatment prevented apoptosis and improved insulin biosynthesis and secretion in -cells and human islets exposed to chronic hyperlipidemia . past studies have established that sustained hyperlipidemia in obese individuals plays an important role in causing -cell apoptosis and dysfunction , thereby contributing to the deterioration of glycemic control and the overt development of t2d [ 2224 ] . this study was conducted to investigate whether long - term dietary intake of kaempferol can promote metabolic homeostasis and thereby prevent diabetic pathogenesis . we show here that dietary intake of kaempferol for 5 months ( mo ) improved insulin sensitivity and glucose tolerances , which were associated with increased glut4 and ampk expression in muscle and adipose tissues in middle - aged mice fed a high - fat ( hf ) diet . in vitro , kaempferol increased lipolysis and restored chronic high fatty acid - impaired glucose uptake and glycogen synthesis in skeletal muscle cells , which were associated with improved ampk activity and glut4 expression . in addition , dietary kaempferol treatment preserved functional pancreatic -cell mass and prevented hyperglycemia and glucose intolerance in stz - induced diabetic mice . male ( 10-month - old ) c57bl/6j mice ( nci , nih ) were individually housed in an animal room maintained on a 12-hour light / dark cycle under constant temperature ( 2225c ) with ad libitum access to food and water . after 1 week of environment acclimation , we conducted the following two animal studies . the institutional animal care and use committee at virginia tech approved the animal study protocols . mice were divided into 4 groups ( n = 12 mice / group ) with blood glucose and body weight balanced and then fed a standard chow ( sd ) diet , with 10% of calories derived from fat , a hf diet ( research diets inc . , new brunswick , nj ) with 58% of calories from fat , or hf diet supplemented with kaempferol ( 0.01% or 0.05% ) for 5 mo . body weight and food intake were recorded weekly throughout the study . to assess fasting blood glucose , mice were fasted for 12 h , and blood glucose was measured in tail vein blood samples using a glucometer ( kroger , cincinnati , oh ) . after 5 mo of dietary treatment , body composition was evaluated using an lf-90 instrument ( bruker optics , inc . , the lf-90 body composition instrument is based on time domain nuclear magnetic resonance ( td - nmr ) technology , which provides an in vivo measurement of lean tissue , body fat , and body fluid in live mice without anesthesia . following this procedure , glucose and insulin tolerance tests were performed . for the glucose tolerance tests ( gtt ) , mice were fasted for 12 h and injected intraperitoneally ( ip ) with a single bolus of glucose ( 2 g / kg bw ) . glucose levels were measured at time points of 0 , 15 , 30 , 60 , and 120 min after glucose administration . for the insulin tolerance tests ( itt ) , mice were injected ip with insulin ( 0.75 units / kg bw ) , and blood glucose levels were measured at 0 , 15 , 30 , 60 , and 120 min after insulin administration . area under the curve ( auc ) was calculated using the trapezoidal rule . at the end of the study , the mice were fasted overnight and euthanized , immediately followed by the collection of blood samples . fasting plasma total cholesterol , hdl - cholesterol , and triacylglycerol were measured by enzymatic methods using a pointer 180 analyzer ( pointe scientific , canton , mi ) as described previously . plasma insulin levels were measured using a mouse insulin elisa kit ( mercodia , inc . , blood hba1c levels were determined using an assay kit ( henry schein , inc . , mice were sacrificed and extensor digitorum longus muscle and abdominal adipose tissues were collected , snap - frozen in liquid nitrogen , and then stored at 80c for the western blot analyses . in a separate experiment , mice were divided into 3 groups ( n = 8 mice / group ) and fed a sd diet or sd diet containing kaempferol ( 0.01% or 0.05% ) for 3 mo . fasting and nonfasting blood glucose levels were measured biweekly . at the end of 3 mo , gtt and itt 10-month - old male mice ( nci , nih ) were divided into 3 groups ( n = 10 mice / group ) with initial fasting blood glucose and body weights balanced among groups . mice were then fed a sd diet , a hf diet ( 58 kcal% fat ) , or hf diet containing 0.05% kaempferol . after 6 weeks of dietary kaempferol supplementation , gtt , itt , and body composition were evaluated as described above . after this procedure , mice received ip injections of stz dissolved in 0.1 m cold sterile sodium citrate buffer ( ph 4.5 ) at 45 mg / kg daily for 3 consecutive days . body weight , food intake , and nonfasting and fasting blood glucose were measured biweekly throughout the study . , mice were euthanized , and the pancreata were dissected , weighed , and then fixed in 4% ( vol / vol ) formaldehyde buffer ( ph 7.2 ) . a series of tissue sections ( 5 m thickness at 200 m interval ) were prepared , mounted on glass slides , and immunofluorescently stained with an insulin antibody and fitc - conjugated secondary antibody ( abcam , cambridge , ma ) for determining -cell mass . pancreatic -cell mass was calculated by dividing the area of insulin - positive cells by the total area of pancreatic tissue and multiplied by the pancreas weight [ 25 , 26 ] . insulin was then extracted from pancreas homogenates with acid - ethanol [ 75% ethanol , 25% acetic acid ( 25% vol / vol ) ] overnight at 4c . the homogenates were centrifuged ( 10 min , 2,000 g , 4c ) and the supernatants were neutralized with tris buffer . pancreatic insulin content was measured by elisa and then normalized to the protein concentration in the same sample . the c2c12 mouse cells ( american type culture collection , manassas , va ) were grown at 37c and 5% co2 in dulbecco 's modified eagle 's medium ( dmem ) , supplemented with penicillin ( 50 iu / ml ) , streptomycin ( 50 g / ml ) , and 10% fetal bovine serum ( fbs ) . the cells were grown to 75% confluence and the growth medium was then switched to dmem supplemented with 2% horse serum and penicillin ( 50 iu / ml ) and streptomycin ( 50 g / ml ) for differentiation . c2c12 myoblasts were grown to ~80% confluence in dmem and then differentiated for 5 days in dmem supplemented with 2% horse serum . on day 5 , the myotubes were treated for 24 h with 10 m kaempferol or vehicle ( dmso ) along with a mixture of fatty acids or vehicle ( bsa ) . the fatty acid mixture contained a 2 : 1 ratio of palmitate to oleate for a final concentration of 0.4 mm complexed with 0.4% bsa in serum - free , low glucose dmem . following the fatty acid and kaempferol treatment , glucose uptake was assessed in krebs - ringer hepes buffer ( in mm : 136 nacl , 4.7 kcl , 1.25 mgso4 , 1.2 cacl , and 20 hepes , ph 7.4 ) with the addition of 10 m 2-deoxyglucose and 1.25 uci / ml 2-deoxy-[3h]glucose . after 15 min of incubation , plates were placed on ice , washed three times with ice - cold pbs , and harvested in 400 l of 0.2 m naoh for cell lysis . c2c12 myotubes were treated for 24 h with 10 m kaempferol or vehicle ( dmso ) along with 0.4 mm fatty acid ( fa ) cocktail or bsa as stated above . after 24 h with fa and kaempferol treatment , cells were further incubated with 1.25 uci / mlc glucose ( american radiolabeled chemicals , saint louis , mo ) in the presence or absence of insulin ( 100 nm ; eli lilly , indianapolis , in ) for 3 h. following this treatment , cells were rinsed twice with pbs at 4c followed by solubilization by adding 250 l of 30% koh . the samples were then mixed with 35 l of 60 mg / ml glycogen ( sigma - aldrich , st . louis , mo ) in distilled water and heated at 80c for 20 min . following centrifugation at 4c ( 10,000 rpm ) for 20 min , the pellet was collected and washed with 70% ethanol and then resuspended in 500 l distilled water . after 20 min of shaking , the glycogen precipitate was counted for the presence of c by liquid scintillation ( ls 6500 , beckman coulter , brea , ca ) . glycogen synthesis was calculated based on specific activity and was expressed relative to protein content . c2c12 muscle cells were preincubated with kaempferol or vehicle ( dmso ) for 30 min followed by addition of 20 mm glucose and 0.5 mm palmitate for 24 h. glycerol released into the medium was then measured by using a free glycerol determination kit ( sigma - aldrich , st . animal tissues or cultured cells were homogenized in lysis buffer ( 50 mm hepes , 0.1% ( v / v ) triton x-100 , 1 mm pmsf , 10 mm e-64 , 10 mm pepstatin a , 10 mm tlck , 100 mm leupeptin , ph 7.4 ) . supernatants of cultured muscle cell or mouse tissue lysates were collected , and protein content was measured using an assay kit . equal amounts of protein extracts from mouse tissues or cells were subjected to western blot analysis as described previously . nitrocellulose membranes were probed with antibody against glut4 , ampk , or phospho - ampk ( cell signaling , danvers , ma ) . the immunoreactive proteins were detected by chemiluminescence ( thermo fischer , rockford , il ) . nitrocellulose membranes were then stripped and reprobed with -actin ( animal tissues ) or ampk ( culture cells ) . the protein bands were digitally imaged for densitometric quantitation with a software program ( image j , nih ) . all proteins levels were normalized to those of -actin or total ampk , where applicable , from the same samples . data were analyzed with one - way anova , using sigmaplot software program , and are expressed as mean standard error ( se ) or mean standard error of mean ( sem ) , where applicable . in this study , we tested the metabolic effects of the long - term dietary supplementation of kaempferol ( 0.01% or 0.05% in the diet ) in middle - aged mice fed a hf diet . the hf diet decreased the accumulative average food intake , but kaempferol supplementation for 5 mo did not alter the food consumption compared with hf diet - fed mice ( figure 1(a ) ) . however , dietary intake of 0.05% kaempferol significantly ameliorated hf diet - induced body weight gain ( figure 1(b ) ) . consistently , mice fed the hf diet developed obesity as determined by measuring their relative percentage of fat ( figure 1(c ) ) and muscle mass ( figure 1(d ) ) . however , kaempferol treatment had no significant effect on adiposity of obese mice . fasting blood levels of cholesterol ( figure 1(e ) ) , ldl - cholesterol ( figure 1(f ) ) , and triglyceride ( figure 1(g ) ) were increased in hf diet - fed obese mice , which were significantly reduced by dietary intake of kaempferol . however , total cholesterol levels in kaempferol - fed mice were still significantly higher as compared to chow diet - fed mice . fasting plasma hdl - cholesterol concentrations were increased by hf feeding but were not further altered by kaempferol treatment ( figure 1(h ) ) . in another study to determine whether kaempferol also improves metabolism and health of sd diet - fed older adult mice , we found that dietary intake of either 0.01% kaempferol ( k1 ) or 0.05% kaempferol ( k2 ) for 3 mo had no effects on body weight gain ( 5.9 0.5 , 6.1 0.4 , and 6.0 0.6 g for control , k1 , and k2 group , resp . ) , food intake ( 4.25 , 4.27 , and 4.23 g / d / mouse ) , fasting ( 136.4 4.1 , 131.6 3.4 , and 127.9 5.8 mg / dl ) and nonfasting ( 185.4 11.1 , 180.5 5.5 , and 174.4 12.2 ) blood glucose levels , glucose tolerance ( 32672 1653 , 31411 1582 , and 31078 1634 auc ) , and insulin sensitivity ( 11073 1065 , 10897 9887 , and 10574 1141 auc ) . after 2 mo of hf diet consumption , mice displayed hyperglycemia throughout this study , as shown by persistently over 50% higher circulating glucose levels as compared with mice that consumed the sd diet . kaempferol supplementation at this point nonsignificantly reduced the hf diet - induced rise in blood glucose . after 3 mo , however , mice fed hf diet supplemented with kaempferol exhibited significantly lower blood glucose levels than those in hf - fed mice ; after 5 mo of treatment , mice fed kaempferol - supplemented diet still had significantly lower blood glucose levels ( 140 10.5 mg / dl ) as compared to hf diet - fed mice ( 197 10.5 mg / dl ) , which were almost reduced to the levels shown in sd - fed mice ( 123.0 4.8 mg / dl ) ( figure 2(a ) ) . data showed that kaempferol ( 0.05% in the diet ) nonsignificantly improved glucose tolerance as demonstrated by lower blood glucose levels at 30 and 60 min ( figure 2(b ) ) as well as reduced auc ( figure 2(c ) ) following ip glucose injection compared to hf - fed mice . consistently , blood levels of hba1c , which reflect an average of blood glucose over a period of two to three mo , were significantly lower in kaempferol - treated mice as compared to those in hf diet - fed mice ( figure 2(d ) ) . insulin resistance is important to the etiology of t2d and usually occurs in obesity . to determine if dietary intake of kaempferol improves insulin sensitivity in obese mice , we performed an ip itt . however , dietary provision of kaempferol as low as 0.01% significantly improved plasma glucose levels ( figure 2(e ) ) and the auc ( figure 2(f ) ) postinsulin injection in hf diet - fed mice , suggesting that kaempferol increases insulin sensitivity . we observed that fasting plasma insulin levels in hf mice were about 6-fold of those in mice that received the sd diet , suggesting that obese mice are insulin resistant ( figure 2(g ) ) . however , plasma insulin levels in 0.05% kaempferol - treated mice were more than 50% lower as compared with hf - fed obese mice ( figure 2(g ) ) . these data strongly suggest that long - term kaempferol supplementation maintained whole body insulin sensitivity in hf diet - induced obese mice . insulin resistance in adipose tissue and skeletal muscle , the primary site of glucose and fatty acid utilization , plays a major role in the development of hf diet - induced t2d . to further determine the molecular events that are associated with improved insulin sensitivity by kaempferol treatment , we measured the expression of ampk and glut4 in skeletal muscle and adipose tissues , which are two critical molecules regulating glucose uptake [ 30 , 31 ] . consistent with impaired peripheral insulin sensitivity , the expression of ampk and glut4 proteins in both skeletal muscle ( figures 3(a)3(c ) ) and adipose tissue ( figures 3(d)3(f ) ) from hf diet - fed mice was significantly attenuated as compared to the control mice . however , treatment with kaempferol completely reversed these detrimental effects caused by feeding the mice with hf diet ( figures 3(a)3(f ) ) . these results further confirmed that long - term intake of kaempferol protects against developing insulin resistance in hf diet - induced obese mice . as skeletal muscle is the major site for fuel metabolism , and obesity and insulin resistance are always accompanied with the impairment in energy metabolism , a leading pathogenic factor for t2d , we performed a series of in vitro assays to determine whether kaempferol has direct beneficial effects on glucose metabolism . in that regard , we cultured skeletal muscle cells with or without kaempferol in the presence of palmitate and oleic acid . we found that exposure of c2c12 myotubes to high fatty acids for 24 h impaired insulin - induced glucose uptake ( figure 4(a ) ) and glycogen synthesis ( figure 4(b ) ) . however , these detrimental effects were partially reversed by treatment with 10 m kaempferol ( figures 4(a ) and 4(b ) ) . to further characterize metabolic and insulin sensitizing effects of kaempferol , we cultured skeletal muscle cells with or without kaempferol in the presence of high glucose ( 20 mm ) and saturated fatty acid palmitate ( 0.5 mm ) , an in vitro environment that is frequently used to simulate diabetic condition in vivo . the results showed that kaempferol at physiologically relevant concentrations significantly improved lipolysis ( figure 5(a ) ) and reversed high fatty acid - impaired ampk activity ( figure 5(b ) ) and glut4 protein expression ( figure 5(c ) ) , a downstream target of activated ampk . while peripheral insulin resistance is common during obesity in rodents and people , its progression to t2d is largely due to insulin secretory dysfunction and significant apoptosis of functional -cells [ 48 ] , leading to an inability to compensate for insulin resistance . past studies have established that sustained hyperlipidemia in obese individuals plays an important role in causing -cell apoptosis and dysfunction , thereby contributing to the deterioration of glycemic control and the overt development of t2d [ 2224 ] . we recently found that kaempferol treatment prevented apoptosis of cultured pancreatic -cells exposed to chronic hyperlipidemic condition . we therefore further assessed whether kaempferol can also protect pancreatic -cell function , thereby preventing diabetes by using a t2d mouse model that was generated through a combination of hf diet feeding and three consecutive injections of low doses of stz . regarding this , c57bl/6 mice ( male , 10-month - old ) were fed a sd diet , a hf diet , or hf diet containing 0.05% kaempferol . consistent with the observations from the animal study described above , treatment with kaempferol for 6 weeks had no effect on body weight gain , food intake , fasting blood glucose levels , glucose intolerance , or insulin resistance of mice fed the hf diet ( data not shown ) . after 6 weeks of dietary treatment , stz ( 40 mg / kg bw ) was administrated ( ip ) for 3 consecutive days to induce diabetes mediated by a destruction of islet -cells in mice . our data showed that dietary ingestion of kaempferol significantly mitigated stz - induced hyperglycemia in diabetic mice as determined by measuring fasting ( figure 6(a ) ) and nonfasting ( figure 6(b ) ) blood glucose levels . consistently , kaempferol ameliorated the loss of body weight secondary to the development of diabetes ( data not shown ) . to determine if the improved glycemic control in mice fed the kaempferol - supplemented diet is the result of preserved islet function , we measured insulin levels in the plasma of the control and kaempferol - fed mice . as shown in figure 6(c ) , plasma insulin levels in mice fed diet containing kaempferol were significantly greater as compared to those in nontreated diabetic mice , suggesting that kaempferol may ameliorate hyperglycemia primarily via preserving islet -cell function . since stz causes diabetes by destroying islet -cells , we then examined whether kaempferol treatment preserved -cell mass in diabetic mice by using an immunohistochemical technique . we observed that hf diet - fed mice exhibited greater pancreas weight as compared with the control mice ( figure 7(a ) ) , which was not modulated by kaempferol supplementation ( figure 7(a ) ) . however , stz administration caused severe destruction of pancreatic -cells as determined by evaluating the area of insulin positive cells ( figure 7(b ) ) and islet mass ( figure 7(c ) ) , which consequently led to the reduction of pancreatic insulin content by nearly 80% ( figure 7(d ) ) . however , dietary provision of kaempferol partially preserved islet -cell mass ( figures 7(b ) and 7(c ) ) and insulin content ( figure 7(d ) ) in diabetic mice . it has been reported that kaempferol elicits a number of health benefits , including antioxidative [ 17 , 37 ] , anti - inflammatory [ 18 , 38 ] , antihypertensive [ 39 , 40 ] , lipolytic [ 19 , 41 ] , and anticarcinogenic effects [ 20 , 21 , 42 ] . however , studies on whether this compound possesses antidiabetic properties are very limited . in this study , we tested the antidiabetic potential of this natural compound by using two mouse models and in vitro studies . it is worthy to note that , unlike many metabolic studies that used young adult mice , we used near middle - aged mice , which may be more clinically relevant , as t2d in humans often occurs during middle and late ages . we provide evidence that long - term dietary intake of kaempferol promoted metabolic homeostasis with the improved fasting blood glucose , hba1c levels , insulin sensitivity , and glucose tolerance in hf diet - induced obese mice , which was associated with increased fuel metabolism , ampk activity , and glut4 expression in skeletal muscle cells . further , dietary kaempferol also ameliorates hyperglycemia in stz - induced diabetic mice by preserving functional -cell mass . importantly , these beneficial metabolic effects , elicited by dietary intake of kaempferol , are not due to alteration in food intake . given that both insulin resistance in peripheral tissues and progressive -cell loss and dysfunction are key components in the pathogenesis of t2d [ 48 ] , kaempferol could be a low - cost and safe natural compound to promote energy metabolism and maintain glucose homeostasis by targeting these two defects . it is well established that obesity is an important risk factor for t2d [ 43 , 44 ] . therefore , hf diets are commonly used as a strategy to induce obesity in animal models , leading to the development of metabolic disorders including hyperlipidemia , impaired insulin sensitivity and glucose tolerance , and elevated blood insulin and glucose [ 4548 ] . consistently , our data showed that consumption of the hf diet induced obesity and elevated plasma concentrations of triglycerides and cholesterol that are typical of obesity , which however were ameliorated in mice fed a hf diet containing kaempferol . given that obesity is a leading pathogenic factor for developing insulin resistance and subsequent glucose intolerance , the improved insulin sensitivity and glucose homeostasis by kaempferol supplementation could be partially due to the secondary effects whereby long - term intake of kaempferol ameliorated obesity in mice fed a hf diet . while it is presently unclear how kaempferol affects lipid metabolism and body weight gain , previous studies demonstrated that several structurally similar flavonoids inhibit lipid absorption and lipogenesis [ 5052 ] . because food intake was not affected by long - term kaempferol supplementation , the decrease in fasting plasma cholesterol and triglycerides in the hf - fed mice could be caused by the regulation of the intestinal digestion and absorption of lipids and/or lipogenesis , which needs further investigation . as kaempferol treatment did not alter calorie intake in mice during the course of this study , it is also possible that the reduced body fat mass as well as body weight in kaempferol - fed mice is due to its effect on energy expenditure , given that obesity results from energy imbalance . several lines of evidence have shown that pharmacological activation of ampk improves blood glucose homeostasis and lipid profile in insulin - resistant rodents . ampk is an energy sensing molecule highly conserved from yeast to all animals , which is increasingly recognized as a master regulator of whole body energy homeostasis . ampk is a heterotrimeric protein kinase composed of a catalytic subunit ( ampk ) and two regulatory subunits ( and ) that sense low cellular energy levels by monitoring changes in the amp : atp ratio . amp binding to the subunit induces a conformational change that allows ampk to be phosphorylated at its threonine residue ( thr 172 ) by the ampk - activating protein kinase ( lkb1 ) . at the whole body level , ampk integrates stress responses , nutrient and hormonal signals to the control of food intake , energy expenditure , and substrate utilization . at the cellular level , activated ampk inhibits hepatic gluconeogenesis , promotes fatty acid oxidation , and regulates mitochondrial biogenesis . in addition , activation of ampk increases glut4 expression and membrane translocation in skeletal muscle , thereby improving glucose uptake . consistent with the impaired insulin sensitivity in hf - fed obese mice , we found that the protein levels of ampk and glut4 in skeletal muscle and adipose tissue from these animals were also considerably attenuated . however , these detrimental effects were completely reversed by supplementation of 0.05% kaempferol in the hf diet . these results suggest that kaempferol may increase peripheral insulin sensitivity via the ampk - mediated pathway . both in experimental animals and people , obesity - related insulin resistance is always associated with the dysfunctions of several metabolic pathways including reduced lipolysis , fatty acid oxidation , glucose uptake , and glycogen synthesis coupled with increased glucose output . specifically , it is believed that elevated intramyocellular lipid accumulation coupled with diminished lipolysis and mitochondrial lipid oxidation play a role in the development of insulin resistance in skeletal muscle . consistently , it was found that inducing lipolysis and fatty acid oxidation in muscle cells protects against lipotoxicity - induced insulin resistance [ 58 , 59 ] . in the present study , we further showed that kaempferol treatment as low as 1 m augmented lipolysis and reversed chronic hyperlipidemia - impaired glucose uptake , glut4 expression , ampk activity , and glycogenesis in skeletal muscle cells , which provides further evidence at cellular levels that kaempferol might be an insulin sensitizing molecule by promoting energy metabolism . however , it is presently unclear how kaempferol exerts these beneficial effects in skeletal muscle cells . kaempferol has been shown to possess antioxidant property . while we found that pharmacological doses of kaempferol ( 1050 m ) showed significant free radical scavenging activity as evaluated by using an oxygen radical absorbance assay , it had no such effect at 1 m . therefore , kaempferol promotion of skeletal muscle cell function may not be due to its potential antioxidant effect . we further explored whether kaempferol directly protects pancreatic -cell function in vivo by using a nongenetic mouse model of t2d , which was generated by employing a combination of feeding a hf diet and administering three mild doses ( 40 mg / kg ) of stz that does not cause diabetes in chow - fed mice , as demonstrated in our recent study . this nongenetic diabetic mouse model manifests the metabolic characteristics of human t2d , including moderate levels of hyperglycemia , hyperlipidemia , insulin resistance , impaired insulin secretion , and reduced -cell mass . the results in the present study show that kaempferol partially preserved -cell mass in stz - induced diabetic mice , which could be primarily attributable to its antidiabetic action , given that diabetes was induced before kaempferol caused changes in body weight , adiposity , or other metabolic parameters . we speculate that kaempferol treatment may protect against -cell apoptosis , thereby improving islet mass , given our recent observations that kaempferol promotes viability of isolated pancreatic islets exposed to chronic hyperglycemia or hyperlipidemia . in summary , we provide evidence that long - term dietary supplementation of kaempferol prevents hf diet - induced metabolic disorders in middle - aged obese mice . on cellular and molecular levels , kaempferol improves glycolysis , glucose uptake , glycogen synthesis , ampk activity , and glut4 expression in skeletal muscle . in addition , dietary supplementation of kaempferol significantly ameliorated hyperglycemia and preserved functional islet mass in old adult obese diabetic mice . these results indicate that phytonutrient kaempferol may be used as a dietary supplement to prevent metabolic disorders that are associated with obesity and aging .

insulin resistance and a progressive decline in functional -cell mass are hallmarks of developing type 2 diabetes ( t2d ) . thus , searching for natural , low - cost compounds to target these two defects could be a promising strategy to prevent the pathogenesis of t2d . here , we show that dietary intake of flavonol kaempferol ( 0.05% in the diet ) significantly ameliorated hyperglycemia , hyperinsulinemia , and circulating lipid profile , which were associated with the improved peripheral insulin sensitivity in middle - aged obese mice fed a high - fat ( hf ) diet . kaempferol treatment reversed hf diet impaired glucose transport-4 ( glut4 ) and amp - dependent protein kinase ( ampk ) expression in both muscle and adipose tissues from obese mice . in vitro , kaempferol increased lipolysis and prevented high fatty acid - impaired glucose uptake , glycogen synthesis , ampk activity , and glut4 expression in skeletal muscle cells . using another mouse model of t2d generated by hf diet feeding and low doses of streptozotocin injection , we found that kaempferol treatment significantly improved hyperglycemia , glucose tolerance , and blood insulin levels in obese diabetic mice , which are associated with the improved islet -cell mass . these results demonstrate that kaempferol may be a naturally occurring anti - diabetic agent by improving peripheral insulin sensitivity and protecting against pancreatic -cell dysfunction .

1. Introduction 2. Methods 3. Results 4. Discussion

while the availability of novel drugs , techniques , and surgical intervention has improved the survival rate of individuals with diabetes , the prevalence of diabetes is still rising in the united states , with the number of people with diabetes projected to double by 2025 . t2d is a result of chronic insulin resistance and loss of -cell mass and function . both in experimental animals and people , obesity is a leading pathogenic factor for developing insulin resistance , which is always associated with the impairment in energy metabolism , causing increased intracellular fat content in skeletal muscle , liver , fat , and pancreatic islets . constant insulin resistance will progress to t2d when -cells are unable to secrete adequate amount of insulin to compensate for decreased insulin sensitivity , which is largely due to insulin secretory dysfunction and significant loss of functional -cells [ 48 ] . as such , the search for novel agents that simultaneously promote insulin sensitivity and -cell survival may provide a more effective strategy to prevent the onset of diabetes . recently , naturally occurring polyphenolic compounds have been the focal point of medicinal research interest due to their pharmacological implications associated with human disease with considerable attention devoted to managing diabetes [ 11 , 12 ] . dietary intake of kaempferol containing foods has been epidemiologically associated with a reduced risk of certain cancers and cardiovascular diseases . it has been reported that kaempferol has antioxidative , antimicrobial , anti - inflammatory , lipolytic , and anticancer [ 20 , 21 ] effects . we show here that dietary intake of kaempferol for 5 months ( mo ) improved insulin sensitivity and glucose tolerances , which were associated with increased glut4 and ampk expression in muscle and adipose tissues in middle - aged mice fed a high - fat ( hf ) diet . in vitro , kaempferol increased lipolysis and restored chronic high fatty acid - impaired glucose uptake and glycogen synthesis in skeletal muscle cells , which were associated with improved ampk activity and glut4 expression . in addition , dietary kaempferol treatment preserved functional pancreatic -cell mass and prevented hyperglycemia and glucose intolerance in stz - induced diabetic mice . mice were divided into 4 groups ( n = 12 mice / group ) with blood glucose and body weight balanced and then fed a standard chow ( sd ) diet , with 10% of calories derived from fat , a hf diet ( research diets inc . , new brunswick , nj ) with 58% of calories from fat , or hf diet supplemented with kaempferol ( 0.01% or 0.05% ) for 5 mo . to assess fasting blood glucose , mice were fasted for 12 h , and blood glucose was measured in tail vein blood samples using a glucometer ( kroger , cincinnati , oh ) . for the insulin tolerance tests ( itt ) , mice were injected ip with insulin ( 0.75 units / kg bw ) , and blood glucose levels were measured at 0 , 15 , 30 , 60 , and 120 min after insulin administration . , mice were sacrificed and extensor digitorum longus muscle and abdominal adipose tissues were collected , snap - frozen in liquid nitrogen , and then stored at 80c for the western blot analyses . in a separate experiment , mice were divided into 3 groups ( n = 8 mice / group ) and fed a sd diet or sd diet containing kaempferol ( 0.01% or 0.05% ) for 3 mo . mice were then fed a sd diet , a hf diet ( 58 kcal% fat ) , or hf diet containing 0.05% kaempferol . a series of tissue sections ( 5 m thickness at 200 m interval ) were prepared , mounted on glass slides , and immunofluorescently stained with an insulin antibody and fitc - conjugated secondary antibody ( abcam , cambridge , ma ) for determining -cell mass . pancreatic -cell mass was calculated by dividing the area of insulin - positive cells by the total area of pancreatic tissue and multiplied by the pancreas weight [ 25 , 26 ] . the fatty acid mixture contained a 2 : 1 ratio of palmitate to oleate for a final concentration of 0.4 mm complexed with 0.4% bsa in serum - free , low glucose dmem . following the fatty acid and kaempferol treatment , glucose uptake was assessed in krebs - ringer hepes buffer ( in mm : 136 nacl , 4.7 kcl , 1.25 mgso4 , 1.2 cacl , and 20 hepes , ph 7.4 ) with the addition of 10 m 2-deoxyglucose and 1.25 uci / ml 2-deoxy-[3h]glucose . in this study , we tested the metabolic effects of the long - term dietary supplementation of kaempferol ( 0.01% or 0.05% in the diet ) in middle - aged mice fed a hf diet . however , dietary intake of 0.05% kaempferol significantly ameliorated hf diet - induced body weight gain ( figure 1(b ) ) . consistently , mice fed the hf diet developed obesity as determined by measuring their relative percentage of fat ( figure 1(c ) ) and muscle mass ( figure 1(d ) ) . however , kaempferol treatment had no significant effect on adiposity of obese mice . fasting blood levels of cholesterol ( figure 1(e ) ) , ldl - cholesterol ( figure 1(f ) ) , and triglyceride ( figure 1(g ) ) were increased in hf diet - fed obese mice , which were significantly reduced by dietary intake of kaempferol . fasting plasma hdl - cholesterol concentrations were increased by hf feeding but were not further altered by kaempferol treatment ( figure 1(h ) ) . in another study to determine whether kaempferol also improves metabolism and health of sd diet - fed older adult mice , we found that dietary intake of either 0.01% kaempferol ( k1 ) or 0.05% kaempferol ( k2 ) for 3 mo had no effects on body weight gain ( 5.9 0.5 , 6.1 0.4 , and 6.0 0.6 g for control , k1 , and k2 group , resp . ) , food intake ( 4.25 , 4.27 , and 4.23 g / d / mouse ) , fasting ( 136.4 4.1 , 131.6 3.4 , and 127.9 5.8 mg / dl ) and nonfasting ( 185.4 11.1 , 180.5 5.5 , and 174.4 12.2 ) blood glucose levels , glucose tolerance ( 32672 1653 , 31411 1582 , and 31078 1634 auc ) , and insulin sensitivity ( 11073 1065 , 10897 9887 , and 10574 1141 auc ) . after 3 mo , however , mice fed hf diet supplemented with kaempferol exhibited significantly lower blood glucose levels than those in hf - fed mice ; after 5 mo of treatment , mice fed kaempferol - supplemented diet still had significantly lower blood glucose levels ( 140 10.5 mg / dl ) as compared to hf diet - fed mice ( 197 10.5 mg / dl ) , which were almost reduced to the levels shown in sd - fed mice ( 123.0 4.8 mg / dl ) ( figure 2(a ) ) . data showed that kaempferol ( 0.05% in the diet ) nonsignificantly improved glucose tolerance as demonstrated by lower blood glucose levels at 30 and 60 min ( figure 2(b ) ) as well as reduced auc ( figure 2(c ) ) following ip glucose injection compared to hf - fed mice . to determine if dietary intake of kaempferol improves insulin sensitivity in obese mice , we performed an ip itt . however , dietary provision of kaempferol as low as 0.01% significantly improved plasma glucose levels ( figure 2(e ) ) and the auc ( figure 2(f ) ) postinsulin injection in hf diet - fed mice , suggesting that kaempferol increases insulin sensitivity . we observed that fasting plasma insulin levels in hf mice were about 6-fold of those in mice that received the sd diet , suggesting that obese mice are insulin resistant ( figure 2(g ) ) . however , plasma insulin levels in 0.05% kaempferol - treated mice were more than 50% lower as compared with hf - fed obese mice ( figure 2(g ) ) . these data strongly suggest that long - term kaempferol supplementation maintained whole body insulin sensitivity in hf diet - induced obese mice . insulin resistance in adipose tissue and skeletal muscle , the primary site of glucose and fatty acid utilization , plays a major role in the development of hf diet - induced t2d . to further determine the molecular events that are associated with improved insulin sensitivity by kaempferol treatment , we measured the expression of ampk and glut4 in skeletal muscle and adipose tissues , which are two critical molecules regulating glucose uptake [ 30 , 31 ] . consistent with impaired peripheral insulin sensitivity , the expression of ampk and glut4 proteins in both skeletal muscle ( figures 3(a)3(c ) ) and adipose tissue ( figures 3(d)3(f ) ) from hf diet - fed mice was significantly attenuated as compared to the control mice . these results further confirmed that long - term intake of kaempferol protects against developing insulin resistance in hf diet - induced obese mice . as skeletal muscle is the major site for fuel metabolism , and obesity and insulin resistance are always accompanied with the impairment in energy metabolism , a leading pathogenic factor for t2d , we performed a series of in vitro assays to determine whether kaempferol has direct beneficial effects on glucose metabolism . in that regard , we cultured skeletal muscle cells with or without kaempferol in the presence of palmitate and oleic acid . we found that exposure of c2c12 myotubes to high fatty acids for 24 h impaired insulin - induced glucose uptake ( figure 4(a ) ) and glycogen synthesis ( figure 4(b ) ) . to further characterize metabolic and insulin sensitizing effects of kaempferol , we cultured skeletal muscle cells with or without kaempferol in the presence of high glucose ( 20 mm ) and saturated fatty acid palmitate ( 0.5 mm ) , an in vitro environment that is frequently used to simulate diabetic condition in vivo . the results showed that kaempferol at physiologically relevant concentrations significantly improved lipolysis ( figure 5(a ) ) and reversed high fatty acid - impaired ampk activity ( figure 5(b ) ) and glut4 protein expression ( figure 5(c ) ) , a downstream target of activated ampk . we recently found that kaempferol treatment prevented apoptosis of cultured pancreatic -cells exposed to chronic hyperlipidemic condition . we therefore further assessed whether kaempferol can also protect pancreatic -cell function , thereby preventing diabetes by using a t2d mouse model that was generated through a combination of hf diet feeding and three consecutive injections of low doses of stz . regarding this , c57bl/6 mice ( male , 10-month - old ) were fed a sd diet , a hf diet , or hf diet containing 0.05% kaempferol . consistent with the observations from the animal study described above , treatment with kaempferol for 6 weeks had no effect on body weight gain , food intake , fasting blood glucose levels , glucose intolerance , or insulin resistance of mice fed the hf diet ( data not shown ) . our data showed that dietary ingestion of kaempferol significantly mitigated stz - induced hyperglycemia in diabetic mice as determined by measuring fasting ( figure 6(a ) ) and nonfasting ( figure 6(b ) ) blood glucose levels . consistently , kaempferol ameliorated the loss of body weight secondary to the development of diabetes ( data not shown ) . to determine if the improved glycemic control in mice fed the kaempferol - supplemented diet is the result of preserved islet function , we measured insulin levels in the plasma of the control and kaempferol - fed mice . as shown in figure 6(c ) , plasma insulin levels in mice fed diet containing kaempferol were significantly greater as compared to those in nontreated diabetic mice , suggesting that kaempferol may ameliorate hyperglycemia primarily via preserving islet -cell function . since stz causes diabetes by destroying islet -cells , we then examined whether kaempferol treatment preserved -cell mass in diabetic mice by using an immunohistochemical technique . we observed that hf diet - fed mice exhibited greater pancreas weight as compared with the control mice ( figure 7(a ) ) , which was not modulated by kaempferol supplementation ( figure 7(a ) ) . however , dietary provision of kaempferol partially preserved islet -cell mass ( figures 7(b ) and 7(c ) ) and insulin content ( figure 7(d ) ) in diabetic mice . it has been reported that kaempferol elicits a number of health benefits , including antioxidative [ 17 , 37 ] , anti - inflammatory [ 18 , 38 ] , antihypertensive [ 39 , 40 ] , lipolytic [ 19 , 41 ] , and anticarcinogenic effects [ 20 , 21 , 42 ] . it is worthy to note that , unlike many metabolic studies that used young adult mice , we used near middle - aged mice , which may be more clinically relevant , as t2d in humans often occurs during middle and late ages . we provide evidence that long - term dietary intake of kaempferol promoted metabolic homeostasis with the improved fasting blood glucose , hba1c levels , insulin sensitivity , and glucose tolerance in hf diet - induced obese mice , which was associated with increased fuel metabolism , ampk activity , and glut4 expression in skeletal muscle cells . further , dietary kaempferol also ameliorates hyperglycemia in stz - induced diabetic mice by preserving functional -cell mass . given that both insulin resistance in peripheral tissues and progressive -cell loss and dysfunction are key components in the pathogenesis of t2d [ 48 ] , kaempferol could be a low - cost and safe natural compound to promote energy metabolism and maintain glucose homeostasis by targeting these two defects . therefore , hf diets are commonly used as a strategy to induce obesity in animal models , leading to the development of metabolic disorders including hyperlipidemia , impaired insulin sensitivity and glucose tolerance , and elevated blood insulin and glucose [ 4548 ] . consistently , our data showed that consumption of the hf diet induced obesity and elevated plasma concentrations of triglycerides and cholesterol that are typical of obesity , which however were ameliorated in mice fed a hf diet containing kaempferol . given that obesity is a leading pathogenic factor for developing insulin resistance and subsequent glucose intolerance , the improved insulin sensitivity and glucose homeostasis by kaempferol supplementation could be partially due to the secondary effects whereby long - term intake of kaempferol ameliorated obesity in mice fed a hf diet . because food intake was not affected by long - term kaempferol supplementation , the decrease in fasting plasma cholesterol and triglycerides in the hf - fed mice could be caused by the regulation of the intestinal digestion and absorption of lipids and/or lipogenesis , which needs further investigation . ampk is a heterotrimeric protein kinase composed of a catalytic subunit ( ampk ) and two regulatory subunits ( and ) that sense low cellular energy levels by monitoring changes in the amp : atp ratio . amp binding to the subunit induces a conformational change that allows ampk to be phosphorylated at its threonine residue ( thr 172 ) by the ampk - activating protein kinase ( lkb1 ) . in addition , activation of ampk increases glut4 expression and membrane translocation in skeletal muscle , thereby improving glucose uptake . consistent with the impaired insulin sensitivity in hf - fed obese mice , we found that the protein levels of ampk and glut4 in skeletal muscle and adipose tissue from these animals were also considerably attenuated . however , these detrimental effects were completely reversed by supplementation of 0.05% kaempferol in the hf diet . these results suggest that kaempferol may increase peripheral insulin sensitivity via the ampk - mediated pathway . both in experimental animals and people , obesity - related insulin resistance is always associated with the dysfunctions of several metabolic pathways including reduced lipolysis , fatty acid oxidation , glucose uptake , and glycogen synthesis coupled with increased glucose output . specifically , it is believed that elevated intramyocellular lipid accumulation coupled with diminished lipolysis and mitochondrial lipid oxidation play a role in the development of insulin resistance in skeletal muscle . consistently , it was found that inducing lipolysis and fatty acid oxidation in muscle cells protects against lipotoxicity - induced insulin resistance [ 58 , 59 ] . in the present study , we further showed that kaempferol treatment as low as 1 m augmented lipolysis and reversed chronic hyperlipidemia - impaired glucose uptake , glut4 expression , ampk activity , and glycogenesis in skeletal muscle cells , which provides further evidence at cellular levels that kaempferol might be an insulin sensitizing molecule by promoting energy metabolism . however , it is presently unclear how kaempferol exerts these beneficial effects in skeletal muscle cells . while we found that pharmacological doses of kaempferol ( 1050 m ) showed significant free radical scavenging activity as evaluated by using an oxygen radical absorbance assay , it had no such effect at 1 m . we further explored whether kaempferol directly protects pancreatic -cell function in vivo by using a nongenetic mouse model of t2d , which was generated by employing a combination of feeding a hf diet and administering three mild doses ( 40 mg / kg ) of stz that does not cause diabetes in chow - fed mice , as demonstrated in our recent study . this nongenetic diabetic mouse model manifests the metabolic characteristics of human t2d , including moderate levels of hyperglycemia , hyperlipidemia , insulin resistance , impaired insulin secretion , and reduced -cell mass . the results in the present study show that kaempferol partially preserved -cell mass in stz - induced diabetic mice , which could be primarily attributable to its antidiabetic action , given that diabetes was induced before kaempferol caused changes in body weight , adiposity , or other metabolic parameters . in summary , we provide evidence that long - term dietary supplementation of kaempferol prevents hf diet - induced metabolic disorders in middle - aged obese mice . on cellular and molecular levels , kaempferol improves glycolysis , glucose uptake , glycogen synthesis , ampk activity , and glut4 expression in skeletal muscle . in addition , dietary supplementation of kaempferol significantly ameliorated hyperglycemia and preserved functional islet mass in old adult obese diabetic mice . these results indicate that phytonutrient kaempferol may be used as a dietary supplement to prevent metabolic disorders that are associated with obesity and aging .

while the availability of novel drugs , techniques , and surgical intervention has improved the survival rate of individuals with diabetes , the prevalence of diabetes is still rising in the united states , with the number of people with diabetes projected to double by 2025 . both in experimental animals and people , obesity is a leading pathogenic factor for developing insulin resistance , which is always associated with the impairment in energy metabolism , causing increased intracellular fat content in skeletal muscle , liver , fat , and pancreatic islets . constant insulin resistance will progress to t2d when -cells are unable to secrete adequate amount of insulin to compensate for decreased insulin sensitivity , which is largely due to insulin secretory dysfunction and significant loss of functional -cells [ 48 ] . as such , the search for novel agents that simultaneously promote insulin sensitivity and -cell survival may provide a more effective strategy to prevent the onset of diabetes . recently , naturally occurring polyphenolic compounds have been the focal point of medicinal research interest due to their pharmacological implications associated with human disease with considerable attention devoted to managing diabetes [ 11 , 12 ] . polyphenols exist naturally as secondary plant metabolites and are the largest source of human dietary antioxidants , with a typical daily intake of roughly 1 g / day . kaempferol ( 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4h-1-benzopyran-4-one ) is a flavonol , of low molecular weight ( 286.2 g / mol ) , that has been identified in many plants used in traditional medicine , including equisetum spp . , sophora japonica , and ginkgo biloba , and edible plants , including beans , broccoli , cabbage , gooseberries , grapes , kale , strawberries , tea , and tomatoes [ 15 , 16 ] . however , to date , the studies regarding its effect on the pathogenesis of diabetes are very limited . we recently demonstrated that kaempferol treatment prevented apoptosis and improved insulin biosynthesis and secretion in -cells and human islets exposed to chronic hyperlipidemia . past studies have established that sustained hyperlipidemia in obese individuals plays an important role in causing -cell apoptosis and dysfunction , thereby contributing to the deterioration of glycemic control and the overt development of t2d [ 2224 ] . this study was conducted to investigate whether long - term dietary intake of kaempferol can promote metabolic homeostasis and thereby prevent diabetic pathogenesis . we show here that dietary intake of kaempferol for 5 months ( mo ) improved insulin sensitivity and glucose tolerances , which were associated with increased glut4 and ampk expression in muscle and adipose tissues in middle - aged mice fed a high - fat ( hf ) diet . in vitro , kaempferol increased lipolysis and restored chronic high fatty acid - impaired glucose uptake and glycogen synthesis in skeletal muscle cells , which were associated with improved ampk activity and glut4 expression . in addition , dietary kaempferol treatment preserved functional pancreatic -cell mass and prevented hyperglycemia and glucose intolerance in stz - induced diabetic mice . mice were divided into 4 groups ( n = 12 mice / group ) with blood glucose and body weight balanced and then fed a standard chow ( sd ) diet , with 10% of calories derived from fat , a hf diet ( research diets inc . to assess fasting blood glucose , mice were fasted for 12 h , and blood glucose was measured in tail vein blood samples using a glucometer ( kroger , cincinnati , oh ) . , the lf-90 body composition instrument is based on time domain nuclear magnetic resonance ( td - nmr ) technology , which provides an in vivo measurement of lean tissue , body fat , and body fluid in live mice without anesthesia . for the insulin tolerance tests ( itt ) , mice were injected ip with insulin ( 0.75 units / kg bw ) , and blood glucose levels were measured at 0 , 15 , 30 , 60 , and 120 min after insulin administration . , mice were sacrificed and extensor digitorum longus muscle and abdominal adipose tissues were collected , snap - frozen in liquid nitrogen , and then stored at 80c for the western blot analyses . in a separate experiment , mice were divided into 3 groups ( n = 8 mice / group ) and fed a sd diet or sd diet containing kaempferol ( 0.01% or 0.05% ) for 3 mo . at the end of 3 mo , gtt and itt 10-month - old male mice ( nci , nih ) were divided into 3 groups ( n = 10 mice / group ) with initial fasting blood glucose and body weights balanced among groups . pancreatic -cell mass was calculated by dividing the area of insulin - positive cells by the total area of pancreatic tissue and multiplied by the pancreas weight [ 25 , 26 ] . the fatty acid mixture contained a 2 : 1 ratio of palmitate to oleate for a final concentration of 0.4 mm complexed with 0.4% bsa in serum - free , low glucose dmem . following the fatty acid and kaempferol treatment , glucose uptake was assessed in krebs - ringer hepes buffer ( in mm : 136 nacl , 4.7 kcl , 1.25 mgso4 , 1.2 cacl , and 20 hepes , ph 7.4 ) with the addition of 10 m 2-deoxyglucose and 1.25 uci / ml 2-deoxy-[3h]glucose . after 24 h with fa and kaempferol treatment , cells were further incubated with 1.25 uci / mlc glucose ( american radiolabeled chemicals , saint louis , mo ) in the presence or absence of insulin ( 100 nm ; eli lilly , indianapolis , in ) for 3 h. following this treatment , cells were rinsed twice with pbs at 4c followed by solubilization by adding 250 l of 30% koh . in this study , we tested the metabolic effects of the long - term dietary supplementation of kaempferol ( 0.01% or 0.05% in the diet ) in middle - aged mice fed a hf diet . the hf diet decreased the accumulative average food intake , but kaempferol supplementation for 5 mo did not alter the food consumption compared with hf diet - fed mice ( figure 1(a ) ) . however , dietary intake of 0.05% kaempferol significantly ameliorated hf diet - induced body weight gain ( figure 1(b ) ) . consistently , mice fed the hf diet developed obesity as determined by measuring their relative percentage of fat ( figure 1(c ) ) and muscle mass ( figure 1(d ) ) . fasting blood levels of cholesterol ( figure 1(e ) ) , ldl - cholesterol ( figure 1(f ) ) , and triglyceride ( figure 1(g ) ) were increased in hf diet - fed obese mice , which were significantly reduced by dietary intake of kaempferol . however , total cholesterol levels in kaempferol - fed mice were still significantly higher as compared to chow diet - fed mice . in another study to determine whether kaempferol also improves metabolism and health of sd diet - fed older adult mice , we found that dietary intake of either 0.01% kaempferol ( k1 ) or 0.05% kaempferol ( k2 ) for 3 mo had no effects on body weight gain ( 5.9 0.5 , 6.1 0.4 , and 6.0 0.6 g for control , k1 , and k2 group , resp . ) , food intake ( 4.25 , 4.27 , and 4.23 g / d / mouse ) , fasting ( 136.4 4.1 , 131.6 3.4 , and 127.9 5.8 mg / dl ) and nonfasting ( 185.4 11.1 , 180.5 5.5 , and 174.4 12.2 ) blood glucose levels , glucose tolerance ( 32672 1653 , 31411 1582 , and 31078 1634 auc ) , and insulin sensitivity ( 11073 1065 , 10897 9887 , and 10574 1141 auc ) . after 3 mo , however , mice fed hf diet supplemented with kaempferol exhibited significantly lower blood glucose levels than those in hf - fed mice ; after 5 mo of treatment , mice fed kaempferol - supplemented diet still had significantly lower blood glucose levels ( 140 10.5 mg / dl ) as compared to hf diet - fed mice ( 197 10.5 mg / dl ) , which were almost reduced to the levels shown in sd - fed mice ( 123.0 4.8 mg / dl ) ( figure 2(a ) ) . data showed that kaempferol ( 0.05% in the diet ) nonsignificantly improved glucose tolerance as demonstrated by lower blood glucose levels at 30 and 60 min ( figure 2(b ) ) as well as reduced auc ( figure 2(c ) ) following ip glucose injection compared to hf - fed mice . consistently , blood levels of hba1c , which reflect an average of blood glucose over a period of two to three mo , were significantly lower in kaempferol - treated mice as compared to those in hf diet - fed mice ( figure 2(d ) ) . however , dietary provision of kaempferol as low as 0.01% significantly improved plasma glucose levels ( figure 2(e ) ) and the auc ( figure 2(f ) ) postinsulin injection in hf diet - fed mice , suggesting that kaempferol increases insulin sensitivity . we observed that fasting plasma insulin levels in hf mice were about 6-fold of those in mice that received the sd diet , suggesting that obese mice are insulin resistant ( figure 2(g ) ) . however , plasma insulin levels in 0.05% kaempferol - treated mice were more than 50% lower as compared with hf - fed obese mice ( figure 2(g ) ) . insulin resistance in adipose tissue and skeletal muscle , the primary site of glucose and fatty acid utilization , plays a major role in the development of hf diet - induced t2d . to further determine the molecular events that are associated with improved insulin sensitivity by kaempferol treatment , we measured the expression of ampk and glut4 in skeletal muscle and adipose tissues , which are two critical molecules regulating glucose uptake [ 30 , 31 ] . consistent with impaired peripheral insulin sensitivity , the expression of ampk and glut4 proteins in both skeletal muscle ( figures 3(a)3(c ) ) and adipose tissue ( figures 3(d)3(f ) ) from hf diet - fed mice was significantly attenuated as compared to the control mice . as skeletal muscle is the major site for fuel metabolism , and obesity and insulin resistance are always accompanied with the impairment in energy metabolism , a leading pathogenic factor for t2d , we performed a series of in vitro assays to determine whether kaempferol has direct beneficial effects on glucose metabolism . in that regard , we cultured skeletal muscle cells with or without kaempferol in the presence of palmitate and oleic acid . we found that exposure of c2c12 myotubes to high fatty acids for 24 h impaired insulin - induced glucose uptake ( figure 4(a ) ) and glycogen synthesis ( figure 4(b ) ) . to further characterize metabolic and insulin sensitizing effects of kaempferol , we cultured skeletal muscle cells with or without kaempferol in the presence of high glucose ( 20 mm ) and saturated fatty acid palmitate ( 0.5 mm ) , an in vitro environment that is frequently used to simulate diabetic condition in vivo . the results showed that kaempferol at physiologically relevant concentrations significantly improved lipolysis ( figure 5(a ) ) and reversed high fatty acid - impaired ampk activity ( figure 5(b ) ) and glut4 protein expression ( figure 5(c ) ) , a downstream target of activated ampk . while peripheral insulin resistance is common during obesity in rodents and people , its progression to t2d is largely due to insulin secretory dysfunction and significant apoptosis of functional -cells [ 48 ] , leading to an inability to compensate for insulin resistance . past studies have established that sustained hyperlipidemia in obese individuals plays an important role in causing -cell apoptosis and dysfunction , thereby contributing to the deterioration of glycemic control and the overt development of t2d [ 2224 ] . we therefore further assessed whether kaempferol can also protect pancreatic -cell function , thereby preventing diabetes by using a t2d mouse model that was generated through a combination of hf diet feeding and three consecutive injections of low doses of stz . consistent with the observations from the animal study described above , treatment with kaempferol for 6 weeks had no effect on body weight gain , food intake , fasting blood glucose levels , glucose intolerance , or insulin resistance of mice fed the hf diet ( data not shown ) . after 6 weeks of dietary treatment , stz ( 40 mg / kg bw ) was administrated ( ip ) for 3 consecutive days to induce diabetes mediated by a destruction of islet -cells in mice . our data showed that dietary ingestion of kaempferol significantly mitigated stz - induced hyperglycemia in diabetic mice as determined by measuring fasting ( figure 6(a ) ) and nonfasting ( figure 6(b ) ) blood glucose levels . to determine if the improved glycemic control in mice fed the kaempferol - supplemented diet is the result of preserved islet function , we measured insulin levels in the plasma of the control and kaempferol - fed mice . as shown in figure 6(c ) , plasma insulin levels in mice fed diet containing kaempferol were significantly greater as compared to those in nontreated diabetic mice , suggesting that kaempferol may ameliorate hyperglycemia primarily via preserving islet -cell function . we observed that hf diet - fed mice exhibited greater pancreas weight as compared with the control mice ( figure 7(a ) ) , which was not modulated by kaempferol supplementation ( figure 7(a ) ) . however , stz administration caused severe destruction of pancreatic -cells as determined by evaluating the area of insulin positive cells ( figure 7(b ) ) and islet mass ( figure 7(c ) ) , which consequently led to the reduction of pancreatic insulin content by nearly 80% ( figure 7(d ) ) . however , dietary provision of kaempferol partially preserved islet -cell mass ( figures 7(b ) and 7(c ) ) and insulin content ( figure 7(d ) ) in diabetic mice . it has been reported that kaempferol elicits a number of health benefits , including antioxidative [ 17 , 37 ] , anti - inflammatory [ 18 , 38 ] , antihypertensive [ 39 , 40 ] , lipolytic [ 19 , 41 ] , and anticarcinogenic effects [ 20 , 21 , 42 ] . it is worthy to note that , unlike many metabolic studies that used young adult mice , we used near middle - aged mice , which may be more clinically relevant , as t2d in humans often occurs during middle and late ages . we provide evidence that long - term dietary intake of kaempferol promoted metabolic homeostasis with the improved fasting blood glucose , hba1c levels , insulin sensitivity , and glucose tolerance in hf diet - induced obese mice , which was associated with increased fuel metabolism , ampk activity , and glut4 expression in skeletal muscle cells . given that both insulin resistance in peripheral tissues and progressive -cell loss and dysfunction are key components in the pathogenesis of t2d [ 48 ] , kaempferol could be a low - cost and safe natural compound to promote energy metabolism and maintain glucose homeostasis by targeting these two defects . therefore , hf diets are commonly used as a strategy to induce obesity in animal models , leading to the development of metabolic disorders including hyperlipidemia , impaired insulin sensitivity and glucose tolerance , and elevated blood insulin and glucose [ 4548 ] . consistently , our data showed that consumption of the hf diet induced obesity and elevated plasma concentrations of triglycerides and cholesterol that are typical of obesity , which however were ameliorated in mice fed a hf diet containing kaempferol . given that obesity is a leading pathogenic factor for developing insulin resistance and subsequent glucose intolerance , the improved insulin sensitivity and glucose homeostasis by kaempferol supplementation could be partially due to the secondary effects whereby long - term intake of kaempferol ameliorated obesity in mice fed a hf diet . because food intake was not affected by long - term kaempferol supplementation , the decrease in fasting plasma cholesterol and triglycerides in the hf - fed mice could be caused by the regulation of the intestinal digestion and absorption of lipids and/or lipogenesis , which needs further investigation . as kaempferol treatment did not alter calorie intake in mice during the course of this study , it is also possible that the reduced body fat mass as well as body weight in kaempferol - fed mice is due to its effect on energy expenditure , given that obesity results from energy imbalance . at the whole body level , ampk integrates stress responses , nutrient and hormonal signals to the control of food intake , energy expenditure , and substrate utilization . consistent with the impaired insulin sensitivity in hf - fed obese mice , we found that the protein levels of ampk and glut4 in skeletal muscle and adipose tissue from these animals were also considerably attenuated . both in experimental animals and people , obesity - related insulin resistance is always associated with the dysfunctions of several metabolic pathways including reduced lipolysis , fatty acid oxidation , glucose uptake , and glycogen synthesis coupled with increased glucose output . specifically , it is believed that elevated intramyocellular lipid accumulation coupled with diminished lipolysis and mitochondrial lipid oxidation play a role in the development of insulin resistance in skeletal muscle . in the present study , we further showed that kaempferol treatment as low as 1 m augmented lipolysis and reversed chronic hyperlipidemia - impaired glucose uptake , glut4 expression , ampk activity , and glycogenesis in skeletal muscle cells , which provides further evidence at cellular levels that kaempferol might be an insulin sensitizing molecule by promoting energy metabolism . we further explored whether kaempferol directly protects pancreatic -cell function in vivo by using a nongenetic mouse model of t2d , which was generated by employing a combination of feeding a hf diet and administering three mild doses ( 40 mg / kg ) of stz that does not cause diabetes in chow - fed mice , as demonstrated in our recent study . this nongenetic diabetic mouse model manifests the metabolic characteristics of human t2d , including moderate levels of hyperglycemia , hyperlipidemia , insulin resistance , impaired insulin secretion , and reduced -cell mass . the results in the present study show that kaempferol partially preserved -cell mass in stz - induced diabetic mice , which could be primarily attributable to its antidiabetic action , given that diabetes was induced before kaempferol caused changes in body weight , adiposity , or other metabolic parameters . in summary , we provide evidence that long - term dietary supplementation of kaempferol prevents hf diet - induced metabolic disorders in middle - aged obese mice .

sensorineural hearing loss is characterized by an elevation of pure tone thresholds and often , difficulty understanding speech , especially in noisy environments . for most patients , hearing aids are effective tools for overcoming sensitivity loss , especially when they incorporate recent technological developments . however , hearing aids are not always capable of helping the patient compensate for difficulties in understanding speech , particularly in a reverberant and/or noisy environment.1 even the most sophisticated hearing aids are unable to improve auditory skills or the comprehension needed for efficient communication . hearing aids provide increased acoustic information , but alone they are not able to directly modify the brain or the patient s behavior.2 furthermore , peripheral hearing loss may coexist with a central auditory processing deficit , thereby contributing to patient dissatisfaction with amplification , even when audiological tests indicate nearly normal aided hearing thresholds . auditory training has been highlighted as part of habilitation / rehabilitation for the hearing impaired , though it has been underutilized . it is primarily based on the belief that the peripheral system benefits from the stimulation provided . however , advances in neuroscience suggest that it is the central auditory system that benefits from auditory stimulation.24 a number of authors have concluded that the auditory processing evaluation of hearing aid candidates / users provides a more complete profile of auditory skills and may be useful in choosing between different amplification options and complementary remediation tools.3,5,6 therefore , auditory processing tests could contribute to and complement the classical peripheral auditory evaluation . detailed clinical research in this area is crucial to determining the best approach and technique so as to achieve maximum success with a rehabilitation program . unfortunately , there do not appear to be any studies in the literature that involve behavioral and electrophysiological measurements of central auditory processing , formal auditory training and self - assessment outcome measures in adult hearing aid users . the aim of this study was to examine the effects of formal auditory training on adult binaural hearing aid users with mild to moderate bilateral sensorineural hearing loss using the following three procedures : behavioral tests of auditory processing , long - latency auditory evoked potentials and a self - assessment questionnaire . behavioral tests of auditory processing were employed to identify and monitor any changes in the central auditory system as a result of the auditory training program . long - latency auditory evoked potentials ( llaep ) were used to plan and monitor rehabilitation outcomes in specific populations . since llaeps are not influenced by the presence of hearing loss , especially in mild and moderate hearing losses , hearing - impaired subjects may be accurately evaluated with this procedure . finally , the inclusion of a self - assessment questionnaire provided the basis for evaluating the success or failure of the auditory training program from an important , objective source the patient . all subjects in this double - blind , randomized study met the following inclusion criteria : age between 16 and 60 years old . no other diagnosed disorders , such as neurological , psychological , cognitive or mental disturbances . fourteen subjects were randomly divided into two equal groups : an experimental group ( with auditory training ) and control group ( without auditory training ) . aided hearing thresholds ranged from 15 to 35 decibels ( db ) for all subjects with the pure tone average ( 500 , 1000 and 2000 hz ) at 35 decibels hearing level ( db hl ) or better . this study was approved by the ethical committee in research of the universidade federal de so paulo , number 0685/05 . all subjects , regardless of group placement , received a behavioral auditory processing evaluation and an electrophysiological evaluation and were asked complete a self - assessment questionnaire , the abbreviated profile of hearing aid benefit ( aphab ) . these procedures were performed before and after the auditory training for the experimental group and at times coinciding with the beginning and end of the study for the control group . in order to characterize this project as a double - blind study , the examiner who performed the second evaluation did not know whether the subject belonged to the experimental or control group and was unaware of the results of the subject s first test procedures . the behavioral auditory processing evaluation was carried out with headphones while subjects wore their hearing aids , since all of them were fitted with in - the - ear ( ite ) hearing aids . as central auditory processing evaluation in hearing aid subjects is a controversial issue , tests were included that have been shown to be more robust in the presence of peripheral auditory damage.5,7 the test battery was designed to ensure maximum use of clinician time during administration as well as to limit the time a subject was exposed to testing procedures , thus offering subjects an optimal opportunity to do well . based on these design elements , the following procedures were selected : sound localization : the patient was instructed to identify the origin of the instrumental sound in five directions ( right , left , above the head , in front of the head and behind the head ) with her / his eyes closed . memory for verbal sounds in sequence : the patient was instructed to repeat the syllables pa , ta , ca , fa in three different sequences without any visual clues . memory for nonverbal sounds in sequence : the patient was asked to reproduce three sequences of four instrumental sounds without any visual clues . word recognition score with recorded stimuli : two lists of 25 recorded monosyllables were presented to each ear separately , and the patient was asked to repeat each of them . speech - in - noise test : two different lists of 25 monosyllables were presented to each ear in the presence of ipsilateral white noise using a signal - to - noise ratio ( snr ) of + 5 , and the patient was asked to repeat each monosyllable while ignoring the noise . synthetic sentence identification ( ssi ) : the patient was instructed to point to the sentence presented in the headphones on a chart displayed in front of him / her while ignoring the competitive stimuli represented by a verbal discourse . binaural integration condition : the patient was asked to repeat twenty series of four digits presented dichotically . all tests , except sound localization and memory for verbal and nonverbal sounds in sequence , which were performed in a sound field using musical instruments , were performed using recorded stimuli from a commercially available brazilian compact disc ( cd)8 . as the subjects of our study exhibited nearly normal aided hearing thresholds , their performance was compared to the expected levels for subjects with normal hearing . the electrophysiological evaluation in this study consisted of measures of long - latency auditory evoked potentials n1 , p2 , n2 and p3 , registered without hearing aids using an odd - ball paradigm with tone bursts . the frequent and rare stimuli were 1000 hz and 2000 hz tones with 80% and 20% of probability , respectively . the intensity level varied from 70 to 85 db hl , according to the residual hearing in the test frequencies involved.9 a four - channel biologic systems unit was used to measure the llaep recordings . inter - electrode impedance of 5 kohms or less was guaranteed during the entire recording . subjects were asked to lie still with their eyes closed while silently counting the number of different ( rare ) stimuli . the latencies and amplitudes of the n1 , p2 and n2 components were marked in the rare tracings . the subtraction of rare from frequent tracings created a waveform from which p3 latency and amplitude were determined . the aphab was used to study the subjective effectiveness of the auditory training.10 this instrument has been translated and validated in portuguese.11 aphab is a self - assessment questionnaire used to quantify auditory difficulties experienced in daily situations involving communication in quiet , noisy and reverberant environments . it is usually administered before and after fitting amplification as a mechanism to verify the benefit provided by hearing aids . all subjects completed an aphab in two different situations , before and after auditory training in the experimental group , and as initial and final evaluations in the control group . as subjects were already wearing hearing aids in both situations , patients were asked to answer the questionnaire using only the column corresponding to with hearing aids . our formal auditory training program was organized into eight one - hour sessions , held twice a week for four weeks . all sessions were performed with hearing aids and designed to provide intensive stimulation and challenge the auditory system . for these purposes , the snr was varied from positive ( easier ) to negative ( more difficult ) during each activity that involved ignoring competitive stimuli . during monaural activities , such as temporal processing training , activities involved pointing to sentences , figures , digits , verbal repetition and humming temporal patterns . right and left ears were trained separately in an attempt to compensate for interaural differences usually observed in behavioral auditory processing tests , except during binaural integration activities and temporal processing training , when sound field presentation was used . the stimulation paradigm of each ear was as follows : the intensity level was fixed for the ear under training while the contralateral intensity level was increased ( snr from positive to negative ) , and the patient s task was to pay attention to the stimuli delivered to the ear under training while ignoring the contralateral messages . this paradigm is similar to one known as dichotic interaural intensity difference ( diid)12 that was proposed by an american audiologist . in order to keep the patients motivated , a 70% correct response rate was required at each step of the training before the patient was permitted to advance to another activity . table 1 summarizes the formal auditory training ( fat ) schedule employed in the present study . the behavioral auditory processing evaluation was carried out with headphones while subjects wore their hearing aids , since all of them were fitted with in - the - ear ( ite ) hearing aids . as central auditory processing evaluation in hearing aid subjects is a controversial issue , tests were included that have been shown to be more robust in the presence of peripheral auditory damage.5,7 the test battery was designed to ensure maximum use of clinician time during administration as well as to limit the time a subject was exposed to testing procedures , thus offering subjects an optimal opportunity to do well . based on these design elements , the following procedures were selected : sound localization : the patient was instructed to identify the origin of the instrumental sound in five directions ( right , left , above the head , in front of the head and behind the head ) with her / his eyes closed . memory for verbal sounds in sequence : the patient was instructed to repeat the syllables pa , ta , ca , fa in three different sequences without any visual clues . memory for nonverbal sounds in sequence : the patient was asked to reproduce three sequences of four instrumental sounds without any visual clues . word recognition score with recorded stimuli : two lists of 25 recorded monosyllables were presented to each ear separately , and the patient was asked to repeat each of them . speech - in - noise test : two different lists of 25 monosyllables were presented to each ear in the presence of ipsilateral white noise using a signal - to - noise ratio ( snr ) of + 5 , and the patient was asked to repeat each monosyllable while ignoring the noise . synthetic sentence identification ( ssi ) : the patient was instructed to point to the sentence presented in the headphones on a chart displayed in front of him / her while ignoring the competitive stimuli represented by a verbal discourse . binaural integration condition : the patient was asked to repeat twenty series of four digits presented dichotically . all tests , except sound localization and memory for verbal and nonverbal sounds in sequence , which were performed in a sound field using musical instruments , were performed using recorded stimuli from a commercially available brazilian compact disc ( cd)8 . as the subjects of our study exhibited nearly normal aided hearing thresholds , their performance was compared to the expected levels for subjects with normal hearing . the electrophysiological evaluation in this study consisted of measures of long - latency auditory evoked potentials n1 , p2 , n2 and p3 , registered without hearing aids using an odd - ball paradigm with tone bursts . the frequent and rare stimuli were 1000 hz and 2000 hz tones with 80% and 20% of probability , respectively . the intensity level varied from 70 to 85 db hl , according to the residual hearing in the test frequencies involved.9 a four - channel biologic systems unit was used to measure the llaep recordings . inter - electrode impedance of 5 kohms or less was guaranteed during the entire recording . subjects were asked to lie still with their eyes closed while silently counting the number of different ( rare ) stimuli . the latencies and amplitudes of the n1 , p2 and n2 components were marked in the rare tracings . the subtraction of rare from frequent tracings created a waveform from which p3 latency and amplitude were determined . the aphab was used to study the subjective effectiveness of the auditory training.10 this instrument has been translated and validated in portuguese.11 aphab is a self - assessment questionnaire used to quantify auditory difficulties experienced in daily situations involving communication in quiet , noisy and reverberant environments . it is usually administered before and after fitting amplification as a mechanism to verify the benefit provided by hearing aids . all subjects completed an aphab in two different situations , before and after auditory training in the experimental group , and as initial and final evaluations in the control group . as subjects were already wearing hearing aids in both situations , patients were asked to answer the questionnaire using only the column corresponding to with hearing aids . our formal auditory training program was organized into eight one - hour sessions , held twice a week for four weeks . all sessions were performed with hearing aids and designed to provide intensive stimulation and challenge the auditory system . for these purposes , the snr was varied from positive ( easier ) to negative ( more difficult ) during each activity that involved ignoring competitive stimuli . during monaural activities , such as temporal processing training , activities involved pointing to sentences , figures , digits , verbal repetition and humming temporal patterns . right and left ears were trained separately in an attempt to compensate for interaural differences usually observed in behavioral auditory processing tests , except during binaural integration activities and temporal processing training , when sound field presentation was used . the stimulation paradigm of each ear was as follows : the intensity level was fixed for the ear under training while the contralateral intensity level was increased ( snr from positive to negative ) , and the patient s task was to pay attention to the stimuli delivered to the ear under training while ignoring the contralateral messages . this paradigm is similar to one known as dichotic interaural intensity difference ( diid)12 that was proposed by an american audiologist . in order to keep the patients motivated , a 70% correct response rate was required at each step of the training before the patient was permitted to advance to another activity . table 1 summarizes the formal auditory training ( fat ) schedule employed in the present study . all data obtained from the behavioral central auditory processing test battery , electrophysiological evaluation and aphab were statistically analyzed . to investigate the effects of the fat program , the student t - test was used to compare the performance variance of subjects from both control and experimental groups , in both evaluations ( pre- and post - training ) , considering behavioral auditory processing tests , the electrophysiological test and the aphab . the significance level was set at 5% ( p = 0.05 ) , and confidence intervals were established at 95% . significant values are highlighted by the symbol ( * ) , and tendency toward statistical significance values are highlighted by the symbol ( # ) . table 2 shows the amount of improvement in behavioral auditory processing tests of the control group comparing pre- and post - training evaluations . no significant differences in performance were observed for the majority of behavioral tests , expect for ssi - icm snrs -10 and -15 , which revealed better results post - training . table 3 shows the amount of improvement in behavioral auditory processing tests of the experimental group by comparing pre- and post - training evaluations . significant differences in performance of the experimental group were observed when comparing the tests results pre- and post - training , demonstrating improved performance following training . table 4 ( variation in latency and amplitude values of llaep for the control group when pre- and post - training evaluations were compared ) shows that significant differences were observed for the latencies of p2 and n2 llaep components . for p2 an increase in latency was observed , while for n2 a decrease in latency was verified when comparing pre- and post - training evaluations . variation in latency and amplitude values of llaep of the experimental group when comparing pre- and post - training evaluations ) it is easily seen that for the experimental group , a significantly lower latency was observed for the p3 component of llaep when comparing pre- and post - training evaluations . as with the control group , no significant differences were seen concerning latency . finally , in table 6 ( benefit observed in aphab of control and experimental groups when comparing pre- and post - training administration ) , we present the comparison of benefit observed through the administration of the aphab to the control and experimental groups . no differences were noted for the control group , while there was a trend toward statistical significance for the experimental group in the reverberation and background noise sub - scales . all patients exhibited abnormal results on at least one of the tests during the pre - training evaluation . thus , everyone demonstrated auditory difficulties with degraded stimuli , as previously reported in different studies.1,13,14 auditory difficulty could not be predicted by either functional gain or word recognition scores in quiet , as results were within normal limits under such conditions . prior to the auditory training program , performance in the control group was generally poorer than in the experimental group the limited amount of improvement in behavioral tests by the control group ( table 2 ) suggest that all behavioral tests used , except ssi - icm ( -10 and -15 ) , were of minimal value . interestingly poorer results were seen during the post - training evaluation after some tests , including word recognition scores and the speech in noise test . when considering ssi - icm ( -10 and -15 ) , significant differences were observed between pre- and post - training evaluations . this improvement , although insufficient to explain the results completely , could be attributed to patients familiarity with the procedure leading them to ignore the competitive message while focusing on the target sentence . this has occurred in one previous study , where the control groups , without training , showed some improvements during the re - evaluations.15 however , these improvements were not of the same magnitude as those observed in the groups which underwent auditory training . the performance of the experimental group during the post - training evaluation was better than the first evaluation ( table 3 ) , since the comparison between post- and pre - training performance resulted in positive values for all tests . statistically significant differences were observed in sound localization , memory for nonverbal sounds in sequence , the speech in noise test , dichotic digits and ssi - icm ( 0 , -10 , -15 ) , with ssi - icm ( -15 ) demonstrating an improvement of almost 70% . this is interpreted to mean that formal auditory training was effective in improving central auditory skills.1618 the post - training evaluation of the experimental group was within established limits for adults with normal hearing . when sound is introduced into an impaired auditory system , spectral and temporal cues in the central auditory nervous system are altered . as a result , patients are forced to combine different spectral and temporal codes as they remember speech sounds . failure to do so culminates in difficulties in understanding , especially in situations with ambient noise . this was confirmed by the results of the present study during the speech in noise test , in which the subjects performance was negatively influenced by the introduction of noise . the stimuli were the same in both quiet and noise ; only the order of presentation was changed . the results indicate that amplification provided by the hearing aids was insufficient to maintain the same quality in both quiet and noisy environments ( table 2 ) . however , this auditory skill may be improved with auditory training ( table 3 ) . finally , it is possible that hearing aid fitting alone fails to produce the ideal environment for the auditory system and its skills.2,6,19,20 when a central auditory processing disorder co - exists with peripheral hearing loss , compensation obtained from hearing aids is at times insufficient to compensate for the auditory processing disorder ( apd ) , and the patient may become dissatisfied and frustrated with the performance of the hearing aids.2,19 based on our results , auditory processing evaluations although there were no significant differences between right and left ears for either group on any of the experimental measures , individual results for some patients in both the control and experimental groups demonstrated asymmetrical results in central auditory processing tests , such as dichotic digits , speech in noise and ssi - icm . such differences were minimized for subjects in the experimental group after the completion of training . the phenomenon of binaural interference is suspected when performance in speech tests with binaural amplification is worse than with monaural or without hearing aids.4,6,2123 in cases of binaural interference , a unilateral hearing aid fitting is advisable . it is important to consider that in the case of a monaural fitting , the unaided ear may suffer the deleterious effects of sensory deprivation , affecting neural plasticity and leading to a progressive degradation of word recognition on the non - aided side . this can occur even when auditory thresholds are stable.14,24 as discussed above , one could question whether it would be more reasonable to document asymmetry in a patient with a central auditory processing disorder and then fit both ears to maximize the advantages of binaural hearing . after waiting a reasonable acclimatization period , the patient would be enrolled in a formal auditory training program , at the end of which the decision would be made as to whether a binaural or a monaural hearing aid fitting is more suitable . one of the most controversial aspects of formal auditory training is the use of the same test for training and evaluation . training for the test , thereby resulting in a positive bias during re - evaluation . nonetheless , our results suggest that generalization in non - trained contexts is possible since , in the tests used only for evaluation , including sound localization , memory for verbal and nonverbal sounds and the speech in noise tests , objective and subjective measures showed improved results for the experimental group after training . such results can be attributed to the auditory training with a considerable degree of confidence . generalization for non - trained situations has been mentioned in several previous studies.16 , 2528 the maintenance of any learned pattern depends on its use.27,29 therefore , it seems reasonable to conclude that benefits accrued from the auditory training program will be maintained if the patient continues to use what he or she has learned . however , future studies are needed to determined if this is the case . concerning llaep , the n1-p2-n2 and p3 complexes were identified in all patients from both the control and experimental groups , and all subjects exhibited latency values within normal limits . these results concur with those of previous authors , who have noted that the presence of peripheral hearing loss , especially to mild and moderate degrees ( which is the case in this study ) , does not prevent the registration of late potentials , since the patient is able to perceive both rare and frequent stimuli.3032 however , also mentioned in other studies is considerable inter - subject variability for both latency and amplitude values.15,27,37 statistical analysis revealed no significant differences between right and left ears . since both ears were considered in the study , statistical analysis was made more reliable , as the sample size was doubled . the control and experimental groups did not differ with regard to llaep components in latency or amplitude in the first evaluation . however , when comparing the variation in latencies values between pre and post training evaluations of the control group ( table 4 ) , significant changes were observed for p2 and n2 latencies . the p2 component showed an increase in latency of 12.86 ms , while a decrease of 5.60 ms was observed for the n2 component . the n1-p2-n2 complex of llaep is influenced by attention.9 if the target stimulus is ignored , waveforms may be attenuated , and possible delays in latency may be observed . for the experimental group , a statistically significant reduction in p3 latency ( 27 ms ) was observed when comparing latency between the pre- and post - training electrophysiological evaluation ( table 5 ) . although no statistical differences were demonstrated in p3 latency for the control group , a slight increase in latency of 6.43 ms was noted ( table 4 ) . latency reduction of evoked potentials after auditory training has been described as a neurophysiologic correlate of neural plasticity . in many cases , this change may precede a behavioral change , which could take longer since it requires the integration of neural modifications and conscious perception and also involves higher cognitive processes.15,33,34 furthermore , latency reduction may be understood as an improvement in electrophysiological function . this improvement may be attributed to the auditory training , as the training was carried out after the acclimatization period.11,14,35,36 finally , as an objective measure of neural change , the improvement appears to influence neural plasticity in adults , as mentioned in a number of other studies.6,28,29,34,3739 since the importance of temporal aspects in maximizing auditory skills is well recognized , the latency reduction observed in the experimental group may be of critical importance for improving communication for hearing aid users , especially in adverse environments . this improvement , along with advanced hearing aid technology , may help patients in daily situations , leading to improved use of hearing aids and better social integration . no statistically significant differences were observed in the variation of amplitude between the pre- and post - training evaluations for both groups ( tables 4 and 5 ) . the increase in amplitude , especially of p3 after auditory training , suggests better synchrony in neural firing and attention improvement and has also been shown to be related to neural plasticity.15,27,28,34,38,40 there is still controversy regarding which parameter , latency or amplitude , is the better indicator of neurophysiologic improvement when determining the efficacy of a specific therapeutic approach.33 we conclude that amplitude , as measured in the present study , does not allow for inferences about the efficiency of the auditory training program described here . our results were the same as those previously noted in different studies in the literature.27,28,31,38 the results from this portion of the study suggest that the behavioral and electrophysiological improvements observed in the experimental group after training support central changes and improved functioning and highlight the importance of establishing the interaction between the central auditory nervous system and amplified sound , as previously recommended.36,39 the aphab self - assessment questionnaire was selected to help determine whether the expected / observed changes in electrophysiological and behavioral evaluations would interfere with the patient s subjective evaluation . there does not appear to be a specific self - assessment questionnaire designed to quantify changes observed in adult hearing aid users following a specific auditory training program . however , aphab has been shown to be a powerful instrument for recording the benefit of a specific therapeutic approach.41 thus , we felt it the most appropriate self - assessment questionnaire for our study . self - assessment questionnaires have played a very important role in the validation stage of hearing aid fitting , as they reveal the benefits perceived in daily situations with amplification.35 the similarity of responses for both groups regarding the ease of communication sub - scale ( ec ) indicates that the patients were well - adapted to their hearing aids , mainly in silent environments and/or in conversations involving small groups in relatively quiet places . however , the vast majority complained of difficulties hearing in noisy environments ( table 6 ) . some admitted they removed their hearing aids in such environments . this strategy frequently put them in embarrassing situations , leaving them feeling uncomfortable with their hearing aids in and missing important parts of conversations , dialogues and lectures without them . as the subjects in our study wore hearing aids in both situations when answering the aphab , the magnitude of benefit should not be the same as was seen during the original application of this instrument . no significant differences were observed in the control group at all sub - scales , when comparing the first and second applications . this suggests that reported difficulties remained stable , as previously reported.42 however for the experimental group , communicating in noisy and reverberant environments was easier than before the training . table 6 shows the comparison of percentage of difficulties experienced by the participants in the sub - scales of aphab for both applications of the questionnaires , pre- and post - training . although the differences observed in the experimental group in reverberation and background noise sub - scales only trended toward statistical significance , all sub - scales revealed negative values , indicating that after auditory training participants in the experimental group reported fewer difficulties in daily situations . this means that communication in adverse environments became easier for the experimental group following auditory training . in another study , 90% of adult hearing aid users felt more capable and more confident in challenging auditory situations after auditory training with a commercial available cd - rom.2 the opposite occurred in the control group , where positive values were seen in the ease of communication , background noise and averseness to loud sound sub - scales ( table 6 ) . improvements for the experimental group were previously noticed in electrophysiological and behavioral evaluations ( tables 2 to 5 ) . although subjects in both groups were similar in peripheral hearing status and hearing aid technology , environmental differences are expected and may account for differences in subjective measures such as self - assessment questionnaires . communication improvement in noisy environments should be the primary goal of any auditory training program , especially since such environments are common in everyday life . however , there is still the chance of having a subject whose hearing thresholds have been made normal but who has an auditory processing disorder . such a patient would not complain of difficulties in receiving sounds but rather in interpreting them , especially in the presence of increasing noise . it is suggested that administering an auditory training program , as described in the present study , and a self - assessment questionnaire , such as the aphab , could make the assessment and identification of hearing - impaired patients with an additional auditory processing disorder easier . the results of the present study suggest that formal auditory training was able to improve the central auditory skills of hearing aid users . improvement was noted in an objective neurophysiologic correlate and perceived by patients , as revealed in a self - assessment questionnaire . therefore , we strongly advocate investing time searching for the presence of a central auditory processing disorder during the course of all hearing aid fittings . furthermore , programs for rehabilitation of these skills are essential for all patients identified , regardless of age . in many cases , the high cost of hearing aids prevents patients from acquiring them , particularly if the patient feels that the benefit will not outweigh the high cost of the investment . including an initial auditory processing evaluation and formal auditory training should provide a basis for such patients to feel more confident about purchasing a sophisticated and expensive hearing aid . audiologists play an important role in this situation by helping patients realize the importance of auditory training and introducing it as a part of the hearing aid fitting process . after a critical analysis of the results , we can conclude that formal auditory training in adult hearing aid users promotes : latency reduction of the p3 component of llaep . improvement in auditory skills for sound localization , memory for nonverbal sounds in sequence , auditory closure and figure - to - ground for verbal sounds .

introductionindividuals with sensorineural hearing loss are often able to regain some lost auditory function with the help of hearing aids . however , hearing aids are not able to overcome auditory distortions such as impaired frequency resolution and speech understanding in noisy environments . the coexistence of peripheral hearing loss and a central auditory deficit may contribute to patient dissatisfaction with amplification , even when audiological tests indicate nearly normal hearing thresholds.objectivethis study was designed to validate the effects of a formal auditory training program in adult hearing aid users with mild to moderate sensorineural hearing loss.methodsfourteen bilateral hearing aid users were divided into two groups : seven who received auditory training and seven who did not . the training program was designed to improve auditory closure , figure - to - ground for verbal and nonverbal sounds and temporal processing ( frequency and duration of sounds ) . pre- and post - training evaluations included measuring electrophysiological and behavioral auditory processing and administration of the abbreviated profile of hearing aid benefit ( aphab ) self - report scale.resultsthe post - training evaluation of the experimental group demonstrated a statistically significant reduction in p3 latency , improved performance in some of the behavioral auditory processing tests and higher hearing aid benefit in noisy situations ( p - value < 0,05 ) . no changes were noted for the control group ( p - value < 0,05).conclusionthe results demonstrated that auditory training in adult hearing aid users can lead to a reduction in p3 latency , improvements in sound localization , memory for nonverbal sounds in sequence , auditory closure , figure - to - ground for verbal sounds and greater benefits in reverberant and noisy environments .

INTRODUCTION MATERIALS AND METHODS Behavioral auditory processing evaluation Electrophysiological Evaluation Self-assessment questionnaire Formal Auditory Training Program RESULTS DISCUSSION CONCLUSIONS

sensorineural hearing loss is characterized by an elevation of pure tone thresholds and often , difficulty understanding speech , especially in noisy environments . for most patients , hearing aids are effective tools for overcoming sensitivity loss , especially when they incorporate recent technological developments . however , hearing aids are not always capable of helping the patient compensate for difficulties in understanding speech , particularly in a reverberant and/or noisy environment.1 even the most sophisticated hearing aids are unable to improve auditory skills or the comprehension needed for efficient communication . hearing aids provide increased acoustic information , but alone they are not able to directly modify the brain or the patient s behavior.2 furthermore , peripheral hearing loss may coexist with a central auditory processing deficit , thereby contributing to patient dissatisfaction with amplification , even when audiological tests indicate nearly normal aided hearing thresholds . however , advances in neuroscience suggest that it is the central auditory system that benefits from auditory stimulation.24 a number of authors have concluded that the auditory processing evaluation of hearing aid candidates / users provides a more complete profile of auditory skills and may be useful in choosing between different amplification options and complementary remediation tools.3,5,6 therefore , auditory processing tests could contribute to and complement the classical peripheral auditory evaluation . unfortunately , there do not appear to be any studies in the literature that involve behavioral and electrophysiological measurements of central auditory processing , formal auditory training and self - assessment outcome measures in adult hearing aid users . the aim of this study was to examine the effects of formal auditory training on adult binaural hearing aid users with mild to moderate bilateral sensorineural hearing loss using the following three procedures : behavioral tests of auditory processing , long - latency auditory evoked potentials and a self - assessment questionnaire . behavioral tests of auditory processing were employed to identify and monitor any changes in the central auditory system as a result of the auditory training program . since llaeps are not influenced by the presence of hearing loss , especially in mild and moderate hearing losses , hearing - impaired subjects may be accurately evaluated with this procedure . finally , the inclusion of a self - assessment questionnaire provided the basis for evaluating the success or failure of the auditory training program from an important , objective source the patient . fourteen subjects were randomly divided into two equal groups : an experimental group ( with auditory training ) and control group ( without auditory training ) . all subjects , regardless of group placement , received a behavioral auditory processing evaluation and an electrophysiological evaluation and were asked complete a self - assessment questionnaire , the abbreviated profile of hearing aid benefit ( aphab ) . these procedures were performed before and after the auditory training for the experimental group and at times coinciding with the beginning and end of the study for the control group . in order to characterize this project as a double - blind study , the examiner who performed the second evaluation did not know whether the subject belonged to the experimental or control group and was unaware of the results of the subject s first test procedures . the behavioral auditory processing evaluation was carried out with headphones while subjects wore their hearing aids , since all of them were fitted with in - the - ear ( ite ) hearing aids . as central auditory processing evaluation in hearing aid subjects is a controversial issue , tests were included that have been shown to be more robust in the presence of peripheral auditory damage.5,7 the test battery was designed to ensure maximum use of clinician time during administration as well as to limit the time a subject was exposed to testing procedures , thus offering subjects an optimal opportunity to do well . memory for verbal sounds in sequence : the patient was instructed to repeat the syllables pa , ta , ca , fa in three different sequences without any visual clues . memory for nonverbal sounds in sequence : the patient was asked to reproduce three sequences of four instrumental sounds without any visual clues . all tests , except sound localization and memory for verbal and nonverbal sounds in sequence , which were performed in a sound field using musical instruments , were performed using recorded stimuli from a commercially available brazilian compact disc ( cd)8 . as the subjects of our study exhibited nearly normal aided hearing thresholds , their performance was compared to the expected levels for subjects with normal hearing . the aphab was used to study the subjective effectiveness of the auditory training.10 this instrument has been translated and validated in portuguese.11 aphab is a self - assessment questionnaire used to quantify auditory difficulties experienced in daily situations involving communication in quiet , noisy and reverberant environments . all subjects completed an aphab in two different situations , before and after auditory training in the experimental group , and as initial and final evaluations in the control group . our formal auditory training program was organized into eight one - hour sessions , held twice a week for four weeks . right and left ears were trained separately in an attempt to compensate for interaural differences usually observed in behavioral auditory processing tests , except during binaural integration activities and temporal processing training , when sound field presentation was used . the behavioral auditory processing evaluation was carried out with headphones while subjects wore their hearing aids , since all of them were fitted with in - the - ear ( ite ) hearing aids . as central auditory processing evaluation in hearing aid subjects is a controversial issue , tests were included that have been shown to be more robust in the presence of peripheral auditory damage.5,7 the test battery was designed to ensure maximum use of clinician time during administration as well as to limit the time a subject was exposed to testing procedures , thus offering subjects an optimal opportunity to do well . based on these design elements , the following procedures were selected : sound localization : the patient was instructed to identify the origin of the instrumental sound in five directions ( right , left , above the head , in front of the head and behind the head ) with her / his eyes closed . memory for verbal sounds in sequence : the patient was instructed to repeat the syllables pa , ta , ca , fa in three different sequences without any visual clues . memory for nonverbal sounds in sequence : the patient was asked to reproduce three sequences of four instrumental sounds without any visual clues . all tests , except sound localization and memory for verbal and nonverbal sounds in sequence , which were performed in a sound field using musical instruments , were performed using recorded stimuli from a commercially available brazilian compact disc ( cd)8 . as the subjects of our study exhibited nearly normal aided hearing thresholds , their performance was compared to the expected levels for subjects with normal hearing . the aphab was used to study the subjective effectiveness of the auditory training.10 this instrument has been translated and validated in portuguese.11 aphab is a self - assessment questionnaire used to quantify auditory difficulties experienced in daily situations involving communication in quiet , noisy and reverberant environments . all subjects completed an aphab in two different situations , before and after auditory training in the experimental group , and as initial and final evaluations in the control group . our formal auditory training program was organized into eight one - hour sessions , held twice a week for four weeks . right and left ears were trained separately in an attempt to compensate for interaural differences usually observed in behavioral auditory processing tests , except during binaural integration activities and temporal processing training , when sound field presentation was used . all data obtained from the behavioral central auditory processing test battery , electrophysiological evaluation and aphab were statistically analyzed . to investigate the effects of the fat program , the student t - test was used to compare the performance variance of subjects from both control and experimental groups , in both evaluations ( pre- and post - training ) , considering behavioral auditory processing tests , the electrophysiological test and the aphab . table 2 shows the amount of improvement in behavioral auditory processing tests of the control group comparing pre- and post - training evaluations . no significant differences in performance were observed for the majority of behavioral tests , expect for ssi - icm snrs -10 and -15 , which revealed better results post - training . table 3 shows the amount of improvement in behavioral auditory processing tests of the experimental group by comparing pre- and post - training evaluations . significant differences in performance of the experimental group were observed when comparing the tests results pre- and post - training , demonstrating improved performance following training . table 4 ( variation in latency and amplitude values of llaep for the control group when pre- and post - training evaluations were compared ) shows that significant differences were observed for the latencies of p2 and n2 llaep components . for p2 an increase in latency was observed , while for n2 a decrease in latency was verified when comparing pre- and post - training evaluations . variation in latency and amplitude values of llaep of the experimental group when comparing pre- and post - training evaluations ) it is easily seen that for the experimental group , a significantly lower latency was observed for the p3 component of llaep when comparing pre- and post - training evaluations . as with the control group , no significant differences were seen concerning latency . finally , in table 6 ( benefit observed in aphab of control and experimental groups when comparing pre- and post - training administration ) , we present the comparison of benefit observed through the administration of the aphab to the control and experimental groups . no differences were noted for the control group , while there was a trend toward statistical significance for the experimental group in the reverberation and background noise sub - scales . all patients exhibited abnormal results on at least one of the tests during the pre - training evaluation . prior to the auditory training program , performance in the control group was generally poorer than in the experimental group the limited amount of improvement in behavioral tests by the control group ( table 2 ) suggest that all behavioral tests used , except ssi - icm ( -10 and -15 ) , were of minimal value . interestingly poorer results were seen during the post - training evaluation after some tests , including word recognition scores and the speech in noise test . when considering ssi - icm ( -10 and -15 ) , significant differences were observed between pre- and post - training evaluations . this has occurred in one previous study , where the control groups , without training , showed some improvements during the re - evaluations.15 however , these improvements were not of the same magnitude as those observed in the groups which underwent auditory training . the performance of the experimental group during the post - training evaluation was better than the first evaluation ( table 3 ) , since the comparison between post- and pre - training performance resulted in positive values for all tests . statistically significant differences were observed in sound localization , memory for nonverbal sounds in sequence , the speech in noise test , dichotic digits and ssi - icm ( 0 , -10 , -15 ) , with ssi - icm ( -15 ) demonstrating an improvement of almost 70% . this is interpreted to mean that formal auditory training was effective in improving central auditory skills.1618 the post - training evaluation of the experimental group was within established limits for adults with normal hearing . the results indicate that amplification provided by the hearing aids was insufficient to maintain the same quality in both quiet and noisy environments ( table 2 ) . finally , it is possible that hearing aid fitting alone fails to produce the ideal environment for the auditory system and its skills.2,6,19,20 when a central auditory processing disorder co - exists with peripheral hearing loss , compensation obtained from hearing aids is at times insufficient to compensate for the auditory processing disorder ( apd ) , and the patient may become dissatisfied and frustrated with the performance of the hearing aids.2,19 based on our results , auditory processing evaluations although there were no significant differences between right and left ears for either group on any of the experimental measures , individual results for some patients in both the control and experimental groups demonstrated asymmetrical results in central auditory processing tests , such as dichotic digits , speech in noise and ssi - icm . such differences were minimized for subjects in the experimental group after the completion of training . the phenomenon of binaural interference is suspected when performance in speech tests with binaural amplification is worse than with monaural or without hearing aids.4,6,2123 in cases of binaural interference , a unilateral hearing aid fitting is advisable . it is important to consider that in the case of a monaural fitting , the unaided ear may suffer the deleterious effects of sensory deprivation , affecting neural plasticity and leading to a progressive degradation of word recognition on the non - aided side . this can occur even when auditory thresholds are stable.14,24 as discussed above , one could question whether it would be more reasonable to document asymmetry in a patient with a central auditory processing disorder and then fit both ears to maximize the advantages of binaural hearing . after waiting a reasonable acclimatization period , the patient would be enrolled in a formal auditory training program , at the end of which the decision would be made as to whether a binaural or a monaural hearing aid fitting is more suitable . one of the most controversial aspects of formal auditory training is the use of the same test for training and evaluation . nonetheless , our results suggest that generalization in non - trained contexts is possible since , in the tests used only for evaluation , including sound localization , memory for verbal and nonverbal sounds and the speech in noise tests , objective and subjective measures showed improved results for the experimental group after training . generalization for non - trained situations has been mentioned in several previous studies.16 , 2528 the maintenance of any learned pattern depends on its use.27,29 therefore , it seems reasonable to conclude that benefits accrued from the auditory training program will be maintained if the patient continues to use what he or she has learned . these results concur with those of previous authors , who have noted that the presence of peripheral hearing loss , especially to mild and moderate degrees ( which is the case in this study ) , does not prevent the registration of late potentials , since the patient is able to perceive both rare and frequent stimuli.3032 however , also mentioned in other studies is considerable inter - subject variability for both latency and amplitude values.15,27,37 statistical analysis revealed no significant differences between right and left ears . however , when comparing the variation in latencies values between pre and post training evaluations of the control group ( table 4 ) , significant changes were observed for p2 and n2 latencies . for the experimental group , a statistically significant reduction in p3 latency ( 27 ms ) was observed when comparing latency between the pre- and post - training electrophysiological evaluation ( table 5 ) . although no statistical differences were demonstrated in p3 latency for the control group , a slight increase in latency of 6.43 ms was noted ( table 4 ) . this improvement may be attributed to the auditory training , as the training was carried out after the acclimatization period.11,14,35,36 finally , as an objective measure of neural change , the improvement appears to influence neural plasticity in adults , as mentioned in a number of other studies.6,28,29,34,3739 since the importance of temporal aspects in maximizing auditory skills is well recognized , the latency reduction observed in the experimental group may be of critical importance for improving communication for hearing aid users , especially in adverse environments . this improvement , along with advanced hearing aid technology , may help patients in daily situations , leading to improved use of hearing aids and better social integration . no statistically significant differences were observed in the variation of amplitude between the pre- and post - training evaluations for both groups ( tables 4 and 5 ) . the increase in amplitude , especially of p3 after auditory training , suggests better synchrony in neural firing and attention improvement and has also been shown to be related to neural plasticity.15,27,28,34,38,40 there is still controversy regarding which parameter , latency or amplitude , is the better indicator of neurophysiologic improvement when determining the efficacy of a specific therapeutic approach.33 we conclude that amplitude , as measured in the present study , does not allow for inferences about the efficiency of the auditory training program described here . our results were the same as those previously noted in different studies in the literature.27,28,31,38 the results from this portion of the study suggest that the behavioral and electrophysiological improvements observed in the experimental group after training support central changes and improved functioning and highlight the importance of establishing the interaction between the central auditory nervous system and amplified sound , as previously recommended.36,39 the aphab self - assessment questionnaire was selected to help determine whether the expected / observed changes in electrophysiological and behavioral evaluations would interfere with the patient s subjective evaluation . there does not appear to be a specific self - assessment questionnaire designed to quantify changes observed in adult hearing aid users following a specific auditory training program . however , aphab has been shown to be a powerful instrument for recording the benefit of a specific therapeutic approach.41 thus , we felt it the most appropriate self - assessment questionnaire for our study . self - assessment questionnaires have played a very important role in the validation stage of hearing aid fitting , as they reveal the benefits perceived in daily situations with amplification.35 the similarity of responses for both groups regarding the ease of communication sub - scale ( ec ) indicates that the patients were well - adapted to their hearing aids , mainly in silent environments and/or in conversations involving small groups in relatively quiet places . however , the vast majority complained of difficulties hearing in noisy environments ( table 6 ) . no significant differences were observed in the control group at all sub - scales , when comparing the first and second applications . this suggests that reported difficulties remained stable , as previously reported.42 however for the experimental group , communicating in noisy and reverberant environments was easier than before the training . table 6 shows the comparison of percentage of difficulties experienced by the participants in the sub - scales of aphab for both applications of the questionnaires , pre- and post - training . although the differences observed in the experimental group in reverberation and background noise sub - scales only trended toward statistical significance , all sub - scales revealed negative values , indicating that after auditory training participants in the experimental group reported fewer difficulties in daily situations . this means that communication in adverse environments became easier for the experimental group following auditory training . in another study , 90% of adult hearing aid users felt more capable and more confident in challenging auditory situations after auditory training with a commercial available cd - rom.2 the opposite occurred in the control group , where positive values were seen in the ease of communication , background noise and averseness to loud sound sub - scales ( table 6 ) . improvements for the experimental group were previously noticed in electrophysiological and behavioral evaluations ( tables 2 to 5 ) . although subjects in both groups were similar in peripheral hearing status and hearing aid technology , environmental differences are expected and may account for differences in subjective measures such as self - assessment questionnaires . communication improvement in noisy environments should be the primary goal of any auditory training program , especially since such environments are common in everyday life . however , there is still the chance of having a subject whose hearing thresholds have been made normal but who has an auditory processing disorder . it is suggested that administering an auditory training program , as described in the present study , and a self - assessment questionnaire , such as the aphab , could make the assessment and identification of hearing - impaired patients with an additional auditory processing disorder easier . the results of the present study suggest that formal auditory training was able to improve the central auditory skills of hearing aid users . therefore , we strongly advocate investing time searching for the presence of a central auditory processing disorder during the course of all hearing aid fittings . in many cases , the high cost of hearing aids prevents patients from acquiring them , particularly if the patient feels that the benefit will not outweigh the high cost of the investment . including an initial auditory processing evaluation and formal auditory training should provide a basis for such patients to feel more confident about purchasing a sophisticated and expensive hearing aid . audiologists play an important role in this situation by helping patients realize the importance of auditory training and introducing it as a part of the hearing aid fitting process . after a critical analysis of the results , we can conclude that formal auditory training in adult hearing aid users promotes : latency reduction of the p3 component of llaep . improvement in auditory skills for sound localization , memory for nonverbal sounds in sequence , auditory closure and figure - to - ground for verbal sounds .

however , hearing aids are not always capable of helping the patient compensate for difficulties in understanding speech , particularly in a reverberant and/or noisy environment.1 even the most sophisticated hearing aids are unable to improve auditory skills or the comprehension needed for efficient communication . hearing aids provide increased acoustic information , but alone they are not able to directly modify the brain or the patient s behavior.2 furthermore , peripheral hearing loss may coexist with a central auditory processing deficit , thereby contributing to patient dissatisfaction with amplification , even when audiological tests indicate nearly normal aided hearing thresholds . auditory training has been highlighted as part of habilitation / rehabilitation for the hearing impaired , though it has been underutilized . however , advances in neuroscience suggest that it is the central auditory system that benefits from auditory stimulation.24 a number of authors have concluded that the auditory processing evaluation of hearing aid candidates / users provides a more complete profile of auditory skills and may be useful in choosing between different amplification options and complementary remediation tools.3,5,6 therefore , auditory processing tests could contribute to and complement the classical peripheral auditory evaluation . unfortunately , there do not appear to be any studies in the literature that involve behavioral and electrophysiological measurements of central auditory processing , formal auditory training and self - assessment outcome measures in adult hearing aid users . the aim of this study was to examine the effects of formal auditory training on adult binaural hearing aid users with mild to moderate bilateral sensorineural hearing loss using the following three procedures : behavioral tests of auditory processing , long - latency auditory evoked potentials and a self - assessment questionnaire . behavioral tests of auditory processing were employed to identify and monitor any changes in the central auditory system as a result of the auditory training program . long - latency auditory evoked potentials ( llaep ) were used to plan and monitor rehabilitation outcomes in specific populations . since llaeps are not influenced by the presence of hearing loss , especially in mild and moderate hearing losses , hearing - impaired subjects may be accurately evaluated with this procedure . finally , the inclusion of a self - assessment questionnaire provided the basis for evaluating the success or failure of the auditory training program from an important , objective source the patient . fourteen subjects were randomly divided into two equal groups : an experimental group ( with auditory training ) and control group ( without auditory training ) . all subjects , regardless of group placement , received a behavioral auditory processing evaluation and an electrophysiological evaluation and were asked complete a self - assessment questionnaire , the abbreviated profile of hearing aid benefit ( aphab ) . these procedures were performed before and after the auditory training for the experimental group and at times coinciding with the beginning and end of the study for the control group . in order to characterize this project as a double - blind study , the examiner who performed the second evaluation did not know whether the subject belonged to the experimental or control group and was unaware of the results of the subject s first test procedures . the behavioral auditory processing evaluation was carried out with headphones while subjects wore their hearing aids , since all of them were fitted with in - the - ear ( ite ) hearing aids . as central auditory processing evaluation in hearing aid subjects is a controversial issue , tests were included that have been shown to be more robust in the presence of peripheral auditory damage.5,7 the test battery was designed to ensure maximum use of clinician time during administration as well as to limit the time a subject was exposed to testing procedures , thus offering subjects an optimal opportunity to do well . based on these design elements , the following procedures were selected : sound localization : the patient was instructed to identify the origin of the instrumental sound in five directions ( right , left , above the head , in front of the head and behind the head ) with her / his eyes closed . memory for verbal sounds in sequence : the patient was instructed to repeat the syllables pa , ta , ca , fa in three different sequences without any visual clues . speech - in - noise test : two different lists of 25 monosyllables were presented to each ear in the presence of ipsilateral white noise using a signal - to - noise ratio ( snr ) of + 5 , and the patient was asked to repeat each monosyllable while ignoring the noise . synthetic sentence identification ( ssi ) : the patient was instructed to point to the sentence presented in the headphones on a chart displayed in front of him / her while ignoring the competitive stimuli represented by a verbal discourse . as the subjects of our study exhibited nearly normal aided hearing thresholds , their performance was compared to the expected levels for subjects with normal hearing . the electrophysiological evaluation in this study consisted of measures of long - latency auditory evoked potentials n1 , p2 , n2 and p3 , registered without hearing aids using an odd - ball paradigm with tone bursts . the intensity level varied from 70 to 85 db hl , according to the residual hearing in the test frequencies involved.9 a four - channel biologic systems unit was used to measure the llaep recordings . the latencies and amplitudes of the n1 , p2 and n2 components were marked in the rare tracings . the aphab was used to study the subjective effectiveness of the auditory training.10 this instrument has been translated and validated in portuguese.11 aphab is a self - assessment questionnaire used to quantify auditory difficulties experienced in daily situations involving communication in quiet , noisy and reverberant environments . all subjects completed an aphab in two different situations , before and after auditory training in the experimental group , and as initial and final evaluations in the control group . as subjects were already wearing hearing aids in both situations , patients were asked to answer the questionnaire using only the column corresponding to with hearing aids . right and left ears were trained separately in an attempt to compensate for interaural differences usually observed in behavioral auditory processing tests , except during binaural integration activities and temporal processing training , when sound field presentation was used . the stimulation paradigm of each ear was as follows : the intensity level was fixed for the ear under training while the contralateral intensity level was increased ( snr from positive to negative ) , and the patient s task was to pay attention to the stimuli delivered to the ear under training while ignoring the contralateral messages . the behavioral auditory processing evaluation was carried out with headphones while subjects wore their hearing aids , since all of them were fitted with in - the - ear ( ite ) hearing aids . as central auditory processing evaluation in hearing aid subjects is a controversial issue , tests were included that have been shown to be more robust in the presence of peripheral auditory damage.5,7 the test battery was designed to ensure maximum use of clinician time during administration as well as to limit the time a subject was exposed to testing procedures , thus offering subjects an optimal opportunity to do well . based on these design elements , the following procedures were selected : sound localization : the patient was instructed to identify the origin of the instrumental sound in five directions ( right , left , above the head , in front of the head and behind the head ) with her / his eyes closed . memory for verbal sounds in sequence : the patient was instructed to repeat the syllables pa , ta , ca , fa in three different sequences without any visual clues . speech - in - noise test : two different lists of 25 monosyllables were presented to each ear in the presence of ipsilateral white noise using a signal - to - noise ratio ( snr ) of + 5 , and the patient was asked to repeat each monosyllable while ignoring the noise . synthetic sentence identification ( ssi ) : the patient was instructed to point to the sentence presented in the headphones on a chart displayed in front of him / her while ignoring the competitive stimuli represented by a verbal discourse . as the subjects of our study exhibited nearly normal aided hearing thresholds , their performance was compared to the expected levels for subjects with normal hearing . the electrophysiological evaluation in this study consisted of measures of long - latency auditory evoked potentials n1 , p2 , n2 and p3 , registered without hearing aids using an odd - ball paradigm with tone bursts . the intensity level varied from 70 to 85 db hl , according to the residual hearing in the test frequencies involved.9 a four - channel biologic systems unit was used to measure the llaep recordings . the latencies and amplitudes of the n1 , p2 and n2 components were marked in the rare tracings . the aphab was used to study the subjective effectiveness of the auditory training.10 this instrument has been translated and validated in portuguese.11 aphab is a self - assessment questionnaire used to quantify auditory difficulties experienced in daily situations involving communication in quiet , noisy and reverberant environments . all subjects completed an aphab in two different situations , before and after auditory training in the experimental group , and as initial and final evaluations in the control group . the stimulation paradigm of each ear was as follows : the intensity level was fixed for the ear under training while the contralateral intensity level was increased ( snr from positive to negative ) , and the patient s task was to pay attention to the stimuli delivered to the ear under training while ignoring the contralateral messages . to investigate the effects of the fat program , the student t - test was used to compare the performance variance of subjects from both control and experimental groups , in both evaluations ( pre- and post - training ) , considering behavioral auditory processing tests , the electrophysiological test and the aphab . table 2 shows the amount of improvement in behavioral auditory processing tests of the control group comparing pre- and post - training evaluations . no significant differences in performance were observed for the majority of behavioral tests , expect for ssi - icm snrs -10 and -15 , which revealed better results post - training . table 3 shows the amount of improvement in behavioral auditory processing tests of the experimental group by comparing pre- and post - training evaluations . significant differences in performance of the experimental group were observed when comparing the tests results pre- and post - training , demonstrating improved performance following training . table 4 ( variation in latency and amplitude values of llaep for the control group when pre- and post - training evaluations were compared ) shows that significant differences were observed for the latencies of p2 and n2 llaep components . variation in latency and amplitude values of llaep of the experimental group when comparing pre- and post - training evaluations ) it is easily seen that for the experimental group , a significantly lower latency was observed for the p3 component of llaep when comparing pre- and post - training evaluations . finally , in table 6 ( benefit observed in aphab of control and experimental groups when comparing pre- and post - training administration ) , we present the comparison of benefit observed through the administration of the aphab to the control and experimental groups . no differences were noted for the control group , while there was a trend toward statistical significance for the experimental group in the reverberation and background noise sub - scales . prior to the auditory training program , performance in the control group was generally poorer than in the experimental group the limited amount of improvement in behavioral tests by the control group ( table 2 ) suggest that all behavioral tests used , except ssi - icm ( -10 and -15 ) , were of minimal value . interestingly poorer results were seen during the post - training evaluation after some tests , including word recognition scores and the speech in noise test . when considering ssi - icm ( -10 and -15 ) , significant differences were observed between pre- and post - training evaluations . this has occurred in one previous study , where the control groups , without training , showed some improvements during the re - evaluations.15 however , these improvements were not of the same magnitude as those observed in the groups which underwent auditory training . the performance of the experimental group during the post - training evaluation was better than the first evaluation ( table 3 ) , since the comparison between post- and pre - training performance resulted in positive values for all tests . statistically significant differences were observed in sound localization , memory for nonverbal sounds in sequence , the speech in noise test , dichotic digits and ssi - icm ( 0 , -10 , -15 ) , with ssi - icm ( -15 ) demonstrating an improvement of almost 70% . this is interpreted to mean that formal auditory training was effective in improving central auditory skills.1618 the post - training evaluation of the experimental group was within established limits for adults with normal hearing . this was confirmed by the results of the present study during the speech in noise test , in which the subjects performance was negatively influenced by the introduction of noise . finally , it is possible that hearing aid fitting alone fails to produce the ideal environment for the auditory system and its skills.2,6,19,20 when a central auditory processing disorder co - exists with peripheral hearing loss , compensation obtained from hearing aids is at times insufficient to compensate for the auditory processing disorder ( apd ) , and the patient may become dissatisfied and frustrated with the performance of the hearing aids.2,19 based on our results , auditory processing evaluations although there were no significant differences between right and left ears for either group on any of the experimental measures , individual results for some patients in both the control and experimental groups demonstrated asymmetrical results in central auditory processing tests , such as dichotic digits , speech in noise and ssi - icm . it is important to consider that in the case of a monaural fitting , the unaided ear may suffer the deleterious effects of sensory deprivation , affecting neural plasticity and leading to a progressive degradation of word recognition on the non - aided side . after waiting a reasonable acclimatization period , the patient would be enrolled in a formal auditory training program , at the end of which the decision would be made as to whether a binaural or a monaural hearing aid fitting is more suitable . one of the most controversial aspects of formal auditory training is the use of the same test for training and evaluation . nonetheless , our results suggest that generalization in non - trained contexts is possible since , in the tests used only for evaluation , including sound localization , memory for verbal and nonverbal sounds and the speech in noise tests , objective and subjective measures showed improved results for the experimental group after training . generalization for non - trained situations has been mentioned in several previous studies.16 , 2528 the maintenance of any learned pattern depends on its use.27,29 therefore , it seems reasonable to conclude that benefits accrued from the auditory training program will be maintained if the patient continues to use what he or she has learned . concerning llaep , the n1-p2-n2 and p3 complexes were identified in all patients from both the control and experimental groups , and all subjects exhibited latency values within normal limits . these results concur with those of previous authors , who have noted that the presence of peripheral hearing loss , especially to mild and moderate degrees ( which is the case in this study ) , does not prevent the registration of late potentials , since the patient is able to perceive both rare and frequent stimuli.3032 however , also mentioned in other studies is considerable inter - subject variability for both latency and amplitude values.15,27,37 statistical analysis revealed no significant differences between right and left ears . however , when comparing the variation in latencies values between pre and post training evaluations of the control group ( table 4 ) , significant changes were observed for p2 and n2 latencies . for the experimental group , a statistically significant reduction in p3 latency ( 27 ms ) was observed when comparing latency between the pre- and post - training electrophysiological evaluation ( table 5 ) . in many cases , this change may precede a behavioral change , which could take longer since it requires the integration of neural modifications and conscious perception and also involves higher cognitive processes.15,33,34 furthermore , latency reduction may be understood as an improvement in electrophysiological function . this improvement may be attributed to the auditory training , as the training was carried out after the acclimatization period.11,14,35,36 finally , as an objective measure of neural change , the improvement appears to influence neural plasticity in adults , as mentioned in a number of other studies.6,28,29,34,3739 since the importance of temporal aspects in maximizing auditory skills is well recognized , the latency reduction observed in the experimental group may be of critical importance for improving communication for hearing aid users , especially in adverse environments . no statistically significant differences were observed in the variation of amplitude between the pre- and post - training evaluations for both groups ( tables 4 and 5 ) . the increase in amplitude , especially of p3 after auditory training , suggests better synchrony in neural firing and attention improvement and has also been shown to be related to neural plasticity.15,27,28,34,38,40 there is still controversy regarding which parameter , latency or amplitude , is the better indicator of neurophysiologic improvement when determining the efficacy of a specific therapeutic approach.33 we conclude that amplitude , as measured in the present study , does not allow for inferences about the efficiency of the auditory training program described here . our results were the same as those previously noted in different studies in the literature.27,28,31,38 the results from this portion of the study suggest that the behavioral and electrophysiological improvements observed in the experimental group after training support central changes and improved functioning and highlight the importance of establishing the interaction between the central auditory nervous system and amplified sound , as previously recommended.36,39 the aphab self - assessment questionnaire was selected to help determine whether the expected / observed changes in electrophysiological and behavioral evaluations would interfere with the patient s subjective evaluation . there does not appear to be a specific self - assessment questionnaire designed to quantify changes observed in adult hearing aid users following a specific auditory training program . however , aphab has been shown to be a powerful instrument for recording the benefit of a specific therapeutic approach.41 thus , we felt it the most appropriate self - assessment questionnaire for our study . self - assessment questionnaires have played a very important role in the validation stage of hearing aid fitting , as they reveal the benefits perceived in daily situations with amplification.35 the similarity of responses for both groups regarding the ease of communication sub - scale ( ec ) indicates that the patients were well - adapted to their hearing aids , mainly in silent environments and/or in conversations involving small groups in relatively quiet places . as the subjects in our study wore hearing aids in both situations when answering the aphab , the magnitude of benefit should not be the same as was seen during the original application of this instrument . table 6 shows the comparison of percentage of difficulties experienced by the participants in the sub - scales of aphab for both applications of the questionnaires , pre- and post - training . although the differences observed in the experimental group in reverberation and background noise sub - scales only trended toward statistical significance , all sub - scales revealed negative values , indicating that after auditory training participants in the experimental group reported fewer difficulties in daily situations . in another study , 90% of adult hearing aid users felt more capable and more confident in challenging auditory situations after auditory training with a commercial available cd - rom.2 the opposite occurred in the control group , where positive values were seen in the ease of communication , background noise and averseness to loud sound sub - scales ( table 6 ) . it is suggested that administering an auditory training program , as described in the present study , and a self - assessment questionnaire , such as the aphab , could make the assessment and identification of hearing - impaired patients with an additional auditory processing disorder easier . the results of the present study suggest that formal auditory training was able to improve the central auditory skills of hearing aid users . therefore , we strongly advocate investing time searching for the presence of a central auditory processing disorder during the course of all hearing aid fittings . after a critical analysis of the results , we can conclude that formal auditory training in adult hearing aid users promotes : latency reduction of the p3 component of llaep .

in addition to the influence of glp-1based medications on those outcomes that typically determine the morbidity and mortality of patients with type 2 diabetes , i.e. , that affect large proportions of such patients , there may be additional safety concerns of special interest . some signals have suggested an untoward influence of such treatment on the risk for certain rare conditions . for glp-1 receptor agonists and for dpp-4 inhibitors , these events of special interest are pancreatitis , pancreatic cancer , and thyroid carcinoma ( table 1 ) . in addition , possible consequences of a rise in pulse rate with glp-1 receptor agonists need to be discussed . contrasting clinical benefits and improved outcomes with adverse outcomes / risks associated with the use of incretin - based glucose - lowering medications ( a , glp-1 receptor agonists ; b , inhibitors of dpp-4 ) cases of pancreatitis have been observed in animals ( 6,7,13 ) and patients ( 14 ) treated with incretin mimetics and dpp-4 inhibitors ( 5 ) . the questions are whether pancreatitis occurs more often in association with treatment using glp-1based medications , and whether it is causally related to such treatment . animal studies describe histological changes compatible with damage to the exocrine pancreas with exenatide ( 6,7 ) and sitagliptin ( 5 ) . a similar study examining liraglutide did not confirm such damages induced by an incretin mimetic ( 13 ) . other studies find an amelioration of the course of experimentally induced acute pancreatitis in mice with exenatide ( 15 ) or an anti - inflammatory pattern of cytokines induced by liraglutide treatment ( 16 ) . another open question is whether these findings are representative of human acute or chronic pancreatitis . attempts to quantify the number of pancreatitis events while patients are treated with glp-1 receptor agonists or dpp-4 inhibitors have found odds ratios ( ors ) around 1 , however with relatively wide cis ( fig . such data have been taken from claims databases and correlating the prescription of drugs used to treat diabetes with a diagnosis of acute pancreatitis . these analyses have made it clear that obese , type 2 diabetic subjects are more prone to developing acute pancreatitis than the nondiabetic population . on the other hand , one single study reports a more than tenfold excess of pancreatitis in patients using exenatide or sitagliptin ( 22 ) . food and drug administration ( fda ) adverse event reporting system ( faers [ formerly known as aers ] ) . it summarizes reports to the faers from a period when publications and changes to drug labels had alerted the medical community to the fact that cases of acute pancreatitis had occurred in patients treated with exenatide and sitagliptin . the standards for the diagnosis of acute pancreatitis ( at least two out of three criteria : 1 ) typical severe abdominal pain , 2 ) elevations in pancreas - specific enzymes such as amylase and lipase , and 3 ) typical findings using appropriate imaging procedures ) may not always have been applied . ors for the diagnosis of ( a and c ) or hospitalization for ( b and d ) acute pancreatitis in association with a medication of exenatide ( glp-1 receptor agonist , upper panels ) or sitagliptin ( dpp-4 inhibitor , lower panels ) . ors and their 95% cis and related p values were obtained directly or calculated ( graphpad prism 5.02 ) from published analysis of claims databases . data have been taken from the references quoted in the figure ( 1721 , 24 , 50 ) . recent exposures : medication prescribed for use between 2 years and 30 days before hospitalization ; current exposures : medication prescribed for use < 30 days before hospitalization . a recent case - control study reported a higher or for the risk of hospitalization for a diagnosis of pancreatitis in patients taking incretin - based medications , since a separate analysis for the use of exenatide ( glp-1 receptor agonist ) or sitagliptin ( dpp-4 inhibitor ) did not yield significant findings ( fig . it can not be excluded at present that a potential combination of stomach cramps , representing gastrointestinal adverse events of glp-1 receptor agonists , and spontaneously elevated serum lipase activities were responsible for the hospitalizations and do not represent true pancreatitis episodes . nevertheless , this small study analyzing only a few patients with pancreatitis is only the second study describing an elevated risk ( 24 ) , however , only by combining exenatide and sitagliptin treatments as incretin - based medications and after adjusting for potential confounders . figure 1b and d presents ors and p values ( all nonsignificant ) calculated without adjustment for potential risk - modifying factors . furthermore , treatment with the glp-1 receptor agonist liraglutide can lead to elevations in lipase without associated symptoms of pancreatitis ( 25 ) . using such enzyme measurements to screen for pancreatitis may have resulted in false diagnoses of pancreatitis because elevations in pancreatic enzymes do not have the degree of specificity that would be necessary to make it a helpful screening instrument . indeed , elevated lipase and amylase activity is found quite frequently in patients with type 2 diabetes with an absence of abdominal pain ( 26 ) . under these circumstances , most elevated amylase or lipase levels would be chance findings without any relationship to inflammatory changes within the exocrine pancreas . however , the nature of the elevation in serum lipase induced by liraglutide treatment needs to be explored so that we can understand its mechanism . at least this phenomenon indicates an interaction of glp-1 receptor agonists with the exocrine pancreas , perhaps indicating the presence of glp-1 receptors in this compartment . effects of glp-1 receptor stimulation on pancreatic enzyme synthesis , potential leakage into the circulation rather than directional secretion into pancreatic digestive juice , and a potential induction of a chronic inflammatory response need to be studied . to date , it certainly can not be taken as a fact that chronic stimulation of the glp-1 receptor ( as occurs during the treatment with incretin mimetics and dpp-4 inhibitors ) induces acute or chronic inflammatory responses in the pancreas , nor that , based on a well - delineated mechanism and supported by convincing epidemiological data , the clinical use of incretin - based glucose - lowering medications would cause pancreatitis . clinically , the development of typical chronic pancreatitis diagnosed because of typical morphological findings and exocrine insufficiency leading to maldigestion , nutritional deficiencies , and weight loss over and above what is expected from continued stimulation of brain glp-1 receptors ( 1,27 ) in patients treated with glp-1based medications has never been described . regarding the related question of chronic changes in the exocrine pancreas leading to pancreatic duct proliferation and the formation of preneoplastic lesions ( like pancreatic intraepithelial neoplasms or pancreatic duct glands ) , data from animal studies are similarly controversial with studies showing alterations of the exocrine pancreatic histology indicative of chronic pancreatitis with exenatide treatment ( 57 ) , while another recent study using liraglutide did describe occasional pancreatitis as a rare finding but not at all related to the dose of liraglutide with similar numbers in placebo - treated rats , mice , and monkeys ( 13 ) . it appears highly unlikely that there should be a difference intrinsic to the two glp-1 receptor agonists used ( exenatide vs. liraglutide ) . a recent finding reported that pancreas specimens from organ donors with type 2 diabetes , who had received treatment with the dpp-4 inhibitor sitagliptin ( n = 7 ) or exenatide ( n = 1 ) , relative to patients with type 2 diabetes treated with other agents , had marked -cell hyperplasia , -cells coexpressing insulin and glucagon , hyperplasia of -cells expressing glucagon , increased expression of proliferation markers , and an increased prevalence of preneoplastic lesions ( 29 ) . this finding needs to be confirmed in a larger , representative sample of pancreas specimens obtained without preceding long - term critical illness , which alone may be responsible for some proliferative responses ( 30 ) . to put the state of this present discussion into perspective , it should be made clear that at most early proliferative or preneoplastic changes have been observed , which as such are not proof that eventually the process described will give rise to pancreatic cancer . thus we have to discuss a potential risk ( and certainly want to learn more about the long - term consequences of stimulating glp-1 receptors for the exocrine pancreas ) , but not an actual threat to patients treated with incretin - based medications , based on a well - characterized mechanism with a risk clearly elevated based on sound epidemiological analyses . it is reassuring that no case of clinically evident chronic pancreatitis has been described after initiating treatment with incretin - based medications . certainly , there is also no case report of pancreatic cancer diagnosed after exposing a patient to glp-1 receptor agonists or dpp-4 inhibitors in a patient in whom there had previously been a morphologically tumor - free pancreas . since pancreatic carcinomas develop slowly ( 31 ) , one would probably not expect to see such a case after at most a few years of treatment , considering the recent introduction of the incretin - based medications , even if there were such a long - term risk . glp-1 receptor agonists have the potential to induce proliferative changes in rodent thyroid c cells . liraglutide increased the number of cases with c - cell hyperplasia , adenomas , and medullary thyroid carcinomas in mice and rats ( 9 ) . in these species such abnormalities are also found spontaneously , i.e. , in the absence of glp-1 receptor stimulation , especially in male rats , in which medullary thyroid carcinoma developed in some animals treated with placebo ( 9 ) . accordingly , rodent c - cell lines in cell culture responded to glp-1 , exenatide , and liraglutide with acutely producing cyclic amp and secreting calcitonin ( 9 ) . similar cell lines of human origin do not show such acute responses when glp-1 receptors are stimulated ( 9 ) . whereas rodent c - cell lines are equipped with glp-1 receptors at a high level of expression , along the same lines , long - term treatment in obese human subjects with high liraglutide doses up to 3-mg per day does not lead to elevations in plasma calcitonin ( 32 ) . based on these results , the ability of glp-1 receptor stimulation to induce proliferative responses in human c cells has been judged as probably absent . medullary thyroid carcinomas are an extremely rare form of thyroid carcinomas in humans ( 33 ) . no case report has been published describing a medullary thyroid carcinoma in a patient receiving a treatment with a glp-1 receptor agonist who prior to such treatment had a morphologically normal thyroid gland and low calcitonin concentrations . given the rare incidence of medullary thyroid carcinoma , 1 ) the consequences of a potential elevation in the risk induced by incretin mimetics would still remain small , and 2 ) to prove or exclude such a relationship , efficient surveillance of extremely large numbers of patients would be needed . the elevated risk for thyroid carcinoma in more general terms described in the study exploring the faers database ( 22 ) is difficult to reconcile . similar reservations apply regarding reporting bias as mentioned for the pancreatitis / pancreatic carcinoma issue raised earlier ( vide supra ) . certainly , this would not be compatible with an explanation through a higher number of medullary carcinomas alone , which would need to increase by more than 30-fold in order to explain such numbers . however , whether follicular cells express glp-1 receptors ( 9 ) or whether malignant cells from thyroid tumors of different histological varieties ( e.g. , papillary thyroid carcinomas ) express the glp-1 receptors ( 34 ) is controversial and may be related to the specificity of the antibody or the radioligand used for immunohistochemistry ( 35 ) . the fact alone that some papillary thyroid carcinomas may show evidence of glp-1 receptor expression ( 34 ) does not prove that such receptors and their stimulation by drugs may contribute to the genesis or proliferation of such tumors . regarding the thyroid issues , certainly more investigations are required , but one hardly can conclude that , based on current knowledge , there is a definitely increased risk for medullary ( or other types of ) thyroid carcinoma with the use of glp-1 receptor agonists . nevertheless , patients with an individually elevate genetic risk should not be treated with such agents . an elevated risk when using dpp-4 inhibitors does not have to considered at all since no such findings have been reported ( 22 ) . in the absence of large - scale cardiovascular outcome trials , summaries of cardiovascular events reported as adverse events in clinical trials with incretin - based glucose - lowering medications and meta - analyses based thereon ( 36 ) are the best available source of information for an overall judgment at present . phase 3 studies have accrued a number of cardiovascular events sufficient for a preliminary judgment based on trends . these trends observed for the incretin mimetics exenatide ( 37 ) and liraglutide ( 38 ) as well the dpp-4 inhibitors sitagliptin ( 39 ) , vildagliptin ( 40 ) , saxagliptin ( 41 ) , linagliptin ( 42 ) , and alogliptin ( 43 ) are surprisingly similar . 2 , in all these analyses the relative risk for a combined end point composed of acute myocardial infarction , stroke , and cardiovascular death is reduced with any of the glp-1based medications relative to placebo or comparator treatment to a value below 1 ( fig . however , the 95% cis ranged to above 1.0 with most compounds , indicating that the number of events available for this analysis was too small to allow the definite conclusion of a significant improvement in cardiovascular prognosis with incretin - based glucose - lowering treatment . relative risk for major cardiovascular events reported as adverse events during phase 3 studies with the glp-1 receptor agonists exenatide and liraglutide ( upper panel ) and with the dpp-4 inhibitors sitagliptin , vildagliptin , saxagliptin , alogliptin , and linagliptin ( lower panel ) compared with pooled comparators ( placebo or active control medications ) . data have been taken from the references quoted in the figure ( 3743 ) . a potential reduction in cardiovascular event rates with linagliptin treatment is further supported by a recent study comparing linagliptin with the sulfonylurea glimepiride ( 44 ) . there is some plausibility based on the influences of glp-1based drugs on cardiovascular risk factors ( 45 ) . they also reduce systolic blood pressure by 25 mmhg , mechanistically explained by improved endothelial function and vasodilation , enhanced natriuresis , and fluid excretion . there is a potential for a reduction in postprandial triglyceride - rich lipoproteins , especially with those agents that have and preserve over time a prominent effect on gastric emptying . effects on nonclassical cardiovascular risk factors point in the same direction . furthermore , glp-1 receptor stimulation has reduced the extent of myocardial necroses in animal experiments inducing acute myocardial infarction by coronary artery ligation . the results have been surprisingly uniform using different agents ( glp-1 , exenatide , liraglutide , sitagliptin ) in various species ( 45 ) . in addition , in animal models of left ventricular failure , glp-1 and incretin mimetics may increase cardiac output by stimulating glucose and oxygen uptake into the myocardium . clinical pilot trials support the notion that glp-1 receptor stimulation may be beneficial in patients with acute coronary syndrome and chronic congestive heart failure ( 45 ) . therefore , one may be optimistic that cardiovascular outcome trials being performed to date , which will report after the year 2015 ( table 2 ) , will at least confirm cardiovascular safety with a potential to substantiate the beneficial effects in this important respect . cases of pancreatitis have been observed in animals ( 6,7,13 ) and patients ( 14 ) treated with incretin mimetics and dpp-4 inhibitors ( 5 ) . the questions are whether pancreatitis occurs more often in association with treatment using glp-1based medications , and whether it is causally related to such treatment . animal studies describe histological changes compatible with damage to the exocrine pancreas with exenatide ( 6,7 ) and sitagliptin ( 5 ) . a similar study examining liraglutide did not confirm such damages induced by an incretin mimetic ( 13 ) . other studies find an amelioration of the course of experimentally induced acute pancreatitis in mice with exenatide ( 15 ) or an anti - inflammatory pattern of cytokines induced by liraglutide treatment ( 16 ) . another open question is whether these findings are representative of human acute or chronic pancreatitis . attempts to quantify the number of pancreatitis events while patients are treated with glp-1 receptor agonists or dpp-4 inhibitors have found odds ratios ( ors ) around 1 , however with relatively wide cis ( fig . such data have been taken from claims databases and correlating the prescription of drugs used to treat diabetes with a diagnosis of acute pancreatitis . these analyses have made it clear that obese , type 2 diabetic subjects are more prone to developing acute pancreatitis than the nondiabetic population . on the other hand , one single study reports a more than tenfold excess of pancreatitis in patients using exenatide or sitagliptin ( 22 ) . food and drug administration ( fda ) adverse event reporting system ( faers [ formerly known as aers ] ) . it summarizes reports to the faers from a period when publications and changes to drug labels had alerted the medical community to the fact that cases of acute pancreatitis had occurred in patients treated with exenatide and sitagliptin . the standards for the diagnosis of acute pancreatitis ( at least two out of three criteria : 1 ) typical severe abdominal pain , 2 ) elevations in pancreas - specific enzymes such as amylase and lipase , and 3 ) typical findings using appropriate imaging procedures ) may not always have been applied . ors for the diagnosis of ( a and c ) or hospitalization for ( b and d ) acute pancreatitis in association with a medication of exenatide ( glp-1 receptor agonist , upper panels ) or sitagliptin ( dpp-4 inhibitor , lower panels ) . ors and their 95% cis and related p values were obtained directly or calculated ( graphpad prism 5.02 ) from published analysis of claims databases . data have been taken from the references quoted in the figure ( 1721 , 24 , 50 ) . recent exposures : medication prescribed for use between 2 years and 30 days before hospitalization ; current exposures : medication prescribed for use < 30 days before hospitalization . a recent case - control study reported a higher or for the risk of hospitalization for a diagnosis of pancreatitis in patients taking incretin - based medications , since a separate analysis for the use of exenatide ( glp-1 receptor agonist ) or sitagliptin ( dpp-4 inhibitor ) did not yield significant findings ( fig . it can not be excluded at present that a potential combination of stomach cramps , representing gastrointestinal adverse events of glp-1 receptor agonists , and spontaneously elevated serum lipase activities were responsible for the hospitalizations and do not represent true pancreatitis episodes . nevertheless , this small study analyzing only a few patients with pancreatitis is only the second study describing an elevated risk ( 24 ) , however , only by combining exenatide and sitagliptin treatments as incretin - based medications and after adjusting for potential confounders . figure 1b and d presents ors and p values ( all nonsignificant ) calculated without adjustment for potential risk - modifying factors . furthermore , treatment with the glp-1 receptor agonist liraglutide can lead to elevations in lipase without associated symptoms of pancreatitis ( 25 ) . using such enzyme measurements to screen for pancreatitis may have resulted in false diagnoses of pancreatitis because elevations in pancreatic enzymes do not have the degree of specificity that would be necessary to make it a helpful screening instrument . indeed , elevated lipase and amylase activity is found quite frequently in patients with type 2 diabetes with an absence of abdominal pain ( 26 ) . under these circumstances , most elevated amylase or lipase levels would be chance findings without any relationship to inflammatory changes within the exocrine pancreas . however , the nature of the elevation in serum lipase induced by liraglutide treatment needs to be explored so that we can understand its mechanism . at least this phenomenon indicates an interaction of glp-1 receptor agonists with the exocrine pancreas , perhaps indicating the presence of glp-1 receptors in this compartment . effects of glp-1 receptor stimulation on pancreatic enzyme synthesis , potential leakage into the circulation rather than directional secretion into pancreatic digestive juice , and a potential induction of a chronic inflammatory response need to be studied . to date , it certainly can not be taken as a fact that chronic stimulation of the glp-1 receptor ( as occurs during the treatment with incretin mimetics and dpp-4 inhibitors ) induces acute or chronic inflammatory responses in the pancreas , nor that , based on a well - delineated mechanism and supported by convincing epidemiological data , the clinical use of incretin - based glucose - lowering medications would cause pancreatitis . clinically , the development of typical chronic pancreatitis diagnosed because of typical morphological findings and exocrine insufficiency leading to maldigestion , nutritional deficiencies , and weight loss over and above what is expected from continued stimulation of brain glp-1 receptors ( 1,27 ) in patients treated with glp-1based medications has never been described . regarding the related question of chronic changes in the exocrine pancreas leading to pancreatic duct proliferation and the formation of preneoplastic lesions ( like pancreatic intraepithelial neoplasms or pancreatic duct glands ) , data from animal studies are similarly controversial with studies showing alterations of the exocrine pancreatic histology indicative of chronic pancreatitis with exenatide treatment ( 57 ) , while another recent study using liraglutide did describe occasional pancreatitis as a rare finding but not at all related to the dose of liraglutide with similar numbers in placebo - treated rats , mice , and monkeys ( 13 ) . it appears highly unlikely that there should be a difference intrinsic to the two glp-1 receptor agonists used ( exenatide vs. liraglutide ) . a recent finding reported that pancreas specimens from organ donors with type 2 diabetes , who had received treatment with the dpp-4 inhibitor sitagliptin ( n = 7 ) or exenatide ( n = 1 ) , relative to patients with type 2 diabetes treated with other agents , had marked -cell hyperplasia , -cells coexpressing insulin and glucagon , hyperplasia of -cells expressing glucagon , increased expression of proliferation markers , and an increased prevalence of preneoplastic lesions ( 29 ) . this finding needs to be confirmed in a larger , representative sample of pancreas specimens obtained without preceding long - term critical illness , which alone may be responsible for some proliferative responses ( 30 ) . to put the state of this present discussion into perspective , it should be made clear that at most early proliferative or preneoplastic changes have been observed , which as such are not proof that eventually the process described will give rise to pancreatic cancer . thus we have to discuss a potential risk ( and certainly want to learn more about the long - term consequences of stimulating glp-1 receptors for the exocrine pancreas ) , but not an actual threat to patients treated with incretin - based medications , based on a well - characterized mechanism with a risk clearly elevated based on sound epidemiological analyses . it is reassuring that no case of clinically evident chronic pancreatitis has been described after initiating treatment with incretin - based medications . certainly , there is also no case report of pancreatic cancer diagnosed after exposing a patient to glp-1 receptor agonists or dpp-4 inhibitors in a patient in whom there had previously been a morphologically tumor - free pancreas . since pancreatic carcinomas develop slowly ( 31 ) , one would probably not expect to see such a case after at most a few years of treatment , considering the recent introduction of the incretin - based medications , even if there were such a long - term risk . glp-1 receptor agonists have the potential to induce proliferative changes in rodent thyroid c cells . liraglutide increased the number of cases with c - cell hyperplasia , adenomas , and medullary thyroid carcinomas in mice and rats ( 9 ) . in these species such abnormalities are also found spontaneously , i.e. , in the absence of glp-1 receptor stimulation , especially in male rats , in which medullary thyroid carcinoma developed in some animals treated with placebo ( 9 ) . accordingly , rodent c - cell lines in cell culture responded to glp-1 , exenatide , and liraglutide with acutely producing cyclic amp and secreting calcitonin ( 9 ) . similar cell lines of human origin do not show such acute responses when glp-1 receptors are stimulated ( 9 ) . whereas rodent c - cell lines are equipped with glp-1 receptors at a high level of expression , along the same lines , long - term treatment in obese human subjects with high liraglutide doses up to 3-mg per day does not lead to elevations in plasma calcitonin ( 32 ) . based on these results , the ability of glp-1 receptor stimulation to induce proliferative responses in human c cells has been judged as probably absent . medullary thyroid carcinomas are an extremely rare form of thyroid carcinomas in humans ( 33 ) . no case report has been published describing a medullary thyroid carcinoma in a patient receiving a treatment with a glp-1 receptor agonist who prior to such treatment had a morphologically normal thyroid gland and low calcitonin concentrations . given the rare incidence of medullary thyroid carcinoma , 1 ) the consequences of a potential elevation in the risk induced by incretin mimetics would still remain small , and 2 ) to prove or exclude such a relationship , efficient surveillance of extremely large numbers of patients would be needed . the elevated risk for thyroid carcinoma in more general terms described in the study exploring the faers database ( 22 ) is difficult to reconcile . similar reservations apply regarding reporting bias as mentioned for the pancreatitis / pancreatic carcinoma issue raised earlier ( vide supra ) . certainly , this would not be compatible with an explanation through a higher number of medullary carcinomas alone , which would need to increase by more than 30-fold in order to explain such numbers . however , whether follicular cells express glp-1 receptors ( 9 ) or whether malignant cells from thyroid tumors of different histological varieties ( e.g. , papillary thyroid carcinomas ) express the glp-1 receptors ( 34 ) is controversial and may be related to the specificity of the antibody or the radioligand used for immunohistochemistry ( 35 ) . the fact alone that some papillary thyroid carcinomas may show evidence of glp-1 receptor expression ( 34 ) does not prove that such receptors and their stimulation by drugs may contribute to the genesis or proliferation of such tumors . certainly more investigations are required , but one hardly can conclude that , based on current knowledge , there is a definitely increased risk for medullary ( or other types of ) thyroid carcinoma with the use of glp-1 receptor agonists . nevertheless , patients with an individually elevate genetic risk should not be treated with such agents . an elevated risk when using dpp-4 inhibitors does not have to considered at all since no such findings have been reported ( 22 ) . in the absence of large - scale cardiovascular outcome trials , summaries of cardiovascular events reported as adverse events in clinical trials with incretin - based glucose - lowering medications and meta - analyses based thereon ( 36 ) are the best available source of information for an overall judgment at present . phase 3 studies have accrued a number of cardiovascular events sufficient for a preliminary judgment based on trends . these trends observed for the incretin mimetics exenatide ( 37 ) and liraglutide ( 38 ) as well the dpp-4 inhibitors sitagliptin ( 39 ) , vildagliptin ( 40 ) , saxagliptin ( 41 ) , linagliptin ( 42 ) , and alogliptin ( 43 ) are surprisingly similar . 2 , in all these analyses the relative risk for a combined end point composed of acute myocardial infarction , stroke , and cardiovascular death is reduced with any of the glp-1based medications relative to placebo or comparator treatment to a value below 1 ( fig . 2 ) . however , the 95% cis ranged to above 1.0 with most compounds , indicating that the number of events available for this analysis was too small to allow the definite conclusion of a significant improvement in cardiovascular prognosis with incretin - based glucose - lowering treatment . relative risk for major cardiovascular events reported as adverse events during phase 3 studies with the glp-1 receptor agonists exenatide and liraglutide ( upper panel ) and with the dpp-4 inhibitors sitagliptin , vildagliptin , saxagliptin , alogliptin , and linagliptin ( lower panel ) compared with pooled comparators ( placebo or active control medications ) . a potential reduction in cardiovascular event rates with linagliptin treatment is further supported by a recent study comparing linagliptin with the sulfonylurea glimepiride ( 44 ) . there is some plausibility based on the influences of glp-1based drugs on cardiovascular risk factors ( 45 ) . they also reduce systolic blood pressure by 25 mmhg , mechanistically explained by improved endothelial function and vasodilation , enhanced natriuresis , and fluid excretion . there is a potential for a reduction in postprandial triglyceride - rich lipoproteins , especially with those agents that have and preserve over time a prominent effect on gastric emptying . effects on nonclassical cardiovascular risk factors point in the same direction . furthermore , glp-1 receptor stimulation has reduced the extent of myocardial necroses in animal experiments inducing acute myocardial infarction by coronary artery ligation . the results have been surprisingly uniform using different agents ( glp-1 , exenatide , liraglutide , sitagliptin ) in various species ( 45 ) . in addition , in animal models of left ventricular failure , glp-1 and incretin mimetics may increase cardiac output by stimulating glucose and oxygen uptake into the myocardium . clinical pilot trials support the notion that glp-1 receptor stimulation may be beneficial in patients with acute coronary syndrome and chronic congestive heart failure ( 45 ) . therefore , one may be optimistic that cardiovascular outcome trials being performed to date , which will report after the year 2015 ( table 2 ) , will at least confirm cardiovascular safety with a potential to substantiate the beneficial effects in this important respect . table 1 summarizes the beneficial effects of incretin - based glucose - lowering agents and their advantages over other antidiabetic pharmaceutical agents , but also the open issues discussed earlier in this article in order to define the balance of benefits on the one hand and the risks and harms on the other . regarding the properties of incretin - based medications as antidiabetic drugs , they are effective in lowering glucose and avoid the problems of some other classes of glucose - lowering medications that are related to the induction of hypoglycemia and weight gain . surrogate parameters indicate an improvement in the cardiovascular risk profile , and preliminary analyses of cardiovascular outcomes suggest the potential for benefit in this respect . critical issues exist , but in many respects they are discussed in a controversial manner with only some data in support of an elevated risk ( table 1 ) . nausea and vomiting may be intolerable and lead to the discontinuation of treatment with glp-1 receptor agonists . putative interference of dpp-4 inhibitors with immune function does not lead to increased rates of common infections ( 39,46 ) . regarding the issues related to the potential short - term induction of acute and the putative long - term risk for chronic pancreatitis and eventually pancreatic cancer , data at hand today do not convincingly prove such risks . thyroid issues related to glp-1 receptors on c cells appear to mainly apply to rodents with a paucity of convincing human data that show a definite risk . the fact that heart rate may increase with glp-1 receptor agonists needs to be understood mechanistically . potential explanations could be a reflex compensating for vasodilation ( 47 ) and lower blood pressure ( 10,11,48 ) , a direct effect on the sinus node , or an increased relationship of sympathetic versus parasympathetic autonomous nervous system tone . epidemiological findings relating higher heart rates to premature cardiovascular morbidity and mortality probably use pulse rate as a surrogate parameter for physical fitness ( 49 ) . there is no reason to assume that incretin - based medications would lead to a reduced cardiorespiratory fitness . thus , while the benefits expected or proven from using incretin - based medications seem to be substantial and address risks central to patients with type 2 diabetes , the potential harms and risks typically refer to rare events and are discussed in a controversial manner , e.g. , without certainty regarding a potential role of incretin - based medications to cause substantial harm . obviously more needs to be learned regarding the open questions , but based on today s available knowledge , incretin - based medications can be considered effective and safe . such considerations should not currently influence our treatment decisions regarding the potential prescription of glp-1 receptor agonists or dpp-4 inhibitors within a treatment regimen for type 2 diabetes .

there is no question that incretin - based glucose - lowering medications have proven to be effective glucose - lowering agents . glucagon - like peptide 1 ( glp-1 ) receptor agonists demonstrate an efficacy comparable to insulin treatment and appear to do so with significant effects to promote weight loss with minimal hypoglycemia . in addition , there are significant data with dipeptidyl peptidase 4 ( dpp-4 ) inhibitors showing efficacy comparable to sulfonylureas but with weight neutral effects and reduced risk for hypoglycemia . however , over the recent past there have been concerns regarding the long - term consequences of using such therapies , and the issues raised are in regard to the potential of both classes to promote acute pancreatitis , to initiate histological changes suggesting chronic pancreatitis including associated preneoplastic lesions , and potentially , in the long run , pancreatic cancer . other issues relate to an increase in thyroid cancer . there are clearly conflicting data that have been presented in preclinical studies and in epidemiologic studies . to provide an understanding of both sides of the argument , we provide a discussion of this topic as part of this two - part point - counterpoint narrative . in the point narrative preceding the counterpoint narrative below , dr . butler and colleagues provide their opinion and review of the data to date and that we need to reconsider use of incretin - based therapies because of the growing concern of potential risk and based on a clearer understanding of the mechanism of action . in the counterpoint narrative provided below , dr . nauck provides a defense of incretin - based therapies and that benefits clearly outweigh any concern of risk.william t. cefalu , md editor in chief , diabetes care

Special issues (rare findings of uncertain clinical importance) Pancreatitis Pancreatitis in animal studies Clinical acute pancreatitis with incretin-based glucose-lowering medications Incretin-based medications and chronic pancreatitis/pancreatic cancer Incretin-based medications and thyroid carcinoma Cardiovascular outcomes The greater pictureweighing benefits against potential risks and harms regarding the clinical use of incretin-based glucose-lowering medications

for glp-1 receptor agonists and for dpp-4 inhibitors , these events of special interest are pancreatitis , pancreatic cancer , and thyroid carcinoma ( table 1 ) . in addition , possible consequences of a rise in pulse rate with glp-1 receptor agonists need to be discussed . contrasting clinical benefits and improved outcomes with adverse outcomes / risks associated with the use of incretin - based glucose - lowering medications ( a , glp-1 receptor agonists ; b , inhibitors of dpp-4 ) cases of pancreatitis have been observed in animals ( 6,7,13 ) and patients ( 14 ) treated with incretin mimetics and dpp-4 inhibitors ( 5 ) . animal studies describe histological changes compatible with damage to the exocrine pancreas with exenatide ( 6,7 ) and sitagliptin ( 5 ) . such data have been taken from claims databases and correlating the prescription of drugs used to treat diabetes with a diagnosis of acute pancreatitis . it summarizes reports to the faers from a period when publications and changes to drug labels had alerted the medical community to the fact that cases of acute pancreatitis had occurred in patients treated with exenatide and sitagliptin . the standards for the diagnosis of acute pancreatitis ( at least two out of three criteria : 1 ) typical severe abdominal pain , 2 ) elevations in pancreas - specific enzymes such as amylase and lipase , and 3 ) typical findings using appropriate imaging procedures ) may not always have been applied . ors for the diagnosis of ( a and c ) or hospitalization for ( b and d ) acute pancreatitis in association with a medication of exenatide ( glp-1 receptor agonist , upper panels ) or sitagliptin ( dpp-4 inhibitor , lower panels ) . data have been taken from the references quoted in the figure ( 1721 , 24 , 50 ) . a recent case - control study reported a higher or for the risk of hospitalization for a diagnosis of pancreatitis in patients taking incretin - based medications , since a separate analysis for the use of exenatide ( glp-1 receptor agonist ) or sitagliptin ( dpp-4 inhibitor ) did not yield significant findings ( fig . it can not be excluded at present that a potential combination of stomach cramps , representing gastrointestinal adverse events of glp-1 receptor agonists , and spontaneously elevated serum lipase activities were responsible for the hospitalizations and do not represent true pancreatitis episodes . nevertheless , this small study analyzing only a few patients with pancreatitis is only the second study describing an elevated risk ( 24 ) , however , only by combining exenatide and sitagliptin treatments as incretin - based medications and after adjusting for potential confounders . however , the nature of the elevation in serum lipase induced by liraglutide treatment needs to be explored so that we can understand its mechanism . effects of glp-1 receptor stimulation on pancreatic enzyme synthesis , potential leakage into the circulation rather than directional secretion into pancreatic digestive juice , and a potential induction of a chronic inflammatory response need to be studied . to date , it certainly can not be taken as a fact that chronic stimulation of the glp-1 receptor ( as occurs during the treatment with incretin mimetics and dpp-4 inhibitors ) induces acute or chronic inflammatory responses in the pancreas , nor that , based on a well - delineated mechanism and supported by convincing epidemiological data , the clinical use of incretin - based glucose - lowering medications would cause pancreatitis . clinically , the development of typical chronic pancreatitis diagnosed because of typical morphological findings and exocrine insufficiency leading to maldigestion , nutritional deficiencies , and weight loss over and above what is expected from continued stimulation of brain glp-1 receptors ( 1,27 ) in patients treated with glp-1based medications has never been described . regarding the related question of chronic changes in the exocrine pancreas leading to pancreatic duct proliferation and the formation of preneoplastic lesions ( like pancreatic intraepithelial neoplasms or pancreatic duct glands ) , data from animal studies are similarly controversial with studies showing alterations of the exocrine pancreatic histology indicative of chronic pancreatitis with exenatide treatment ( 57 ) , while another recent study using liraglutide did describe occasional pancreatitis as a rare finding but not at all related to the dose of liraglutide with similar numbers in placebo - treated rats , mice , and monkeys ( 13 ) . this finding needs to be confirmed in a larger , representative sample of pancreas specimens obtained without preceding long - term critical illness , which alone may be responsible for some proliferative responses ( 30 ) . to put the state of this present discussion into perspective , it should be made clear that at most early proliferative or preneoplastic changes have been observed , which as such are not proof that eventually the process described will give rise to pancreatic cancer . thus we have to discuss a potential risk ( and certainly want to learn more about the long - term consequences of stimulating glp-1 receptors for the exocrine pancreas ) , but not an actual threat to patients treated with incretin - based medications , based on a well - characterized mechanism with a risk clearly elevated based on sound epidemiological analyses . it is reassuring that no case of clinically evident chronic pancreatitis has been described after initiating treatment with incretin - based medications . certainly , there is also no case report of pancreatic cancer diagnosed after exposing a patient to glp-1 receptor agonists or dpp-4 inhibitors in a patient in whom there had previously been a morphologically tumor - free pancreas . since pancreatic carcinomas develop slowly ( 31 ) , one would probably not expect to see such a case after at most a few years of treatment , considering the recent introduction of the incretin - based medications , even if there were such a long - term risk . glp-1 receptor agonists have the potential to induce proliferative changes in rodent thyroid c cells . , in the absence of glp-1 receptor stimulation , especially in male rats , in which medullary thyroid carcinoma developed in some animals treated with placebo ( 9 ) . whereas rodent c - cell lines are equipped with glp-1 receptors at a high level of expression , along the same lines , long - term treatment in obese human subjects with high liraglutide doses up to 3-mg per day does not lead to elevations in plasma calcitonin ( 32 ) . given the rare incidence of medullary thyroid carcinoma , 1 ) the consequences of a potential elevation in the risk induced by incretin mimetics would still remain small , and 2 ) to prove or exclude such a relationship , efficient surveillance of extremely large numbers of patients would be needed . the elevated risk for thyroid carcinoma in more general terms described in the study exploring the faers database ( 22 ) is difficult to reconcile . , papillary thyroid carcinomas ) express the glp-1 receptors ( 34 ) is controversial and may be related to the specificity of the antibody or the radioligand used for immunohistochemistry ( 35 ) . regarding the thyroid issues , certainly more investigations are required , but one hardly can conclude that , based on current knowledge , there is a definitely increased risk for medullary ( or other types of ) thyroid carcinoma with the use of glp-1 receptor agonists . in the absence of large - scale cardiovascular outcome trials , summaries of cardiovascular events reported as adverse events in clinical trials with incretin - based glucose - lowering medications and meta - analyses based thereon ( 36 ) are the best available source of information for an overall judgment at present . 2 , in all these analyses the relative risk for a combined end point composed of acute myocardial infarction , stroke , and cardiovascular death is reduced with any of the glp-1based medications relative to placebo or comparator treatment to a value below 1 ( fig . however , the 95% cis ranged to above 1.0 with most compounds , indicating that the number of events available for this analysis was too small to allow the definite conclusion of a significant improvement in cardiovascular prognosis with incretin - based glucose - lowering treatment . relative risk for major cardiovascular events reported as adverse events during phase 3 studies with the glp-1 receptor agonists exenatide and liraglutide ( upper panel ) and with the dpp-4 inhibitors sitagliptin , vildagliptin , saxagliptin , alogliptin , and linagliptin ( lower panel ) compared with pooled comparators ( placebo or active control medications ) . the results have been surprisingly uniform using different agents ( glp-1 , exenatide , liraglutide , sitagliptin ) in various species ( 45 ) . in addition , in animal models of left ventricular failure , glp-1 and incretin mimetics may increase cardiac output by stimulating glucose and oxygen uptake into the myocardium . other studies find an amelioration of the course of experimentally induced acute pancreatitis in mice with exenatide ( 15 ) or an anti - inflammatory pattern of cytokines induced by liraglutide treatment ( 16 ) . such data have been taken from claims databases and correlating the prescription of drugs used to treat diabetes with a diagnosis of acute pancreatitis . it summarizes reports to the faers from a period when publications and changes to drug labels had alerted the medical community to the fact that cases of acute pancreatitis had occurred in patients treated with exenatide and sitagliptin . the standards for the diagnosis of acute pancreatitis ( at least two out of three criteria : 1 ) typical severe abdominal pain , 2 ) elevations in pancreas - specific enzymes such as amylase and lipase , and 3 ) typical findings using appropriate imaging procedures ) may not always have been applied . ors for the diagnosis of ( a and c ) or hospitalization for ( b and d ) acute pancreatitis in association with a medication of exenatide ( glp-1 receptor agonist , upper panels ) or sitagliptin ( dpp-4 inhibitor , lower panels ) . data have been taken from the references quoted in the figure ( 1721 , 24 , 50 ) . a recent case - control study reported a higher or for the risk of hospitalization for a diagnosis of pancreatitis in patients taking incretin - based medications , since a separate analysis for the use of exenatide ( glp-1 receptor agonist ) or sitagliptin ( dpp-4 inhibitor ) did not yield significant findings ( fig . it can not be excluded at present that a potential combination of stomach cramps , representing gastrointestinal adverse events of glp-1 receptor agonists , and spontaneously elevated serum lipase activities were responsible for the hospitalizations and do not represent true pancreatitis episodes . nevertheless , this small study analyzing only a few patients with pancreatitis is only the second study describing an elevated risk ( 24 ) , however , only by combining exenatide and sitagliptin treatments as incretin - based medications and after adjusting for potential confounders . however , the nature of the elevation in serum lipase induced by liraglutide treatment needs to be explored so that we can understand its mechanism . effects of glp-1 receptor stimulation on pancreatic enzyme synthesis , potential leakage into the circulation rather than directional secretion into pancreatic digestive juice , and a potential induction of a chronic inflammatory response need to be studied . to date , it certainly can not be taken as a fact that chronic stimulation of the glp-1 receptor ( as occurs during the treatment with incretin mimetics and dpp-4 inhibitors ) induces acute or chronic inflammatory responses in the pancreas , nor that , based on a well - delineated mechanism and supported by convincing epidemiological data , the clinical use of incretin - based glucose - lowering medications would cause pancreatitis . clinically , the development of typical chronic pancreatitis diagnosed because of typical morphological findings and exocrine insufficiency leading to maldigestion , nutritional deficiencies , and weight loss over and above what is expected from continued stimulation of brain glp-1 receptors ( 1,27 ) in patients treated with glp-1based medications has never been described . regarding the related question of chronic changes in the exocrine pancreas leading to pancreatic duct proliferation and the formation of preneoplastic lesions ( like pancreatic intraepithelial neoplasms or pancreatic duct glands ) , data from animal studies are similarly controversial with studies showing alterations of the exocrine pancreatic histology indicative of chronic pancreatitis with exenatide treatment ( 57 ) , while another recent study using liraglutide did describe occasional pancreatitis as a rare finding but not at all related to the dose of liraglutide with similar numbers in placebo - treated rats , mice , and monkeys ( 13 ) . a recent finding reported that pancreas specimens from organ donors with type 2 diabetes , who had received treatment with the dpp-4 inhibitor sitagliptin ( n = 7 ) or exenatide ( n = 1 ) , relative to patients with type 2 diabetes treated with other agents , had marked -cell hyperplasia , -cells coexpressing insulin and glucagon , hyperplasia of -cells expressing glucagon , increased expression of proliferation markers , and an increased prevalence of preneoplastic lesions ( 29 ) . this finding needs to be confirmed in a larger , representative sample of pancreas specimens obtained without preceding long - term critical illness , which alone may be responsible for some proliferative responses ( 30 ) . to put the state of this present discussion into perspective , it should be made clear that at most early proliferative or preneoplastic changes have been observed , which as such are not proof that eventually the process described will give rise to pancreatic cancer . thus we have to discuss a potential risk ( and certainly want to learn more about the long - term consequences of stimulating glp-1 receptors for the exocrine pancreas ) , but not an actual threat to patients treated with incretin - based medications , based on a well - characterized mechanism with a risk clearly elevated based on sound epidemiological analyses . it is reassuring that no case of clinically evident chronic pancreatitis has been described after initiating treatment with incretin - based medications . certainly , there is also no case report of pancreatic cancer diagnosed after exposing a patient to glp-1 receptor agonists or dpp-4 inhibitors in a patient in whom there had previously been a morphologically tumor - free pancreas . since pancreatic carcinomas develop slowly ( 31 ) , one would probably not expect to see such a case after at most a few years of treatment , considering the recent introduction of the incretin - based medications , even if there were such a long - term risk . whereas rodent c - cell lines are equipped with glp-1 receptors at a high level of expression , along the same lines , long - term treatment in obese human subjects with high liraglutide doses up to 3-mg per day does not lead to elevations in plasma calcitonin ( 32 ) . given the rare incidence of medullary thyroid carcinoma , 1 ) the consequences of a potential elevation in the risk induced by incretin mimetics would still remain small , and 2 ) to prove or exclude such a relationship , efficient surveillance of extremely large numbers of patients would be needed . the elevated risk for thyroid carcinoma in more general terms described in the study exploring the faers database ( 22 ) is difficult to reconcile . certainly more investigations are required , but one hardly can conclude that , based on current knowledge , there is a definitely increased risk for medullary ( or other types of ) thyroid carcinoma with the use of glp-1 receptor agonists . in the absence of large - scale cardiovascular outcome trials , summaries of cardiovascular events reported as adverse events in clinical trials with incretin - based glucose - lowering medications and meta - analyses based thereon ( 36 ) are the best available source of information for an overall judgment at present . 2 , in all these analyses the relative risk for a combined end point composed of acute myocardial infarction , stroke , and cardiovascular death is reduced with any of the glp-1based medications relative to placebo or comparator treatment to a value below 1 ( fig . however , the 95% cis ranged to above 1.0 with most compounds , indicating that the number of events available for this analysis was too small to allow the definite conclusion of a significant improvement in cardiovascular prognosis with incretin - based glucose - lowering treatment . relative risk for major cardiovascular events reported as adverse events during phase 3 studies with the glp-1 receptor agonists exenatide and liraglutide ( upper panel ) and with the dpp-4 inhibitors sitagliptin , vildagliptin , saxagliptin , alogliptin , and linagliptin ( lower panel ) compared with pooled comparators ( placebo or active control medications ) . there is a potential for a reduction in postprandial triglyceride - rich lipoproteins , especially with those agents that have and preserve over time a prominent effect on gastric emptying . the results have been surprisingly uniform using different agents ( glp-1 , exenatide , liraglutide , sitagliptin ) in various species ( 45 ) . in addition , in animal models of left ventricular failure , glp-1 and incretin mimetics may increase cardiac output by stimulating glucose and oxygen uptake into the myocardium . table 1 summarizes the beneficial effects of incretin - based glucose - lowering agents and their advantages over other antidiabetic pharmaceutical agents , but also the open issues discussed earlier in this article in order to define the balance of benefits on the one hand and the risks and harms on the other . regarding the properties of incretin - based medications as antidiabetic drugs , they are effective in lowering glucose and avoid the problems of some other classes of glucose - lowering medications that are related to the induction of hypoglycemia and weight gain . surrogate parameters indicate an improvement in the cardiovascular risk profile , and preliminary analyses of cardiovascular outcomes suggest the potential for benefit in this respect . regarding the issues related to the potential short - term induction of acute and the putative long - term risk for chronic pancreatitis and eventually pancreatic cancer , data at hand today do not convincingly prove such risks . thyroid issues related to glp-1 receptors on c cells appear to mainly apply to rodents with a paucity of convincing human data that show a definite risk . the fact that heart rate may increase with glp-1 receptor agonists needs to be understood mechanistically . there is no reason to assume that incretin - based medications would lead to a reduced cardiorespiratory fitness . thus , while the benefits expected or proven from using incretin - based medications seem to be substantial and address risks central to patients with type 2 diabetes , the potential harms and risks typically refer to rare events and are discussed in a controversial manner , e.g. , without certainty regarding a potential role of incretin - based medications to cause substantial harm . obviously more needs to be learned regarding the open questions , but based on today s available knowledge , incretin - based medications can be considered effective and safe . such considerations should not currently influence our treatment decisions regarding the potential prescription of glp-1 receptor agonists or dpp-4 inhibitors within a treatment regimen for type 2 diabetes .

in addition to the influence of glp-1based medications on those outcomes that typically determine the morbidity and mortality of patients with type 2 diabetes , i.e. for glp-1 receptor agonists and for dpp-4 inhibitors , these events of special interest are pancreatitis , pancreatic cancer , and thyroid carcinoma ( table 1 ) . contrasting clinical benefits and improved outcomes with adverse outcomes / risks associated with the use of incretin - based glucose - lowering medications ( a , glp-1 receptor agonists ; b , inhibitors of dpp-4 ) cases of pancreatitis have been observed in animals ( 6,7,13 ) and patients ( 14 ) treated with incretin mimetics and dpp-4 inhibitors ( 5 ) . other studies find an amelioration of the course of experimentally induced acute pancreatitis in mice with exenatide ( 15 ) or an anti - inflammatory pattern of cytokines induced by liraglutide treatment ( 16 ) . attempts to quantify the number of pancreatitis events while patients are treated with glp-1 receptor agonists or dpp-4 inhibitors have found odds ratios ( ors ) around 1 , however with relatively wide cis ( fig . it summarizes reports to the faers from a period when publications and changes to drug labels had alerted the medical community to the fact that cases of acute pancreatitis had occurred in patients treated with exenatide and sitagliptin . the standards for the diagnosis of acute pancreatitis ( at least two out of three criteria : 1 ) typical severe abdominal pain , 2 ) elevations in pancreas - specific enzymes such as amylase and lipase , and 3 ) typical findings using appropriate imaging procedures ) may not always have been applied . ors for the diagnosis of ( a and c ) or hospitalization for ( b and d ) acute pancreatitis in association with a medication of exenatide ( glp-1 receptor agonist , upper panels ) or sitagliptin ( dpp-4 inhibitor , lower panels ) . a recent case - control study reported a higher or for the risk of hospitalization for a diagnosis of pancreatitis in patients taking incretin - based medications , since a separate analysis for the use of exenatide ( glp-1 receptor agonist ) or sitagliptin ( dpp-4 inhibitor ) did not yield significant findings ( fig . it can not be excluded at present that a potential combination of stomach cramps , representing gastrointestinal adverse events of glp-1 receptor agonists , and spontaneously elevated serum lipase activities were responsible for the hospitalizations and do not represent true pancreatitis episodes . nevertheless , this small study analyzing only a few patients with pancreatitis is only the second study describing an elevated risk ( 24 ) , however , only by combining exenatide and sitagliptin treatments as incretin - based medications and after adjusting for potential confounders . however , the nature of the elevation in serum lipase induced by liraglutide treatment needs to be explored so that we can understand its mechanism . effects of glp-1 receptor stimulation on pancreatic enzyme synthesis , potential leakage into the circulation rather than directional secretion into pancreatic digestive juice , and a potential induction of a chronic inflammatory response need to be studied . to date , it certainly can not be taken as a fact that chronic stimulation of the glp-1 receptor ( as occurs during the treatment with incretin mimetics and dpp-4 inhibitors ) induces acute or chronic inflammatory responses in the pancreas , nor that , based on a well - delineated mechanism and supported by convincing epidemiological data , the clinical use of incretin - based glucose - lowering medications would cause pancreatitis . clinically , the development of typical chronic pancreatitis diagnosed because of typical morphological findings and exocrine insufficiency leading to maldigestion , nutritional deficiencies , and weight loss over and above what is expected from continued stimulation of brain glp-1 receptors ( 1,27 ) in patients treated with glp-1based medications has never been described . regarding the related question of chronic changes in the exocrine pancreas leading to pancreatic duct proliferation and the formation of preneoplastic lesions ( like pancreatic intraepithelial neoplasms or pancreatic duct glands ) , data from animal studies are similarly controversial with studies showing alterations of the exocrine pancreatic histology indicative of chronic pancreatitis with exenatide treatment ( 57 ) , while another recent study using liraglutide did describe occasional pancreatitis as a rare finding but not at all related to the dose of liraglutide with similar numbers in placebo - treated rats , mice , and monkeys ( 13 ) . a recent finding reported that pancreas specimens from organ donors with type 2 diabetes , who had received treatment with the dpp-4 inhibitor sitagliptin ( n = 7 ) or exenatide ( n = 1 ) , relative to patients with type 2 diabetes treated with other agents , had marked -cell hyperplasia , -cells coexpressing insulin and glucagon , hyperplasia of -cells expressing glucagon , increased expression of proliferation markers , and an increased prevalence of preneoplastic lesions ( 29 ) . to put the state of this present discussion into perspective , it should be made clear that at most early proliferative or preneoplastic changes have been observed , which as such are not proof that eventually the process described will give rise to pancreatic cancer . thus we have to discuss a potential risk ( and certainly want to learn more about the long - term consequences of stimulating glp-1 receptors for the exocrine pancreas ) , but not an actual threat to patients treated with incretin - based medications , based on a well - characterized mechanism with a risk clearly elevated based on sound epidemiological analyses . certainly , there is also no case report of pancreatic cancer diagnosed after exposing a patient to glp-1 receptor agonists or dpp-4 inhibitors in a patient in whom there had previously been a morphologically tumor - free pancreas . since pancreatic carcinomas develop slowly ( 31 ) , one would probably not expect to see such a case after at most a few years of treatment , considering the recent introduction of the incretin - based medications , even if there were such a long - term risk . liraglutide increased the number of cases with c - cell hyperplasia , adenomas , and medullary thyroid carcinomas in mice and rats ( 9 ) . , in the absence of glp-1 receptor stimulation , especially in male rats , in which medullary thyroid carcinoma developed in some animals treated with placebo ( 9 ) . accordingly , rodent c - cell lines in cell culture responded to glp-1 , exenatide , and liraglutide with acutely producing cyclic amp and secreting calcitonin ( 9 ) . whereas rodent c - cell lines are equipped with glp-1 receptors at a high level of expression , along the same lines , long - term treatment in obese human subjects with high liraglutide doses up to 3-mg per day does not lead to elevations in plasma calcitonin ( 32 ) . based on these results , the ability of glp-1 receptor stimulation to induce proliferative responses in human c cells has been judged as probably absent . no case report has been published describing a medullary thyroid carcinoma in a patient receiving a treatment with a glp-1 receptor agonist who prior to such treatment had a morphologically normal thyroid gland and low calcitonin concentrations . given the rare incidence of medullary thyroid carcinoma , 1 ) the consequences of a potential elevation in the risk induced by incretin mimetics would still remain small , and 2 ) to prove or exclude such a relationship , efficient surveillance of extremely large numbers of patients would be needed . the elevated risk for thyroid carcinoma in more general terms described in the study exploring the faers database ( 22 ) is difficult to reconcile . , papillary thyroid carcinomas ) express the glp-1 receptors ( 34 ) is controversial and may be related to the specificity of the antibody or the radioligand used for immunohistochemistry ( 35 ) . regarding the thyroid issues , certainly more investigations are required , but one hardly can conclude that , based on current knowledge , there is a definitely increased risk for medullary ( or other types of ) thyroid carcinoma with the use of glp-1 receptor agonists . in the absence of large - scale cardiovascular outcome trials , summaries of cardiovascular events reported as adverse events in clinical trials with incretin - based glucose - lowering medications and meta - analyses based thereon ( 36 ) are the best available source of information for an overall judgment at present . these trends observed for the incretin mimetics exenatide ( 37 ) and liraglutide ( 38 ) as well the dpp-4 inhibitors sitagliptin ( 39 ) , vildagliptin ( 40 ) , saxagliptin ( 41 ) , linagliptin ( 42 ) , and alogliptin ( 43 ) are surprisingly similar . 2 , in all these analyses the relative risk for a combined end point composed of acute myocardial infarction , stroke , and cardiovascular death is reduced with any of the glp-1based medications relative to placebo or comparator treatment to a value below 1 ( fig . however , the 95% cis ranged to above 1.0 with most compounds , indicating that the number of events available for this analysis was too small to allow the definite conclusion of a significant improvement in cardiovascular prognosis with incretin - based glucose - lowering treatment . relative risk for major cardiovascular events reported as adverse events during phase 3 studies with the glp-1 receptor agonists exenatide and liraglutide ( upper panel ) and with the dpp-4 inhibitors sitagliptin , vildagliptin , saxagliptin , alogliptin , and linagliptin ( lower panel ) compared with pooled comparators ( placebo or active control medications ) . therefore , one may be optimistic that cardiovascular outcome trials being performed to date , which will report after the year 2015 ( table 2 ) , will at least confirm cardiovascular safety with a potential to substantiate the beneficial effects in this important respect . other studies find an amelioration of the course of experimentally induced acute pancreatitis in mice with exenatide ( 15 ) or an anti - inflammatory pattern of cytokines induced by liraglutide treatment ( 16 ) . attempts to quantify the number of pancreatitis events while patients are treated with glp-1 receptor agonists or dpp-4 inhibitors have found odds ratios ( ors ) around 1 , however with relatively wide cis ( fig . it summarizes reports to the faers from a period when publications and changes to drug labels had alerted the medical community to the fact that cases of acute pancreatitis had occurred in patients treated with exenatide and sitagliptin . the standards for the diagnosis of acute pancreatitis ( at least two out of three criteria : 1 ) typical severe abdominal pain , 2 ) elevations in pancreas - specific enzymes such as amylase and lipase , and 3 ) typical findings using appropriate imaging procedures ) may not always have been applied . ors for the diagnosis of ( a and c ) or hospitalization for ( b and d ) acute pancreatitis in association with a medication of exenatide ( glp-1 receptor agonist , upper panels ) or sitagliptin ( dpp-4 inhibitor , lower panels ) . a recent case - control study reported a higher or for the risk of hospitalization for a diagnosis of pancreatitis in patients taking incretin - based medications , since a separate analysis for the use of exenatide ( glp-1 receptor agonist ) or sitagliptin ( dpp-4 inhibitor ) did not yield significant findings ( fig . it can not be excluded at present that a potential combination of stomach cramps , representing gastrointestinal adverse events of glp-1 receptor agonists , and spontaneously elevated serum lipase activities were responsible for the hospitalizations and do not represent true pancreatitis episodes . nevertheless , this small study analyzing only a few patients with pancreatitis is only the second study describing an elevated risk ( 24 ) , however , only by combining exenatide and sitagliptin treatments as incretin - based medications and after adjusting for potential confounders . however , the nature of the elevation in serum lipase induced by liraglutide treatment needs to be explored so that we can understand its mechanism . effects of glp-1 receptor stimulation on pancreatic enzyme synthesis , potential leakage into the circulation rather than directional secretion into pancreatic digestive juice , and a potential induction of a chronic inflammatory response need to be studied . to date , it certainly can not be taken as a fact that chronic stimulation of the glp-1 receptor ( as occurs during the treatment with incretin mimetics and dpp-4 inhibitors ) induces acute or chronic inflammatory responses in the pancreas , nor that , based on a well - delineated mechanism and supported by convincing epidemiological data , the clinical use of incretin - based glucose - lowering medications would cause pancreatitis . clinically , the development of typical chronic pancreatitis diagnosed because of typical morphological findings and exocrine insufficiency leading to maldigestion , nutritional deficiencies , and weight loss over and above what is expected from continued stimulation of brain glp-1 receptors ( 1,27 ) in patients treated with glp-1based medications has never been described . regarding the related question of chronic changes in the exocrine pancreas leading to pancreatic duct proliferation and the formation of preneoplastic lesions ( like pancreatic intraepithelial neoplasms or pancreatic duct glands ) , data from animal studies are similarly controversial with studies showing alterations of the exocrine pancreatic histology indicative of chronic pancreatitis with exenatide treatment ( 57 ) , while another recent study using liraglutide did describe occasional pancreatitis as a rare finding but not at all related to the dose of liraglutide with similar numbers in placebo - treated rats , mice , and monkeys ( 13 ) . a recent finding reported that pancreas specimens from organ donors with type 2 diabetes , who had received treatment with the dpp-4 inhibitor sitagliptin ( n = 7 ) or exenatide ( n = 1 ) , relative to patients with type 2 diabetes treated with other agents , had marked -cell hyperplasia , -cells coexpressing insulin and glucagon , hyperplasia of -cells expressing glucagon , increased expression of proliferation markers , and an increased prevalence of preneoplastic lesions ( 29 ) . to put the state of this present discussion into perspective , it should be made clear that at most early proliferative or preneoplastic changes have been observed , which as such are not proof that eventually the process described will give rise to pancreatic cancer . thus we have to discuss a potential risk ( and certainly want to learn more about the long - term consequences of stimulating glp-1 receptors for the exocrine pancreas ) , but not an actual threat to patients treated with incretin - based medications , based on a well - characterized mechanism with a risk clearly elevated based on sound epidemiological analyses . certainly , there is also no case report of pancreatic cancer diagnosed after exposing a patient to glp-1 receptor agonists or dpp-4 inhibitors in a patient in whom there had previously been a morphologically tumor - free pancreas . , in the absence of glp-1 receptor stimulation , especially in male rats , in which medullary thyroid carcinoma developed in some animals treated with placebo ( 9 ) . accordingly , rodent c - cell lines in cell culture responded to glp-1 , exenatide , and liraglutide with acutely producing cyclic amp and secreting calcitonin ( 9 ) . whereas rodent c - cell lines are equipped with glp-1 receptors at a high level of expression , along the same lines , long - term treatment in obese human subjects with high liraglutide doses up to 3-mg per day does not lead to elevations in plasma calcitonin ( 32 ) . given the rare incidence of medullary thyroid carcinoma , 1 ) the consequences of a potential elevation in the risk induced by incretin mimetics would still remain small , and 2 ) to prove or exclude such a relationship , efficient surveillance of extremely large numbers of patients would be needed . certainly more investigations are required , but one hardly can conclude that , based on current knowledge , there is a definitely increased risk for medullary ( or other types of ) thyroid carcinoma with the use of glp-1 receptor agonists . in the absence of large - scale cardiovascular outcome trials , summaries of cardiovascular events reported as adverse events in clinical trials with incretin - based glucose - lowering medications and meta - analyses based thereon ( 36 ) are the best available source of information for an overall judgment at present . these trends observed for the incretin mimetics exenatide ( 37 ) and liraglutide ( 38 ) as well the dpp-4 inhibitors sitagliptin ( 39 ) , vildagliptin ( 40 ) , saxagliptin ( 41 ) , linagliptin ( 42 ) , and alogliptin ( 43 ) are surprisingly similar . 2 , in all these analyses the relative risk for a combined end point composed of acute myocardial infarction , stroke , and cardiovascular death is reduced with any of the glp-1based medications relative to placebo or comparator treatment to a value below 1 ( fig . however , the 95% cis ranged to above 1.0 with most compounds , indicating that the number of events available for this analysis was too small to allow the definite conclusion of a significant improvement in cardiovascular prognosis with incretin - based glucose - lowering treatment . relative risk for major cardiovascular events reported as adverse events during phase 3 studies with the glp-1 receptor agonists exenatide and liraglutide ( upper panel ) and with the dpp-4 inhibitors sitagliptin , vildagliptin , saxagliptin , alogliptin , and linagliptin ( lower panel ) compared with pooled comparators ( placebo or active control medications ) . therefore , one may be optimistic that cardiovascular outcome trials being performed to date , which will report after the year 2015 ( table 2 ) , will at least confirm cardiovascular safety with a potential to substantiate the beneficial effects in this important respect . table 1 summarizes the beneficial effects of incretin - based glucose - lowering agents and their advantages over other antidiabetic pharmaceutical agents , but also the open issues discussed earlier in this article in order to define the balance of benefits on the one hand and the risks and harms on the other . regarding the properties of incretin - based medications as antidiabetic drugs , they are effective in lowering glucose and avoid the problems of some other classes of glucose - lowering medications that are related to the induction of hypoglycemia and weight gain . regarding the issues related to the potential short - term induction of acute and the putative long - term risk for chronic pancreatitis and eventually pancreatic cancer , data at hand today do not convincingly prove such risks . thus , while the benefits expected or proven from using incretin - based medications seem to be substantial and address risks central to patients with type 2 diabetes , the potential harms and risks typically refer to rare events and are discussed in a controversial manner , e.g.

a report , published in september 2009 , from the world health organization ( who)-sponsored study includes tuberculosis ( tb ) and aids with a share of 5.5% each in the list of top 10 causes of death in all 1024 year olds , males and females combined , globally . this has revealed an urgency to act on these issues with all our available resources and an approach to improve upon them . stop tb partnership announced the theme for world tuberculosis day 2010 as on the move against tuberculosis : innovate to accelerate action . this calls for ideas and projects to come up with something other than what we have been practicing for long . improving the research in tb has been a neglected area for long , in part due to the complex characteristics of the causal organism mycobacterium tuberculosis , the variable host response to infection , and , perhaps , complacency as this disease was nearly eradicated in high - income settings . with increasing hiv tb co - infections , tb has become a high priority for action and research in international health again . looking into this , who and the world bank , with international union against tuberculosis and lung disease ( iuatld ) , centre for disease control and prevention ( cdc ) , and other organizations , are reassessing their approaches to the prevention and control of tb . of late , agencies such as the foundation for innovative new diagnostics ( find ) , stop tb partnership 's new diagnostics working group ( ndwg ) , global laboratory initiative ( gli , another stop tb partnership working group ) , who , and the special program for research and training in tropical diseases ( tdr ) have shown increased interest toward the development of better diagnostics for tb . funding agencies such as the bill and melinda gates foundation , the global fund to fight aids , tb and malaria ( gfatm ) , and unitaid have emerged as resource providers for work on this disease that was being neglected in terms of sponsorship and interest of the private sector till now . with this , lots of efforts and funds are being brought up for the development of newer diagnostic methods in mycobacteriology . there are multiple hurdles to cross before we reach the ultimate goal of having an efficacious vaccine , easy and affordable diagnosis , and short - term treatment regimens with minimal side effects . in the field of tb , problem starts at the very first step , i.e. , diagnosing the case . conventional tb diagnosis has been relying on medical history , tuberculin skin test , chest x - rays , and bacteriological examination . m. tuberculosis grows slowly , and clinical specimens submitted to the tb microscopy and culture are contaminated to varying degrees by more rapidly growing unwanted normal flora . . moreover , the hydrophobic nature of the cell wall due to a high concentration of mycolic acid makes it more difficult to stain mycobaterial cells . the poor sensitivity of conventional smear microscopy has been a major concern . with various samples and sample processing methods , it has been found to be around 3643% sensitive . in general , direct smear reportedly detects afb only at concentrations of around 10,000 bacilli / ml of the specimen . conversely , as few as 100 bacilli / ml may be required for a positive culture . various modifications to the technique and higher level of sophistications have been introduced in this field to get the highest level of sensitivity . with certain variations in the sample processing methods for smear preparation , samples were subjected to different centrifugation forces and centrifugation times after decontamination and liquefaction and the centrifuged deposits were examined by smear and culture . the sensitivity of detection at an rcf of 4000 g for 15 min was 5000 organisms / ml for a smear . various workers have demonstrated the improved detection of direct smear - negative cases by universal sample processing ( usp ) smear microscopy . the highest limit of detection in smear microscopy after employing the usp methodology and observing 400500 fields was experimentally found to be 250300 bacilli / ml of the sample . under the best conditions for culture on solid and liquid media with usp - treated spiked sputum , the detection limit was found to be 400 cfu / ml after 8 weeks of incubation . hence , although it could reduce the bacilli count needed to be detected using smear , it has not helped improving detection by culture . however , the researchers felt a need for a more sensitive test to detect samples with a low bacterial load . steingart and co - workers did a systematic review to assess the ability of different processing methods to improve the sensitivity of microscopy . their search suggested that centrifugation with any of several chemical methods ( including bleach ) is more sensitive , that overnight sedimentation preceded by chemical processing is more sensitive , and that the specificity is similar . recently , a pilot study has compared the diagnostic accuracy and incremental yield of two short - duration ( < 1 h ) sputum pretreatment procedures involving pretreatment with bleach and usp centrifugation and concluded that both did not increase yield as compared to direct sputum smears . people tried hard for developing field - based methods to avoid the use of centrifugation during decontamination . but it was found to have a lower sensitivity as compared to the conventional method of centrifuge decontamination . fluorescence microscopy is credited with increased sensitivity and lower work effort but has a rider of increased cost and technical complexity . pai and co - workers have reported an average 10% increment in sensitivity of microscopy using fluorescence microscopy with no difference in specificity . lange and mori have given an update on the developments being made in the field of fluorescent microscopy . these include light - emitting diode ( led)-based fluorescent microscopy , mobile phone - based microscopy , and automated detection systems using image processing . with many more other studies , it is very apparent that we are still struggling hard to get a sensitive , easy , cost - effective , and faster method for smear microscopy . it seems possible to decrease mortality and morbidity due to tb using smear microscopy diagnosis and dots coverage , as being done in most of the high prevalence countries like india . but as many smear negative cases may remain undiagnosed , cutting transmission of infection does not seem to be an achievable target without having better diagnostic amenities . just the presence of acid - fast bacilli ( afb ) on a sputum smear or other specimens does not confirm a diagnosis of tb as all acid - fast - bacilli are not m. tuberculosis . also , bacterial isolation for drug - susceptibility testing and genotyping is required using a solid or liquid medium . therefore , a culture is expected on all initial samples to confirm the diagnosis followed by biochemical tests like catalase test at 68c , nitrate reduction , and niacin accumulation for speciation . although culture - based diagnosis of tb is recommended in international standards of tuberculosis care , lack of resources and technical expertise poses as a major limitation in most of the high prevalence countries . traditionally , primary isolation and culture of mycobacteria is performed on agar or lowenstein jensen ( lj ) media . drug susceptibility testing of isolates is done using the same medium following the proportion method . this method compares the mycobacterial growth levels in clinical isolates to be tested with known standard culture in the presence of different concentrations of anti - tb drugs in a proportional way . this method enables a determination of the concentration that inhibits more than 99% of the inoculum and is reported as the minimal inhibitory concentration ( mic ) of the given drug . this is considered as a gold standard . but it is cumbersome , technically demanding , lacks reproducibility , and it generally takes at least 21 days for a result after assay set - up . liquid culturing with radioisotopic detection or with the incorporation of fluorescent dyes was introduced in the past as a confirmatory method ( bactec 460 , bactec mgit 960 system , mb / bact , and versa trek system ) . however , looking at the sophistication and issues like contamination , the struggle again started to search something better . microscopic observation drug susceptibility ( mods ) testing developed recently allows both rapid and low - cost tb diagnosis in liquid culture with the simultaneous determination of drug susceptibilities.[1618 ] however , mods requires higher biosafety level facilities , an expert and experienced microbiologist , and the samples are very prone to contaminations . thus , it needs addition of various antibiotics in culture media and the samples need to be processed before inoculation involving decontamination and centrifugation steps . some other unconventional methods like thin - layer agar ( tla ) and the direct nitrate reductase assay ( nra ) have attempted to address the problem of multiple point processing and hence the generation of aerosols by incorporating visual inspection of results in the form of typical colony morphology or color change to identify tb growth . although they have tried to make things simple , they could not avoid the most cumbersome step of specimen processing . microbiological detection is possible only once the microbial load in the sample reaches a substantial number . some other situations like immune suppression and in the case of children , getting a good quality sample and getting the required microbial load in the samples is tough . hence , indirect approaches to trace the causal organism / byproducts or to look into the host response against the infection are also being considered . the pcr - based amplification of various target nucleic acids has been tried extensively that allows rapid and sensitive detection of target dna sequences . ideally , pcr can detect a single copy of the gene being targeted for amplification but with all the processing to avoid interference , the detection of as few as 410 copies has been made possible by several workers . various targets have been tried and tested to get better identification or speciation till date . although the 16s rrna gene is the most commonly used target , other targets have also provided a high - sensitivity and representative species - specific differentiation . the pcr amplification of the entire 16s23s rrna spacer region and use of a secondary technique of randomly amplified polymorphic dna ( rapd ) fingerprinting to differentiate strains belonging to the mycobacterium species has been reported . the amplification of the entire 16s23s intergenic region , and diagnostic tests for bacterial organisms using probes targeted for sequences within the 16s23s intergenic region have also been described . other targets include the 16s rrna gene , the 16s23s internal transcribed spacer , the 65-kda heat shock protein , reca , rpob , and gyrb.[232629 ] the 16s rrna gene - based methods are presently widely used for the identification and differentiation of mycobacteria.[3034 ] however , some species can not be differentiated by their 16s sequences because the number of polymorphic sites in the 16s rrna gene in the genus mycobacterium is rather small ( e.g. , mycobacterium kansasii and mycobacterium gastri ) , while others possess a very high degree of sequence similarity ( e.g. , mycobacterium marinum and mycobacterium ulcerans , mycobacterium abscessus , and mycobacterium chelonae ) . the 16s23s rrna gene internal transcribed spacer ( its ) region contains both conserved and highly variable signatures and is rather small . the 16s23s rrna gene its - based pcr produces a relatively small pcr product ( 200350 bp ) . this sequence of the 16s23s rrna its can distinguish between m. kansasii and m gastri ; however , it fails to distinguish between m. marinum and m. ulcerans and needs secondary methods like rflp or line blot hybridization to get conclusive results as demonstrated by various workers . the first marketed test was inno - lipa mycobacteria ( innogenetics ) in which the 16s23s intergenic region was amplified and then hybridized to a membrane on which were attached probes that recognized mycobacterial species revealed by colorimetry . the same principle was used to develop genotype mtbc tests ( hain lifescience gmbh , nehrin , germany ) , which use multiplex amplification of dna fragments ( 23s rrna gene , rd1 region , and gyrb gene ) to differentiate m. tuberculosis complex species ( mycobacterium tuberculosis , mycobacterium bovis ssp bovis , m. bovis bcg , bovis ssp caprae , mycobacterium africanum , mycobacterium canettii , and mycobacterium microti ) . the hsp65 gene - based pcr restriction pattern analysis ( pra ) mcnabb and co - workers assessed the use of partial sequences of the hsp65 gene for the routine identification of mycobacteria and reported an overall agreement of 85.2% with other identification methods ; discrepancies were most frequently encountered with isolates of m. chelonae , mycobacterium fortuitum , mycobacterium gordonae , mycobacterium scrofulaceum , and mycobacterium terrae . the amplification of the seca1 gene that codes for the essential protein seca1 , a key component of the major pathway of protein secretion across the cytoplasmic membrane , has also been tried . it was demonstrated that the seca1 gene of the genus mycobacterium can be used for species - level or complex - level ( for the mtb complex ) identification . it was also found that seca1 is a suitable target for diagnostics as the degree of interspecies variation for gene sequences among mycobacteria was observed to be moderate . these conclusions came from nonexhaustive studies and a need for further inputs for the improvement of the same was felt . apart from costly and cumbersome probe - based methods , combining the detection of various characteristic features or the multiple species identification using multiplex pcr also seems to be promising . for instance , to detect three mycobacterial species , the multiplex pcr is estimated to cost at least half that of a commercial dna probe which is used to identify only one of the three pathogens that can be identified by a multiplex pcr assay . in this regard , the use of the multiplex pcr could provide large savings in time , costs , and laboratory resources compared with the use of the expensive commercial dna probes , subcloning procedures , and biochemical tests . the use of multiplex pcr to detect members of the mycobacterium genus and to detect and differentiate m. tuberculosis , mycobacterium avium , and mycobacterium intracellulare has also been investigated . however , there exist a few limitations with respect to dna isolation , species identification , and obtaining cultures from a sophisticated system like bactec as a source to begin with . the recent development of the real - time pcr - based instrument named genexpert system , recently endorsed by the who , has led to the rapid detection of tb and drug resistance at the point of care . the mtb / rif test using this system provided sensitive detection of tb and rifampin resistance directly from the untreated sputum in less than 2 h with minimal hands - on time . who has recently issued a policy statement for the xpert mtb / rif system . this statement provides a brief description of the results obtained from various analytical studies , controlled clinical validation trials , field demonstration studies , etc . analysis with this system reports the detection of five genomic copies and 131 cfu / ml mtb spiked in sputum . based on the results , it has been recommended that xpert mtb / rif should be used as the initial diagnostic test in individuals suspected of mdr - tb or hiv - associated tb ( strong recommendation).xpert mtb / rif may be used as a follow - on test to microscopy in settings where mdr and/or hiv is of lesser concern , especially in smear - negative specimens ( conditional recommendation , recognizing major resource implications ) . xpert mtb / rif should be used as the initial diagnostic test in individuals suspected of mdr - tb or hiv - associated tb ( strong recommendation ) . xpert mtb / rif may be used as a follow - on test to microscopy in settings where mdr and/or hiv is of lesser concern , especially in smear - negative specimens ( conditional recommendation , recognizing major resource implications ) . however , conventional microscopy , culture , and dst , which are required to monitor treatment progress and to detect resistance to drugs other than rifampicin were found to be indispensible at this time . also several operational conditions need to be maintained for proper functioning of the instrument . apart from this , the cost of the instrument and the cartridges used is another concern that is being negotiated and taken care of by agencies like find . currently , there is an urgent need for a highly sensitive and specific diagnostic method for the identification of active m. tuberculosis disease that can be performed at the point of care , i.e. , outside the traditional laboratory setting . as serological methods have never shown to be very consistent in terms of their sensitivity and specificity for the detection of m. tuberculosis infection , research has again focused on finding new biomarkers for better detection . developing a method that uses alternate clues like biomarkers is desperately needed as all available diagnostic methods require a sputum sample limiting applicability to patients with pulmonary disease who are able to provide sputum for analysis . various molecules from mycobacterial as well as the host origin are being explored and tested for the same to get the desired speed , accuracy , and consistency in diagnosing the disease and restricting it for the benefit of patient and their environment . in addition to the biomarkers used in the currently available diagnostic tests , the literature is rich with candidate biomarkers like early secretary proteins such as early secretory antigenic 6 kda ( esat-6 ) and culture filtrate protein 10 ( cfp-10 ) , malate synthase , etc . , and a few molecules emerging from the host in response to infection with varying degrees of validation with the potential for development into new diagnostic tests.[4346 ] the emergence of new knowledge about interactions of m. tuberculosis with its host with the advancing technology has provided new concepts of the clinical phenotypes , pathogenesis , and host immune responses in tuberculosis and the resulting opportunities for biomarker discovery . the use of omics approaches may be needed for biomarker discovery in individuals who are exposed but not infected , who get infected but do not get the disease and in comparing them with those who succumb to the infection . also , it is expected that these new biomarkers would be able to differentiate between active and latent tuberculosis . the omics approach involves fetching out the signatures from transcriptomic , proteomic , or metabolomic profiling . hopefully , some biomarkers would provide us with a reliably consistent indication of infection and would help us detect the same before the infection proceeds to a level wherein we could easily detect afb in sputum or otherwise but late . also , with a significantly higher number of hiv patients ( 2.3 million prevalent cases ) , it complicates the management of tb in hiv - infected individuals . with about 50% lifetime risk of developing tb disease in hiv - infected people , it is projected that 50%60% of the hiv - infected persons in india will develop tb disease during their lifetime . comparable data from various indian studies conducted between 1994 and 2006 also report the prevalence of hiv tb co - infection in the range of 3%55% . scientific efforts have been put in by academia and research institutes in india for the development of better diagnostic tools . india has been a big market for in vitro diagnostics but has been dominated by imported and generic products , mostly serological , with virtually no innovations . examples of the development in the field of hepatitis and others from the indian industry have given a hope that indian diagnostic companies could also become world 's hub for high - quality generic diagnostics in the field of tb diagnostics . for this to happen , the indian industry needs to venture into genuine innovation in the field . this requires supportive policies , enhanced and timely funding , and greater collaboration between the workers and the funders from the public and private sector . rntcp , being an official caretaker in india for tb control , has been very active in the recent past . in line with the who 12-point policy package , rntcp has also adopted strategies to diagnose and manage tb in hiv - infected patients . the program has immediate priorities of restricting tb infection by providing treatment to all infected individuals . for diagnosis , there exist the guidelines for intensive case finding at the community level , but for early diagnosis of tb in the indian population , not many efforts could be made . this is very justifiable in light of huge numbers of already existing cases of tb . indian council of medical research ( icmr ) has also been working extensively on disease control programs with a support of the continued exploitation of scientific and technological advances from basic to applied research , from biomedical to health sciences , and from laboratory to field research . icmr is providing significant information through its laboratories engaged in tb research and also provides funding to various academic and research institutions for research in this area . they are not only discussing the problem and its solutions among themselves , but also consulting the international community for disseminating their work and getting updates from all over the world . an international symposium on tb diagnostics held at international centre for genetic engineering and biotechnology ( icgeb ) , new delhi , india , in december 2010 titled innovating to make an impact discussed multiple aspects regarding the challenges in tb diagnostics . a very positive feel for support in the field of diagnostic development came out of this . a consultative meeting held in january 2011 at national aids research institute , india , on galvanizing evidence for hiv management also incorporated a full session on tb supported by who . policy makers , subject experts , program implementers , researchers , hiv and tb physicians from different parts of india , researchers from another resource - limited setting like south africa , and researchers from usa took part in the discussions . exclusive discussions on diagnosing extra - pulmonary tb , childhood tb , and hiv tb were conducted as these pose serious challenges to developing universally applicable diagnostic tools for tb . the willingness and determination for better diagnosis and management of tb from laboratory workers to the policy makers this systematic review has not tried to incorporate the strategy of meta - analysis as the reports or publications being considered here are not sufficient to cover the entire range of efforts being contributed . recently , private enterprises are putting in a lot for the development of newer and better diagnostic tools in the field of tb . but the data from this end are not available to statistically assemble the results of studies into a single estimate . although the review has not taken into view all upcoming and sophisticated technologies which are not evidence based , the aim is to land upon an idea of basic problems being identified in due course of diagnostic development . table 1 reviews the frequently used techniques , problems associated with them , and an approach to solve these problems . also , in normal diagnostic settings which use conventional methods of diagnosis , it is always difficult to maintain quality and ensuring biosafety in the absence of recommended equipments . in a very recent policy recommendation by who , a warning has been issued against the use of inaccurate blood tests for active tb , and these tests have been defined as substandard tests with unreliable results . who has advised most of the countries which use these methods to ban the inaccurate and unapproved blood tests and instead rely on accurate microbiological or molecular tests , as recommended by who . as there is a big market for these tests in india , the focus is now going to be toward alternatives which can be placed in the market for active tb diagnosis . this is going to generate a huge demand of better diagnostic methods . in the light of a recent report totally drug resistant tuberculosis in india , followed by a big controversy about the origin and proper identification , it becomes indispensible for a clinician to confirm about the status of infecting species along with the exact nature of drug resistance . at the same time , a clinician can not wait for culture - based reports to initiate the treatment . in such cases , pcr / marker - based techniques would help getting concrete evidences of the etiology and would direct the treatment options on the right track . overview of diagnostic methods and their limitations looking on the other side , wherein commercial automated liquid cultures are recommended for better quality with minimum handling and manipulation of cultures by laboratory technicians , the expense and sophistication required is not a bearable load for all . in such conditions , alternative and novel culture - based approaches with a justifiable balance of cost , biosafety , and sensitivity are highly solicited . nucleic acid amplification test ( naat)-based methods seem to be the best and fastest options among all . the big question that arises then is how to facilitate the required resources . we need to upgrade all research and diagnostic units all over the affected regions of the world to equip themselves with basic set - ups of pcr assays and to make confirmative diagnosis a routine and feasible option . in designing assays , sometimes the worker is faced with a significant challenge in selecting the primer and probe combinations that detect all of the organisms to cover a broader range of significant organisms , avoiding cross - reactivity , sequence analysis , etc . anticipation and removal of inhibitory substances from the samples before the assay set - up is also a major issue to consider . for this , basic knowledge and training about molecular assays can be made a part of the basic training for technicians and scientists working in the field . also naa tests are not considered for the evaluation of patients receiving therapy as the technology can not distinguish between live and dead organisms . this can still be used with the same specificity if followed by short - term culture - based enrichment . a possibility of having some method that incorporates both culture and modern methods of detection has been visualized during this review . a culture enriching the initial viable loads followed by a sensitive and specific naa test with a minimized need of containment can make identification safer , faster , and more reliable . with the recent enthusiasm of various policy makers , funding agencies , and grants , the need is now to foster innovations that deliver better tools to diagnose tb with confidence using affordable approaches . after this only , the next step to treat and eradicate the menace of tb and associated risks can be taken with confidence . this review has been written with an intention to make readers aware of the kind of methodological challenges involved in such diagnosis . the author hopes that this will raise inquisitiveness in the brains of true scientific warriors to strive for newer ideas in making things simple and affordable .

tuberculosis ( tb ) has been a disease affecting almost all parts of the world since ages . lot many efforts came in the past for improving diagnosis and treatment . also , an effective vaccine has been sought after for long . with the emergence of resistant strains of mycobacterium tuberculosis , the causal organisms of tuberculosis , and complexities emerging due to other associated infections and disease conditions , there is a desperate need for further research input in the field . be it the better medication and care or better resistance management , proper diagnostics holds the key to success . it has been observed that a high burden of the disease was accompanied by resource limitations and poor research set - up . the scenario remained like this for several decades . with the refreshed vision of resourceful countries and funding agencies , funding is being provided in many areas of research in tuberculosis diagnosis and treatment . this review has been written with an aim to bring forth the limitations of available methods in the field of diagnostics and making researchers aware about the changing scenario with better funding opportunities and support . the author visualizes an enthusiasm from all over the world for the development of better modalities and urges scientists to join the struggle at this very perfect time to take the challenge and come forward with innovations in this field .

INTRODUCTION SMEAR MICROSCOPY THE CULTURE OF CULTURING THE EVOLUTION CONTINUES THE INDIAN PERSPECTIVE CONCLUSIONS

a report , published in september 2009 , from the world health organization ( who)-sponsored study includes tuberculosis ( tb ) and aids with a share of 5.5% each in the list of top 10 causes of death in all 1024 year olds , males and females combined , globally . this calls for ideas and projects to come up with something other than what we have been practicing for long . improving the research in tb has been a neglected area for long , in part due to the complex characteristics of the causal organism mycobacterium tuberculosis , the variable host response to infection , and , perhaps , complacency as this disease was nearly eradicated in high - income settings . with increasing hiv tb co - infections , tb has become a high priority for action and research in international health again . looking into this , who and the world bank , with international union against tuberculosis and lung disease ( iuatld ) , centre for disease control and prevention ( cdc ) , and other organizations , are reassessing their approaches to the prevention and control of tb . of late , agencies such as the foundation for innovative new diagnostics ( find ) , stop tb partnership 's new diagnostics working group ( ndwg ) , global laboratory initiative ( gli , another stop tb partnership working group ) , who , and the special program for research and training in tropical diseases ( tdr ) have shown increased interest toward the development of better diagnostics for tb . funding agencies such as the bill and melinda gates foundation , the global fund to fight aids , tb and malaria ( gfatm ) , and unitaid have emerged as resource providers for work on this disease that was being neglected in terms of sponsorship and interest of the private sector till now . with this , lots of efforts and funds are being brought up for the development of newer diagnostic methods in mycobacteriology . there are multiple hurdles to cross before we reach the ultimate goal of having an efficacious vaccine , easy and affordable diagnosis , and short - term treatment regimens with minimal side effects . in the field of tb , problem starts at the very first step , i.e. conventional tb diagnosis has been relying on medical history , tuberculin skin test , chest x - rays , and bacteriological examination . m. tuberculosis grows slowly , and clinical specimens submitted to the tb microscopy and culture are contaminated to varying degrees by more rapidly growing unwanted normal flora . moreover , the hydrophobic nature of the cell wall due to a high concentration of mycolic acid makes it more difficult to stain mycobaterial cells . the poor sensitivity of conventional smear microscopy has been a major concern . with various samples and sample processing methods , it has been found to be around 3643% sensitive . in general , direct smear reportedly detects afb only at concentrations of around 10,000 bacilli / ml of the specimen . various modifications to the technique and higher level of sophistications have been introduced in this field to get the highest level of sensitivity . the highest limit of detection in smear microscopy after employing the usp methodology and observing 400500 fields was experimentally found to be 250300 bacilli / ml of the sample . under the best conditions for culture on solid and liquid media with usp - treated spiked sputum , the detection limit was found to be 400 cfu / ml after 8 weeks of incubation . hence , although it could reduce the bacilli count needed to be detected using smear , it has not helped improving detection by culture . however , the researchers felt a need for a more sensitive test to detect samples with a low bacterial load . lange and mori have given an update on the developments being made in the field of fluorescent microscopy . these include light - emitting diode ( led)-based fluorescent microscopy , mobile phone - based microscopy , and automated detection systems using image processing . with many more other studies , it is very apparent that we are still struggling hard to get a sensitive , easy , cost - effective , and faster method for smear microscopy . it seems possible to decrease mortality and morbidity due to tb using smear microscopy diagnosis and dots coverage , as being done in most of the high prevalence countries like india . also , bacterial isolation for drug - susceptibility testing and genotyping is required using a solid or liquid medium . therefore , a culture is expected on all initial samples to confirm the diagnosis followed by biochemical tests like catalase test at 68c , nitrate reduction , and niacin accumulation for speciation . although culture - based diagnosis of tb is recommended in international standards of tuberculosis care , lack of resources and technical expertise poses as a major limitation in most of the high prevalence countries . this method compares the mycobacterial growth levels in clinical isolates to be tested with known standard culture in the presence of different concentrations of anti - tb drugs in a proportional way . this method enables a determination of the concentration that inhibits more than 99% of the inoculum and is reported as the minimal inhibitory concentration ( mic ) of the given drug . but it is cumbersome , technically demanding , lacks reproducibility , and it generally takes at least 21 days for a result after assay set - up . liquid culturing with radioisotopic detection or with the incorporation of fluorescent dyes was introduced in the past as a confirmatory method ( bactec 460 , bactec mgit 960 system , mb / bact , and versa trek system ) . however , looking at the sophistication and issues like contamination , the struggle again started to search something better . microscopic observation drug susceptibility ( mods ) testing developed recently allows both rapid and low - cost tb diagnosis in liquid culture with the simultaneous determination of drug susceptibilities. [1618 ] however , mods requires higher biosafety level facilities , an expert and experienced microbiologist , and the samples are very prone to contaminations . some other situations like immune suppression and in the case of children , getting a good quality sample and getting the required microbial load in the samples is tough . hence , indirect approaches to trace the causal organism / byproducts or to look into the host response against the infection are also being considered . the pcr - based amplification of various target nucleic acids has been tried extensively that allows rapid and sensitive detection of target dna sequences . ideally , pcr can detect a single copy of the gene being targeted for amplification but with all the processing to avoid interference , the detection of as few as 410 copies has been made possible by several workers . although the 16s rrna gene is the most commonly used target , other targets have also provided a high - sensitivity and representative species - specific differentiation . the pcr amplification of the entire 16s23s rrna spacer region and use of a secondary technique of randomly amplified polymorphic dna ( rapd ) fingerprinting to differentiate strains belonging to the mycobacterium species has been reported . the amplification of the entire 16s23s intergenic region , and diagnostic tests for bacterial organisms using probes targeted for sequences within the 16s23s intergenic region have also been described . other targets include the 16s rrna gene , the 16s23s internal transcribed spacer , the 65-kda heat shock protein , reca , rpob , and gyrb. [232629 ] the 16s rrna gene - based methods are presently widely used for the identification and differentiation of mycobacteria. [3034 ] however , some species can not be differentiated by their 16s sequences because the number of polymorphic sites in the 16s rrna gene in the genus mycobacterium is rather small ( e.g. , mycobacterium marinum and mycobacterium ulcerans , mycobacterium abscessus , and mycobacterium chelonae ) . this sequence of the 16s23s rrna its can distinguish between m. kansasii and m gastri ; however , it fails to distinguish between m. marinum and m. ulcerans and needs secondary methods like rflp or line blot hybridization to get conclusive results as demonstrated by various workers . the same principle was used to develop genotype mtbc tests ( hain lifescience gmbh , nehrin , germany ) , which use multiplex amplification of dna fragments ( 23s rrna gene , rd1 region , and gyrb gene ) to differentiate m. tuberculosis complex species ( mycobacterium tuberculosis , mycobacterium bovis ssp bovis , m. bovis bcg , bovis ssp caprae , mycobacterium africanum , mycobacterium canettii , and mycobacterium microti ) . the hsp65 gene - based pcr restriction pattern analysis ( pra ) mcnabb and co - workers assessed the use of partial sequences of the hsp65 gene for the routine identification of mycobacteria and reported an overall agreement of 85.2% with other identification methods ; discrepancies were most frequently encountered with isolates of m. chelonae , mycobacterium fortuitum , mycobacterium gordonae , mycobacterium scrofulaceum , and mycobacterium terrae . the amplification of the seca1 gene that codes for the essential protein seca1 , a key component of the major pathway of protein secretion across the cytoplasmic membrane , has also been tried . it was demonstrated that the seca1 gene of the genus mycobacterium can be used for species - level or complex - level ( for the mtb complex ) identification . these conclusions came from nonexhaustive studies and a need for further inputs for the improvement of the same was felt . for instance , to detect three mycobacterial species , the multiplex pcr is estimated to cost at least half that of a commercial dna probe which is used to identify only one of the three pathogens that can be identified by a multiplex pcr assay . in this regard , the use of the multiplex pcr could provide large savings in time , costs , and laboratory resources compared with the use of the expensive commercial dna probes , subcloning procedures , and biochemical tests . the use of multiplex pcr to detect members of the mycobacterium genus and to detect and differentiate m. tuberculosis , mycobacterium avium , and mycobacterium intracellulare has also been investigated . however , there exist a few limitations with respect to dna isolation , species identification , and obtaining cultures from a sophisticated system like bactec as a source to begin with . the recent development of the real - time pcr - based instrument named genexpert system , recently endorsed by the who , has led to the rapid detection of tb and drug resistance at the point of care . who has recently issued a policy statement for the xpert mtb / rif system . this statement provides a brief description of the results obtained from various analytical studies , controlled clinical validation trials , field demonstration studies , etc . based on the results , it has been recommended that xpert mtb / rif should be used as the initial diagnostic test in individuals suspected of mdr - tb or hiv - associated tb ( strong recommendation).xpert mtb / rif may be used as a follow - on test to microscopy in settings where mdr and/or hiv is of lesser concern , especially in smear - negative specimens ( conditional recommendation , recognizing major resource implications ) . however , conventional microscopy , culture , and dst , which are required to monitor treatment progress and to detect resistance to drugs other than rifampicin were found to be indispensible at this time . also several operational conditions need to be maintained for proper functioning of the instrument . apart from this , the cost of the instrument and the cartridges used is another concern that is being negotiated and taken care of by agencies like find . currently , there is an urgent need for a highly sensitive and specific diagnostic method for the identification of active m. tuberculosis disease that can be performed at the point of care , i.e. as serological methods have never shown to be very consistent in terms of their sensitivity and specificity for the detection of m. tuberculosis infection , research has again focused on finding new biomarkers for better detection . various molecules from mycobacterial as well as the host origin are being explored and tested for the same to get the desired speed , accuracy , and consistency in diagnosing the disease and restricting it for the benefit of patient and their environment . in addition to the biomarkers used in the currently available diagnostic tests , the literature is rich with candidate biomarkers like early secretary proteins such as early secretory antigenic 6 kda ( esat-6 ) and culture filtrate protein 10 ( cfp-10 ) , malate synthase , etc . , and a few molecules emerging from the host in response to infection with varying degrees of validation with the potential for development into new diagnostic tests. [4346 ] the emergence of new knowledge about interactions of m. tuberculosis with its host with the advancing technology has provided new concepts of the clinical phenotypes , pathogenesis , and host immune responses in tuberculosis and the resulting opportunities for biomarker discovery . the use of omics approaches may be needed for biomarker discovery in individuals who are exposed but not infected , who get infected but do not get the disease and in comparing them with those who succumb to the infection . also , with a significantly higher number of hiv patients ( 2.3 million prevalent cases ) , it complicates the management of tb in hiv - infected individuals . with about 50% lifetime risk of developing tb disease in hiv - infected people , it is projected that 50%60% of the hiv - infected persons in india will develop tb disease during their lifetime . comparable data from various indian studies conducted between 1994 and 2006 also report the prevalence of hiv tb co - infection in the range of 3%55% . scientific efforts have been put in by academia and research institutes in india for the development of better diagnostic tools . india has been a big market for in vitro diagnostics but has been dominated by imported and generic products , mostly serological , with virtually no innovations . examples of the development in the field of hepatitis and others from the indian industry have given a hope that indian diagnostic companies could also become world 's hub for high - quality generic diagnostics in the field of tb diagnostics . for this to happen , the indian industry needs to venture into genuine innovation in the field . this requires supportive policies , enhanced and timely funding , and greater collaboration between the workers and the funders from the public and private sector . rntcp , being an official caretaker in india for tb control , has been very active in the recent past . for diagnosis , there exist the guidelines for intensive case finding at the community level , but for early diagnosis of tb in the indian population , not many efforts could be made . indian council of medical research ( icmr ) has also been working extensively on disease control programs with a support of the continued exploitation of scientific and technological advances from basic to applied research , from biomedical to health sciences , and from laboratory to field research . icmr is providing significant information through its laboratories engaged in tb research and also provides funding to various academic and research institutions for research in this area . they are not only discussing the problem and its solutions among themselves , but also consulting the international community for disseminating their work and getting updates from all over the world . a very positive feel for support in the field of diagnostic development came out of this . policy makers , subject experts , program implementers , researchers , hiv and tb physicians from different parts of india , researchers from another resource - limited setting like south africa , and researchers from usa took part in the discussions . the willingness and determination for better diagnosis and management of tb from laboratory workers to the policy makers this systematic review has not tried to incorporate the strategy of meta - analysis as the reports or publications being considered here are not sufficient to cover the entire range of efforts being contributed . recently , private enterprises are putting in a lot for the development of newer and better diagnostic tools in the field of tb . although the review has not taken into view all upcoming and sophisticated technologies which are not evidence based , the aim is to land upon an idea of basic problems being identified in due course of diagnostic development . table 1 reviews the frequently used techniques , problems associated with them , and an approach to solve these problems . also , in normal diagnostic settings which use conventional methods of diagnosis , it is always difficult to maintain quality and ensuring biosafety in the absence of recommended equipments . in a very recent policy recommendation by who , a warning has been issued against the use of inaccurate blood tests for active tb , and these tests have been defined as substandard tests with unreliable results . who has advised most of the countries which use these methods to ban the inaccurate and unapproved blood tests and instead rely on accurate microbiological or molecular tests , as recommended by who . as there is a big market for these tests in india , the focus is now going to be toward alternatives which can be placed in the market for active tb diagnosis . in the light of a recent report totally drug resistant tuberculosis in india , followed by a big controversy about the origin and proper identification , it becomes indispensible for a clinician to confirm about the status of infecting species along with the exact nature of drug resistance . overview of diagnostic methods and their limitations looking on the other side , wherein commercial automated liquid cultures are recommended for better quality with minimum handling and manipulation of cultures by laboratory technicians , the expense and sophistication required is not a bearable load for all . in such conditions , alternative and novel culture - based approaches with a justifiable balance of cost , biosafety , and sensitivity are highly solicited . we need to upgrade all research and diagnostic units all over the affected regions of the world to equip themselves with basic set - ups of pcr assays and to make confirmative diagnosis a routine and feasible option . in designing assays , sometimes the worker is faced with a significant challenge in selecting the primer and probe combinations that detect all of the organisms to cover a broader range of significant organisms , avoiding cross - reactivity , sequence analysis , etc . anticipation and removal of inhibitory substances from the samples before the assay set - up is also a major issue to consider . for this , basic knowledge and training about molecular assays can be made a part of the basic training for technicians and scientists working in the field . also naa tests are not considered for the evaluation of patients receiving therapy as the technology can not distinguish between live and dead organisms . this can still be used with the same specificity if followed by short - term culture - based enrichment . a possibility of having some method that incorporates both culture and modern methods of detection has been visualized during this review . a culture enriching the initial viable loads followed by a sensitive and specific naa test with a minimized need of containment can make identification safer , faster , and more reliable . with the recent enthusiasm of various policy makers , funding agencies , and grants , the need is now to foster innovations that deliver better tools to diagnose tb with confidence using affordable approaches . after this only , the next step to treat and eradicate the menace of tb and associated risks can be taken with confidence . this review has been written with an intention to make readers aware of the kind of methodological challenges involved in such diagnosis . the author hopes that this will raise inquisitiveness in the brains of true scientific warriors to strive for newer ideas in making things simple and affordable .

a report , published in september 2009 , from the world health organization ( who)-sponsored study includes tuberculosis ( tb ) and aids with a share of 5.5% each in the list of top 10 causes of death in all 1024 year olds , males and females combined , globally . stop tb partnership announced the theme for world tuberculosis day 2010 as on the move against tuberculosis : innovate to accelerate action . improving the research in tb has been a neglected area for long , in part due to the complex characteristics of the causal organism mycobacterium tuberculosis , the variable host response to infection , and , perhaps , complacency as this disease was nearly eradicated in high - income settings . with increasing hiv tb co - infections , tb has become a high priority for action and research in international health again . looking into this , who and the world bank , with international union against tuberculosis and lung disease ( iuatld ) , centre for disease control and prevention ( cdc ) , and other organizations , are reassessing their approaches to the prevention and control of tb . of late , agencies such as the foundation for innovative new diagnostics ( find ) , stop tb partnership 's new diagnostics working group ( ndwg ) , global laboratory initiative ( gli , another stop tb partnership working group ) , who , and the special program for research and training in tropical diseases ( tdr ) have shown increased interest toward the development of better diagnostics for tb . funding agencies such as the bill and melinda gates foundation , the global fund to fight aids , tb and malaria ( gfatm ) , and unitaid have emerged as resource providers for work on this disease that was being neglected in terms of sponsorship and interest of the private sector till now . with this , lots of efforts and funds are being brought up for the development of newer diagnostic methods in mycobacteriology . there are multiple hurdles to cross before we reach the ultimate goal of having an efficacious vaccine , easy and affordable diagnosis , and short - term treatment regimens with minimal side effects . in the field of tb , problem starts at the very first step , i.e. conventional tb diagnosis has been relying on medical history , tuberculin skin test , chest x - rays , and bacteriological examination . m. tuberculosis grows slowly , and clinical specimens submitted to the tb microscopy and culture are contaminated to varying degrees by more rapidly growing unwanted normal flora . moreover , the hydrophobic nature of the cell wall due to a high concentration of mycolic acid makes it more difficult to stain mycobaterial cells . in general , direct smear reportedly detects afb only at concentrations of around 10,000 bacilli / ml of the specimen . various modifications to the technique and higher level of sophistications have been introduced in this field to get the highest level of sensitivity . with certain variations in the sample processing methods for smear preparation , samples were subjected to different centrifugation forces and centrifugation times after decontamination and liquefaction and the centrifuged deposits were examined by smear and culture . various workers have demonstrated the improved detection of direct smear - negative cases by universal sample processing ( usp ) smear microscopy . the highest limit of detection in smear microscopy after employing the usp methodology and observing 400500 fields was experimentally found to be 250300 bacilli / ml of the sample . under the best conditions for culture on solid and liquid media with usp - treated spiked sputum , the detection limit was found to be 400 cfu / ml after 8 weeks of incubation . steingart and co - workers did a systematic review to assess the ability of different processing methods to improve the sensitivity of microscopy . their search suggested that centrifugation with any of several chemical methods ( including bleach ) is more sensitive , that overnight sedimentation preceded by chemical processing is more sensitive , and that the specificity is similar . recently , a pilot study has compared the diagnostic accuracy and incremental yield of two short - duration ( < 1 h ) sputum pretreatment procedures involving pretreatment with bleach and usp centrifugation and concluded that both did not increase yield as compared to direct sputum smears . people tried hard for developing field - based methods to avoid the use of centrifugation during decontamination . but it was found to have a lower sensitivity as compared to the conventional method of centrifuge decontamination . fluorescence microscopy is credited with increased sensitivity and lower work effort but has a rider of increased cost and technical complexity . pai and co - workers have reported an average 10% increment in sensitivity of microscopy using fluorescence microscopy with no difference in specificity . these include light - emitting diode ( led)-based fluorescent microscopy , mobile phone - based microscopy , and automated detection systems using image processing . with many more other studies , it is very apparent that we are still struggling hard to get a sensitive , easy , cost - effective , and faster method for smear microscopy . it seems possible to decrease mortality and morbidity due to tb using smear microscopy diagnosis and dots coverage , as being done in most of the high prevalence countries like india . but as many smear negative cases may remain undiagnosed , cutting transmission of infection does not seem to be an achievable target without having better diagnostic amenities . just the presence of acid - fast bacilli ( afb ) on a sputum smear or other specimens does not confirm a diagnosis of tb as all acid - fast - bacilli are not m. tuberculosis . therefore , a culture is expected on all initial samples to confirm the diagnosis followed by biochemical tests like catalase test at 68c , nitrate reduction , and niacin accumulation for speciation . although culture - based diagnosis of tb is recommended in international standards of tuberculosis care , lack of resources and technical expertise poses as a major limitation in most of the high prevalence countries . this method compares the mycobacterial growth levels in clinical isolates to be tested with known standard culture in the presence of different concentrations of anti - tb drugs in a proportional way . this method enables a determination of the concentration that inhibits more than 99% of the inoculum and is reported as the minimal inhibitory concentration ( mic ) of the given drug . liquid culturing with radioisotopic detection or with the incorporation of fluorescent dyes was introduced in the past as a confirmatory method ( bactec 460 , bactec mgit 960 system , mb / bact , and versa trek system ) . however , looking at the sophistication and issues like contamination , the struggle again started to search something better . microscopic observation drug susceptibility ( mods ) testing developed recently allows both rapid and low - cost tb diagnosis in liquid culture with the simultaneous determination of drug susceptibilities. thus , it needs addition of various antibiotics in culture media and the samples need to be processed before inoculation involving decontamination and centrifugation steps . some other unconventional methods like thin - layer agar ( tla ) and the direct nitrate reductase assay ( nra ) have attempted to address the problem of multiple point processing and hence the generation of aerosols by incorporating visual inspection of results in the form of typical colony morphology or color change to identify tb growth . some other situations like immune suppression and in the case of children , getting a good quality sample and getting the required microbial load in the samples is tough . hence , indirect approaches to trace the causal organism / byproducts or to look into the host response against the infection are also being considered . the pcr - based amplification of various target nucleic acids has been tried extensively that allows rapid and sensitive detection of target dna sequences . ideally , pcr can detect a single copy of the gene being targeted for amplification but with all the processing to avoid interference , the detection of as few as 410 copies has been made possible by several workers . although the 16s rrna gene is the most commonly used target , other targets have also provided a high - sensitivity and representative species - specific differentiation . the pcr amplification of the entire 16s23s rrna spacer region and use of a secondary technique of randomly amplified polymorphic dna ( rapd ) fingerprinting to differentiate strains belonging to the mycobacterium species has been reported . the amplification of the entire 16s23s intergenic region , and diagnostic tests for bacterial organisms using probes targeted for sequences within the 16s23s intergenic region have also been described . other targets include the 16s rrna gene , the 16s23s internal transcribed spacer , the 65-kda heat shock protein , reca , rpob , and gyrb. [232629 ] the 16s rrna gene - based methods are presently widely used for the identification and differentiation of mycobacteria. [3034 ] however , some species can not be differentiated by their 16s sequences because the number of polymorphic sites in the 16s rrna gene in the genus mycobacterium is rather small ( e.g. this sequence of the 16s23s rrna its can distinguish between m. kansasii and m gastri ; however , it fails to distinguish between m. marinum and m. ulcerans and needs secondary methods like rflp or line blot hybridization to get conclusive results as demonstrated by various workers . the first marketed test was inno - lipa mycobacteria ( innogenetics ) in which the 16s23s intergenic region was amplified and then hybridized to a membrane on which were attached probes that recognized mycobacterial species revealed by colorimetry . the same principle was used to develop genotype mtbc tests ( hain lifescience gmbh , nehrin , germany ) , which use multiplex amplification of dna fragments ( 23s rrna gene , rd1 region , and gyrb gene ) to differentiate m. tuberculosis complex species ( mycobacterium tuberculosis , mycobacterium bovis ssp bovis , m. bovis bcg , bovis ssp caprae , mycobacterium africanum , mycobacterium canettii , and mycobacterium microti ) . the hsp65 gene - based pcr restriction pattern analysis ( pra ) mcnabb and co - workers assessed the use of partial sequences of the hsp65 gene for the routine identification of mycobacteria and reported an overall agreement of 85.2% with other identification methods ; discrepancies were most frequently encountered with isolates of m. chelonae , mycobacterium fortuitum , mycobacterium gordonae , mycobacterium scrofulaceum , and mycobacterium terrae . the amplification of the seca1 gene that codes for the essential protein seca1 , a key component of the major pathway of protein secretion across the cytoplasmic membrane , has also been tried . it was demonstrated that the seca1 gene of the genus mycobacterium can be used for species - level or complex - level ( for the mtb complex ) identification . it was also found that seca1 is a suitable target for diagnostics as the degree of interspecies variation for gene sequences among mycobacteria was observed to be moderate . apart from costly and cumbersome probe - based methods , combining the detection of various characteristic features or the multiple species identification using multiplex pcr also seems to be promising . for instance , to detect three mycobacterial species , the multiplex pcr is estimated to cost at least half that of a commercial dna probe which is used to identify only one of the three pathogens that can be identified by a multiplex pcr assay . in this regard , the use of the multiplex pcr could provide large savings in time , costs , and laboratory resources compared with the use of the expensive commercial dna probes , subcloning procedures , and biochemical tests . the use of multiplex pcr to detect members of the mycobacterium genus and to detect and differentiate m. tuberculosis , mycobacterium avium , and mycobacterium intracellulare has also been investigated . however , there exist a few limitations with respect to dna isolation , species identification , and obtaining cultures from a sophisticated system like bactec as a source to begin with . the recent development of the real - time pcr - based instrument named genexpert system , recently endorsed by the who , has led to the rapid detection of tb and drug resistance at the point of care . the mtb / rif test using this system provided sensitive detection of tb and rifampin resistance directly from the untreated sputum in less than 2 h with minimal hands - on time . analysis with this system reports the detection of five genomic copies and 131 cfu / ml mtb spiked in sputum . based on the results , it has been recommended that xpert mtb / rif should be used as the initial diagnostic test in individuals suspected of mdr - tb or hiv - associated tb ( strong recommendation).xpert mtb / rif may be used as a follow - on test to microscopy in settings where mdr and/or hiv is of lesser concern , especially in smear - negative specimens ( conditional recommendation , recognizing major resource implications ) . xpert mtb / rif should be used as the initial diagnostic test in individuals suspected of mdr - tb or hiv - associated tb ( strong recommendation ) . xpert mtb / rif may be used as a follow - on test to microscopy in settings where mdr and/or hiv is of lesser concern , especially in smear - negative specimens ( conditional recommendation , recognizing major resource implications ) . however , conventional microscopy , culture , and dst , which are required to monitor treatment progress and to detect resistance to drugs other than rifampicin were found to be indispensible at this time . apart from this , the cost of the instrument and the cartridges used is another concern that is being negotiated and taken care of by agencies like find . currently , there is an urgent need for a highly sensitive and specific diagnostic method for the identification of active m. tuberculosis disease that can be performed at the point of care , i.e. as serological methods have never shown to be very consistent in terms of their sensitivity and specificity for the detection of m. tuberculosis infection , research has again focused on finding new biomarkers for better detection . various molecules from mycobacterial as well as the host origin are being explored and tested for the same to get the desired speed , accuracy , and consistency in diagnosing the disease and restricting it for the benefit of patient and their environment . in addition to the biomarkers used in the currently available diagnostic tests , the literature is rich with candidate biomarkers like early secretary proteins such as early secretory antigenic 6 kda ( esat-6 ) and culture filtrate protein 10 ( cfp-10 ) , malate synthase , etc . , and a few molecules emerging from the host in response to infection with varying degrees of validation with the potential for development into new diagnostic tests. [4346 ] the emergence of new knowledge about interactions of m. tuberculosis with its host with the advancing technology has provided new concepts of the clinical phenotypes , pathogenesis , and host immune responses in tuberculosis and the resulting opportunities for biomarker discovery . the use of omics approaches may be needed for biomarker discovery in individuals who are exposed but not infected , who get infected but do not get the disease and in comparing them with those who succumb to the infection . hopefully , some biomarkers would provide us with a reliably consistent indication of infection and would help us detect the same before the infection proceeds to a level wherein we could easily detect afb in sputum or otherwise but late . also , with a significantly higher number of hiv patients ( 2.3 million prevalent cases ) , it complicates the management of tb in hiv - infected individuals . with about 50% lifetime risk of developing tb disease in hiv - infected people , it is projected that 50%60% of the hiv - infected persons in india will develop tb disease during their lifetime . comparable data from various indian studies conducted between 1994 and 2006 also report the prevalence of hiv tb co - infection in the range of 3%55% . examples of the development in the field of hepatitis and others from the indian industry have given a hope that indian diagnostic companies could also become world 's hub for high - quality generic diagnostics in the field of tb diagnostics . this requires supportive policies , enhanced and timely funding , and greater collaboration between the workers and the funders from the public and private sector . for diagnosis , there exist the guidelines for intensive case finding at the community level , but for early diagnosis of tb in the indian population , not many efforts could be made . indian council of medical research ( icmr ) has also been working extensively on disease control programs with a support of the continued exploitation of scientific and technological advances from basic to applied research , from biomedical to health sciences , and from laboratory to field research . an international symposium on tb diagnostics held at international centre for genetic engineering and biotechnology ( icgeb ) , new delhi , india , in december 2010 titled innovating to make an impact discussed multiple aspects regarding the challenges in tb diagnostics . policy makers , subject experts , program implementers , researchers , hiv and tb physicians from different parts of india , researchers from another resource - limited setting like south africa , and researchers from usa took part in the discussions . exclusive discussions on diagnosing extra - pulmonary tb , childhood tb , and hiv tb were conducted as these pose serious challenges to developing universally applicable diagnostic tools for tb . the willingness and determination for better diagnosis and management of tb from laboratory workers to the policy makers this systematic review has not tried to incorporate the strategy of meta - analysis as the reports or publications being considered here are not sufficient to cover the entire range of efforts being contributed . recently , private enterprises are putting in a lot for the development of newer and better diagnostic tools in the field of tb . although the review has not taken into view all upcoming and sophisticated technologies which are not evidence based , the aim is to land upon an idea of basic problems being identified in due course of diagnostic development . table 1 reviews the frequently used techniques , problems associated with them , and an approach to solve these problems . as there is a big market for these tests in india , the focus is now going to be toward alternatives which can be placed in the market for active tb diagnosis . in the light of a recent report totally drug resistant tuberculosis in india , followed by a big controversy about the origin and proper identification , it becomes indispensible for a clinician to confirm about the status of infecting species along with the exact nature of drug resistance . in such conditions , alternative and novel culture - based approaches with a justifiable balance of cost , biosafety , and sensitivity are highly solicited . we need to upgrade all research and diagnostic units all over the affected regions of the world to equip themselves with basic set - ups of pcr assays and to make confirmative diagnosis a routine and feasible option . in designing assays , sometimes the worker is faced with a significant challenge in selecting the primer and probe combinations that detect all of the organisms to cover a broader range of significant organisms , avoiding cross - reactivity , sequence analysis , etc . for this , basic knowledge and training about molecular assays can be made a part of the basic training for technicians and scientists working in the field .

interferon- ( ifn- ) is an essential cytokine for immunity against intracellular pathogens and cancer . mice with targeted disruptions of the ifng gene or ifng gene receptor 1 are highly susceptible to a variety of bacteria , protozoans , and virus infection . furthermore , when mice lacking sensitivity to ifn- were challenged with chemical carcinogens , they developed tumor more rapidly and with higher frequency than wild type animals [ 2 , 3 ] . ifn- is produced by cells that mediate both innate and adaptative immune responses . natural killer ( nk ) and natural killer t ( nkt ) cells are the innate cells sources of this cytokine and rapidly produce ifn- upon activation . on the other hand significant increase in ifn- expression by cd4 t lymphocytes is observed only after t helper ( th ) 1 differentiation . in fact , upon activation , cd4 t cells can differentiate into several effector lineages , of which th1 is the only one that produce high levels of ifn-. nave cd8 t lymphocytes do not produce large amounts of ifn- , but after tcr stimulation these cells undergo differentiation into cytotoxic t lymphocytes ( ctl ) and memory cells , which are capable of producing high levels of this cytokine in response to tcr activation or interleukin- ( il- ) 12 and il-18 . the best characterized role of ifn- in cd8 t cell immunity is in enhancing class i antigen presentation pathway , which facilitates cytotoxic t cells to recognize infected cells . ifn- signaling upregulation leads to expression of mhc class i and the tap transporter , as well as chaperones such as tapasin . ifn- also induces a replacement of the constitutive proteasome subunits , 1 , 2 and 5 , for the immunoproteasome subunits lmp2 , mecl-1 , and lmp7 , an essential feature for increasing the quantity and repertoire of peptides presented in the context of class i mhc ( reviewed in ) . ifn- also plays an important role in cd8 t cell homeostasis that is independent from its function in clearance of infection . several studies have suggested that ifn- is a key determinant of immunodominance [ 68 ] . badovinac and colleagues have shown that ifn- deficient mice infected with an attenuated listeria monocytogenes strain exhibited an altered immunodominance hierarchy due to an increased expansion of cd8 t cells specific for a subdominant epitope of l. monocytogenes . furthermore , ifn- seems to be required for subdominant cd8 t cells response suppression by dominant cd8 t cell response and the cd8 t cells that promptly produce ifn- after stimulation are preferentially expanded . this cytokine is important for the differentiation of tcrcd4cd8 intraepithelial lymphocytes ( iels ) in the gut . iels are lymphocytes considered activated yet resting and their regulation is crucial role in the maintenance of the epithelial cell barrier and gut physiological inflammation . ifn- also acts directly on cd8 t cells by stimulating their abundance in an acute lymphocytic choriomeningitis virus ( lcmv ) infection and enhancing the development of memory cells . interestingly , sercan and colleagues show that ifn- produced by innate immune cells contributes to antigen - specific cd8 t cell homeostasis . however , badovinac and colleagues also have shown that ifn- deficiency resulted in a delayed contraction of antigen - specific cd8 t cell populations from both listeria monocytogenes and lcmv infections , which suggests an important role of this cytokine in control of death phase of antigen - specific cd8 t cells . therefore , this cytokine has both positive and negative effects on cd8 t cell abundance depending on the phase of the cd8 response and also the biology model evaluated . this dual role is clearly evident in ifn- deficient mice , in which both the expansion and contraction of cd8 t cell response are impaired . cd8 t cells are usually characterized by their cytolytic activities involving perforin or fas mechanisms to kill targeted cells . however cytokine secretion by cd8 t cells also has an important role in the control of intracellular infections . in 1990 , fong and mosmann suggested that th1 cells and cd8 t cells could share cytokine mediated functions , like combating intracellular pathogens and tumors cells . they observed that alloreactive murine cd8 t cell clones produced both mrna and protein profile characteristic of th1 clones , which include high levels of ifn-. it is well known that the clearance of several infections also depends on noncytolytic functions of cd8 t cells . the control of m. tuberculosis infections in mice requires the ability of the cd8 t cells to produce ifn- . furthermore , ifn- produced by cd8 t cells is essential to clear numerous viral infections such as measles virus , herpes simplex virus type 1 , lcmv , and borna disease virus . this ifn- mediated response seems to be important to avoid tissue damage and inflammation , which is normally observed in cytotoxic cd8 t cell response ( reviewed in ) . indeed , hiv control is associated with polyfunctional cd8 t cells , that is , epitope - specific cells expressing several effector functions , which includes the expression of cytokines such as tnf and ifn- . the ifn- produced by cd8 t cells seems to have other immunomodulatory roles , acting on cd4 t cells and b cells and also on cd8 t cells themselves . the differentiation of cd4 t cells in t helper subsets depends on the cytokine milieu where these primary t cells were stimulated . the main sources of these cytokines are activated innate immune cells . for th1 differentiation , the cytokines il-12 , produced by apcs , and ifn- , mainly produced by nk cells , are critical to induce and reinforce th1 commitment . several studies have also suggested that cd8 t lymphocytes could have a role in the generation of th1 immunity and in the inhibition of th2 response [ 1820 ] . uzonna and colleagues have shown that ifn- produced by murine cd8 t cells , in response to low doses of l. major , downregulates an initial th2 response and enhances th1 commitment . data from our group suggest that , besides nk and dendritic cells , cd8 t lymphocytes are also another source of ifn- that enhances cd4 th1 phenotype development . we have shown that , after tcr activation of primary lymph node cells , cd8 t lymphocytes are the major source of ifn- production . furthermore , cd4 t cells cocultured with ifn--competent cd8 t cells clearly produce more ifn- and less il-4 than cd4 t cells cultured with ifn--deficient cd8 t cells . this work also suggested that nfat1 transcription factor - dependent ifn- production by cd8 t cell is important during eosinophil migration to pleura in a pleurisy model , which suggests an important role for ifn- produced by cd8 t cells in the control of allergic diseases . together these studies reinforce the role of ifn- produced by cd8 t cells in regulation of th immune responses in vivo . the autocrine ifn- signaling is important for th1 differentiation , and recently it has been suggested that ifn- is an autocrine / paracrine factor to promote nave t cd8 cell differentiation . low levels of ifn- produced by cd8 t cells promote the upregulation of t - bet , granzyme b , and ifn- also promotes a week cytolytic activity . ifn- alone does not support strong differentiation but can synergize with ifn- in driving effector differentiation of these cd8 t cells . a previous paper has also shown that nave cd8 t cells receive an ifn- signal few hours after l. monocytogenes infection . these studies suggest that the ifn- signals for cd8 t cell differentiation are delivered early in the immune response . when nave t cd4 and cd8 cells are compared for the ifn- production , it is clear that , after tcr activation , cd8 t cells produce more levels of ifn- than cd4 t cells , and the requirements for tcr induced ifn- production are different between these primary cells . in fact , carter and murphy have shown that cd4 t cells require stat4 activation beside tcr signalization to produce ifn- , while cd8 t cells need only tcr activation . furthermore , the main difference in transcriptional requirements for ifng expression between cd4 and cd8 t cells seems to be the differential role of members of the t - box family of transcription factors , t - bet and eomesodermin ( eomes ) . t - bet is the master regulator of ifng and th1 commitment of cd4 t cells and sufficient for induction of ifng expression in these cells . on the other hand , additionally , eomes and t - bet play important roles during cd8 t cell differentiation to effector and memory t cells , where t - bet is associated with effector phenotype whereas expression of eomes increases in memory cd8 t cells . there are several studies about ifn- production by th1 cells and the molecular mechanisms that control ifng expression in these cells are widely investigated and extensively reviewed . however , regulation of ifng expression in cd8 t cells is not fairly explored and discussed . an effective cellular immune response is characterized by robust stimulation of nave lymphocytes to undergo differentiation into effector cells , which provides pathogen clearance while promoting the development of long - lived memory cells that can respond to reinfection faster than nave cells . the cd8 differentiation is accompanied by large - scale changes in the coordinate expression of genes associated with effector function , survival , and self - renewal and recently it has been recognized that epigenetic modification is an integral part of this process . this section will focus on the chromatin status of ifng promoter in cd8 t cells . however , due to the scarcity of studies exploring cd8 t cells and the higher availability of data investigating cd4 t cells , some aspects of cd4 regulation will be mentioned for comparison . differential gene expression can be regulated from the selective action of transcriptional factors and also from epigenetic mechanisms , such as dna methylation or posttranslational histone modifications . these epigenetic modifications could be heritable and occur without affecting the dna sequence , which makes the epigenetic information potentially plastic . it is the whole set of epigenetic modifications at a given locus , including the interaction of atp - dependent nucleosomal remodeling complexes with dna methylation and histone modifications that play a key role in regulating gene expression and chromatin organization . methylation of cytosine residues within cpg dinucleotides is an efficient epigenetic mechanism for gene silencing . the methyl group addition at the 5 carbon of the pyrimidine ring of cytosine is catalyzed by enzymes called dna methyltransferases ( dnmts ) . dna methylation in the vicinity of transcriptional start sites results in repression and gene silencing by direct and indirect mechanisms . the direct mechanism is done by affecting the binding of transcription factors that do not recognize methylated cpg sites . these proteins prevent the binding of transcription factors to dna and can recruit several enzymes that catalyze transcriptionally silent histone modifications and other factors that make the chromatin more compact and consequently less accessible to transcription machinery . the importance of cpg methylation for ifng expression is supported by experiments of cd8 t cell cultures stimulated via tcr in the presence of 5-azacytidine ( 5-aza ) , a drug that causes dna demethylation upon proliferation . upon aza treatment , increased levels of this cytokine in culture supernatants were described , as well as an increment in the number of nave cells able to produce ifn- when compared to control . several genomic regions could be involved in this regulation and a possible role of ifng mouse promoter was particularly investigated . numeration of these sites varies among different publications , but here we will denominate it according to the initial transcription site from the refseq sequence identifier nm_008337 . cd8 t cell clonal analysis of ifng promoter methylation by bisulfite genomic dna sequencing and mrna expression by quantitative competitive pcr ( qcpcr ) revealed an overall association between demethylation of ifn- promoter and expression of mrna . cpg sites located at 212 , 198 , and 58 were methylated in most ifn--negative mrna clones and their demethylation was closely related to ifn- expression . however , there is a clonal heterogeneity , with clone- and site - specific differences across the promoter . interestingly much more variability was observed when clones derived from nave cd8cd44 t cells were assayed . on the other hand , most of the cpg sites in ifng promoter of clones derived from cd8cd44 t cells were demethylated and these clones expressed high levels of ifn-. in line with that winders and colleagues showed by southern blot analysis with methylation - sensitive enzymes that ifn- promoter is mostly unmethylated at the 301 , 212 , and 58 sites in cd8 t cells from ot - i mice . similar patterns were observed in cd4 t cells from 5c.c7 mice , and earlier stages of t cell development also revealed hypomethylation at the 212 to 50 cpg sites . but the upstream site 380 was hypermethylated in both double positive and double negative thymocytes . in accord with the hypothesis that cpg methylation occurs in ifn- nonproducers , also , stimulation of cd4 t cells in th2 polarizing conditions leads to a pronounced increase of methylation at particular sites . kersh and colleagues performed by bisulfite sequencing an ex vivo analysis of nave and in vivo generated effector and memory cd8 t cells from p14 tcr - transgenic mice , which are specific for gp33 - 41 epitope of lcmv glycoprotein . but these cells have posttranscriptional sites ( + 12 , + 91 , and + 114 ) mainly methylated . although the average numbers of methyl - cpg sites in nave and memory cells were the same ( 2.8 and 2.7 , resp . ) similar to data from northrop and colleagues , effector cd8 cells had a completely unmethylated promoter . the discrete differences in dna methylation between nave and memory cd8 cells may represent a differential regulation in these cell types , because after 5 hours of antigenic - stimulation , a demethylation independent of dna replication was observed in memory cells , but this is not true for nave p14 cells . furthermore , treatment of nave p14 cells with 5-aza led to an increase in the number of cells able to produce ifn- , and no difference was observed when memory cells were used suggesting that ifng is silenced by dna methylation in nave , but not cd8 , memory cells . not only the number but also the amount of intracellular ifn- produced by nave p14 cells treated with 5-aza has increased , when compared to control . in line with that , makar and wilson reported that when nave cd8 t cells deficient in the maintenance dnmt 1 ( dnmt1 ) are stimulated for 3 days they increase ifng expression 510-fold after restimulation . but it is important to note that even in dnmt1 deficient mice the ifn- production is higher in cd8 than cd4 lymphocytes , suggesting that maintenance of ifn- expression in specific t cell subsets is not dependent on cpg methylation . in opposition , th2-related cytokines ( il-4 , il-5 , and il-13 ) were significantly expressed only when cd8 t cells lacks dnmt1 . the other study characterizing cpg methylation of ifn- promoter in several cell types investigated the methylation status of the 212 , 198 , 178 , 58 , 50 , 39 , + 12 , and + 91 cpg sites . all the cpgs were almost completely methylated in kidney and heart tissues , and entirely unmethylated in nk cells . consistent with other reports [ 32 , 33 ] , cpgs located at untranscribed regions were hypomethylated and those at transcribed regions were hypermethylated in cd8 nave cells from c57/bl6 mice . although a different mouse lineage was used to obtain memory cd8 t cells , they presented 06% and 3070% methylation in untranscribed and transcribed region , respectively . as previously reported , earlier stages of t cell development ( dn , dp , cd4 , and cd8 thymocytes ) have also hypomethylated cpg sites at ifn- promoter . cd4 t lymphocytes from the same mice presented a similar cpg methylation pattern of c57/bl6 cd8 nave cells , but polarization to th1 results in a significant level of demethylation in transcribed region . like other reports , th2 underwent some level of methylation at untranscribed region , with the position 58 being more methylated than other promoter sites , while no detectable change was observed when differentiation occurred at neutralizing conditions ( th0 ) . taken together , all these data suggest that nave cd8 t cells exhibit a low methylation profile in untranscribed region and a hypermethylation in transcribed region , and , following tcr stimulation , methylation decreases in both regions . the functional significance of ifn- promoter cpg methylation was assayed by luciferase reporter assays showing that methylation of the whole ifn- promoter vector inhibits its transcriptional activity [ 33 , 36 ] . methylation of the 212 , 198 , and 178 sites individually did not affect the activity of ifng promoter , and little effect was observed when the 50 and 39 sites were exclusively methylated , but modification of 58 cpg site significantly reduced the activity to a level similar to that of the observed for the completely methylated vector . interestingly , methylation of 58 site occurred faster and more completely than the other sites during th1 and th2 polarization , but a more accentuated outcome was observed in th2 [ 33 , 36 ] . the versatile profile of the 58 site could potentially interfere with transcription . the use of oligonucleotide probes with methylated 58 cpg , but not 50 and 39 , abolished the formation of 2 complexes verified when unmethylated probes were used in emsa with nuclear extracts of th1 cell line ae7 . in supershift assays c - jun and atf2 were identified in the upper band and creb and atf1/creb in the lower one , and this was also observed when th2 nuclear extracts from d10 lineage were used . chromatin immunoprecipitation ( chip ) assays confirmed the creb and creb / atf1 binding and identified fosb , junb , c - jun , and atf2 interacting with ifn- promoter in th1 cell lineage in vivo . concerning the th2 cell lineage , the mentioned functional and binding assays were performed in th cells , and although they are related to cd8 lymphocytes , several data suggest that ifng regulation may have particular characteristics in each t cell subset , like data obtained from transgenic mice model which express the luciferase gene under the control of proximal ( 70 to 44 ) and distal ( 98 to 78 ) regulatory elements from the ifn- promoter . in vitro primed cd4 t cells express reporters under control of both elements , while cd8 cells do so only under the distal element . in addition , elevated cyclic amp inhibited transcriptional activity directed by the proximal regulatory element in primed cd4 + t cells but enhanced transcriptional activity directed by the distal in primed cd8 + t cells . this differential gene expression depends on the selective action of transcriptional factors , but also from epigenetic modifications that could change the chromatin accessibility to transcriptional machinery . depending on both covalent modification type and the modified residue , these modifications could result in gene activation or silencing . ( ach3 and ach4 , resp . ) and methylation of lysine 4 of histone h3 are histone modifications associated with poised or transcriptionally active genes . on the other hand , trimethylation of lysines 9 or 27 of histone h3 in fact , reduced repressive h3k27me3 and h3k27me2 throughout ifng after primary infection and persistence in memory cd8 t lymphocytes was reported , as well as a detection of minor h3k4me3 peaks near ifng tss in effector and memory t cd8 cells . in addition to the loss of dna methylation at ifn- promoter in the differentiation of nave p14 cd8 t cells to effector , northrop and colleagues reported a more pronounced demethylation of the ifn- enhancer at the first intron . investigation of ach3 by chip and detection by real - time pcr revealed a significant increase in ifn- promoter after stimulation , and similar increase was observed in the comparison of nave cd8 t cells with effector and memory cd8 t cells in promoter and enhancer . nevertheless , when cd8 memory and effector cells were generated in cd4 deficient mice ( b6 cd4 ) , they produced considerably less ifn- per cell and the shift in histone acetylation is no longer seen , suggesting that demethylation of cpg sites within ifn- promoter and enhancer in cd8 effector and memory cells occurred independently of cd4 t help , while histone acetylation at these same regions was highly dependent upon the presence of cd4 help . this effect is cytokine specific , because hypoacetylation of il-2 does not change in differentiated cd8 cells . although the above data suggest that epigenetic regulation of the ifng promoter may interfere in its expression , transgenes containing the 8.6-kb fragment of human genomic dna containing the full length ifn- gene ( promoter , introns , and up to 3.4 kb of 5 flanking sequence ) do not confer proper t cell subset - specific expression in vivo [ 4143 ] . indeed , a transgenic model containing the human ifng gene and 9095 kb of flanking sequence results in high - level , th1-specific ifn- production . therefore , the search for conserved noncoding sequences ( cns ) among different species has been proved as a good method for identification of relevant cis - regulatory elements for ifn- gene . in silico searches for cnss across human , mouse , and rat genomes allowed the identification of 2 sequences located at 5.27 kb [ 44 , 45 ] and 17.36 kb of murine ifng translational start site . the first one is referred to as cns-1 or ifn- 5 cns and the second , cns-2 . both of them exhibited enhancer like function in luciferase reporter assays in response to ionomycin in t cell lineages and correspond to a dnase i hypersensitive site in th1 cells but not in th2 cells [ 44 , 45 ] . similar pattern of ach3 ( and more slightly ach4 ) and h3k4me2 is observed in cd8 and th1 cells , which are good sources of ifn- [ 40 , 44 ] . interestingly , despite the fact that in vitro primed effector cd8 t cells have more levels of ach3 at intron 3 compared to cns-1 , tcr tg effector cd8 t cells primed in vivo displayed greater ach3 at cns-1 compared to intron 3 . increased levels of mentioned modifications were detected at promoter , intron 3 , and cns-2 regions while th2 has more discrete peaks . interestingly , h3ac chip analysis of th1 , th2 , and cd8 cells from mice deficient in t - bet revealed that this transcription factor is required for induction of histone modifications in th1 , but not t cd8 cells . despite some similarities between cd4 and cd8 t lymphocytes , cd8 cells seem to have particular regulation of ifn production . therefore , more research is needed to characterize possible pathways engaged in this control . hatton and colleagues identified a cns located at 22 kb from ifng that when deleted blocks ifng reporter expression in th1 and cd8 t cells . transgenic mouse model also suggests that this element is required for ifn- expression in cd4 and cd8 t cells . in 2007 , schoenborn and colleagues also performed a comparative genomic analysis that revealed eight highly conserved noncoding sequences ( cnss ) in a ~100 kb region surrounding ifng . previously identified cns-1 ( also called cns-6 ) , 22 , 34 , 54 , and + 18/20 ( also called cns-2 ) were among them , but they further identify the cns+29 , + 46 , and + 55 . although the aim of the study was to characterize regulatory elements that govern ifng expression in cd4 t cells , functional elements identified were also investigated in primary cd8 t cells : cns-6 enhanced expression in stimulated cd8 cells as well as in th0 or th1 cells , but the authors report that cns-34 did so only in cd8 . on the other hand , cns22 enhanced expression in response to il-12 plus il-18 in th0 and th1 cells , but this effect was not consistently evident in cd8 + t cells . these results suggest that some sequences like cns-22 may be necessary for ifng expression in both cd4 and cd8 t cells , while others , as cns-34 , may have regulatory role only in cd8 t lymphocytes . moreover , more studies are needed to explore cnss functions and epigenetic marks in cd8 t cells . there are relatively concordant data concerning cpg methylation of ifng promoter , but fewer studies investigated the status of other relevant sequences for ifn- expression in cd8 lymphocytes . there is also a relative lack of data concerning other epigenetic marks , as histone modifications , and the interplay between factors that may determine or influence this status in cd8 t cells . it would be of particular interest to investigate if epigenetic events can influence heterogeneous features of cd8 t cell populations , like the capacity of polyfunctional cells to express several cytokines , and if specific microenvironments could modulate the ifng expression through epigenetic marks , as in iels . these observations reinforce that more studies are needed to understand the transcriptional regulation of ifng in cd8 t cell lineage .

interferon- ( ifn- ) is an essential cytokine for immunity against intracellular pathogens and cancer . ifn- expression by cd4 t lymphocytes is observed only after t helper ( th ) 1 differentiation and there are several studies about the molecular mechanisms that control ifng expression in these cells . however , nave cd8 t lymphocytes do not produce large amounts of ifn- , but after tcr stimulation there is a progressive acquisition of ifn- expression during differentiation into cytotoxic t lymphocytes ( ctl ) and memory cells , which are capable of producing high levels of this cytokine . differential gene expression can be regulated from the selective action of transcriptional factors and also from epigenetic mechanisms , such as dna cpg methylation or posttranslational histone modifications . recently it has been recognized that epigenetic modification is an integral part of cd8 lymphocyte differentiation . this review will focus on the chromatin status of ifng promoter in cd8 t cells and possible influences of epigenetic modifications in ifng gene and conserved noncoding sequences ( cnss ) in regulation of ifn- production by cd8 t lymphocytes .

1. Interferon- (IFN-) 2. IFN- 3. Epigenetic of Ifng in CD8 T Cells 4. Concluding Remarks and Perspectives

interferon- ( ifn- ) is an essential cytokine for immunity against intracellular pathogens and cancer . natural killer ( nk ) and natural killer t ( nkt ) cells are the innate cells sources of this cytokine and rapidly produce ifn- upon activation . on the other hand significant increase in ifn- expression by cd4 t lymphocytes is observed only after t helper ( th ) 1 differentiation . in fact , upon activation , cd4 t cells can differentiate into several effector lineages , of which th1 is the only one that produce high levels of ifn-. nave cd8 t lymphocytes do not produce large amounts of ifn- , but after tcr stimulation these cells undergo differentiation into cytotoxic t lymphocytes ( ctl ) and memory cells , which are capable of producing high levels of this cytokine in response to tcr activation or interleukin- ( il- ) 12 and il-18 . the best characterized role of ifn- in cd8 t cell immunity is in enhancing class i antigen presentation pathway , which facilitates cytotoxic t cells to recognize infected cells . several studies have suggested that ifn- is a key determinant of immunodominance [ 68 ] . badovinac and colleagues have shown that ifn- deficient mice infected with an attenuated listeria monocytogenes strain exhibited an altered immunodominance hierarchy due to an increased expansion of cd8 t cells specific for a subdominant epitope of l. monocytogenes . furthermore , ifn- seems to be required for subdominant cd8 t cells response suppression by dominant cd8 t cell response and the cd8 t cells that promptly produce ifn- after stimulation are preferentially expanded . this cytokine is important for the differentiation of tcrcd4cd8 intraepithelial lymphocytes ( iels ) in the gut . ifn- also acts directly on cd8 t cells by stimulating their abundance in an acute lymphocytic choriomeningitis virus ( lcmv ) infection and enhancing the development of memory cells . however , badovinac and colleagues also have shown that ifn- deficiency resulted in a delayed contraction of antigen - specific cd8 t cell populations from both listeria monocytogenes and lcmv infections , which suggests an important role of this cytokine in control of death phase of antigen - specific cd8 t cells . therefore , this cytokine has both positive and negative effects on cd8 t cell abundance depending on the phase of the cd8 response and also the biology model evaluated . this dual role is clearly evident in ifn- deficient mice , in which both the expansion and contraction of cd8 t cell response are impaired . cd8 t cells are usually characterized by their cytolytic activities involving perforin or fas mechanisms to kill targeted cells . however cytokine secretion by cd8 t cells also has an important role in the control of intracellular infections . in 1990 , fong and mosmann suggested that th1 cells and cd8 t cells could share cytokine mediated functions , like combating intracellular pathogens and tumors cells . they observed that alloreactive murine cd8 t cell clones produced both mrna and protein profile characteristic of th1 clones , which include high levels of ifn-. it is well known that the clearance of several infections also depends on noncytolytic functions of cd8 t cells . the control of m. tuberculosis infections in mice requires the ability of the cd8 t cells to produce ifn- . furthermore , ifn- produced by cd8 t cells is essential to clear numerous viral infections such as measles virus , herpes simplex virus type 1 , lcmv , and borna disease virus . indeed , hiv control is associated with polyfunctional cd8 t cells , that is , epitope - specific cells expressing several effector functions , which includes the expression of cytokines such as tnf and ifn- . the ifn- produced by cd8 t cells seems to have other immunomodulatory roles , acting on cd4 t cells and b cells and also on cd8 t cells themselves . the differentiation of cd4 t cells in t helper subsets depends on the cytokine milieu where these primary t cells were stimulated . several studies have also suggested that cd8 t lymphocytes could have a role in the generation of th1 immunity and in the inhibition of th2 response [ 1820 ] . uzonna and colleagues have shown that ifn- produced by murine cd8 t cells , in response to low doses of l. major , downregulates an initial th2 response and enhances th1 commitment . data from our group suggest that , besides nk and dendritic cells , cd8 t lymphocytes are also another source of ifn- that enhances cd4 th1 phenotype development . we have shown that , after tcr activation of primary lymph node cells , cd8 t lymphocytes are the major source of ifn- production . furthermore , cd4 t cells cocultured with ifn--competent cd8 t cells clearly produce more ifn- and less il-4 than cd4 t cells cultured with ifn--deficient cd8 t cells . this work also suggested that nfat1 transcription factor - dependent ifn- production by cd8 t cell is important during eosinophil migration to pleura in a pleurisy model , which suggests an important role for ifn- produced by cd8 t cells in the control of allergic diseases . together these studies reinforce the role of ifn- produced by cd8 t cells in regulation of th immune responses in vivo . the autocrine ifn- signaling is important for th1 differentiation , and recently it has been suggested that ifn- is an autocrine / paracrine factor to promote nave t cd8 cell differentiation . low levels of ifn- produced by cd8 t cells promote the upregulation of t - bet , granzyme b , and ifn- also promotes a week cytolytic activity . a previous paper has also shown that nave cd8 t cells receive an ifn- signal few hours after l. monocytogenes infection . when nave t cd4 and cd8 cells are compared for the ifn- production , it is clear that , after tcr activation , cd8 t cells produce more levels of ifn- than cd4 t cells , and the requirements for tcr induced ifn- production are different between these primary cells . in fact , carter and murphy have shown that cd4 t cells require stat4 activation beside tcr signalization to produce ifn- , while cd8 t cells need only tcr activation . furthermore , the main difference in transcriptional requirements for ifng expression between cd4 and cd8 t cells seems to be the differential role of members of the t - box family of transcription factors , t - bet and eomesodermin ( eomes ) . t - bet is the master regulator of ifng and th1 commitment of cd4 t cells and sufficient for induction of ifng expression in these cells . on the other hand , additionally , eomes and t - bet play important roles during cd8 t cell differentiation to effector and memory t cells , where t - bet is associated with effector phenotype whereas expression of eomes increases in memory cd8 t cells . there are several studies about ifn- production by th1 cells and the molecular mechanisms that control ifng expression in these cells are widely investigated and extensively reviewed . however , regulation of ifng expression in cd8 t cells is not fairly explored and discussed . an effective cellular immune response is characterized by robust stimulation of nave lymphocytes to undergo differentiation into effector cells , which provides pathogen clearance while promoting the development of long - lived memory cells that can respond to reinfection faster than nave cells . the cd8 differentiation is accompanied by large - scale changes in the coordinate expression of genes associated with effector function , survival , and self - renewal and recently it has been recognized that epigenetic modification is an integral part of this process . this section will focus on the chromatin status of ifng promoter in cd8 t cells . however , due to the scarcity of studies exploring cd8 t cells and the higher availability of data investigating cd4 t cells , some aspects of cd4 regulation will be mentioned for comparison . differential gene expression can be regulated from the selective action of transcriptional factors and also from epigenetic mechanisms , such as dna methylation or posttranslational histone modifications . it is the whole set of epigenetic modifications at a given locus , including the interaction of atp - dependent nucleosomal remodeling complexes with dna methylation and histone modifications that play a key role in regulating gene expression and chromatin organization . these proteins prevent the binding of transcription factors to dna and can recruit several enzymes that catalyze transcriptionally silent histone modifications and other factors that make the chromatin more compact and consequently less accessible to transcription machinery . the importance of cpg methylation for ifng expression is supported by experiments of cd8 t cell cultures stimulated via tcr in the presence of 5-azacytidine ( 5-aza ) , a drug that causes dna demethylation upon proliferation . upon aza treatment , increased levels of this cytokine in culture supernatants were described , as well as an increment in the number of nave cells able to produce ifn- when compared to control . cd8 t cell clonal analysis of ifng promoter methylation by bisulfite genomic dna sequencing and mrna expression by quantitative competitive pcr ( qcpcr ) revealed an overall association between demethylation of ifn- promoter and expression of mrna . however , there is a clonal heterogeneity , with clone- and site - specific differences across the promoter . on the other hand , most of the cpg sites in ifng promoter of clones derived from cd8cd44 t cells were demethylated and these clones expressed high levels of ifn-. in line with that winders and colleagues showed by southern blot analysis with methylation - sensitive enzymes that ifn- promoter is mostly unmethylated at the 301 , 212 , and 58 sites in cd8 t cells from ot - i mice . similar patterns were observed in cd4 t cells from 5c.c7 mice , and earlier stages of t cell development also revealed hypomethylation at the 212 to 50 cpg sites . in accord with the hypothesis that cpg methylation occurs in ifn- nonproducers , also , stimulation of cd4 t cells in th2 polarizing conditions leads to a pronounced increase of methylation at particular sites . kersh and colleagues performed by bisulfite sequencing an ex vivo analysis of nave and in vivo generated effector and memory cd8 t cells from p14 tcr - transgenic mice , which are specific for gp33 - 41 epitope of lcmv glycoprotein . although the average numbers of methyl - cpg sites in nave and memory cells were the same ( 2.8 and 2.7 , resp . ) the discrete differences in dna methylation between nave and memory cd8 cells may represent a differential regulation in these cell types , because after 5 hours of antigenic - stimulation , a demethylation independent of dna replication was observed in memory cells , but this is not true for nave p14 cells . furthermore , treatment of nave p14 cells with 5-aza led to an increase in the number of cells able to produce ifn- , and no difference was observed when memory cells were used suggesting that ifng is silenced by dna methylation in nave , but not cd8 , memory cells . in line with that , makar and wilson reported that when nave cd8 t cells deficient in the maintenance dnmt 1 ( dnmt1 ) are stimulated for 3 days they increase ifng expression 510-fold after restimulation . but it is important to note that even in dnmt1 deficient mice the ifn- production is higher in cd8 than cd4 lymphocytes , suggesting that maintenance of ifn- expression in specific t cell subsets is not dependent on cpg methylation . in opposition , th2-related cytokines ( il-4 , il-5 , and il-13 ) were significantly expressed only when cd8 t cells lacks dnmt1 . the other study characterizing cpg methylation of ifn- promoter in several cell types investigated the methylation status of the 212 , 198 , 178 , 58 , 50 , 39 , + 12 , and + 91 cpg sites . although a different mouse lineage was used to obtain memory cd8 t cells , they presented 06% and 3070% methylation in untranscribed and transcribed region , respectively . cd4 t lymphocytes from the same mice presented a similar cpg methylation pattern of c57/bl6 cd8 nave cells , but polarization to th1 results in a significant level of demethylation in transcribed region . taken together , all these data suggest that nave cd8 t cells exhibit a low methylation profile in untranscribed region and a hypermethylation in transcribed region , and , following tcr stimulation , methylation decreases in both regions . the functional significance of ifn- promoter cpg methylation was assayed by luciferase reporter assays showing that methylation of the whole ifn- promoter vector inhibits its transcriptional activity [ 33 , 36 ] . methylation of the 212 , 198 , and 178 sites individually did not affect the activity of ifng promoter , and little effect was observed when the 50 and 39 sites were exclusively methylated , but modification of 58 cpg site significantly reduced the activity to a level similar to that of the observed for the completely methylated vector . concerning the th2 cell lineage , the mentioned functional and binding assays were performed in th cells , and although they are related to cd8 lymphocytes , several data suggest that ifng regulation may have particular characteristics in each t cell subset , like data obtained from transgenic mice model which express the luciferase gene under the control of proximal ( 70 to 44 ) and distal ( 98 to 78 ) regulatory elements from the ifn- promoter . this differential gene expression depends on the selective action of transcriptional factors , but also from epigenetic modifications that could change the chromatin accessibility to transcriptional machinery . on the other hand , trimethylation of lysines 9 or 27 of histone h3 in fact , reduced repressive h3k27me3 and h3k27me2 throughout ifng after primary infection and persistence in memory cd8 t lymphocytes was reported , as well as a detection of minor h3k4me3 peaks near ifng tss in effector and memory t cd8 cells . in addition to the loss of dna methylation at ifn- promoter in the differentiation of nave p14 cd8 t cells to effector , northrop and colleagues reported a more pronounced demethylation of the ifn- enhancer at the first intron . investigation of ach3 by chip and detection by real - time pcr revealed a significant increase in ifn- promoter after stimulation , and similar increase was observed in the comparison of nave cd8 t cells with effector and memory cd8 t cells in promoter and enhancer . nevertheless , when cd8 memory and effector cells were generated in cd4 deficient mice ( b6 cd4 ) , they produced considerably less ifn- per cell and the shift in histone acetylation is no longer seen , suggesting that demethylation of cpg sites within ifn- promoter and enhancer in cd8 effector and memory cells occurred independently of cd4 t help , while histone acetylation at these same regions was highly dependent upon the presence of cd4 help . although the above data suggest that epigenetic regulation of the ifng promoter may interfere in its expression , transgenes containing the 8.6-kb fragment of human genomic dna containing the full length ifn- gene ( promoter , introns , and up to 3.4 kb of 5 flanking sequence ) do not confer proper t cell subset - specific expression in vivo [ 4143 ] . indeed , a transgenic model containing the human ifng gene and 9095 kb of flanking sequence results in high - level , th1-specific ifn- production . therefore , the search for conserved noncoding sequences ( cns ) among different species has been proved as a good method for identification of relevant cis - regulatory elements for ifn- gene . similar pattern of ach3 ( and more slightly ach4 ) and h3k4me2 is observed in cd8 and th1 cells , which are good sources of ifn- [ 40 , 44 ] . interestingly , despite the fact that in vitro primed effector cd8 t cells have more levels of ach3 at intron 3 compared to cns-1 , tcr tg effector cd8 t cells primed in vivo displayed greater ach3 at cns-1 compared to intron 3 . interestingly , h3ac chip analysis of th1 , th2 , and cd8 cells from mice deficient in t - bet revealed that this transcription factor is required for induction of histone modifications in th1 , but not t cd8 cells . despite some similarities between cd4 and cd8 t lymphocytes , cd8 cells seem to have particular regulation of ifn production . hatton and colleagues identified a cns located at 22 kb from ifng that when deleted blocks ifng reporter expression in th1 and cd8 t cells . transgenic mouse model also suggests that this element is required for ifn- expression in cd4 and cd8 t cells . in 2007 , schoenborn and colleagues also performed a comparative genomic analysis that revealed eight highly conserved noncoding sequences ( cnss ) in a ~100 kb region surrounding ifng . although the aim of the study was to characterize regulatory elements that govern ifng expression in cd4 t cells , functional elements identified were also investigated in primary cd8 t cells : cns-6 enhanced expression in stimulated cd8 cells as well as in th0 or th1 cells , but the authors report that cns-34 did so only in cd8 . on the other hand , cns22 enhanced expression in response to il-12 plus il-18 in th0 and th1 cells , but this effect was not consistently evident in cd8 + t cells . these results suggest that some sequences like cns-22 may be necessary for ifng expression in both cd4 and cd8 t cells , while others , as cns-34 , may have regulatory role only in cd8 t lymphocytes . moreover , more studies are needed to explore cnss functions and epigenetic marks in cd8 t cells . there are relatively concordant data concerning cpg methylation of ifng promoter , but fewer studies investigated the status of other relevant sequences for ifn- expression in cd8 lymphocytes . there is also a relative lack of data concerning other epigenetic marks , as histone modifications , and the interplay between factors that may determine or influence this status in cd8 t cells . it would be of particular interest to investigate if epigenetic events can influence heterogeneous features of cd8 t cell populations , like the capacity of polyfunctional cells to express several cytokines , and if specific microenvironments could modulate the ifng expression through epigenetic marks , as in iels . these observations reinforce that more studies are needed to understand the transcriptional regulation of ifng in cd8 t cell lineage .

mice with targeted disruptions of the ifng gene or ifng gene receptor 1 are highly susceptible to a variety of bacteria , protozoans , and virus infection . natural killer ( nk ) and natural killer t ( nkt ) cells are the innate cells sources of this cytokine and rapidly produce ifn- upon activation . in fact , upon activation , cd4 t cells can differentiate into several effector lineages , of which th1 is the only one that produce high levels of ifn-. nave cd8 t lymphocytes do not produce large amounts of ifn- , but after tcr stimulation these cells undergo differentiation into cytotoxic t lymphocytes ( ctl ) and memory cells , which are capable of producing high levels of this cytokine in response to tcr activation or interleukin- ( il- ) 12 and il-18 . the best characterized role of ifn- in cd8 t cell immunity is in enhancing class i antigen presentation pathway , which facilitates cytotoxic t cells to recognize infected cells . ifn- signaling upregulation leads to expression of mhc class i and the tap transporter , as well as chaperones such as tapasin . ifn- also induces a replacement of the constitutive proteasome subunits , 1 , 2 and 5 , for the immunoproteasome subunits lmp2 , mecl-1 , and lmp7 , an essential feature for increasing the quantity and repertoire of peptides presented in the context of class i mhc ( reviewed in ) . ifn- also plays an important role in cd8 t cell homeostasis that is independent from its function in clearance of infection . badovinac and colleagues have shown that ifn- deficient mice infected with an attenuated listeria monocytogenes strain exhibited an altered immunodominance hierarchy due to an increased expansion of cd8 t cells specific for a subdominant epitope of l. monocytogenes . furthermore , ifn- seems to be required for subdominant cd8 t cells response suppression by dominant cd8 t cell response and the cd8 t cells that promptly produce ifn- after stimulation are preferentially expanded . this cytokine is important for the differentiation of tcrcd4cd8 intraepithelial lymphocytes ( iels ) in the gut . iels are lymphocytes considered activated yet resting and their regulation is crucial role in the maintenance of the epithelial cell barrier and gut physiological inflammation . ifn- also acts directly on cd8 t cells by stimulating their abundance in an acute lymphocytic choriomeningitis virus ( lcmv ) infection and enhancing the development of memory cells . interestingly , sercan and colleagues show that ifn- produced by innate immune cells contributes to antigen - specific cd8 t cell homeostasis . however , badovinac and colleagues also have shown that ifn- deficiency resulted in a delayed contraction of antigen - specific cd8 t cell populations from both listeria monocytogenes and lcmv infections , which suggests an important role of this cytokine in control of death phase of antigen - specific cd8 t cells . therefore , this cytokine has both positive and negative effects on cd8 t cell abundance depending on the phase of the cd8 response and also the biology model evaluated . this dual role is clearly evident in ifn- deficient mice , in which both the expansion and contraction of cd8 t cell response are impaired . however cytokine secretion by cd8 t cells also has an important role in the control of intracellular infections . in 1990 , fong and mosmann suggested that th1 cells and cd8 t cells could share cytokine mediated functions , like combating intracellular pathogens and tumors cells . it is well known that the clearance of several infections also depends on noncytolytic functions of cd8 t cells . the control of m. tuberculosis infections in mice requires the ability of the cd8 t cells to produce ifn- . furthermore , ifn- produced by cd8 t cells is essential to clear numerous viral infections such as measles virus , herpes simplex virus type 1 , lcmv , and borna disease virus . this ifn- mediated response seems to be important to avoid tissue damage and inflammation , which is normally observed in cytotoxic cd8 t cell response ( reviewed in ) . indeed , hiv control is associated with polyfunctional cd8 t cells , that is , epitope - specific cells expressing several effector functions , which includes the expression of cytokines such as tnf and ifn- . the ifn- produced by cd8 t cells seems to have other immunomodulatory roles , acting on cd4 t cells and b cells and also on cd8 t cells themselves . the differentiation of cd4 t cells in t helper subsets depends on the cytokine milieu where these primary t cells were stimulated . for th1 differentiation , the cytokines il-12 , produced by apcs , and ifn- , mainly produced by nk cells , are critical to induce and reinforce th1 commitment . several studies have also suggested that cd8 t lymphocytes could have a role in the generation of th1 immunity and in the inhibition of th2 response [ 1820 ] . data from our group suggest that , besides nk and dendritic cells , cd8 t lymphocytes are also another source of ifn- that enhances cd4 th1 phenotype development . we have shown that , after tcr activation of primary lymph node cells , cd8 t lymphocytes are the major source of ifn- production . furthermore , cd4 t cells cocultured with ifn--competent cd8 t cells clearly produce more ifn- and less il-4 than cd4 t cells cultured with ifn--deficient cd8 t cells . this work also suggested that nfat1 transcription factor - dependent ifn- production by cd8 t cell is important during eosinophil migration to pleura in a pleurisy model , which suggests an important role for ifn- produced by cd8 t cells in the control of allergic diseases . together these studies reinforce the role of ifn- produced by cd8 t cells in regulation of th immune responses in vivo . low levels of ifn- produced by cd8 t cells promote the upregulation of t - bet , granzyme b , and ifn- also promotes a week cytolytic activity . these studies suggest that the ifn- signals for cd8 t cell differentiation are delivered early in the immune response . when nave t cd4 and cd8 cells are compared for the ifn- production , it is clear that , after tcr activation , cd8 t cells produce more levels of ifn- than cd4 t cells , and the requirements for tcr induced ifn- production are different between these primary cells . furthermore , the main difference in transcriptional requirements for ifng expression between cd4 and cd8 t cells seems to be the differential role of members of the t - box family of transcription factors , t - bet and eomesodermin ( eomes ) . t - bet is the master regulator of ifng and th1 commitment of cd4 t cells and sufficient for induction of ifng expression in these cells . on the other hand , additionally , eomes and t - bet play important roles during cd8 t cell differentiation to effector and memory t cells , where t - bet is associated with effector phenotype whereas expression of eomes increases in memory cd8 t cells . there are several studies about ifn- production by th1 cells and the molecular mechanisms that control ifng expression in these cells are widely investigated and extensively reviewed . an effective cellular immune response is characterized by robust stimulation of nave lymphocytes to undergo differentiation into effector cells , which provides pathogen clearance while promoting the development of long - lived memory cells that can respond to reinfection faster than nave cells . the cd8 differentiation is accompanied by large - scale changes in the coordinate expression of genes associated with effector function , survival , and self - renewal and recently it has been recognized that epigenetic modification is an integral part of this process . however , due to the scarcity of studies exploring cd8 t cells and the higher availability of data investigating cd4 t cells , some aspects of cd4 regulation will be mentioned for comparison . differential gene expression can be regulated from the selective action of transcriptional factors and also from epigenetic mechanisms , such as dna methylation or posttranslational histone modifications . it is the whole set of epigenetic modifications at a given locus , including the interaction of atp - dependent nucleosomal remodeling complexes with dna methylation and histone modifications that play a key role in regulating gene expression and chromatin organization . the methyl group addition at the 5 carbon of the pyrimidine ring of cytosine is catalyzed by enzymes called dna methyltransferases ( dnmts ) . dna methylation in the vicinity of transcriptional start sites results in repression and gene silencing by direct and indirect mechanisms . these proteins prevent the binding of transcription factors to dna and can recruit several enzymes that catalyze transcriptionally silent histone modifications and other factors that make the chromatin more compact and consequently less accessible to transcription machinery . the importance of cpg methylation for ifng expression is supported by experiments of cd8 t cell cultures stimulated via tcr in the presence of 5-azacytidine ( 5-aza ) , a drug that causes dna demethylation upon proliferation . upon aza treatment , increased levels of this cytokine in culture supernatants were described , as well as an increment in the number of nave cells able to produce ifn- when compared to control . several genomic regions could be involved in this regulation and a possible role of ifng mouse promoter was particularly investigated . cd8 t cell clonal analysis of ifng promoter methylation by bisulfite genomic dna sequencing and mrna expression by quantitative competitive pcr ( qcpcr ) revealed an overall association between demethylation of ifn- promoter and expression of mrna . on the other hand , most of the cpg sites in ifng promoter of clones derived from cd8cd44 t cells were demethylated and these clones expressed high levels of ifn-. in line with that winders and colleagues showed by southern blot analysis with methylation - sensitive enzymes that ifn- promoter is mostly unmethylated at the 301 , 212 , and 58 sites in cd8 t cells from ot - i mice . similar patterns were observed in cd4 t cells from 5c.c7 mice , and earlier stages of t cell development also revealed hypomethylation at the 212 to 50 cpg sites . in accord with the hypothesis that cpg methylation occurs in ifn- nonproducers , also , stimulation of cd4 t cells in th2 polarizing conditions leads to a pronounced increase of methylation at particular sites . kersh and colleagues performed by bisulfite sequencing an ex vivo analysis of nave and in vivo generated effector and memory cd8 t cells from p14 tcr - transgenic mice , which are specific for gp33 - 41 epitope of lcmv glycoprotein . although the average numbers of methyl - cpg sites in nave and memory cells were the same ( 2.8 and 2.7 , resp . ) the discrete differences in dna methylation between nave and memory cd8 cells may represent a differential regulation in these cell types , because after 5 hours of antigenic - stimulation , a demethylation independent of dna replication was observed in memory cells , but this is not true for nave p14 cells . furthermore , treatment of nave p14 cells with 5-aza led to an increase in the number of cells able to produce ifn- , and no difference was observed when memory cells were used suggesting that ifng is silenced by dna methylation in nave , but not cd8 , memory cells . in line with that , makar and wilson reported that when nave cd8 t cells deficient in the maintenance dnmt 1 ( dnmt1 ) are stimulated for 3 days they increase ifng expression 510-fold after restimulation . but it is important to note that even in dnmt1 deficient mice the ifn- production is higher in cd8 than cd4 lymphocytes , suggesting that maintenance of ifn- expression in specific t cell subsets is not dependent on cpg methylation . the other study characterizing cpg methylation of ifn- promoter in several cell types investigated the methylation status of the 212 , 198 , 178 , 58 , 50 , 39 , + 12 , and + 91 cpg sites . as previously reported , earlier stages of t cell development ( dn , dp , cd4 , and cd8 thymocytes ) have also hypomethylated cpg sites at ifn- promoter . cd4 t lymphocytes from the same mice presented a similar cpg methylation pattern of c57/bl6 cd8 nave cells , but polarization to th1 results in a significant level of demethylation in transcribed region . taken together , all these data suggest that nave cd8 t cells exhibit a low methylation profile in untranscribed region and a hypermethylation in transcribed region , and , following tcr stimulation , methylation decreases in both regions . methylation of the 212 , 198 , and 178 sites individually did not affect the activity of ifng promoter , and little effect was observed when the 50 and 39 sites were exclusively methylated , but modification of 58 cpg site significantly reduced the activity to a level similar to that of the observed for the completely methylated vector . interestingly , methylation of 58 site occurred faster and more completely than the other sites during th1 and th2 polarization , but a more accentuated outcome was observed in th2 [ 33 , 36 ] . the use of oligonucleotide probes with methylated 58 cpg , but not 50 and 39 , abolished the formation of 2 complexes verified when unmethylated probes were used in emsa with nuclear extracts of th1 cell line ae7 . in supershift assays c - jun and atf2 were identified in the upper band and creb and atf1/creb in the lower one , and this was also observed when th2 nuclear extracts from d10 lineage were used . chromatin immunoprecipitation ( chip ) assays confirmed the creb and creb / atf1 binding and identified fosb , junb , c - jun , and atf2 interacting with ifn- promoter in th1 cell lineage in vivo . concerning the th2 cell lineage , the mentioned functional and binding assays were performed in th cells , and although they are related to cd8 lymphocytes , several data suggest that ifng regulation may have particular characteristics in each t cell subset , like data obtained from transgenic mice model which express the luciferase gene under the control of proximal ( 70 to 44 ) and distal ( 98 to 78 ) regulatory elements from the ifn- promoter . in addition , elevated cyclic amp inhibited transcriptional activity directed by the proximal regulatory element in primed cd4 + t cells but enhanced transcriptional activity directed by the distal in primed cd8 + t cells . this differential gene expression depends on the selective action of transcriptional factors , but also from epigenetic modifications that could change the chromatin accessibility to transcriptional machinery . on the other hand , trimethylation of lysines 9 or 27 of histone h3 in fact , reduced repressive h3k27me3 and h3k27me2 throughout ifng after primary infection and persistence in memory cd8 t lymphocytes was reported , as well as a detection of minor h3k4me3 peaks near ifng tss in effector and memory t cd8 cells . in addition to the loss of dna methylation at ifn- promoter in the differentiation of nave p14 cd8 t cells to effector , northrop and colleagues reported a more pronounced demethylation of the ifn- enhancer at the first intron . investigation of ach3 by chip and detection by real - time pcr revealed a significant increase in ifn- promoter after stimulation , and similar increase was observed in the comparison of nave cd8 t cells with effector and memory cd8 t cells in promoter and enhancer . nevertheless , when cd8 memory and effector cells were generated in cd4 deficient mice ( b6 cd4 ) , they produced considerably less ifn- per cell and the shift in histone acetylation is no longer seen , suggesting that demethylation of cpg sites within ifn- promoter and enhancer in cd8 effector and memory cells occurred independently of cd4 t help , while histone acetylation at these same regions was highly dependent upon the presence of cd4 help . although the above data suggest that epigenetic regulation of the ifng promoter may interfere in its expression , transgenes containing the 8.6-kb fragment of human genomic dna containing the full length ifn- gene ( promoter , introns , and up to 3.4 kb of 5 flanking sequence ) do not confer proper t cell subset - specific expression in vivo [ 4143 ] . therefore , the search for conserved noncoding sequences ( cns ) among different species has been proved as a good method for identification of relevant cis - regulatory elements for ifn- gene . both of them exhibited enhancer like function in luciferase reporter assays in response to ionomycin in t cell lineages and correspond to a dnase i hypersensitive site in th1 cells but not in th2 cells [ 44 , 45 ] . interestingly , despite the fact that in vitro primed effector cd8 t cells have more levels of ach3 at intron 3 compared to cns-1 , tcr tg effector cd8 t cells primed in vivo displayed greater ach3 at cns-1 compared to intron 3 . interestingly , h3ac chip analysis of th1 , th2 , and cd8 cells from mice deficient in t - bet revealed that this transcription factor is required for induction of histone modifications in th1 , but not t cd8 cells . previously identified cns-1 ( also called cns-6 ) , 22 , 34 , 54 , and + 18/20 ( also called cns-2 ) were among them , but they further identify the cns+29 , + 46 , and + 55 . although the aim of the study was to characterize regulatory elements that govern ifng expression in cd4 t cells , functional elements identified were also investigated in primary cd8 t cells : cns-6 enhanced expression in stimulated cd8 cells as well as in th0 or th1 cells , but the authors report that cns-34 did so only in cd8 . on the other hand , cns22 enhanced expression in response to il-12 plus il-18 in th0 and th1 cells , but this effect was not consistently evident in cd8 + t cells . these results suggest that some sequences like cns-22 may be necessary for ifng expression in both cd4 and cd8 t cells , while others , as cns-34 , may have regulatory role only in cd8 t lymphocytes . it would be of particular interest to investigate if epigenetic events can influence heterogeneous features of cd8 t cell populations , like the capacity of polyfunctional cells to express several cytokines , and if specific microenvironments could modulate the ifng expression through epigenetic marks , as in iels .

increase in the number of the elderly and in the prevalence of chronic diseases presses healthcare systems to offer individuals with chronic diseases more comprehensive care through primary healthcare ( phc ) [ 13 ] . to meet these needs , different models of chronic disease management the most widely known is the chronic care model that advocates an integrated approach to care at all levels of the healthcare system for comprehensive and multidisciplinary management of chronic diseases . it also advocates for the establishment of organizational mechanisms to support provision of services and interorganizational linkage to achieve better coordination and integration of services . related to the chronic care model , other models have been proposed for the delivery of primary healthcare , including the patient - centered care medical home all proposed models focus on common elements , including sharing of responsibilities between healthcare professionals in a multidisciplinary teamwork perspective , active role of individuals in monitoring of their diseases , importance of communication facilitated by a clinical information system , and integration of services in an organizational framework that supports clinical practices and fosters linkages with other components of healthcare systems [ 3 , 10 ] . based on these models , two new forms of phc practices were created in the early 2000s in quebec : family medicine groups ( fmgs ) aimed at improving continuity of care and network clinics ( ncs ) intended to provide greater accessibility of services . a fmg is composed of 6 to 10 physicians with no geographical catchment area for patients they can register ( between 10,000 and 15,000 patients by fmg ) . the fmg can count on a grant from the ministry of health and social services to support its operations , in exchange for a contractual engagement to register a predetermined number of patients and to provide a minimum of specified services . it also provides greater accessibility through extended hours and participation in a regional on - call system . as of november 2010 , there were 217 accredited fmgs , including 25 in montral and 35 in montrgie , the two regions that participated in this study . the nc is a complementary phc practice model implemented in both regions , but mainly in montral at the initiative of the regional health and social services agency . it specifically aimed to improve accessibility , through providing walk - in services and ensuring access to radiology and laboratory tests as well as medical specialist services . it receives financial support from the regional agency . in august 2010 , there were 33 ncs in montral and 4 in montrgie . the distinction between fmg and nc is often blurred , especially as some phc practices have a dual fmg - nc status , thus benefiting from two sources of funding . the complementarity between fmgs and ncs was to allow the provision of more complete and better integrated care , particularly for individuals with chronic diseases [ 1113 ] . in november 2010 , 19 phc practices had a fmg - nc status in montral and none in montrgie . several studies have explored the association between structural features of phc practices and experience of care [ 1418 ] . studies have also assessed the impact of implementing specific components of the chronic care model on various care processes and outcomes [ 1921 ] . few studies have looked at phc practice organizational attributes to assess their potential for managing chronic diseases [ 3 , 4 ] . an ontario study found that chronic disease management was superior in community health centers compared to other types of practices mainly due to the presence of nurse practitioners and interdisciplinary teams . in the context of a large study conducted in 2005 in two quebec regions , we examined the overall patient experience of care in phc practices and found a better continuity of care among practices that were more integrated and coordinated , but the model that focused on walk - in visits provided more accessible care [ 13 , 23 ] . this study reported on the situation prevailing in 2005 and did not address the impact of the new phc practices as it was too early to assess the change . a related cohort study found that fmgs represented an integrated model of phc delivery associated with higher levels of achievement in chronic care . in close collaboration with the regional health and social services agencies of the two most heavily populated quebec regions , montral and montrgie , we conducted a second study in 2010 to assess the evolution of the phc reform and its impacts on patients ' experience of care , unmet needs , and use of services . the main objective of this paper is to assess the extent to which new phc practice models implemented in the two regions ( fmg - nc , fmg , and nc ) have improved patient experience of care , unmet needs , and use of services for individuals with and without chronic diseases , compared with other forms of phc practices that did not undergo such a change . the study design corresponds to a before - after natural experiment in which fmgs , ncs , and fmg - ncs constituted the experimental group and the other practices formed the control group ( figure 1 ) . the study consisted of population and organization surveys conducted in 2005 and 2010 in montral and montrgie . at that time , the two regions were divided into 23 local territories ( 12 in montral and 11 in montrgie ) under the governance of health and social services centres . the population surveys were carried out on independent samples of the 2005 and 2010 populations . they included 9,206 adults aged 18 or more in 2005 with a response rate of 64% and 9,180 in 2010 with a response rate of 56% . for the purpose of this study , we excluded respondents who did not have a regular source of care since only those who had a regular source of care reported on their experience of care . we also excluded those whose usual source of care was not identified at both time periods . short telephone surveys of all phc organizations were also carried out : 659 organizations in 2005 and 606 organizations in 2010 . basic information such as type of practice ( solo , group , fmg , and nc ) , number of practicing family physicians , having a nurse in the clinic , and offering walk - in services was gathered through telephone calls to the receptionists of all practices . population survey samples were stratified with approximatively 400 respondents in each of the 23 local territories regardless of their total population . the questionnaire was constructed drawing mainly on two validated instruments : the primary care assessment survey and the primary care assessment tool , to which we added questions when the topic had not been addressed [ 2628 ] . it focused on respondents ' attachment to a phc practice , experience of care , and use of services in the two years preceding the surveys as well as unmet needs in the six months preceding the survey . the population questionnaire documented also individual characteristics such as gender , age , level of education , economic status , perceived health , and presence of morbidities . population surveys data were linked with information gathered on phc organization through respondents ' identification of their regular source of primary care in the two years preceding the survey ( 20032005 and 20082010 ) . failing to precisely identify a usual source of primary care , respondents identified the phc practice most frequently attended in the two years preceding the survey . phc organizations were classified into two groups : an experimental group that included new model practices ( fmgs , ncs , and fmg - ncs ) and a control group that included all other practices ( local community services centres ( lcscs ) , family medicine units ( fmus ) , and solo and group practices that were not fmg or nc ) . fmus are academic training units that are likely to reflect current practice of family medicine and thus espouse hosting practices ' dominant philosophy of care . for the purposes of this study , fmgs implemented in lcsc and fmu were included in the experimental group . these represented 11 lcscs out of 49 and 9 fmus out of 12 . in 2010 , among the different phc organization models in montral and montrgie , group and solo practices that were not fmg or nc were dominant , as they represented 73% of all organizations and were the usual source of care for 50% of all service users . regardless of their status ( fmg , nc , or none ) , they are privately run by physicians , who are paid on a fee - for - service basis . information concerning patients ' experience of care , use of services , and unmet needs was gathered from population surveys . experience of care and utilization referred to the two years preceding the survey and unmet needs to the six months preceding the survey . variables of experience of care were accessibility , continuity , comprehensiveness , responsiveness , and perceived care outcomes . the 28 selected indicators of experience of care were grouped under five dimensions : accessibility ( 6 items ) , continuity ( 5 items ) , comprehensiveness ( 5 items ) , responsiveness ( 7 items ) , and perceived care outcomes ( 5 items ) . we carried out factor analysis within each of the five dimensions and calculated cronbach 's alpha which reached values of 0.60 or more for continuity ( 0.61 ) , comprehensiveness ( 0.79 ) , responsiveness ( 0.63 ) , and care outcomes ( 0.82 ) but was low for accessibility ( 0.30 ) , presumably reflecting the formative nature of this index . respondents were asked if a doctor had told them they were suffering from specific chronic diseases . we then classified chronic diseases as follows : heart disease ( coronary artery disease , heart failure ) , respiratory disease ( chronic obstructive pulmonary disease , asthma ) , musculoskeletal disease ( arthritis , osteoarthritis , and rheumatism ) , cardiometabolic diseases ( hypertension , diabetes , and hypercholesterolemia ) , and other chronic diseases which was a residual and more heterogeneous category that included all remaining chronic diseases such as cancer , anemia , and gastrointestinal disorders . mental illnesses were not excluded ; they were considered as comorbidities associated with all the above categories , including the no chronic disease one ; to be included in the broad category with chronic disease , all others were included in the no chronic disease category . to test for differences in the evolution of experimental and control groups between 2003 and 2010 , we applied the difference - in - differences ( dd ) technique and matched individuals with the propensity score method to ensure better comparability of the two groups [ 3236 ] . this method is particularly well suited to compare change in outcomes over time between individuals exposed to an intervention ( the experimental group ) and individuals that were not ( control group ) . individuals were matched on the basis of propensity scores that estimate the probability for an individual with given characteristics to be attached to a phc organization of the experimental group . the propensity score acts as a balancing score that renders the distribution of observed baseline covariates similar between the experimental group and the control group . the propensity score was calculated with logistic regression using sex , age , level of education , economic situation , immigration status , perception of health status , and morbidities as predictors . the subjects were then matched applying the kernel method , in which treated subjects are matched with a weighted average of all controls with weights that are inversely proportional to the distance between the propensity scores of treated individuals and controls . as the groups were very similar at the onset , nearly all subjects could be correctly matched . difference - in - differences analyses were conducted separately on respondents with and without chronic diseases . effect size , which expresses the magnitude of change , was measured in percentage and calculated by dividing dd scores by the average value of the indicators in the experimental and control groups at the baseline time period . table 1 presents the percentage of respondents with a regular source of primary care . as expected , the percentage is higher for those with than for those without chronic diseases at both time periods . for both groups table 2 shows , among those who had a regular source of primary care , percentages of users with different chronic diseases in the 2005 and 2010 surveys . table 3 presents for each category of chronic diseases in the two time periods the percentage of those who , aside from that disease , also present other chronic morbidities . data for the two time periods are generally comparable except for cardiometabolic and other chronic diseases . tables 4 and 5 present data on other users ' characteristics for differences between control and experimental groups of the two samples . the impact of the introduction of new phc models for individuals with chronic diseases was tested by comparing , in 20032005 and 20082010 , individuals attached to fmg - nc , fmg , and nc ( experimental group ) to those attached to other practices that had not changed ( control group ) with regard to experience of care , unmet needs , and use of services . as shown in table 6 , accessibility presents the lowest score among indices of experience of care . it decreased in both control and experimental groups over the years , but the experimental group remained with higher score and statistically significant differences at both times ( p = 0.048 ; p = 0.004 ) . however , the change was not sufficient to yield a significant dd value ( p = 0.640 ) . continuity was high in both groups . at both times , scores were higher for the control group , and the difference even increased in 20082010 ( p = 0.001 ; p < 0.001 ) . the dd value approached the 0.05 level of significance ( p = 0.08 ) with a small negative effect size of 1.5% . the pattern for comprehensiveness and responsiveness is very similar to that of continuity with nonsignificant dd values . this resulted in a significant dd value of 1.6 in favor of the control group ( p = 0.044 ) and an effect size of 1.8% . for unmet needs and utilization indicators , the results show no significant results except for high utilization of the usual source of primary care that decreased in both groups , but more so in the experimental group ( 7.9 ) than in the control group ( 4.0 ) , resulting in a significant and negative dd value ( p = 0.049 ) , corresponding to a 12.7% decrease . data presented in table 7 show overall decline in accessibility over time in both experimental and control groups . however , the decline was larger in the control than in the experimental group while it was very small in the experimental group , yielding a positive and significant dd value ( p < 0.001 ) with a 5.5% increase . as regards continuity , the differences between groups remain constant over time in favor of the control group and this lack of change resulted in a nonsignificant dd value ( p = 0.563 ) . the difference between groups for responsiveness turned in favor of the experimental group in 20082010 , resulting in a positive and significant dd value ( p = 0.007 ) with a positive 2.3% change . aside from these results , the evolution in both groups for all other indicators has been comparable , with no significant dd values . patients with chronic diseases in the experimental group had a worse experience of care than those in the control group in 20082010 on all indicators , except accessibility that was better and responsiveness that showed no difference . the percentage of users who reported unmet needs was lower in the control group and hospitalization was slightly higher in the experimental group , but these differences were not significant . looking at the evolution of experience of care for patients with chronic diseases in the two groups , measured by dd values , we observed that continuity increased more in the experimental than in the control group although the dd value failed to reach the 0.05 level of significance ( p = 0.081 ) . perceived care outcomes increased more in the control than in the experimental group resulting in a negative and significant dd value . the percentage of users attending more often their usual source of primary care also declined more in the experimental than in the control group and these results are statistically significant ( p = 0.049 ) with a percentage of change close to 13% . the findings for patients with no chronic disease contrast with those of patients with chronic diseases . in 20082010 , accessibility was better in the experimental group than in control group ( p < 0.001 ) , but continuity was worse ( p < 0.001 ) . dd values are positive and statistically significant for accessibility with a 5% effect size , indicating less unfavorable evolution for the experimental group ( p < 0.001 ) . continuity shows no difference between groups in its evolution , nor with all the other indicators of experience of care , unmet needs , and utilization . first , it is disappointing to see that while continuity improved in the population it did so more in the control than in the experimental groups . the main objective pursued by the reform in providing additional resources to new model practices was precisely to improve continuity of care , especially among individuals with chronic diseases [ 11 , 12 ] . a possible explanation for this result could be that financial incentives to enrollment of patients with chronic diseases were extended to all phc organizations , thus offsetting the advantage enjoyed up to that point by fmgs . another explanation may be increased provision of services by nurses in fmgs and ncs that are not included in our measures of continuity . these findings regarding continuity do not support those of an earlier study conducted by tourigny et al . among 1,275 patients followed up in five fmgs . that study found an increase in continuity among patients 18 months after their enrollment in fmgs . . 's study did not have a control group and was limited to only five fmgs . a second unanticipated result concerns accessibility which showed less unfavorable evolution for patients without chronic diseases in the experimental group than for patients with chronic diseases . again , the establishment of new models of phc organizations , and particularly of ncs , was aimed at improving accessibility to care for the general population , but in priority to the neediest individuals . it is rather ironic that the ones more in need were more affected by the unfavorable evolution of accessibility . another finding that raises concerns is the lack of impact of the reform on unmet needs and use of services . conversely , the larger decrease in the percentage of the high users of services at the usual source of primary care in the experimental group can be considered as a positive outcome , possibly associated with more comprehensive and preventive care . it could also result from substitution between services provided by physicians and those provided by nurses or other health professionals . however , the impact of this outcome remains marginal if not coupled to decrease in er attendance and hospitalization that represent the most costly services . our findings regarding accessibility and utilization corroborate studies reporting on fmgs . in an ecological study of the entire quebec population , dunkley - hickin found that a higher degree of physicians ' participation in fgm in a geographical area was not associated with improved accessibility among users of services living in that area . in a large scale cohort study involving 79 fmgs , strumpf observed no improvement in accessibility but a slight reduction in use of primary care and specialist services . . followed up a cohort of 122,722 patients enrolled in fmgs compared to 675,102 who were not . they found a small reduction in number of emergency visits for those attached to fmgs , but , as in our study , no change in hospitalization . none of those studies has reported specifically on patients with chronic diseases compared to those without chronic diseases . in an earlier study conducted on the 2005 sample we concluded that the coordination - integrated model , which matched the attributes of the ideal type of fmgs that were to be implemented at that time , emerged as the model with the greatest potential to concomitantly achieve high levels of accessibility and continuity for patients with chronic diseases . it could be that the reform had an effect on both phc organizations that have become fmgs or ncs and those that have not changed their status . it was not possible with our data to test that spillover effect hypothesis . according to rothman and wagner , the future of primary healthcare largely depends on ability to improve healthcare delivery to meet the needs of chronically ill patients . the chronic care model provides six interrelated components for achieving such change among which is delivery system redesign . this last condition does not seem to have been met in the implementation of new phc models in quebec . a recent report of the auditor general of quebec to the national assembly casts a critical look at the implantation of fmgs and ncs . it explains the relative failure of these phc organizations to fully attain the objectives established by the ministry of health and social services by many factors , notably the absence of clear rules , guidelines , and incentives and , above all , the lack of control of the ministry and the regional agencies on the process of implementation . this means that several new phc practices received complementary funding without respecting all the components of the contractual engagement . howe institute underlines that important factors associated with the slow implementation of fmgs are the low registration rate of patients and the deficient development of teamwork . the findings of our study concur with those of other studies and support concerns expressed by analysts . the use of difference - in - differences ( dd ) method for isolating the specific effect of the reform is fully justified because it provides good internal validity . however , when carried out in a complex system such as the healthcare system , dd analyses can not completely rule out the possibility of spillover effects . for example , it is possible that some aspects of the healthcare policies spilled over and influenced phc practices of the unexposed control group or that new phc model practices exerted a mimetic influence on the other practices . surveys lend themselves to possible recall bias by respondents . concerning experience of care , unmet needs , and use of services , if present , biases should be equally distributed among respondents . recall biases are less likely to occur when reporting events extending over a certain period of time rather than single events taking place at a given point in time . responses are always limited by respondents ' subjective understanding and their capacity to report accurately medical information . the wording of the questions attenuated this possible effect by referring to validation of the diagnosis by a doctor ( i.e. , has a doctor ever told you that you have diabetes ? ) . categories of chronic diseases were defined and operationalized in the same way in both 2005 and 2010 surveys . however , for the category other chronic diseases , it is possible that our redefinition and classification were more inclusive in 2005 , which may explain the higher percentage of respondents reporting this type of morbidity in 2005 . as pointed out in methods , mental illnesses were not excluded but left in all categories of physical chronic conditions , including the no disease category . the questions concerning mental health problems were slightly different in 2005 and 2010 as well as the time period reference : have you ever had in 2005 and in the last 2 years , did you see your doctor in 2010 . this may explain the higher percentage of individuals reporting mental health problems in 2005 than in 2010 . aside from this difference between the two surveys , mental health problems happened to be evenly distributed among our categories of chronic physical diseases , including the no disease category . consequently , we thought that their inclusion would minimize potential biases while increasing statistical power . they are subjective but still most appropriate to use in a patient - centered perspective . our questionnaire largely draws from two validated instruments ( pcas and pcat ) [ 2628 ] . we constructed new scales of experience of care , more adapted to our context . following the classical theory of measurement , we carried out factor analysis and calculated cronbach 's alpha coefficients which have values close to the commonly accepted level ( 0.70 ) except for accessibility ( 0.30 ) . in contrast to the classical reflective approach , in the formative approach items composing an index are not necessarily correlated or substitutable with each other , as is the case in reflective scales [ 30 , 31 ] . as mentioned earlier , our scales of experience of care may fail to include processes of care rendered by health professionals other than doctors . in that sense , they may underestimate experience of care in contexts of multidisciplinary work and thus fail to fully account for the effect of introducing new types of professionals , as is the case in fmgs and ncs . the possibility of such a bias has been suggested , particularly by researchers using large administrative data banks based on physicians ' reimbursement claims [ 47 , 48 ] . the first fmgs were accredited in march 2003 . since questions related to experience of care and use of services referred to the two years preceding the population surveys , we are confident that the 20032005 time period was a valid baseline in the context of a natural experiment . furthermore , as expected , changes took some time before being fully implemented in phc practices after their accreditation . our study 's findings raise questions concerning benefits resulting from the introduction of new phc practice models in qubec . the aim of the phc reform was mainly to improve accessibility and continuity of care for the entire population , but especially for individuals in greater needs such as those with chronic diseases . our findings show that , for patients with chronic diseases attached to new phc model practices , continuity increased less than for those attached to other practices . accessibility decreased for all users of services but much less for those with no chronic disease in the new model phc practices but did not change for those with chronic diseases . these results raise equity concerns since the ones with more needs seem to have benefitted less from the reform than those less in needs . furthermore , the reform did not reduce the use of the most costly services , er attendance , and hospitalization , nor did it reduce the reporting of unmet needs . based on those findings , it is difficult to conclude that the qubec healthcare reform has been so far successful .

objectives . to assess the extent to which new primary healthcare ( phc ) models implemented in two regions of quebec have improved patient experience of care , unmet needs , and use of services for individuals with and without chronic diseases , compared with other forms of phc practices . methods . in 2005 and 2010 , we carried out population and organization surveys . we divided phc organizations into new model practices and other practices and followed the evolution over time of patient experience of care . results . patients with chronic diseases had better accessibility but worse continuity of care in the new model practices than in the other practices at both time periods . through the reform , accessibility decreased evenly in both groups , but continuity and perceived outcomes improved more in the other practices . use of primary care services decreased more in the new model practices . among patients without chronic disease , accessibility decreased much less in the new models and responsiveness increased more . there was no significant change in er attendance and hospitalization . conclusion . the evolution of patient experience of care has been more favorable for patients without chronic diseases . these findings raise concerns about equity since the aim of the phc reform was targeting in priority individuals with the greatest needs .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Limitations 6. Conclusion

increase in the number of the elderly and in the prevalence of chronic diseases presses healthcare systems to offer individuals with chronic diseases more comprehensive care through primary healthcare ( phc ) [ 13 ] . to meet these needs , different models of chronic disease management the most widely known is the chronic care model that advocates an integrated approach to care at all levels of the healthcare system for comprehensive and multidisciplinary management of chronic diseases . related to the chronic care model , other models have been proposed for the delivery of primary healthcare , including the patient - centered care medical home all proposed models focus on common elements , including sharing of responsibilities between healthcare professionals in a multidisciplinary teamwork perspective , active role of individuals in monitoring of their diseases , importance of communication facilitated by a clinical information system , and integration of services in an organizational framework that supports clinical practices and fosters linkages with other components of healthcare systems [ 3 , 10 ] . based on these models , two new forms of phc practices were created in the early 2000s in quebec : family medicine groups ( fmgs ) aimed at improving continuity of care and network clinics ( ncs ) intended to provide greater accessibility of services . a fmg is composed of 6 to 10 physicians with no geographical catchment area for patients they can register ( between 10,000 and 15,000 patients by fmg ) . as of november 2010 , there were 217 accredited fmgs , including 25 in montral and 35 in montrgie , the two regions that participated in this study . the nc is a complementary phc practice model implemented in both regions , but mainly in montral at the initiative of the regional health and social services agency . in august 2010 , there were 33 ncs in montral and 4 in montrgie . the distinction between fmg and nc is often blurred , especially as some phc practices have a dual fmg - nc status , thus benefiting from two sources of funding . the complementarity between fmgs and ncs was to allow the provision of more complete and better integrated care , particularly for individuals with chronic diseases [ 1113 ] . in november 2010 , 19 phc practices had a fmg - nc status in montral and none in montrgie . several studies have explored the association between structural features of phc practices and experience of care [ 1418 ] . studies have also assessed the impact of implementing specific components of the chronic care model on various care processes and outcomes [ 1921 ] . few studies have looked at phc practice organizational attributes to assess their potential for managing chronic diseases [ 3 , 4 ] . an ontario study found that chronic disease management was superior in community health centers compared to other types of practices mainly due to the presence of nurse practitioners and interdisciplinary teams . in the context of a large study conducted in 2005 in two quebec regions , we examined the overall patient experience of care in phc practices and found a better continuity of care among practices that were more integrated and coordinated , but the model that focused on walk - in visits provided more accessible care [ 13 , 23 ] . this study reported on the situation prevailing in 2005 and did not address the impact of the new phc practices as it was too early to assess the change . in close collaboration with the regional health and social services agencies of the two most heavily populated quebec regions , montral and montrgie , we conducted a second study in 2010 to assess the evolution of the phc reform and its impacts on patients ' experience of care , unmet needs , and use of services . the main objective of this paper is to assess the extent to which new phc practice models implemented in the two regions ( fmg - nc , fmg , and nc ) have improved patient experience of care , unmet needs , and use of services for individuals with and without chronic diseases , compared with other forms of phc practices that did not undergo such a change . the study design corresponds to a before - after natural experiment in which fmgs , ncs , and fmg - ncs constituted the experimental group and the other practices formed the control group ( figure 1 ) . the study consisted of population and organization surveys conducted in 2005 and 2010 in montral and montrgie . at that time , the two regions were divided into 23 local territories ( 12 in montral and 11 in montrgie ) under the governance of health and social services centres . the population surveys were carried out on independent samples of the 2005 and 2010 populations . they included 9,206 adults aged 18 or more in 2005 with a response rate of 64% and 9,180 in 2010 with a response rate of 56% . for the purpose of this study , we excluded respondents who did not have a regular source of care since only those who had a regular source of care reported on their experience of care . we also excluded those whose usual source of care was not identified at both time periods . short telephone surveys of all phc organizations were also carried out : 659 organizations in 2005 and 606 organizations in 2010 . basic information such as type of practice ( solo , group , fmg , and nc ) , number of practicing family physicians , having a nurse in the clinic , and offering walk - in services was gathered through telephone calls to the receptionists of all practices . the questionnaire was constructed drawing mainly on two validated instruments : the primary care assessment survey and the primary care assessment tool , to which we added questions when the topic had not been addressed [ 2628 ] . it focused on respondents ' attachment to a phc practice , experience of care , and use of services in the two years preceding the surveys as well as unmet needs in the six months preceding the survey . population surveys data were linked with information gathered on phc organization through respondents ' identification of their regular source of primary care in the two years preceding the survey ( 20032005 and 20082010 ) . failing to precisely identify a usual source of primary care , respondents identified the phc practice most frequently attended in the two years preceding the survey . phc organizations were classified into two groups : an experimental group that included new model practices ( fmgs , ncs , and fmg - ncs ) and a control group that included all other practices ( local community services centres ( lcscs ) , family medicine units ( fmus ) , and solo and group practices that were not fmg or nc ) . fmus are academic training units that are likely to reflect current practice of family medicine and thus espouse hosting practices ' dominant philosophy of care . for the purposes of this study , fmgs implemented in lcsc and fmu were included in the experimental group . in 2010 , among the different phc organization models in montral and montrgie , group and solo practices that were not fmg or nc were dominant , as they represented 73% of all organizations and were the usual source of care for 50% of all service users . information concerning patients ' experience of care , use of services , and unmet needs was gathered from population surveys . experience of care and utilization referred to the two years preceding the survey and unmet needs to the six months preceding the survey . variables of experience of care were accessibility , continuity , comprehensiveness , responsiveness , and perceived care outcomes . the 28 selected indicators of experience of care were grouped under five dimensions : accessibility ( 6 items ) , continuity ( 5 items ) , comprehensiveness ( 5 items ) , responsiveness ( 7 items ) , and perceived care outcomes ( 5 items ) . we carried out factor analysis within each of the five dimensions and calculated cronbach 's alpha which reached values of 0.60 or more for continuity ( 0.61 ) , comprehensiveness ( 0.79 ) , responsiveness ( 0.63 ) , and care outcomes ( 0.82 ) but was low for accessibility ( 0.30 ) , presumably reflecting the formative nature of this index . we then classified chronic diseases as follows : heart disease ( coronary artery disease , heart failure ) , respiratory disease ( chronic obstructive pulmonary disease , asthma ) , musculoskeletal disease ( arthritis , osteoarthritis , and rheumatism ) , cardiometabolic diseases ( hypertension , diabetes , and hypercholesterolemia ) , and other chronic diseases which was a residual and more heterogeneous category that included all remaining chronic diseases such as cancer , anemia , and gastrointestinal disorders . mental illnesses were not excluded ; they were considered as comorbidities associated with all the above categories , including the no chronic disease one ; to be included in the broad category with chronic disease , all others were included in the no chronic disease category . to test for differences in the evolution of experimental and control groups between 2003 and 2010 , we applied the difference - in - differences ( dd ) technique and matched individuals with the propensity score method to ensure better comparability of the two groups [ 3236 ] . this method is particularly well suited to compare change in outcomes over time between individuals exposed to an intervention ( the experimental group ) and individuals that were not ( control group ) . individuals were matched on the basis of propensity scores that estimate the probability for an individual with given characteristics to be attached to a phc organization of the experimental group . the propensity score was calculated with logistic regression using sex , age , level of education , economic situation , immigration status , perception of health status , and morbidities as predictors . difference - in - differences analyses were conducted separately on respondents with and without chronic diseases . effect size , which expresses the magnitude of change , was measured in percentage and calculated by dividing dd scores by the average value of the indicators in the experimental and control groups at the baseline time period . table 1 presents the percentage of respondents with a regular source of primary care . as expected , the percentage is higher for those with than for those without chronic diseases at both time periods . for both groups table 2 shows , among those who had a regular source of primary care , percentages of users with different chronic diseases in the 2005 and 2010 surveys . table 3 presents for each category of chronic diseases in the two time periods the percentage of those who , aside from that disease , also present other chronic morbidities . data for the two time periods are generally comparable except for cardiometabolic and other chronic diseases . tables 4 and 5 present data on other users ' characteristics for differences between control and experimental groups of the two samples . the impact of the introduction of new phc models for individuals with chronic diseases was tested by comparing , in 20032005 and 20082010 , individuals attached to fmg - nc , fmg , and nc ( experimental group ) to those attached to other practices that had not changed ( control group ) with regard to experience of care , unmet needs , and use of services . as shown in table 6 , accessibility presents the lowest score among indices of experience of care . it decreased in both control and experimental groups over the years , but the experimental group remained with higher score and statistically significant differences at both times ( p = 0.048 ; p = 0.004 ) . continuity was high in both groups . at both times , scores were higher for the control group , and the difference even increased in 20082010 ( p = 0.001 ; p < 0.001 ) . for unmet needs and utilization indicators , the results show no significant results except for high utilization of the usual source of primary care that decreased in both groups , but more so in the experimental group ( 7.9 ) than in the control group ( 4.0 ) , resulting in a significant and negative dd value ( p = 0.049 ) , corresponding to a 12.7% decrease . data presented in table 7 show overall decline in accessibility over time in both experimental and control groups . however , the decline was larger in the control than in the experimental group while it was very small in the experimental group , yielding a positive and significant dd value ( p < 0.001 ) with a 5.5% increase . as regards continuity , the differences between groups remain constant over time in favor of the control group and this lack of change resulted in a nonsignificant dd value ( p = 0.563 ) . aside from these results , the evolution in both groups for all other indicators has been comparable , with no significant dd values . patients with chronic diseases in the experimental group had a worse experience of care than those in the control group in 20082010 on all indicators , except accessibility that was better and responsiveness that showed no difference . the percentage of users who reported unmet needs was lower in the control group and hospitalization was slightly higher in the experimental group , but these differences were not significant . looking at the evolution of experience of care for patients with chronic diseases in the two groups , measured by dd values , we observed that continuity increased more in the experimental than in the control group although the dd value failed to reach the 0.05 level of significance ( p = 0.081 ) . perceived care outcomes increased more in the control than in the experimental group resulting in a negative and significant dd value . the percentage of users attending more often their usual source of primary care also declined more in the experimental than in the control group and these results are statistically significant ( p = 0.049 ) with a percentage of change close to 13% . the findings for patients with no chronic disease contrast with those of patients with chronic diseases . in 20082010 , accessibility was better in the experimental group than in control group ( p < 0.001 ) , but continuity was worse ( p < 0.001 ) . continuity shows no difference between groups in its evolution , nor with all the other indicators of experience of care , unmet needs , and utilization . first , it is disappointing to see that while continuity improved in the population it did so more in the control than in the experimental groups . the main objective pursued by the reform in providing additional resources to new model practices was precisely to improve continuity of care , especially among individuals with chronic diseases [ 11 , 12 ] . a possible explanation for this result could be that financial incentives to enrollment of patients with chronic diseases were extended to all phc organizations , thus offsetting the advantage enjoyed up to that point by fmgs . a second unanticipated result concerns accessibility which showed less unfavorable evolution for patients without chronic diseases in the experimental group than for patients with chronic diseases . again , the establishment of new models of phc organizations , and particularly of ncs , was aimed at improving accessibility to care for the general population , but in priority to the neediest individuals . it is rather ironic that the ones more in need were more affected by the unfavorable evolution of accessibility . another finding that raises concerns is the lack of impact of the reform on unmet needs and use of services . conversely , the larger decrease in the percentage of the high users of services at the usual source of primary care in the experimental group can be considered as a positive outcome , possibly associated with more comprehensive and preventive care . however , the impact of this outcome remains marginal if not coupled to decrease in er attendance and hospitalization that represent the most costly services . in an ecological study of the entire quebec population , dunkley - hickin found that a higher degree of physicians ' participation in fgm in a geographical area was not associated with improved accessibility among users of services living in that area . in a large scale cohort study involving 79 fmgs , strumpf observed no improvement in accessibility but a slight reduction in use of primary care and specialist services . they found a small reduction in number of emergency visits for those attached to fmgs , but , as in our study , no change in hospitalization . none of those studies has reported specifically on patients with chronic diseases compared to those without chronic diseases . in an earlier study conducted on the 2005 sample we concluded that the coordination - integrated model , which matched the attributes of the ideal type of fmgs that were to be implemented at that time , emerged as the model with the greatest potential to concomitantly achieve high levels of accessibility and continuity for patients with chronic diseases . it could be that the reform had an effect on both phc organizations that have become fmgs or ncs and those that have not changed their status . according to rothman and wagner , the future of primary healthcare largely depends on ability to improve healthcare delivery to meet the needs of chronically ill patients . a recent report of the auditor general of quebec to the national assembly casts a critical look at the implantation of fmgs and ncs . it explains the relative failure of these phc organizations to fully attain the objectives established by the ministry of health and social services by many factors , notably the absence of clear rules , guidelines , and incentives and , above all , the lack of control of the ministry and the regional agencies on the process of implementation . this means that several new phc practices received complementary funding without respecting all the components of the contractual engagement . the use of difference - in - differences ( dd ) method for isolating the specific effect of the reform is fully justified because it provides good internal validity . for example , it is possible that some aspects of the healthcare policies spilled over and influenced phc practices of the unexposed control group or that new phc model practices exerted a mimetic influence on the other practices . concerning experience of care , unmet needs , and use of services , if present , biases should be equally distributed among respondents . the wording of the questions attenuated this possible effect by referring to validation of the diagnosis by a doctor ( i.e. categories of chronic diseases were defined and operationalized in the same way in both 2005 and 2010 surveys . however , for the category other chronic diseases , it is possible that our redefinition and classification were more inclusive in 2005 , which may explain the higher percentage of respondents reporting this type of morbidity in 2005 . the questions concerning mental health problems were slightly different in 2005 and 2010 as well as the time period reference : have you ever had in 2005 and in the last 2 years , did you see your doctor in 2010 . this may explain the higher percentage of individuals reporting mental health problems in 2005 than in 2010 . we constructed new scales of experience of care , more adapted to our context . following the classical theory of measurement , we carried out factor analysis and calculated cronbach 's alpha coefficients which have values close to the commonly accepted level ( 0.70 ) except for accessibility ( 0.30 ) . as mentioned earlier , our scales of experience of care may fail to include processes of care rendered by health professionals other than doctors . in that sense , they may underestimate experience of care in contexts of multidisciplinary work and thus fail to fully account for the effect of introducing new types of professionals , as is the case in fmgs and ncs . the possibility of such a bias has been suggested , particularly by researchers using large administrative data banks based on physicians ' reimbursement claims [ 47 , 48 ] . since questions related to experience of care and use of services referred to the two years preceding the population surveys , we are confident that the 20032005 time period was a valid baseline in the context of a natural experiment . furthermore , as expected , changes took some time before being fully implemented in phc practices after their accreditation . the aim of the phc reform was mainly to improve accessibility and continuity of care for the entire population , but especially for individuals in greater needs such as those with chronic diseases . our findings show that , for patients with chronic diseases attached to new phc model practices , continuity increased less than for those attached to other practices . accessibility decreased for all users of services but much less for those with no chronic disease in the new model phc practices but did not change for those with chronic diseases . these results raise equity concerns since the ones with more needs seem to have benefitted less from the reform than those less in needs . furthermore , the reform did not reduce the use of the most costly services , er attendance , and hospitalization , nor did it reduce the reporting of unmet needs .

increase in the number of the elderly and in the prevalence of chronic diseases presses healthcare systems to offer individuals with chronic diseases more comprehensive care through primary healthcare ( phc ) [ 13 ] . to meet these needs , different models of chronic disease management the most widely known is the chronic care model that advocates an integrated approach to care at all levels of the healthcare system for comprehensive and multidisciplinary management of chronic diseases . it also advocates for the establishment of organizational mechanisms to support provision of services and interorganizational linkage to achieve better coordination and integration of services . related to the chronic care model , other models have been proposed for the delivery of primary healthcare , including the patient - centered care medical home all proposed models focus on common elements , including sharing of responsibilities between healthcare professionals in a multidisciplinary teamwork perspective , active role of individuals in monitoring of their diseases , importance of communication facilitated by a clinical information system , and integration of services in an organizational framework that supports clinical practices and fosters linkages with other components of healthcare systems [ 3 , 10 ] . based on these models , two new forms of phc practices were created in the early 2000s in quebec : family medicine groups ( fmgs ) aimed at improving continuity of care and network clinics ( ncs ) intended to provide greater accessibility of services . a fmg is composed of 6 to 10 physicians with no geographical catchment area for patients they can register ( between 10,000 and 15,000 patients by fmg ) . the fmg can count on a grant from the ministry of health and social services to support its operations , in exchange for a contractual engagement to register a predetermined number of patients and to provide a minimum of specified services . as of november 2010 , there were 217 accredited fmgs , including 25 in montral and 35 in montrgie , the two regions that participated in this study . the nc is a complementary phc practice model implemented in both regions , but mainly in montral at the initiative of the regional health and social services agency . it specifically aimed to improve accessibility , through providing walk - in services and ensuring access to radiology and laboratory tests as well as medical specialist services . in august 2010 , there were 33 ncs in montral and 4 in montrgie . the distinction between fmg and nc is often blurred , especially as some phc practices have a dual fmg - nc status , thus benefiting from two sources of funding . the complementarity between fmgs and ncs was to allow the provision of more complete and better integrated care , particularly for individuals with chronic diseases [ 1113 ] . in november 2010 , 19 phc practices had a fmg - nc status in montral and none in montrgie . several studies have explored the association between structural features of phc practices and experience of care [ 1418 ] . studies have also assessed the impact of implementing specific components of the chronic care model on various care processes and outcomes [ 1921 ] . an ontario study found that chronic disease management was superior in community health centers compared to other types of practices mainly due to the presence of nurse practitioners and interdisciplinary teams . in the context of a large study conducted in 2005 in two quebec regions , we examined the overall patient experience of care in phc practices and found a better continuity of care among practices that were more integrated and coordinated , but the model that focused on walk - in visits provided more accessible care [ 13 , 23 ] . this study reported on the situation prevailing in 2005 and did not address the impact of the new phc practices as it was too early to assess the change . a related cohort study found that fmgs represented an integrated model of phc delivery associated with higher levels of achievement in chronic care . in close collaboration with the regional health and social services agencies of the two most heavily populated quebec regions , montral and montrgie , we conducted a second study in 2010 to assess the evolution of the phc reform and its impacts on patients ' experience of care , unmet needs , and use of services . the main objective of this paper is to assess the extent to which new phc practice models implemented in the two regions ( fmg - nc , fmg , and nc ) have improved patient experience of care , unmet needs , and use of services for individuals with and without chronic diseases , compared with other forms of phc practices that did not undergo such a change . the study design corresponds to a before - after natural experiment in which fmgs , ncs , and fmg - ncs constituted the experimental group and the other practices formed the control group ( figure 1 ) . at that time , the two regions were divided into 23 local territories ( 12 in montral and 11 in montrgie ) under the governance of health and social services centres . they included 9,206 adults aged 18 or more in 2005 with a response rate of 64% and 9,180 in 2010 with a response rate of 56% . for the purpose of this study , we excluded respondents who did not have a regular source of care since only those who had a regular source of care reported on their experience of care . basic information such as type of practice ( solo , group , fmg , and nc ) , number of practicing family physicians , having a nurse in the clinic , and offering walk - in services was gathered through telephone calls to the receptionists of all practices . population survey samples were stratified with approximatively 400 respondents in each of the 23 local territories regardless of their total population . the questionnaire was constructed drawing mainly on two validated instruments : the primary care assessment survey and the primary care assessment tool , to which we added questions when the topic had not been addressed [ 2628 ] . it focused on respondents ' attachment to a phc practice , experience of care , and use of services in the two years preceding the surveys as well as unmet needs in the six months preceding the survey . the population questionnaire documented also individual characteristics such as gender , age , level of education , economic status , perceived health , and presence of morbidities . population surveys data were linked with information gathered on phc organization through respondents ' identification of their regular source of primary care in the two years preceding the survey ( 20032005 and 20082010 ) . failing to precisely identify a usual source of primary care , respondents identified the phc practice most frequently attended in the two years preceding the survey . phc organizations were classified into two groups : an experimental group that included new model practices ( fmgs , ncs , and fmg - ncs ) and a control group that included all other practices ( local community services centres ( lcscs ) , family medicine units ( fmus ) , and solo and group practices that were not fmg or nc ) . fmus are academic training units that are likely to reflect current practice of family medicine and thus espouse hosting practices ' dominant philosophy of care . for the purposes of this study , fmgs implemented in lcsc and fmu were included in the experimental group . in 2010 , among the different phc organization models in montral and montrgie , group and solo practices that were not fmg or nc were dominant , as they represented 73% of all organizations and were the usual source of care for 50% of all service users . regardless of their status ( fmg , nc , or none ) , they are privately run by physicians , who are paid on a fee - for - service basis . information concerning patients ' experience of care , use of services , and unmet needs was gathered from population surveys . experience of care and utilization referred to the two years preceding the survey and unmet needs to the six months preceding the survey . variables of experience of care were accessibility , continuity , comprehensiveness , responsiveness , and perceived care outcomes . the 28 selected indicators of experience of care were grouped under five dimensions : accessibility ( 6 items ) , continuity ( 5 items ) , comprehensiveness ( 5 items ) , responsiveness ( 7 items ) , and perceived care outcomes ( 5 items ) . we carried out factor analysis within each of the five dimensions and calculated cronbach 's alpha which reached values of 0.60 or more for continuity ( 0.61 ) , comprehensiveness ( 0.79 ) , responsiveness ( 0.63 ) , and care outcomes ( 0.82 ) but was low for accessibility ( 0.30 ) , presumably reflecting the formative nature of this index . we then classified chronic diseases as follows : heart disease ( coronary artery disease , heart failure ) , respiratory disease ( chronic obstructive pulmonary disease , asthma ) , musculoskeletal disease ( arthritis , osteoarthritis , and rheumatism ) , cardiometabolic diseases ( hypertension , diabetes , and hypercholesterolemia ) , and other chronic diseases which was a residual and more heterogeneous category that included all remaining chronic diseases such as cancer , anemia , and gastrointestinal disorders . mental illnesses were not excluded ; they were considered as comorbidities associated with all the above categories , including the no chronic disease one ; to be included in the broad category with chronic disease , all others were included in the no chronic disease category . to test for differences in the evolution of experimental and control groups between 2003 and 2010 , we applied the difference - in - differences ( dd ) technique and matched individuals with the propensity score method to ensure better comparability of the two groups [ 3236 ] . this method is particularly well suited to compare change in outcomes over time between individuals exposed to an intervention ( the experimental group ) and individuals that were not ( control group ) . individuals were matched on the basis of propensity scores that estimate the probability for an individual with given characteristics to be attached to a phc organization of the experimental group . the propensity score acts as a balancing score that renders the distribution of observed baseline covariates similar between the experimental group and the control group . the propensity score was calculated with logistic regression using sex , age , level of education , economic situation , immigration status , perception of health status , and morbidities as predictors . the subjects were then matched applying the kernel method , in which treated subjects are matched with a weighted average of all controls with weights that are inversely proportional to the distance between the propensity scores of treated individuals and controls . effect size , which expresses the magnitude of change , was measured in percentage and calculated by dividing dd scores by the average value of the indicators in the experimental and control groups at the baseline time period . table 1 presents the percentage of respondents with a regular source of primary care . as expected , the percentage is higher for those with than for those without chronic diseases at both time periods . for both groups table 2 shows , among those who had a regular source of primary care , percentages of users with different chronic diseases in the 2005 and 2010 surveys . table 3 presents for each category of chronic diseases in the two time periods the percentage of those who , aside from that disease , also present other chronic morbidities . tables 4 and 5 present data on other users ' characteristics for differences between control and experimental groups of the two samples . the impact of the introduction of new phc models for individuals with chronic diseases was tested by comparing , in 20032005 and 20082010 , individuals attached to fmg - nc , fmg , and nc ( experimental group ) to those attached to other practices that had not changed ( control group ) with regard to experience of care , unmet needs , and use of services . as shown in table 6 , accessibility presents the lowest score among indices of experience of care . it decreased in both control and experimental groups over the years , but the experimental group remained with higher score and statistically significant differences at both times ( p = 0.048 ; p = 0.004 ) . at both times , scores were higher for the control group , and the difference even increased in 20082010 ( p = 0.001 ; p < 0.001 ) . this resulted in a significant dd value of 1.6 in favor of the control group ( p = 0.044 ) and an effect size of 1.8% . for unmet needs and utilization indicators , the results show no significant results except for high utilization of the usual source of primary care that decreased in both groups , but more so in the experimental group ( 7.9 ) than in the control group ( 4.0 ) , resulting in a significant and negative dd value ( p = 0.049 ) , corresponding to a 12.7% decrease . data presented in table 7 show overall decline in accessibility over time in both experimental and control groups . however , the decline was larger in the control than in the experimental group while it was very small in the experimental group , yielding a positive and significant dd value ( p < 0.001 ) with a 5.5% increase . as regards continuity , the differences between groups remain constant over time in favor of the control group and this lack of change resulted in a nonsignificant dd value ( p = 0.563 ) . the difference between groups for responsiveness turned in favor of the experimental group in 20082010 , resulting in a positive and significant dd value ( p = 0.007 ) with a positive 2.3% change . aside from these results , the evolution in both groups for all other indicators has been comparable , with no significant dd values . patients with chronic diseases in the experimental group had a worse experience of care than those in the control group in 20082010 on all indicators , except accessibility that was better and responsiveness that showed no difference . the percentage of users who reported unmet needs was lower in the control group and hospitalization was slightly higher in the experimental group , but these differences were not significant . looking at the evolution of experience of care for patients with chronic diseases in the two groups , measured by dd values , we observed that continuity increased more in the experimental than in the control group although the dd value failed to reach the 0.05 level of significance ( p = 0.081 ) . perceived care outcomes increased more in the control than in the experimental group resulting in a negative and significant dd value . the percentage of users attending more often their usual source of primary care also declined more in the experimental than in the control group and these results are statistically significant ( p = 0.049 ) with a percentage of change close to 13% . in 20082010 , accessibility was better in the experimental group than in control group ( p < 0.001 ) , but continuity was worse ( p < 0.001 ) . dd values are positive and statistically significant for accessibility with a 5% effect size , indicating less unfavorable evolution for the experimental group ( p < 0.001 ) . continuity shows no difference between groups in its evolution , nor with all the other indicators of experience of care , unmet needs , and utilization . first , it is disappointing to see that while continuity improved in the population it did so more in the control than in the experimental groups . the main objective pursued by the reform in providing additional resources to new model practices was precisely to improve continuity of care , especially among individuals with chronic diseases [ 11 , 12 ] . a second unanticipated result concerns accessibility which showed less unfavorable evolution for patients without chronic diseases in the experimental group than for patients with chronic diseases . again , the establishment of new models of phc organizations , and particularly of ncs , was aimed at improving accessibility to care for the general population , but in priority to the neediest individuals . another finding that raises concerns is the lack of impact of the reform on unmet needs and use of services . conversely , the larger decrease in the percentage of the high users of services at the usual source of primary care in the experimental group can be considered as a positive outcome , possibly associated with more comprehensive and preventive care . however , the impact of this outcome remains marginal if not coupled to decrease in er attendance and hospitalization that represent the most costly services . in an ecological study of the entire quebec population , dunkley - hickin found that a higher degree of physicians ' participation in fgm in a geographical area was not associated with improved accessibility among users of services living in that area . in a large scale cohort study involving 79 fmgs , strumpf observed no improvement in accessibility but a slight reduction in use of primary care and specialist services . they found a small reduction in number of emergency visits for those attached to fmgs , but , as in our study , no change in hospitalization . in an earlier study conducted on the 2005 sample we concluded that the coordination - integrated model , which matched the attributes of the ideal type of fmgs that were to be implemented at that time , emerged as the model with the greatest potential to concomitantly achieve high levels of accessibility and continuity for patients with chronic diseases . according to rothman and wagner , the future of primary healthcare largely depends on ability to improve healthcare delivery to meet the needs of chronically ill patients . this last condition does not seem to have been met in the implementation of new phc models in quebec . a recent report of the auditor general of quebec to the national assembly casts a critical look at the implantation of fmgs and ncs . it explains the relative failure of these phc organizations to fully attain the objectives established by the ministry of health and social services by many factors , notably the absence of clear rules , guidelines , and incentives and , above all , the lack of control of the ministry and the regional agencies on the process of implementation . howe institute underlines that important factors associated with the slow implementation of fmgs are the low registration rate of patients and the deficient development of teamwork . the use of difference - in - differences ( dd ) method for isolating the specific effect of the reform is fully justified because it provides good internal validity . for example , it is possible that some aspects of the healthcare policies spilled over and influenced phc practices of the unexposed control group or that new phc model practices exerted a mimetic influence on the other practices . however , for the category other chronic diseases , it is possible that our redefinition and classification were more inclusive in 2005 , which may explain the higher percentage of respondents reporting this type of morbidity in 2005 . the questions concerning mental health problems were slightly different in 2005 and 2010 as well as the time period reference : have you ever had in 2005 and in the last 2 years , did you see your doctor in 2010 . aside from this difference between the two surveys , mental health problems happened to be evenly distributed among our categories of chronic physical diseases , including the no disease category . following the classical theory of measurement , we carried out factor analysis and calculated cronbach 's alpha coefficients which have values close to the commonly accepted level ( 0.70 ) except for accessibility ( 0.30 ) . in contrast to the classical reflective approach , in the formative approach items composing an index are not necessarily correlated or substitutable with each other , as is the case in reflective scales [ 30 , 31 ] . in that sense , they may underestimate experience of care in contexts of multidisciplinary work and thus fail to fully account for the effect of introducing new types of professionals , as is the case in fmgs and ncs . since questions related to experience of care and use of services referred to the two years preceding the population surveys , we are confident that the 20032005 time period was a valid baseline in the context of a natural experiment . the aim of the phc reform was mainly to improve accessibility and continuity of care for the entire population , but especially for individuals in greater needs such as those with chronic diseases . our findings show that , for patients with chronic diseases attached to new phc model practices , continuity increased less than for those attached to other practices . accessibility decreased for all users of services but much less for those with no chronic disease in the new model phc practices but did not change for those with chronic diseases . furthermore , the reform did not reduce the use of the most costly services , er attendance , and hospitalization , nor did it reduce the reporting of unmet needs .

reconstruction surgery is very common to restore a rupture of an anterior cruciate ligament ( acl ) . there is currently a multiplicity of functional performance tests and patient reported outcome measures to determine the success of this surgery and rehabilitation [ 14 ] . for instance , the review done by garratt et al . found more than 15 patient - assessed health instruments specific to the knee in the 31 studies that were included . also , wang et al . identified twenty - four unique instrument outcomes measurements for the knee . regarding functional performance tests the review done by clark reported that more than 18 tests were used to evaluate the function of lower extremity following an acl deficiency or acl reconstruction . in light of the abundance of tests available , there appears no consensus regarding which test or combination of tests is most appropriate for evaluating recovery following acl reconstruction . it has been recommended that a multiplicity of assessments , incorporating both functional performance testing and patient reported tools , is important to evaluate functional ability and outcome for patients following acl reconstruction , but which of these tests or combination of tests provides the most rigorous test for outcome remains unclear . as no single instrument or functional performance test is currently capable of measuring all the multitude of parameters believed to relate to outcome , it is rational to accept that a range of tests should be administered to facilitate a full comprehensive evaluation of outcome . functional performance testing is likely to indicate the outcome of the neuromuscular training and appears to consist of two components . the first component is the quantity of movement or the capabilities of the production of the force , for example , muscle strength measurements and hop tests . the second component is the quality of movement , for example , the total knee flexion when landing from a jump or the occurrence of dynamic knee valgus [ 9 , 10 ] . these two components are important in rehabilitation and prevention of acl recurrent injuries or surgery failure [ 8 , 11 , 12 ] . most papers describing the functional performance following acl reconstruction are using the limb symmetry index ( lsi ) and thus are limited to quantitative measurements [ 13 , 14 ] . functional performance testing using qualitative methods evaluates compensation , or asymmetry , through clinical observation . the limb symmetry index ( lsi ) the lsi is the percentage deficit of the distance hopped on the involved leg compared with the contralateral noninvolved leg . the use of the lsi minimizes the probable confounding variable of the biological variation between people , which may affects the results . the work of munro and herrington showed lsi needs to be in excess of 90% to be deemed normal . a functional outcome is a predicted result of care that is meaningful and practical for the patients and sustainable beyond the rehabilitation environment . there are advantages and limitations to each measure used independently or in conjunction with other measures . the practicality of functional outcome measures employed in the clinical / research setting is an important consideration . functional or performance tests provide an objective assessment of components of the patients ' ability in a structured , controlled setting . combining several tests to assess function may serve to minimize any trade - offs between specificity and sensitivity . regardless of which tests are selected , it is imperative that they be standardised , reliable , valid , and responsive to change with time as well as being clinically relevant [ 2022 ] . ideally , outcome measures in research and clinical practice should be low - cost , take an acceptable length of time to administer , be convenient for researcher and clinicians to use , and be acceptable to the participants under investigation [ 21 , 22 ] . therefore , the purpose of this scoping review was to identify and explore a number of commonly used outcome measures for patients following acl reconstruction and postoperative rehabilitation to assess both aspects ( quantitative and qualitative ) of functional performance tests and self - reported questionnaires that have been used in last decade . we adopted a systematic scoping review approach this is a combination of a scoping review methodology to ensure the inclusion of broad areas of research and study designs and a systematic review of the methodology of the reviews . a scoping review is a relatively new type of study providing an assessment of available evidence from the literature in a broad area of research such as the compliance in the reporting of clinical studies to established guidelines . it also serves to identify information gaps in the field and provide recommendations for implementation . the methodology of scoping reviews was first described in detail by arksey and o'malley in their pivotal paper published in 2005 , which provided the foundation for carrying out a scoping review . this framework was further refined , and five stages were proposed to be followed when conducting a scoping review , including ( 1 ) the identification of a research question ; ( 2 ) finding the relevant studies ; ( 3 ) the selection of studies to be included in the review ; ( 4 ) data extraction from the included studies ; and ( 5 ) assembling , summarizing , and reporting the results of the review . a prisma compliant search strategy was used for study selection . the inclusion criteria of studies were as follows : ( 1 ) at least one lower extremity / knee functional performance test used as an outcome measurement of the article and/or patient reported outcomes , ( 2 ) subjects who were post - acl reconstruction , ( 3 ) studies which were either randomised control trial ( rct ) , cross - sectional , or cohort designs , and ( 4 ) studies published in english between april 2004 and april 2014 . the electronic databases used were medline ( mesh terms ) , pubmed , cochrane library ( systematic reviews and controlled trials registers ) , embase , cinahl , sportdiscus , pedro ( physiotherapy evidence database ) , and amed ( allied and complementary medicine index ) . in order to capture as many relevant references as possible , an expanded search was performed , including hand - searching the reference lists of all relevant articles , texts , and systematic reviews . search was conducted using the terms knee and acl injuries or functional performance and measure or test or screen or assessment or patient reported . the keyword search was also performed on pubmed utilising the key terms anterior cruciate ligament and surgery and injury and physical performance outcome measurements to ensure a detailed and comprehensive search strategy , and the additional search was performed in academic textbook that contained an extensive review of functional performance tests ( see table 1 ) . two reviewers ( adel almangoush and lee herrington ) independently reviewed all titles and abstracts that were identified from the search strategy . in accordance with the predefined eligibility criteria the full - text manuscripts for all potentially eligible studies were obtained , and then in accordance with the predefined eligibility criteria the reviewers independently reviewed them a second time . data extraction for each eligible paper was performed independently by two reviewers ( adel almangoush and lee herrington ) using a predefined spreadsheet . the standardised data extraction form included details on ( a ) focus of study , study design , participant details , outcome measure ( functional performance tests and patient reported outcomes ) , and results . in cases where insufficient data were provided within the publication , attempts were made to contact all corresponding authors to identify such data . each study 's methodological quality was assessed by using an appraisal tool devised to specifically evaluate functional performance testing and patients ' reported questionnaires of studies that included those patients following acl reconstruction . this was based on the critical appraisal skills programme ( casp ) critical appraisal tool ( casp , 2007 ) , which has been widely used and employed in previous systematic reviews to evaluate the methodological quality of clinical studies [ 4345 ] . the tool assessed domains such as the identification of the research questions , appropriateness of the research design , surgery and rehabilitation outcomes , the accuracy of description of methodology and population , appropriateness of analysis methods , and interpretation of findings . the appraisal was independently undertaken by two aforementioned reviewers ( adel almangoush and lee herrington ) . if any disagreements arose regarding the study selection , data extraction , or appraisal score , these were sorted out through discussion between the two reviewers until a consensus was met . studies were excluded if they achieved a very low methodological score of less than 50% through the casp scoring system . all analyses were initially undertaken by one reviewer ( adel almangoush ) and verified by the other reviewer ( lee herrington ) . an assessment of the quantity and quality of functional performance testing and patient reported tools of those patients following acl reconstruction by means of a meta - analysis was planned . however , unfortunately due to the heterogeneity of the studies , in particular the information regarding surgery and rehabilitation outcomes , it was not possible to complete this analysis . a prisma compliant search strategy was used , and results are presented in a prisma flow diagram ( figure 1 ) . as figure 1 demonstrates , eleven studies assessed knee laxity using a variety of instrumented laxity tests . nine studies used a kt-1000 arthrometer ( medmetric , san diego , ca , usa ) [ 26 , 27 , 29 , 31 , 3337 ] . one study used a manual maximum test with a rolimeter ( aircast , summit , nj , usa ) . all studies assessed the anterior displacement of the tibia relative to the femur , except one study which used medial joint space opening on manual valgus stress testing . only six papers ( 38% ) justified their sample sizes based on power calculations . whilst the surgery management strategies undertaken were clearly described in most of these papers , only four publications presented sufficient information to reproduce their methodologies for physiotherapy treatments and described the rehabilitation programs undertaken ( 25% ) . furthermore , whilst all studies reviewed used appropriate outcome measures to evaluate their participants , only a few of them defined the presence of an observer . whilst inferential statistics were presented in all included publications , confidence intervals however , all authors interpreted their findings appropriately and related these results in a suitable manner to clinical practice and the existing evidence base . outcome measures . a variety of different functional performance tests and patient reported outcomes measures have been reported in patients following acl reconstruction . a number of different assessment methods were used to determine the functional performance of patients following acl reconstruction . these methods included the one - leg hope for distance ; this is a commonly used functional performance test of both strength and confidence in the tested leg ; it correlates positively with muscle strength and power [ 7 , 47 ] . the one - leg hope for distance was assessed in fourteen studies ( 88% ) of the papers included . triple hop test for distance was evaluated in four papers [ 31 , 32 , 34 , 37 ] . three studies described a 6-meter timed hop test for speed [ 31 , 32 , 34 ] ; crossover hop of distance was assessed in two studies [ 31 , 34 ] ; side hop and vertical jump were also assessed in two studies [ 21 , 38 ] ; triple - jump test and stair hop test were evaluated in one study only ; and functional squat test was assessed in only one study also . more than 50% of studies used the hop tests as a measurement of function within the battery of different tests completed . only seven studies used multiple hop tests ( 44% ) , and only seven papers ( less than 50% ) reported limb symmetry index ( lsi ) comparing the injured with uninjured leg . only one study described the quality of movement whilst carrying out the test ( e.g. , dynamic knee valgus or knee flexion angle ) . postural stability of patients following acl reconstruction was assessed in four studies by using different measurement methods . used the modified star excursion balance test ( sebt ) to evaluate the postural control of their patients [ 24 , 28 ] . balance was recorded using static and dynamic balance tests on an instrumented unstable platform ( kat2000 ) . several reported questionnaires presented in the papers were evaluated in this scoping review , whereas koos and ikdc were assessed in most of the selected papers . only four studies used lysholm score [ 29 , 32 , 33 , 36 ] , three papers assessed the tegner activity level rating scale [ 23 , 27 , 32 ] , and only two studies per each score evaluated the global rating scale [ 31 , 34 ] , the kos - adls questionnaire [ 31 , 34 ] , and the cincinnati knee score [ 35 , 37 ] . they concluded that subjective variables are more important for evaluation of patient reported outcomes than objective findings . they found 7 key symptoms that together accounted for 83% of the variability in patients reported outcomes . the authors of the current review aimed to identify existing functional performance testing and patient reported outcomes for patients following acl reconstruction in the last decade . the most important finding of the present study was that all included articles used limited quantitative measurements to determine functional performance , except the study done by trulsson et al . . in the last decade most of the studies included in this review were focusing on the hop tests especially the single - leg hop test and few of these studies looked at a postural stability . regarding the reported outcomes the focus was on the koos and ikdc questionnaires . although the included articles reported the use of several hop tests , fourteen studies used a single - leg hop for distance as the gold standard for measuring functional performance after acl reconstruction because the reliability of this test is high ( icc ranging from 0.86 to 0.95 ) [ 49 , 50 ] . the relative reliability of the single hop for distance test in patients 1 to 2 years following acl reconstruction has previously been reported . however , several studies showed that the sensitivity increases when two or more different hop tests are performed [ 5052 ] . by using multiple hop tests , their qualities can be assessed and thereby the opportunity to detect discrepancies in hop performance increases . there is a strong relationship between crossover hop performance and functional outcome correlating significantly to ikdc subjective and koos questionnaire scores . the most reliable and valid of the multitude of hop tests in relation to the aclr patient would appear to be the single hop for distance and the crossover hop tests [ 7 , 50 , 54 ] . the ability of the aclr patient to perform well during hop tests is of paramount importance when judging functional performance . hop testing has frequently been proposed as a practical performance - based outcome measure that reflects the integrated effect of neuromuscular control , strength ( force - generating capacity ) , and confidence in the limb and requires minimal equipment and time to administer . based on a review of the potential use of hop tests as measures of dynamic knee stability , fitzgerald et al . suggested that hopping may be appropriate for use as a predictive tool for identifying patients who may have future problems as a result of knee injury or pathology and as an evaluative tool to reflect change in the patient status in response to treatment . within the published literature , the gold standard is often regarded as having a limb symmetry index ( lsi ) of greater than 85% , indicating that anything less than a 15% deficit in strength between the operated and nonoperated limb is acceptable . a study conducted in has shown that the contralateral ( noninjured ) leg is significantly weaker than matched controls . therefore , this assumption of normality should be viewed with caution , as the period of time during both preoperative and postoperative rehabilitation is likely to have caused atrophy of the noninjured leg . however , using the lsi is debatable because recent studies have shown that an acl injury could lead to a crossover effect in the uninvolved leg resulting in strength and function loss based on biomechanical and neuromuscular changes . to the best of the knowledge of this study 's researchers , there are few published studies that search for postural stability following acl reconstruction . for example , the sebt outcome measure offers a simple , reliable , valid , and low - cost alternative to more sophisticated instrumented methods , to assess dynamic balance ability [ 59 , 60 ] ; unlike force plates or electronically controlled balance platforms , it is an easy and highly portable test that could be employed in a range of clinical environments . according to logerstedt et al . the grid required testing for acl deficiency patients , three lines are positioned on the grid ( anterior , medial , and lateral reach distance ) which are labelled according to the direction of excursion relative to the stance leg . high intertester reliability of the sebt has previously been reported . whilst previous studies have evaluated intratester reliability , only one study has evaluated between - session reliability of the sebt with normalised scores with icc values ranging from 0.89 to 0.93 . however , only 3 reach distances , anterior , posteromedial , and posterolateral , were evaluated . therefore , further study of between - session reliability of all reach directions is warranted . previous research has suggested that the sebt is reliable and sensitive enough to detect dynamic postural control deficits in patients with an acl - deficient ( acl - d ) limb [ 62 , 63 ] . in these studies , patients who were injured were shown to have lower sebt scores compared to those of their uninjured limb and those of healthy participants . in particular , herrington et al . found that patients with acl deficiency showed functional deficits in the anterior , medial , lateral , and posteromedial reach directions . functional tests are a quick and inexpensive method of obtaining an objective measure of lower limb function following surgery . these tests are thought to provide an indication of muscle strength and power , neuromuscular control and confidence [ 64 , 65 ] . additionally , a number of authors have highlighted that a single functional test may not be sensitive enough to detect performance limitations and that at least two functional tests should be used [ 51 , 52 , 55 ] . only one study described the quality of movement whilst carrying out the test ( e.g. , dynamic knee valgus or knee flexion angle ) . studies focusing on prevention showed that the risk for acl injuries was reduced when training was done before high quality trials [ 6668 ] . for acl injury screening , ekegren and his colleagues examined dynamic knee valgus during a drop - jump task . the drop jump turned out to be a reliable and valid instrument in observing the dynamic knee valgus . investigated videotaped functional performance tests in acl injured subjects , and they reported that observation is a reliable and valid instrument for assessing knee flexion angles of the one - leg hop for a distance . the single - leg squat ( sls ) test is a cost - effective and simple movement to determine lower extremity alignment in the coronal plane . carried out with a single camera in any setting , this procedure can visibly identify a valgus lower extremity alignment on landing , which is considered to be a potential risk factor for a possible noncontact acl injury . the sls test has been described in a number of studies as a useful clinical measure to identify hip muscle function and dynamic knee control . patient reported instruments are normally related to signs and symptoms experienced by the patient and/or the functional tasks that individuals are able to achieve during their activities of daily living . a commonly used knee outcome instrument is the cincinnati knee scoring scale , and although it has been demonstrated to be an adequate tool to evaluate knee function following acl reconstruction , it also includes manual and instrumented stability testing to assess symptoms and function ; thus it becomes more difficult to separate various aspects of knee function following acl injury . the international knee documentation committee ( ikdc ) developed a scoring system for knees with acl injuries . the ikdc , the cincinnati knee scoring scale , and the first version of the lysholm score are assessor reported scores , which have been exposed to be biased when applied to individuals with an acl injury . on the other hand , carlos argued that for those clinicians and researchers considering using only the ikdc as their patient reported outcomes for acl reconstruction , they should include as a minimum the koos subscales that address broader areas of concern , including quality of life and emotional health that are most important to patients following acl reconstruction and are not wholly represented in ikdc . moreover , there is a suggestion that the koos is perhaps more suitable for the assessment of patients in the longer term unlike the ikdc . the koos has shown good validity and demonstrated that it is responsive to acl reconstruction and rehabilitation ; it shows that it is a reliable instrument for patients undergoing acl surgery and rehabilitation . koos has been used in an extensive amount of current research protocols and it has been translated and culturally adapted into various languages . clinicians and researchers looking to use a patient - based score measure of outcomes must consider the specific patient population in which it has been evaluated . using a diagnostic algorithm that measures the anatomic parts of the knee as separate constructs may solve this dilemma , allowing for the measurement of treatment outcomes across patient groups and the selection of the optimal clinical intervention . in general , the papers in this literature review included poorly described sample sizes and whether or not the sample size was based on power calculations . accordingly , the samples recruited may not necessarily have been big enough to identify a difference in outcome following a rehabilitation programme , irrespective of whether or not a difference existed . accordingly , it was not possible to determine whether measurement error influenced the results obtained or whether the experiences or training of the assessors was a variable which may have accounted for any between - study differences . limitations . for instance , the authors established very specific inclusion / exclusion criteria for selection of functional performance tests included inthis review . many tests were excluded because the studies were performed on healthy people or subjects with various neurological or debilitating comorbidities . this may modify the interpretation of the values attained for a specific functional performance test , and this was also the reason for the small number of studies included . future studies are required to establish the reliability and validity of existing functional performance tests or explore new , relevant quality measurements of the functional performance tests to be used in patients following acl surgery . this review shows that , following the acl reconstruction , the one - leg hop for distance or a combination of different hops and the limb symmetry index ( lsi ) of functional performance tests was used as a main outcome parameter of several studies . no extensive research has been carried out over the past 10 years to measure the control stability of patients following acl reconstruction . furthermore , no observation or videotaping was used to assess the quality of any test of any functional performance and control stability of acl patients following surgery except for one study . because previous studies discuss additional important parameters , a more extensive battery of tests is suggested to measure both the quantitative and qualitative aspects of functional performance after the acl reconstruction . the koos and the ikdc are both measures that are increasingly being used for acl reconstruction during the last 10 years .

objective . a systematic scoping review of the literature to identify functional performance tests and patient reported outcomes for patients who undergo anterior cruciate ligament ( acl ) reconstruction and rehabilitation that are used in clinical practice and research during the last decade . methods . a literature search was conducted . electronic databases used included medline , pubmed , cochrane library , embase , cinahl , sportdiscus , pedro , and amed . the inclusion criteria were english language , publication between april 2004 and april 2014 , and primary acl reconstruction with objective and/or subjective outcomes used . two authors screened the selected papers for title , abstract , and full - text in accordance with predefined inclusion and exclusion criteria . the methodological quality of all papers was assessed by a checklist of the critical appraisal skills programme ( casp ) . results . a total of 16 papers were included with full - text . different authors used different study designs for functional performance testing which led to different outcomes that could not be compared . all papers used a measurement for quantity of functional performance except one study which used both quantity and quality outcomes . several functional performance tests and patient reported outcomes were identified in this review . conclusion . no extensive research has been carried out over the past 10 years to measure the quality of functional performance testing and control stability of patients following acl reconstruction . however this study found that the measurement of functional performance following acl reconstruction consisting of a one - leg hop for a set distance or a combination of different hops using limb symmetry index ( lsi ) was a main outcome parameter of several studies . a more extensive series of tests is suggested to measure both the quantitative and qualitative aspects of functional performance after the acl reconstruction . the koos and the ikdc questionnaires are both measures that are increasingly being used for acl reconstruction throughout the last decade .

1. Introduction 2. Methodology 3. Results 4. Discussion 5. Conclusion

reconstruction surgery is very common to restore a rupture of an anterior cruciate ligament ( acl ) . there is currently a multiplicity of functional performance tests and patient reported outcome measures to determine the success of this surgery and rehabilitation [ 14 ] . found more than 15 patient - assessed health instruments specific to the knee in the 31 studies that were included . also , wang et al . regarding functional performance tests the review done by clark reported that more than 18 tests were used to evaluate the function of lower extremity following an acl deficiency or acl reconstruction . in light of the abundance of tests available , there appears no consensus regarding which test or combination of tests is most appropriate for evaluating recovery following acl reconstruction . it has been recommended that a multiplicity of assessments , incorporating both functional performance testing and patient reported tools , is important to evaluate functional ability and outcome for patients following acl reconstruction , but which of these tests or combination of tests provides the most rigorous test for outcome remains unclear . as no single instrument or functional performance test is currently capable of measuring all the multitude of parameters believed to relate to outcome , it is rational to accept that a range of tests should be administered to facilitate a full comprehensive evaluation of outcome . functional performance testing is likely to indicate the outcome of the neuromuscular training and appears to consist of two components . the first component is the quantity of movement or the capabilities of the production of the force , for example , muscle strength measurements and hop tests . the second component is the quality of movement , for example , the total knee flexion when landing from a jump or the occurrence of dynamic knee valgus [ 9 , 10 ] . most papers describing the functional performance following acl reconstruction are using the limb symmetry index ( lsi ) and thus are limited to quantitative measurements [ 13 , 14 ] . functional performance testing using qualitative methods evaluates compensation , or asymmetry , through clinical observation . the limb symmetry index ( lsi ) the lsi is the percentage deficit of the distance hopped on the involved leg compared with the contralateral noninvolved leg . the use of the lsi minimizes the probable confounding variable of the biological variation between people , which may affects the results . the practicality of functional outcome measures employed in the clinical / research setting is an important consideration . functional or performance tests provide an objective assessment of components of the patients ' ability in a structured , controlled setting . regardless of which tests are selected , it is imperative that they be standardised , reliable , valid , and responsive to change with time as well as being clinically relevant [ 2022 ] . ideally , outcome measures in research and clinical practice should be low - cost , take an acceptable length of time to administer , be convenient for researcher and clinicians to use , and be acceptable to the participants under investigation [ 21 , 22 ] . therefore , the purpose of this scoping review was to identify and explore a number of commonly used outcome measures for patients following acl reconstruction and postoperative rehabilitation to assess both aspects ( quantitative and qualitative ) of functional performance tests and self - reported questionnaires that have been used in last decade . we adopted a systematic scoping review approach this is a combination of a scoping review methodology to ensure the inclusion of broad areas of research and study designs and a systematic review of the methodology of the reviews . a scoping review is a relatively new type of study providing an assessment of available evidence from the literature in a broad area of research such as the compliance in the reporting of clinical studies to established guidelines . it also serves to identify information gaps in the field and provide recommendations for implementation . the methodology of scoping reviews was first described in detail by arksey and o'malley in their pivotal paper published in 2005 , which provided the foundation for carrying out a scoping review . this framework was further refined , and five stages were proposed to be followed when conducting a scoping review , including ( 1 ) the identification of a research question ; ( 2 ) finding the relevant studies ; ( 3 ) the selection of studies to be included in the review ; ( 4 ) data extraction from the included studies ; and ( 5 ) assembling , summarizing , and reporting the results of the review . a prisma compliant search strategy was used for study selection . the inclusion criteria of studies were as follows : ( 1 ) at least one lower extremity / knee functional performance test used as an outcome measurement of the article and/or patient reported outcomes , ( 2 ) subjects who were post - acl reconstruction , ( 3 ) studies which were either randomised control trial ( rct ) , cross - sectional , or cohort designs , and ( 4 ) studies published in english between april 2004 and april 2014 . the electronic databases used were medline ( mesh terms ) , pubmed , cochrane library ( systematic reviews and controlled trials registers ) , embase , cinahl , sportdiscus , pedro ( physiotherapy evidence database ) , and amed ( allied and complementary medicine index ) . in order to capture as many relevant references as possible , an expanded search was performed , including hand - searching the reference lists of all relevant articles , texts , and systematic reviews . search was conducted using the terms knee and acl injuries or functional performance and measure or test or screen or assessment or patient reported . the keyword search was also performed on pubmed utilising the key terms anterior cruciate ligament and surgery and injury and physical performance outcome measurements to ensure a detailed and comprehensive search strategy , and the additional search was performed in academic textbook that contained an extensive review of functional performance tests ( see table 1 ) . two reviewers ( adel almangoush and lee herrington ) independently reviewed all titles and abstracts that were identified from the search strategy . in accordance with the predefined eligibility criteria the full - text manuscripts for all potentially eligible studies were obtained , and then in accordance with the predefined eligibility criteria the reviewers independently reviewed them a second time . the standardised data extraction form included details on ( a ) focus of study , study design , participant details , outcome measure ( functional performance tests and patient reported outcomes ) , and results . in cases where insufficient data were provided within the publication , attempts were made to contact all corresponding authors to identify such data . each study 's methodological quality was assessed by using an appraisal tool devised to specifically evaluate functional performance testing and patients ' reported questionnaires of studies that included those patients following acl reconstruction . this was based on the critical appraisal skills programme ( casp ) critical appraisal tool ( casp , 2007 ) , which has been widely used and employed in previous systematic reviews to evaluate the methodological quality of clinical studies [ 4345 ] . the tool assessed domains such as the identification of the research questions , appropriateness of the research design , surgery and rehabilitation outcomes , the accuracy of description of methodology and population , appropriateness of analysis methods , and interpretation of findings . the appraisal was independently undertaken by two aforementioned reviewers ( adel almangoush and lee herrington ) . if any disagreements arose regarding the study selection , data extraction , or appraisal score , these were sorted out through discussion between the two reviewers until a consensus was met . an assessment of the quantity and quality of functional performance testing and patient reported tools of those patients following acl reconstruction by means of a meta - analysis was planned . however , unfortunately due to the heterogeneity of the studies , in particular the information regarding surgery and rehabilitation outcomes , it was not possible to complete this analysis . a prisma compliant search strategy was used , and results are presented in a prisma flow diagram ( figure 1 ) . nine studies used a kt-1000 arthrometer ( medmetric , san diego , ca , usa ) [ 26 , 27 , 29 , 31 , 3337 ] . one study used a manual maximum test with a rolimeter ( aircast , summit , nj , usa ) . all studies assessed the anterior displacement of the tibia relative to the femur , except one study which used medial joint space opening on manual valgus stress testing . whilst inferential statistics were presented in all included publications , confidence intervals however , all authors interpreted their findings appropriately and related these results in a suitable manner to clinical practice and the existing evidence base . a variety of different functional performance tests and patient reported outcomes measures have been reported in patients following acl reconstruction . a number of different assessment methods were used to determine the functional performance of patients following acl reconstruction . these methods included the one - leg hope for distance ; this is a commonly used functional performance test of both strength and confidence in the tested leg ; it correlates positively with muscle strength and power [ 7 , 47 ] . the one - leg hope for distance was assessed in fourteen studies ( 88% ) of the papers included . triple hop test for distance was evaluated in four papers [ 31 , 32 , 34 , 37 ] . three studies described a 6-meter timed hop test for speed [ 31 , 32 , 34 ] ; crossover hop of distance was assessed in two studies [ 31 , 34 ] ; side hop and vertical jump were also assessed in two studies [ 21 , 38 ] ; triple - jump test and stair hop test were evaluated in one study only ; and functional squat test was assessed in only one study also . more than 50% of studies used the hop tests as a measurement of function within the battery of different tests completed . only seven studies used multiple hop tests ( 44% ) , and only seven papers ( less than 50% ) reported limb symmetry index ( lsi ) comparing the injured with uninjured leg . only one study described the quality of movement whilst carrying out the test ( e.g. postural stability of patients following acl reconstruction was assessed in four studies by using different measurement methods . several reported questionnaires presented in the papers were evaluated in this scoping review , whereas koos and ikdc were assessed in most of the selected papers . only four studies used lysholm score [ 29 , 32 , 33 , 36 ] , three papers assessed the tegner activity level rating scale [ 23 , 27 , 32 ] , and only two studies per each score evaluated the global rating scale [ 31 , 34 ] , the kos - adls questionnaire [ 31 , 34 ] , and the cincinnati knee score [ 35 , 37 ] . they concluded that subjective variables are more important for evaluation of patient reported outcomes than objective findings . they found 7 key symptoms that together accounted for 83% of the variability in patients reported outcomes . the authors of the current review aimed to identify existing functional performance testing and patient reported outcomes for patients following acl reconstruction in the last decade . the most important finding of the present study was that all included articles used limited quantitative measurements to determine functional performance , except the study done by trulsson et al . in the last decade most of the studies included in this review were focusing on the hop tests especially the single - leg hop test and few of these studies looked at a postural stability . regarding the reported outcomes the focus was on the koos and ikdc questionnaires . although the included articles reported the use of several hop tests , fourteen studies used a single - leg hop for distance as the gold standard for measuring functional performance after acl reconstruction because the reliability of this test is high ( icc ranging from 0.86 to 0.95 ) [ 49 , 50 ] . the relative reliability of the single hop for distance test in patients 1 to 2 years following acl reconstruction has previously been reported . however , several studies showed that the sensitivity increases when two or more different hop tests are performed [ 5052 ] . there is a strong relationship between crossover hop performance and functional outcome correlating significantly to ikdc subjective and koos questionnaire scores . the most reliable and valid of the multitude of hop tests in relation to the aclr patient would appear to be the single hop for distance and the crossover hop tests [ 7 , 50 , 54 ] . the ability of the aclr patient to perform well during hop tests is of paramount importance when judging functional performance . hop testing has frequently been proposed as a practical performance - based outcome measure that reflects the integrated effect of neuromuscular control , strength ( force - generating capacity ) , and confidence in the limb and requires minimal equipment and time to administer . based on a review of the potential use of hop tests as measures of dynamic knee stability , fitzgerald et al . suggested that hopping may be appropriate for use as a predictive tool for identifying patients who may have future problems as a result of knee injury or pathology and as an evaluative tool to reflect change in the patient status in response to treatment . within the published literature , the gold standard is often regarded as having a limb symmetry index ( lsi ) of greater than 85% , indicating that anything less than a 15% deficit in strength between the operated and nonoperated limb is acceptable . a study conducted in has shown that the contralateral ( noninjured ) leg is significantly weaker than matched controls . therefore , this assumption of normality should be viewed with caution , as the period of time during both preoperative and postoperative rehabilitation is likely to have caused atrophy of the noninjured leg . to the best of the knowledge of this study 's researchers , there are few published studies that search for postural stability following acl reconstruction . for example , the sebt outcome measure offers a simple , reliable , valid , and low - cost alternative to more sophisticated instrumented methods , to assess dynamic balance ability [ 59 , 60 ] ; unlike force plates or electronically controlled balance platforms , it is an easy and highly portable test that could be employed in a range of clinical environments . the grid required testing for acl deficiency patients , three lines are positioned on the grid ( anterior , medial , and lateral reach distance ) which are labelled according to the direction of excursion relative to the stance leg . high intertester reliability of the sebt has previously been reported . whilst previous studies have evaluated intratester reliability , only one study has evaluated between - session reliability of the sebt with normalised scores with icc values ranging from 0.89 to 0.93 . however , only 3 reach distances , anterior , posteromedial , and posterolateral , were evaluated . therefore , further study of between - session reliability of all reach directions is warranted . previous research has suggested that the sebt is reliable and sensitive enough to detect dynamic postural control deficits in patients with an acl - deficient ( acl - d ) limb [ 62 , 63 ] . in these studies , patients who were injured were shown to have lower sebt scores compared to those of their uninjured limb and those of healthy participants . found that patients with acl deficiency showed functional deficits in the anterior , medial , lateral , and posteromedial reach directions . additionally , a number of authors have highlighted that a single functional test may not be sensitive enough to detect performance limitations and that at least two functional tests should be used [ 51 , 52 , 55 ] . only one study described the quality of movement whilst carrying out the test ( e.g. studies focusing on prevention showed that the risk for acl injuries was reduced when training was done before high quality trials [ 6668 ] . for acl injury screening , ekegren and his colleagues examined dynamic knee valgus during a drop - jump task . the drop jump turned out to be a reliable and valid instrument in observing the dynamic knee valgus . investigated videotaped functional performance tests in acl injured subjects , and they reported that observation is a reliable and valid instrument for assessing knee flexion angles of the one - leg hop for a distance . the single - leg squat ( sls ) test is a cost - effective and simple movement to determine lower extremity alignment in the coronal plane . carried out with a single camera in any setting , this procedure can visibly identify a valgus lower extremity alignment on landing , which is considered to be a potential risk factor for a possible noncontact acl injury . the sls test has been described in a number of studies as a useful clinical measure to identify hip muscle function and dynamic knee control . patient reported instruments are normally related to signs and symptoms experienced by the patient and/or the functional tasks that individuals are able to achieve during their activities of daily living . a commonly used knee outcome instrument is the cincinnati knee scoring scale , and although it has been demonstrated to be an adequate tool to evaluate knee function following acl reconstruction , it also includes manual and instrumented stability testing to assess symptoms and function ; thus it becomes more difficult to separate various aspects of knee function following acl injury . the international knee documentation committee ( ikdc ) developed a scoring system for knees with acl injuries . the ikdc , the cincinnati knee scoring scale , and the first version of the lysholm score are assessor reported scores , which have been exposed to be biased when applied to individuals with an acl injury . on the other hand , carlos argued that for those clinicians and researchers considering using only the ikdc as their patient reported outcomes for acl reconstruction , they should include as a minimum the koos subscales that address broader areas of concern , including quality of life and emotional health that are most important to patients following acl reconstruction and are not wholly represented in ikdc . moreover , there is a suggestion that the koos is perhaps more suitable for the assessment of patients in the longer term unlike the ikdc . the koos has shown good validity and demonstrated that it is responsive to acl reconstruction and rehabilitation ; it shows that it is a reliable instrument for patients undergoing acl surgery and rehabilitation . koos has been used in an extensive amount of current research protocols and it has been translated and culturally adapted into various languages . clinicians and researchers looking to use a patient - based score measure of outcomes must consider the specific patient population in which it has been evaluated . using a diagnostic algorithm that measures the anatomic parts of the knee as separate constructs may solve this dilemma , allowing for the measurement of treatment outcomes across patient groups and the selection of the optimal clinical intervention . in general , the papers in this literature review included poorly described sample sizes and whether or not the sample size was based on power calculations . accordingly , the samples recruited may not necessarily have been big enough to identify a difference in outcome following a rehabilitation programme , irrespective of whether or not a difference existed . accordingly , it was not possible to determine whether measurement error influenced the results obtained or whether the experiences or training of the assessors was a variable which may have accounted for any between - study differences . for instance , the authors established very specific inclusion / exclusion criteria for selection of functional performance tests included inthis review . this may modify the interpretation of the values attained for a specific functional performance test , and this was also the reason for the small number of studies included . future studies are required to establish the reliability and validity of existing functional performance tests or explore new , relevant quality measurements of the functional performance tests to be used in patients following acl surgery . this review shows that , following the acl reconstruction , the one - leg hop for distance or a combination of different hops and the limb symmetry index ( lsi ) of functional performance tests was used as a main outcome parameter of several studies . no extensive research has been carried out over the past 10 years to measure the control stability of patients following acl reconstruction . furthermore , no observation or videotaping was used to assess the quality of any test of any functional performance and control stability of acl patients following surgery except for one study . because previous studies discuss additional important parameters , a more extensive battery of tests is suggested to measure both the quantitative and qualitative aspects of functional performance after the acl reconstruction . the koos and the ikdc are both measures that are increasingly being used for acl reconstruction during the last 10 years .

there is currently a multiplicity of functional performance tests and patient reported outcome measures to determine the success of this surgery and rehabilitation [ 14 ] . found more than 15 patient - assessed health instruments specific to the knee in the 31 studies that were included . regarding functional performance tests the review done by clark reported that more than 18 tests were used to evaluate the function of lower extremity following an acl deficiency or acl reconstruction . in light of the abundance of tests available , there appears no consensus regarding which test or combination of tests is most appropriate for evaluating recovery following acl reconstruction . it has been recommended that a multiplicity of assessments , incorporating both functional performance testing and patient reported tools , is important to evaluate functional ability and outcome for patients following acl reconstruction , but which of these tests or combination of tests provides the most rigorous test for outcome remains unclear . as no single instrument or functional performance test is currently capable of measuring all the multitude of parameters believed to relate to outcome , it is rational to accept that a range of tests should be administered to facilitate a full comprehensive evaluation of outcome . functional performance testing is likely to indicate the outcome of the neuromuscular training and appears to consist of two components . the first component is the quantity of movement or the capabilities of the production of the force , for example , muscle strength measurements and hop tests . the second component is the quality of movement , for example , the total knee flexion when landing from a jump or the occurrence of dynamic knee valgus [ 9 , 10 ] . these two components are important in rehabilitation and prevention of acl recurrent injuries or surgery failure [ 8 , 11 , 12 ] . most papers describing the functional performance following acl reconstruction are using the limb symmetry index ( lsi ) and thus are limited to quantitative measurements [ 13 , 14 ] . the limb symmetry index ( lsi ) the lsi is the percentage deficit of the distance hopped on the involved leg compared with the contralateral noninvolved leg . the use of the lsi minimizes the probable confounding variable of the biological variation between people , which may affects the results . the work of munro and herrington showed lsi needs to be in excess of 90% to be deemed normal . a functional outcome is a predicted result of care that is meaningful and practical for the patients and sustainable beyond the rehabilitation environment . the practicality of functional outcome measures employed in the clinical / research setting is an important consideration . functional or performance tests provide an objective assessment of components of the patients ' ability in a structured , controlled setting . combining several tests to assess function may serve to minimize any trade - offs between specificity and sensitivity . regardless of which tests are selected , it is imperative that they be standardised , reliable , valid , and responsive to change with time as well as being clinically relevant [ 2022 ] . ideally , outcome measures in research and clinical practice should be low - cost , take an acceptable length of time to administer , be convenient for researcher and clinicians to use , and be acceptable to the participants under investigation [ 21 , 22 ] . therefore , the purpose of this scoping review was to identify and explore a number of commonly used outcome measures for patients following acl reconstruction and postoperative rehabilitation to assess both aspects ( quantitative and qualitative ) of functional performance tests and self - reported questionnaires that have been used in last decade . we adopted a systematic scoping review approach this is a combination of a scoping review methodology to ensure the inclusion of broad areas of research and study designs and a systematic review of the methodology of the reviews . a scoping review is a relatively new type of study providing an assessment of available evidence from the literature in a broad area of research such as the compliance in the reporting of clinical studies to established guidelines . it also serves to identify information gaps in the field and provide recommendations for implementation . the methodology of scoping reviews was first described in detail by arksey and o'malley in their pivotal paper published in 2005 , which provided the foundation for carrying out a scoping review . this framework was further refined , and five stages were proposed to be followed when conducting a scoping review , including ( 1 ) the identification of a research question ; ( 2 ) finding the relevant studies ; ( 3 ) the selection of studies to be included in the review ; ( 4 ) data extraction from the included studies ; and ( 5 ) assembling , summarizing , and reporting the results of the review . the inclusion criteria of studies were as follows : ( 1 ) at least one lower extremity / knee functional performance test used as an outcome measurement of the article and/or patient reported outcomes , ( 2 ) subjects who were post - acl reconstruction , ( 3 ) studies which were either randomised control trial ( rct ) , cross - sectional , or cohort designs , and ( 4 ) studies published in english between april 2004 and april 2014 . the electronic databases used were medline ( mesh terms ) , pubmed , cochrane library ( systematic reviews and controlled trials registers ) , embase , cinahl , sportdiscus , pedro ( physiotherapy evidence database ) , and amed ( allied and complementary medicine index ) . in order to capture as many relevant references as possible , an expanded search was performed , including hand - searching the reference lists of all relevant articles , texts , and systematic reviews . search was conducted using the terms knee and acl injuries or functional performance and measure or test or screen or assessment or patient reported . the keyword search was also performed on pubmed utilising the key terms anterior cruciate ligament and surgery and injury and physical performance outcome measurements to ensure a detailed and comprehensive search strategy , and the additional search was performed in academic textbook that contained an extensive review of functional performance tests ( see table 1 ) . two reviewers ( adel almangoush and lee herrington ) independently reviewed all titles and abstracts that were identified from the search strategy . in accordance with the predefined eligibility criteria the full - text manuscripts for all potentially eligible studies were obtained , and then in accordance with the predefined eligibility criteria the reviewers independently reviewed them a second time . data extraction for each eligible paper was performed independently by two reviewers ( adel almangoush and lee herrington ) using a predefined spreadsheet . the standardised data extraction form included details on ( a ) focus of study , study design , participant details , outcome measure ( functional performance tests and patient reported outcomes ) , and results . each study 's methodological quality was assessed by using an appraisal tool devised to specifically evaluate functional performance testing and patients ' reported questionnaires of studies that included those patients following acl reconstruction . this was based on the critical appraisal skills programme ( casp ) critical appraisal tool ( casp , 2007 ) , which has been widely used and employed in previous systematic reviews to evaluate the methodological quality of clinical studies [ 4345 ] . the tool assessed domains such as the identification of the research questions , appropriateness of the research design , surgery and rehabilitation outcomes , the accuracy of description of methodology and population , appropriateness of analysis methods , and interpretation of findings . the appraisal was independently undertaken by two aforementioned reviewers ( adel almangoush and lee herrington ) . if any disagreements arose regarding the study selection , data extraction , or appraisal score , these were sorted out through discussion between the two reviewers until a consensus was met . an assessment of the quantity and quality of functional performance testing and patient reported tools of those patients following acl reconstruction by means of a meta - analysis was planned . however , unfortunately due to the heterogeneity of the studies , in particular the information regarding surgery and rehabilitation outcomes , it was not possible to complete this analysis . a prisma compliant search strategy was used , and results are presented in a prisma flow diagram ( figure 1 ) . as figure 1 demonstrates , eleven studies assessed knee laxity using a variety of instrumented laxity tests . all studies assessed the anterior displacement of the tibia relative to the femur , except one study which used medial joint space opening on manual valgus stress testing . whilst the surgery management strategies undertaken were clearly described in most of these papers , only four publications presented sufficient information to reproduce their methodologies for physiotherapy treatments and described the rehabilitation programs undertaken ( 25% ) . furthermore , whilst all studies reviewed used appropriate outcome measures to evaluate their participants , only a few of them defined the presence of an observer . whilst inferential statistics were presented in all included publications , confidence intervals however , all authors interpreted their findings appropriately and related these results in a suitable manner to clinical practice and the existing evidence base . a variety of different functional performance tests and patient reported outcomes measures have been reported in patients following acl reconstruction . a number of different assessment methods were used to determine the functional performance of patients following acl reconstruction . these methods included the one - leg hope for distance ; this is a commonly used functional performance test of both strength and confidence in the tested leg ; it correlates positively with muscle strength and power [ 7 , 47 ] . the one - leg hope for distance was assessed in fourteen studies ( 88% ) of the papers included . triple hop test for distance was evaluated in four papers [ 31 , 32 , 34 , 37 ] . three studies described a 6-meter timed hop test for speed [ 31 , 32 , 34 ] ; crossover hop of distance was assessed in two studies [ 31 , 34 ] ; side hop and vertical jump were also assessed in two studies [ 21 , 38 ] ; triple - jump test and stair hop test were evaluated in one study only ; and functional squat test was assessed in only one study also . more than 50% of studies used the hop tests as a measurement of function within the battery of different tests completed . only seven studies used multiple hop tests ( 44% ) , and only seven papers ( less than 50% ) reported limb symmetry index ( lsi ) comparing the injured with uninjured leg . only one study described the quality of movement whilst carrying out the test ( e.g. postural stability of patients following acl reconstruction was assessed in four studies by using different measurement methods . used the modified star excursion balance test ( sebt ) to evaluate the postural control of their patients [ 24 , 28 ] . several reported questionnaires presented in the papers were evaluated in this scoping review , whereas koos and ikdc were assessed in most of the selected papers . only four studies used lysholm score [ 29 , 32 , 33 , 36 ] , three papers assessed the tegner activity level rating scale [ 23 , 27 , 32 ] , and only two studies per each score evaluated the global rating scale [ 31 , 34 ] , the kos - adls questionnaire [ 31 , 34 ] , and the cincinnati knee score [ 35 , 37 ] . they concluded that subjective variables are more important for evaluation of patient reported outcomes than objective findings . they found 7 key symptoms that together accounted for 83% of the variability in patients reported outcomes . the authors of the current review aimed to identify existing functional performance testing and patient reported outcomes for patients following acl reconstruction in the last decade . the most important finding of the present study was that all included articles used limited quantitative measurements to determine functional performance , except the study done by trulsson et al . in the last decade most of the studies included in this review were focusing on the hop tests especially the single - leg hop test and few of these studies looked at a postural stability . regarding the reported outcomes the focus was on the koos and ikdc questionnaires . although the included articles reported the use of several hop tests , fourteen studies used a single - leg hop for distance as the gold standard for measuring functional performance after acl reconstruction because the reliability of this test is high ( icc ranging from 0.86 to 0.95 ) [ 49 , 50 ] . the relative reliability of the single hop for distance test in patients 1 to 2 years following acl reconstruction has previously been reported . by using multiple hop tests , their qualities can be assessed and thereby the opportunity to detect discrepancies in hop performance increases . there is a strong relationship between crossover hop performance and functional outcome correlating significantly to ikdc subjective and koos questionnaire scores . the most reliable and valid of the multitude of hop tests in relation to the aclr patient would appear to be the single hop for distance and the crossover hop tests [ 7 , 50 , 54 ] . the ability of the aclr patient to perform well during hop tests is of paramount importance when judging functional performance . hop testing has frequently been proposed as a practical performance - based outcome measure that reflects the integrated effect of neuromuscular control , strength ( force - generating capacity ) , and confidence in the limb and requires minimal equipment and time to administer . based on a review of the potential use of hop tests as measures of dynamic knee stability , fitzgerald et al . suggested that hopping may be appropriate for use as a predictive tool for identifying patients who may have future problems as a result of knee injury or pathology and as an evaluative tool to reflect change in the patient status in response to treatment . within the published literature , the gold standard is often regarded as having a limb symmetry index ( lsi ) of greater than 85% , indicating that anything less than a 15% deficit in strength between the operated and nonoperated limb is acceptable . a study conducted in has shown that the contralateral ( noninjured ) leg is significantly weaker than matched controls . therefore , this assumption of normality should be viewed with caution , as the period of time during both preoperative and postoperative rehabilitation is likely to have caused atrophy of the noninjured leg . however , using the lsi is debatable because recent studies have shown that an acl injury could lead to a crossover effect in the uninvolved leg resulting in strength and function loss based on biomechanical and neuromuscular changes . to the best of the knowledge of this study 's researchers , there are few published studies that search for postural stability following acl reconstruction . for example , the sebt outcome measure offers a simple , reliable , valid , and low - cost alternative to more sophisticated instrumented methods , to assess dynamic balance ability [ 59 , 60 ] ; unlike force plates or electronically controlled balance platforms , it is an easy and highly portable test that could be employed in a range of clinical environments . the grid required testing for acl deficiency patients , three lines are positioned on the grid ( anterior , medial , and lateral reach distance ) which are labelled according to the direction of excursion relative to the stance leg . whilst previous studies have evaluated intratester reliability , only one study has evaluated between - session reliability of the sebt with normalised scores with icc values ranging from 0.89 to 0.93 . however , only 3 reach distances , anterior , posteromedial , and posterolateral , were evaluated . therefore , further study of between - session reliability of all reach directions is warranted . previous research has suggested that the sebt is reliable and sensitive enough to detect dynamic postural control deficits in patients with an acl - deficient ( acl - d ) limb [ 62 , 63 ] . in these studies , patients who were injured were shown to have lower sebt scores compared to those of their uninjured limb and those of healthy participants . found that patients with acl deficiency showed functional deficits in the anterior , medial , lateral , and posteromedial reach directions . these tests are thought to provide an indication of muscle strength and power , neuromuscular control and confidence [ 64 , 65 ] . additionally , a number of authors have highlighted that a single functional test may not be sensitive enough to detect performance limitations and that at least two functional tests should be used [ 51 , 52 , 55 ] . studies focusing on prevention showed that the risk for acl injuries was reduced when training was done before high quality trials [ 6668 ] . for acl injury screening , ekegren and his colleagues examined dynamic knee valgus during a drop - jump task . the drop jump turned out to be a reliable and valid instrument in observing the dynamic knee valgus . investigated videotaped functional performance tests in acl injured subjects , and they reported that observation is a reliable and valid instrument for assessing knee flexion angles of the one - leg hop for a distance . the single - leg squat ( sls ) test is a cost - effective and simple movement to determine lower extremity alignment in the coronal plane . carried out with a single camera in any setting , this procedure can visibly identify a valgus lower extremity alignment on landing , which is considered to be a potential risk factor for a possible noncontact acl injury . the sls test has been described in a number of studies as a useful clinical measure to identify hip muscle function and dynamic knee control . patient reported instruments are normally related to signs and symptoms experienced by the patient and/or the functional tasks that individuals are able to achieve during their activities of daily living . a commonly used knee outcome instrument is the cincinnati knee scoring scale , and although it has been demonstrated to be an adequate tool to evaluate knee function following acl reconstruction , it also includes manual and instrumented stability testing to assess symptoms and function ; thus it becomes more difficult to separate various aspects of knee function following acl injury . the ikdc , the cincinnati knee scoring scale , and the first version of the lysholm score are assessor reported scores , which have been exposed to be biased when applied to individuals with an acl injury . on the other hand , carlos argued that for those clinicians and researchers considering using only the ikdc as their patient reported outcomes for acl reconstruction , they should include as a minimum the koos subscales that address broader areas of concern , including quality of life and emotional health that are most important to patients following acl reconstruction and are not wholly represented in ikdc . moreover , there is a suggestion that the koos is perhaps more suitable for the assessment of patients in the longer term unlike the ikdc . the koos has shown good validity and demonstrated that it is responsive to acl reconstruction and rehabilitation ; it shows that it is a reliable instrument for patients undergoing acl surgery and rehabilitation . clinicians and researchers looking to use a patient - based score measure of outcomes must consider the specific patient population in which it has been evaluated . using a diagnostic algorithm that measures the anatomic parts of the knee as separate constructs may solve this dilemma , allowing for the measurement of treatment outcomes across patient groups and the selection of the optimal clinical intervention . in general , the papers in this literature review included poorly described sample sizes and whether or not the sample size was based on power calculations . accordingly , the samples recruited may not necessarily have been big enough to identify a difference in outcome following a rehabilitation programme , irrespective of whether or not a difference existed . accordingly , it was not possible to determine whether measurement error influenced the results obtained or whether the experiences or training of the assessors was a variable which may have accounted for any between - study differences . this may modify the interpretation of the values attained for a specific functional performance test , and this was also the reason for the small number of studies included . future studies are required to establish the reliability and validity of existing functional performance tests or explore new , relevant quality measurements of the functional performance tests to be used in patients following acl surgery . this review shows that , following the acl reconstruction , the one - leg hop for distance or a combination of different hops and the limb symmetry index ( lsi ) of functional performance tests was used as a main outcome parameter of several studies . no extensive research has been carried out over the past 10 years to measure the control stability of patients following acl reconstruction . because previous studies discuss additional important parameters , a more extensive battery of tests is suggested to measure both the quantitative and qualitative aspects of functional performance after the acl reconstruction .

one important goal of the chromosome - centric human proteome project ( c - hpp ) , an international consortium , is to completely map the human proteome by identification of proteins in selected tissues and cells . as an articulated goal , the c - hpp is also determined to capture biological features of gene variation , gene regulation , and protein expression mapped by chromosome localization . consequently , all c - hpp projects are generating and reporting protein data in a format that is aligned with the dna sequence of individual chromosomes and with the output of transcriptome data ( rna sequencing ) . in addition to sequential data derived from the most frequent proteoforms ( consensus or canonical sequences ) , it is desirable to characterize additional major proteoforms , such as alternative splicing transcript ( ast ) , single amino acid variants ( sav ) , and post - translational modifications ( ptms ) . a complementary hpp activity , the biology / disease - driven hpp ( b / d - hpp ) , is focused on generation of knowledge from studies of cellular mechanisms and biochemical processes , analyzing proteomes associated with human diseases . the results are expected to facilitate routine determinations of processes and disease relevant proteins in life science research . at present , human protein sequence data is collected at a rapid pace , predominantly due to the technological advances in mass spectrometry ( ms ) . however , roughly 10% is still missing , due to the lack of quality observations of certain proteins , incorrect gene annotation , very low abundance or absence of expression in most tissues , or unfavorable structure ( or cleavage sites ) for bottom - up ms studies . on the other hand , two recent publications have set new milestones in providing the most complete draft of the human proteome to date . according to the proteomics db ( http://www.proteomicsdb.org ) , the current state of the chromosome 19 is at 96.4% completion ( 1352 genes and 1304 proteins ) and includes details about a high number of asts ( isoforms ) . nevertheless , if all proteoforms with different sequences that have biological functions are considered , then the exact size of the human proteome is still unknown today and may reach extremely high numbers , up to several million . because the hpp s directive is to identify at least one ast and one sav of each consensus proteins as well as three major ptms ( phosphorylation , glycosylation and acetylation ) , the number of proteoform entries in the complete human proteome is estimated in the range of 100 000 to 1 000 000 . the latest downloadable version of the nextprot database ( http://www.nextprot.org ) includes 20 055 protein entries ( released on 19 september 2014 ) . there are 1430 genes and 1426 protein entries reported for chromosome 19 , which are identified at the protein level ( 1129 entries ) , transcript level ( 248 entries ) , uncertain ( 36 entries ) , homology ( 10 entries ) , and predicted ( 7 entries ) . although this database fasta file does not include any information about savs ( nor do the uniprot databases ) , references to mutant proteoforms are listed on the nextprot web site when certain proteins are selected . however , the level of identification for these savs is not indicated otherwise . genomic databases are typically more detailed , providing a list of missense single nucleotide variations ( missense snvs ) of each human gene . databases such as 1000 genomes ( http://www.1000genomes.org ) provide population - based frequency information as well . other genomic databases such as the catalogue of somatic mutations in cancer ( cosmic at http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/ ) have accumulated gene expression variants and revealed disease relation . genome investigations have identified a large number of gene mutations , including missense snvs that are currently used for risk analysis of cancer , for example , brca1 and brca2 . however , it is important to underline that not the genes but the proteins have biological function and are the working units of cell machinery . consequently , their expression patterns in both qualitative and quantitative aspects should be characterized when the altered biology of diseases are in the focus of study . despite this fact , the majority of protein identifications are based upon ms / ms data from shotgun proteomics experiments searched against protein databases such as uniprotkb and nextprot , which are typically restricted to proteoforms with consensus and ast sequences only . thus , direct searches of savs can not be performed with tandem spectra and such data is seldom reported , despite the fact that savs may play important role in disease biology . some search engines are constructed by use of various approaches and algorithms in order to provide tools to recognize savs . as an example , peaks ( bioinformatics solutions inc . , waterloo , on , canada ) offers an algorithm ( spider ) that can utilize the match of peptide spectrum matches ( psms ) with replaced amino acids . others , like the freely available x!tandem ( http://www.thegpm.org ) employs another strategy that systematically changes each residue in a peptide for all other possible amino acids and score the mutated peptide ( and all potential modifications ) against all of the available ms / ms spectra . therefore , bioinformatic tools do exist that aid in the process of identifying savs in shotgun proteomics data , and these tools can prove to be valuable in the characterization of savs associated with a particular disease . glioblastomas are among the deadliest of cancers ; fewer than 10% of patients survive five years after diagnosis . even with standard - of - care treatments , tumors nearly always recur . recurrence is at least in part due the existence of glioma stem cells ( gscs ) , which are resistant to radiation and chemotherapy . we have acquired comprehensive , quantitative data sets from thirty - six gsc lines , including gene activity and protein expression . we have previously examined patterns of global as well as chromosome-19-specific protein and gene expression in a subset of the 36 gsc lines . our hypothesis is that gscs , as a rare type of diseased cell type , harbor protein savs due to germline or somatic mutations . those proteins may normally have cell protective properties , but proteins with amino acid substitutions have the potential to be transformed into promoters of genetic instability or invasivity , as well as ineffective regulators of epigenetic or metabolic control . through the use of a customized database and bioinformatic tools , we provide the first comparative data of chromosome 19 snp products on 36 glioma stem cell lines and compelling evidence for the expression of a sav in branched - chain aminotransferase 2 ( bcat2 ) , a protein encoded by chromosome 19 , in six of the gsc lines . we have devised a systematic workflow to address the second level of protein expression complexity ( represented by the savs ) and have applied this workflow in the identification of missense snv products at the protein level in glioma samples based on high resolution ms / ms data . we used a custom protein database to widen the search window for mutant proteoforms . we have identified and further verified novel mutant proteoforms that might be strongly associated with glioma . isolation of gscs from patient tumors was performed as previously described in accordance with the institutional review board of the university of texas m.d . downstream proteomic analyses were performed on identical cell culture batches in order to reduce the influence of batch variance in the comparative assays . cell lysates from 2 10 gscs were reduced , alkylated , and analyzed in triplicate by lc ms / ms on an orbitrap elite equipped with an easy nanolc 1000 pump ( thermo fisher scientific , waltham , ma ) as described in a previous publication . briefly , peptide mixtures were separated on a c18 column ( proteopep ii , new objective , 10 cm 75 m ) using a 240 min gradient ( solvent a , 0.1% formic acid in water ; solvent b , 0.1% formic acid in acetonitrile ) . data were acquired using high - resolution data - driven analysis ( dda ) , with the survey scan ( ms ) acquired in the orbitrap at 60 000 resolution ( at m / z 400 ) in profile mode . the survey scan was followed by ten hcd ms / ms spectra , acquired in centroid mode at 15 000 resolution in the orbitrap . for database searching the technical replicates were combined and searched against a combination of the uniprotkb / swissprot - human database ( 2014_06 version , 40 548 protein entries ) with all known chromosome 19 sav sequences ( 132 264 ) together with 115 sequences of the common repository of adventitious proteins ( crap ) contaminant database ( http://www.thegpm.org/crap/index.html ) . searches were performed using peaks 7.0 ( bioinformatics solutions ) with 10 ppm parent mass error tolerance and 0.025 fragment mass tolerance , allowing for a maximum of two missed cleavages and one nonspecific cleavage . carbamidomethyl cysteine was set as a fixed modification , and oxidation ( m ) and phosphorylation ( sty ) were set as variable modifications . peptides assigned as savs with a 10log p score > 30 were selected for further validation . homology searching was performed using the blast utility ( www.uniprot.org ) against the uniprot - human database in order to confirm that each peptide sequence was unique . for those peptides that passed these initial filters , manual verification of ms / ms spectral assignments of sav peptides was performed by comparing m / z values for ions observed in the ms / ms spectrum with a theoretical m / z list generated using the ms - product utility on the protein prospector web site ( http://prospector.ucsf.edu ) . for optimization of the assay , isotopically labeled peptides were mixed and diluted with 5% acetonitrile ( acn ) at a concentration of 325 pmol/l for each synthetic peptide . ms / ms using a tsq vantage triple quadrupole mass spectrometer equipped with an easy n - lc ii pump ( thermo scientific , waltham , ma ) . samples ( 2 l ) were injected onto an easy c18-a1 precolumn ( thermo scientific , waltham , ma ) , and following online desalting and trapping at a pressure of 280 bar , the peptides were separated on a 75 m 150 mm fused silica column packed with reprosil c18 ( 3 m , 120 from dr . separations were performed in a 55 min linear gradient from 5 to 40% acn containing 0.1% formic acid at the flow rate of 300 nl / min . the ms analysis was conducted in positive ion mode at 1750 v applied spray voltage . the transfer capillary temperature was 270 c and tuned s - lens value was used . selected reaction monitoring ( srm ) transitions were acquired in q1 and q3 operated at unit resolution ( 0.7 fwhm ) , the collision gas pressure in q2 was set to 1.2 mtorr . scheduled method was used for data acquisition with 4 min time windows and the cycle time was set to 1.5 s , whereas the maximum number of consecutive transitions was 50 . the srm assay optimization was done with the aid of skyline v2.5.0.6079 software ( maccoss lab ) . primarily , high numbers of transitions , including b- and y - ion series , were chosen for each peptide at both 2 + and 3 + charge states . the five transitions , which produced the most abundant signals without observed interferences in the glioma samples , were selected for further analysis and utilized for unambiguous identification . for the sample analysis , the same chromatographic conditions were used as described above for the assay development . identical srm parameters were used for the heavy and native forms of each peptide . all raw data generated on the triple quadrupole mass spectrometer were imported to skyline for data analysis . paired - end whole transcriptome sequencing was performed on the illumina hiseq platform after random priming and rrna reduction . transcript reads were aligned to human reference transcriptome ( ensembl version 64 ) using prada . downstream data analyses including variant calling were performed using burroughs - wheeler alignment , samtools , and genome analysis toolkit . more details on transcriptomic data acquisition and analysis are available in lichti et al . the bcat2 sav was modeled using a combination of two crystal structures : the substrate , l - isoleucine covalently bound to the cofactor pyridoxamine phosphate ( pmp ) , bound form of the enzyme ( pdb code : 1kt8 ) , and the pmp bound form ( pdb code : 1kta ) . both were downloaded from www.pdb.org and prepared using the visual molecular dynamics ( vmd ) software . as 1kt8 is missing residues near the mutation site , it was used as a reference structure onto which the coordinates of 1kta were superimposed in order to include the coordinates of residues 175 to 191 . residue 186 ( please , note that our residue numbering scheme in this paper follows the recommendations of human genome variation society and thus may differ by six from other authors ) was then mutated from threonine to arginine , and hydrogens were added . the resulting coordinates were stored and from them an additional starting structure was generated in which the arg186 backbone dihedral was rotated from + 132 to 60 by rotating parts of both arg186 and pro187 , while was held constant at 80 . after rotation , residues 185188 were minimized with the remainder of the protein fixed . both these final coordinates and the original unrotated structure were then minimized , holding all atoms of the protein fixed except for those in residues 175191 . the final coordinates of each were stored and used as the basis for side chain rotamer generation . for both the original and rotated backbone structures , the side chain angles 1 and 2 of arg186 were subsequently rotated in six increments of 60 , yielding a total of 72 structures . from the original structure , the side chain was rotated about the 1 bond without altering the original 2 torsion , generating an additional six structures . two independent minimizations were performed on each of the 78 structures : one in which the entire system was minimized , and one in which only residues 177188 were minimized while all else was held fixed . the final coordinates of each minimization were used for electrostatic free energy calculations and structure analysis . electrostatic free energy calculations were conducted on the first monomer ( chain a ) of each of the 156 bcat2 structures . the pdb 2pqr software was used to generate pqr files for each structure . here , the charmm27 parameter set was used to obtain partial atomic charges and atomic radii , and a ph of 7.0 was used in assigning protonation states for titratable residues . the total electrostatic free energy of the system was calculated using the apbs software package . the structures of each minimization set were ranked by their corresponding energies and the best ten structures of both sets were analyzed using vmd . we addressed the determination of mutant proteoforms in biological samples , a high priority within the c - hpp . a novel systematic approach was developed that is based on searching shotgun proteome tandem spectra , collected via data dependent analysis , against a customized protein sequence database containing chromosome 19 savs . the new database used for identification of mutant proteoforms was compiled with all human consensus proteins together with a new set of missense snv - derived sequences of chromosome 19 . to improve search quality , the frequently used crap contaminant protein database was also included , resulting in a database of 172 927 protein sequences in total . when search space is confined to short sequences derived from only one chromosome , a problem is expected that is similar to one observed when a search is performed against a database for a partially sequenced organism . in a standard target - decoy approach , a number of high quality spectra will be wrongly matched to decoy sequences simply due to the absence of correct peptide sequences in the target database . this makes adequate significance estimation , often represented in proteomics as global false discovery rate , rather challenging . because many proteins exist in multiple isoforms , for example , structural proteins such as myosin or tubulin , there is a high risk that a spectrum may be also erroneously assigned to a sav - containing peptide . those issues can be addressed by creating a custom decoy database , a task that is not trivial in the case of short , low complexity sequences . alternatively , protein sequences from the other homologous species , or in our case the rest of the canonical proteome from the same species , could be appended to a database of interest , adding more possibilities for the correct peptide - spectrum matches in a target data set and , therefore , enabling target - decoy approach implemented by standard search engines . because protein databases for shotgun proteomic searches do not commonly include sav sequences , and different search engines may perform differently with the same database ; three search engines ( sequest , peaks , and mascot ) were employed and the corresponding search results were compared . triplicate data files for each cell line were used as a single input for database searching , initially performed with nearly identical search parameters . an initial search and validation on a subset of the gscs revealed several savs . manual verification of spectra containing savs led to confirmation of a t186r substitution of branched - chain aminotransferase 2 ( bcat2 ) ( see figure 1 ) , a known natural variant of the protein , in gsc28 . this finding was further validated at the transcript level and by srm ( see sections 3.2 and 3.3 , respectively ) . hcd - ms / ms spectrum of pvlignepslgvsqpr , supporting assignment of the p.thr186arg proteoform of bcat2 . a complete list of theoretical and observed m / z values for ions observed in this spectrum can be found in supporting information table 4 . due to the high confidence in this finding , identification of the sav - containing peptide of bcat2 ( pvlignepslgvsqpr ) in gsc 28 was used as a positive control for all database searches . according to the snap database of gpmdb , this tryptic peptide of bcat2 is less frequently observed compared with its longer , miscleaved forms ( http://snap.thegpm.org/%7e/dblist_protein_mut/label=ensp00000322991 ) . however , this may be a biased result of search algorithms that consider such peptides as false . interestingly , peaks db was the only search engine that successfully identified the peptide containing bcat2 p.thr186arg , with a 10log p score of 74 . neither sequest nor mascot identified this peptide , despite the high confidence of the assignment in peaks . our initial thought was that both failed to identify the peptide due to trypsin cleavage specificity assigned in the search parameters , as the n - terminus of the sav - containing peptide arises from cleavage between arginine and proline . therefore , we adjusted the trypsin specificity in mascot to allow for cleavage n - terminal to proline and repeated the search using gsc 28 as a positive control . because mascot again failed to identify the peptide containing bcat2 p.thr186arg , we focused on peaks db to identify savs in the remaining cell lines ( see table 1 for a verified list and supporting information table 1 for a complete list of sav peptides identified by peaks ) . annotated hcd ms / ms spectra and the corresponding ion tables can be found in supporting information figure 1 . conflict in dbsnp and ensembl databases , which indicates this mutation q h. for those peptides that were confirmed to be truly unique by homology searching , manual verification of spectra was performed in order to ensure that the assigned savs were correct . because our data were acquired using high resolution ms in an orbitrap , mass error for the precursor ions was used as an initial filter of quality . because the mass errors for peptide assignments in each lc ms / ms file fall into a normal distribution , and 95% of correct assignments fall within two standard deviations of the mean error , we removed peptides whose mass error was dramatically higher than for other peptides within the same analytical run . in a similar manner , high mass accuracy ms / ms data facilitates spectral verification . leucine and isoleucine are indistinguishable from one another by exact mass and chemical deamidation can convert glutamine and asparagine to glutamic acid and aspartic acid , respectively . furthermore , the carbamidomethyl group ( 57.02146 ) has the same exact mass as a glycine residue . therefore , species formed due to overalkylation ( addition to nucleophilic amino acid side chains or the peptide n - terminus ) can be assigned incorrectly as being due to savs . therefore , it is often critical to have orthogonal validation of savs at the transcript level . ms / ms is presented in table 1 . a further list of sav - containing peptides that require additional transcript level validation is provided in supporting information table 2 . each identified peptide containing , we evaluated the individual reads from the transcriptome sequencing to quantitate the frequency of c ( acg coding thr ) and g ( agg coding arg ) alleles . the c g variant occurs at position 613 in the reference mrna sequence of bcat2 nm_001190 ( rs11548193 in dbsnp ) . a total of 8 gsc lines contained reads of > 50% with c613 g variant , including gscs 28 , 274 , 711 , 275 , 17 , 280 , 289 , and 293 ( data not shown ) . the successful identification of sav proteoforms in gscs was further validated by srm . selected unique peptide sequences with a single mutation were synthesized and srm assays were developed using multiple transitions for each peptide ( supporting information table 3 ) . a subset of ten gsc samples ( gscs 28 , 627 , 20 , 112 , 129 , 522 , 275 , 711 , 274 , and 72 ) was tested for four newly identified mutant proteoforms . the primary target of srm confirmation was the bcat2 p.thr186arg mutation that was unambiguously verified in samples gsc 28 , gsc 711 , and gsc 275 by perfectly matching transitions and retention times of heavy labeled internal standard and native peptides ( see figure 2a ) . ( a ) bcat2 p.thr186arg was confirmed by matching heavy - isotope - labeled synthetic peptide ( blue ) with the endogenous pvlignepslgvsqpr ( red ) , whereas ( b ) the endogenous signals of elongation factor 2 p.ile665leu and endophilin - a2 p.leu95ile could not agree with the internal standards . the specificity of srm assays was utilized in investigation of leu ile and ile leu mutations that were initially identified in database search despite of the isobaric nature of these savs . the difference in retention times observed in reversed phase chromatography due to the lower hydrophobicity of peptides containing isoleucine provided a utility . a sav of elongation factor 2 ( p13639 - 1 p.ile665leu ) was expected with longer retention time but the endogenous signal with identical transitions eluted earlier than the synthetic isotope labeled standard . a reversed case was also observed when an endophilin - a2 mutation ( q99961 - 2 p.leu95ile ) was investigated ( see figure 2b ) . besides their validation power , srm assays can be further utilized in quantitative analysis of mutant allele expressions in biological samples . this information is necessary in the case of heterozygous samples , in particular when the expression of the mutant allele has a strong concordance with biological activity of cells . the p.thr186arg mutation on bcat2 , confirmed at the rna level to be in eight of the 36 gsc lines , is located along a flexible loop in close proximity to the cxxc center ( figure 3 ) . to study the possible effects of this mutation , we generated 78 p.thr186arg bcat2 variant structures by rotating the arginine side chain about the 1 and 2 and scanning the backbond . the structures that where highest in electrostatic free energy or contained severe violations of the chemical constrains were removed from the data set . of the structures lowest in electrostatic free energy , which satisfied the packing and stereochemical constraints in minimization , four were grouped within a few kilocalories per meters , whereas the rest were separated by over 100 kcal / m in electrostatic free energy and so were not considered for further analysis . best four bcat2 p.thr186arg structures are superimposed with arg186 , glu229 , and the cxxc ( cys321 and cys324 ) highlighted . among rotamers with residues 177188 minimized , the four overlaid here were hundreds of kcal / m lower than the average of all rotamers . the arginine side chain is found in a salt bridge with glu229 and toward the inside of the flexible arm , where the arginine side chain has the most stable hydrogen bonding . these lower energy positions of the arginine side chain all greatly change the electrostatic environment as compared with the thr186 wild type , and this may influence the cxxc oxidation reaction that regulates enzyme activity . as seen by the four lowest energy structures ( figure 3 ) , the arg186 side chain gravitates toward the carboxylate group of glu229 , as well as inward toward the cxxc site , both of which provide stability through hydrogen bonding pairs . compared with the thr186 wild type , the arginine side chain significantly changes the electrostatic environment near the cxxc , potentially shifting the pka of the cysteine residues . the electrostatically induced pka shift could influence the cysteine protonation state and therefore the equilibrium of the oxidation reaction ( figure 4 ) , consequently altering enzyme activity . although direct interaction , for example , through hydrogen bonding , is not sterically forbidden , it is also not required . the electrostatic shift of the intrinsic pka of the cysteine would be sufficient to explain a change in activity . it has been demonstrated that the cxxc center ( cys321 and cys324 ) contributes to enzyme activity through reversible disulfide bond formation that prevents substrate from correctly orienting with the pyridoxal phosphate ( plp ) cofactor . reaction mechanism for bcat2 , which catalyzes the transfer of an amino group from leucine , isoleucine , and valine to -ketoglutarate to form glutamate . we demonstrated the utility of our custom protein database in the identification of chromosome 19 savs in gscs . through a combination of homology searching and manual verification of spectra identified by peaks db one of these savs , bcat2 p.thr186arg , was validated orthogonally in multiple cell lines by srm and by rna - seq future studies will be directed toward verification of the remainder of these savs and a study of the biological implications of these savs . the identification and characterization of savs in glioma has already yielded the identity proteins that will be of further interest to study as potential modulators of gliomagenesis , contributors to glioma pathology , and resistance to standard of care treatments . we expect that our methods are readily transferrable to other cancer cells and tissues as well . to improve visibility of savs , we strongly suggest extending the registry of protein sequences in nextprot with information about the mutant proteoforms pointing to identification levels as well as expression sites .

novel proteoforms with single amino acid variations represent proteins that often have altered biological functions but are less explored in the human proteome . we have developed an approach , searching high quality shotgun proteomic data against an extended protein database , to identify expressed mutant proteoforms in glioma stem cell ( gsc ) lines . the systematic search of ms / ms spectra using peaks 7.0 as the search engine has recognized 17 chromosome 19 proteins in gscs with altered amino acid sequences . the results were further verified by manual spectral examination , validating 19 proteoforms . one of the novel findings , a mutant form of branched - chain aminotransferase 2 ( p.thr186arg ) , was verified at the transcript level and by targeted proteomics in several glioma stem cell lines . the structure of this proteoform was examined by molecular modeling in order to estimate conformational changes due to mutation that might lead to functional modifications potentially linked to glioma . based on our initial findings , we believe that our approach presented could contribute to construct a more complete map of the human functional proteome .

Introduction Experimental Section Results and Discussion Conclusions

one important goal of the chromosome - centric human proteome project ( c - hpp ) , an international consortium , is to completely map the human proteome by identification of proteins in selected tissues and cells . in addition to sequential data derived from the most frequent proteoforms ( consensus or canonical sequences ) , it is desirable to characterize additional major proteoforms , such as alternative splicing transcript ( ast ) , single amino acid variants ( sav ) , and post - translational modifications ( ptms ) . the results are expected to facilitate routine determinations of processes and disease relevant proteins in life science research . at present , human protein sequence data is collected at a rapid pace , predominantly due to the technological advances in mass spectrometry ( ms ) . however , roughly 10% is still missing , due to the lack of quality observations of certain proteins , incorrect gene annotation , very low abundance or absence of expression in most tissues , or unfavorable structure ( or cleavage sites ) for bottom - up ms studies . on the other hand , two recent publications have set new milestones in providing the most complete draft of the human proteome to date . according to the proteomics db ( http://www.proteomicsdb.org ) , the current state of the chromosome 19 is at 96.4% completion ( 1352 genes and 1304 proteins ) and includes details about a high number of asts ( isoforms ) . nevertheless , if all proteoforms with different sequences that have biological functions are considered , then the exact size of the human proteome is still unknown today and may reach extremely high numbers , up to several million . because the hpp s directive is to identify at least one ast and one sav of each consensus proteins as well as three major ptms ( phosphorylation , glycosylation and acetylation ) , the number of proteoform entries in the complete human proteome is estimated in the range of 100 000 to 1 000 000 . there are 1430 genes and 1426 protein entries reported for chromosome 19 , which are identified at the protein level ( 1129 entries ) , transcript level ( 248 entries ) , uncertain ( 36 entries ) , homology ( 10 entries ) , and predicted ( 7 entries ) . although this database fasta file does not include any information about savs ( nor do the uniprot databases ) , references to mutant proteoforms are listed on the nextprot web site when certain proteins are selected . other genomic databases such as the catalogue of somatic mutations in cancer ( cosmic at http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/ ) have accumulated gene expression variants and revealed disease relation . despite this fact , the majority of protein identifications are based upon ms / ms data from shotgun proteomics experiments searched against protein databases such as uniprotkb and nextprot , which are typically restricted to proteoforms with consensus and ast sequences only . some search engines are constructed by use of various approaches and algorithms in order to provide tools to recognize savs . others , like the freely available x!tandem ( http://www.thegpm.org ) employs another strategy that systematically changes each residue in a peptide for all other possible amino acids and score the mutated peptide ( and all potential modifications ) against all of the available ms / ms spectra . therefore , bioinformatic tools do exist that aid in the process of identifying savs in shotgun proteomics data , and these tools can prove to be valuable in the characterization of savs associated with a particular disease . recurrence is at least in part due the existence of glioma stem cells ( gscs ) , which are resistant to radiation and chemotherapy . we have acquired comprehensive , quantitative data sets from thirty - six gsc lines , including gene activity and protein expression . we have previously examined patterns of global as well as chromosome-19-specific protein and gene expression in a subset of the 36 gsc lines . our hypothesis is that gscs , as a rare type of diseased cell type , harbor protein savs due to germline or somatic mutations . those proteins may normally have cell protective properties , but proteins with amino acid substitutions have the potential to be transformed into promoters of genetic instability or invasivity , as well as ineffective regulators of epigenetic or metabolic control . through the use of a customized database and bioinformatic tools , we provide the first comparative data of chromosome 19 snp products on 36 glioma stem cell lines and compelling evidence for the expression of a sav in branched - chain aminotransferase 2 ( bcat2 ) , a protein encoded by chromosome 19 , in six of the gsc lines . we have devised a systematic workflow to address the second level of protein expression complexity ( represented by the savs ) and have applied this workflow in the identification of missense snv products at the protein level in glioma samples based on high resolution ms / ms data . we used a custom protein database to widen the search window for mutant proteoforms . we have identified and further verified novel mutant proteoforms that might be strongly associated with glioma . isolation of gscs from patient tumors was performed as previously described in accordance with the institutional review board of the university of texas m.d . downstream proteomic analyses were performed on identical cell culture batches in order to reduce the influence of batch variance in the comparative assays . cell lysates from 2 10 gscs were reduced , alkylated , and analyzed in triplicate by lc ms / ms on an orbitrap elite equipped with an easy nanolc 1000 pump ( thermo fisher scientific , waltham , ma ) as described in a previous publication . data were acquired using high - resolution data - driven analysis ( dda ) , with the survey scan ( ms ) acquired in the orbitrap at 60 000 resolution ( at m / z 400 ) in profile mode . the survey scan was followed by ten hcd ms / ms spectra , acquired in centroid mode at 15 000 resolution in the orbitrap . for database searching the technical replicates were combined and searched against a combination of the uniprotkb / swissprot - human database ( 2014_06 version , 40 548 protein entries ) with all known chromosome 19 sav sequences ( 132 264 ) together with 115 sequences of the common repository of adventitious proteins ( crap ) contaminant database ( http://www.thegpm.org/crap/index.html ) . searches were performed using peaks 7.0 ( bioinformatics solutions ) with 10 ppm parent mass error tolerance and 0.025 fragment mass tolerance , allowing for a maximum of two missed cleavages and one nonspecific cleavage . homology searching was performed using the blast utility ( www.uniprot.org ) against the uniprot - human database in order to confirm that each peptide sequence was unique . for those peptides that passed these initial filters , manual verification of ms / ms spectral assignments of sav peptides was performed by comparing m / z values for ions observed in the ms / ms spectrum with a theoretical m / z list generated using the ms - product utility on the protein prospector web site ( http://prospector.ucsf.edu ) . for optimization of the assay , isotopically labeled peptides were mixed and diluted with 5% acetonitrile ( acn ) at a concentration of 325 pmol/l for each synthetic peptide . ms / ms using a tsq vantage triple quadrupole mass spectrometer equipped with an easy n - lc ii pump ( thermo scientific , waltham , ma ) . separations were performed in a 55 min linear gradient from 5 to 40% acn containing 0.1% formic acid at the flow rate of 300 nl / min . the bcat2 sav was modeled using a combination of two crystal structures : the substrate , l - isoleucine covalently bound to the cofactor pyridoxamine phosphate ( pmp ) , bound form of the enzyme ( pdb code : 1kt8 ) , and the pmp bound form ( pdb code : 1kta ) . as 1kt8 is missing residues near the mutation site , it was used as a reference structure onto which the coordinates of 1kta were superimposed in order to include the coordinates of residues 175 to 191 . after rotation , residues 185188 were minimized with the remainder of the protein fixed . here , the charmm27 parameter set was used to obtain partial atomic charges and atomic radii , and a ph of 7.0 was used in assigning protonation states for titratable residues . the total electrostatic free energy of the system was calculated using the apbs software package . we addressed the determination of mutant proteoforms in biological samples , a high priority within the c - hpp . a novel systematic approach was developed that is based on searching shotgun proteome tandem spectra , collected via data dependent analysis , against a customized protein sequence database containing chromosome 19 savs . the new database used for identification of mutant proteoforms was compiled with all human consensus proteins together with a new set of missense snv - derived sequences of chromosome 19 . to improve search quality , the frequently used crap contaminant protein database was also included , resulting in a database of 172 927 protein sequences in total . when search space is confined to short sequences derived from only one chromosome , a problem is expected that is similar to one observed when a search is performed against a database for a partially sequenced organism . in a standard target - decoy approach , a number of high quality spectra will be wrongly matched to decoy sequences simply due to the absence of correct peptide sequences in the target database . those issues can be addressed by creating a custom decoy database , a task that is not trivial in the case of short , low complexity sequences . alternatively , protein sequences from the other homologous species , or in our case the rest of the canonical proteome from the same species , could be appended to a database of interest , adding more possibilities for the correct peptide - spectrum matches in a target data set and , therefore , enabling target - decoy approach implemented by standard search engines . because protein databases for shotgun proteomic searches do not commonly include sav sequences , and different search engines may perform differently with the same database ; three search engines ( sequest , peaks , and mascot ) were employed and the corresponding search results were compared . manual verification of spectra containing savs led to confirmation of a t186r substitution of branched - chain aminotransferase 2 ( bcat2 ) ( see figure 1 ) , a known natural variant of the protein , in gsc28 . this finding was further validated at the transcript level and by srm ( see sections 3.2 and 3.3 , respectively ) . hcd - ms / ms spectrum of pvlignepslgvsqpr , supporting assignment of the p.thr186arg proteoform of bcat2 . due to the high confidence in this finding , identification of the sav - containing peptide of bcat2 ( pvlignepslgvsqpr ) in gsc 28 was used as a positive control for all database searches . interestingly , peaks db was the only search engine that successfully identified the peptide containing bcat2 p.thr186arg , with a 10log p score of 74 . neither sequest nor mascot identified this peptide , despite the high confidence of the assignment in peaks . our initial thought was that both failed to identify the peptide due to trypsin cleavage specificity assigned in the search parameters , as the n - terminus of the sav - containing peptide arises from cleavage between arginine and proline . therefore , we adjusted the trypsin specificity in mascot to allow for cleavage n - terminal to proline and repeated the search using gsc 28 as a positive control . because mascot again failed to identify the peptide containing bcat2 p.thr186arg , we focused on peaks db to identify savs in the remaining cell lines ( see table 1 for a verified list and supporting information table 1 for a complete list of sav peptides identified by peaks ) . annotated hcd ms / ms spectra and the corresponding ion tables can be found in supporting information figure 1 . conflict in dbsnp and ensembl databases , which indicates this mutation q h. for those peptides that were confirmed to be truly unique by homology searching , manual verification of spectra was performed in order to ensure that the assigned savs were correct . because the mass errors for peptide assignments in each lc ms / ms file fall into a normal distribution , and 95% of correct assignments fall within two standard deviations of the mean error , we removed peptides whose mass error was dramatically higher than for other peptides within the same analytical run . in a similar manner , high mass accuracy ms / ms data facilitates spectral verification . therefore , species formed due to overalkylation ( addition to nucleophilic amino acid side chains or the peptide n - terminus ) can be assigned incorrectly as being due to savs . therefore , it is often critical to have orthogonal validation of savs at the transcript level . ms / ms is presented in table 1 . a further list of sav - containing peptides that require additional transcript level validation is provided in supporting information table 2 . each identified peptide containing , we evaluated the individual reads from the transcriptome sequencing to quantitate the frequency of c ( acg coding thr ) and g ( agg coding arg ) alleles . the c g variant occurs at position 613 in the reference mrna sequence of bcat2 nm_001190 ( rs11548193 in dbsnp ) . the successful identification of sav proteoforms in gscs was further validated by srm . a subset of ten gsc samples ( gscs 28 , 627 , 20 , 112 , 129 , 522 , 275 , 711 , 274 , and 72 ) was tested for four newly identified mutant proteoforms . the primary target of srm confirmation was the bcat2 p.thr186arg mutation that was unambiguously verified in samples gsc 28 , gsc 711 , and gsc 275 by perfectly matching transitions and retention times of heavy labeled internal standard and native peptides ( see figure 2a ) . ( a ) bcat2 p.thr186arg was confirmed by matching heavy - isotope - labeled synthetic peptide ( blue ) with the endogenous pvlignepslgvsqpr ( red ) , whereas ( b ) the endogenous signals of elongation factor 2 p.ile665leu and endophilin - a2 p.leu95ile could not agree with the internal standards . this information is necessary in the case of heterozygous samples , in particular when the expression of the mutant allele has a strong concordance with biological activity of cells . the p.thr186arg mutation on bcat2 , confirmed at the rna level to be in eight of the 36 gsc lines , is located along a flexible loop in close proximity to the cxxc center ( figure 3 ) . to study the possible effects of this mutation , we generated 78 p.thr186arg bcat2 variant structures by rotating the arginine side chain about the 1 and 2 and scanning the backbond . of the structures lowest in electrostatic free energy , which satisfied the packing and stereochemical constraints in minimization , four were grouped within a few kilocalories per meters , whereas the rest were separated by over 100 kcal / m in electrostatic free energy and so were not considered for further analysis . best four bcat2 p.thr186arg structures are superimposed with arg186 , glu229 , and the cxxc ( cys321 and cys324 ) highlighted . these lower energy positions of the arginine side chain all greatly change the electrostatic environment as compared with the thr186 wild type , and this may influence the cxxc oxidation reaction that regulates enzyme activity . as seen by the four lowest energy structures ( figure 3 ) , the arg186 side chain gravitates toward the carboxylate group of glu229 , as well as inward toward the cxxc site , both of which provide stability through hydrogen bonding pairs . the electrostatically induced pka shift could influence the cysteine protonation state and therefore the equilibrium of the oxidation reaction ( figure 4 ) , consequently altering enzyme activity . we demonstrated the utility of our custom protein database in the identification of chromosome 19 savs in gscs . through a combination of homology searching and manual verification of spectra identified by peaks db one of these savs , bcat2 p.thr186arg , was validated orthogonally in multiple cell lines by srm and by rna - seq future studies will be directed toward verification of the remainder of these savs and a study of the biological implications of these savs . the identification and characterization of savs in glioma has already yielded the identity proteins that will be of further interest to study as potential modulators of gliomagenesis , contributors to glioma pathology , and resistance to standard of care treatments . we expect that our methods are readily transferrable to other cancer cells and tissues as well . to improve visibility of savs , we strongly suggest extending the registry of protein sequences in nextprot with information about the mutant proteoforms pointing to identification levels as well as expression sites .

one important goal of the chromosome - centric human proteome project ( c - hpp ) , an international consortium , is to completely map the human proteome by identification of proteins in selected tissues and cells . as an articulated goal , the c - hpp is also determined to capture biological features of gene variation , gene regulation , and protein expression mapped by chromosome localization . consequently , all c - hpp projects are generating and reporting protein data in a format that is aligned with the dna sequence of individual chromosomes and with the output of transcriptome data ( rna sequencing ) . in addition to sequential data derived from the most frequent proteoforms ( consensus or canonical sequences ) , it is desirable to characterize additional major proteoforms , such as alternative splicing transcript ( ast ) , single amino acid variants ( sav ) , and post - translational modifications ( ptms ) . a complementary hpp activity , the biology / disease - driven hpp ( b / d - hpp ) , is focused on generation of knowledge from studies of cellular mechanisms and biochemical processes , analyzing proteomes associated with human diseases . the results are expected to facilitate routine determinations of processes and disease relevant proteins in life science research . at present , human protein sequence data is collected at a rapid pace , predominantly due to the technological advances in mass spectrometry ( ms ) . however , roughly 10% is still missing , due to the lack of quality observations of certain proteins , incorrect gene annotation , very low abundance or absence of expression in most tissues , or unfavorable structure ( or cleavage sites ) for bottom - up ms studies . on the other hand , two recent publications have set new milestones in providing the most complete draft of the human proteome to date . according to the proteomics db ( http://www.proteomicsdb.org ) , the current state of the chromosome 19 is at 96.4% completion ( 1352 genes and 1304 proteins ) and includes details about a high number of asts ( isoforms ) . nevertheless , if all proteoforms with different sequences that have biological functions are considered , then the exact size of the human proteome is still unknown today and may reach extremely high numbers , up to several million . because the hpp s directive is to identify at least one ast and one sav of each consensus proteins as well as three major ptms ( phosphorylation , glycosylation and acetylation ) , the number of proteoform entries in the complete human proteome is estimated in the range of 100 000 to 1 000 000 . there are 1430 genes and 1426 protein entries reported for chromosome 19 , which are identified at the protein level ( 1129 entries ) , transcript level ( 248 entries ) , uncertain ( 36 entries ) , homology ( 10 entries ) , and predicted ( 7 entries ) . other genomic databases such as the catalogue of somatic mutations in cancer ( cosmic at http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/ ) have accumulated gene expression variants and revealed disease relation . genome investigations have identified a large number of gene mutations , including missense snvs that are currently used for risk analysis of cancer , for example , brca1 and brca2 . however , it is important to underline that not the genes but the proteins have biological function and are the working units of cell machinery . consequently , their expression patterns in both qualitative and quantitative aspects should be characterized when the altered biology of diseases are in the focus of study . despite this fact , the majority of protein identifications are based upon ms / ms data from shotgun proteomics experiments searched against protein databases such as uniprotkb and nextprot , which are typically restricted to proteoforms with consensus and ast sequences only . thus , direct searches of savs can not be performed with tandem spectra and such data is seldom reported , despite the fact that savs may play important role in disease biology . others , like the freely available x!tandem ( http://www.thegpm.org ) employs another strategy that systematically changes each residue in a peptide for all other possible amino acids and score the mutated peptide ( and all potential modifications ) against all of the available ms / ms spectra . therefore , bioinformatic tools do exist that aid in the process of identifying savs in shotgun proteomics data , and these tools can prove to be valuable in the characterization of savs associated with a particular disease . glioblastomas are among the deadliest of cancers ; fewer than 10% of patients survive five years after diagnosis . recurrence is at least in part due the existence of glioma stem cells ( gscs ) , which are resistant to radiation and chemotherapy . we have acquired comprehensive , quantitative data sets from thirty - six gsc lines , including gene activity and protein expression . we have previously examined patterns of global as well as chromosome-19-specific protein and gene expression in a subset of the 36 gsc lines . our hypothesis is that gscs , as a rare type of diseased cell type , harbor protein savs due to germline or somatic mutations . those proteins may normally have cell protective properties , but proteins with amino acid substitutions have the potential to be transformed into promoters of genetic instability or invasivity , as well as ineffective regulators of epigenetic or metabolic control . through the use of a customized database and bioinformatic tools , we provide the first comparative data of chromosome 19 snp products on 36 glioma stem cell lines and compelling evidence for the expression of a sav in branched - chain aminotransferase 2 ( bcat2 ) , a protein encoded by chromosome 19 , in six of the gsc lines . we have devised a systematic workflow to address the second level of protein expression complexity ( represented by the savs ) and have applied this workflow in the identification of missense snv products at the protein level in glioma samples based on high resolution ms / ms data . we have identified and further verified novel mutant proteoforms that might be strongly associated with glioma . briefly , peptide mixtures were separated on a c18 column ( proteopep ii , new objective , 10 cm 75 m ) using a 240 min gradient ( solvent a , 0.1% formic acid in water ; solvent b , 0.1% formic acid in acetonitrile ) . data were acquired using high - resolution data - driven analysis ( dda ) , with the survey scan ( ms ) acquired in the orbitrap at 60 000 resolution ( at m / z 400 ) in profile mode . for database searching the technical replicates were combined and searched against a combination of the uniprotkb / swissprot - human database ( 2014_06 version , 40 548 protein entries ) with all known chromosome 19 sav sequences ( 132 264 ) together with 115 sequences of the common repository of adventitious proteins ( crap ) contaminant database ( http://www.thegpm.org/crap/index.html ) . for those peptides that passed these initial filters , manual verification of ms / ms spectral assignments of sav peptides was performed by comparing m / z values for ions observed in the ms / ms spectrum with a theoretical m / z list generated using the ms - product utility on the protein prospector web site ( http://prospector.ucsf.edu ) . for optimization of the assay , isotopically labeled peptides were mixed and diluted with 5% acetonitrile ( acn ) at a concentration of 325 pmol/l for each synthetic peptide . samples ( 2 l ) were injected onto an easy c18-a1 precolumn ( thermo scientific , waltham , ma ) , and following online desalting and trapping at a pressure of 280 bar , the peptides were separated on a 75 m 150 mm fused silica column packed with reprosil c18 ( 3 m , 120 from dr . the five transitions , which produced the most abundant signals without observed interferences in the glioma samples , were selected for further analysis and utilized for unambiguous identification . the bcat2 sav was modeled using a combination of two crystal structures : the substrate , l - isoleucine covalently bound to the cofactor pyridoxamine phosphate ( pmp ) , bound form of the enzyme ( pdb code : 1kt8 ) , and the pmp bound form ( pdb code : 1kta ) . as 1kt8 is missing residues near the mutation site , it was used as a reference structure onto which the coordinates of 1kta were superimposed in order to include the coordinates of residues 175 to 191 . residue 186 ( please , note that our residue numbering scheme in this paper follows the recommendations of human genome variation society and thus may differ by six from other authors ) was then mutated from threonine to arginine , and hydrogens were added . for both the original and rotated backbone structures , the side chain angles 1 and 2 of arg186 were subsequently rotated in six increments of 60 , yielding a total of 72 structures . from the original structure , the side chain was rotated about the 1 bond without altering the original 2 torsion , generating an additional six structures . here , the charmm27 parameter set was used to obtain partial atomic charges and atomic radii , and a ph of 7.0 was used in assigning protonation states for titratable residues . we addressed the determination of mutant proteoforms in biological samples , a high priority within the c - hpp . a novel systematic approach was developed that is based on searching shotgun proteome tandem spectra , collected via data dependent analysis , against a customized protein sequence database containing chromosome 19 savs . the new database used for identification of mutant proteoforms was compiled with all human consensus proteins together with a new set of missense snv - derived sequences of chromosome 19 . to improve search quality , the frequently used crap contaminant protein database was also included , resulting in a database of 172 927 protein sequences in total . in a standard target - decoy approach , a number of high quality spectra will be wrongly matched to decoy sequences simply due to the absence of correct peptide sequences in the target database . alternatively , protein sequences from the other homologous species , or in our case the rest of the canonical proteome from the same species , could be appended to a database of interest , adding more possibilities for the correct peptide - spectrum matches in a target data set and , therefore , enabling target - decoy approach implemented by standard search engines . because protein databases for shotgun proteomic searches do not commonly include sav sequences , and different search engines may perform differently with the same database ; three search engines ( sequest , peaks , and mascot ) were employed and the corresponding search results were compared . manual verification of spectra containing savs led to confirmation of a t186r substitution of branched - chain aminotransferase 2 ( bcat2 ) ( see figure 1 ) , a known natural variant of the protein , in gsc28 . hcd - ms / ms spectrum of pvlignepslgvsqpr , supporting assignment of the p.thr186arg proteoform of bcat2 . due to the high confidence in this finding , identification of the sav - containing peptide of bcat2 ( pvlignepslgvsqpr ) in gsc 28 was used as a positive control for all database searches . according to the snap database of gpmdb , this tryptic peptide of bcat2 is less frequently observed compared with its longer , miscleaved forms ( http://snap.thegpm.org/%7e/dblist_protein_mut/label=ensp00000322991 ) . interestingly , peaks db was the only search engine that successfully identified the peptide containing bcat2 p.thr186arg , with a 10log p score of 74 . our initial thought was that both failed to identify the peptide due to trypsin cleavage specificity assigned in the search parameters , as the n - terminus of the sav - containing peptide arises from cleavage between arginine and proline . therefore , we adjusted the trypsin specificity in mascot to allow for cleavage n - terminal to proline and repeated the search using gsc 28 as a positive control . because mascot again failed to identify the peptide containing bcat2 p.thr186arg , we focused on peaks db to identify savs in the remaining cell lines ( see table 1 for a verified list and supporting information table 1 for a complete list of sav peptides identified by peaks ) . conflict in dbsnp and ensembl databases , which indicates this mutation q h. for those peptides that were confirmed to be truly unique by homology searching , manual verification of spectra was performed in order to ensure that the assigned savs were correct . because the mass errors for peptide assignments in each lc ms / ms file fall into a normal distribution , and 95% of correct assignments fall within two standard deviations of the mean error , we removed peptides whose mass error was dramatically higher than for other peptides within the same analytical run . each identified peptide containing , we evaluated the individual reads from the transcriptome sequencing to quantitate the frequency of c ( acg coding thr ) and g ( agg coding arg ) alleles . a total of 8 gsc lines contained reads of > 50% with c613 g variant , including gscs 28 , 274 , 711 , 275 , 17 , 280 , 289 , and 293 ( data not shown ) . the primary target of srm confirmation was the bcat2 p.thr186arg mutation that was unambiguously verified in samples gsc 28 , gsc 711 , and gsc 275 by perfectly matching transitions and retention times of heavy labeled internal standard and native peptides ( see figure 2a ) . ( a ) bcat2 p.thr186arg was confirmed by matching heavy - isotope - labeled synthetic peptide ( blue ) with the endogenous pvlignepslgvsqpr ( red ) , whereas ( b ) the endogenous signals of elongation factor 2 p.ile665leu and endophilin - a2 p.leu95ile could not agree with the internal standards . the specificity of srm assays was utilized in investigation of leu ile and ile leu mutations that were initially identified in database search despite of the isobaric nature of these savs . this information is necessary in the case of heterozygous samples , in particular when the expression of the mutant allele has a strong concordance with biological activity of cells . the p.thr186arg mutation on bcat2 , confirmed at the rna level to be in eight of the 36 gsc lines , is located along a flexible loop in close proximity to the cxxc center ( figure 3 ) . to study the possible effects of this mutation , we generated 78 p.thr186arg bcat2 variant structures by rotating the arginine side chain about the 1 and 2 and scanning the backbond . of the structures lowest in electrostatic free energy , which satisfied the packing and stereochemical constraints in minimization , four were grouped within a few kilocalories per meters , whereas the rest were separated by over 100 kcal / m in electrostatic free energy and so were not considered for further analysis . the arginine side chain is found in a salt bridge with glu229 and toward the inside of the flexible arm , where the arginine side chain has the most stable hydrogen bonding . these lower energy positions of the arginine side chain all greatly change the electrostatic environment as compared with the thr186 wild type , and this may influence the cxxc oxidation reaction that regulates enzyme activity . as seen by the four lowest energy structures ( figure 3 ) , the arg186 side chain gravitates toward the carboxylate group of glu229 , as well as inward toward the cxxc site , both of which provide stability through hydrogen bonding pairs . compared with the thr186 wild type , the arginine side chain significantly changes the electrostatic environment near the cxxc , potentially shifting the pka of the cysteine residues . the electrostatically induced pka shift could influence the cysteine protonation state and therefore the equilibrium of the oxidation reaction ( figure 4 ) , consequently altering enzyme activity . it has been demonstrated that the cxxc center ( cys321 and cys324 ) contributes to enzyme activity through reversible disulfide bond formation that prevents substrate from correctly orienting with the pyridoxal phosphate ( plp ) cofactor . through a combination of homology searching and manual verification of spectra identified by peaks db one of these savs , bcat2 p.thr186arg , was validated orthogonally in multiple cell lines by srm and by rna - seq future studies will be directed toward verification of the remainder of these savs and a study of the biological implications of these savs . the identification and characterization of savs in glioma has already yielded the identity proteins that will be of further interest to study as potential modulators of gliomagenesis , contributors to glioma pathology , and resistance to standard of care treatments . to improve visibility of savs , we strongly suggest extending the registry of protein sequences in nextprot with information about the mutant proteoforms pointing to identification levels as well as expression sites .

two experiments were carried out with exposure of mushrooms to uv - b doses in the range of 0250mj cm over three days ( experiment a ) , and exposure of mushrooms to uv - b doses of 2202,400 mj cm during one day ( experiment b ) . a transportable and adjustable uv - b unit was constructed using six uv - b tubes ( medical therapy/12 , philips , eindhoven , the netherlands ) giving a homogeneous distribution of uv - b over an area of 0.60.4 m . the intensity of uv - b was 1.88 mw cm at a distance of 0.25 m between the mushrooms and the uv - b tubes . exposure to uv - b for 1 min 46 sec provided a dose of 200 mj cm . the spectral distribution of the uv - b unit was measured by a spectrometer ( avaspec-2048 , avantes , eerbeek , the netherlands ) that was calibrated with a standard of spectral irradiance ol fel - c ( optronic laboratories , orlando , fa , usa ) , a lamp that includes wavelengths down to 250 nm . the spectral composition was measured at 0.25 m distance from the uv - b tubes , with a black screen surrounding the unit to avoid stray light . the uv - b doses in the different treatments were measured by the accumulated intensity over time by use of a uv - b photometer ( il1400 sel005/tls312/td ; international light technologies , ma , usa ) . the spectral distribution of the uv - b unit used for treatments of supplementary uv - b during the growth phase and post - harvest . white button mushrooms ( agaricus bisporus ( lange ) imbach ) were grown at 18c day and night temperature and 88% relative humidity on horse manure / barley straw compost at the commercial production facility of egehj champignon a / s , veflinge , denmark in 2009 . experiment a was initiated on the second sampling day of the second flush ( 7-day harvest cycle ) , 36 days after inoculation of the compost with mycelium . experiment b was carried out on the first day of the third flush , 45 days after inoculation . in both experiments , the mushrooms were sampled for yield and vitamin d2 analyses at harvest , according to the sampling practice for commercial harvest - ready mushrooms . in experiment a , 28 plots of 0.40.4 m were established on the shelves with growing mushrooms in a complete randomised statistical design with four blocks , two in each of two growth chambers giving four replicate plots per treatment . by varying the duration of exposure , the treatments were 0 , 100 , 150 , 200 and 250 mj m supplementary uv - b on the first day ; and 200 mj cm given repeatedly once a day on the first , second and third day of the experiment ( 3200 mj cm ) . all plots were sampled immediately after exposure and also on the three consecutive days ( at 24 hour periods ) after the first day exposure to 0 or 200 mj cm and after the repeated exposure to 200 mj cm . additional samples were taken for post - harvest treatments of exposure to 200 mj cm natural uv - b in sunlight for approximately 20 min at mid day , or following overnight cooling ( 2c ) , re - adjustment to 18c and exposure to 200 mj cm supplementary uv - b . additional production areas from the four blocks were sampled the three days forestimation ofbiomass of small mushrooms , thatremained after harvest to grow for next day harvest . this was to calculate vitamin d2 recovery during the experiment . in experiment b , seven plots of 0.60.4 m were established on the shelves with growing mushrooms in a growth chamber . treatments at 220 , 500 , 1,000 and 2,400 mj m supplementary uv - b were given to the plots in a complete randomised statistical design with no replications in the range of 2201,000 mj m and six replications of the 2,400 mj m treatment . to october 2009 , four composite samples of each 23 wild growing mushrooms ( agaricus sp . ) were sampled in an orchard at aarslev , denmark ( 1027e , 5518n ) for determination of vitamin d2 content . in addition , four samples of a. bisporus of miscellaneous origin ( strains and producers ) were purchased for determination of vitamin d2 content . all fresh mushroom samples were weighed , cooled ( 1c , 100% relative humidity ) and mushrooms counted , cleaned and base removed . each sample of at least 200 g material was frozen at 18c for subsequent freeze drying for determination of dry matter and vitamin d2 content . prior to analysis , the frozen samples of mushrooms were thawed to room temperature , homogenised for 30 sec ( type 320 , moulinex , group seb , f ) and freeze - dried ( christ beta 18 , martin christ gefriertrochnungsanlagen gnbh , osterode , d ) . to achieve a homogenised sub - sample , the freeze - dried mushrooms were homogenised again for 30 sec ( type 320 , moulinex , group seb , f ) . the analytical method and the equipment used to determine vitamin d2 in the mushrooms were a modification of a method used for meat ( 7 ) . in short , 1 g freeze - dried mushrooms were taken for analysis , and vitamin d3 was added as an internal standard according to en12821 . the samples were extracted by alkaline saponification followed by clean - up by silica solid phase extraction . due to the extremely high amount of ergosterol in the samples , the combination of a silica and an amino column was replaced by a shorter silica column ( luna , si 60 , 3 m , 1504.6 mm , phenomenex , torrance , ca , usa ) in combination with an isocratic mobile phase consisting of 2-propanol : methyl - tert - butyl - ether : cyclohexan : n - heptan ( 0.7:2:48.65:48.65 ) . a flow rate of 1.2 ml min gave a retention time for vitamin d2 of 11.2 min ; sample collection was made during the interval 10.412.2 min . the retention time for ergosterol was 16.0 min , which was baseline separated from vitamin d2 . for separation , a c18 column combined with a mobile phase consisting of acetonitrile : methanol ( 80:20 ) was used , whereas detection was performed by photo - diode array detector ( 220320 nm ) and quantification at 265 nm . a house - reference material consisting of freeze - dried mushrooms was analysed on each day of analyses , showing an internal reproducibility of 5.8% . the analysis was run in a laboratory accredited after iso 17025 for the original analytical method , and the correctness for the method was controlled by participation in the food analysis performance assessment scheme ( www.fapas.com ) . recovery of vitamin d2 produced after the first day exposure to uv - b was calculated per unit production area over the three days of experiment based on the biomass of large harvested and small remaining mushrooms . for this , it was assumed that vitamin d2 content in small mushrooms equalled that was measured in harvested mushrooms . multiple comparisons were based on values of the least significant difference derived from analysis of variance ( proc glm , sas institute inc . , a transportable and adjustable uv - b unit was constructed using six uv - b tubes ( medical therapy/12 , philips , eindhoven , the netherlands ) giving a homogeneous distribution of uv - b over an area of 0.60.4 m . the intensity of uv - b was 1.88 mw cm at a distance of 0.25 m between the mushrooms and the uv - b tubes . exposure to uv - b for 1 min 46 sec provided a dose of 200 mj cm . the spectral distribution of the uv - b unit was measured by a spectrometer ( avaspec-2048 , avantes , eerbeek , the netherlands ) that was calibrated with a standard of spectral irradiance ol fel - c ( optronic laboratories , orlando , fa , usa ) , a lamp that includes wavelengths down to 250 nm . the spectral composition was measured at 0.25 m distance from the uv - b tubes , with a black screen surrounding the unit to avoid stray light . the uv - b doses in the different treatments were measured by the accumulated intensity over time by use of a uv - b photometer ( il1400 sel005/tls312/td ; international light technologies , ma , usa ) . the spectral distribution of the uv - b unit used for treatments of supplementary uv - b during the growth phase and post - harvest . white button mushrooms ( agaricus bisporus ( lange ) imbach ) were grown at 18c day and night temperature and 88% relative humidity on horse manure / barley straw compost at the commercial production facility of egehj champignon a / s , veflinge , denmark in 2009 . experiment a was initiated on the second sampling day of the second flush ( 7-day harvest cycle ) , 36 days after inoculation of the compost with mycelium . experiment b was carried out on the first day of the third flush , 45 days after inoculation . in both experiments , the mushrooms were sampled for yield and vitamin d2 analyses at harvest , according to the sampling practice for commercial harvest - ready mushrooms . in experiment a , 28 plots of 0.40.4 m were established on the shelves with growing mushrooms in a complete randomised statistical design with four blocks , two in each of two growth chambers giving four replicate plots per treatment . by varying the duration of exposure , the treatments were 0 , 100 , 150 , 200 and 250 mj m supplementary uv - b on the first day ; and 200 mj cm given repeatedly once a day on the first , second and third day of the experiment ( 3200 mj cm ) . all plots were sampled immediately after exposure and also on the three consecutive days ( at 24 hour periods ) after the first day exposure to 0 or 200 mj cm and after the repeated exposure to 200 mj cm . additional samples were taken for post - harvest treatments of exposure to 200 mj cm natural uv - b in sunlight for approximately 20 min at mid day , or following overnight cooling ( 2c ) , re - adjustment to 18c and exposure to 200 mj cm supplementary uv - b . additional production areas from the four blocks were sampled the three days forestimation ofbiomass of small mushrooms , thatremained after harvest to grow for next day harvest . b , seven plots of 0.60.4 m were established on the shelves with growing mushrooms in a growth chamber . treatments at 220 , 500 , 1,000 and 2,400 mj m supplementary uv - b were given to the plots in a complete randomised statistical design with no replications in the range of 2201,000 mj m and six replications of the 2,400 mj m treatment . all plots were sampled immediately after exposure . in the period of late july to october 2009 , four composite samples of each 23 wild growing mushrooms ( agaricus sp . ) were sampled in an orchard at aarslev , denmark ( 1027e , 5518n ) for determination of vitamin d2 content . in addition , four samples of a. bisporus of miscellaneous origin ( strains and producers ) were purchased for determination of vitamin d2 content . all fresh mushroom samples were weighed , cooled ( 1c , 100% relative humidity ) and mushrooms counted , cleaned and base removed . each sample of at least 200 g material was frozen at 18c for subsequent freeze drying for determination of dry matter and vitamin d2 content . prior to analysis , the frozen samples of mushrooms were thawed to room temperature , homogenised for 30 sec ( type 320 , moulinex , group seb , f ) and freeze - dried ( christ beta 18 , martin christ gefriertrochnungsanlagen gnbh , osterode , d ) . to achieve a homogenised sub - sample , the freeze - dried mushrooms were homogenised again for 30 sec ( type 320 , moulinex , group seb , f ) . the analytical method and the equipment used to determine vitamin d2 in the mushrooms were a modification of a method used for meat ( 7 ) . in short , 1 g freeze - dried mushrooms were taken for analysis , and vitamin d3 was added as an internal standard according to en12821 . the samples were extracted by alkaline saponification followed by clean - up by silica solid phase extraction . due to the extremely high amount of ergosterol in the samples , the combination of a silica and an amino column was replaced by a shorter silica column ( luna , si 60 , 3 m , 1504.6 mm , phenomenex , torrance , ca , usa ) in combination with an isocratic mobile phase consisting of 2-propanol : methyl - tert - butyl - ether : cyclohexan : n - heptan ( 0.7:2:48.65:48.65 ) . a flow rate of 1.2 ml min gave a retention time for vitamin d2 of 11.2 min ; sample collection was made during the interval 10.412.2 min . the retention time for ergosterol was 16.0 min , which was baseline separated from vitamin d2 . for separation , a c18 column combined with a mobile phase consisting of acetonitrile : methanol ( 80:20 ) was used , whereas detection was performed by photo - diode array detector ( 220320 nm ) and quantification at 265 nm . a house - reference material consisting of freeze - dried mushrooms was analysed on each day of analyses , showing an internal reproducibility of 5.8% . the analysis was run in a laboratory accredited after iso 17025 for the original analytical method , and the correctness for the method was controlled by participation in the food analysis performance assessment scheme ( www.fapas.com ) . recovery of vitamin d2 produced after the first day exposure to uv - b was calculated per unit production area over the three days of experiment based on the biomass of large harvested and small remaining mushrooms . for this , it was assumed that vitamin d2 content in small mushrooms equalled that was measured in harvested mushrooms . multiple comparisons were based on values of the least significant difference derived from analysis of variance ( proc glm , sas institute inc . , results from experiments a and b showed a close to linear relationship between dose of uv - b and formation of vitamin d2 at 01,000 mj cm supplementary uv - b during the growth phase . the outputs from the linear regressions were very similar with a - factors of 0.118 and 0.108 for the range of 0250 mj cm and 01,000 mj cm , respectively , and high correlation coefficients ( fig . 2a and b ) . the linearity of the relationship found in these two experiments was previously reported by roberts et al . 2008 ( 6 ) following post - harvest exposure in the range of 5001,000 mj cm supplementary uv - b after overnight cooling of white button mushrooms . however , in the present study , the a - factor of the linear regression was 0.11 at exposure during the growth phase , compared to 0.06 calculated from roberts et al . thus , exposure , for example , to 500 mj cm during the growth phase formed vitamin d2 of approximately 57 g 100 g fresh weight compared to 37 g 100 g at post - harvest exposure ( 6 ) . however , the difference between the two studies can not be ascribed to a higher efficiency of vitamin d2 conversion at exposure during the growth phase . in the present study , mushrooms sampled after overnight cooling and post - harvest exposure were found to have a higher vitamin d2 content compared to those exposed during the growth phase , and this difference was not caused by effects of overnight cooling on dry matter content ( table 1 ) . the higher conversion of vitamin d2 in the present study compared with that of roberts et al . ( 2008 ) ( 6 ) was probably caused by differences in spectral distribution of the uv - b from different types of lamps . also , experimental conditions , such as temperature and moisture content , have been shown to affect the formation of vitamin d2 ( 5 ) . orientation of the mushrooms to the uv source has also been a factor that contributes to vitamin d2 level , that is , the content of vitamin d2 varies within an individual mushroom ( 8) ; however , in this study and that of roberts et al . finally , also the difference between our hplc - dad and the lc - ms / ms methods used in ( 6 ) may give a bias in the results . the relationship between vitamin d2 concentration in mushrooms and exposure to uv - b dose up to 250 mj cm in experiment a and up to 2,400 mj cm in experiment b. error bars indicate standard errors in experiment a ( n=4 ) , and in experiment b for the highest dose ( n=6 ) . the effect of source of uv - b and timing of exposure in relation to harvest on vitamin d2 in wild and cultivated agaricus mushrooms within columns means followed by different letters are significantly different . means followed by the same letter are not ( p<0.05 ; n=4 ) ; samples taken from other flush . the formation of vitamin d2 levelled off at a content of 164 g 100 g at the high dose of 2,400 mj cm supplementary uv - b as also reported for doses beyond 1,200 mj cm by others ( 5 , 6 ) . in this study , a clear browning effect was observed on the surface of the exposed mushrooms indicating that at high doses of uv - b , there was damage to the surface cells ( 8) . it has been suggested that high doses of uv cause photodegradation of vitamin d2 ( 3 ) . thus , very high doses of uv - b are relatively less efficient for conversion of ergosterol to vitamin d2 and cause surface discolouration . uv - b exposure did not affect the growth rate of the mushrooms in experiment a ( fig . the average yields were 2.25 , 4.83 and 3.36 kg m , and the number of mushrooms were 236 , 322 and 163 pieces m on the first , second and third day of the flush , respectively . the dry matter contents were 8.4 , 8.3 and 9.3 g 100 g , respectively . over the three days , the vitamin d2 content of mushrooms decreased sharply from 24 to 3 g 100 g when measured after one exposure at 200 mj cm of supplementary uv - b ( fig . this decrease was observed mainly from the first to the second day of the experiment . by day 3 , 94% of the vitamin d2 formed during one exposure to uv - b on day 1 could be accounted for in the large harvested and the small remaining mushrooms ( table 2 ) . thus , the fast growth rate of the mushrooms diluted the vitamin d2 formed after the exposure on day 1 , as the high recovery of vitamin d2 indicated that there was no significant degradation or transport of vitamin d2 from the mushrooms . the yield of mushrooms obtained during the three days of experiment a after ( a ) no exposure to uv - b , exposure to supplementary uv - b of 200 mj cm , ( b ) the first day and ( c ) repeated every day . the vitamin d2 concentration in mushrooms found during the three days of experiment a after exposure to supplementary uv - b of 200 mj cm ( a ) the first day , and ( b ) repeated every day . recovery of vitamin d2 calculated on basis of production area for three days of experiment a after one exposure to supplementary uv - b of 200 mj cm the first day based on the biomass of small remaining mushrooms and the assumption that their vitamin d2 content equalled that measured in large harvested mushrooms . when the uv - b exposure was repeated at daily intervals ( day 13 ) , the vitamin d2 content increased to 33 g 100 g. these results suggest that repeated exposure to uv - b is needed just prior to harvest and during all days of a flush to ensure a high level of vitamin d2 in the final product . the increase in the vitamin d2 content after repeated exposure may be explained by a carryover of vitamin d2 formed in the small mushrooms left growing for the next day . there was no indication that one exposure to uv - b stimulated or inhibited the formation of vitamin d2 at later exposures . the vitamin d2 content was much lower in cultivated mushrooms exposed to sunlight of 200 mj cm uv - b or in wild mushrooms samples compared to mushrooms exposed to supplementary uv - b ( table 1 ) . the samples of cultivated mushrooms without exposure to uv - b or of miscellaneous origin ( strains and producers ) were found to have the lowest content of vitamin d2 . to our knowledge , the vitamin d2 content of 1.5 g 100 g is the first report on wild agaricus sp . , while higher contents have been reported for other wild grown species such as cantharellus cibarius and c. tubaeformis ( 1013 and 2130 g 100 g , respectively ) as well as for boletus edulis ( 359 g 100 g ) ( 1 , 2 ) . the low vitamin d2 contents of wild agaricus sp . and cultivated a. bisporus exposed to 200 mj cm uv - b in sunlight were surprising considering the results of other reports and the present study of cultivated a. bisporus forming high amounts of vitamin d2 when exposed to supplementary uv - b ( 6 , 4 , 8) . even though a. bisporus does not produce as high levels of vitamin d2 after uv exposure as wild c. tubaeformis ( 2 ) and cultivated pleurotus ostreatus ( 5 ) . the difference between the effect of uv - b from sunlight and from the supplementary uv - b unit may have been caused by differences in the distribution of wavelengths within the range of uv - b that are integrated in the measurement of the uv - b sensor . the tubes of the uv - b unit emit some radiation of wavelengths shorter than 290 nm ( fig . 1 ) , which is the part of the uv - b range that is almost absent in sunlight after absorption in the earth 's atmosphere ( 9 ) . this short - waved uv - b is close to the uv - c range that has , however , been found to be less effective than uv - b in the conversion of ergosterol to vitamin d2 ( 5 ) . lentinus edodes was found to have higher vitamin d2 content when harvested on sunny as compared to cloudy days ( 10 as reported in ( 4 ) ) . the uv - exposure is similar to the exposure of wild mushrooms that is not reported to cause toxic elements in mushrooms . the level of vitamin d2 content in wild and cultivated mushrooms with or without exposure to sunlight ( table 1 ) is comparable to that of , for example , 0.40.7 g of vitamin d3 in 100 g of pork and beef meat and 0.20.4 g of vitamin d3 in 100 g of a variety of cheeses ( www.foodcomp.dk ) . commercial growers may consider using uv - b during the growth phase for vitamin d2 formation in white button mushrooms . in this case , the need for prolonged exposure time is less critical as treatment times do not have to be confined to the time available during the post - harvest processing on a conveyor belt . instead , an automatic moving uv - b unit can be constructed , for example , for overnight exposure of mushrooms on the growing shelves just prior to harvest . with the high intensities used in the present study , doses of 200 mj cm could be obtained with an exposure time of < 2 min and resulting in vitamin d2 contents of around 24 g 100 g. at this vitamin d2 level , approximately 35 g of mushroom is equivalent to the recommended daily intake of 7.5 g vitamin d2 for adults in denmark . the upper safe limit for daily intake of 50 g vitamin d2 should be taken into consideration ( 11 ) . mushrooms with a vitamin d2 content of 24 g 100 g would contribute to 26% of the recommended daily intake of 7.5 g based on the average intake of mushrooms by adults of 3 kg yr in 2006 in denmark ( 12 ) . therefore , uv - b - enhanced vitamin d2 levels in mushrooms provide a potentially important food source of vitamin d. the close to linear relationship between uv - b dose and formation of vitamin d2 at exposure just prior to harvest makes it possible to produce mushrooms with a well - defined content of vitamin d2 . therefore , mushroom products will be a rich source for increased intake of vitamin d2 in food . due to the dilution of vitamin d2 by the fast growth of the mushrooms , uv - b exposure needs to be repeated daily during the harvest flush to maintain a high content of vitamin d2 in the end product . supplementary uv - b from tubes is a more effective source of uv - b for the formation of vitamin d2 in mushrooms as compared to natural sunlight that gave a low content of vitamin d2 equivalent to that found in wild agaricus sp . mushrooms . this work was supported by the danish food industry agency , the committee for horticultural sale and egehej champignon a / s .

backgroundmushrooms are the only non - animal food source of vitamin d. wild mushrooms have naturally high vitamin d2 content , and cultivated mushrooms produce vitamin d2 from ergosterol when exposed to supplementary uv - b during the post - harvest phase.objectivesthis study investigated the effects of providing supplementary uv - b during the growth phase on vitamin d2 formation and the interactions with growth of mushrooms , as compared to supplementary uv - b during the post - harvest phase or exposure to sunlight for both cultivated and wild mushrooms.methodsexperiments were carried out with exposure to supplementary uv - b just prior to harvest in the range of 02,400 mj cm2 . mushrooms grew for 2 days with or without repeated uv - b exposure each day . vitamin d2 and growth rate were determined . in addition , some mushrooms were post - harvest treated by exposure at 200 mj cm2 supplementary uv - b or natural sunlight , prior to vitamin d2 determination.resultsthe content of vitamin d2 was 0.2164 g 100 g1 fresh weight , and there was a linear relationship between uv - dose up to 1,000 mj cm2 and vitamin d2 content . the fast growth rate of the mushrooms diluted the vitamin d2 from 24 to 3 g 100 g1 within 2 days of exposure at 200 mj cm2 . following repeated uv - b exposure , vitamin d2 increased to 33 g vitamin d2 100 g1 . growth was unaffected by uv - b . post - harvest exposure to supplementary uv - b resulted in a higher vitamin d2 content of 32 g 100 g1 compared to the 24 g 100 g1 obtained from exposure to uv - b during the growth phase . in contrast , wild and cultivated mushrooms with and without exposure to sunlight had vitamin d2 content in the range of 0.21.5 g vitamin d2 100 g1.conclusionsthis study showed that mushrooms with a well - defined content of vitamin d2 can be obtained by exposure to supplementary uv - b just prior to harvest .

Materials and methods The UV-B equipment Experimental design Vitamin D Data and statistical analysis Results and discussion Conclusion Conflict of interest and funding

two experiments were carried out with exposure of mushrooms to uv - b doses in the range of 0250mj cm over three days ( experiment a ) , and exposure of mushrooms to uv - b doses of 2202,400 mj cm during one day ( experiment b ) . a transportable and adjustable uv - b unit was constructed using six uv - b tubes ( medical therapy/12 , philips , eindhoven , the netherlands ) giving a homogeneous distribution of uv - b over an area of 0.60.4 m . the intensity of uv - b was 1.88 mw cm at a distance of 0.25 m between the mushrooms and the uv - b tubes . exposure to uv - b for 1 min 46 sec provided a dose of 200 mj cm . the spectral distribution of the uv - b unit was measured by a spectrometer ( avaspec-2048 , avantes , eerbeek , the netherlands ) that was calibrated with a standard of spectral irradiance ol fel - c ( optronic laboratories , orlando , fa , usa ) , a lamp that includes wavelengths down to 250 nm . the spectral composition was measured at 0.25 m distance from the uv - b tubes , with a black screen surrounding the unit to avoid stray light . the uv - b doses in the different treatments were measured by the accumulated intensity over time by use of a uv - b photometer ( il1400 sel005/tls312/td ; international light technologies , ma , usa ) . the spectral distribution of the uv - b unit used for treatments of supplementary uv - b during the growth phase and post - harvest . in both experiments , the mushrooms were sampled for yield and vitamin d2 analyses at harvest , according to the sampling practice for commercial harvest - ready mushrooms . by varying the duration of exposure , the treatments were 0 , 100 , 150 , 200 and 250 mj m supplementary uv - b on the first day ; and 200 mj cm given repeatedly once a day on the first , second and third day of the experiment ( 3200 mj cm ) . additional samples were taken for post - harvest treatments of exposure to 200 mj cm natural uv - b in sunlight for approximately 20 min at mid day , or following overnight cooling ( 2c ) , re - adjustment to 18c and exposure to 200 mj cm supplementary uv - b . in experiment b , seven plots of 0.60.4 m were established on the shelves with growing mushrooms in a growth chamber . treatments at 220 , 500 , 1,000 and 2,400 mj m supplementary uv - b were given to the plots in a complete randomised statistical design with no replications in the range of 2201,000 mj m and six replications of the 2,400 mj m treatment . were sampled in an orchard at aarslev , denmark ( 1027e , 5518n ) for determination of vitamin d2 content . in addition , four samples of a. bisporus of miscellaneous origin ( strains and producers ) were purchased for determination of vitamin d2 content . each sample of at least 200 g material was frozen at 18c for subsequent freeze drying for determination of dry matter and vitamin d2 content . prior to analysis , the frozen samples of mushrooms were thawed to room temperature , homogenised for 30 sec ( type 320 , moulinex , group seb , f ) and freeze - dried ( christ beta 18 , martin christ gefriertrochnungsanlagen gnbh , osterode , d ) . the analytical method and the equipment used to determine vitamin d2 in the mushrooms were a modification of a method used for meat ( 7 ) . in short , 1 g freeze - dried mushrooms were taken for analysis , and vitamin d3 was added as an internal standard according to en12821 . a flow rate of 1.2 ml min gave a retention time for vitamin d2 of 11.2 min ; sample collection was made during the interval 10.412.2 min . the analysis was run in a laboratory accredited after iso 17025 for the original analytical method , and the correctness for the method was controlled by participation in the food analysis performance assessment scheme ( www.fapas.com ) . recovery of vitamin d2 produced after the first day exposure to uv - b was calculated per unit production area over the three days of experiment based on the biomass of large harvested and small remaining mushrooms . for this , it was assumed that vitamin d2 content in small mushrooms equalled that was measured in harvested mushrooms . , a transportable and adjustable uv - b unit was constructed using six uv - b tubes ( medical therapy/12 , philips , eindhoven , the netherlands ) giving a homogeneous distribution of uv - b over an area of 0.60.4 m . the intensity of uv - b was 1.88 mw cm at a distance of 0.25 m between the mushrooms and the uv - b tubes . exposure to uv - b for 1 min 46 sec provided a dose of 200 mj cm . the spectral distribution of the uv - b unit was measured by a spectrometer ( avaspec-2048 , avantes , eerbeek , the netherlands ) that was calibrated with a standard of spectral irradiance ol fel - c ( optronic laboratories , orlando , fa , usa ) , a lamp that includes wavelengths down to 250 nm . the spectral composition was measured at 0.25 m distance from the uv - b tubes , with a black screen surrounding the unit to avoid stray light . the uv - b doses in the different treatments were measured by the accumulated intensity over time by use of a uv - b photometer ( il1400 sel005/tls312/td ; international light technologies , ma , usa ) . the spectral distribution of the uv - b unit used for treatments of supplementary uv - b during the growth phase and post - harvest . experiment b was carried out on the first day of the third flush , 45 days after inoculation . in both experiments , the mushrooms were sampled for yield and vitamin d2 analyses at harvest , according to the sampling practice for commercial harvest - ready mushrooms . in experiment a , 28 plots of 0.40.4 m were established on the shelves with growing mushrooms in a complete randomised statistical design with four blocks , two in each of two growth chambers giving four replicate plots per treatment . by varying the duration of exposure , the treatments were 0 , 100 , 150 , 200 and 250 mj m supplementary uv - b on the first day ; and 200 mj cm given repeatedly once a day on the first , second and third day of the experiment ( 3200 mj cm ) . additional samples were taken for post - harvest treatments of exposure to 200 mj cm natural uv - b in sunlight for approximately 20 min at mid day , or following overnight cooling ( 2c ) , re - adjustment to 18c and exposure to 200 mj cm supplementary uv - b . treatments at 220 , 500 , 1,000 and 2,400 mj m supplementary uv - b were given to the plots in a complete randomised statistical design with no replications in the range of 2201,000 mj m and six replications of the 2,400 mj m treatment . were sampled in an orchard at aarslev , denmark ( 1027e , 5518n ) for determination of vitamin d2 content . in addition , four samples of a. bisporus of miscellaneous origin ( strains and producers ) were purchased for determination of vitamin d2 content . each sample of at least 200 g material was frozen at 18c for subsequent freeze drying for determination of dry matter and vitamin d2 content . prior to analysis , the frozen samples of mushrooms were thawed to room temperature , homogenised for 30 sec ( type 320 , moulinex , group seb , f ) and freeze - dried ( christ beta 18 , martin christ gefriertrochnungsanlagen gnbh , osterode , d ) . the analytical method and the equipment used to determine vitamin d2 in the mushrooms were a modification of a method used for meat ( 7 ) . in short , 1 g freeze - dried mushrooms were taken for analysis , and vitamin d3 was added as an internal standard according to en12821 . due to the extremely high amount of ergosterol in the samples , the combination of a silica and an amino column was replaced by a shorter silica column ( luna , si 60 , 3 m , 1504.6 mm , phenomenex , torrance , ca , usa ) in combination with an isocratic mobile phase consisting of 2-propanol : methyl - tert - butyl - ether : cyclohexan : n - heptan ( 0.7:2:48.65:48.65 ) . a flow rate of 1.2 ml min gave a retention time for vitamin d2 of 11.2 min ; sample collection was made during the interval 10.412.2 min . the analysis was run in a laboratory accredited after iso 17025 for the original analytical method , and the correctness for the method was controlled by participation in the food analysis performance assessment scheme ( www.fapas.com ) . recovery of vitamin d2 produced after the first day exposure to uv - b was calculated per unit production area over the three days of experiment based on the biomass of large harvested and small remaining mushrooms . for this , it was assumed that vitamin d2 content in small mushrooms equalled that was measured in harvested mushrooms . , results from experiments a and b showed a close to linear relationship between dose of uv - b and formation of vitamin d2 at 01,000 mj cm supplementary uv - b during the growth phase . the outputs from the linear regressions were very similar with a - factors of 0.118 and 0.108 for the range of 0250 mj cm and 01,000 mj cm , respectively , and high correlation coefficients ( fig . 2008 ( 6 ) following post - harvest exposure in the range of 5001,000 mj cm supplementary uv - b after overnight cooling of white button mushrooms . however , in the present study , the a - factor of the linear regression was 0.11 at exposure during the growth phase , compared to 0.06 calculated from roberts et al . thus , exposure , for example , to 500 mj cm during the growth phase formed vitamin d2 of approximately 57 g 100 g fresh weight compared to 37 g 100 g at post - harvest exposure ( 6 ) . however , the difference between the two studies can not be ascribed to a higher efficiency of vitamin d2 conversion at exposure during the growth phase . in the present study , mushrooms sampled after overnight cooling and post - harvest exposure were found to have a higher vitamin d2 content compared to those exposed during the growth phase , and this difference was not caused by effects of overnight cooling on dry matter content ( table 1 ) . the higher conversion of vitamin d2 in the present study compared with that of roberts et al . ( 2008 ) ( 6 ) was probably caused by differences in spectral distribution of the uv - b from different types of lamps . also , experimental conditions , such as temperature and moisture content , have been shown to affect the formation of vitamin d2 ( 5 ) . orientation of the mushrooms to the uv source has also been a factor that contributes to vitamin d2 level , that is , the content of vitamin d2 varies within an individual mushroom ( 8) ; however , in this study and that of roberts et al . the relationship between vitamin d2 concentration in mushrooms and exposure to uv - b dose up to 250 mj cm in experiment a and up to 2,400 mj cm in experiment b. error bars indicate standard errors in experiment a ( n=4 ) , and in experiment b for the highest dose ( n=6 ) . the effect of source of uv - b and timing of exposure in relation to harvest on vitamin d2 in wild and cultivated agaricus mushrooms within columns means followed by different letters are significantly different . the formation of vitamin d2 levelled off at a content of 164 g 100 g at the high dose of 2,400 mj cm supplementary uv - b as also reported for doses beyond 1,200 mj cm by others ( 5 , 6 ) . in this study , a clear browning effect was observed on the surface of the exposed mushrooms indicating that at high doses of uv - b , there was damage to the surface cells ( 8) . thus , very high doses of uv - b are relatively less efficient for conversion of ergosterol to vitamin d2 and cause surface discolouration . uv - b exposure did not affect the growth rate of the mushrooms in experiment a ( fig . the average yields were 2.25 , 4.83 and 3.36 kg m , and the number of mushrooms were 236 , 322 and 163 pieces m on the first , second and third day of the flush , respectively . the dry matter contents were 8.4 , 8.3 and 9.3 g 100 g , respectively . over the three days , the vitamin d2 content of mushrooms decreased sharply from 24 to 3 g 100 g when measured after one exposure at 200 mj cm of supplementary uv - b ( fig . this decrease was observed mainly from the first to the second day of the experiment . by day 3 , 94% of the vitamin d2 formed during one exposure to uv - b on day 1 could be accounted for in the large harvested and the small remaining mushrooms ( table 2 ) . thus , the fast growth rate of the mushrooms diluted the vitamin d2 formed after the exposure on day 1 , as the high recovery of vitamin d2 indicated that there was no significant degradation or transport of vitamin d2 from the mushrooms . the yield of mushrooms obtained during the three days of experiment a after ( a ) no exposure to uv - b , exposure to supplementary uv - b of 200 mj cm , ( b ) the first day and ( c ) repeated every day . the vitamin d2 concentration in mushrooms found during the three days of experiment a after exposure to supplementary uv - b of 200 mj cm ( a ) the first day , and ( b ) repeated every day . recovery of vitamin d2 calculated on basis of production area for three days of experiment a after one exposure to supplementary uv - b of 200 mj cm the first day based on the biomass of small remaining mushrooms and the assumption that their vitamin d2 content equalled that measured in large harvested mushrooms . when the uv - b exposure was repeated at daily intervals ( day 13 ) , the vitamin d2 content increased to 33 g 100 g. these results suggest that repeated exposure to uv - b is needed just prior to harvest and during all days of a flush to ensure a high level of vitamin d2 in the final product . the increase in the vitamin d2 content after repeated exposure may be explained by a carryover of vitamin d2 formed in the small mushrooms left growing for the next day . there was no indication that one exposure to uv - b stimulated or inhibited the formation of vitamin d2 at later exposures . the vitamin d2 content was much lower in cultivated mushrooms exposed to sunlight of 200 mj cm uv - b or in wild mushrooms samples compared to mushrooms exposed to supplementary uv - b ( table 1 ) . the samples of cultivated mushrooms without exposure to uv - b or of miscellaneous origin ( strains and producers ) were found to have the lowest content of vitamin d2 . to our knowledge , the vitamin d2 content of 1.5 g 100 g is the first report on wild agaricus sp . and cultivated a. bisporus exposed to 200 mj cm uv - b in sunlight were surprising considering the results of other reports and the present study of cultivated a. bisporus forming high amounts of vitamin d2 when exposed to supplementary uv - b ( 6 , 4 , 8) . even though a. bisporus does not produce as high levels of vitamin d2 after uv exposure as wild c. tubaeformis ( 2 ) and cultivated pleurotus ostreatus ( 5 ) . the difference between the effect of uv - b from sunlight and from the supplementary uv - b unit may have been caused by differences in the distribution of wavelengths within the range of uv - b that are integrated in the measurement of the uv - b sensor . the tubes of the uv - b unit emit some radiation of wavelengths shorter than 290 nm ( fig . 1 ) , which is the part of the uv - b range that is almost absent in sunlight after absorption in the earth 's atmosphere ( 9 ) . this short - waved uv - b is close to the uv - c range that has , however , been found to be less effective than uv - b in the conversion of ergosterol to vitamin d2 ( 5 ) . lentinus edodes was found to have higher vitamin d2 content when harvested on sunny as compared to cloudy days ( 10 as reported in ( 4 ) ) . the uv - exposure is similar to the exposure of wild mushrooms that is not reported to cause toxic elements in mushrooms . the level of vitamin d2 content in wild and cultivated mushrooms with or without exposure to sunlight ( table 1 ) is comparable to that of , for example , 0.40.7 g of vitamin d3 in 100 g of pork and beef meat and 0.20.4 g of vitamin d3 in 100 g of a variety of cheeses ( www.foodcomp.dk ) . commercial growers may consider using uv - b during the growth phase for vitamin d2 formation in white button mushrooms . in this case , the need for prolonged exposure time is less critical as treatment times do not have to be confined to the time available during the post - harvest processing on a conveyor belt . instead , an automatic moving uv - b unit can be constructed , for example , for overnight exposure of mushrooms on the growing shelves just prior to harvest . with the high intensities used in the present study , doses of 200 mj cm could be obtained with an exposure time of < 2 min and resulting in vitamin d2 contents of around 24 g 100 g. at this vitamin d2 level , approximately 35 g of mushroom is equivalent to the recommended daily intake of 7.5 g vitamin d2 for adults in denmark . the upper safe limit for daily intake of 50 g vitamin d2 should be taken into consideration ( 11 ) . mushrooms with a vitamin d2 content of 24 g 100 g would contribute to 26% of the recommended daily intake of 7.5 g based on the average intake of mushrooms by adults of 3 kg yr in 2006 in denmark ( 12 ) . therefore , uv - b - enhanced vitamin d2 levels in mushrooms provide a potentially important food source of vitamin d. the close to linear relationship between uv - b dose and formation of vitamin d2 at exposure just prior to harvest makes it possible to produce mushrooms with a well - defined content of vitamin d2 . therefore , mushroom products will be a rich source for increased intake of vitamin d2 in food . due to the dilution of vitamin d2 by the fast growth of the mushrooms , uv - b exposure needs to be repeated daily during the harvest flush to maintain a high content of vitamin d2 in the end product . supplementary uv - b from tubes is a more effective source of uv - b for the formation of vitamin d2 in mushrooms as compared to natural sunlight that gave a low content of vitamin d2 equivalent to that found in wild agaricus sp .

two experiments were carried out with exposure of mushrooms to uv - b doses in the range of 0250mj cm over three days ( experiment a ) , and exposure of mushrooms to uv - b doses of 2202,400 mj cm during one day ( experiment b ) . a transportable and adjustable uv - b unit was constructed using six uv - b tubes ( medical therapy/12 , philips , eindhoven , the netherlands ) giving a homogeneous distribution of uv - b over an area of 0.60.4 m . the intensity of uv - b was 1.88 mw cm at a distance of 0.25 m between the mushrooms and the uv - b tubes . the spectral distribution of the uv - b unit was measured by a spectrometer ( avaspec-2048 , avantes , eerbeek , the netherlands ) that was calibrated with a standard of spectral irradiance ol fel - c ( optronic laboratories , orlando , fa , usa ) , a lamp that includes wavelengths down to 250 nm . the uv - b doses in the different treatments were measured by the accumulated intensity over time by use of a uv - b photometer ( il1400 sel005/tls312/td ; international light technologies , ma , usa ) . the spectral distribution of the uv - b unit used for treatments of supplementary uv - b during the growth phase and post - harvest . white button mushrooms ( agaricus bisporus ( lange ) imbach ) were grown at 18c day and night temperature and 88% relative humidity on horse manure / barley straw compost at the commercial production facility of egehj champignon a / s , veflinge , denmark in 2009 . experiment a was initiated on the second sampling day of the second flush ( 7-day harvest cycle ) , 36 days after inoculation of the compost with mycelium . in both experiments , the mushrooms were sampled for yield and vitamin d2 analyses at harvest , according to the sampling practice for commercial harvest - ready mushrooms . in experiment a , 28 plots of 0.40.4 m were established on the shelves with growing mushrooms in a complete randomised statistical design with four blocks , two in each of two growth chambers giving four replicate plots per treatment . by varying the duration of exposure , the treatments were 0 , 100 , 150 , 200 and 250 mj m supplementary uv - b on the first day ; and 200 mj cm given repeatedly once a day on the first , second and third day of the experiment ( 3200 mj cm ) . all plots were sampled immediately after exposure and also on the three consecutive days ( at 24 hour periods ) after the first day exposure to 0 or 200 mj cm and after the repeated exposure to 200 mj cm . additional samples were taken for post - harvest treatments of exposure to 200 mj cm natural uv - b in sunlight for approximately 20 min at mid day , or following overnight cooling ( 2c ) , re - adjustment to 18c and exposure to 200 mj cm supplementary uv - b . additional production areas from the four blocks were sampled the three days forestimation ofbiomass of small mushrooms , thatremained after harvest to grow for next day harvest . in experiment b , seven plots of 0.60.4 m were established on the shelves with growing mushrooms in a growth chamber . treatments at 220 , 500 , 1,000 and 2,400 mj m supplementary uv - b were given to the plots in a complete randomised statistical design with no replications in the range of 2201,000 mj m and six replications of the 2,400 mj m treatment . in addition , four samples of a. bisporus of miscellaneous origin ( strains and producers ) were purchased for determination of vitamin d2 content . prior to analysis , the frozen samples of mushrooms were thawed to room temperature , homogenised for 30 sec ( type 320 , moulinex , group seb , f ) and freeze - dried ( christ beta 18 , martin christ gefriertrochnungsanlagen gnbh , osterode , d ) . the analytical method and the equipment used to determine vitamin d2 in the mushrooms were a modification of a method used for meat ( 7 ) . in short , 1 g freeze - dried mushrooms were taken for analysis , and vitamin d3 was added as an internal standard according to en12821 . due to the extremely high amount of ergosterol in the samples , the combination of a silica and an amino column was replaced by a shorter silica column ( luna , si 60 , 3 m , 1504.6 mm , phenomenex , torrance , ca , usa ) in combination with an isocratic mobile phase consisting of 2-propanol : methyl - tert - butyl - ether : cyclohexan : n - heptan ( 0.7:2:48.65:48.65 ) . the analysis was run in a laboratory accredited after iso 17025 for the original analytical method , and the correctness for the method was controlled by participation in the food analysis performance assessment scheme ( www.fapas.com ) . recovery of vitamin d2 produced after the first day exposure to uv - b was calculated per unit production area over the three days of experiment based on the biomass of large harvested and small remaining mushrooms . , a transportable and adjustable uv - b unit was constructed using six uv - b tubes ( medical therapy/12 , philips , eindhoven , the netherlands ) giving a homogeneous distribution of uv - b over an area of 0.60.4 m . the intensity of uv - b was 1.88 mw cm at a distance of 0.25 m between the mushrooms and the uv - b tubes . the spectral distribution of the uv - b unit was measured by a spectrometer ( avaspec-2048 , avantes , eerbeek , the netherlands ) that was calibrated with a standard of spectral irradiance ol fel - c ( optronic laboratories , orlando , fa , usa ) , a lamp that includes wavelengths down to 250 nm . the uv - b doses in the different treatments were measured by the accumulated intensity over time by use of a uv - b photometer ( il1400 sel005/tls312/td ; international light technologies , ma , usa ) . the spectral distribution of the uv - b unit used for treatments of supplementary uv - b during the growth phase and post - harvest . white button mushrooms ( agaricus bisporus ( lange ) imbach ) were grown at 18c day and night temperature and 88% relative humidity on horse manure / barley straw compost at the commercial production facility of egehj champignon a / s , veflinge , denmark in 2009 . experiment a was initiated on the second sampling day of the second flush ( 7-day harvest cycle ) , 36 days after inoculation of the compost with mycelium . in both experiments , the mushrooms were sampled for yield and vitamin d2 analyses at harvest , according to the sampling practice for commercial harvest - ready mushrooms . in experiment a , 28 plots of 0.40.4 m were established on the shelves with growing mushrooms in a complete randomised statistical design with four blocks , two in each of two growth chambers giving four replicate plots per treatment . by varying the duration of exposure , the treatments were 0 , 100 , 150 , 200 and 250 mj m supplementary uv - b on the first day ; and 200 mj cm given repeatedly once a day on the first , second and third day of the experiment ( 3200 mj cm ) . all plots were sampled immediately after exposure and also on the three consecutive days ( at 24 hour periods ) after the first day exposure to 0 or 200 mj cm and after the repeated exposure to 200 mj cm . additional samples were taken for post - harvest treatments of exposure to 200 mj cm natural uv - b in sunlight for approximately 20 min at mid day , or following overnight cooling ( 2c ) , re - adjustment to 18c and exposure to 200 mj cm supplementary uv - b . additional production areas from the four blocks were sampled the three days forestimation ofbiomass of small mushrooms , thatremained after harvest to grow for next day harvest . b , seven plots of 0.60.4 m were established on the shelves with growing mushrooms in a growth chamber . treatments at 220 , 500 , 1,000 and 2,400 mj m supplementary uv - b were given to the plots in a complete randomised statistical design with no replications in the range of 2201,000 mj m and six replications of the 2,400 mj m treatment . in the period of late july to october 2009 , four composite samples of each 23 wild growing mushrooms ( agaricus sp . ) in addition , four samples of a. bisporus of miscellaneous origin ( strains and producers ) were purchased for determination of vitamin d2 content . prior to analysis , the frozen samples of mushrooms were thawed to room temperature , homogenised for 30 sec ( type 320 , moulinex , group seb , f ) and freeze - dried ( christ beta 18 , martin christ gefriertrochnungsanlagen gnbh , osterode , d ) . the analytical method and the equipment used to determine vitamin d2 in the mushrooms were a modification of a method used for meat ( 7 ) . due to the extremely high amount of ergosterol in the samples , the combination of a silica and an amino column was replaced by a shorter silica column ( luna , si 60 , 3 m , 1504.6 mm , phenomenex , torrance , ca , usa ) in combination with an isocratic mobile phase consisting of 2-propanol : methyl - tert - butyl - ether : cyclohexan : n - heptan ( 0.7:2:48.65:48.65 ) . the analysis was run in a laboratory accredited after iso 17025 for the original analytical method , and the correctness for the method was controlled by participation in the food analysis performance assessment scheme ( www.fapas.com ) . recovery of vitamin d2 produced after the first day exposure to uv - b was calculated per unit production area over the three days of experiment based on the biomass of large harvested and small remaining mushrooms . , results from experiments a and b showed a close to linear relationship between dose of uv - b and formation of vitamin d2 at 01,000 mj cm supplementary uv - b during the growth phase . the outputs from the linear regressions were very similar with a - factors of 0.118 and 0.108 for the range of 0250 mj cm and 01,000 mj cm , respectively , and high correlation coefficients ( fig . 2008 ( 6 ) following post - harvest exposure in the range of 5001,000 mj cm supplementary uv - b after overnight cooling of white button mushrooms . however , in the present study , the a - factor of the linear regression was 0.11 at exposure during the growth phase , compared to 0.06 calculated from roberts et al . thus , exposure , for example , to 500 mj cm during the growth phase formed vitamin d2 of approximately 57 g 100 g fresh weight compared to 37 g 100 g at post - harvest exposure ( 6 ) . however , the difference between the two studies can not be ascribed to a higher efficiency of vitamin d2 conversion at exposure during the growth phase . in the present study , mushrooms sampled after overnight cooling and post - harvest exposure were found to have a higher vitamin d2 content compared to those exposed during the growth phase , and this difference was not caused by effects of overnight cooling on dry matter content ( table 1 ) . the higher conversion of vitamin d2 in the present study compared with that of roberts et al . orientation of the mushrooms to the uv source has also been a factor that contributes to vitamin d2 level , that is , the content of vitamin d2 varies within an individual mushroom ( 8) ; however , in this study and that of roberts et al . finally , also the difference between our hplc - dad and the lc - ms / ms methods used in ( 6 ) may give a bias in the results . the relationship between vitamin d2 concentration in mushrooms and exposure to uv - b dose up to 250 mj cm in experiment a and up to 2,400 mj cm in experiment b. error bars indicate standard errors in experiment a ( n=4 ) , and in experiment b for the highest dose ( n=6 ) . the effect of source of uv - b and timing of exposure in relation to harvest on vitamin d2 in wild and cultivated agaricus mushrooms within columns means followed by different letters are significantly different . the formation of vitamin d2 levelled off at a content of 164 g 100 g at the high dose of 2,400 mj cm supplementary uv - b as also reported for doses beyond 1,200 mj cm by others ( 5 , 6 ) . in this study , a clear browning effect was observed on the surface of the exposed mushrooms indicating that at high doses of uv - b , there was damage to the surface cells ( 8) . thus , very high doses of uv - b are relatively less efficient for conversion of ergosterol to vitamin d2 and cause surface discolouration . uv - b exposure did not affect the growth rate of the mushrooms in experiment a ( fig . the average yields were 2.25 , 4.83 and 3.36 kg m , and the number of mushrooms were 236 , 322 and 163 pieces m on the first , second and third day of the flush , respectively . over the three days , the vitamin d2 content of mushrooms decreased sharply from 24 to 3 g 100 g when measured after one exposure at 200 mj cm of supplementary uv - b ( fig . by day 3 , 94% of the vitamin d2 formed during one exposure to uv - b on day 1 could be accounted for in the large harvested and the small remaining mushrooms ( table 2 ) . thus , the fast growth rate of the mushrooms diluted the vitamin d2 formed after the exposure on day 1 , as the high recovery of vitamin d2 indicated that there was no significant degradation or transport of vitamin d2 from the mushrooms . the yield of mushrooms obtained during the three days of experiment a after ( a ) no exposure to uv - b , exposure to supplementary uv - b of 200 mj cm , ( b ) the first day and ( c ) repeated every day . the vitamin d2 concentration in mushrooms found during the three days of experiment a after exposure to supplementary uv - b of 200 mj cm ( a ) the first day , and ( b ) repeated every day . recovery of vitamin d2 calculated on basis of production area for three days of experiment a after one exposure to supplementary uv - b of 200 mj cm the first day based on the biomass of small remaining mushrooms and the assumption that their vitamin d2 content equalled that measured in large harvested mushrooms . when the uv - b exposure was repeated at daily intervals ( day 13 ) , the vitamin d2 content increased to 33 g 100 g. these results suggest that repeated exposure to uv - b is needed just prior to harvest and during all days of a flush to ensure a high level of vitamin d2 in the final product . the increase in the vitamin d2 content after repeated exposure may be explained by a carryover of vitamin d2 formed in the small mushrooms left growing for the next day . there was no indication that one exposure to uv - b stimulated or inhibited the formation of vitamin d2 at later exposures . the vitamin d2 content was much lower in cultivated mushrooms exposed to sunlight of 200 mj cm uv - b or in wild mushrooms samples compared to mushrooms exposed to supplementary uv - b ( table 1 ) . the samples of cultivated mushrooms without exposure to uv - b or of miscellaneous origin ( strains and producers ) were found to have the lowest content of vitamin d2 . , while higher contents have been reported for other wild grown species such as cantharellus cibarius and c. tubaeformis ( 1013 and 2130 g 100 g , respectively ) as well as for boletus edulis ( 359 g 100 g ) ( 1 , 2 ) . and cultivated a. bisporus exposed to 200 mj cm uv - b in sunlight were surprising considering the results of other reports and the present study of cultivated a. bisporus forming high amounts of vitamin d2 when exposed to supplementary uv - b ( 6 , 4 , 8) . even though a. bisporus does not produce as high levels of vitamin d2 after uv exposure as wild c. tubaeformis ( 2 ) and cultivated pleurotus ostreatus ( 5 ) . the difference between the effect of uv - b from sunlight and from the supplementary uv - b unit may have been caused by differences in the distribution of wavelengths within the range of uv - b that are integrated in the measurement of the uv - b sensor . 1 ) , which is the part of the uv - b range that is almost absent in sunlight after absorption in the earth 's atmosphere ( 9 ) . this short - waved uv - b is close to the uv - c range that has , however , been found to be less effective than uv - b in the conversion of ergosterol to vitamin d2 ( 5 ) . the level of vitamin d2 content in wild and cultivated mushrooms with or without exposure to sunlight ( table 1 ) is comparable to that of , for example , 0.40.7 g of vitamin d3 in 100 g of pork and beef meat and 0.20.4 g of vitamin d3 in 100 g of a variety of cheeses ( www.foodcomp.dk ) . in this case , the need for prolonged exposure time is less critical as treatment times do not have to be confined to the time available during the post - harvest processing on a conveyor belt . with the high intensities used in the present study , doses of 200 mj cm could be obtained with an exposure time of < 2 min and resulting in vitamin d2 contents of around 24 g 100 g. at this vitamin d2 level , approximately 35 g of mushroom is equivalent to the recommended daily intake of 7.5 g vitamin d2 for adults in denmark . mushrooms with a vitamin d2 content of 24 g 100 g would contribute to 26% of the recommended daily intake of 7.5 g based on the average intake of mushrooms by adults of 3 kg yr in 2006 in denmark ( 12 ) . therefore , uv - b - enhanced vitamin d2 levels in mushrooms provide a potentially important food source of vitamin d. the close to linear relationship between uv - b dose and formation of vitamin d2 at exposure just prior to harvest makes it possible to produce mushrooms with a well - defined content of vitamin d2 . due to the dilution of vitamin d2 by the fast growth of the mushrooms , uv - b exposure needs to be repeated daily during the harvest flush to maintain a high content of vitamin d2 in the end product . supplementary uv - b from tubes is a more effective source of uv - b for the formation of vitamin d2 in mushrooms as compared to natural sunlight that gave a low content of vitamin d2 equivalent to that found in wild agaricus sp .

hepatitis c virus ( hcv ) is one of the main causes of liver cirrhosis and hepatocellular carcinoma , where approximately 350,000 deaths occur each year due to hcv - related liver diseases . the latest estimation indicates that more than 185 million people around the world ( 2.8% of the world population ) have been infected with hcv . in china , approximately 2.2% of the population on average were infected with hcv ( most of which were genotype 1b ) , with a distribution range from 2.1% in fujian province to 9.6% in henan province . to inhibit the global prevalence of hcv infection , various strategies with regard to therapeutic medication the traditional standard of care contains pegylated interferon plus ribavirin ( rbv ) , which could help genotype 1b patients to achieve sustained virological response ( svr ) rates of 45% , further up to 6575% for genotype 2/3 patients . although patients chronically infected with hepatitis c virus ( chc ) in asia carries more beneficial il28b genotypes , nearly 20% of them could not achieve svr . in this regard , more than 30 newly developed direct - acting antivirals ( daas ) have readily been approved in the europe , north american , and japan or are currently being in phase 2/3 clinical trials . daas mainly encompass ns3/4a protease inhibitors ( pis , such as telaprevir , boceprevir , simeprevir , mk-8742 , asunaprevir , etc . ) , ns5a inhibitors ( such as daclatasvir , ledipasvir [ ldv ] , mk-5172 , etc . ) , and ns5b polymerase inhibitors ( such as sofosbuvir [ sof ] , dasabuvir , etc . ) . in general , they are effective toward pan - genotypic hcv by helping patients to achieve high svr rates up to 100% , especially genotype 1b chc patients . the high replication rate of hcv , along with the low fidelity and poor proof - reading of its polymerase , generates a highly variable virus population denoted as quasispecies . the creation of variants encoding amino acid substitutions may result in reduced susceptibility to antiviral agents . compared with hiv , the rate of emergence of resistant variants is much higher for hcv . it is estimated that the error rate of the hcv polymerase is 10-fold higher than that of hiv , and the rate of production of rna virus is 100-fold higher than that of hiv . data from clinical trials have revealed that baseline resistance - associated variants ( ravs ) were correlated with treatment outcomes of daas . for example , patients presented pis ravs , such as q80k , d168e / v , ns5a ravs l31m / v , y93h , and ns5b polymerase inhibitors ravs l159f , c316n , v321a achieved svr rates much lower than those who did not . ravs could exist even as minor variants at baseline , which would rapidly become the main strain under selective pressure , subsequently leading to a treatment - failure . however , compared with population sequencing , the expense for ultra - deep sequencing was relatively high . since none of the daas has been approved in china , there have been seldom reports by far on baseline daas ravs in our country . in turn , it remains unclear if it is necessary to detect baseline daas ravs before daas treatment . in this study , we perform both population sequencing and deep sequencing to detect baseline daas ravs in genotype 1b chc patients in china . serum samples were derived from 160 treatment - nave patients chronically infected with genotype 1b hcv . population sequencing was carried out in 160 patients to figure out the prevalence of ravs in gt1b treatment - nave chc patients . ultra - deep sequencing was carried out in 23 patients to find out minor variants with low mutation frequencies . the study was conducted in accordance with the international society for pharmacoepidemiology guidelines for good epidemiology practices and applicable regulatory requirements . hcv viral load ( abbott real time hcv ; abbott laboratories , des plaines , il , usa ) , hcv genotype ( versant hcv genotype 2.0 [ lipa ] ; siemens healthcare diagnostics , tarrytown , ny , usa ) , and il28b genotype ( iplex gold ; sequenom , san diego , ca , usa ) , were conducted . hcv rna was extracted from 140 l ethylenediaminetetraacetic acid anticoagulated plasma using the qiaamp viral rna mini kit ( qiagen , hilden , germany ) after centrifugation at 24,000 g for 1 h at 4c . the extracted rna was transcribed to cdna and the ns3 , ns5a , and ns5b fragments were amplified by polymerase chain reaction ( pcr ) in a one - step process ( superscript iii one - step rt - pcr with platinum taq kit ; invitrogen , carlsbad , ca , usa ) following the manufacturers instructions . the primers used are listed in table 1 . cycling conditions included an initial cdna synthesis step at 55c for 30 min , followed by a denaturation step at 94c for 2 min , 40 cycles of pcr amplification ( 94c for 15 s , 58c for 30 s , 68c for 2 min ) , and a final 10 min extension step at 68c . the pcr mix contained 25 l of 2 reaction mix , 1 l of each primer , 8 l of extracted rna as template , and nuclease - free h2o to a final volume of 50 l . the pcr reaction was carried out with thermal cycler pcr machine ( thermo , ca , usa ) . the pcr products were purified using the qiaquick pcr purification kit ( qiagen , hilden , germany ) . in the deep sequencing study , the amplified ns3 , ns5a , and ns5b fragments were modified by the multiplexing sample preparation kit ( illumina , san diego , ca , usa ) , and sequence analysis was performed by illumina hiseq 2000 . in the population study , the amplified fragments were subjected to direct sequencing by abi 3730xl dna sequencer ( abi , usa ) . primers used for amplifying the ns3 , ns5a and ns5b regions the bioedit 7.09 software ( borland company , usa ) was used for editing the sequences . translation from the nucleic acids sequences into amino acids sequences was performed with revtrans 2.0 ( technical university of denmark , denmark ) . the sequence alignment was performed with clustal x 2.0 ( university college dublin , irland ) . the reported amino acid substitutions associated resistance to daas according to previously reported data were scored : v36a / m , f43s , t54s , q80k , s122 g , r155k , d168e / v , v170i , etc . , for ns3/4a pis ; and l23f , l28 t , r30q , l31m / v , p32l , q54h , p58s , q62h , y93h , etc . , for ns5a protein inhibitors ; t19s , n142 t , s282 t , a442 t , s556 g , etc . , for ns5b ravs . nonparameters mann whitney u - test , chi - square test with yates correction , or fisher exact test was performed . serum samples were derived from 160 treatment - nave patients chronically infected with genotype 1b hcv . population sequencing was carried out in 160 patients to figure out the prevalence of ravs in gt1b treatment - nave chc patients . ultra - deep sequencing was carried out in 23 patients to find out minor variants with low mutation frequencies . the study was conducted in accordance with the international society for pharmacoepidemiology guidelines for good epidemiology practices and applicable regulatory requirements . hcv viral load ( abbott real time hcv ; abbott laboratories , des plaines , il , usa ) , hcv genotype ( versant hcv genotype 2.0 [ lipa ] ; siemens healthcare diagnostics , tarrytown , ny , usa ) , and il28b genotype ( iplex gold ; sequenom , san diego , ca , usa ) , were conducted . hcv rna was extracted from 140 l ethylenediaminetetraacetic acid anticoagulated plasma using the qiaamp viral rna mini kit ( qiagen , hilden , germany ) after centrifugation at 24,000 g for 1 h at 4c . the extracted rna was transcribed to cdna and the ns3 , ns5a , and ns5b fragments were amplified by polymerase chain reaction ( pcr ) in a one - step process ( superscript iii one - step rt - pcr with platinum taq kit ; invitrogen , carlsbad , ca , usa ) following the manufacturers instructions . cycling conditions included an initial cdna synthesis step at 55c for 30 min , followed by a denaturation step at 94c for 2 min , 40 cycles of pcr amplification ( 94c for 15 s , 58c for 30 s , 68c for 2 min ) , and a final 10 min extension step at 68c . the pcr mix contained 25 l of 2 reaction mix , 1 l of each primer , 8 l of extracted rna as template , and nuclease - free h2o to a final volume of 50 l . the pcr reaction was carried out with thermal cycler pcr machine ( thermo , ca , usa ) . the pcr products were purified using the qiaquick pcr purification kit ( qiagen , hilden , germany ) . in the deep sequencing study , the amplified ns3 , ns5a , and ns5b fragments were modified by the multiplexing sample preparation kit ( illumina , san diego , ca , usa ) , and sequence analysis was performed by illumina hiseq 2000 . in the population study , the amplified fragments were subjected to direct sequencing by abi 3730xl dna sequencer ( abi , usa ) . the bioedit 7.09 software ( borland company , usa ) was used for editing the sequences . translation from the nucleic acids sequences into amino acids sequences was performed with revtrans 2.0 ( technical university of denmark , denmark ) . the sequence alignment was performed with clustal x 2.0 ( university college dublin , irland ) . the reported amino acid substitutions associated resistance to daas according to previously reported data were scored : v36a / m , f43s , t54s , q80k , s122 g , r155k , d168e / v , v170i , etc . , for ns3/4a pis ; and l23f , l28 t , r30q , l31m / v , p32l , q54h , p58s , q62h , y93h , etc . , for ns5a protein inhibitors ; t19s , n142 t , s282 t , a442 t , s556 g , etc . , for ns5b ravs . nonparameters mann whitney u - test , chi - square test with yates correction , or fisher exact test was performed . in total , 160 treatment - nave patients chc genotype 1b were enrolled in this study . fifty - two percent of patients were male , and the median age at testing was 46.5 ( range , 2765 ) years [ table 2 ] . baseline characteristics of the study population bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; hcv : hepatitis c virus . one hundred and forty - five patients ( 90.6% ) were successfully amplified with the ns3 fragments , 71% ( 103/145 ) of whom presented at least one pis ravs . about 56.6% ( 82/145 ) of the patients presented s122 g variant , 33.1% ( 48/145 ) of the patients presented v132i variant , 13.1% ( 19/145 ) of the patients presented v170i variant , and 5.5% ( 8/145 ) of the patients presented t54s variant [ table 3 ] . nucleoside changes at codon position 36 , 55 , and 80 were detected but could cause only synonymous substitutions . number of patients harboring ns3/4a pis and ns5a protein inhibitors ravs daas : direct - acting antivirals ; ravs : resistance - associated variants ; pi : protease inhibitor . as results by ultra - deep sequencing , the average mutation frequencies were 23.6% for s122 g variants , 75.4% for v132i and 33.8% for v170i . all of the ravs detected by ultra - deep sequencing could be detected by population sequencing . about 92.5% ( 148/160 ) of the patients were successfully amplified with the ns5a fragments , 14.9% ( 22/148 ) of whom presented at least one ns5a protein inhibitors ravs . about 6.1% ( 9/148 ) of the patients presented q30r variant , 8.8% ( 13/148 ) of the patients presented q54h variant , 10.1% ( 14/148 ) of the patients presented y93h variant . only synonymous substitutions were detected at amino acid sites 23 , 28 , and 32 . nucleoside substitutions from a to g at 6347 site were detected in 42.6% ( 63/148 ) patients , which could only cause synonymous substitutions at amino acid site 31 [ table 3 ] . according to the results of ultra - deep sequencing , the average mutation frequencies were 61.0% for q30r , 13.5% for q54h , 95.7% for q62h , 14% for y93h . interestingly , the daclatasvir , ldv , and mk-8742 ravs l31 m have not been detected by population sequencing in any one of the 148 patients but have been detected by ultra - deep sequencing in 3 patients , with average mutation frequencies of 4.7% . the fragments of ns5b were successfully amplified in 85.6% ( 137/160 ) patients . nearly , all patients presented at least one rav to polymerase inhibitors at baseline , except for one . when considering ravs of nucleoside inhibitors , 94.2% ( 129/137 ) of the patients were detected harboring the c316n variant . when il28b genotype was regarded , the prevalence of c316n variant between patients who carried cc and non - cc genotype was identical . there were also no significant differences between the patients with and without c316n variant for clinical characteristics , except for the hemoglobin levels ( p = 0.004 ) . , l159f variant , which was always detected with c316n variant simultaneously , has not been detected in any of patients in our study . at the codon position 282 of ns5b , the primary sof resistant associated variant , only synonymous variant ( agc<->agt ) was detected . other important ravs to ns5b polymerase inhibitors , such as l320f , v321a , and v499a , were detected in our study . in addition , only three patients presented s142 at baseline , whereas none of the patients exhibited the resistant type n142 t [ tables 4 and 5 ] . number of patients harboring ns5b polymerase inhibitors ravs ( n = 137 ) ravs : resistance - associated variants . baseline characteristics of the patients with and without c316n variant bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; peg - ifn : pegylated interferon ; rbv : ribavirin ; svr : sustained virological response ; hcv : hepatitis c virus . it is worth - mentioning that some ravs of nonnucleoside inhibitors were also detected in our study , such as v494a / t / l , v499a ( thumb i ) ; m423i / t , m424v , m426 t ( thumb ii ) ; h95q , m414i / l , s556g / n ( palm i ) ; s365a / l / s / t ( palm ii ) , etc . , regarding the cross - resistance between these four categories , we have worked out the possible overlap of ravs of different sorts of nonnucleoside inhibitors . most of the patients presented ravs belonging to certain one category , for instance , the most epidemic rav c316n pertained to palm ii . as results by ultra - deep sequencing , the average mutation frequencies were 41.7% for c316n , 24.8% for a338v , 26.5% for a442 t , and 31.4% for s556 g . interestingly , l419 m variant , which was reported to be resistant to vch-759 and vch-916 , had not been detected by population sequencing and were detected in two patients as minor variants ( with average mutation frequencies of 1.6% ) . in total , 160 treatment - nave patients chc genotype 1b were enrolled in this study . fifty - two percent of patients were male , and the median age at testing was 46.5 ( range , 2765 ) years [ table 2 ] . baseline characteristics of the study population bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; hcv : hepatitis c virus . one hundred and forty - five patients ( 90.6% ) were successfully amplified with the ns3 fragments , 71% ( 103/145 ) of whom presented at least one pis ravs . about 56.6% ( 82/145 ) of the patients presented s122 g variant , 33.1% ( 48/145 ) of the patients presented v132i variant , 13.1% ( 19/145 ) of the patients presented v170i variant , and 5.5% ( 8/145 ) of the patients presented t54s variant [ table 3 ] . nucleoside changes at codon position 36 , 55 , and 80 were detected but could cause only synonymous substitutions . number of patients harboring ns3/4a pis and ns5a protein inhibitors ravs daas : direct - acting antivirals ; ravs : resistance - associated variants ; pi : protease inhibitor . as results by ultra - deep sequencing , the average mutation frequencies were 23.6% for s122 g variants , 75.4% for v132i and 33.8% for v170i . all of the ravs detected by ultra - deep sequencing could be detected by population sequencing . about 92.5% ( 148/160 ) of the patients were successfully amplified with the ns5a fragments , 14.9% ( 22/148 ) of whom presented at least one ns5a protein inhibitors ravs . about 6.1% ( 9/148 ) of the patients presented q30r variant , 8.8% ( 13/148 ) of the patients presented q54h variant , 10.1% ( 14/148 ) of the patients presented y93h variant . only 3 patients presented p58s variant , and only 5 patients presented q62h variant . only synonymous substitutions were detected at amino acid sites 23 , 28 , and 32 . nucleoside substitutions from a to g at 6347 site were detected in 42.6% ( 63/148 ) patients , which could only cause synonymous substitutions at amino acid site 31 [ table 3 ] . according to the results of ultra - deep sequencing , the average mutation frequencies were 61.0% for q30r , 13.5% for q54h , 95.7% for q62h , 14% for y93h . interestingly , the daclatasvir , ldv , and mk-8742 ravs l31 m have not been detected by population sequencing in any one of the 148 patients but have been detected by ultra - deep sequencing in 3 patients , with average mutation frequencies of 4.7% . the fragments of ns5b were successfully amplified in 85.6% ( 137/160 ) patients . nearly , all patients presented at least one rav to polymerase inhibitors at baseline , except for one . when considering ravs of nucleoside inhibitors , 94.2% ( 129/137 ) of the patients were detected harboring the c316n variant . when il28b genotype was regarded , the prevalence of c316n variant between patients who carried cc and non - cc genotype was identical . there were also no significant differences between the patients with and without c316n variant for clinical characteristics , except for the hemoglobin levels ( p = 0.004 ) . , l159f variant , which was always detected with c316n variant simultaneously , has not been detected in any of patients in our study . at the codon position 282 of ns5b , the primary sof resistant associated variant , only synonymous variant ( agc<->agt ) was detected . other important ravs to ns5b polymerase inhibitors , such as l320f , v321a , and v499a , were detected in our study . in addition , only three patients presented s142 at baseline , whereas none of the patients exhibited the resistant type n142 t [ tables 4 and 5 ] . number of patients harboring ns5b polymerase inhibitors ravs ( n = 137 ) ravs : resistance - associated variants . baseline characteristics of the patients with and without c316n variant bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; peg - ifn : pegylated interferon ; rbv : ribavirin ; svr : sustained virological response ; hcv : hepatitis c virus . it is worth - mentioning that some ravs of nonnucleoside inhibitors were also detected in our study , such as v494a / t / l , v499a ( thumb i ) ; m423i / t , m424v , m426 t ( thumb ii ) ; h95q , m414i / l , s556g / n ( palm i ) ; s365a / l / s / t ( palm ii ) , etc . , regarding the cross - resistance between these four categories , we have worked out the possible overlap of ravs of different sorts of nonnucleoside inhibitors . most of the patients presented ravs belonging to certain one category , for instance , the most epidemic rav c316n pertained to palm ii . as results by ultra - deep sequencing , the average mutation frequencies were 41.7% for c316n , 24.8% for a338v , 26.5% for a442 t , and 31.4% for s556 g . interestingly , l419 m variant , which was reported to be resistant to vch-759 and vch-916 , had not been detected by population sequencing and were detected in two patients as minor variants ( with average mutation frequencies of 1.6% ) . in our study , as many as 71% of genotype 1b treatment - nave patients presented at least one ns3/4a pis rav . the most prevalent variant was s122 g ( 56.6% ) , which has been confirmed as one of the major ravs of simeprevir ( one of the first - generation second - wave pis ) . in concerto trial conducted in japan , the baseline prevalence of s122 g variant was relatively lower ( 34.9% ) than that of our findings , while the baseline prevalence of v170i variant was relatively higher ( 39.6% vs. 13.1% ) than our findings . t54s variant detected in our study was also correlated with resistance to simeprevir , with relatively low prevalence ( 5.5% ) . mutations associated with first - generation first - wave pis detected in this study were t54s ( telaprevir and boceprevir ) , s122 g ( telaprevir ) , and v170i ( telaprevir ) . however , these ravs only conferred low - level resistance to these two licensed daas . with respect to other reports , none of other baseline polymorphisms correlated with treatment outcomes of pis were detected in our study . for example , r155k and d168e / v variants were ravs of asunaprevir ( had licensed in combination with daclatasvir as interferon [ ifn]-free regimens in japan ) and mk-5172 ( one of the second generation pis ) , none of which were found . this discrepancy might be due to the fact that virus of different quasispecies even within the same subtype has different mutations . since all of the ravs detected by ultra - deep sequencing could be detected by population sequencing , we can conclude that population sequencing could qualified for baseline pis ravs screening in china . as one of the most important ravs of most of the ns5a protein inhibitors , y93h variants were detected in 10.1% of patients included in our study . l31 m variants , another mutation associated with treatment outcomes of ldv , daclatasvir , and abt-267 , were only detected by ultra - deep sequencing in three patients with low mutation frequencies . several in vitro studies have reported that single l31 m variant could only lead to 3-fold change with ec50 of daclatasvir . single y93h variant could lead to 24-fold change of it while combination mutations of l31 m plus y93h could lead to 7105-fold change with ec50 of daclatasvir . in addition , single y93h variant could lead to 77-fold change of ec50 of abt-267 while l31 m plus y93h could lead to 142-fold change of it . what 's more , y93h variant could lead to extremely high - level ( 994-fold ) fold - change with ec50 of ldv . although ns5a protein inhibitors were usually used in combination with pis ( such as asunaprevir ) , baseline polymorphisms of l31 m or y93h could make patients to experience virologic relapse more easily and achieved much lower svr rates . data by ultra - deep sequencing revealed that most of the ns5a protein inhibitors ravs detected here were with relatively high mutation frequencies , except for l31 m variants . nevertheless , l31 m variants occurred only in three patients with low mutation frequencies . in this way , population sequencing might be qualified for baseline ns5a protein inhibitors ravs screening in china . the detection of naturally occurring ravs of ns5b polymerase inhibitors at baseline has been reported in numerous studies . some of them may influence the treatment outcomes of daas - containing regimens . in the clinical trials of sof - containing regimens , the primary concern lies in the s282 t , an important variant associated with treatment - failure . however , a representative study conducted by fda from the us suggested that ns5b - c316n was found to be associated with treatment failure when patients chc genotype 1b receiving sof plus rbv therapy , throughout re - systemizing all the data from sof - containing trials where ultra - deep sequencing was performed . in the clinical trial p7977 - 2025 , 16/61 posttransplantation patients failed therapy of sof plus rbv , where 6/16 patients harbored c316n at baseline , in other words , all of the six patients did not achieve svr . on the contrary , for the other 45 patients who achieved svr12 , eighty - two percent ( 45/55 ) of the patients who did not carry c316n pretreatment achieved svr12 . however , 66.7% ( 4/6 ) of patients harbored l159f at the same time . in clinical trial neutrino , 4/66 of the patients harbored c316n variant at baseline , 50% of which ( 2/4 ) experienced virologic relapse after receiving treatment of sof plus rbv . in this trial , rav c316n is located in palm ii ( allosteric ) . despite the fact that the majority of ravs of sof reported are at the active sites ( such as s282 t and n142 t ) , position 316 was deemed as the only different residue within 8 a in the ns5b polymerase between hcv genotype 1a and 1b . n316 in hcv gt1b polymerase structure is much larger than c316 , which might lead to the block of the active site of sof . however , c316 has been reported to be relatively conserved in gt1a , but polymorphic in gt1b . data from los alamos databank showed that the prevalence of c316n was much higher in asia than worldwide ( 91.6% vs. 36% ) as well as american continent ( from 0% to 18% ) . in our study , both the ultra - deep sequencing and population sequencing revealed that the prevalence of c316n variant in treatment - nave chinese gt1b chc patients was quite high , which is in good agreement with previous findings . as part of ifn - free regimens , sof in combination with ldv with and without rbv has been one of the most important regimens nowadays . combination treatment could help patients to overcome antiviral resistance more easily . , data showed that the patient presented ns5a - l31m - y93h joined variants as previously stated , simultaneously harbored ns5b - c316n . even in the combination regimens of sof plus ldv , baseline polymorphisms of y93h results by ultra - deep sequencing showed that the mutation frequencies increased dramatically at the time of relapse . both rav s282 t and n142 t were at the active site of ns5b polymerase , but none of the 137 patients exhibited the existence of these two variants . since most of the patients presented ravs of one category , cross - resistance seems less likely to occur . data by ultra - deep sequencing revealed that most of the ns5b polymerase inhibitors ravs detected here were with high mutation frequencies , especially for c316n variants . nevertheless , l419 m variants occurred only in two patients with low mutation frequencies . in this way , population sequencing might qualify for baseline ns5b polymerase inhibitors ravs screening in china . in conclusion , we have employed both the ultra - deep sequencing and population sequencing methods to detect daas ravs in genotype 1b treatment - nave patients , among which the prevalence of variants related with treatment - failure of ns3/4a pis ( s122 g ) , ns5b polymerase inhibitors ( c316n ) and ns5a inhibitors ( y93h ) were quite high , in particular , c316n . currently high prevalence and high mutation frequencies of rav c316n in china might impede the virologic response when distributing sof - containing regimens in the near future . our findings suggest that pretreatment sequencing of hcv genome might need to be performed when patients infected with gt1b hcv receiving daas - containing regimens in china . this work was supported by grants from the china national science and technology major project for infectious diseases control during the 11th 5-year plan period ( no . 2008zx10002 - 013 , 2008zx10002 - 012 ) and 12th 5-year plan period ( no . this work was supported by grants from the china national science and technology major project for infectious diseases control during the 11th 5-year plan period ( no . 2008zx10002 - 013 , 2008zx10002 - 012 ) and 12th 5-year plan period ( no .

background : it has been reported that several baseline polymorphisms of direct - acting antivirals ( daas ) agents resistance - associated variants ( ravs ) would affect the treatment outcomes of patients chronically infected with hepatitis c virus ( chc ) . the aim of this study is to investigate the prevalence of daas ravs in treatment - nave gt1b chc patients.methods:direct sequencing and ultra - deep sequencing of the hcv ns3 , ns5a , and ns5b gene were performed in baseline serum samples of treatment - nave patients infected with genotype 1b hepatitis c virus ( hcvs).results : one hundred and sixty chc patients were studied . complete sequence information was obtained for 145 patients ( ns3 ) , 148 patients ( ns5a ) , and 137 patients ( ns5b ) . treatment - failure associated variants of daas were detected : 56.6% ( 82/145 ) of the patients presented s122 g for simeprevir ( ns3 protease inhibitor ) ; 10.1% ( 14/148 ) of the patients presented y93h for daclatasvir and ledipasvir ( ns5a protein inhibitors ) ; 94.2% ( 129/137 ) of the patients presented c316n for sofosbuvir ( ns5b polymerase inhibitor ) . nearly , all of the daas ravs detected by ultra - deep sequencing could be detected by direct sequencing.conclusions:the majority of genotype 1b chc patients in china present a virus population carrying hcv daas ravs . pretreatment sequencing of hcv genome might need to be performed when patients infected with gt1b hcv receiving daas - containing regimens in china . population sequencing would be quite quantified for the work .

I M Study design RNA extraction, polymerase chain reaction, and sequencing Sequence alignment and analysis Statistical analysis R Patient characteristics Over 50% of patients presented PI resistance-associated variants NS3-S122G NS5A PI resistance-associated variants Y93H were found, and L31M variants were detected only by deep sequencing High percentage of patients presented NS5B polymerase inhibitors resistance-associated variants C316N D Financial support and sponsorship Conflicts of interest

hepatitis c virus ( hcv ) is one of the main causes of liver cirrhosis and hepatocellular carcinoma , where approximately 350,000 deaths occur each year due to hcv - related liver diseases . in china , approximately 2.2% of the population on average were infected with hcv ( most of which were genotype 1b ) , with a distribution range from 2.1% in fujian province to 9.6% in henan province . to inhibit the global prevalence of hcv infection , various strategies with regard to therapeutic medication the traditional standard of care contains pegylated interferon plus ribavirin ( rbv ) , which could help genotype 1b patients to achieve sustained virological response ( svr ) rates of 45% , further up to 6575% for genotype 2/3 patients . although patients chronically infected with hepatitis c virus ( chc ) in asia carries more beneficial il28b genotypes , nearly 20% of them could not achieve svr . in this regard , more than 30 newly developed direct - acting antivirals ( daas ) have readily been approved in the europe , north american , and japan or are currently being in phase 2/3 clinical trials . , and ns5b polymerase inhibitors ( such as sofosbuvir [ sof ] , dasabuvir , etc . ) in general , they are effective toward pan - genotypic hcv by helping patients to achieve high svr rates up to 100% , especially genotype 1b chc patients . the high replication rate of hcv , along with the low fidelity and poor proof - reading of its polymerase , generates a highly variable virus population denoted as quasispecies . it is estimated that the error rate of the hcv polymerase is 10-fold higher than that of hiv , and the rate of production of rna virus is 100-fold higher than that of hiv . data from clinical trials have revealed that baseline resistance - associated variants ( ravs ) were correlated with treatment outcomes of daas . for example , patients presented pis ravs , such as q80k , d168e / v , ns5a ravs l31m / v , y93h , and ns5b polymerase inhibitors ravs l159f , c316n , v321a achieved svr rates much lower than those who did not . however , compared with population sequencing , the expense for ultra - deep sequencing was relatively high . since none of the daas has been approved in china , there have been seldom reports by far on baseline daas ravs in our country . in this study , we perform both population sequencing and deep sequencing to detect baseline daas ravs in genotype 1b chc patients in china . serum samples were derived from 160 treatment - nave patients chronically infected with genotype 1b hcv . population sequencing was carried out in 160 patients to figure out the prevalence of ravs in gt1b treatment - nave chc patients . ultra - deep sequencing was carried out in 23 patients to find out minor variants with low mutation frequencies . the extracted rna was transcribed to cdna and the ns3 , ns5a , and ns5b fragments were amplified by polymerase chain reaction ( pcr ) in a one - step process ( superscript iii one - step rt - pcr with platinum taq kit ; invitrogen , carlsbad , ca , usa ) following the manufacturers instructions . in the deep sequencing study , the amplified ns3 , ns5a , and ns5b fragments were modified by the multiplexing sample preparation kit ( illumina , san diego , ca , usa ) , and sequence analysis was performed by illumina hiseq 2000 . primers used for amplifying the ns3 , ns5a and ns5b regions the bioedit 7.09 software ( borland company , usa ) was used for editing the sequences . serum samples were derived from 160 treatment - nave patients chronically infected with genotype 1b hcv . population sequencing was carried out in 160 patients to figure out the prevalence of ravs in gt1b treatment - nave chc patients . ultra - deep sequencing was carried out in 23 patients to find out minor variants with low mutation frequencies . the extracted rna was transcribed to cdna and the ns3 , ns5a , and ns5b fragments were amplified by polymerase chain reaction ( pcr ) in a one - step process ( superscript iii one - step rt - pcr with platinum taq kit ; invitrogen , carlsbad , ca , usa ) following the manufacturers instructions . cycling conditions included an initial cdna synthesis step at 55c for 30 min , followed by a denaturation step at 94c for 2 min , 40 cycles of pcr amplification ( 94c for 15 s , 58c for 30 s , 68c for 2 min ) , and a final 10 min extension step at 68c . in the deep sequencing study , the amplified ns3 , ns5a , and ns5b fragments were modified by the multiplexing sample preparation kit ( illumina , san diego , ca , usa ) , and sequence analysis was performed by illumina hiseq 2000 . in total , 160 treatment - nave patients chc genotype 1b were enrolled in this study . fifty - two percent of patients were male , and the median age at testing was 46.5 ( range , 2765 ) years [ table 2 ] . baseline characteristics of the study population bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; hcv : hepatitis c virus . one hundred and forty - five patients ( 90.6% ) were successfully amplified with the ns3 fragments , 71% ( 103/145 ) of whom presented at least one pis ravs . about 56.6% ( 82/145 ) of the patients presented s122 g variant , 33.1% ( 48/145 ) of the patients presented v132i variant , 13.1% ( 19/145 ) of the patients presented v170i variant , and 5.5% ( 8/145 ) of the patients presented t54s variant [ table 3 ] . number of patients harboring ns3/4a pis and ns5a protein inhibitors ravs daas : direct - acting antivirals ; ravs : resistance - associated variants ; pi : protease inhibitor . as results by ultra - deep sequencing , the average mutation frequencies were 23.6% for s122 g variants , 75.4% for v132i and 33.8% for v170i . all of the ravs detected by ultra - deep sequencing could be detected by population sequencing . about 92.5% ( 148/160 ) of the patients were successfully amplified with the ns5a fragments , 14.9% ( 22/148 ) of whom presented at least one ns5a protein inhibitors ravs . about 6.1% ( 9/148 ) of the patients presented q30r variant , 8.8% ( 13/148 ) of the patients presented q54h variant , 10.1% ( 14/148 ) of the patients presented y93h variant . according to the results of ultra - deep sequencing , the average mutation frequencies were 61.0% for q30r , 13.5% for q54h , 95.7% for q62h , 14% for y93h . interestingly , the daclatasvir , ldv , and mk-8742 ravs l31 m have not been detected by population sequencing in any one of the 148 patients but have been detected by ultra - deep sequencing in 3 patients , with average mutation frequencies of 4.7% . nearly , all patients presented at least one rav to polymerase inhibitors at baseline , except for one . when considering ravs of nucleoside inhibitors , 94.2% ( 129/137 ) of the patients were detected harboring the c316n variant . other important ravs to ns5b polymerase inhibitors , such as l320f , v321a , and v499a , were detected in our study . in addition , only three patients presented s142 at baseline , whereas none of the patients exhibited the resistant type n142 t [ tables 4 and 5 ] . number of patients harboring ns5b polymerase inhibitors ravs ( n = 137 ) ravs : resistance - associated variants . baseline characteristics of the patients with and without c316n variant bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; peg - ifn : pegylated interferon ; rbv : ribavirin ; svr : sustained virological response ; hcv : hepatitis c virus . most of the patients presented ravs belonging to certain one category , for instance , the most epidemic rav c316n pertained to palm ii . as results by ultra - deep sequencing , the average mutation frequencies were 41.7% for c316n , 24.8% for a338v , 26.5% for a442 t , and 31.4% for s556 g . interestingly , l419 m variant , which was reported to be resistant to vch-759 and vch-916 , had not been detected by population sequencing and were detected in two patients as minor variants ( with average mutation frequencies of 1.6% ) . in total , 160 treatment - nave patients chc genotype 1b were enrolled in this study . fifty - two percent of patients were male , and the median age at testing was 46.5 ( range , 2765 ) years [ table 2 ] . baseline characteristics of the study population bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; hcv : hepatitis c virus . one hundred and forty - five patients ( 90.6% ) were successfully amplified with the ns3 fragments , 71% ( 103/145 ) of whom presented at least one pis ravs . about 56.6% ( 82/145 ) of the patients presented s122 g variant , 33.1% ( 48/145 ) of the patients presented v132i variant , 13.1% ( 19/145 ) of the patients presented v170i variant , and 5.5% ( 8/145 ) of the patients presented t54s variant [ table 3 ] . number of patients harboring ns3/4a pis and ns5a protein inhibitors ravs daas : direct - acting antivirals ; ravs : resistance - associated variants ; pi : protease inhibitor . as results by ultra - deep sequencing , the average mutation frequencies were 23.6% for s122 g variants , 75.4% for v132i and 33.8% for v170i . all of the ravs detected by ultra - deep sequencing could be detected by population sequencing . about 92.5% ( 148/160 ) of the patients were successfully amplified with the ns5a fragments , 14.9% ( 22/148 ) of whom presented at least one ns5a protein inhibitors ravs . about 6.1% ( 9/148 ) of the patients presented q30r variant , 8.8% ( 13/148 ) of the patients presented q54h variant , 10.1% ( 14/148 ) of the patients presented y93h variant . according to the results of ultra - deep sequencing , the average mutation frequencies were 61.0% for q30r , 13.5% for q54h , 95.7% for q62h , 14% for y93h . interestingly , the daclatasvir , ldv , and mk-8742 ravs l31 m have not been detected by population sequencing in any one of the 148 patients but have been detected by ultra - deep sequencing in 3 patients , with average mutation frequencies of 4.7% . nearly , all patients presented at least one rav to polymerase inhibitors at baseline , except for one . when considering ravs of nucleoside inhibitors , 94.2% ( 129/137 ) of the patients were detected harboring the c316n variant . when il28b genotype was regarded , the prevalence of c316n variant between patients who carried cc and non - cc genotype was identical . other important ravs to ns5b polymerase inhibitors , such as l320f , v321a , and v499a , were detected in our study . in addition , only three patients presented s142 at baseline , whereas none of the patients exhibited the resistant type n142 t [ tables 4 and 5 ] . number of patients harboring ns5b polymerase inhibitors ravs ( n = 137 ) ravs : resistance - associated variants . baseline characteristics of the patients with and without c316n variant bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; peg - ifn : pegylated interferon ; rbv : ribavirin ; svr : sustained virological response ; hcv : hepatitis c virus . most of the patients presented ravs belonging to certain one category , for instance , the most epidemic rav c316n pertained to palm ii . as results by ultra - deep sequencing , the average mutation frequencies were 41.7% for c316n , 24.8% for a338v , 26.5% for a442 t , and 31.4% for s556 g . interestingly , l419 m variant , which was reported to be resistant to vch-759 and vch-916 , had not been detected by population sequencing and were detected in two patients as minor variants ( with average mutation frequencies of 1.6% ) . in our study , as many as 71% of genotype 1b treatment - nave patients presented at least one ns3/4a pis rav . the most prevalent variant was s122 g ( 56.6% ) , which has been confirmed as one of the major ravs of simeprevir ( one of the first - generation second - wave pis ) . mutations associated with first - generation first - wave pis detected in this study were t54s ( telaprevir and boceprevir ) , s122 g ( telaprevir ) , and v170i ( telaprevir ) . with respect to other reports , none of other baseline polymorphisms correlated with treatment outcomes of pis were detected in our study . for example , r155k and d168e / v variants were ravs of asunaprevir ( had licensed in combination with daclatasvir as interferon [ ifn]-free regimens in japan ) and mk-5172 ( one of the second generation pis ) , none of which were found . since all of the ravs detected by ultra - deep sequencing could be detected by population sequencing , we can conclude that population sequencing could qualified for baseline pis ravs screening in china . as one of the most important ravs of most of the ns5a protein inhibitors , y93h variants were detected in 10.1% of patients included in our study . l31 m variants , another mutation associated with treatment outcomes of ldv , daclatasvir , and abt-267 , were only detected by ultra - deep sequencing in three patients with low mutation frequencies . although ns5a protein inhibitors were usually used in combination with pis ( such as asunaprevir ) , baseline polymorphisms of l31 m or y93h could make patients to experience virologic relapse more easily and achieved much lower svr rates . data by ultra - deep sequencing revealed that most of the ns5a protein inhibitors ravs detected here were with relatively high mutation frequencies , except for l31 m variants . in this way , population sequencing might be qualified for baseline ns5a protein inhibitors ravs screening in china . the detection of naturally occurring ravs of ns5b polymerase inhibitors at baseline has been reported in numerous studies . some of them may influence the treatment outcomes of daas - containing regimens . in the clinical trials of sof - containing regimens , the primary concern lies in the s282 t , an important variant associated with treatment - failure . however , a representative study conducted by fda from the us suggested that ns5b - c316n was found to be associated with treatment failure when patients chc genotype 1b receiving sof plus rbv therapy , throughout re - systemizing all the data from sof - containing trials where ultra - deep sequencing was performed . in the clinical trial p7977 - 2025 , 16/61 posttransplantation patients failed therapy of sof plus rbv , where 6/16 patients harbored c316n at baseline , in other words , all of the six patients did not achieve svr . on the contrary , for the other 45 patients who achieved svr12 , eighty - two percent ( 45/55 ) of the patients who did not carry c316n pretreatment achieved svr12 . however , 66.7% ( 4/6 ) of patients harbored l159f at the same time . despite the fact that the majority of ravs of sof reported are at the active sites ( such as s282 t and n142 t ) , position 316 was deemed as the only different residue within 8 a in the ns5b polymerase between hcv genotype 1a and 1b . however , c316 has been reported to be relatively conserved in gt1a , but polymorphic in gt1b . in our study , both the ultra - deep sequencing and population sequencing revealed that the prevalence of c316n variant in treatment - nave chinese gt1b chc patients was quite high , which is in good agreement with previous findings . even in the combination regimens of sof plus ldv , baseline polymorphisms of y93h results by ultra - deep sequencing showed that the mutation frequencies increased dramatically at the time of relapse . both rav s282 t and n142 t were at the active site of ns5b polymerase , but none of the 137 patients exhibited the existence of these two variants . since most of the patients presented ravs of one category , cross - resistance seems less likely to occur . data by ultra - deep sequencing revealed that most of the ns5b polymerase inhibitors ravs detected here were with high mutation frequencies , especially for c316n variants . in this way , population sequencing might qualify for baseline ns5b polymerase inhibitors ravs screening in china . in conclusion , we have employed both the ultra - deep sequencing and population sequencing methods to detect daas ravs in genotype 1b treatment - nave patients , among which the prevalence of variants related with treatment - failure of ns3/4a pis ( s122 g ) , ns5b polymerase inhibitors ( c316n ) and ns5a inhibitors ( y93h ) were quite high , in particular , c316n . currently high prevalence and high mutation frequencies of rav c316n in china might impede the virologic response when distributing sof - containing regimens in the near future . our findings suggest that pretreatment sequencing of hcv genome might need to be performed when patients infected with gt1b hcv receiving daas - containing regimens in china .

hepatitis c virus ( hcv ) is one of the main causes of liver cirrhosis and hepatocellular carcinoma , where approximately 350,000 deaths occur each year due to hcv - related liver diseases . in china , approximately 2.2% of the population on average were infected with hcv ( most of which were genotype 1b ) , with a distribution range from 2.1% in fujian province to 9.6% in henan province . to inhibit the global prevalence of hcv infection , various strategies with regard to therapeutic medication the traditional standard of care contains pegylated interferon plus ribavirin ( rbv ) , which could help genotype 1b patients to achieve sustained virological response ( svr ) rates of 45% , further up to 6575% for genotype 2/3 patients . although patients chronically infected with hepatitis c virus ( chc ) in asia carries more beneficial il28b genotypes , nearly 20% of them could not achieve svr . in this regard , more than 30 newly developed direct - acting antivirals ( daas ) have readily been approved in the europe , north american , and japan or are currently being in phase 2/3 clinical trials . in general , they are effective toward pan - genotypic hcv by helping patients to achieve high svr rates up to 100% , especially genotype 1b chc patients . the high replication rate of hcv , along with the low fidelity and poor proof - reading of its polymerase , generates a highly variable virus population denoted as quasispecies . compared with hiv , the rate of emergence of resistant variants is much higher for hcv . it is estimated that the error rate of the hcv polymerase is 10-fold higher than that of hiv , and the rate of production of rna virus is 100-fold higher than that of hiv . data from clinical trials have revealed that baseline resistance - associated variants ( ravs ) were correlated with treatment outcomes of daas . for example , patients presented pis ravs , such as q80k , d168e / v , ns5a ravs l31m / v , y93h , and ns5b polymerase inhibitors ravs l159f , c316n , v321a achieved svr rates much lower than those who did not . since none of the daas has been approved in china , there have been seldom reports by far on baseline daas ravs in our country . in this study , we perform both population sequencing and deep sequencing to detect baseline daas ravs in genotype 1b chc patients in china . population sequencing was carried out in 160 patients to figure out the prevalence of ravs in gt1b treatment - nave chc patients . hcv viral load ( abbott real time hcv ; abbott laboratories , des plaines , il , usa ) , hcv genotype ( versant hcv genotype 2.0 [ lipa ] ; siemens healthcare diagnostics , tarrytown , ny , usa ) , and il28b genotype ( iplex gold ; sequenom , san diego , ca , usa ) , were conducted . the extracted rna was transcribed to cdna and the ns3 , ns5a , and ns5b fragments were amplified by polymerase chain reaction ( pcr ) in a one - step process ( superscript iii one - step rt - pcr with platinum taq kit ; invitrogen , carlsbad , ca , usa ) following the manufacturers instructions . in the deep sequencing study , the amplified ns3 , ns5a , and ns5b fragments were modified by the multiplexing sample preparation kit ( illumina , san diego , ca , usa ) , and sequence analysis was performed by illumina hiseq 2000 . the reported amino acid substitutions associated resistance to daas according to previously reported data were scored : v36a / m , f43s , t54s , q80k , s122 g , r155k , d168e / v , v170i , etc . population sequencing was carried out in 160 patients to figure out the prevalence of ravs in gt1b treatment - nave chc patients . hcv viral load ( abbott real time hcv ; abbott laboratories , des plaines , il , usa ) , hcv genotype ( versant hcv genotype 2.0 [ lipa ] ; siemens healthcare diagnostics , tarrytown , ny , usa ) , and il28b genotype ( iplex gold ; sequenom , san diego , ca , usa ) , were conducted . the extracted rna was transcribed to cdna and the ns3 , ns5a , and ns5b fragments were amplified by polymerase chain reaction ( pcr ) in a one - step process ( superscript iii one - step rt - pcr with platinum taq kit ; invitrogen , carlsbad , ca , usa ) following the manufacturers instructions . in the deep sequencing study , the amplified ns3 , ns5a , and ns5b fragments were modified by the multiplexing sample preparation kit ( illumina , san diego , ca , usa ) , and sequence analysis was performed by illumina hiseq 2000 . the reported amino acid substitutions associated resistance to daas according to previously reported data were scored : v36a / m , f43s , t54s , q80k , s122 g , r155k , d168e / v , v170i , etc . , for ns5a protein inhibitors ; t19s , n142 t , s282 t , a442 t , s556 g , etc . fifty - two percent of patients were male , and the median age at testing was 46.5 ( range , 2765 ) years [ table 2 ] . one hundred and forty - five patients ( 90.6% ) were successfully amplified with the ns3 fragments , 71% ( 103/145 ) of whom presented at least one pis ravs . about 56.6% ( 82/145 ) of the patients presented s122 g variant , 33.1% ( 48/145 ) of the patients presented v132i variant , 13.1% ( 19/145 ) of the patients presented v170i variant , and 5.5% ( 8/145 ) of the patients presented t54s variant [ table 3 ] . number of patients harboring ns3/4a pis and ns5a protein inhibitors ravs daas : direct - acting antivirals ; ravs : resistance - associated variants ; pi : protease inhibitor . as results by ultra - deep sequencing , the average mutation frequencies were 23.6% for s122 g variants , 75.4% for v132i and 33.8% for v170i . about 92.5% ( 148/160 ) of the patients were successfully amplified with the ns5a fragments , 14.9% ( 22/148 ) of whom presented at least one ns5a protein inhibitors ravs . about 6.1% ( 9/148 ) of the patients presented q30r variant , 8.8% ( 13/148 ) of the patients presented q54h variant , 10.1% ( 14/148 ) of the patients presented y93h variant . according to the results of ultra - deep sequencing , the average mutation frequencies were 61.0% for q30r , 13.5% for q54h , 95.7% for q62h , 14% for y93h . interestingly , the daclatasvir , ldv , and mk-8742 ravs l31 m have not been detected by population sequencing in any one of the 148 patients but have been detected by ultra - deep sequencing in 3 patients , with average mutation frequencies of 4.7% . when considering ravs of nucleoside inhibitors , 94.2% ( 129/137 ) of the patients were detected harboring the c316n variant . when il28b genotype was regarded , the prevalence of c316n variant between patients who carried cc and non - cc genotype was identical . there were also no significant differences between the patients with and without c316n variant for clinical characteristics , except for the hemoglobin levels ( p = 0.004 ) . , l159f variant , which was always detected with c316n variant simultaneously , has not been detected in any of patients in our study . at the codon position 282 of ns5b , the primary sof resistant associated variant , only synonymous variant ( agc<->agt ) was detected . in addition , only three patients presented s142 at baseline , whereas none of the patients exhibited the resistant type n142 t [ tables 4 and 5 ] . baseline characteristics of the patients with and without c316n variant bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; peg - ifn : pegylated interferon ; rbv : ribavirin ; svr : sustained virological response ; hcv : hepatitis c virus . it is worth - mentioning that some ravs of nonnucleoside inhibitors were also detected in our study , such as v494a / t / l , v499a ( thumb i ) ; m423i / t , m424v , m426 t ( thumb ii ) ; h95q , m414i / l , s556g / n ( palm i ) ; s365a / l / s / t ( palm ii ) , etc . , regarding the cross - resistance between these four categories , we have worked out the possible overlap of ravs of different sorts of nonnucleoside inhibitors . as results by ultra - deep sequencing , the average mutation frequencies were 41.7% for c316n , 24.8% for a338v , 26.5% for a442 t , and 31.4% for s556 g . interestingly , l419 m variant , which was reported to be resistant to vch-759 and vch-916 , had not been detected by population sequencing and were detected in two patients as minor variants ( with average mutation frequencies of 1.6% ) . fifty - two percent of patients were male , and the median age at testing was 46.5 ( range , 2765 ) years [ table 2 ] . about 56.6% ( 82/145 ) of the patients presented s122 g variant , 33.1% ( 48/145 ) of the patients presented v132i variant , 13.1% ( 19/145 ) of the patients presented v170i variant , and 5.5% ( 8/145 ) of the patients presented t54s variant [ table 3 ] . number of patients harboring ns3/4a pis and ns5a protein inhibitors ravs daas : direct - acting antivirals ; ravs : resistance - associated variants ; pi : protease inhibitor . about 92.5% ( 148/160 ) of the patients were successfully amplified with the ns5a fragments , 14.9% ( 22/148 ) of whom presented at least one ns5a protein inhibitors ravs . about 6.1% ( 9/148 ) of the patients presented q30r variant , 8.8% ( 13/148 ) of the patients presented q54h variant , 10.1% ( 14/148 ) of the patients presented y93h variant . according to the results of ultra - deep sequencing , the average mutation frequencies were 61.0% for q30r , 13.5% for q54h , 95.7% for q62h , 14% for y93h . interestingly , the daclatasvir , ldv , and mk-8742 ravs l31 m have not been detected by population sequencing in any one of the 148 patients but have been detected by ultra - deep sequencing in 3 patients , with average mutation frequencies of 4.7% . when il28b genotype was regarded , the prevalence of c316n variant between patients who carried cc and non - cc genotype was identical . there were also no significant differences between the patients with and without c316n variant for clinical characteristics , except for the hemoglobin levels ( p = 0.004 ) . , l159f variant , which was always detected with c316n variant simultaneously , has not been detected in any of patients in our study . at the codon position 282 of ns5b , the primary sof resistant associated variant , only synonymous variant ( agc<->agt ) was detected . in addition , only three patients presented s142 at baseline , whereas none of the patients exhibited the resistant type n142 t [ tables 4 and 5 ] . baseline characteristics of the patients with and without c316n variant bmi : body mass index ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; plt : platelet ; peg - ifn : pegylated interferon ; rbv : ribavirin ; svr : sustained virological response ; hcv : hepatitis c virus . it is worth - mentioning that some ravs of nonnucleoside inhibitors were also detected in our study , such as v494a / t / l , v499a ( thumb i ) ; m423i / t , m424v , m426 t ( thumb ii ) ; h95q , m414i / l , s556g / n ( palm i ) ; s365a / l / s / t ( palm ii ) , etc . , regarding the cross - resistance between these four categories , we have worked out the possible overlap of ravs of different sorts of nonnucleoside inhibitors . as results by ultra - deep sequencing , the average mutation frequencies were 41.7% for c316n , 24.8% for a338v , 26.5% for a442 t , and 31.4% for s556 g . interestingly , l419 m variant , which was reported to be resistant to vch-759 and vch-916 , had not been detected by population sequencing and were detected in two patients as minor variants ( with average mutation frequencies of 1.6% ) . the most prevalent variant was s122 g ( 56.6% ) , which has been confirmed as one of the major ravs of simeprevir ( one of the first - generation second - wave pis ) . in concerto trial conducted in japan , the baseline prevalence of s122 g variant was relatively lower ( 34.9% ) than that of our findings , while the baseline prevalence of v170i variant was relatively higher ( 39.6% vs. 13.1% ) than our findings . mutations associated with first - generation first - wave pis detected in this study were t54s ( telaprevir and boceprevir ) , s122 g ( telaprevir ) , and v170i ( telaprevir ) . with respect to other reports , none of other baseline polymorphisms correlated with treatment outcomes of pis were detected in our study . for example , r155k and d168e / v variants were ravs of asunaprevir ( had licensed in combination with daclatasvir as interferon [ ifn]-free regimens in japan ) and mk-5172 ( one of the second generation pis ) , none of which were found . since all of the ravs detected by ultra - deep sequencing could be detected by population sequencing , we can conclude that population sequencing could qualified for baseline pis ravs screening in china . as one of the most important ravs of most of the ns5a protein inhibitors , y93h variants were detected in 10.1% of patients included in our study . l31 m variants , another mutation associated with treatment outcomes of ldv , daclatasvir , and abt-267 , were only detected by ultra - deep sequencing in three patients with low mutation frequencies . data by ultra - deep sequencing revealed that most of the ns5a protein inhibitors ravs detected here were with relatively high mutation frequencies , except for l31 m variants . in the clinical trials of sof - containing regimens , the primary concern lies in the s282 t , an important variant associated with treatment - failure . however , a representative study conducted by fda from the us suggested that ns5b - c316n was found to be associated with treatment failure when patients chc genotype 1b receiving sof plus rbv therapy , throughout re - systemizing all the data from sof - containing trials where ultra - deep sequencing was performed . in the clinical trial p7977 - 2025 , 16/61 posttransplantation patients failed therapy of sof plus rbv , where 6/16 patients harbored c316n at baseline , in other words , all of the six patients did not achieve svr . on the contrary , for the other 45 patients who achieved svr12 , eighty - two percent ( 45/55 ) of the patients who did not carry c316n pretreatment achieved svr12 . in clinical trial neutrino , 4/66 of the patients harbored c316n variant at baseline , 50% of which ( 2/4 ) experienced virologic relapse after receiving treatment of sof plus rbv . despite the fact that the majority of ravs of sof reported are at the active sites ( such as s282 t and n142 t ) , position 316 was deemed as the only different residue within 8 a in the ns5b polymerase between hcv genotype 1a and 1b . data from los alamos databank showed that the prevalence of c316n was much higher in asia than worldwide ( 91.6% vs. 36% ) as well as american continent ( from 0% to 18% ) . in our study , both the ultra - deep sequencing and population sequencing revealed that the prevalence of c316n variant in treatment - nave chinese gt1b chc patients was quite high , which is in good agreement with previous findings . as part of ifn - free regimens , sof in combination with ldv with and without rbv has been one of the most important regimens nowadays . even in the combination regimens of sof plus ldv , baseline polymorphisms of y93h results by ultra - deep sequencing showed that the mutation frequencies increased dramatically at the time of relapse . both rav s282 t and n142 t were at the active site of ns5b polymerase , but none of the 137 patients exhibited the existence of these two variants . data by ultra - deep sequencing revealed that most of the ns5b polymerase inhibitors ravs detected here were with high mutation frequencies , especially for c316n variants . in conclusion , we have employed both the ultra - deep sequencing and population sequencing methods to detect daas ravs in genotype 1b treatment - nave patients , among which the prevalence of variants related with treatment - failure of ns3/4a pis ( s122 g ) , ns5b polymerase inhibitors ( c316n ) and ns5a inhibitors ( y93h ) were quite high , in particular , c316n . currently high prevalence and high mutation frequencies of rav c316n in china might impede the virologic response when distributing sof - containing regimens in the near future . our findings suggest that pretreatment sequencing of hcv genome might need to be performed when patients infected with gt1b hcv receiving daas - containing regimens in china .

whilst both natural evolutionary processes and modern genetic selection result in phenotypic changes , the mechanisms that determine the changes are quite different . the environmental pressures are managed through intervention strategies such as providing high quality feeds , artificial housing or drugs to restrict disease . these intervention strategies are energy dense and enable agriculture to maximize outputs ( productivism ) ; however , they result in populations that are unable to withstand the pressure of more natural environments . in some cases , the extreme selection for a single trait can result in physiological breakdown as resources are channelled towards particular production proteins . this study explores how evolutionary genetics might help inform modern cattle breeding programmes and shows some examples from tropically adapted cattle . we consider the importance of environmental stress for identifying robust genotypes in production systems that will increasingly need to focus on efficiency rather than total productivity . since the 1950s in the developed countries , the era of agricultural productivism emerged with a corresponding demise in the influence of natural selection in livestock production systems . livestock productivism was characterized by a significant use of technology , both mechanization and biotechnology , to maximize productive output . this productivism was facilitated by relatively abundant cheap fossil fuels ; environmental regulation removed environmental stressors to allow intensification , concentration and specialization of the systems ( ilbery and bowler 1998 ) . the genetic diversity of both bos taurus and bos indicus cattle was reduced with the concentration on breeds and sires within breeds that had the highest production potential . more recently , there has been a trend for a measured increase in productivity and growing pressure for cattle production systems to manage their total carbon budgets , which will require a reduction in their greenhouse gas emissions . the reduction in the use of fossil fuel derived energy and nutrients ( direct : fuel and fertilizer ; indirect : acaricide , anthelmintics and vaccine ) will reduce the ability to manage directly environmental stress . a paradigm shift in priorities is therefore required more diverse genotypes that are productive in a range of environments will need to be developed . the evolutionary path of cattle since domestication provides insight into the current fitness levels as well as a better understanding of the interactions between cattle genotypes , environments and management intervention strategies ( hence forth called g e m interactions ) . there has been a long history of domestication and implicit genetic selection ; however , systematic breeding programmes have only been used over the last 60 years . these breeding programmes have enabled cattle to meet specific roles : predominantly supplying meat and milk for human consumption . however , a recent article published in science ( the bovine hapmap consortium 2009 ) looked at the process of cattle domestication . their analyses ( based on mutation rate , inbreeding rate , linkage disequilibrium levels , etc . ) revealed that cattle have undergone a rapid recent decrease in effective population size from a very large ancestral population , possibly due to bottlenecks associated with domestication , selection and breed formation . hence , any future proposal for evolutionary management of cattle domestication should consider this worldwide structure of the breeds to design conservation programmes and mating schemes . the reduction in the ancestral population diversity has important implications for breeding programmes in the 21st century . the opportunity for modern cattle breeds to evolve in response to environmental stress is restricted as the diversity of the gene pool of cattle populations diminishes . however , there are examples of cattle breeds that have demonstrated rapid evolutionary change : in particular where b. taurus cattle have been reared in challenging tropical environments . shorthorn ) that has undergone a relatively rapid transition from extreme susceptibility to extreme resistance to cattle tick via a synergy between natural and artificial selection for tick resistance ( frisch 1981 ; hetzel et al . 1990 ; frisch et al . 2000 ) . the evolution of the adaptaur under tropical stress provides a model approach to cattle breeding , which , if applied within the broader livestock industries might determine more favourable genotypes especially in the advent of escalating environmental stress due to climate change . this study explores the consequences of selection for production traits and demonstrates evolutionary genetics of taurine cattle in both temperate ( favourable ) and tropical ( unfavourable ) environments . as an example of evolutionary adaptation we focus on the adaptaur from northern australia ; however , we also draw on examples from three other b. taurus breeds that all have evolutionary backgrounds in unfavourable environments : senepol from the caribbean , the criollo breed group from central and south america and the n'dama from west africa . we show how exposure to a specific environmental stress ( cattle tick infestation in a tropical environment ) demonstrates the potential for experimental evolution to generate new more robust genotypes that can perform in environmentally stressful environments . genetic adaptation of cattle for a successful transition into the postproductivism era must incorporate alleles of economic importance from genotypes adapted to potentially more stressful environments . the principles of evolutionary genetics can be used to facilitate darwinian fitness in future livestock breeding programmes to generate optimal genotypes for a low - cost and environmentally sustainable production system . in essence , there are complex interactions between genotype , environment and management ( g e m ) in the livestock systems and the implications relate to : ( i ) fitting the right genotypes to the right environments and production systems and ( ii ) determining the optimal genotype across a range of environments and production systems . we provide some perspectives on g e m and how this could contribute to evolution of future strains or breeds of cattle . about 10 000 years bp ( before present ) , neolithic hunter - gathers began to domesticate simultaneously both b. taurus ( fertile crescent and saharan africa ) and b. indicus ( indian subcontinent ) ( bradley et al . 2010 ) . the history of b. indicus in asia ( harris 1992 ) , and more recent introduction to africa via the african east coast about 3500 years bp ( hanotte et al . 2002 ) , is notable for the high levels of environmental stress associated with indicine evolution . the heterosis achieved from b. taurus b. indicus crossbreeding is well documented ( e.g. mcdowell et al . 1996 ) . however , some african taurine breeds , such as n'dama , were able to survive in stressful tropical environments with little or no b. indicus hybridization1 ( frisch et al . 2007 ) . in europe , b. taurus accompanied neolithic man 's conversion from nomadic hunter - gatherer to a sedentary agriculturalist and cattle evolved adaptations to temperate climates . under various celtic , germanic and nordic tribes , there was a general divergence of the larger continental breeds ( e.g. charolais , simmental ) from the medium - sized british breeds ( e.g. hereford , red poll , murray grey ) and specialization into dairy ( e.g. holstein , jersey ) , beef ( e.g. angus , shorthorn ) and dual purpose ( e.g. brown swiss ) . in the developed regions of europe , cattle increased their propensity to supply meat and milk through increased industrialization and dedicated breeding programmes . european taurine cattle were also subjected to the stressors of tropical and semi - arid regions as these cattle were a significant component of the human colonization of central and south america by the spanish and portuguese during the 16th century . cattle from the colonization were collectively known as creole or criollo,2 and adapted to the environments that ranged from semi - arid to the wet tropics ( russell et al . also regarded as tropically adapted is the senepol , a two - breed composite of n'dama and red poll cattle , bred on the caribbean island of st croix in 1918 ( hammond et al . herefords and shorthorns , first imported into australia from united kingdom in 1826 , along with the south african cattle breed , africander imported from king ranch , texas , usa in 1953 formed part of a csiro research programme at the belmont research station , rockhampton in the 1950s ( kennedy and turner 1959 ) . selection lines within a four - breed composite of africander , brahman , hereford and shorthorn , commercialized as belmont red , and a two - breed composite of hereford and shorthorn ( hs line ) were established . modern genetic selection programmes are capable of delivering cumulative gains of 12% per annum for the trait ( or combination of traits ) under selection ( simm et al . quantitative genetic methods have been a crucial component of productivism : development of selection index principles in animal breeding programmes in early 1940s ( hazel and lush 1943 ) followed by application of best linear unbiased prediction ( blup ) of animal 's genetic merit from early 1970s : the animal model ( henderson 1975 ) . these methods accurately quantify breeding potential ( estimated breeding values or ebvs ; expected progeny differences or epds ) for economically important traits and are expressed in real units of measurement ( e.g. kg for growth rate ) . to assess the relative superiority of a bull or a cow , the animal 's ebv is compared with those reported by a breed average . with continuing advances in statistical and computing technology , quantitative geneticists successfully refined and modified blup methodology as new performance records ( e.g. on daily growth ) and associated new traits became available . utilizing these sophisticated levels of technologies , livestock industries of the developed countries have achieved significant increases in the product quantity and quality within the optimal ( i.e. stress - free ) environments ( rauw et al . high performance under these stress - free environments does not , however , imply high performance in all environments . whilst quantitative genetic techniques have successfully identified superior performing individuals and selectively bred from them , the selection process has been largely under increasingly optimal environments ( i.e. stress - free ) . there is an emerging question of the costs of stress - free selection as genotypes are exposed to more stressful environments . whilst management intervention has increasingly reduced environmental stress the ability for an individual to respond has decreased as genetic selection has refined metabolic pathways towards product output at the expense of other traits such as disease resistance . production diseases ( e.g. lameness and metabolic and reproductive disorders ) , in parallel with increasing levels of milk production ( lucy 2001 ; mulligan and doherty 2008 ; ward 2009 ) . however , research and breeding programmes of dairy cattle have started to shift emphasis from production - only traits in the breeding goal to traits that are associated with robustness ( e.g. resistance to clinical mastitis , lameness and milk fever ) and sustainability ( e.g. fertility , feed efficiency , conformation , optimal behaviour ) ( distl 2000 ; kadarmideen et al . 2000 ; kadarmideen and pryce 2001 ; neuenschwander et al . 2005 ) . beef cattle breed societies have gone down a similar path to the dairy industries with the emphasis on performance recording production traits . in beef cattle breeding , growth , calving ease , reproduction and meat quality traits such as muscle and/or fat depth and tenderness are the main drivers of commercial operations . as with the dairy industries there has been an increase in persistent production diseases in the beef industries ; namely respiratory diseases and metabolic disorders in the feedlot systems ( e.g. owens et al . continuing outbreaks of the ocular disease called infectious bovine keratoconjunctivitis ( ibk ) also generates significant economic and welfare issues for the cattle industries ( brown et al . these problems have been addressed through research to develop animal management protocols , feed additives , pharmacological agents and vaccines to combat the diseases ( quigley et al . environment control strategy . however , genetic methods for the identification of host resistance / susceptibility for diseases such as ibk in the us department of agriculture reference cattle population ( casas and stone 2006 ) and australian adaptaur cattle ( ali et al . the animal model can be used to evaluate genetic merit of animals for traits associated with the production diseases described above ( heringstad et al . it is not only possible to select animals for just one trait , but rather a combination of traits . hence , animals ebvs for different traits ( e.g. growth , reproductive success and some measures of adaptability like ibk resistance ) are combined to form an aggregate or total breeding value ( tbv ) . therefore , a multi - trait breeding objective would appear as : where is the total breeding value , bi is an economic weight for the ith trait in breeding objective , ebvi is the ebv for the ith trait and n is the number of traits in the breeding goal . breeders can modify their herd 's genetics based on assigning different economic weights to different traits ; for instance if adaptability or disease resistance is more important than other traits in tropical environments , then there needs to be higher weights for those traits in the breeding goal . application of these novel methods has had major beneficial effects on the cost and quality of food , and on the efficiency , competitiveness and welfare status of the livestock industries concerned . the incorporation of resistance traits ensures that the tbv is also driven by evolutionary principles of adaptation . however , in any breeding programme the animal 's physiological limits must be considered . in production systems of negligible environmental stress , the need for both energy and physiological mechanisms to alleviate stress are not required . in theory , the metabolic energy is therefore available for production traits but with continued selection for high production the dairy cow is unable to consume enough energy to meet this production potential and the cow 's fertility ( butler and smith 1989 ; kadarmideen et al . 2003 ; wathes et al . 2009 ) and health ( ingvartsen et al . 2003 ; zuerner et al . 2007 ; mulligan and doherty 2008 ) suffer as a consequence . moreover , a number of studies have shown a negative relationship between metabolic rate and resistance to stress ( see parsons 1990 and hoffmann and parsons 1991 ) . animals of low resistance to environmental stress have inherently higher rates of growth , appetite and fasting metabolism than animals relatively resistant to environmental stress ( frisch and vercoe 1984 ) . thus , selection for production traits , disregarding physiological limits , fertility and resistance to environmental stress , would have serious negative consequences for future cattle herds as cows can only be maintained with high inputs that are unsustainable in the longer term . the three main genetic processes involved in the evolution of animals during domestication are selection , genetic drift and inbreeding ( falconer and mackay 1996 ) , and inbreeding is posing additional problems for the intensive cattle industries . inbreeding can lead to loss of fitness and even survival of a breed or species . for the dairy industry in the usa , ( 1998 ) found that the effects of inbreeding in the holstein herd , years 19831993 , were cumulative and larger on lifetime profit functions than on lactation traits . techniques are now available to control inbreeding using conventional blup methods ( villanueva et al . 2006 ) or using molecular genetic markers in genetic improvement programmes ( e.g. li et al . 2008 ) . hence , any future breeding programmes must be designed to manage inbreeding depression ( kearney et al . cattle breeds of the developed countries have been subjected to breeding programmes that are not sustainable and inappropriate for determining genotypes that are productive in unfavourable environments . stern and orgogozo ( 2009 ) suggest that there is a need to identify and select specific mutations without negative consequences . we argue that for cattle of the 21st century it will be necessary to also explore the evolution of cattle in unfavourable environments , where there has been selection for alleles at many loci offering specific environmental adaptations . the next section provides some examples of breeding programmes that have identified desirable genetics contributing to adaptations of temperate cattle breeds to unfavourable tropical environments . modern agricultural populations of plants and animals have shown rapid evolutionary adaptation to environmental stress in timeframes of decades ( or less ) ( thompson 1998 ; bell and collins 2008 ; campbell et al . environmental stress is defined as an environmental force , from either biotic or abiotic factors , that impairs an animal 's regulatory mechanism to such an extent that fitness is compromised . fitness , an animal 's ability to grow and reproduce viable offspring will be adversely affected if the animal is unable to mount an appropriate response to the stressor via means of its physiology and/or behaviour . the degree of sensitivity to these stressors , or homeostasis , will be regarded as an aspect of fitness ( falconer and mackay 1996 ) . in a relatively short period of time , european b. taurus relocated from temperate to tropical regions and had to achieve higher fitness in the presence of tropical stressors . this higher fitness was achieved through the application of evolutionary principles within a genetic selection program . the response to environmental stress involves a synergy between behaviour and physiological adaptation ( hart 1990 ; hoffmann and parsons 1991 ; lomborg et al . 2008 ) . documenting the plethora of tropical environmental stressors and the corresponding responses of b. taurus to these stressors is beyond the scope of this study . therefore , we present data from experiments that aimed to assess the evolutionary adaptation of b. taurus cattle to stress from infestations of the ectoparasite cattle tick rhipicephalus ( boophilus ) microplus . cattle tick infestations have a substantial negative impact on host fitness ( frisch and vercoe 1984 ; lehmann 1993 ) . by the end of last century , estimates of the yearly global costs of cattle tick and tick - borne diseases were between us$13.9 and us$18.7 billion ( de castro 1997 ) . the global costs of ticks are set to escalate ; increasing levels of acaricide resistance ( sangster 2001 ) , limited efficacy of anti - tick vaccines and emergence of nonresponders to vaccination ( outteridge 1993 ; sonenshine et al . 2006 ) , the genetic antagonism on fitness traits from biased artificial selection for production traits ( frisch 1981 ; kadarmideen et al . 2000 ) and the potential negative impact of climate change ( white et al . 2003 ) . exploring the full repertoire of the animal 's anti - tick mechanisms is warranted if genetic technologies that utilize naturally occurring adaptation to parasitic stress are going to contribute to sustainable livestock production . european taurine cattle , such as the criollo in the 1500s , were relocated to their respective tropical environments from temperate environments that were free of parasites endemic in the tropics . a similar case occurred in 1952 with the introduction of herefords and shorthorns ( hs line ) on the csiro belmont research station , a property located 26 km nnw of rockhampton , in north - eastern australia . as a research station located in the tropics , acaricide was initially used routinely to treat tick infestations but from 1966 it was used only as an experimental treatment or to prevent mortalities from tick infestations ( frisch 1981 ) . thus on belmont , there was a need to select european b. taurus cattle which had anti - parasitic mechanisms to enable them to survive and maintain fitness . cattle , as with other herbivores , have evolved various anti - tick mechanisms : multiple immunological processors , hypersensitivity and exudate and grooming behaviour ( riek 1962 ; roberts 1968a ; schleger et al . 1981 ; brossard and wikel 2004 ) . roberts ( 1968b ) also showed that rejection of larvae on b. taurus occurred within 24 h of infestation and the effect was greater in animals resistant to tick . as pointed out by roberts ( 1968b ) the early mortality of larvae has implications for the restricted transmission of such tick - borne diseases as babesia bigemina . a family within the hs line on belmont did acquire extreme resistance to infestations of cattle tick ( frisch et al . 2000 ) . the origins of the adaptaur began on belmont in 1980 with the identification of a heifer ( animal identity : 790546 ) of the 1979 calf crop of the hs line and after 1980 selection for both high growth tick resistance within the hs line focused on this family . the heifer acquired extreme resistance to field infestations of cattle tick soon after weaning ( subsequent counts of zero or one regardless of tick counts of cohorts see fig . 1 for tick counts of cohorts ) . there was no evidence of hypersensitivity ( j. e. frisch and c. j. o'neill , unpublished data ) and a grooming response to remove larvae within 24 h of infestation ( c. j. o'neill and j. e. frisch , unpublished data ) . figure 1 presents a compilation of tick count data from various studies of the 1980s on adaptation , production or heterosis [ see frisch and vercoe ( 1984 ) , frisch ( 1987 ) , frisch et al . ( 2000 ) for details of environment , breeding programmes experimental designs and various analyses of data ] . these studies were conducted as a precursor to a major heterosis study conducted during the 1990s , involving breeds of european , indian and african origins , to identify the optimal genotype for herd productivity in a stressful tropical environment ( frisch and o'neill 1998a , b , c ) . figure 1 illustrates that there is a strong tendency in low tick counts of progeny from sires of the 790546 family compared to progeny from sires of the remaining adaptaur herd and progeny from the brahman sires . the focus on the maternal lineage of 790546 not only assisted in rapid reduction in the phenotypic trend in tick count ( fig . 1 ) , but also a corresponding linear ( r = 0.93 ) genetic trend from 1983 to 1998 of a mean rate of 7 ticks / year of the adaptaur herd ( frisch et al . arithmetic means ( three highest counts for the calf crop ) ( sem ) of field tick ( rhipicephalus microplus ) infestations of heifer progeny of brahman and adaptaur sires for calf crops 1979 , 19831984 and 19861988 on belmont research station . number of sires and progeny per group : brahman , 24 sires with 159 progeny , adaptaur maternal lineage 790546 , five sires with 55 progeny , and remaining adaptaur , 20 sires with 109 progeny . twenty - seven years after the identification of heifer 790546 , in 2007 descendents from this heifer exhibited high tick resistance ( table 1 ) and with some straightbred animals ( e.g. animal identities : 060017 and 060031 ) resembling the extreme phenotype of 790546 ( table 2 ) . tick count data from an experiment conducted on belmont during november and december 2007 , involving 2-year - old heifers are presented in tables 1 and 2 . this experiment was aimed at studying tick resistance of different breed types and observing host hypersensitivity to tick larvae . animals were sourced for this experiment as follows : there were 15 brahman heifers chosen at random from a calf crop of 140 heifers from belmont research station and the adaptaur and adaptaur crossbreds were sourced locally from commercial seedstock producers vineree and ben m'cree . to restrict the immediate host grooming to remove tick larvae , we fitted a collar to the animal and taped a tube of larvae onto the collar . these cattle were artificially infested with 10 000 two - week - old tick larvae ( r. microplus , queensland department of primary industries and fisheries , yeerongpilly , australia ) . at 48-h postinfestation , the adaptaur straightbred has not lost its high resistance to cattle tick , even after 27 years ( table 1 ) , and this high tick resistance appears to be conferred to other taurine breeds regardless of their genetic background for tick resistance ( table 2 ) . the finding that some heifers with high counts at 6 months of age have progressively lower counts at 12 months of age and 24 months of age ( table 2 ) confirms that resistance is acquired . although general hypersensitivity was not observed in this experiment , five of the most tick resistant b. taurus heifers had a small area ( approx . 5 cm in diameter ) of exudate ranging from minor to excessive at the site of larval release ( table 2 ) , indicating a host immune response to the parasite . by day 15 the relatively high weight gain on pasture of the adaptaur straightbred and f1 compared to the brahman ( table 1 ) would suggest that neither the imposition of 10 000 tick larvae nor the adaptaur anti - tick mechanism was detrimental to their productivity . the results presented in tables 1 and 2 suggest a relative advantage of adaptaur and adaptaur cross - over brahmans in tick resistance at the same level of growth . however , if strong conclusions are to be made , a larger sample size in each of the breed classes would be required and a statistical analysis conducted . number of bos indicus ( straightbred brahman ) and bos taurus ( adaptaur and f1 adaptaur cross ) 24-month - old heifers in each class of tick resistance from an artificial infestation of cattle tick ( rhipicephalus microplus ) on belmont research station november 2007 * to confirm the viability of the tick larvae a straightbred murray grey heifer from ben m'cree of known high susceptibility ( field counts > 100 ticks / side at both 6 and 12 months of age at ben m'cree , personal communication p and l quayle ) of the same cohort was pastured with the above heifers at belmont and produced a count of 233 mature ticks by day 21 of the artificial infestation . tick resistance class was based on number of maturing ticks from cumulative tick counts ( /side ) on days 1921 from an artificial infestation of 10 000 larvae . bos taurus ( adaptaur straightbred and f1 adaptaur cross ) heifers with individual tick counts ( rhipicephalus microplus ) at 6 , 12 and 24 months of age and an assessment of the degree of exudate at the site of larval release from an artificial infestation of cattle tick at 24 months of age on belmont research station , november 2007 tick counts ( /side ) courtesy of p and l quayle from field infestations at ben m'cree , peachester , queensland , australia . cumulative tick counts ( /side ) on days 1921 from the artificial infestation of 10 000 larvae . exudate assessed at the site of larval release 48-h postinfestation of the 10 000 larvae . just as the african b. taurus evolved mechanisms for resistance to tropical stress so did the european b. taurus . there are a number of adaptation mechanisms that could explain the relatively high levels of resistance to tropical stressors for the adaptaur , n'dama , senepol and criollo breeds . the following scenarios highlight the complexity of disease resistance and pertain to evolutionary genetics : chance mutations , a synergy between natural selection and artificial selection and phenotypic plasticity . ( 2005 ) contend that the host 's optimum immune response to parasites and pathogens is one of immunosufficiency , because the former is a balance between energy loss due to pathogen infection and energy expenditure mounting an immune response that is conducive to fitness . naessens ( 2006 ) has shown that it is the inappropriate response of the immune system that results in the mortality and morbidity of trypanosome - infected cattle . n'dama not only possesses a genetic capacity to control parasitism ( claxton and leperre 1991 ) , but also to avoid anaemia and the severe pathology associated with trypanosome infection ( murray and dexter 1988 ; naessens 2006 ) . a major gene for hair length slick hair that contributes to improving the animal 's thermoregulatory ability , has been described in both senepol and venezuelan carora a two - breed composite of brown swiss and local criollo ( olson et al . 2003 ) . it could be speculated that the senepol also has the potential for high tick resistance given its n'dama origin and slick coat ( claxton and leperre 1991 ; hammond et al . o'kelly and spiers ( 1983 ) using animals from the hs line , among other breeds , demonstrated that fewer ticks matured on clipped areas of coat compared to unclipped areas . relative to their temperate origins , the environmental assault on the hs line on belmont was extreme , from solar radiation , heat stress , parasites and periods of poor nutrition , thus creating strong natural selection . a livestock population surviving with minimal intervention to remove environmental stress , such as not treating the adaptaur with acaricide to remove parasites , means that the population was under natural selection pressure . using both calf survival data and a multi - trait mixed model genetic analysis of growth rates from 1966 to 1981 , hetzel et al . ( 1990 ) concluded that the fitness of this population was increasing under natural selection . moreover , with the artificial selection protocol in place , selecting sires for high growth rate in a stressful environment ( frisch 1981 ) , was working in synergy with natural selection ( hetzel et al . frisch ( 1981 ) also concluded that the increasing level of resistance to environmental stress was solely responsible for the improved growth rates for this population . since 1983 , the additional selection protocol of high tick resistance focusing on a single family with selection for high growth rate , confirmed the value of targeting components that positively impact on productivity in a stressful tropical environment . the 33% improvement in calving rates of adaptaur cows from 1992 to 1996 , compared to adaptaurs before selection 19641972 ( o'neill and frisch 1998 ) , is further evidence that high selection intensity ( high growth and high tick resistance ) on fitness traits was successful for european b. taurus in a tropical environment . in a large australian purebred - crossbreeding study from the cooperative research centre for cattle and beef quality , newman et al . ( 2002 ) found that progeny from belmont red sires produced carcasses with the lowest sub - cutaneous fat and highest retail beef yield , desirable traits for human consumption . the above - mentioned research work with adapted genotypes has highlighted the importance of the synergy between natural and artificial selection for both adaptation and production traits . the transition from unadapted to an adapted state for the n'dama , adaptaur , senepol and criollo could also involve elements of phenotypic plasticity , defined as the ability of a genotype to produce more than one phenotype in response to differing environments ( scheiner 1993 ; de jong 2005 ; fordyce 2006 ) . garland and kelly ( 2006 ) point out that directional selection should support alleles that increase phenotypic plasticity in the direction of selection . when animal breeders are faced with multiple environments , models of phenotypic plasticity could contribute to the development of appropriate selection programmes for their production system ( de jong and bijma 2002 ) . it is conceivable that the mexican criollo from the chihuahuan desert3 possess alleles pertinent to plasticity for variations in water consumption , extreme fluctuations in ambient temperature , metabolism of a variety of herbaceous plants , and alternating between browsing and grazing . existence of such alleles has implications for other semi - arid regions such as the livestock component in the management of dryland salinity of southern australia as described by masters et al . ( 2006 ) . given the extreme range of environments involved , plus the simultaneous natural and artificial selection pressure applied to achieve fitness , phenotypic plasticity could have contributed to the adaptation of the b. taurus to extreme and highly variable environmental parameters , and therefore future breeding programmes in variable challenging environments . extreme environmental conditions that result in high selection intensity on traits directly related to fitness have been shown to impact directly on the evolutionary process . such high selection intensity tends to rapidly fix pleiotropic genes with predominantly positive effects ( lande 1982 ) . hoffmann and parsons ( 1991 ) also point out that short periods ( years , decades ) of high selection intensity , rather than long periods ( century or more ) of low selection intensity , favour genes of major effect in the evolutionary process hence the emergence of a major gene for slick hair that contributes to heat resistance in the senepol and carora breeds ( olson et al . 2003 ) . the identification and evidence of a putative major gene for tick resistance in the adaptaur ( frisch 1994 ; kerr et al . 1994 ) was not confirmed by henshall ( 2004 ) with additional data and inclusion of polygenic effects in the statistical model . ( 2009 ) have shown evidence of major gene effects in this breed . the first large - scale gene expression profiling study ( skin cdna microarrays ) was undertaken using resistant / susceptible adaptaur cattle and identified several candidate mechanisms ( e.g. antibody immune response day 1 of the infestation ) that were involved in adaptaur tick resistance ( wang et al . 2007 ) . nevertheless , because complex response mechanisms to multiple and variable stressors are involved , the contribution of quantitative genetics and molecular biology for the improvement of b. taurus to a stressful tropical and/or semi - arid environment remains to be fully assessed . the completion of the bovine genome assembly ( the bovine genome sequencing and analysis consortium 2009 ) provides an opportunity to understand how cattle evolved and adapted to different geographical regions and production systems . the biological systems most affected by changes in the number and organization of genes in the cattle lineage include reproduction , immunity , lactation and digestion , all of which are important to our discussion on tropical adaptation . they concluded that these changes in the cattle lineage probably reflect metabolic , physiologic and immune adaptations due to microbial fermentation in the rumen , the environment and its influence on disease transmission , and the reproductive strategy of cattle . indeed , understanding the taurine evolutionary trajectories also provides an insight into the predictability of cattle performance in cattle industries of the 21st century . awareness of the taurine evolutionary trajectories in favourable environments will provide a guide to future breeding experiments . breeding goals are enhanced by not only knowledge about the diseases associated with production and the stress they cause but also mechanisms of resistance to diseases . global warming , with elevated ambient temperatures , altered rainfall patterns and changed distributions of parasites and pathogens ( sutherst 2001 ; howden et al . 2009 ) , will also impact livestock evolutionary trajectories ( burdon and thrall 2008 ) and provide new challenges for livestock producers . in the previous sections , we reviewed and discussed the evolutionary history and genetics of b. taurus cattle and the selection pressures imposed by different environments and/or production systems using tick resistance as a case study . in a systems framework , it is the interactions between genotype , environment and management that contribute to evolution of different genotypes . compared to the tropically adapted b. taurus , the evolutionary history of temperate ( productivism ) b. taurus possess inheritantly higher levels of production potential , the production achieved in the absence of all environmental stress , and attributed to traits such as higher metabolic rates and appetites of the temperate taurine breeds . conversely , the evolution of b. taurus in environmentally stressful regions has ensured that those genotypes possess mechanisms of adaptation ( e.g. resistance to cattle tick ) unlike their temperate counterpart ( see hoffmann and parsons 1991 ) . hence , the metabolic rates of breeds such as charolais and angus would be higher than the tropically adapted breeds n'dama and criollo . however , in terms of adaptation to environmental stress the reverse is true and the n'dama and criollo possess relatively higher levels of environmental adaptation than breeds synonymous with productivism . historically in commercial livestock production systems environmental stress is alleviated with strategies such as parasite control and supplementary feeding . figure 2 shows the g e m interactions in terms of relative production potential of temperate and tropical b. taurus ( g ) in the presence and absence of management ( m ) to negate the effect of increasing levels of environmental stress ( e ) . the relative levels of production potential are shown on the vertical axis whilst the increasing levels of environmental stress are shown on the horizontal axis . thus , as the level of environmental stress increases , the decline in production of the tropically adapted b. taurus is substantially less than the decline of the temperate b. taurus but if there is intervention to remove environmental stress then the response on production by the temperate taurine breeds would be greater than the response by the tropically adapted taurine breeds ( fig . this implies that the costs associated with maintaining high productivity in temperate b. taurus might negate the profit obtained via high production . diagrammatic representation of the g e m for bos taurus adapted to a temperate environment ( e.g. angus ) and b. taurus adapted to a tropical environment ( e.g. n'dama ) showing the proportional decline from optimal production potential for growth when there is no intervention by management to alleviate increasing levels of tropical environmental stress . the two shaded horizontal lines indicate the level of management required to maintain production potential . we have specifically avoided discussing g e m interactions that exist within breed between animals at the level of genes which respond to different environments and/or different management interventions . ( 2006a , b ) and are outside the scope of this study . in practice , the major hurdle to achieving economic sustainability will be the acknowledgement that no single genotype will outperform all other genotypes regardless of environmental conditions . moreover , interactions between genotype and management ( e.g. use of acaricide to control cattle tick ) occur when performances of different genotypes are not equally affected by different management inputs . if n'dama and angus cattle are given the same dosage of acaricide in a region of tick infestation , angus cattle responds better to treatment than n'dama cattle ( response as in growth and fertility ) . if , however , n'dama and angus cattle are not given any treatment of acaricide in the same region , then n'dama cattle are likely to achieve higher production performance than angus cattle ( fig . there is g m. hence , a livestock system in essence is defined by the g e m interactions and understanding g e m and its impact on genetic , environmental and managerial strategies is crucial in future livestock systems . livestock production in the 21st century will be characterized by systems where both market ( food and fibre output ) and nonmarket ( social , environmental and climate change ) issues are given consideration . thus , livestock producers will maintain their drive for maximization of output but with diminished reliance on industrialization and therapeutic agents to alleviate environmental stress and rely more on the genetic make - up of their animals to combat stress from heat , parasites , pathogens and poor nutrition . various evolutionary trajectories of b. taurus since domestication suggest that the environmentally adapted breeds , along with b. indicus breeds , will play a pivotal role in the transition to sustainable production systems . the review of work on such breeds as the n'dama , senepol , criollo and adaptaur implies that both natural and artificial selection should be incorporated into selection decisions once there is an understanding of the beef and dairy production diseases and the mechanisms of resistance to environmental stress then , as with the plant industries , molecular biology technology may be successfully applied to cattle . corresponding breeding objectives for both production and adaptation traits could also be established via a combination of well - established quantitative genetic methodologies with emerging molecular biology technologies such as functional genomics . we also conclude that the four adapted taurine breeds noted above , along with breeds that have undergone similar evolutionary trajectories , would possess candidate genes that could contribute to improving the productivity of cattle in challenging environments . however , the physiological antagonism between production potential and adaptation ensures no genotype achieves highest performance in all environments . the evolutionary history and knowledge of the genetic architecture will provide estimates of production potential ( in a favourable environment ) and level of resistance to environmental stress ( in an unfavourable environment ) and therefore estimates of how much production is realized when environmental stressors are controlled . hence , the performance of a breed or combination of breeds in a given environment or degree of intervention by management to alleviate environmental stressors , are able to be predicted . utilizing the full compliment of global cattle genetics and knowledge of g e m interactions will help ensure sustainable levels of beef and milk production in the 21st century .

the evolutionary processes that have enabled bos taurus cattle to establish around the globe are at the core to the future success of livestock production . our study focuses on the history of cattle domestication including the last 60 years of b. taurus breeding programmes in both favourable and unfavourable environments and its consequences on evolution and fitness of cattle . we discuss the emergence of production diseases in temperate production systems and consider the evolutionary genetics of tropical adaptation in cattle and conclude that the senepol , n'dama , adaptaur and criollo breeds , among others with similar evolutionary trajectories , would possess genes capable of improving the productivity of cattle in challenging environments . using our own experimental evidence from northern australia , we review the evolution of the adaptaur cattle breed which has become resistant to cattle tick . we emphasize that the knowledge of interactions between genotype , environment and management in the livestock systems will be required to generate genotypes for efficient livestock production that are both economically and environmentally sustainable . livestock producers in the 21st century will have less reliance on infrastructure and veterinary products to alleviate environmental stress and more on the animal 's ability to achieve fitness in a given production environment .

Introduction Historical movement of African and European None None Evolutionary genetics of adaptation to environmental stressors in the tropics Genotype by environment by management interaction in cattle Conclusion

we consider the importance of environmental stress for identifying robust genotypes in production systems that will increasingly need to focus on efficiency rather than total productivity . since the 1950s in the developed countries , the era of agricultural productivism emerged with a corresponding demise in the influence of natural selection in livestock production systems . the reduction in the use of fossil fuel derived energy and nutrients ( direct : fuel and fertilizer ; indirect : acaricide , anthelmintics and vaccine ) will reduce the ability to manage directly environmental stress . a paradigm shift in priorities is therefore required more diverse genotypes that are productive in a range of environments will need to be developed . the evolutionary path of cattle since domestication provides insight into the current fitness levels as well as a better understanding of the interactions between cattle genotypes , environments and management intervention strategies ( hence forth called g e m interactions ) . there has been a long history of domestication and implicit genetic selection ; however , systematic breeding programmes have only been used over the last 60 years . these breeding programmes have enabled cattle to meet specific roles : predominantly supplying meat and milk for human consumption . however , a recent article published in science ( the bovine hapmap consortium 2009 ) looked at the process of cattle domestication . hence , any future proposal for evolutionary management of cattle domestication should consider this worldwide structure of the breeds to design conservation programmes and mating schemes . the reduction in the ancestral population diversity has important implications for breeding programmes in the 21st century . the opportunity for modern cattle breeds to evolve in response to environmental stress is restricted as the diversity of the gene pool of cattle populations diminishes . however , there are examples of cattle breeds that have demonstrated rapid evolutionary change : in particular where b. taurus cattle have been reared in challenging tropical environments . the evolution of the adaptaur under tropical stress provides a model approach to cattle breeding , which , if applied within the broader livestock industries might determine more favourable genotypes especially in the advent of escalating environmental stress due to climate change . this study explores the consequences of selection for production traits and demonstrates evolutionary genetics of taurine cattle in both temperate ( favourable ) and tropical ( unfavourable ) environments . as an example of evolutionary adaptation we focus on the adaptaur from northern australia ; however , we also draw on examples from three other b. taurus breeds that all have evolutionary backgrounds in unfavourable environments : senepol from the caribbean , the criollo breed group from central and south america and the n'dama from west africa . we show how exposure to a specific environmental stress ( cattle tick infestation in a tropical environment ) demonstrates the potential for experimental evolution to generate new more robust genotypes that can perform in environmentally stressful environments . the principles of evolutionary genetics can be used to facilitate darwinian fitness in future livestock breeding programmes to generate optimal genotypes for a low - cost and environmentally sustainable production system . in essence , there are complex interactions between genotype , environment and management ( g e m ) in the livestock systems and the implications relate to : ( i ) fitting the right genotypes to the right environments and production systems and ( ii ) determining the optimal genotype across a range of environments and production systems . the history of b. indicus in asia ( harris 1992 ) , and more recent introduction to africa via the african east coast about 3500 years bp ( hanotte et al . european taurine cattle were also subjected to the stressors of tropical and semi - arid regions as these cattle were a significant component of the human colonization of central and south america by the spanish and portuguese during the 16th century . also regarded as tropically adapted is the senepol , a two - breed composite of n'dama and red poll cattle , bred on the caribbean island of st croix in 1918 ( hammond et al . herefords and shorthorns , first imported into australia from united kingdom in 1826 , along with the south african cattle breed , africander imported from king ranch , texas , usa in 1953 formed part of a csiro research programme at the belmont research station , rockhampton in the 1950s ( kennedy and turner 1959 ) . quantitative genetic methods have been a crucial component of productivism : development of selection index principles in animal breeding programmes in early 1940s ( hazel and lush 1943 ) followed by application of best linear unbiased prediction ( blup ) of animal 's genetic merit from early 1970s : the animal model ( henderson 1975 ) . utilizing these sophisticated levels of technologies , livestock industries of the developed countries have achieved significant increases in the product quantity and quality within the optimal ( i.e. whilst management intervention has increasingly reduced environmental stress the ability for an individual to respond has decreased as genetic selection has refined metabolic pathways towards product output at the expense of other traits such as disease resistance . however , research and breeding programmes of dairy cattle have started to shift emphasis from production - only traits in the breeding goal to traits that are associated with robustness ( e.g. beef cattle breed societies have gone down a similar path to the dairy industries with the emphasis on performance recording production traits . as with the dairy industries there has been an increase in persistent production diseases in the beef industries ; namely respiratory diseases and metabolic disorders in the feedlot systems ( e.g. however , genetic methods for the identification of host resistance / susceptibility for diseases such as ibk in the us department of agriculture reference cattle population ( casas and stone 2006 ) and australian adaptaur cattle ( ali et al . application of these novel methods has had major beneficial effects on the cost and quality of food , and on the efficiency , competitiveness and welfare status of the livestock industries concerned . in production systems of negligible environmental stress , the need for both energy and physiological mechanisms to alleviate stress are not required . animals of low resistance to environmental stress have inherently higher rates of growth , appetite and fasting metabolism than animals relatively resistant to environmental stress ( frisch and vercoe 1984 ) . thus , selection for production traits , disregarding physiological limits , fertility and resistance to environmental stress , would have serious negative consequences for future cattle herds as cows can only be maintained with high inputs that are unsustainable in the longer term . the three main genetic processes involved in the evolution of animals during domestication are selection , genetic drift and inbreeding ( falconer and mackay 1996 ) , and inbreeding is posing additional problems for the intensive cattle industries . cattle breeds of the developed countries have been subjected to breeding programmes that are not sustainable and inappropriate for determining genotypes that are productive in unfavourable environments . we argue that for cattle of the 21st century it will be necessary to also explore the evolution of cattle in unfavourable environments , where there has been selection for alleles at many loci offering specific environmental adaptations . the next section provides some examples of breeding programmes that have identified desirable genetics contributing to adaptations of temperate cattle breeds to unfavourable tropical environments . environmental stress is defined as an environmental force , from either biotic or abiotic factors , that impairs an animal 's regulatory mechanism to such an extent that fitness is compromised . fitness , an animal 's ability to grow and reproduce viable offspring will be adversely affected if the animal is unable to mount an appropriate response to the stressor via means of its physiology and/or behaviour . in a relatively short period of time , european b. taurus relocated from temperate to tropical regions and had to achieve higher fitness in the presence of tropical stressors . documenting the plethora of tropical environmental stressors and the corresponding responses of b. taurus to these stressors is beyond the scope of this study . therefore , we present data from experiments that aimed to assess the evolutionary adaptation of b. taurus cattle to stress from infestations of the ectoparasite cattle tick rhipicephalus ( boophilus ) microplus . by the end of last century , estimates of the yearly global costs of cattle tick and tick - borne diseases were between us$13.9 and us$18.7 billion ( de castro 1997 ) . exploring the full repertoire of the animal 's anti - tick mechanisms is warranted if genetic technologies that utilize naturally occurring adaptation to parasitic stress are going to contribute to sustainable livestock production . thus on belmont , there was a need to select european b. taurus cattle which had anti - parasitic mechanisms to enable them to survive and maintain fitness . roberts ( 1968b ) also showed that rejection of larvae on b. taurus occurred within 24 h of infestation and the effect was greater in animals resistant to tick . a family within the hs line on belmont did acquire extreme resistance to infestations of cattle tick ( frisch et al . the heifer acquired extreme resistance to field infestations of cattle tick soon after weaning ( subsequent counts of zero or one regardless of tick counts of cohorts see fig . the focus on the maternal lineage of 790546 not only assisted in rapid reduction in the phenotypic trend in tick count ( fig . 1 ) , but also a corresponding linear ( r = 0.93 ) genetic trend from 1983 to 1998 of a mean rate of 7 ticks / year of the adaptaur herd ( frisch et al . animals were sourced for this experiment as follows : there were 15 brahman heifers chosen at random from a calf crop of 140 heifers from belmont research station and the adaptaur and adaptaur crossbreds were sourced locally from commercial seedstock producers vineree and ben m'cree . to restrict the immediate host grooming to remove tick larvae , we fitted a collar to the animal and taped a tube of larvae onto the collar . at 48-h postinfestation , the adaptaur straightbred has not lost its high resistance to cattle tick , even after 27 years ( table 1 ) , and this high tick resistance appears to be conferred to other taurine breeds regardless of their genetic background for tick resistance ( table 2 ) . although general hypersensitivity was not observed in this experiment , five of the most tick resistant b. taurus heifers had a small area ( approx . 5 cm in diameter ) of exudate ranging from minor to excessive at the site of larval release ( table 2 ) , indicating a host immune response to the parasite . by day 15 the relatively high weight gain on pasture of the adaptaur straightbred and f1 compared to the brahman ( table 1 ) would suggest that neither the imposition of 10 000 tick larvae nor the adaptaur anti - tick mechanism was detrimental to their productivity . the results presented in tables 1 and 2 suggest a relative advantage of adaptaur and adaptaur cross - over brahmans in tick resistance at the same level of growth . number of bos indicus ( straightbred brahman ) and bos taurus ( adaptaur and f1 adaptaur cross ) 24-month - old heifers in each class of tick resistance from an artificial infestation of cattle tick ( rhipicephalus microplus ) on belmont research station november 2007 * to confirm the viability of the tick larvae a straightbred murray grey heifer from ben m'cree of known high susceptibility ( field counts > 100 ticks / side at both 6 and 12 months of age at ben m'cree , personal communication p and l quayle ) of the same cohort was pastured with the above heifers at belmont and produced a count of 233 mature ticks by day 21 of the artificial infestation . bos taurus ( adaptaur straightbred and f1 adaptaur cross ) heifers with individual tick counts ( rhipicephalus microplus ) at 6 , 12 and 24 months of age and an assessment of the degree of exudate at the site of larval release from an artificial infestation of cattle tick at 24 months of age on belmont research station , november 2007 tick counts ( /side ) courtesy of p and l quayle from field infestations at ben m'cree , peachester , queensland , australia . there are a number of adaptation mechanisms that could explain the relatively high levels of resistance to tropical stressors for the adaptaur , n'dama , senepol and criollo breeds . a major gene for hair length slick hair that contributes to improving the animal 's thermoregulatory ability , has been described in both senepol and venezuelan carora a two - breed composite of brown swiss and local criollo ( olson et al . it could be speculated that the senepol also has the potential for high tick resistance given its n'dama origin and slick coat ( claxton and leperre 1991 ; hammond et al . o'kelly and spiers ( 1983 ) using animals from the hs line , among other breeds , demonstrated that fewer ticks matured on clipped areas of coat compared to unclipped areas . a livestock population surviving with minimal intervention to remove environmental stress , such as not treating the adaptaur with acaricide to remove parasites , means that the population was under natural selection pressure . frisch ( 1981 ) also concluded that the increasing level of resistance to environmental stress was solely responsible for the improved growth rates for this population . the 33% improvement in calving rates of adaptaur cows from 1992 to 1996 , compared to adaptaurs before selection 19641972 ( o'neill and frisch 1998 ) , is further evidence that high selection intensity ( high growth and high tick resistance ) on fitness traits was successful for european b. taurus in a tropical environment . in a large australian purebred - crossbreeding study from the cooperative research centre for cattle and beef quality , newman et al . the transition from unadapted to an adapted state for the n'dama , adaptaur , senepol and criollo could also involve elements of phenotypic plasticity , defined as the ability of a genotype to produce more than one phenotype in response to differing environments ( scheiner 1993 ; de jong 2005 ; fordyce 2006 ) . existence of such alleles has implications for other semi - arid regions such as the livestock component in the management of dryland salinity of southern australia as described by masters et al . given the extreme range of environments involved , plus the simultaneous natural and artificial selection pressure applied to achieve fitness , phenotypic plasticity could have contributed to the adaptation of the b. taurus to extreme and highly variable environmental parameters , and therefore future breeding programmes in variable challenging environments . extreme environmental conditions that result in high selection intensity on traits directly related to fitness have been shown to impact directly on the evolutionary process . hoffmann and parsons ( 1991 ) also point out that short periods ( years , decades ) of high selection intensity , rather than long periods ( century or more ) of low selection intensity , favour genes of major effect in the evolutionary process hence the emergence of a major gene for slick hair that contributes to heat resistance in the senepol and carora breeds ( olson et al . nevertheless , because complex response mechanisms to multiple and variable stressors are involved , the contribution of quantitative genetics and molecular biology for the improvement of b. taurus to a stressful tropical and/or semi - arid environment remains to be fully assessed . the completion of the bovine genome assembly ( the bovine genome sequencing and analysis consortium 2009 ) provides an opportunity to understand how cattle evolved and adapted to different geographical regions and production systems . they concluded that these changes in the cattle lineage probably reflect metabolic , physiologic and immune adaptations due to microbial fermentation in the rumen , the environment and its influence on disease transmission , and the reproductive strategy of cattle . indeed , understanding the taurine evolutionary trajectories also provides an insight into the predictability of cattle performance in cattle industries of the 21st century . in the previous sections , we reviewed and discussed the evolutionary history and genetics of b. taurus cattle and the selection pressures imposed by different environments and/or production systems using tick resistance as a case study . in a systems framework , it is the interactions between genotype , environment and management that contribute to evolution of different genotypes . compared to the tropically adapted b. taurus , the evolutionary history of temperate ( productivism ) b. taurus possess inheritantly higher levels of production potential , the production achieved in the absence of all environmental stress , and attributed to traits such as higher metabolic rates and appetites of the temperate taurine breeds . conversely , the evolution of b. taurus in environmentally stressful regions has ensured that those genotypes possess mechanisms of adaptation ( e.g. historically in commercial livestock production systems environmental stress is alleviated with strategies such as parasite control and supplementary feeding . figure 2 shows the g e m interactions in terms of relative production potential of temperate and tropical b. taurus ( g ) in the presence and absence of management ( m ) to negate the effect of increasing levels of environmental stress ( e ) . the relative levels of production potential are shown on the vertical axis whilst the increasing levels of environmental stress are shown on the horizontal axis . thus , as the level of environmental stress increases , the decline in production of the tropically adapted b. taurus is substantially less than the decline of the temperate b. taurus but if there is intervention to remove environmental stress then the response on production by the temperate taurine breeds would be greater than the response by the tropically adapted taurine breeds ( fig . this implies that the costs associated with maintaining high productivity in temperate b. taurus might negate the profit obtained via high production . diagrammatic representation of the g e m for bos taurus adapted to a temperate environment ( e.g. n'dama ) showing the proportional decline from optimal production potential for growth when there is no intervention by management to alleviate increasing levels of tropical environmental stress . moreover , interactions between genotype and management ( e.g. if , however , n'dama and angus cattle are not given any treatment of acaricide in the same region , then n'dama cattle are likely to achieve higher production performance than angus cattle ( fig . livestock production in the 21st century will be characterized by systems where both market ( food and fibre output ) and nonmarket ( social , environmental and climate change ) issues are given consideration . thus , livestock producers will maintain their drive for maximization of output but with diminished reliance on industrialization and therapeutic agents to alleviate environmental stress and rely more on the genetic make - up of their animals to combat stress from heat , parasites , pathogens and poor nutrition . various evolutionary trajectories of b. taurus since domestication suggest that the environmentally adapted breeds , along with b. indicus breeds , will play a pivotal role in the transition to sustainable production systems . the review of work on such breeds as the n'dama , senepol , criollo and adaptaur implies that both natural and artificial selection should be incorporated into selection decisions once there is an understanding of the beef and dairy production diseases and the mechanisms of resistance to environmental stress then , as with the plant industries , molecular biology technology may be successfully applied to cattle . we also conclude that the four adapted taurine breeds noted above , along with breeds that have undergone similar evolutionary trajectories , would possess candidate genes that could contribute to improving the productivity of cattle in challenging environments . the evolutionary history and knowledge of the genetic architecture will provide estimates of production potential ( in a favourable environment ) and level of resistance to environmental stress ( in an unfavourable environment ) and therefore estimates of how much production is realized when environmental stressors are controlled . hence , the performance of a breed or combination of breeds in a given environment or degree of intervention by management to alleviate environmental stressors , are able to be predicted . utilizing the full compliment of global cattle genetics and knowledge of g e m interactions will help ensure sustainable levels of beef and milk production in the 21st century .

since the 1950s in the developed countries , the era of agricultural productivism emerged with a corresponding demise in the influence of natural selection in livestock production systems . the genetic diversity of both bos taurus and bos indicus cattle was reduced with the concentration on breeds and sires within breeds that had the highest production potential . more recently , there has been a trend for a measured increase in productivity and growing pressure for cattle production systems to manage their total carbon budgets , which will require a reduction in their greenhouse gas emissions . the reduction in the use of fossil fuel derived energy and nutrients ( direct : fuel and fertilizer ; indirect : acaricide , anthelmintics and vaccine ) will reduce the ability to manage directly environmental stress . the evolutionary path of cattle since domestication provides insight into the current fitness levels as well as a better understanding of the interactions between cattle genotypes , environments and management intervention strategies ( hence forth called g e m interactions ) . there has been a long history of domestication and implicit genetic selection ; however , systematic breeding programmes have only been used over the last 60 years . however , a recent article published in science ( the bovine hapmap consortium 2009 ) looked at the process of cattle domestication . hence , any future proposal for evolutionary management of cattle domestication should consider this worldwide structure of the breeds to design conservation programmes and mating schemes . the opportunity for modern cattle breeds to evolve in response to environmental stress is restricted as the diversity of the gene pool of cattle populations diminishes . however , there are examples of cattle breeds that have demonstrated rapid evolutionary change : in particular where b. taurus cattle have been reared in challenging tropical environments . the evolution of the adaptaur under tropical stress provides a model approach to cattle breeding , which , if applied within the broader livestock industries might determine more favourable genotypes especially in the advent of escalating environmental stress due to climate change . this study explores the consequences of selection for production traits and demonstrates evolutionary genetics of taurine cattle in both temperate ( favourable ) and tropical ( unfavourable ) environments . as an example of evolutionary adaptation we focus on the adaptaur from northern australia ; however , we also draw on examples from three other b. taurus breeds that all have evolutionary backgrounds in unfavourable environments : senepol from the caribbean , the criollo breed group from central and south america and the n'dama from west africa . we show how exposure to a specific environmental stress ( cattle tick infestation in a tropical environment ) demonstrates the potential for experimental evolution to generate new more robust genotypes that can perform in environmentally stressful environments . the principles of evolutionary genetics can be used to facilitate darwinian fitness in future livestock breeding programmes to generate optimal genotypes for a low - cost and environmentally sustainable production system . in essence , there are complex interactions between genotype , environment and management ( g e m ) in the livestock systems and the implications relate to : ( i ) fitting the right genotypes to the right environments and production systems and ( ii ) determining the optimal genotype across a range of environments and production systems . we provide some perspectives on g e m and how this could contribute to evolution of future strains or breeds of cattle . about 10 000 years bp ( before present ) , neolithic hunter - gathers began to domesticate simultaneously both b. taurus ( fertile crescent and saharan africa ) and b. indicus ( indian subcontinent ) ( bradley et al . the history of b. indicus in asia ( harris 1992 ) , and more recent introduction to africa via the african east coast about 3500 years bp ( hanotte et al . european taurine cattle were also subjected to the stressors of tropical and semi - arid regions as these cattle were a significant component of the human colonization of central and south america by the spanish and portuguese during the 16th century . also regarded as tropically adapted is the senepol , a two - breed composite of n'dama and red poll cattle , bred on the caribbean island of st croix in 1918 ( hammond et al . herefords and shorthorns , first imported into australia from united kingdom in 1826 , along with the south african cattle breed , africander imported from king ranch , texas , usa in 1953 formed part of a csiro research programme at the belmont research station , rockhampton in the 1950s ( kennedy and turner 1959 ) . selection lines within a four - breed composite of africander , brahman , hereford and shorthorn , commercialized as belmont red , and a two - breed composite of hereford and shorthorn ( hs line ) were established . quantitative genetic methods have been a crucial component of productivism : development of selection index principles in animal breeding programmes in early 1940s ( hazel and lush 1943 ) followed by application of best linear unbiased prediction ( blup ) of animal 's genetic merit from early 1970s : the animal model ( henderson 1975 ) . utilizing these sophisticated levels of technologies , livestock industries of the developed countries have achieved significant increases in the product quantity and quality within the optimal ( i.e. there is an emerging question of the costs of stress - free selection as genotypes are exposed to more stressful environments . whilst management intervention has increasingly reduced environmental stress the ability for an individual to respond has decreased as genetic selection has refined metabolic pathways towards product output at the expense of other traits such as disease resistance . however , research and breeding programmes of dairy cattle have started to shift emphasis from production - only traits in the breeding goal to traits that are associated with robustness ( e.g. as with the dairy industries there has been an increase in persistent production diseases in the beef industries ; namely respiratory diseases and metabolic disorders in the feedlot systems ( e.g. however , genetic methods for the identification of host resistance / susceptibility for diseases such as ibk in the us department of agriculture reference cattle population ( casas and stone 2006 ) and australian adaptaur cattle ( ali et al . therefore , a multi - trait breeding objective would appear as : where is the total breeding value , bi is an economic weight for the ith trait in breeding objective , ebvi is the ebv for the ith trait and n is the number of traits in the breeding goal . application of these novel methods has had major beneficial effects on the cost and quality of food , and on the efficiency , competitiveness and welfare status of the livestock industries concerned . in theory , the metabolic energy is therefore available for production traits but with continued selection for high production the dairy cow is unable to consume enough energy to meet this production potential and the cow 's fertility ( butler and smith 1989 ; kadarmideen et al . animals of low resistance to environmental stress have inherently higher rates of growth , appetite and fasting metabolism than animals relatively resistant to environmental stress ( frisch and vercoe 1984 ) . thus , selection for production traits , disregarding physiological limits , fertility and resistance to environmental stress , would have serious negative consequences for future cattle herds as cows can only be maintained with high inputs that are unsustainable in the longer term . the three main genetic processes involved in the evolution of animals during domestication are selection , genetic drift and inbreeding ( falconer and mackay 1996 ) , and inbreeding is posing additional problems for the intensive cattle industries . for the dairy industry in the usa , ( 1998 ) found that the effects of inbreeding in the holstein herd , years 19831993 , were cumulative and larger on lifetime profit functions than on lactation traits . we argue that for cattle of the 21st century it will be necessary to also explore the evolution of cattle in unfavourable environments , where there has been selection for alleles at many loci offering specific environmental adaptations . documenting the plethora of tropical environmental stressors and the corresponding responses of b. taurus to these stressors is beyond the scope of this study . therefore , we present data from experiments that aimed to assess the evolutionary adaptation of b. taurus cattle to stress from infestations of the ectoparasite cattle tick rhipicephalus ( boophilus ) microplus . by the end of last century , estimates of the yearly global costs of cattle tick and tick - borne diseases were between us$13.9 and us$18.7 billion ( de castro 1997 ) . the global costs of ticks are set to escalate ; increasing levels of acaricide resistance ( sangster 2001 ) , limited efficacy of anti - tick vaccines and emergence of nonresponders to vaccination ( outteridge 1993 ; sonenshine et al . exploring the full repertoire of the animal 's anti - tick mechanisms is warranted if genetic technologies that utilize naturally occurring adaptation to parasitic stress are going to contribute to sustainable livestock production . a similar case occurred in 1952 with the introduction of herefords and shorthorns ( hs line ) on the csiro belmont research station , a property located 26 km nnw of rockhampton , in north - eastern australia . the origins of the adaptaur began on belmont in 1980 with the identification of a heifer ( animal identity : 790546 ) of the 1979 calf crop of the hs line and after 1980 selection for both high growth tick resistance within the hs line focused on this family . figure 1 presents a compilation of tick count data from various studies of the 1980s on adaptation , production or heterosis [ see frisch and vercoe ( 1984 ) , frisch ( 1987 ) , frisch et al . these studies were conducted as a precursor to a major heterosis study conducted during the 1990s , involving breeds of european , indian and african origins , to identify the optimal genotype for herd productivity in a stressful tropical environment ( frisch and o'neill 1998a , b , c ) . figure 1 illustrates that there is a strong tendency in low tick counts of progeny from sires of the 790546 family compared to progeny from sires of the remaining adaptaur herd and progeny from the brahman sires . the focus on the maternal lineage of 790546 not only assisted in rapid reduction in the phenotypic trend in tick count ( fig . arithmetic means ( three highest counts for the calf crop ) ( sem ) of field tick ( rhipicephalus microplus ) infestations of heifer progeny of brahman and adaptaur sires for calf crops 1979 , 19831984 and 19861988 on belmont research station . twenty - seven years after the identification of heifer 790546 , in 2007 descendents from this heifer exhibited high tick resistance ( table 1 ) and with some straightbred animals ( e.g. at 48-h postinfestation , the adaptaur straightbred has not lost its high resistance to cattle tick , even after 27 years ( table 1 ) , and this high tick resistance appears to be conferred to other taurine breeds regardless of their genetic background for tick resistance ( table 2 ) . the finding that some heifers with high counts at 6 months of age have progressively lower counts at 12 months of age and 24 months of age ( table 2 ) confirms that resistance is acquired . by day 15 the relatively high weight gain on pasture of the adaptaur straightbred and f1 compared to the brahman ( table 1 ) would suggest that neither the imposition of 10 000 tick larvae nor the adaptaur anti - tick mechanism was detrimental to their productivity . the results presented in tables 1 and 2 suggest a relative advantage of adaptaur and adaptaur cross - over brahmans in tick resistance at the same level of growth . number of bos indicus ( straightbred brahman ) and bos taurus ( adaptaur and f1 adaptaur cross ) 24-month - old heifers in each class of tick resistance from an artificial infestation of cattle tick ( rhipicephalus microplus ) on belmont research station november 2007 * to confirm the viability of the tick larvae a straightbred murray grey heifer from ben m'cree of known high susceptibility ( field counts > 100 ticks / side at both 6 and 12 months of age at ben m'cree , personal communication p and l quayle ) of the same cohort was pastured with the above heifers at belmont and produced a count of 233 mature ticks by day 21 of the artificial infestation . bos taurus ( adaptaur straightbred and f1 adaptaur cross ) heifers with individual tick counts ( rhipicephalus microplus ) at 6 , 12 and 24 months of age and an assessment of the degree of exudate at the site of larval release from an artificial infestation of cattle tick at 24 months of age on belmont research station , november 2007 tick counts ( /side ) courtesy of p and l quayle from field infestations at ben m'cree , peachester , queensland , australia . there are a number of adaptation mechanisms that could explain the relatively high levels of resistance to tropical stressors for the adaptaur , n'dama , senepol and criollo breeds . the following scenarios highlight the complexity of disease resistance and pertain to evolutionary genetics : chance mutations , a synergy between natural selection and artificial selection and phenotypic plasticity . ( 2005 ) contend that the host 's optimum immune response to parasites and pathogens is one of immunosufficiency , because the former is a balance between energy loss due to pathogen infection and energy expenditure mounting an immune response that is conducive to fitness . naessens ( 2006 ) has shown that it is the inappropriate response of the immune system that results in the mortality and morbidity of trypanosome - infected cattle . a major gene for hair length slick hair that contributes to improving the animal 's thermoregulatory ability , has been described in both senepol and venezuelan carora a two - breed composite of brown swiss and local criollo ( olson et al . the 33% improvement in calving rates of adaptaur cows from 1992 to 1996 , compared to adaptaurs before selection 19641972 ( o'neill and frisch 1998 ) , is further evidence that high selection intensity ( high growth and high tick resistance ) on fitness traits was successful for european b. taurus in a tropical environment . the transition from unadapted to an adapted state for the n'dama , adaptaur , senepol and criollo could also involve elements of phenotypic plasticity , defined as the ability of a genotype to produce more than one phenotype in response to differing environments ( scheiner 1993 ; de jong 2005 ; fordyce 2006 ) . it is conceivable that the mexican criollo from the chihuahuan desert3 possess alleles pertinent to plasticity for variations in water consumption , extreme fluctuations in ambient temperature , metabolism of a variety of herbaceous plants , and alternating between browsing and grazing . given the extreme range of environments involved , plus the simultaneous natural and artificial selection pressure applied to achieve fitness , phenotypic plasticity could have contributed to the adaptation of the b. taurus to extreme and highly variable environmental parameters , and therefore future breeding programmes in variable challenging environments . hoffmann and parsons ( 1991 ) also point out that short periods ( years , decades ) of high selection intensity , rather than long periods ( century or more ) of low selection intensity , favour genes of major effect in the evolutionary process hence the emergence of a major gene for slick hair that contributes to heat resistance in the senepol and carora breeds ( olson et al . the first large - scale gene expression profiling study ( skin cdna microarrays ) was undertaken using resistant / susceptible adaptaur cattle and identified several candidate mechanisms ( e.g. nevertheless , because complex response mechanisms to multiple and variable stressors are involved , the contribution of quantitative genetics and molecular biology for the improvement of b. taurus to a stressful tropical and/or semi - arid environment remains to be fully assessed . the completion of the bovine genome assembly ( the bovine genome sequencing and analysis consortium 2009 ) provides an opportunity to understand how cattle evolved and adapted to different geographical regions and production systems . the biological systems most affected by changes in the number and organization of genes in the cattle lineage include reproduction , immunity , lactation and digestion , all of which are important to our discussion on tropical adaptation . they concluded that these changes in the cattle lineage probably reflect metabolic , physiologic and immune adaptations due to microbial fermentation in the rumen , the environment and its influence on disease transmission , and the reproductive strategy of cattle . indeed , understanding the taurine evolutionary trajectories also provides an insight into the predictability of cattle performance in cattle industries of the 21st century . in the previous sections , we reviewed and discussed the evolutionary history and genetics of b. taurus cattle and the selection pressures imposed by different environments and/or production systems using tick resistance as a case study . compared to the tropically adapted b. taurus , the evolutionary history of temperate ( productivism ) b. taurus possess inheritantly higher levels of production potential , the production achieved in the absence of all environmental stress , and attributed to traits such as higher metabolic rates and appetites of the temperate taurine breeds . however , in terms of adaptation to environmental stress the reverse is true and the n'dama and criollo possess relatively higher levels of environmental adaptation than breeds synonymous with productivism . figure 2 shows the g e m interactions in terms of relative production potential of temperate and tropical b. taurus ( g ) in the presence and absence of management ( m ) to negate the effect of increasing levels of environmental stress ( e ) . thus , as the level of environmental stress increases , the decline in production of the tropically adapted b. taurus is substantially less than the decline of the temperate b. taurus but if there is intervention to remove environmental stress then the response on production by the temperate taurine breeds would be greater than the response by the tropically adapted taurine breeds ( fig . if n'dama and angus cattle are given the same dosage of acaricide in a region of tick infestation , angus cattle responds better to treatment than n'dama cattle ( response as in growth and fertility ) . there is g m. hence , a livestock system in essence is defined by the g e m interactions and understanding g e m and its impact on genetic , environmental and managerial strategies is crucial in future livestock systems . thus , livestock producers will maintain their drive for maximization of output but with diminished reliance on industrialization and therapeutic agents to alleviate environmental stress and rely more on the genetic make - up of their animals to combat stress from heat , parasites , pathogens and poor nutrition . the review of work on such breeds as the n'dama , senepol , criollo and adaptaur implies that both natural and artificial selection should be incorporated into selection decisions once there is an understanding of the beef and dairy production diseases and the mechanisms of resistance to environmental stress then , as with the plant industries , molecular biology technology may be successfully applied to cattle . we also conclude that the four adapted taurine breeds noted above , along with breeds that have undergone similar evolutionary trajectories , would possess candidate genes that could contribute to improving the productivity of cattle in challenging environments . the evolutionary history and knowledge of the genetic architecture will provide estimates of production potential ( in a favourable environment ) and level of resistance to environmental stress ( in an unfavourable environment ) and therefore estimates of how much production is realized when environmental stressors are controlled . utilizing the full compliment of global cattle genetics and knowledge of g e m interactions will help ensure sustainable levels of beef and milk production in the 21st century .

western flower thrips ( wft ) , frankliniella occidentalis , is a highly polyphagous insect that causes both direct and indirect effects on plant development and health . the adults and larvae feed on epidermal and subepidermal cells of both meristematic and mature leaf and flower tissues , inhibiting plant growth and development and causing necrotic or light - reflective blotches on the tissue . furthermore , they indirectly damage plants by transmitting tospoviruses such as tomato spotted wilt virus ( reitz , 2009 ) . as a result , wft has become the most serious pest in several vegetable and flower crops world - wide ( daughtrey et al . , 1997 ; reitz , 2009 ) . the widespread use of chemical insecticides to control wft has led to increasing resistance against the major classes of synthetic insecticides ( broadbent and pree , 1997 ; flanders et al . , 2000 ; broughton and herron , 2009 ) . the growing awareness and demand for insecticides that are not environmentally hazardous has stimulated the study of plant - derived compounds for pest control ( boeke et al . , 2004 ) . such compounds could be used as natural pesticides , and in theory , genes responsible for the biosynthesis of those compounds could be isolated and transferred to crops to improve plant defense against wft ( annadana et al . , 2002 ; among the sources of botanical pesticides , pyrethrins from pyrethrum plants ( tanacetum cinerariifolium ) represent the economically most important class of compounds with broad usage both in homes and organic agriculture ( casida , 1973 ) . pyrethrins are neurotoxins that bind to voltage - gated sodium channels of neuronal cells , causing the channels to remain open ( davies et al . , 2007 ) . pyrethrins comprise a group of six closely related esters , named pyrethrin i and ii , cinerin i and ii , and jasmolin i and ii . they are found in all aboveground parts of the pyrethrum plants , but predominantly in the ovaries of the flower heads ( brewer , 1973 ) . on average , the concentration of pyrethrins is about 0.1 % ( dry weight ) in leaves and 1 - 2 % ( dry weight ) in flowers ( baldwin et al . , 1993 ) . assuming a water content of 90 % , pyrethrins account for around 0.01 % of the fresh weight of leaves and 0.1 - 0.2 % of the fresh weight of flowers . pyrethrins are effective against a broad spectrum of insects , while their toxicity for mammals is very low , allowing their use as a preharvest spray ( casida and quistad , 1995 ; schoenig , 1995 ) . western flower thrips are sensitive to synthetic pyrethroids ( thalavaisundaram et al . , 2008 ) , but there is no report on the effect of natural pyrethrins against wft . pyrethrins might provide pyrethrum with a broad range protection against many different insect pests , but the role of pyrethrins in pyrethrum defense has not been studied . in initial experiments , we observed that wft adults died within one day when fed pyrethrum leaves , but that they were abundant in open flowers . here , we tested the hypothesis that pyrethrins are responsible for protecting pyrethrum leaves against wft by assessing adult and embryo toxicity , and by examining feeding and oviposition deterrence both in vitro and in planta . a pyrethrum field close to luxi , yunnan province , china , was used for surveying thrips populations ( 2427'10.34"n-10332'21.01"e ) . the field was 0.5 ha in size , and the presence of insect species was monitored during the flowering period of spring 2010 , when the flowers were predominantly in developmental stages 25 [ numbered according to casida ( 1973 ) ] . to assess populations of small resident insects including thrips , flowers at each developmental stage each flower was taken by the stem and turned upside down into a jar containing 75 % alcohol . the procedure was repeated until each jar contained the insects from 100 flowers from a single block and at a particular stage . the number of insects of each species for each stage was scored . in the case of thrips , the number of adults and larvae were scored separately . among all collected thrips , 30 were randomly picked and identified , where possible to the species level . a population of wft was mass - reared on flowering chrysanthemum ( chrysanthemum morifolium ramat . ) sunny casa in a greenhouse under a photoperiod of l16:d8 at 25 2c . in this study , the chrysanthemum plants used for bioassays were from the same cultivar , but were grown in an insect - free compartment of the greenhouse under the same light and temperature conditions . all bioassays were conducted in a climate room at 2022c with a l16:d8 photo regime . pyrethrum oil ( 70 % w / w ) had been extracted from dried and ground pyrethrum flower heads with liquid co2 leaving no solvent residue ( honghe senju biological co. ltd . , yunnan , china ) . butylated hydroxytoluene ( bht ) had been added to the oil ( 1 % ) to prevent oxidation . we confirmed the concentration and composition of the oil by gas chromatography mass spectrometry comparison to a pyrethrin standard ( nguyen et al . , 1998 ) . since the major insecticidal compounds in pyrethrum have long been known as pyrethrins ( casida , 1973 ) , the effect of pyrethrum oil was considered to be the effect of pyrethrins . when calculating the concentrations of pyrethrins in different solutions , the percentage of pyrethrins in the oil ( 70 % ) was taken into account . for example , 1 % ( w / v ) pyrethrins was prepared by dissolving 14.3 mg pyrethrum oil in 1 ml solvent . the toxicity of pyrethrins was evaluated by topical application to thrips ( robb et al . , 1995 ) . pyrethrum oil was dissolved in acetone to achieve a concentration range of 1 to 30 mg pyrethrins per ml , and the solutions were applied to the thorax with a 10-l glass syringe at 1 l per thrips . the droplet briefly covered the thorax of the insect and also the paper support before evaporating in a few seconds , leaving a residue both on the insect and the support . after treatment , all thrips were transferred to petri dishes containing a piece of chrysanthemum leaf embedded in an agar substrate . mortality was assayed after 24 h by counting the number of insects that did not respond to prodding with a fine brush . six replicates were used for each concentration , and 10 thrips were used per replicate . percent mortality was corrected for mortality observed in acetone control using schneider - orelli s formula ( schneider - orelli , 1947 ) . a dual - choice leaf disk assay was used to determine the deterrent effect of pyrethrins on wft . all leaf disks ( diam 1.6 cm ) were punched from chrysanthemum leaves of similar leaf age . pyrethrum oil was dissolved in 0.2 % ( v / v ) aqueous tween-80 to achieve 3 concentrations of pyrethrins : 0.01 , 0.1 , and 1 % ( w / v ) . control leaf disks were sprayed with solvent solution ( 0.2 % tween-80 ) , and test leaf disks were sprayed with the pyrethrin solutions using a potter precision laboratory spray tower , which produced a uniform deposit ( 3 l / cm ) of solution on the leaf disks . groups of 10 wft were positioned between a control and a test leaf disk placed abaxial side up and 2 cm apart on a 1.5 % ( w / v ) agar - bed in a petri dish ( 7 cm diam ) . after positioning the thrips , the petri dish was covered by a 120 m mesh size nylon mesh lid to prevent condensation . the number of wft on each leaf disk was recorded 0.25 , 1 , 2 , 4 , 20 , and 28 h after the release of the wft . each concentration was replicated with 12 leaf disks . at each time point , a student 's paired t - test was used to assess the significance of the differences in the mean number of wft between test and control . oviposition - deterrent effects were assayed with a non - choice method slightly modified from annadana et al . the assay was conducted in perspex ring cages ( 3 cm in length and 3.5 cm diam ) , which were closed with a nylon mesh at the bottom . pollen of scotch pine ( pinus sylvestris l. ) was supplied in a small open tube as food source for wft . after placing 10 wft in a cage , the top was sealed with two layers of stretched parafilm , with 300 l aqueous solution in between the layers . the solutions used were water , 0.2 % tween-80 , or pyrethrins at 0.01 , 0.1 , or 1 % dissolved in 0.2 % tween-80 . wft were allowed to adapt to the diet ( pollen and water ) for 3 d , and then every day for 5 d fresh test solution was provided . all eggs were deposited in the solutions , and were counted daily under a binocular microscope . data were analyzed by a one - way anova and a mean separation test was conducted using lsd ( = 0.05 ) . around 200 wft were kept in a perspex ring cage ( 7 cm in length and 9 cm diam ) to allow oviposition in a water solution as described above . eggs laid on the same day were collected with a fine brush under a binocular microscope and then transferred to 2 layers of filter paper in petri dishes ( 3.5 cm diam ) . the filter papers were drenched in 300 l of assay solution ( water , 0.2 % tween-80 or pyrethrins at 0.01 , 0.1 , or 1 % in 0.2 % tween-80 ) so that each paper was fully wetted but had no excess solution . after transferring the eggs , the developmental status of eggs was monitored every day for 6 d. to facilitate the observations , the bottoms of the petri dishes were marked with lines that could be seen through the filter paper from the top , and the eggs were placed on filter paper along these lines . this facilitated finding the eggs under the microscope , and the viability of hatched larvae was assessed in terms of their ability to move away ( > 0.5 cm ) from the hatch position . data were analyzed by a one - way anova and mean separation test was conducted using lsd ( = 0.05 ) . mature pyrethrum leaves were harvested in november from a field in the netherlands when they were still flowering ( 5159'22.08"n-539'44.75"e , wageningen ) . two or three pieces of leaves were placed , abaxial side up , on 1 % ( w / v ) agar in a petri dish ( 7 cm diam ) . after transferring 10 wft to each petri dish , petri dishes with two leaf disks ( 1.6 cm diam ) of chrysanthemum leaves , with a total mass similar to the mass of the pyrethrum leaf samples , or with only agar were used as controls . the mortality of wft was recorded daily for 3 d. to test the in planta activity of pyrethrins against wft , pyrethrins were infiltrated into whole chrysanthemum leaves as described by ratcliff et al . leaf disks ( diam 1.6 cm ) were punched from the infiltrated leaves , avoiding the infiltration points so that wft would not contact pyrethrins directly except at the edge of the disk . in the initial experiments , we infiltrated only water into the leaves and determined that on average 29.1 mg ( 2.1 mg ) water could be infiltrated into each leaf disk ( 6 replicates ) . as the fresh weight of each leaf disk was on average 45.3 mg ( 1.2 mg ) , we infiltrated 0.025 , 0.25 , or 2.5 % pyrethrins solution to bring the concentrations to 0.01 , 0.1 , or 1 % pyrethrins . the assay and data analysis were conducted as described above for the choice assays with topically applied pyrethrins . the number of wft on each leaf disk was recorded 0.25 , 1 , 2 , 4 , 20 , and 28 h after the release of the wft . to test the effects of pyrethrins on oviposition and hatching of larvae , wft were assayed with chrysanthemum leaf disks as described by de kogel et al . leaf disks were punched from untreated leaves , from leaves infiltrated with 0.2 % tween-80 , or from leaves containing 0.01 , 0.1 , or 1 % pyrethrins in tween solution . wft were placed on leaf disks ( 1.2 cm diam , 2 wft / disk ) , which were embedded , abaxial side up , on agar in wells of 24-well greiner plates . plates were covered with parafilm , and every well was carefully sealed by pressing the parafilm on the edge of each well . wft were allowed to oviposit for 48 h and were then removed , with simultaneous assessment of mortality . subsequently , half of the leaf disks from each plate were used to determine the number of eggs , and the other half of the leaf disks were used to determine the number of hatched larvae . to determine the number of eggs , the leaf disks were boiled in water for 3 min so that the eggs were clearly visible under a binocular microscope with transmitting light . to determine the number of hatched larvae , the leaf disks were transferred to petri dishes containing water and incubated in a climate chamber ( 25c , l16:d8 ) for 5 d to allow the larvae to hatch . data were analyzed by a one - way anova and mean separation test was conducted using lsd ( = 0.05 ) . our field survey in china showed that several thrips species were the most abundant ( 98 % ) insects on pyrethrum flowers ( table 1 ) . in addition , a few nysius species ( heteroptera : lygaeidae ) ( 1.9 % ) and lacewing larvae ( neuroptera ) ( 0.05 % ) were found . a total of 30 individuals were identified to species level ; the thrips species found were mainly thrips tabaci ( 44 % ) , frankliniella occidentalis ( western flower thrips , or wft , 25 % ) , and thrips flavus ( 22 % ) . the number of thrips in flowers was dependent on the flower s developmental stage ( fig . 1 ) . the number of thrips increased until stage 3 ( the first row of disk florets are open ) , and then decreased in later stages . larvae accounted for 7 - 26 % of the total number of thrips per flower , depending on the flower developmental stage ( fig . 1).table 1frequencies of small insect species living on pyrethrum flowers in the fieldinsectsfrequency ( % ) speciesfrequency ( % ) thripidae ( thrips)98thrips tabaci43frankliniella occidentalis25thrips flavus21thrips palmi3other species6nysius sp.1.9n.d.1.9chrysoperla / chrysopa sp . ( lacewing larva)0.05n.d.0.05a total of 1200 insects were collected to count the frequencies of different insects . 1distribution of thrips adults and larvae across different developemental stages of pyrethrum flowers in the field . error bars indicate se ( n = 300 per stage ) . stage 2 , vertical ray florets ; stage 3 , horizontal ray florets and first row of disk florets open ; stage 4 , 3 rows of disk florets open ; stage 5 , all disk florets open frequencies of small insect species living on pyrethrum flowers in the field a total of 1200 insects were collected to count the frequencies of different insects . , not determined distribution of thrips adults and larvae across different developemental stages of pyrethrum flowers in the field . stage 2 , vertical ray florets ; stage 3 , horizontal ray florets and first row of disk florets open ; stage 4 , 3 rows of disk florets open ; stage 5 , all disk florets open we assayed the suitability of pyrethrum leaves as a food substrate for wft . mortality could be as high as 80 % within 3 days , although the degree of mortality depended on the plant source ( data not shown ) . when only water and agar were provided , with no plant - based food , only 20 - 30 % wft died in 3 days . this showed that the mortality of wft was caused by a toxic principle of pyrethrum leaves rather than deterrence or starvation . the toxic principle of pyrethrum plants against insects has long been known to be a group of 6 pyrethrin esters ( casida , 1973 ) . we were , therefore , interested in specifically testing the effect of pyrethrins against wft . to determine the effects of pyrethrins against wft , pyrethrins were tested in vitro at different concentrations on adult mortality , feeding , oviposition , and embryo development . the mortality of wft female adults increased with the concentration of topically applied pyrethrins in the range of 1 to 30 mg / ml . probit analysis showed that the lc50 and lc90 of pyrethrins was 12.9 mg / ml ( with 95 % confidence limit of 10.9 - 14.8 mg / ml ) and 39.0 mg / ml ( with 95 % confidence limit from 30.7 to 50.4 mg / ml ) , respectively . thrips were significantly deterred from feeding by 0.1 % and 1 % pyrethrins ( fig . 2 ) . when given a choice between chrysanthemum leaf disks coated with 0.2 % tween ( control ) or 0.1 % added pyrethrins , after 2 h significantly more ( 61 - 77 % of thrips ) settled on control leaf disks . within 1 h , 72 - 90 % of thrips chose control leaf disks . for both concentrations of pyrethrins , the maximum deterrent effect was reached at 4 h. application of 0.01 % pyrethrins on leaf disks did not show significant deterrent effects except at the 4 h time point ( fig . 2).fig . 2dual choice assays of western flower thrips on chrysanthemum leaf disks sprayed with 0.2 % tween ( control ) or 0.2 % tween with 0.01 % , 0.1 % or 1 % pyrethrins . the presence on either leaf disk was visually recorded 0.25 , 1 , 2 , 4 , 20 and 28 h after wft release . asterisks indicate significant differences to the control ( * : p < 0.05 ; * * : p < 0.01 ) . error bars indicate se ( n = 120 per treatment ) dual choice assays of western flower thrips on chrysanthemum leaf disks sprayed with 0.2 % tween ( control ) or 0.2 % tween with 0.01 % , 0.1 % or 1 % pyrethrins . the presence on either leaf disk was visually recorded 0.25 , 1 , 2 , 4 , 20 and 28 h after wft release . asterisks indicate significant differences to the control ( * : p < 0.05 ; * * : p < 0.01 ) . error bars indicate se ( n = 120 per treatment ) pyrethrins negatively affected oviposition by wft ( fig . 3 ) . the carrier , 0.2 % tween-80 , did not affect the oviposition of thrips compared to water throughout the experiment , but wft oviposited significantly fewer eggs with increasing pyrethrin concentrations during the 5-day experiment ( fig . 3the number of eggs deposited by western flower thrips when supplied with different concentrations of pyrethrins starting on day 1 . data points with the same letter within days are not significantly different , p < 0.05 . error bars indicate se ( n = 60 per treatment ) the number of eggs deposited by western flower thrips when supplied with different concentrations of pyrethrins starting on day 1 . data points with the same letter within days are not significantly different , p < 0.05 . error bars indicate se ( n = 60 per treatment ) the development of eggs was inhibited by 0.1 % and 1 % pyrethrins . about 80 % of larvae hatched when the eggs were incubated with water , 0.2 % tween , or 0.01 % pyrethrins , while only 28 % or 6 % of the larvae hatched when the eggs were incubated with 0.1 % or 1 % pyrethrins , respectively ( fig . the embryos that did not develop into larvae had severely stunted and abnormal shapes ( fig . 4percentage of larvae hatching from western flower thrips eggs during incubation with different concentrations of pyrethrins . data points with the same letter within days are not significantly different , p < 0.05 . ( a ) , larva hatched in solutions of water , 0.2 % tween or 0.01 % pyrethrins at day 5 ; ( b ) , abnormally developed embryos in solutions of 0.1 % and 1 % pyrethrins at day 5 ; ( c ) and ( d ) , embryos before treatment percentage of larvae hatching from western flower thrips eggs during incubation with different concentrations of pyrethrins . data points with the same letter within days are not significantly different , p < 0.05 . error bars indicate se ( n = 40 per treatment ) effects of pyrethrins on embryo development of western flower thrips at day 5 . ( a ) , larva hatched in solutions of water , 0.2 % tween or 0.01 % pyrethrins at day 5 ; ( b ) , abnormally developed embryos in solutions of 0.1 % and 1 % pyrethrins at day 5 ; ( c ) and ( d ) , embryos before treatment to study in planta activity of pyrethrins against wft , thrips were assayed with chrysanthemum leaves that had been infiltrated with pyrethrins to contain 0.01 , 0.1 , or 1 % pyrethrins . in this experiment , the pyrethrins could not be contacted directly by thrips except by feeding , and the source of nutrition consisted of leaves instead of pollen . in the reproduction assay , thrips fed with chrysanthemum leaf disks containing pyrethrins exhibited higher mortality and lower reproduction rates compared to those fed with untreated leaf disks or leaf disks containing 0.2 % tween ( table 2).table 2mortality , number of eggs and hatched western flower thrips larvae per leaf disk on chrysanthemum leaf disks infiltrated with different concentrations of pyrethrinstreatment of leaf disksmortality ( % ) eggshatched larvaeuntreated leaf disks0 a2.0 0.4 a1.4 0.3 aleaf disks containing 0.2 % tween0 a1.7 0.5 a1.3 0.3 ableaf disks containing 0.01 % pyrethrins25.0 6.7 b1.3 0.3 ab0.8 0.2 bleaf disks containing 0.1 % pyrethrins29.2 7.9 b0.7 0.2 bc0.1 0.1 cleaf disks containing 1 % pyrethrins68.8 9.3 c0 c0 cvalues ( mean se , n = 48 per treatment ) followed by the same letter within a column are not significantly different ( anova : p > 0.05 ) mortality , number of eggs and hatched western flower thrips larvae per leaf disk on chrysanthemum leaf disks infiltrated with different concentrations of pyrethrins values ( mean se , n = 48 per treatment ) followed by the same letter within a column are not significantly different ( anova : p > 0.05 ) in the dual - choice assay , chrysanthemum leaves containing 0.1 % and 1 % pyrethrins showed significant deterrent effects on thrips within 15 min of release ( fig . a total of 74 - 93 % of the thrips settled on the control leaf disk when the other leaf disk contained 0.1 % pyrethrins , and 85 - 95 % thrips settled on the control leaf disk when the other leaf disk contained 1 % pyrethrins . 6percentage of western flower thrips settled on the control chrysanthemum leaf disk in dual choice assays of leaf disks containing 0.2 % tween with or without 0.01 % , 0.1 % , or 1 % pyrethrins . the choices were recorded 0.25 , 1 , 2 , 4 , 20 , and 28 h after wft release . asterisks indicate significant differences to the control ( * : p < 0.05 ; * * : p < 0.01 ) . error bars indicate se ( n = 120 per treatment ) percentage of western flower thrips settled on the control chrysanthemum leaf disk in dual choice assays of leaf disks containing 0.2 % tween with or without 0.01 % , 0.1 % , or 1 % pyrethrins . the choices were recorded 0.25 , 1 , 2 , 4 , 20 , and 28 h after wft release . asterisks indicate significant differences to the control ( * : p < 0.05 ; * * : p < 0.01 ) . pyrethrins , well - known natural insecticidal compounds , are found exclusively in and extracted from the composite flowers of pyrethrum ( tanacetum cinerariifolium ) , which belongs to anthemideae tribe within the astaraceae family ( casida and quistad , 1995 ) . remarkably , the potential role of pyrethrins in pyrethrum plant defense has not been studied . here , we report that western flower thrips ( wft ) adults thrive on pyrethrum flowers , but die within a few days on pyrethrum leaves . the hypothesis that pyrethrins are responsible for protecting pyrethrum leaves against wft was tested by spraying or infiltrating pyrethrins to leaves of chrysanthemum , a related pyrethrins - free species belonging to the same tribe . we assessed toxicity to the adult and embryo stages of wft , and negative effects on feeding and oviposition both in vitro and in planta , and found that the natural concentrations of pyrethrins present in leaves have strong negative effects on wft . we speculate that the thrips found on pyrethrum flowers survive on pollen that is devoid of pyrethrins ( t. yang , unpublished data ) . for many populations of wft , resistance has been reported for some synthetic insecticides ( espinosa et al . , 2005 ) . previously , several other plant - derived compounds were tested for their insecticidal effects against wft adults . for example , carvacrol at 1 % and thymol at 0.1 % and 1 % significantly reduced the oviposition rate of wft when these compounds were sprayed on leaf disks , but neither compound affected the feeding activity of wft ( sedy and kosehier , 2003 ) . salicylaldehyde ( 0.1 % and 1 % ) and methyl salicylate ( 0.1 % and 1 % ) were also tested . within 24 h of applying 1 % methyl salicylate to bean or cucumber , the feeding and oviposition activities of thrips females the insect could be a result of changes in the plant induced by methyl salicylate , since it is a plant hormone involved in induced resistance ( pieterse et al . , 2009 ) . a series of commercially available plant - derived essential oils tested at recommended concentrations ( 0.02 - 0.5 % ) , including neem oil , rosemary oil , peppermint oil , garlic oil , and cottonseed oil , caused less than 30 % mortality within 7 days ( cloyd et al . , 2009 ) . our results showed that 0.1 % and 1 % pyrethrin solutions sprayed on leaf disks significantly deterred wft at 4 h , and topically applied pyrethrins were toxic to adults at an lc50 value of 12.9 mg / ml ( 1.3 % ) . by mimicking the natural site of pyrethrin accumulation by infiltration of leaves , we found that 1 % pyrethrins caused 69 % mortality and completely inhibited oviposition . furthermore , 0.1 % pyrethrins was strongly deterrent and resulted in abortion of 95 % of the embryos , while as little as 0.01 % pyrethrins caused 25 % mortality in 2 days . we propose , therefore , that the natural concentrations of pyrethrin in pyrethrum leaves , around 0.01 % by fresh weight , accounts for the observed high mortality of thrips adults on this plant . wft eggs are embedded in plant tissues ( childers , 1997 ) , and as a result they are unlikely to be affected by non - systemic chemicals that are applied on the surface of plants . however , pyrethrins naturally accumulate inside pyrethrum tissues , stored in what appear to be unstructured intercellular cavities ( m.a . jongsma , unpublished observations ) . therefore , besides feeding and oviposition deterrence , the embryo - toxic effect of pyrethrins is a third component that contributes to their effect for plant defense against wft ( figs . 4 and 5 ) . compared to some synthetic insecticides , the toxicity of natural pyrethrins against wft in the absence of synergists was not high . in previous studies using topical application methods , the lc50 values of insecticides tested against susceptible wft strains ranged from 10 to 83 g / ml for pyrethroids , 20 to 960 g / ml for carbamates , and 49 to 522 g / ml for organophosphates ( espinosa et al . , 2005 the lc50 value of pyrethrins against wft by topical application was determined as 12.9 mg / ml , and the action of pyrethrins was , therefore , 10 to 1000-fold weaker than for these synthetic pesticides . on the other hand , pyrethrins did show much stronger negative effects on feeding behavior and reproduction , which may be explained by the action of pyrethrins on the nervous system , resulting in disordered function of excitable ( nerve and muscle ) cells ( bradberry et al . , pyrethrins not only caused mortality of adults and embryos , but also significantly reduced oviposition ( table 2 ) . all these factors together cumulatively affect the life history parameters . as a result wft damage on pyrethrin - containing leaves we hypothesize that if plants such as closely related chrysanthemum species , which do not contain any pyrethrins , were genetically engineered to produce pyrethrins , their resistance to wft in leaves could be significantly improved .

pyrethrins are active ingredients extracted from pyrethrum flowers ( tanacetum cinerariifolium ) , and are the most widely used botanical insecticide . however , several thrips species are commonly found on pyrethrum flowers in the field , and are the dominant insects found inside the flowers . up to 80 % of western flower thrips ( wft , frankliniella occidentalis ) adults died within 3 days of initiating feeding on leaves of pyrethrum , leading us to evaluate the role of pyrethrins in the defense of pyrethrum leaves against wft . the effects of pyrethrins on wft survival , feeding behavior , and reproduction were measured both in vitro and in planta ( infiltrated leaves ) . the lethal concentration value ( lc50 ) for pyrethrins against wft adults was 12.9 mg / ml , and pyrethrins at 0.1 % ( w / v ) and 1 % ( w / v ) had significantly negative effects on feeding , embryo development , and oviposition . about 20 - 70 % of wft were killed within 2 days when they were fed chrysanthemum leaves containing 0.01 - 1 % pyrethrins . chrysanthemum leaves containing 0.1 % or 1 % pyrethrins were significantly deterrent to wft . in a no - choice assay , the reproduction of wft was reduced significantly when the insects were fed leaves containing 0.1 % pyrethrins , and no eggs were found in leaves containing 1 % pyrethrins . our results suggest that the natural concentrations of pyrethrins in the leaves may be responsible for the observed high mortality of wft on pyrethrum .

Introduction Methods and Materials Results Discussion

western flower thrips ( wft ) , frankliniella occidentalis , is a highly polyphagous insect that causes both direct and indirect effects on plant development and health . such compounds could be used as natural pesticides , and in theory , genes responsible for the biosynthesis of those compounds could be isolated and transferred to crops to improve plant defense against wft ( annadana et al . , 2002 ; among the sources of botanical pesticides , pyrethrins from pyrethrum plants ( tanacetum cinerariifolium ) represent the economically most important class of compounds with broad usage both in homes and organic agriculture ( casida , 1973 ) . on average , the concentration of pyrethrins is about 0.1 % ( dry weight ) in leaves and 1 - 2 % ( dry weight ) in flowers ( baldwin et al . assuming a water content of 90 % , pyrethrins account for around 0.01 % of the fresh weight of leaves and 0.1 - 0.2 % of the fresh weight of flowers . pyrethrins might provide pyrethrum with a broad range protection against many different insect pests , but the role of pyrethrins in pyrethrum defense has not been studied . in initial experiments , we observed that wft adults died within one day when fed pyrethrum leaves , but that they were abundant in open flowers . here , we tested the hypothesis that pyrethrins are responsible for protecting pyrethrum leaves against wft by assessing adult and embryo toxicity , and by examining feeding and oviposition deterrence both in vitro and in planta . the field was 0.5 ha in size , and the presence of insect species was monitored during the flowering period of spring 2010 , when the flowers were predominantly in developmental stages 25 [ numbered according to casida ( 1973 ) ] . pyrethrum oil ( 70 % w / w ) had been extracted from dried and ground pyrethrum flower heads with liquid co2 leaving no solvent residue ( honghe senju biological co. ltd . since the major insecticidal compounds in pyrethrum have long been known as pyrethrins ( casida , 1973 ) , the effect of pyrethrum oil was considered to be the effect of pyrethrins . when calculating the concentrations of pyrethrins in different solutions , the percentage of pyrethrins in the oil ( 70 % ) was taken into account . for example , 1 % ( w / v ) pyrethrins was prepared by dissolving 14.3 mg pyrethrum oil in 1 ml solvent . a dual - choice leaf disk assay was used to determine the deterrent effect of pyrethrins on wft . pyrethrum oil was dissolved in 0.2 % ( v / v ) aqueous tween-80 to achieve 3 concentrations of pyrethrins : 0.01 , 0.1 , and 1 % ( w / v ) . groups of 10 wft were positioned between a control and a test leaf disk placed abaxial side up and 2 cm apart on a 1.5 % ( w / v ) agar - bed in a petri dish ( 7 cm diam ) . the number of wft on each leaf disk was recorded 0.25 , 1 , 2 , 4 , 20 , and 28 h after the release of the wft . after placing 10 wft in a cage , the top was sealed with two layers of stretched parafilm , with 300 l aqueous solution in between the layers . the solutions used were water , 0.2 % tween-80 , or pyrethrins at 0.01 , 0.1 , or 1 % dissolved in 0.2 % tween-80 . wft were allowed to adapt to the diet ( pollen and water ) for 3 d , and then every day for 5 d fresh test solution was provided . the filter papers were drenched in 300 l of assay solution ( water , 0.2 % tween-80 or pyrethrins at 0.01 , 0.1 , or 1 % in 0.2 % tween-80 ) so that each paper was fully wetted but had no excess solution . mature pyrethrum leaves were harvested in november from a field in the netherlands when they were still flowering ( 5159'22.08"n-539'44.75"e , wageningen ) . two or three pieces of leaves were placed , abaxial side up , on 1 % ( w / v ) agar in a petri dish ( 7 cm diam ) . the mortality of wft was recorded daily for 3 d. to test the in planta activity of pyrethrins against wft , pyrethrins were infiltrated into whole chrysanthemum leaves as described by ratcliff et al . as the fresh weight of each leaf disk was on average 45.3 mg ( 1.2 mg ) , we infiltrated 0.025 , 0.25 , or 2.5 % pyrethrins solution to bring the concentrations to 0.01 , 0.1 , or 1 % pyrethrins . the number of wft on each leaf disk was recorded 0.25 , 1 , 2 , 4 , 20 , and 28 h after the release of the wft . to test the effects of pyrethrins on oviposition and hatching of larvae , wft were assayed with chrysanthemum leaf disks as described by de kogel et al . leaf disks were punched from untreated leaves , from leaves infiltrated with 0.2 % tween-80 , or from leaves containing 0.01 , 0.1 , or 1 % pyrethrins in tween solution . to determine the number of hatched larvae , the leaf disks were transferred to petri dishes containing water and incubated in a climate chamber ( 25c , l16:d8 ) for 5 d to allow the larvae to hatch . our field survey in china showed that several thrips species were the most abundant ( 98 % ) insects on pyrethrum flowers ( table 1 ) . a total of 30 individuals were identified to species level ; the thrips species found were mainly thrips tabaci ( 44 % ) , frankliniella occidentalis ( western flower thrips , or wft , 25 % ) , and thrips flavus ( 22 % ) . 1).table 1frequencies of small insect species living on pyrethrum flowers in the fieldinsectsfrequency ( % ) speciesfrequency ( % ) thripidae ( thrips)98thrips tabaci43frankliniella occidentalis25thrips flavus21thrips palmi3other species6nysius sp.1.9n.d.1.9chrysoperla / chrysopa sp . 1distribution of thrips adults and larvae across different developemental stages of pyrethrum flowers in the field . stage 2 , vertical ray florets ; stage 3 , horizontal ray florets and first row of disk florets open ; stage 4 , 3 rows of disk florets open ; stage 5 , all disk florets open frequencies of small insect species living on pyrethrum flowers in the field a total of 1200 insects were collected to count the frequencies of different insects . , not determined distribution of thrips adults and larvae across different developemental stages of pyrethrum flowers in the field . mortality could be as high as 80 % within 3 days , although the degree of mortality depended on the plant source ( data not shown ) . this showed that the mortality of wft was caused by a toxic principle of pyrethrum leaves rather than deterrence or starvation . we were , therefore , interested in specifically testing the effect of pyrethrins against wft . to determine the effects of pyrethrins against wft , pyrethrins were tested in vitro at different concentrations on adult mortality , feeding , oviposition , and embryo development . the mortality of wft female adults increased with the concentration of topically applied pyrethrins in the range of 1 to 30 mg / ml . probit analysis showed that the lc50 and lc90 of pyrethrins was 12.9 mg / ml ( with 95 % confidence limit of 10.9 - 14.8 mg / ml ) and 39.0 mg / ml ( with 95 % confidence limit from 30.7 to 50.4 mg / ml ) , respectively . thrips were significantly deterred from feeding by 0.1 % and 1 % pyrethrins ( fig . when given a choice between chrysanthemum leaf disks coated with 0.2 % tween ( control ) or 0.1 % added pyrethrins , after 2 h significantly more ( 61 - 77 % of thrips ) settled on control leaf disks . for both concentrations of pyrethrins , the maximum deterrent effect was reached at 4 h. application of 0.01 % pyrethrins on leaf disks did not show significant deterrent effects except at the 4 h time point ( fig . 2dual choice assays of western flower thrips on chrysanthemum leaf disks sprayed with 0.2 % tween ( control ) or 0.2 % tween with 0.01 % , 0.1 % or 1 % pyrethrins . error bars indicate se ( n = 120 per treatment ) dual choice assays of western flower thrips on chrysanthemum leaf disks sprayed with 0.2 % tween ( control ) or 0.2 % tween with 0.01 % , 0.1 % or 1 % pyrethrins . 3the number of eggs deposited by western flower thrips when supplied with different concentrations of pyrethrins starting on day 1 . error bars indicate se ( n = 60 per treatment ) the number of eggs deposited by western flower thrips when supplied with different concentrations of pyrethrins starting on day 1 . error bars indicate se ( n = 60 per treatment ) the development of eggs was inhibited by 0.1 % and 1 % pyrethrins . about 80 % of larvae hatched when the eggs were incubated with water , 0.2 % tween , or 0.01 % pyrethrins , while only 28 % or 6 % of the larvae hatched when the eggs were incubated with 0.1 % or 1 % pyrethrins , respectively ( fig . 4percentage of larvae hatching from western flower thrips eggs during incubation with different concentrations of pyrethrins . ( a ) , larva hatched in solutions of water , 0.2 % tween or 0.01 % pyrethrins at day 5 ; ( b ) , abnormally developed embryos in solutions of 0.1 % and 1 % pyrethrins at day 5 ; ( c ) and ( d ) , embryos before treatment percentage of larvae hatching from western flower thrips eggs during incubation with different concentrations of pyrethrins . error bars indicate se ( n = 40 per treatment ) effects of pyrethrins on embryo development of western flower thrips at day 5 . ( a ) , larva hatched in solutions of water , 0.2 % tween or 0.01 % pyrethrins at day 5 ; ( b ) , abnormally developed embryos in solutions of 0.1 % and 1 % pyrethrins at day 5 ; ( c ) and ( d ) , embryos before treatment to study in planta activity of pyrethrins against wft , thrips were assayed with chrysanthemum leaves that had been infiltrated with pyrethrins to contain 0.01 , 0.1 , or 1 % pyrethrins . in this experiment , the pyrethrins could not be contacted directly by thrips except by feeding , and the source of nutrition consisted of leaves instead of pollen . in the reproduction assay , thrips fed with chrysanthemum leaf disks containing pyrethrins exhibited higher mortality and lower reproduction rates compared to those fed with untreated leaf disks or leaf disks containing 0.2 % tween ( table 2).table 2mortality , number of eggs and hatched western flower thrips larvae per leaf disk on chrysanthemum leaf disks infiltrated with different concentrations of pyrethrinstreatment of leaf disksmortality ( % ) eggshatched larvaeuntreated leaf disks0 a2.0 0.4 a1.4 0.3 aleaf disks containing 0.2 % tween0 a1.7 0.5 a1.3 0.3 ableaf disks containing 0.01 % pyrethrins25.0 6.7 b1.3 0.3 ab0.8 0.2 bleaf disks containing 0.1 % pyrethrins29.2 7.9 b0.7 0.2 bc0.1 0.1 cleaf disks containing 1 % pyrethrins68.8 9.3 c0 c0 cvalues ( mean se , n = 48 per treatment ) followed by the same letter within a column are not significantly different ( anova : p > 0.05 ) mortality , number of eggs and hatched western flower thrips larvae per leaf disk on chrysanthemum leaf disks infiltrated with different concentrations of pyrethrins values ( mean se , n = 48 per treatment ) followed by the same letter within a column are not significantly different ( anova : p > 0.05 ) in the dual - choice assay , chrysanthemum leaves containing 0.1 % and 1 % pyrethrins showed significant deterrent effects on thrips within 15 min of release ( fig . a total of 74 - 93 % of the thrips settled on the control leaf disk when the other leaf disk contained 0.1 % pyrethrins , and 85 - 95 % thrips settled on the control leaf disk when the other leaf disk contained 1 % pyrethrins . 6percentage of western flower thrips settled on the control chrysanthemum leaf disk in dual choice assays of leaf disks containing 0.2 % tween with or without 0.01 % , 0.1 % , or 1 % pyrethrins . error bars indicate se ( n = 120 per treatment ) percentage of western flower thrips settled on the control chrysanthemum leaf disk in dual choice assays of leaf disks containing 0.2 % tween with or without 0.01 % , 0.1 % , or 1 % pyrethrins . pyrethrins , well - known natural insecticidal compounds , are found exclusively in and extracted from the composite flowers of pyrethrum ( tanacetum cinerariifolium ) , which belongs to anthemideae tribe within the astaraceae family ( casida and quistad , 1995 ) . remarkably , the potential role of pyrethrins in pyrethrum plant defense has not been studied . here , we report that western flower thrips ( wft ) adults thrive on pyrethrum flowers , but die within a few days on pyrethrum leaves . the hypothesis that pyrethrins are responsible for protecting pyrethrum leaves against wft was tested by spraying or infiltrating pyrethrins to leaves of chrysanthemum , a related pyrethrins - free species belonging to the same tribe . we assessed toxicity to the adult and embryo stages of wft , and negative effects on feeding and oviposition both in vitro and in planta , and found that the natural concentrations of pyrethrins present in leaves have strong negative effects on wft . we speculate that the thrips found on pyrethrum flowers survive on pollen that is devoid of pyrethrins ( t. yang , unpublished data ) . for example , carvacrol at 1 % and thymol at 0.1 % and 1 % significantly reduced the oviposition rate of wft when these compounds were sprayed on leaf disks , but neither compound affected the feeding activity of wft ( sedy and kosehier , 2003 ) . salicylaldehyde ( 0.1 % and 1 % ) and methyl salicylate ( 0.1 % and 1 % ) were also tested . within 24 h of applying 1 % methyl salicylate to bean or cucumber , the feeding and oviposition activities of thrips females the insect could be a result of changes in the plant induced by methyl salicylate , since it is a plant hormone involved in induced resistance ( pieterse et al . our results showed that 0.1 % and 1 % pyrethrin solutions sprayed on leaf disks significantly deterred wft at 4 h , and topically applied pyrethrins were toxic to adults at an lc50 value of 12.9 mg / ml ( 1.3 % ) . by mimicking the natural site of pyrethrin accumulation by infiltration of leaves , we found that 1 % pyrethrins caused 69 % mortality and completely inhibited oviposition . furthermore , 0.1 % pyrethrins was strongly deterrent and resulted in abortion of 95 % of the embryos , while as little as 0.01 % pyrethrins caused 25 % mortality in 2 days . we propose , therefore , that the natural concentrations of pyrethrin in pyrethrum leaves , around 0.01 % by fresh weight , accounts for the observed high mortality of thrips adults on this plant . therefore , besides feeding and oviposition deterrence , the embryo - toxic effect of pyrethrins is a third component that contributes to their effect for plant defense against wft ( figs . compared to some synthetic insecticides , the toxicity of natural pyrethrins against wft in the absence of synergists was not high . in previous studies using topical application methods , the lc50 values of insecticides tested against susceptible wft strains ranged from 10 to 83 g / ml for pyrethroids , 20 to 960 g / ml for carbamates , and 49 to 522 g / ml for organophosphates ( espinosa et al . , 2005 the lc50 value of pyrethrins against wft by topical application was determined as 12.9 mg / ml , and the action of pyrethrins was , therefore , 10 to 1000-fold weaker than for these synthetic pesticides . on the other hand , pyrethrins did show much stronger negative effects on feeding behavior and reproduction , which may be explained by the action of pyrethrins on the nervous system , resulting in disordered function of excitable ( nerve and muscle ) cells ( bradberry et al . as a result wft damage on pyrethrin - containing leaves we hypothesize that if plants such as closely related chrysanthemum species , which do not contain any pyrethrins , were genetically engineered to produce pyrethrins , their resistance to wft in leaves could be significantly improved .

the widespread use of chemical insecticides to control wft has led to increasing resistance against the major classes of synthetic insecticides ( broadbent and pree , 1997 ; flanders et al . the growing awareness and demand for insecticides that are not environmentally hazardous has stimulated the study of plant - derived compounds for pest control ( boeke et al . such compounds could be used as natural pesticides , and in theory , genes responsible for the biosynthesis of those compounds could be isolated and transferred to crops to improve plant defense against wft ( annadana et al . , 2002 ; among the sources of botanical pesticides , pyrethrins from pyrethrum plants ( tanacetum cinerariifolium ) represent the economically most important class of compounds with broad usage both in homes and organic agriculture ( casida , 1973 ) . pyrethrins are neurotoxins that bind to voltage - gated sodium channels of neuronal cells , causing the channels to remain open ( davies et al . pyrethrins comprise a group of six closely related esters , named pyrethrin i and ii , cinerin i and ii , and jasmolin i and ii . they are found in all aboveground parts of the pyrethrum plants , but predominantly in the ovaries of the flower heads ( brewer , 1973 ) . on average , the concentration of pyrethrins is about 0.1 % ( dry weight ) in leaves and 1 - 2 % ( dry weight ) in flowers ( baldwin et al . assuming a water content of 90 % , pyrethrins account for around 0.01 % of the fresh weight of leaves and 0.1 - 0.2 % of the fresh weight of flowers . pyrethrins might provide pyrethrum with a broad range protection against many different insect pests , but the role of pyrethrins in pyrethrum defense has not been studied . here , we tested the hypothesis that pyrethrins are responsible for protecting pyrethrum leaves against wft by assessing adult and embryo toxicity , and by examining feeding and oviposition deterrence both in vitro and in planta . the field was 0.5 ha in size , and the presence of insect species was monitored during the flowering period of spring 2010 , when the flowers were predominantly in developmental stages 25 [ numbered according to casida ( 1973 ) ] . to assess populations of small resident insects including thrips , flowers at each developmental stage each flower was taken by the stem and turned upside down into a jar containing 75 % alcohol . in this study , the chrysanthemum plants used for bioassays were from the same cultivar , but were grown in an insect - free compartment of the greenhouse under the same light and temperature conditions . we confirmed the concentration and composition of the oil by gas chromatography mass spectrometry comparison to a pyrethrin standard ( nguyen et al . since the major insecticidal compounds in pyrethrum have long been known as pyrethrins ( casida , 1973 ) , the effect of pyrethrum oil was considered to be the effect of pyrethrins . when calculating the concentrations of pyrethrins in different solutions , the percentage of pyrethrins in the oil ( 70 % ) was taken into account . pyrethrum oil was dissolved in acetone to achieve a concentration range of 1 to 30 mg pyrethrins per ml , and the solutions were applied to the thorax with a 10-l glass syringe at 1 l per thrips . the droplet briefly covered the thorax of the insect and also the paper support before evaporating in a few seconds , leaving a residue both on the insect and the support . a dual - choice leaf disk assay was used to determine the deterrent effect of pyrethrins on wft . control leaf disks were sprayed with solvent solution ( 0.2 % tween-80 ) , and test leaf disks were sprayed with the pyrethrin solutions using a potter precision laboratory spray tower , which produced a uniform deposit ( 3 l / cm ) of solution on the leaf disks . groups of 10 wft were positioned between a control and a test leaf disk placed abaxial side up and 2 cm apart on a 1.5 % ( w / v ) agar - bed in a petri dish ( 7 cm diam ) . the number of wft on each leaf disk was recorded 0.25 , 1 , 2 , 4 , 20 , and 28 h after the release of the wft . at each time point , a student 's paired t - test was used to assess the significance of the differences in the mean number of wft between test and control . the filter papers were drenched in 300 l of assay solution ( water , 0.2 % tween-80 or pyrethrins at 0.01 , 0.1 , or 1 % in 0.2 % tween-80 ) so that each paper was fully wetted but had no excess solution . after transferring the eggs , the developmental status of eggs was monitored every day for 6 d. to facilitate the observations , the bottoms of the petri dishes were marked with lines that could be seen through the filter paper from the top , and the eggs were placed on filter paper along these lines . this facilitated finding the eggs under the microscope , and the viability of hatched larvae was assessed in terms of their ability to move away ( > 0.5 cm ) from the hatch position . after transferring 10 wft to each petri dish , petri dishes with two leaf disks ( 1.6 cm diam ) of chrysanthemum leaves , with a total mass similar to the mass of the pyrethrum leaf samples , or with only agar were used as controls . the mortality of wft was recorded daily for 3 d. to test the in planta activity of pyrethrins against wft , pyrethrins were infiltrated into whole chrysanthemum leaves as described by ratcliff et al . in the initial experiments , we infiltrated only water into the leaves and determined that on average 29.1 mg ( 2.1 mg ) water could be infiltrated into each leaf disk ( 6 replicates ) . as the fresh weight of each leaf disk was on average 45.3 mg ( 1.2 mg ) , we infiltrated 0.025 , 0.25 , or 2.5 % pyrethrins solution to bring the concentrations to 0.01 , 0.1 , or 1 % pyrethrins . to test the effects of pyrethrins on oviposition and hatching of larvae , wft were assayed with chrysanthemum leaf disks as described by de kogel et al . subsequently , half of the leaf disks from each plate were used to determine the number of eggs , and the other half of the leaf disks were used to determine the number of hatched larvae . to determine the number of eggs , the leaf disks were boiled in water for 3 min so that the eggs were clearly visible under a binocular microscope with transmitting light . to determine the number of hatched larvae , the leaf disks were transferred to petri dishes containing water and incubated in a climate chamber ( 25c , l16:d8 ) for 5 d to allow the larvae to hatch . our field survey in china showed that several thrips species were the most abundant ( 98 % ) insects on pyrethrum flowers ( table 1 ) . a total of 30 individuals were identified to species level ; the thrips species found were mainly thrips tabaci ( 44 % ) , frankliniella occidentalis ( western flower thrips , or wft , 25 % ) , and thrips flavus ( 22 % ) . the number of thrips in flowers was dependent on the flower s developmental stage ( fig . larvae accounted for 7 - 26 % of the total number of thrips per flower , depending on the flower developmental stage ( fig . 1).table 1frequencies of small insect species living on pyrethrum flowers in the fieldinsectsfrequency ( % ) speciesfrequency ( % ) thripidae ( thrips)98thrips tabaci43frankliniella occidentalis25thrips flavus21thrips palmi3other species6nysius sp.1.9n.d.1.9chrysoperla / chrysopa sp . 1distribution of thrips adults and larvae across different developemental stages of pyrethrum flowers in the field . stage 2 , vertical ray florets ; stage 3 , horizontal ray florets and first row of disk florets open ; stage 4 , 3 rows of disk florets open ; stage 5 , all disk florets open frequencies of small insect species living on pyrethrum flowers in the field a total of 1200 insects were collected to count the frequencies of different insects . stage 2 , vertical ray florets ; stage 3 , horizontal ray florets and first row of disk florets open ; stage 4 , 3 rows of disk florets open ; stage 5 , all disk florets open we assayed the suitability of pyrethrum leaves as a food substrate for wft . to determine the effects of pyrethrins against wft , pyrethrins were tested in vitro at different concentrations on adult mortality , feeding , oviposition , and embryo development . the mortality of wft female adults increased with the concentration of topically applied pyrethrins in the range of 1 to 30 mg / ml . probit analysis showed that the lc50 and lc90 of pyrethrins was 12.9 mg / ml ( with 95 % confidence limit of 10.9 - 14.8 mg / ml ) and 39.0 mg / ml ( with 95 % confidence limit from 30.7 to 50.4 mg / ml ) , respectively . when given a choice between chrysanthemum leaf disks coated with 0.2 % tween ( control ) or 0.1 % added pyrethrins , after 2 h significantly more ( 61 - 77 % of thrips ) settled on control leaf disks . for both concentrations of pyrethrins , the maximum deterrent effect was reached at 4 h. application of 0.01 % pyrethrins on leaf disks did not show significant deterrent effects except at the 4 h time point ( fig . 2dual choice assays of western flower thrips on chrysanthemum leaf disks sprayed with 0.2 % tween ( control ) or 0.2 % tween with 0.01 % , 0.1 % or 1 % pyrethrins . error bars indicate se ( n = 120 per treatment ) dual choice assays of western flower thrips on chrysanthemum leaf disks sprayed with 0.2 % tween ( control ) or 0.2 % tween with 0.01 % , 0.1 % or 1 % pyrethrins . the carrier , 0.2 % tween-80 , did not affect the oviposition of thrips compared to water throughout the experiment , but wft oviposited significantly fewer eggs with increasing pyrethrin concentrations during the 5-day experiment ( fig . about 80 % of larvae hatched when the eggs were incubated with water , 0.2 % tween , or 0.01 % pyrethrins , while only 28 % or 6 % of the larvae hatched when the eggs were incubated with 0.1 % or 1 % pyrethrins , respectively ( fig . ( a ) , larva hatched in solutions of water , 0.2 % tween or 0.01 % pyrethrins at day 5 ; ( b ) , abnormally developed embryos in solutions of 0.1 % and 1 % pyrethrins at day 5 ; ( c ) and ( d ) , embryos before treatment percentage of larvae hatching from western flower thrips eggs during incubation with different concentrations of pyrethrins . ( a ) , larva hatched in solutions of water , 0.2 % tween or 0.01 % pyrethrins at day 5 ; ( b ) , abnormally developed embryos in solutions of 0.1 % and 1 % pyrethrins at day 5 ; ( c ) and ( d ) , embryos before treatment to study in planta activity of pyrethrins against wft , thrips were assayed with chrysanthemum leaves that had been infiltrated with pyrethrins to contain 0.01 , 0.1 , or 1 % pyrethrins . in the reproduction assay , thrips fed with chrysanthemum leaf disks containing pyrethrins exhibited higher mortality and lower reproduction rates compared to those fed with untreated leaf disks or leaf disks containing 0.2 % tween ( table 2).table 2mortality , number of eggs and hatched western flower thrips larvae per leaf disk on chrysanthemum leaf disks infiltrated with different concentrations of pyrethrinstreatment of leaf disksmortality ( % ) eggshatched larvaeuntreated leaf disks0 a2.0 0.4 a1.4 0.3 aleaf disks containing 0.2 % tween0 a1.7 0.5 a1.3 0.3 ableaf disks containing 0.01 % pyrethrins25.0 6.7 b1.3 0.3 ab0.8 0.2 bleaf disks containing 0.1 % pyrethrins29.2 7.9 b0.7 0.2 bc0.1 0.1 cleaf disks containing 1 % pyrethrins68.8 9.3 c0 c0 cvalues ( mean se , n = 48 per treatment ) followed by the same letter within a column are not significantly different ( anova : p > 0.05 ) mortality , number of eggs and hatched western flower thrips larvae per leaf disk on chrysanthemum leaf disks infiltrated with different concentrations of pyrethrins values ( mean se , n = 48 per treatment ) followed by the same letter within a column are not significantly different ( anova : p > 0.05 ) in the dual - choice assay , chrysanthemum leaves containing 0.1 % and 1 % pyrethrins showed significant deterrent effects on thrips within 15 min of release ( fig . a total of 74 - 93 % of the thrips settled on the control leaf disk when the other leaf disk contained 0.1 % pyrethrins , and 85 - 95 % thrips settled on the control leaf disk when the other leaf disk contained 1 % pyrethrins . 6percentage of western flower thrips settled on the control chrysanthemum leaf disk in dual choice assays of leaf disks containing 0.2 % tween with or without 0.01 % , 0.1 % , or 1 % pyrethrins . error bars indicate se ( n = 120 per treatment ) percentage of western flower thrips settled on the control chrysanthemum leaf disk in dual choice assays of leaf disks containing 0.2 % tween with or without 0.01 % , 0.1 % , or 1 % pyrethrins . pyrethrins , well - known natural insecticidal compounds , are found exclusively in and extracted from the composite flowers of pyrethrum ( tanacetum cinerariifolium ) , which belongs to anthemideae tribe within the astaraceae family ( casida and quistad , 1995 ) . here , we report that western flower thrips ( wft ) adults thrive on pyrethrum flowers , but die within a few days on pyrethrum leaves . we assessed toxicity to the adult and embryo stages of wft , and negative effects on feeding and oviposition both in vitro and in planta , and found that the natural concentrations of pyrethrins present in leaves have strong negative effects on wft . for example , carvacrol at 1 % and thymol at 0.1 % and 1 % significantly reduced the oviposition rate of wft when these compounds were sprayed on leaf disks , but neither compound affected the feeding activity of wft ( sedy and kosehier , 2003 ) . within 24 h of applying 1 % methyl salicylate to bean or cucumber , the feeding and oviposition activities of thrips females the insect could be a result of changes in the plant induced by methyl salicylate , since it is a plant hormone involved in induced resistance ( pieterse et al . a series of commercially available plant - derived essential oils tested at recommended concentrations ( 0.02 - 0.5 % ) , including neem oil , rosemary oil , peppermint oil , garlic oil , and cottonseed oil , caused less than 30 % mortality within 7 days ( cloyd et al . our results showed that 0.1 % and 1 % pyrethrin solutions sprayed on leaf disks significantly deterred wft at 4 h , and topically applied pyrethrins were toxic to adults at an lc50 value of 12.9 mg / ml ( 1.3 % ) . by mimicking the natural site of pyrethrin accumulation by infiltration of leaves , we found that 1 % pyrethrins caused 69 % mortality and completely inhibited oviposition . furthermore , 0.1 % pyrethrins was strongly deterrent and resulted in abortion of 95 % of the embryos , while as little as 0.01 % pyrethrins caused 25 % mortality in 2 days . we propose , therefore , that the natural concentrations of pyrethrin in pyrethrum leaves , around 0.01 % by fresh weight , accounts for the observed high mortality of thrips adults on this plant . therefore , besides feeding and oviposition deterrence , the embryo - toxic effect of pyrethrins is a third component that contributes to their effect for plant defense against wft ( figs . in previous studies using topical application methods , the lc50 values of insecticides tested against susceptible wft strains ranged from 10 to 83 g / ml for pyrethroids , 20 to 960 g / ml for carbamates , and 49 to 522 g / ml for organophosphates ( espinosa et al . , 2005 the lc50 value of pyrethrins against wft by topical application was determined as 12.9 mg / ml , and the action of pyrethrins was , therefore , 10 to 1000-fold weaker than for these synthetic pesticides . on the other hand , pyrethrins did show much stronger negative effects on feeding behavior and reproduction , which may be explained by the action of pyrethrins on the nervous system , resulting in disordered function of excitable ( nerve and muscle ) cells ( bradberry et al . as a result wft damage on pyrethrin - containing leaves we hypothesize that if plants such as closely related chrysanthemum species , which do not contain any pyrethrins , were genetically engineered to produce pyrethrins , their resistance to wft in leaves could be significantly improved .

a plausible abiotic chemical route to the canonical nucleotides is a major goal in origins of life research . recently , we demonstrated the first chemical steps toward a divergent pyrimidine and purine ribonucleotide synthesis . a one - pot multicomponent reaction was demonstrated to stereoselectively tether and consequently regiospecifically glycosylate purine precursors and masked pentose sugars , while concurrently furnishing known pyrimidine precursors ( scheme 1 ) . although rna has often been considered as a candidate for the first biopolymer of life , extant biology utilizes two chemically distinct , but related , nucleotide polymers , rna and dna . dna is usually viewed as a late evolutionary adaptation of earlier rna - based life . however , it would not be possible to make dna without deoxyribonucleotides , and yet in the absence of dna there is no obvious reason for the evolution of the biochemical pathways for the synthesis of deoxyribonucleotides . only after the emergence of dna as an important cellular component would there have been a strong selective pressure favoring the emergence of biochemical pathways for the synthesis of deoxyribonucleotides from ribonucleotides . although the advantages of dna as a medium for information storage are clear , there has not until now been any reasonable hypothesis for how dna could be invented by primitive cells in the absence of pre - existing deoxyribonucleotides . in this paper biochemically , 2-deoxyribonucleotides are synthesized by chemo- and regio - specific reduction of ribonucleotides . in the absence of the complex and energetically costly enzymatic resources required to regiospecifically deoxygenate ribonucleotides , abiological 2-deoxygenation of nucleotides requires site - specific ribo- or arabino - nucleoside deoxygenation . in light of the difficulty of regioselective nucleotide deoxygenation in the absence of sophisticated enzymatic control , it would seem that regiospecific positioning of a group / atom with the appropriate latent potential reactivity could form the basis of an alternative pathway to the 2-deoxyribonucleotides . in principle , a sufficiently chemoselective reactivity difference could be exploited to generate both ribonucleotides and 2-deoxyribonucleotides , simultaneously . constitutional analysis of dna , with respect to rna , in light of the low dissociation energy of c s bonds , suggests that regiospecific positioning of sulfur at c2 could result in the chemical differentiation required for the divergent synthesis of dna and rna monomers from common precursors ( scheme 2 ) . oxazole 1a key ribonucleotide precursor is derived from cyanamide 2 and glycolaldehyde 3 . the c2-carbon atom of aldehyde 3 is formally delivered to a ribonucleotide as the c2 sugar carbon . it is of note that aldehyde 3 is also the aldehyde precursor of serine 4 , a proteinogenic amino acid , via strecker synthesis with ammonium cyanide ( scheme 3 ) . the constitutional similarity of serine 4 and cysteine 5 , and their aldehyde precursors 3 and -mercapto - acetaldehyde 6 , suggests that both 3 and 6 must be considered within our exploration of prebiotic azole synthesis . specifically , we propose that the divergent reactivity of glycolaldehyde 3 and -mercapto - acetaldehyde 6 with cyanamide 2 could lead , via 2-aminooxazole 1 and 2-aminothiazole 7 , to ribonucleotides and 2-deoxyribonucleotides , by desulfurization of c2 ( scheme 2 ) . the importance of 2-aminothiazole 7 , and its derivatives , has been recognized in medicinal chemistry , but though the synthesis of thiazole 7 in water has been reported , the potential relevance of thiazole 7 to the origins of life remains unexplored . the formation of oxazole 1 from cyanamide 2 and glycolaldehyde 3 at neutral ph requires stoichiometric ( or excess ) cyanamide 2 and general acid aldehyde 6 , upon imine formation with cyanamide 2 , has , like 3 , the potential to undergo 5-exo - dig cyclization to furnish imine 14 ( or hemiaminal 15 ) as shown in scheme 3 . furthermore , we expected the reaction to proceed more rapidly at neutral ph and be less prone to stall at intermediates en route to thiazole 7 due to the increased nucleophilicity of sulfur and greater aromaticity of thiazoles with respect to oxazoles . to test this supposition , -mercapto - acetaldehyde 6 ( 8.5 mm ) and 2 ( 15 , 26 , or 50 mm ) were incubated in d2o at pd 7 and 20 c and a rapid ( ca . 10 min ) quantitative conversion of aldehyde 6 to hemiaminal 15 was observed ( see the supporting information figure s1 ) . over the course of several hours , 15 dehydrated to furnish thiazole 7 ( see supporting information figures s2 and s3 ) . it is of note that excess aldehyde 6 did not lead to further reaction of thiazole 7 . moreover , at elevated concentration ( > 150 mm ) 2-aminothiazole 7 was observed to directly crystallize from water , and a crystal structure of thiazole 7 is shown in scheme 3 . next we investigated the concomitant synthesis of oxazole 1 and thiazole 7 . in the presence of excess cyanamide 2 ( 2.25 equiv ) , rapid and stoichiometric conversion of glycolaldehyde 3 ( 1 equiv ) and -mercapto - acetaldehyde 6 ( 1 equiv ) to hemiaminals 11 and 15 , respectively , was observed in water . at high ph / pd ( ph / pd > 9 ) specific base catalysis or in 1 m phosphate solution ( ph / pd 6.57.5 ) general acid base catalysis furnish oxazole 1 and thiazole 7 over the course of 2436 h at ambient temperature ( scheme 3 ; see supporting information figure s8s10 ) . at neutral pd , selective dehydration of 15 to liberate thiazole 7 was observed . limiting cyanamide 2 ( 2/3/6 0.8:1:1 ) also resulted in the specific generation of thiazole 7 , even in 1 m phosphate solution ( ph 7 ; see supporting information figure s9 ) . it is likely that the rapid 5-exo - dig cyclization of imine 12/ hemiaminal 13 leads to the selective sequestration of cyanamide 2 in thiazole 7 . we did not observe significant homo- and hetero - aldol reaction during the reaction of glycolaldehyde 3 and mercaptoacetaldehyde 6 with either excess or limiting cyanamide 2 between ambient temperature and 60 c in 1 m phosphate solution at ph 7 . interestingly , we also found that , like oxazole 1 , thiazole 7 is easily sublimed ( see supporting information figure s4 ) , suggesting possible geochemical routes to the distribution , separation , and purification of thiazole 7 under abiotically plausible conditions . sublimation from a mixture of oxazole 1 and thiazole 7 resulted in the isolation of both azoles as colorless crystalline solids . partial sublimation of a mixture of oxazole 1 and thiazole 7 ( 1:1 ) resulted in the isolation 4:12:1 oxazole 1/thiazole 7 , at 50 c between 1 and 24 h at ambient pressure , indicating the more rapid but comparable sublimation rate of oxazole 1 relative to thiazole 7 . complete sublimation was observed after 2 d at 60 c to afford 1:1 mixture of crystalline azoles at ambient pressure . we then examined the multicomponent reaction of thiazole 7 , 4-amino - imidazole 5-carboxamide 16 , and a series of aldehydes . equimolar quantities of thiazole 7 and aliphatic aldehydes ( including formaldehyde , acetaldehyde , propionaldehyde , and cyclobutaldehyde ) with excess amino - imidazole 16 ( 1.23 equiv ) furnished high yields ( 7590% ) of thiazolines 17 , 18 , 19 , and 20 , respectively , at ph 5 . the multicomponent reactions were found to be robust at multi - gram scale and comparable diastereoselectivity was observed in the reaction of aliphatic aldehydes with oxazole 1 or thiazole 7 and amino - imidazole 16 ( via imine 21 r = alkyl ; d.r.= 2.5:14:1 threo / erythro ) . compounds 17 , threo-18 and erythro-18 , and the major isomers of 19 and 20 were isolated by fractional crystallization , and the crystal structures are shown in scheme 4 . to continue our investigation into the potential of multicomponent assembly of nucleotides we studied the participation of thiazole 7 in carbon carbon bond - forming reaction with -hydroxyaldehydes and imines derived from -hydroxyaldehydes in water . incubation of equimolar aldehyde 3 , 2-aminothiazole 7 , and amino - imidazole 16 for 5 d in water at ph 46 at 20 c gave a high yield of c4-thiazoline 23 ( 80% isolated yield ) as 1.2:1 erythro / threo mixture of diastereomeric products . surprisingly , this signified that the facial selectivity of thiazole 7 is significantly relaxed with respect to oxazole 1 and imine 21 ( r = ch2oh ) . however , both erythro-23 and threo-23 were formed with complete annulation control ( > 99% , see h nmr spectrum shown in supporting information figure s28 ; single crystal x - ray structures of erythro-23 and threo-23 are shown in scheme 5 ) . upon investigation of the parallel mcr with the c3-synthon glyceraldehyde 22 , comparable efficiency in the synthesis of c5-thiazoline 24 ( 70% isolated yield ) was observed . again , exclusive 6-exo - trig annulation is observed ; however , in this case , 24 is furnished with significant diastereoselectivity ( d.r.= 9:5:1.1:1 , see scheme 5 and supporting information figure s35 ) . isolation of the two major isomers , crystallization , and x - ray diffraction proved their xylo- and lyxo - configuration . the diastereoselectivity obtained , upon carbon carbon bond formation is proposed to result from intramolecular hydrogen - bonding and concomitant steric impedance of thiazole addition to the si - face of imine 21 ( see supporting information scheme s1 ) . although the facial selectivity of the nucleophile 7 has been relaxed with respect to the comparable reaction of 1 , the facial selectivity of the glyceraldehyde - imine 21 ( r = ch2oh ) is retained . this is important with respect to purine nucleotide synthesis by 3-purine tethering , which necessarily requires n3-displacement and c3-inversion . furthermore , as well as regiospecific tethering of 16 and consequent delivery of n1 to the anomeric carbon atom , reaction of thiazole 7 with imine 21 regiospecifically delivers sulfur to c2 , which is of significant interest with regards to the potential synthesis of 2-deoxynucleotides by c2-desulfurization . the multicomponent tricyclic thiazoline products 1724 were uncontaminated with homoaldolization and homo - mannich byproducts . additionally , oxazole 1 is known to react efficiently with -hydroxyaldehydes in a ph - dependent bimolecular domino - reaction . in striking contrast , carbon bond formation was observed by h nmr analysis upon incubation of thiazole 7 with either glycolaldehyde 3 or rac - glyceraldehdye 22 in d2o ( pd 39 , 4100 c ) . instead , over a period of 4 days at pd 7 , crystal formation was observed from an aqueous solution containing 3 ( 100 mm ) and thiazole 7 ( 100 mm ) at 20 c . single crystal x - ray diffraction of a crystal isolated directly from the reaction supernatant proved that the structure was aminal 25 ( scheme 6 ) . though nmr studies confirmed that rac-22 formed hemiaminal 26 and aminal 27 with thiazole 7 in d2o , no aminal crystallization was observed in solutions of rac-22 and 7 , even at significantly elevated concentrations ( 500 mm to 2 m ) or with substoichiometric quantities of rac-22 . to test whether the change in solubility properties was due to the increased number of hydroxyl moieties or perhaps due to the differential solubility and packing properties of rac-27 with respect to the achiral aminal 25 , we investigated the solubility properties of the aminal structures formed in a mixture of d - glyceraldehyde 22 and thiazole 7 . intriguingly , we found that at 100 mm concentration , d-27 now crystallized from water at 20 c . nmr analysis showed a 1:2 ratio of 22/7 and recrystallization ( 10:1 h2o / etoh ) allowed for crystallographic confirmation of the aminal structure ( scheme 6 ) . in summary , our results suggest that in a sulfur - rich prebiotic environment , oxazole 1 and thiazole 7 could be formed together under the same conditions , as long as both glycolaldehyde 3 and its thiol analogue , -mercapto - acetaldehyde 6 , were present along with cyanamide 2 . in such mixed reactions , the absence of any interference between oxazole and thiazole synthesis shows that these potential precursors of deoxynucleotides and ribonucleotides could indeed be formed at the same time in the same conditions . we have also found that two abiotically plausible methods for the purification of oxazole 1 , namely , crystallization and sublimation , also apply to thiazole 7 . however , the chemical reactivities of oxazole 1 and thiazole 7 are quite different , suggesting that the subsequent pathways would diverge . the most striking difference between the reactivity of oxazole 1 and thiazole 7 is the failure of the thiazole to react with aldehydes to form the thiazoline precursors of the ( thio)pyrimidine nucleotides . the most likely explanation for this difference is the increased aromaticity of the thiazole relative to the oxazole , and thus decreased nucleophilicity of c5 . we are currently investigating an alternative synthesis of 2-deoxyribo - pyrimidine nucleotides mediated by c2-inversion of anhydronucleotide 28 ( scheme 1 ) . however , during the course of efforts to observe two - component reactivity of thiazole 7 with aldehydes , we observed instead the crystallization of glycolaldehyde 3 and homochiral ( but not racemic ) glyceraldehyde 22 ( two known ribonucleotide precursors ) as aminals directly from water . the facile reversibility of aminal formation suggests that the chemical sequestration and protection of c2- and c3-aldehyde nucleotide synthons by another important class of nucleotide synthon ( azoles ) may have provided a route to the concentration , purification , and stabilization of the necessary aldehyde precursors of nucleotides . although these aldehydes can in principle be readily synthesized by a variety of routes , their high reactivity has until now made it difficult to conceive of means for accumulating large reservoirs of these essential starting materials in a prebiotic context . by exploring the multicomponent reactivity of thiazole 7 , together with aldehydes and the purine precursor amino - imidazole 16 , we have demonstrated a selective and high yielding 3-component carbon carbon bond - forming reaction that chemospecifically furnishes masked-2-thiosugars regiospecifically tethered to an amino - imidazole purine - precursor . the facial selectivity of thiazole 7 , upon addition to -hydroxyaldehyde - imines , is quite relaxed ( relative to oxazole 1 ) , resulting in low diastereoselectivity at c2 ; however , this is inconsequential with regards to the proposed deoxynucleotide synthesis because c2 would be rendered achiral following desulfurization . in contrast , we observed very high diastereoselectivity of nucleophilic addition of thiazole 7 to imine 21 , generating almost exclusively the lyxo and xylo isomers with controlled n9c1 annulation . the conversion of the resulting c5-thiazolines to 2-deoxyribo - purine nucleotides requires three additional steps : c3-inversion to release the purine - precursor from its tethered attachment to c3 , completion of the purine heterocycle , and chemospecific desulfurization of c2. these steps are currently under investigation in our laboratories as part of our continuing studies aimed at the abiotic synthesis of 2-deoxyribonucleotides . finally , it is of note that if life emerged in an environment containing both ribo- and 2-deoxyribonucleotides , it is likely that the first biopolymers were heterogeneous in composition , that is , composed of a mixture of ribo- and deoxyribonucleotides , and perhaps other compatible nucleotides . recent work has shown that functional biopolymers , in the form of aptamers with highly specific molecular recognition properties , can be derived by in vitro evolution from libraries of polynucleotides composed of randomly interspersed ribo- and deoxyribo - nucleotides . thus , chimeric rna / dna polymers may have been sufficient for the emergence of life . the primordial biochemical exploitation of a mixed rna and dna genetic system could eliminate the requirement for a genetic takeover ( of rna by dna ) , and would arguably result in a simplification of the transition from chemistry to biology . however , such mixed - composition polymers have neither the advantageous stability of dna , nor the optimal functional characteristics of rna . thus , rna and dna may have emerged as early and contemporaneous specializations of a primitive mixed biopolymer . reagents and solvents were purchased from sigma - aldrich , tci america , frontier scientific , or cambridge isotope laboratories . flash column chromatography was carried out on merck 9385 silica gel 60 ( 230400 mesh ) . nmr spectroscopy was carried out on a varian nmr spectrometer ( oxford as-400 ) or a bruker nmr spectrometer ( avanceiii 600 ) operating at 25 c probe temperature ( unless otherwise specified ) . when possible , the chemical shift of the corresponding solvent was used as a reference . chemical shift values are reported in parts per million ( ppm ) and j - couplings are recorded in hertz ( hz ) . electrospray mass spectrometry was recorded on a bruker daltonics esquire 6000 esi - ms . high resolution mass spectrometry was carried out on a waters q - tof micro lc / ms / ms system . infrared spectra were recorded as manually pressed kbr discs on a perkinelmer spectrum 100 series ft - ir spectrometer . single crystal x - ray crystallography was carried out with a bruker apex ii ccd diffractometer ( mo k radiation , = 0.71073 ) , equipped with an oxford cryosystems nitrogen flow apparatus , at 100 k. data integration down to 0.76 resolution was carried out using saint v7.46 a ( bruker diffractometer , 2009 ) with reflection spot size optimization . absorption corrections were made with the program sadabs ( bruker diffractometer , 2009 ) . the structure was solved by the direct methods procedure and refined by least - squares methods against f by using shelxs-97 and shelxl-97 ( sheldrick , 2008 ) . mercaptoacetaldehyde 6 and cyanamide 2 ( 0.51.5 equiv ) were dissolved in h2o , d2o or phosphate buffer ( 1 ml ) at ph / pd 7.0 . reaction progress was monitored by the h nmr spectroscopy ( see the supporting information , figure s1 ) . an initial concentration of 100 mm led to direct crystallization of 7 ( 60% isolated yield of crystals ) . alternatively , upon complete conversion ( by nmr spectroscopy ) , the solution was concentrated and 7 recovered by sublimation . compound 7 ( 15 g ) was placed in a covered beaker or erlenmeyer flask and warmed from beneath to 40 c . sublimate was collected on flask / beaker sides and cover at ambient temperature ( see figure s4 ) or on a water - cooled coldfinger ( 5 c ) . ir ( solid , cm ) : 3407 , 3285 , 3120 , 3086 , 1622 , 1518 , 1488 . h nmr ( 400 mhz ; dmso ) : h 6.90 ( d , j = 3.7 , 1h , h(c4 ) ) , 6.84 ( s , 2h , nh2 ) , 6.51 ( d , j = 3.7 , 1h , h(c5 ) ) . c nmr ( 101 mhz ; dmso ) : c 169.5 , 139.3 , 107.1 . ms esi ( pos . ) 101 ( 100% , [ m + h ] ) . glycolaldehyde 3 ( 1 equiv ) , mercaptoacetaldehyde 6 ( 1 equiv ) , and cyanamide 2 ( 0.55 equiv ) were dissolved in h2o / d2o ( 1 ml ) at ph / pd 7 , 10 or 12 , or 1 m phosphate buffer ( 1 ml ) at ph / pd 6.57.0 . reaction progress was monitored by the h nmr spectroscopy ( see the supporting information , figure s810 ) . an initial concentration of 200 mm of 2 , 3 and 6 led to direct crystallization of 15 ( 510% isolated yield of crystals ) . alternatively , upon complete conversion ( by nmr spectroscopy ) , the solution was concentrated and 7 and 1 were recovered by sublimation . compounds 7 and 1 ( 1:1 ; 100 mg ) as fine powder or film ( evaporated water ) were deposited in a covered glass tube ( 10 cm 1 cm ) . a temperature gradient ( 40 , 50 , or 60 c to ambient temperature ) was set up across the tube and a mixture of crystalline thiazole 7 and oxazole 1 were isolated at ambient temperature as a deposit upon the glass wall of the tube . aldehyde ( 0.491.0 equiv ) was added to a solution of 7 in h2o at ph 7 0.2 . the solution was then incubated at room temperature for 510 d ; the resultant solid was then isolated by filtration or trituration and washed twice with ethanol and once with ice cold water . d - glyceraldehyde 22/thiazole 7 crystals were then recrystallized from aqueous ethanol ( 10:1 h2o / etoh ) . ir ( solid , cm ) : 3213 ( br ) , 2966 , 2934 , 2869 , 1589 , 1539 , 1497 . ir ( solid , cm ) : 3326 ( br ) , 3244 , 3113 , 2948 , 2933 , 2901 , 2882 , 1536 , 1489 . aldehyde ( 50250 mm , 1 equiv ) was added to a solution of 7 ( 1.1 equiv ) and 5-aminoimidazole-4-carboxamide 16 ( 1.53.0 equiv ) in h2o at ph 5.0 0.2 . the reaction was stirred under an argon atmosphere until no residual aldehyde was detectable following lyophilization and analysis by h nmr . the reaction was lyophilized and purified by silica gel column chromatography ( ch2cl2/meoh , 10:0 to 6:4 ) . the major product(s ) were concentrated and recrystallized from ethanol / water or dichloromethane / methanol mixtures . ir ( solid , cm ) : 3333 , 3179 , 2977 , 2922 , 2860 , 1636 , 1607 , 1580 , 1558 . h nmr ( 400 mhz ; d2o ) : h 7.22 ( s , 1h , ar ) , 5.85 ( d , j = 6.0 , 1h , h(c1 ) ) , 4.18 ( ddd , j = 7.8 , 6.0 , 4.1 , 1h , h(c2 ) ) , 3.39 ( abx , j = 12.8 , 4.1 , 1h , h(c3 ) ) , 3.20 ( abx , j = 7.8 , 1h , h(c3 ) ) . h nmr ( 400 mhz ; dmso ) : h 7.26 ( d , j = 1.1 , 1h , ar ) , 5.91 ( d , j = 6.1 , 1h , h(c1 ) ) , 4.294.25 ( ddd , j = 7.0 , 6.1 , 3.8 , 1h , h(c2 ) ) , 3.41 ( abx , j = 13.0 , 3.8 , 1h , h(c3 ) ) , 3.18 ( abx , j = 13.0 , 7.0 , 1h , h(c3 ) ) . c nmr ( 101 mhz ; dmso ) : c 166.6 , 161.4 , 141.6 , 129.4 , 111.4 , 79.5 , 48.0 , 42.2 . cambridge crystallographic data centre deposition number 864226 . 18 : yield > 85% d.r.= 2.5:1 erythro-18 ( minor ) : rf = 0.25 ( ch2cl2/meoh 7:3 ) . ir ( solid , cm ) : 3437 , 3387 , 3292 , 3123 , 2975 , 2856 , 1638 , 1613 , 1571 . h nmr ( 400 mhz ; dmso ) : h 7.05 ( s , 1h , ar ) , 6.97 ( s , 2h , nh2 ) , 6.68 ( br d , j = 19.9 , 2h , nh2 ) , 5.95 ( d , j = 6.4 , 1h , h(c1 ) ) , 5.89 ( s , 1h , hn ) , 4.54 ( ddd , j = 6.4 , 2.5 , 1.7 , 1h , h(c2 ) ) , 3.78 ( qd , j = 6.4 , 2.6 , 1h , h(c3 ) ) , 1.25 ( d , j = 6.4 , 3h , h(c4 ) ) . c nmr ( 101 mhz ; dmso ) : c 166.7 , 165.2 , 141.4 , 128.4 , 111.6 , 79.4 , 58.6 , 46.7 , 22.7 . hrms esi [ m + na ] calc . for c9h12n6osna 275.0691 ; obs . ir ( solid , cm ) : 3391 , 3302 , 3103 , 2972 , 2865 , 1651 , 1621 , 1582 . h nmr ( 400 mhz ; dmso ) : h 7.33 ( s , 1h ) , 6.96 ( s , 1h ) , 6.76 ( d , j = 27.2 , 1h ) , 6.20 ( s , 1h ) , 6.00 ( d , j = 6.1 , 1h ) , 3.85 ( dd , j = 9.5 , 6.3 , 1h ) , 3.243.17 ( m , 1h ) , 1.31 ( d , j = 6.2 , 1h ) . c nmr ( 101 mhz ; dmso ) : c 166.7 , 159.2 , 141.3 , 129.7 , 111.3 , 79.1 , 54.6 , 48.9 , 20.4 . hrms esi [ m + na ] calc . for c9h12n6osna 275.0691 ; obs . 275.0686 . 19 : yield > 85% d.r.= 3:1 rf = 0.25 ( ch2cl2/meoh 7:3 ) . threo-19 ( major ) : ir ( solid , cm ) : 3432 , 3371 , 3314 , 3105 , 2966 , 2930 , 1645 , 1604 , 1575 . h nmr ( 400 mhz ; dmso ) : h 7.04 ( s , 1h , ar ) , 6.97 ( s , 2h , nh2 ) , 6.67 ( br.s , 2h , nh2 ) , 5.95 ( d , j = 6.4 , 1h , h(c1 ) ) , 5.85 ( s , 1h , nh ) , 4.60 ( d , j = 6.3 , 1h , h(c2 ) ) , 3.60 ( t , j = 6.9 , 1h , h(c3 ) ) , 1.741.63 ( m , 1h , h(c4h ) ) , 1.461.35 ( m , 1h , h(c4h ) ) , 0.94 ( t , j = 7.3 , 3h , me ) . c nmr ( 101 mhz ; dmso ) : c 166.8 , 165.3 , 141.3 , 128.3 , 111.6 , 79.6 , 56.5 , 52.5 , 29.8 , 10.6 . hrms esi [ m + na ] calc . for c10h14n6osna 289.0848 ; obs . threo-20 ( major ) : ir ( solid , cm ) : 3404 , 3109 , 2923 , 2525 , 2293 , 1605 , 1581 , 1548 , 1512 . h nmr ( 400 mhz ; dmso ) : h 6.70 ( s , 1h , ar ) , 5.58 ( d , j = 6.4 , 1h , h(c1 ) ) , 4.28 ( dd , j = 6.3 , 2.1 , 1h , nh ) ) , 2.57 ( dd , j = 9.0 , 2.2 , 1h , h(c2 ) ) , 0.39 ( qt , j = 8.1 , 4.2 , 1h , h(c3 ) ) , 0.18 ( quintet , j = 8.7 , 2h , h2(c4 ) ) , 0.00 ( d , j = 3.9 , 2h , h2(c4 ) ) . c nmr ( 101 mhz ; dmso ) : c 164.4 , 163.1 , 138.8 , 126.1 , 109.2 , 77.2 , 54.9 , 53.6 , 15.1 , 2.0 , 0.0 hrms esi [ m+na ] calc . for c11h14n6osna 301.0846 ; obs . ir ( solid , cm ) : 3363 , 3213 , 3192 , 2920 , 2844 , 2375 , 1626 , 1592 , 1547 , 1519 . h nmr ( 400 mhz ; d2o ) : h 7.23 ( s , 1h , ar ) , 5.81 ( d , j = 7.5 , 1h , h(c1 ) ) , 4.99 ( dd , j = 7.5 , 2.5 , 1h , h(c2 ) ) , 3.75 ( ddd , j = 2.5 , 5.9 , 5.2 1h , h(c3 ) ) , 3.53 ( abx , j = 12.5 , 5.2 , 1h , h(c4 ) ) , 3.49 ( abx , j = 12.1 , 5.9 , 1h , h(c4 ) ) . h nmr ( 400 mhz ; dmso - d6 ) : h 7.05 ( s , 1h , ar ) , 6.97 ( s , 2h , conh2 ) , 6.66 ( bs , 2h , nh2 ) , 6.05 ( s , 1h , nh ) , 6.02 ( d , j = 6.3 , 1h , h(c1 ) ) , 5.29 ( t , j = 0.5 , 1h , oh ) , 4.48 ( d , j = 5.9 , 1h , h(c2 ) ) , 3.78 ( m , 1h , h(c3 ) ) , 3.60 ( m , 1h , h(c4 ) ) , 3.27 ( m , 1h , h(c4 ) partially obscured by dmso residual solvent signal ) . c nmr ( 101 mhz ; dmso ) : c 166.8 , 165.9 , 141.2 , 128.3 , 111.8 , 80.2 , 65.3 , 53.6 , 53.0 . hrms esi [ m + na ] calc . for c9h12n6o2sna 291.0640 ; obs . 291.0640 . ir ( solid , cm ) : 3330 , 3212 , 3111 , 2950 , 2926 , 2853 , 2394 , 1631 , 1568 . h nmr ( 400 mhz ; d2o ) : h 7.35 ( s , 1h , ar ) , 5.98 ( d , j = 5.9 , 1h , h(c1 ) ) , 4.09 ( dd , j = 7.9 , 6.5 , 1h , h(c2 ) ) , 3.80 ( abx , j = 12.0 , 2.4 , 1h , h(c4 ) ) , 3.62 ( abx , j = 11.9 , 5.7 , 1h , h(c4 ) ) , 3.453.41 ( m , 1h , h(c3 ) ) . h nmr ( 400 mhz ; dmso ) : h 7.31 ( s , 1h , ar ) , 6.99 ( s , 2h , nh2 ) , 6.76 ( bd , j = 10.1 , 2h , c(o)nh2 ) , 6.35 ( s , 1h , nh ) , 6.02 ( d , j = 6.2 , 1h , h(c1 ) ) , 5.21 ( dd , j = 6.5 , 4.6 , 1h , ho ) , 3.95 ( dd , j = 9.7 , 6.2 , 1h , h(c2 ) ) , 3.73 ( dt , j = 11.1 , 3.8 , 1h , h(c4 ) ) , 3.463.41 ( m , inc . j = 11.1 , 1h , h(c4 ) partially obscured by h2o signal ) , 3.18 ( m , inc . c nmr ( 101 mhz ; dmso ) : c 166.5 , 159.2 , 140.9 , 129.3 , 111.0 , 79.0 , 63.2 , 54.4 , 49.0 . ir ( solid , cm ) : 3344 , 3106 , 2917 , 2833 , 1627 , 1553 . h nmr ( 400 mhz ; dmso ) : h 7.14 ( s , 1h , ar ) , 6.90 ( s , 2h , nh2 ) , 6.59 ( bs , 1h , nh2 ) , 6.35 ( s , 1h , nh ) , 5.93 ( d , j = 6.1 , 1h , h(c1 ) ) , 4.98 ( d , j = 6.1 , 1h , ho ) , 4.93 ( t , j = 5.4 , 1h , ho ) , 4.19 ( dd , j = 7.6 , 6.2 , 1h , h(c2 ) ) , 3.593.54 ( m , 1h , h(c4 ) ) , 3.49 ( abx , j = 10.9 , 5.0 , 1h , h(c5 ) ) , 3.463.41 ( m , inc . j = 10.9 , 1h , h(c5 ) ) , 3.363.34 ( m , inc . j = 7.6 hz , 1h , h(c3 ) ) . c nmr ( 100 mhz ; dmso ) : c 166.6 , 160.5 , 148.7 , 141.2 , 128.6 , 110.8 , 78.38 , 78.35 , 71.2 , 63.2 , 53.3 , 50.5 . ir ( solid , cm ) : 3309 , 3125 , 2936 , 2835 , 1632 , 1565 . h nmr ( 400 mhz ; d2o ) : h 7.26 ( s , 1h , ar ) , 5.94 ( d , j = 5.8 , 1h , h(c1 ) ) , 4.21 ( t , j = 6.7 , 1h , h(c2 ) ) , 3.73 ( m , 1h , h(c4 ) ) , 3.69 ( abx , j = 11.8 , 4.5 , 1h , h(c5 ) ) , 3.64 ( abx , j = 11.8 , 4.5 , 1h , h(c5 ) ) , 3.52 ( dd , j = 7.5 , 1.9 , 1h , h(c3 ) ) . h nmr ( 400 mhz ; dmso ) : h 7.05 ( s , 1h , ar ) , 6.99 ( s , 1h , nh2 ) , 6.67 ( bs , 1h , nh2 ) , 6.18 ( s , 1h , nh ) , 6.01 ( d , j = 6.3 , 1h , h(c1 ) ) , 5.44 ( d , j = 6.0 , 1h , ho ) , 4.85 ( t , j = 5.5 , 1h , ho ) , 4.55 ( d , j = 6.1 , 1h , h(c2 ) ) , 3.69 ( d , j = 8.3 , 1h , h(c4 ) ) , 3.54 ( abx(oh ) , j = 11.3 , 5.5 , 1h , h(c5 ) ) , 3.47 ( abx(oh ) , j = 11.3 , 5.5 , 1h , h(c5 ) ) , 3.20 ( m , 1h , h(c3 ) ) . c nmr ( 101 mhz ; dmso ) : c 166.5 , 165.9 , 141.0 , 127.9 , 111.5 , 100.0 , 79.7 , 75.1 , 63.5 , 53.5 . hrms esi [ m + na ] calc . for c10h14n6o3sna 321.0746 ; obs . 321.0750 .

we propose a novel pathway for the prebiotic synthesis of 2-deoxynucleotides . consideration of the constitutional chemical relationships between glycolaldehyde and -mercapto - acetaldehyde , and the corresponding proteinogenic amino acids , serine and cysteine , led us to explore the consequences of the corresponding sulfur substitution for our previously proposed pathways leading to the canonical ribonucleotides . we demonstrate that just as 2-aminooxazole an important prebiotic ribonucleotide precursor is readily formed from glycolaldehyde and cyanamide , so is 2-aminothiazole formed from -mercapto - acetaldehyde and cyanamide in water at neutral ph . indeed , both the oxazole and the thiazole can be formed together in a one - pot reaction , and can be co - purified by crystallization or sublimation . we then show that 2-aminothiazole can take part in a 3-component carbon carbon bond - forming reaction in water that leads to the diastereoselective synthesis of masked 2-thiosugars regiospecifically tethered to purine precursors , which would lead to 2-deoxynucleotides upon desulfurization . the possibility of an abiotic route to the 2-deoxynucleotides provides a new perspective on the evolutionary origins of dna . we also show that 2-aminothiazole is able to sequester , through reversible aminal formation , the important nucleotide precursors glycolaldehyde and glyceraldehyde in a stable , crystalline form .

Introduction Results and Discussion Summary Experimental Section

a plausible abiotic chemical route to the canonical nucleotides is a major goal in origins of life research . recently , we demonstrated the first chemical steps toward a divergent pyrimidine and purine ribonucleotide synthesis . a one - pot multicomponent reaction was demonstrated to stereoselectively tether and consequently regiospecifically glycosylate purine precursors and masked pentose sugars , while concurrently furnishing known pyrimidine precursors ( scheme 1 ) . although rna has often been considered as a candidate for the first biopolymer of life , extant biology utilizes two chemically distinct , but related , nucleotide polymers , rna and dna . however , it would not be possible to make dna without deoxyribonucleotides , and yet in the absence of dna there is no obvious reason for the evolution of the biochemical pathways for the synthesis of deoxyribonucleotides . only after the emergence of dna as an important cellular component would there have been a strong selective pressure favoring the emergence of biochemical pathways for the synthesis of deoxyribonucleotides from ribonucleotides . although the advantages of dna as a medium for information storage are clear , there has not until now been any reasonable hypothesis for how dna could be invented by primitive cells in the absence of pre - existing deoxyribonucleotides . in the absence of the complex and energetically costly enzymatic resources required to regiospecifically deoxygenate ribonucleotides , abiological 2-deoxygenation of nucleotides requires site - specific ribo- or arabino - nucleoside deoxygenation . in light of the difficulty of regioselective nucleotide deoxygenation in the absence of sophisticated enzymatic control , it would seem that regiospecific positioning of a group / atom with the appropriate latent potential reactivity could form the basis of an alternative pathway to the 2-deoxyribonucleotides . constitutional analysis of dna , with respect to rna , in light of the low dissociation energy of c s bonds , suggests that regiospecific positioning of sulfur at c2 could result in the chemical differentiation required for the divergent synthesis of dna and rna monomers from common precursors ( scheme 2 ) . oxazole 1a key ribonucleotide precursor is derived from cyanamide 2 and glycolaldehyde 3 . it is of note that aldehyde 3 is also the aldehyde precursor of serine 4 , a proteinogenic amino acid , via strecker synthesis with ammonium cyanide ( scheme 3 ) . the constitutional similarity of serine 4 and cysteine 5 , and their aldehyde precursors 3 and -mercapto - acetaldehyde 6 , suggests that both 3 and 6 must be considered within our exploration of prebiotic azole synthesis . specifically , we propose that the divergent reactivity of glycolaldehyde 3 and -mercapto - acetaldehyde 6 with cyanamide 2 could lead , via 2-aminooxazole 1 and 2-aminothiazole 7 , to ribonucleotides and 2-deoxyribonucleotides , by desulfurization of c2 ( scheme 2 ) . the importance of 2-aminothiazole 7 , and its derivatives , has been recognized in medicinal chemistry , but though the synthesis of thiazole 7 in water has been reported , the potential relevance of thiazole 7 to the origins of life remains unexplored . the formation of oxazole 1 from cyanamide 2 and glycolaldehyde 3 at neutral ph requires stoichiometric ( or excess ) cyanamide 2 and general acid aldehyde 6 , upon imine formation with cyanamide 2 , has , like 3 , the potential to undergo 5-exo - dig cyclization to furnish imine 14 ( or hemiaminal 15 ) as shown in scheme 3 . furthermore , we expected the reaction to proceed more rapidly at neutral ph and be less prone to stall at intermediates en route to thiazole 7 due to the increased nucleophilicity of sulfur and greater aromaticity of thiazoles with respect to oxazoles . to test this supposition , -mercapto - acetaldehyde 6 ( 8.5 mm ) and 2 ( 15 , 26 , or 50 mm ) were incubated in d2o at pd 7 and 20 c and a rapid ( ca . it is of note that excess aldehyde 6 did not lead to further reaction of thiazole 7 . moreover , at elevated concentration ( > 150 mm ) 2-aminothiazole 7 was observed to directly crystallize from water , and a crystal structure of thiazole 7 is shown in scheme 3 . next we investigated the concomitant synthesis of oxazole 1 and thiazole 7 . in the presence of excess cyanamide 2 ( 2.25 equiv ) , rapid and stoichiometric conversion of glycolaldehyde 3 ( 1 equiv ) and -mercapto - acetaldehyde 6 ( 1 equiv ) to hemiaminals 11 and 15 , respectively , was observed in water . at neutral pd , selective dehydration of 15 to liberate thiazole 7 was observed . it is likely that the rapid 5-exo - dig cyclization of imine 12/ hemiaminal 13 leads to the selective sequestration of cyanamide 2 in thiazole 7 . interestingly , we also found that , like oxazole 1 , thiazole 7 is easily sublimed ( see supporting information figure s4 ) , suggesting possible geochemical routes to the distribution , separation , and purification of thiazole 7 under abiotically plausible conditions . we then examined the multicomponent reaction of thiazole 7 , 4-amino - imidazole 5-carboxamide 16 , and a series of aldehydes . equimolar quantities of thiazole 7 and aliphatic aldehydes ( including formaldehyde , acetaldehyde , propionaldehyde , and cyclobutaldehyde ) with excess amino - imidazole 16 ( 1.23 equiv ) furnished high yields ( 7590% ) of thiazolines 17 , 18 , 19 , and 20 , respectively , at ph 5 . compounds 17 , threo-18 and erythro-18 , and the major isomers of 19 and 20 were isolated by fractional crystallization , and the crystal structures are shown in scheme 4 . to continue our investigation into the potential of multicomponent assembly of nucleotides we studied the participation of thiazole 7 in carbon carbon bond - forming reaction with -hydroxyaldehydes and imines derived from -hydroxyaldehydes in water . incubation of equimolar aldehyde 3 , 2-aminothiazole 7 , and amino - imidazole 16 for 5 d in water at ph 46 at 20 c gave a high yield of c4-thiazoline 23 ( 80% isolated yield ) as 1.2:1 erythro / threo mixture of diastereomeric products . however , both erythro-23 and threo-23 were formed with complete annulation control ( > 99% , see h nmr spectrum shown in supporting information figure s28 ; single crystal x - ray structures of erythro-23 and threo-23 are shown in scheme 5 ) . upon investigation of the parallel mcr with the c3-synthon glyceraldehyde 22 , comparable efficiency in the synthesis of c5-thiazoline 24 ( 70% isolated yield ) was observed . isolation of the two major isomers , crystallization , and x - ray diffraction proved their xylo- and lyxo - configuration . the diastereoselectivity obtained , upon carbon carbon bond formation is proposed to result from intramolecular hydrogen - bonding and concomitant steric impedance of thiazole addition to the si - face of imine 21 ( see supporting information scheme s1 ) . although the facial selectivity of the nucleophile 7 has been relaxed with respect to the comparable reaction of 1 , the facial selectivity of the glyceraldehyde - imine 21 ( r = ch2oh ) is retained . this is important with respect to purine nucleotide synthesis by 3-purine tethering , which necessarily requires n3-displacement and c3-inversion . furthermore , as well as regiospecific tethering of 16 and consequent delivery of n1 to the anomeric carbon atom , reaction of thiazole 7 with imine 21 regiospecifically delivers sulfur to c2 , which is of significant interest with regards to the potential synthesis of 2-deoxynucleotides by c2-desulfurization . additionally , oxazole 1 is known to react efficiently with -hydroxyaldehydes in a ph - dependent bimolecular domino - reaction . in striking contrast , carbon bond formation was observed by h nmr analysis upon incubation of thiazole 7 with either glycolaldehyde 3 or rac - glyceraldehdye 22 in d2o ( pd 39 , 4100 c ) . to test whether the change in solubility properties was due to the increased number of hydroxyl moieties or perhaps due to the differential solubility and packing properties of rac-27 with respect to the achiral aminal 25 , we investigated the solubility properties of the aminal structures formed in a mixture of d - glyceraldehyde 22 and thiazole 7 . intriguingly , we found that at 100 mm concentration , d-27 now crystallized from water at 20 c . nmr analysis showed a 1:2 ratio of 22/7 and recrystallization ( 10:1 h2o / etoh ) allowed for crystallographic confirmation of the aminal structure ( scheme 6 ) . in summary , our results suggest that in a sulfur - rich prebiotic environment , oxazole 1 and thiazole 7 could be formed together under the same conditions , as long as both glycolaldehyde 3 and its thiol analogue , -mercapto - acetaldehyde 6 , were present along with cyanamide 2 . in such mixed reactions , the absence of any interference between oxazole and thiazole synthesis shows that these potential precursors of deoxynucleotides and ribonucleotides could indeed be formed at the same time in the same conditions . we have also found that two abiotically plausible methods for the purification of oxazole 1 , namely , crystallization and sublimation , also apply to thiazole 7 . however , the chemical reactivities of oxazole 1 and thiazole 7 are quite different , suggesting that the subsequent pathways would diverge . the most striking difference between the reactivity of oxazole 1 and thiazole 7 is the failure of the thiazole to react with aldehydes to form the thiazoline precursors of the ( thio)pyrimidine nucleotides . the most likely explanation for this difference is the increased aromaticity of the thiazole relative to the oxazole , and thus decreased nucleophilicity of c5 . we are currently investigating an alternative synthesis of 2-deoxyribo - pyrimidine nucleotides mediated by c2-inversion of anhydronucleotide 28 ( scheme 1 ) . the facile reversibility of aminal formation suggests that the chemical sequestration and protection of c2- and c3-aldehyde nucleotide synthons by another important class of nucleotide synthon ( azoles ) may have provided a route to the concentration , purification , and stabilization of the necessary aldehyde precursors of nucleotides . although these aldehydes can in principle be readily synthesized by a variety of routes , their high reactivity has until now made it difficult to conceive of means for accumulating large reservoirs of these essential starting materials in a prebiotic context . by exploring the multicomponent reactivity of thiazole 7 , together with aldehydes and the purine precursor amino - imidazole 16 , we have demonstrated a selective and high yielding 3-component carbon carbon bond - forming reaction that chemospecifically furnishes masked-2-thiosugars regiospecifically tethered to an amino - imidazole purine - precursor . the facial selectivity of thiazole 7 , upon addition to -hydroxyaldehyde - imines , is quite relaxed ( relative to oxazole 1 ) , resulting in low diastereoselectivity at c2 ; however , this is inconsequential with regards to the proposed deoxynucleotide synthesis because c2 would be rendered achiral following desulfurization . the conversion of the resulting c5-thiazolines to 2-deoxyribo - purine nucleotides requires three additional steps : c3-inversion to release the purine - precursor from its tethered attachment to c3 , completion of the purine heterocycle , and chemospecific desulfurization of c2. these steps are currently under investigation in our laboratories as part of our continuing studies aimed at the abiotic synthesis of 2-deoxyribonucleotides . finally , it is of note that if life emerged in an environment containing both ribo- and 2-deoxyribonucleotides , it is likely that the first biopolymers were heterogeneous in composition , that is , composed of a mixture of ribo- and deoxyribonucleotides , and perhaps other compatible nucleotides . recent work has shown that functional biopolymers , in the form of aptamers with highly specific molecular recognition properties , can be derived by in vitro evolution from libraries of polynucleotides composed of randomly interspersed ribo- and deoxyribo - nucleotides . thus , chimeric rna / dna polymers may have been sufficient for the emergence of life . the primordial biochemical exploitation of a mixed rna and dna genetic system could eliminate the requirement for a genetic takeover ( of rna by dna ) , and would arguably result in a simplification of the transition from chemistry to biology . however , such mixed - composition polymers have neither the advantageous stability of dna , nor the optimal functional characteristics of rna . when possible , the chemical shift of the corresponding solvent was used as a reference . mercaptoacetaldehyde 6 and cyanamide 2 ( 0.51.5 equiv ) were dissolved in h2o , d2o or phosphate buffer ( 1 ml ) at ph / pd 7.0 . alternatively , upon complete conversion ( by nmr spectroscopy ) , the solution was concentrated and 7 recovered by sublimation . compound 7 ( 15 g ) was placed in a covered beaker or erlenmeyer flask and warmed from beneath to 40 c . c nmr ( 101 mhz ; dmso ) : c 169.5 , 139.3 , 107.1 . glycolaldehyde 3 ( 1 equiv ) , mercaptoacetaldehyde 6 ( 1 equiv ) , and cyanamide 2 ( 0.55 equiv ) were dissolved in h2o / d2o ( 1 ml ) at ph / pd 7 , 10 or 12 , or 1 m phosphate buffer ( 1 ml ) at ph / pd 6.57.0 . alternatively , upon complete conversion ( by nmr spectroscopy ) , the solution was concentrated and 7 and 1 were recovered by sublimation . compounds 7 and 1 ( 1:1 ; 100 mg ) as fine powder or film ( evaporated water ) were deposited in a covered glass tube ( 10 cm 1 cm ) . a temperature gradient ( 40 , 50 , or 60 c to ambient temperature ) was set up across the tube and a mixture of crystalline thiazole 7 and oxazole 1 were isolated at ambient temperature as a deposit upon the glass wall of the tube . the solution was then incubated at room temperature for 510 d ; the resultant solid was then isolated by filtration or trituration and washed twice with ethanol and once with ice cold water . the reaction was lyophilized and purified by silica gel column chromatography ( ch2cl2/meoh , 10:0 to 6:4 ) . the major product(s ) were concentrated and recrystallized from ethanol / water or dichloromethane / methanol mixtures . 18 : yield > 85% d.r.= 2.5:1 erythro-18 ( minor ) : rf = 0.25 ( ch2cl2/meoh 7:3 ) . ir ( solid , cm ) : 3437 , 3387 , 3292 , 3123 , 2975 , 2856 , 1638 , 1613 , 1571 . c nmr ( 101 mhz ; dmso ) : c 166.7 , 165.2 , 141.4 , 128.4 , 111.6 , 79.4 , 58.6 , 46.7 , 22.7 . h nmr ( 400 mhz ; dmso ) : h 7.33 ( s , 1h ) , 6.96 ( s , 1h ) , 6.76 ( d , j = 27.2 , 1h ) , 6.20 ( s , 1h ) , 6.00 ( d , j = 6.1 , 1h ) , 3.85 ( dd , j = 9.5 , 6.3 , 1h ) , 3.243.17 ( m , 1h ) , 1.31 ( d , j = 6.2 , 1h ) . hrms esi [ m + na ] calc . threo-19 ( major ) : ir ( solid , cm ) : 3432 , 3371 , 3314 , 3105 , 2966 , 2930 , 1645 , 1604 , 1575 . hrms esi [ m + na ] calc . for c10h14n6osna 289.0848 ; obs . 291.0640 . j = 7.6 hz , 1h , h(c3 ) ) . for c10h14n6o3sna 321.0746 ; obs .

a one - pot multicomponent reaction was demonstrated to stereoselectively tether and consequently regiospecifically glycosylate purine precursors and masked pentose sugars , while concurrently furnishing known pyrimidine precursors ( scheme 1 ) . although rna has often been considered as a candidate for the first biopolymer of life , extant biology utilizes two chemically distinct , but related , nucleotide polymers , rna and dna . however , it would not be possible to make dna without deoxyribonucleotides , and yet in the absence of dna there is no obvious reason for the evolution of the biochemical pathways for the synthesis of deoxyribonucleotides . only after the emergence of dna as an important cellular component would there have been a strong selective pressure favoring the emergence of biochemical pathways for the synthesis of deoxyribonucleotides from ribonucleotides . although the advantages of dna as a medium for information storage are clear , there has not until now been any reasonable hypothesis for how dna could be invented by primitive cells in the absence of pre - existing deoxyribonucleotides . in the absence of the complex and energetically costly enzymatic resources required to regiospecifically deoxygenate ribonucleotides , abiological 2-deoxygenation of nucleotides requires site - specific ribo- or arabino - nucleoside deoxygenation . in light of the difficulty of regioselective nucleotide deoxygenation in the absence of sophisticated enzymatic control , it would seem that regiospecific positioning of a group / atom with the appropriate latent potential reactivity could form the basis of an alternative pathway to the 2-deoxyribonucleotides . constitutional analysis of dna , with respect to rna , in light of the low dissociation energy of c s bonds , suggests that regiospecific positioning of sulfur at c2 could result in the chemical differentiation required for the divergent synthesis of dna and rna monomers from common precursors ( scheme 2 ) . the constitutional similarity of serine 4 and cysteine 5 , and their aldehyde precursors 3 and -mercapto - acetaldehyde 6 , suggests that both 3 and 6 must be considered within our exploration of prebiotic azole synthesis . specifically , we propose that the divergent reactivity of glycolaldehyde 3 and -mercapto - acetaldehyde 6 with cyanamide 2 could lead , via 2-aminooxazole 1 and 2-aminothiazole 7 , to ribonucleotides and 2-deoxyribonucleotides , by desulfurization of c2 ( scheme 2 ) . the importance of 2-aminothiazole 7 , and its derivatives , has been recognized in medicinal chemistry , but though the synthesis of thiazole 7 in water has been reported , the potential relevance of thiazole 7 to the origins of life remains unexplored . the formation of oxazole 1 from cyanamide 2 and glycolaldehyde 3 at neutral ph requires stoichiometric ( or excess ) cyanamide 2 and general acid aldehyde 6 , upon imine formation with cyanamide 2 , has , like 3 , the potential to undergo 5-exo - dig cyclization to furnish imine 14 ( or hemiaminal 15 ) as shown in scheme 3 . furthermore , we expected the reaction to proceed more rapidly at neutral ph and be less prone to stall at intermediates en route to thiazole 7 due to the increased nucleophilicity of sulfur and greater aromaticity of thiazoles with respect to oxazoles . moreover , at elevated concentration ( > 150 mm ) 2-aminothiazole 7 was observed to directly crystallize from water , and a crystal structure of thiazole 7 is shown in scheme 3 . next we investigated the concomitant synthesis of oxazole 1 and thiazole 7 . in the presence of excess cyanamide 2 ( 2.25 equiv ) , rapid and stoichiometric conversion of glycolaldehyde 3 ( 1 equiv ) and -mercapto - acetaldehyde 6 ( 1 equiv ) to hemiaminals 11 and 15 , respectively , was observed in water . at high ph / pd ( ph / pd > 9 ) specific base catalysis or in 1 m phosphate solution ( ph / pd 6.57.5 ) general acid base catalysis furnish oxazole 1 and thiazole 7 over the course of 2436 h at ambient temperature ( scheme 3 ; see supporting information figure s8s10 ) . we did not observe significant homo- and hetero - aldol reaction during the reaction of glycolaldehyde 3 and mercaptoacetaldehyde 6 with either excess or limiting cyanamide 2 between ambient temperature and 60 c in 1 m phosphate solution at ph 7 . interestingly , we also found that , like oxazole 1 , thiazole 7 is easily sublimed ( see supporting information figure s4 ) , suggesting possible geochemical routes to the distribution , separation , and purification of thiazole 7 under abiotically plausible conditions . partial sublimation of a mixture of oxazole 1 and thiazole 7 ( 1:1 ) resulted in the isolation 4:12:1 oxazole 1/thiazole 7 , at 50 c between 1 and 24 h at ambient pressure , indicating the more rapid but comparable sublimation rate of oxazole 1 relative to thiazole 7 . equimolar quantities of thiazole 7 and aliphatic aldehydes ( including formaldehyde , acetaldehyde , propionaldehyde , and cyclobutaldehyde ) with excess amino - imidazole 16 ( 1.23 equiv ) furnished high yields ( 7590% ) of thiazolines 17 , 18 , 19 , and 20 , respectively , at ph 5 . the multicomponent reactions were found to be robust at multi - gram scale and comparable diastereoselectivity was observed in the reaction of aliphatic aldehydes with oxazole 1 or thiazole 7 and amino - imidazole 16 ( via imine 21 r = alkyl ; d.r.= 2.5:14:1 threo / erythro ) . compounds 17 , threo-18 and erythro-18 , and the major isomers of 19 and 20 were isolated by fractional crystallization , and the crystal structures are shown in scheme 4 . to continue our investigation into the potential of multicomponent assembly of nucleotides we studied the participation of thiazole 7 in carbon carbon bond - forming reaction with -hydroxyaldehydes and imines derived from -hydroxyaldehydes in water . incubation of equimolar aldehyde 3 , 2-aminothiazole 7 , and amino - imidazole 16 for 5 d in water at ph 46 at 20 c gave a high yield of c4-thiazoline 23 ( 80% isolated yield ) as 1.2:1 erythro / threo mixture of diastereomeric products . surprisingly , this signified that the facial selectivity of thiazole 7 is significantly relaxed with respect to oxazole 1 and imine 21 ( r = ch2oh ) . however , both erythro-23 and threo-23 were formed with complete annulation control ( > 99% , see h nmr spectrum shown in supporting information figure s28 ; single crystal x - ray structures of erythro-23 and threo-23 are shown in scheme 5 ) . upon investigation of the parallel mcr with the c3-synthon glyceraldehyde 22 , comparable efficiency in the synthesis of c5-thiazoline 24 ( 70% isolated yield ) was observed . again , exclusive 6-exo - trig annulation is observed ; however , in this case , 24 is furnished with significant diastereoselectivity ( d.r.= 9:5:1.1:1 , see scheme 5 and supporting information figure s35 ) . isolation of the two major isomers , crystallization , and x - ray diffraction proved their xylo- and lyxo - configuration . the diastereoselectivity obtained , upon carbon carbon bond formation is proposed to result from intramolecular hydrogen - bonding and concomitant steric impedance of thiazole addition to the si - face of imine 21 ( see supporting information scheme s1 ) . although the facial selectivity of the nucleophile 7 has been relaxed with respect to the comparable reaction of 1 , the facial selectivity of the glyceraldehyde - imine 21 ( r = ch2oh ) is retained . furthermore , as well as regiospecific tethering of 16 and consequent delivery of n1 to the anomeric carbon atom , reaction of thiazole 7 with imine 21 regiospecifically delivers sulfur to c2 , which is of significant interest with regards to the potential synthesis of 2-deoxynucleotides by c2-desulfurization . in striking contrast , carbon bond formation was observed by h nmr analysis upon incubation of thiazole 7 with either glycolaldehyde 3 or rac - glyceraldehdye 22 in d2o ( pd 39 , 4100 c ) . single crystal x - ray diffraction of a crystal isolated directly from the reaction supernatant proved that the structure was aminal 25 ( scheme 6 ) . though nmr studies confirmed that rac-22 formed hemiaminal 26 and aminal 27 with thiazole 7 in d2o , no aminal crystallization was observed in solutions of rac-22 and 7 , even at significantly elevated concentrations ( 500 mm to 2 m ) or with substoichiometric quantities of rac-22 . to test whether the change in solubility properties was due to the increased number of hydroxyl moieties or perhaps due to the differential solubility and packing properties of rac-27 with respect to the achiral aminal 25 , we investigated the solubility properties of the aminal structures formed in a mixture of d - glyceraldehyde 22 and thiazole 7 . in summary , our results suggest that in a sulfur - rich prebiotic environment , oxazole 1 and thiazole 7 could be formed together under the same conditions , as long as both glycolaldehyde 3 and its thiol analogue , -mercapto - acetaldehyde 6 , were present along with cyanamide 2 . in such mixed reactions , the absence of any interference between oxazole and thiazole synthesis shows that these potential precursors of deoxynucleotides and ribonucleotides could indeed be formed at the same time in the same conditions . we have also found that two abiotically plausible methods for the purification of oxazole 1 , namely , crystallization and sublimation , also apply to thiazole 7 . the most striking difference between the reactivity of oxazole 1 and thiazole 7 is the failure of the thiazole to react with aldehydes to form the thiazoline precursors of the ( thio)pyrimidine nucleotides . the most likely explanation for this difference is the increased aromaticity of the thiazole relative to the oxazole , and thus decreased nucleophilicity of c5 . however , during the course of efforts to observe two - component reactivity of thiazole 7 with aldehydes , we observed instead the crystallization of glycolaldehyde 3 and homochiral ( but not racemic ) glyceraldehyde 22 ( two known ribonucleotide precursors ) as aminals directly from water . the facile reversibility of aminal formation suggests that the chemical sequestration and protection of c2- and c3-aldehyde nucleotide synthons by another important class of nucleotide synthon ( azoles ) may have provided a route to the concentration , purification , and stabilization of the necessary aldehyde precursors of nucleotides . although these aldehydes can in principle be readily synthesized by a variety of routes , their high reactivity has until now made it difficult to conceive of means for accumulating large reservoirs of these essential starting materials in a prebiotic context . by exploring the multicomponent reactivity of thiazole 7 , together with aldehydes and the purine precursor amino - imidazole 16 , we have demonstrated a selective and high yielding 3-component carbon carbon bond - forming reaction that chemospecifically furnishes masked-2-thiosugars regiospecifically tethered to an amino - imidazole purine - precursor . the facial selectivity of thiazole 7 , upon addition to -hydroxyaldehyde - imines , is quite relaxed ( relative to oxazole 1 ) , resulting in low diastereoselectivity at c2 ; however , this is inconsequential with regards to the proposed deoxynucleotide synthesis because c2 would be rendered achiral following desulfurization . in contrast , we observed very high diastereoselectivity of nucleophilic addition of thiazole 7 to imine 21 , generating almost exclusively the lyxo and xylo isomers with controlled n9c1 annulation . the conversion of the resulting c5-thiazolines to 2-deoxyribo - purine nucleotides requires three additional steps : c3-inversion to release the purine - precursor from its tethered attachment to c3 , completion of the purine heterocycle , and chemospecific desulfurization of c2. finally , it is of note that if life emerged in an environment containing both ribo- and 2-deoxyribonucleotides , it is likely that the first biopolymers were heterogeneous in composition , that is , composed of a mixture of ribo- and deoxyribonucleotides , and perhaps other compatible nucleotides . recent work has shown that functional biopolymers , in the form of aptamers with highly specific molecular recognition properties , can be derived by in vitro evolution from libraries of polynucleotides composed of randomly interspersed ribo- and deoxyribo - nucleotides . the primordial biochemical exploitation of a mixed rna and dna genetic system could eliminate the requirement for a genetic takeover ( of rna by dna ) , and would arguably result in a simplification of the transition from chemistry to biology . however , such mixed - composition polymers have neither the advantageous stability of dna , nor the optimal functional characteristics of rna . single crystal x - ray crystallography was carried out with a bruker apex ii ccd diffractometer ( mo k radiation , = 0.71073 ) , equipped with an oxford cryosystems nitrogen flow apparatus , at 100 k. data integration down to 0.76 resolution was carried out using saint v7.46 a ( bruker diffractometer , 2009 ) with reflection spot size optimization . glycolaldehyde 3 ( 1 equiv ) , mercaptoacetaldehyde 6 ( 1 equiv ) , and cyanamide 2 ( 0.55 equiv ) were dissolved in h2o / d2o ( 1 ml ) at ph / pd 7 , 10 or 12 , or 1 m phosphate buffer ( 1 ml ) at ph / pd 6.57.0 . a temperature gradient ( 40 , 50 , or 60 c to ambient temperature ) was set up across the tube and a mixture of crystalline thiazole 7 and oxazole 1 were isolated at ambient temperature as a deposit upon the glass wall of the tube . h nmr ( 400 mhz ; dmso ) : h 7.05 ( s , 1h , ar ) , 6.97 ( s , 2h , nh2 ) , 6.68 ( br d , j = 19.9 , 2h , nh2 ) , 5.95 ( d , j = 6.4 , 1h , h(c1 ) ) , 5.89 ( s , 1h , hn ) , 4.54 ( ddd , j = 6.4 , 2.5 , 1.7 , 1h , h(c2 ) ) , 3.78 ( qd , j = 6.4 , 2.6 , 1h , h(c3 ) ) , 1.25 ( d , j = 6.4 , 3h , h(c4 ) ) . h nmr ( 400 mhz ; dmso ) : h 7.04 ( s , 1h , ar ) , 6.97 ( s , 2h , nh2 ) , 6.67 ( br.s , 2h , nh2 ) , 5.95 ( d , j = 6.4 , 1h , h(c1 ) ) , 5.85 ( s , 1h , nh ) , 4.60 ( d , j = 6.3 , 1h , h(c2 ) ) , 3.60 ( t , j = 6.9 , 1h , h(c3 ) ) , 1.741.63 ( m , 1h , h(c4h ) ) , 1.461.35 ( m , 1h , h(c4h ) ) , 0.94 ( t , j = 7.3 , 3h , me ) . h nmr ( 400 mhz ; d2o ) : h 7.26 ( s , 1h , ar ) , 5.94 ( d , j = 5.8 , 1h , h(c1 ) ) , 4.21 ( t , j = 6.7 , 1h , h(c2 ) ) , 3.73 ( m , 1h , h(c4 ) ) , 3.69 ( abx , j = 11.8 , 4.5 , 1h , h(c5 ) ) , 3.64 ( abx , j = 11.8 , 4.5 , 1h , h(c5 ) ) , 3.52 ( dd , j = 7.5 , 1.9 , 1h , h(c3 ) ) .

periodontal therapy involves controlling of periodontal infection and aims at regeneration of the lost periodontium . the two techniques with the most successful documentation of periodontal regeneration are osseous grafting and guided - tissue regeneration ( gtr ) . the regeneration of the periodontium with new connective fiber insertion , new cementum , and new bone formation constitutes ideal healing . the regeneration process can only be initiated by periodontal ligament cells derived from the remaining periodontium as these are the cells capable of differentiating into new fibroblasts , cementoblasts , and osteoblasts . nyman et al . suggested the placement of a physical barrier between the flap and the root surface to exclude gingival connective tissue and epithelium from the healing process , giving the periodontal ligament cells the opportunity to repopulate the coagulum on the root surface . the barriers recommended for the use in gtr , regardless of the material used , must be safe , biocompatible , and non - toxic , not induce any inflammatory response , and be designed for clinical applicability based on the morphology of the osseous defects . furthermore , gingival recession , device exposure , infection , and inflammation are frequently experienced with nonresorbable membranes . among resorbable barriers , investigators have examined type - i collagen for the use in gtr procedures as it is a major extracellular macromolecule of the periodontal connective tissue . also collagen is known to be a weak immunogen , has a chemotactic property for fibroblasts , and acts as a barrier for migrating epithelial cells in vitro . collagen from mammalian sources , primarily bovine skin , has been utilized in foods , cosmetics , and biomaterials and has the advantage of biodegradability and low toxicity . therefore , there have been various attempts to use mammalian collagen in the medical field as a scaffold for developing artificial organs . however , the use of bovine collagen has been reconsidered , as some reports have shown the risk of transmission of bovine spongiform encephalopathy ( bse ) to human beings . recently , there has been interest in non - mammalian collagen sources , primarily in fish collagen such as that of shark and salmon , as it is known to have low risk for transmission of infectious disease to humans than bovine collagen . hence , an attempt was made to assess , a bioresorbable collagen membrane of fish origin ( periocol , eucare pharmaceuticals private limited , chennai , india ) in the treatment of periodontal intrabony defects . the study was planned with the following objectives . to assess the clinical parameters like probing pocket depth ( ppd ) , relative attachment level ( ral ) and recession ( r ) with and without a bioresorbable collagen barrier membrane in the treatment of intrabony defectsto assess the alveolar crestal bone level ( abl ) and percentage of defect fill ( df ) radiographically in intrabony defects with and without a bioresorbable collagen barrier membrane . to assess the clinical parameters like probing pocket depth ( ppd ) , relative attachment level ( ral ) and recession ( r ) with and without a bioresorbable collagen barrier membrane in the treatment of intrabony defects to assess the alveolar crestal bone level ( abl ) and percentage of defect fill ( df ) radiographically in intrabony defects with and without a bioresorbable collagen barrier membrane . the study employed a total of 10 patients ( 6 males and 4 females ) aged between 21 and 50 years in a split mouth manner to assess the collagen membrane both clinically and radiographically . subjects having bilateral isolated intra osseous defects in the mandibular posterior teeth were screened based on the following inclusion and exclusion criteria . inclusion criteria : ( a ) patients of either sex having chronic periodontitis , ( b ) patients who were systemically healthy with no contraindication to periodontal surgery , ( c ) non smokers , ( d ) presence of an intrabony defect with residual ppd of five millimeters ( mm ) and with radiographic evidence of the affected site , ( e ) patients having 2 to 3 mm band of keratinized tissue to allow surgical manipulation and suturing , and ( f ) patients who were co - operative and able to come for regular follow up . exclusion criteria : ( a ) patients allergic or sensitive to any medication or any ingredient of the test material , ( b ) patients showing unacceptable oral hygiene compliance during or after phase i therapy , and ( c ) pregnant and lactating mothers . following selection of subjects based on inclusion criteria , patients were given an explanation of the study , and an informed consent was obtained . the study was analyzed and approved by the institutional review board and the ethical clearance committee of the institution . all patients underwent initial therapy consisting of oral hygiene instructions and scaling and root planing . randomization was done by a coin toss to select the sites to be treated by gtr using a bioresorbable membrane ( i.e. , test group ) and control sites treated by conventional open flap debridement ( ofd ) . all the surgeries were performed by the same operator , and the measurements were recorded by a blinded examiner . plaque index , gingival bleeding index , ral , ppd , and r were recorded from baseline , 3 , 6 , and at 9 months using university of north carolina-15 ( unc ) probe . a customized acrylic occlusal stent with a vertical groove was made for proper guidance and orientation of the periodontal probe in the same plane [ figure 1 ] . the stents were preserved in the study casts to minimize distortion for follow - up measurements . ral was measured from the reference point ( rp ) on the stent to the base of the pocket ( bop ) . ppd was recorded by noting the difference between measurements from rp to fgm and rp to the bop . baseline clinical parameters were assessed by using unc-15 probe on a custom - made acrylic stent in relation to 46 radiographs were taken at baseline , 6 , and 9 months postoperatively . intra - oral periapical radiographs were taken using a long cone / extension cone paralleling technique with the positioning device ( rinn xcp , dentsply , il , usa . ) and a size 2e speed intra - oral periapical radiographic film ( kodak x - ray film , usa ) was used in a roentgen machine operating at 70 kilo voltage power ( kvp ) , 0.6 milli amperes ( ma ) . bite registration was done using rubber base impression material and stored for follow - up assessment . radiographs were scanned using a scanner ( hp 3010 g ) at an input of 400 dpi and 100% scale . radiographic parameters were recorded as follows : ( 1 ) distance from cementoenamel junction to base of the defect ( cej to bd ) = a , ( 2 ) distance from cementoenamel junction to alveolar crest ( cej to ac ) = b , ( 3 ) defect depth = a - b , ( 4 ) df was measured as the difference in defect depth from baseline to 6 months and 9 months . intracrevicular incisions were given both buccally and lingually and full thickness mucoperiosteal flap was elevated . after the reflection of the flap and exposure of the osseous defect , a thorough surgical debridement was carried out to remove sub - gingival plaque , calculus , diseased granulation tissue , and pocket epithelium [ figure 2 ] . the surgical sites were irrigated with sterile saline , and care was taken to keep the area free of saliva . the gtr membrane was removed from the sterile package and hydrated in normal saline for few seconds before placement on to the defect to improve adhesion properties and malleability . the membrane design was custom prepared chair side to receive for an intrabony defect and then carefully tweezed through the interproximal contact area [ figure 3 ] and extended 2 to 3 mm beyond the bony margin , to provide a broad base during placement . prior to closure of mucoperiosteal flaps , decortication of the osseous defect was done to induce bleeding . surgical flaps were repositioned to the pre - surgical level and sutured with 3 - 0 silk suture ( mersilk -ethicon , division of johnson and johnson ltd , aurangabad , india ) utilizing an interrupted direct loop suturing technique thereby achieving primary closure [ figure 4 ] . care was taken not to displace the gtr membrane during suturing . a non - eugenol periodontal dressing ( coe - pack - gc america inc . alsip , il , usa ) was placed on the surgical area following which the post - operative instructions were given . following incision , a full thickness mucoperiosteal flap was reflected and the defect debridement was done the collagen barrier membrane was trimmed to the required size and shape and placed over the interproximal vertical defect suturing was done using 3 - 0 silk with a simple interrupted direct loop technique post - operative medication included amoxicillin ( novomox - cipla ltd , india ) 500 mg three times daily for 5 days , non - steroidal anti - inflammatory agent ; three times daily for 5 days and 0.2% chlorhexidine gluconate ( hexidine - icpa health products ltd , mumbai , india ) mouth rinse for 3 to 4 weeks . all patients were recalled at a 1-month interval initially to monitor wound healing and to reinforce oral hygiene instructions . later , they were followed up at 3 , 6 , and at 9 months to assess clinical and radiographic parameters postoperatively [ figure 5 ] . postoperative follow up at 9 months in relation to the test site the statistical software spss 15.0 , stata 8.0 , medcalc 9.0.1 and systat 11.0 were used for the analysis of the data . . student t test ( two - tailed , dependent ) was used to find the significance of study parameters on a continuous scale within each group . wilcoxon signed - rank test was used to find the significance of percentage change of df . plaque index , gingival bleeding index , ral , ppd , and r were recorded from baseline , 3 , 6 , and at 9 months using university of north carolina-15 ( unc ) probe . a customized acrylic occlusal stent with a vertical groove was made for proper guidance and orientation of the periodontal probe in the same plane [ figure 1 ] . the stents were preserved in the study casts to minimize distortion for follow - up measurements . ral was measured from the reference point ( rp ) on the stent to the base of the pocket ( bop ) . ppd was recorded by noting the difference between measurements from rp to fgm and rp to the bop . baseline clinical parameters were assessed by using unc-15 probe on a custom - made acrylic stent in relation to 46 intra - oral periapical radiographs were taken using a long cone / extension cone paralleling technique with the positioning device ( rinn xcp , dentsply , il , usa . ) and a size 2e speed intra - oral periapical radiographic film ( kodak x - ray film , usa ) was used in a roentgen machine operating at 70 kilo voltage power ( kvp ) , 0.6 milli amperes ( ma ) . bite registration was done using rubber base impression material and stored for follow - up assessment . radiographs were scanned using a scanner ( hp 3010 g ) at an input of 400 dpi and 100% scale . radiographic parameters were recorded as follows : ( 1 ) distance from cementoenamel junction to base of the defect ( cej to bd ) = a , ( 2 ) distance from cementoenamel junction to alveolar crest ( cej to ac ) = b , ( 3 ) defect depth = a - b , ( 4 ) df was measured as the difference in defect depth from baseline to 6 months and 9 months . intracrevicular incisions were given both buccally and lingually and full thickness mucoperiosteal flap was elevated . after the reflection of the flap and exposure of the osseous defect , a thorough surgical debridement was carried out to remove sub - gingival plaque , calculus , diseased granulation tissue , and pocket epithelium [ figure 2 ] . the surgical sites were irrigated with sterile saline , and care was taken to keep the area free of saliva . the gtr membrane was removed from the sterile package and hydrated in normal saline for few seconds before placement on to the defect to improve adhesion properties and malleability . the membrane design was custom prepared chair side to receive for an intrabony defect and then carefully tweezed through the interproximal contact area [ figure 3 ] and extended 2 to 3 mm beyond the bony margin , to provide a broad base during placement . prior to closure of mucoperiosteal flaps , decortication of the osseous defect was done to induce bleeding . surgical flaps were repositioned to the pre - surgical level and sutured with 3 - 0 silk suture ( mersilk -ethicon , division of johnson and johnson ltd , aurangabad , india ) utilizing an interrupted direct loop suturing technique thereby achieving primary closure [ figure 4 ] . care was taken not to displace the gtr membrane during suturing . a non - eugenol periodontal dressing ( coe - pack - gc america inc . alsip , il , usa ) was placed on the surgical area following which the post - operative instructions were given . following incision , a full thickness mucoperiosteal flap was reflected and the defect debridement was done the collagen barrier membrane was trimmed to the required size and shape and placed over the interproximal vertical defect suturing was done using 3 - 0 silk with a simple interrupted direct loop technique post - operative medication included amoxicillin ( novomox - cipla ltd , india ) 500 mg three times daily for 5 days , non - steroidal anti - inflammatory agent ; three times daily for 5 days and 0.2% chlorhexidine gluconate ( hexidine - icpa health products ltd , mumbai , india ) mouth rinse for 3 to 4 weeks . all patients were recalled at a 1-month interval initially to monitor wound healing and to reinforce oral hygiene instructions . later , they were followed up at 3 , 6 , and at 9 months to assess clinical and radiographic parameters postoperatively [ figure 5 ] . the statistical software spss 15.0 , stata 8.0 , medcalc 9.0.1 and systat 11.0 were used for the analysis of the data . student t test ( two - tailed , dependent ) was used to find the significance of study parameters on a continuous scale within each group . wilcoxon signed - rank test was used to find the significance of percentage change of df . 1 patient failed to return for the 9 months recall , and 9 patients completed the study . over the course of the study , there was no membrane exposure or dehiscence or infectious episodes , or any other adverse complications in sites treated with gtr . the mean plaque index scores in test sites at baseline were 0.79 0.37 , which reduced to 0.49 0.31 at 3 months , and further reduced to 0.35 0.24 and 0.32 0.11 at 6 and 9 months respectively . this difference was found to be highly statistically significant with p = 0.008 , p < 0.001 , the mean plaque index scores in control sites at baseline were 0.91 0.35 , which reduced to 0.54 0.36 at 3 months and further reduced to 0.29 0.23 and 0.32 0.11 at 6 and 9 months , respectively . this difference was found to be highly statistically significant with p = 0.011 , p = 0.001 , p < 0.001 at 3 , 6 , and 9 months , respectively . the mean gingival bleeding index scores in test sites at baseline were 0.72 0.22 , reduced to 0.28 0.17 at 3 months and further to 0.18 0.11 and 0.22 0.44 at 6 and 9 months , respectively . this difference was found to be highly statistically significant with p = 0.003 , p = 0.001 , p = 0.001 at 3 , 6 , and 9 months , respectively . similarly , in control sites the mean gingival bleeding index scores at baseline were 0.69 0.22 , reduced to 0.30 0.26 at 3 months and further reduced to 0.18 0.14 at 6 months . this difference was found to be highly statistically significant with p = 0.016 , p = 0.001 , p = 0.002 at 3 , 6 , and 9 months , respectively . the mean ral scores in test sites at baseline were 12.33 2.83 mm , reduced to 9.67 3.00 mm at 3 months and further reduced to 9.00 2.00 mm at 6 months . this difference was found to be highly statistically significant with p = 0.011 , p = 0.004 , p = 0.006 at 3 , 6 , and 9 months , respectively . the mean ral scores in control sites at baseline were 12.44 3.00 mm , reduced to 11.00 3.08 mm at 3 months and further reduced to 10.22 2.91 mm at 6 months . this difference was found to be statistically significant with p = 0.089 , p = 0.017 , p = 0.071 at 3 , 6 , and 9 months , respectively . the mean ppd scores in test sites at baseline were 7.22 2.28 mm , reduced to 4.33 3.16 mm at 3 months . at 6 months this difference was found to be highly statistically significant with p = 0.007 , p = 0.001 , p < 0.001 at 3 , 6 , and 9 months , respectively . the mean ppd in the control site at baseline was 7.00 2.55 mm , reduced to 4.33 2.12 mm and 4.11 2.15 mm at 3 and 6 months , respectively . there was a slight increase in ppd to 4.33 1.73 mm at 9 months . this difference was found to be highly statistically significant with p = 0.018 , p = 0.020 , p = 0.008 at 3 , 6 , and 9 months , respectively . the mean distance from the lower border of the stent to the gingival margin was 7.22 4.24 mm at baseline in test sites . at the end of 3 months , it increased to 7.33 4.44 mm and then at 6 and 9 months it reduced to 7.11 3.44 mm and 6.89 3.29 mm , respectively . the mean distance from the lower border of the stent to the gingival margin was 5.44 1.94 mm at baseline in control sites . at the end of 3 months , it increased to 6.89 2.52 mm and then at 6 and 9 months ; it reduced to 6.55 2.35 mm , and 6.56 2.55 mm , respectively . the comparison from baseline to 3 months , 6 months , and 9 months with respect to clinical parameters showed significant statistical difference within the test and control groups ( p < 0.05 ) , but the inter group comparison did not show significant statistical differences [ table 1 ] . clinical parameters assessed ( meanstandard deviation ) at baseline , 3 months , 6 months , and 9 months for test and control sites the percent of df in test sites at 6 months was 51.85 median % and 46.66 median % at 9 months . at control sites , the percent of df at 6 months was 46.03 median % and 78.46 median % at 9 months . radiographical evaluation with respect to abl and percentage of df from baseline to 6 months and at 9 months did not show significant statistical differences both within and between the groups ( p > 0.05 ) [ table 2 ] . radiographic parameters assessed ( meanstandard deviation ) at baseline , 6 months , and 9 months for test and control sites the regeneration of the periodontium is the result of elective cellular events that are facilitated by tissue exclusion using bioabsorbable or non - resorbable barriers . the results of our study demonstrated that no significant differences were found either by using a bioabsorbable membrane or ofd in the treatment of intrabony defects both clinically and radiographically . this study was designed as a spilt - mouth investigation to facilitate the comparison of both gtr and ofd under similar healing conditions by eliminating patient - specific characteristics , which might impact on the results of the conventional and regenerative surgeries . the clinical methods used to evaluate therapeutic end points include various assessments of gingival inflammation , periodontal probing , radiographs , and re - entry procedures . the true endpoint is determined by histology . however , due to ethical reasons and patient concerns , re - entry procedures and histological analysis are not feasible in clinical trials . the plaque and gingival bleeding indices were assessed to monitor patient 's oral hygiene and its effect on soft tissues . there was a reduction in mean plaque index and gingival bleeding index for the test and control group from baseline to 9 months with a high statistical significance ( p < 0.05 ) . there was no statistically significant difference between test and control groups with respect to plaque and gingival scores thereby showing that there was a good maintenance of oral hygiene throughout the study . cortellini and pini - prato et al . have reported the clinical effect of plaque control and the influence of increased bacterial contamination on the outcomes to gtr . the clinical parameters on the sites treated with the barrier were similar to those of open flap surgery . ppd in the test group at baseline was 7.22 2.28 mm which reduced to 4.00 1.94 mm at the end of 9 months . in the control group , the mean ppd reduced from 7.00 2.55 to 4.33 1.73 mm at 9 months . the clinical attachment level or ral has become widely accepted as the primary clinical endpoint of regenerative attempts around natural teeth . significant loss in clinical attachment levels is reflected in histological loss of the tooth 's attachment apparatus . the mean ral in the test group reduced from 12.33 2.83 to 9.78 2.28 mm at 9 months . at the control sites , the mean ral reduced from 12.44 3.00 to 10.89 2.47 mm at the end of 9 months . although these clinical changes were statistically significant between baseline and final measurements within the groups , no statistically significant differences were found between the test and control groups [ table 1 ] . cortellini and falk et al . have reported greater gain in the clinical attachment level after gtr ; however , these authors were dealing with deeper initial intrabony defects . this may suggest that clinical attachment level after gtr is dependent up on the initial defect depth . during the surgical procedures , efforts were made to preserve the interproximal soft tissues . in relation to gingival recession there was a decrease of 0.3 mm of recession from baseline to 9 months in gtr - treated sites . the possible explanation could be due to non - exposure of the barrier membrane following placement in any of the test sites . this would have prevented undue exposure of the barrier to oral environment , thereby preventing infection or soft - tissue inflammation , which would lead to faster resorption of membrane and cause recession . in contrast , studies by cortellini and becker et al . in 1996 have shown that sites treated with gtr have increased amount of recession between 1.8 and 2 mm . has postulated that the greater the degree of gingival recession , shorter the root surface area that is provided for the repopulation of the periodontal ligament cells thereby negatively influences new attachment formation . new bone formation is frequently used as a primary outcome variable in controlled clinical trials of regenerative therapy . radiographic monitoring of alveolar bone changes following regenerative procedures is a non - invasive painless alternative to direct bone measurement . most of the alveolar bone changes following regenerative therapy of intrabony defects occur in the intrabony component while crestal resorption may be minimal or may not occur at all . in our study , no significant change in the level of the alveolar crest was seen in both test and control groups after 6 and 9 months . where in 0.33 mm of crestal resorption was noted in gtr - treated sites . there was a significant difference in defect depth from baseline to 9 months in test and control groups ( p < 0.05 ) while an inter group comparison did not show any significant differences in defect depth . similarly , no significant difference was found in percentage of df both within and between the groups [ table 2 and figures 611 ] . this observation is in accordance to the findings by gottlow et al . in 1986 , becker et al . in 1993 , and micheal et al . in 2000 . in our study , conventional radiography with image analysis software ( auto cad 2007 ) was used to assess radiographic parameters as applicability and reliability of the image analysis system in alveolar bone measurement have been studied previously by micheal , hausmann et al . , and verdonschot et al . however , advanced radiographic techniques like subtraction digital radiography remain the method of choice if subtle changes in mineralization of alveolar bone need to be detected . radiograph of test site at baseline in relation to 46 and 47 radiograph of control site at baseline in relation to 36 and 37 radiograph of test site at 6 months in relation to 46 and 47 radiograph of control site at 6 months in relation to 36 and 37 radiograph of test site at 9 months in relation to 46 and 47 radiograph of control site at 9 months in relation to 36 and 37 the gtr material utilized in this study is an orange - brown color type - i fish collagen . it is available in dimensions of 1 1 cm and 1 2 cm and can be easily manipulated and adapted to the root surface . the largest concentration of collagen is found in the skeleton , fins , skin , and air bladder . use of this barrier in our study did not show any case reporting with any form of allergy or hypersensitivity reaction . the results of the study is difficult to compare due to a number of possible differences like in small sample size , study design , patient / defect selection , percentage of one / two / three wall defect components , baseline depth of defects , probing forces , and evaluation method . hence , the regenerative potential and beneficial effects of this gtr membrane should be further evaluated with larger sample size , longer follow ups , use of advanced radiographic aids ( like cone - beam - computed tomography , etc . ) and with combination therapy involving bone grafts . within the limits of the study , it can be inferred that no significant differences were found either by using a collagen membrane of fish origin or open flap debridement in the treatment of periodontal intrabony defects both clinically and radiographically .

background : recently , there has been interest in non - mammalian collagen sources such as fish collagen in periodontal regeneration . in the present study , collagen barrier membrane of fish origin was assessed in the treatment of periodontal intrabony defects.materials and methods : ten systemically healthy chronic periodontitis patients having a paired osseous defect in the mandibular posterior teeth were selected and randomly assigned to receive a collagen membrane ( test ) or open flap debridement ( control ) in a split mouth design . clinical parameters such as plaque index , gingival bleeding index , probing pocket depth , relative attachment level , and recession were recorded at baseline , 3 , 6 , and at 9 months , while radiographic evaluation was done to assess alveolar crestal bone level and percentage of defect fill at 6 and 9 months using autocad 2007 software . student 's t test ( two - tailed , dependent ) was used to find the significance of study parameters on continuous scale . significance was set at 5% level of significance . wilcoxon signed rank test was used to find the significance of percentage change of defect fill.results:the comparison between the two groups did not show any statistically significant differences in the parameters assessed ( p > 0.05 ) but , within each group , clinical parameters showed statistically significant differences from baseline to 9 months ( p < 0.05).conclusion : within the limits of the study , it can be inferred that no significant differences were found either by using collagen membrane of fish origin or open flap debridement in the treatment of periodontal intrabony defects .

INTRODUCTION MATERIALS AND METHODS Clinical parameters assessment Radiographic parameters assessment Surgical procedure Statistical analysis RESULTS DISCUSSION CONCLUSION

periodontal therapy involves controlling of periodontal infection and aims at regeneration of the lost periodontium . the two techniques with the most successful documentation of periodontal regeneration are osseous grafting and guided - tissue regeneration ( gtr ) . the regeneration of the periodontium with new connective fiber insertion , new cementum , and new bone formation constitutes ideal healing . the barriers recommended for the use in gtr , regardless of the material used , must be safe , biocompatible , and non - toxic , not induce any inflammatory response , and be designed for clinical applicability based on the morphology of the osseous defects . furthermore , gingival recession , device exposure , infection , and inflammation are frequently experienced with nonresorbable membranes . collagen from mammalian sources , primarily bovine skin , has been utilized in foods , cosmetics , and biomaterials and has the advantage of biodegradability and low toxicity . therefore , there have been various attempts to use mammalian collagen in the medical field as a scaffold for developing artificial organs . recently , there has been interest in non - mammalian collagen sources , primarily in fish collagen such as that of shark and salmon , as it is known to have low risk for transmission of infectious disease to humans than bovine collagen . hence , an attempt was made to assess , a bioresorbable collagen membrane of fish origin ( periocol , eucare pharmaceuticals private limited , chennai , india ) in the treatment of periodontal intrabony defects . to assess the clinical parameters like probing pocket depth ( ppd ) , relative attachment level ( ral ) and recession ( r ) with and without a bioresorbable collagen barrier membrane in the treatment of intrabony defectsto assess the alveolar crestal bone level ( abl ) and percentage of defect fill ( df ) radiographically in intrabony defects with and without a bioresorbable collagen barrier membrane . to assess the clinical parameters like probing pocket depth ( ppd ) , relative attachment level ( ral ) and recession ( r ) with and without a bioresorbable collagen barrier membrane in the treatment of intrabony defects to assess the alveolar crestal bone level ( abl ) and percentage of defect fill ( df ) radiographically in intrabony defects with and without a bioresorbable collagen barrier membrane . the study employed a total of 10 patients ( 6 males and 4 females ) aged between 21 and 50 years in a split mouth manner to assess the collagen membrane both clinically and radiographically . subjects having bilateral isolated intra osseous defects in the mandibular posterior teeth were screened based on the following inclusion and exclusion criteria . inclusion criteria : ( a ) patients of either sex having chronic periodontitis , ( b ) patients who were systemically healthy with no contraindication to periodontal surgery , ( c ) non smokers , ( d ) presence of an intrabony defect with residual ppd of five millimeters ( mm ) and with radiographic evidence of the affected site , ( e ) patients having 2 to 3 mm band of keratinized tissue to allow surgical manipulation and suturing , and ( f ) patients who were co - operative and able to come for regular follow up . exclusion criteria : ( a ) patients allergic or sensitive to any medication or any ingredient of the test material , ( b ) patients showing unacceptable oral hygiene compliance during or after phase i therapy , and ( c ) pregnant and lactating mothers . following selection of subjects based on inclusion criteria , patients were given an explanation of the study , and an informed consent was obtained . the study was analyzed and approved by the institutional review board and the ethical clearance committee of the institution . randomization was done by a coin toss to select the sites to be treated by gtr using a bioresorbable membrane ( i.e. , test group ) and control sites treated by conventional open flap debridement ( ofd ) . all the surgeries were performed by the same operator , and the measurements were recorded by a blinded examiner . plaque index , gingival bleeding index , ral , ppd , and r were recorded from baseline , 3 , 6 , and at 9 months using university of north carolina-15 ( unc ) probe . a customized acrylic occlusal stent with a vertical groove was made for proper guidance and orientation of the periodontal probe in the same plane [ figure 1 ] . the stents were preserved in the study casts to minimize distortion for follow - up measurements . baseline clinical parameters were assessed by using unc-15 probe on a custom - made acrylic stent in relation to 46 radiographs were taken at baseline , 6 , and 9 months postoperatively . and a size 2e speed intra - oral periapical radiographic film ( kodak x - ray film , usa ) was used in a roentgen machine operating at 70 kilo voltage power ( kvp ) , 0.6 milli amperes ( ma ) . radiographic parameters were recorded as follows : ( 1 ) distance from cementoenamel junction to base of the defect ( cej to bd ) = a , ( 2 ) distance from cementoenamel junction to alveolar crest ( cej to ac ) = b , ( 3 ) defect depth = a - b , ( 4 ) df was measured as the difference in defect depth from baseline to 6 months and 9 months . after the reflection of the flap and exposure of the osseous defect , a thorough surgical debridement was carried out to remove sub - gingival plaque , calculus , diseased granulation tissue , and pocket epithelium [ figure 2 ] . the surgical sites were irrigated with sterile saline , and care was taken to keep the area free of saliva . prior to closure of mucoperiosteal flaps , decortication of the osseous defect was done to induce bleeding . surgical flaps were repositioned to the pre - surgical level and sutured with 3 - 0 silk suture ( mersilk -ethicon , division of johnson and johnson ltd , aurangabad , india ) utilizing an interrupted direct loop suturing technique thereby achieving primary closure [ figure 4 ] . following incision , a full thickness mucoperiosteal flap was reflected and the defect debridement was done the collagen barrier membrane was trimmed to the required size and shape and placed over the interproximal vertical defect suturing was done using 3 - 0 silk with a simple interrupted direct loop technique post - operative medication included amoxicillin ( novomox - cipla ltd , india ) 500 mg three times daily for 5 days , non - steroidal anti - inflammatory agent ; three times daily for 5 days and 0.2% chlorhexidine gluconate ( hexidine - icpa health products ltd , mumbai , india ) mouth rinse for 3 to 4 weeks . later , they were followed up at 3 , 6 , and at 9 months to assess clinical and radiographic parameters postoperatively [ figure 5 ] . postoperative follow up at 9 months in relation to the test site the statistical software spss 15.0 , stata 8.0 , medcalc 9.0.1 and systat 11.0 were used for the analysis of the data . student t test ( two - tailed , dependent ) was used to find the significance of study parameters on a continuous scale within each group . wilcoxon signed - rank test was used to find the significance of percentage change of df . plaque index , gingival bleeding index , ral , ppd , and r were recorded from baseline , 3 , 6 , and at 9 months using university of north carolina-15 ( unc ) probe . a customized acrylic occlusal stent with a vertical groove was made for proper guidance and orientation of the periodontal probe in the same plane [ figure 1 ] . the stents were preserved in the study casts to minimize distortion for follow - up measurements . baseline clinical parameters were assessed by using unc-15 probe on a custom - made acrylic stent in relation to 46 intra - oral periapical radiographs were taken using a long cone / extension cone paralleling technique with the positioning device ( rinn xcp , dentsply , il , usa . ) and a size 2e speed intra - oral periapical radiographic film ( kodak x - ray film , usa ) was used in a roentgen machine operating at 70 kilo voltage power ( kvp ) , 0.6 milli amperes ( ma ) . radiographic parameters were recorded as follows : ( 1 ) distance from cementoenamel junction to base of the defect ( cej to bd ) = a , ( 2 ) distance from cementoenamel junction to alveolar crest ( cej to ac ) = b , ( 3 ) defect depth = a - b , ( 4 ) df was measured as the difference in defect depth from baseline to 6 months and 9 months . after the reflection of the flap and exposure of the osseous defect , a thorough surgical debridement was carried out to remove sub - gingival plaque , calculus , diseased granulation tissue , and pocket epithelium [ figure 2 ] . prior to closure of mucoperiosteal flaps , decortication of the osseous defect was done to induce bleeding . following incision , a full thickness mucoperiosteal flap was reflected and the defect debridement was done the collagen barrier membrane was trimmed to the required size and shape and placed over the interproximal vertical defect suturing was done using 3 - 0 silk with a simple interrupted direct loop technique post - operative medication included amoxicillin ( novomox - cipla ltd , india ) 500 mg three times daily for 5 days , non - steroidal anti - inflammatory agent ; three times daily for 5 days and 0.2% chlorhexidine gluconate ( hexidine - icpa health products ltd , mumbai , india ) mouth rinse for 3 to 4 weeks . later , they were followed up at 3 , 6 , and at 9 months to assess clinical and radiographic parameters postoperatively [ figure 5 ] . student t test ( two - tailed , dependent ) was used to find the significance of study parameters on a continuous scale within each group . wilcoxon signed - rank test was used to find the significance of percentage change of df . 1 patient failed to return for the 9 months recall , and 9 patients completed the study . over the course of the study , there was no membrane exposure or dehiscence or infectious episodes , or any other adverse complications in sites treated with gtr . the mean plaque index scores in test sites at baseline were 0.79 0.37 , which reduced to 0.49 0.31 at 3 months , and further reduced to 0.35 0.24 and 0.32 0.11 at 6 and 9 months respectively . this difference was found to be highly statistically significant with p = 0.008 , p < 0.001 , the mean plaque index scores in control sites at baseline were 0.91 0.35 , which reduced to 0.54 0.36 at 3 months and further reduced to 0.29 0.23 and 0.32 0.11 at 6 and 9 months , respectively . this difference was found to be highly statistically significant with p = 0.011 , p = 0.001 , p < 0.001 at 3 , 6 , and 9 months , respectively . the mean gingival bleeding index scores in test sites at baseline were 0.72 0.22 , reduced to 0.28 0.17 at 3 months and further to 0.18 0.11 and 0.22 0.44 at 6 and 9 months , respectively . this difference was found to be highly statistically significant with p = 0.003 , p = 0.001 , p = 0.001 at 3 , 6 , and 9 months , respectively . similarly , in control sites the mean gingival bleeding index scores at baseline were 0.69 0.22 , reduced to 0.30 0.26 at 3 months and further reduced to 0.18 0.14 at 6 months . this difference was found to be highly statistically significant with p = 0.016 , p = 0.001 , p = 0.002 at 3 , 6 , and 9 months , respectively . the mean ral scores in test sites at baseline were 12.33 2.83 mm , reduced to 9.67 3.00 mm at 3 months and further reduced to 9.00 2.00 mm at 6 months . this difference was found to be highly statistically significant with p = 0.011 , p = 0.004 , p = 0.006 at 3 , 6 , and 9 months , respectively . the mean ral scores in control sites at baseline were 12.44 3.00 mm , reduced to 11.00 3.08 mm at 3 months and further reduced to 10.22 2.91 mm at 6 months . this difference was found to be statistically significant with p = 0.089 , p = 0.017 , p = 0.071 at 3 , 6 , and 9 months , respectively . at 6 months this difference was found to be highly statistically significant with p = 0.007 , p = 0.001 , p < 0.001 at 3 , 6 , and 9 months , respectively . the mean ppd in the control site at baseline was 7.00 2.55 mm , reduced to 4.33 2.12 mm and 4.11 2.15 mm at 3 and 6 months , respectively . there was a slight increase in ppd to 4.33 1.73 mm at 9 months . this difference was found to be highly statistically significant with p = 0.018 , p = 0.020 , p = 0.008 at 3 , 6 , and 9 months , respectively . the mean distance from the lower border of the stent to the gingival margin was 7.22 4.24 mm at baseline in test sites . at the end of 3 months , it increased to 7.33 4.44 mm and then at 6 and 9 months it reduced to 7.11 3.44 mm and 6.89 3.29 mm , respectively . the mean distance from the lower border of the stent to the gingival margin was 5.44 1.94 mm at baseline in control sites . at the end of 3 months , it increased to 6.89 2.52 mm and then at 6 and 9 months ; it reduced to 6.55 2.35 mm , and 6.56 2.55 mm , respectively . the comparison from baseline to 3 months , 6 months , and 9 months with respect to clinical parameters showed significant statistical difference within the test and control groups ( p < 0.05 ) , but the inter group comparison did not show significant statistical differences [ table 1 ] . clinical parameters assessed ( meanstandard deviation ) at baseline , 3 months , 6 months , and 9 months for test and control sites the percent of df in test sites at 6 months was 51.85 median % and 46.66 median % at 9 months . at control sites , the percent of df at 6 months was 46.03 median % and 78.46 median % at 9 months . radiographical evaluation with respect to abl and percentage of df from baseline to 6 months and at 9 months did not show significant statistical differences both within and between the groups ( p > 0.05 ) [ table 2 ] . radiographic parameters assessed ( meanstandard deviation ) at baseline , 6 months , and 9 months for test and control sites the regeneration of the periodontium is the result of elective cellular events that are facilitated by tissue exclusion using bioabsorbable or non - resorbable barriers . the results of our study demonstrated that no significant differences were found either by using a bioabsorbable membrane or ofd in the treatment of intrabony defects both clinically and radiographically . the clinical methods used to evaluate therapeutic end points include various assessments of gingival inflammation , periodontal probing , radiographs , and re - entry procedures . there was a reduction in mean plaque index and gingival bleeding index for the test and control group from baseline to 9 months with a high statistical significance ( p < 0.05 ) . there was no statistically significant difference between test and control groups with respect to plaque and gingival scores thereby showing that there was a good maintenance of oral hygiene throughout the study . the clinical parameters on the sites treated with the barrier were similar to those of open flap surgery . ppd in the test group at baseline was 7.22 2.28 mm which reduced to 4.00 1.94 mm at the end of 9 months . in the control group , the mean ppd reduced from 7.00 2.55 to 4.33 1.73 mm at 9 months . the mean ral in the test group reduced from 12.33 2.83 to 9.78 2.28 mm at 9 months . although these clinical changes were statistically significant between baseline and final measurements within the groups , no statistically significant differences were found between the test and control groups [ table 1 ] . have reported greater gain in the clinical attachment level after gtr ; however , these authors were dealing with deeper initial intrabony defects . in relation to gingival recession there was a decrease of 0.3 mm of recession from baseline to 9 months in gtr - treated sites . the possible explanation could be due to non - exposure of the barrier membrane following placement in any of the test sites . radiographic monitoring of alveolar bone changes following regenerative procedures is a non - invasive painless alternative to direct bone measurement . most of the alveolar bone changes following regenerative therapy of intrabony defects occur in the intrabony component while crestal resorption may be minimal or may not occur at all . in our study , no significant change in the level of the alveolar crest was seen in both test and control groups after 6 and 9 months . there was a significant difference in defect depth from baseline to 9 months in test and control groups ( p < 0.05 ) while an inter group comparison did not show any significant differences in defect depth . similarly , no significant difference was found in percentage of df both within and between the groups [ table 2 and figures 611 ] . in our study , conventional radiography with image analysis software ( auto cad 2007 ) was used to assess radiographic parameters as applicability and reliability of the image analysis system in alveolar bone measurement have been studied previously by micheal , hausmann et al . radiograph of test site at baseline in relation to 46 and 47 radiograph of control site at baseline in relation to 36 and 37 radiograph of test site at 6 months in relation to 46 and 47 radiograph of control site at 6 months in relation to 36 and 37 radiograph of test site at 9 months in relation to 46 and 47 radiograph of control site at 9 months in relation to 36 and 37 the gtr material utilized in this study is an orange - brown color type - i fish collagen . the largest concentration of collagen is found in the skeleton , fins , skin , and air bladder . use of this barrier in our study did not show any case reporting with any form of allergy or hypersensitivity reaction . the results of the study is difficult to compare due to a number of possible differences like in small sample size , study design , patient / defect selection , percentage of one / two / three wall defect components , baseline depth of defects , probing forces , and evaluation method . within the limits of the study , it can be inferred that no significant differences were found either by using a collagen membrane of fish origin or open flap debridement in the treatment of periodontal intrabony defects both clinically and radiographically .

periodontal therapy involves controlling of periodontal infection and aims at regeneration of the lost periodontium . the two techniques with the most successful documentation of periodontal regeneration are osseous grafting and guided - tissue regeneration ( gtr ) . the regeneration of the periodontium with new connective fiber insertion , new cementum , and new bone formation constitutes ideal healing . the regeneration process can only be initiated by periodontal ligament cells derived from the remaining periodontium as these are the cells capable of differentiating into new fibroblasts , cementoblasts , and osteoblasts . suggested the placement of a physical barrier between the flap and the root surface to exclude gingival connective tissue and epithelium from the healing process , giving the periodontal ligament cells the opportunity to repopulate the coagulum on the root surface . the barriers recommended for the use in gtr , regardless of the material used , must be safe , biocompatible , and non - toxic , not induce any inflammatory response , and be designed for clinical applicability based on the morphology of the osseous defects . furthermore , gingival recession , device exposure , infection , and inflammation are frequently experienced with nonresorbable membranes . among resorbable barriers , investigators have examined type - i collagen for the use in gtr procedures as it is a major extracellular macromolecule of the periodontal connective tissue . also collagen is known to be a weak immunogen , has a chemotactic property for fibroblasts , and acts as a barrier for migrating epithelial cells in vitro . collagen from mammalian sources , primarily bovine skin , has been utilized in foods , cosmetics , and biomaterials and has the advantage of biodegradability and low toxicity . therefore , there have been various attempts to use mammalian collagen in the medical field as a scaffold for developing artificial organs . however , the use of bovine collagen has been reconsidered , as some reports have shown the risk of transmission of bovine spongiform encephalopathy ( bse ) to human beings . recently , there has been interest in non - mammalian collagen sources , primarily in fish collagen such as that of shark and salmon , as it is known to have low risk for transmission of infectious disease to humans than bovine collagen . hence , an attempt was made to assess , a bioresorbable collagen membrane of fish origin ( periocol , eucare pharmaceuticals private limited , chennai , india ) in the treatment of periodontal intrabony defects . to assess the clinical parameters like probing pocket depth ( ppd ) , relative attachment level ( ral ) and recession ( r ) with and without a bioresorbable collagen barrier membrane in the treatment of intrabony defectsto assess the alveolar crestal bone level ( abl ) and percentage of defect fill ( df ) radiographically in intrabony defects with and without a bioresorbable collagen barrier membrane . to assess the clinical parameters like probing pocket depth ( ppd ) , relative attachment level ( ral ) and recession ( r ) with and without a bioresorbable collagen barrier membrane in the treatment of intrabony defects to assess the alveolar crestal bone level ( abl ) and percentage of defect fill ( df ) radiographically in intrabony defects with and without a bioresorbable collagen barrier membrane . the study employed a total of 10 patients ( 6 males and 4 females ) aged between 21 and 50 years in a split mouth manner to assess the collagen membrane both clinically and radiographically . subjects having bilateral isolated intra osseous defects in the mandibular posterior teeth were screened based on the following inclusion and exclusion criteria . inclusion criteria : ( a ) patients of either sex having chronic periodontitis , ( b ) patients who were systemically healthy with no contraindication to periodontal surgery , ( c ) non smokers , ( d ) presence of an intrabony defect with residual ppd of five millimeters ( mm ) and with radiographic evidence of the affected site , ( e ) patients having 2 to 3 mm band of keratinized tissue to allow surgical manipulation and suturing , and ( f ) patients who were co - operative and able to come for regular follow up . exclusion criteria : ( a ) patients allergic or sensitive to any medication or any ingredient of the test material , ( b ) patients showing unacceptable oral hygiene compliance during or after phase i therapy , and ( c ) pregnant and lactating mothers . plaque index , gingival bleeding index , ral , ppd , and r were recorded from baseline , 3 , 6 , and at 9 months using university of north carolina-15 ( unc ) probe . a customized acrylic occlusal stent with a vertical groove was made for proper guidance and orientation of the periodontal probe in the same plane [ figure 1 ] . ral was measured from the reference point ( rp ) on the stent to the base of the pocket ( bop ) . ppd was recorded by noting the difference between measurements from rp to fgm and rp to the bop . baseline clinical parameters were assessed by using unc-15 probe on a custom - made acrylic stent in relation to 46 radiographs were taken at baseline , 6 , and 9 months postoperatively . intra - oral periapical radiographs were taken using a long cone / extension cone paralleling technique with the positioning device ( rinn xcp , dentsply , il , usa . ) and a size 2e speed intra - oral periapical radiographic film ( kodak x - ray film , usa ) was used in a roentgen machine operating at 70 kilo voltage power ( kvp ) , 0.6 milli amperes ( ma ) . radiographic parameters were recorded as follows : ( 1 ) distance from cementoenamel junction to base of the defect ( cej to bd ) = a , ( 2 ) distance from cementoenamel junction to alveolar crest ( cej to ac ) = b , ( 3 ) defect depth = a - b , ( 4 ) df was measured as the difference in defect depth from baseline to 6 months and 9 months . after the reflection of the flap and exposure of the osseous defect , a thorough surgical debridement was carried out to remove sub - gingival plaque , calculus , diseased granulation tissue , and pocket epithelium [ figure 2 ] . the gtr membrane was removed from the sterile package and hydrated in normal saline for few seconds before placement on to the defect to improve adhesion properties and malleability . surgical flaps were repositioned to the pre - surgical level and sutured with 3 - 0 silk suture ( mersilk -ethicon , division of johnson and johnson ltd , aurangabad , india ) utilizing an interrupted direct loop suturing technique thereby achieving primary closure [ figure 4 ] . following incision , a full thickness mucoperiosteal flap was reflected and the defect debridement was done the collagen barrier membrane was trimmed to the required size and shape and placed over the interproximal vertical defect suturing was done using 3 - 0 silk with a simple interrupted direct loop technique post - operative medication included amoxicillin ( novomox - cipla ltd , india ) 500 mg three times daily for 5 days , non - steroidal anti - inflammatory agent ; three times daily for 5 days and 0.2% chlorhexidine gluconate ( hexidine - icpa health products ltd , mumbai , india ) mouth rinse for 3 to 4 weeks . later , they were followed up at 3 , 6 , and at 9 months to assess clinical and radiographic parameters postoperatively [ figure 5 ] . postoperative follow up at 9 months in relation to the test site the statistical software spss 15.0 , stata 8.0 , medcalc 9.0.1 and systat 11.0 were used for the analysis of the data . plaque index , gingival bleeding index , ral , ppd , and r were recorded from baseline , 3 , 6 , and at 9 months using university of north carolina-15 ( unc ) probe . a customized acrylic occlusal stent with a vertical groove was made for proper guidance and orientation of the periodontal probe in the same plane [ figure 1 ] . ral was measured from the reference point ( rp ) on the stent to the base of the pocket ( bop ) . baseline clinical parameters were assessed by using unc-15 probe on a custom - made acrylic stent in relation to 46 intra - oral periapical radiographs were taken using a long cone / extension cone paralleling technique with the positioning device ( rinn xcp , dentsply , il , usa . ) and a size 2e speed intra - oral periapical radiographic film ( kodak x - ray film , usa ) was used in a roentgen machine operating at 70 kilo voltage power ( kvp ) , 0.6 milli amperes ( ma ) . radiographic parameters were recorded as follows : ( 1 ) distance from cementoenamel junction to base of the defect ( cej to bd ) = a , ( 2 ) distance from cementoenamel junction to alveolar crest ( cej to ac ) = b , ( 3 ) defect depth = a - b , ( 4 ) df was measured as the difference in defect depth from baseline to 6 months and 9 months . after the reflection of the flap and exposure of the osseous defect , a thorough surgical debridement was carried out to remove sub - gingival plaque , calculus , diseased granulation tissue , and pocket epithelium [ figure 2 ] . the gtr membrane was removed from the sterile package and hydrated in normal saline for few seconds before placement on to the defect to improve adhesion properties and malleability . surgical flaps were repositioned to the pre - surgical level and sutured with 3 - 0 silk suture ( mersilk -ethicon , division of johnson and johnson ltd , aurangabad , india ) utilizing an interrupted direct loop suturing technique thereby achieving primary closure [ figure 4 ] . following incision , a full thickness mucoperiosteal flap was reflected and the defect debridement was done the collagen barrier membrane was trimmed to the required size and shape and placed over the interproximal vertical defect suturing was done using 3 - 0 silk with a simple interrupted direct loop technique post - operative medication included amoxicillin ( novomox - cipla ltd , india ) 500 mg three times daily for 5 days , non - steroidal anti - inflammatory agent ; three times daily for 5 days and 0.2% chlorhexidine gluconate ( hexidine - icpa health products ltd , mumbai , india ) mouth rinse for 3 to 4 weeks . over the course of the study , there was no membrane exposure or dehiscence or infectious episodes , or any other adverse complications in sites treated with gtr . the mean plaque index scores in test sites at baseline were 0.79 0.37 , which reduced to 0.49 0.31 at 3 months , and further reduced to 0.35 0.24 and 0.32 0.11 at 6 and 9 months respectively . this difference was found to be highly statistically significant with p = 0.008 , p < 0.001 , the mean plaque index scores in control sites at baseline were 0.91 0.35 , which reduced to 0.54 0.36 at 3 months and further reduced to 0.29 0.23 and 0.32 0.11 at 6 and 9 months , respectively . this difference was found to be highly statistically significant with p = 0.011 , p = 0.001 , p < 0.001 at 3 , 6 , and 9 months , respectively . the mean gingival bleeding index scores in test sites at baseline were 0.72 0.22 , reduced to 0.28 0.17 at 3 months and further to 0.18 0.11 and 0.22 0.44 at 6 and 9 months , respectively . this difference was found to be highly statistically significant with p = 0.003 , p = 0.001 , p = 0.001 at 3 , 6 , and 9 months , respectively . similarly , in control sites the mean gingival bleeding index scores at baseline were 0.69 0.22 , reduced to 0.30 0.26 at 3 months and further reduced to 0.18 0.14 at 6 months . this difference was found to be highly statistically significant with p = 0.016 , p = 0.001 , p = 0.002 at 3 , 6 , and 9 months , respectively . the mean ral scores in test sites at baseline were 12.33 2.83 mm , reduced to 9.67 3.00 mm at 3 months and further reduced to 9.00 2.00 mm at 6 months . this difference was found to be highly statistically significant with p = 0.011 , p = 0.004 , p = 0.006 at 3 , 6 , and 9 months , respectively . the mean ral scores in control sites at baseline were 12.44 3.00 mm , reduced to 11.00 3.08 mm at 3 months and further reduced to 10.22 2.91 mm at 6 months . this difference was found to be statistically significant with p = 0.089 , p = 0.017 , p = 0.071 at 3 , 6 , and 9 months , respectively . at 6 months this difference was found to be highly statistically significant with p = 0.007 , p = 0.001 , p < 0.001 at 3 , 6 , and 9 months , respectively . the mean ppd in the control site at baseline was 7.00 2.55 mm , reduced to 4.33 2.12 mm and 4.11 2.15 mm at 3 and 6 months , respectively . this difference was found to be highly statistically significant with p = 0.018 , p = 0.020 , p = 0.008 at 3 , 6 , and 9 months , respectively . at the end of 3 months , it increased to 6.89 2.52 mm and then at 6 and 9 months ; it reduced to 6.55 2.35 mm , and 6.56 2.55 mm , respectively . the comparison from baseline to 3 months , 6 months , and 9 months with respect to clinical parameters showed significant statistical difference within the test and control groups ( p < 0.05 ) , but the inter group comparison did not show significant statistical differences [ table 1 ] . clinical parameters assessed ( meanstandard deviation ) at baseline , 3 months , 6 months , and 9 months for test and control sites the percent of df in test sites at 6 months was 51.85 median % and 46.66 median % at 9 months . at control sites , the percent of df at 6 months was 46.03 median % and 78.46 median % at 9 months . radiographical evaluation with respect to abl and percentage of df from baseline to 6 months and at 9 months did not show significant statistical differences both within and between the groups ( p > 0.05 ) [ table 2 ] . radiographic parameters assessed ( meanstandard deviation ) at baseline , 6 months , and 9 months for test and control sites the regeneration of the periodontium is the result of elective cellular events that are facilitated by tissue exclusion using bioabsorbable or non - resorbable barriers . the results of our study demonstrated that no significant differences were found either by using a bioabsorbable membrane or ofd in the treatment of intrabony defects both clinically and radiographically . this study was designed as a spilt - mouth investigation to facilitate the comparison of both gtr and ofd under similar healing conditions by eliminating patient - specific characteristics , which might impact on the results of the conventional and regenerative surgeries . the clinical methods used to evaluate therapeutic end points include various assessments of gingival inflammation , periodontal probing , radiographs , and re - entry procedures . there was a reduction in mean plaque index and gingival bleeding index for the test and control group from baseline to 9 months with a high statistical significance ( p < 0.05 ) . there was no statistically significant difference between test and control groups with respect to plaque and gingival scores thereby showing that there was a good maintenance of oral hygiene throughout the study . have reported the clinical effect of plaque control and the influence of increased bacterial contamination on the outcomes to gtr . in the control group , the mean ppd reduced from 7.00 2.55 to 4.33 1.73 mm at 9 months . significant loss in clinical attachment levels is reflected in histological loss of the tooth 's attachment apparatus . although these clinical changes were statistically significant between baseline and final measurements within the groups , no statistically significant differences were found between the test and control groups [ table 1 ] . in relation to gingival recession there was a decrease of 0.3 mm of recession from baseline to 9 months in gtr - treated sites . has postulated that the greater the degree of gingival recession , shorter the root surface area that is provided for the repopulation of the periodontal ligament cells thereby negatively influences new attachment formation . most of the alveolar bone changes following regenerative therapy of intrabony defects occur in the intrabony component while crestal resorption may be minimal or may not occur at all . in our study , no significant change in the level of the alveolar crest was seen in both test and control groups after 6 and 9 months . there was a significant difference in defect depth from baseline to 9 months in test and control groups ( p < 0.05 ) while an inter group comparison did not show any significant differences in defect depth . similarly , no significant difference was found in percentage of df both within and between the groups [ table 2 and figures 611 ] . in our study , conventional radiography with image analysis software ( auto cad 2007 ) was used to assess radiographic parameters as applicability and reliability of the image analysis system in alveolar bone measurement have been studied previously by micheal , hausmann et al . radiograph of test site at baseline in relation to 46 and 47 radiograph of control site at baseline in relation to 36 and 37 radiograph of test site at 6 months in relation to 46 and 47 radiograph of control site at 6 months in relation to 36 and 37 radiograph of test site at 9 months in relation to 46 and 47 radiograph of control site at 9 months in relation to 36 and 37 the gtr material utilized in this study is an orange - brown color type - i fish collagen . the results of the study is difficult to compare due to a number of possible differences like in small sample size , study design , patient / defect selection , percentage of one / two / three wall defect components , baseline depth of defects , probing forces , and evaluation method . hence , the regenerative potential and beneficial effects of this gtr membrane should be further evaluated with larger sample size , longer follow ups , use of advanced radiographic aids ( like cone - beam - computed tomography , etc . ) within the limits of the study , it can be inferred that no significant differences were found either by using a collagen membrane of fish origin or open flap debridement in the treatment of periodontal intrabony defects both clinically and radiographically .

growth regulation is a critical developmental process whose dysfunction can lead to many diseases , including cancer ( conlon and raff , 1999 ) . the salvador ( sav)/warts ( wts)/hippo ( hpo ) ( swh ) network , identified in drosophila and conserved in mammals , plays a major role in limiting growth by inhibiting cell proliferation and promoting apoptosis ( harvey and tapon , 2007 ; reddy and irvine , 2008 ) . activation of the upstream kinase hpo allows it to phosphorylate the downstream kinase wts , which in turn phosphorylates and inhibits the transcription coactivator yorkie ( yki ) . scaffold proteins , such as salvador ( sav ) and mats , potentiate the activity of the hpo / wts complex ( harvey and tapon , 2007 ) . one upstream input of the pathway is mediated via merlin ( mer ) and expanded ( ex ) , two ferm ( four point one , ezrin , moesin , radixin ) domain proteins ( hamaratoglu et al . , 2006 ) . recently , ex was shown to bind yki , and new experiments hint at the existence of an ex / hpo / wts - containing apical complex anchoring yki at the cortex ( badouel et al . , 2009 ; oh et al . , 2009 ) . however , though some upstream members are known , how the swh network is activated remains unclear . here , we identify the ww - domain - containing protein kibra as a regulator of the swh network . human kibra ( kremerskothen et al . , 2003 ) is known to be phosphorylated by protein kinase c ( pkc ) ( buther et al . , 2004 ) and has recently been reported to have a role in cell migration ( duning et al . , 2008 ; rosse et al . , 2009 ) . in drosophila , kibra had previously been recovered as a minor hit in several screens for growth regulators ( boutros et al . , 2004 ; muller et al . , 2005 ; ringrose et al . , 2003 ; tseng and hariharan , 2002 ) but has not been further studied . our experiments show that kibra associates with mer , ex , and wts and stabilizes the mer / ex interaction . this suggests that kibra is a component of an apical scaffold that controls swh pathway activation . we performed an in vivo screen in the fly wing in order to identify genes implicated in growth control . transgenic flies bearing rna interference ( rnai ) constructs generated by the vienna drosophila rnai centre ( vdrc ) ( dietzl et al . , 2007 ) were crossed to the hedgehog - gal4 ( hh - gal4 ) driver , leading to target gene silencing in the posterior compartment of the wing . the results of this screen will be described elsewhere . in this context , expressing an rnai line directed against kibra induced overgrowth of the posterior wing compartment ( figure 1b ) compared to control flies ( figure 1a ) . this phenotype was also observed upon wts depletion ( figure 1c ) . driving the same kibra rnai line in the eye also led to increased organ size ( figures 1d and 1e ) , similarly to a wts rnai line ( figure 1f ) . to exclude off - target effects , we generated a transgenic line expressing a nonoverlapping rnai construct and observed identical overgrowth phenotypes ( data not shown ) . furthermore , adult eye sections revealed that kibra knockdown retinas present an excess of interommatidial cells ( iocs ) ( figures 1 g and 1h ) . the iocs , the last population of cells to differentiate in the eye primordium , give rise to the secondary and tertiary pigment cells that optically isolate the ommatidia in the compound eye from each other . extra iocs are produced during normal development but are then eliminated by apoptosis at the pupal stage to give rise to the adult lattice ( wolff and ready , 1993 ) . the presence of extra iocs is a hallmark of swh network loss of function ( kango - singh et al . , 2002 ; 2002 ) , which reduces retinal apoptosis , as seen in wts rnai adult eye sections ( figure 1i ) . thus , depletion of kibra elicits a similar phenotype to swh network mutants , suggesting a potential role for kibra in hpo signaling . to study kibra loss of function , we generated the kibra allele by imprecise excision of the ep747 transposon ( see figure 1j and experimental procedures ) . this deletion allele , which removes the translation initiation site , is homozygous lethal and may be a null allele for kibra . kibra flp / frt mutant clones in 40 hr after - puparium - formation ( apf ) retinas present extra iocs ( figure 1k1 m ) , similarly to what was observed in adult eyes with kibra knockdown ( figures 1g1i ) . we determined apoptotic indexes ( colombani et al . , 2006 ) during the retinal apoptosis wave ( 28 hr apf ) in pupal retinas containing kibra mutant clones stained with an anti - active caspase-3 antibody . kibra mutant tissue presents a reduced apoptotic index compared with wild - type ( wt ) areas in the same retinas ( figures 1n1p ; see figures s1a we assessed the proliferation rate of kibra mutant cells in imaginal discs , the larval precursors to the adult appendages . by using the flp / frt system under the control of the heat - shock promoter , kibra mutant cells and their wt sister clones were generated through single recombination events from heterozygous mother cells ( brumby and richardson , 2005 ) . after several rounds of divisions , the sizes of mutant clones ( no gfp ) and wt twin spots ( two copies of gfp ) were compared , allowing us to estimate the relative proliferation rates of mutant versus wt cells . the total kibra clone area is 1.57 ( 0.12)-fold larger than the control twin spot area , compared to a ratio of 0.98 ( 0.09 ) when both clones and twin spots are wt ( figures 1q1s ) , indicating that kibra mutant cells grow 1.6 times faster than wt cells . in addition to cell cycle rates , the timing of cell cycle exit can readily be measured in the eye disc , where cell divisions follow a spatially determined pattern ( wolff and ready , 1993 ) . during the third larval instar , the morphogenetic furrow anterior to the furrow cells still proliferate asynchronously , while in the furrow cells synchronize in g1 . immediately posterior to the furrow , cells enter a final round of synchronous s phases , the second mitotic wave ( smw ) . thus , in wt discs , no s phases ( marked by edu incorporation ) can be observed posterior to the smw ( figure 1 t ) . as expected , hpo mutant cells fail to exit from the cell cycle in a timely manner and present ectopic edu - positive staining posterior to the swm ( figures s1b and s1b ) . kibra mutant cells exhibit a less pronounced but similar phenotype ( figures 1u and 1v ) . thus , kibra mutant tissues have a proliferative advantage and an apoptosis defect , consistent with an involvement in the swh network . the overgrowth defect appears more subtle than that of core pathway members such as wts and is more akin to upstream regulators ( e.g. , ex and mer ) . several transcriptional targets of the swh network have been identified , such as the drosophila inhibitor of apoptosis 1 ( diap1 ) gene , the cell cycle regulator cyce , the mirna bantam , as well as ex ( harvey and tapon , 2007 ; saucedo and edgar , 2007 ) . in kibra mutant wing or eye discs , we could not detect a strong change in diap1 , cyce , or ex - lacz reporter levels ( data not shown ) . since overgrowth of kibra mutant cells in the wing is subtle compared to wts mutants , it is possible that kibra plays a relatively minor role in swh signaling in the wing . accordingly , using an anti - kibra antibody we generated ( figures s2a s2c ) , we noted that kibra staining in the wing disc is weak and consists of a punctate apical staining which can clearly be observed when kibra is overexpressed in a stripe of cells . we and others have previously reported that ovarian posterior follicle cells ( pfcs ) are particularly sensitive to swh loss of function ( meignin et al . , 2007 ; polesello and tapon , 2007 ; yu et al . , 2008 ) , leading us to study the kibra phenotype in the ovary . first , we noted that kibra protein levels are higher in follicle cells than in the wing discs ( figure s2d kibra staining is mainly apical and is severely reduced in kibra clones . similarly to hpo or wts loss of function , kibra loss of function in the pfcs induces an upregulation of the ex - lacz reporter ( figures 2a2b , compare with figures 2c2c ) . hpo or wts mutant pfcs also show a misregulation of the notch ( n ) pathway and ectopic cell divisions ( meignin et al . the n target hindsight ( hnt ) is normally repressed in all follicle cells up to stage 6 and switched on from stage 7 to stage 10b ( figures 2d2e ) ( poulton and deng , 2007 ) . cut , which is repressed by hnt , presents an opposite pattern of expression ( figures 2h2i ) . in kibra mutant pfcs from stage 710b egg chambers , hnt expression is lost ( figures 2d2d and 2f2g ) , while cut is ectopically expressed ( figures 2j2k ) . loss of kibra also leads to perturbation of epithelial integrity , as mutant pfcs show an accumulation of the apical polarity protein apkc and the n receptor ( figures s2f s2g ) as well as multilayering of the follicular epithelium ( figures 2i2i and 2k2k ) . ectopic mitotic divisions are also observed in pfcs clones after stage 6 , as detected by phospho - histone h3 ( ph3 ) staining ( figures s2h s2h ) . together , these phenotypes are identical to those observed in hpo or wts loss of function , suggesting that kibra is indeed a member of the swh network . to further explore the role of kibra in the swh network , genetic interaction and epistasis experiments were performed . overexpressing kibra in the eye under the gmr ( glass multimer reporter ) promoter elicits the formation of a small rough eye with frequent ommatidial fusions ( figures s3a s3b ) . this phenotype can be partially rescued by removing one copy of the hpo gene ( figures s3c and s3c ) . in contrast , overexpressing kibra could not rescue the hpo - like overgrowth phenotype induced by yki overexpression ( figures s3d and s3e ) , suggesting that kibra may be an upstream regulator of the pathway . to conduct epistasis experiments between kibra and yki , we used the marcm system to generate clones of mutant cells while simultaneously overexpressing or depleting other pathway components ( lee and luo , 1999 ) . marcm clones expressing yki rnai generated with eyflp lead to the formation of a normal eye , because yki - depleted cells are eliminated by apoptosis ( data not shown ) and replaced by wt cells ( figure s3f ) . as expected , eyflp kibra marcm clones cause eye overgrowth ( figure s3 g ) . , overexpressing kibra in the eye under the gmr promoter induces apoptosis in third instar eye discs , which is suppressed by loss of hpo ( figures 3a3a ) . together , these epistasis experiments are consistent with kibra being a member of the swh network acting upstream of yki and hpo . genetic interactions between kibra , mer , and ex , upstream members of the swh network , were then investigated . expressing a kibra , an ex , or a mer rnai line in the eye under the gmr promoter induces eye overgrowth ( figures s3i s3l ) . combined depletion of either ex / kibra or mer / kibra shows stronger phenotypes than individual depletion of these proteins ( figures s3 m and s3n ) . hsflp marcm clones of various genotypes were generated and scored according to the severity of the wing overgrowth phenotypes , with type 0 representing normal wings and type 4 the strongest overgrowth ( figures 3b and 3c ) . overexpressing ex or mer in kibra mutant clones significantly rescues the overgrowth of kibra mutant clones ( p < 0.0001 for both genotypes ) . reciprocally , kibra overexpression was also able to suppress the ex overgrowth phenotype ( p < 0.0001 ) . thus , we could not determine a strict epistatic relationship between kibra , ex , and mer , consistent with a model whereby kibra , ex , and mer cooperate to control swh pathway activity . as well as being an upstream regulator of the swh network , ex is also one of its transcriptional targets ( hamaratoglu et al . , 2006 ) , as are other upstream regulators ( e.g. , mer , four - jointed , dachsous ) . since epistasis experiments place kibra at the level of mer and ex , we wished to test whether this is also the case for kibra . kibra levels were highly upregulated in mer;ex or hpo clones ( figures 3d3e ) , showing an apical localization ( figures 3f3g ) . similarly , hpo - depleted cultured drosophila s2r+ cells have increased kibra levels ( figure 3j ) . to determine whether kibra is a transcriptional swh network target , quantitative rt - pcr experiments were performed on yki - overexpressing and control wing imaginal discs ( figure 3k ) . as expected , ex mrna levels were increased ( 2.97 0.25-fold ) in yki - expressing discs compared to control discs . interestingly , kibra mrna levels were also upregulated in yki - expressing discs ( 6.24 2.12-fold ) , confirming that kibra is a yki transcriptional target and suggesting the existence of a possible negative feedback loop regulating kibra expression . because hpo clones present increased levels of kibra as well as mer and ex , these constitute a good system to evaluate the colocalization of those proteins . thus , kibra , mer , and ex colocalize apically in imaginal disc cells , but are dispensable for each other 's apical sorting , because kibra is still apical in mer;ex clones and mer / ex are normally localized in kibra clones ( figures 3d , 3d , 3f , and 3f and data not shown ) . because kibra colocalizes with mer / ex , a possible association between those proteins was examined by conducting coimmunoprecipitation ( co - ip ) assays in s2r+ cells . kibra was found to co - ip with ex and mer , but not with hpo or with the negative regulator of hpo , drassf ( polesello et al . , 2006 ) interestingly , kibra was reported to interact with mer in a large - scale yeast two - hybrid screening study ( formstecher et al . , 2005 ) . kibra possesses two ww domains , which are predicted to mediate protein - protein interactions by binding to ppxy motifs . furthermore , the first ww domain of human kibra was shown to recognize the consensus motif rxppxy in vitro ( kremerskothen et al . , 2003 ) . in flies , mer does not contain any ppxy sites , while ex has two ppxy sites ( pppy and pppy ) and an rxppxy site ( rdpppy ) . we therefore further investigated the association between kibra and ex by mutating amino acids that are known to be required for ww domains and ppxy sites to interact ( kremerskothen et al . a kibra protein mutant for its first ww domain ( p85a ) could no longer co - ip wt ex . reciprocally , wt kibra could not co - ip an ex protein deficient for its rxppxy site ( p845a ) ( figure 4b ) . thus , kibra associates with ex through its first ww domain and the ex rxppxy motif . in contrast , mutating either one or both kibra ww domains does not affect kibra / mer association ( figure s4a ) . further assays reveal that both kibra n- and c - terminal fragments are sufficient for the association with mer , but a central stretch ( aa 484857 ) is not ( figure s4a ) . because the ww motifs , which are required for the ex / kibra association , are located in the n - terminal fragment , this suggests that mer can complex with kibra both through ex and independently of ex . to further test this possibility , coimmunoprecipitation assays between mer and kibra were performed in ex - depleted cells ( figure s4b ) . the kibra / mer immunoprecipitation is not affected by ex depletion , suggesting that ex is not required for the kibra / mer association . because the hpo pathway is highly conserved from drosophila to humans , we tested for potential interactions between human kibra and the human orthologs of ex ( frmd6 ) , mer ( nf2/mer ) , hpo ( mst2 ) , and drassf ( rassf6 ) . we used split - tev as readout , which is based on tev protease complementation and represents a sensitive method for detecting interactions between membrane - associated proteins ( wehr et al . we found that kibra associates with nf2/mer but did not interact with frmd6 , mst2 , or rassf6 ( figure s4c ) . interestingly , frmd6 contains only an n - terminally conserved sequence of ex but lacks the entire c - terminal part , which in ex harbors the ppxy motifs . thus , the missing ppxy motifs and the generally limited level of sequence conservation in frmd6 likely explain the absence of interaction between kibra and frmd6 . these results imply that the ability to associate with kibra evolved in an ancestral ex / mer - like ferm domain protein and was later lost in frmd6 but retained in nf2/mer . alternatively , as kibra complexes with both ex and mer and ex / mer have been reported to directly interact ( mccartney et al . , 2000 ) , we tested the possibility that kibra could affect the mer / ex interaction . we performed co - ip assays between mer and ex in cells expressing different levels of kibra protein ( figure 4c ) . the mer / ex interaction is reduced in kibra - depleted cells compared to wt cells , whereas the interaction is strengthened in cells that express a myc - kibra construct . thus , the presence of kibra is required to fine - tune the stability of the mer / ex interaction . because kibra complexes with ex and a yki / ex interaction has recently been described ( badouel et al . , 2009 ) , we sought to determine whether kibra can affect yki activity . s2r+ cells were treated with rnai against several swh pathway components , and yki phophorylation on ser168 was monitored by western blotting ( figure 4d ) . the phosphorylation of yki by wts at ser168 leads to yki inactivation and sequestration in the cytoplasm , where it has been reported to bind ex , wts , hpo , and 14.3.3 ( badouel et al . , 2009 ; huang et al . , 2005 ; oh and irvine , 2008 ; oh et al . , 2009 ) . lacz rnai - treated cells show a high basal level of phospho - yki ( p - yki ) . as expected , yki phosphorylation is abolished when wts is depleted , and mildly reduced when the wts cofactor mats ( lai et al . , 2005 ) treated rnai cells , a yki downward shift can also be observed using a pan - yki antibody ( figure 4d , second row ) . when depleted in conjunction with ex , the p - yki signal becomes even further reduced . this suggests that kibra and ex are required for wts activity on yki , which prompted us to investigate whether kibra could associate with wts . co - ip assays reveal that kibra interacts with wts ( figure 4e ) . wts does not seem to compete with ex for kibra association , because it could still complex with a form of kibra mutant for its first ww domain . because kibra associates with wts and ex interacts with yki , we investigated whether wts requires kibra / ex to bind yki . endogenous ips between yki and wts were performed in s2 cells treated with various dsrnas ( figure 4f ) . in these conditions , the effect of kibra and ex depletion on yki phosphorylation can also be observed ( see input ) . in control cells , this endogenous interaction is unaffected by the individual or combined depletion of ex and kibra . these results suggest that ex and kibra are required to activate the swh pathway by nucleating an active hpo / wts kinase cassette , rather than promoting the wts / yki interaction . our data identify kibra as a regulator of the swh network that associates with ex and mer , with which it is colocalized apically and transcriptionally coregulated . given that the apical surface of epithelial cells is instrumental in both cell - cell signaling and tissue morphogenesis , we speculate that kibra may cooperate with ex and mer to transduce an extracellular signal , or relay information about epithelial architecture , via the swh network , to control tissue growth and morphogenesis . recent data have suggested that an apical scaffold machinery containing hpo , wts , and ex recruits yki to the apical membrane , facilitating its inhibitory phosphorylation by wts ( badouel et al . , 2009 ; oh et al . , 2009 since kibra associates with ex and is also apically localized , we can hypothesize that kibra is also part of this scaffold and participates in nucleating an active hpo / wts complex and recruiting yki for inactivation . this view is supported by our finding that kibra complexes with wts and that combined depletion of kibra and ex leads to a strong decrease in yki phosphorylation , but does not disrupt the wts / yki interaction . our data also suggest that the importance of kibra may be tissue - specific since we observe robust phenotypes in ovaries and hemocyte - derived s2r+ cells , but weaker effects in imaginal discs . thus , considering the relative levels of expression of ex , mer , and kibra may be important in determining pathway activation . finally , since mammalian kibra complexes with the nf2/mer tumor suppressor , our findings raise the possibility that human kibra may contribute to tumor suppression in human neurofibromas and potentially other tumors . for drosophila genotypes , primer sequences , and further experimental details , see supplemental experimental procedures . the p element of ep747 ( bloomington stock center ) was mobilized using standard genetic techniques , and excisions were screened by pcr ( see supplemental experimental procedures for details ) . mouse -galactosidase ( promega ) , rabbit anti - cleaved caspase-3 ( asp175 ) ( cell signaling technology ) , rabbit anti - apkc ( santa cruz ) , and mouse nicd ( c17.9c6 , development studies hybridoma bank ) antibodies were used at 1/500 . mouse anti - arm , anti - cut , and anti - hnt ( n2 7a1 , 2b10 and 1g9 , dshb ) were used at 1/10 and 1/20 . the edu staining was performed as described in the click - it edu alexa fluor imaging kit ( invitrogen ) . guinea pig anti - mer , a gift from r. fehon , was used at 1/7500 , and rabbit anti - ex , a gift from a. laughon , at 1/400 . rabbit anti - kibra antibody ( kib18 , 1/100 ) was generated by eurogentec sa ( seraing , belgium ) against a peptide corresponding to the last 15 amino acids of kibra . ms1096 > > ( control ) and ms1096 > > yki wing discs were dissected in pbs and snap - frozen in liquid nitrogen . rna isolation and subsequent qrt - pcr reactions were performed as described in genevet et al . all statistical analyses performed ( except epistasis analysis ) were assessed by mann - whitney nonparametric tests using the website http://elegans.swmed.edu/leon/stats/utest.html . the epistasis analysis was made by pairwise comparison after correction for the batch effect on 42 to 310 flies of each genotype , divided in 4 to 6 cohorts . the approach used was a three - way log - linear model , against a null - hypothesis of no interaction between phenotype and population . the p value indicates whether the pair of populations differ in their phenotype profiles : p value(kibra ; kibra + uas ex ) = 2.69 10 , p value(kibra ; kibra + uas mer ) = 6.07 10 , p value(ex ; ex + uas kibra ) = 1.76 10 .

summarythe salvador ( sav)/warts ( wts)/hippo ( hpo ) ( swh ) network controls tissue growth by inhibiting cell proliferation and promoting apoptosis . the core of the pathway consists of a mst and lats family kinase cascade that ultimately phosphorylates and inactivates the yap / yorkie ( yki ) transcription coactivator . the ferm domain proteins merlin ( mer ) and expanded ( ex ) represent one mode of upstream regulation controlling pathway activity . here , we identify kibra as a member of the swh network . kibra , which colocalizes and associates with mer and ex , also promotes the mer / ex association . furthermore , the kibra / mer association is conserved in human cells . finally , kibra complexes with wts and kibra depletion in tissue culture cells induces a marked reduction in yki phosphorylation without affecting the yki / wts interaction . we suggest that kibra is part of an apical scaffold that promotes swh pathway activity .

Introduction Results Discussion Experimental Procedures

the salvador ( sav)/warts ( wts)/hippo ( hpo ) ( swh ) network , identified in drosophila and conserved in mammals , plays a major role in limiting growth by inhibiting cell proliferation and promoting apoptosis ( harvey and tapon , 2007 ; reddy and irvine , 2008 ) . activation of the upstream kinase hpo allows it to phosphorylate the downstream kinase wts , which in turn phosphorylates and inhibits the transcription coactivator yorkie ( yki ) . scaffold proteins , such as salvador ( sav ) and mats , potentiate the activity of the hpo / wts complex ( harvey and tapon , 2007 ) . one upstream input of the pathway is mediated via merlin ( mer ) and expanded ( ex ) , two ferm ( four point one , ezrin , moesin , radixin ) domain proteins ( hamaratoglu et al . recently , ex was shown to bind yki , and new experiments hint at the existence of an ex / hpo / wts - containing apical complex anchoring yki at the cortex ( badouel et al . however , though some upstream members are known , how the swh network is activated remains unclear . here , we identify the ww - domain - containing protein kibra as a regulator of the swh network . , 2003 ) is known to be phosphorylated by protein kinase c ( pkc ) ( buther et al . in drosophila , kibra had previously been recovered as a minor hit in several screens for growth regulators ( boutros et al . our experiments show that kibra associates with mer , ex , and wts and stabilizes the mer / ex interaction . this suggests that kibra is a component of an apical scaffold that controls swh pathway activation . , 2007 ) were crossed to the hedgehog - gal4 ( hh - gal4 ) driver , leading to target gene silencing in the posterior compartment of the wing . in this context , expressing an rnai line directed against kibra induced overgrowth of the posterior wing compartment ( figure 1b ) compared to control flies ( figure 1a ) . to exclude off - target effects , we generated a transgenic line expressing a nonoverlapping rnai construct and observed identical overgrowth phenotypes ( data not shown ) . furthermore , adult eye sections revealed that kibra knockdown retinas present an excess of interommatidial cells ( iocs ) ( figures 1 g and 1h ) . the iocs , the last population of cells to differentiate in the eye primordium , give rise to the secondary and tertiary pigment cells that optically isolate the ommatidia in the compound eye from each other . , 2002 ; 2002 ) , which reduces retinal apoptosis , as seen in wts rnai adult eye sections ( figure 1i ) . thus , depletion of kibra elicits a similar phenotype to swh network mutants , suggesting a potential role for kibra in hpo signaling . to study kibra loss of function , we generated the kibra allele by imprecise excision of the ep747 transposon ( see figure 1j and experimental procedures ) . this deletion allele , which removes the translation initiation site , is homozygous lethal and may be a null allele for kibra . kibra mutant tissue presents a reduced apoptotic index compared with wild - type ( wt ) areas in the same retinas ( figures 1n1p ; see figures s1a we assessed the proliferation rate of kibra mutant cells in imaginal discs , the larval precursors to the adult appendages . by using the flp / frt system under the control of the heat - shock promoter , kibra mutant cells and their wt sister clones were generated through single recombination events from heterozygous mother cells ( brumby and richardson , 2005 ) . after several rounds of divisions , the sizes of mutant clones ( no gfp ) and wt twin spots ( two copies of gfp ) were compared , allowing us to estimate the relative proliferation rates of mutant versus wt cells . the total kibra clone area is 1.57 ( 0.12)-fold larger than the control twin spot area , compared to a ratio of 0.98 ( 0.09 ) when both clones and twin spots are wt ( figures 1q1s ) , indicating that kibra mutant cells grow 1.6 times faster than wt cells . in addition to cell cycle rates , the timing of cell cycle exit can readily be measured in the eye disc , where cell divisions follow a spatially determined pattern ( wolff and ready , 1993 ) . during the third larval instar , the morphogenetic furrow anterior to the furrow cells still proliferate asynchronously , while in the furrow cells synchronize in g1 . thus , kibra mutant tissues have a proliferative advantage and an apoptosis defect , consistent with an involvement in the swh network . the overgrowth defect appears more subtle than that of core pathway members such as wts and is more akin to upstream regulators ( e.g. , ex and mer ) . several transcriptional targets of the swh network have been identified , such as the drosophila inhibitor of apoptosis 1 ( diap1 ) gene , the cell cycle regulator cyce , the mirna bantam , as well as ex ( harvey and tapon , 2007 ; saucedo and edgar , 2007 ) . in kibra mutant wing or eye discs , we could not detect a strong change in diap1 , cyce , or ex - lacz reporter levels ( data not shown ) . since overgrowth of kibra mutant cells in the wing is subtle compared to wts mutants , it is possible that kibra plays a relatively minor role in swh signaling in the wing . accordingly , using an anti - kibra antibody we generated ( figures s2a s2c ) , we noted that kibra staining in the wing disc is weak and consists of a punctate apical staining which can clearly be observed when kibra is overexpressed in a stripe of cells . , 2008 ) , leading us to study the kibra phenotype in the ovary . first , we noted that kibra protein levels are higher in follicle cells than in the wing discs ( figure s2d kibra staining is mainly apical and is severely reduced in kibra clones . similarly to hpo or wts loss of function , kibra loss of function in the pfcs induces an upregulation of the ex - lacz reporter ( figures 2a2b , compare with figures 2c2c ) . hpo or wts mutant pfcs also show a misregulation of the notch ( n ) pathway and ectopic cell divisions ( meignin et al . loss of kibra also leads to perturbation of epithelial integrity , as mutant pfcs show an accumulation of the apical polarity protein apkc and the n receptor ( figures s2f s2g ) as well as multilayering of the follicular epithelium ( figures 2i2i and 2k2k ) . together , these phenotypes are identical to those observed in hpo or wts loss of function , suggesting that kibra is indeed a member of the swh network . to further explore the role of kibra in the swh network , genetic interaction and epistasis experiments were performed . in contrast , overexpressing kibra could not rescue the hpo - like overgrowth phenotype induced by yki overexpression ( figures s3d and s3e ) , suggesting that kibra may be an upstream regulator of the pathway . to conduct epistasis experiments between kibra and yki , we used the marcm system to generate clones of mutant cells while simultaneously overexpressing or depleting other pathway components ( lee and luo , 1999 ) . marcm clones expressing yki rnai generated with eyflp lead to the formation of a normal eye , because yki - depleted cells are eliminated by apoptosis ( data not shown ) and replaced by wt cells ( figure s3f ) . , overexpressing kibra in the eye under the gmr promoter induces apoptosis in third instar eye discs , which is suppressed by loss of hpo ( figures 3a3a ) . together , these epistasis experiments are consistent with kibra being a member of the swh network acting upstream of yki and hpo . genetic interactions between kibra , mer , and ex , upstream members of the swh network , were then investigated . expressing a kibra , an ex , or a mer rnai line in the eye under the gmr promoter induces eye overgrowth ( figures s3i s3l ) . combined depletion of either ex / kibra or mer / kibra shows stronger phenotypes than individual depletion of these proteins ( figures s3 m and s3n ) . hsflp marcm clones of various genotypes were generated and scored according to the severity of the wing overgrowth phenotypes , with type 0 representing normal wings and type 4 the strongest overgrowth ( figures 3b and 3c ) . reciprocally , kibra overexpression was also able to suppress the ex overgrowth phenotype ( p < 0.0001 ) . thus , we could not determine a strict epistatic relationship between kibra , ex , and mer , consistent with a model whereby kibra , ex , and mer cooperate to control swh pathway activity . as well as being an upstream regulator of the swh network , ex is also one of its transcriptional targets ( hamaratoglu et al . since epistasis experiments place kibra at the level of mer and ex , we wished to test whether this is also the case for kibra . kibra levels were highly upregulated in mer;ex or hpo clones ( figures 3d3e ) , showing an apical localization ( figures 3f3g ) . to determine whether kibra is a transcriptional swh network target , quantitative rt - pcr experiments were performed on yki - overexpressing and control wing imaginal discs ( figure 3k ) . as expected , ex mrna levels were increased ( 2.97 0.25-fold ) in yki - expressing discs compared to control discs . interestingly , kibra mrna levels were also upregulated in yki - expressing discs ( 6.24 2.12-fold ) , confirming that kibra is a yki transcriptional target and suggesting the existence of a possible negative feedback loop regulating kibra expression . because hpo clones present increased levels of kibra as well as mer and ex , these constitute a good system to evaluate the colocalization of those proteins . thus , kibra , mer , and ex colocalize apically in imaginal disc cells , but are dispensable for each other 's apical sorting , because kibra is still apical in mer;ex clones and mer / ex are normally localized in kibra clones ( figures 3d , 3d , 3f , and 3f and data not shown ) . because kibra colocalizes with mer / ex , a possible association between those proteins was examined by conducting coimmunoprecipitation ( co - ip ) assays in s2r+ cells . , 2006 ) interestingly , kibra was reported to interact with mer in a large - scale yeast two - hybrid screening study ( formstecher et al . furthermore , the first ww domain of human kibra was shown to recognize the consensus motif rxppxy in vitro ( kremerskothen et al . in flies , mer does not contain any ppxy sites , while ex has two ppxy sites ( pppy and pppy ) and an rxppxy site ( rdpppy ) . we therefore further investigated the association between kibra and ex by mutating amino acids that are known to be required for ww domains and ppxy sites to interact ( kremerskothen et al . thus , kibra associates with ex through its first ww domain and the ex rxppxy motif . in contrast , mutating either one or both kibra ww domains does not affect kibra / mer association ( figure s4a ) . further assays reveal that both kibra n- and c - terminal fragments are sufficient for the association with mer , but a central stretch ( aa 484857 ) is not ( figure s4a ) . because the ww motifs , which are required for the ex / kibra association , are located in the n - terminal fragment , this suggests that mer can complex with kibra both through ex and independently of ex . to further test this possibility , coimmunoprecipitation assays between mer and kibra were performed in ex - depleted cells ( figure s4b ) . the kibra / mer immunoprecipitation is not affected by ex depletion , suggesting that ex is not required for the kibra / mer association . because the hpo pathway is highly conserved from drosophila to humans , we tested for potential interactions between human kibra and the human orthologs of ex ( frmd6 ) , mer ( nf2/mer ) , hpo ( mst2 ) , and drassf ( rassf6 ) . we found that kibra associates with nf2/mer but did not interact with frmd6 , mst2 , or rassf6 ( figure s4c ) . interestingly , frmd6 contains only an n - terminally conserved sequence of ex but lacks the entire c - terminal part , which in ex harbors the ppxy motifs . thus , the missing ppxy motifs and the generally limited level of sequence conservation in frmd6 likely explain the absence of interaction between kibra and frmd6 . these results imply that the ability to associate with kibra evolved in an ancestral ex / mer - like ferm domain protein and was later lost in frmd6 but retained in nf2/mer . alternatively , as kibra complexes with both ex and mer and ex / mer have been reported to directly interact ( mccartney et al . , 2000 ) , we tested the possibility that kibra could affect the mer / ex interaction . we performed co - ip assays between mer and ex in cells expressing different levels of kibra protein ( figure 4c ) . the mer / ex interaction is reduced in kibra - depleted cells compared to wt cells , whereas the interaction is strengthened in cells that express a myc - kibra construct . thus , the presence of kibra is required to fine - tune the stability of the mer / ex interaction . because kibra complexes with ex and a yki / ex interaction has recently been described ( badouel et al . , 2009 ) , we sought to determine whether kibra can affect yki activity . s2r+ cells were treated with rnai against several swh pathway components , and yki phophorylation on ser168 was monitored by western blotting ( figure 4d ) . lacz rnai - treated cells show a high basal level of phospho - yki ( p - yki ) . as expected , yki phosphorylation is abolished when wts is depleted , and mildly reduced when the wts cofactor mats ( lai et al . when depleted in conjunction with ex , the p - yki signal becomes even further reduced . this suggests that kibra and ex are required for wts activity on yki , which prompted us to investigate whether kibra could associate with wts . co - ip assays reveal that kibra interacts with wts ( figure 4e ) . because kibra associates with wts and ex interacts with yki , we investigated whether wts requires kibra / ex to bind yki . in these conditions , the effect of kibra and ex depletion on yki phosphorylation can also be observed ( see input ) . in control cells , this endogenous interaction is unaffected by the individual or combined depletion of ex and kibra . these results suggest that ex and kibra are required to activate the swh pathway by nucleating an active hpo / wts kinase cassette , rather than promoting the wts / yki interaction . our data identify kibra as a regulator of the swh network that associates with ex and mer , with which it is colocalized apically and transcriptionally coregulated . given that the apical surface of epithelial cells is instrumental in both cell - cell signaling and tissue morphogenesis , we speculate that kibra may cooperate with ex and mer to transduce an extracellular signal , or relay information about epithelial architecture , via the swh network , to control tissue growth and morphogenesis . recent data have suggested that an apical scaffold machinery containing hpo , wts , and ex recruits yki to the apical membrane , facilitating its inhibitory phosphorylation by wts ( badouel et al . , 2009 since kibra associates with ex and is also apically localized , we can hypothesize that kibra is also part of this scaffold and participates in nucleating an active hpo / wts complex and recruiting yki for inactivation . this view is supported by our finding that kibra complexes with wts and that combined depletion of kibra and ex leads to a strong decrease in yki phosphorylation , but does not disrupt the wts / yki interaction . our data also suggest that the importance of kibra may be tissue - specific since we observe robust phenotypes in ovaries and hemocyte - derived s2r+ cells , but weaker effects in imaginal discs . thus , considering the relative levels of expression of ex , mer , and kibra may be important in determining pathway activation . finally , since mammalian kibra complexes with the nf2/mer tumor suppressor , our findings raise the possibility that human kibra may contribute to tumor suppression in human neurofibromas and potentially other tumors . mouse -galactosidase ( promega ) , rabbit anti - cleaved caspase-3 ( asp175 ) ( cell signaling technology ) , rabbit anti - apkc ( santa cruz ) , and mouse nicd ( c17.9c6 , development studies hybridoma bank ) antibodies were used at 1/500 . guinea pig anti - mer , a gift from r. fehon , was used at 1/7500 , and rabbit anti - ex , a gift from a. laughon , at 1/400 . ms1096 > > ( control ) and ms1096 > > yki wing discs were dissected in pbs and snap - frozen in liquid nitrogen . the p value indicates whether the pair of populations differ in their phenotype profiles : p value(kibra ; kibra + uas ex ) = 2.69 10 , p value(kibra ; kibra + uas mer ) = 6.07 10 , p value(ex ; ex + uas kibra ) = 1.76 10 .

growth regulation is a critical developmental process whose dysfunction can lead to many diseases , including cancer ( conlon and raff , 1999 ) . the salvador ( sav)/warts ( wts)/hippo ( hpo ) ( swh ) network , identified in drosophila and conserved in mammals , plays a major role in limiting growth by inhibiting cell proliferation and promoting apoptosis ( harvey and tapon , 2007 ; reddy and irvine , 2008 ) . activation of the upstream kinase hpo allows it to phosphorylate the downstream kinase wts , which in turn phosphorylates and inhibits the transcription coactivator yorkie ( yki ) . scaffold proteins , such as salvador ( sav ) and mats , potentiate the activity of the hpo / wts complex ( harvey and tapon , 2007 ) . one upstream input of the pathway is mediated via merlin ( mer ) and expanded ( ex ) , two ferm ( four point one , ezrin , moesin , radixin ) domain proteins ( hamaratoglu et al . recently , ex was shown to bind yki , and new experiments hint at the existence of an ex / hpo / wts - containing apical complex anchoring yki at the cortex ( badouel et al . here , we identify the ww - domain - containing protein kibra as a regulator of the swh network . in drosophila , kibra had previously been recovered as a minor hit in several screens for growth regulators ( boutros et al . our experiments show that kibra associates with mer , ex , and wts and stabilizes the mer / ex interaction . this suggests that kibra is a component of an apical scaffold that controls swh pathway activation . we performed an in vivo screen in the fly wing in order to identify genes implicated in growth control . transgenic flies bearing rna interference ( rnai ) constructs generated by the vienna drosophila rnai centre ( vdrc ) ( dietzl et al . , 2007 ) were crossed to the hedgehog - gal4 ( hh - gal4 ) driver , leading to target gene silencing in the posterior compartment of the wing . in this context , expressing an rnai line directed against kibra induced overgrowth of the posterior wing compartment ( figure 1b ) compared to control flies ( figure 1a ) . driving the same kibra rnai line in the eye also led to increased organ size ( figures 1d and 1e ) , similarly to a wts rnai line ( figure 1f ) . to exclude off - target effects , we generated a transgenic line expressing a nonoverlapping rnai construct and observed identical overgrowth phenotypes ( data not shown ) . furthermore , adult eye sections revealed that kibra knockdown retinas present an excess of interommatidial cells ( iocs ) ( figures 1 g and 1h ) . the iocs , the last population of cells to differentiate in the eye primordium , give rise to the secondary and tertiary pigment cells that optically isolate the ommatidia in the compound eye from each other . extra iocs are produced during normal development but are then eliminated by apoptosis at the pupal stage to give rise to the adult lattice ( wolff and ready , 1993 ) . the presence of extra iocs is a hallmark of swh network loss of function ( kango - singh et al . , 2002 ; 2002 ) , which reduces retinal apoptosis , as seen in wts rnai adult eye sections ( figure 1i ) . thus , depletion of kibra elicits a similar phenotype to swh network mutants , suggesting a potential role for kibra in hpo signaling . to study kibra loss of function , we generated the kibra allele by imprecise excision of the ep747 transposon ( see figure 1j and experimental procedures ) . kibra flp / frt mutant clones in 40 hr after - puparium - formation ( apf ) retinas present extra iocs ( figure 1k1 m ) , similarly to what was observed in adult eyes with kibra knockdown ( figures 1g1i ) . , 2006 ) during the retinal apoptosis wave ( 28 hr apf ) in pupal retinas containing kibra mutant clones stained with an anti - active caspase-3 antibody . kibra mutant tissue presents a reduced apoptotic index compared with wild - type ( wt ) areas in the same retinas ( figures 1n1p ; see figures s1a we assessed the proliferation rate of kibra mutant cells in imaginal discs , the larval precursors to the adult appendages . by using the flp / frt system under the control of the heat - shock promoter , kibra mutant cells and their wt sister clones were generated through single recombination events from heterozygous mother cells ( brumby and richardson , 2005 ) . after several rounds of divisions , the sizes of mutant clones ( no gfp ) and wt twin spots ( two copies of gfp ) were compared , allowing us to estimate the relative proliferation rates of mutant versus wt cells . the total kibra clone area is 1.57 ( 0.12)-fold larger than the control twin spot area , compared to a ratio of 0.98 ( 0.09 ) when both clones and twin spots are wt ( figures 1q1s ) , indicating that kibra mutant cells grow 1.6 times faster than wt cells . in addition to cell cycle rates , the timing of cell cycle exit can readily be measured in the eye disc , where cell divisions follow a spatially determined pattern ( wolff and ready , 1993 ) . during the third larval instar , the morphogenetic furrow anterior to the furrow cells still proliferate asynchronously , while in the furrow cells synchronize in g1 . immediately posterior to the furrow , cells enter a final round of synchronous s phases , the second mitotic wave ( smw ) . thus , in wt discs , no s phases ( marked by edu incorporation ) can be observed posterior to the smw ( figure 1 t ) . as expected , hpo mutant cells fail to exit from the cell cycle in a timely manner and present ectopic edu - positive staining posterior to the swm ( figures s1b and s1b ) . thus , kibra mutant tissues have a proliferative advantage and an apoptosis defect , consistent with an involvement in the swh network . the overgrowth defect appears more subtle than that of core pathway members such as wts and is more akin to upstream regulators ( e.g. several transcriptional targets of the swh network have been identified , such as the drosophila inhibitor of apoptosis 1 ( diap1 ) gene , the cell cycle regulator cyce , the mirna bantam , as well as ex ( harvey and tapon , 2007 ; saucedo and edgar , 2007 ) . in kibra mutant wing or eye discs , we could not detect a strong change in diap1 , cyce , or ex - lacz reporter levels ( data not shown ) . since overgrowth of kibra mutant cells in the wing is subtle compared to wts mutants , it is possible that kibra plays a relatively minor role in swh signaling in the wing . accordingly , using an anti - kibra antibody we generated ( figures s2a s2c ) , we noted that kibra staining in the wing disc is weak and consists of a punctate apical staining which can clearly be observed when kibra is overexpressed in a stripe of cells . we and others have previously reported that ovarian posterior follicle cells ( pfcs ) are particularly sensitive to swh loss of function ( meignin et al . first , we noted that kibra protein levels are higher in follicle cells than in the wing discs ( figure s2d kibra staining is mainly apical and is severely reduced in kibra clones . similarly to hpo or wts loss of function , kibra loss of function in the pfcs induces an upregulation of the ex - lacz reporter ( figures 2a2b , compare with figures 2c2c ) . hpo or wts mutant pfcs also show a misregulation of the notch ( n ) pathway and ectopic cell divisions ( meignin et al . the n target hindsight ( hnt ) is normally repressed in all follicle cells up to stage 6 and switched on from stage 7 to stage 10b ( figures 2d2e ) ( poulton and deng , 2007 ) . in kibra mutant pfcs from stage 710b egg chambers , hnt expression is lost ( figures 2d2d and 2f2g ) , while cut is ectopically expressed ( figures 2j2k ) . loss of kibra also leads to perturbation of epithelial integrity , as mutant pfcs show an accumulation of the apical polarity protein apkc and the n receptor ( figures s2f s2g ) as well as multilayering of the follicular epithelium ( figures 2i2i and 2k2k ) . ectopic mitotic divisions are also observed in pfcs clones after stage 6 , as detected by phospho - histone h3 ( ph3 ) staining ( figures s2h s2h ) . together , these phenotypes are identical to those observed in hpo or wts loss of function , suggesting that kibra is indeed a member of the swh network . to further explore the role of kibra in the swh network , genetic interaction and epistasis experiments were performed . overexpressing kibra in the eye under the gmr ( glass multimer reporter ) promoter elicits the formation of a small rough eye with frequent ommatidial fusions ( figures s3a s3b ) . in contrast , overexpressing kibra could not rescue the hpo - like overgrowth phenotype induced by yki overexpression ( figures s3d and s3e ) , suggesting that kibra may be an upstream regulator of the pathway . to conduct epistasis experiments between kibra and yki , we used the marcm system to generate clones of mutant cells while simultaneously overexpressing or depleting other pathway components ( lee and luo , 1999 ) . marcm clones expressing yki rnai generated with eyflp lead to the formation of a normal eye , because yki - depleted cells are eliminated by apoptosis ( data not shown ) and replaced by wt cells ( figure s3f ) . , overexpressing kibra in the eye under the gmr promoter induces apoptosis in third instar eye discs , which is suppressed by loss of hpo ( figures 3a3a ) . together , these epistasis experiments are consistent with kibra being a member of the swh network acting upstream of yki and hpo . genetic interactions between kibra , mer , and ex , upstream members of the swh network , were then investigated . expressing a kibra , an ex , or a mer rnai line in the eye under the gmr promoter induces eye overgrowth ( figures s3i s3l ) . hsflp marcm clones of various genotypes were generated and scored according to the severity of the wing overgrowth phenotypes , with type 0 representing normal wings and type 4 the strongest overgrowth ( figures 3b and 3c ) . overexpressing ex or mer in kibra mutant clones significantly rescues the overgrowth of kibra mutant clones ( p < 0.0001 for both genotypes ) . reciprocally , kibra overexpression was also able to suppress the ex overgrowth phenotype ( p < 0.0001 ) . thus , we could not determine a strict epistatic relationship between kibra , ex , and mer , consistent with a model whereby kibra , ex , and mer cooperate to control swh pathway activity . since epistasis experiments place kibra at the level of mer and ex , we wished to test whether this is also the case for kibra . to determine whether kibra is a transcriptional swh network target , quantitative rt - pcr experiments were performed on yki - overexpressing and control wing imaginal discs ( figure 3k ) . interestingly , kibra mrna levels were also upregulated in yki - expressing discs ( 6.24 2.12-fold ) , confirming that kibra is a yki transcriptional target and suggesting the existence of a possible negative feedback loop regulating kibra expression . because hpo clones present increased levels of kibra as well as mer and ex , these constitute a good system to evaluate the colocalization of those proteins . thus , kibra , mer , and ex colocalize apically in imaginal disc cells , but are dispensable for each other 's apical sorting , because kibra is still apical in mer;ex clones and mer / ex are normally localized in kibra clones ( figures 3d , 3d , 3f , and 3f and data not shown ) . because kibra colocalizes with mer / ex , a possible association between those proteins was examined by conducting coimmunoprecipitation ( co - ip ) assays in s2r+ cells . kibra was found to co - ip with ex and mer , but not with hpo or with the negative regulator of hpo , drassf ( polesello et al . furthermore , the first ww domain of human kibra was shown to recognize the consensus motif rxppxy in vitro ( kremerskothen et al . in flies , mer does not contain any ppxy sites , while ex has two ppxy sites ( pppy and pppy ) and an rxppxy site ( rdpppy ) . we therefore further investigated the association between kibra and ex by mutating amino acids that are known to be required for ww domains and ppxy sites to interact ( kremerskothen et al . further assays reveal that both kibra n- and c - terminal fragments are sufficient for the association with mer , but a central stretch ( aa 484857 ) is not ( figure s4a ) . because the ww motifs , which are required for the ex / kibra association , are located in the n - terminal fragment , this suggests that mer can complex with kibra both through ex and independently of ex . because the hpo pathway is highly conserved from drosophila to humans , we tested for potential interactions between human kibra and the human orthologs of ex ( frmd6 ) , mer ( nf2/mer ) , hpo ( mst2 ) , and drassf ( rassf6 ) . we used split - tev as readout , which is based on tev protease complementation and represents a sensitive method for detecting interactions between membrane - associated proteins ( wehr et al . interestingly , frmd6 contains only an n - terminally conserved sequence of ex but lacks the entire c - terminal part , which in ex harbors the ppxy motifs . thus , the missing ppxy motifs and the generally limited level of sequence conservation in frmd6 likely explain the absence of interaction between kibra and frmd6 . these results imply that the ability to associate with kibra evolved in an ancestral ex / mer - like ferm domain protein and was later lost in frmd6 but retained in nf2/mer . we performed co - ip assays between mer and ex in cells expressing different levels of kibra protein ( figure 4c ) . the mer / ex interaction is reduced in kibra - depleted cells compared to wt cells , whereas the interaction is strengthened in cells that express a myc - kibra construct . thus , the presence of kibra is required to fine - tune the stability of the mer / ex interaction . the phosphorylation of yki by wts at ser168 leads to yki inactivation and sequestration in the cytoplasm , where it has been reported to bind ex , wts , hpo , and 14.3.3 ( badouel et al . lacz rnai - treated cells show a high basal level of phospho - yki ( p - yki ) . this suggests that kibra and ex are required for wts activity on yki , which prompted us to investigate whether kibra could associate with wts . because kibra associates with wts and ex interacts with yki , we investigated whether wts requires kibra / ex to bind yki . these results suggest that ex and kibra are required to activate the swh pathway by nucleating an active hpo / wts kinase cassette , rather than promoting the wts / yki interaction . our data identify kibra as a regulator of the swh network that associates with ex and mer , with which it is colocalized apically and transcriptionally coregulated . given that the apical surface of epithelial cells is instrumental in both cell - cell signaling and tissue morphogenesis , we speculate that kibra may cooperate with ex and mer to transduce an extracellular signal , or relay information about epithelial architecture , via the swh network , to control tissue growth and morphogenesis . , 2009 since kibra associates with ex and is also apically localized , we can hypothesize that kibra is also part of this scaffold and participates in nucleating an active hpo / wts complex and recruiting yki for inactivation . this view is supported by our finding that kibra complexes with wts and that combined depletion of kibra and ex leads to a strong decrease in yki phosphorylation , but does not disrupt the wts / yki interaction . our data also suggest that the importance of kibra may be tissue - specific since we observe robust phenotypes in ovaries and hemocyte - derived s2r+ cells , but weaker effects in imaginal discs . thus , considering the relative levels of expression of ex , mer , and kibra may be important in determining pathway activation . finally , since mammalian kibra complexes with the nf2/mer tumor suppressor , our findings raise the possibility that human kibra may contribute to tumor suppression in human neurofibromas and potentially other tumors . mouse -galactosidase ( promega ) , rabbit anti - cleaved caspase-3 ( asp175 ) ( cell signaling technology ) , rabbit anti - apkc ( santa cruz ) , and mouse nicd ( c17.9c6 , development studies hybridoma bank ) antibodies were used at 1/500 . the p value indicates whether the pair of populations differ in their phenotype profiles : p value(kibra ; kibra + uas ex ) = 2.69 10 , p value(kibra ; kibra + uas mer ) = 6.07 10 , p value(ex ; ex + uas kibra ) = 1.76 10 .

a growing body of evidence shows that prediabetic condition is a risk factor for type-2-diabetes and cardiovascular complications.13 prediabetes is defined as impaired glucose tolerance ( igt ) and/or impaired fasting glucose.2 igt is associated with metabolic syndrome and is a strong predictor of atherosclerotic cardiovascular disease , whereas impaired fasting glucose is a much weaker predictor.4 there are no less than 300 million people with prediabetic condition worldwide , and the number is predicted to rise up to 418 million by 2025 . every year , 2%11% of the prediabetic population are converting to type-2-diabetics.1,2,4,5 therefore , early intervention in the prediabetic stage is a valuable approach to reduce diabetes development and prevent related complications.3,6,7 insulin resistance , -cell dysfunction , and uncontrolled calorie consumption have been the crucial contributors to acute postprandial hyperglycemia and are the potential agents that convert prediabetes to diabetes.4,8,9 the acute postprandial hyperglycemia is responsible for glucotoxicity in the pathophysiology of type-2-diabetes even at the earlier stage ( ie , prediabetes).10 further in a vicious cycle , the impairment of -cell function and exacerbation of insulin resistance due to glucotoxicity play a crucial role in the progression of diabetes and development of its cardiovascular complications.4,10 interventions that preserve -cell function and ameliorate insulin resistance have been proven to be most effective in preventing progression to diabetes . many studies on lifestyle modification , including diet control and regular physical exercise,1114 and pharmacological interventions using -glucosidase inhibitors,15,16 insulin sensitizers , such as metformin17 and thiazolidinediones,15,18 and incretin - mimetic agents,19 have been reported to have beneficial effects on inhibition of diabetes progression and complication . indeed , lifestyle approach is safe , and in the long run ( 24 years ) , effectively reduces type-2-diabetes incidence by 28%58%.1113 however , lifestyle interventions are notoriously difficult to maintain over a long period . among the antidiabetic agents , thiazolidinediones , particularly pioglitazone , showed the most remarkable risk reduction ( ie , up to 72% ) of igt conversion to type-2-diabetes.3,4,18 however , long - term use of pioglitazone ( > 2 years ) was associated with discernible weight gain and edema,18 both of which are not favorable with respect to reducing cardiovascular risks . because of these limitations of currently available interventions , there is a need to introduce a new substance for the treatment of prediabetes . dlbs3233 , a novel bioactive fraction derived from the plants cinnamomum burmanii and lagerstroemia speciosa , has previously been studied for its insulin - sensitizing activity.20,21 c. burmanii was obtained from kerinci , jambi , indonesia , and l. speciosa from cianjur , west java , indonesia . both the plants have been identified by herbarium bogoriense , research center for biology , indonesian institute of sciences , with reference no 1261/iph.1.02/if.8/xii/2009 . a previous study demonstrated the safety and tolerability of the bioactive fraction dlbs3233 in healthy volunteers.22 an unpublished preliminary study by suastika et al23 also demonstrated the favorable effects of dlbs3233 on glycemic control in newly diagnosed type-2-diabetes patients . in this study , dlbs3233 was preliminarily evaluated for its efficacy and safety in improving insulin resistance and preserving -cell performance as well as for its clinical impacts in patients with igt . this study was conducted in compliance with the declaration of helsinki , good clinical practice , and relevant local regulatory requirements . the study protocol was reviewed and approved by the independent ethics committee of andalas university , padang , indonesia , prior to trial initiation . written informed consent from each subject was obtained prior to screening . subjects with igt , defined as 2-hour postprandial glucose ( 2h - pg ) level of 140199 mg / dl assessed by the oral glucose tolerance test ( ogtt),2 were recruited from the clinic of internal medicine department , dr m djamil padang hospital , and enrolled in this two - arm , prospective , double - blind , randomized , and controlled study . pregnant or nursing women , subjects with diabetes mellitus , history of symptomatic coronary arterial disease , stroke , and any cardiovascular events , impaired liver function with serum alanine aminotransferase ( alt ) > 2.5 times the upper limit of normal ( uln ) , impaired renal function with serum creatinine 1.5 times uln , uncontrolled chronic diseases , and acute or chronic infections , and those undergoing treatment with systemic corticosteroids or herbal ( alternative ) medicines were excluded . eligible subjects were randomly allocated to receive either dlbs3233 or dlbs3233-matched placebo for a total of 12 weeks of therapy . on the day of enrollment ( at baseline ) , in the morning after a 10-hour overnight fast , subjects underwent a standard 75 g ogtt . plasma glucose and insulin concentrations were determined from venous blood samples drawn at time points of 0 , 15 , and 120 minutes during ogtt . all clinical and laboratory examinations measuring the investigational product s efficacy were performed at baseline , week 8 , and week 12 ( end ) of study treatment . in addition , fasting plasma glucose and 2h - pg were also measured at week 4 . safety laboratory examinations ( listed in last paragraph of subjects and study design section ) were performed at baseline and the end of study , while vital signs and body weight were measured at baseline and at intervals of 4 weeks over the study period . insulin sensitivity was quantified as a measure of hepatic insulin sensitivity during ogtt reflected by homeostatic model assessment insulin resistance ( homa - ir ) index : ( homa - ir = ins0(u / ml)glc0(mmol / l)22.5)24.(1 ) in order to indicate -cell performance , plasma insulin levels at 15 minutes ( early phase ) and 2 hours ( second phase ) during ogtt were measured , and the oral disposition index ( dio ) of the early phase during ogtt : ( dio=ins015(u / ml)glc015(mg / dl)homa - ir)4,25(2)was also calculated . further , other metabolic end points , such as reduction of 15-minute postprandial glucose and 2h - pg , glucose metabolic clearance rate ( mcr ) which was estimated from fasting and 2-hour plasma insulin and glucose levels during ogtt as described by stumvoll et al ( mcr = 13,273 ( 0.00384 ins120 ) ( 0.0232 ins0 ) ( 0.463 glc120))26,27 , and fasting triglyceride level , were also measured . safety end points were the following : body weight , vital signs , and electrocardiography ; serum alt , -glutamyl transferase , and alkaline phosphatase ; and serum creatinine . the study product , dlbs3233 , a bioactive fraction ( dexa laboratories of biomolecular sciences , pt dexa medica , cikarang , indonesia ) , contained natural components extracted from the indonesian native plants : c. burmanii and l. speciosa . the details of preparation and extraction procedure as well as the phytochemical characterization of dlbs3233 were as previously described.20,22 dlbs3233 was pharmaceutically formulated in the dosage form of capsules , each of which contained 50 mg of the bioactive fraction . the study medication was given at the dose of one capsule once daily for the first 4 weeks . based on the evaluation of subjects response to treatment at week 4 , subjects in both groups who were good responders , defined as those who achieved 2h - pg level < 140 mg / dl or a reduction of 2h - pg 10% from baseline , remained at the same regimen , while the poor responders received a titrated dose of two capsules once daily , for the subsequent 8 weeks . at each study visit , subjects were provided with education on lifestyle modification , such as regular exercise and dietary advice , by the assigned nutritionist . any systemic medications other than the study product that were considered to interfere with the study evaluation , such as antidiabetic agents , systemic corticosteroids , or herbal ( alternative ) medicines , were not allowed to be taken during the study . efficacy analyses were carried out on intent - to - treat population , consisting of all patients who were randomized , exposed to at least one dose of the study product , and then attended at least one follow - up after treatment initiation . safety analyses were based on data of all patients who received at least one dose of study product . characteristic comparability between groups at baseline was assessed by independent t - test for continuous data , or by pearson chi - square test for categorical data . changes from baseline of homa - ir , plasma insulin levels , dio , other measured metabolic parameters , vital signs , as well as body weight at each visit were analyzed individually between groups by independent t - test . in addition , each individual variable was analyzed within group by repeated - measures analysis of variance , followed by contrast analysis . laboratory safety variables at baseline and end of study were statistically analyzed within group by paired t - test , and between groups by independent t - test . throughout the analysis , the underlying distributional assumptions of the statistical models should have been met ; otherwise , the corresponding nonparametric tests were used . adverse events were presented by system organ class and world health organization adverse reaction dictionary - preferred term . this study was conducted in compliance with the declaration of helsinki , good clinical practice , and relevant local regulatory requirements . the study protocol was reviewed and approved by the independent ethics committee of andalas university , padang , indonesia , prior to trial initiation . written informed consent from each subject was obtained prior to screening . subjects with igt , defined as 2-hour postprandial glucose ( 2h - pg ) level of 140199 mg / dl assessed by the oral glucose tolerance test ( ogtt),2 were recruited from the clinic of internal medicine department , dr m djamil padang hospital , and enrolled in this two - arm , prospective , double - blind , randomized , and controlled study . pregnant or nursing women , subjects with diabetes mellitus , history of symptomatic coronary arterial disease , stroke , and any cardiovascular events , impaired liver function with serum alanine aminotransferase ( alt ) > 2.5 times the upper limit of normal ( uln ) , impaired renal function with serum creatinine 1.5 times uln , uncontrolled chronic diseases , and acute or chronic infections , and those undergoing treatment with systemic corticosteroids or herbal ( alternative ) medicines were excluded . eligible subjects were randomly allocated to receive either dlbs3233 or dlbs3233-matched placebo for a total of 12 weeks of therapy . on the day of enrollment ( at baseline ) , in the morning after a 10-hour overnight fast , subjects underwent a standard 75 g ogtt . plasma glucose and insulin concentrations were determined from venous blood samples drawn at time points of 0 , 15 , and 120 minutes during ogtt . all clinical and laboratory examinations measuring the investigational product s efficacy were performed at baseline , week 8 , and week 12 ( end ) of study treatment . in addition , fasting plasma glucose and 2h - pg were also measured at week 4 . safety laboratory examinations ( listed in last paragraph of subjects and study design section ) were performed at baseline and the end of study , while vital signs and body weight were measured at baseline and at intervals of 4 weeks over the study period . insulin sensitivity was quantified as a measure of hepatic insulin sensitivity during ogtt reflected by homeostatic model assessment insulin resistance ( homa - ir ) index : ( homa - ir = ins0(u / ml)glc0(mmol / l)22.5)24.(1 ) in order to indicate -cell performance , plasma insulin levels at 15 minutes ( early phase ) and 2 hours ( second phase ) during ogtt were measured , and the oral disposition index ( dio ) of the early phase during ogtt : ( dio=ins015(u / ml)glc015(mg / dl)homa - ir)4,25(2)was also calculated . further , other metabolic end points , such as reduction of 15-minute postprandial glucose and 2h - pg , glucose metabolic clearance rate ( mcr ) which was estimated from fasting and 2-hour plasma insulin and glucose levels during ogtt as described by stumvoll et al ( mcr = 13,273 ( 0.00384 ins120 ) ( 0.0232 ins0 ) ( 0.463 glc120))26,27 , and fasting triglyceride level , were also measured . safety end points were the following : body weight , vital signs , and electrocardiography ; serum alt , -glutamyl transferase , and alkaline phosphatase ; and serum creatinine . the study product , dlbs3233 , a bioactive fraction ( dexa laboratories of biomolecular sciences , pt dexa medica , cikarang , indonesia ) , contained natural components extracted from the indonesian native plants : c. burmanii and l. speciosa . the details of preparation and extraction procedure as well as the phytochemical characterization of dlbs3233 were as previously described.20,22 dlbs3233 was pharmaceutically formulated in the dosage form of capsules , each of which contained 50 mg of the bioactive fraction . the study medication was given at the dose of one capsule once daily for the first 4 weeks . based on the evaluation of subjects response to treatment at week 4 , subjects in both groups who were good responders , defined as those who achieved 2h - pg level < 140 mg / dl or a reduction of 2h - pg 10% from baseline , remained at the same regimen , while the poor responders received a titrated dose of two capsules once daily , for the subsequent 8 weeks . at each study visit , subjects were provided with education on lifestyle modification , such as regular exercise and dietary advice , by the assigned nutritionist . any systemic medications other than the study product that were considered to interfere with the study evaluation , such as antidiabetic agents , systemic corticosteroids , or herbal ( alternative ) medicines , were not allowed to be taken during the study . efficacy analyses were carried out on intent - to - treat population , consisting of all patients who were randomized , exposed to at least one dose of the study product , and then attended at least one follow - up after treatment initiation . safety analyses were based on data of all patients who received at least one dose of study product . characteristic comparability between groups at baseline was assessed by independent t - test for continuous data , or by pearson chi - square test for categorical data . changes from baseline of homa - ir , plasma insulin levels , dio , other measured metabolic parameters , vital signs , as well as body weight at each visit were analyzed individually between groups by independent t - test . in addition , each individual variable was analyzed within group by repeated - measures analysis of variance , followed by contrast analysis . laboratory safety variables at baseline and end of study were statistically analyzed within group by paired t - test , and between groups by independent t - test . throughout the analysis , the underlying distributional assumptions of the statistical models should have been met ; otherwise , the corresponding nonparametric tests were used . adverse events were presented by system organ class and world health organization adverse reaction dictionary - preferred term . the number of events and subjects with each event were tabulated . 74 subjects ( 92.5% ) completed the study and were available for intent - to - treat analysis , with a mean age of 46.97.6 and 50.57.3 years , in dlbs3233 and placebo groups , respectively , and both groups were of female predominance ( 92.1% and 58.3% , respectively ) . three subjects ( two in dlbs3233 and one in placebo groups ) were withdrawn due to noncompliance with the study protocol , one ( in placebo group ) due to pregnancy , one ( in placebo group ) due to adverse event ( abdominal pain ) , and the remaining one patient ( in placebo group ) moved to another town and did not return for evaluation . those six subjects could not be evaluated for efficacy and laboratory safety since they had no available posttreatment data . with respect to cardiovascular risk factors and metabolic disorders , 50.0% and 63.9% of subjects in dlbs3233 and placebo groups , respectively , had hypertension ; 31.6% and 38.9% , respectively , had dyslipidemia ; and 18.4% and 13.9% , respectively , were obese . all subjects received adequate treatment for such relevant concomitant illnesses during the study participation , with captopril and amlodipine as the predominant antihypertensive agents and simvastatin as the antihyperlipidemic agent used by study subjects in both groups . the mean blood pressures ( bps ) were not significantly different between groups , with systolic bp of dlbs3233 and placebo group at 125.316.0 and 133.216.7 mmhg , respectively ( p=0.326 ) , and diastolic bp at 81.310.4 and 85.314.2 mmhg , respectively ( p=0.181 ) . baseline levels of the observed efficacy and laboratory safety parameters ( tables 1 and 2 , respectively ) were all comparable between groups . sixteen subjects ( 42.1% ) in dlbs3233 group were poor responders and had their dose regimen titrated from 50 to 100 mg at week 4 till the end of study , while 15 ( 41.7% ) subjects in placebo group who met the nonresponders criteria also had their regimen titrated in order to maintain the blinding fashion . the remaining subjects were all maintained at the same regimen ( 50 mg daily ) until the end of study . in contrast , fasting insulin in both groups decreased significantly . when fasting glucose and fasting insulin were combined as an index of hepatic insulin resistance ( homa - ir ) , a more marked improvement was found in dlbs3233 group than in placebo group ( figure 1a ) . a significant reduction of homa - ir from baseline in dlbs3233 group was evident , from 3.001.76 to 2.321.32 ( p=0.002 ) and to 2.161.17 ( p=0.001 ) at week 8 and week 12 , respectively . in placebo group , a significant reduction from baseline was found at week 8 ( from 2.762.28 to 2.030.99 , p=0.005 ) , but it was then diminished at week 12 ( to 2.281.24 , p=0.158 vs baseline ) . at the end of study , the percentage of homa - ir reduction from baseline was significantly greater in dlbs3233 group than that in placebo group ( 27.0429.41 vs 4.9041.27 , p=0.013 ) ( figure 1a ) . at baseline , an elevated first - phase insulin was observed at comparable level ( p=0.088 ) in both groups ( figure 1b ) . a significant improvement ( normalization ) of the insulin level was observed in dlbs3233 group , from 435.27223.19 pmol / l at baseline , to 301.47185.57 pmol / l ( p<0.001 ) at week 8 , and then to 290.48132.44 pmol / l ( p<0.001 ) at week 12 . the changes observed in placebo group were not statistically significant ( p>0.05 ) , from 378.08167.22 to 288.85188.94 pmol / l , and then to 306.87184.92 pmol / l , respectively . the sizes of improvement at week 8 and week 12 were also significantly greater in dlbs3233 group than that in placebo group ( figure 1b ) . aligned with the improvement of first - phase insulin level , the second - phase insulin level at week 8 and week 12 was also found to improve markedly in dlbs3233 group , from 1,113.72550.47 to 752.31550.24 pmol / l ( p<0.001 ) , and then to 658.70493.67 pmol / l ( p<0.001 ) , respectively . in contrast , we found no significant improvement in placebo group ( p>0.05 ) , where the level went down from 1,004.34467.01 to 758.35459.89 pmol / l , and then to 734.86517.54 pmol / l , respectively . at week 12 , the amount of improvement by dlbs3233 was significantly greater than that by placebo ( figure 1c ) . the preservation of -cell performance calculated as the dio increased significantly only with dlbs3233 but was absent in placebo group ( table 1 ) , with the increase at week 8 being greater in dlbs3233 group than in placebo group ( 0.250.81 vs 0.161.03 , p=0.032 ) . in line with the augmentation of insulin sensitivity ( reduced homa - ir ) , we also found an increase of mcr of glucose at week 8 and week 12 , in both groups ( table 1 ) , with the enhancement of mcr at week 12 being higher in dlbs3233 group than in placebo group ( 2.852.79 ml / kg / min vs 1.703.05 ml / kg / min , p=0.044 ) . in terms of 15-minute postprandial glucose and 2h - pg during ogtt , despite the significant reduction observed in dlbs3233 group , the difference between groups was not statistically significant ( table 1 ) . a significantly lower level of triglyceride after 8 and 12 weeks of treatment was seen in dlbs3233 group , from 1.490.70 mmol / l at baseline , to 1.200.39 mmol / l ( p=0.008 ) , and then to 1.170.43 mmol / l ( p=0.004 ) , respectively . in comparison with the placebo , the reductions of triglyceride level from baseline showed a trend in favor of dlbs3233 ( table 1 ) . interestingly , after 12 weeks of treatment , a greater reduction of systolic and diastolic bps was found with dlbs3233 ( table 2 ) . in dlbs3233 and placebo groups , the systolic bps were reduced from 125.316.0 to 118.913.9 mmhg ( p=0.035 ) vs 133.226.7 to 129.018.3 mmhg ( p=0.600 ) , and the diastolic bps from 81.310.4 to 76.69.4 mmhg ( p=0.026 ) vs 85.314.2 to 81.98.2 mmhg ( p=0.200 ) . all study subjects with hypertension in both groups had controlled bps at baseline and received adequate and generally similar antihypertensive treatment during the study . reductions in alt and alkaline phosphatase after 12-week treatment ( at end of study ) were found with dlbs3233 , as compared to their respective baseline levels . body weight was significantly reduced in both groups , indicating that dlbs3233 was not associated with weight gain . further , other than bps , the changes of each of the safety parameters from baseline to the end of treatment were not significantly different between groups . a total frequency of 180 adverse events occurred in 20 ( of 40 ) subjects in dlbs3233 group and 19 ( of 40 ) subjects in placebo group ( table 3 ) . of them , adverse events that were regarded possibly associated with the study product were dizziness , headache , tremor , and palpitation . all adverse events were mild in severity and subjects had fully recovered or stabilized at the end of study . 74 subjects ( 92.5% ) completed the study and were available for intent - to - treat analysis , with a mean age of 46.97.6 and 50.57.3 years , in dlbs3233 and placebo groups , respectively , and both groups were of female predominance ( 92.1% and 58.3% , respectively ) . three subjects ( two in dlbs3233 and one in placebo groups ) were withdrawn due to noncompliance with the study protocol , one ( in placebo group ) due to pregnancy , one ( in placebo group ) due to adverse event ( abdominal pain ) , and the remaining one patient ( in placebo group ) moved to another town and did not return for evaluation . those six subjects could not be evaluated for efficacy and laboratory safety since they had no available posttreatment data . with respect to cardiovascular risk factors and metabolic disorders , 50.0% and 63.9% of subjects in dlbs3233 and placebo groups , respectively , had hypertension ; 31.6% and 38.9% , respectively , had dyslipidemia ; and 18.4% and 13.9% , respectively , were obese . all subjects received adequate treatment for such relevant concomitant illnesses during the study participation , with captopril and amlodipine as the predominant antihypertensive agents and simvastatin as the antihyperlipidemic agent used by study subjects in both groups . the mean blood pressures ( bps ) were not significantly different between groups , with systolic bp of dlbs3233 and placebo group at 125.316.0 and 133.216.7 mmhg , respectively ( p=0.326 ) , and diastolic bp at 81.310.4 and 85.314.2 mmhg , respectively ( p=0.181 ) . baseline levels of the observed efficacy and laboratory safety parameters ( tables 1 and 2 , respectively ) were all comparable between groups . sixteen subjects ( 42.1% ) in dlbs3233 group were poor responders and had their dose regimen titrated from 50 to 100 mg at week 4 till the end of study , while 15 ( 41.7% ) subjects in placebo group who met the nonresponders criteria also had their regimen titrated in order to maintain the blinding fashion . the remaining subjects were all maintained at the same regimen ( 50 mg daily ) until the end of study . in contrast , fasting insulin in both groups decreased significantly . when fasting glucose and fasting insulin were combined as an index of hepatic insulin resistance ( homa - ir ) , a more marked improvement was found in dlbs3233 group than in placebo group ( figure 1a ) . a significant reduction of homa - ir from baseline in dlbs3233 group was evident , from 3.001.76 to 2.321.32 ( p=0.002 ) and to 2.161.17 ( p=0.001 ) at week 8 and week 12 , respectively . in placebo group , a significant reduction from baseline was found at week 8 ( from 2.762.28 to 2.030.99 , p=0.005 ) , but it was then diminished at week 12 ( to 2.281.24 , p=0.158 vs baseline ) . at the end of study , the percentage of homa - ir reduction from baseline was significantly greater in dlbs3233 group than that in placebo group ( 27.0429.41 vs 4.9041.27 , p=0.013 ) ( figure 1a ) . at baseline , an elevated first - phase insulin was observed at comparable level ( p=0.088 ) in both groups ( figure 1b ) . a significant improvement ( normalization ) of the insulin level was observed in dlbs3233 group , from 435.27223.19 pmol / l at baseline , to 301.47185.57 pmol / l ( p<0.001 ) at week 8 , and then to 290.48132.44 pmol / l ( p<0.001 ) at week 12 . the changes observed in placebo group were not statistically significant ( p>0.05 ) , from 378.08167.22 to 288.85188.94 pmol / l , and then to 306.87184.92 pmol / l , respectively . the sizes of improvement at week 8 and week 12 were also significantly greater in dlbs3233 group than that in placebo group ( figure 1b ) . aligned with the improvement of first - phase insulin level , the second - phase insulin level at week 8 and week 12 was also found to improve markedly in dlbs3233 group , from 1,113.72550.47 to 752.31550.24 pmol / l ( p<0.001 ) , and then to 658.70493.67 pmol / l ( p<0.001 ) , respectively . in contrast , we found no significant improvement in placebo group ( p>0.05 ) , where the level went down from 1,004.34467.01 to 758.35459.89 pmol / l , and then to 734.86517.54 pmol / l , respectively . at week 12 , the amount of improvement by dlbs3233 was significantly greater than that by placebo ( figure 1c ) . the preservation of -cell performance calculated as the dio increased significantly only with dlbs3233 but was absent in placebo group ( table 1 ) , with the increase at week 8 being greater in dlbs3233 group than in placebo group ( 0.250.81 vs 0.161.03 , p=0.032 ) . in line with the augmentation of insulin sensitivity ( reduced homa - ir ) , we also found an increase of mcr of glucose at week 8 and week 12 , in both groups ( table 1 ) , with the enhancement of mcr at week 12 being higher in dlbs3233 group than in placebo group ( 2.852.79 ml / kg / min vs 1.703.05 ml / kg / min , p=0.044 ) . in terms of 15-minute postprandial glucose and 2h - pg during ogtt , despite the significant reduction observed in dlbs3233 group , the difference between groups was not statistically significant ( table 1 ) . a significantly lower level of triglyceride after 8 and 12 weeks of treatment was seen in dlbs3233 group , from 1.490.70 mmol / l at baseline , to 1.200.39 mmol / l ( p=0.008 ) , and then to 1.170.43 mmol / l ( p=0.004 ) , respectively . in comparison with the placebo , the reductions of triglyceride level from baseline showed a trend in favor of dlbs3233 ( table 1 ) . interestingly , after 12 weeks of treatment , a greater reduction of systolic and diastolic bps was found with dlbs3233 ( table 2 ) . in dlbs3233 and placebo groups , the systolic bps were reduced from 125.316.0 to 118.913.9 mmhg ( p=0.035 ) vs 133.226.7 to 129.018.3 mmhg ( p=0.600 ) , and the diastolic bps from 81.310.4 to 76.69.4 mmhg ( p=0.026 ) vs 85.314.2 to 81.98.2 mmhg ( p=0.200 ) . all study subjects with hypertension in both groups had controlled bps at baseline and received adequate and generally similar antihypertensive treatment during the study . reductions in alt and alkaline phosphatase after 12-week treatment ( at end of study ) were found with dlbs3233 , as compared to their respective baseline levels . body weight was significantly reduced in both groups , indicating that dlbs3233 was not associated with weight gain . further , other than bps , the changes of each of the safety parameters from baseline to the end of treatment were not significantly different between groups . a total frequency of 180 adverse events occurred in 20 ( of 40 ) subjects in dlbs3233 group and 19 ( of 40 ) subjects in placebo group ( table 3 ) . of them , adverse events that were regarded possibly associated with the study product were dizziness , headache , tremor , and palpitation . all adverse events were mild in severity and subjects had fully recovered or stabilized at the end of study . patients with igt are at high risk of developing type-2-diabetes since they already have insulin resistance and may have lost a large amount of -cell function.4 igt is also a strong predictor of metabolic syndrome and arteriosclerotic heart disease . approximately 22% of individuals with igt have at least one microvascular complication.28,29 therefore , nowadays , igt has been attracting more attention for primary prevention of type-2-diabetes and its macrovascular complications . a growing body of evidence demonstrates that both non - pharmacological and pharmacological interventions in igt have been effective in preventing or delaying macrovascular complications and conversion to overt diabetes . our study preliminarily demonstrated the benefits of dlbs3233 in improving insulin resistance and preserving -cell performance in subjects with igt . in this study , subjects were diagnosed to have igt based on the ogtt . -cells respond to an increment in glucose ( glc ) with an increment in insulin ( ins ) , and this response is modulated by the severity of insulin resistance . therefore , -cell performance is more appropriately measured as a function of both insulin secretory response to glucose stimulus and insulin resistance . simply measuring plasma insulin response to assess -cell function may be misleading.4 in our study , we included both the insulin level and insulin resistance to estimate -cell performance as reflected by the dio . after 12 weeks of treatment , the insulin resistance was ameliorated in dlbs3233 group as demonstrated by a significant homa - ir reduction ( figure 1a ) . our study showed that augmentation of insulin sensitivity by dlbs3233 , through its insulin - sensitizing properties,20,21 was finally compensated by a significant reduction which can also be translated as normalization of the first- and second - phase insulin levels as shown in dlbs3233-treated group after 8 and 12 weeks . a former study by kahn30 showed a similar finding that when insulin sensitivity was improved by exercise , a reduced insulin secretion was observed . another study by ferrannini et al31 and gastaldelli et al32 demonstrated that improvement of insulin sensitivity by insulin sensitizers was associated with a decline in plasma insulin response . the decline in the first- and second - phase insulin response has been generally interpreted to reflect the normal compensatory response of -cells to the improvement of insulin sensitivity and/or reduced plasma glucose level . guldstrand et al33 demonstrated that the reduction in insulin secretion was quantitatively not as prominent as the increase in insulin sensitivity , which increased the disposition index along with weight reduction . in our study , we also observed that the increased insulin sensitivity and reduced insulin demand by dlbs3233 treatment simultaneously led to the enhanced performance of -cells , as confirmed by a significant increase of the dio . increased however , whether dlbs3233 exerts a direct effect on the pancreatic -cells to enhance insulin secretion remains to be studied . in line with the augmentation of insulin sensitivity , the rate of clearance ( mcr ) of the glucose from circulation was promoted , at a significantly greater extent than placebo . nevertheless , the reduction of both 15-minute and 2-hour postprandial glucose levels by dlbs3233 was not significantly different than that of placebo . yet , as has been elaborately described earlier , the finding that the required 15-minute and 2-hour plasma insulin secretion to maintain the normal level of postprandial glucose was normalized ( lowered ) was of clinical importance ; it consistently indicated an improvement of insulin sensitivity . bergman et al34 and kahn et al35 identified the hyperbolic relationship between insulin sensitivity and secretion . the clinical consequence of the hyperbolic relationship is that increased insulin sensitivity will be compensated by reduction , or downregulation , of -cell function , which may be a mechanism to avoid hypoglycemic episodes.36 such an inverse relationship between insulin sensitivity and insulin secretion indicates that it is impossible to judge insulin secretion in a given individual without knowing the ambient insulin sensitivity , since both increases and decreases of insulin sensitivity are associated with compensatory reciprocal changes in insulin secretion . in brief , the present study demonstrated that the dlbs3233 treatment augmented insulin sensitivity and simultaneously preserved the performance of -cell in regulating insulin secretion . the finding also indicated a more effective utilization of insulin by both the liver and peripheral tissues . we also noted that even though preservation or improvement of -cell performance did not occur in placebo group , the insulin sensitivity index ( homa - ir ) was better than the corresponding baseline . the improvement observed in placebo group was possibly attributed to the lifestyle education given to all study subjects during their participation in the study . lifestyle changes have been proven to delay the progression of igt to type-2-diabetes.37 however , non - adherence to the lifestyle modification for a long period is a major constraint . therefore , the use of pharmacological therapy , such as dlbs3233 , is likely to offset , at least in part , the adverse impacts of non - adherence to lifestyle changes . about one - third of subjects in dlbs3233 and placebo groups had dyslipidemia , but all had been adequately treated before screening . therefore , at study entry , the average triglyceride levels of both groups were within the normal range . however , since the insulin resistance was markedly improved with dlbs3233 , it was not surprising that a substantial reduction of triglyceride level from baseline was still observed in that group , after 8 and 12 weeks of treatment . at both time points , the findings of this study were consistent with the results of our previous study in insulin - resistant wistar rats.20 the augmentation of insulin sensitivity found in this study can be explained by the action of dlbs3233 in modulating the expression of genes associated with insulin signaling and sensitivity , such as peroxisome proliferators - activator receptor ( ppar)- and ppar-. in vitro studies with 3t3-swiss - albino preadipocyte cells showed that stimulation of insulin - signaling transduction by dlbs3233 occurred through the promotion of tyrosine phosphorylation of the insulin - receptor - substrate and upregulation of the expression of phosphatidylinositol-3-kinase , ppar- , ppar- , and glucose transporter-4 at the mrna level.20,21 activation of ppar- and ppar- by dlbs3233 leads to a series of beneficial metabolic effects on glucose and lipids , suggesting that the product possesses the potential capacity to decrease the incidence of insulin resistance - associated diseases . the mechanism underlying the beneficial effect of dlbs3233 on bp is probably associated with the reduced plasma insulin level and improvement of insulin resistance . it has been widely known that insulin resistance , through a complex physiological interaction with hyperinsulinemia makes an independent contribution to the elevation of bp.38 therefore , improvement in insulin sensitivity will produce a certain reduction in bp , independent of other factors.39 other drugs that improve insulin sensitivity also demonstrated their effects in lowering bp , such as rosiglitazone,39 pioglitazone,18 and metformin.40 the data suggest that along with its virtues on glucose metabolism , dlbs3233 might also provide protection against the development of cardiovascular disease in prediabetic subjects . whether or not dlbs3233 has direct effects on the vascular system with respect to the safety parameters , the reductions in serum alt and alkaline phosphatase observed with dlbs3233 were all not clinically significant , indicating the safety of treatment , with a trend of protective effect on the liver . a slight increase of the level of serum creatinine observed at the end of treatment with dlbs3233 was still far below the uln , and the increment was not clinically significant either . no serious or severe vascular - related adverse events occurred in either group during the study . even edema which was commonly found with use of insulin sensitizers was not found with dlbs3233 treatment . significant reduction of body weight from baseline was observed in both groups , but still , there was no significant difference between groups . other than appendicitis which was unlikely to have causal relationship with the study product , all adverse events were mild in severity . first , we had limited control of the implementation of lifestyle modification during the study , where only lifestyle education and motivation were provided on a regular basis . however , employing random allocation in the study should ensure the equal distribution between groups , of adherent and non - adherent subjects to lifestyle modification . the second limitation is that the duration of this study was too short for us to evaluate the ultimate goal of treating igt subjects , that is , the prevention of conversion to type-2-diabetes . however , this study may serve as a sound ground to justify a larger outcome research with a longer duration of treatment and more refined study design to evaluate the risk reduction of type-2-diabetes by dlbs3233 treatment in prediabetic subjects and to confirm the real benefits of dlbs3233 treatment in prediabetes . this study demonstrated that dlbs3233 treatment at the dose of 50100 mg once daily was promisingly efficacious in improving insulin sensitivity and preserving the performance of -cells . the product was also very well tolerated without unfavorable effects on then cardiovascular system , such as weight gain and edema .

backgroundthe aim of this paper is to evaluate the efficacy and safety of dlbs3233 , a novel bioactive fraction derived from cinnamomum burmanii and lagerstroemia speciosa , in improving insulin resistance and preserving -cell performance in patients with impaired glucose tolerance ( igt).patients and methodseighty adult subjects with igt , defined as 2-hour postprandial glucose level of 140199 mg / dl , were enrolled in this two - arm , 12-week , double - blind , randomized , placebo - controlled preliminary study . eligible subjects were randomly allocated to receive either dlbs3233 at a dose of 50100 mg daily or placebo for 12 weeks . the study mainly assessed the improvement of homeostatic model - assessed insulin resistance ( homa - ir ) , the 15-minute and 2-hour plasma insulin levels , and the oral disposition index.resultsafter 12 weeks , dlbs3233 improved insulin resistance better than placebo as reflected by a reduced homa - ir ( 27.04%29.41% vs 4.90%41.27% , p=0.013 ) . the improvement of the first- and second - phase insulin secretion was consistently greater in dlbs3233 group than placebo group ( 144.78194.06 vs 71.21157.19 , p=0.022 , and 455.03487.56 vs 269.49467.77 , p=0.033 , respectively ) . further , dlbs3233 also significantly better improved oral disposition index than placebo . no serious hypoglycemia , edema , or cardiovascular - related adverse events were found in either groups.conclusionthis study has shown that dlbs3233 at the dose of 50100 mg once daily was well tolerated , and promisingly efficacious in improving insulin sensitivity as well as preserving -cell performance in subjects with igt .

Introduction Materials and methods Subjects and study design Study treatment Statistical analysis Results Participants and baseline characteristics Improved insulin sensitivity Improved -cell performance Improved metabolic parameters: glucose MCR, postprandial plasma glucose, and triglyceride levels Additional beneficial effect on the BP Safety to organ functions Adverse events Discussion Conclusion

a growing body of evidence shows that prediabetic condition is a risk factor for type-2-diabetes and cardiovascular complications.13 prediabetes is defined as impaired glucose tolerance ( igt ) and/or impaired fasting glucose.2 igt is associated with metabolic syndrome and is a strong predictor of atherosclerotic cardiovascular disease , whereas impaired fasting glucose is a much weaker predictor.4 there are no less than 300 million people with prediabetic condition worldwide , and the number is predicted to rise up to 418 million by 2025 . every year , 2%11% of the prediabetic population are converting to type-2-diabetics.1,2,4,5 therefore , early intervention in the prediabetic stage is a valuable approach to reduce diabetes development and prevent related complications.3,6,7 insulin resistance , -cell dysfunction , and uncontrolled calorie consumption have been the crucial contributors to acute postprandial hyperglycemia and are the potential agents that convert prediabetes to diabetes.4,8,9 the acute postprandial hyperglycemia is responsible for glucotoxicity in the pathophysiology of type-2-diabetes even at the earlier stage ( ie , prediabetes).10 further in a vicious cycle , the impairment of -cell function and exacerbation of insulin resistance due to glucotoxicity play a crucial role in the progression of diabetes and development of its cardiovascular complications.4,10 interventions that preserve -cell function and ameliorate insulin resistance have been proven to be most effective in preventing progression to diabetes . dlbs3233 , a novel bioactive fraction derived from the plants cinnamomum burmanii and lagerstroemia speciosa , has previously been studied for its insulin - sensitizing activity.20,21 c. burmanii was obtained from kerinci , jambi , indonesia , and l. speciosa from cianjur , west java , indonesia . in this study , dlbs3233 was preliminarily evaluated for its efficacy and safety in improving insulin resistance and preserving -cell performance as well as for its clinical impacts in patients with igt . subjects with igt , defined as 2-hour postprandial glucose ( 2h - pg ) level of 140199 mg / dl assessed by the oral glucose tolerance test ( ogtt),2 were recruited from the clinic of internal medicine department , dr m djamil padang hospital , and enrolled in this two - arm , prospective , double - blind , randomized , and controlled study . eligible subjects were randomly allocated to receive either dlbs3233 or dlbs3233-matched placebo for a total of 12 weeks of therapy . insulin sensitivity was quantified as a measure of hepatic insulin sensitivity during ogtt reflected by homeostatic model assessment insulin resistance ( homa - ir ) index : ( homa - ir = ins0(u / ml)glc0(mmol / l)22.5)24. (1 ) in order to indicate -cell performance , plasma insulin levels at 15 minutes ( early phase ) and 2 hours ( second phase ) during ogtt were measured , and the oral disposition index ( dio ) of the early phase during ogtt : ( dio=ins015(u / ml)glc015(mg / dl)homa - ir)4,25(2)was also calculated . further , other metabolic end points , such as reduction of 15-minute postprandial glucose and 2h - pg , glucose metabolic clearance rate ( mcr ) which was estimated from fasting and 2-hour plasma insulin and glucose levels during ogtt as described by stumvoll et al ( mcr = 13,273 ( 0.00384 ins120 ) ( 0.0232 ins0 ) ( 0.463 glc120))26,27 , and fasting triglyceride level , were also measured . the study product , dlbs3233 , a bioactive fraction ( dexa laboratories of biomolecular sciences , pt dexa medica , cikarang , indonesia ) , contained natural components extracted from the indonesian native plants : c. burmanii and l. speciosa . the study medication was given at the dose of one capsule once daily for the first 4 weeks . based on the evaluation of subjects response to treatment at week 4 , subjects in both groups who were good responders , defined as those who achieved 2h - pg level < 140 mg / dl or a reduction of 2h - pg 10% from baseline , remained at the same regimen , while the poor responders received a titrated dose of two capsules once daily , for the subsequent 8 weeks . efficacy analyses were carried out on intent - to - treat population , consisting of all patients who were randomized , exposed to at least one dose of the study product , and then attended at least one follow - up after treatment initiation . changes from baseline of homa - ir , plasma insulin levels , dio , other measured metabolic parameters , vital signs , as well as body weight at each visit were analyzed individually between groups by independent t - test . subjects with igt , defined as 2-hour postprandial glucose ( 2h - pg ) level of 140199 mg / dl assessed by the oral glucose tolerance test ( ogtt),2 were recruited from the clinic of internal medicine department , dr m djamil padang hospital , and enrolled in this two - arm , prospective , double - blind , randomized , and controlled study . eligible subjects were randomly allocated to receive either dlbs3233 or dlbs3233-matched placebo for a total of 12 weeks of therapy . insulin sensitivity was quantified as a measure of hepatic insulin sensitivity during ogtt reflected by homeostatic model assessment insulin resistance ( homa - ir ) index : ( homa - ir = ins0(u / ml)glc0(mmol / l)22.5)24. (1 ) in order to indicate -cell performance , plasma insulin levels at 15 minutes ( early phase ) and 2 hours ( second phase ) during ogtt were measured , and the oral disposition index ( dio ) of the early phase during ogtt : ( dio=ins015(u / ml)glc015(mg / dl)homa - ir)4,25(2)was also calculated . further , other metabolic end points , such as reduction of 15-minute postprandial glucose and 2h - pg , glucose metabolic clearance rate ( mcr ) which was estimated from fasting and 2-hour plasma insulin and glucose levels during ogtt as described by stumvoll et al ( mcr = 13,273 ( 0.00384 ins120 ) ( 0.0232 ins0 ) ( 0.463 glc120))26,27 , and fasting triglyceride level , were also measured . the study product , dlbs3233 , a bioactive fraction ( dexa laboratories of biomolecular sciences , pt dexa medica , cikarang , indonesia ) , contained natural components extracted from the indonesian native plants : c. burmanii and l. speciosa . the details of preparation and extraction procedure as well as the phytochemical characterization of dlbs3233 were as previously described.20,22 dlbs3233 was pharmaceutically formulated in the dosage form of capsules , each of which contained 50 mg of the bioactive fraction . based on the evaluation of subjects response to treatment at week 4 , subjects in both groups who were good responders , defined as those who achieved 2h - pg level < 140 mg / dl or a reduction of 2h - pg 10% from baseline , remained at the same regimen , while the poor responders received a titrated dose of two capsules once daily , for the subsequent 8 weeks . efficacy analyses were carried out on intent - to - treat population , consisting of all patients who were randomized , exposed to at least one dose of the study product , and then attended at least one follow - up after treatment initiation . changes from baseline of homa - ir , plasma insulin levels , dio , other measured metabolic parameters , vital signs , as well as body weight at each visit were analyzed individually between groups by independent t - test . 74 subjects ( 92.5% ) completed the study and were available for intent - to - treat analysis , with a mean age of 46.97.6 and 50.57.3 years , in dlbs3233 and placebo groups , respectively , and both groups were of female predominance ( 92.1% and 58.3% , respectively ) . three subjects ( two in dlbs3233 and one in placebo groups ) were withdrawn due to noncompliance with the study protocol , one ( in placebo group ) due to pregnancy , one ( in placebo group ) due to adverse event ( abdominal pain ) , and the remaining one patient ( in placebo group ) moved to another town and did not return for evaluation . the mean blood pressures ( bps ) were not significantly different between groups , with systolic bp of dlbs3233 and placebo group at 125.316.0 and 133.216.7 mmhg , respectively ( p=0.326 ) , and diastolic bp at 81.310.4 and 85.314.2 mmhg , respectively ( p=0.181 ) . when fasting glucose and fasting insulin were combined as an index of hepatic insulin resistance ( homa - ir ) , a more marked improvement was found in dlbs3233 group than in placebo group ( figure 1a ) . a significant reduction of homa - ir from baseline in dlbs3233 group was evident , from 3.001.76 to 2.321.32 ( p=0.002 ) and to 2.161.17 ( p=0.001 ) at week 8 and week 12 , respectively . at the end of study , the percentage of homa - ir reduction from baseline was significantly greater in dlbs3233 group than that in placebo group ( 27.0429.41 vs 4.9041.27 , p=0.013 ) ( figure 1a ) . the sizes of improvement at week 8 and week 12 were also significantly greater in dlbs3233 group than that in placebo group ( figure 1b ) . aligned with the improvement of first - phase insulin level , the second - phase insulin level at week 8 and week 12 was also found to improve markedly in dlbs3233 group , from 1,113.72550.47 to 752.31550.24 pmol / l ( p<0.001 ) , and then to 658.70493.67 pmol / l ( p<0.001 ) , respectively . in contrast , we found no significant improvement in placebo group ( p>0.05 ) , where the level went down from 1,004.34467.01 to 758.35459.89 pmol / l , and then to 734.86517.54 pmol / l , respectively . the preservation of -cell performance calculated as the dio increased significantly only with dlbs3233 but was absent in placebo group ( table 1 ) , with the increase at week 8 being greater in dlbs3233 group than in placebo group ( 0.250.81 vs 0.161.03 , p=0.032 ) . in line with the augmentation of insulin sensitivity ( reduced homa - ir ) , we also found an increase of mcr of glucose at week 8 and week 12 , in both groups ( table 1 ) , with the enhancement of mcr at week 12 being higher in dlbs3233 group than in placebo group ( 2.852.79 ml / kg / min vs 1.703.05 ml / kg / min , p=0.044 ) . a significantly lower level of triglyceride after 8 and 12 weeks of treatment was seen in dlbs3233 group , from 1.490.70 mmol / l at baseline , to 1.200.39 mmol / l ( p=0.008 ) , and then to 1.170.43 mmol / l ( p=0.004 ) , respectively . in dlbs3233 and placebo groups , the systolic bps were reduced from 125.316.0 to 118.913.9 mmhg ( p=0.035 ) vs 133.226.7 to 129.018.3 mmhg ( p=0.600 ) , and the diastolic bps from 81.310.4 to 76.69.4 mmhg ( p=0.026 ) vs 85.314.2 to 81.98.2 mmhg ( p=0.200 ) . a total frequency of 180 adverse events occurred in 20 ( of 40 ) subjects in dlbs3233 group and 19 ( of 40 ) subjects in placebo group ( table 3 ) . 74 subjects ( 92.5% ) completed the study and were available for intent - to - treat analysis , with a mean age of 46.97.6 and 50.57.3 years , in dlbs3233 and placebo groups , respectively , and both groups were of female predominance ( 92.1% and 58.3% , respectively ) . three subjects ( two in dlbs3233 and one in placebo groups ) were withdrawn due to noncompliance with the study protocol , one ( in placebo group ) due to pregnancy , one ( in placebo group ) due to adverse event ( abdominal pain ) , and the remaining one patient ( in placebo group ) moved to another town and did not return for evaluation . the mean blood pressures ( bps ) were not significantly different between groups , with systolic bp of dlbs3233 and placebo group at 125.316.0 and 133.216.7 mmhg , respectively ( p=0.326 ) , and diastolic bp at 81.310.4 and 85.314.2 mmhg , respectively ( p=0.181 ) . when fasting glucose and fasting insulin were combined as an index of hepatic insulin resistance ( homa - ir ) , a more marked improvement was found in dlbs3233 group than in placebo group ( figure 1a ) . a significant reduction of homa - ir from baseline in dlbs3233 group was evident , from 3.001.76 to 2.321.32 ( p=0.002 ) and to 2.161.17 ( p=0.001 ) at week 8 and week 12 , respectively . at the end of study , the percentage of homa - ir reduction from baseline was significantly greater in dlbs3233 group than that in placebo group ( 27.0429.41 vs 4.9041.27 , p=0.013 ) ( figure 1a ) . the sizes of improvement at week 8 and week 12 were also significantly greater in dlbs3233 group than that in placebo group ( figure 1b ) . aligned with the improvement of first - phase insulin level , the second - phase insulin level at week 8 and week 12 was also found to improve markedly in dlbs3233 group , from 1,113.72550.47 to 752.31550.24 pmol / l ( p<0.001 ) , and then to 658.70493.67 pmol / l ( p<0.001 ) , respectively . in contrast , we found no significant improvement in placebo group ( p>0.05 ) , where the level went down from 1,004.34467.01 to 758.35459.89 pmol / l , and then to 734.86517.54 pmol / l , respectively . the preservation of -cell performance calculated as the dio increased significantly only with dlbs3233 but was absent in placebo group ( table 1 ) , with the increase at week 8 being greater in dlbs3233 group than in placebo group ( 0.250.81 vs 0.161.03 , p=0.032 ) . in line with the augmentation of insulin sensitivity ( reduced homa - ir ) , we also found an increase of mcr of glucose at week 8 and week 12 , in both groups ( table 1 ) , with the enhancement of mcr at week 12 being higher in dlbs3233 group than in placebo group ( 2.852.79 ml / kg / min vs 1.703.05 ml / kg / min , p=0.044 ) . a significantly lower level of triglyceride after 8 and 12 weeks of treatment was seen in dlbs3233 group , from 1.490.70 mmol / l at baseline , to 1.200.39 mmol / l ( p=0.008 ) , and then to 1.170.43 mmol / l ( p=0.004 ) , respectively . in dlbs3233 and placebo groups , the systolic bps were reduced from 125.316.0 to 118.913.9 mmhg ( p=0.035 ) vs 133.226.7 to 129.018.3 mmhg ( p=0.600 ) , and the diastolic bps from 81.310.4 to 76.69.4 mmhg ( p=0.026 ) vs 85.314.2 to 81.98.2 mmhg ( p=0.200 ) . our study preliminarily demonstrated the benefits of dlbs3233 in improving insulin resistance and preserving -cell performance in subjects with igt . simply measuring plasma insulin response to assess -cell function may be misleading.4 in our study , we included both the insulin level and insulin resistance to estimate -cell performance as reflected by the dio . after 12 weeks of treatment , the insulin resistance was ameliorated in dlbs3233 group as demonstrated by a significant homa - ir reduction ( figure 1a ) . our study showed that augmentation of insulin sensitivity by dlbs3233 , through its insulin - sensitizing properties,20,21 was finally compensated by a significant reduction which can also be translated as normalization of the first- and second - phase insulin levels as shown in dlbs3233-treated group after 8 and 12 weeks . the decline in the first- and second - phase insulin response has been generally interpreted to reflect the normal compensatory response of -cells to the improvement of insulin sensitivity and/or reduced plasma glucose level . nevertheless , the reduction of both 15-minute and 2-hour postprandial glucose levels by dlbs3233 was not significantly different than that of placebo . yet , as has been elaborately described earlier , the finding that the required 15-minute and 2-hour plasma insulin secretion to maintain the normal level of postprandial glucose was normalized ( lowered ) was of clinical importance ; it consistently indicated an improvement of insulin sensitivity . we also noted that even though preservation or improvement of -cell performance did not occur in placebo group , the insulin sensitivity index ( homa - ir ) was better than the corresponding baseline . at both time points , the findings of this study were consistent with the results of our previous study in insulin - resistant wistar rats.20 the augmentation of insulin sensitivity found in this study can be explained by the action of dlbs3233 in modulating the expression of genes associated with insulin signaling and sensitivity , such as peroxisome proliferators - activator receptor ( ppar)- and ppar-. no serious or severe vascular - related adverse events occurred in either group during the study . this study demonstrated that dlbs3233 treatment at the dose of 50100 mg once daily was promisingly efficacious in improving insulin sensitivity and preserving the performance of -cells .

a growing body of evidence shows that prediabetic condition is a risk factor for type-2-diabetes and cardiovascular complications.13 prediabetes is defined as impaired glucose tolerance ( igt ) and/or impaired fasting glucose.2 igt is associated with metabolic syndrome and is a strong predictor of atherosclerotic cardiovascular disease , whereas impaired fasting glucose is a much weaker predictor.4 there are no less than 300 million people with prediabetic condition worldwide , and the number is predicted to rise up to 418 million by 2025 . every year , 2%11% of the prediabetic population are converting to type-2-diabetics.1,2,4,5 therefore , early intervention in the prediabetic stage is a valuable approach to reduce diabetes development and prevent related complications.3,6,7 insulin resistance , -cell dysfunction , and uncontrolled calorie consumption have been the crucial contributors to acute postprandial hyperglycemia and are the potential agents that convert prediabetes to diabetes.4,8,9 the acute postprandial hyperglycemia is responsible for glucotoxicity in the pathophysiology of type-2-diabetes even at the earlier stage ( ie , prediabetes).10 further in a vicious cycle , the impairment of -cell function and exacerbation of insulin resistance due to glucotoxicity play a crucial role in the progression of diabetes and development of its cardiovascular complications.4,10 interventions that preserve -cell function and ameliorate insulin resistance have been proven to be most effective in preventing progression to diabetes . among the antidiabetic agents , thiazolidinediones , particularly pioglitazone , showed the most remarkable risk reduction ( ie , up to 72% ) of igt conversion to type-2-diabetes.3,4,18 however , long - term use of pioglitazone ( > 2 years ) was associated with discernible weight gain and edema,18 both of which are not favorable with respect to reducing cardiovascular risks . dlbs3233 , a novel bioactive fraction derived from the plants cinnamomum burmanii and lagerstroemia speciosa , has previously been studied for its insulin - sensitizing activity.20,21 c. burmanii was obtained from kerinci , jambi , indonesia , and l. speciosa from cianjur , west java , indonesia . a previous study demonstrated the safety and tolerability of the bioactive fraction dlbs3233 in healthy volunteers.22 an unpublished preliminary study by suastika et al23 also demonstrated the favorable effects of dlbs3233 on glycemic control in newly diagnosed type-2-diabetes patients . subjects with igt , defined as 2-hour postprandial glucose ( 2h - pg ) level of 140199 mg / dl assessed by the oral glucose tolerance test ( ogtt),2 were recruited from the clinic of internal medicine department , dr m djamil padang hospital , and enrolled in this two - arm , prospective , double - blind , randomized , and controlled study . pregnant or nursing women , subjects with diabetes mellitus , history of symptomatic coronary arterial disease , stroke , and any cardiovascular events , impaired liver function with serum alanine aminotransferase ( alt ) > 2.5 times the upper limit of normal ( uln ) , impaired renal function with serum creatinine 1.5 times uln , uncontrolled chronic diseases , and acute or chronic infections , and those undergoing treatment with systemic corticosteroids or herbal ( alternative ) medicines were excluded . (1 ) in order to indicate -cell performance , plasma insulin levels at 15 minutes ( early phase ) and 2 hours ( second phase ) during ogtt were measured , and the oral disposition index ( dio ) of the early phase during ogtt : ( dio=ins015(u / ml)glc015(mg / dl)homa - ir)4,25(2)was also calculated . further , other metabolic end points , such as reduction of 15-minute postprandial glucose and 2h - pg , glucose metabolic clearance rate ( mcr ) which was estimated from fasting and 2-hour plasma insulin and glucose levels during ogtt as described by stumvoll et al ( mcr = 13,273 ( 0.00384 ins120 ) ( 0.0232 ins0 ) ( 0.463 glc120))26,27 , and fasting triglyceride level , were also measured . based on the evaluation of subjects response to treatment at week 4 , subjects in both groups who were good responders , defined as those who achieved 2h - pg level < 140 mg / dl or a reduction of 2h - pg 10% from baseline , remained at the same regimen , while the poor responders received a titrated dose of two capsules once daily , for the subsequent 8 weeks . subjects with igt , defined as 2-hour postprandial glucose ( 2h - pg ) level of 140199 mg / dl assessed by the oral glucose tolerance test ( ogtt),2 were recruited from the clinic of internal medicine department , dr m djamil padang hospital , and enrolled in this two - arm , prospective , double - blind , randomized , and controlled study . pregnant or nursing women , subjects with diabetes mellitus , history of symptomatic coronary arterial disease , stroke , and any cardiovascular events , impaired liver function with serum alanine aminotransferase ( alt ) > 2.5 times the upper limit of normal ( uln ) , impaired renal function with serum creatinine 1.5 times uln , uncontrolled chronic diseases , and acute or chronic infections , and those undergoing treatment with systemic corticosteroids or herbal ( alternative ) medicines were excluded . (1 ) in order to indicate -cell performance , plasma insulin levels at 15 minutes ( early phase ) and 2 hours ( second phase ) during ogtt were measured , and the oral disposition index ( dio ) of the early phase during ogtt : ( dio=ins015(u / ml)glc015(mg / dl)homa - ir)4,25(2)was also calculated . further , other metabolic end points , such as reduction of 15-minute postprandial glucose and 2h - pg , glucose metabolic clearance rate ( mcr ) which was estimated from fasting and 2-hour plasma insulin and glucose levels during ogtt as described by stumvoll et al ( mcr = 13,273 ( 0.00384 ins120 ) ( 0.0232 ins0 ) ( 0.463 glc120))26,27 , and fasting triglyceride level , were also measured . based on the evaluation of subjects response to treatment at week 4 , subjects in both groups who were good responders , defined as those who achieved 2h - pg level < 140 mg / dl or a reduction of 2h - pg 10% from baseline , remained at the same regimen , while the poor responders received a titrated dose of two capsules once daily , for the subsequent 8 weeks . 74 subjects ( 92.5% ) completed the study and were available for intent - to - treat analysis , with a mean age of 46.97.6 and 50.57.3 years , in dlbs3233 and placebo groups , respectively , and both groups were of female predominance ( 92.1% and 58.3% , respectively ) . three subjects ( two in dlbs3233 and one in placebo groups ) were withdrawn due to noncompliance with the study protocol , one ( in placebo group ) due to pregnancy , one ( in placebo group ) due to adverse event ( abdominal pain ) , and the remaining one patient ( in placebo group ) moved to another town and did not return for evaluation . with respect to cardiovascular risk factors and metabolic disorders , 50.0% and 63.9% of subjects in dlbs3233 and placebo groups , respectively , had hypertension ; 31.6% and 38.9% , respectively , had dyslipidemia ; and 18.4% and 13.9% , respectively , were obese . the mean blood pressures ( bps ) were not significantly different between groups , with systolic bp of dlbs3233 and placebo group at 125.316.0 and 133.216.7 mmhg , respectively ( p=0.326 ) , and diastolic bp at 81.310.4 and 85.314.2 mmhg , respectively ( p=0.181 ) . a significant reduction of homa - ir from baseline in dlbs3233 group was evident , from 3.001.76 to 2.321.32 ( p=0.002 ) and to 2.161.17 ( p=0.001 ) at week 8 and week 12 , respectively . at the end of study , the percentage of homa - ir reduction from baseline was significantly greater in dlbs3233 group than that in placebo group ( 27.0429.41 vs 4.9041.27 , p=0.013 ) ( figure 1a ) . a significant improvement ( normalization ) of the insulin level was observed in dlbs3233 group , from 435.27223.19 pmol / l at baseline , to 301.47185.57 pmol / l ( p<0.001 ) at week 8 , and then to 290.48132.44 pmol / l ( p<0.001 ) at week 12 . aligned with the improvement of first - phase insulin level , the second - phase insulin level at week 8 and week 12 was also found to improve markedly in dlbs3233 group , from 1,113.72550.47 to 752.31550.24 pmol / l ( p<0.001 ) , and then to 658.70493.67 pmol / l ( p<0.001 ) , respectively . in contrast , we found no significant improvement in placebo group ( p>0.05 ) , where the level went down from 1,004.34467.01 to 758.35459.89 pmol / l , and then to 734.86517.54 pmol / l , respectively . the preservation of -cell performance calculated as the dio increased significantly only with dlbs3233 but was absent in placebo group ( table 1 ) , with the increase at week 8 being greater in dlbs3233 group than in placebo group ( 0.250.81 vs 0.161.03 , p=0.032 ) . in line with the augmentation of insulin sensitivity ( reduced homa - ir ) , we also found an increase of mcr of glucose at week 8 and week 12 , in both groups ( table 1 ) , with the enhancement of mcr at week 12 being higher in dlbs3233 group than in placebo group ( 2.852.79 ml / kg / min vs 1.703.05 ml / kg / min , p=0.044 ) . a significantly lower level of triglyceride after 8 and 12 weeks of treatment was seen in dlbs3233 group , from 1.490.70 mmol / l at baseline , to 1.200.39 mmol / l ( p=0.008 ) , and then to 1.170.43 mmol / l ( p=0.004 ) , respectively . in dlbs3233 and placebo groups , the systolic bps were reduced from 125.316.0 to 118.913.9 mmhg ( p=0.035 ) vs 133.226.7 to 129.018.3 mmhg ( p=0.600 ) , and the diastolic bps from 81.310.4 to 76.69.4 mmhg ( p=0.026 ) vs 85.314.2 to 81.98.2 mmhg ( p=0.200 ) . 74 subjects ( 92.5% ) completed the study and were available for intent - to - treat analysis , with a mean age of 46.97.6 and 50.57.3 years , in dlbs3233 and placebo groups , respectively , and both groups were of female predominance ( 92.1% and 58.3% , respectively ) . three subjects ( two in dlbs3233 and one in placebo groups ) were withdrawn due to noncompliance with the study protocol , one ( in placebo group ) due to pregnancy , one ( in placebo group ) due to adverse event ( abdominal pain ) , and the remaining one patient ( in placebo group ) moved to another town and did not return for evaluation . with respect to cardiovascular risk factors and metabolic disorders , 50.0% and 63.9% of subjects in dlbs3233 and placebo groups , respectively , had hypertension ; 31.6% and 38.9% , respectively , had dyslipidemia ; and 18.4% and 13.9% , respectively , were obese . the mean blood pressures ( bps ) were not significantly different between groups , with systolic bp of dlbs3233 and placebo group at 125.316.0 and 133.216.7 mmhg , respectively ( p=0.326 ) , and diastolic bp at 81.310.4 and 85.314.2 mmhg , respectively ( p=0.181 ) . a significant reduction of homa - ir from baseline in dlbs3233 group was evident , from 3.001.76 to 2.321.32 ( p=0.002 ) and to 2.161.17 ( p=0.001 ) at week 8 and week 12 , respectively . in placebo group , a significant reduction from baseline was found at week 8 ( from 2.762.28 to 2.030.99 , p=0.005 ) , but it was then diminished at week 12 ( to 2.281.24 , p=0.158 vs baseline ) . at the end of study , the percentage of homa - ir reduction from baseline was significantly greater in dlbs3233 group than that in placebo group ( 27.0429.41 vs 4.9041.27 , p=0.013 ) ( figure 1a ) . a significant improvement ( normalization ) of the insulin level was observed in dlbs3233 group , from 435.27223.19 pmol / l at baseline , to 301.47185.57 pmol / l ( p<0.001 ) at week 8 , and then to 290.48132.44 pmol / l ( p<0.001 ) at week 12 . aligned with the improvement of first - phase insulin level , the second - phase insulin level at week 8 and week 12 was also found to improve markedly in dlbs3233 group , from 1,113.72550.47 to 752.31550.24 pmol / l ( p<0.001 ) , and then to 658.70493.67 pmol / l ( p<0.001 ) , respectively . in contrast , we found no significant improvement in placebo group ( p>0.05 ) , where the level went down from 1,004.34467.01 to 758.35459.89 pmol / l , and then to 734.86517.54 pmol / l , respectively . the preservation of -cell performance calculated as the dio increased significantly only with dlbs3233 but was absent in placebo group ( table 1 ) , with the increase at week 8 being greater in dlbs3233 group than in placebo group ( 0.250.81 vs 0.161.03 , p=0.032 ) . in line with the augmentation of insulin sensitivity ( reduced homa - ir ) , we also found an increase of mcr of glucose at week 8 and week 12 , in both groups ( table 1 ) , with the enhancement of mcr at week 12 being higher in dlbs3233 group than in placebo group ( 2.852.79 ml / kg / min vs 1.703.05 ml / kg / min , p=0.044 ) . a significantly lower level of triglyceride after 8 and 12 weeks of treatment was seen in dlbs3233 group , from 1.490.70 mmol / l at baseline , to 1.200.39 mmol / l ( p=0.008 ) , and then to 1.170.43 mmol / l ( p=0.004 ) , respectively . in dlbs3233 and placebo groups , the systolic bps were reduced from 125.316.0 to 118.913.9 mmhg ( p=0.035 ) vs 133.226.7 to 129.018.3 mmhg ( p=0.600 ) , and the diastolic bps from 81.310.4 to 76.69.4 mmhg ( p=0.026 ) vs 85.314.2 to 81.98.2 mmhg ( p=0.200 ) . our study showed that augmentation of insulin sensitivity by dlbs3233 , through its insulin - sensitizing properties,20,21 was finally compensated by a significant reduction which can also be translated as normalization of the first- and second - phase insulin levels as shown in dlbs3233-treated group after 8 and 12 weeks . the clinical consequence of the hyperbolic relationship is that increased insulin sensitivity will be compensated by reduction , or downregulation , of -cell function , which may be a mechanism to avoid hypoglycemic episodes.36 such an inverse relationship between insulin sensitivity and insulin secretion indicates that it is impossible to judge insulin secretion in a given individual without knowing the ambient insulin sensitivity , since both increases and decreases of insulin sensitivity are associated with compensatory reciprocal changes in insulin secretion . at both time points , the findings of this study were consistent with the results of our previous study in insulin - resistant wistar rats.20 the augmentation of insulin sensitivity found in this study can be explained by the action of dlbs3233 in modulating the expression of genes associated with insulin signaling and sensitivity , such as peroxisome proliferators - activator receptor ( ppar)- and ppar-. in vitro studies with 3t3-swiss - albino preadipocyte cells showed that stimulation of insulin - signaling transduction by dlbs3233 occurred through the promotion of tyrosine phosphorylation of the insulin - receptor - substrate and upregulation of the expression of phosphatidylinositol-3-kinase , ppar- , ppar- , and glucose transporter-4 at the mrna level.20,21 activation of ppar- and ppar- by dlbs3233 leads to a series of beneficial metabolic effects on glucose and lipids , suggesting that the product possesses the potential capacity to decrease the incidence of insulin resistance - associated diseases . it has been widely known that insulin resistance , through a complex physiological interaction with hyperinsulinemia makes an independent contribution to the elevation of bp.38 therefore , improvement in insulin sensitivity will produce a certain reduction in bp , independent of other factors.39 other drugs that improve insulin sensitivity also demonstrated their effects in lowering bp , such as rosiglitazone,39 pioglitazone,18 and metformin.40 the data suggest that along with its virtues on glucose metabolism , dlbs3233 might also provide protection against the development of cardiovascular disease in prediabetic subjects . however , this study may serve as a sound ground to justify a larger outcome research with a longer duration of treatment and more refined study design to evaluate the risk reduction of type-2-diabetes by dlbs3233 treatment in prediabetic subjects and to confirm the real benefits of dlbs3233 treatment in prediabetes .

humans tend to assume that our vision represents the world as it is ; that , for example , an object appears red because that object is red . however , the colours that objects appear depend on the colour vision of the observer , and humans actually have a rare form of colour vision among all animals ( e.g. bowmaker , 1998 ; jacobs , 1993 ) . the early vertebrates were probably tetrachromatic and many fish , reptiles and birds remain so meaning they have four types of cone receptor . early mammals lost this multidimensional colour vision and became dichromatic , retaining only the cone classes sensitive to the longest and shortest wavelengths . an ancestor to african and asian primates , with which we share our type of colour vision , reinvented full trichromatic vision from these dichromatic ancestors , so that we now have three types of cone shortwave ( s ) , mediumwave ( m ) and longwave ( l)two of which , l and m , remain similar to each other in many ways because they are the offspring of the same ancestral mammalian longwave cone . most placental mammals have remained dichromatic , possessing s and l cones , while some groups have reduced to just l cones ( and rods ) . the reason why primates bucked this trend has been the focus of a long - running debate . the main theories have suggested that the key selective advantage of trichromacy for primates lay in foraging , either for fruit ( allen , 1879 ; mollon , 1989 ; osorio & vorobyev , 1996 ; polyak , 1957 ; regan et al . , 1998 , 2001 ; riba - hernandez , stoner , & osorio , 2004 ; steward & cole , 1989 ) or for young leaves ( dominy & lucas , 2001 ; sumner & mollon , 2000a ) among the ubiquitous forest background of mature leaves . , we ask whether the key task might have been spotting food from a distance , or efficiently picking food once it is within reach ( i.e. food detection or food selection ; lucas et al . , 2003 ) . parraga , troscianko , and tolhurst ( 2002 ) compared the spatiochromatic properties of calibrated natural images , including those of fruit on leafy backgrounds with the properties of the three early channels of human vision : the lm colour pathway , which compares l cone signals with m cone signals ( this pathway is often called red green , but this is slightly misleading ) ; the s - lm pathway comparing s cone signals with pooled l and m signals ( this pathway is often called blue yellow , but this is highly misleading ) , and an achromatic pathway . it is the ability to compare l with m signals that was new in primate trichromacy , and parraga et al . ( 2002 ) found that the spatiochromatic properties of this channel are optimized for encoding reddish or yellowish fruit or leaves on a background of foliage at relatively small viewing distances . in other words , our lm system is optimized for picking fruit or edible leaves , rather than for spotting them from a distance . does it follow that picking food was the critical task driving the natural selection of primate trichromacy ? we can not necessarily make this inference because natural selection is not driven by what a phenotype might be best at , but by the advantage that phenotype offers over other competing phenotypes . even though the lm pathway may be best at selecting food close up , the biggest advantage over dichromacy might still be at spotting fruit at a distance . in fact , other authors have explicitly assumed that the biggest advantage lay in spotting food from a distance ( dominy , lucas , osorio , & yamashita , 2001 ; lucas et al . , 2003 ; regan et al . , 1998 , 2001 ; sumner & mollon , 2000a , 2000b ) . for example , regan et al . and sumner & mollon modelled the task of finding fruit or young leaves in foliage for all plausible sensitivity functions that the l and m cones might take , in order to work out which combination of potential l and m cones would be optimal . it turned out that the optimal pair are very close to what we actually possess , supporting the argument that finding important objects amongst foliage was a driving force in the evolution of the lm colour channel . this approach did not incorporate the spatial properties of the system , unlike parraga et al . ( 2002 ) but a component of the modelling the estimation of quantum noise did require the viewing distance of the stimuli to be specified . these studies explicitly assumed the key task was spotting food from a distance rather than picking food close up , and thus chose distances of about 10 m. behavioural evidence in favour of either task is circumstantial . south american monkeys have been the main focus of study because they are polymorphic in their colour vision ( mollon , bowmaker , & jacobs , 1984 ) ; within the same species , some individuals can have trichromacy , while others have dichromacy ( we will return to this in the discussion section ) . these species offer the opportunity of natural experiments , comparing the behaviour of trichromatic individuals with dichromatic individuals , either in zoos or in the wild . caine and mundy ( 2000 ) tested marmosets in visual search tasks in a naturalized captive setting . in one task , they scattered edible orange or green cereal balls in the cage , which required viewing from up to 6 m away , while in the other task the targets were placed on a tray among green wood shavings and viewed close up ( < 0.5 m ) . an advantage for trichromats was evident only for the longer distance task , but the backgrounds for the visual search were not matched across tasks ( the primary aim of the study was to establish whether the trichromats had an advantage in any task rather than to formally test the distance manipulation ) . since then , there has been no distance manipulation within the same study , and it has been surprisingly difficult for researchers to observe any significant behavioural differences between trichromatic and dichromatic monkeys in the wild . in a captive environment , the foraging advantage of trichromats has been replicated with tamarins finding simulated fruits in simulated leaves , but there was no distance manipulation ( smith , buchanan - smith , surridge , osorio , & mundy , 2003b ) . in the wild , smith et al . ( 2003b ) found a hint that trichromatic moustached tamarins might be more likely to lead the troop into fruiting trees than dichromats , consistent with an advantage in spotting fruit , but this was not consistent and it was not the case that all troops of tamarins were led by trichromats ( saddleback tamarins were mainly male - led , who are all dichromats ) . similarly , bunce , isbell , grote , and jacobs ( 2011 ) found a hint that wild trichromatic titi monkeys found small patches of red or yellow fruit more often than their dichromatic conspecifics . however , the only significant observed advantage in wild monkeys has been capuchins selecting fruit at arms ' reach ( melin et al . , 2009 ) , where trichromats accepted ( i.e. ate ) more of the fruit they picked , sniffed fruit less , and had shorter foraging sequences than dichromats did . however , this apparent advantage for picking fruit does not appear to translate into consuming more food overall in trichromatic capuchins or spider monkeys ( hiramatsu et al . , 2008 ; melin et al . , 2009 ; vogel , neitz , & dominy , 2007 ) . moreover , hiramatsu et al . ( 2008 ) found that luminance , rather than colour , was the cue that best predicts foraging efficiency at grasping distance ( for the fruits in their study at least ) . additionally , olfaction and tactile cues can be available , reducing the importance of trichromacy ( hiramatsu et al . , 2009 ; lucas et al . , 2003 ; steward & cole , 1989 ) . thus , it remains unclear whether we should expect the greatest advantage of human - like trichromacy to be in the longer distance task of spotting food , or the close - up task of picking it . the aim of our experiment was to simulate the competition to find fruit between two primates ( in this case humans ) in a naturalistic visual search task of real fruit in real foliage . we tested normal trichromats , dichromats and anomalous trichromats who have less separation between the l and m cone sensitivity functions than do we included anomalous trichromats both to boost colour - deficient participant numbers ( dichromats are relatively rare , 2% of the male population ) , and because evolutionarily the competing state to what we now consider normal trichromacy has almost certainly been anomalous trichromacy as well as dichromacy . the task was to count the number of ripe fruit hanging in a real bush from a distance of 1 , 4 , 8 or 12 m ( figure 1 ) . we used segments of yellow pepper as targets because they display spectral and thus chromatic properties representative of many fruit favoured by primates in a ugandan rainforest ( figures 2 and 3 ) . the task was performed simultaneously by a pair of participants , one dichromat or anomalous trichromat and one normal trichromat matched as closely as possible for age , sex , experience of visual experiments and knowledge of colour vision . each pair performed the task simultaneously so that they were exposed to the identical trial sequence and lighting conditions ( cloudy or sunny ) . this set - up also engendered some aspect of competition between the pair , simulating the competition for fruit of two primates simultaneously foraging in the same tree . the upper panel shows two participants during a trial at 4 m with three targets . both have removed their blindfolds and opened their eyes in order to search the shrub . the lower panel shows the shrub with the eight potential target positions marked with red circles . e.g. panel a : samples of unripe , green , and ripe , yellow , chrysophyllum albidum ) , we chose segments yellow pepper as targets and green pepper as distractors ( b ) , because they have similar reflectance spectra ( c ) and chromatic properties ( figure 3 ) . stimuli ( b ) subtended approximately 2 , 0.5 , 0.25 and 0.16 degrees of visual angle for distances 1 , 4 , 8 , 12 m. in panel ( c ) , thin orange and green lines are ripe and unripe ugandan fruit chrysophyllum albidum , orange and green thick lines are ripe and unripe segments of yellow / orange and green pepper used as targets and distractors overlap in their chromatic properties ( orange and green filled circles ) with some fruits eaten by primates in uganda ( unfilled circles ) . the leaves from the experimental bush ( grey squares ) also show similar properties to leaves in a ugandan rainforest ( open squares ) . normal trichromats made faster responses and fewer errors than their colour - deficient competitors , as expected ( mean rts 4.7 vs. 6.4 s ; error score 0.17 vs. 0.54 ; all errors were misses , our unit of analysis is the pairwise comparison between the members of each matched pair . as shown in figures 4(a and b ) , the relative advantage increased with distance , both for response time and errors [ one - way anovas , f(3,21 ) = 9.7 , p < 0.001 and f(3,30 ) = 4.0 , p < 0.02 ; see the methods section for subject numbers and other details ] . in figures 4(a and b ) , the difference in performance ( rt or error ) between the colour - deficient member and the colour - normal member of each pair was divided by the overall mean rt or error of that pair , in order to control for different overall performance and speed accuracy trade - offs ( range in mean rt , 3.412.8 s and in mean error score 0.150.57 ; the behaviour of each member was correlated , r = 0.76 for rt and r = 0.32 for errors , probably because they influenced each other in this regard , as would be expected , and/or because they had correlated age , experience and testing conditions ) . a and b ) , the difference in performance ( rt or error ) between the colour - deficient member and the colour - normal member of each pair was divided by the overall mean rt or error of that pair , in order to control for different overall performance and speed accuracy trade - offs . in panel ( c ) , the rt for the colour - deficient member of each pair in each condition is simply divided by the rt of the colour - normal member ( which is a simpler analysis and also controls for differences in baseline speed with distance ) . this analysis could not be done reliably for error scores because there were many instances of dividing by zero or near - zero bins . the dashed and dotted lines show the subsets of results for dichromats and anomalous trichromats ( each compared with their matched normal trichromat ) . figure 4(c ) shows that the same pattern [ f(3,21 ) = 5.6 , p = 0.005 ] occurred for the mean ratio of response times of the colour - normal and colour - deficient members of each pair . the dashed and dotted lines in figures 4(a c ) show the subsets of results for dichromats and anomalous trichromats ( each compared with their matched normal trichromat ) , indicating that the effect on response time is greater for full dichromats [ interaction f(3,18 ) = 3.4 , p < 0.05 ] , as would be expected . the same pattern is not evident in the error scores , but this could be a simple lack of power or a difference in speed accuracy strategy between groups . note that the key comparison was between the colour - deficient and normal trichromatic member of each pair , not between dichromats and anomalous trichromats , because although the dichromats and anomalous trichromats were each matched in age and relevant experience to their control trichromats , dichromats and anomalous trichromats could not be matched in age or experience with each other . the mean age of the dichromats was 10 years older than that of the anomalous trichromats . our results show that in our naturalistic visual search task with human observers , the advantage of normal trichromacy over dichromacy or anomalous trichromacy increased with distance ( even without the additional cues of smell and touch available to foraging monkeys ) . the advantage was greatest when the stimuli subtended no more than 0.2 degrees at the eye . this is likely to be because visual cues such as shape and the s/(l+m ) dimension of colour vision decrease with distance for small targets . for very small stimuli , the absence of s cones in the very centre of the fovea ( about the central 1/3 of a degree of visual angle ) , precludes information in the s/(l+m ) pathway for fixated targets ( known as small field tritanopia ) , although the effect of this is less when observers scan a scene , as in the present task ( e.g. mccree , 1960 ) . although we used fruit not young ( reddish or yellowish ) leaves as targets , we see no reason that our findings would not extend to such leaves , which are also important sources of food for some species of primate . our results imply that the more important task for the natural selection of primate trichromacy may have been spotting food from a distance rather than picking it at arms ' length . this in no way challenges the previous finding that the lm colour channel is optimal at the close - up task ( parraga et al . , 2002 ) . the biggest advantage of one phenotype over competing phenotypes does not have to arise for the task that the phenotype is best at . however , while the spatial - frequency sensitivity of the lm system undoubtedly makes it optimal at the close - up task , our results do raise the question of whether it has been optimized by that task . rather , its optimality may have emerged as a side effect of the combined pressures of the longer distance task and other constraints , such as the random layout of l and m cones on the retina ( deeb , 2006 ) and the unavoidable fact that both colour resolution and achromatic resolution can not be equally high because they are conflated in the most fine scale comparison of neighbouring cones . one general puzzle is why it has been so difficult to observe any behavioural advantages for trichromats over dichromats in the wild , when they have been apparent as expected in experiments such as this one and those on captive tamarins and marmosets ( caine & mundy , 2000 ; smith et al . , 2003b ) . most studies in the wild have concentrated on observing monkeys picking fruit ( e.g. hiramatsu et al . , 2008 ; melin et al . , 2009 ; vogel et al . , 2007 ) , where our results predict the advantage is smallest , even without the extra tactile and olfactory cues available in the wild but not in our experiment . unfortunately , it is very difficult to discern and quantify the longer distance task except by observing troop leadership , which is known to be influenced by other social factors which might mask any effect of colour vision ( smith , buchanan - smith , surridge , & mundy , 2003a ) . another possibility is that the colour properties of fruits consumed in the forests where these studies have taken place are not fully representative of the gamut of fruits eaten by our ancestors . it is difficult to do more than speculate here , but the chromaticity diagrams in hiramatsu et al . ( 2008 ) , who found no differences in feeding behaviour between dichromatic and trichromatic spider monkeys , show a higher ratio of cryptic fruits ( those that stay green ; see also de araujo , lima , & pessoa , 2006 ) and fruits that turn dark brownish purple ( as do many types of fig ) , and fewer orange or yellow fruit than are found in the diet of primates in uganda ( sumner & mollon , 2000a , 2000b ) . cryptic fruits are detected from afar by smell or shape ( and memory ) , and close up by shape , position , smell or feel ( e.g. hiramatsu et al . , 2009 ; melin et al . , 2009 ) , all of which dichromats can do at least as well as trichromats . dark fruits have , by definition , a strong achromatic signal , which again dichromats can detect at least as well as trichromats ( and possibly are better attuned to ) . however , viewed amongst foliage from a distance , dark fruits ( unless in a clump ) could easily be mistaken for shadow or gaps , so the use of achromatic cues in fruit foraging remains unclear ( as does the extra cue of glossiness often present in dark fruits ) . it would be interesting to test human dichromats and trichromats with fruits showing these properties , such as green and red grapes . note that our targets did also contain a strong luminance signal relative to the foliage background , and of course wild monkeys have a lifetime of practice in fruit picking which acts to diminish any behavioural differences between them . field studies have concentrated on polymorphic species of monkey where some individuals are trichromatic and some are dichromatic , both because this situation makes an ideal natural experiment and because the polymorphism is interesting and rare in itself . it arises through there being different alleles of the gene coding the l cone pigment , which give the pigment different sensitivities ( akin to our l and m cones ) . if a monkey is homozygous for this gene , they can only have one type of l cone ( in addition to s cones ) , and are thus dichromatic . but if they are heterozygous , and the alleles are expressed in different cones cells , then they possess two types of l cone ( i.e. l and m cones ) and can be trichromatic ( mollon et al . , 1984 ) . the polymorphic gene is on the x chromosome , which means that only females get the opportunity to be heterozygous and thus trichromatic . in many species , there are three alleles present in different frequencies , which means that approximately 60% of females are trichromatic ( jacobs , neitz , & neitz , 1993 ; williams , hunt , bowmaker , & mollon , 1992 ) . because trichromacy arises from heterozygosity , rather than from possessing a single advantageous allele , it can not be simply passed on to offspring . in fact , there is about equal chance of a dichromatic or trichromatic mother having a trichromatic daughter ( in - breeding avoidance can complicate this , and possibly make dichromats more likely to have trichromatic daughters ; surridge , suarez , buchanan - smith , & mundy , 2005 ) . however , rare alleles tend to be lost to populations if their presence provides no advantage ( e.g. surridge & mundy , 2002 ) , so it is assumed that to maintain the polymorphism there must be some advantage to trichromacy in the wild that translates into improved fitness ( the potential advantage of having different types of dichromacy in a population has not been given any support ; riba - hernandez et al . , 2004 ) . on the other hand , if this advantage was very large , there is the obvious question of why full trichromacy has not emerged in these species , as it did for asian and african primates , and has done also for howler monkeys ( jacobs , neitz , deegan , & neitz , 1996 ) . one possibility is that the genetic event required to allow this is vanishingly rare and simply never happened in those species that remain polymorphic ( regan et al . , 2001)a gene duplication is required that puts two different alleles onto the same chromosome alongside working promoter regions and a mechanism for expressing these genes differentially in different cones . the other possibility is that the advantage of trichromacy is much subtler than originally expected when combined with social factors within a polymorphic population ( e.g. bunce et al . , 2011 ; , 2003a ) , and may also be offset by some advantages for dichromacy , such as breaking camouflage of cryptic insects ( melin , fedigan , hiramatsu , sendall , & kawamura , 2007 ; regan et al . , 2001 ; saito et al . , 2005 ) . either way , given that many species of monkey have successfully survived and diversified over millions of years in which the majority of individuals ( and all the males ) have been dichromatic ( e.g. surridge & mundy , 2002 ) , it should not be surprising that dichromats are not glaringly deficient at any crucial task in the wild that would dramatically harm their chances of survival . from this perspective , the advantage of trichromacy is bound to be quite subtle and hard to measure in free - ranging wild monkeys . lastly , some authors have suggested that the selective advantage of trichromacy might have been in signalling ( changizi , zhang , & shimojo , 2006 ) . many monkeys have red faces ( and sometimes red sexual signals ) or orange fur markings ( sumner & mollon , 2003 ) . more recent evidence suggests that such signalling post - dates the invention of trichromacy , and thus took advantage of trichromacy once it had arisen , rather than driving its natural selection initially ( fernandez & morris , 2007 ) . however , it may form part of the selection pressure maintaining it in current populations , as might predator detection ( smith , buchanan - smith , surridge , & mundy , 2005 ; sumner & mollon , 2003 ) . in conclusion , we found that in a naturalistic visual search task of finding food in foliage , humans with normal colour vision diverged in performance from dichromats or anomalous trichromats most for the longest viewing distance ( 12 m ) . this implies that in the natural selection of trichromacy , the task of spotting fruit from a distance was a greater driving force than picking fruit close up . colour - deficient participants were recruited through advert and tested using the ishihara test ( 24 plate edition ) and nagel anomaloscope to determine their type of colour vision . following this screening , 12 participants with clearly defined colour deficiency ( 3 protanopes , 4 deuteranopes , 2 protanomalous and 3 deuteranomalous ) performed the visual search task . each was paired with control participant with normal trichromacy ( tested using the ishihara plates ) , matching as closely as possible for age , sex , experience of visual experiments and knowledge of colour vision . each pair performed the task simultaneously so that they were exposed to the identical trial sequence in identical lighting conditions ( cloudy or sunny ) . this set - up also engendered some aspect of competition between the pair , simulating the competition for fruit of two primates simultaneously foraging in the same tree . for the first three pairs of participants , accuracy was recorded , but not reaction time due to a technical error . one participant opened his eyes prematurely on some of the visual search trials , and so that pair was excluded from all analyses . therefore , there were 16 participants ( 8 pairs ) for the analysis of response time and 22 participants ( 11 pairs ) for the analysis of error scores . participants had to count the number of ripe fruit hanging in a bush from a distance of 1 , 4 , 8 or 12 m ( figure 1 ) . on each trial , the two participants were positioned at the appropriate distance and blindfolded while the experimenter positioned four fruit between zero and four of these stimuli were targets ( ripe fruit ) and the rest were distractors ( unripe fruit ) . when the stimuli were positioned , the experimenter instructed the participants to remove their blindfold keeping their eyes closed ( compliance was monitored by the experimenter ) . then , on the instruction go the participants simultaneously opened their eyes and viewed the stimuli . their task was to press one of the five keys ( labelled 0 , 1 , 2 , 3 , 4 ) on a keyboard as quickly and accurately as possible to indicate the number of targets they could see . the keyboards were positioned on a trolley between the participants , which also contained the computer and could be moved to each distance as appropriate ( figure 1 , lower panel ) . the side of the trolley taken by the colour - deficient participant was counterbalanced across pairs . rt was recorded ( except for the first three participants ) as well as the difference between the answer given and the correct answer ( error score ) . while there was no barrier to entirely rule out participants seeing each other 's responses , behaviour was watched by the experimenter . furthermore , if a participant based their response on that of their competitor , by definition they would have longer reaction times , but consistently across distances . the effect this might have on results there were 40 trials per pair ( 10 per distance ) , taking approximately one hour to complete . on each trial , the number of each stimulus type and their positions , as well as the viewing distance , all followed a randomized sequence that ensured two trials with each number of targets ( 04 ) were conducted for each distance . the aim was to use real fruit as target and distractor stimuli , positioned amongst real leaves . we located a suitable bush ( aucuba japonica ) in an unused courtyard on the university campus , which could be viewed from between 1 and 12 m. we measured the reflected spectra ( spectrocal , cambridge research systems ) from 25 of the leaves in situ under natural overcast conditions in order to compare their chromaticity coordinates ( figure 3 ) with leaves measured in a ugandan rainforest , also under overcast conditions , by sumner and mollon ( 2000a ) . to calculate chromaticities , we used cone fundamentals from stockman and sharpe ( 2000 ) and corresponding scaling factors ( http://www.cvrl.org ) . to choose target and distractor stimuli , we measured the reflectance spectra of a range of fruits obtained from local supermarkets and specialist shops , in order to compare them with the fruit in a ugandan forest ( sumner and mollon ( 2000a , b ) . highly favoured by the primates in the kibale forest , including monkeys and chimpanzees , from the cambridge database of natural spectra ( see figure 2 ) . to be used in our task , target and distractor fruit had to satisfy three conditions : ( 1 ) the chromaticities had to be representative of the rainforest fruit ( to calculate chromaticities , we used a natural illuminant in overcast conditions from near the experimental bush for experimental stimuli , and from uganda for ugandan fruit , http://vision.psychol.cam.ac.uk/spectra/ ) ; ( 2 ) the stimuli had to be light enough to hang on the bush ; ( 3 ) the stimuli must not change ( e.g. change colour through oxidization ) during the course of an experimental session . the fruit that best satisfied these criteria were green ( unripe ) and yellow / orange ( ripe ) peppers , which could be cut into equal - sized segments and hung on the bush with small green hooks . although peppers are not indigenous to africa , natural spectra are constrained by the pigments available to plants . finding segments of pepper amongst leaves is a task highly representative of the chromatic visual search task facing primates in uganda , which in turn is the environment most likely to represent that of our shared ancestors . segments 3 5 cm were used ( figure 2 ) , subtending the following degrees of visual angle for distances 1 , 4 , 8 , 12 : 1.7 2.3 , 0.43 0.57 , 0.21 0.29 , 0.14 0.19 . before performing the visual search trials , each participant was shown the yellow and green pepper segments intermingled on a plate and asked to categorize them into two piles . they were then informed that the yellow peppers , but not the green , were the targets for the visual search . colour - deficient participants were recruited through advert and tested using the ishihara test ( 24 plate edition ) and nagel anomaloscope to determine their type of colour vision . following this screening , 12 participants with clearly defined colour deficiency ( 3 protanopes , 4 deuteranopes , 2 protanomalous and 3 deuteranomalous ) performed the visual search task . each was paired with control participant with normal trichromacy ( tested using the ishihara plates ) , matching as closely as possible for age , sex , experience of visual experiments and knowledge of colour vision . each pair performed the task simultaneously so that they were exposed to the identical trial sequence in identical lighting conditions ( cloudy or sunny ) . this set - up also engendered some aspect of competition between the pair , simulating the competition for fruit of two primates simultaneously foraging in the same tree . for the first three pairs of participants , accuracy was recorded , but not reaction time due to a technical error . one participant opened his eyes prematurely on some of the visual search trials , and so that pair was excluded from all analyses . therefore , there were 16 participants ( 8 pairs ) for the analysis of response time and 22 participants ( 11 pairs ) for the analysis of error scores . participants had to count the number of ripe fruit hanging in a bush from a distance of 1 , 4 , 8 or 12 m ( figure 1 ) . on each trial , the two participants were positioned at the appropriate distance and blindfolded while the experimenter positioned four fruit ( see the stimuli section below ) amongst the leaves of the bush . between zero and four of these stimuli were targets ( ripe fruit ) and the rest were distractors ( unripe fruit ) . when the stimuli were positioned , the experimenter instructed the participants to remove their blindfold keeping their eyes closed ( compliance was monitored by the experimenter ) . then , on the instruction go the participants simultaneously opened their eyes and viewed the stimuli . their task was to press one of the five keys ( labelled 0 , 1 , 2 , 3 , 4 ) on a keyboard as quickly and accurately as possible to indicate the number of targets they could see . the keyboards were positioned on a trolley between the participants , which also contained the computer and could be moved to each distance as appropriate ( figure 1 , lower panel ) . the side of the trolley taken by the colour - deficient participant was counterbalanced across pairs . rt was recorded ( except for the first three participants ) as well as the difference between the answer given and the correct answer ( error score ) . while there was no barrier to entirely rule out participants seeing each other 's responses , behaviour was watched by the experimenter . furthermore , if a participant based their response on that of their competitor , by definition they would have longer reaction times , but consistently across distances . the effect this might have on results there were 40 trials per pair ( 10 per distance ) , taking approximately one hour to complete . on each trial , the number of each stimulus type and their positions , as well as the viewing distance , all followed a randomized sequence that ensured two trials with each number of targets ( 04 ) were conducted for each distance . the aim was to use real fruit as target and distractor stimuli , positioned amongst real leaves . we located a suitable bush ( aucuba japonica ) in an unused courtyard on the university campus , which could be viewed from between 1 and 12 m. we measured the reflected spectra ( spectrocal , cambridge research systems ) from 25 of the leaves in situ under natural overcast conditions in order to compare their chromaticity coordinates ( figure 3 ) with leaves measured in a ugandan rainforest , also under overcast conditions , by sumner and mollon ( 2000a ) . to calculate chromaticities , we used cone fundamentals from stockman and sharpe ( 2000 ) and corresponding scaling factors ( http://www.cvrl.org ) . to choose target and distractor stimuli , we measured the reflectance spectra of a range of fruits obtained from local supermarkets and specialist shops , in order to compare them with the fruit in a ugandan forest ( sumner and mollon ( 2000a , b ) . for this comparison highly favoured by the primates in the kibale forest , including monkeys and chimpanzees , from the cambridge database of natural spectra ( see figure 2 ) . to be used in our task , target and distractor fruit had to satisfy three conditions : ( 1 ) the chromaticities had to be representative of the rainforest fruit ( to calculate chromaticities , we used a natural illuminant in overcast conditions from near the experimental bush for experimental stimuli , and from uganda for ugandan fruit , http://vision.psychol.cam.ac.uk/spectra/ ) ; ( 2 ) the stimuli had to be light enough to hang on the bush ; ( 3 ) the stimuli must not change ( e.g. change colour through oxidization ) during the course of an experimental session . the fruit that best satisfied these criteria were green ( unripe ) and yellow / orange ( ripe ) peppers , which could be cut into equal - sized segments and hung on the bush with small green hooks . although peppers are not indigenous to africa , natural spectra are constrained by the pigments available to plants . finding segments of pepper amongst leaves is a task highly representative of the chromatic visual search task facing primates in uganda , which in turn is the environment most likely to represent that of our shared ancestors . segments 3 5 cm were used ( figure 2 ) , subtending the following degrees of visual angle for distances 1 , 4 , 8 , 12 : 1.7 2.3 , 0.43 0.57 , 0.21 0.29 , 0.14 0.19 . before performing the visual search trials , each participant was shown the yellow and green pepper segments intermingled on a plate and asked to categorize them into two piles . they were then informed that the yellow peppers , but not the green , were the targets for the visual search .

the spatiochromatic properties of the red green dimension of human colour vision appear to be optimized for picking fruit in leaves at about arms ' reach . however , other evidence suggests that the task of spotting fruit from a distance might be more important . this discrepancy may arise because the task a system ( e.g. human trichromacy ) is best at is not necessarily the same task where the largest advantage occurs over the evolutionary alternatives ( dichromacy or anomalous trichromacy ) . we tested human dichromats , anomalous trichromats and normal trichromats in a naturalistic visual search task in which they had to find fruit pieces in a bush at 1 , 4 , 8 or 12 m viewing distance . we found that the largest advantage ( in terms of either performance ratio or performance difference ) of normal trichromacy over both types of colour deficiency was for the largest viewing distance . we infer that in the evolution of human colour vision , spotting fruit from a distance was a more important selective advantage than picking fruit at arms ' reach .

Introduction Results Discussion Conclusion Methods Participants Visual search task Stimuli

however , the colours that objects appear depend on the colour vision of the observer , and humans actually have a rare form of colour vision among all animals ( e.g. an ancestor to african and asian primates , with which we share our type of colour vision , reinvented full trichromatic vision from these dichromatic ancestors , so that we now have three types of cone shortwave ( s ) , mediumwave ( m ) and longwave ( l)two of which , l and m , remain similar to each other in many ways because they are the offspring of the same ancestral mammalian longwave cone . the main theories have suggested that the key selective advantage of trichromacy for primates lay in foraging , either for fruit ( allen , 1879 ; mollon , 1989 ; osorio & vorobyev , 1996 ; polyak , 1957 ; regan et al . , we ask whether the key task might have been spotting food from a distance , or efficiently picking food once it is within reach ( i.e. parraga , troscianko , and tolhurst ( 2002 ) compared the spatiochromatic properties of calibrated natural images , including those of fruit on leafy backgrounds with the properties of the three early channels of human vision : the lm colour pathway , which compares l cone signals with m cone signals ( this pathway is often called red green , but this is slightly misleading ) ; the s - lm pathway comparing s cone signals with pooled l and m signals ( this pathway is often called blue yellow , but this is highly misleading ) , and an achromatic pathway . ( 2002 ) found that the spatiochromatic properties of this channel are optimized for encoding reddish or yellowish fruit or leaves on a background of foliage at relatively small viewing distances . in other words , our lm system is optimized for picking fruit or edible leaves , rather than for spotting them from a distance . we can not necessarily make this inference because natural selection is not driven by what a phenotype might be best at , but by the advantage that phenotype offers over other competing phenotypes . even though the lm pathway may be best at selecting food close up , the biggest advantage over dichromacy might still be at spotting fruit at a distance . in fact , other authors have explicitly assumed that the biggest advantage lay in spotting food from a distance ( dominy , lucas , osorio , & yamashita , 2001 ; lucas et al . and sumner & mollon modelled the task of finding fruit or young leaves in foliage for all plausible sensitivity functions that the l and m cones might take , in order to work out which combination of potential l and m cones would be optimal . it turned out that the optimal pair are very close to what we actually possess , supporting the argument that finding important objects amongst foliage was a driving force in the evolution of the lm colour channel . this approach did not incorporate the spatial properties of the system , unlike parraga et al . ( 2002 ) but a component of the modelling the estimation of quantum noise did require the viewing distance of the stimuli to be specified . these studies explicitly assumed the key task was spotting food from a distance rather than picking food close up , and thus chose distances of about 10 m. behavioural evidence in favour of either task is circumstantial . south american monkeys have been the main focus of study because they are polymorphic in their colour vision ( mollon , bowmaker , & jacobs , 1984 ) ; within the same species , some individuals can have trichromacy , while others have dichromacy ( we will return to this in the discussion section ) . caine and mundy ( 2000 ) tested marmosets in visual search tasks in a naturalized captive setting . an advantage for trichromats was evident only for the longer distance task , but the backgrounds for the visual search were not matched across tasks ( the primary aim of the study was to establish whether the trichromats had an advantage in any task rather than to formally test the distance manipulation ) . since then , there has been no distance manipulation within the same study , and it has been surprisingly difficult for researchers to observe any significant behavioural differences between trichromatic and dichromatic monkeys in the wild . ( 2003b ) found a hint that trichromatic moustached tamarins might be more likely to lead the troop into fruiting trees than dichromats , consistent with an advantage in spotting fruit , but this was not consistent and it was not the case that all troops of tamarins were led by trichromats ( saddleback tamarins were mainly male - led , who are all dichromats ) . however , the only significant observed advantage in wild monkeys has been capuchins selecting fruit at arms ' reach ( melin et al . however , this apparent advantage for picking fruit does not appear to translate into consuming more food overall in trichromatic capuchins or spider monkeys ( hiramatsu et al . thus , it remains unclear whether we should expect the greatest advantage of human - like trichromacy to be in the longer distance task of spotting food , or the close - up task of picking it . the aim of our experiment was to simulate the competition to find fruit between two primates ( in this case humans ) in a naturalistic visual search task of real fruit in real foliage . we tested normal trichromats , dichromats and anomalous trichromats who have less separation between the l and m cone sensitivity functions than do we included anomalous trichromats both to boost colour - deficient participant numbers ( dichromats are relatively rare , 2% of the male population ) , and because evolutionarily the competing state to what we now consider normal trichromacy has almost certainly been anomalous trichromacy as well as dichromacy . the task was to count the number of ripe fruit hanging in a real bush from a distance of 1 , 4 , 8 or 12 m ( figure 1 ) . the task was performed simultaneously by a pair of participants , one dichromat or anomalous trichromat and one normal trichromat matched as closely as possible for age , sex , experience of visual experiments and knowledge of colour vision . this set - up also engendered some aspect of competition between the pair , simulating the competition for fruit of two primates simultaneously foraging in the same tree . stimuli ( b ) subtended approximately 2 , 0.5 , 0.25 and 0.16 degrees of visual angle for distances 1 , 4 , 8 , 12 m. in panel ( c ) , thin orange and green lines are ripe and unripe ugandan fruit chrysophyllum albidum , orange and green thick lines are ripe and unripe segments of yellow / orange and green pepper used as targets and distractors overlap in their chromatic properties ( orange and green filled circles ) with some fruits eaten by primates in uganda ( unfilled circles ) . in panel ( c ) , the rt for the colour - deficient member of each pair in each condition is simply divided by the rt of the colour - normal member ( which is a simpler analysis and also controls for differences in baseline speed with distance ) . figure 4(c ) shows that the same pattern [ f(3,21 ) = 5.6 , p = 0.005 ] occurred for the mean ratio of response times of the colour - normal and colour - deficient members of each pair . the dashed and dotted lines in figures 4(a c ) show the subsets of results for dichromats and anomalous trichromats ( each compared with their matched normal trichromat ) , indicating that the effect on response time is greater for full dichromats [ interaction f(3,18 ) = 3.4 , p < 0.05 ] , as would be expected . the same pattern is not evident in the error scores , but this could be a simple lack of power or a difference in speed accuracy strategy between groups . note that the key comparison was between the colour - deficient and normal trichromatic member of each pair , not between dichromats and anomalous trichromats , because although the dichromats and anomalous trichromats were each matched in age and relevant experience to their control trichromats , dichromats and anomalous trichromats could not be matched in age or experience with each other . the mean age of the dichromats was 10 years older than that of the anomalous trichromats . our results show that in our naturalistic visual search task with human observers , the advantage of normal trichromacy over dichromacy or anomalous trichromacy increased with distance ( even without the additional cues of smell and touch available to foraging monkeys ) . this is likely to be because visual cues such as shape and the s/(l+m ) dimension of colour vision decrease with distance for small targets . for very small stimuli , the absence of s cones in the very centre of the fovea ( about the central 1/3 of a degree of visual angle ) , precludes information in the s/(l+m ) pathway for fixated targets ( known as small field tritanopia ) , although the effect of this is less when observers scan a scene , as in the present task ( e.g. our results imply that the more important task for the natural selection of primate trichromacy may have been spotting food from a distance rather than picking it at arms ' length . the biggest advantage of one phenotype over competing phenotypes does not have to arise for the task that the phenotype is best at . however , while the spatial - frequency sensitivity of the lm system undoubtedly makes it optimal at the close - up task , our results do raise the question of whether it has been optimized by that task . rather , its optimality may have emerged as a side effect of the combined pressures of the longer distance task and other constraints , such as the random layout of l and m cones on the retina ( deeb , 2006 ) and the unavoidable fact that both colour resolution and achromatic resolution can not be equally high because they are conflated in the most fine scale comparison of neighbouring cones . most studies in the wild have concentrated on observing monkeys picking fruit ( e.g. unfortunately , it is very difficult to discern and quantify the longer distance task except by observing troop leadership , which is known to be influenced by other social factors which might mask any effect of colour vision ( smith , buchanan - smith , surridge , & mundy , 2003a ) . another possibility is that the colour properties of fruits consumed in the forests where these studies have taken place are not fully representative of the gamut of fruits eaten by our ancestors . ( 2008 ) , who found no differences in feeding behaviour between dichromatic and trichromatic spider monkeys , show a higher ratio of cryptic fruits ( those that stay green ; see also de araujo , lima , & pessoa , 2006 ) and fruits that turn dark brownish purple ( as do many types of fig ) , and fewer orange or yellow fruit than are found in the diet of primates in uganda ( sumner & mollon , 2000a , 2000b ) . cryptic fruits are detected from afar by smell or shape ( and memory ) , and close up by shape , position , smell or feel ( e.g. however , viewed amongst foliage from a distance , dark fruits ( unless in a clump ) could easily be mistaken for shadow or gaps , so the use of achromatic cues in fruit foraging remains unclear ( as does the extra cue of glossiness often present in dark fruits ) . however , rare alleles tend to be lost to populations if their presence provides no advantage ( e.g. surridge & mundy , 2002 ) , so it is assumed that to maintain the polymorphism there must be some advantage to trichromacy in the wild that translates into improved fitness ( the potential advantage of having different types of dichromacy in a population has not been given any support ; riba - hernandez et al . the other possibility is that the advantage of trichromacy is much subtler than originally expected when combined with social factors within a polymorphic population ( e.g. either way , given that many species of monkey have successfully survived and diversified over millions of years in which the majority of individuals ( and all the males ) have been dichromatic ( e.g. surridge & mundy , 2002 ) , it should not be surprising that dichromats are not glaringly deficient at any crucial task in the wild that would dramatically harm their chances of survival . lastly , some authors have suggested that the selective advantage of trichromacy might have been in signalling ( changizi , zhang , & shimojo , 2006 ) . more recent evidence suggests that such signalling post - dates the invention of trichromacy , and thus took advantage of trichromacy once it had arisen , rather than driving its natural selection initially ( fernandez & morris , 2007 ) . however , it may form part of the selection pressure maintaining it in current populations , as might predator detection ( smith , buchanan - smith , surridge , & mundy , 2005 ; sumner & mollon , 2003 ) . in conclusion , we found that in a naturalistic visual search task of finding food in foliage , humans with normal colour vision diverged in performance from dichromats or anomalous trichromats most for the longest viewing distance ( 12 m ) . this implies that in the natural selection of trichromacy , the task of spotting fruit from a distance was a greater driving force than picking fruit close up . colour - deficient participants were recruited through advert and tested using the ishihara test ( 24 plate edition ) and nagel anomaloscope to determine their type of colour vision . following this screening , 12 participants with clearly defined colour deficiency ( 3 protanopes , 4 deuteranopes , 2 protanomalous and 3 deuteranomalous ) performed the visual search task . each was paired with control participant with normal trichromacy ( tested using the ishihara plates ) , matching as closely as possible for age , sex , experience of visual experiments and knowledge of colour vision . this set - up also engendered some aspect of competition between the pair , simulating the competition for fruit of two primates simultaneously foraging in the same tree . one participant opened his eyes prematurely on some of the visual search trials , and so that pair was excluded from all analyses . participants had to count the number of ripe fruit hanging in a bush from a distance of 1 , 4 , 8 or 12 m ( figure 1 ) . their task was to press one of the five keys ( labelled 0 , 1 , 2 , 3 , 4 ) on a keyboard as quickly and accurately as possible to indicate the number of targets they could see . we located a suitable bush ( aucuba japonica ) in an unused courtyard on the university campus , which could be viewed from between 1 and 12 m. we measured the reflected spectra ( spectrocal , cambridge research systems ) from 25 of the leaves in situ under natural overcast conditions in order to compare their chromaticity coordinates ( figure 3 ) with leaves measured in a ugandan rainforest , also under overcast conditions , by sumner and mollon ( 2000a ) . to be used in our task , target and distractor fruit had to satisfy three conditions : ( 1 ) the chromaticities had to be representative of the rainforest fruit ( to calculate chromaticities , we used a natural illuminant in overcast conditions from near the experimental bush for experimental stimuli , and from uganda for ugandan fruit , http://vision.psychol.cam.ac.uk/spectra/ ) ; ( 2 ) the stimuli had to be light enough to hang on the bush ; ( 3 ) the stimuli must not change ( e.g. finding segments of pepper amongst leaves is a task highly representative of the chromatic visual search task facing primates in uganda , which in turn is the environment most likely to represent that of our shared ancestors . segments 3 5 cm were used ( figure 2 ) , subtending the following degrees of visual angle for distances 1 , 4 , 8 , 12 : 1.7 2.3 , 0.43 0.57 , 0.21 0.29 , 0.14 0.19 . they were then informed that the yellow peppers , but not the green , were the targets for the visual search . colour - deficient participants were recruited through advert and tested using the ishihara test ( 24 plate edition ) and nagel anomaloscope to determine their type of colour vision . following this screening , 12 participants with clearly defined colour deficiency ( 3 protanopes , 4 deuteranopes , 2 protanomalous and 3 deuteranomalous ) performed the visual search task . each was paired with control participant with normal trichromacy ( tested using the ishihara plates ) , matching as closely as possible for age , sex , experience of visual experiments and knowledge of colour vision . this set - up also engendered some aspect of competition between the pair , simulating the competition for fruit of two primates simultaneously foraging in the same tree . one participant opened his eyes prematurely on some of the visual search trials , and so that pair was excluded from all analyses . participants had to count the number of ripe fruit hanging in a bush from a distance of 1 , 4 , 8 or 12 m ( figure 1 ) . their task was to press one of the five keys ( labelled 0 , 1 , 2 , 3 , 4 ) on a keyboard as quickly and accurately as possible to indicate the number of targets they could see . we located a suitable bush ( aucuba japonica ) in an unused courtyard on the university campus , which could be viewed from between 1 and 12 m. we measured the reflected spectra ( spectrocal , cambridge research systems ) from 25 of the leaves in situ under natural overcast conditions in order to compare their chromaticity coordinates ( figure 3 ) with leaves measured in a ugandan rainforest , also under overcast conditions , by sumner and mollon ( 2000a ) . to choose target and distractor stimuli , we measured the reflectance spectra of a range of fruits obtained from local supermarkets and specialist shops , in order to compare them with the fruit in a ugandan forest ( sumner and mollon ( 2000a , b ) . to be used in our task , target and distractor fruit had to satisfy three conditions : ( 1 ) the chromaticities had to be representative of the rainforest fruit ( to calculate chromaticities , we used a natural illuminant in overcast conditions from near the experimental bush for experimental stimuli , and from uganda for ugandan fruit , http://vision.psychol.cam.ac.uk/spectra/ ) ; ( 2 ) the stimuli had to be light enough to hang on the bush ; ( 3 ) the stimuli must not change ( e.g. finding segments of pepper amongst leaves is a task highly representative of the chromatic visual search task facing primates in uganda , which in turn is the environment most likely to represent that of our shared ancestors . segments 3 5 cm were used ( figure 2 ) , subtending the following degrees of visual angle for distances 1 , 4 , 8 , 12 : 1.7 2.3 , 0.43 0.57 , 0.21 0.29 , 0.14 0.19 . they were then informed that the yellow peppers , but not the green , were the targets for the visual search .

however , the colours that objects appear depend on the colour vision of the observer , and humans actually have a rare form of colour vision among all animals ( e.g. an ancestor to african and asian primates , with which we share our type of colour vision , reinvented full trichromatic vision from these dichromatic ancestors , so that we now have three types of cone shortwave ( s ) , mediumwave ( m ) and longwave ( l)two of which , l and m , remain similar to each other in many ways because they are the offspring of the same ancestral mammalian longwave cone . the main theories have suggested that the key selective advantage of trichromacy for primates lay in foraging , either for fruit ( allen , 1879 ; mollon , 1989 ; osorio & vorobyev , 1996 ; polyak , 1957 ; regan et al . parraga , troscianko , and tolhurst ( 2002 ) compared the spatiochromatic properties of calibrated natural images , including those of fruit on leafy backgrounds with the properties of the three early channels of human vision : the lm colour pathway , which compares l cone signals with m cone signals ( this pathway is often called red green , but this is slightly misleading ) ; the s - lm pathway comparing s cone signals with pooled l and m signals ( this pathway is often called blue yellow , but this is highly misleading ) , and an achromatic pathway . it is the ability to compare l with m signals that was new in primate trichromacy , and parraga et al . does it follow that picking food was the critical task driving the natural selection of primate trichromacy ? and sumner & mollon modelled the task of finding fruit or young leaves in foliage for all plausible sensitivity functions that the l and m cones might take , in order to work out which combination of potential l and m cones would be optimal . it turned out that the optimal pair are very close to what we actually possess , supporting the argument that finding important objects amongst foliage was a driving force in the evolution of the lm colour channel . ( 2002 ) but a component of the modelling the estimation of quantum noise did require the viewing distance of the stimuli to be specified . these studies explicitly assumed the key task was spotting food from a distance rather than picking food close up , and thus chose distances of about 10 m. behavioural evidence in favour of either task is circumstantial . south american monkeys have been the main focus of study because they are polymorphic in their colour vision ( mollon , bowmaker , & jacobs , 1984 ) ; within the same species , some individuals can have trichromacy , while others have dichromacy ( we will return to this in the discussion section ) . these species offer the opportunity of natural experiments , comparing the behaviour of trichromatic individuals with dichromatic individuals , either in zoos or in the wild . in one task , they scattered edible orange or green cereal balls in the cage , which required viewing from up to 6 m away , while in the other task the targets were placed on a tray among green wood shavings and viewed close up ( < 0.5 m ) . an advantage for trichromats was evident only for the longer distance task , but the backgrounds for the visual search were not matched across tasks ( the primary aim of the study was to establish whether the trichromats had an advantage in any task rather than to formally test the distance manipulation ) . since then , there has been no distance manipulation within the same study , and it has been surprisingly difficult for researchers to observe any significant behavioural differences between trichromatic and dichromatic monkeys in the wild . in a captive environment , the foraging advantage of trichromats has been replicated with tamarins finding simulated fruits in simulated leaves , but there was no distance manipulation ( smith , buchanan - smith , surridge , osorio , & mundy , 2003b ) . ( 2003b ) found a hint that trichromatic moustached tamarins might be more likely to lead the troop into fruiting trees than dichromats , consistent with an advantage in spotting fruit , but this was not consistent and it was not the case that all troops of tamarins were led by trichromats ( saddleback tamarins were mainly male - led , who are all dichromats ) . ( 2008 ) found that luminance , rather than colour , was the cue that best predicts foraging efficiency at grasping distance ( for the fruits in their study at least ) . thus , it remains unclear whether we should expect the greatest advantage of human - like trichromacy to be in the longer distance task of spotting food , or the close - up task of picking it . the aim of our experiment was to simulate the competition to find fruit between two primates ( in this case humans ) in a naturalistic visual search task of real fruit in real foliage . we tested normal trichromats , dichromats and anomalous trichromats who have less separation between the l and m cone sensitivity functions than do we included anomalous trichromats both to boost colour - deficient participant numbers ( dichromats are relatively rare , 2% of the male population ) , and because evolutionarily the competing state to what we now consider normal trichromacy has almost certainly been anomalous trichromacy as well as dichromacy . the task was to count the number of ripe fruit hanging in a real bush from a distance of 1 , 4 , 8 or 12 m ( figure 1 ) . we used segments of yellow pepper as targets because they display spectral and thus chromatic properties representative of many fruit favoured by primates in a ugandan rainforest ( figures 2 and 3 ) . the task was performed simultaneously by a pair of participants , one dichromat or anomalous trichromat and one normal trichromat matched as closely as possible for age , sex , experience of visual experiments and knowledge of colour vision . this set - up also engendered some aspect of competition between the pair , simulating the competition for fruit of two primates simultaneously foraging in the same tree . panel a : samples of unripe , green , and ripe , yellow , chrysophyllum albidum ) , we chose segments yellow pepper as targets and green pepper as distractors ( b ) , because they have similar reflectance spectra ( c ) and chromatic properties ( figure 3 ) . stimuli ( b ) subtended approximately 2 , 0.5 , 0.25 and 0.16 degrees of visual angle for distances 1 , 4 , 8 , 12 m. in panel ( c ) , thin orange and green lines are ripe and unripe ugandan fruit chrysophyllum albidum , orange and green thick lines are ripe and unripe segments of yellow / orange and green pepper used as targets and distractors overlap in their chromatic properties ( orange and green filled circles ) with some fruits eaten by primates in uganda ( unfilled circles ) . normal trichromats made faster responses and fewer errors than their colour - deficient competitors , as expected ( mean rts 4.7 vs. 6.4 s ; error score 0.17 vs. 0.54 ; all errors were misses , our unit of analysis is the pairwise comparison between the members of each matched pair . as shown in figures 4(a and b ) , the relative advantage increased with distance , both for response time and errors [ one - way anovas , f(3,21 ) = 9.7 , p < 0.001 and f(3,30 ) = 4.0 , p < 0.02 ; see the methods section for subject numbers and other details ] . in figures 4(a and b ) , the difference in performance ( rt or error ) between the colour - deficient member and the colour - normal member of each pair was divided by the overall mean rt or error of that pair , in order to control for different overall performance and speed accuracy trade - offs ( range in mean rt , 3.412.8 s and in mean error score 0.150.57 ; the behaviour of each member was correlated , r = 0.76 for rt and r = 0.32 for errors , probably because they influenced each other in this regard , as would be expected , and/or because they had correlated age , experience and testing conditions ) . a and b ) , the difference in performance ( rt or error ) between the colour - deficient member and the colour - normal member of each pair was divided by the overall mean rt or error of that pair , in order to control for different overall performance and speed accuracy trade - offs . in panel ( c ) , the rt for the colour - deficient member of each pair in each condition is simply divided by the rt of the colour - normal member ( which is a simpler analysis and also controls for differences in baseline speed with distance ) . figure 4(c ) shows that the same pattern [ f(3,21 ) = 5.6 , p = 0.005 ] occurred for the mean ratio of response times of the colour - normal and colour - deficient members of each pair . the dashed and dotted lines in figures 4(a c ) show the subsets of results for dichromats and anomalous trichromats ( each compared with their matched normal trichromat ) , indicating that the effect on response time is greater for full dichromats [ interaction f(3,18 ) = 3.4 , p < 0.05 ] , as would be expected . note that the key comparison was between the colour - deficient and normal trichromatic member of each pair , not between dichromats and anomalous trichromats , because although the dichromats and anomalous trichromats were each matched in age and relevant experience to their control trichromats , dichromats and anomalous trichromats could not be matched in age or experience with each other . our results show that in our naturalistic visual search task with human observers , the advantage of normal trichromacy over dichromacy or anomalous trichromacy increased with distance ( even without the additional cues of smell and touch available to foraging monkeys ) . for very small stimuli , the absence of s cones in the very centre of the fovea ( about the central 1/3 of a degree of visual angle ) , precludes information in the s/(l+m ) pathway for fixated targets ( known as small field tritanopia ) , although the effect of this is less when observers scan a scene , as in the present task ( e.g. our results imply that the more important task for the natural selection of primate trichromacy may have been spotting food from a distance rather than picking it at arms ' length . this in no way challenges the previous finding that the lm colour channel is optimal at the close - up task ( parraga et al . however , while the spatial - frequency sensitivity of the lm system undoubtedly makes it optimal at the close - up task , our results do raise the question of whether it has been optimized by that task . rather , its optimality may have emerged as a side effect of the combined pressures of the longer distance task and other constraints , such as the random layout of l and m cones on the retina ( deeb , 2006 ) and the unavoidable fact that both colour resolution and achromatic resolution can not be equally high because they are conflated in the most fine scale comparison of neighbouring cones . one general puzzle is why it has been so difficult to observe any behavioural advantages for trichromats over dichromats in the wild , when they have been apparent as expected in experiments such as this one and those on captive tamarins and marmosets ( caine & mundy , 2000 ; smith et al . , 2007 ) , where our results predict the advantage is smallest , even without the extra tactile and olfactory cues available in the wild but not in our experiment . unfortunately , it is very difficult to discern and quantify the longer distance task except by observing troop leadership , which is known to be influenced by other social factors which might mask any effect of colour vision ( smith , buchanan - smith , surridge , & mundy , 2003a ) . ( 2008 ) , who found no differences in feeding behaviour between dichromatic and trichromatic spider monkeys , show a higher ratio of cryptic fruits ( those that stay green ; see also de araujo , lima , & pessoa , 2006 ) and fruits that turn dark brownish purple ( as do many types of fig ) , and fewer orange or yellow fruit than are found in the diet of primates in uganda ( sumner & mollon , 2000a , 2000b ) . however , viewed amongst foliage from a distance , dark fruits ( unless in a clump ) could easily be mistaken for shadow or gaps , so the use of achromatic cues in fruit foraging remains unclear ( as does the extra cue of glossiness often present in dark fruits ) . note that our targets did also contain a strong luminance signal relative to the foliage background , and of course wild monkeys have a lifetime of practice in fruit picking which acts to diminish any behavioural differences between them . it arises through there being different alleles of the gene coding the l cone pigment , which give the pigment different sensitivities ( akin to our l and m cones ) . in many species , there are three alleles present in different frequencies , which means that approximately 60% of females are trichromatic ( jacobs , neitz , & neitz , 1993 ; williams , hunt , bowmaker , & mollon , 1992 ) . surridge & mundy , 2002 ) , so it is assumed that to maintain the polymorphism there must be some advantage to trichromacy in the wild that translates into improved fitness ( the potential advantage of having different types of dichromacy in a population has not been given any support ; riba - hernandez et al . on the other hand , if this advantage was very large , there is the obvious question of why full trichromacy has not emerged in these species , as it did for asian and african primates , and has done also for howler monkeys ( jacobs , neitz , deegan , & neitz , 1996 ) . either way , given that many species of monkey have successfully survived and diversified over millions of years in which the majority of individuals ( and all the males ) have been dichromatic ( e.g. from this perspective , the advantage of trichromacy is bound to be quite subtle and hard to measure in free - ranging wild monkeys . more recent evidence suggests that such signalling post - dates the invention of trichromacy , and thus took advantage of trichromacy once it had arisen , rather than driving its natural selection initially ( fernandez & morris , 2007 ) . in conclusion , we found that in a naturalistic visual search task of finding food in foliage , humans with normal colour vision diverged in performance from dichromats or anomalous trichromats most for the longest viewing distance ( 12 m ) . this implies that in the natural selection of trichromacy , the task of spotting fruit from a distance was a greater driving force than picking fruit close up . therefore , there were 16 participants ( 8 pairs ) for the analysis of response time and 22 participants ( 11 pairs ) for the analysis of error scores . on each trial , the two participants were positioned at the appropriate distance and blindfolded while the experimenter positioned four fruit between zero and four of these stimuli were targets ( ripe fruit ) and the rest were distractors ( unripe fruit ) . we located a suitable bush ( aucuba japonica ) in an unused courtyard on the university campus , which could be viewed from between 1 and 12 m. we measured the reflected spectra ( spectrocal , cambridge research systems ) from 25 of the leaves in situ under natural overcast conditions in order to compare their chromaticity coordinates ( figure 3 ) with leaves measured in a ugandan rainforest , also under overcast conditions , by sumner and mollon ( 2000a ) . to choose target and distractor stimuli , we measured the reflectance spectra of a range of fruits obtained from local supermarkets and specialist shops , in order to compare them with the fruit in a ugandan forest ( sumner and mollon ( 2000a , b ) . to be used in our task , target and distractor fruit had to satisfy three conditions : ( 1 ) the chromaticities had to be representative of the rainforest fruit ( to calculate chromaticities , we used a natural illuminant in overcast conditions from near the experimental bush for experimental stimuli , and from uganda for ugandan fruit , http://vision.psychol.cam.ac.uk/spectra/ ) ; ( 2 ) the stimuli had to be light enough to hang on the bush ; ( 3 ) the stimuli must not change ( e.g. finding segments of pepper amongst leaves is a task highly representative of the chromatic visual search task facing primates in uganda , which in turn is the environment most likely to represent that of our shared ancestors . segments 3 5 cm were used ( figure 2 ) , subtending the following degrees of visual angle for distances 1 , 4 , 8 , 12 : 1.7 2.3 , 0.43 0.57 , 0.21 0.29 , 0.14 0.19 . therefore , there were 16 participants ( 8 pairs ) for the analysis of response time and 22 participants ( 11 pairs ) for the analysis of error scores . we located a suitable bush ( aucuba japonica ) in an unused courtyard on the university campus , which could be viewed from between 1 and 12 m. we measured the reflected spectra ( spectrocal , cambridge research systems ) from 25 of the leaves in situ under natural overcast conditions in order to compare their chromaticity coordinates ( figure 3 ) with leaves measured in a ugandan rainforest , also under overcast conditions , by sumner and mollon ( 2000a ) . to choose target and distractor stimuli , we measured the reflectance spectra of a range of fruits obtained from local supermarkets and specialist shops , in order to compare them with the fruit in a ugandan forest ( sumner and mollon ( 2000a , b ) . to be used in our task , target and distractor fruit had to satisfy three conditions : ( 1 ) the chromaticities had to be representative of the rainforest fruit ( to calculate chromaticities , we used a natural illuminant in overcast conditions from near the experimental bush for experimental stimuli , and from uganda for ugandan fruit , http://vision.psychol.cam.ac.uk/spectra/ ) ; ( 2 ) the stimuli had to be light enough to hang on the bush ; ( 3 ) the stimuli must not change ( e.g. finding segments of pepper amongst leaves is a task highly representative of the chromatic visual search task facing primates in uganda , which in turn is the environment most likely to represent that of our shared ancestors . segments 3 5 cm were used ( figure 2 ) , subtending the following degrees of visual angle for distances 1 , 4 , 8 , 12 : 1.7 2.3 , 0.43 0.57 , 0.21 0.29 , 0.14 0.19 .

the er participates in folding and posttranslational processing of proteins destined for secretion or membrane insertion . several signaling pathways , collectively referred to as the unfolded protein response ( upr ) , * maintain an equilibrium between the capacity of the er to process its client proteins and the physiological demand on the organelle ( kaufman , 1999 ; mori , 2000 ; patil and walter , 2001 ) . inactivation of upr signaling has deleterious consequences on cell survival and organ function ( bertolotti et al . , 2001 ; ; shen et al . , 2001 ) , and accumulation of malfolded proteins in the er may play an important role in human diseases ( carrell and lomas , 1997 ; aridor and balch , 1999 ) . in yeast , er stress activates the er resident transmembrane protein kinase and endonuclease ire1p , leading to the posttranscriptional processing of a downstream mrna encoding the transcription factor hac1p . these target genes encode proteins involved in processing , trafficking , and degradation of er client proteins ( for reviews see mori , 2000 ; patil and walter , 2001 ) . in addition to a conserved signaling pathway of ire1 homologues and a downstream hac1-like transcription factor , xbp-1 ( yoshida et al . , 2001 ; calfon et al . , pancreatic - enriched er kinase ( perk ) phosphorylates eukaryotic translation initiation factor 2 to attenuate protein synthesis and activate specific gene expression during er stress ( for review see ron and harding , 2000 ) , and the er stress activated transcription factor atf6 directly activates upr target genes ( haze et al . , 1999 ; ye et al . , 2000 ; yoshida et al . , 2001 ) . the coordination of these three pathways and their specific contribution to the metazoan er stress response are unclear , though in caenorhabditis elegans , the ire1 and perk pathways provide redundant protection against er stress ( shen et al . , 2001 ) . we used cdna microarrays to characterize the transcriptional response to er stress in c. elegans , a simple metazoan that has counterparts of all three known components of the mammalian upr ( shen et al . we find that in c. elegans , the ire-1 and xbp-1 pathway has retained its essential role in upregulating expression of many upr target genes that are similarly upregulated by the homologous pathway in yeast . we also discovered a novel family of highly related genes that protect against er stress when the ire-1 and xbp-1 signaling pathway is defective . we compared the gene expression profile in unstressed and er - stressed wild - type young adult c. elegans with the profile in an xbp-1 mutant strain that is defective in the ire-1 and xbp-1 signaling pathway ( calfon et al . , 2002 ) . exposure of wild - type animals to the er stress inducing glycosylation inhibitor tunicamycin resulted in a 2.5-fold or greater statistically significant activation ( p < 0.05 ) of 34 of the 18,000 genes on the array . of these 34 genes , 26 were uninduced or induced to significantly lower levels ( p < 0.05 ) in the xbp-1 mutant strain . all but 2 of the 26 xbp-1regulated genes encode familiar proteins such as er or cytosolic chaperones , proteins involved in vesicular transport or other aspects of posttranslational processing of er client proteins ( table i ) . therefore , in c. elegans , as in yeast , the upr entails induction of genes involved in many different aspects of posttranslational protein processing in the secretory pathway . we confined our presentation only to genes whose induction withstood stringent statistical criteria that took into account both variation among replicate points in our experiments ( so - called local variation ) and historical information on variation between replicates in other experiments performed on the same array ( so - called global variation ) . among the genes these observations suggest that robustly induced upr genes are significantly controlled by xbp-1 , however we have not addressed the role of xbp-1 in the activation of genes with more variable levels of induction by er stress . shown is the mean sem ( n = 3 ) of the base2 log fold increase in tunicamycin - treated animals over untreated animals for each genotype . the induction of lin-15 ( zk662.4 ) is likely to be an artifact of the hsp-4::gfp;lin-15 array present in both wild - type and mutant animals . 19 genes were induced to higher levels in xbp-1 mutant animals than in wild - type animals ( table ii ) . nine of these encode highly similar , novel proteins with a hydrophobic nh2-terminal signal sequence , a potential transmembrane domain , and a short cooh - terminal cytoplasmic domain ( fig . 1 , a , b , and c ) . we refer to members of this family as activated in blocked upr ( abu ) . two other similarly regulated genes encode proteins that are more distantly related to the nine abu family members ( table ii ) . a representative family member , abu-1 ( ac3.3 ) , was chosen for further study based on its central location in the family tree ( fig . northern blot analysis confirmed the induction of abu-1 by tunicamycin treatment of xbp-1 mutant animals but not wild - type animals ( fig . shown is the mean sem ( n = 3 ) of the base2 log fold increase in hybridization signal in tunicamycin - treated animals over untreated animals for each genotype . the linkage group for the nine abu genes is also shown . the c. elegans activated in blocked upr ( abu ) gene family . ( a ) the amino acid sequence identity relationship of the abu family members , c. elegans ced-1 ( zhou et al . , 2001 ) , and human scavenger receptor of endothelial cells ( adachi et al . , 1997 ) is displayed as a radial tree ( page , 1998 ) . ( b ) amino acid sequence alignment of the nine abu proteins using the clustal w ( 1.81 ) program . ( d ) northern blot analysis of total rna from untreated and tunicamycin - treated wild - type and xbp-1(zc12)iii mutant c. elegans . the blot was hybridized sequentially to labeled abu-1 and hsp-4 cdna . the ethidium bromide stained gel ( bottom panel ) reports on the integrity of the mrna in all samples . abu proteins have a distant similarity to a mammalian scavenger receptor of endothelial cells and to the c. elegans cell corpse engulfment protein ced-1 , which are transmembrane cell surface proteins ( adachi et al . , 1997 ; zhou et al . , 2001 ) . to study the subcellular localization of abu-1 , the protein was tagged at its cooh terminus with gfp and expressed in the intestine of c. elegans from a transgene driven by the ges-1 promoter . ges-1::abu-1gfp confocal microscopy showed that the puncta corresponded to vesicular structures that were present throughout the large intestinal cell and tended to cluster near its apical surface ( fig . in contrast , ges-1::gfp fluorescence was observed throughout the cytoplasm and nucleus of the intestinal cell ( fig . the expression level of this transgene was much lower than that of ges-1::abu-1gfp and was noted in the pharynx and not the intestine ( also see fig . 2 , d and e ) , suggesting that this pattern of expression is not an artifact of unphysiologically high expression levels of the fusion proteins . however , given that we were not able to detect the endogenous abu-1 protein , it remains possible that the apparent localization to intracellular vesicles is due to misfolding of the fusion proteins . ( a ) ges-1::abu-1::gfp(zcex7 ) expressing abu-1 with gfp fused to its cooh terminus in the intestinal cell . the arrows point to the vesicular structures positive for abu-1gfp and the arrowheads point to the background autofluorescence that is present in worms with no transgene . shown are representative images obtained using the same settings . ( d ) the abu-1::gfp(zcex8 ) transgene expresses an abu-1gfp fusion protein from the endogenous abu-1 promoter . inactivation of abu-1 causes er stress and enhances the phenotype of c. elegans with defective er - associated protein degradation . ( a ) fluorescence micrographs of untreated ( ut ) or cadmium - treated ( cd ) wild - type or xbp-1(zc12)iii mutant c. elegans transgenic for an abu-1::gfp(zcis8)x reporter . ( b ) fluorescence micrographs of untreated ( ut ) , abu-1(rnai ) , or sel-1(rnai ) animals transgenic for the er stress reporter gene hsp-4::gfp(zcis4)v . ( c ) lethality at 72 h of wild - type and xbp-1(zc12)iii mutant c. elegans untreated ( ut ) , abu-1(rnai ) , sel-1(rnai ) , or compound abu-1(rnai ) ; sel-1(rnai ) . shown are mean sem of a typical experiment performed in duplicate on 30 animals of each genotype and reproduced four times . ( d ) photomicrographs using nomarski optics ( x400 ) of the intestine of living young adult c. elegans of the following genotypes : ( 1 ) wild type , ( 2 ) abu-1(rnai);sel-1(rnai ) , ( 3 ) xbp-1(zc12)iii , ( 4 ) xbp-1(zc12)iii ; abu-1(rnai ) , ( 5 ) xbp-1(zc12)iii ; sel-1(rnai ) , and ( 6 ) xbp-1(zc12)iii;abu-1(rnai);sel-1(rnai ) . the arrow points to the dark large granules that accumulate in the cytoplasm of the compound mutant animals . the fluorescence pattern of abu-1gfp suggested that the protein was associated with the intracellular endomembrane system . to extend these observations , we studied the subcellular localization of c. elegans abu-1 in mammalian cells , an experimental system in which we could make use of established markers for the various organelles and compartments . in transfected mammalian cos1 cells , metabolically - labeled abu-1 tagged at its cooh terminus with the flag epitope remained associated with the cell pellet , from which it could be extracted only by detergent solubilization ( fig . deletion of the predicted transmembrane domain led to secretion of the protein into the culture media ( fig . 3 a ) . immunostaining of the flag - tagged abu-1expressing cos1 cells with anti - flag antibodies showed a diffuse reticular pattern that colocalized with the er marker ribophorin i ( fig . these results suggest that abu-1 encodes an integral membrane protein that is retained in the er by its transmembrane domain . the secretion of transmembrane domain deleted abu-1 suggests that the lumenal portion of the protein folds properly in mammalian cells and that the full - length abu-1 is unlikely to be retained in the er as a consequence of malfolding in mammalian cells . ( a ) autoradiogram of metabolically - labeled wild - type abu-1 ( wt ) or abu-1 lacking the transmembrane domain ( tm ) expressed in cos1 cells and immunoprecipitated immediately after the labeling period ( lanes 1 and 3 ) or after 4 h of cold chase ( lanes 2 and 46 ) . the protein in lanes 14 was extracted by detergent from disrupted cells , whereas the protein in lanes 5 and 6 was recovered from the culture supernatant . ( b ) immunocytochemical staining of flag - tagged abu-1 expressed in cos1 cells using an anti - flag monoclonal antibody . staining of the same cells with ribophorin i outlines the er and the merged image reveals the colocalization of abu-1 ( anti - flag ) and ribophorin 1 . the protein - coding region of the nine abu genes is > 89% identical at the nucleotide level over a stretch of 100 residues in the 3 end of the genes . therefore , we considered it possible to simultaneously inactivate several members of this gene family by rna - mediated interference ( rnai ) , using sequences derived from this conserved region . to study the impact of this genetic manipulation , we first identified a pharmacological treatment that would cause er stress but could be tolerated by both wild - type and ire-1 or xbp-1 mutant animals . the heavy metal cadmium activated the hsp-4::gfp reporter ( a marker of er stress ) in an ire-1 and xbp-1dependent manner ( fig . 4 a ) , indicating that it causes er stress . both wild - type and xbp-1 mutant animals tolerated cadmium exposure for extended periods of time ( fig . feeding of double - stranded gfp rna ( gfp[rnai ] ) had little impact on the viability of wild - type or mutant animals whether exposed to cadmium or not ( < 5% lethality ) . however , feeding of double - stranded abu-1 rna markedly impaired survival of cadmium - treated xbp-1 and ire-1 mutant animals , but had minimal impact on survival of cadmium - treated wild - type animals or on survival of untreated xbp-1 and ire-1 mutant animals ( fig . 4 b ) . the inhibition of abu-1 protein expression by abu-1(rnai ) was revealed by the observation that feeding double - stranded abu-1 rna to ges-1::abu-1gfp animals strongly inhibited gfp fluorescence ( fig . 4 c ) . these observations are consistent with a role for abu-1 ( and possibly other abu genes ) in protecting animals with a defective upr against er stress . inactivation of abu-1 by rnai reduces the viability of c. elegans with a blocked upr . ( a ) fluorescence micrographs of untreated ( ut ) or cadmium - treated ( cd ) wild - type , xbp-1(zc12)iii mutant , or ire-1(zc14)ii mutant c. elegans transgenic for the er stress reporter gene hsp-4::gfp(zcis4)v . ( b ) survival at 48 h of untreated ( ut ) or cadmium - treated ( cd ) wild - type , xbp-1(zc12)iii mutant , or ire-1(zc14)ii mutant c. elegans at the l4 stage that had been subjected to abu-1(rnai ) , gfp(rnai ) , or no rnai . shown are mean sem of a typical experiment performed in duplicate on 30 animals in each group and reproduced four times . ( c ) fluorescence micrograph of untreated ( ut ) and abu-1(rnai)treated ges-1::abu-1 : : gfp(zcex7 ) animals . to determine the expression pattern of abu-1 in er - stressed animals abu-1::gfp was expressed constitutively in the pharynx and head region of wild - type and xbp-1 mutant adult animals . intestinal expression was induced by er stress , most notably in xbp-1 mutant animals ( fig . 5 a ) . the induction of abu-1::gfp in the intestine of cadmium- and tunicamycin - treated animals is consistent with observations that the intestine is active in protein secretion and is a major target for er stress in c. elegans ( fig . 4 a ) ( shen et al . , 2001 ; calfon et al . , abu-1(rnai ) upregulated the er stress reporter gene hsp-4::gfp in the intestine ( fig . 5 b ) , implying that abu-1 ( and possibly other abu genes ) may also be active in the intestine under basal conditions and that interfering with abu function causes er stress . genes active in degradation of malfolded er proteins ( er - associated degradation [ erad ] genes ) are not essential for yeast viability under normal growth conditions but are essential during er stress or when the upr is blocked . furthermore , disruption of erad genes in yeast activates the upr ( casagrande et al . , 2000 ; friedlander et al . , 2000 ; we considered the possibility that abu genes might interact with the process of malfolded protein disposal from the c. elegans er . first we confirmed that in c. elegans , like yeast , inactivation of erad genes causes er stress . rnai of sel-1 , the c. elegans homologue of the yeast erad gene hrd3 ( hampton et al . , 1996 ) , resulted in marked upregulation of the er stress indicator hsp-4::gfp ( fig . the induction of hsp-4::gfp by sel-1(rnai ) and by rnai of a homologue of another yeast erad gene , hrd1 ( encoded in c. elegans by f55a11.3 ) , were both xbp-1 dependent ( unpublished data ) . inactivation of sel-1 by rnai had no impact on the viability of wild - type animals and only modestly reduced the viability of xbp-1 mutants . however , when combined with inactivation of abu-1 , sel-1(rnai ) increased lethality of xbp-1 mutant animals ( fig . viewed under nomarski optics , the intestine of wild - type , xbp-1 mutant , abu-1(rnai ) , or sel-1(rnai ) young adults had a similar , fine , granular appearance . animals with compound xbp-1;abu-1(rnai ) or xbp-1;sel-1(rnai ) genotypes had more dark staining large intestinal granules . the xbp-1;abu-1(rnai);sel-1(rnai ) animal had a marked increase in such granules ( fig . 5 d ) . these observations suggest that abu-1 ( and possibly other abu genes ) and sel-1 perform partially redundant functions in animals with a blocked upr . our search for genes activated in the c. elegans upr revealed similarities to the yeast s. cerevisiae . in both species the upr induces genes that function at many levels of the secretory pathway and encode proteins with diverse and unrelated biochemical activities . in both species , the signaling pathway initiated by ire1 exerts nearly complete control over the induction of well - characterized components of er client protein processing machinery , such as bip ( hsp-4 ) , protein disulfide isomerases , and 4-prolyl hydroxylases . the application of these stringent statistical criteria eliminated many genes that probably are true upr targets and therefore the list presented in table i is only representative of the most reliably induced upr genes in c. elegans . heterogeneity in developmental stage and variation in uptake of drug stand out as likely contributors to the lower power of the whole animal array performed here compared with yeast arrays ( casagrande et al . , 2000 ; travers et al . , 2000 ) , and as fig . 1 d shows , the microarray hybridization procedure is also less sensitive than northern blot analysis in detecting fold changes in expression of hsp-4 . despite these limitations , we can firmly conclude that most of the reliably induced upr genes in c. elegans are xbp-1 dependent . our genome - wide survey also identified 19 genes that were activated by er stress in xbp-1 mutant animals but not in wild - type animals . among these were nine genes predicted to encode a family of highly related abu proteins . these abu genes are coregulated across different physiological and developmental perturbation ; clustering together in a single , small , isolated and well - defined expression mountain , mount 29 , in the recently established gene expression map of c. elegans ( kim et al . , 2001 ) . gfp driven by the promoter of a representative family member , abu-1::gfp , was expressed strongly in the pharynx and head region of transgenic animals from larval stages l3l4 to young adult ( fig . 5 a ) . a similar expression pattern was also observed in animals transgenic for a transcriptional fusion of gfp to a different abu gene f19g12.7 ( unpublished data ) . furthermore , the basal expression pattern of the abu-1 transcriptional reporters correlated nicely with the in situ hybridization pattern of another abu family member , c03a7.7 . together , these observations suggest that the abu-1::gfp reporter reflects the activity of the endogenous gene . in older animals , we noted low levels of basal expression of abu-1::gfp in the intestine that was markedly upregulated by er stress in xbp-1 mutant animals ( fig . 5 a ) . this last observation is consistent with the northern blot analysis of abu-1 expression ( fig . although the significance of basal expression of abu genes in the pharynx is currently not understood , the intestinal expression correlates with the phenotype of animals in which abu-1 had been inactivated by rnai . we do not know if abu-1 induction by er stress in xbp-1 mutant animals is dependent on perk ( pek-1 ) or atf6 ( atf-6 ) signaling , as compound xbp-1;pek-1 and xbp-1;atf-6(rnai ) mutant animals arrest at early larval stages before we could reliably examine abu-1::gfp expression in the gut ( shen et al . inactivation of abu-1 by rnai led to marked induction of the er stress indicator hsp-4::gfp in the intestine ( fig . this induction was completely blocked by the xbp-1 mutation ( unpublished data ) , indicating the development of er stress in the gut of abu-1(rnai ) animals and implying that abu-1 plays a role in an er function , which , when inactivated , causes er stress . this er stress appeared to be well tolerated , as the abu-1(rnai ) animals developed at a normal rate and survived exposure to cd , a toxin that induced further er stress . however , in xbp-1 mutant animals , abu-1(rnai ) had serious consequences , significantly reducing their viability after exposure to additional er stress ( fig . 4 b ) . the abu genes have a very high level of sequence identity at the nucleotide level , and the double - stranded rna used to inactivate abu-1 could also initiate rna inactivation of other family members . it is therefore possible that the strong phenotype observed in the abu-1(rnai ) animals reflects the simultaneous inactivation of other abu family members . collectively , these results point to a role for abu-1 , and possibly other abu genes , in some aspect of er function that is also active under basal conditions . reduced levels of this abu - dependent activity can be compensated by a normal upr . however , when the upr is compromised , loss of this abu - dependent activity becomes limiting , killing the affected animals . in yeast , the ability to dispose of misfolded proteins in the secretory pathway ( erad ) plays an important role in adapting to high levels of er stress , but is dispensable for viability under normal conditions ( casagrande et al . , 2000 ; given that abu-1(rnai ) is well tolerated under normal conditions , but not in animals experiencing high levels of er stress , we considered the possibility that the abu genes may interact genetically with components of the c. elegans erad apparatus . an interaction was uncovered between sel-1 , a c. elegans gene encoding an er - localized component of the erad apparatus , and abu-1 . animals with diminished xbp-1 , sel-1 , and abu-1 activity had reduced viability and accumulated unusually large vesicles with dark content in their intestinal cells ( fig . 5 d ) . sel-1 and abu-1 are unlikely to function in the exact same pathway , as abu-1(rnai ) did not affect the phenotype of a hypomorphic allele of lin-12 , whereas sel-1(rnai ) suppressed the egg - laying defect of lin-12(n676 ; n930)iii ( unpublished data ) . however , the observation that inactivating either gene leads to er stress and the synthetic interactions between them suggest that sel-1 and abu-1 function in interacting cellular processes . abu-1 is a transmembrane protein that likely functions within the endomembrane system ( figs . 2 and 3 ) . the lumenal domain of some abu proteins resembles the extracellular domains of a mammalian scavenger receptor and c. elegans ced-1 . both the scavenger receptors and ced-1 are believed to serve as cell surface receptors for chemically modified macromolecules , oxidized lipoproteins , and other altered plasma proteins in the case of the scavenger receptors ( van berkel et al . , 2000 ) and corpses of apoptotic cells in the case of ced-1 ( zhou et al . , 2001 ) . scavenger receptors and ced-1 are therefore believed to play a role in recycling the building blocks of damaged macromolecules that are present outside the cell . it is possible therefore that the abu proteins may be playing a similar role within the endomembrane system , perhaps by binding to altered er client proteins and modulating their intracellular fate . whole genome hybridization experiments were conducted as previously described ( reinke et al . , 2000 ) . in brief , rna was harvested from untreated and tunicamycin - treated ( 5 g / ml , 5 h ) hsp-4::gfp(zcis4)v ( wild type ) and hsp-4::gfp(zcis4)v ; xbp-1(zc12)iii ( mutant ) animals . poly(a ) rna was purified from total rna by oligo ( dt ) affinity chromatography . labeled cdna probe for dna microarray hybridization was prepared from 10 g of poly(a ) rna . cdna from untreated and tunicamycin - treated animals was labeled with cy5 and cy3 , respectively . microarrays were hybridized for 20 h , scanned using an axon scanner , and the expression level for each gene was determined using genepix software . the base2 log of the ratio of cy3/cy5 signal for each gene was determined in each of three hybridization experiments with a pair of untreated and tunicamycin - treated mrna samples with wild - type or xbp-1(zc12)iii genotypes ( for a total of six hybridization experiments ) . a t test was used to determine the probability that differences in treatment - induced levels of mrna between the wild type and xbp-1 mutant were due to chance . a 95% confidence limit the data were analyzed twice , first using the standard deviation resulting from the experiments presented in this paper and then using the global standard deviation derived from the large set of combined experiments in the stanford microarray database . differences in levels of expression were reported only if they were significant using both measurements of standard deviation . a 1.3-kb fragment of c. elegans genomic dna immediately 5 of the predicted initiation atg codon of abu-1 ( ac3.3 ) was amplified by pcr using the oligonucleotides ac3.1s ( 5-ggcattgtggcacgcattgaactg-3 ) and ac3bam.2as ( 5-gataggatccattgttaatatgcttgaagagctgc-3 ) and ligated in frame with the gfp coding region in the plasmid of ppd95.75 ( gift of andy fire , carnegie institute of washington , baltimore , md ) . the abu-1::gfp(zcex8 ) strain was created by coinjecting the ac3.3.ppd95.75 plasmid ( 25 g / ml ) with a lin-15 rescuing plasmid , psk1 ( 25 g / ml ) , into lin-15(n765ts ) strain . the extrachromosomal array was integrated into the chromosome with ultraviolet / trimethylpsoraren treatment , yielding the abu-1::gfp(zcis8)x reporter strain . to produce the ges-1::gfp(zcex6 ) strain , the ges-1 promoter was amplified by pcr from c. elegans genomic dna using the primers ges-1.1s ( 5-ctcatacatcattgtcaagtgacg-3 ) and ges-1.pst.2s ( 5-agtactgcagagacaaggaatatccgcatct-3 ) and ligated into the hindiii - psti sites of ppd95.81 ( gift of andy fire ) . the abu-1 coding region fragment encoding abu-1 amino acids 7425 was ligated in frame with gfp into psti - bamhi sites of this plasmid to produce the ges-1::abu-1::gfp(zxex7 ) strain . the promoter and coding region of abu-1 was ligated in frame with gfp to produce abu1::abu-1::gfp(zcex8 ) strain . the animals containing transmissible extrachromosomal arrays were analyzed using a 510 laser scanning confocal microscope ( zeiss ) . interference with gene function by rnai followed an established protocol ( timmons et al . , 2001 ) . in brief , double - stranded rna was produced in ht115 strain of escherichia coli transformed with ppd129 plasmids containing cdna fragments of genes being studied ( nucleotides 10321278 of abu-1 cdna and nucleotides 4781631 of sel-1 cdna ) . iptg ( 1 mm ) was added to the bacterial growth media to induce transcription of the double - stranded rna , and l4-staged animals of defined genotype were added to plates individually and produced their brood . rnai phenotypes were evaluated in these progeny . where indicated , 30 l4-stage progeny from the rnai plate or from a control plate were transferred to a new plate seeded with fresh e. coli where they were exposed to tunicamycin ( 5 g / ml ) for 5 h or cdcl2 ( 10 mm ) for the indicated period . animals were scored as dead when pharyngeal pumping had ceased and they failed to move after being tapped on the nose with a platinum wire . all cells were grown in dme containing 10% fbs ( cellgro ) at 5% co2 and 37c . abu-1 was tagged with a flag epitope at its cooh terminus by overlapping pcr using the primers ac3.bam.7s ( 5-gcgcggatccgcaatgcgctttatcgcaattgcag-3 ) , ac3.flag.16as ( 5-atcgtcgtccttgtagtcctttctcttgcaacactg-3 ) , and flag3u.not ( 5-ctag - cggccgctcacttgtcatcgtcgtccttgtagtc-3 ) . the full - length coding region of abu-1 with the cooh - terminal flag tag was cloned as a bamhi - noti fragment into pcdna3 ( invitrogen ) . cos-1 cells were transfected with this plasmid and stained with anti - flag antibody ( sigma - aldrich ) . gert kreibich , new york university [ nyu ] school of medicine ) was used to visualize the er staining pattern . the abu-1 coding region fragment encoding abu-1 amino acids 22425 was amplified by pcr using primers ac3.ecori.5s ( 5-ggcaacaagaattcgcgataaacg-3 ) and ac3.xbai.4as ( 5-gcgctctagatgtctactttctcttgcaac-3 ) . the abu-1 coding region fragment coding abu-1 amino acids 22331 was amplified by pcr using ac3.ecori.5s and ac3.xbai.6as ( 5-ggcatctagagcttggcatgcagattggc-3 ) , deleting the transmembrane domain and cooh terminus of abu-1 . cos-1 cells were transfected with wild - type abu-1 or abu-1 lacking the transmembrane domain with a flag tag . newly synthesized proteins were labeled in vivo with s - methionine and s - cysteine ( 500 ci / ml ; icn biochemicals ) . after removal of the labeling media , cells were washed and incubated in complete media for cold chase for the indicated times . cells were washed and lysed with 1% triton buffer ( 20 mm hepes , ph 7.5 , 150 mm nacl , 1% triton x-100 , 10% glycerol , 1 mm edta , 1 mm pmsf , 4 mg / ml aprotinin , and 2 mg / ml pepstatin a ) . samples were clarified by centrifugation at 14,000 g for 10 min and by preincubation for 1 h with 10 l protein a sepharose . soluble proteins from the cells and secreted proteins in the media were immunoprecipitated by anti - flag antibody bound to 10 l protein a sepharose and washed three times with ripa buffer . bound proteins were resolved by 10% sds - page under reducing conditions and revealed by autoradiography . whole genome hybridization experiments were conducted as previously described ( reinke et al . , 2000 ) . in brief , rna was harvested from untreated and tunicamycin - treated ( 5 g / ml , 5 h ) hsp-4::gfp(zcis4)v ( wild type ) and hsp-4::gfp(zcis4)v ; xbp-1(zc12)iii ( mutant ) animals . poly(a ) rna was purified from total rna by oligo ( dt ) affinity chromatography . labeled cdna probe for dna microarray hybridization was prepared from 10 g of poly(a ) rna . cdna from untreated and tunicamycin - treated animals was labeled with cy5 and cy3 , respectively . microarrays were hybridized for 20 h , scanned using an axon scanner , and the expression level for each gene was determined using genepix software . the base2 log of the ratio of cy3/cy5 signal for each gene was determined in each of three hybridization experiments with a pair of untreated and tunicamycin - treated mrna samples with wild - type or xbp-1(zc12)iii genotypes ( for a total of six hybridization experiments ) . a t test was used to determine the probability that differences in treatment - induced levels of mrna between the wild type and xbp-1 mutant were due to chance . a 95% confidence limit the data were analyzed twice , first using the standard deviation resulting from the experiments presented in this paper and then using the global standard deviation derived from the large set of combined experiments in the stanford microarray database . differences in levels of expression were reported only if they were significant using both measurements of standard deviation . a 1.3-kb fragment of c. elegans genomic dna immediately 5 of the predicted initiation atg codon of abu-1 ( ac3.3 ) was amplified by pcr using the oligonucleotides ac3.1s ( 5-ggcattgtggcacgcattgaactg-3 ) and ac3bam.2as ( 5-gataggatccattgttaatatgcttgaagagctgc-3 ) and ligated in frame with the gfp coding region in the plasmid of ppd95.75 ( gift of andy fire , carnegie institute of washington , baltimore , md ) . the abu-1::gfp(zcex8 ) strain was created by coinjecting the ac3.3.ppd95.75 plasmid ( 25 g / ml ) with a lin-15 rescuing plasmid , psk1 ( 25 g / ml ) , into lin-15(n765ts ) strain . the extrachromosomal array was integrated into the chromosome with ultraviolet / trimethylpsoraren treatment , yielding the abu-1::gfp(zcis8)x reporter strain . to produce the ges-1::gfp(zcex6 ) strain , the ges-1 promoter was amplified by pcr from c. elegans genomic dna using the primers ges-1.1s ( 5-ctcatacatcattgtcaagtgacg-3 ) and ges-1.pst.2s ( 5-agtactgcagagacaaggaatatccgcatct-3 ) and ligated into the hindiii - psti sites of ppd95.81 ( gift of andy fire ) . the abu-1 coding region fragment encoding abu-1 amino acids 7425 was ligated in frame with gfp into psti - bamhi sites of this plasmid to produce the ges-1::abu-1::gfp(zxex7 ) strain . the promoter and coding region of abu-1 was ligated in frame with gfp to produce abu1::abu-1::gfp(zcex8 ) strain . the animals containing transmissible extrachromosomal arrays were analyzed using a 510 laser scanning confocal microscope ( zeiss ) . interference with gene function by rnai followed an established protocol ( timmons et al . , 2001 ) . in brief , double - stranded rna was produced in ht115 strain of escherichia coli transformed with ppd129 plasmids containing cdna fragments of genes being studied ( nucleotides 10321278 of abu-1 cdna and nucleotides 4781631 of sel-1 cdna ) . iptg ( 1 mm ) was added to the bacterial growth media to induce transcription of the double - stranded rna , and l4-staged animals of defined genotype were added to plates individually and produced their brood . rnai phenotypes were evaluated in these progeny . where indicated , 30 l4-stage progeny from the rnai plate or from a control plate were transferred to a new plate seeded with fresh e. coli where they were exposed to tunicamycin ( 5 g / ml ) for 5 h or cdcl2 ( 10 mm ) for the indicated period . animals were scored as dead when pharyngeal pumping had ceased and they failed to move after being tapped on the nose with a platinum wire . cos-1 cells were obtained from american type culture collection . all cells were grown in dme containing 10% fbs ( cellgro ) at 5% co2 and 37c . abu-1 was tagged with a flag epitope at its cooh terminus by overlapping pcr using the primers ac3.bam.7s ( 5-gcgcggatccgcaatgcgctttatcgcaattgcag-3 ) , ac3.flag.16as ( 5-atcgtcgtccttgtagtcctttctcttgcaacactg-3 ) , and flag3u.not ( 5-ctag - cggccgctcacttgtcatcgtcgtccttgtagtc-3 ) . the full - length coding region of abu-1 with the cooh - terminal flag tag was cloned as a bamhi - noti fragment into pcdna3 ( invitrogen ) . cos-1 cells were transfected with this plasmid and stained with anti - flag antibody ( sigma - aldrich ) . gert kreibich , new york university [ nyu ] school of medicine ) was used to visualize the er staining pattern . the abu-1 coding region fragment encoding abu-1 amino acids 22425 was amplified by pcr using primers ac3.ecori.5s ( 5-ggcaacaagaattcgcgataaacg-3 ) and ac3.xbai.4as ( 5-gcgctctagatgtctactttctcttgcaac-3 ) . the abu-1 coding region fragment coding abu-1 amino acids 22331 was amplified by pcr using ac3.ecori.5s and ac3.xbai.6as ( 5-ggcatctagagcttggcatgcagattggc-3 ) , deleting the transmembrane domain and cooh terminus of abu-1 . cos-1 cells were transfected with wild - type abu-1 or abu-1 lacking the transmembrane domain with a flag tag . newly synthesized proteins were labeled in vivo with s - methionine and s - cysteine ( 500 ci / ml ; icn biochemicals ) . after removal of the labeling media , cells were washed and incubated in complete media for cold chase for the indicated times . cells were washed and lysed with 1% triton buffer ( 20 mm hepes , ph 7.5 , 150 mm nacl , 1% triton x-100 , 10% glycerol , 1 mm edta , 1 mm pmsf , 4 mg / ml aprotinin , and 2 mg / ml pepstatin a ) . samples were clarified by centrifugation at 14,000 g for 10 min and by preincubation for 1 h with 10 l protein a soluble proteins from the cells and secreted proteins in the media were immunoprecipitated by anti - flag antibody bound to 10 l protein a sepharose and washed three times with ripa buffer . bound proteins were resolved by 10% sds - page under reducing conditions and revealed by autoradiography .

the unfolded protein response ( upr ) counteracts stress caused by unprocessed er client proteins . a genome - wide survey showed impaired induction of many upr target genes in xbp-1 mutant caenorhabditis elegans that are unable to signal in the highly conserved ire1-dependent upr pathway . however a family of genes , abu ( activated in blocked upr ) , was induced to higher levels in er - stressed xbp-1 mutant animals than in er - stressed wild - type animals . rna - mediated interference ( rnai ) inactivation of a representative abu family member , abu-1 ( ac3.3 ) , activated the er stress marker hsp-4::gfp in otherwise normal animals and killed 50% of er - stressed ire-1 and xbp-1 mutant animals . abu-1(rnai ) also enhanced the effect of inactivation of sel-1 , an er - associated protein degradation gene . the nine abu genes encode highly related type i transmembrane proteins whose lumenal domains have sequence similarity to a mammalian cell surface scavenger receptor of endothelial cells that binds chemically modified extracellular proteins and directs their lysosomal degradation . our findings that abu-1 is an intracellular protein located within the endomembrane system that is induced by er stress in xbp-1 mutant animals suggest that abu proteins may interact with abnormal er client proteins and this function may be particularly important in animals with an impaired upr .

Introduction Results Discussion Materials and methods RNA isolation, cDNA synthesis, microarray hybridization, and data analysis Transgenic RNAi feeding and activation of ER stress by pharmacological means Cell culture, transfection, immunocytochemistry, and pulse-chase analysis

several signaling pathways , collectively referred to as the unfolded protein response ( upr ) , * maintain an equilibrium between the capacity of the er to process its client proteins and the physiological demand on the organelle ( kaufman , 1999 ; mori , 2000 ; patil and walter , 2001 ) . , 2001 ) , and accumulation of malfolded proteins in the er may play an important role in human diseases ( carrell and lomas , 1997 ; aridor and balch , 1999 ) . in yeast , er stress activates the er resident transmembrane protein kinase and endonuclease ire1p , leading to the posttranscriptional processing of a downstream mrna encoding the transcription factor hac1p . these target genes encode proteins involved in processing , trafficking , and degradation of er client proteins ( for reviews see mori , 2000 ; patil and walter , 2001 ) . , pancreatic - enriched er kinase ( perk ) phosphorylates eukaryotic translation initiation factor 2 to attenuate protein synthesis and activate specific gene expression during er stress ( for review see ron and harding , 2000 ) , and the er stress activated transcription factor atf6 directly activates upr target genes ( haze et al . we find that in c. elegans , the ire-1 and xbp-1 pathway has retained its essential role in upregulating expression of many upr target genes that are similarly upregulated by the homologous pathway in yeast . we also discovered a novel family of highly related genes that protect against er stress when the ire-1 and xbp-1 signaling pathway is defective . we compared the gene expression profile in unstressed and er - stressed wild - type young adult c. elegans with the profile in an xbp-1 mutant strain that is defective in the ire-1 and xbp-1 signaling pathway ( calfon et al . exposure of wild - type animals to the er stress inducing glycosylation inhibitor tunicamycin resulted in a 2.5-fold or greater statistically significant activation ( p < 0.05 ) of 34 of the 18,000 genes on the array . of these 34 genes , 26 were uninduced or induced to significantly lower levels ( p < 0.05 ) in the xbp-1 mutant strain . all but 2 of the 26 xbp-1regulated genes encode familiar proteins such as er or cytosolic chaperones , proteins involved in vesicular transport or other aspects of posttranslational processing of er client proteins ( table i ) . therefore , in c. elegans , as in yeast , the upr entails induction of genes involved in many different aspects of posttranslational protein processing in the secretory pathway . among the genes these observations suggest that robustly induced upr genes are significantly controlled by xbp-1 , however we have not addressed the role of xbp-1 in the activation of genes with more variable levels of induction by er stress . the induction of lin-15 ( zk662.4 ) is likely to be an artifact of the hsp-4::gfp;lin-15 array present in both wild - type and mutant animals . 19 genes were induced to higher levels in xbp-1 mutant animals than in wild - type animals ( table ii ) . we refer to members of this family as activated in blocked upr ( abu ) . two other similarly regulated genes encode proteins that are more distantly related to the nine abu family members ( table ii ) . a representative family member , abu-1 ( ac3.3 ) , was chosen for further study based on its central location in the family tree ( fig . northern blot analysis confirmed the induction of abu-1 by tunicamycin treatment of xbp-1 mutant animals but not wild - type animals ( fig . the linkage group for the nine abu genes is also shown . the c. elegans activated in blocked upr ( abu ) gene family . , 2001 ) , and human scavenger receptor of endothelial cells ( adachi et al . ( b ) amino acid sequence alignment of the nine abu proteins using the clustal w ( 1.81 ) program . abu proteins have a distant similarity to a mammalian scavenger receptor of endothelial cells and to the c. elegans cell corpse engulfment protein ced-1 , which are transmembrane cell surface proteins ( adachi et al . inactivation of abu-1 causes er stress and enhances the phenotype of c. elegans with defective er - associated protein degradation . ( b ) fluorescence micrographs of untreated ( ut ) , abu-1(rnai ) , or sel-1(rnai ) animals transgenic for the er stress reporter gene hsp-4::gfp(zcis4)v . ( c ) lethality at 72 h of wild - type and xbp-1(zc12)iii mutant c. elegans untreated ( ut ) , abu-1(rnai ) , sel-1(rnai ) , or compound abu-1(rnai ) ; sel-1(rnai ) . ( d ) photomicrographs using nomarski optics ( x400 ) of the intestine of living young adult c. elegans of the following genotypes : ( 1 ) wild type , ( 2 ) abu-1(rnai);sel-1(rnai ) , ( 3 ) xbp-1(zc12)iii , ( 4 ) xbp-1(zc12)iii ; abu-1(rnai ) , ( 5 ) xbp-1(zc12)iii ; sel-1(rnai ) , and ( 6 ) xbp-1(zc12)iii;abu-1(rnai);sel-1(rnai ) . these results suggest that abu-1 encodes an integral membrane protein that is retained in the er by its transmembrane domain . the secretion of transmembrane domain deleted abu-1 suggests that the lumenal portion of the protein folds properly in mammalian cells and that the full - length abu-1 is unlikely to be retained in the er as a consequence of malfolding in mammalian cells . ( a ) autoradiogram of metabolically - labeled wild - type abu-1 ( wt ) or abu-1 lacking the transmembrane domain ( tm ) expressed in cos1 cells and immunoprecipitated immediately after the labeling period ( lanes 1 and 3 ) or after 4 h of cold chase ( lanes 2 and 46 ) . the protein - coding region of the nine abu genes is > 89% identical at the nucleotide level over a stretch of 100 residues in the 3 end of the genes . therefore , we considered it possible to simultaneously inactivate several members of this gene family by rna - mediated interference ( rnai ) , using sequences derived from this conserved region . to study the impact of this genetic manipulation , we first identified a pharmacological treatment that would cause er stress but could be tolerated by both wild - type and ire-1 or xbp-1 mutant animals . the heavy metal cadmium activated the hsp-4::gfp reporter ( a marker of er stress ) in an ire-1 and xbp-1dependent manner ( fig . both wild - type and xbp-1 mutant animals tolerated cadmium exposure for extended periods of time ( fig . feeding of double - stranded gfp rna ( gfp[rnai ] ) had little impact on the viability of wild - type or mutant animals whether exposed to cadmium or not ( < 5% lethality ) . however , feeding of double - stranded abu-1 rna markedly impaired survival of cadmium - treated xbp-1 and ire-1 mutant animals , but had minimal impact on survival of cadmium - treated wild - type animals or on survival of untreated xbp-1 and ire-1 mutant animals ( fig . these observations are consistent with a role for abu-1 ( and possibly other abu genes ) in protecting animals with a defective upr against er stress . ( a ) fluorescence micrographs of untreated ( ut ) or cadmium - treated ( cd ) wild - type , xbp-1(zc12)iii mutant , or ire-1(zc14)ii mutant c. elegans transgenic for the er stress reporter gene hsp-4::gfp(zcis4)v . ( b ) survival at 48 h of untreated ( ut ) or cadmium - treated ( cd ) wild - type , xbp-1(zc12)iii mutant , or ire-1(zc14)ii mutant c. elegans at the l4 stage that had been subjected to abu-1(rnai ) , gfp(rnai ) , or no rnai . to determine the expression pattern of abu-1 in er - stressed animals abu-1::gfp was expressed constitutively in the pharynx and head region of wild - type and xbp-1 mutant adult animals . intestinal expression was induced by er stress , most notably in xbp-1 mutant animals ( fig . the induction of abu-1::gfp in the intestine of cadmium- and tunicamycin - treated animals is consistent with observations that the intestine is active in protein secretion and is a major target for er stress in c. elegans ( fig . , abu-1(rnai ) upregulated the er stress reporter gene hsp-4::gfp in the intestine ( fig . 5 b ) , implying that abu-1 ( and possibly other abu genes ) may also be active in the intestine under basal conditions and that interfering with abu function causes er stress . genes active in degradation of malfolded er proteins ( er - associated degradation [ erad ] genes ) are not essential for yeast viability under normal growth conditions but are essential during er stress or when the upr is blocked . , 2000 ; we considered the possibility that abu genes might interact with the process of malfolded protein disposal from the c. elegans er . first we confirmed that in c. elegans , like yeast , inactivation of erad genes causes er stress . , 1996 ) , resulted in marked upregulation of the er stress indicator hsp-4::gfp ( fig . the induction of hsp-4::gfp by sel-1(rnai ) and by rnai of a homologue of another yeast erad gene , hrd1 ( encoded in c. elegans by f55a11.3 ) , were both xbp-1 dependent ( unpublished data ) . inactivation of sel-1 by rnai had no impact on the viability of wild - type animals and only modestly reduced the viability of xbp-1 mutants . however , when combined with inactivation of abu-1 , sel-1(rnai ) increased lethality of xbp-1 mutant animals ( fig . viewed under nomarski optics , the intestine of wild - type , xbp-1 mutant , abu-1(rnai ) , or sel-1(rnai ) young adults had a similar , fine , granular appearance . these observations suggest that abu-1 ( and possibly other abu genes ) and sel-1 perform partially redundant functions in animals with a blocked upr . in both species , the signaling pathway initiated by ire1 exerts nearly complete control over the induction of well - characterized components of er client protein processing machinery , such as bip ( hsp-4 ) , protein disulfide isomerases , and 4-prolyl hydroxylases . our genome - wide survey also identified 19 genes that were activated by er stress in xbp-1 mutant animals but not in wild - type animals . among these were nine genes predicted to encode a family of highly related abu proteins . gfp driven by the promoter of a representative family member , abu-1::gfp , was expressed strongly in the pharynx and head region of transgenic animals from larval stages l3l4 to young adult ( fig . a similar expression pattern was also observed in animals transgenic for a transcriptional fusion of gfp to a different abu gene f19g12.7 ( unpublished data ) . furthermore , the basal expression pattern of the abu-1 transcriptional reporters correlated nicely with the in situ hybridization pattern of another abu family member , c03a7.7 . in older animals , we noted low levels of basal expression of abu-1::gfp in the intestine that was markedly upregulated by er stress in xbp-1 mutant animals ( fig . although the significance of basal expression of abu genes in the pharynx is currently not understood , the intestinal expression correlates with the phenotype of animals in which abu-1 had been inactivated by rnai . we do not know if abu-1 induction by er stress in xbp-1 mutant animals is dependent on perk ( pek-1 ) or atf6 ( atf-6 ) signaling , as compound xbp-1;pek-1 and xbp-1;atf-6(rnai ) mutant animals arrest at early larval stages before we could reliably examine abu-1::gfp expression in the gut ( shen et al . inactivation of abu-1 by rnai led to marked induction of the er stress indicator hsp-4::gfp in the intestine ( fig . this induction was completely blocked by the xbp-1 mutation ( unpublished data ) , indicating the development of er stress in the gut of abu-1(rnai ) animals and implying that abu-1 plays a role in an er function , which , when inactivated , causes er stress . however , in xbp-1 mutant animals , abu-1(rnai ) had serious consequences , significantly reducing their viability after exposure to additional er stress ( fig . the abu genes have a very high level of sequence identity at the nucleotide level , and the double - stranded rna used to inactivate abu-1 could also initiate rna inactivation of other family members . it is therefore possible that the strong phenotype observed in the abu-1(rnai ) animals reflects the simultaneous inactivation of other abu family members . collectively , these results point to a role for abu-1 , and possibly other abu genes , in some aspect of er function that is also active under basal conditions . in yeast , the ability to dispose of misfolded proteins in the secretory pathway ( erad ) plays an important role in adapting to high levels of er stress , but is dispensable for viability under normal conditions ( casagrande et al . , 2000 ; given that abu-1(rnai ) is well tolerated under normal conditions , but not in animals experiencing high levels of er stress , we considered the possibility that the abu genes may interact genetically with components of the c. elegans erad apparatus . an interaction was uncovered between sel-1 , a c. elegans gene encoding an er - localized component of the erad apparatus , and abu-1 . animals with diminished xbp-1 , sel-1 , and abu-1 activity had reduced viability and accumulated unusually large vesicles with dark content in their intestinal cells ( fig . sel-1 and abu-1 are unlikely to function in the exact same pathway , as abu-1(rnai ) did not affect the phenotype of a hypomorphic allele of lin-12 , whereas sel-1(rnai ) suppressed the egg - laying defect of lin-12(n676 ; n930)iii ( unpublished data ) . abu-1 is a transmembrane protein that likely functions within the endomembrane system ( figs . the lumenal domain of some abu proteins resembles the extracellular domains of a mammalian scavenger receptor and c. elegans ced-1 . both the scavenger receptors and ced-1 are believed to serve as cell surface receptors for chemically modified macromolecules , oxidized lipoproteins , and other altered plasma proteins in the case of the scavenger receptors ( van berkel et al . it is possible therefore that the abu proteins may be playing a similar role within the endomembrane system , perhaps by binding to altered er client proteins and modulating their intracellular fate . the base2 log of the ratio of cy3/cy5 signal for each gene was determined in each of three hybridization experiments with a pair of untreated and tunicamycin - treated mrna samples with wild - type or xbp-1(zc12)iii genotypes ( for a total of six hybridization experiments ) . a t test was used to determine the probability that differences in treatment - induced levels of mrna between the wild type and xbp-1 mutant were due to chance . a 1.3-kb fragment of c. elegans genomic dna immediately 5 of the predicted initiation atg codon of abu-1 ( ac3.3 ) was amplified by pcr using the oligonucleotides ac3.1s ( 5-ggcattgtggcacgcattgaactg-3 ) and ac3bam.2as ( 5-gataggatccattgttaatatgcttgaagagctgc-3 ) and ligated in frame with the gfp coding region in the plasmid of ppd95.75 ( gift of andy fire , carnegie institute of washington , baltimore , md ) . cos-1 cells were transfected with wild - type abu-1 or abu-1 lacking the transmembrane domain with a flag tag . the base2 log of the ratio of cy3/cy5 signal for each gene was determined in each of three hybridization experiments with a pair of untreated and tunicamycin - treated mrna samples with wild - type or xbp-1(zc12)iii genotypes ( for a total of six hybridization experiments ) . a t test was used to determine the probability that differences in treatment - induced levels of mrna between the wild type and xbp-1 mutant were due to chance . a 1.3-kb fragment of c. elegans genomic dna immediately 5 of the predicted initiation atg codon of abu-1 ( ac3.3 ) was amplified by pcr using the oligonucleotides ac3.1s ( 5-ggcattgtggcacgcattgaactg-3 ) and ac3bam.2as ( 5-gataggatccattgttaatatgcttgaagagctgc-3 ) and ligated in frame with the gfp coding region in the plasmid of ppd95.75 ( gift of andy fire , carnegie institute of washington , baltimore , md ) . the abu-1::gfp(zcex8 ) strain was created by coinjecting the ac3.3.ppd95.75 plasmid ( 25 g / ml ) with a lin-15 rescuing plasmid , psk1 ( 25 g / ml ) , into lin-15(n765ts ) strain . in brief , double - stranded rna was produced in ht115 strain of escherichia coli transformed with ppd129 plasmids containing cdna fragments of genes being studied ( nucleotides 10321278 of abu-1 cdna and nucleotides 4781631 of sel-1 cdna ) . cos-1 cells were transfected with wild - type abu-1 or abu-1 lacking the transmembrane domain with a flag tag .

several signaling pathways , collectively referred to as the unfolded protein response ( upr ) , * maintain an equilibrium between the capacity of the er to process its client proteins and the physiological demand on the organelle ( kaufman , 1999 ; mori , 2000 ; patil and walter , 2001 ) . , 2001 ) , and accumulation of malfolded proteins in the er may play an important role in human diseases ( carrell and lomas , 1997 ; aridor and balch , 1999 ) . in yeast , er stress activates the er resident transmembrane protein kinase and endonuclease ire1p , leading to the posttranscriptional processing of a downstream mrna encoding the transcription factor hac1p . these target genes encode proteins involved in processing , trafficking , and degradation of er client proteins ( for reviews see mori , 2000 ; patil and walter , 2001 ) . , pancreatic - enriched er kinase ( perk ) phosphorylates eukaryotic translation initiation factor 2 to attenuate protein synthesis and activate specific gene expression during er stress ( for review see ron and harding , 2000 ) , and the er stress activated transcription factor atf6 directly activates upr target genes ( haze et al . the coordination of these three pathways and their specific contribution to the metazoan er stress response are unclear , though in caenorhabditis elegans , the ire1 and perk pathways provide redundant protection against er stress ( shen et al . we used cdna microarrays to characterize the transcriptional response to er stress in c. elegans , a simple metazoan that has counterparts of all three known components of the mammalian upr ( shen et al . we find that in c. elegans , the ire-1 and xbp-1 pathway has retained its essential role in upregulating expression of many upr target genes that are similarly upregulated by the homologous pathway in yeast . we also discovered a novel family of highly related genes that protect against er stress when the ire-1 and xbp-1 signaling pathway is defective . we compared the gene expression profile in unstressed and er - stressed wild - type young adult c. elegans with the profile in an xbp-1 mutant strain that is defective in the ire-1 and xbp-1 signaling pathway ( calfon et al . exposure of wild - type animals to the er stress inducing glycosylation inhibitor tunicamycin resulted in a 2.5-fold or greater statistically significant activation ( p < 0.05 ) of 34 of the 18,000 genes on the array . of these 34 genes , 26 were uninduced or induced to significantly lower levels ( p < 0.05 ) in the xbp-1 mutant strain . all but 2 of the 26 xbp-1regulated genes encode familiar proteins such as er or cytosolic chaperones , proteins involved in vesicular transport or other aspects of posttranslational processing of er client proteins ( table i ) . therefore , in c. elegans , as in yeast , the upr entails induction of genes involved in many different aspects of posttranslational protein processing in the secretory pathway . among the genes these observations suggest that robustly induced upr genes are significantly controlled by xbp-1 , however we have not addressed the role of xbp-1 in the activation of genes with more variable levels of induction by er stress . the induction of lin-15 ( zk662.4 ) is likely to be an artifact of the hsp-4::gfp;lin-15 array present in both wild - type and mutant animals . 19 genes were induced to higher levels in xbp-1 mutant animals than in wild - type animals ( table ii ) . nine of these encode highly similar , novel proteins with a hydrophobic nh2-terminal signal sequence , a potential transmembrane domain , and a short cooh - terminal cytoplasmic domain ( fig . shown is the mean sem ( n = 3 ) of the base2 log fold increase in hybridization signal in tunicamycin - treated animals over untreated animals for each genotype . ( a ) the amino acid sequence identity relationship of the abu family members , c. elegans ced-1 ( zhou et al . ( d ) northern blot analysis of total rna from untreated and tunicamycin - treated wild - type and xbp-1(zc12)iii mutant c. elegans . abu proteins have a distant similarity to a mammalian scavenger receptor of endothelial cells and to the c. elegans cell corpse engulfment protein ced-1 , which are transmembrane cell surface proteins ( adachi et al . to study the subcellular localization of abu-1 , the protein was tagged at its cooh terminus with gfp and expressed in the intestine of c. elegans from a transgene driven by the ges-1 promoter . ges-1::abu-1gfp confocal microscopy showed that the puncta corresponded to vesicular structures that were present throughout the large intestinal cell and tended to cluster near its apical surface ( fig . 2 , d and e ) , suggesting that this pattern of expression is not an artifact of unphysiologically high expression levels of the fusion proteins . however , given that we were not able to detect the endogenous abu-1 protein , it remains possible that the apparent localization to intracellular vesicles is due to misfolding of the fusion proteins . ( a ) fluorescence micrographs of untreated ( ut ) or cadmium - treated ( cd ) wild - type or xbp-1(zc12)iii mutant c. elegans transgenic for an abu-1::gfp(zcis8)x reporter . ( c ) lethality at 72 h of wild - type and xbp-1(zc12)iii mutant c. elegans untreated ( ut ) , abu-1(rnai ) , sel-1(rnai ) , or compound abu-1(rnai ) ; sel-1(rnai ) . ( d ) photomicrographs using nomarski optics ( x400 ) of the intestine of living young adult c. elegans of the following genotypes : ( 1 ) wild type , ( 2 ) abu-1(rnai);sel-1(rnai ) , ( 3 ) xbp-1(zc12)iii , ( 4 ) xbp-1(zc12)iii ; abu-1(rnai ) , ( 5 ) xbp-1(zc12)iii ; sel-1(rnai ) , and ( 6 ) xbp-1(zc12)iii;abu-1(rnai);sel-1(rnai ) . to extend these observations , we studied the subcellular localization of c. elegans abu-1 in mammalian cells , an experimental system in which we could make use of established markers for the various organelles and compartments . in transfected mammalian cos1 cells , metabolically - labeled abu-1 tagged at its cooh terminus with the flag epitope remained associated with the cell pellet , from which it could be extracted only by detergent solubilization ( fig . the secretion of transmembrane domain deleted abu-1 suggests that the lumenal portion of the protein folds properly in mammalian cells and that the full - length abu-1 is unlikely to be retained in the er as a consequence of malfolding in mammalian cells . ( a ) autoradiogram of metabolically - labeled wild - type abu-1 ( wt ) or abu-1 lacking the transmembrane domain ( tm ) expressed in cos1 cells and immunoprecipitated immediately after the labeling period ( lanes 1 and 3 ) or after 4 h of cold chase ( lanes 2 and 46 ) . staining of the same cells with ribophorin i outlines the er and the merged image reveals the colocalization of abu-1 ( anti - flag ) and ribophorin 1 . the protein - coding region of the nine abu genes is > 89% identical at the nucleotide level over a stretch of 100 residues in the 3 end of the genes . therefore , we considered it possible to simultaneously inactivate several members of this gene family by rna - mediated interference ( rnai ) , using sequences derived from this conserved region . to study the impact of this genetic manipulation , we first identified a pharmacological treatment that would cause er stress but could be tolerated by both wild - type and ire-1 or xbp-1 mutant animals . the heavy metal cadmium activated the hsp-4::gfp reporter ( a marker of er stress ) in an ire-1 and xbp-1dependent manner ( fig . however , feeding of double - stranded abu-1 rna markedly impaired survival of cadmium - treated xbp-1 and ire-1 mutant animals , but had minimal impact on survival of cadmium - treated wild - type animals or on survival of untreated xbp-1 and ire-1 mutant animals ( fig . ( a ) fluorescence micrographs of untreated ( ut ) or cadmium - treated ( cd ) wild - type , xbp-1(zc12)iii mutant , or ire-1(zc14)ii mutant c. elegans transgenic for the er stress reporter gene hsp-4::gfp(zcis4)v . ( b ) survival at 48 h of untreated ( ut ) or cadmium - treated ( cd ) wild - type , xbp-1(zc12)iii mutant , or ire-1(zc14)ii mutant c. elegans at the l4 stage that had been subjected to abu-1(rnai ) , gfp(rnai ) , or no rnai . to determine the expression pattern of abu-1 in er - stressed animals abu-1::gfp was expressed constitutively in the pharynx and head region of wild - type and xbp-1 mutant adult animals . the induction of abu-1::gfp in the intestine of cadmium- and tunicamycin - treated animals is consistent with observations that the intestine is active in protein secretion and is a major target for er stress in c. elegans ( fig . genes active in degradation of malfolded er proteins ( er - associated degradation [ erad ] genes ) are not essential for yeast viability under normal growth conditions but are essential during er stress or when the upr is blocked . the induction of hsp-4::gfp by sel-1(rnai ) and by rnai of a homologue of another yeast erad gene , hrd1 ( encoded in c. elegans by f55a11.3 ) , were both xbp-1 dependent ( unpublished data ) . inactivation of sel-1 by rnai had no impact on the viability of wild - type animals and only modestly reduced the viability of xbp-1 mutants . our search for genes activated in the c. elegans upr revealed similarities to the yeast s. cerevisiae . in both species the upr induces genes that function at many levels of the secretory pathway and encode proteins with diverse and unrelated biochemical activities . in both species , the signaling pathway initiated by ire1 exerts nearly complete control over the induction of well - characterized components of er client protein processing machinery , such as bip ( hsp-4 ) , protein disulfide isomerases , and 4-prolyl hydroxylases . the application of these stringent statistical criteria eliminated many genes that probably are true upr targets and therefore the list presented in table i is only representative of the most reliably induced upr genes in c. elegans . heterogeneity in developmental stage and variation in uptake of drug stand out as likely contributors to the lower power of the whole animal array performed here compared with yeast arrays ( casagrande et al . despite these limitations , we can firmly conclude that most of the reliably induced upr genes in c. elegans are xbp-1 dependent . our genome - wide survey also identified 19 genes that were activated by er stress in xbp-1 mutant animals but not in wild - type animals . these abu genes are coregulated across different physiological and developmental perturbation ; clustering together in a single , small , isolated and well - defined expression mountain , mount 29 , in the recently established gene expression map of c. elegans ( kim et al . gfp driven by the promoter of a representative family member , abu-1::gfp , was expressed strongly in the pharynx and head region of transgenic animals from larval stages l3l4 to young adult ( fig . furthermore , the basal expression pattern of the abu-1 transcriptional reporters correlated nicely with the in situ hybridization pattern of another abu family member , c03a7.7 . in older animals , we noted low levels of basal expression of abu-1::gfp in the intestine that was markedly upregulated by er stress in xbp-1 mutant animals ( fig . although the significance of basal expression of abu genes in the pharynx is currently not understood , the intestinal expression correlates with the phenotype of animals in which abu-1 had been inactivated by rnai . we do not know if abu-1 induction by er stress in xbp-1 mutant animals is dependent on perk ( pek-1 ) or atf6 ( atf-6 ) signaling , as compound xbp-1;pek-1 and xbp-1;atf-6(rnai ) mutant animals arrest at early larval stages before we could reliably examine abu-1::gfp expression in the gut ( shen et al . this induction was completely blocked by the xbp-1 mutation ( unpublished data ) , indicating the development of er stress in the gut of abu-1(rnai ) animals and implying that abu-1 plays a role in an er function , which , when inactivated , causes er stress . in yeast , the ability to dispose of misfolded proteins in the secretory pathway ( erad ) plays an important role in adapting to high levels of er stress , but is dispensable for viability under normal conditions ( casagrande et al . , 2000 ; given that abu-1(rnai ) is well tolerated under normal conditions , but not in animals experiencing high levels of er stress , we considered the possibility that the abu genes may interact genetically with components of the c. elegans erad apparatus . sel-1 and abu-1 are unlikely to function in the exact same pathway , as abu-1(rnai ) did not affect the phenotype of a hypomorphic allele of lin-12 , whereas sel-1(rnai ) suppressed the egg - laying defect of lin-12(n676 ; n930)iii ( unpublished data ) . however , the observation that inactivating either gene leads to er stress and the synthetic interactions between them suggest that sel-1 and abu-1 function in interacting cellular processes . both the scavenger receptors and ced-1 are believed to serve as cell surface receptors for chemically modified macromolecules , oxidized lipoproteins , and other altered plasma proteins in the case of the scavenger receptors ( van berkel et al . the base2 log of the ratio of cy3/cy5 signal for each gene was determined in each of three hybridization experiments with a pair of untreated and tunicamycin - treated mrna samples with wild - type or xbp-1(zc12)iii genotypes ( for a total of six hybridization experiments ) . a t test was used to determine the probability that differences in treatment - induced levels of mrna between the wild type and xbp-1 mutant were due to chance . a 95% confidence limit the data were analyzed twice , first using the standard deviation resulting from the experiments presented in this paper and then using the global standard deviation derived from the large set of combined experiments in the stanford microarray database . a 1.3-kb fragment of c. elegans genomic dna immediately 5 of the predicted initiation atg codon of abu-1 ( ac3.3 ) was amplified by pcr using the oligonucleotides ac3.1s ( 5-ggcattgtggcacgcattgaactg-3 ) and ac3bam.2as ( 5-gataggatccattgttaatatgcttgaagagctgc-3 ) and ligated in frame with the gfp coding region in the plasmid of ppd95.75 ( gift of andy fire , carnegie institute of washington , baltimore , md ) . to produce the ges-1::gfp(zcex6 ) strain , the ges-1 promoter was amplified by pcr from c. elegans genomic dna using the primers ges-1.1s ( 5-ctcatacatcattgtcaagtgacg-3 ) and ges-1.pst.2s ( 5-agtactgcagagacaaggaatatccgcatct-3 ) and ligated into the hindiii - psti sites of ppd95.81 ( gift of andy fire ) . the base2 log of the ratio of cy3/cy5 signal for each gene was determined in each of three hybridization experiments with a pair of untreated and tunicamycin - treated mrna samples with wild - type or xbp-1(zc12)iii genotypes ( for a total of six hybridization experiments ) . a t test was used to determine the probability that differences in treatment - induced levels of mrna between the wild type and xbp-1 mutant were due to chance . a 1.3-kb fragment of c. elegans genomic dna immediately 5 of the predicted initiation atg codon of abu-1 ( ac3.3 ) was amplified by pcr using the oligonucleotides ac3.1s ( 5-ggcattgtggcacgcattgaactg-3 ) and ac3bam.2as ( 5-gataggatccattgttaatatgcttgaagagctgc-3 ) and ligated in frame with the gfp coding region in the plasmid of ppd95.75 ( gift of andy fire , carnegie institute of washington , baltimore , md ) . to produce the ges-1::gfp(zcex6 ) strain , the ges-1 promoter was amplified by pcr from c. elegans genomic dna using the primers ges-1.1s ( 5-ctcatacatcattgtcaagtgacg-3 ) and ges-1.pst.2s ( 5-agtactgcagagacaaggaatatccgcatct-3 ) and ligated into the hindiii - psti sites of ppd95.81 ( gift of andy fire ) . samples were clarified by centrifugation at 14,000 g for 10 min and by preincubation for 1 h with 10 l protein a soluble proteins from the cells and secreted proteins in the media were immunoprecipitated by anti - flag antibody bound to 10 l protein a sepharose and washed three times with ripa buffer .

this study regards itself as a supplement to the common melancholy of failed expectations , contrary effects , unfulfilled promises and the generally deplorable state of affairs lamented in much of the mainstream anthropological and historical literature . it aims to achieve a more dynamic perspective by asking when , how and why the initial optimism was fuelled or frustrated in the course of the projects . instead of focusing exclusively on the constructions of the periphery , ie . the african part and its agency , as is the tendency among established african scholars of history and anthropology due to their professional perspective , this paper seeks to explore more of the european activities both within and outside europe . since studies of medical science in european countries after the second world war rarely touch on relations with former colonies , there seems to be an imbalance between the number of historical and anthropological studies devoted to the european activities compared to those devoted to the african side in these encounters . furthermore , a renewed examination of the institutions and personnel of the northern hemisphere that dominated the early triumphant histories of colonial medicine and science can shed new light on these : the emphasis on african agency in more recent studies calls for a reversed and complementary perspective focusing on the europeans , who would then have to be seen as being greatly changed in the encounter , often learning as much themselves as they were attempting to teach the africans . thus these two shifts a procedural turn from the rather static description of results to the dynamics of transfer and interaction , and a recipients to the modifications of the donor side will be some of the primary perspectives of this article . the particular scenario to be examined is that of transfer processes between two countries in western europe and west africa . regarding this binational focus , it is important to note that , unlike most studies on white protagonists in africa after independence , this paper has not chosen a recent european colonial power with many continuing ties to african countries . a considerable number of academics and academic institutions in the federal republic of germany ( frg ) were among those involved in the re - establishment of a german role in health care in tropical and sub - tropical countries within the new framework of western development aid. ( an eastern european state like the german democratic republic ( gdr ) , by contrast , called its more modest efforts international solidarity ) . during the first un development was still mainly understood in economic terms , 8% of the west german budget for technical aid was allotted to the health sector , at a total of 130 million deutschmarks for 176 projects . africa became the main regional focus of west german technical and personnel aid in health , whereas the main focus of aid in the middle east , for example , was vocational training . this was due to a variety of reasons , notably extraordinary local need , the absence of infrastructure necessary for immediate industrialisation , previous colonial , diplomatic , academic and medical relations as well as contemporary power politics . the african recipients included countries north and south of the sahara , with a focus in the 1960s on ethiopia , algeria , senegal , niger and togo . west german expert medical academics were involved in most of the major health projects . in view of this , the german togolese relationship in microbiology within the framework of development aid in the 1960s and 1970s was chosen as an example for closer study . the article will first give a short summary of post - colonial , pre-1945 german academic interest in health outside the north atlantic world , continuing with an overview of west german academic activities in health in non - european countries and of the relationship between the academic world and west german political and administrative structures in the main part of the article will consist of an extensive case study concerning togo . this includes an examination of international co - operation on the part of several institutes at west german universities which did not have a long pre - war tradition of medical research in the tropics : the institute of comparative tropical medicine in the veterinary faculty at munich university , the institutes of hygiene and of parasitology at bonn university and the hygiene institute of wrzburg university . the directors of these institutes responded to the idea that political independence should be followed by scientific independence and they actively supported the creation of a microbiological institute in a sub - saharan state , the institut national dhygine in togo . inevitably , the dynamics of this project were rooted in the earlier background of german academic activities in tropical medicine as well as in later policies for west german thus refraining from the contested issue of the positive or negative impact on the host country s health situation the second major question to be examined in this study will be the relationship between was it one of mutual benefit and understanding , or did one of the two have to suffer due to pressure from the other s special interests ? resources for each other has been explicitly posed and widely discussed with regard to national socialism in germany . a similar question although with much less emphasis on eventual guilt for crimes and failures is present in much writing on colonial medicine . a study on the british colonial medical research committee until 1950 , for example , recently addressed this issue for a scenario not unlike this west german case without exploring moral connotations . being sceptical towards theoretical monism , the approach chosen here will combine several perspectives from different discourses . in the light of functionalist systems theory , medical academia ( as part of science ) and development aid ( as part of politics and administration ) will be treated as functional social systems of communications with their own specific objectives , rewards and procedures which render their coupling potentially difficult . but it is also important to question and deconstruct these notions since neither academia nor development aid in their new african settings were fixed entities in accordance with an official definition but had to be re - invented in the course of their establishment . this looks at practices such as scientific research and training with their core material means of biological samples , laboratory results and publications on the one hand , ie . practices such as planning , agreements , recruiting and practical training on the other hand , ie . the developmental side . as these different functional systems and sets of practices often exist side by side within the same projects , institutions and persons , their interaction can be studied in multiple ways . apart from the focus on medical research , the additional aims of this article include further investigation of the general relationship between development ( aid) and health , as this subject has been the focus of increased attention over recent years . as this article focuses on the german side in the encounter , the sources used were from four german archives and from german and international journals only . unfortunately , none of the main protagonists mentioned could be interviewed as eyewitnesses as they are already deceased . the academic part of the history is documented in the archives of the university of bonn and the two grant - giving institutions for academic activities , the german research foundation ( dfg ) for research and the german academic exchange service ( daad ) for international co - operation between academic institutions . the governmental contribution , mainly in the form of the then ministry of economic co - operation ( bmz ) , was examined on the basis of documents in the german federal archives ( barch ) . it was possible to gain an instructive insight into the different goals , concepts and strategies of these unequal partners by contrasting sources from academia and government . the common temptation to treat the european side in north south relations as rather monolithic in nature could be countered here by indicating the effects of classic conflicts of interest , especially those between the intellectual , universal and long - term perspective of science and the more practical , national and short - term aims of politics . for several reasons germany occupies a somewhat exceptional place in the history of the interaction between medical academia and health politics at an international level . german academia achieved scientific prestige in the early years of research in tropical medicine with authorities such as robert koch in berlin or renowned institutions such as the institute for naval and tropical diseases in hamburg , founded in 1900 . but after the first world war , german research was affected by the loss of colonies and by restricted access to tropical territories . consequently , the activities of german experts outside germany consisted of the participation in expeditions and in international commissions of the league of nations . under national socialist rule and during the second world war , temporary plans to restore a german empire in africa and the military campaigns in north africa and southern europe led to renewed political interest in tropical medicine and hygiene . parasitology at the university of bonn in a department of the institute of pharmacology in 1943 . after the war , the situation was again different from that of the colonial powers of the period . there was no special interest in work on other continents in any of the german research councils , let alone a new colonial research service such as existed in britain . in tropical agricultural research , the purpose of the discipline was quickly shifted from supporting german settlers and those of other nationalities to development aid. in medicine , decolonisation provided new opportunities for academic specialists dependent on contact with tropical regions , notably parasitologists or bacteriological experts on cholera and other diarrhoeal diseases . in addition , the decline of smallpox , tuberculosis and other infectious diseases in germany freed up capacity for the support of anti - infectious hygiene and vaccination initiatives outside europe . west germany thus regained the necessary preconditions for a role in international medicine and health , ie . a sufficient supply of well - qualified scientists and the renewal of economic and diplomatic influence that had been lost after the first world war . medical scientists now readily seized the opportunities offered by the bilateral technical assistance for under - developed countries ( technische hilfe fr unterentwickelte lnder ) started by west germany in 1956 . thus the complex relationship between academia and development aid ( as parts of the differing social systems of science and politics ) began in the context of cold war . development aid when the first west german university institutes and hospitals started their engagement in asia , africa and latin america in the early 1960s . government funds for health had first been used to support the german christian medical missions , which continued and even increased their pre - war programmes of sending health workers , equipment and material overseas and of training local health staff . the first so - called entwicklungshelfer [ development aid workers ] of the new deutscher entwicklungsdienst ( ded ) [ german development service , a volunteer service for professional staff modelled on the us peace corps , did not leave germany until 1964 . in the period up to october 1965 , just ten of these members of staff fewer than five per cent held a university degree . this prompted ded managers to think about job opportunities for university graduates at universities in developing countries. another agency of the west german government in charge of development issues , the gawi ( garantieabwicklungsgesellschaft ) , also sent some medical specialists . requested by friendly governments , they worked in major hospitals , mainly in ethiopia , algeria and togo . meanwhile , several hundred other german physicians had continued to work in government health facilities in decolonised countries , especially in the middle east and in south - east asia without their work being organised or supported by west german government agencies . in the 1960s , application , non - attachment and project. these principles meant that aid would only be granted at the explicit request of the national government in the form of a project proposal , that financial aid would not commit recipients to buy the required equipment in germany and that the aid was tied to the agreed projects and could not consist of grants to the general budget or of the mere delivery of goods . while these principles were often handled in a rather cavalier manner in practice , they did nevertheless establish the basis required for scientific and medical co - operation : new hospitals or institutes were not offered , but had to be requested by the governments of the partner countries ; the means granted were bound to these institutions and could not be used for other health care or research purposes ; and the construction , equipment and supply was not necessarily commissioned with german companies . although the west german government also financed multilateral organizations such as the who , it systemically prioritised aid to individual states , as this offered better opportunities for pursuing its interests in foreign politics . the west german report for the development assistance committee ( dac ) of the organization for economic co - operation and development ( oecd ) of 1962 stated bluntly that the federal government prefers bilateral assistance. within health , it especially supported medical education , training and research . typically , health projects for research and practice were negotiated and operated between two governments , with an international agency fulfilling an advisory function . following the wishes of its member states , the who itself had to refrain from establishing its own research institutes . instead , it worked with collaborating centres in several member countries and received support from its technical departments in geneva as a kind of substitute for proper research institutes of its own . restriction on multilateral activities remained a feature of german development aid beyond political parties and coalitions . in the 1970s , the west german preference for unilaterally sending experts and development workers even increased dramatically when erhard eppler , the minister responsible for development co - operation , and chancellor willy brandt demanded a doubling of their numbers within five years . despite opposition from the ministries of health and foreign affairs , the ministry of development co - operation , which had been created for managing development aid in 1961 , was finally given responsibility for all projects of technical assistance in 1965 . it relied on the gawi , later renamed the gtz , for the planning and implementation of the projects . technical assistance was even more unclear , however , since this also touched on responsibilities which were located within yet another area . this was the area of the federal ministry of education and science ( bmbw ) founded in 1969 , which in turn sponsored two further agencies , the daad and dfg . thus various different parties were involved : not only the donor and recipient countries as such , but also various public donors in germany and various recipients the project partners in germany and in the foreign country . the situation was highly complex , mirroring german federalism and thereby fundamentally differing from the centralism of france and britain , where the pasteurians and orstom ( office de la recherche scientifique et technique outre mer ) or the medical research council respectively controlled overseas microbiological work in terms of staffing , funding and research direction . universities had to find their position within this particular and rather complicated framework of west german development aid and research funding . at the time , curative medicine in rural hospitals in africa and south or south - east asia had been left to the german christian missions . the fields of hygiene and public health were regarded by experts as most important for solving the health problems of developing countries. hygiene institutes in addition to medical education and teaching hospitals therefore became a major focus of early west german development aid. there are several examples of this kind of co - operation with areas of focus quite characteristic of the respective countries . in kabul , professor fritz fhner of the hygiene institute of hamburg first oversaw the establishment of a hygiene institute at the university from 1960 to 1963 and , from 1964 onwards , the creation of a similar service at national level . for this purpose , he headed a team of ten german expatriates who were responsible for virology , clinical chemistry and haematology as well as for the inspection of food , water and drugs . the microbiology , health education and epidemiology units , on the other hand , were organised by the who . in bangkok , the public health research institute received its equipment from the frg and had four west german staff members in 1969 . the hamburg institute for naval and tropical diseases , when asked for advice on laboratory equipment , established a programme of co - operation with the tropical institute at the university of lima in the early 1960s . the hamburg institute also founded a research centre in bong mine , liberia in 1968 . an example of early academic co - operation well known to the west german public due to its tragic ending was a project by the medical faculty of freiburg university , which created a medical school in hu , the ancient imperial city of vietnam , in 1961 . it was devoted to medical education only and succeeded in qualifying its first medical doctors before the project was discontinued due to the murder of three german physicians and the head of mission s wife by the vietcong in 1968 . however , such co - operation was also often affected by side - effects of the cold war , as in the case of the theodor bilharz institute in egypt funded by the frg . here , construction was delayed , though not completely interrupted , for several years following the suspension of diplomatic relations between egypt and the frg from 1965 to 1972 , which had occurred as a result of the state visit of the east german head of state walter ulbricht to cairo . in overall terms it can be said that several west german institutes and medical scientists had already received government or other public support to develop health - related academic infrastructure in african , asian and latin american countries before joint scientific research was explicitly included in development aid. it was only in the late 1960s that germany publicly looked at british practice as a possible role model for international co - operation between universities , extending an invitation to professor c. t. ingold , vice - chair of the london - based inter - university council for higher education overseas to speak in germany . ingold , who was a renowned mycologist himself ( and father of the later famous anthropologist tim ingold ) , presented british policies and experiences to a german audience in 1969 and emphasised the need for political independence on both sides as well as flexibility and the avoidance of paternalistic neocolonialism. two years later , in 1971 , co - operation in science and higher education became an official part of west german one of the earlier , in many ways pioneering , projects linking the german government focus on public health to german academia was already in place in the small west african state of togo . german medical relations with togo had already had a long tradition before the country became independent of france in 1960 . having previously been part of the west african region known as the slave coast and of the portuguese trade network , the territory of the present republic of togo and of the upper volta region of ghana was proclaimed as the german protectorate of togo in 1884 by a medical doctor , gustav nachtigal ( 183485 ) , who was then also german consul in tunis and german imperial commissioner for west africa . being a so - called model colony ( musterkolonie ) , togo experienced various german activities in the fields of health care and medical research , amongst them the first german government hospital in tropical africa . internationally famous german doctors like the discoverer of the smallpox virus , the pathologist enrique paschen ( 18601936 ; in togo 191112 ) , and the hygienist ernst rodenwaldt ( 18781965 ; in togo 190913 ) worked in the colony before it came under french and british control during the first world war . the official division into british togoland and french togoland as a league of nations mandate in 1922 finally led to the present situation , the british part being incorporated into the gold coast , later ghana , and the french part retaining the name and forming the republic of togo . although france had also introduced its common west african health system here , french togoland had not received institutions for the higher education of health professionals or for medical research in the same way as other french territories in west africa such as senegal , cameroon , congo - brazzaville or the central african republic . immediately after independence in 1960 , the new republic of togo and the frg signed a bilateral agreement , which also included health . the first prime minister and subsequent president of togo , sylvio olympio ( 190263 ) , was especially interested in these relations with the first colonial power . his cousin pedro olympio ( 18981969 ) , after education at secondary schools in the netherlands and germany run by a german missionary society , had studied medicine at the universities of bonn and munich and had worked temporarily at the hamburg institute for naval and tropical diseases before undertaking further studies in paris and returning to his native country as the first togolese physician educated and trained in europe . it seems to have been this special interest in germany which led to the official agreement on technical co - operation , including first ideas for a national institute of hygiene , more than a year before west germany even had a ministry responsible for development co - operation . given the early contacts in the area of hygiene , the government of togo requested west german assistance in this particular field while ignoring conflicts and repression the german colonial administration had caused , especially in the field of public health . the absence of an institut pasteur , however , was probably a more important actual reason than mere colonial nostalgia or anti - french sentiment . the co - operation between the federal government and medical academia in germany which became necessary for the agreed german intervention in the field of hygiene in togo was facilitated by the contacts of professor dr med . horst habs ( 190287 ) , who had been director of the hygiene institute at the university of bonn since 1958 . at that time , this city was the provisional capital of the frg as well as the seat of the west german parliament , government and ministries . habs had a long record in politics related to medicine and academia . during national socialism , he had been a member of the nsdap , of the national socialist association for physicians ( ns - rztebund ) and of the national socialist association for lecturers ( ns - dozentenbund ) , and he had also been an officer in the ss . he had taken part in a confidential meeting in 1943 , which among other topics discussed inhumane experiments concerning sulfonamide and typhus on inmates of german concentration camps . after the war , he advised the new west german army ( bundeswehr ) on ways to ( re-)establish its medical service . in 1960 , heinz peter rudolf seeliger ( 192097 ) , was commissioned with a consultancy on the health situation in togo . strangely enough an initiative by the west german ministry of the interior , where a health department was in charge of all medical and health affairs before the creation of a ministry of health in 1961 . due to his previous work in the laboratory of the us forces in heidelberg , seeliger was far more internationally informed and minded than most of his german colleagues , although he had not worked outside the western world at that point . seeliger visited togo for two months in 1961 and wrote a survey in close collaboration with international agencies such as the who . formulated in both a german and french version , this then served as the basis for planning the agreed institute of hygiene . in a later publication on the trip , seeliger described the togolese expectations of germany as an often touching affection towards the former protective power in the hope of a decisive contribution to building the young state. when the official additional bilateral agreement on the establishment of an institute of hygiene in lom was signed on 31 may 1965 , construction of the institute building had already been underway for a year . completed in 1967 , the republic of togo provided the construction site , locally produced furniture , tax exemption for the import of necessary materials from abroad , transport and storage within the country , and various kinds of support for the accommodation of the german staff . upon completion of the building , seeliger , then director of the institute for hygiene and microbiology at wrzburg university since 1965 and thereafter consultant to the gawi , referred once again to the colonial legacy : on the whole , it should be noted that the building now makes an excellent impression . ( ) simple and functional and like a typical purpose - built structure , it fits beautifully into the government quarters of the capital of lom . ( ) nevertheless , compared to the french institutes in abidjan and dakar , visited by the undersigned , the interior looks rather modest . however , it thereby achieves the required degree of frugality , while retaining a certain class . this state of affairs should be especially emphasised when informing the german public , as is desirable . on the whole ( ) simple and functional and like a typical purpose - built structure , it fits beautifully into the government quarters of the capital of lom . ( ) nevertheless , compared to the french institutes in abidjan and dakar , visited by the undersigned , the interior looks rather modest . however , it thereby achieves the required degree of frugality , while retaining a certain class . this state of affairs should be especially emphasised when informing the german public , as is desirable . such remarks reveal a search for the right balance between self - confidence and modesty when approaching the new tasks , especially in comparison with well - established previous colonial nations . the operation of the institute was regulated in an additional agreement in 1967 , granting german financial support for four years and german staff for three years . running the institute was a joint german togolese enterprise and obviously functioned smoothly , apart from very rare german complaints such as in 1969 : despite all the goodwill , the togolese have difficulties to provide their contributions. the first german team , all of whom had previous ties to the institute of hygiene at bonn university , consisted of two doctors the project leader and head of the department of applied hygiene , and the head of the department of parasitology as well as two medico - technical assistants , a mechanic and an electro - technician ; a typist was later added . upon its opening in 1967 , mentoring by german university institutes or faculties of their counterparts in developing countries had already been recommended by a german expert meeting on experiences in establishing health care in developing countries in 1962 , where seeliger had been one of the few university - affiliated participants . habs also became involved in efforts to control disease in togo by organising a polio immunisation programme , together with his wife , and outside africa in efforts to eradicate malaria in greece . among the german mentors , he had been director of the bavarian state institute for vaccination , including an institute for infectious and tropical medicine , since 1948 , and had held the chair for comparative tropical medicine in the faculty of veterinary medicine at the university of munich since 1964 . herrlich had spent most of his time from 1933 to 1939 in east africa , afghanistan and india , studying the genealogy of wheat in the hindukush region in 1935 and visiting most countries of eastern and southern africa between 1936 and 1937 . after 1950 , he devoted several trips to the study of acute problems of tropical medicine , eg . investigating a smallpox epidemic in india in 1958 . herrlich s participation in mentoring the lom institute is remarkable , since he had , in 1963 , been an outspoken opponent of the establishment of permanent outposts of european institutes of tropical medicine in former colonial territories . he had considered these places as mainly suitable for training academic novices and not for long - term research . the high costs and the narrow focus on a single region instead of the broader information to be gained by expeditions had been his main arguments against fixed research facilities ; instead , he had proposed commissioning research to varying regions and for limited periods . after herrlich s death in 1970 , the parasitologist professor dr gerhard piekarski ( 191092 ) from bonn university replaced him as mentor . in september 1969 , the inter - ministerial committee of the west german government responsible for the project prolonged it for another two years and increased the expatriate staff . the hygienist , who was still also the project leader , and the parasitologist were supplemented by a bacteriologist while the number of medico - technical assistants was increased from two to four . a laboratory assistant for animals after these two years , the co - operation was formalised in an additional treaty of 15 september 1971 . as early as 1968 the west german ministry of economic co - operation had praised the institute in almost lyrical terms : the institute of hygiene as the centre of public hygiene excels by its enormous impact [ ausstrahlungskraft ] . it is one of the most important projects of german technical aid in togo , which essentially functions in education and training beyond its diagnostic tasks . the institute of hygiene as the centre of public hygiene excels by its enormous impact [ ausstrahlungskraft ] . it is one of the most important projects of german technical aid in togo , which essentially functions in education and training beyond its diagnostic tasks . its actual functioning in these different areas , however , needs to be looked at in more detail since the whole project was still far ahead of the general development of medical infrastructure in togo . in his report of 1973 , dr med . peter scheiber , project leader since 1969 , criticised the fact that , at the time of the establishment of the institute , the place and the tasks for a central laboratory with the range of duties described were neither prepared nor even projected ( ) in the public health system of togo. only after 1970 did conditions regarding the supply of water and power , roads and self - confidence improve . nevertheless , in the report which they published in the journal of the german medical association , scheiber and his predecessor dr med . georg quincke , later a staff member of the who from 1969 onwards and finally its director of the division of food and nutrition , described the start as being marked by a rapid increase in the number of samples and smooth collaboration with national authorities and international agencies such as the who . the same was said about the immunisations organised by the institute between 1967 and 1969 . according to habs , the provision of such vaccines and the less frequently needed anti - sera made the pioneering function of an institute for the future organization of the health system especially visible . the national institute seems to have worked well from the very beginning as a laboratory , vaccination service and advisory body . with regard to the long - term perspective seen in the categories of german governmental budgets and public funds , the project remained one of technical aid rather than financial aid. its main aim was thus specific capacity - building rather than the production of scientific knowledge or an increase in the financial resources available for health care and research . co - operating german academics were well aware of this task : it can not be overemphasised that there is long - term educational work ahead of us which concerns all kinds of staff at the institute. the initial togolese staff consisted of an administrator , two medico - technical assistants trained in germany , but still inexperienced , three workers for the garden , stables and cleaning , a driver , a servant for the scullery and a porter . a co - director and an administrator from togo assisted the german academics in their organisational and bureaucratic tasks . perhaps it was also this model of prolonged joint work that contributed to the prevention of struggles for power in the transition period . such conflicts had occurred at the pasteur institute of cameroon in the same year of 1967 , where a parting french director and his colleagues blocked an african successor who was just returning from france . concerning the main , capacity - building task , seeliger had to disappoint expectations : employing togolese counterparts ( homologues ) who can take over duties as collaborators of the heads of department can not be expected in the near future , as only a few togolese medical students have returned home after completing their education , and they were immediately charged with medical care . employing togolese counterparts ( homologues ) who can take over duties as collaborators of the heads of department can not be expected in the near future , as only a few togolese medical students have returned home after completing their education , and they were immediately charged with medical care . between 1965 and 1970 , university education for citizens of togo was mainly provided at the institut suprieur du benin . again in 1969 , a note in the german records referred to the seemingly hopeless counterpart situation due to the medical students lack of interest in these jobs . as late as 1972 , the german project leaders still complained about the lack of togolese academic staff , as there were hardly any candidates that were interested in questions of theoretical medicine although the number of togolese doctors in the country had nearly doubled since the start of the project . an improvement could be hoped for from the most important togolese development for the academic side of the project , ie . the foundation of a university at lom in 1970 , which included a medical school . the academic staff of the institute provided practical instruction at the school in co - operation with the hospitals of ophthalmology and gynaecology / obstetrics and also offered scientific training for possible future researchers . as early as 1961 , prior to the german togolese co - operation via the hygiene institute , german technical aid had supported the hospital in tokoin , a suburb of lom . five german doctors and two medico - technical assistants were employed there in 1971 , when the hospital was becoming the clinical part of the new university s medical school . further assistance through mentoring ( patenschaft ) by german universities was explicitly requested from the togolese side . inter - university co - operation seemed to be regarded as a successful model for capacity building . the capacity - building activities of the institute also went beyond its own needs and medical academia . besides training on the job for the togolese academic staff of the institute and the contribution to medical education at the university , the institute organised special courses for laboratory assistants , hygiene assistants in the public health service and who fellows . the question as to whether and to what extent research was to be part of the institute s activities was far less clearly regulated than might be expected of plans involving german university professors . after all , they had been brought up in the humboldtian tradition of a unity of teaching and research , complemented in the medical faculty by practical health care via clinical work and public health . in contrast to other institutes of medical schools or to medical institutes outside universities , institutes of hygiene had always been characterised by their combination of research and public health , ie . their dual function for science and for current medico - political or administrative purposes . in the nineteenth century , they had often been established as laboratories by local authorities and had only later been incorporated into universities , as occurred in bonn about 1900 . or they remained independent of a university despite their strong research activities , as in the case of hamburg , which was for a time the largest institute of hygiene in germany . thus german academic hygienists were well acquainted with the hybrid position between laboratory research and routine work , between the production of new scientific knowledge and serving the country , eg . in vaccination and epidemiological surveillance . it remained unclear as to how far this double function was now also intended for the national institute in lom . the chances for the two fields were judged quite differently . in his expert opinion on the institute of 1967 , seeliger stated bluntly : a research activity of its own can not be considered , things given as they are , since for this activity there were other existing opportunities in lom. obviously , research was not even planned in the official description of the tasks of the institute , revealing a slight difference of views between the german development aid agencies and german academia . this tension becomes visible in publications too , sometimes even occurring within the same article , as in the above - mentioned 1972 journal of the german medical association . here , diverging lists regarding the duties of the institute can be found in the information box on the one hand , which is obviously based on official sources , and in the report by the two german scientists on the other hand . the box only speaks of the institute s function as a reference laboratory for samples sent in , as a central health office of the republic and as a central institution for a system of outposts under construction , by which a network of the health system shall cover the whole country. the authors , however , risked minor contradictions when they wrote that the institute took over the tasks of a laboratory for the public health service and , as such , shall perform examinations as well as research work in the field of public health [ volksgesundheit]. furthermore they stated that these tasks equalled those of a public health office [ medizinalbro ] for examination , responsible for the whole of togo , which has an advisory function in the control of diseases and delivers the necessary documents for this purpose. solely a public health office or also responsible for research work , what were the major tasks ? the scientists explicitly emphasised research work as an equal task , whereas the institute s only official function in the view of the german donor , ie . development aid and the republic of togo was public health . as far as can be surmised from the sources , this diversity of aims did not hinder the work of the institute , perhaps even resulting in the contrary . the backbone provided by the german contribution was the fact that staff stayed for at least a year , the minimum requirement suggested by wilhelm schulze , an army hygienist , bacteriologist and specialist for tropical medicine at the institute in 1969 . physical fitness , self - initiative and independence in working and identifying problems , and the readiness to establish contacts. mentor habs appealed to the german public health service to supply experts of this kind by dispatching them to development aid missions and reintegrating them afterwards , which would make a career in the public health service more attractive for some doctors. but the opportunity to do research also attracted promising junior scientists to the institute . among these were the later directors of the hygiene institutes in hamburg and stuttgart , jochen bockemhl and gerhard schrter respectively , who probably would not have come as ordinary development aid workers. coming for research , they brought their scientific expertise to the institute with its increasingly togolese academic staff . project assistants had already been provided for in article 4 of the additional treaty of 1965 . the mentoring senior scientists , all of them holders of university chairs or directors of institutes , were certainly more prepared to take on co - responsibility for the institute because it promised additional research opportunities . this was especially true of a parasitologist such as piekarsky , who only had very minor public health tasks at home in germany and was therefore much more of a pure academic than a practitioner of hygiene . nevertheless , the academic merits garnered by research remained almost completely on the german side , even when the scientific publications were in languages other than german , including french as the official language of togo . to date , it has only been possible to identify three articles by the german researchers where a co - author has an obviously african surname . one of these two co - authors , a. amdom , was not even a staff member at the institute , but was instead in the service des contagieux , centre hospitalier universitaire , lom ( togo ) , and had already published in france and africa several times previously , once even on the issue of cholera under investigation in the article . the other author , k. n. agbo , belonged to the academic staff of the national institute , as medcin - chef of the parasitology section . the article in question was the first and only authorship for nearly a decade under the name of k. agbo in an internationally listed medical journal . the next occurrence was in 1984 , when a k. agbo , technicien suprieur at the laboratory and parasitological services of the university hospital lom , started a long list of publications on parasitological issues with francophone and anglophone research partners . perhaps k. n. agbo s early co - authorship was also facilitated by his newly arrived european co - authors : the german reny anneliese braun - munzinger ( 194890 ) , who was accustomed to the co - operative practice of local co - authorship through her previous work with indian researchers in india , and the british scientist brian a. southgate ( 19302011 ) , who became well known for his support of non - european researchers and who obviously saw this practice as an international standard . yet on the whole , junior african researchers with an initial scientific training at the national institute could not further their potential academic careers by means of publications arising from the research activities of the german scientists . most probably , the initiative , the design of the studies and the writing of the manuscripts are mirrored in this result : that almost exclusively germans appear as authors . even in the archival documents and apart from the occasional acknowledgement of technical assistance in the publications , it usually remains unclear as to how far the togolese counterparts were actually involved in , eg . there is just one very elaborate acknowledgment from 1976 and 1977 by bockemhl in the context of his research on salmonella and shigella , conducted between 1971 and 1973 : the examinations reported in this paper have been performed under somewhat difficult field conditions and were only possible thanks to the sympathy and collaboration of the local authorities : the regional representatives of the government , health officers , nurses , sanitary inspectors , school teachers , and village headmen.special thanks are due to my team , who worked together for 16 months : mr v. kpelevi , mr l. lawson , mr l. pr . agbodjan , mr r. apedoh , and mr m. sessou.serotyping of strains was performed at the institute of hygiene and microbiology , university of wrzburg , by mrs r. miethke , mrs d. legat , miss i. voigt , and the national reference centre for salmonella , hamburg , by priv .- doz . [ the examinations reported in this paper have been performed under somewhat difficult field conditions and were only possible thanks to the sympathy and collaboration of the local authorities : the regional representatives of the government , health officers , nurses , sanitary inspectors , school teachers , and village headmen . special thanks are due to my team , who worked together for 16 months : mr v. kpelevi , mr l. lawson , mr l. pr . serotyping of strains was performed at the institute of hygiene and microbiology , university of wrzburg , by mrs r. miethke , mrs d. legat , miss i. voigt , and the national reference centre for salmonella , hamburg , by priv .- doz . the three female german collaborators without a doctoral degree were probably medico - technical assistants and had been co - authors in two previous publications . the names of the african collaborators were not mentioned again in the second part of the publication and never recurred as authors of articles in international medical journals . thus , even when a german scientist clearly wanted to acknowledge the essential work of african staff members , german academic conventions seemed to exclude their function as co - authors . this story of academic publishing serves as a further telling example of how medico - scientific practices may crystallise post - colonial obscenity , by defining , incorporating , and exploiting socio - racial inequities , as formulated concisely by the french historian guillaume lachenal . the final plan had been that the institute could be handed over to the authorities of togo in early 1976 after a year under the responsibility of a togolese director advised by his german predecessor . thus the whole project , from the start to the transition , was intended to last less than ten years , a goal which was , in fact , ultimately achieved . when the institute was handed over to the togolese ministry of health and social services on 9 january 1976 , the high esteem for the institute from both sides was indicated by the presence of several togolese ministers and several german representatives , including the two mentors seeliger and piekarski . there was no evidence of conflicts during the transition of power such as those which again paralysed the previously mentioned institut pasteur in cameroon between 1974 and 1980 . considering contemporary german journalism s widespread criticism of german development aid or emergency aid in other places and its particular , politically motivated attention to german relations with the dictatorial ruler eyadma ( president , 19672005 ) , it should be mentioned that the hygiene institute in lom remained without unfavourable media coverage in germany . the institute had been requested by a still legitimate government and was clearly in the interests of the whole country ; it was in line with the german shift towards preventative medicine and academic research in development co - operation ; its construction was not delayed in any notable way ; collaboration occurred without major conflicts ; and the aim of capacity - building by training counterparts was achieved in so far as togolese heads of departments were able to take over the responsibilities in routine work at the end of the project . the institute was not a foreign body , but formed part of the national health system . in fact , the institutes in lom and bangkok were the only two positive exceptions explicitly mentioned in an otherwise devastating evaluation of health projects by a senior official in the german ministry of economic co - operation in 1968 , their success being particularly attributed to continuous mentorship leading to new forms of partnership . beyond the routine work of the institute , togo profited from at least some of the data gathered by german scientists during research . the studies mostly supported by the dfg were largely epidemiological , primarily giving information on the health situation in the country concerning the frequency of certain germs and , to a lesser degree , contributing to universal medical knowledge such as bacteriological taxonomy . research especially concerned common bacterial diseases such as cholera and salmonellosis , as well as shigellosis and gonorrhoea . the academic publications were often a side - effect of routine activities at the national institute such as health examinations of prostitutes and hygiene checks of schools , wells and food . emphasis was placed on the importance of unbiased , critical parasitological examinations , as opposed to simple routine diagnostics for expected findings . only in this way was it possible to establish the presence of certain worm eggs which had not previously been described for west africa or to identify a cholera - like epidemic due to vibrio parahaemolyticus . the parasitologist piekarski did not publish any research work himself on the health situation in togo . but the institute s research interest was extended from bacteriology to parasitological diseases , such as bancroftian filariasis , onchocercosis , malaria , bilharzia and sleeping sickness , during the his post-1970 mentoring period and even for many years after the completion of the development project in 1976 . reny braun - munzinger s joining of the national institute gave it an expatriate medico - technical assistant who had previously worked at the university of heidelberg and would do so again after her time in togo . she thus broadened the institute s scientific connections beyond the initial mentoring universities of bonn , wrzburg and munich . in these studies towards the end of the project , acknowledgement is made of financial support other than that provided by the dfg with its exclusive responsibility for research , eg . by the ministry of public health and social affairs , togo and by the agency for technical co - operation ( gtz ) , eschborn , federal republic of germany. this indicates a full integration of scientific research into the development project and the national health system . meanwhile , however , ideas on scientific relations were changing in germany . in 1971 , the professor of economics peter meyer - dohm ( * 1930 ) , who was later head of bochum university ( 19759 ) , demanded that the common transfer of knowledge must be supplemented or even replaced by independent academic colonialism. scientists of the developing country should have their share in the research work and its results . as noted above several factors stood in the way of achieving more than training for the routine tasks of a central national laboratory : less than a decade was probably far too short ; the mentor herrlich had doubted the chances of fruitful research work in a permanent place in the tropics anyway ; the project leaders hinted at the difficulties of keeping up the standards to which they were accustomed at home under these conditions ; researchers preferred serological testing at home in germany to the difficulties and expenses involved in establishing further bacteriological methods in togo ; and probably most important togolese staff with academic qualifications were scarce and were already overburdened with routine tasks . obviously , the project was ill - suited for capacity - building in scientific research . this would have demanded far more commitment from all sides and perhaps also conditions within the country that would not be achieved for quite some time . a further task and development project which was started two years after the completion of the original development project brought increased importance to research at the institute . in 1978 , the gtz supported the establishment of an onchocerciasis reference laboratory to be directed by the parasitologist hartwig schulz - key from the hamburg institute for naval and tropical diseases . schulz - key retained this responsibility even after moving from hamburg to the university of tuebingen . however , the site for future research was moved from the capital to the regional hospital of sokod , the centre of onchocerciasis control . the institute s research centre flourished within this large project , which had already been started by the who in 1974 and received support from the world bank and the european economic community ( later european union ) , among others , offering research opportunities in a tropical place over the years to more than a hundred , mostly german , junior scientists in the fields of medicine and biology until the present day . following the loss of these research opportunities to the regional hospital of sokod , the institute in lom also had to give up the name it had adopted in 1967 to honour its german donor and early german work in togo . when the west german army decided to remove the name of ernst rodenwaldt , incidentally one of piekarski s early mentors in the late 1930s , from the name of an institute in koblenz in germany in march 1998 , after his connivance with inhumane human experimentation during national socialism had become widely known , the togolese authorities followed suit in october of the same year . the close relations between germany and togo thus even influenced issues not related to the african continent and development co - operation . the two major research questions the relationship between west germany and togo and between medical academia and development aid have been answered to quite different extents due to the respective content of the german sources . the eurocentric reports and correspondence reveal more about the interaction between development politics and biomedical science than that between the germans and togolese . the political administration invited academia to play a role in what were both old and new fields . there appeared to be a necessary connection between the progress and modernity that development aid claimed to bring and the promises of science despite the obvious fact that the first was directed towards what were regarded as the most backward regions and the latter strived to be at the most advanced edge of human progress . unequal partners had to negotiate their terms and seem to have done so to a certain degree of satisfaction on both sides . however , the area of expertise for which university members were sought remained limited in the west german scenario during the first two decades of development co - operation . while non - medical academic experts in britain such as health economists richard titmuss and brian abel - smith were also commissioned with recommendations on comprehensive health care services in countries of the global south , west german professors were asked only for infectious disease control and clinical work . this mirrors two characteristics of germany : its previous pivotal role in hygiene and bacteriology ( which , of course , equally holds for british and french institutions on tropical health ) and its lack of a continuous and recent colonial experience in medical administration . in view of the interwar german approach of social hygiene , a move towards social medicine as known from late british colonial policy the case studied here is thus typical of west germany of the period , since academic expertise was not expected for health care in general . medical academics were expected to contribute only to medical education and to establishing institutes on viral , bacterial and parasitological diseases or general hygiene , including toxicology , while returning german doctors were invited to contribute their expertise on the practical level of health care only . the first task of academics was consultancy to the west german government , since the government agencies for technical aid did not yet have relevant experts of their own in these early years and benefited from temporarily hiring academics from university institutes without having to offer permanent jobs in germany . thus these experts travelled to the tropics in order to identify health problems that would need special academic expertise in the relevant country . a decade later , by 1973 , the west german press maintained that the method practised by the ministry of economic co - operation in sending ad hoc consultants , usually travel - happy university professors , on exploratory trips for a week has proved to be a failure since permanent qualified observation and administrative support on the spot was lacking. however , this accusation typical of the increasingly sceptical attitude of the early 1970s towards development aid even before the professors visited the country , the host country had expressed the desire for the institute , and its establishment was principally agreed between the governments . the diseases to be addressed , the methods required , and the staff and equipment needed . as togo did not have any specialists in these fields , the project itself was mainly based in this case on the plan drawn up by the german academic who first visited the country . after the planning and construction of the institute , the next task for the universities was the provision of both technical and academic german staff . this was a further advantage for the federal ministries , which thereby gained privileged access to suitable staff . but the german institutes also profited from being able to offer what were often well - paid and attractive jobs as well as research opportunities to their post - doctoral members . here , two different groups of biomedical experts emerged , reflecting the continuing tension between science and the health services , or academia and development co - operation : the doctors who held jobs at the institutes in africa for a year or more did not usually pursue academic careers later , but rather stayed in international or development organisations , whereas those who stayed in africa for research projects only became professors or directors afterwards for the most part . thus the increasing west german split between academic experts on communicable diseases at universities and other research institutes on the one hand and public health officers in the civil service on the other was also made manifest in international co - operation . in togo , the main task , so - called capacity - building , was fulfilled for routine activities through collaboration between the german and togolese counterparts . periods spent in europe by togolese students , mainly as part of their doctoral studies , seem to have been a precondition for acquiring the skills necessary to carry out research independently . long - standing co - operation which continued after the end of the development project was the decisive condition for building up research capacities . professor fhner , head of the similar project in kabul mentioned above , had demanded three phases , each of about three years : ( 1 ) establishing routine work , ( 2 ) sending the counterparts to germany for further training , and ( 3 ) transferring responsibility for research to them by reducing german staff . development decade , beginning in 1990 , development projects usually envisaged a short timeline that was not suitable for developing research capacities . hyperoptimism of development workers regarding the feasibility of transferring the knowledge , skills and necessary approaches within , eg . a two - year stay , seemed to be especially ill suited to the long formation period of researchers . its common timescale of decades instead of years , its post - graduate training or research in german institutions and its mutual visits financed by funding agencies for research and higher learning enabled it to follow and complement the initial development project. while the story documented in this paper ends in that decade , academic relations between ( west ) germany and developing countries , especially in africa , continued . they will therefore be the subject of the following short overview , which introduces further comparisons and aspects . since the 1970s , these relations received regular attention and financial support in both the academic and development sectors . the institute for tropical hygiene and public health at heidelberg university became a centre for north south co - operation for more than just infectious diseases , as its major topics included midwifery and nutrition , the latter also being found at the university of giessen . research on tropical medicine in tropical countries gained new momentum even beyond governmental interests . in 1981 , the hamburg institute of naval and tropical diseases opened a research laboratory in lambarene , gabon , the former place of work of the famous doctor albert schweitzer . this was done without german public funding and was entirely based on private donations . on the whole , links between development co - operation and academia decreased despite , or perhaps because of , special funding that the dfg and the daad started by programmes for academic co - operation with developing countries. meanwhile the german agencies for technical and financial co - operation ( the gtz and the west german development bank ) had their own health experts and professional external consultants , so that they no longer always needed professors and lecturers for expertise in this area . at the same time , the german universities could rely on previously established institutions for research and higher education in their partner countries . such institutions no longer had to be built from scratch as part of development projects and probably would no longer have been built anyway within the new international health policies of the late 1970s directed towards primary health care . this tacit institutional separation of medical academia and development co - operation was partly stopped with a series of new initiatives after 2000 , when increased funding for global health emerged . funds from implementing the millennium development goals ( mdgs ) , the global fund to fight aids , tuberculosis and malaria ( gfatm ) and the global alliance for vaccines and immunisation ( gavi ) boosted research on neglected health issues and operational research in particular in the countries concerned alongside these initiatives dominant activities with more immediate effects . at the 31st g8 summit in gleneagles in 2005 , co - operation with africa was declared a task for all ministries of the participating countries , including those with responsibility for education and research and for health . it was thus taken beyond the narrower focus of development co - operation . the ( re-)united germany reacted to these tasks by also providing additional funding and new medical research programmes . thus , the challenges of the 1960s in integrating public health and academic research on communicable diseases within the framework of international co - operation returned under the new conditions of twenty - first century globalisation . it will be interesting to see to what extent history repeats itself or whether fundamental changes in national policies , scientific attitudes and international co - operation since the 1960s lead to different results in the future .

after losing the importance it had held around 1900 both as a colonial power and in the field of tropical medicine , germany searched for a new place in international health care during decolonisation . under the aegis of early government development aid , which started in 1956 , medical academics from west german universities became involved in several asian , african and south american countries . the example selected for closer study is the support for the national hygiene institute in togo , a former german model colony and now a stout ally of the west . positioned between public health and scientific research , between development aid and academia and between west german and west african interests , the project required multiple arrangements that are analysed for their impact on the co - operation between the two countries . in a country like togo , where higher education had been neglected under colonial rule , having qualified national staff became the decisive factor for the project . while routine services soon worked well , research required more sustained capacity building and did not lead to joint work on equal terms . in west germany , the arrangement with the universities was a mutual benefit deal for government officials and medical academics . west german development aid did not have to create permanent jobs at home for the consulting experts it needed ; it improved its chances to find sufficiently qualified german staff to work abroad and it profited from the academic renown of its consultants . the medical scientists secured jobs and research opportunities for their postgraduates , received grants for foreign doctoral students , gained additional expertise and enjoyed international prestige . independence from foreign politics was not an issue for most west german medical academics in the 1960s .

Research Questions and Material: Medical Science through Development Aid Background: Tropical Medicine, Development Aid and Academia inGermany The Setting: Institut National dHygine in Lom, Togo The German Contribution: Staff, Mentoring, Materials and Finances Opportunities for Togo: Capacity-Building, Training and Education Academic Output: Scientific Research at the Institute The Impact of the Project Conclusions and Outlook

furthermore , a renewed examination of the institutions and personnel of the northern hemisphere that dominated the early triumphant histories of colonial medicine and science can shed new light on these : the emphasis on african agency in more recent studies calls for a reversed and complementary perspective focusing on the europeans , who would then have to be seen as being greatly changed in the encounter , often learning as much themselves as they were attempting to teach the africans . a considerable number of academics and academic institutions in the federal republic of germany ( frg ) were among those involved in the re - establishment of a german role in health care in tropical and sub - tropical countries within the new framework of western development aid. in view of this , the german togolese relationship in microbiology within the framework of development aid in the 1960s and 1970s was chosen as an example for closer study . the article will first give a short summary of post - colonial , pre-1945 german academic interest in health outside the north atlantic world , continuing with an overview of west german academic activities in health in non - european countries and of the relationship between the academic world and west german political and administrative structures in the main part of the article will consist of an extensive case study concerning togo . this includes an examination of international co - operation on the part of several institutes at west german universities which did not have a long pre - war tradition of medical research in the tropics : the institute of comparative tropical medicine in the veterinary faculty at munich university , the institutes of hygiene and of parasitology at bonn university and the hygiene institute of wrzburg university . inevitably , the dynamics of this project were rooted in the earlier background of german academic activities in tropical medicine as well as in later policies for west german thus refraining from the contested issue of the positive or negative impact on the host country s health situation the second major question to be examined in this study will be the relationship between was it one of mutual benefit and understanding , or did one of the two have to suffer due to pressure from the other s special interests ? a study on the british colonial medical research committee until 1950 , for example , recently addressed this issue for a scenario not unlike this west german case without exploring moral connotations . apart from the focus on medical research , the additional aims of this article include further investigation of the general relationship between development ( aid) and health , as this subject has been the focus of increased attention over recent years . as this article focuses on the german side in the encounter , the sources used were from four german archives and from german and international journals only . the academic part of the history is documented in the archives of the university of bonn and the two grant - giving institutions for academic activities , the german research foundation ( dfg ) for research and the german academic exchange service ( daad ) for international co - operation between academic institutions . the governmental contribution , mainly in the form of the then ministry of economic co - operation ( bmz ) , was examined on the basis of documents in the german federal archives ( barch ) . thus the complex relationship between academia and development aid ( as parts of the differing social systems of science and politics ) began in the context of cold war . the first so - called entwicklungshelfer [ development aid workers ] of the new deutscher entwicklungsdienst ( ded ) [ german development service , a volunteer service for professional staff modelled on the us peace corps , did not leave germany until 1964 . while these principles were often handled in a rather cavalier manner in practice , they did nevertheless establish the basis required for scientific and medical co - operation : new hospitals or institutes were not offered , but had to be requested by the governments of the partner countries ; the means granted were bound to these institutions and could not be used for other health care or research purposes ; and the construction , equipment and supply was not necessarily commissioned with german companies . the west german report for the development assistance committee ( dac ) of the organization for economic co - operation and development ( oecd ) of 1962 stated bluntly that the federal government prefers bilateral assistance. in the 1970s , the west german preference for unilaterally sending experts and development workers even increased dramatically when erhard eppler , the minister responsible for development co - operation , and chancellor willy brandt demanded a doubling of their numbers within five years . despite opposition from the ministries of health and foreign affairs , the ministry of development co - operation , which had been created for managing development aid in 1961 , was finally given responsibility for all projects of technical assistance in 1965 . the situation was highly complex , mirroring german federalism and thereby fundamentally differing from the centralism of france and britain , where the pasteurians and orstom ( office de la recherche scientifique et technique outre mer ) or the medical research council respectively controlled overseas microbiological work in terms of staffing , funding and research direction . universities had to find their position within this particular and rather complicated framework of west german development aid and research funding . hygiene institutes in addition to medical education and teaching hospitals therefore became a major focus of early west german development aid. in bangkok , the public health research institute received its equipment from the frg and had four west german staff members in 1969 . the hamburg institute for naval and tropical diseases , when asked for advice on laboratory equipment , established a programme of co - operation with the tropical institute at the university of lima in the early 1960s . an example of early academic co - operation well known to the west german public due to its tragic ending was a project by the medical faculty of freiburg university , which created a medical school in hu , the ancient imperial city of vietnam , in 1961 . however , such co - operation was also often affected by side - effects of the cold war , as in the case of the theodor bilharz institute in egypt funded by the frg . in overall terms it can be said that several west german institutes and medical scientists had already received government or other public support to develop health - related academic infrastructure in african , asian and latin american countries before joint scientific research was explicitly included in development aid. it was only in the late 1960s that germany publicly looked at british practice as a possible role model for international co - operation between universities , extending an invitation to professor c. t. ingold , vice - chair of the london - based inter - university council for higher education overseas to speak in germany . two years later , in 1971 , co - operation in science and higher education became an official part of west german one of the earlier , in many ways pioneering , projects linking the german government focus on public health to german academia was already in place in the small west african state of togo . being a so - called model colony ( musterkolonie ) , togo experienced various german activities in the fields of health care and medical research , amongst them the first german government hospital in tropical africa . internationally famous german doctors like the discoverer of the smallpox virus , the pathologist enrique paschen ( 18601936 ; in togo 191112 ) , and the hygienist ernst rodenwaldt ( 18781965 ; in togo 190913 ) worked in the colony before it came under french and british control during the first world war . although france had also introduced its common west african health system here , french togoland had not received institutions for the higher education of health professionals or for medical research in the same way as other french territories in west africa such as senegal , cameroon , congo - brazzaville or the central african republic . given the early contacts in the area of hygiene , the government of togo requested west german assistance in this particular field while ignoring conflicts and repression the german colonial administration had caused , especially in the field of public health . the co - operation between the federal government and medical academia in germany which became necessary for the agreed german intervention in the field of hygiene in togo was facilitated by the contacts of professor dr med . during national socialism , he had been a member of the nsdap , of the national socialist association for physicians ( ns - rztebund ) and of the national socialist association for lecturers ( ns - dozentenbund ) , and he had also been an officer in the ss . completed in 1967 , the republic of togo provided the construction site , locally produced furniture , tax exemption for the import of necessary materials from abroad , transport and storage within the country , and various kinds of support for the accommodation of the german staff . upon its opening in 1967 , mentoring by german university institutes or faculties of their counterparts in developing countries had already been recommended by a german expert meeting on experiences in establishing health care in developing countries in 1962 , where seeliger had been one of the few university - affiliated participants . among the german mentors , he had been director of the bavarian state institute for vaccination , including an institute for infectious and tropical medicine , since 1948 , and had held the chair for comparative tropical medicine in the faculty of veterinary medicine at the university of munich since 1964 . in september 1969 , the inter - ministerial committee of the west german government responsible for the project prolonged it for another two years and increased the expatriate staff . as early as 1968 the west german ministry of economic co - operation had praised the institute in almost lyrical terms : the institute of hygiene as the centre of public hygiene excels by its enormous impact [ ausstrahlungskraft ] . it is one of the most important projects of german technical aid in togo , which essentially functions in education and training beyond its diagnostic tasks . it is one of the most important projects of german technical aid in togo , which essentially functions in education and training beyond its diagnostic tasks . peter scheiber , project leader since 1969 , criticised the fact that , at the time of the establishment of the institute , the place and the tasks for a central laboratory with the range of duties described were neither prepared nor even projected ( ) in the public health system of togo. an improvement could be hoped for from the most important togolese development for the academic side of the project , ie . the academic staff of the institute provided practical instruction at the school in co - operation with the hospitals of ophthalmology and gynaecology / obstetrics and also offered scientific training for possible future researchers . as early as 1961 , prior to the german togolese co - operation via the hygiene institute , german technical aid had supported the hospital in tokoin , a suburb of lom . besides training on the job for the togolese academic staff of the institute and the contribution to medical education at the university , the institute organised special courses for laboratory assistants , hygiene assistants in the public health service and who fellows . after all , they had been brought up in the humboldtian tradition of a unity of teaching and research , complemented in the medical faculty by practical health care via clinical work and public health . obviously , research was not even planned in the official description of the tasks of the institute , revealing a slight difference of views between the german development aid agencies and german academia . here , diverging lists regarding the duties of the institute can be found in the information box on the one hand , which is obviously based on official sources , and in the report by the two german scientists on the other hand . the authors , however , risked minor contradictions when they wrote that the institute took over the tasks of a laboratory for the public health service and , as such , shall perform examinations as well as research work in the field of public health [ volksgesundheit]. furthermore they stated that these tasks equalled those of a public health office [ medizinalbro ] for examination , responsible for the whole of togo , which has an advisory function in the control of diseases and delivers the necessary documents for this purpose. one of these two co - authors , a. amdom , was not even a staff member at the institute , but was instead in the service des contagieux , centre hospitalier universitaire , lom ( togo ) , and had already published in france and africa several times previously , once even on the issue of cholera under investigation in the article . when the institute was handed over to the togolese ministry of health and social services on 9 january 1976 , the high esteem for the institute from both sides was indicated by the presence of several togolese ministers and several german representatives , including the two mentors seeliger and piekarski . considering contemporary german journalism s widespread criticism of german development aid or emergency aid in other places and its particular , politically motivated attention to german relations with the dictatorial ruler eyadma ( president , 19672005 ) , it should be mentioned that the hygiene institute in lom remained without unfavourable media coverage in germany . the institute had been requested by a still legitimate government and was clearly in the interests of the whole country ; it was in line with the german shift towards preventative medicine and academic research in development co - operation ; its construction was not delayed in any notable way ; collaboration occurred without major conflicts ; and the aim of capacity - building by training counterparts was achieved in so far as togolese heads of departments were able to take over the responsibilities in routine work at the end of the project . in fact , the institutes in lom and bangkok were the only two positive exceptions explicitly mentioned in an otherwise devastating evaluation of health projects by a senior official in the german ministry of economic co - operation in 1968 , their success being particularly attributed to continuous mentorship leading to new forms of partnership . as noted above several factors stood in the way of achieving more than training for the routine tasks of a central national laboratory : less than a decade was probably far too short ; the mentor herrlich had doubted the chances of fruitful research work in a permanent place in the tropics anyway ; the project leaders hinted at the difficulties of keeping up the standards to which they were accustomed at home under these conditions ; researchers preferred serological testing at home in germany to the difficulties and expenses involved in establishing further bacteriological methods in togo ; and probably most important togolese staff with academic qualifications were scarce and were already overburdened with routine tasks . the institute s research centre flourished within this large project , which had already been started by the who in 1974 and received support from the world bank and the european economic community ( later european union ) , among others , offering research opportunities in a tropical place over the years to more than a hundred , mostly german , junior scientists in the fields of medicine and biology until the present day . following the loss of these research opportunities to the regional hospital of sokod , the institute in lom also had to give up the name it had adopted in 1967 to honour its german donor and early german work in togo . when the west german army decided to remove the name of ernst rodenwaldt , incidentally one of piekarski s early mentors in the late 1930s , from the name of an institute in koblenz in germany in march 1998 , after his connivance with inhumane human experimentation during national socialism had become widely known , the togolese authorities followed suit in october of the same year . the two major research questions the relationship between west germany and togo and between medical academia and development aid have been answered to quite different extents due to the respective content of the german sources . however , the area of expertise for which university members were sought remained limited in the west german scenario during the first two decades of development co - operation . in view of the interwar german approach of social hygiene , a move towards social medicine as known from late british colonial policy the case studied here is thus typical of west germany of the period , since academic expertise was not expected for health care in general . the first task of academics was consultancy to the west german government , since the government agencies for technical aid did not yet have relevant experts of their own in these early years and benefited from temporarily hiring academics from university institutes without having to offer permanent jobs in germany . a decade later , by 1973 , the west german press maintained that the method practised by the ministry of economic co - operation in sending ad hoc consultants , usually travel - happy university professors , on exploratory trips for a week has proved to be a failure since permanent qualified observation and administrative support on the spot was lacking. however , this accusation typical of the increasingly sceptical attitude of the early 1970s towards development aid even before the professors visited the country , the host country had expressed the desire for the institute , and its establishment was principally agreed between the governments . as togo did not have any specialists in these fields , the project itself was mainly based in this case on the plan drawn up by the german academic who first visited the country . after the planning and construction of the institute , the next task for the universities was the provision of both technical and academic german staff . here , two different groups of biomedical experts emerged , reflecting the continuing tension between science and the health services , or academia and development co - operation : the doctors who held jobs at the institutes in africa for a year or more did not usually pursue academic careers later , but rather stayed in international or development organisations , whereas those who stayed in africa for research projects only became professors or directors afterwards for the most part . thus the increasing west german split between academic experts on communicable diseases at universities and other research institutes on the one hand and public health officers in the civil service on the other was also made manifest in international co - operation . in togo , the main task , so - called capacity - building , was fulfilled for routine activities through collaboration between the german and togolese counterparts . long - standing co - operation which continued after the end of the development project was the decisive condition for building up research capacities . the institute for tropical hygiene and public health at heidelberg university became a centre for north south co - operation for more than just infectious diseases , as its major topics included midwifery and nutrition , the latter also being found at the university of giessen . on the whole , links between development co - operation and academia decreased despite , or perhaps because of , special funding that the dfg and the daad started by programmes for academic co - operation with developing countries. meanwhile the german agencies for technical and financial co - operation ( the gtz and the west german development bank ) had their own health experts and professional external consultants , so that they no longer always needed professors and lecturers for expertise in this area . at the 31st g8 summit in gleneagles in 2005 , co - operation with africa was declared a task for all ministries of the participating countries , including those with responsibility for education and research and for health . thus , the challenges of the 1960s in integrating public health and academic research on communicable diseases within the framework of international co - operation returned under the new conditions of twenty - first century globalisation . it will be interesting to see to what extent history repeats itself or whether fundamental changes in national policies , scientific attitudes and international co - operation since the 1960s lead to different results in the future .

the african part and its agency , as is the tendency among established african scholars of history and anthropology due to their professional perspective , this paper seeks to explore more of the european activities both within and outside europe . since studies of medical science in european countries after the second world war rarely touch on relations with former colonies , there seems to be an imbalance between the number of historical and anthropological studies devoted to the european activities compared to those devoted to the african side in these encounters . furthermore , a renewed examination of the institutions and personnel of the northern hemisphere that dominated the early triumphant histories of colonial medicine and science can shed new light on these : the emphasis on african agency in more recent studies calls for a reversed and complementary perspective focusing on the europeans , who would then have to be seen as being greatly changed in the encounter , often learning as much themselves as they were attempting to teach the africans . thus these two shifts a procedural turn from the rather static description of results to the dynamics of transfer and interaction , and a recipients to the modifications of the donor side will be some of the primary perspectives of this article . a considerable number of academics and academic institutions in the federal republic of germany ( frg ) were among those involved in the re - establishment of a german role in health care in tropical and sub - tropical countries within the new framework of western development aid. during the first un development was still mainly understood in economic terms , 8% of the west german budget for technical aid was allotted to the health sector , at a total of 130 million deutschmarks for 176 projects . in view of this , the german togolese relationship in microbiology within the framework of development aid in the 1960s and 1970s was chosen as an example for closer study . the article will first give a short summary of post - colonial , pre-1945 german academic interest in health outside the north atlantic world , continuing with an overview of west german academic activities in health in non - european countries and of the relationship between the academic world and west german political and administrative structures in the main part of the article will consist of an extensive case study concerning togo . this includes an examination of international co - operation on the part of several institutes at west german universities which did not have a long pre - war tradition of medical research in the tropics : the institute of comparative tropical medicine in the veterinary faculty at munich university , the institutes of hygiene and of parasitology at bonn university and the hygiene institute of wrzburg university . inevitably , the dynamics of this project were rooted in the earlier background of german academic activities in tropical medicine as well as in later policies for west german thus refraining from the contested issue of the positive or negative impact on the host country s health situation the second major question to be examined in this study will be the relationship between was it one of mutual benefit and understanding , or did one of the two have to suffer due to pressure from the other s special interests ? in the light of functionalist systems theory , medical academia ( as part of science ) and development aid ( as part of politics and administration ) will be treated as functional social systems of communications with their own specific objectives , rewards and procedures which render their coupling potentially difficult . apart from the focus on medical research , the additional aims of this article include further investigation of the general relationship between development ( aid) and health , as this subject has been the focus of increased attention over recent years . the academic part of the history is documented in the archives of the university of bonn and the two grant - giving institutions for academic activities , the german research foundation ( dfg ) for research and the german academic exchange service ( daad ) for international co - operation between academic institutions . the governmental contribution , mainly in the form of the then ministry of economic co - operation ( bmz ) , was examined on the basis of documents in the german federal archives ( barch ) . the common temptation to treat the european side in north south relations as rather monolithic in nature could be countered here by indicating the effects of classic conflicts of interest , especially those between the intellectual , universal and long - term perspective of science and the more practical , national and short - term aims of politics . the first so - called entwicklungshelfer [ development aid workers ] of the new deutscher entwicklungsdienst ( ded ) [ german development service , a volunteer service for professional staff modelled on the us peace corps , did not leave germany until 1964 . these principles meant that aid would only be granted at the explicit request of the national government in the form of a project proposal , that financial aid would not commit recipients to buy the required equipment in germany and that the aid was tied to the agreed projects and could not consist of grants to the general budget or of the mere delivery of goods . while these principles were often handled in a rather cavalier manner in practice , they did nevertheless establish the basis required for scientific and medical co - operation : new hospitals or institutes were not offered , but had to be requested by the governments of the partner countries ; the means granted were bound to these institutions and could not be used for other health care or research purposes ; and the construction , equipment and supply was not necessarily commissioned with german companies . in the 1970s , the west german preference for unilaterally sending experts and development workers even increased dramatically when erhard eppler , the minister responsible for development co - operation , and chancellor willy brandt demanded a doubling of their numbers within five years . despite opposition from the ministries of health and foreign affairs , the ministry of development co - operation , which had been created for managing development aid in 1961 , was finally given responsibility for all projects of technical assistance in 1965 . the situation was highly complex , mirroring german federalism and thereby fundamentally differing from the centralism of france and britain , where the pasteurians and orstom ( office de la recherche scientifique et technique outre mer ) or the medical research council respectively controlled overseas microbiological work in terms of staffing , funding and research direction . here , construction was delayed , though not completely interrupted , for several years following the suspension of diplomatic relations between egypt and the frg from 1965 to 1972 , which had occurred as a result of the state visit of the east german head of state walter ulbricht to cairo . it was only in the late 1960s that germany publicly looked at british practice as a possible role model for international co - operation between universities , extending an invitation to professor c. t. ingold , vice - chair of the london - based inter - university council for higher education overseas to speak in germany . ingold , who was a renowned mycologist himself ( and father of the later famous anthropologist tim ingold ) , presented british policies and experiences to a german audience in 1969 and emphasised the need for political independence on both sides as well as flexibility and the avoidance of paternalistic neocolonialism. two years later , in 1971 , co - operation in science and higher education became an official part of west german one of the earlier , in many ways pioneering , projects linking the german government focus on public health to german academia was already in place in the small west african state of togo . having previously been part of the west african region known as the slave coast and of the portuguese trade network , the territory of the present republic of togo and of the upper volta region of ghana was proclaimed as the german protectorate of togo in 1884 by a medical doctor , gustav nachtigal ( 183485 ) , who was then also german consul in tunis and german imperial commissioner for west africa . internationally famous german doctors like the discoverer of the smallpox virus , the pathologist enrique paschen ( 18601936 ; in togo 191112 ) , and the hygienist ernst rodenwaldt ( 18781965 ; in togo 190913 ) worked in the colony before it came under french and british control during the first world war . the official division into british togoland and french togoland as a league of nations mandate in 1922 finally led to the present situation , the british part being incorporated into the gold coast , later ghana , and the french part retaining the name and forming the republic of togo . although france had also introduced its common west african health system here , french togoland had not received institutions for the higher education of health professionals or for medical research in the same way as other french territories in west africa such as senegal , cameroon , congo - brazzaville or the central african republic . during national socialism , he had been a member of the nsdap , of the national socialist association for physicians ( ns - rztebund ) and of the national socialist association for lecturers ( ns - dozentenbund ) , and he had also been an officer in the ss . completed in 1967 , the republic of togo provided the construction site , locally produced furniture , tax exemption for the import of necessary materials from abroad , transport and storage within the country , and various kinds of support for the accommodation of the german staff . the first german team , all of whom had previous ties to the institute of hygiene at bonn university , consisted of two doctors the project leader and head of the department of applied hygiene , and the head of the department of parasitology as well as two medico - technical assistants , a mechanic and an electro - technician ; a typist was later added . herrlich had spent most of his time from 1933 to 1939 in east africa , afghanistan and india , studying the genealogy of wheat in the hindukush region in 1935 and visiting most countries of eastern and southern africa between 1936 and 1937 . peter scheiber , project leader since 1969 , criticised the fact that , at the time of the establishment of the institute , the place and the tasks for a central laboratory with the range of duties described were neither prepared nor even projected ( ) in the public health system of togo. concerning the main , capacity - building task , seeliger had to disappoint expectations : employing togolese counterparts ( homologues ) who can take over duties as collaborators of the heads of department can not be expected in the near future , as only a few togolese medical students have returned home after completing their education , and they were immediately charged with medical care . employing togolese counterparts ( homologues ) who can take over duties as collaborators of the heads of department can not be expected in the near future , as only a few togolese medical students have returned home after completing their education , and they were immediately charged with medical care . as late as 1972 , the german project leaders still complained about the lack of togolese academic staff , as there were hardly any candidates that were interested in questions of theoretical medicine although the number of togolese doctors in the country had nearly doubled since the start of the project . besides training on the job for the togolese academic staff of the institute and the contribution to medical education at the university , the institute organised special courses for laboratory assistants , hygiene assistants in the public health service and who fellows . here , diverging lists regarding the duties of the institute can be found in the information box on the one hand , which is obviously based on official sources , and in the report by the two german scientists on the other hand . the box only speaks of the institute s function as a reference laboratory for samples sent in , as a central health office of the republic and as a central institution for a system of outposts under construction , by which a network of the health system shall cover the whole country. perhaps k. n. agbo s early co - authorship was also facilitated by his newly arrived european co - authors : the german reny anneliese braun - munzinger ( 194890 ) , who was accustomed to the co - operative practice of local co - authorship through her previous work with indian researchers in india , and the british scientist brian a. southgate ( 19302011 ) , who became well known for his support of non - european researchers and who obviously saw this practice as an international standard . there is just one very elaborate acknowledgment from 1976 and 1977 by bockemhl in the context of his research on salmonella and shigella , conducted between 1971 and 1973 : the examinations reported in this paper have been performed under somewhat difficult field conditions and were only possible thanks to the sympathy and collaboration of the local authorities : the regional representatives of the government , health officers , nurses , sanitary inspectors , school teachers , and village headmen.special thanks are due to my team , who worked together for 16 months : mr v. kpelevi , mr l. lawson , mr l. pr . agbodjan , mr r. apedoh , and mr m. sessou.serotyping of strains was performed at the institute of hygiene and microbiology , university of wrzburg , by mrs r. miethke , mrs d. legat , miss i. voigt , and the national reference centre for salmonella , hamburg , by priv .- doz . [ the examinations reported in this paper have been performed under somewhat difficult field conditions and were only possible thanks to the sympathy and collaboration of the local authorities : the regional representatives of the government , health officers , nurses , sanitary inspectors , school teachers , and village headmen . when the institute was handed over to the togolese ministry of health and social services on 9 january 1976 , the high esteem for the institute from both sides was indicated by the presence of several togolese ministers and several german representatives , including the two mentors seeliger and piekarski . considering contemporary german journalism s widespread criticism of german development aid or emergency aid in other places and its particular , politically motivated attention to german relations with the dictatorial ruler eyadma ( president , 19672005 ) , it should be mentioned that the hygiene institute in lom remained without unfavourable media coverage in germany . the institute had been requested by a still legitimate government and was clearly in the interests of the whole country ; it was in line with the german shift towards preventative medicine and academic research in development co - operation ; its construction was not delayed in any notable way ; collaboration occurred without major conflicts ; and the aim of capacity - building by training counterparts was achieved in so far as togolese heads of departments were able to take over the responsibilities in routine work at the end of the project . in fact , the institutes in lom and bangkok were the only two positive exceptions explicitly mentioned in an otherwise devastating evaluation of health projects by a senior official in the german ministry of economic co - operation in 1968 , their success being particularly attributed to continuous mentorship leading to new forms of partnership . as noted above several factors stood in the way of achieving more than training for the routine tasks of a central national laboratory : less than a decade was probably far too short ; the mentor herrlich had doubted the chances of fruitful research work in a permanent place in the tropics anyway ; the project leaders hinted at the difficulties of keeping up the standards to which they were accustomed at home under these conditions ; researchers preferred serological testing at home in germany to the difficulties and expenses involved in establishing further bacteriological methods in togo ; and probably most important togolese staff with academic qualifications were scarce and were already overburdened with routine tasks . the institute s research centre flourished within this large project , which had already been started by the who in 1974 and received support from the world bank and the european economic community ( later european union ) , among others , offering research opportunities in a tropical place over the years to more than a hundred , mostly german , junior scientists in the fields of medicine and biology until the present day . when the west german army decided to remove the name of ernst rodenwaldt , incidentally one of piekarski s early mentors in the late 1930s , from the name of an institute in koblenz in germany in march 1998 , after his connivance with inhumane human experimentation during national socialism had become widely known , the togolese authorities followed suit in october of the same year . there appeared to be a necessary connection between the progress and modernity that development aid claimed to bring and the promises of science despite the obvious fact that the first was directed towards what were regarded as the most backward regions and the latter strived to be at the most advanced edge of human progress . while non - medical academic experts in britain such as health economists richard titmuss and brian abel - smith were also commissioned with recommendations on comprehensive health care services in countries of the global south , west german professors were asked only for infectious disease control and clinical work . in view of the interwar german approach of social hygiene , a move towards social medicine as known from late british colonial policy the case studied here is thus typical of west germany of the period , since academic expertise was not expected for health care in general . however , this accusation typical of the increasingly sceptical attitude of the early 1970s towards development aid even before the professors visited the country , the host country had expressed the desire for the institute , and its establishment was principally agreed between the governments . here , two different groups of biomedical experts emerged , reflecting the continuing tension between science and the health services , or academia and development co - operation : the doctors who held jobs at the institutes in africa for a year or more did not usually pursue academic careers later , but rather stayed in international or development organisations , whereas those who stayed in africa for research projects only became professors or directors afterwards for the most part . professor fhner , head of the similar project in kabul mentioned above , had demanded three phases , each of about three years : ( 1 ) establishing routine work , ( 2 ) sending the counterparts to germany for further training , and ( 3 ) transferring responsibility for research to them by reducing german staff . meanwhile the german agencies for technical and financial co - operation ( the gtz and the west german development bank ) had their own health experts and professional external consultants , so that they no longer always needed professors and lecturers for expertise in this area . funds from implementing the millennium development goals ( mdgs ) , the global fund to fight aids , tuberculosis and malaria ( gfatm ) and the global alliance for vaccines and immunisation ( gavi ) boosted research on neglected health issues and operational research in particular in the countries concerned alongside these initiatives dominant activities with more immediate effects . thus , the challenges of the 1960s in integrating public health and academic research on communicable diseases within the framework of international co - operation returned under the new conditions of twenty - first century globalisation .

plasmatic viral load strongly predicts t cd4 cell count decline , aids progression and death . anyway , disease prognosis in hiv infected subjects is more rigorously defined by the combination of plasmatic viral load quantification and t cd4 cell count.1 it is internationally noted that the main predictive factors for failure of antiretroviral therapy are t cd4 cells low count and elevated viral load before commencing treatment.2 the efficacy of the highly active antiretroviral therapy ( haart ) and the pattern of therapy management could be evaluated based on plasmatic rna viral load assessement . in hiv / aids treatment the response is considered successful when the hiv rna levels remain undetectable by certified commercial assays , notwithstanding in a variable proportion of haart submitted patients , the viral replication and evolution goes on which could eventually contribute to the development of antiretroviral drug resistance and therapeutic failure.3 unsuccessful antiretroviral therapy could occur due to virologic and immunologic failure and clinical manifestations that can appear during the course of hiv infection . the identification of therapeutic failure is based on patient follow - up during treatment , taking into account the initial level of t cd4 , plasmatic hiv rna load and the patient clinical evolution . also , many factors could be related to therapeutic failure such as low treatment adhesion , insufficient drug dosage , mal - absorption , hiv antiretroviral resistance and drug interactions that could reduce the efficacy and hiv resistance to antiretroviral medications . patients that failed to respond to antiretroviral therapy confirmed by the genotype resistant assay will be guided to choose new drugs . this choice is based on the knowledge of previous treatments and yet the reason to remove a drug from the antiretroviral regimen is mainly justified by the in vitro antiretroviral resistance profile . the antiretroviral therapeutic suspension as an alternative therapy is based on the reemergence of the initial virus population before treatment initiation , which would be susceptible to the antiretroviral drugs earlier prescribed , so this proposal is presently under evaluation . the essential objective of treatment in patients presenting therapeutic failure is to maintain t cd4 acceptable cell population density , whilst new therapeutic options are awaited , contrary to the priority aim to reach and keep up an undetectable viral load.4 according to moore et al,5 virological failure is characterized by viral load higher than 400 copies / ml after 48 weeks of initial treatment or among subjects that had complete viral suppression , but later on the viral load will recrudesce . the viral failure precedes the immunologic failure which is defined by a decline of more than 25.0% in the subsequent count of t cd4 lymphocytes or regresion to the initial t cd4 cell count before treatment . these conditions are mainly suggestive of immunologic failure but laboratory analysis confirmation is mandatory.6 cozzi - lepri et al7 concluded that patients remaining in haart failure presented viral load slowly progressing during the next 12 months , differently to t cd4 cell count which remained stable . the impact of haart on t cd4 cell count and viral load of hiv infected patients has been demonstrated to improve the patient s immunological status and it also diminished the viral load , interrupting the aids progression.8 however there are crucial limitations of haart regimen , as it does not eradicate viral infection despite long and permanent antiretroviral therapy . consequently , a significant number of these patients on haart therapy develop viral resistance to the drugs besides diverse side effects including metabolic disorders . therefore new approaches are necessary to control and/or eradicate hiv infection.9,10 this research work aimed to analyze t cd4 cell density and viral load response in hiv infected patients undergoing different therapeutic regimens which failed virologically , and also the associated factors to it during 20022008 in brasilia , federal district , brazil . a cohort study was conducted in the health center number 01 attached to the secretary of health , federal district ( ses / df ) ( centro de sade nmero 01 , secretaria de sade do distrito federal ) which included 139 hiv-1 infected patients . the patients had clinical and laboratory diagnosis as defined by the 1993 aids clinical course criteria and by the us center for disease control ( cdc)11 and also by the recommendations of the brazilian consensus of antiretroviral therapy in infected adults and adolescents by the ministry of health , secretary of health surveillance , brazilian national program of sexually transmitted diseases and aids.12 patients selected for the study were identified in the records of logistic system of the ministry of health , siclom13 utilized by the hospital dia , ses / df . the plasmatic hiv rna load was quantified by the bdna system 340 presenting sensitivity of less than 50 copies / ml . the t cd4 helper lymphocytes count was carried out by automatized flow cytometry utilizing the facscalibur count system . the sampling utilized for patient selection was by convenience whose inclusion criteria were : ( a ) age higher than 18 years old and of both sexes ; ( b ) hiv infection diagnosis defined by the brazillian ministry of health standardized patterns ; ( c ) detectable viral load previous to haart beginning > 400 copies / ml ; ( d ) t cd4 cell count < 500 cells / mm before haart regimen initiation ; ( e ) present laboratory analysis for viral load and t cd4 count between 6 and 12 months in order to compare the former ones with those of baseline ; ( f ) patients have had clinical and immunological follow up during 3 months intervals . if undetectable viral load of 50 copies / ml was not sustained during 24 weeks of treatment or higher than 400 copies / ml after 48 weeks treatment , it was considered virological failure to the initial antiretroviral therapeutic regimen.5 immunological failure was considered by the declining of t cd4 cell counting 25.0% of the absolute values6 or by returning to the t cd4 cell count initial values before antiretroviral therapy initiation . the viral load previous to haart regimen initiation and t cd4 cell count were evaluated as possible predictors of viral failure leading us to establish the cut off values to viral load > 100.000 copies / ml or 100.000 copies / ml as also t cd4 > 200 cells / mm or 200 cells / mm . the antiretroviral therapy regimen were : ( a ) double with two nucleoside reverse transcriptase inhibitor and non nucleoside reverse transcriptase inhibitor ; ( b ) triple with two nucleoside reverse transcriptase inhibitor and one protease inhibitor and ( c ) three nucleoside reverse transcriptase inhibitor , according to cdc classification and the brazilian ministry of health guidelines as previously described . in the population here studied , the chosen antiretroviral regimen was decided by the physician in charge without interference by the authors of this research work . the criteria for the diagnosis of hepatitis b and c virus infection followed the guidelines established by the manual of viral hepatitis consultancy , secretary of health surveillance , department of epidemiological surveillance , braslia , ministry of health14 and also for the tuberculosis diagnosis , the technical manual for tuberculosis control , notebook of basic attention , secretary of health politics , department of basic attention , braslia . ministry of health.15 the exclusion criteria for this research were : ( a ) be pregnant or minor under 18 years old ; ( b ) be prescribed with medications which could metabolically interact with antiretroviral drugs , including phytotherapics ; ( c ) have had last clinical appointment in a period longer than 6 months which was considered lost to follow - up . the analysis of baseline characteristics ( initial data previous to initiation of treatment referring to t cd4 cell count and viral load ) included frequency tables of categorical variables and their descriptive statistics ( median , arithmetic mean and standard deviation ) as also as continuous variables . in order to measure the dependent association between two categorial variables it was utilized the chi - square test ( x ) or when necessary the exact fisher test . to compare before and after results the macnemar test was applied . for the analysis of logistic regression and to predict the event of virological failure16 ( depedent variable ) , the wald method was applied . the association measurement calculated from the logistic model is the adjusted odds ratio ( or ) . the patients data were confidential and consentment to utilize information were obtained at the health center number 01 direction according to the federal district secretary ( ses / df ) of health agreement . the research work protocol was approved by the federal district secretary of health researh ethics committee , foundation of teaching and research in health sciences ( fepecs ) . a cohort study was conducted in the health center number 01 attached to the secretary of health , federal district ( ses / df ) ( centro de sade nmero 01 , secretaria de sade do distrito federal ) which included 139 hiv-1 infected patients . the patients had clinical and laboratory diagnosis as defined by the 1993 aids clinical course criteria and by the us center for disease control ( cdc)11 and also by the recommendations of the brazilian consensus of antiretroviral therapy in infected adults and adolescents by the ministry of health , secretary of health surveillance , brazilian national program of sexually transmitted diseases and aids.12 patients selected for the study were identified in the records of logistic system of the ministry of health , siclom13 utilized by the hospital dia , ses / df . the plasmatic hiv rna load was quantified by the bdna system 340 presenting sensitivity of less than 50 copies / ml . the t cd4 helper lymphocytes count was carried out by automatized flow cytometry utilizing the facscalibur count system . the sampling utilized for patient selection was by convenience whose inclusion criteria were : ( a ) age higher than 18 years old and of both sexes ; ( b ) hiv infection diagnosis defined by the brazillian ministry of health standardized patterns ; ( c ) detectable viral load previous to haart beginning > 400 copies / ml ; ( d ) t cd4 cell count < 500 cells / mm before haart regimen initiation ; ( e ) present laboratory analysis for viral load and t cd4 count between 6 and 12 months in order to compare the former ones with those of baseline ; ( f ) patients have had clinical and immunological follow up during 3 months intervals . if undetectable viral load of 50 copies / ml was not sustained during 24 weeks of treatment or higher than 400 copies / ml after 48 weeks treatment , it was considered virological failure to the initial antiretroviral therapeutic regimen.5 immunological failure was considered by the declining of t cd4 cell counting 25.0% of the absolute values6 or by returning to the t cd4 cell count initial values before antiretroviral therapy initiation . the viral load previous to haart regimen initiation and t cd4 cell count were evaluated as possible predictors of viral failure leading us to establish the cut off values to viral load > 100.000 copies / ml or 100.000 copies / ml as also t cd4 > 200 cells / mm or 200 cells / mm . the antiretroviral therapy regimen were : ( a ) double with two nucleoside reverse transcriptase inhibitor and non nucleoside reverse transcriptase inhibitor ; ( b ) triple with two nucleoside reverse transcriptase inhibitor and one protease inhibitor and ( c ) three nucleoside reverse transcriptase inhibitor , according to cdc classification and the brazilian ministry of health guidelines as previously described . in the population here studied , the chosen antiretroviral regimen was decided by the physician in charge without interference by the authors of this research work . the criteria for the diagnosis of hepatitis b and c virus infection followed the guidelines established by the manual of viral hepatitis consultancy , secretary of health surveillance , department of epidemiological surveillance , braslia , ministry of health14 and also for the tuberculosis diagnosis , the technical manual for tuberculosis control , notebook of basic attention , secretary of health politics , department of basic attention , braslia . ministry of health.15 the exclusion criteria for this research were : ( a ) be pregnant or minor under 18 years old ; ( b ) be prescribed with medications which could metabolically interact with antiretroviral drugs , including phytotherapics ; ( c ) have had last clinical appointment in a period longer than 6 months which was considered lost to follow - up . the analysis of baseline characteristics ( initial data previous to initiation of treatment referring to t cd4 cell count and viral load ) included frequency tables of categorical variables and their descriptive statistics ( median , arithmetic mean and standard deviation ) as also as continuous variables . in order to measure the dependent association between two categorial variables it was utilized the chi - square test ( x ) or when necessary the exact fisher test . to compare before and after results the macnemar test was applied . for the analysis of logistic regression and to predict the event of virological failure16 ( depedent variable ) , the wald method was applied . the association measurement calculated from the logistic model is the adjusted odds ratio ( or ) . the patients data were confidential and consentment to utilize information were obtained at the health center number 01 direction according to the federal district secretary ( ses / df ) of health agreement . the research work protocol was approved by the federal district secretary of health researh ethics committee , foundation of teaching and research in health sciences ( fepecs ) . initially , 165 patients were eligible to participate in the study for starting the treatment in the period 20022008 . however , only 139 patients met the inclusion criteria . among them ( table 1a and b ) , males predominated ( 74.1% ) . ages ranged from 2065 years old ( mean = 39.7 years , median = 40.0 ) . there was a slight predominance of patients in the range of 4165 years old ( 51.1% ) compared to younger patients . as for race / ethnical background , there was a predominance of mestizos ( 54.6% ) . regarding to the marital status , there was a predominance of single people ( 48.2% ) . people living in taguatinga ( 13.7% ) , a satellite city located in the surrounding areas of braslia , represented the majority of the patients enrolled in this study . the place of birth showed that patients predominated from the midwest ( 40.3% ) and the northeast of brazil ( 34.5% ) . hetrosexual relationships predominated ( 51.8% ) , followed by the homosexual relationships ( 23.7% ) . the initial viral load ranged between 1.643 copies / ml to 500.000 copies / ml ( mean = 180.721 copies / ml ; arithmetic mean = 126.417 copies / ml ) presenting more than half of the patients ( 55.4% ) cell density higher than 100.000 copies / ml ( table 2 ) . after antiretroviral therapy , the initial profile changed substantially more than 70% of the patients had viral load lower than 50 copies / ml . in reference to the initial t cd4 cell count , 68.3% of the patients were in the range of 0200 cells / mm . after treatment a relevant change was observed in the patients profile concerning t cd4 cell counts , remaining less than 37% of the cases in this stage . in the following cell density interval , there was an expressive increment in the number of cases , representing more than 43.0% of the patients ( table 2 ) . initially the most utilized antiretroviral regimen included two drugs , the nucleoside reverse transcriptase inhibitor and non nucleoside reverse transcriptase inhibitor in 71.2% of the cases and the remaining 28.1% included two nucleoside reverse transcriptase inhibitor and one protease inhibitor . during the course of the treatment , the presented regimen distribution slightly changed to 66.9% to the first drug combination and 32.4% to the second one , there remaining only one utilizing three nucleoside reverse transcriptase inhibitor ( table 2 ) . virologic failure was 12.2% ( table 3 ) , or antiretroviral treatment success was 88.8% , exceeding the results obtained by johnson and way of 80%.17 among the socioeconomic and demographic variables , just the birthplace and the fact that originating in the midwest region which includes the federal district , was associated with the occurrence of virologic failure in the study . cole et al18 stated that hiv infection is sufficiently widespread , suggesting that it is a multidimensional epidemic , with demographic , residential , social , biological and behavioral significance . perhaps this is a natural social structural marker as the study was conducted in the midwest region compared to those migrating people from other regions to the federal district . there was a trend toward a higher incidence of virologic failure being male , over 40 years old , mestizo , unmarried or separate and also heterossexual exposure category . patients from the midwestern had 2.7 times greater risk of virologic failure than those from other regions of brazil , especially because the study was conducted in brasilia , which is part of this region . in this study , the initial viral load is not statistically associated and significant to the occurrence of virologic failure . however , there is the presence of a higher incidence of virologic failure among those who had viral load greater than 100.000 copies / ml after the introduction of haart . the virologic target for patients on haart is to achieve viral load plasma levels below 50 copies / ml when two or more potent drugs are used . these results highlight the importance of compliance with primary success and reinforces the need to work on the accession of such patients . some authors19 interpret and agree that those with viral load higher than the baseline level of 100.000 copies / ml had a slower pace to achieve viral suppression . however , a potent and well tolerated prophylactic regimen with haart can improve cd4 t cell count at the beginning , during and after treatment . kantor et al2 concluded that plasma viral load strongly predicts the rate of cd4 lymphocyte count decrease , progression to aids and death . but the prognosis for people infected with hiv is more strictly defined by the combination of plasma viral load measurement and cd4 lymphocytes counts . some patients with viral load higher than 100.000 copies / ml remained in treatment failure after haart regimen in contrast to t cd4 lymphocyte counts decrease at a slower pace , leaving some in virologic failure , probably due to the emergence of hiv resistant strains . cozzi - lepri et al7 advocate that patients who remained in haart regimen failure , the viral load in the next twelve months was growing at a relatively slow pace , the cd4 t cell count was stable and the time course of viral replication in patients with virological failure had not been fully elucidated . asjo and langeland20 suggest that the lack of complete suppression of viral replication allows the continued development of hiv variants with different degrees of resistance . it results not only in treatment failure , but it also increases the risk of hiv primary resistance and dissemination . patients with initial cd4 t cell count 200 cells / mm had a tendency to virologic failure , however , there was not any statistical significance . tuberculosis coinfection was associated with virologic failure demonstrated with statistical significance , p = 0.019 . patients with a previous diagnosis of tuberculosis infection had 2.9 times great risk of virologic failure . it is observed that the t cd4 cell count baseline above 200 cells / mm was not statistically associated with virological failure , but the incidence of failure in this group was noticeably lower than in those with initial t cd4 less than 200 cells / mm . studies conducted by skowron et al21 demonstrated that t cd4 cell count is a better predictor of viral suppression . however , in order to achieve viral load suppression to undetectable levels , it is necessary to have an optimal response of t cd4 cell count in patients under antiretroviral therapy . piliero22 comments that the maximum suppression of viral replication remains the primary goal of therapy after haart regimen , therefore t cd4 cell count and hiv plasma viral load values are the prognostic markers for treatment success after four , eight or twelve weeks post treatment , as the changes are predictive of favorable long - term success in six months or more . according to moreno et al23 in multifailed patients , at least two active drugs can not be used , the therapeutic scheme should be kept in use until new drugs become available , assuming that there is an immunologic and clinical stability in order to avoid the use of a drug from a common chemical group which usually leads to a rapid viral resistance development , further limiting future treatment options . geretti et al24 stated that patients in first haart line who maintained consistently undetectable plasma viral load for a year , had a low risk of virologic failure . in this study , special attention should be given to early tuberculosis diagnosis during the early haart regimen prescription . studies reported by bekker et al25 and von reyn et al26 showed that the mycobacterial disease was a major contributor to hiv mortality . in addition , the infection increases the risk of latent tuberculosis reactivation , a new infection progression or re - infection to active disease , increasing the risk of the emergence of hiv resistant strains to the usual antiretroviral therapy . tuberculosis also accelerates the course of hiv induced disease by activating viral replication and accentuating the decline of t cd4 cells . in the logistic regresion analysis the best model to explain the event of virological failure is the one that includes variables such as origin and infection by m. tuberculosis , yielding or values of 3.2 and 3.8 respectively despite the enormous progress as a result of the impact of haart - related morbidity and mortality in patients with hiv / aids continue and will continue to fail in the face of the therapeutic classes of haart currently available . the presented results reinforce the importance of monitoring the biological markers t cd4 cells and viral load in patients living with and without aids , both to ensure that viral replication is under control and to reassure the maintenance of immune reconstitution compatible with life , and to predict the risk of developing resistance and therapeutic failures in the course of the treatment of hiv - infected people . it should be noted that the sample size limited the analysis of this study , as the retrospective information taken from the medical records , excluded some data from the laboratory and the clinical follow up . the sample obtained in this way may have had a limited ability to highlight the predictive differences of risk for both t cd4 cell count and viral load in naive patients . it is possible that working with a larger cohort of patients and longer follow - up of these predictors probably would show more consistent evidence that is statistically supported . initial treatment with any nnrti - based regimen or an ip , but not both , is a good strategy for managing the long - term antiretroviral treatment in naive hiv patients . moreover , the recent availability of new antiretroviral agents for the treatment of hiv has increased treatment options and improved durability , tolerability and efficacy in the long - term haart regimen . other limitations that should be highlighted refer to the possibility of verifying the association of hbv and hcv coinfections which do not have evolutionary studies of these hiv comorbidities . it is possible that if we had a homogeneous group of patients already in advanced stages of these pathologies , the risk of virologic failure would be high . the sociodemographic data were collected from medical records and not through interviews , this fact may also have skewed the results of the analysis of association between these variables and treatment failure . however , this service data and variables accounted are limitations imposed by real situations in nosocomial institutions in the federal district in brazil . it is of great relevance to the findings in this study considering the limitations and difficulties of conducting studies in routine service , as it can be concluded that even in the non - ideal conditions of a clinical trial , patients treated in this unit had virological success of 12.2% up to one year of treatment , a fact that can predict the durability of the first scheme which is better than expected in many other international centers.17 also tuberculosis infection should be traced as a priority as early as possible . as the low values of t cd4 cell counts and high viral load pre - treatment should be considered to implement the prescribed antiretroviral therapy , especially when new classes are available and have superior performance , as much viral suppression and immune reconstitution could be achieved .

little data exists concerning the efficacy of the antiretroviral therapy in the federal district in brazil , therefore in order to improve hiv / aids patients therapy and to pinpoint hot spots in the treatment , this research work was conducted . of 139 hiv / aids patients submitted to the highly active antiretroviral therapy , 12.2% failed virologically . the significant associated factors related to unresponsiveness to the lentiviral treatment were : patients place of origin ( or = 3.28 ; ic95% = 1.09.73 ; p = 0.032 ) and mycobacterium tuberculosis infection ( rr = 2.90 ; ic95% = 1.197.02 ; p = 0.019 ) . in the logistic regression analysis , the remaining variables in the model were : patients birthplace ( or = 3.28 ; ic95% = 1.109.73 ; p = 0.032 ) and tuberculosis comorbidity ( or = 3.82 ; ic95% = 1.1912.22 ; p = 0.024 ) . the patients enrolled in this survey had an 88.0% therapeutic success rate for the maximum period of one year of treatment , predicting that t cd4 + low values and elevated viral loads at pretreatment should be particularly considered in tuberculosis coinfection , besides the availability of new antiretroviral drugs displaying optimal activity both in viral suppression and immunological reconstitution .

Introduction Material and Methods Patients Results and Discussion Conclusion

anyway , disease prognosis in hiv infected subjects is more rigorously defined by the combination of plasmatic viral load quantification and t cd4 cell count.1 it is internationally noted that the main predictive factors for failure of antiretroviral therapy are t cd4 cells low count and elevated viral load before commencing treatment.2 the efficacy of the highly active antiretroviral therapy ( haart ) and the pattern of therapy management could be evaluated based on plasmatic rna viral load assessement . in hiv / aids treatment the response is considered successful when the hiv rna levels remain undetectable by certified commercial assays , notwithstanding in a variable proportion of haart submitted patients , the viral replication and evolution goes on which could eventually contribute to the development of antiretroviral drug resistance and therapeutic failure.3 unsuccessful antiretroviral therapy could occur due to virologic and immunologic failure and clinical manifestations that can appear during the course of hiv infection . the identification of therapeutic failure is based on patient follow - up during treatment , taking into account the initial level of t cd4 , plasmatic hiv rna load and the patient clinical evolution . also , many factors could be related to therapeutic failure such as low treatment adhesion , insufficient drug dosage , mal - absorption , hiv antiretroviral resistance and drug interactions that could reduce the efficacy and hiv resistance to antiretroviral medications . patients that failed to respond to antiretroviral therapy confirmed by the genotype resistant assay will be guided to choose new drugs . this choice is based on the knowledge of previous treatments and yet the reason to remove a drug from the antiretroviral regimen is mainly justified by the in vitro antiretroviral resistance profile . the antiretroviral therapeutic suspension as an alternative therapy is based on the reemergence of the initial virus population before treatment initiation , which would be susceptible to the antiretroviral drugs earlier prescribed , so this proposal is presently under evaluation . the essential objective of treatment in patients presenting therapeutic failure is to maintain t cd4 acceptable cell population density , whilst new therapeutic options are awaited , contrary to the priority aim to reach and keep up an undetectable viral load.4 according to moore et al,5 virological failure is characterized by viral load higher than 400 copies / ml after 48 weeks of initial treatment or among subjects that had complete viral suppression , but later on the viral load will recrudesce . the viral failure precedes the immunologic failure which is defined by a decline of more than 25.0% in the subsequent count of t cd4 lymphocytes or regresion to the initial t cd4 cell count before treatment . the impact of haart on t cd4 cell count and viral load of hiv infected patients has been demonstrated to improve the patient s immunological status and it also diminished the viral load , interrupting the aids progression.8 however there are crucial limitations of haart regimen , as it does not eradicate viral infection despite long and permanent antiretroviral therapy . therefore new approaches are necessary to control and/or eradicate hiv infection.9,10 this research work aimed to analyze t cd4 cell density and viral load response in hiv infected patients undergoing different therapeutic regimens which failed virologically , and also the associated factors to it during 20022008 in brasilia , federal district , brazil . a cohort study was conducted in the health center number 01 attached to the secretary of health , federal district ( ses / df ) ( centro de sade nmero 01 , secretaria de sade do distrito federal ) which included 139 hiv-1 infected patients . the patients had clinical and laboratory diagnosis as defined by the 1993 aids clinical course criteria and by the us center for disease control ( cdc)11 and also by the recommendations of the brazilian consensus of antiretroviral therapy in infected adults and adolescents by the ministry of health , secretary of health surveillance , brazilian national program of sexually transmitted diseases and aids.12 patients selected for the study were identified in the records of logistic system of the ministry of health , siclom13 utilized by the hospital dia , ses / df . the sampling utilized for patient selection was by convenience whose inclusion criteria were : ( a ) age higher than 18 years old and of both sexes ; ( b ) hiv infection diagnosis defined by the brazillian ministry of health standardized patterns ; ( c ) detectable viral load previous to haart beginning > 400 copies / ml ; ( d ) t cd4 cell count < 500 cells / mm before haart regimen initiation ; ( e ) present laboratory analysis for viral load and t cd4 count between 6 and 12 months in order to compare the former ones with those of baseline ; ( f ) patients have had clinical and immunological follow up during 3 months intervals . if undetectable viral load of 50 copies / ml was not sustained during 24 weeks of treatment or higher than 400 copies / ml after 48 weeks treatment , it was considered virological failure to the initial antiretroviral therapeutic regimen.5 immunological failure was considered by the declining of t cd4 cell counting 25.0% of the absolute values6 or by returning to the t cd4 cell count initial values before antiretroviral therapy initiation . the antiretroviral therapy regimen were : ( a ) double with two nucleoside reverse transcriptase inhibitor and non nucleoside reverse transcriptase inhibitor ; ( b ) triple with two nucleoside reverse transcriptase inhibitor and one protease inhibitor and ( c ) three nucleoside reverse transcriptase inhibitor , according to cdc classification and the brazilian ministry of health guidelines as previously described . in the population here studied , the chosen antiretroviral regimen was decided by the physician in charge without interference by the authors of this research work . the criteria for the diagnosis of hepatitis b and c virus infection followed the guidelines established by the manual of viral hepatitis consultancy , secretary of health surveillance , department of epidemiological surveillance , braslia , ministry of health14 and also for the tuberculosis diagnosis , the technical manual for tuberculosis control , notebook of basic attention , secretary of health politics , department of basic attention , braslia . ministry of health.15 the exclusion criteria for this research were : ( a ) be pregnant or minor under 18 years old ; ( b ) be prescribed with medications which could metabolically interact with antiretroviral drugs , including phytotherapics ; ( c ) have had last clinical appointment in a period longer than 6 months which was considered lost to follow - up . the analysis of baseline characteristics ( initial data previous to initiation of treatment referring to t cd4 cell count and viral load ) included frequency tables of categorical variables and their descriptive statistics ( median , arithmetic mean and standard deviation ) as also as continuous variables . in order to measure the dependent association between two categorial variables it was utilized the chi - square test ( x ) or when necessary the exact fisher test . for the analysis of logistic regression and to predict the event of virological failure16 ( depedent variable ) , the wald method was applied . the association measurement calculated from the logistic model is the adjusted odds ratio ( or ) . the patients data were confidential and consentment to utilize information were obtained at the health center number 01 direction according to the federal district secretary ( ses / df ) of health agreement . the research work protocol was approved by the federal district secretary of health researh ethics committee , foundation of teaching and research in health sciences ( fepecs ) . a cohort study was conducted in the health center number 01 attached to the secretary of health , federal district ( ses / df ) ( centro de sade nmero 01 , secretaria de sade do distrito federal ) which included 139 hiv-1 infected patients . the patients had clinical and laboratory diagnosis as defined by the 1993 aids clinical course criteria and by the us center for disease control ( cdc)11 and also by the recommendations of the brazilian consensus of antiretroviral therapy in infected adults and adolescents by the ministry of health , secretary of health surveillance , brazilian national program of sexually transmitted diseases and aids.12 patients selected for the study were identified in the records of logistic system of the ministry of health , siclom13 utilized by the hospital dia , ses / df . the sampling utilized for patient selection was by convenience whose inclusion criteria were : ( a ) age higher than 18 years old and of both sexes ; ( b ) hiv infection diagnosis defined by the brazillian ministry of health standardized patterns ; ( c ) detectable viral load previous to haart beginning > 400 copies / ml ; ( d ) t cd4 cell count < 500 cells / mm before haart regimen initiation ; ( e ) present laboratory analysis for viral load and t cd4 count between 6 and 12 months in order to compare the former ones with those of baseline ; ( f ) patients have had clinical and immunological follow up during 3 months intervals . if undetectable viral load of 50 copies / ml was not sustained during 24 weeks of treatment or higher than 400 copies / ml after 48 weeks treatment , it was considered virological failure to the initial antiretroviral therapeutic regimen.5 immunological failure was considered by the declining of t cd4 cell counting 25.0% of the absolute values6 or by returning to the t cd4 cell count initial values before antiretroviral therapy initiation . the viral load previous to haart regimen initiation and t cd4 cell count were evaluated as possible predictors of viral failure leading us to establish the cut off values to viral load > 100.000 copies / ml or 100.000 copies / ml as also t cd4 > 200 cells / mm or 200 cells / mm . the antiretroviral therapy regimen were : ( a ) double with two nucleoside reverse transcriptase inhibitor and non nucleoside reverse transcriptase inhibitor ; ( b ) triple with two nucleoside reverse transcriptase inhibitor and one protease inhibitor and ( c ) three nucleoside reverse transcriptase inhibitor , according to cdc classification and the brazilian ministry of health guidelines as previously described . in the population here studied , the chosen antiretroviral regimen was decided by the physician in charge without interference by the authors of this research work . the criteria for the diagnosis of hepatitis b and c virus infection followed the guidelines established by the manual of viral hepatitis consultancy , secretary of health surveillance , department of epidemiological surveillance , braslia , ministry of health14 and also for the tuberculosis diagnosis , the technical manual for tuberculosis control , notebook of basic attention , secretary of health politics , department of basic attention , braslia . ministry of health.15 the exclusion criteria for this research were : ( a ) be pregnant or minor under 18 years old ; ( b ) be prescribed with medications which could metabolically interact with antiretroviral drugs , including phytotherapics ; ( c ) have had last clinical appointment in a period longer than 6 months which was considered lost to follow - up . the analysis of baseline characteristics ( initial data previous to initiation of treatment referring to t cd4 cell count and viral load ) included frequency tables of categorical variables and their descriptive statistics ( median , arithmetic mean and standard deviation ) as also as continuous variables . in order to measure the dependent association between two categorial variables it was utilized the chi - square test ( x ) or when necessary the exact fisher test . for the analysis of logistic regression and to predict the event of virological failure16 ( depedent variable ) , the wald method was applied . the association measurement calculated from the logistic model is the adjusted odds ratio ( or ) . the patients data were confidential and consentment to utilize information were obtained at the health center number 01 direction according to the federal district secretary ( ses / df ) of health agreement . the research work protocol was approved by the federal district secretary of health researh ethics committee , foundation of teaching and research in health sciences ( fepecs ) . initially , 165 patients were eligible to participate in the study for starting the treatment in the period 20022008 . people living in taguatinga ( 13.7% ) , a satellite city located in the surrounding areas of braslia , represented the majority of the patients enrolled in this study . the place of birth showed that patients predominated from the midwest ( 40.3% ) and the northeast of brazil ( 34.5% ) . the initial viral load ranged between 1.643 copies / ml to 500.000 copies / ml ( mean = 180.721 copies / ml ; arithmetic mean = 126.417 copies / ml ) presenting more than half of the patients ( 55.4% ) cell density higher than 100.000 copies / ml ( table 2 ) . after antiretroviral therapy , the initial profile changed substantially more than 70% of the patients had viral load lower than 50 copies / ml . in reference to the initial t cd4 cell count , 68.3% of the patients were in the range of 0200 cells / mm . after treatment a relevant change was observed in the patients profile concerning t cd4 cell counts , remaining less than 37% of the cases in this stage . in the following cell density interval , there was an expressive increment in the number of cases , representing more than 43.0% of the patients ( table 2 ) . initially the most utilized antiretroviral regimen included two drugs , the nucleoside reverse transcriptase inhibitor and non nucleoside reverse transcriptase inhibitor in 71.2% of the cases and the remaining 28.1% included two nucleoside reverse transcriptase inhibitor and one protease inhibitor . during the course of the treatment , the presented regimen distribution slightly changed to 66.9% to the first drug combination and 32.4% to the second one , there remaining only one utilizing three nucleoside reverse transcriptase inhibitor ( table 2 ) . virologic failure was 12.2% ( table 3 ) , or antiretroviral treatment success was 88.8% , exceeding the results obtained by johnson and way of 80%.17 among the socioeconomic and demographic variables , just the birthplace and the fact that originating in the midwest region which includes the federal district , was associated with the occurrence of virologic failure in the study . perhaps this is a natural social structural marker as the study was conducted in the midwest region compared to those migrating people from other regions to the federal district . patients from the midwestern had 2.7 times greater risk of virologic failure than those from other regions of brazil , especially because the study was conducted in brasilia , which is part of this region . in this study , the initial viral load is not statistically associated and significant to the occurrence of virologic failure . some authors19 interpret and agree that those with viral load higher than the baseline level of 100.000 copies / ml had a slower pace to achieve viral suppression . some patients with viral load higher than 100.000 copies / ml remained in treatment failure after haart regimen in contrast to t cd4 lymphocyte counts decrease at a slower pace , leaving some in virologic failure , probably due to the emergence of hiv resistant strains . cozzi - lepri et al7 advocate that patients who remained in haart regimen failure , the viral load in the next twelve months was growing at a relatively slow pace , the cd4 t cell count was stable and the time course of viral replication in patients with virological failure had not been fully elucidated . tuberculosis coinfection was associated with virologic failure demonstrated with statistical significance , p = 0.019 . patients with a previous diagnosis of tuberculosis infection had 2.9 times great risk of virologic failure . it is observed that the t cd4 cell count baseline above 200 cells / mm was not statistically associated with virological failure , but the incidence of failure in this group was noticeably lower than in those with initial t cd4 less than 200 cells / mm . studies conducted by skowron et al21 demonstrated that t cd4 cell count is a better predictor of viral suppression . however , in order to achieve viral load suppression to undetectable levels , it is necessary to have an optimal response of t cd4 cell count in patients under antiretroviral therapy . piliero22 comments that the maximum suppression of viral replication remains the primary goal of therapy after haart regimen , therefore t cd4 cell count and hiv plasma viral load values are the prognostic markers for treatment success after four , eight or twelve weeks post treatment , as the changes are predictive of favorable long - term success in six months or more . according to moreno et al23 in multifailed patients , at least two active drugs can not be used , the therapeutic scheme should be kept in use until new drugs become available , assuming that there is an immunologic and clinical stability in order to avoid the use of a drug from a common chemical group which usually leads to a rapid viral resistance development , further limiting future treatment options . in this study , special attention should be given to early tuberculosis diagnosis during the early haart regimen prescription . in addition , the infection increases the risk of latent tuberculosis reactivation , a new infection progression or re - infection to active disease , increasing the risk of the emergence of hiv resistant strains to the usual antiretroviral therapy . tuberculosis also accelerates the course of hiv induced disease by activating viral replication and accentuating the decline of t cd4 cells . in the logistic regresion analysis the best model to explain the event of virological failure is the one that includes variables such as origin and infection by m. tuberculosis , yielding or values of 3.2 and 3.8 respectively despite the enormous progress as a result of the impact of haart - related morbidity and mortality in patients with hiv / aids continue and will continue to fail in the face of the therapeutic classes of haart currently available . the presented results reinforce the importance of monitoring the biological markers t cd4 cells and viral load in patients living with and without aids , both to ensure that viral replication is under control and to reassure the maintenance of immune reconstitution compatible with life , and to predict the risk of developing resistance and therapeutic failures in the course of the treatment of hiv - infected people . the sample obtained in this way may have had a limited ability to highlight the predictive differences of risk for both t cd4 cell count and viral load in naive patients . moreover , the recent availability of new antiretroviral agents for the treatment of hiv has increased treatment options and improved durability , tolerability and efficacy in the long - term haart regimen . other limitations that should be highlighted refer to the possibility of verifying the association of hbv and hcv coinfections which do not have evolutionary studies of these hiv comorbidities . the sociodemographic data were collected from medical records and not through interviews , this fact may also have skewed the results of the analysis of association between these variables and treatment failure . however , this service data and variables accounted are limitations imposed by real situations in nosocomial institutions in the federal district in brazil . it is of great relevance to the findings in this study considering the limitations and difficulties of conducting studies in routine service , as it can be concluded that even in the non - ideal conditions of a clinical trial , patients treated in this unit had virological success of 12.2% up to one year of treatment , a fact that can predict the durability of the first scheme which is better than expected in many other international centers.17 also tuberculosis infection should be traced as a priority as early as possible . as the low values of t cd4 cell counts and high viral load pre - treatment should be considered to implement the prescribed antiretroviral therapy , especially when new classes are available and have superior performance , as much viral suppression and immune reconstitution could be achieved .

anyway , disease prognosis in hiv infected subjects is more rigorously defined by the combination of plasmatic viral load quantification and t cd4 cell count.1 it is internationally noted that the main predictive factors for failure of antiretroviral therapy are t cd4 cells low count and elevated viral load before commencing treatment.2 the efficacy of the highly active antiretroviral therapy ( haart ) and the pattern of therapy management could be evaluated based on plasmatic rna viral load assessement . in hiv / aids treatment the response is considered successful when the hiv rna levels remain undetectable by certified commercial assays , notwithstanding in a variable proportion of haart submitted patients , the viral replication and evolution goes on which could eventually contribute to the development of antiretroviral drug resistance and therapeutic failure.3 unsuccessful antiretroviral therapy could occur due to virologic and immunologic failure and clinical manifestations that can appear during the course of hiv infection . the identification of therapeutic failure is based on patient follow - up during treatment , taking into account the initial level of t cd4 , plasmatic hiv rna load and the patient clinical evolution . also , many factors could be related to therapeutic failure such as low treatment adhesion , insufficient drug dosage , mal - absorption , hiv antiretroviral resistance and drug interactions that could reduce the efficacy and hiv resistance to antiretroviral medications . this choice is based on the knowledge of previous treatments and yet the reason to remove a drug from the antiretroviral regimen is mainly justified by the in vitro antiretroviral resistance profile . the antiretroviral therapeutic suspension as an alternative therapy is based on the reemergence of the initial virus population before treatment initiation , which would be susceptible to the antiretroviral drugs earlier prescribed , so this proposal is presently under evaluation . the essential objective of treatment in patients presenting therapeutic failure is to maintain t cd4 acceptable cell population density , whilst new therapeutic options are awaited , contrary to the priority aim to reach and keep up an undetectable viral load.4 according to moore et al,5 virological failure is characterized by viral load higher than 400 copies / ml after 48 weeks of initial treatment or among subjects that had complete viral suppression , but later on the viral load will recrudesce . the viral failure precedes the immunologic failure which is defined by a decline of more than 25.0% in the subsequent count of t cd4 lymphocytes or regresion to the initial t cd4 cell count before treatment . these conditions are mainly suggestive of immunologic failure but laboratory analysis confirmation is mandatory.6 cozzi - lepri et al7 concluded that patients remaining in haart failure presented viral load slowly progressing during the next 12 months , differently to t cd4 cell count which remained stable . the impact of haart on t cd4 cell count and viral load of hiv infected patients has been demonstrated to improve the patient s immunological status and it also diminished the viral load , interrupting the aids progression.8 however there are crucial limitations of haart regimen , as it does not eradicate viral infection despite long and permanent antiretroviral therapy . consequently , a significant number of these patients on haart therapy develop viral resistance to the drugs besides diverse side effects including metabolic disorders . therefore new approaches are necessary to control and/or eradicate hiv infection.9,10 this research work aimed to analyze t cd4 cell density and viral load response in hiv infected patients undergoing different therapeutic regimens which failed virologically , and also the associated factors to it during 20022008 in brasilia , federal district , brazil . a cohort study was conducted in the health center number 01 attached to the secretary of health , federal district ( ses / df ) ( centro de sade nmero 01 , secretaria de sade do distrito federal ) which included 139 hiv-1 infected patients . the patients had clinical and laboratory diagnosis as defined by the 1993 aids clinical course criteria and by the us center for disease control ( cdc)11 and also by the recommendations of the brazilian consensus of antiretroviral therapy in infected adults and adolescents by the ministry of health , secretary of health surveillance , brazilian national program of sexually transmitted diseases and aids.12 patients selected for the study were identified in the records of logistic system of the ministry of health , siclom13 utilized by the hospital dia , ses / df . the sampling utilized for patient selection was by convenience whose inclusion criteria were : ( a ) age higher than 18 years old and of both sexes ; ( b ) hiv infection diagnosis defined by the brazillian ministry of health standardized patterns ; ( c ) detectable viral load previous to haart beginning > 400 copies / ml ; ( d ) t cd4 cell count < 500 cells / mm before haart regimen initiation ; ( e ) present laboratory analysis for viral load and t cd4 count between 6 and 12 months in order to compare the former ones with those of baseline ; ( f ) patients have had clinical and immunological follow up during 3 months intervals . if undetectable viral load of 50 copies / ml was not sustained during 24 weeks of treatment or higher than 400 copies / ml after 48 weeks treatment , it was considered virological failure to the initial antiretroviral therapeutic regimen.5 immunological failure was considered by the declining of t cd4 cell counting 25.0% of the absolute values6 or by returning to the t cd4 cell count initial values before antiretroviral therapy initiation . the viral load previous to haart regimen initiation and t cd4 cell count were evaluated as possible predictors of viral failure leading us to establish the cut off values to viral load > 100.000 copies / ml or 100.000 copies / ml as also t cd4 > 200 cells / mm or 200 cells / mm . the antiretroviral therapy regimen were : ( a ) double with two nucleoside reverse transcriptase inhibitor and non nucleoside reverse transcriptase inhibitor ; ( b ) triple with two nucleoside reverse transcriptase inhibitor and one protease inhibitor and ( c ) three nucleoside reverse transcriptase inhibitor , according to cdc classification and the brazilian ministry of health guidelines as previously described . in the population here studied , the chosen antiretroviral regimen was decided by the physician in charge without interference by the authors of this research work . the criteria for the diagnosis of hepatitis b and c virus infection followed the guidelines established by the manual of viral hepatitis consultancy , secretary of health surveillance , department of epidemiological surveillance , braslia , ministry of health14 and also for the tuberculosis diagnosis , the technical manual for tuberculosis control , notebook of basic attention , secretary of health politics , department of basic attention , braslia . ministry of health.15 the exclusion criteria for this research were : ( a ) be pregnant or minor under 18 years old ; ( b ) be prescribed with medications which could metabolically interact with antiretroviral drugs , including phytotherapics ; ( c ) have had last clinical appointment in a period longer than 6 months which was considered lost to follow - up . the analysis of baseline characteristics ( initial data previous to initiation of treatment referring to t cd4 cell count and viral load ) included frequency tables of categorical variables and their descriptive statistics ( median , arithmetic mean and standard deviation ) as also as continuous variables . for the analysis of logistic regression and to predict the event of virological failure16 ( depedent variable ) , the wald method was applied . the patients data were confidential and consentment to utilize information were obtained at the health center number 01 direction according to the federal district secretary ( ses / df ) of health agreement . a cohort study was conducted in the health center number 01 attached to the secretary of health , federal district ( ses / df ) ( centro de sade nmero 01 , secretaria de sade do distrito federal ) which included 139 hiv-1 infected patients . the patients had clinical and laboratory diagnosis as defined by the 1993 aids clinical course criteria and by the us center for disease control ( cdc)11 and also by the recommendations of the brazilian consensus of antiretroviral therapy in infected adults and adolescents by the ministry of health , secretary of health surveillance , brazilian national program of sexually transmitted diseases and aids.12 patients selected for the study were identified in the records of logistic system of the ministry of health , siclom13 utilized by the hospital dia , ses / df . the sampling utilized for patient selection was by convenience whose inclusion criteria were : ( a ) age higher than 18 years old and of both sexes ; ( b ) hiv infection diagnosis defined by the brazillian ministry of health standardized patterns ; ( c ) detectable viral load previous to haart beginning > 400 copies / ml ; ( d ) t cd4 cell count < 500 cells / mm before haart regimen initiation ; ( e ) present laboratory analysis for viral load and t cd4 count between 6 and 12 months in order to compare the former ones with those of baseline ; ( f ) patients have had clinical and immunological follow up during 3 months intervals . if undetectable viral load of 50 copies / ml was not sustained during 24 weeks of treatment or higher than 400 copies / ml after 48 weeks treatment , it was considered virological failure to the initial antiretroviral therapeutic regimen.5 immunological failure was considered by the declining of t cd4 cell counting 25.0% of the absolute values6 or by returning to the t cd4 cell count initial values before antiretroviral therapy initiation . the viral load previous to haart regimen initiation and t cd4 cell count were evaluated as possible predictors of viral failure leading us to establish the cut off values to viral load > 100.000 copies / ml or 100.000 copies / ml as also t cd4 > 200 cells / mm or 200 cells / mm . the antiretroviral therapy regimen were : ( a ) double with two nucleoside reverse transcriptase inhibitor and non nucleoside reverse transcriptase inhibitor ; ( b ) triple with two nucleoside reverse transcriptase inhibitor and one protease inhibitor and ( c ) three nucleoside reverse transcriptase inhibitor , according to cdc classification and the brazilian ministry of health guidelines as previously described . in the population here studied , the chosen antiretroviral regimen was decided by the physician in charge without interference by the authors of this research work . the criteria for the diagnosis of hepatitis b and c virus infection followed the guidelines established by the manual of viral hepatitis consultancy , secretary of health surveillance , department of epidemiological surveillance , braslia , ministry of health14 and also for the tuberculosis diagnosis , the technical manual for tuberculosis control , notebook of basic attention , secretary of health politics , department of basic attention , braslia . ministry of health.15 the exclusion criteria for this research were : ( a ) be pregnant or minor under 18 years old ; ( b ) be prescribed with medications which could metabolically interact with antiretroviral drugs , including phytotherapics ; ( c ) have had last clinical appointment in a period longer than 6 months which was considered lost to follow - up . the analysis of baseline characteristics ( initial data previous to initiation of treatment referring to t cd4 cell count and viral load ) included frequency tables of categorical variables and their descriptive statistics ( median , arithmetic mean and standard deviation ) as also as continuous variables . for the analysis of logistic regression and to predict the event of virological failure16 ( depedent variable ) , the wald method was applied . the patients data were confidential and consentment to utilize information were obtained at the health center number 01 direction according to the federal district secretary ( ses / df ) of health agreement . initially , 165 patients were eligible to participate in the study for starting the treatment in the period 20022008 . there was a slight predominance of patients in the range of 4165 years old ( 51.1% ) compared to younger patients . people living in taguatinga ( 13.7% ) , a satellite city located in the surrounding areas of braslia , represented the majority of the patients enrolled in this study . the place of birth showed that patients predominated from the midwest ( 40.3% ) and the northeast of brazil ( 34.5% ) . the initial viral load ranged between 1.643 copies / ml to 500.000 copies / ml ( mean = 180.721 copies / ml ; arithmetic mean = 126.417 copies / ml ) presenting more than half of the patients ( 55.4% ) cell density higher than 100.000 copies / ml ( table 2 ) . after antiretroviral therapy , the initial profile changed substantially more than 70% of the patients had viral load lower than 50 copies / ml . in reference to the initial t cd4 cell count , 68.3% of the patients were in the range of 0200 cells / mm . after treatment a relevant change was observed in the patients profile concerning t cd4 cell counts , remaining less than 37% of the cases in this stage . in the following cell density interval , there was an expressive increment in the number of cases , representing more than 43.0% of the patients ( table 2 ) . initially the most utilized antiretroviral regimen included two drugs , the nucleoside reverse transcriptase inhibitor and non nucleoside reverse transcriptase inhibitor in 71.2% of the cases and the remaining 28.1% included two nucleoside reverse transcriptase inhibitor and one protease inhibitor . during the course of the treatment , the presented regimen distribution slightly changed to 66.9% to the first drug combination and 32.4% to the second one , there remaining only one utilizing three nucleoside reverse transcriptase inhibitor ( table 2 ) . virologic failure was 12.2% ( table 3 ) , or antiretroviral treatment success was 88.8% , exceeding the results obtained by johnson and way of 80%.17 among the socioeconomic and demographic variables , just the birthplace and the fact that originating in the midwest region which includes the federal district , was associated with the occurrence of virologic failure in the study . perhaps this is a natural social structural marker as the study was conducted in the midwest region compared to those migrating people from other regions to the federal district . there was a trend toward a higher incidence of virologic failure being male , over 40 years old , mestizo , unmarried or separate and also heterossexual exposure category . patients from the midwestern had 2.7 times greater risk of virologic failure than those from other regions of brazil , especially because the study was conducted in brasilia , which is part of this region . in this study , the initial viral load is not statistically associated and significant to the occurrence of virologic failure . however , there is the presence of a higher incidence of virologic failure among those who had viral load greater than 100.000 copies / ml after the introduction of haart . these results highlight the importance of compliance with primary success and reinforces the need to work on the accession of such patients . however , a potent and well tolerated prophylactic regimen with haart can improve cd4 t cell count at the beginning , during and after treatment . some patients with viral load higher than 100.000 copies / ml remained in treatment failure after haart regimen in contrast to t cd4 lymphocyte counts decrease at a slower pace , leaving some in virologic failure , probably due to the emergence of hiv resistant strains . cozzi - lepri et al7 advocate that patients who remained in haart regimen failure , the viral load in the next twelve months was growing at a relatively slow pace , the cd4 t cell count was stable and the time course of viral replication in patients with virological failure had not been fully elucidated . asjo and langeland20 suggest that the lack of complete suppression of viral replication allows the continued development of hiv variants with different degrees of resistance . it results not only in treatment failure , but it also increases the risk of hiv primary resistance and dissemination . it is observed that the t cd4 cell count baseline above 200 cells / mm was not statistically associated with virological failure , but the incidence of failure in this group was noticeably lower than in those with initial t cd4 less than 200 cells / mm . however , in order to achieve viral load suppression to undetectable levels , it is necessary to have an optimal response of t cd4 cell count in patients under antiretroviral therapy . piliero22 comments that the maximum suppression of viral replication remains the primary goal of therapy after haart regimen , therefore t cd4 cell count and hiv plasma viral load values are the prognostic markers for treatment success after four , eight or twelve weeks post treatment , as the changes are predictive of favorable long - term success in six months or more . according to moreno et al23 in multifailed patients , at least two active drugs can not be used , the therapeutic scheme should be kept in use until new drugs become available , assuming that there is an immunologic and clinical stability in order to avoid the use of a drug from a common chemical group which usually leads to a rapid viral resistance development , further limiting future treatment options . in addition , the infection increases the risk of latent tuberculosis reactivation , a new infection progression or re - infection to active disease , increasing the risk of the emergence of hiv resistant strains to the usual antiretroviral therapy . in the logistic regresion analysis the best model to explain the event of virological failure is the one that includes variables such as origin and infection by m. tuberculosis , yielding or values of 3.2 and 3.8 respectively despite the enormous progress as a result of the impact of haart - related morbidity and mortality in patients with hiv / aids continue and will continue to fail in the face of the therapeutic classes of haart currently available . the presented results reinforce the importance of monitoring the biological markers t cd4 cells and viral load in patients living with and without aids , both to ensure that viral replication is under control and to reassure the maintenance of immune reconstitution compatible with life , and to predict the risk of developing resistance and therapeutic failures in the course of the treatment of hiv - infected people . it should be noted that the sample size limited the analysis of this study , as the retrospective information taken from the medical records , excluded some data from the laboratory and the clinical follow up . moreover , the recent availability of new antiretroviral agents for the treatment of hiv has increased treatment options and improved durability , tolerability and efficacy in the long - term haart regimen . other limitations that should be highlighted refer to the possibility of verifying the association of hbv and hcv coinfections which do not have evolutionary studies of these hiv comorbidities . it is possible that if we had a homogeneous group of patients already in advanced stages of these pathologies , the risk of virologic failure would be high . it is of great relevance to the findings in this study considering the limitations and difficulties of conducting studies in routine service , as it can be concluded that even in the non - ideal conditions of a clinical trial , patients treated in this unit had virological success of 12.2% up to one year of treatment , a fact that can predict the durability of the first scheme which is better than expected in many other international centers.17 also tuberculosis infection should be traced as a priority as early as possible . as the low values of t cd4 cell counts and high viral load pre - treatment should be considered to implement the prescribed antiretroviral therapy , especially when new classes are available and have superior performance , as much viral suppression and immune reconstitution could be achieved .

the dichloromethane - soluble extract from roots and rhizomes of n. incisum exerted significant ppar activation in a ppar-driven luciferase reporter gene assay ( 2.5 0.28-fold activation , p < 0.001 ) . fractionation of the extract by several chromatographic separation steps on normal- and reversed- phase silica gel yielded 11 new polyyne derivatives ( 111 ) . notoethers a h ( 18 ) are four pairs of isomeric ethers , each consisting of a falcarindiol unit and a sesquiterpene unit . this is the first report of polyynes fused with sesquiterpenoids , and also the second type of polyacetylene adducts connected through an ether bond , besides reported polyacetylene coumarin adducts . notoincisols a c ( 911 ) are adducts of a polyacetylene and a phenylpropanoid unit , with 10 and 11 representing two new carbon skeletons . the hresims , c nmr , and hsqc data indicated a molecular formula of c32h50o3 . four acetylene carbon signals , at 80.0 , 79.9 , 69.6 , and 69.3 ppm , and four alkene carbons , at 116.1 , 136.3 , 128.1 , and 134.4 , together with a characteristic alkene proton at 5.79 ( ddd , j = 16.9 , 10.2 , 4.7 hz ) and a pair of terminal alkene protons at 5.58 and 5.50 suggested the presence of a falcarindiol unit . the remaining 15 carbon resonances including four tertiary methyl groups indicated an additional sesquiterpene unit . complete assignments of the 1d and 2d nmr signals and comparison with literature data revealed the second part of the molecule to be a 4,11-eudesmane diol . the hmbc correlation between c-4 ( 80.0 ) of the eudesmane and h-3 ( 4.84 ) of the falcarindiol unit indicated an ether linkage between these two parts , which was also supported by a noesy correlation between me-15 ( 1.11 ) of the eudesmane and h-3 ( 4.84 ) of the falcarindiol unit . the relative configuration of the eudesmane diol was determined by analyzing noesy correlations , together with coupling constants from the 1d proton spectrum and cross - peak intensities in the dqf - cosy spectrum . the observed noes , typical for a trans - fused decalin system in a chair conformation , indicated the -orientations of me-14 and me-15. the quadruplet signal of the axial h-6 ( 1.01 ) in combination with the large j coupling ( 12.3 hz ) required axial orientations of both the h-7 and h-5 methine protons . therefore , the relative configuration at the stereogenic centers was determined as depicted . the unsubstituted sesquiterpene , cryptomeridol , has been previously obtained by chemical modification of -eudesmane . the hresims , c nmr , and hsqc data indicated a molecular formula of c32h50o3 . four acetylene and four alkene carbons with chemical shifts like those of compound 1 , as well as the corresponding alkene protons , revealed the presence of a falcarindiol unit . correlations from the 2d nmr spectra confirmed a cryptomeridiol structure . a comparison with reference spectroscopic data obtained for falcarindiol showed different chemical shifts of the carbons centered on c-8 , including upfield shifts of c-8 ( 1.3 ppm ) , c-6 ( 0.9 ppm ) , and c-10 ( 3.1 ppm ) , as well as downfield shifts of c-7 ( 1.8 ppm ) and c-9 ( 0.9 ppm ) . further evidence came from the hmbc correlation between c-4 ( 79.5 ) and h-8 ( 5.01 ) and the noesy correlation between me-15 and h-8 . on the basis of these results , the structure of 2 was elucidated as a falcarindiol unit connected at c-8 via an ether bond to c-4 of cryptomeridiol . the hresims , c nmr , and hsqc data indicated a molecular formula of c32h50o4 . a falcarindiol subunit was identified from four acetylene signals and four alkene carbon signals , as well as from three alkene protons and two oxygenated methine proton ( h-3 , h-8 ) signals . complete nmr resonance assignments revealed that compound 3 consists of a falcarindiol unit attached to a trihydroxyeudesmane moiety . an hmbc correlation between h-3 ( 4.86 ) and c-4 ( 79.3 ) as well as a noesy correlation between h-3 and me-14 indicated that these two parts are connected via an ether bond between c-3 and c-4. the relative configuration of the sesquiterpene unit was determined by analyzing noe and coupling constant data . in contrast , the h-1 , h-5 , and h-7 protons were assigned with an -orientation , which corresponded to these protons with an axial orientation in a trans - decalin structure . the h-5 singlet and the missing cross - peak in the dqf - cosy spectrum for h-6 indicated an equatorial orientation of h-6. therefore , an axial orientation of the hydroxy group at c-6 could be suggested . an acetylated derivative of this sesquiterpene has been isolated but not fully characterized by onorato and co - workers . the hresims , c nmr , and hsqc data indicated a molecular formula of c32h50o4 . a falcarindiol unit was identified by its specific carbon and proton shifts , for which the chemical shift values correlated closely with those of compound 2 and therefore indicated c-8 substitution . assignment of the 2d nmr spectra confirmed the presence of a 1,4,6-trihydroxyeudesmane residue substituted at position c-4. an hmbc correlation between h-8 and c-4 , as well as a noesy correlation between h-8 and me-14 , indicated these two parts to be connected through an ether bond between c-8 and c-4. notoether e ( 5 ) was obtained as colorless needles . the hresims , c nmr , and hsqc data indicated a molecular formula of c32h50o4 . the falcarindiol unit was recognized again by its characteristic set of carbon and proton signals , for which the chemical shift values correlated closely with those of compounds 1 and 3 , and hence indicated a c-3 substitution of the falcarindiol moiety . assignments of the 1d and 2d nmr spectra were used to further identify a 1,4,6-trioxygenated eudesmane unit substituted at c-4. the absence of a noesy correlation between h-5 and the angular me-14 supported the occurrence of a trans - fused a / b ring system . coupling of h-1 ( dd , j = 10.9 , 4.1 hz ) and the noesy correlation between h-5 and h-1 suggested the -orientation of the c-1 hydroxy group . coupling of the bridgehead h-5 ( d , j = 10.9 hz ) with the h-6 of hydroxy methine ( dt , j = 10.4 , 3.3 hz ) revealed their vicinal diaxial relationship and , hence , an equatorial arrangement for the c-6 hydroxy group , which was also confirmed by the noesy correlation observed between me-14 and h-6. a noesy correlation between h-6 and the angular me-15 , as well as between me-14 and me-15 , suggested the c-4 oxy group to be also -oriented . it is noteworthy that the two free hydroxy group proton signals of the sesquiterpenoid moiety showed different patterns . the more low - field signal showed a doublet ( 4.76 , j = 2.6 hz , oh-6 ) , while a singlet could be found at high field ( 2.36 , oh-1 ) . the low - field pattern of 6-oh supported a syn - equatorial configuration for -o-4 and oh-6 , which allows an intramolecular hydrogen bonding that may resist hydrogen exchange with the residual solvent water indicated by a flat oh-1. thus , with one of the two small couplings of h-6 ( 2.6 hz ) assigned to the oh-6 hydroxy group proton , the remaining small coupling of h-6 ( 3.7 hz ) requires a cis relationship to the adjacent h-7 , suggesting the axial orientation of the isopropyl group . the axial -orientation of the isopropyl group was supported also by strong noes between h-11 , h-5 , and h-9 , respectively . when comparing its c-7 chemical shift ( 46.2 ) with the counterpart carbon of zingibertriol ( 51.5 ) , the difference ( = 5.3 ppm ) was close to that between equatorial and axial isopropylcyclohexane ( = 3.4 ppm , 150 c ) . the sesquiterpenoid moiety of compound 5 was therefore identified as the c-7 epimer of zingibertriol . accordingly , compound 5 was elucidated as a falcarindiol unit substituted at c-3 through an ether bond by 7-epizingibertriol ( c-4 ) . the hresims , c nmr , and hsqc data indicated a molecular formula of c32h50o4 . through assignments of the 1d and 2d nmr spectra , a c-8-substituted falcarindiol moiety was identified in the same way as in the case of compounds 2 and 4 . the other unit of this molecule was also found to be a c-4-substituted 7s isomer of zingibertriol . therefore , compound 6 was established as a falcarindiol unit substituted at c-8 through an ether bond by the 7s isomer of zingibertriol ( c-4).figure 1noesy correlations and intramolecular hydrogen bond ( dashed ) of the sesquiterpenoid moiety of 5 and 6 . noesy correlations and intramolecular hydrogen bond ( dashed ) of the sesquiterpenoid moiety of 5 and 6 . the hresims , c nmr , and hsqc data indicated a molecular formula of c32h50o4 . the falcarindiol moiety and its substitution at c-3 were apparent as described for earlier compounds . unlike for compounds 16 , nmr experiments revealed the presence of a different type of sesquiterpenoid . the most obvious difference was that both tertiary methyl groups of compound 7 showed hmbc correlations with three carbons , including one methylene , one methane , and one oxygenated tertiary carbon . assignment of the 1d and 2d nmr data observed and comparison with literature data revealed that the c nmr data were consistent with those previously reported for teuclatriol ( guaiane-4,6,10-triol ) , except for chemical shift changes of c-10 ( = 7.4 ppm ) and its vicinal c-1 ( = 2.1 ppm ) , c-9 ( = 5.3 ppm ) , and c-14 ( = 2.9 ppm ) , which suggested substitution at c-10. two broad singlets ( 2.38 , 2.24 ) , each integrating for one proton , assigned to oh-4 and oh-6 , were observed , with their shapes due to proton exchange . these data , together with hmbc correlation between c-4 and h-3 , as well as the noesy correlation between h-3 and me-14 , were used to establish compound 7 as a falcarindiol unit substituted at c-3 by teuclatriol ( c-10 ) via an ether bond . notoether h ( 8) was obtained as a colorless gum . the hresims , c nmr , and hsqc data indicated a molecular formula of c32h50o4 . 1d and 2d nmr spectroscopic data analysis revealed the presence of a falcarindiol moiety substituted at the c-8 position . the two hydroxy group singlets ( 2.37 , oh-4 ; 2.23 , oh-6 ) exhibited similar chemical shifts to those of 7 . furthermore , their sharp signal shape indicated the absence of any proton exchange . for the same reason , the oh-3 ( 1.91 ) proton of falcarindiol , which was absent in the other compounds isolated , was also observed . in contrast to oh-6 of compounds 5 and 6 , no downfield shift of the hydroxy group proton caused by intramolecular hydrogen bonding was observed , although oh-4 and oh-6 are both syn - equatorial in 7 and 8 . this is due to the distance between the two hydroxy group protons , which is larger in the guaiane skeleton when compared to the eudesmane skeleton . these arguments together with an hmbc correlation between c-4 and h-8 , as well as a noesy correlation between me-14 and h-8 , confirmed 8 as a falcarindiol unit substituted by teuclatriol via a ( c-8)o(c-10 ) ether bond . the hresims , c nmr , and hsqc data indicated a molecular formula of c27h32o5 . the falcarindiol moiety and its substitution at c-8 were identified through assignments of 1d and 2d nmr signals . the proton signals observed included three abx coupled benzene protons at 7.03 ( s ) , 6.92 ( d , j = 8.2 hz ) , and 7.07 ( br d , j = 8.3 hz ) , two trans vinyl protons at 7.65 ( d , j = 15.8 hz ) and 6.28 ( d , j = 15.4 hz ) , and a methoxy singlet at 3.93 . these h nmr data and 10 remaining carbon signals suggested the presence of a ferulic acid ester unit . therefore , compound 9 was elucidated as a falcarindiol moiety esterified at c-8 with ferulic acid . a biogenic pathway to form compound 9 from falcarindiol and ferulic acid is proposed ( figure 2 ) . compound 9 was found to be unstable , as it visibly changed color when exposed to elevated temperatures ( 45 c ) or was kept under direct sunlight at ambient temperature for an extended time period . possible biogenetic pathway for notoincisols a ( 9 ) and c ( 11 ) . notoincisol b ( 10 ) was obtained as a colorless gum . the hresims , c nmr , and hsqc data indicated a molecular formula of c27h32o4 . the h nmr spectrum showed three alkene signals at 6.11 ( m ) , 5.70 ( t , j = 9.4 hz ) , and 5.65 ( m ) , with similar chemical shifts and identical splitting patterns to that of h-2 , h-9 , and h-10 of a falcarindiol unit . the 2d nmr data further revealed two terminal alkene protons at 5.49 ( d , j = 18.0 hz ) and 5.24 ( d , j = 10.7 hz ) , as well as protons of two oxygenated methines at 5.80 ( d , j = 6.1 hz ) and 5.49 ( d , j = 8.7 hz ) , which corresponded to h-1cis , h-1trans , h-3 , and h-8 , respectively . furthermore , a seven - membered aliphatic chain connected to the alkene c-10 was identified . two alkylene carbons at c-6 ( 80.5 ) and c-7 ( 99.0 ) , both showing an hmbc correlation with h-8 , showed significant downfield shifts when compared to model compounds . instead of two alkyne carbons at the c-4 and c-5 positions , two quaternary aromatic carbons were observed , with hmbc correlations to h-3 and weak hmbc correlations to h-8 . the h-3 proton showed hmbc correlations with six carbons , including a pair of olefinic carbons ( c-1 , c-2 ) and a pair of aromatic carbons ( c-4 , c-5 ) , originating from falcarindiol . the other two hmbc correlations were between h-3 and the oxygenated methylene group c-9 and a quaternary aromatic carbon ( c-8 ) . the h-9 proton showed hmbc correlations with the c-7 , c-4 , and c-3 , indicating a five - membered ring . in turn , h-7 showed hmbc correlations with the aromatic carbons c-4 and c-6. the remaining two aromatic proton resonances of h-2 and h-5 were observed as singlets and were both correlated in the hmbc spectrum with the two quaternary aryl carbons c-4 ( 146.5 ) and c-3 ( 147.7 ) , for which the c nmr chemical shift values indicated an ortho - relationship of the phenolic carbons . in addition , h-5 correlated with c-1 and c-3 , while h-2 correlated with c-6 and c-7. the position of a methoxy group was determined by the hmbc correlation of its protons with c-3. analysis of all the aforementioned correlations led to the assignment of compound 10 as depicted , which is a cyclization and oxidation product of a falcarindiol and a hydroxy - methoxy phenyl propane unit . in this molecule , a second aromatic ring is formed , comprising carbons c-4 and c-5 of the falcarindiol and carbons c-7 and c-8 of the phenyl propane unit . this is the first time that this type of carbon skeleton has been described . the hresims , c nmr , and hsqc data indicated a molecular formula of c27h32o4 . the h nmr spectrum showed three alkene signals at 6.10 ( m ) , 5.53 ( t , j = 10.2 hz ) , and 5.74 ( m ) , with similar chemical shifts and identical splitting patterns to that of h-2 , h-9 , and h-10 of a falcarindiol unit . a 2d nmr experiment further revealed two terminal alkene protons at 5.57 ( d , j = 17.5 hz ) and 5.30 ( d , j = 10.1 hz ) , as well as protons of two oxygenated methines at 5.18 ( br s ) and 6.13 ( d , j = 9.2 hz ) , which correspond to h-1cis , h-1trans , h-3 , and h-8 , respectively . also identified was a seven - membered aliphatic chain connected to alkene c-10 . a major downfield shift occurred for c-8 , and two alkylene carbons at c-4 ( 97.2 ) and c-5 ( 81.9 ) , which both showed hmbc correlations with h-3 , experienced considerable downfield shift when compared to model compounds . instead of two alkyne carbons , which are supposed to be c-6 and c-7 , two quaternary aromatic carbons were apparent , which both showed hmbc correlations with h-8 and weak hmbc correlations with h-3 . as in the case of compound 10 , these data suggested a conversion of the alkyne carbons c-6 and c-7 into aromatic ones . c-7 showed hmbc correlations with three protons , including one aryl proton ( h-7 ) , one oxygenated methylene ( h-9 ) , and one oxygenated methane ( h-8 ) . the h-9 signal gave hmbc correlations with four carbons , including one now aromatic carbon ( c-7 ) of falcarindiol origin , an oxygenated methine ( c-8 ) , a quaternary aromatic carbon ( c-8 ) , and an unsubstituted aromatic carbon ( c-7 ) , indicating a five - membered ring , which resembled that of compound 10 . in turn , h-7 showed hmbc correlations with the aromatic carbons c-2 , c-6 , c-7 , and oxygenated methylene c-9. the remaining two aromatic proton resonances , h-2 and h-5 , gave clear singlet structures , and both showed hmbc correlations with the two quaternary carbons c-4 ( 146.5 ) and c-3 ( 147.7 ) , for which the c nmr chemical shift values indicated an ortho - position of the phenolic carbons . in addition , h-5 correlated with c-1 and c-3 , while h-2 correlated with c-6 and c-7. the position of a methoxy group was determined by the hmbc correlation of its protons with c-3. the aforementioned correlations led to the structure of compound 11 as depicted . like compound 10 , this is also a cyclization and oxidation product of a falcarindiol and a hydroxy - methoxy phenyl propane unit . a biogenic pathway to form compound 11 from falcarindiol and ferulic acid via compound 9 is proposed ( figure 2 ) . notoethers a c ( 13 ) , notoincisol a ( 9 ) , and notoincisol b ( 10 ) were the most potent partial ppar agonists among the tested compounds , with ec50 values ranging from 1.7 to 2.3 m and a maximal fold activation ranging from 1.6 to 2.8 ( see table 5 for comparison of the ec50 values and maximal fold activation induced by 13 , 9 , and 10 , as well as figure 3 for comparison of the effectiveness of all tested compounds at 10 m ) . for comparison , the full ppar agonist pioglitazone used as positive control activated 6.6-fold at 5 m ( figure 3 ) with an ec50 value of 0.21 m ( not shown ) . ( * * * p 0.001 , n.s . : not significant ; n = 3 , anova ) . in a previous study , molecular details for the ppar binding mode of falcarindiol falcarindiol was observed to occupy parts of the entrance region of the ppar ligand binding domain and established interactions with the mainly hydrophobic binding site arms i and ii . since the active compounds from this study have some structural features in common with falcarindiol but are significantly larger , it was investigated how the proposed binding mode would shift within the binding site and if the docking could distinguish between active and inactive polyyne hybrid compounds . in general , the ppar ligand binding domain is y - shaped and is divided into three parts : the entrance domain , arm i , and arm ii ( figure 4 ) . while the ppar ligand binding site entrance is lined by several polar residues ( e.g. , arg288 , glu291 , glu295 , glu343 ) , the two branches of the binding pocket arm i and arm ii are formed by mainly hydrophobic amino acids . arm i , however , accommodates some moderately hydrophobic residues ( e.g. , cys285 , ser289 , his323 , his449 , and tyr473 ) . falcarindiol - type polyacetylenes were supposed to form hydrophobic contacts with residues of arm i ( e.g. , ile326 , tyr327 , phe363 ) , arm ii ( e.g. , ile281 ) , and the entrance region ( e.g. , ala292 , met329 , leu330 , and leu333 ) . the hydroxy groups formed hydrogen bonds with the backbone amide of cys285 in arm i and the carboxy group of glu295 at the entrance . amino acids from the entrance region are marked in green , from arm i in blue , and from arm ii in pink . analyzing the protein ligand interactions of all active compounds and comparing them to the ones of the inactive molecules revealed that hydrogen bonding with ser289 was associated with the activity ( figure 5 ) . while all active compounds formed this hydrogen bond in their best - ranked docking pose , none of the inactive structures did so . ser289 is one amino acid in the core of the ligand binding site , where it is involved in the molecular recognition of many ppar agonists , as observed in x - ray crystal structures . the docking results rationalized the observed in vitro ppar activation by compounds 1 , 2 , 3 , 9 , and 10 and could serve as an inspiration for synthetic optimization to develop more potent partial agonists . representative docking poses of compounds 2 ( a ) and 10 ( b ) fitted into the ppar ligand binding site . both compounds occupied parts of the entrance region ( green ) and extend into one of the hydrophobic arms ( blue and pink ) with their long alkyl chains . the hydrogen bond with ser289 , which was only observed with the active compounds 1 , 2 , 3 , 9 , and 10 , is depicted as a green arrow . hydrophobic contacts with the binding site are shown as yellow spheres ; those with the hydrogen bonds , as arrows . the new polyacetylene derivatives were tested for inhibition of no production in stimulated raw 264.7 macrophages . while most of the compounds were inactive at the concentrations tested ( data not shown ) , notoincisol a ( 9 ) inhibited no production with an ic50 value ( 14.6 0.7 m ) comparable to those of polyacetylenes previously isolated from n. incisum ( around 10 to 30 m ) . the search for constituents with ppar agonistic activity of n. incisum led to the identification of active novel polyacetylene adducts ( 1 , 2 , 3 , 9 , and 10 ) . their preliminary structure notoincisol a ( 9 ) was also able to inhibit no production in lps - induced raw 264.7 macrophages . these compounds can serve as starting points for chemical modifications in order to optimize potency , selectivity , safety , and pharmacokinetic parameters , thereby offering new scaffolds for the development of compounds to treat inflammatory processes and possibly the related metabolic syndrome . melting points were determined with a reichert melting point apparatus and are uncorrected . h , c , and 2d nmr spectra were recorded in cdcl3 on unity 600 ( varian , palo alto , ca , usa ) and avance 700 ( bruker , billerica , ma , usa ) spectrometers . lc - esims were carried out using a thermo finnigan lcq deca xp plus mass spectrometer connected to a surveyor hplc system ( thermo fisher , waltham , ma , usa ) , with a zorbax sb - c18 narrow bore ( 3.5 m ) 2.1 150 mm column ( agilent , santa clara , ca , usa ) . open column chromatography was carried out using mci ch - p 20p resin ( mitsubishi chemical , tokyo , japan ) , octadecyl silica gel ( 2540 m , fuji silysia , kasugai , japan ) , sephadex lh-20 ( ge healthcare , little chalfont , uk ) , and silica gel ( 1540 m , merck kgaa , darmstadt , germany ) as stationary phase . tlc was conducted on silica gel 60 f254 and silica gel 60 rp-18 f254s plates ( merck kgaa ) . all chemical reagents ( ar grade ) were purchased from carl roth gmbh + co. kg ( karlsruhe , germany ) . accurate mass determinations were performed using an lc / ftms system consisting of an exactive orbitrap mass spectrometer , equipped with a heated esi ii source ( thermo fisher ) and operated in ultra - high - resolution mode ( 100.000 ) coupled to a u - hplc system ( thermo fisher ) . operating conditions for the esi source used in the positive ionization mode were as follows : 3.5 kv spray voltage , 325 c capillary temperature , 300 c heater temperature , sheath gas flow rate 45 units , and auxiliary gas flow 10 units ( units refer to arbitrary values set by the exactive software ) . u - hplc separations were performed on a hypersil gold c18 ( thermo fisher ) , 1.9 m , 2.1 50 mm i.d . each 10 min chromatographic run was carried out at a flow rate of 0.3 ml / min with a binary mobile phase consisting of acetonitrile ( a ) and 0.1% formic acid ( b ) using a step gradient profile of 50% a for 0.5 min and increased up to 100% a in 5 min , kept isocratic at 100% for 0.5 min , then decreased down to 50% a in 0.1 min . after re - equilibration at 50% a for 3.9 min , the next sample was injected . rhizoma et radix notopterygii ( 2 kg ) were purchased in 2008 from plantasia , oberndorf , austria , and authenticated as notopterygium incisum via dna - based identification . a voucher specimen ( no . 650107 ) has been deposited at the department of pharmacognosy , institute of pharmaceutical sciences , karl - franzens - university graz . the plant material ( 2 kg ) was ground and percolated with ch2cl2 ( 17.5 l ) , to produce 250 g of an extract . then , 145 g of the extract was partitioned twice between n - hexane and meoh ( 1.5:1 ) , to obtain n - hexane- ( 39 g ) and meoh - soluble ( 104 g ) portions . water solution ( 1:1 ) to obtain a ch2cl2 layer ( 94 g ) and an aqueous meoh layer ( 1.5 g ) . the dried ch2cl2 layer ( 94 g ) was fractionated using mci chp-20p resin , with a meoh h2o gradient ( 40% to 100% meoh ) as mobile phase , to afford 313 fractions . to facilitate subsequent biological testing , 0.3% of each of these fractions was sampled and recombined according to their tlc profile to afford 10 pooled fractions ( fraction pools first to 10th ) , which were assayed together with the ch2cl2 layer for ppar activation . the most active fraction pools , 8 and 9 ( eluting with 6590% meoh ) , were subjected to successive column chromatography , including rp-18 ( meoh h2o , 85:15 ) , sephadex lh-20 ( meoh h2o , 60:40 ) , silica gel ( n - hexane ethyl acetate , 3:1 or 2.2:1 ) , and preparative hplc ( acetonitrile h2o , 68:32 , 80:20 , or 95:5 ) , to afford 11 compounds : 1 ( 6.3 mg ) , 2 ( 11.2 mg ) , 3 ( 1.5 mg ) , 4 ( 1.1 mg ) , 5 ( 10.9 mg ) , 6 ( 9.6 mg ) , 7 ( 3.6 mg ) , 8 ( 2.3 mg ) , 9 ( 4.7 mg ) , 10 ( 3.1 mg ) , and 11 ( 0.68 mg ) . c32h50o3 , colorless oil ; [ ]d20 + 155.1 ( c 0.2 , meoh ) ; uv ( mecn h2o ) max 246 , 260 nm ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 150 mhz , cdcl3 ) data , see tables 1 and 2 ; positive esims m / z 505 [ m + na ] , 987 [ 2 m + na ] ; hresims m / z 505.3652 [ m + na ] ( calcd for c32h50o3na , 505.3658 ) . signals are interchangeable . c32h50o3 , colorless oil ; [ ]d20 + 68.4 ( c 0.4 , meoh ) ; uv ( mecn h2o ) max 246 , 260 nm ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 150 mhz , cdcl3 ) data , see tables 1 and 2 ; positive esims m / z 505 [ m + na ] ; hresims m / z 505.3652 [ m + na ] ( calcd for c32h50o3na , 505.3658 ) . c32h50o4 , colorless gum ; [ ]d20 + 155.3 ( c 0.05 , meoh ) ; uv ( mecn h2o ) max 246 , 260 nm ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 150 mhz , cdcl3 ) data , see tables 1 and 2 ; positive esims m / z 499 [ m + h ] , 997 [ 2 m + h ] ; hresims m / z 521.3607 [ m + na ] ( calcd for c32h50o4na , 521.3607 ) . c32h50o4 , colorless gum ; [ ]d20 + 8.6 ( c 0.04 , meoh ) ; uv ( mecn h2o ) max 246 , 260 nm ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 150 mhz , cdcl3 ) data , see tables 1 and 2 ; positive esims m / z 521 [ m + na ] ; hresims m / z 521.3602 [ m + na ] ( calcd for c32h50o4na , 521.3607 ) . c32h50o4 , colorless needles , mp : 9598 c ; [ ]d20 + 144.9 ( c 0.4 , meoh ) ; uv ( mecn h2o ) max 246 , 260 nm ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 150 mhz , cdcl3 ) data , see tables 1 and 3 ; positive esims m / z 521 [ m + na ] , 1019 [ 2 m + na ] ; hresims m / z 521.3600 [ m + na ] ( calcd for c32h50o4na , 521.3607 ) . signals are interchangeable . c32h50o4 , colorless gum ; [ ]d20 + 108.9 ( c 0.3 , meoh ) ; uv ( mecn h2o ) max 246 , 260 nm ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 150 mhz , cdcl3 ) data , see tables 1 and 3 ; positive esims m / z 499 [ m + h ] , 997 [ 2 m + h ] , 1019 [ 2 m + na ] ; hresims m / z 521.3600 [ m + na ] ( calcd for c32h50o4na , 521.3607 ) . c32h50o4 , colorless gum ; [ ]d20 + 148.5 ( c 0.07 , meoh ) ; uv ( mecn / h2o ) max 246 , 260 nm ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 150 mhz , cdcl3 ) data , see tables 1 and 3 ; positive esims m / z 521 [ m + na ] ; hresims m / z 521.3600 [ m + na ] ( calcd for c32h50o4na , 521.3607 ) . c32h50o4 , colorless gum ; [ ]d20 + 34.8 ( c 0.08 , meoh ) ; uv ( mecn h2o ) max 246 , 260 nm ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 150 mhz , cdcl3 ) data , see tables 1 and 3 ; positive esims m / z 521 [ m + na ] ; hresims m / z 521.3600 [ m + na ] ( calcd for c32h50o4na , 521.3607 ) . c27h32o5 , light green oil ; [ ]d20 + 85.5 ( c 0.09 , meoh ) ; uv ( mecn h2o ) max 237 , 328 nm ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 150 mhz , cdcl3 ) data , see tables 1 and 4 ; positive esims m / z 437 [ m + h ] ; hresims m / z 437.2353 [ m + h ] ( calcd for c27h33o5 , 436.2328 ) . signals are interchangeable . c27h32o4 , colorless gum ; [ ]d20 + 268.9 ( c 0.1 , meoh ) ; uv ( mecn h2o ) max 244 , 315 , 346 ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 150 mhz , cdcl3 ) data , see tables 1 and 4 ; positive esims m / z 421 [ m + h ] ; hresims m / z 421.2370 [ m + h ] ( calcd for c27h33o4 , 421.2379 ) . c27h32o4 , colorless gum ; [ ]d20 + 25.8 ( c 0.03 , meoh ) ; uv ( mecn h2o ) max 244 , 316 , 346 ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 150 mhz , cdcl3 ) data , see tables 1 and 4 ; positive esims m / z 421 [ m + h ] ; hresims m / z 421.2370 [ m + h ] ( calcd for c27h33o4 , 421.2379 ) . dulbecco s modified eagle s medium ( dmem ) and fetal calf serum ( fcs ) were supplied by lonza ( basel , switzerland ) . pioglitazone , a full ppar agonist , was used as positive control and was purchased from molekula ltd . for evaluation of ppar activation the test compounds were first dissolved in dmso , divided into aliquots , and frozen at 20 c until used . in all experiments , dmso was applied as solvent control , and the final concentration of dmso was always kept at 0.1% . the ppar luciferase reporter plasmid ( tk - pprex3-luc ) was a kind gift from prof . ronald m. evans ( howard hughes medical institute , la jolla , ca , usa ) , the plasmid encoding enhanced green fluorescent protein ( pegfp - n1 ) was supplied by clontech ( mountain view , ca , usa ) , and the plasmid encoding human ppar ( psg5-pl - hppar-1 ) was kindly supplied by prof . beatrice desvergne ( center for integrative genomics , university of lausanne , switzerland ) . briefly , hek-293 cells ( atcc , manassas , va , usa ) were grown in dmem with 2 mm glutamine , 10% fbs , 100 u / ml benzylpenicillin , and 100 g / ml streptomycin . cells were grown in 10 cm dishes ( 6 10 cells / dish ) for 18 h and then transfected by the calcium phosphate precipitation method with 4 g of plasmid encoding human ppar ( psg5-pl - hppar-1 ) , 4 g of reporter plasmid ( tk - pprex3-luc ) , and 2 g of pegfp - n1 as control for internal normalization . after 6 h , the cells were transferred to 96-well plates ( 5 10 cells / well ) in dmem without phenol red supplemented with charcoal - stripped fbs , glutamine , and antibiotics . the cells were treated with different concentrations of the indicated compounds and incubated for 18 h. cells were then lysed , and the luminescence of the firefly luciferase and the fluorescence of egfp were quantified with a geniospro microplate reader ( tecan , grdig , austria ) . the luminescence was finally normalized to the egfp - derived fluorescence from each well to account for differences in transfection efficiency or cell number . raw 264.7 macrophages were stimulated with lipopolysaccharides ( lps ) and interferon- ( ifn ) for induction of inos gene expression . the effects on no production were determined by photometric quantification of nitrite accumulation in cell culture supernatants using the griess assay compared with a sodium nitrite standard curve after 16 h of incubation with the respective sample as described by baer et al . with slight modifications . activity is referred to nitrite accumulation of cells treated with lps / ifn-/dmso ( final concentration of 0.1% dmso serves as solvent control ) . test compounds were first dissolved in dmso and then diluted with pbs to obtain respective concentrations . l - nmma ( n - monomethyl - l - arginine ) , a known nonselective inhibitor of all nos isoforms , was used as a positive control . ic50 determination was performed in eight concentrations , each in at least three independent experiments , every time in duplicate . statistical analyses for effects on ppar were performed with the graphpad prism software version 4.03 . ( with sigmoidal dose response ) was used to calculate the ec50 values and maximal fold activation . ic50 values for effects on no production were calculated with the sigmaplot program package employing the four - parameter logistic regression model . the prediction of binding modes for the investigated compounds was accomplished in a docking study . a quantum mechanics - polarized ligand docking ( qpld ) workflow available within maestro version 9.2.112 ( schrdinger , llc , new york , ny , 2011,www.schrodinger.com ) was applied as described previously . for each molecule , up to 10 docking poses were calculated . for further investigations , the highest ranked docking solution as determined by the emodel scoring function was used . visual inspection and analysis of the retrieved binding poses were performed with ligandscout 3.1 ( inte : ligand gmbh , maria enzersdorf , austria , 2012 , www.inteligand.com ) .

in the search for peroxisome proliferator - activated receptor gamma ( ppar ) active constituents from the roots and rhizomes of notopterygium incisum , 11 new polyacetylene derivatives ( 111 ) were isolated . their structures were elucidated by nmr and hresims as new polyyne hybrid molecules of falcarindiol with sesquiterpenoid or phenylpropanoid moieties , named notoethers a h ( 18 ) and notoincisols a c ( 911 ) , respectively . notoincisol b ( 10 ) and notoincisol c ( 11 ) represent two new carbon skeletons . when tested for ppar activation in a luciferase reporter assay with hek-293 cells , notoethers a c ( 13 ) , notoincisol a ( 9 ) , and notoincisol b ( 10 ) showed promising agonistic activity ( ec50 values of 1.7 to 2.3 m ) . in addition , notoincisol a ( 9 ) exhibited inhibitory activity on no production of stimulated raw 264.7 macrophages .

Results and Discussion Experimental Section

the dichloromethane - soluble extract from roots and rhizomes of n. incisum exerted significant ppar activation in a ppar-driven luciferase reporter gene assay ( 2.5 0.28-fold activation , p < 0.001 ) . fractionation of the extract by several chromatographic separation steps on normal- and reversed- phase silica gel yielded 11 new polyyne derivatives ( 111 ) . notoethers a h ( 18 ) are four pairs of isomeric ethers , each consisting of a falcarindiol unit and a sesquiterpene unit . notoincisols a c ( 911 ) are adducts of a polyacetylene and a phenylpropanoid unit , with 10 and 11 representing two new carbon skeletons . four acetylene carbon signals , at 80.0 , 79.9 , 69.6 , and 69.3 ppm , and four alkene carbons , at 116.1 , 136.3 , 128.1 , and 134.4 , together with a characteristic alkene proton at 5.79 ( ddd , j = 16.9 , 10.2 , 4.7 hz ) and a pair of terminal alkene protons at 5.58 and 5.50 suggested the presence of a falcarindiol unit . the hmbc correlation between c-4 ( 80.0 ) of the eudesmane and h-3 ( 4.84 ) of the falcarindiol unit indicated an ether linkage between these two parts , which was also supported by a noesy correlation between me-15 ( 1.11 ) of the eudesmane and h-3 ( 4.84 ) of the falcarindiol unit . the relative configuration of the eudesmane diol was determined by analyzing noesy correlations , together with coupling constants from the 1d proton spectrum and cross - peak intensities in the dqf - cosy spectrum . correlations from the 2d nmr spectra confirmed a cryptomeridiol structure . a comparison with reference spectroscopic data obtained for falcarindiol showed different chemical shifts of the carbons centered on c-8 , including upfield shifts of c-8 ( 1.3 ppm ) , c-6 ( 0.9 ppm ) , and c-10 ( 3.1 ppm ) , as well as downfield shifts of c-7 ( 1.8 ppm ) and c-9 ( 0.9 ppm ) . further evidence came from the hmbc correlation between c-4 ( 79.5 ) and h-8 ( 5.01 ) and the noesy correlation between me-15 and h-8 . in contrast , the h-1 , h-5 , and h-7 protons were assigned with an -orientation , which corresponded to these protons with an axial orientation in a trans - decalin structure . the axial -orientation of the isopropyl group was supported also by strong noes between h-11 , h-5 , and h-9 , respectively . the hresims , c nmr , and hsqc data indicated a molecular formula of c32h50o4 . assignment of the 1d and 2d nmr data observed and comparison with literature data revealed that the c nmr data were consistent with those previously reported for teuclatriol ( guaiane-4,6,10-triol ) , except for chemical shift changes of c-10 ( = 7.4 ppm ) and its vicinal c-1 ( = 2.1 ppm ) , c-9 ( = 5.3 ppm ) , and c-14 ( = 2.9 ppm ) , which suggested substitution at c-10. the hresims , c nmr , and hsqc data indicated a molecular formula of c32h50o4 . for the same reason , the oh-3 ( 1.91 ) proton of falcarindiol , which was absent in the other compounds isolated , was also observed . the proton signals observed included three abx coupled benzene protons at 7.03 ( s ) , 6.92 ( d , j = 8.2 hz ) , and 7.07 ( br d , j = 8.3 hz ) , two trans vinyl protons at 7.65 ( d , j = 15.8 hz ) and 6.28 ( d , j = 15.4 hz ) , and a methoxy singlet at 3.93 . possible biogenetic pathway for notoincisols a ( 9 ) and c ( 11 ) . notoincisol b ( 10 ) was obtained as a colorless gum . the h nmr spectrum showed three alkene signals at 6.11 ( m ) , 5.70 ( t , j = 9.4 hz ) , and 5.65 ( m ) , with similar chemical shifts and identical splitting patterns to that of h-2 , h-9 , and h-10 of a falcarindiol unit . the 2d nmr data further revealed two terminal alkene protons at 5.49 ( d , j = 18.0 hz ) and 5.24 ( d , j = 10.7 hz ) , as well as protons of two oxygenated methines at 5.80 ( d , j = 6.1 hz ) and 5.49 ( d , j = 8.7 hz ) , which corresponded to h-1cis , h-1trans , h-3 , and h-8 , respectively . two alkylene carbons at c-6 ( 80.5 ) and c-7 ( 99.0 ) , both showing an hmbc correlation with h-8 , showed significant downfield shifts when compared to model compounds . the h-3 proton showed hmbc correlations with six carbons , including a pair of olefinic carbons ( c-1 , c-2 ) and a pair of aromatic carbons ( c-4 , c-5 ) , originating from falcarindiol . the remaining two aromatic proton resonances of h-2 and h-5 were observed as singlets and were both correlated in the hmbc spectrum with the two quaternary aryl carbons c-4 ( 146.5 ) and c-3 ( 147.7 ) , for which the c nmr chemical shift values indicated an ortho - relationship of the phenolic carbons . in addition , h-5 correlated with c-1 and c-3 , while h-2 correlated with c-6 and c-7. the h nmr spectrum showed three alkene signals at 6.10 ( m ) , 5.53 ( t , j = 10.2 hz ) , and 5.74 ( m ) , with similar chemical shifts and identical splitting patterns to that of h-2 , h-9 , and h-10 of a falcarindiol unit . a 2d nmr experiment further revealed two terminal alkene protons at 5.57 ( d , j = 17.5 hz ) and 5.30 ( d , j = 10.1 hz ) , as well as protons of two oxygenated methines at 5.18 ( br s ) and 6.13 ( d , j = 9.2 hz ) , which correspond to h-1cis , h-1trans , h-3 , and h-8 , respectively . a major downfield shift occurred for c-8 , and two alkylene carbons at c-4 ( 97.2 ) and c-5 ( 81.9 ) , which both showed hmbc correlations with h-3 , experienced considerable downfield shift when compared to model compounds . c-7 showed hmbc correlations with three protons , including one aryl proton ( h-7 ) , one oxygenated methylene ( h-9 ) , and one oxygenated methane ( h-8 ) . the h-9 signal gave hmbc correlations with four carbons , including one now aromatic carbon ( c-7 ) of falcarindiol origin , an oxygenated methine ( c-8 ) , a quaternary aromatic carbon ( c-8 ) , and an unsubstituted aromatic carbon ( c-7 ) , indicating a five - membered ring , which resembled that of compound 10 . the remaining two aromatic proton resonances , h-2 and h-5 , gave clear singlet structures , and both showed hmbc correlations with the two quaternary carbons c-4 ( 146.5 ) and c-3 ( 147.7 ) , for which the c nmr chemical shift values indicated an ortho - position of the phenolic carbons . in addition , h-5 correlated with c-1 and c-3 , while h-2 correlated with c-6 and c-7. notoethers a c ( 13 ) , notoincisol a ( 9 ) , and notoincisol b ( 10 ) were the most potent partial ppar agonists among the tested compounds , with ec50 values ranging from 1.7 to 2.3 m and a maximal fold activation ranging from 1.6 to 2.8 ( see table 5 for comparison of the ec50 values and maximal fold activation induced by 13 , 9 , and 10 , as well as figure 3 for comparison of the effectiveness of all tested compounds at 10 m ) . in a previous study , molecular details for the ppar binding mode of falcarindiol falcarindiol was observed to occupy parts of the entrance region of the ppar ligand binding domain and established interactions with the mainly hydrophobic binding site arms i and ii . in general , the ppar ligand binding domain is y - shaped and is divided into three parts : the entrance domain , arm i , and arm ii ( figure 4 ) . , ile281 ) , and the entrance region ( e.g. amino acids from the entrance region are marked in green , from arm i in blue , and from arm ii in pink . the docking results rationalized the observed in vitro ppar activation by compounds 1 , 2 , 3 , 9 , and 10 and could serve as an inspiration for synthetic optimization to develop more potent partial agonists . the new polyacetylene derivatives were tested for inhibition of no production in stimulated raw 264.7 macrophages . while most of the compounds were inactive at the concentrations tested ( data not shown ) , notoincisol a ( 9 ) inhibited no production with an ic50 value ( 14.6 0.7 m ) comparable to those of polyacetylenes previously isolated from n. incisum ( around 10 to 30 m ) . the search for constituents with ppar agonistic activity of n. incisum led to the identification of active novel polyacetylene adducts ( 1 , 2 , 3 , 9 , and 10 ) . their preliminary structure notoincisol a ( 9 ) was also able to inhibit no production in lps - induced raw 264.7 macrophages . h , c , and 2d nmr spectra were recorded in cdcl3 on unity 600 ( varian , palo alto , ca , usa ) and avance 700 ( bruker , billerica , ma , usa ) spectrometers . lc - esims were carried out using a thermo finnigan lcq deca xp plus mass spectrometer connected to a surveyor hplc system ( thermo fisher , waltham , ma , usa ) , with a zorbax sb - c18 narrow bore ( 3.5 m ) 2.1 150 mm column ( agilent , santa clara , ca , usa ) . open column chromatography was carried out using mci ch - p 20p resin ( mitsubishi chemical , tokyo , japan ) , octadecyl silica gel ( 2540 m , fuji silysia , kasugai , japan ) , sephadex lh-20 ( ge healthcare , little chalfont , uk ) , and silica gel ( 1540 m , merck kgaa , darmstadt , germany ) as stationary phase . operating conditions for the esi source used in the positive ionization mode were as follows : 3.5 kv spray voltage , 325 c capillary temperature , 300 c heater temperature , sheath gas flow rate 45 units , and auxiliary gas flow 10 units ( units refer to arbitrary values set by the exactive software ) . u - hplc separations were performed on a hypersil gold c18 ( thermo fisher ) , 1.9 m , 2.1 50 mm i.d . rhizoma et radix notopterygii ( 2 kg ) were purchased in 2008 from plantasia , oberndorf , austria , and authenticated as notopterygium incisum via dna - based identification . then , 145 g of the extract was partitioned twice between n - hexane and meoh ( 1.5:1 ) , to obtain n - hexane- ( 39 g ) and meoh - soluble ( 104 g ) portions . to facilitate subsequent biological testing , 0.3% of each of these fractions was sampled and recombined according to their tlc profile to afford 10 pooled fractions ( fraction pools first to 10th ) , which were assayed together with the ch2cl2 layer for ppar activation . the most active fraction pools , 8 and 9 ( eluting with 6590% meoh ) , were subjected to successive column chromatography , including rp-18 ( meoh h2o , 85:15 ) , sephadex lh-20 ( meoh h2o , 60:40 ) , silica gel ( n - hexane ethyl acetate , 3:1 or 2.2:1 ) , and preparative hplc ( acetonitrile h2o , 68:32 , 80:20 , or 95:5 ) , to afford 11 compounds : 1 ( 6.3 mg ) , 2 ( 11.2 mg ) , 3 ( 1.5 mg ) , 4 ( 1.1 mg ) , 5 ( 10.9 mg ) , 6 ( 9.6 mg ) , 7 ( 3.6 mg ) , 8 ( 2.3 mg ) , 9 ( 4.7 mg ) , 10 ( 3.1 mg ) , and 11 ( 0.68 mg ) . c32h50o4 , colorless gum ; [ ]d20 + 108.9 ( c 0.3 , meoh ) ; uv ( mecn h2o ) max 246 , 260 nm ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 150 mhz , cdcl3 ) data , see tables 1 and 3 ; positive esims m / z 499 [ m + h ] , 997 [ 2 m + h ] , 1019 [ 2 m + na ] ; hresims m / z 521.3600 [ m + na ] ( calcd for c32h50o4na , 521.3607 ) . c27h32o4 , colorless gum ; [ ]d20 + 268.9 ( c 0.1 , meoh ) ; uv ( mecn h2o ) max 244 , 315 , 346 ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 150 mhz , cdcl3 ) data , see tables 1 and 4 ; positive esims m / z 421 [ m + h ] ; hresims m / z 421.2370 [ m + h ] ( calcd for c27h33o4 , 421.2379 ) . dulbecco s modified eagle s medium ( dmem ) and fetal calf serum ( fcs ) were supplied by lonza ( basel , switzerland ) . for evaluation of ppar activation the test compounds were first dissolved in dmso , divided into aliquots , and frozen at 20 c until used . ronald m. evans ( howard hughes medical institute , la jolla , ca , usa ) , the plasmid encoding enhanced green fluorescent protein ( pegfp - n1 ) was supplied by clontech ( mountain view , ca , usa ) , and the plasmid encoding human ppar ( psg5-pl - hppar-1 ) was kindly supplied by prof . briefly , hek-293 cells ( atcc , manassas , va , usa ) were grown in dmem with 2 mm glutamine , 10% fbs , 100 u / ml benzylpenicillin , and 100 g / ml streptomycin . cells were grown in 10 cm dishes ( 6 10 cells / dish ) for 18 h and then transfected by the calcium phosphate precipitation method with 4 g of plasmid encoding human ppar ( psg5-pl - hppar-1 ) , 4 g of reporter plasmid ( tk - pprex3-luc ) , and 2 g of pegfp - n1 as control for internal normalization . after 6 h , the cells were transferred to 96-well plates ( 5 10 cells / well ) in dmem without phenol red supplemented with charcoal - stripped fbs , glutamine , and antibiotics . raw 264.7 macrophages were stimulated with lipopolysaccharides ( lps ) and interferon- ( ifn ) for induction of inos gene expression . the effects on no production were determined by photometric quantification of nitrite accumulation in cell culture supernatants using the griess assay compared with a sodium nitrite standard curve after 16 h of incubation with the respective sample as described by baer et al . ic50 values for effects on no production were calculated with the sigmaplot program package employing the four - parameter logistic regression model .

the dichloromethane - soluble extract from roots and rhizomes of n. incisum exerted significant ppar activation in a ppar-driven luciferase reporter gene assay ( 2.5 0.28-fold activation , p < 0.001 ) . fractionation of the extract by several chromatographic separation steps on normal- and reversed- phase silica gel yielded 11 new polyyne derivatives ( 111 ) . this is the first report of polyynes fused with sesquiterpenoids , and also the second type of polyacetylene adducts connected through an ether bond , besides reported polyacetylene coumarin adducts . four acetylene carbon signals , at 80.0 , 79.9 , 69.6 , and 69.3 ppm , and four alkene carbons , at 116.1 , 136.3 , 128.1 , and 134.4 , together with a characteristic alkene proton at 5.79 ( ddd , j = 16.9 , 10.2 , 4.7 hz ) and a pair of terminal alkene protons at 5.58 and 5.50 suggested the presence of a falcarindiol unit . complete assignments of the 1d and 2d nmr signals and comparison with literature data revealed the second part of the molecule to be a 4,11-eudesmane diol . the hmbc correlation between c-4 ( 80.0 ) of the eudesmane and h-3 ( 4.84 ) of the falcarindiol unit indicated an ether linkage between these two parts , which was also supported by a noesy correlation between me-15 ( 1.11 ) of the eudesmane and h-3 ( 4.84 ) of the falcarindiol unit . the relative configuration of the eudesmane diol was determined by analyzing noesy correlations , together with coupling constants from the 1d proton spectrum and cross - peak intensities in the dqf - cosy spectrum . the observed noes , typical for a trans - fused decalin system in a chair conformation , indicated the -orientations of me-14 and me-15. the quadruplet signal of the axial h-6 ( 1.01 ) in combination with the large j coupling ( 12.3 hz ) required axial orientations of both the h-7 and h-5 methine protons . four acetylene and four alkene carbons with chemical shifts like those of compound 1 , as well as the corresponding alkene protons , revealed the presence of a falcarindiol unit . a comparison with reference spectroscopic data obtained for falcarindiol showed different chemical shifts of the carbons centered on c-8 , including upfield shifts of c-8 ( 1.3 ppm ) , c-6 ( 0.9 ppm ) , and c-10 ( 3.1 ppm ) , as well as downfield shifts of c-7 ( 1.8 ppm ) and c-9 ( 0.9 ppm ) . further evidence came from the hmbc correlation between c-4 ( 79.5 ) and h-8 ( 5.01 ) and the noesy correlation between me-15 and h-8 . on the basis of these results , the structure of 2 was elucidated as a falcarindiol unit connected at c-8 via an ether bond to c-4 of cryptomeridiol . in contrast , the h-1 , h-5 , and h-7 protons were assigned with an -orientation , which corresponded to these protons with an axial orientation in a trans - decalin structure . the h-5 singlet and the missing cross - peak in the dqf - cosy spectrum for h-6 indicated an equatorial orientation of h-6. a falcarindiol unit was identified by its specific carbon and proton shifts , for which the chemical shift values correlated closely with those of compound 2 and therefore indicated c-8 substitution . the falcarindiol unit was recognized again by its characteristic set of carbon and proton signals , for which the chemical shift values correlated closely with those of compounds 1 and 3 , and hence indicated a c-3 substitution of the falcarindiol moiety . coupling of h-1 ( dd , j = 10.9 , 4.1 hz ) and the noesy correlation between h-5 and h-1 suggested the -orientation of the c-1 hydroxy group . coupling of the bridgehead h-5 ( d , j = 10.9 hz ) with the h-6 of hydroxy methine ( dt , j = 10.4 , 3.3 hz ) revealed their vicinal diaxial relationship and , hence , an equatorial arrangement for the c-6 hydroxy group , which was also confirmed by the noesy correlation observed between me-14 and h-6. a noesy correlation between h-6 and the angular me-15 , as well as between me-14 and me-15 , suggested the c-4 oxy group to be also -oriented . the low - field pattern of 6-oh supported a syn - equatorial configuration for -o-4 and oh-6 , which allows an intramolecular hydrogen bonding that may resist hydrogen exchange with the residual solvent water indicated by a flat oh-1. thus , with one of the two small couplings of h-6 ( 2.6 hz ) assigned to the oh-6 hydroxy group proton , the remaining small coupling of h-6 ( 3.7 hz ) requires a cis relationship to the adjacent h-7 , suggesting the axial orientation of the isopropyl group . the axial -orientation of the isopropyl group was supported also by strong noes between h-11 , h-5 , and h-9 , respectively . when comparing its c-7 chemical shift ( 46.2 ) with the counterpart carbon of zingibertriol ( 51.5 ) , the difference ( = 5.3 ppm ) was close to that between equatorial and axial isopropylcyclohexane ( = 3.4 ppm , 150 c ) . through assignments of the 1d and 2d nmr spectra , a c-8-substituted falcarindiol moiety was identified in the same way as in the case of compounds 2 and 4 . therefore , compound 6 was established as a falcarindiol unit substituted at c-8 through an ether bond by the 7s isomer of zingibertriol ( c-4).figure 1noesy correlations and intramolecular hydrogen bond ( dashed ) of the sesquiterpenoid moiety of 5 and 6 . assignment of the 1d and 2d nmr data observed and comparison with literature data revealed that the c nmr data were consistent with those previously reported for teuclatriol ( guaiane-4,6,10-triol ) , except for chemical shift changes of c-10 ( = 7.4 ppm ) and its vicinal c-1 ( = 2.1 ppm ) , c-9 ( = 5.3 ppm ) , and c-14 ( = 2.9 ppm ) , which suggested substitution at c-10. these data , together with hmbc correlation between c-4 and h-3 , as well as the noesy correlation between h-3 and me-14 , were used to establish compound 7 as a falcarindiol unit substituted at c-3 by teuclatriol ( c-10 ) via an ether bond . in contrast to oh-6 of compounds 5 and 6 , no downfield shift of the hydroxy group proton caused by intramolecular hydrogen bonding was observed , although oh-4 and oh-6 are both syn - equatorial in 7 and 8 . these arguments together with an hmbc correlation between c-4 and h-8 , as well as a noesy correlation between me-14 and h-8 , confirmed 8 as a falcarindiol unit substituted by teuclatriol via a ( c-8)o(c-10 ) ether bond . the proton signals observed included three abx coupled benzene protons at 7.03 ( s ) , 6.92 ( d , j = 8.2 hz ) , and 7.07 ( br d , j = 8.3 hz ) , two trans vinyl protons at 7.65 ( d , j = 15.8 hz ) and 6.28 ( d , j = 15.4 hz ) , and a methoxy singlet at 3.93 . the h nmr spectrum showed three alkene signals at 6.11 ( m ) , 5.70 ( t , j = 9.4 hz ) , and 5.65 ( m ) , with similar chemical shifts and identical splitting patterns to that of h-2 , h-9 , and h-10 of a falcarindiol unit . the 2d nmr data further revealed two terminal alkene protons at 5.49 ( d , j = 18.0 hz ) and 5.24 ( d , j = 10.7 hz ) , as well as protons of two oxygenated methines at 5.80 ( d , j = 6.1 hz ) and 5.49 ( d , j = 8.7 hz ) , which corresponded to h-1cis , h-1trans , h-3 , and h-8 , respectively . instead of two alkyne carbons at the c-4 and c-5 positions , two quaternary aromatic carbons were observed , with hmbc correlations to h-3 and weak hmbc correlations to h-8 . the h-3 proton showed hmbc correlations with six carbons , including a pair of olefinic carbons ( c-1 , c-2 ) and a pair of aromatic carbons ( c-4 , c-5 ) , originating from falcarindiol . the remaining two aromatic proton resonances of h-2 and h-5 were observed as singlets and were both correlated in the hmbc spectrum with the two quaternary aryl carbons c-4 ( 146.5 ) and c-3 ( 147.7 ) , for which the c nmr chemical shift values indicated an ortho - relationship of the phenolic carbons . analysis of all the aforementioned correlations led to the assignment of compound 10 as depicted , which is a cyclization and oxidation product of a falcarindiol and a hydroxy - methoxy phenyl propane unit . in this molecule , a second aromatic ring is formed , comprising carbons c-4 and c-5 of the falcarindiol and carbons c-7 and c-8 of the phenyl propane unit . the h nmr spectrum showed three alkene signals at 6.10 ( m ) , 5.53 ( t , j = 10.2 hz ) , and 5.74 ( m ) , with similar chemical shifts and identical splitting patterns to that of h-2 , h-9 , and h-10 of a falcarindiol unit . a 2d nmr experiment further revealed two terminal alkene protons at 5.57 ( d , j = 17.5 hz ) and 5.30 ( d , j = 10.1 hz ) , as well as protons of two oxygenated methines at 5.18 ( br s ) and 6.13 ( d , j = 9.2 hz ) , which correspond to h-1cis , h-1trans , h-3 , and h-8 , respectively . a major downfield shift occurred for c-8 , and two alkylene carbons at c-4 ( 97.2 ) and c-5 ( 81.9 ) , which both showed hmbc correlations with h-3 , experienced considerable downfield shift when compared to model compounds . instead of two alkyne carbons , which are supposed to be c-6 and c-7 , two quaternary aromatic carbons were apparent , which both showed hmbc correlations with h-8 and weak hmbc correlations with h-3 . the h-9 signal gave hmbc correlations with four carbons , including one now aromatic carbon ( c-7 ) of falcarindiol origin , an oxygenated methine ( c-8 ) , a quaternary aromatic carbon ( c-8 ) , and an unsubstituted aromatic carbon ( c-7 ) , indicating a five - membered ring , which resembled that of compound 10 . the remaining two aromatic proton resonances , h-2 and h-5 , gave clear singlet structures , and both showed hmbc correlations with the two quaternary carbons c-4 ( 146.5 ) and c-3 ( 147.7 ) , for which the c nmr chemical shift values indicated an ortho - position of the phenolic carbons . notoethers a c ( 13 ) , notoincisol a ( 9 ) , and notoincisol b ( 10 ) were the most potent partial ppar agonists among the tested compounds , with ec50 values ranging from 1.7 to 2.3 m and a maximal fold activation ranging from 1.6 to 2.8 ( see table 5 for comparison of the ec50 values and maximal fold activation induced by 13 , 9 , and 10 , as well as figure 3 for comparison of the effectiveness of all tested compounds at 10 m ) . in a previous study , molecular details for the ppar binding mode of falcarindiol falcarindiol was observed to occupy parts of the entrance region of the ppar ligand binding domain and established interactions with the mainly hydrophobic binding site arms i and ii . since the active compounds from this study have some structural features in common with falcarindiol but are significantly larger , it was investigated how the proposed binding mode would shift within the binding site and if the docking could distinguish between active and inactive polyyne hybrid compounds . in general , the ppar ligand binding domain is y - shaped and is divided into three parts : the entrance domain , arm i , and arm ii ( figure 4 ) . , arg288 , glu291 , glu295 , glu343 ) , the two branches of the binding pocket arm i and arm ii are formed by mainly hydrophobic amino acids . analyzing the protein ligand interactions of all active compounds and comparing them to the ones of the inactive molecules revealed that hydrogen bonding with ser289 was associated with the activity ( figure 5 ) . ser289 is one amino acid in the core of the ligand binding site , where it is involved in the molecular recognition of many ppar agonists , as observed in x - ray crystal structures . the docking results rationalized the observed in vitro ppar activation by compounds 1 , 2 , 3 , 9 , and 10 and could serve as an inspiration for synthetic optimization to develop more potent partial agonists . while most of the compounds were inactive at the concentrations tested ( data not shown ) , notoincisol a ( 9 ) inhibited no production with an ic50 value ( 14.6 0.7 m ) comparable to those of polyacetylenes previously isolated from n. incisum ( around 10 to 30 m ) . the search for constituents with ppar agonistic activity of n. incisum led to the identification of active novel polyacetylene adducts ( 1 , 2 , 3 , 9 , and 10 ) . these compounds can serve as starting points for chemical modifications in order to optimize potency , selectivity , safety , and pharmacokinetic parameters , thereby offering new scaffolds for the development of compounds to treat inflammatory processes and possibly the related metabolic syndrome . operating conditions for the esi source used in the positive ionization mode were as follows : 3.5 kv spray voltage , 325 c capillary temperature , 300 c heater temperature , sheath gas flow rate 45 units , and auxiliary gas flow 10 units ( units refer to arbitrary values set by the exactive software ) . then , 145 g of the extract was partitioned twice between n - hexane and meoh ( 1.5:1 ) , to obtain n - hexane- ( 39 g ) and meoh - soluble ( 104 g ) portions . cells were grown in 10 cm dishes ( 6 10 cells / dish ) for 18 h and then transfected by the calcium phosphate precipitation method with 4 g of plasmid encoding human ppar ( psg5-pl - hppar-1 ) , 4 g of reporter plasmid ( tk - pprex3-luc ) , and 2 g of pegfp - n1 as control for internal normalization . the cells were treated with different concentrations of the indicated compounds and incubated for 18 h. cells were then lysed , and the luminescence of the firefly luciferase and the fluorescence of egfp were quantified with a geniospro microplate reader ( tecan , grdig , austria ) . the effects on no production were determined by photometric quantification of nitrite accumulation in cell culture supernatants using the griess assay compared with a sodium nitrite standard curve after 16 h of incubation with the respective sample as described by baer et al . a quantum mechanics - polarized ligand docking ( qpld ) workflow available within maestro version 9.2.112 ( schrdinger , llc , new york , ny , 2011,www.schrodinger.com ) was applied as described previously . visual inspection and analysis of the retrieved binding poses were performed with ligandscout 3.1 ( inte : ligand gmbh , maria enzersdorf , austria , 2012 , www.inteligand.com ) .

ovarian suppression by treatment with goserelin had been shown to reduce the risk of mortality in premenopausal breast cancer patients , but data on the long - term benefit of this treatment were lacking . the analysis was based on a randomized trial with 2 2 factorial assignment of breast cancer patients younger than 50 years to no hormonal therapy or 2 years of treatment with tamoxifen alone , goserelin alone , or goserelin plus tamoxifen . meier curves were constructed and hazard ratios and absolute risk reductions were calculated to compare treatment groups in terms of survival and recurrence outcomes . this study quantified the absolute risk reduction of mortality and recurrence conferred by goserelin treatment among women who did and did not take tamoxifen based on long - term follow - up ( mean follow - up time = 11 years ) . for every 100 women younger than 50 years and not treated with tamoxifen , 2 years of goserelin treatment would result in 8.5 fewer breast cancer deaths compared to those not given goserelin . the benefit of goserelin treatment in women treated with tamoxifen , if any , would be smaller ( possibly 2.6 fewer deaths ) . women in the trial were treated with tamoxifen for 2 years , which is currently not standard practice . the trial did not address the question of the optimal frequency and duration of goserelin treatment . ovarian suppression by treatment with goserelin had been shown to reduce the risk of mortality in premenopausal breast cancer patients , but data on the long - term benefit of this treatment were lacking . the analysis was based on a randomized trial with 2 2 factorial assignment of breast cancer patients younger than 50 years to no hormonal therapy or 2 years of treatment with tamoxifen alone , goserelin alone , or goserelin plus tamoxifen . meier curves were constructed and hazard ratios and absolute risk reductions were calculated to compare treatment groups in terms of survival and recurrence outcomes . this study quantified the absolute risk reduction of mortality and recurrence conferred by goserelin treatment among women who did and did not take tamoxifen based on long - term follow - up ( mean follow - up time = 11 years ) . for every 100 women younger than 50 years and not treated with tamoxifen , 2 years of goserelin treatment would result in 8.5 fewer breast cancer deaths compared to those not given goserelin . the benefit of goserelin treatment in women treated with tamoxifen , if any , would be smaller ( possibly 2.6 fewer deaths ) . women in the trial were treated with tamoxifen for 2 years , which is currently not standard practice . the trial did not address the question of the optimal frequency and duration of goserelin treatment . the zipp trial was conducted jointly by 4 breast cancer study groups from cancer research uk ( cruk , formerly cancer research campaign ) , gruppo interdisciplinare valutazione interventi in oncologia ( givio ) italy , south east sweden , and stockholm . the aim was to determine whether the addition of goserelin ( zoladex ) to local treatment , with or without tamoxifen , provided a benefit in survival or recurrence among pre- or perimenopausal women with operable , early breast cancer . the investigators in each location used the same protocol and worked together when setting up and conducting the trial . the local ethics committee for each center taking part in the trial approved the protocol , and all patients provided informed consent . a total of 2710 premenopausal women were recruited from august 27 , 1987 , to march 22 , 1999 , and randomly assigned to receive goserelin alone ( 3.6-mg subcutaneous depot injection into the abdominal wall every 4 weeks ) , tamoxifen alone ( 20 or 40 mg daily ) , both treatments , or no endocrine therapy ( figure 1 ) . most women were younger than 50 years , with invasive , operable breast cancer confined to one breast without evidence of metastatic disease . women received primary therapy : surgery with or without radiotherapy and adjuvant systemic chemotherapy , where appropriate . chemotherapy was perioperative cyclophosphamide , or six cycles of cyclophosphamide , methotrexate , and 5-fluorouracil ( cmf ) or 5-fluorouracil , epirubicin , and cyclophosphamide ( fec ) . the proportions of patients who received surgery , radiotherapy , or chemotherapy were evenly distributed between the goserelin groups ( supplementary table 1 , available online ) , so these treatments would not be expected to affect the results . distribution of patients in the zoladex in premenopausal patients ( zipp ) trial ( consort flow diagram ) . all patients in the stockholm and givio trials were included in the 2 2 factorial random assignment . patients in the cruk and south east sweden trials were initially randomly assigned into four arms , but with the publication of the data on tamoxifen in younger patients ( 6 ) , investigators were permitted to give tamoxifen electively , following random assignment to goserelin or no goserelin . this did not affect the results because the numbers of women treated and not treated with tamoxifen were balanced between the trial arms : of those electively given tamoxifen , 432 were randomly assigned to goserelin and 428 to no goserelin , and of those electively not given tamoxifen , 25 were randomly assigned to goserelin and 25 to no goserelin . four patients were subsequently found to have been ineligible at the time of random assignment ( two had contralateral breast cancer , one had metastatic disease , and one had recurrent disease ) and were excluded from this analysis . the numbers of women analyzed in the control , tamoxifen - only , goserelin - only , and both therapies arms were 476 , 879 , 469 , and 882 , respectively ( 2706 in total ) . the following four outcome measures were examined : ( 1 ) event - free survival ( efs ) , the main trial endpoint and defined as a recurrence , new tumor , or death . ( 2 ) overall survival ( os , death from any cause ) . ( 3 ) risk of recurrence [ recurrence could be local or distant and among 10 women who died from breast cancer with no previously recorded recurrence , it was assumed that they had a distant recurrence , assumed to occur on the date of death ; consistent with the assumptions of the ebctcg overview ( 3 ) ] . there were also 110 whose cause of death was unknown , but these deaths also were assumed to be due to breast cancer because among those with a known cause of death , 90% were due to breast cancer . in addition , there were 13 women who died from other known causes but in whom breast cancer was present at death ; these women were censored at the date of first recurrence because according to the ebctcg overview ( 3 ) , cause of death may not be reliably recorded after a recurrence . the sample size target was set at 2700 women to detect an absolute difference in the 5-year efs rate of 5% ( 70% vs 75% in the control and treated group , respectively ) , with 83% power and alpha set at .05 . simple randomization was used in each of the four trial groups to allocate patients to the trial arms with a block size of four . meier survival curves and hazard ratios ( hrs ) were used to compare treatment groups based on intention - to - treat analyses . we examined the interaction between goserelin and tamoxifen and whether the effect of goserelin differed between subgroups of patients for four prognostic factors : age ( < 40 or 40 years ) , nodal status ( positive or negative ) , er status ( positive , negative , or unknown ) , and previous adjuvant systemic chemotherapy ( yes or no ) . although age was not prespecified in the original trial protocol , the intention was to include it in the long - term follow - up analysis , particularly because it was examined in detail in the recent lhrh overview ( 4 ) . the assumption of proportional hazards was verified using a plot of the schoenfeld residuals ( 7 ) . statistical analyses were performed using sas 9.1 ( 8) . because tamoxifen is the current standard of treatment , most analyses were presented according to whether women received tamoxifen or not , allowing an examination of the effect of goserelin in addition to tamoxifen . relative or proportional risk reductions , based on hazard ratios , are indicated by percentages ( % ) , whereas absolute risk differences at specified time points are indicated by percentage points ( ie , the number of events prevented among every 100 treated women ) . all statistical tests were two - sided , and p values less than .05 were considered statistically significant . a total of 2710 premenopausal women were recruited from august 27 , 1987 , to march 22 , 1999 , and randomly assigned to receive goserelin alone ( 3.6-mg subcutaneous depot injection into the abdominal wall every 4 weeks ) , tamoxifen alone ( 20 or 40 mg daily ) , both treatments , or no endocrine therapy ( figure 1 ) . most women were younger than 50 years , with invasive , operable breast cancer confined to one breast without evidence of metastatic disease . women received primary therapy : surgery with or without radiotherapy and adjuvant systemic chemotherapy , where appropriate . chemotherapy was perioperative cyclophosphamide , or six cycles of cyclophosphamide , methotrexate , and 5-fluorouracil ( cmf ) or 5-fluorouracil , epirubicin , and cyclophosphamide ( fec ) . the proportions of patients who received surgery , radiotherapy , or chemotherapy were evenly distributed between the goserelin groups ( supplementary table 1 , available online ) , so these treatments would not be expected to affect the results . distribution of patients in the zoladex in premenopausal patients ( zipp ) trial ( consort flow diagram ) . all patients in the stockholm and givio trials were included in the 2 2 factorial random assignment . patients in the cruk and south east sweden trials were initially randomly assigned into four arms , but with the publication of the data on tamoxifen in younger patients ( 6 ) , investigators were permitted to give tamoxifen electively , following random assignment to goserelin or no goserelin . this did not affect the results because the numbers of women treated and not treated with tamoxifen were balanced between the trial arms : of those electively given tamoxifen , 432 were randomly assigned to goserelin and 428 to no goserelin , and of those electively not given tamoxifen , 25 were randomly assigned to goserelin and 25 to no goserelin . four patients were subsequently found to have been ineligible at the time of random assignment ( two had contralateral breast cancer , one had metastatic disease , and one had recurrent disease ) and were excluded from this analysis . the numbers of women analyzed in the control , tamoxifen - only , goserelin - only , and both therapies arms were 476 , 879 , 469 , and 882 , respectively ( 2706 in total ) . the following four outcome measures were examined : ( 1 ) event - free survival ( efs ) , the main trial endpoint and defined as a recurrence , new tumor , or death . ( 2 ) overall survival ( os , death from any cause ) . ( 3 ) risk of recurrence [ recurrence could be local or distant and among 10 women who died from breast cancer with no previously recorded recurrence , it was assumed that they had a distant recurrence , assumed to occur on the date of death ; consistent with the assumptions of the ebctcg overview ( 3 ) ] . there were also 110 whose cause of death was unknown , but these deaths also were assumed to be due to breast cancer because among those with a known cause of death , 90% were due to breast cancer . in addition , there were 13 women who died from other known causes but in whom breast cancer was present at death ; these women were censored at the date of first recurrence because according to the ebctcg overview ( 3 ) , cause of death may not be reliably recorded after a recurrence . the sample size target was set at 2700 women to detect an absolute difference in the 5-year efs rate of 5% ( 70% vs 75% in the control and treated group , respectively ) , with 83% power and alpha set at .05 . simple randomization was used in each of the four trial groups to allocate patients to the trial arms with a block size of four . meier survival curves and hazard ratios ( hrs ) were used to compare treatment groups based on intention - to - treat analyses . we examined the interaction between goserelin and tamoxifen and whether the effect of goserelin differed between subgroups of patients for four prognostic factors : age ( < 40 or 40 years ) , nodal status ( positive or negative ) , er status ( positive , negative , or unknown ) , and previous adjuvant systemic chemotherapy ( yes or no ) . although age was not prespecified in the original trial protocol , the intention was to include it in the long - term follow - up analysis , particularly because it was examined in detail in the recent lhrh overview ( 4 ) . the assumption of proportional hazards was verified using a plot of the schoenfeld residuals ( 7 ) . statistical analyses were performed using sas 9.1 ( 8) . because tamoxifen is the current standard of treatment , most analyses were presented according to whether women received tamoxifen or not , allowing an examination of the effect of goserelin in addition to tamoxifen . relative or proportional risk reductions , based on hazard ratios , are indicated by percentages ( % ) , whereas absolute risk differences at specified time points are indicated by percentage points ( ie , the number of events prevented among every 100 treated women ) . all statistical tests were two - sided , and p values less than .05 were considered statistically significant . the median length of follow - up was 12 years , with 26 545 person - years in total . the numbers of efs events ( recurrence , second primary tumor , or death ) , deaths , breast cancer recurrences , and breast cancer deaths were 1148 , 690 , 941 , and 630 , respectively ( supplementary table 2 , available online ) . hrs associated with goserelin treatment according to trial group and overall for the four outcome measures are shown in ( supplementary table 3 , available online . goserelin was associated with a risk reduction in all four endpoints : the risk of having an efs event ( hr = 0.82 , 95% confidence interval [ ci ] 0.73 to 0.92 , p = .001 ) , overall mortality ( hr = 0.83 , 95% ci 0.71 to 0.96 , p = .013 ) , the risk of recurrence ( hr = 0.81 , 95% ci 0.71 to 0.92 , p = .001 ) , and breast cancer mortality ( hr = 0.82 , 95% ci 0.70 to 0.96 , p = 0.03 ) . although the point estimates of hr for efs , os , recurrence , and breast cancer mortality in the givio trial appeared to differ from those of the other trials , the confidence intervals contained the overall estimate for each outcome , suggesting that the differences may have been due to chance . there was a clear benefit on survival and recurrence 10 or 15 years after randomization ( figure 2 ) . a ) the percentages ( 95% confidence interval [ ci ] ) of women treated with goserelin who experienced death , had recurrence , or had a new tumor at 5 , 10 , and 15 years were 25 ( 23 to 27 ) , 38 ( 35 to 41 ) , and 47 ( 43 to 51 ) , respectively ; corresponding percentages in control women were 32 ( 29 to 35 ) , 43 ( 40 to 46 ) , and 52 ( 48 to 55 ) . b ) the percentages ( 95% ci ) of women treated with goserelin who died at 5 , 10 , and 15 years were 12 ( 10 to 14 ) , 21 ( 19 to 23 ) , and 29 ( 26 to 32 ) , respectively ; the corresponding percentages in control women were 15 ( 13 to 17 ) , 26 ( 23 to 28 ) , and 33 ( 30 to 36 ) . c ) the percentages ( 95% ci ) of women treated with goserelin who had a recurrence at 5 , 10 , and 15 years were 22 ( 20 to 24 ) , 31 ( 28 to 34 ) , and 36 ( 33 to 39 ) , respectively ; the corresponding percentages in control women were 27 ( 24 to 29 ) , 37 ( 34 to 40 ) , and 42 ( 39 to 45 ) . d ) the percentages ( 95% ci ) of women treated with goserelin who died from breast cancer at 5 , 10 , and 15 years were 11 ( 9 to 13 ) , 20 ( 18 to 22 ) , and 26 ( 23 to 29 ) , respectively ; the corresponding percentages in control women were 14 ( 12 to 16 ) , 24 ( 22 to 26 ) , and 30 ( 27 to 33 ) . two - sided p values from log - rank tests are shown . the effect of goserelin depended on whether women received tamoxifen ( table 1 ) . the test for interaction was statistically significant for efs and recurrence , and although the p values were greater than .05 for os and breast cancer mortality , the hrs were in a direction similar to the other endpoints , that is , women who did not receive tamoxifen experienced a greater benefit from goserelin than those who received tamoxifen . thus , in women who did not receive tamoxifen , goserelin was associated with a 33% reduction in the risk of having an efs event , a 29% reduction in risk of overall mortality , a 34% reduction in risk of recurrence , and a 29% reduction in risk of breast cancer mortality . in women who received tamoxifen , there was a much smaller benefit due to goserelin : 8% reduction in risk of efs events , 10% reduction in risk of overall mortality , 9% reduction in risk of recurrence , and 11% reduction in risk of breast cancer mortality . table 1 also shows that goserelin was as effective as tamoxifen ; the hazard ratios for each treatment on its own were similar , when compared with the control group . examination of the interaction between goserelin and tamoxifen the hazard ratios can be converted to percentage reduction or increase in risk by subtracting 1 and multiplying by 100 . the p values for the test of interaction between goserelin and tamoxifen were .01 ( any event ) , .13 ( death from any cause ) , .016 ( breast cancer recurrence ) , and .17 ( death from breast cancer ) . taking either tamoxifen or goserelin had similar and large effects , but using both was associated with a possible small additional benefit that was not statistically significant . we calculated absolute risk differences associated with goserelin treatment at 5 , 10 , and 15 years after random assignment among women who did or did not take tamoxifen ( table 2 ) . among women who did not take tamoxifen , there was a clear and substantial benefit associated with goserelin treatment for all four endpoints . for example , the estimated efs absolute risk difference at 15 years indicates that for every 100 such women treated with goserelin , there could be 13.9 fewer with an event ( recurrence , new tumor or death ) , compared with those not given goserelin ( risk difference 13.9 percentage points , 95% ci 19.4 to 7.5 ) . this corresponds to a number needed to treat of 7 . among women who took tamoxifen , the benefit was smaller and not statistically significant : for every 100 such women treated with goserelin , there could be 2.8 fewer with an event ( 95% ci 7.7 to 2.0 ) . with regards to the effect on breast cancer mortality , 12 patients needed to be treated with goserelin ( and not tamoxifen ) to avoid one woman dying ( risk difference 8.5 percentage points , 95% ci 13.7 to 2.2 ) , compared with no goserelin , whereas 38 needed to be treated with both goserelin and tamoxifen to avoid one death ( risk difference 2.6 percentage points , 95% ci 6.6 to 2.1 ) . risk differences and nnts are likely to be different in populations with much lower or higher baseline risks than in the zipp trial . estimated absolute risk difference ( percentage points ) between goserelin and no goserelin for four outcomes if p is the event rate in the no goserelin arm ( eg , efs rate or risk of recurrence ) at a specified time point , then the absolute difference at this time is p exp[hazard ratio logep ] . the hazard ratios were taken from table 1 ( tamoxifen = no or yes ) . number needed to treat = 100% ( absolute risk difference ) . the expected number of women who need to be treated with goserelin to avoid one event at the specified time point . outcomes considered were death , recurrence , or new tumor ( a ) ; death from any cause ( b ) ; breast cancer recurrence ( c ) ; and death due to breast cancer ( d ) . older women ( 40 years ) who did not take tamoxifen benefited the most : the absolute risk difference was 14.8 percentage points after 15 years . in younger women ( < 40 years ) , goserelin decreased the event rate by 45 percentage points at 15 years , but the confidence intervals were wide , which may partly reflect the smaller sample size in these subgroups . the least benefit associated with goserelin treatment appeared to be among older women who took tamoxifen ( the absolute risk was 1.7 percentage points lower ) . estimated absolute risk difference ( percentage points ) for any event ( recurrence , new tumour or death ) between women treated with goserelin and those not treated with goserelin , according to age and whether women took tamoxifen or not . if p = the event rate in the no goserelin arm at a specified time point , then the absolute risk difference at this time is p minus exp[hazard ratio x logep ] . among younger women who had prior chemotherapy , the hazard ratio for goserelin treatment ( compared with those not given goserelin ) in relation to breast cancer mortality was 0.66 ( 95% ci 0.44 to 0.98 ) , and this reduction in risk did not seem to depend on whether they took tamoxifen or not ( hrs 0.66 and 0.63 , respectively ) . the corresponding hazard ratio in younger women without prior chemotherapy was 0.86 ( 95% ci 0.55 to 1.34 ) . these observations could be partly explained by the fact that younger women derive greater benefits from adjuvant chemotherapy . among older women who had prior chemotherapy , the hr was 0.87 ( 95% ci 0.68 to 1.13 ) , although the effect might be greater when they also took tamoxifen ( hrs of 0.80 and 0.93 , with and without tamoxifen , respectively ) . among older women who did not have prior chemotherapy , the effect of goserelin depended on whether they took tamoxifen ( hr for the effect of goserelin for older women who did or did not receive tamoxifen was 1.06 [ 95% ci = 0.75 to 1.52 ] and 0.60 [ 95% ci = 0.39 to 0.93 ] , respectively ) . we investigated interactions between goserelin and four prognostic factors ( supplementary table 4 , available online ) . the effect of goserelin was greatest among women who were node negative , those with er - positive tumors , and those who had received no prior chemotherapy ( although age and tamoxifen use influenced this ) . similar to other studies , a benefit of goserelin in women with er - positive tumors was observed , whether they took tamoxifen or not . it is possible that the interaction between goserelin and nodal status might have been influenced by prior chemotherapy because women with node - positive tumors were more likely to receive chemotherapy . however , when patients were stratified by nodal status and prior chemotherapy , there was still a tendency for the effect of goserelin to be greater among node - negative women , although those who were node positive who had prior chemotherapy did not appear to benefit from goserelin ( supplementary table 5 , available online ) . to our knowledge , the zipp trial is the largest that has investigated an lhrh agonist ( 2706 women ) and the only factorial study that has evaluated goserelin and tamoxifen simultaneously with a control group of women who received no endocrine therapy . recurrence and breast cancer mortality rates in both the tamoxifen and control arms were similar to those from the ebctcg overview ( 3 ) . the overview of lhrh agonists ( 4 ) , based on an average follow - up of 6.8 years , reported data on recurrence , death from any cause , and death after recurrence . the main conclusions were that lhrh agonists had an effect on reducing the risk of these events similar to that for chemotherapy ( such as fec or cmf ) , and could be used as an effective treatment in women with er - positive tumors , either alone or in combination with chemotherapy or tamoxifen . furthermore , there seemed to be a particular benefit among younger women ( < 40 years ) who had received chemotherapy . although our earlier data were included in the overview ( 4 ) , our results from the long - term follow - up ( average follow - up is 12 years ) add further information on the effect of lhrh agonists and allow estimates of the absolute risk difference at 10 and 15 years after the start of treatment . in addition to information on recurrence and death from any cause , we analyzed the effect of goserelin on the chance of dying from breast cancer and the chance of having any event ( recurrence , new tumor , or death ) . our analysis also quantified the long - term effect of goserelin separately among women who did or did not have tamoxifen . because tamoxifen is now offered routinely to many women , it is important to know what the additional benefit of goserelin is 10 and 15 years after the initiation of treatment . among women who did not receive tamoxifen , goserelin was associated with large reductions in the event rate , the chance of dying from any cause or from breast cancer , and the risk of having a recurrence ; the effect remained substantial 15 years later . although the effect of goserelin was smaller ( and not statistically significant ) among women who took tamoxifen , a difference in risk of 23 percentage points in absolute risk at 15 years might be important , given the high incidence of early breast cancer . this would correspond to treating 3350 women with tamoxifen and goserelin to avoid one woman having a recurrence , new tumor , or death . indeed , the number needed to treat at 15 years was 18 in women younger than 40 years who also took tamoxifen ( table 3 ) . the effect of goserelin was greatest in women who were node negative and in those with er - positive tumors . our results were also consistent with the observation that the effect of goserelin may be greater in younger women who had prior chemotherapy ( 4 ) . a limitation of our trial is that it was based on 2 years of tamoxifen treatment , like many other studies at the time , though it is now standard practice to treat women for 5 years . data from the int 0101 trial , in which all women received chemotherapy , showed that the combination of 5 years of goserelin and tamoxifen was associated with a 9-year disease - free rate that was 9 and 7 percentage points higher among younger ( < 40 years ) and older ( 40 ) women , respectively , compared with those treated with goserelin alone ( 9 ) . furthermore , there is evidence that a large proportion of women discontinue tamoxifen before 5 years . in a cohort of 2816 women in ireland , 20% therefore , the effect of 2 years of goserelin or tamoxifen therapy might still be relevant in these women . data on the tolerability of goserelin were presented in our first report ( 5 ) . the most common side effect was hot flashes : this was experienced by none of the control patients , 26% of patients treated with goserelin , 17% of those treated with tamoxifen , and 44% of those who received goserelin plus tamoxifen . in a trial of 874 women with lymph node negative breast cancer randomly assigned to receive six courses of cmf chemotherapy , goserelin for 2 years , or cmf followed by 18 months of goserelin , a greater proportion of women who received goserelin alone had hot flashes compared with those treated with cmf alone ( 11 ) . however , in the goserelin group , the incidence 3 years later was similar to that in baseline , but not in those who received cmf . this effect is expected , given that amenorrhea can be induced by either chemotherapy or goserelin , but is reversible after stopping goserelin but permanent after chemotherapy . goserelin was also associated with an improvement in quality - of - life measures such as mood , coping effort , tiredness , nausea and/or vomiting , and overall subjective assessment of health , compared with cmf alone ( 11 ) . treatment - induced bone loss is a side effect of ovarian ablation or aromatase inhibitor therapy , which increases the risk of fractures or spinal cord compression ( 12 ) . in a subgroup of patients randomly assigned to receive goserelin ( n = 53 ) for 2 years or cmf chemotherapy for 6 months ( n = 43 ) , loss of bone mineral density ( bmd ) was greater in the goserelin group ( 13 ) . although there was partial recovery 1 year after stopping goserelin , the bone loss in the cmf group persisted . although the combination of goserelin and tamoxifen may have a small additional benefit compared with either alone , there may be a further case for recommending both therapies because of the possible mitigating effect of tamoxifen on bmd . markers of bone health were not measured in all women in the zipp trial , but the effects of 2 years of treatment with goserelin and tamoxifen on bmd were examined in a subgroup ( n = 89 ) of patients in the stockholm group ( 14 ) . the reduction in bmd was 5% in the patients treated with goserelin ( with partial recovery 1 year after stopping therapy ) and 1.4% in the patients treated with tamoxifen and goserelin . there is accumulating evidence that zoledronic acid combined with endocrine therapy can also help prevent bmd loss . in a trial of 401 patients treated with goserelin , women were randomly assigned to receive 3 years of either tamoxifen or anastrazole , each with or without zoledronic acid ( 15 ) . there were reductions in lumbar spine and trochanter bmd when tamoxifen or anastrazole was used alone , but no material reduction when zoledronic acid was added . a combined interim analysis of two recent randomized trials , z - fast and zo - fast ( total from both trials , n = 1667 ) , in which all women received adjuvant letrozole , compared giving zoledronic acid either at the time of random assignment or only if their bone density fell below a prespecified cutoff or they suffered a nontraumatic fracture ( 16 ) . after 1 year , the score for lumbar spine bmd was 5.2% higher in the patients given zoledronic acid at the time of random assignment , and the total hip score was 3.5% higher . the effect of zoledronic acid on survival and recurrence has been confirmed in a large trial of 1801 premenopausal women randomly assigned to receive endocrine therapy ( tamoxifen or anastrozole ) , with or without zoledronic acid , with all women receiving goserelin ( 17 ) . the risk of death or recurrence was reduced by 35% ( 95% ci = 8% to 54% ) among women who were given zoledronic acid . given its benefits in terms of both bone health and clinical outcome , zoledronic acid could be considered as an additional treatment to goserelin . the optimal frequency and duration of treatment with lhrh agonists are unknown . in the zipp trial , evidence from a randomized trial of 599 women with node - positive breast cancer indicated that a less frequent schedule ( 3-monthly depot of the lhrh agonist leuprorelin acetate , 11.25 mg ) may be as beneficial as cmf chemotherapy in terms of the effect5-year recurrence - free survival ( hr 0.97 ) and after 2 years os was better in patients given leuprorelin acetate ( hr 0.64 , 95% ci 0.46 to 0.90 ) ( 18 ) . nevertheless , no trial has specifically addressed the question of the optimal frequency and duration of goserelin treatment . in summary , long - term follow - up of our large trial showed that goserelin had a demonstrable effect on survival and recurrence 15 years after starting treatment and is as effective as tamoxifen when each are given for 2 years . the benefit was greatest among women who did not receive tamoxifen ( among every 100 women treated with goserelin , there could be 8.5 fewer breast cancer deaths ) , and there was a possible gain in those who did receive tamoxifen ( among every 100 women treated with goserelin , there could be 2.6 fewer breast cancer deaths ) . it may be that women who are unlikely to complete 5 years of tamoxifen tablets may prefer 2 years of goserelin injections . it may also be reasonable to recommend both therapies to minimize the reduction in bmd associated with endocrine treatment . free drug was supplied by ici ( now astrazeneca ) ( givio ) for the uk and gruppo interdisciplinare valutazione interventi in oncologia trials . the uk trial was supported by a grant from cancer research uk ( formerly cancer research campaign ) . in italy , the coordination of the trial was supported by an educational grant from astrazeneca ( the manufacturer of goserelin ) . the stockholm trial had received funding from the king gustaf v jubilee fund and an unrestricted research grant from astrazeneca . in 2007 , astrazeneca provided a grant to collect and collate the long - term follow - up data from the four study groups , and the statistical analysis was performed in the cancer research uk & ucl cancer trials centre . ( grant number 45034 ) m. baum has been a principal investigator for other trials sponsored by astrazeneca and spoken at events funded by the company . astrazeneca was not involved in either the analysis or the writing of the paper , or the decision to publish .

backgroundsystematic reviews have found that luteinizing hormone releasing hormone ( lhrh ) agonists are effective in treating premenopausal women with early breast cancer.methodswe conducted long - term follow - up ( median 12 years ) of 2706 women in the zoladex in premenopausal patients ( zipp ) , which evaluated the lhrh agonist goserelin ( 3.6 mg injection every 4 weeks ) and tamoxifen ( 20 or 40 mg daily ) , given for 2 years . women were randomly assigned to receive each therapy alone , both , or neither , after primary therapy ( surgery with or without radiotherapy / chemotherapy ) . hazard ratios and absolute risk differences were used to assess the effect of goserelin treatment on event - free survival ( breast cancer recurrence , new tumor or death ) , overall survival , risk of recurrence of breast cancer , and risk of dying from breast cancer , in the presence or absence of tamoxifen.resultsfifteen years after the initiation of treatment , for every 100 women not given tamoxifen , there were 13.9 ( 95% confidence interval [ ci ] = 17.5 to 19.4 ) fewer events among those who were treated with goserelin compared with those who were not treated with goserelin . however , among women who did take tamoxifen , there were 2.8 fewer events ( 95% ci = 7.7 fewer to 2.0 more ) per 100 women treated with goserelin compared with those not treated with goserelin . the risk of dying from breast cancer was also reduced at 15 years : for every 100 women given goserelin , the number of breast cancer deaths was lower by 2.6 ( 95% ci = 6.6 fewer to 2.1 more ) and 8.5 ( 95% ci = 2.2 to 13.7 ) in those who did and did not take tamoxifen , respectively , although in the former group the difference was not statistically significant.conclusionstwo years of goserelin treatment was as effective as 2 years of tamoxifen treatment 15 years after starting therapy . in women who did not take tamoxifen , there was a large benefit of goserelin treatment on survival and recurrence , and in women who did take tamoxifen , there was a marginal potential benefit on these outcomes when goserelin was added .

Context and Caveats Prior knowledge Study design Contribution Implications Limitations Methods Trial Design and Subsequent Modifications Outcomes Statistical Analysis Results Discussion Funding Supplementary Material

ovarian suppression by treatment with goserelin had been shown to reduce the risk of mortality in premenopausal breast cancer patients , but data on the long - term benefit of this treatment were lacking . the analysis was based on a randomized trial with 2 2 factorial assignment of breast cancer patients younger than 50 years to no hormonal therapy or 2 years of treatment with tamoxifen alone , goserelin alone , or goserelin plus tamoxifen . this study quantified the absolute risk reduction of mortality and recurrence conferred by goserelin treatment among women who did and did not take tamoxifen based on long - term follow - up ( mean follow - up time = 11 years ) . for every 100 women younger than 50 years and not treated with tamoxifen , 2 years of goserelin treatment would result in 8.5 fewer breast cancer deaths compared to those not given goserelin . the benefit of goserelin treatment in women treated with tamoxifen , if any , would be smaller ( possibly 2.6 fewer deaths ) . ovarian suppression by treatment with goserelin had been shown to reduce the risk of mortality in premenopausal breast cancer patients , but data on the long - term benefit of this treatment were lacking . the analysis was based on a randomized trial with 2 2 factorial assignment of breast cancer patients younger than 50 years to no hormonal therapy or 2 years of treatment with tamoxifen alone , goserelin alone , or goserelin plus tamoxifen . this study quantified the absolute risk reduction of mortality and recurrence conferred by goserelin treatment among women who did and did not take tamoxifen based on long - term follow - up ( mean follow - up time = 11 years ) . for every 100 women younger than 50 years and not treated with tamoxifen , 2 years of goserelin treatment would result in 8.5 fewer breast cancer deaths compared to those not given goserelin . the benefit of goserelin treatment in women treated with tamoxifen , if any , would be smaller ( possibly 2.6 fewer deaths ) . the aim was to determine whether the addition of goserelin ( zoladex ) to local treatment , with or without tamoxifen , provided a benefit in survival or recurrence among pre- or perimenopausal women with operable , early breast cancer . a total of 2710 premenopausal women were recruited from august 27 , 1987 , to march 22 , 1999 , and randomly assigned to receive goserelin alone ( 3.6-mg subcutaneous depot injection into the abdominal wall every 4 weeks ) , tamoxifen alone ( 20 or 40 mg daily ) , both treatments , or no endocrine therapy ( figure 1 ) . distribution of patients in the zoladex in premenopausal patients ( zipp ) trial ( consort flow diagram ) . this did not affect the results because the numbers of women treated and not treated with tamoxifen were balanced between the trial arms : of those electively given tamoxifen , 432 were randomly assigned to goserelin and 428 to no goserelin , and of those electively not given tamoxifen , 25 were randomly assigned to goserelin and 25 to no goserelin . the following four outcome measures were examined : ( 1 ) event - free survival ( efs ) , the main trial endpoint and defined as a recurrence , new tumor , or death . ( 3 ) risk of recurrence [ recurrence could be local or distant and among 10 women who died from breast cancer with no previously recorded recurrence , it was assumed that they had a distant recurrence , assumed to occur on the date of death ; consistent with the assumptions of the ebctcg overview ( 3 ) ] . in addition , there were 13 women who died from other known causes but in whom breast cancer was present at death ; these women were censored at the date of first recurrence because according to the ebctcg overview ( 3 ) , cause of death may not be reliably recorded after a recurrence . we examined the interaction between goserelin and tamoxifen and whether the effect of goserelin differed between subgroups of patients for four prognostic factors : age ( < 40 or 40 years ) , nodal status ( positive or negative ) , er status ( positive , negative , or unknown ) , and previous adjuvant systemic chemotherapy ( yes or no ) . although age was not prespecified in the original trial protocol , the intention was to include it in the long - term follow - up analysis , particularly because it was examined in detail in the recent lhrh overview ( 4 ) . relative or proportional risk reductions , based on hazard ratios , are indicated by percentages ( % ) , whereas absolute risk differences at specified time points are indicated by percentage points ( ie , the number of events prevented among every 100 treated women ) . a total of 2710 premenopausal women were recruited from august 27 , 1987 , to march 22 , 1999 , and randomly assigned to receive goserelin alone ( 3.6-mg subcutaneous depot injection into the abdominal wall every 4 weeks ) , tamoxifen alone ( 20 or 40 mg daily ) , both treatments , or no endocrine therapy ( figure 1 ) . distribution of patients in the zoladex in premenopausal patients ( zipp ) trial ( consort flow diagram ) . this did not affect the results because the numbers of women treated and not treated with tamoxifen were balanced between the trial arms : of those electively given tamoxifen , 432 were randomly assigned to goserelin and 428 to no goserelin , and of those electively not given tamoxifen , 25 were randomly assigned to goserelin and 25 to no goserelin . the following four outcome measures were examined : ( 1 ) event - free survival ( efs ) , the main trial endpoint and defined as a recurrence , new tumor , or death . ( 3 ) risk of recurrence [ recurrence could be local or distant and among 10 women who died from breast cancer with no previously recorded recurrence , it was assumed that they had a distant recurrence , assumed to occur on the date of death ; consistent with the assumptions of the ebctcg overview ( 3 ) ] . in addition , there were 13 women who died from other known causes but in whom breast cancer was present at death ; these women were censored at the date of first recurrence because according to the ebctcg overview ( 3 ) , cause of death may not be reliably recorded after a recurrence . we examined the interaction between goserelin and tamoxifen and whether the effect of goserelin differed between subgroups of patients for four prognostic factors : age ( < 40 or 40 years ) , nodal status ( positive or negative ) , er status ( positive , negative , or unknown ) , and previous adjuvant systemic chemotherapy ( yes or no ) . although age was not prespecified in the original trial protocol , the intention was to include it in the long - term follow - up analysis , particularly because it was examined in detail in the recent lhrh overview ( 4 ) . relative or proportional risk reductions , based on hazard ratios , are indicated by percentages ( % ) , whereas absolute risk differences at specified time points are indicated by percentage points ( ie , the number of events prevented among every 100 treated women ) . the numbers of efs events ( recurrence , second primary tumor , or death ) , deaths , breast cancer recurrences , and breast cancer deaths were 1148 , 690 , 941 , and 630 , respectively ( supplementary table 2 , available online ) . goserelin was associated with a risk reduction in all four endpoints : the risk of having an efs event ( hr = 0.82 , 95% confidence interval [ ci ] 0.73 to 0.92 , p = .001 ) , overall mortality ( hr = 0.83 , 95% ci 0.71 to 0.96 , p = .013 ) , the risk of recurrence ( hr = 0.81 , 95% ci 0.71 to 0.92 , p = .001 ) , and breast cancer mortality ( hr = 0.82 , 95% ci 0.70 to 0.96 , p = 0.03 ) . there was a clear benefit on survival and recurrence 10 or 15 years after randomization ( figure 2 ) . a ) the percentages ( 95% confidence interval [ ci ] ) of women treated with goserelin who experienced death , had recurrence , or had a new tumor at 5 , 10 , and 15 years were 25 ( 23 to 27 ) , 38 ( 35 to 41 ) , and 47 ( 43 to 51 ) , respectively ; corresponding percentages in control women were 32 ( 29 to 35 ) , 43 ( 40 to 46 ) , and 52 ( 48 to 55 ) . b ) the percentages ( 95% ci ) of women treated with goserelin who died at 5 , 10 , and 15 years were 12 ( 10 to 14 ) , 21 ( 19 to 23 ) , and 29 ( 26 to 32 ) , respectively ; the corresponding percentages in control women were 15 ( 13 to 17 ) , 26 ( 23 to 28 ) , and 33 ( 30 to 36 ) . c ) the percentages ( 95% ci ) of women treated with goserelin who had a recurrence at 5 , 10 , and 15 years were 22 ( 20 to 24 ) , 31 ( 28 to 34 ) , and 36 ( 33 to 39 ) , respectively ; the corresponding percentages in control women were 27 ( 24 to 29 ) , 37 ( 34 to 40 ) , and 42 ( 39 to 45 ) . d ) the percentages ( 95% ci ) of women treated with goserelin who died from breast cancer at 5 , 10 , and 15 years were 11 ( 9 to 13 ) , 20 ( 18 to 22 ) , and 26 ( 23 to 29 ) , respectively ; the corresponding percentages in control women were 14 ( 12 to 16 ) , 24 ( 22 to 26 ) , and 30 ( 27 to 33 ) . the test for interaction was statistically significant for efs and recurrence , and although the p values were greater than .05 for os and breast cancer mortality , the hrs were in a direction similar to the other endpoints , that is , women who did not receive tamoxifen experienced a greater benefit from goserelin than those who received tamoxifen . thus , in women who did not receive tamoxifen , goserelin was associated with a 33% reduction in the risk of having an efs event , a 29% reduction in risk of overall mortality , a 34% reduction in risk of recurrence , and a 29% reduction in risk of breast cancer mortality . in women who received tamoxifen , there was a much smaller benefit due to goserelin : 8% reduction in risk of efs events , 10% reduction in risk of overall mortality , 9% reduction in risk of recurrence , and 11% reduction in risk of breast cancer mortality . the p values for the test of interaction between goserelin and tamoxifen were .01 ( any event ) , .13 ( death from any cause ) , .016 ( breast cancer recurrence ) , and .17 ( death from breast cancer ) . we calculated absolute risk differences associated with goserelin treatment at 5 , 10 , and 15 years after random assignment among women who did or did not take tamoxifen ( table 2 ) . among women who did not take tamoxifen , there was a clear and substantial benefit associated with goserelin treatment for all four endpoints . for example , the estimated efs absolute risk difference at 15 years indicates that for every 100 such women treated with goserelin , there could be 13.9 fewer with an event ( recurrence , new tumor or death ) , compared with those not given goserelin ( risk difference 13.9 percentage points , 95% ci 19.4 to 7.5 ) . among women who took tamoxifen , the benefit was smaller and not statistically significant : for every 100 such women treated with goserelin , there could be 2.8 fewer with an event ( 95% ci 7.7 to 2.0 ) . with regards to the effect on breast cancer mortality , 12 patients needed to be treated with goserelin ( and not tamoxifen ) to avoid one woman dying ( risk difference 8.5 percentage points , 95% ci 13.7 to 2.2 ) , compared with no goserelin , whereas 38 needed to be treated with both goserelin and tamoxifen to avoid one death ( risk difference 2.6 percentage points , 95% ci 6.6 to 2.1 ) . older women ( 40 years ) who did not take tamoxifen benefited the most : the absolute risk difference was 14.8 percentage points after 15 years . estimated absolute risk difference ( percentage points ) for any event ( recurrence , new tumour or death ) between women treated with goserelin and those not treated with goserelin , according to age and whether women took tamoxifen or not . among younger women who had prior chemotherapy , the hazard ratio for goserelin treatment ( compared with those not given goserelin ) in relation to breast cancer mortality was 0.66 ( 95% ci 0.44 to 0.98 ) , and this reduction in risk did not seem to depend on whether they took tamoxifen or not ( hrs 0.66 and 0.63 , respectively ) . among older women who had prior chemotherapy , the hr was 0.87 ( 95% ci 0.68 to 1.13 ) , although the effect might be greater when they also took tamoxifen ( hrs of 0.80 and 0.93 , with and without tamoxifen , respectively ) . among older women who did not have prior chemotherapy , the effect of goserelin depended on whether they took tamoxifen ( hr for the effect of goserelin for older women who did or did not receive tamoxifen was 1.06 [ 95% ci = 0.75 to 1.52 ] and 0.60 [ 95% ci = 0.39 to 0.93 ] , respectively ) . the effect of goserelin was greatest among women who were node negative , those with er - positive tumors , and those who had received no prior chemotherapy ( although age and tamoxifen use influenced this ) . however , when patients were stratified by nodal status and prior chemotherapy , there was still a tendency for the effect of goserelin to be greater among node - negative women , although those who were node positive who had prior chemotherapy did not appear to benefit from goserelin ( supplementary table 5 , available online ) . although our earlier data were included in the overview ( 4 ) , our results from the long - term follow - up ( average follow - up is 12 years ) add further information on the effect of lhrh agonists and allow estimates of the absolute risk difference at 10 and 15 years after the start of treatment . in addition to information on recurrence and death from any cause , we analyzed the effect of goserelin on the chance of dying from breast cancer and the chance of having any event ( recurrence , new tumor , or death ) . our analysis also quantified the long - term effect of goserelin separately among women who did or did not have tamoxifen . because tamoxifen is now offered routinely to many women , it is important to know what the additional benefit of goserelin is 10 and 15 years after the initiation of treatment . among women who did not receive tamoxifen , goserelin was associated with large reductions in the event rate , the chance of dying from any cause or from breast cancer , and the risk of having a recurrence ; the effect remained substantial 15 years later . although the effect of goserelin was smaller ( and not statistically significant ) among women who took tamoxifen , a difference in risk of 23 percentage points in absolute risk at 15 years might be important , given the high incidence of early breast cancer . the effect of goserelin was greatest in women who were node negative and in those with er - positive tumors . data from the int 0101 trial , in which all women received chemotherapy , showed that the combination of 5 years of goserelin and tamoxifen was associated with a 9-year disease - free rate that was 9 and 7 percentage points higher among younger ( < 40 years ) and older ( 40 ) women , respectively , compared with those treated with goserelin alone ( 9 ) . in a trial of 874 women with lymph node negative breast cancer randomly assigned to receive six courses of cmf chemotherapy , goserelin for 2 years , or cmf followed by 18 months of goserelin , a greater proportion of women who received goserelin alone had hot flashes compared with those treated with cmf alone ( 11 ) . in a subgroup of patients randomly assigned to receive goserelin ( n = 53 ) for 2 years or cmf chemotherapy for 6 months ( n = 43 ) , loss of bone mineral density ( bmd ) was greater in the goserelin group ( 13 ) . markers of bone health were not measured in all women in the zipp trial , but the effects of 2 years of treatment with goserelin and tamoxifen on bmd were examined in a subgroup ( n = 89 ) of patients in the stockholm group ( 14 ) . in a trial of 401 patients treated with goserelin , women were randomly assigned to receive 3 years of either tamoxifen or anastrazole , each with or without zoledronic acid ( 15 ) . the effect of zoledronic acid on survival and recurrence has been confirmed in a large trial of 1801 premenopausal women randomly assigned to receive endocrine therapy ( tamoxifen or anastrozole ) , with or without zoledronic acid , with all women receiving goserelin ( 17 ) . the risk of death or recurrence was reduced by 35% ( 95% ci = 8% to 54% ) among women who were given zoledronic acid . in the zipp trial , evidence from a randomized trial of 599 women with node - positive breast cancer indicated that a less frequent schedule ( 3-monthly depot of the lhrh agonist leuprorelin acetate , 11.25 mg ) may be as beneficial as cmf chemotherapy in terms of the effect5-year recurrence - free survival ( hr 0.97 ) and after 2 years os was better in patients given leuprorelin acetate ( hr 0.64 , 95% ci 0.46 to 0.90 ) ( 18 ) . in summary , long - term follow - up of our large trial showed that goserelin had a demonstrable effect on survival and recurrence 15 years after starting treatment and is as effective as tamoxifen when each are given for 2 years . the benefit was greatest among women who did not receive tamoxifen ( among every 100 women treated with goserelin , there could be 8.5 fewer breast cancer deaths ) , and there was a possible gain in those who did receive tamoxifen ( among every 100 women treated with goserelin , there could be 2.6 fewer breast cancer deaths ) .

the analysis was based on a randomized trial with 2 2 factorial assignment of breast cancer patients younger than 50 years to no hormonal therapy or 2 years of treatment with tamoxifen alone , goserelin alone , or goserelin plus tamoxifen . this study quantified the absolute risk reduction of mortality and recurrence conferred by goserelin treatment among women who did and did not take tamoxifen based on long - term follow - up ( mean follow - up time = 11 years ) . ovarian suppression by treatment with goserelin had been shown to reduce the risk of mortality in premenopausal breast cancer patients , but data on the long - term benefit of this treatment were lacking . the analysis was based on a randomized trial with 2 2 factorial assignment of breast cancer patients younger than 50 years to no hormonal therapy or 2 years of treatment with tamoxifen alone , goserelin alone , or goserelin plus tamoxifen . this study quantified the absolute risk reduction of mortality and recurrence conferred by goserelin treatment among women who did and did not take tamoxifen based on long - term follow - up ( mean follow - up time = 11 years ) . the zipp trial was conducted jointly by 4 breast cancer study groups from cancer research uk ( cruk , formerly cancer research campaign ) , gruppo interdisciplinare valutazione interventi in oncologia ( givio ) italy , south east sweden , and stockholm . the aim was to determine whether the addition of goserelin ( zoladex ) to local treatment , with or without tamoxifen , provided a benefit in survival or recurrence among pre- or perimenopausal women with operable , early breast cancer . a total of 2710 premenopausal women were recruited from august 27 , 1987 , to march 22 , 1999 , and randomly assigned to receive goserelin alone ( 3.6-mg subcutaneous depot injection into the abdominal wall every 4 weeks ) , tamoxifen alone ( 20 or 40 mg daily ) , both treatments , or no endocrine therapy ( figure 1 ) . patients in the cruk and south east sweden trials were initially randomly assigned into four arms , but with the publication of the data on tamoxifen in younger patients ( 6 ) , investigators were permitted to give tamoxifen electively , following random assignment to goserelin or no goserelin . this did not affect the results because the numbers of women treated and not treated with tamoxifen were balanced between the trial arms : of those electively given tamoxifen , 432 were randomly assigned to goserelin and 428 to no goserelin , and of those electively not given tamoxifen , 25 were randomly assigned to goserelin and 25 to no goserelin . the numbers of women analyzed in the control , tamoxifen - only , goserelin - only , and both therapies arms were 476 , 879 , 469 , and 882 , respectively ( 2706 in total ) . ( 3 ) risk of recurrence [ recurrence could be local or distant and among 10 women who died from breast cancer with no previously recorded recurrence , it was assumed that they had a distant recurrence , assumed to occur on the date of death ; consistent with the assumptions of the ebctcg overview ( 3 ) ] . in addition , there were 13 women who died from other known causes but in whom breast cancer was present at death ; these women were censored at the date of first recurrence because according to the ebctcg overview ( 3 ) , cause of death may not be reliably recorded after a recurrence . the sample size target was set at 2700 women to detect an absolute difference in the 5-year efs rate of 5% ( 70% vs 75% in the control and treated group , respectively ) , with 83% power and alpha set at .05 . we examined the interaction between goserelin and tamoxifen and whether the effect of goserelin differed between subgroups of patients for four prognostic factors : age ( < 40 or 40 years ) , nodal status ( positive or negative ) , er status ( positive , negative , or unknown ) , and previous adjuvant systemic chemotherapy ( yes or no ) . a total of 2710 premenopausal women were recruited from august 27 , 1987 , to march 22 , 1999 , and randomly assigned to receive goserelin alone ( 3.6-mg subcutaneous depot injection into the abdominal wall every 4 weeks ) , tamoxifen alone ( 20 or 40 mg daily ) , both treatments , or no endocrine therapy ( figure 1 ) . patients in the cruk and south east sweden trials were initially randomly assigned into four arms , but with the publication of the data on tamoxifen in younger patients ( 6 ) , investigators were permitted to give tamoxifen electively , following random assignment to goserelin or no goserelin . this did not affect the results because the numbers of women treated and not treated with tamoxifen were balanced between the trial arms : of those electively given tamoxifen , 432 were randomly assigned to goserelin and 428 to no goserelin , and of those electively not given tamoxifen , 25 were randomly assigned to goserelin and 25 to no goserelin . the numbers of women analyzed in the control , tamoxifen - only , goserelin - only , and both therapies arms were 476 , 879 , 469 , and 882 , respectively ( 2706 in total ) . ( 3 ) risk of recurrence [ recurrence could be local or distant and among 10 women who died from breast cancer with no previously recorded recurrence , it was assumed that they had a distant recurrence , assumed to occur on the date of death ; consistent with the assumptions of the ebctcg overview ( 3 ) ] . in addition , there were 13 women who died from other known causes but in whom breast cancer was present at death ; these women were censored at the date of first recurrence because according to the ebctcg overview ( 3 ) , cause of death may not be reliably recorded after a recurrence . the sample size target was set at 2700 women to detect an absolute difference in the 5-year efs rate of 5% ( 70% vs 75% in the control and treated group , respectively ) , with 83% power and alpha set at .05 . we examined the interaction between goserelin and tamoxifen and whether the effect of goserelin differed between subgroups of patients for four prognostic factors : age ( < 40 or 40 years ) , nodal status ( positive or negative ) , er status ( positive , negative , or unknown ) , and previous adjuvant systemic chemotherapy ( yes or no ) . although age was not prespecified in the original trial protocol , the intention was to include it in the long - term follow - up analysis , particularly because it was examined in detail in the recent lhrh overview ( 4 ) . the numbers of efs events ( recurrence , second primary tumor , or death ) , deaths , breast cancer recurrences , and breast cancer deaths were 1148 , 690 , 941 , and 630 , respectively ( supplementary table 2 , available online ) . goserelin was associated with a risk reduction in all four endpoints : the risk of having an efs event ( hr = 0.82 , 95% confidence interval [ ci ] 0.73 to 0.92 , p = .001 ) , overall mortality ( hr = 0.83 , 95% ci 0.71 to 0.96 , p = .013 ) , the risk of recurrence ( hr = 0.81 , 95% ci 0.71 to 0.92 , p = .001 ) , and breast cancer mortality ( hr = 0.82 , 95% ci 0.70 to 0.96 , p = 0.03 ) . although the point estimates of hr for efs , os , recurrence , and breast cancer mortality in the givio trial appeared to differ from those of the other trials , the confidence intervals contained the overall estimate for each outcome , suggesting that the differences may have been due to chance . a ) the percentages ( 95% confidence interval [ ci ] ) of women treated with goserelin who experienced death , had recurrence , or had a new tumor at 5 , 10 , and 15 years were 25 ( 23 to 27 ) , 38 ( 35 to 41 ) , and 47 ( 43 to 51 ) , respectively ; corresponding percentages in control women were 32 ( 29 to 35 ) , 43 ( 40 to 46 ) , and 52 ( 48 to 55 ) . b ) the percentages ( 95% ci ) of women treated with goserelin who died at 5 , 10 , and 15 years were 12 ( 10 to 14 ) , 21 ( 19 to 23 ) , and 29 ( 26 to 32 ) , respectively ; the corresponding percentages in control women were 15 ( 13 to 17 ) , 26 ( 23 to 28 ) , and 33 ( 30 to 36 ) . c ) the percentages ( 95% ci ) of women treated with goserelin who had a recurrence at 5 , 10 , and 15 years were 22 ( 20 to 24 ) , 31 ( 28 to 34 ) , and 36 ( 33 to 39 ) , respectively ; the corresponding percentages in control women were 27 ( 24 to 29 ) , 37 ( 34 to 40 ) , and 42 ( 39 to 45 ) . d ) the percentages ( 95% ci ) of women treated with goserelin who died from breast cancer at 5 , 10 , and 15 years were 11 ( 9 to 13 ) , 20 ( 18 to 22 ) , and 26 ( 23 to 29 ) , respectively ; the corresponding percentages in control women were 14 ( 12 to 16 ) , 24 ( 22 to 26 ) , and 30 ( 27 to 33 ) . the test for interaction was statistically significant for efs and recurrence , and although the p values were greater than .05 for os and breast cancer mortality , the hrs were in a direction similar to the other endpoints , that is , women who did not receive tamoxifen experienced a greater benefit from goserelin than those who received tamoxifen . thus , in women who did not receive tamoxifen , goserelin was associated with a 33% reduction in the risk of having an efs event , a 29% reduction in risk of overall mortality , a 34% reduction in risk of recurrence , and a 29% reduction in risk of breast cancer mortality . in women who received tamoxifen , there was a much smaller benefit due to goserelin : 8% reduction in risk of efs events , 10% reduction in risk of overall mortality , 9% reduction in risk of recurrence , and 11% reduction in risk of breast cancer mortality . the p values for the test of interaction between goserelin and tamoxifen were .01 ( any event ) , .13 ( death from any cause ) , .016 ( breast cancer recurrence ) , and .17 ( death from breast cancer ) . for example , the estimated efs absolute risk difference at 15 years indicates that for every 100 such women treated with goserelin , there could be 13.9 fewer with an event ( recurrence , new tumor or death ) , compared with those not given goserelin ( risk difference 13.9 percentage points , 95% ci 19.4 to 7.5 ) . with regards to the effect on breast cancer mortality , 12 patients needed to be treated with goserelin ( and not tamoxifen ) to avoid one woman dying ( risk difference 8.5 percentage points , 95% ci 13.7 to 2.2 ) , compared with no goserelin , whereas 38 needed to be treated with both goserelin and tamoxifen to avoid one death ( risk difference 2.6 percentage points , 95% ci 6.6 to 2.1 ) . estimated absolute risk difference ( percentage points ) between goserelin and no goserelin for four outcomes if p is the event rate in the no goserelin arm ( eg , efs rate or risk of recurrence ) at a specified time point , then the absolute difference at this time is p exp[hazard ratio logep ] . among younger women who had prior chemotherapy , the hazard ratio for goserelin treatment ( compared with those not given goserelin ) in relation to breast cancer mortality was 0.66 ( 95% ci 0.44 to 0.98 ) , and this reduction in risk did not seem to depend on whether they took tamoxifen or not ( hrs 0.66 and 0.63 , respectively ) . among older women who had prior chemotherapy , the hr was 0.87 ( 95% ci 0.68 to 1.13 ) , although the effect might be greater when they also took tamoxifen ( hrs of 0.80 and 0.93 , with and without tamoxifen , respectively ) . among older women who did not have prior chemotherapy , the effect of goserelin depended on whether they took tamoxifen ( hr for the effect of goserelin for older women who did or did not receive tamoxifen was 1.06 [ 95% ci = 0.75 to 1.52 ] and 0.60 [ 95% ci = 0.39 to 0.93 ] , respectively ) . however , when patients were stratified by nodal status and prior chemotherapy , there was still a tendency for the effect of goserelin to be greater among node - negative women , although those who were node positive who had prior chemotherapy did not appear to benefit from goserelin ( supplementary table 5 , available online ) . to our knowledge , the zipp trial is the largest that has investigated an lhrh agonist ( 2706 women ) and the only factorial study that has evaluated goserelin and tamoxifen simultaneously with a control group of women who received no endocrine therapy . the main conclusions were that lhrh agonists had an effect on reducing the risk of these events similar to that for chemotherapy ( such as fec or cmf ) , and could be used as an effective treatment in women with er - positive tumors , either alone or in combination with chemotherapy or tamoxifen . although our earlier data were included in the overview ( 4 ) , our results from the long - term follow - up ( average follow - up is 12 years ) add further information on the effect of lhrh agonists and allow estimates of the absolute risk difference at 10 and 15 years after the start of treatment . in addition to information on recurrence and death from any cause , we analyzed the effect of goserelin on the chance of dying from breast cancer and the chance of having any event ( recurrence , new tumor , or death ) . among women who did not receive tamoxifen , goserelin was associated with large reductions in the event rate , the chance of dying from any cause or from breast cancer , and the risk of having a recurrence ; the effect remained substantial 15 years later . although the effect of goserelin was smaller ( and not statistically significant ) among women who took tamoxifen , a difference in risk of 23 percentage points in absolute risk at 15 years might be important , given the high incidence of early breast cancer . data from the int 0101 trial , in which all women received chemotherapy , showed that the combination of 5 years of goserelin and tamoxifen was associated with a 9-year disease - free rate that was 9 and 7 percentage points higher among younger ( < 40 years ) and older ( 40 ) women , respectively , compared with those treated with goserelin alone ( 9 ) . the most common side effect was hot flashes : this was experienced by none of the control patients , 26% of patients treated with goserelin , 17% of those treated with tamoxifen , and 44% of those who received goserelin plus tamoxifen . in a trial of 874 women with lymph node negative breast cancer randomly assigned to receive six courses of cmf chemotherapy , goserelin for 2 years , or cmf followed by 18 months of goserelin , a greater proportion of women who received goserelin alone had hot flashes compared with those treated with cmf alone ( 11 ) . goserelin was also associated with an improvement in quality - of - life measures such as mood , coping effort , tiredness , nausea and/or vomiting , and overall subjective assessment of health , compared with cmf alone ( 11 ) . in a subgroup of patients randomly assigned to receive goserelin ( n = 53 ) for 2 years or cmf chemotherapy for 6 months ( n = 43 ) , loss of bone mineral density ( bmd ) was greater in the goserelin group ( 13 ) . markers of bone health were not measured in all women in the zipp trial , but the effects of 2 years of treatment with goserelin and tamoxifen on bmd were examined in a subgroup ( n = 89 ) of patients in the stockholm group ( 14 ) . the reduction in bmd was 5% in the patients treated with goserelin ( with partial recovery 1 year after stopping therapy ) and 1.4% in the patients treated with tamoxifen and goserelin . a combined interim analysis of two recent randomized trials , z - fast and zo - fast ( total from both trials , n = 1667 ) , in which all women received adjuvant letrozole , compared giving zoledronic acid either at the time of random assignment or only if their bone density fell below a prespecified cutoff or they suffered a nontraumatic fracture ( 16 ) . after 1 year , the score for lumbar spine bmd was 5.2% higher in the patients given zoledronic acid at the time of random assignment , and the total hip score was 3.5% higher . the effect of zoledronic acid on survival and recurrence has been confirmed in a large trial of 1801 premenopausal women randomly assigned to receive endocrine therapy ( tamoxifen or anastrozole ) , with or without zoledronic acid , with all women receiving goserelin ( 17 ) . in the zipp trial , evidence from a randomized trial of 599 women with node - positive breast cancer indicated that a less frequent schedule ( 3-monthly depot of the lhrh agonist leuprorelin acetate , 11.25 mg ) may be as beneficial as cmf chemotherapy in terms of the effect5-year recurrence - free survival ( hr 0.97 ) and after 2 years os was better in patients given leuprorelin acetate ( hr 0.64 , 95% ci 0.46 to 0.90 ) ( 18 ) . in summary , long - term follow - up of our large trial showed that goserelin had a demonstrable effect on survival and recurrence 15 years after starting treatment and is as effective as tamoxifen when each are given for 2 years . the benefit was greatest among women who did not receive tamoxifen ( among every 100 women treated with goserelin , there could be 8.5 fewer breast cancer deaths ) , and there was a possible gain in those who did receive tamoxifen ( among every 100 women treated with goserelin , there could be 2.6 fewer breast cancer deaths ) .

stable transfectants , the lac repressor expression vector , p3ss ( stratagene , la jolla , ca ) , was transfected into nih3t3 cells by calcium phosphate coprecipitation . stable transfectants were selected in 250 g / ml hygromycin and clonal cell lines were isolated . lac repressor expression was measured on western blots reacted to anti - lac repressor antibody ( stratagene ) . cell lines expressing high levels of lac repressor were transfected with the isopropyl -d - thiogalactopyranoside ( iptg ) inducible pop13 ( stratagene ) plasmid containing the human rnase l cdna in the sense orientation , cloned into the not 1 site . stable transfectants were selected in 500 g / ml g418 and clonal cell lines were isolated for analysis . for rnase l activity measurements , after indicated treatments , cells were washed with pbs and then incubated in serum - containing medium for an additional 3.5 h before harvesting . total cellular rna was isolated using trizol ( gibco brl , gaithersburg , md ) and 5 g was electrophoresed on 1.4% agarose , stained with ethidium bromide , and visualized under uv fluorescence . alternatively , rna ( 10 g ) was separated by glyoxal - agarose gel electrophoresis and then transferred to nytran membranes ( schleicher and schuell , inc . , keene , nh ) before hybridization to p - labeled 18s rrna cdna ( 12 ) . the cdna probe was radiolabeled with -p - deoxycytidine 5triphosphate using prime - a - gene system ( promega corp . , cells were cultured overnight at 5 10 cells / ml in eagle 's minimum essential medium ( emem ) , containing 10% fcs , penicillin / streptomycin , gentamycin , and for stably transfected cells 500 g / ml g418 ( gibco brl ) and 250 g / ml hygromycin . for protein synthesis inhibition assays , cells , in 96-well flat - bottomed plates , were incubated in leucine - free rpmi media containing 0.5 ci [ c]leucine for 1.25 h. cells were harvested onto glass fiber filters with a cell harvester and radioactivity was counted . for in situ dna fragmentation end - labeling , cells were cultured to 5 10 cells / well on glass coverslips precoated with poly - l - lysine ( 0.1 mg / ml in distilled water ) . after 24 h of treatment , cells were fixed for 10 min in 4% formaldehyde , washed twice , incubated for 15 min in methanol , and permeabilized in 1% bsa , 0.1% saponin , and 1.5 mg / ml glycine . cells were stained for dna fragmentation using either t7 dna polymerase ( 13 ) or tdt ( 14 ) ( trevigen , gaithersburg , md ) , then counterstained with methyl green . for transient cotransfections with gfp , hela cells and l929 cells were plated at 3 10 cells / well and incubated overnight . the gfp plasmid ( 1 g ) , cloned into pcdna3 ( invitrogen corp . , carlsbad , ca ) , was mixed with 4 g of either empty vector pcdna3 , rnase lzb1 in pcdna1 , or bcl - xl in pcdna3 and 10 l of lipofectamine ( gibco brl ) in a volume of 1 ml/ transfection for 5 h. for viral studies , poliovirus type i at 10 pfu / well was added to hela cells 36 h after transfection and was allowed to adsorb for 30 min . after incubation , cells were washed twice and incubated in serum - containing media . cells expressing gfp were counted at each of the indicated time points using fluorescent microscopy and calculated as a percentage of untreated gfp - positive cells ( 15 ) . to investigate the potential role of rnase l in apoptosis , we generated nih3t3 cell lines stably transfected with the human rnase l gene under the control of an iptg - inducible lac promoter . one representative clone of seven independent clones characterized , 3t3/rnasels , expressed the human rnase l protein constitutively ( fig . 1 a , lane 2 ) and 24 h after induction with iptg , expression levels increased by severalfold ( fig . 1 a , lane 3 ) . to examine rnase l activity in the 3t3/rnasels cells , the rnase l activator , trimeric 25a [ ppp5(a2p5)2a ] was introduced into 3t3/rnasels cells . specific 18s and 28s rrna degradation products , characteristic of 25a - dependent rnase l cleavage ( 16 ) , were detected in uninduced 3t3/rnasels cells ( fig . 1 b and c , lane 2 ) and this ribonuclease activity increased after iptg induction ( fig . 1 b and c , lane 3 ) . degradation of 28s rrna , a characteristic of human rnase l ( 16 ) , was not observed in 3t3/neo control cells expressing only the endogenous murine rnase l ( data not shown ) or in 3t3/rnasels cells induced by iptg in the absence of 25a . active human rnase l is expressed and inducible in 3t3/rnasels cells . in the absence of iptg , 3t3/rnasels cells grew more slowly than control 3t3/neo cells ( table 1 ) . the rate of [ h]thymidine incorporation was identical in the two cell lines ( table 1 ) , suggesting that the 3t3/rnasels cells , chronically expressing human rnase l , had a higher cell death rate rather than a slower cell division rate . l in 3t3/rnasels cells by exposure to iptg for 72 h decreased the protein synthesis rate to 20% of uninduced cells ( fig . 2 ) and decreased cell viability measured by trypan blue dye exclusion to 38% . similar results were found in two of two independent clones examined . using in situ dna fragmentation detection ( 13 , 14 ) ( fig . 3 ) , we quantitated the number of cells that died with the characteristic dna cleavage of apoptosis . the 3t3/ rnasels fibroblasts spontaneously underwent apoptosis at a low rate ( 02.2% ) and induction of higher levels of rnase l expression for 48 h increased the number of cells staining for dna fragmentation to 8.813.5% of the total cells in four separate experiments ( p < 0.003 ) . vector control cell lines displayed either considerably less or undetectable apoptosis either in the absence ( 0 cells of 1,116 counted ) or presence ( 0 cells of 1,105 counted ) of iptg ( fig . 3 , a and c ) . interestingly , almost all of the 3t3/ rnasels cells had pyknotic nuclei when stained with methyl green after induction of high rnase l levels with iptg whereas vector control cells did not show the pyknotic morphology upon iptg induction ( fig . 3 , c and d ) . thus , increasing rnase l expression increases the rate of nih3t3 cell apoptosis . the only known biological activity of 25a is to bind rnase l. rnase l binds 25a with high specificity , resulting in a large increase in enzyme activity . trimeric 25a ( ppp5a2p5a2p5a ) introduced into l929 cells directly triggered apoptosis whereas mock transfection of cells did not ( table 2 ) . moreover , apoptosis was not triggered by the structurally related analogue - inhibitor , ppp5a2p5 a2p5u ( 17 ) ( table 2 ) , which can bind to rnase l but is 10-fold less effective as an activator due to the missing n1/n6 domain of the third adenine ring of parent 25a ( 18 ) . these results correlate an increase in rnase l enzymatic activity with an induction in apoptosis . poly ( i)poly ( c ) has been shown to activate oas , to increase levels of endogenous 25a , and to cause death of interferon - treated fibroblasts ( 6 ) . neither interferon nor dsrna alone significantly reduced l929 cell viability or induced apoptosis ( fig . however , the combination of interferon and dsrna caused an 8891% decrease in cell viability and caused a large increase in the number of apoptotic cells ( fig . 4 , c and f ) . to determine whether rnase l was required for this apoptosis pathway , as opposed to the interferon - inducible protein kinase ( 19 ) , we transiently transfected l929 cells with a dominant negative inhibitor of rnase l. the inhibitor is a truncated version of rnase l , designated rnase lzb1 , which lacks 89 cooh - terminal amino acids and lacks ribonuclease activity ( 12 , 20 ) . this truncated protein can function as a potent inhibitor of the catalytic activity of wild - type rnase l both in cell - free systems and in intact cells ( 12 ) . cells expressing the rnase l inhibitor remained viable significantly longer than the vector control cells after interferon and poly ( i)poly ( c ) treatment ( fig . thus , dsrna can directly induce apoptosis in interferon - treated cells and inhibition of rnase l inhibits this cell death . interestingly , transfection of cells with the bcl - xl gene did not protect cells from poly ( i)poly ( c)induced apoptosis ( fig . 5 a ) . either poly ( i)poly ( c ) induces an apoptosis pathway which is not blocked by bcl - xl or it activates apoptosis downstream of the bcl - xl regulatory point . these results suggested that rnase l activity and the 25a pathway may mediate viral - induced apoptosis . we therefore examined poliovirus , a single - stranded rna virus classified in the picornavirus family , which has been shown recently to induce apoptosis in hela cells ( 21 ) . hela cells were transiently transfected with rnase lzb1 , the dominant negative mutant , and then infected with poliovirus . after 45 h , only 22% of the vector control transfected hela cells remained viable , whereas 80% of the rnase lzb1 transfected cells remained viable ( fig . interestingly , and in contrast to poly ( i)poly ( c)induced apoptosis , the poliovirus - induced cell death was blocked by transfection with the bcl - xl gene ( fig . thus , rnase l activity was required for the poliovirus induced , bcl - xl sensitive pathway of apoptosis . rnase l was initially discovered as a mediator of the 25a dependent rna breakdown stimulated by interferon and viral infections ( 4 , 6 ) . however , the precise mechanism for the antiviral effect of the 25a system in vivo is unknown . we show here that overexpression of rnase l triggers apoptosis , that allosteric activation of rnase l with 25a triggers apoptosis , and that a dominant negative inhibitor of rnase l blocks apoptosis induced by both dsrna and poliovirus infection . this suggests that activation of rnase l by viral infection could serve to eliminate infected cells by apoptosis , preventing viral spread through the cell population . this is a particularly intriguing hypothesis because several studies indicate that apoptosis is a cellular mechanism of viral defense ; for example , virus infection itself can trigger apoptosis ( 22 , 23 ) , and many viruses express virulence factors which block apoptosis ( 24 ) . the results presented here suggest that rnase l activates apoptosis of virus - infected cells to mediate the established antiviral activity of the 25a system . to investigate the potential role of rnase l in apoptosis , we generated nih3t3 cell lines stably transfected with the human rnase l gene under the control of an iptg - inducible lac promoter . one representative clone of seven independent clones characterized , 3t3/rnasels , expressed the human rnase l protein constitutively ( fig . 1 a , lane 2 ) and 24 h after induction with iptg , expression levels increased by severalfold ( fig . 1 a , lane 3 ) . to examine rnase l activity in the 3t3/rnasels cells , the rnase l activator , trimeric 25a [ ppp5(a2p5)2a ] was introduced into 3t3/rnasels cells . specific 18s and 28s rrna degradation products , characteristic of 25a - dependent rnase l cleavage ( 16 ) , were detected in uninduced 3t3/rnasels cells ( fig . 1 b and c , lane 2 ) and this ribonuclease activity increased after iptg induction ( fig . 1 b and c , lane 3 ) . degradation of 28s rrna , a characteristic of human rnase l ( 16 ) , was not observed in 3t3/neo control cells expressing only the endogenous murine rnase l ( data not shown ) or in 3t3/rnasels cells induced by iptg in the absence of 25a . active human rnase l is expressed and inducible in 3t3/rnasels cells . in the absence of iptg , 3t3/rnasels cells grew more slowly than control 3t3/neo cells ( table 1 ) . the rate of [ h]thymidine incorporation was identical in the two cell lines ( table 1 ) , suggesting that the 3t3/rnasels cells , chronically expressing human rnase l , had a higher cell death rate rather than a slower cell division rate . l in 3t3/rnasels cells by exposure to iptg for 72 h decreased the protein synthesis rate to 20% of uninduced cells ( fig . 2 ) and decreased cell viability measured by trypan blue dye exclusion to 38% . similar results were found in two of two independent clones examined . using in situ dna fragmentation detection ( 13 , 14 ) ( fig . 3 ) , we quantitated the number of cells that died with the characteristic dna cleavage of apoptosis . the 3t3/ rnasels fibroblasts spontaneously underwent apoptosis at a low rate ( 02.2% ) and induction of higher levels of rnase l expression for 48 h increased the number of cells staining for dna fragmentation to 8.813.5% of the total cells in four separate experiments ( p < 0.003 ) . vector control cell lines displayed either considerably less or undetectable apoptosis either in the absence ( 0 cells of 1,116 counted ) or presence ( 0 cells of 1,105 counted ) of iptg ( fig . 3 , a and c ) . interestingly , almost all of the 3t3/ rnasels cells had pyknotic nuclei when stained with methyl green after induction of high rnase l levels with iptg whereas vector control cells did not show the pyknotic morphology upon iptg induction ( fig . 3 , c and d ) . thus , increasing rnase l expression increases the rate of nih3t3 cell apoptosis . the only known biological activity of 25a is to bind rnase l. rnase l binds 25a with high specificity , resulting in a large increase in enzyme activity . trimeric 25a ( ppp5a2p5a2p5a ) introduced into l929 cells directly triggered apoptosis whereas mock transfection of cells did not ( table 2 ) . moreover , apoptosis was not triggered by the structurally related analogue - inhibitor , ppp5a2p5 a2p5u ( 17 ) ( table 2 ) , which can bind to rnase l but is 10-fold less effective as an activator due to the missing n1/n6 domain of the third adenine ring of parent 25a ( 18 ) . these results correlate an increase in rnase l enzymatic activity with an induction in apoptosis . poly ( i)poly ( c ) has been shown to activate oas , to increase levels of endogenous 25a , and to cause death of interferon - treated fibroblasts ( 6 ) . neither interferon nor dsrna alone significantly reduced l929 cell viability or induced apoptosis ( fig . however , the combination of interferon and dsrna caused an 8891% decrease in cell viability and caused a large increase in the number of apoptotic cells ( fig . whether rnase l was required for this apoptosis pathway , as opposed to the interferon - inducible protein kinase ( 19 ) , we transiently transfected l929 cells with a dominant negative inhibitor of rnase l. the inhibitor is a truncated version of rnase l , designated rnase lzb1 , which lacks 89 cooh - terminal amino acids and lacks ribonuclease activity ( 12 , 20 ) . this truncated protein can function as a potent inhibitor of the catalytic activity of wild - type rnase l both in cell - free systems and in intact cells ( 12 ) . cells expressing the rnase l inhibitor remained viable significantly longer than the vector control cells after interferon and poly ( i)poly ( c ) treatment ( fig . thus , dsrna can directly induce apoptosis in interferon - treated cells and inhibition of rnase l inhibits this cell death . interestingly , transfection of cells with the bcl - xl gene did not protect cells from poly ( i)poly ( c)induced apoptosis ( fig . 5 a ) . either poly ( i)poly ( c ) induces an apoptosis pathway which is not blocked by bcl - xl or it activates apoptosis downstream of the bcl - xl regulatory point . these results suggested that rnase l activity and the 25a pathway may mediate viral - induced apoptosis . we therefore examined poliovirus , a single - stranded rna virus classified in the picornavirus family , which has been shown recently to induce apoptosis in hela cells ( 21 ) . hela cells were transiently transfected with rnase lzb1 , the dominant negative mutant , and then infected with poliovirus . after 45 h , only 22% of the vector control transfected hela cells remained viable , whereas 80% of the rnase lzb1 transfected cells remained viable ( fig . thus , inhibition of rnase l activity blocked apoptosis due to poliovirus . interestingly , and in contrast to poly ( i)poly ( c)induced apoptosis , the poliovirus - induced cell death was blocked by transfection with the bcl - xl gene ( fig . thus , rnase l activity was required for the poliovirus induced , bcl - xl sensitive pathway of apoptosis . rnase l was initially discovered as a mediator of the 25a dependent rna breakdown stimulated by interferon and viral infections ( 4 , 6 ) . however , the precise mechanism for the antiviral effect of the 25a system in vivo is unknown . we show here that overexpression of rnase l triggers apoptosis , that allosteric activation of rnase l with 25a triggers apoptosis , and that a dominant negative inhibitor of rnase l blocks apoptosis induced by both dsrna and poliovirus infection . this suggests that activation of rnase l by viral infection could serve to eliminate infected cells by apoptosis , preventing viral spread through the cell population . this is a particularly intriguing hypothesis because several studies indicate that apoptosis is a cellular mechanism of viral defense ; for example , virus infection itself can trigger apoptosis ( 22 , 23 ) , and many viruses express virulence factors which block apoptosis ( 24 ) . the results presented here suggest that rnase l activates apoptosis of virus - infected cells to mediate the established antiviral activity of the 25a system . a ) western blot analysis was performed for the detection of human rnase l expression in vector control 3t3/neo cells ( lane 1 ) and in nih3t3 cells transfected with a lac inducible vector containing the human rnase l gene ( 3t3/rnasels ) incubated 24 h in the absence ( lane 2 ) or in the presence ( lane 3 ) of 3 mm iptg . western blot analysis was performed as previously described ( 25 ) using a 1:2,500 dilution of a monoclonal antibody specific for the human rnase l enzyme which did not detect the endogenous murine rnase l ( lane 1 ) . proteins were detected using ecl reagents ( amersham , arlington heights , il ) . rna degradation after induction of rnase l. ( b ) 3t3/rnasels cells were incubated in the absence ( lanes 1 and 2 ) and presence ( lane 3 ) of 3 mm iptg for 24 h then transfected with 1 m of ppp5(a2p5)2a ( lanes 2 and 3 ) by calcium phosphate coprecipitation as previously described ( 12 ) . ( c ) northern blot analysis using an 18s rrna probe is shown after incubation of cells in the absence ( lane 2 ) and presence ( lanes 1 and 3 ) of 3 mm iptg for 24 h followed by transfection with 1 m of ppp5(a2p5)2a ( lanes 2 and 3 ) . growth rates of 3t3/neo and 3t3/rnasels cell lines cells were plated at 10 cells / well and immediately after adherence they were washed twice and incubated in leucine - free rpmi media containing 0.5 ci [ h]thymidine for 1.25 h. cells were harvested onto glass fiber filters and radioactivity was determined by scintillation counting . cells were plated at 1.5 10 cells / well and after 24 h of incubation , trypsinized , resuspended in trypan blue dye , and cells excluding dye were counted . after iptg treatment , cellular protein synthesis was determined in 3t3/neo cells ( circles ) and 3t3/rnasels cells ( squares ) and plotted as a percentage of untreated cells . 3t3/neo ( a and c ) and 3t3/rnasels ( b and d ) cells were incubated for 24 h in the absence ( a and b ) and presence ( c and d ) of 3 mm iptg . apoptotic cells were detected in situ by using t7 dna polymerase with methyl green counterstaining . induction of apoptosis by 25a l929 cells were transfected by calcium phosphate coprecipitation for 75 min with a buffer control ( mock ) , with 1 m ppp2a5p2a5pu as a negative control or with 1 m ppp2a5p2a5pa(25a ) . cells were washed , incubated for 24 h , and then processed for apoptosis . apoptosis was measured by counting cells in random fields positive for dna fragmentation detected by tdt in situ labeling . apoptosis of interferon - treated fibroblasts after activation with dsrna . in situ dna end - labeling ( a c ) and hoechst dye no . 33342 staining ( d f ) of l929 cells . ( a and d ) after 48 h incubation in interferon-/ ( 1,000 u / ml ) ( sigma chemical co. , st . ( b and e ) 24 h incubation in synthetic dsrna poly ( i)(c ) ( 25g / ml ) . ( c and f ) , 24 h preincubation in interferon-/ followed by an additional 24 h in poly ( i)poly ( c ) . apoptotic cells were detected in situ by using terminal deoxynucleotidyl transferase with methyl green counterstaining . cells were stained with hoechst dye ( 0.1 mg / ml in pbs for 15 min ) and photographed using fluorescence on an axiovert microscope ( carl zeiss , inc . , thornwood ( g ) l929 cells were transiently cotransfected with the gfp ( green fluorescent protein ) gene and either control vector ( open circles ) or the rnase lzb1 gene ( closed squares ) then incubated in interferon-/ ( 1,000 u / ml ) for 24 h. cells were then treated with poly ( i)poly ( c ) ( 25 g / ml ) and green fluorescent cells were quantified as a measure of cell viability at each of the indicated time points . after transient cotransfection of the gfp gene with control vector , ( gray bars ) , rnase lzb1 ( black bars ) , or bcl - xl ( hatched bars ) genes , l929 cells were treated with ( a ) interferon-/ ( 1,000 u / ml ) and poly ( i)poly ( c ) ( 25 g / ml ) for 24 h and hela cells were treated with ( b ) poliovirus at 10 pfu for 45 h. cell viability was assessed by quantitation of green fluorescent cells and presented as a percentage of untreated transfected cells . hela cells were transiently cotransfected with gfp gene and either control vector ( gray bars ) or the rnase lzb1 gene ( black bars ) . after incubation in 10 pfu type i poliovirus , gfp - positive cells were quantitated at each of the indicated time points . cell numbers were calculated as a percentage of uninfected cells transfected with each of the vectors .

the 25a system contributes to the antiviral effect of interferons through the synthesis of 25a and its activation of the ribonuclease , rnase l. rnase l degrades viral and cellular rna after activation by unique , 25 phosphodiester - linked , oligoadenylates [ 25a , ( pp)p5 a2(p5a2)]n , n 2 . because both the 25a system and apoptosis can serve as viral defense mechanisms and rna degradation occurs during both processes , we investigated the potential role of rnase l in apoptosis . overexpression of human rnase l by an inducible promoter in nih3t3 fibroblasts decreased cell viability and triggered apoptosis . activation of endogenous rnase l , specifically with 25a or with dsrna , induced apoptosis . inhibition of rnase l with a dominant negative mutant suppressed poly ( i)poly ( c)induced apoptosis in interferon - primed fibroblasts . moreover , inhibition of rnase l suppressed apoptosis induced by poliovirus . thus , increased rnase l levels induced apoptosis and inhibition of rnase l activity blocked viral - induced apoptosis . apoptosis may be one of the antiviral mechanisms regulated by the 25a system .

Materials and Methods Results and Discussion Increased RNase L Expression Induces Apoptosis in Fibroblasts. Allosteric Activation of RNase L Induces Apoptosis. Inhibition of RNase L Blocks Poliovirus-induced Apoptosis. Figures and Tables

cell lines expressing high levels of lac repressor were transfected with the isopropyl -d - thiogalactopyranoside ( iptg ) inducible pop13 ( stratagene ) plasmid containing the human rnase l cdna in the sense orientation , cloned into the not 1 site . for rnase l activity measurements , after indicated treatments , cells were washed with pbs and then incubated in serum - containing medium for an additional 3.5 h before harvesting . for protein synthesis inhibition assays , cells , in 96-well flat - bottomed plates , were incubated in leucine - free rpmi media containing 0.5 ci [ c]leucine for 1.25 h. cells were harvested onto glass fiber filters with a cell harvester and radioactivity was counted . cells expressing gfp were counted at each of the indicated time points using fluorescent microscopy and calculated as a percentage of untreated gfp - positive cells ( 15 ) . to investigate the potential role of rnase l in apoptosis , we generated nih3t3 cell lines stably transfected with the human rnase l gene under the control of an iptg - inducible lac promoter . one representative clone of seven independent clones characterized , 3t3/rnasels , expressed the human rnase l protein constitutively ( fig . to examine rnase l activity in the 3t3/rnasels cells , the rnase l activator , trimeric 25a [ ppp5(a2p5)2a ] was introduced into 3t3/rnasels cells . specific 18s and 28s rrna degradation products , characteristic of 25a - dependent rnase l cleavage ( 16 ) , were detected in uninduced 3t3/rnasels cells ( fig . degradation of 28s rrna , a characteristic of human rnase l ( 16 ) , was not observed in 3t3/neo control cells expressing only the endogenous murine rnase l ( data not shown ) or in 3t3/rnasels cells induced by iptg in the absence of 25a . active human rnase l is expressed and inducible in 3t3/rnasels cells . the rate of [ h]thymidine incorporation was identical in the two cell lines ( table 1 ) , suggesting that the 3t3/rnasels cells , chronically expressing human rnase l , had a higher cell death rate rather than a slower cell division rate . l in 3t3/rnasels cells by exposure to iptg for 72 h decreased the protein synthesis rate to 20% of uninduced cells ( fig . 2 ) and decreased cell viability measured by trypan blue dye exclusion to 38% . the 3t3/ rnasels fibroblasts spontaneously underwent apoptosis at a low rate ( 02.2% ) and induction of higher levels of rnase l expression for 48 h increased the number of cells staining for dna fragmentation to 8.813.5% of the total cells in four separate experiments ( p < 0.003 ) . interestingly , almost all of the 3t3/ rnasels cells had pyknotic nuclei when stained with methyl green after induction of high rnase l levels with iptg whereas vector control cells did not show the pyknotic morphology upon iptg induction ( fig . thus , increasing rnase l expression increases the rate of nih3t3 cell apoptosis . the only known biological activity of 25a is to bind rnase l. rnase l binds 25a with high specificity , resulting in a large increase in enzyme activity . trimeric 25a ( ppp5a2p5a2p5a ) introduced into l929 cells directly triggered apoptosis whereas mock transfection of cells did not ( table 2 ) . moreover , apoptosis was not triggered by the structurally related analogue - inhibitor , ppp5a2p5 a2p5u ( 17 ) ( table 2 ) , which can bind to rnase l but is 10-fold less effective as an activator due to the missing n1/n6 domain of the third adenine ring of parent 25a ( 18 ) . these results correlate an increase in rnase l enzymatic activity with an induction in apoptosis . poly ( i)poly ( c ) has been shown to activate oas , to increase levels of endogenous 25a , and to cause death of interferon - treated fibroblasts ( 6 ) . neither interferon nor dsrna alone significantly reduced l929 cell viability or induced apoptosis ( fig . however , the combination of interferon and dsrna caused an 8891% decrease in cell viability and caused a large increase in the number of apoptotic cells ( fig . to determine whether rnase l was required for this apoptosis pathway , as opposed to the interferon - inducible protein kinase ( 19 ) , we transiently transfected l929 cells with a dominant negative inhibitor of rnase l. the inhibitor is a truncated version of rnase l , designated rnase lzb1 , which lacks 89 cooh - terminal amino acids and lacks ribonuclease activity ( 12 , 20 ) . this truncated protein can function as a potent inhibitor of the catalytic activity of wild - type rnase l both in cell - free systems and in intact cells ( 12 ) . cells expressing the rnase l inhibitor remained viable significantly longer than the vector control cells after interferon and poly ( i)poly ( c ) treatment ( fig . thus , dsrna can directly induce apoptosis in interferon - treated cells and inhibition of rnase l inhibits this cell death . interestingly , transfection of cells with the bcl - xl gene did not protect cells from poly ( i)poly ( c)induced apoptosis ( fig . either poly ( i)poly ( c ) induces an apoptosis pathway which is not blocked by bcl - xl or it activates apoptosis downstream of the bcl - xl regulatory point . these results suggested that rnase l activity and the 25a pathway may mediate viral - induced apoptosis . hela cells were transiently transfected with rnase lzb1 , the dominant negative mutant , and then infected with poliovirus . interestingly , and in contrast to poly ( i)poly ( c)induced apoptosis , the poliovirus - induced cell death was blocked by transfection with the bcl - xl gene ( fig . thus , rnase l activity was required for the poliovirus induced , bcl - xl sensitive pathway of apoptosis . rnase l was initially discovered as a mediator of the 25a dependent rna breakdown stimulated by interferon and viral infections ( 4 , 6 ) . however , the precise mechanism for the antiviral effect of the 25a system in vivo is unknown . we show here that overexpression of rnase l triggers apoptosis , that allosteric activation of rnase l with 25a triggers apoptosis , and that a dominant negative inhibitor of rnase l blocks apoptosis induced by both dsrna and poliovirus infection . this suggests that activation of rnase l by viral infection could serve to eliminate infected cells by apoptosis , preventing viral spread through the cell population . this is a particularly intriguing hypothesis because several studies indicate that apoptosis is a cellular mechanism of viral defense ; for example , virus infection itself can trigger apoptosis ( 22 , 23 ) , and many viruses express virulence factors which block apoptosis ( 24 ) . the results presented here suggest that rnase l activates apoptosis of virus - infected cells to mediate the established antiviral activity of the 25a system . to investigate the potential role of rnase l in apoptosis , we generated nih3t3 cell lines stably transfected with the human rnase l gene under the control of an iptg - inducible lac promoter . one representative clone of seven independent clones characterized , 3t3/rnasels , expressed the human rnase l protein constitutively ( fig . to examine rnase l activity in the 3t3/rnasels cells , the rnase l activator , trimeric 25a [ ppp5(a2p5)2a ] was introduced into 3t3/rnasels cells . specific 18s and 28s rrna degradation products , characteristic of 25a - dependent rnase l cleavage ( 16 ) , were detected in uninduced 3t3/rnasels cells ( fig . degradation of 28s rrna , a characteristic of human rnase l ( 16 ) , was not observed in 3t3/neo control cells expressing only the endogenous murine rnase l ( data not shown ) or in 3t3/rnasels cells induced by iptg in the absence of 25a . active human rnase l is expressed and inducible in 3t3/rnasels cells . the rate of [ h]thymidine incorporation was identical in the two cell lines ( table 1 ) , suggesting that the 3t3/rnasels cells , chronically expressing human rnase l , had a higher cell death rate rather than a slower cell division rate . 2 ) and decreased cell viability measured by trypan blue dye exclusion to 38% . 3 ) , we quantitated the number of cells that died with the characteristic dna cleavage of apoptosis . the 3t3/ rnasels fibroblasts spontaneously underwent apoptosis at a low rate ( 02.2% ) and induction of higher levels of rnase l expression for 48 h increased the number of cells staining for dna fragmentation to 8.813.5% of the total cells in four separate experiments ( p < 0.003 ) . interestingly , almost all of the 3t3/ rnasels cells had pyknotic nuclei when stained with methyl green after induction of high rnase l levels with iptg whereas vector control cells did not show the pyknotic morphology upon iptg induction ( fig . thus , increasing rnase l expression increases the rate of nih3t3 cell apoptosis . the only known biological activity of 25a is to bind rnase l. rnase l binds 25a with high specificity , resulting in a large increase in enzyme activity . trimeric 25a ( ppp5a2p5a2p5a ) introduced into l929 cells directly triggered apoptosis whereas mock transfection of cells did not ( table 2 ) . moreover , apoptosis was not triggered by the structurally related analogue - inhibitor , ppp5a2p5 a2p5u ( 17 ) ( table 2 ) , which can bind to rnase l but is 10-fold less effective as an activator due to the missing n1/n6 domain of the third adenine ring of parent 25a ( 18 ) . these results correlate an increase in rnase l enzymatic activity with an induction in apoptosis . poly ( i)poly ( c ) has been shown to activate oas , to increase levels of endogenous 25a , and to cause death of interferon - treated fibroblasts ( 6 ) . neither interferon nor dsrna alone significantly reduced l929 cell viability or induced apoptosis ( fig . however , the combination of interferon and dsrna caused an 8891% decrease in cell viability and caused a large increase in the number of apoptotic cells ( fig . whether rnase l was required for this apoptosis pathway , as opposed to the interferon - inducible protein kinase ( 19 ) , we transiently transfected l929 cells with a dominant negative inhibitor of rnase l. the inhibitor is a truncated version of rnase l , designated rnase lzb1 , which lacks 89 cooh - terminal amino acids and lacks ribonuclease activity ( 12 , 20 ) . this truncated protein can function as a potent inhibitor of the catalytic activity of wild - type rnase l both in cell - free systems and in intact cells ( 12 ) . cells expressing the rnase l inhibitor remained viable significantly longer than the vector control cells after interferon and poly ( i)poly ( c ) treatment ( fig . thus , dsrna can directly induce apoptosis in interferon - treated cells and inhibition of rnase l inhibits this cell death . interestingly , transfection of cells with the bcl - xl gene did not protect cells from poly ( i)poly ( c)induced apoptosis ( fig . either poly ( i)poly ( c ) induces an apoptosis pathway which is not blocked by bcl - xl or it activates apoptosis downstream of the bcl - xl regulatory point . these results suggested that rnase l activity and the 25a pathway may mediate viral - induced apoptosis . hela cells were transiently transfected with rnase lzb1 , the dominant negative mutant , and then infected with poliovirus . after 45 h , only 22% of the vector control transfected hela cells remained viable , whereas 80% of the rnase lzb1 transfected cells remained viable ( fig . thus , inhibition of rnase l activity blocked apoptosis due to poliovirus . interestingly , and in contrast to poly ( i)poly ( c)induced apoptosis , the poliovirus - induced cell death was blocked by transfection with the bcl - xl gene ( fig . thus , rnase l activity was required for the poliovirus induced , bcl - xl sensitive pathway of apoptosis . rnase l was initially discovered as a mediator of the 25a dependent rna breakdown stimulated by interferon and viral infections ( 4 , 6 ) . however , the precise mechanism for the antiviral effect of the 25a system in vivo is unknown . we show here that overexpression of rnase l triggers apoptosis , that allosteric activation of rnase l with 25a triggers apoptosis , and that a dominant negative inhibitor of rnase l blocks apoptosis induced by both dsrna and poliovirus infection . this suggests that activation of rnase l by viral infection could serve to eliminate infected cells by apoptosis , preventing viral spread through the cell population . this is a particularly intriguing hypothesis because several studies indicate that apoptosis is a cellular mechanism of viral defense ; for example , virus infection itself can trigger apoptosis ( 22 , 23 ) , and many viruses express virulence factors which block apoptosis ( 24 ) . the results presented here suggest that rnase l activates apoptosis of virus - infected cells to mediate the established antiviral activity of the 25a system . a ) western blot analysis was performed for the detection of human rnase l expression in vector control 3t3/neo cells ( lane 1 ) and in nih3t3 cells transfected with a lac inducible vector containing the human rnase l gene ( 3t3/rnasels ) incubated 24 h in the absence ( lane 2 ) or in the presence ( lane 3 ) of 3 mm iptg . western blot analysis was performed as previously described ( 25 ) using a 1:2,500 dilution of a monoclonal antibody specific for the human rnase l enzyme which did not detect the endogenous murine rnase l ( lane 1 ) . rna degradation after induction of rnase l. ( b ) 3t3/rnasels cells were incubated in the absence ( lanes 1 and 2 ) and presence ( lane 3 ) of 3 mm iptg for 24 h then transfected with 1 m of ppp5(a2p5)2a ( lanes 2 and 3 ) by calcium phosphate coprecipitation as previously described ( 12 ) . induction of apoptosis by 25a l929 cells were transfected by calcium phosphate coprecipitation for 75 min with a buffer control ( mock ) , with 1 m ppp2a5p2a5pu as a negative control or with 1 m ppp2a5p2a5pa(25a ) . apoptosis of interferon - treated fibroblasts after activation with dsrna . ( b and e ) 24 h incubation in synthetic dsrna poly ( i)(c ) ( 25g / ml ) . ( c and f ) , 24 h preincubation in interferon-/ followed by an additional 24 h in poly ( i)poly ( c ) . , thornwood ( g ) l929 cells were transiently cotransfected with the gfp ( green fluorescent protein ) gene and either control vector ( open circles ) or the rnase lzb1 gene ( closed squares ) then incubated in interferon-/ ( 1,000 u / ml ) for 24 h. cells were then treated with poly ( i)poly ( c ) ( 25 g / ml ) and green fluorescent cells were quantified as a measure of cell viability at each of the indicated time points . after transient cotransfection of the gfp gene with control vector , ( gray bars ) , rnase lzb1 ( black bars ) , or bcl - xl ( hatched bars ) genes , l929 cells were treated with ( a ) interferon-/ ( 1,000 u / ml ) and poly ( i)poly ( c ) ( 25 g / ml ) for 24 h and hela cells were treated with ( b ) poliovirus at 10 pfu for 45 h. cell viability was assessed by quantitation of green fluorescent cells and presented as a percentage of untreated transfected cells . cell numbers were calculated as a percentage of uninfected cells transfected with each of the vectors .

cell lines expressing high levels of lac repressor were transfected with the isopropyl -d - thiogalactopyranoside ( iptg ) inducible pop13 ( stratagene ) plasmid containing the human rnase l cdna in the sense orientation , cloned into the not 1 site . , cells were cultured overnight at 5 10 cells / ml in eagle 's minimum essential medium ( emem ) , containing 10% fcs , penicillin / streptomycin , gentamycin , and for stably transfected cells 500 g / ml g418 ( gibco brl ) and 250 g / ml hygromycin . for protein synthesis inhibition assays , cells , in 96-well flat - bottomed plates , were incubated in leucine - free rpmi media containing 0.5 ci [ c]leucine for 1.25 h. cells were harvested onto glass fiber filters with a cell harvester and radioactivity was counted . for in situ dna fragmentation end - labeling , cells were cultured to 5 10 cells / well on glass coverslips precoated with poly - l - lysine ( 0.1 mg / ml in distilled water ) . after 24 h of treatment , cells were fixed for 10 min in 4% formaldehyde , washed twice , incubated for 15 min in methanol , and permeabilized in 1% bsa , 0.1% saponin , and 1.5 mg / ml glycine . , carlsbad , ca ) , was mixed with 4 g of either empty vector pcdna3 , rnase lzb1 in pcdna1 , or bcl - xl in pcdna3 and 10 l of lipofectamine ( gibco brl ) in a volume of 1 ml/ transfection for 5 h. for viral studies , poliovirus type i at 10 pfu / well was added to hela cells 36 h after transfection and was allowed to adsorb for 30 min . to investigate the potential role of rnase l in apoptosis , we generated nih3t3 cell lines stably transfected with the human rnase l gene under the control of an iptg - inducible lac promoter . one representative clone of seven independent clones characterized , 3t3/rnasels , expressed the human rnase l protein constitutively ( fig . 1 a , lane 2 ) and 24 h after induction with iptg , expression levels increased by severalfold ( fig . to examine rnase l activity in the 3t3/rnasels cells , the rnase l activator , trimeric 25a [ ppp5(a2p5)2a ] was introduced into 3t3/rnasels cells . specific 18s and 28s rrna degradation products , characteristic of 25a - dependent rnase l cleavage ( 16 ) , were detected in uninduced 3t3/rnasels cells ( fig . 1 b and c , lane 2 ) and this ribonuclease activity increased after iptg induction ( fig . degradation of 28s rrna , a characteristic of human rnase l ( 16 ) , was not observed in 3t3/neo control cells expressing only the endogenous murine rnase l ( data not shown ) or in 3t3/rnasels cells induced by iptg in the absence of 25a . active human rnase l is expressed and inducible in 3t3/rnasels cells . in the absence of iptg , 3t3/rnasels cells grew more slowly than control 3t3/neo cells ( table 1 ) . the rate of [ h]thymidine incorporation was identical in the two cell lines ( table 1 ) , suggesting that the 3t3/rnasels cells , chronically expressing human rnase l , had a higher cell death rate rather than a slower cell division rate . l in 3t3/rnasels cells by exposure to iptg for 72 h decreased the protein synthesis rate to 20% of uninduced cells ( fig . 3 ) , we quantitated the number of cells that died with the characteristic dna cleavage of apoptosis . the 3t3/ rnasels fibroblasts spontaneously underwent apoptosis at a low rate ( 02.2% ) and induction of higher levels of rnase l expression for 48 h increased the number of cells staining for dna fragmentation to 8.813.5% of the total cells in four separate experiments ( p < 0.003 ) . vector control cell lines displayed either considerably less or undetectable apoptosis either in the absence ( 0 cells of 1,116 counted ) or presence ( 0 cells of 1,105 counted ) of iptg ( fig . interestingly , almost all of the 3t3/ rnasels cells had pyknotic nuclei when stained with methyl green after induction of high rnase l levels with iptg whereas vector control cells did not show the pyknotic morphology upon iptg induction ( fig . thus , increasing rnase l expression increases the rate of nih3t3 cell apoptosis . the only known biological activity of 25a is to bind rnase l. rnase l binds 25a with high specificity , resulting in a large increase in enzyme activity . moreover , apoptosis was not triggered by the structurally related analogue - inhibitor , ppp5a2p5 a2p5u ( 17 ) ( table 2 ) , which can bind to rnase l but is 10-fold less effective as an activator due to the missing n1/n6 domain of the third adenine ring of parent 25a ( 18 ) . these results correlate an increase in rnase l enzymatic activity with an induction in apoptosis . poly ( i)poly ( c ) has been shown to activate oas , to increase levels of endogenous 25a , and to cause death of interferon - treated fibroblasts ( 6 ) . neither interferon nor dsrna alone significantly reduced l929 cell viability or induced apoptosis ( fig . however , the combination of interferon and dsrna caused an 8891% decrease in cell viability and caused a large increase in the number of apoptotic cells ( fig . to determine whether rnase l was required for this apoptosis pathway , as opposed to the interferon - inducible protein kinase ( 19 ) , we transiently transfected l929 cells with a dominant negative inhibitor of rnase l. the inhibitor is a truncated version of rnase l , designated rnase lzb1 , which lacks 89 cooh - terminal amino acids and lacks ribonuclease activity ( 12 , 20 ) . this truncated protein can function as a potent inhibitor of the catalytic activity of wild - type rnase l both in cell - free systems and in intact cells ( 12 ) . cells expressing the rnase l inhibitor remained viable significantly longer than the vector control cells after interferon and poly ( i)poly ( c ) treatment ( fig . thus , dsrna can directly induce apoptosis in interferon - treated cells and inhibition of rnase l inhibits this cell death . interestingly , transfection of cells with the bcl - xl gene did not protect cells from poly ( i)poly ( c)induced apoptosis ( fig . these results suggested that rnase l activity and the 25a pathway may mediate viral - induced apoptosis . we therefore examined poliovirus , a single - stranded rna virus classified in the picornavirus family , which has been shown recently to induce apoptosis in hela cells ( 21 ) . after 45 h , only 22% of the vector control transfected hela cells remained viable , whereas 80% of the rnase lzb1 transfected cells remained viable ( fig . interestingly , and in contrast to poly ( i)poly ( c)induced apoptosis , the poliovirus - induced cell death was blocked by transfection with the bcl - xl gene ( fig . thus , rnase l activity was required for the poliovirus induced , bcl - xl sensitive pathway of apoptosis . however , the precise mechanism for the antiviral effect of the 25a system in vivo is unknown . we show here that overexpression of rnase l triggers apoptosis , that allosteric activation of rnase l with 25a triggers apoptosis , and that a dominant negative inhibitor of rnase l blocks apoptosis induced by both dsrna and poliovirus infection . this suggests that activation of rnase l by viral infection could serve to eliminate infected cells by apoptosis , preventing viral spread through the cell population . this is a particularly intriguing hypothesis because several studies indicate that apoptosis is a cellular mechanism of viral defense ; for example , virus infection itself can trigger apoptosis ( 22 , 23 ) , and many viruses express virulence factors which block apoptosis ( 24 ) . the results presented here suggest that rnase l activates apoptosis of virus - infected cells to mediate the established antiviral activity of the 25a system . to investigate the potential role of rnase l in apoptosis , we generated nih3t3 cell lines stably transfected with the human rnase l gene under the control of an iptg - inducible lac promoter . one representative clone of seven independent clones characterized , 3t3/rnasels , expressed the human rnase l protein constitutively ( fig . 1 a , lane 2 ) and 24 h after induction with iptg , expression levels increased by severalfold ( fig . to examine rnase l activity in the 3t3/rnasels cells , the rnase l activator , trimeric 25a [ ppp5(a2p5)2a ] was introduced into 3t3/rnasels cells . specific 18s and 28s rrna degradation products , characteristic of 25a - dependent rnase l cleavage ( 16 ) , were detected in uninduced 3t3/rnasels cells ( fig . degradation of 28s rrna , a characteristic of human rnase l ( 16 ) , was not observed in 3t3/neo control cells expressing only the endogenous murine rnase l ( data not shown ) or in 3t3/rnasels cells induced by iptg in the absence of 25a . in the absence of iptg , 3t3/rnasels cells grew more slowly than control 3t3/neo cells ( table 1 ) . the rate of [ h]thymidine incorporation was identical in the two cell lines ( table 1 ) , suggesting that the 3t3/rnasels cells , chronically expressing human rnase l , had a higher cell death rate rather than a slower cell division rate . l in 3t3/rnasels cells by exposure to iptg for 72 h decreased the protein synthesis rate to 20% of uninduced cells ( fig . 3 ) , we quantitated the number of cells that died with the characteristic dna cleavage of apoptosis . the 3t3/ rnasels fibroblasts spontaneously underwent apoptosis at a low rate ( 02.2% ) and induction of higher levels of rnase l expression for 48 h increased the number of cells staining for dna fragmentation to 8.813.5% of the total cells in four separate experiments ( p < 0.003 ) . vector control cell lines displayed either considerably less or undetectable apoptosis either in the absence ( 0 cells of 1,116 counted ) or presence ( 0 cells of 1,105 counted ) of iptg ( fig . interestingly , almost all of the 3t3/ rnasels cells had pyknotic nuclei when stained with methyl green after induction of high rnase l levels with iptg whereas vector control cells did not show the pyknotic morphology upon iptg induction ( fig . moreover , apoptosis was not triggered by the structurally related analogue - inhibitor , ppp5a2p5 a2p5u ( 17 ) ( table 2 ) , which can bind to rnase l but is 10-fold less effective as an activator due to the missing n1/n6 domain of the third adenine ring of parent 25a ( 18 ) . poly ( i)poly ( c ) has been shown to activate oas , to increase levels of endogenous 25a , and to cause death of interferon - treated fibroblasts ( 6 ) . however , the combination of interferon and dsrna caused an 8891% decrease in cell viability and caused a large increase in the number of apoptotic cells ( fig . whether rnase l was required for this apoptosis pathway , as opposed to the interferon - inducible protein kinase ( 19 ) , we transiently transfected l929 cells with a dominant negative inhibitor of rnase l. the inhibitor is a truncated version of rnase l , designated rnase lzb1 , which lacks 89 cooh - terminal amino acids and lacks ribonuclease activity ( 12 , 20 ) . this truncated protein can function as a potent inhibitor of the catalytic activity of wild - type rnase l both in cell - free systems and in intact cells ( 12 ) . cells expressing the rnase l inhibitor remained viable significantly longer than the vector control cells after interferon and poly ( i)poly ( c ) treatment ( fig . thus , dsrna can directly induce apoptosis in interferon - treated cells and inhibition of rnase l inhibits this cell death . interestingly , transfection of cells with the bcl - xl gene did not protect cells from poly ( i)poly ( c)induced apoptosis ( fig . we therefore examined poliovirus , a single - stranded rna virus classified in the picornavirus family , which has been shown recently to induce apoptosis in hela cells ( 21 ) . interestingly , and in contrast to poly ( i)poly ( c)induced apoptosis , the poliovirus - induced cell death was blocked by transfection with the bcl - xl gene ( fig . thus , rnase l activity was required for the poliovirus induced , bcl - xl sensitive pathway of apoptosis . however , the precise mechanism for the antiviral effect of the 25a system in vivo is unknown . we show here that overexpression of rnase l triggers apoptosis , that allosteric activation of rnase l with 25a triggers apoptosis , and that a dominant negative inhibitor of rnase l blocks apoptosis induced by both dsrna and poliovirus infection . the results presented here suggest that rnase l activates apoptosis of virus - infected cells to mediate the established antiviral activity of the 25a system . a ) western blot analysis was performed for the detection of human rnase l expression in vector control 3t3/neo cells ( lane 1 ) and in nih3t3 cells transfected with a lac inducible vector containing the human rnase l gene ( 3t3/rnasels ) incubated 24 h in the absence ( lane 2 ) or in the presence ( lane 3 ) of 3 mm iptg . rna degradation after induction of rnase l. ( b ) 3t3/rnasels cells were incubated in the absence ( lanes 1 and 2 ) and presence ( lane 3 ) of 3 mm iptg for 24 h then transfected with 1 m of ppp5(a2p5)2a ( lanes 2 and 3 ) by calcium phosphate coprecipitation as previously described ( 12 ) . ( c ) northern blot analysis using an 18s rrna probe is shown after incubation of cells in the absence ( lane 2 ) and presence ( lanes 1 and 3 ) of 3 mm iptg for 24 h followed by transfection with 1 m of ppp5(a2p5)2a ( lanes 2 and 3 ) . growth rates of 3t3/neo and 3t3/rnasels cell lines cells were plated at 10 cells / well and immediately after adherence they were washed twice and incubated in leucine - free rpmi media containing 0.5 ci [ h]thymidine for 1.25 h. cells were harvested onto glass fiber filters and radioactivity was determined by scintillation counting . 3t3/neo ( a and c ) and 3t3/rnasels ( b and d ) cells were incubated for 24 h in the absence ( a and b ) and presence ( c and d ) of 3 mm iptg . , thornwood ( g ) l929 cells were transiently cotransfected with the gfp ( green fluorescent protein ) gene and either control vector ( open circles ) or the rnase lzb1 gene ( closed squares ) then incubated in interferon-/ ( 1,000 u / ml ) for 24 h. cells were then treated with poly ( i)poly ( c ) ( 25 g / ml ) and green fluorescent cells were quantified as a measure of cell viability at each of the indicated time points . after transient cotransfection of the gfp gene with control vector , ( gray bars ) , rnase lzb1 ( black bars ) , or bcl - xl ( hatched bars ) genes , l929 cells were treated with ( a ) interferon-/ ( 1,000 u / ml ) and poly ( i)poly ( c ) ( 25 g / ml ) for 24 h and hela cells were treated with ( b ) poliovirus at 10 pfu for 45 h. cell viability was assessed by quantitation of green fluorescent cells and presented as a percentage of untreated transfected cells .

patients with copd are characterized by dyspnea and physical exercise intolerance , which impair their ability to participate in physical activities and contribute to poor health - related quality of life ( hrqol).1,2 the effectiveness of pulmonary rehabilitation ( pr ) for these patients has been amply demonstrated,3 regardless of where it is performed ( hospital , outpatient clinic , or home ) . a recent meta - analysis including 733 patients with copd confirmed that home - based pr is a valuable intervention strategy to relieve dyspnea and improve exercise tolerance and hrqol.4 yet , to optimize self - care for these patients , a global management program is recommended that combines physical exercise , therapeutic patient education , and self - management.3,5 although the usual endurance physical exercises ( uepes ) are often part of these global management programs,3 emerging therapies like transcutaneous neuromuscular electrical stimulation ( nmes ) can also be used.3,6 nmes involves the application of a low - level electrical current to targeted muscles through surface cutaneous electrodes , which depolarizes the motor neurons and induces involuntary muscle contractions.7 the main advantages of this therapy are that it does not provoke dyspnea ( which can reinforce sedentary lifestyles),8 it is useful for severely disabled patients with copd,9 and it can be used at home.10 nmes may therefore have the potential to break the vicious circle of negative emotions , unpleasant respiratory sensations , and poor exercise performance . very severe copd improved muscle strength and endurance , exercise tolerance , and perceived dyspnea during daily living activities.10 home - based nmes is thus considered a nonpharmacological treatment for lower limb dysfunction and exercise intolerance in patients with severe or very severe copd . to the best of the authors knowledge , only napolis et al assessed home - based nmes in patients with moderate copd.11 the authors reported no significant improvement in quadriceps strength , symptom - limited peak oxygen uptake , endurance , or 6-minute walking distance after this home - based treatment in patients with moderate the authors concluded that , contrary to previously reported data from more severe patients , home - based nmes was not efficient for improving lower limb dysfunction and exercise intolerance in the group as a whole,11 thus suggesting a possible effect of copd severity . as previously indicated by vivodtzev et al,7 success with nmes may vary with disease severity , with less beneficial effects in patients with moderate however , the effect of home - based nmes on hrqol has not , yet , been examined in patients with moderate copd.10 this retrospective observational study of a routine clinical practice was conducted in the north of france to compare the effects of home - based pr including nmes and pr with uepe on exercise tolerance , anxiety / depression , and hrqol in patients with copd . we hypothesized that home - based nmes would be feasible and effective in improving exercise tolerance , anxiety / depression , and hrqol in patients with copd , but that the degree of improvements would depend on the disease severity.7 between 2012 and 2014 , a home - based pr with nmes was proposed to 71 patients with copd experiencing dyspnea during daily living activities ( group nmes : gnmes ) . nmes was proposed when patient performance of a 6-minute stepper test ( 6mst ) was less than or equal to 250 strokes . patients with higher performances were offered home - based pr with the uepes ( group uepe : guepe ) . one hundred and seventeen patients were eligible for this routine clinical practice . whatever the group , the patients chose home - based pr because of preference and/or because no pr center was nearby . copd was confirmed in each patient by persistent post - bronchodilator airflow limitation ( forced expiratory volume in 1 second / forced vital capacity : fev1/fvc < 0.70 ) , according to rabe et al.12 exclusion criteria were dementia or poorly controlled psychiatric illness , neurological sequelae , or bone and joint diseases preventing physical activity . patients receiving long - term oxygen therapy and/or noninvasive ventilation and/or continuous positive airways pressure and/or with multiple stable comorbidities were included in the study . moreover , six patients in gnmes were dropped from the program for the following reasons : death ( n=2 ) , exacerbation ( n=2 ) , aggravation of ischemic heart disease ( n=1 ) , and deterioration of the general status ( n=1 ) . for guepe , the reasons were : death ( n=1 ) , exacerbation ( n=4 ) , lack of motivation ( n=2 ) , and hospitalization ( n=2 ) . the study protocol was approved by the observational research protocol evaluation committee of the french language society of pulmonology , france . as the study data was collected as part of a pr prior to this retrospective study commencing the observational research protocol evaluation committee advised written informed consent did not need to be obtained from the patients . the intervention program is detailed in figure 1 . briefly , as described elsewhere,5 this individual home - based pr consisted of 90-minute sessions once a week based on an educational needs assessment . it comprised endurance physical exercise for guepe , nmes for gnmes and resumption of the physical activities of daily living , therapeutic patient education , and psychosocial support to facilitate health behavior changes for both the groups . each weekly session was conducted under the direct supervision of a team member , although patients were expected to continue performing endurance physical exercise or nmes on their own on the other days of the week , according to a personalized action plan . this home - based pr lasted for 6 weeks for gnmes13 nmes and 8 weeks for guepe.5,14 in gnmes , the patients self - administered nmes to the bilateral quadriceps with surface electrodes . for practical reasons , quadriceps was selected because deficits in strength of this muscle are frequently observed in the patients with copd.6 a portable , user - friendly , dual - channel nmes stimulator was used ( cefar rehab x2 ; djo incorporated , guildford , uk ) . based on the practical recommendations of gloeckl et al,15 the nmes training program comprised : 1 ) a symmetrical biphasic square pulsed current at 50 hz , 2 ) a duty cycle of 5 seconds on and 8 seconds off , and 3 ) pulses of 300 microseconds . the patients were asked to lie down with the knees positioned at a fixed angle ( ~45 ) and not to contract the muscles during nmes . in guepe , individual endurance exercise on a cycle ergometer was performed at the target heart rate , initially in 10-minute sequences , at least 5 days per week , with the goal of reaching 3045 minutes per sequence , in one or several sessions.5 for both groups , three upper and lower limb muscle strengthening exercises were systematically proposed ( with instruction sheets ) , lasting 1015 minutes per day , using weights and dumbbells and/or elastic bands . warm - up and stretching exercises were also recommended , together with balance exercises whenever necessary . the patients performed the timed up - and - go test,16 which requires no specific equipment and is easy to execute at home , to quantify functional mobility , and the 6mst to evaluate physical exercise capacity . to circumvent the environmental constraints of the 6-minute walking test , which is difficult to carry out in the patient s home , the 6mst was performed as previously described.1719 this field exercise test is feasible for patients with pulmonary disease,14,17,19 and it detects improved functional capacity after pr in patients with copd.5,19 the stepper ( go sport , grenoble , france ) , with a step height fixed at 20 cm , was placed near a wall to support patients if they became unbalanced or exhausted . during the 6mst , the patients could freely regulate their stepping rate to reach the highest number of strokes . at the end of the test , al , a minimal clinically important difference ( mcid ) of 40 steps during 6mst has been proposed.5 each evaluation also included the administration of three questionnaires ( hospital anxiety and depression [ had ] questionnaire,20 maugeri foundation respiratory failure [ mrf-28 ] questionnaire,21,22 and the visual simplified respiratory questionnaire [ vsrq]).23 the had questionnaire determined the patient s psychological state in terms of anxiety and depression.20 this questionnaire has a total of 14 items ( seven items for each psychological state ) , with responses being scored on a scale of 03 ( the higher the score , the higher is the frequency ) . for both scores ( ie , anxiety and depression ) , a change of 1.5 units or greater in magnitude was considered to be the threshold for an mcid.24 the mrf-28 questionnaire is composed of 28 items covering three theoretical components ( daily activity , cognitive function , and false and scored as 1 or 0 , respectively.22 the components and total score are then summed and expressed as a percentage of the maximum possible score . the final scores range from 0 to 100 , higher scores reflecting a higher degree of impairment.22 the mcid of mrf-28 is actually unknown.5,14 the vsrq is a valid and reliable questionnaire to assess hrqol , and it was especially developed for patients with copd.23 it comprises eight visual analog scales ranging from 0 to 10 , with a total score ranging from 0 to 80 . the mcid of vsrq is equal to 3.4 units.23 the questionnaires and exercise tests were chosen in light of recent studies in which home - based pr was evaluated in patients with pulmonary disease.5,14 data are reported as mean and standard deviation . for all data , normal gaussian distributions were verified by the shapiro wilk test and homogeneity of variance by the levene test . when the data did not pass the test for normality and/or homogeneity of variance , they were log - transformed . for anthropometric and spirometric data , a general linear model with a two - way design ( groups : gnmes vs guepe , and severity of airflow limitation : moderate vs severe vs very severe ) was used to compare the groups and examine the effect of the severity of airflow limitation . given the initial difference in 6mst performance between the groups , effects after the intervention period were separately tested in each group ( gnmes or guepe ) from the general linear model ( severity of airflow limitation ) for repeated measures ( before and after the intervention period ) . the sphericity was checked by the mauchley test , and when it was not met , the significance of f - ratios was adjusted according to the greenhouse when significant differences were obtained , a bonferroni post hoc test was conducted . to compare the gnmes and guepe prs , the data after the intervention period were expressed in percentage of baseline values , and then analyzed from the general linear model with a two - way design ( groups and severity ) . statistical significance was set at p<0.05 and all analyses were performed with the statistical package for the social sciences ( release 18.0 ; spss inc . , chicago , il , usa ) . between 2012 and 2014 , a home - based pr with nmes was proposed to 71 patients with copd experiencing dyspnea during daily living activities ( group nmes : gnmes ) . nmes was proposed when patient performance of a 6-minute stepper test ( 6mst ) was less than or equal to 250 strokes . patients with higher performances were offered home - based pr with the uepes ( group uepe : guepe ) . one hundred and seventeen patients were eligible for this routine clinical practice . whatever the group , the patients chose home - based pr because of preference and/or because no pr center was nearby . copd was confirmed in each patient by persistent post - bronchodilator airflow limitation ( forced expiratory volume in 1 second / forced vital capacity : fev1/fvc < 0.70 ) , according to rabe et al.12 exclusion criteria were dementia or poorly controlled psychiatric illness , neurological sequelae , or bone and joint diseases preventing physical activity . patients receiving long - term oxygen therapy and/or noninvasive ventilation and/or continuous positive airways pressure and/or with multiple stable comorbidities were included in the study . moreover , six patients in gnmes were dropped from the program for the following reasons : death ( n=2 ) , exacerbation ( n=2 ) , aggravation of ischemic heart disease ( n=1 ) , and deterioration of the general status ( n=1 ) . for guepe , the reasons were : death ( n=1 ) , exacerbation ( n=4 ) , lack of motivation ( n=2 ) , and hospitalization ( n=2 ) . the study protocol was approved by the observational research protocol evaluation committee of the french language society of pulmonology , france . as the study data was collected as part of a pr prior to this retrospective study commencing the observational research protocol evaluation committee advised written informed consent did not need to be obtained from the patients . the intervention program is detailed in figure 1 . briefly , as described elsewhere,5 this individual home - based pr consisted of 90-minute sessions once a week based on an educational needs assessment . it comprised endurance physical exercise for guepe , nmes for gnmes and resumption of the physical activities of daily living , therapeutic patient education , and psychosocial support to facilitate health behavior changes for both the groups . each weekly session was conducted under the direct supervision of a team member , although patients were expected to continue performing endurance physical exercise or nmes on their own on the other days of the week , according to a personalized action plan . this home - based pr lasted for 6 weeks for gnmes13 nmes and 8 weeks for guepe.5,14 in gnmes , the patients self - administered nmes to the bilateral quadriceps with surface electrodes . for practical reasons quadriceps was selected because deficits in strength of this muscle are frequently observed in the patients with copd.6 a portable , user - friendly , dual - channel nmes stimulator was used ( cefar rehab x2 ; djo incorporated , guildford , uk ) . based on the practical recommendations of gloeckl et al,15 the nmes training program comprised : 1 ) a symmetrical biphasic square pulsed current at 50 hz , 2 ) a duty cycle of 5 seconds on and 8 seconds off , and 3 ) pulses of 300 microseconds . the patients were asked to lie down with the knees positioned at a fixed angle ( ~45 ) and not to contract the muscles during nmes . in guepe , individual endurance exercise on a cycle ergometer was performed at the target heart rate , initially in 10-minute sequences , at least 5 days per week , with the goal of reaching 3045 minutes per sequence , in one or several sessions.5 for both groups , three upper and lower limb muscle strengthening exercises were systematically proposed ( with instruction sheets ) , lasting 1015 minutes per day , using weights and dumbbells and/or elastic bands . warm - up and stretching exercises were also recommended , together with balance exercises whenever necessary . the patients performed the timed up - and - go test,16 which requires no specific equipment and is easy to execute at home , to quantify functional mobility , and the 6mst to evaluate physical exercise capacity . to circumvent the environmental constraints of the 6-minute walking test , which is difficult to carry out in the patient s home , the 6mst was performed as previously described.1719 this field exercise test is feasible for patients with pulmonary disease,14,17,19 and it detects improved functional capacity after pr in patients with copd.5,19 the stepper ( go sport , grenoble , france ) , with a step height fixed at 20 cm , was placed near a wall to support patients if they became unbalanced or exhausted . during the 6mst , the patients could freely regulate their stepping rate to reach the highest number of strokes . at the end of the test , et al , a minimal clinically important difference ( mcid ) of 40 steps during 6mst has been proposed.5 each evaluation also included the administration of three questionnaires ( hospital anxiety and depression [ had ] questionnaire,20 maugeri foundation respiratory failure [ mrf-28 ] questionnaire,21,22 and the visual simplified respiratory questionnaire [ vsrq]).23 the had questionnaire determined the patient s psychological state in terms of anxiety and depression.20 this questionnaire has a total of 14 items ( seven items for each psychological state ) , with responses being scored on a scale of 03 ( the higher the score , the higher is the frequency ) . for both scores ( ie , anxiety and depression ) , a change of 1.5 units or greater in magnitude was considered to be the threshold for an mcid.24 the mrf-28 questionnaire is composed of 28 items covering three theoretical components ( daily activity , cognitive function , and false and scored as 1 or 0 , respectively.22 the components and total score are then summed and expressed as a percentage of the maximum possible score . the final scores range from 0 to 100 , higher scores reflecting a higher degree of impairment.22 the mcid of mrf-28 is actually unknown.5,14 the vsrq is a valid and reliable questionnaire to assess hrqol , and it was especially developed for patients with copd.23 it comprises eight visual analog scales ranging from 0 to 10 , with a total score ranging from 0 to 80 . the mcid of vsrq is equal to 3.4 units.23 the questionnaires and exercise tests were chosen in light of recent studies in which home - based pr was evaluated in patients with pulmonary disease.5,14 normal gaussian distributions were verified by the shapiro wilk test and homogeneity of variance by the levene test . when the data did not pass the test for normality and/or homogeneity of variance , they were log - transformed . for anthropometric and spirometric data , a general linear model with a two - way design ( groups : gnmes vs guepe , and severity of airflow limitation : moderate vs severe vs very severe ) was used to compare the groups and examine the effect of the severity of airflow limitation . given the initial difference in 6mst performance between the groups , effects after the intervention period were separately tested in each group ( gnmes or guepe ) from the general linear model ( severity of airflow limitation ) for repeated measures ( before and after the intervention period ) . the sphericity was checked by the mauchley test , and when it was not met , the significance of f - ratios was adjusted according to the greenhouse geisser procedure or the huyn feldt procedure . when significant differences were obtained , a bonferroni post hoc test was conducted . to compare the gnmes and guepe prs , the data after the intervention period were expressed in percentage of baseline values , and then analyzed from the general linear model with a two - way design ( groups and severity ) . statistical significance was set at p<0.05 and all analyses were performed with the statistical package for the social sciences ( release 18.0 ; spss inc . , chicago , il , usa ) . at baseline , fev1 was 41.1%18.4% and 40.7%17.4% of predicted fev1 in gnmes and guepe , respectively . the severity of airflow limitation in gnmes and guepe was regarded as moderate in 31.0% and 28.2% patients , severe in 31.0% and 40.2% patients , and very severe in 38.0% and 31.6% patients , respectively ( table 1 ) . three or more comorbidities were found for 82% and 76% patients in gnmes and guepe , respectively . the main comorbidities were cardiovascular disease ( hypertensive and ischemic heart disease , arrhythmia , and peripheral artery disease ) , metabolic disease ( obesity , type 2 diabetes , and hypothyroidism ) , rheumatic disease ( osteoporosis and osteoarthritis ) , and anxiety and depression . moreover , the patients with severe copd were older compared with patients with moderate airflow limitation ( p=0.027 ) , regardless of the group ( gnmes or guepe ) . body mass index ( bmi ) was significantly lower in patients with very severe copd , regardless of the group ( p<0.001 ) . the maximum tolerated intensity level from nmes group was 19.56.5 ma during the first week , and 30.110.9 ma during the last week . timed up - and - go and 6mst performances were improved after the intervention period in both the groups ( p0.01 , tables 2 and 3 ) . moreover , the number of strokes for the patients with moderate airflow limitation in guepe was significantly higher than that observed in patients with severe or very severe copd ( p<0.001 , table 3 ) . for 6mst , the number of responders was 62% and 57% in gnmes and guepe , respectively ( figure 2 ) . a significant reduction in the depression score was noted in both the groups ( p<0.05 , tables 2 and 3 ) , and the anxiety score was decreased in guepe after the intervention period ( p=0.001 ) . the number of responders was similar between the groups for anxiety ( gnmes = 45% vs guepe = 43% ) . however , this percentage was higher in guepe compared to gnmes for depression ( 50% vs 45% , figure 2 ) . although overall hrqol was significantly improved in gnmes and guepe ( p<0.05 , tables 2 and 3 ) , only the scores from two subscales of the mrf-28 ( quality of life in daily activity and invalidity ) in guepe were reduced ( p<0.05 ) , with a lower invalidity score for patients with moderate copd ( p<0.05 ) . for the vsrq , the hrqol improved in both the groups ( p<0.05 ) , with similar number of responders in gnmes and guepe ( 66% for both groups , figure 2 ) . table 4 compares the changes in hrqol and performances between the groups ( gnmes and guepe ) according to the severity of airflow limitation . the reduction in the invalidity score of mrf-28 was significantly greater in guepe than in gnmes ( p=0.020 ) . no other significant difference was noted for hrqol or performances between the groups ( table 4 ) . the current study shows that nmes during home - based pr is feasible and effective , and can significantly improve timed up - and - go and 6mst performances , depression , and overall hrqol in copd patients with severe exercise intolerance , regardless of the severity of airflow obstruction . in line with most previous studies , our results showed that nmes at home improves exercise tolerance8,10,13,25 and hrqol in a large population with copd,8,10 including patients with moderate airflow obstruction . napolis et al reported no significant effect of home - based nmes on exercise tolerance in 30 patients with moderate to very severe airflow obstruction,11 which led to the hypothesis that responses might differ according to the copd severity . however , in addition to including a larger sample , the discrepancies between our findings and those of napolis et al might be explained by the population characteristics of our study,11 since gnmes included only patients with severe exercise intolerance ( performing 250 strokes in 6mst ) . thus , the current results suggest that nmes may be recommended even to copd patients with severe exercise intolerance regardless of the airflow obstruction or the value of bmi . our results showed no significant difference between gnmes and guepe in the improvement of performances or hrqol ( except the invalidity score of the mrf-28 ) during home - based pr . consequently , self - monitored nmes seemed to be as effective as the uepes for these copd patients . nevertheless , nmes s main advantage is that it is feasible for severely disabled individuals,9 who are unable to endure exercise . it should be kept in mind that gnmes performed both nmes and voluntary strengthening exercises ( similarly to guepe ) , two training modalities known to increase strength but through different neural mechanisms.26 in healthy human skeletal muscles , the neural adaptations induced by nmes seem to be mainly supraspinal , rather than the spinal and supraspinal adaptations from voluntary strengthening exercises . in severely disabled patients with copd ( performance < 400 m in 6-minute walking test ) , vivodtzev et al showed an increase in muscle strength and endurance ( on the quadriceps and calf muscles ) after an nmes program ( improved by + 11% and + 37% , respectively ; p<0.03).13 they also noted a nonsignificant increase in the walking distance during an endurance shuttle test ( + 174 m ; p=0.08 ) . even more interesting , the authors noted that nmes increased the mid - thigh and calf muscle cross - sectional areas ( improved by + 6% and + 6% , respectively ; p<0.05 ) . furthermore , the atrogin-1 protein content was downregulated after the nmes program , whereas the 70 kda ribosomal s6 kinase content was upregulated . collectively , these major findings suggest that nmes reduces muscle protein degradation and enhances protein synthesis , which promotes a net gain in muscle mass . as the lower limb muscles are particularly vulnerable to atrophy in copd,6 nmes should be recommended especially to severely disabled patients as a way to gain muscle mass . it has been well documented that home - based pr is an equivalent alternative to hospital - based pr in patients with copd.3,4,14,27,28 our study revealed that a personal educational intervention including either self - monitored and home - based pr with nmes ( gnmes ) or the uepes ( guepe ) was efficient to improve exercise tolerance and hrqol . however , based on the literature , we optimized patient care by also including weekly self - management sessions5,29 and motivational interviews.5,30 it has been shown that self - management increases program engagement , improves hrqol , reduces respiratory - related and all - cause hospital admissions , and even improves dyspnea,3133 and that motivational interviews are more efficient when performed individually and repeatedly.34 this global management program combining nmes , therapeutic patient education , and self - management was also effective in copd patients with lower exercise tolerance , regardless of the copd severity . patients were thus not randomized and they were distributed into gnmes or guepe based on their performance in the 6mst . the patients in gnmes thus had lower exercise intolerance ( performing 250 strokes in 6mst ) than those in guepe . a controlled trial randomized for physical capacity regardless of the copd severity is recommended to confirm our results . moreover , because of the retrospective nature of the data analysis , it was not possible to evaluate the compliance of patients in both the groups . the current observational study , in the real life , reveals that nmes significantly improves exercise tolerance , depression , and overall hrqol in patients with copd , regardless of the severity of airflow obstruction . home - based pr including self - monitored nmes seems to be feasible and effective in severely disabled individuals who are unable to perform the uepes .

backgroundthis retrospective , observational study of a routine clinical practice reports the feasibility and efficiency of home - based pulmonary rehabilitation ( pr ) , including transcutaneous neuromuscular electrical stimulation ( nmes ) or usual endurance physical exercise ( uepe ) , on exercise tolerance , anxiety / depression , and health - related quality of life ( hrqol ) in patients with copd.methodsseventy-one patients with copd participated in home - based pr with nmes ( group nmes [ gnmes ] ) , while 117 patients participated in home - based pr with the uepes ( group uepe [ guepe ] ) . nmes was applied for 30 minutes twice a day , every day . the endurance exercises in guepe began with a minimum 10-minute session at least 5 days a week , with the goal being 3045 minutes per session . three upper and lower limb muscle strengthening exercises lasting 1015 minutes were also proposed to both the groups for daily practice . moreover , pr in both the groups included a weekly 90-minute session based on an educational needs assessment . the sessions comprised endurance physical exercise for guepe , nmes for gnmes , resumption of physical daily living activities , therapeutic patient education , and psychosocial support to facilitate health behavior changes . before and after pr , functional mobility and physical exercise capacity , anxiety , depression , and hrqol were evaluated at home.resultsthe study revealed that nmes significantly improved functional mobility ( 18.8% in gnmes and 20.6% in guepe ) , exercise capacity ( + 20.8% in gnmes and + 21.8% in guepe ) , depression ( 15.8% in gnmes and 30.1% in guepe ) , and overall hrqol ( 7.0% in gnmes and 18.5% in guepe ) in the patients with copd , regardless of the group ( gnmes or guepe ) or severity of airflow obstruction . moreover , no significant difference was observed between the groups with respect to these data ( p>0.05).conclusionhome - based pr including self - monitored nmes seems feasible and effective for severely disabled copd patients with severe exercise intolerance .

Introduction Materials and methods Patients Global management program Statistical analysis Results Discussion Conclusion

patients with copd are characterized by dyspnea and physical exercise intolerance , which impair their ability to participate in physical activities and contribute to poor health - related quality of life ( hrqol).1,2 the effectiveness of pulmonary rehabilitation ( pr ) for these patients has been amply demonstrated,3 regardless of where it is performed ( hospital , outpatient clinic , or home ) . a recent meta - analysis including 733 patients with copd confirmed that home - based pr is a valuable intervention strategy to relieve dyspnea and improve exercise tolerance and hrqol.4 yet , to optimize self - care for these patients , a global management program is recommended that combines physical exercise , therapeutic patient education , and self - management.3,5 although the usual endurance physical exercises ( uepes ) are often part of these global management programs,3 emerging therapies like transcutaneous neuromuscular electrical stimulation ( nmes ) can also be used.3,6 nmes involves the application of a low - level electrical current to targeted muscles through surface cutaneous electrodes , which depolarizes the motor neurons and induces involuntary muscle contractions.7 the main advantages of this therapy are that it does not provoke dyspnea ( which can reinforce sedentary lifestyles),8 it is useful for severely disabled patients with copd,9 and it can be used at home.10 nmes may therefore have the potential to break the vicious circle of negative emotions , unpleasant respiratory sensations , and poor exercise performance . very severe copd improved muscle strength and endurance , exercise tolerance , and perceived dyspnea during daily living activities.10 home - based nmes is thus considered a nonpharmacological treatment for lower limb dysfunction and exercise intolerance in patients with severe or very severe copd . to the best of the authors knowledge , only napolis et al assessed home - based nmes in patients with moderate copd.11 the authors reported no significant improvement in quadriceps strength , symptom - limited peak oxygen uptake , endurance , or 6-minute walking distance after this home - based treatment in patients with moderate the authors concluded that , contrary to previously reported data from more severe patients , home - based nmes was not efficient for improving lower limb dysfunction and exercise intolerance in the group as a whole,11 thus suggesting a possible effect of copd severity . as previously indicated by vivodtzev et al,7 success with nmes may vary with disease severity , with less beneficial effects in patients with moderate however , the effect of home - based nmes on hrqol has not , yet , been examined in patients with moderate copd.10 this retrospective observational study of a routine clinical practice was conducted in the north of france to compare the effects of home - based pr including nmes and pr with uepe on exercise tolerance , anxiety / depression , and hrqol in patients with copd . we hypothesized that home - based nmes would be feasible and effective in improving exercise tolerance , anxiety / depression , and hrqol in patients with copd , but that the degree of improvements would depend on the disease severity.7 between 2012 and 2014 , a home - based pr with nmes was proposed to 71 patients with copd experiencing dyspnea during daily living activities ( group nmes : gnmes ) . patients with higher performances were offered home - based pr with the uepes ( group uepe : guepe ) . whatever the group , the patients chose home - based pr because of preference and/or because no pr center was nearby . moreover , six patients in gnmes were dropped from the program for the following reasons : death ( n=2 ) , exacerbation ( n=2 ) , aggravation of ischemic heart disease ( n=1 ) , and deterioration of the general status ( n=1 ) . for guepe , the reasons were : death ( n=1 ) , exacerbation ( n=4 ) , lack of motivation ( n=2 ) , and hospitalization ( n=2 ) . briefly , as described elsewhere,5 this individual home - based pr consisted of 90-minute sessions once a week based on an educational needs assessment . it comprised endurance physical exercise for guepe , nmes for gnmes and resumption of the physical activities of daily living , therapeutic patient education , and psychosocial support to facilitate health behavior changes for both the groups . each weekly session was conducted under the direct supervision of a team member , although patients were expected to continue performing endurance physical exercise or nmes on their own on the other days of the week , according to a personalized action plan . this home - based pr lasted for 6 weeks for gnmes13 nmes and 8 weeks for guepe.5,14 in gnmes , the patients self - administered nmes to the bilateral quadriceps with surface electrodes . for practical reasons , quadriceps was selected because deficits in strength of this muscle are frequently observed in the patients with copd.6 a portable , user - friendly , dual - channel nmes stimulator was used ( cefar rehab x2 ; djo incorporated , guildford , uk ) . in guepe , individual endurance exercise on a cycle ergometer was performed at the target heart rate , initially in 10-minute sequences , at least 5 days per week , with the goal of reaching 3045 minutes per sequence , in one or several sessions.5 for both groups , three upper and lower limb muscle strengthening exercises were systematically proposed ( with instruction sheets ) , lasting 1015 minutes per day , using weights and dumbbells and/or elastic bands . the patients performed the timed up - and - go test,16 which requires no specific equipment and is easy to execute at home , to quantify functional mobility , and the 6mst to evaluate physical exercise capacity . to circumvent the environmental constraints of the 6-minute walking test , which is difficult to carry out in the patient s home , the 6mst was performed as previously described.1719 this field exercise test is feasible for patients with pulmonary disease,14,17,19 and it detects improved functional capacity after pr in patients with copd.5,19 the stepper ( go sport , grenoble , france ) , with a step height fixed at 20 cm , was placed near a wall to support patients if they became unbalanced or exhausted . for both scores ( ie , anxiety and depression ) , a change of 1.5 units or greater in magnitude was considered to be the threshold for an mcid.24 the mrf-28 questionnaire is composed of 28 items covering three theoretical components ( daily activity , cognitive function , and false and scored as 1 or 0 , respectively.22 the components and total score are then summed and expressed as a percentage of the maximum possible score . the final scores range from 0 to 100 , higher scores reflecting a higher degree of impairment.22 the mcid of mrf-28 is actually unknown.5,14 the vsrq is a valid and reliable questionnaire to assess hrqol , and it was especially developed for patients with copd.23 it comprises eight visual analog scales ranging from 0 to 10 , with a total score ranging from 0 to 80 . the mcid of vsrq is equal to 3.4 units.23 the questionnaires and exercise tests were chosen in light of recent studies in which home - based pr was evaluated in patients with pulmonary disease.5,14 data are reported as mean and standard deviation . for anthropometric and spirometric data , a general linear model with a two - way design ( groups : gnmes vs guepe , and severity of airflow limitation : moderate vs severe vs very severe ) was used to compare the groups and examine the effect of the severity of airflow limitation . given the initial difference in 6mst performance between the groups , effects after the intervention period were separately tested in each group ( gnmes or guepe ) from the general linear model ( severity of airflow limitation ) for repeated measures ( before and after the intervention period ) . between 2012 and 2014 , a home - based pr with nmes was proposed to 71 patients with copd experiencing dyspnea during daily living activities ( group nmes : gnmes ) . patients with higher performances were offered home - based pr with the uepes ( group uepe : guepe ) . whatever the group , the patients chose home - based pr because of preference and/or because no pr center was nearby . moreover , six patients in gnmes were dropped from the program for the following reasons : death ( n=2 ) , exacerbation ( n=2 ) , aggravation of ischemic heart disease ( n=1 ) , and deterioration of the general status ( n=1 ) . for guepe , the reasons were : death ( n=1 ) , exacerbation ( n=4 ) , lack of motivation ( n=2 ) , and hospitalization ( n=2 ) . briefly , as described elsewhere,5 this individual home - based pr consisted of 90-minute sessions once a week based on an educational needs assessment . it comprised endurance physical exercise for guepe , nmes for gnmes and resumption of the physical activities of daily living , therapeutic patient education , and psychosocial support to facilitate health behavior changes for both the groups . each weekly session was conducted under the direct supervision of a team member , although patients were expected to continue performing endurance physical exercise or nmes on their own on the other days of the week , according to a personalized action plan . this home - based pr lasted for 6 weeks for gnmes13 nmes and 8 weeks for guepe.5,14 in gnmes , the patients self - administered nmes to the bilateral quadriceps with surface electrodes . for practical reasons quadriceps was selected because deficits in strength of this muscle are frequently observed in the patients with copd.6 a portable , user - friendly , dual - channel nmes stimulator was used ( cefar rehab x2 ; djo incorporated , guildford , uk ) . in guepe , individual endurance exercise on a cycle ergometer was performed at the target heart rate , initially in 10-minute sequences , at least 5 days per week , with the goal of reaching 3045 minutes per sequence , in one or several sessions.5 for both groups , three upper and lower limb muscle strengthening exercises were systematically proposed ( with instruction sheets ) , lasting 1015 minutes per day , using weights and dumbbells and/or elastic bands . the patients performed the timed up - and - go test,16 which requires no specific equipment and is easy to execute at home , to quantify functional mobility , and the 6mst to evaluate physical exercise capacity . to circumvent the environmental constraints of the 6-minute walking test , which is difficult to carry out in the patient s home , the 6mst was performed as previously described.1719 this field exercise test is feasible for patients with pulmonary disease,14,17,19 and it detects improved functional capacity after pr in patients with copd.5,19 the stepper ( go sport , grenoble , france ) , with a step height fixed at 20 cm , was placed near a wall to support patients if they became unbalanced or exhausted . at the end of the test , et al , a minimal clinically important difference ( mcid ) of 40 steps during 6mst has been proposed.5 each evaluation also included the administration of three questionnaires ( hospital anxiety and depression [ had ] questionnaire,20 maugeri foundation respiratory failure [ mrf-28 ] questionnaire,21,22 and the visual simplified respiratory questionnaire [ vsrq]).23 the had questionnaire determined the patient s psychological state in terms of anxiety and depression.20 this questionnaire has a total of 14 items ( seven items for each psychological state ) , with responses being scored on a scale of 03 ( the higher the score , the higher is the frequency ) . for both scores ( ie , anxiety and depression ) , a change of 1.5 units or greater in magnitude was considered to be the threshold for an mcid.24 the mrf-28 questionnaire is composed of 28 items covering three theoretical components ( daily activity , cognitive function , and false and scored as 1 or 0 , respectively.22 the components and total score are then summed and expressed as a percentage of the maximum possible score . the final scores range from 0 to 100 , higher scores reflecting a higher degree of impairment.22 the mcid of mrf-28 is actually unknown.5,14 the vsrq is a valid and reliable questionnaire to assess hrqol , and it was especially developed for patients with copd.23 it comprises eight visual analog scales ranging from 0 to 10 , with a total score ranging from 0 to 80 . the mcid of vsrq is equal to 3.4 units.23 the questionnaires and exercise tests were chosen in light of recent studies in which home - based pr was evaluated in patients with pulmonary disease.5,14 normal gaussian distributions were verified by the shapiro wilk test and homogeneity of variance by the levene test . for anthropometric and spirometric data , a general linear model with a two - way design ( groups : gnmes vs guepe , and severity of airflow limitation : moderate vs severe vs very severe ) was used to compare the groups and examine the effect of the severity of airflow limitation . given the initial difference in 6mst performance between the groups , effects after the intervention period were separately tested in each group ( gnmes or guepe ) from the general linear model ( severity of airflow limitation ) for repeated measures ( before and after the intervention period ) . at baseline , fev1 was 41.1%18.4% and 40.7%17.4% of predicted fev1 in gnmes and guepe , respectively . the severity of airflow limitation in gnmes and guepe was regarded as moderate in 31.0% and 28.2% patients , severe in 31.0% and 40.2% patients , and very severe in 38.0% and 31.6% patients , respectively ( table 1 ) . three or more comorbidities were found for 82% and 76% patients in gnmes and guepe , respectively . moreover , the patients with severe copd were older compared with patients with moderate airflow limitation ( p=0.027 ) , regardless of the group ( gnmes or guepe ) . body mass index ( bmi ) was significantly lower in patients with very severe copd , regardless of the group ( p<0.001 ) . timed up - and - go and 6mst performances were improved after the intervention period in both the groups ( p0.01 , tables 2 and 3 ) . moreover , the number of strokes for the patients with moderate airflow limitation in guepe was significantly higher than that observed in patients with severe or very severe copd ( p<0.001 , table 3 ) . a significant reduction in the depression score was noted in both the groups ( p<0.05 , tables 2 and 3 ) , and the anxiety score was decreased in guepe after the intervention period ( p=0.001 ) . the number of responders was similar between the groups for anxiety ( gnmes = 45% vs guepe = 43% ) . although overall hrqol was significantly improved in gnmes and guepe ( p<0.05 , tables 2 and 3 ) , only the scores from two subscales of the mrf-28 ( quality of life in daily activity and invalidity ) in guepe were reduced ( p<0.05 ) , with a lower invalidity score for patients with moderate copd ( p<0.05 ) . for the vsrq , the hrqol improved in both the groups ( p<0.05 ) , with similar number of responders in gnmes and guepe ( 66% for both groups , figure 2 ) . table 4 compares the changes in hrqol and performances between the groups ( gnmes and guepe ) according to the severity of airflow limitation . no other significant difference was noted for hrqol or performances between the groups ( table 4 ) . the current study shows that nmes during home - based pr is feasible and effective , and can significantly improve timed up - and - go and 6mst performances , depression , and overall hrqol in copd patients with severe exercise intolerance , regardless of the severity of airflow obstruction . in line with most previous studies , our results showed that nmes at home improves exercise tolerance8,10,13,25 and hrqol in a large population with copd,8,10 including patients with moderate airflow obstruction . napolis et al reported no significant effect of home - based nmes on exercise tolerance in 30 patients with moderate to very severe airflow obstruction,11 which led to the hypothesis that responses might differ according to the copd severity . however , in addition to including a larger sample , the discrepancies between our findings and those of napolis et al might be explained by the population characteristics of our study,11 since gnmes included only patients with severe exercise intolerance ( performing 250 strokes in 6mst ) . thus , the current results suggest that nmes may be recommended even to copd patients with severe exercise intolerance regardless of the airflow obstruction or the value of bmi . our results showed no significant difference between gnmes and guepe in the improvement of performances or hrqol ( except the invalidity score of the mrf-28 ) during home - based pr . consequently , self - monitored nmes seemed to be as effective as the uepes for these copd patients . in severely disabled patients with copd ( performance < 400 m in 6-minute walking test ) , vivodtzev et al showed an increase in muscle strength and endurance ( on the quadriceps and calf muscles ) after an nmes program ( improved by + 11% and + 37% , respectively ; p<0.03).13 they also noted a nonsignificant increase in the walking distance during an endurance shuttle test ( + 174 m ; p=0.08 ) . it has been well documented that home - based pr is an equivalent alternative to hospital - based pr in patients with copd.3,4,14,27,28 our study revealed that a personal educational intervention including either self - monitored and home - based pr with nmes ( gnmes ) or the uepes ( guepe ) was efficient to improve exercise tolerance and hrqol . however , based on the literature , we optimized patient care by also including weekly self - management sessions5,29 and motivational interviews.5,30 it has been shown that self - management increases program engagement , improves hrqol , reduces respiratory - related and all - cause hospital admissions , and even improves dyspnea,3133 and that motivational interviews are more efficient when performed individually and repeatedly.34 this global management program combining nmes , therapeutic patient education , and self - management was also effective in copd patients with lower exercise tolerance , regardless of the copd severity . patients were thus not randomized and they were distributed into gnmes or guepe based on their performance in the 6mst . the patients in gnmes thus had lower exercise intolerance ( performing 250 strokes in 6mst ) than those in guepe . moreover , because of the retrospective nature of the data analysis , it was not possible to evaluate the compliance of patients in both the groups . the current observational study , in the real life , reveals that nmes significantly improves exercise tolerance , depression , and overall hrqol in patients with copd , regardless of the severity of airflow obstruction . home - based pr including self - monitored nmes seems to be feasible and effective in severely disabled individuals who are unable to perform the uepes .

patients with copd are characterized by dyspnea and physical exercise intolerance , which impair their ability to participate in physical activities and contribute to poor health - related quality of life ( hrqol).1,2 the effectiveness of pulmonary rehabilitation ( pr ) for these patients has been amply demonstrated,3 regardless of where it is performed ( hospital , outpatient clinic , or home ) . a recent meta - analysis including 733 patients with copd confirmed that home - based pr is a valuable intervention strategy to relieve dyspnea and improve exercise tolerance and hrqol.4 yet , to optimize self - care for these patients , a global management program is recommended that combines physical exercise , therapeutic patient education , and self - management.3,5 although the usual endurance physical exercises ( uepes ) are often part of these global management programs,3 emerging therapies like transcutaneous neuromuscular electrical stimulation ( nmes ) can also be used.3,6 nmes involves the application of a low - level electrical current to targeted muscles through surface cutaneous electrodes , which depolarizes the motor neurons and induces involuntary muscle contractions.7 the main advantages of this therapy are that it does not provoke dyspnea ( which can reinforce sedentary lifestyles),8 it is useful for severely disabled patients with copd,9 and it can be used at home.10 nmes may therefore have the potential to break the vicious circle of negative emotions , unpleasant respiratory sensations , and poor exercise performance . very severe copd improved muscle strength and endurance , exercise tolerance , and perceived dyspnea during daily living activities.10 home - based nmes is thus considered a nonpharmacological treatment for lower limb dysfunction and exercise intolerance in patients with severe or very severe copd . to the best of the authors knowledge , only napolis et al assessed home - based nmes in patients with moderate copd.11 the authors reported no significant improvement in quadriceps strength , symptom - limited peak oxygen uptake , endurance , or 6-minute walking distance after this home - based treatment in patients with moderate the authors concluded that , contrary to previously reported data from more severe patients , home - based nmes was not efficient for improving lower limb dysfunction and exercise intolerance in the group as a whole,11 thus suggesting a possible effect of copd severity . as previously indicated by vivodtzev et al,7 success with nmes may vary with disease severity , with less beneficial effects in patients with moderate however , the effect of home - based nmes on hrqol has not , yet , been examined in patients with moderate copd.10 this retrospective observational study of a routine clinical practice was conducted in the north of france to compare the effects of home - based pr including nmes and pr with uepe on exercise tolerance , anxiety / depression , and hrqol in patients with copd . we hypothesized that home - based nmes would be feasible and effective in improving exercise tolerance , anxiety / depression , and hrqol in patients with copd , but that the degree of improvements would depend on the disease severity.7 between 2012 and 2014 , a home - based pr with nmes was proposed to 71 patients with copd experiencing dyspnea during daily living activities ( group nmes : gnmes ) . moreover , six patients in gnmes were dropped from the program for the following reasons : death ( n=2 ) , exacerbation ( n=2 ) , aggravation of ischemic heart disease ( n=1 ) , and deterioration of the general status ( n=1 ) . for guepe , the reasons were : death ( n=1 ) , exacerbation ( n=4 ) , lack of motivation ( n=2 ) , and hospitalization ( n=2 ) . it comprised endurance physical exercise for guepe , nmes for gnmes and resumption of the physical activities of daily living , therapeutic patient education , and psychosocial support to facilitate health behavior changes for both the groups . each weekly session was conducted under the direct supervision of a team member , although patients were expected to continue performing endurance physical exercise or nmes on their own on the other days of the week , according to a personalized action plan . this home - based pr lasted for 6 weeks for gnmes13 nmes and 8 weeks for guepe.5,14 in gnmes , the patients self - administered nmes to the bilateral quadriceps with surface electrodes . for practical reasons , quadriceps was selected because deficits in strength of this muscle are frequently observed in the patients with copd.6 a portable , user - friendly , dual - channel nmes stimulator was used ( cefar rehab x2 ; djo incorporated , guildford , uk ) . based on the practical recommendations of gloeckl et al,15 the nmes training program comprised : 1 ) a symmetrical biphasic square pulsed current at 50 hz , 2 ) a duty cycle of 5 seconds on and 8 seconds off , and 3 ) pulses of 300 microseconds . in guepe , individual endurance exercise on a cycle ergometer was performed at the target heart rate , initially in 10-minute sequences , at least 5 days per week , with the goal of reaching 3045 minutes per sequence , in one or several sessions.5 for both groups , three upper and lower limb muscle strengthening exercises were systematically proposed ( with instruction sheets ) , lasting 1015 minutes per day , using weights and dumbbells and/or elastic bands . the patients performed the timed up - and - go test,16 which requires no specific equipment and is easy to execute at home , to quantify functional mobility , and the 6mst to evaluate physical exercise capacity . to circumvent the environmental constraints of the 6-minute walking test , which is difficult to carry out in the patient s home , the 6mst was performed as previously described.1719 this field exercise test is feasible for patients with pulmonary disease,14,17,19 and it detects improved functional capacity after pr in patients with copd.5,19 the stepper ( go sport , grenoble , france ) , with a step height fixed at 20 cm , was placed near a wall to support patients if they became unbalanced or exhausted . at the end of the test , al , a minimal clinically important difference ( mcid ) of 40 steps during 6mst has been proposed.5 each evaluation also included the administration of three questionnaires ( hospital anxiety and depression [ had ] questionnaire,20 maugeri foundation respiratory failure [ mrf-28 ] questionnaire,21,22 and the visual simplified respiratory questionnaire [ vsrq]).23 the had questionnaire determined the patient s psychological state in terms of anxiety and depression.20 this questionnaire has a total of 14 items ( seven items for each psychological state ) , with responses being scored on a scale of 03 ( the higher the score , the higher is the frequency ) . for both scores ( ie , anxiety and depression ) , a change of 1.5 units or greater in magnitude was considered to be the threshold for an mcid.24 the mrf-28 questionnaire is composed of 28 items covering three theoretical components ( daily activity , cognitive function , and false and scored as 1 or 0 , respectively.22 the components and total score are then summed and expressed as a percentage of the maximum possible score . the final scores range from 0 to 100 , higher scores reflecting a higher degree of impairment.22 the mcid of mrf-28 is actually unknown.5,14 the vsrq is a valid and reliable questionnaire to assess hrqol , and it was especially developed for patients with copd.23 it comprises eight visual analog scales ranging from 0 to 10 , with a total score ranging from 0 to 80 . the mcid of vsrq is equal to 3.4 units.23 the questionnaires and exercise tests were chosen in light of recent studies in which home - based pr was evaluated in patients with pulmonary disease.5,14 data are reported as mean and standard deviation . for anthropometric and spirometric data , a general linear model with a two - way design ( groups : gnmes vs guepe , and severity of airflow limitation : moderate vs severe vs very severe ) was used to compare the groups and examine the effect of the severity of airflow limitation . given the initial difference in 6mst performance between the groups , effects after the intervention period were separately tested in each group ( gnmes or guepe ) from the general linear model ( severity of airflow limitation ) for repeated measures ( before and after the intervention period ) . the sphericity was checked by the mauchley test , and when it was not met , the significance of f - ratios was adjusted according to the greenhouse when significant differences were obtained , a bonferroni post hoc test was conducted . to compare the gnmes and guepe prs , the data after the intervention period were expressed in percentage of baseline values , and then analyzed from the general linear model with a two - way design ( groups and severity ) . between 2012 and 2014 , a home - based pr with nmes was proposed to 71 patients with copd experiencing dyspnea during daily living activities ( group nmes : gnmes ) . moreover , six patients in gnmes were dropped from the program for the following reasons : death ( n=2 ) , exacerbation ( n=2 ) , aggravation of ischemic heart disease ( n=1 ) , and deterioration of the general status ( n=1 ) . for guepe , the reasons were : death ( n=1 ) , exacerbation ( n=4 ) , lack of motivation ( n=2 ) , and hospitalization ( n=2 ) . it comprised endurance physical exercise for guepe , nmes for gnmes and resumption of the physical activities of daily living , therapeutic patient education , and psychosocial support to facilitate health behavior changes for both the groups . this home - based pr lasted for 6 weeks for gnmes13 nmes and 8 weeks for guepe.5,14 in gnmes , the patients self - administered nmes to the bilateral quadriceps with surface electrodes . for practical reasons quadriceps was selected because deficits in strength of this muscle are frequently observed in the patients with copd.6 a portable , user - friendly , dual - channel nmes stimulator was used ( cefar rehab x2 ; djo incorporated , guildford , uk ) . based on the practical recommendations of gloeckl et al,15 the nmes training program comprised : 1 ) a symmetrical biphasic square pulsed current at 50 hz , 2 ) a duty cycle of 5 seconds on and 8 seconds off , and 3 ) pulses of 300 microseconds . in guepe , individual endurance exercise on a cycle ergometer was performed at the target heart rate , initially in 10-minute sequences , at least 5 days per week , with the goal of reaching 3045 minutes per sequence , in one or several sessions.5 for both groups , three upper and lower limb muscle strengthening exercises were systematically proposed ( with instruction sheets ) , lasting 1015 minutes per day , using weights and dumbbells and/or elastic bands . the patients performed the timed up - and - go test,16 which requires no specific equipment and is easy to execute at home , to quantify functional mobility , and the 6mst to evaluate physical exercise capacity . to circumvent the environmental constraints of the 6-minute walking test , which is difficult to carry out in the patient s home , the 6mst was performed as previously described.1719 this field exercise test is feasible for patients with pulmonary disease,14,17,19 and it detects improved functional capacity after pr in patients with copd.5,19 the stepper ( go sport , grenoble , france ) , with a step height fixed at 20 cm , was placed near a wall to support patients if they became unbalanced or exhausted . at the end of the test , et al , a minimal clinically important difference ( mcid ) of 40 steps during 6mst has been proposed.5 each evaluation also included the administration of three questionnaires ( hospital anxiety and depression [ had ] questionnaire,20 maugeri foundation respiratory failure [ mrf-28 ] questionnaire,21,22 and the visual simplified respiratory questionnaire [ vsrq]).23 the had questionnaire determined the patient s psychological state in terms of anxiety and depression.20 this questionnaire has a total of 14 items ( seven items for each psychological state ) , with responses being scored on a scale of 03 ( the higher the score , the higher is the frequency ) . for both scores ( ie , anxiety and depression ) , a change of 1.5 units or greater in magnitude was considered to be the threshold for an mcid.24 the mrf-28 questionnaire is composed of 28 items covering three theoretical components ( daily activity , cognitive function , and false and scored as 1 or 0 , respectively.22 the components and total score are then summed and expressed as a percentage of the maximum possible score . the final scores range from 0 to 100 , higher scores reflecting a higher degree of impairment.22 the mcid of mrf-28 is actually unknown.5,14 the vsrq is a valid and reliable questionnaire to assess hrqol , and it was especially developed for patients with copd.23 it comprises eight visual analog scales ranging from 0 to 10 , with a total score ranging from 0 to 80 . the mcid of vsrq is equal to 3.4 units.23 the questionnaires and exercise tests were chosen in light of recent studies in which home - based pr was evaluated in patients with pulmonary disease.5,14 normal gaussian distributions were verified by the shapiro wilk test and homogeneity of variance by the levene test . for anthropometric and spirometric data , a general linear model with a two - way design ( groups : gnmes vs guepe , and severity of airflow limitation : moderate vs severe vs very severe ) was used to compare the groups and examine the effect of the severity of airflow limitation . given the initial difference in 6mst performance between the groups , effects after the intervention period were separately tested in each group ( gnmes or guepe ) from the general linear model ( severity of airflow limitation ) for repeated measures ( before and after the intervention period ) . to compare the gnmes and guepe prs , the data after the intervention period were expressed in percentage of baseline values , and then analyzed from the general linear model with a two - way design ( groups and severity ) . the severity of airflow limitation in gnmes and guepe was regarded as moderate in 31.0% and 28.2% patients , severe in 31.0% and 40.2% patients , and very severe in 38.0% and 31.6% patients , respectively ( table 1 ) . the main comorbidities were cardiovascular disease ( hypertensive and ischemic heart disease , arrhythmia , and peripheral artery disease ) , metabolic disease ( obesity , type 2 diabetes , and hypothyroidism ) , rheumatic disease ( osteoporosis and osteoarthritis ) , and anxiety and depression . moreover , the patients with severe copd were older compared with patients with moderate airflow limitation ( p=0.027 ) , regardless of the group ( gnmes or guepe ) . body mass index ( bmi ) was significantly lower in patients with very severe copd , regardless of the group ( p<0.001 ) . moreover , the number of strokes for the patients with moderate airflow limitation in guepe was significantly higher than that observed in patients with severe or very severe copd ( p<0.001 , table 3 ) . a significant reduction in the depression score was noted in both the groups ( p<0.05 , tables 2 and 3 ) , and the anxiety score was decreased in guepe after the intervention period ( p=0.001 ) . although overall hrqol was significantly improved in gnmes and guepe ( p<0.05 , tables 2 and 3 ) , only the scores from two subscales of the mrf-28 ( quality of life in daily activity and invalidity ) in guepe were reduced ( p<0.05 ) , with a lower invalidity score for patients with moderate copd ( p<0.05 ) . for the vsrq , the hrqol improved in both the groups ( p<0.05 ) , with similar number of responders in gnmes and guepe ( 66% for both groups , figure 2 ) . table 4 compares the changes in hrqol and performances between the groups ( gnmes and guepe ) according to the severity of airflow limitation . the current study shows that nmes during home - based pr is feasible and effective , and can significantly improve timed up - and - go and 6mst performances , depression , and overall hrqol in copd patients with severe exercise intolerance , regardless of the severity of airflow obstruction . napolis et al reported no significant effect of home - based nmes on exercise tolerance in 30 patients with moderate to very severe airflow obstruction,11 which led to the hypothesis that responses might differ according to the copd severity . thus , the current results suggest that nmes may be recommended even to copd patients with severe exercise intolerance regardless of the airflow obstruction or the value of bmi . our results showed no significant difference between gnmes and guepe in the improvement of performances or hrqol ( except the invalidity score of the mrf-28 ) during home - based pr . it should be kept in mind that gnmes performed both nmes and voluntary strengthening exercises ( similarly to guepe ) , two training modalities known to increase strength but through different neural mechanisms.26 in healthy human skeletal muscles , the neural adaptations induced by nmes seem to be mainly supraspinal , rather than the spinal and supraspinal adaptations from voluntary strengthening exercises . in severely disabled patients with copd ( performance < 400 m in 6-minute walking test ) , vivodtzev et al showed an increase in muscle strength and endurance ( on the quadriceps and calf muscles ) after an nmes program ( improved by + 11% and + 37% , respectively ; p<0.03).13 they also noted a nonsignificant increase in the walking distance during an endurance shuttle test ( + 174 m ; p=0.08 ) . even more interesting , the authors noted that nmes increased the mid - thigh and calf muscle cross - sectional areas ( improved by + 6% and + 6% , respectively ; p<0.05 ) . it has been well documented that home - based pr is an equivalent alternative to hospital - based pr in patients with copd.3,4,14,27,28 our study revealed that a personal educational intervention including either self - monitored and home - based pr with nmes ( gnmes ) or the uepes ( guepe ) was efficient to improve exercise tolerance and hrqol . however , based on the literature , we optimized patient care by also including weekly self - management sessions5,29 and motivational interviews.5,30 it has been shown that self - management increases program engagement , improves hrqol , reduces respiratory - related and all - cause hospital admissions , and even improves dyspnea,3133 and that motivational interviews are more efficient when performed individually and repeatedly.34 this global management program combining nmes , therapeutic patient education , and self - management was also effective in copd patients with lower exercise tolerance , regardless of the copd severity . the current observational study , in the real life , reveals that nmes significantly improves exercise tolerance , depression , and overall hrqol in patients with copd , regardless of the severity of airflow obstruction .

as genomes have been sequenced and sequence databases have been compiled , these databases have been used to identify proteins present in biological samples using tandem mass spectrometry data . in a process called shotgun proteomics , mixtures of proteins from biological samples are digested to peptides using proteases , and liquid chromatography coupled to tandem mass spectrometry is used to directly identify the peptides present . identification of a peptide is used to infer the presence of the protein it is derived from . as more complex samples , such as cellular components and whole cells , have become targets of analysis , more comprehensive separations have become necessary to resolve complex peptide mixtures and increase dynamic range . giddings described multidimensional chromatography as a means to increase peak capacity by combining orthogonal separations . for the analysis of complex peptide mixtures , several combinations of separation media have been used . the last phase in multidimensional liquid chromatography ( mdlc ) separations used for mass spectrometry is typically reversed - phase ( rp ) , which separates peptides by hydrophobicity and is effective at removing salts or other small molecule contaminants prior to introduction of peptides into the mass spectrometer . many different forms of mdlc have appeared over the years , from combinations of ion exchange ( iex ) with rp separations to combinations of capillary electrophoresis and liquid chromatography . in any multidimensional separation , how material is transferred from one separation stage to another is critical for maximizing peak capacity and optimizing sample recovery . in proteomics , link et al . employed a biphasic column of strong cation exchange ( scx ) and rp packing material in a single column . in this arrangement , a multidimensional separation is created by running buffer containing a set concentration of salt across the column to elute peptides from the scx phase onto the rp column . once the salt pulse is completed , the rp buffer is applied to the column at 0% b to remove salt from the column prior to running the gradient . after a rp gradient is completed , a second salt pulse with a higher concentration of salt is applied to the column to move a new population of peptides to the rp material . the process of salt pulse / rp gradient is repeated until all peptides are removed from the scx phase . a single column contains both the iex phase and the rp phase , and all solvents flow over both phases . the sample is loaded directly onto the column from an eppendorf tube using a pressurized device , and the column is placed in line with the ion source with the voltage placed on a waste line at the backend of the column . this method is commonly referred to as online mdlc and may employ a bi- or triphasic column . this method usually employs scx , but strong anion exchange ( sax ) has also been used . advantages to off - line fractionation include the ability to add a high organic phase to the salt buffer ( e.g. , 25% organic in the iex buffer ) to minimize mixed - mode interactions , the capability to collect many fractions , and the capacity to load large amounts of material onto the column . in a clever use of off - line fractionation , wang et al . showed improvement in peptide identifications by combining fractions from different parts of a high ph rp separation to produce collections of peptides with different physical characteristics like hydrophobicity for the second - dimension , low - ph rp separation . after rp separation , excess solvent in the collected fractions is removed , and each fraction is loaded into an autosampler for introduction into the mass spectrometer . a third strategy for multidimensional separation is also an online method that employs a valving system to direct solvents to an iex column , an enrichment column , a rp column , and waste . this system represents a compromise between the direct online and the off - line approaches . the valving system is used to redirect flow to shunt salt solutions used to elute peptides from an iex column to waste rather than have it run through the rp analytical column , or in the case of a rp - rp lclc system , the valves are used to direct peptides to the enrichment column to alter the ph of the buffer before the analytical separation . sample loading is a critical part of capillary chromatography , as these systems involve small diameter openings that must be aligned and low solvent flows , for which dead space can have a great impact . kennedy and jorgenson developed a pressurized device for both packing and loading capillary columns . the end of the column was placed directly into a slurry of packing material and when the device was pressurized the packing material was driven into the column . this same strategy could be used as a means to load samples directly from eppendorf tubes into a column . this method has been adopted by others as a means to load small quantities of samples . an important consideration when analyzing peptides and proteins is sample loss associated with sample handling . proteins and peptides can easily adhere to surfaces resulting in losses . a carrier protein is often used to minimize adherence to active surfaces during sample manipulations to protect low abundance proteins from losses . an advantage of shotgun proteomics is the manipulation of complex protein mixtures where the more abundant proteins may presumably act as carrier proteins to protect lower abundance proteins from loss . because losses can occur on active surfaces such a glass and metal , efforts two recent papers showed that peptides can be lost when analyzed from autosamplers , and peptide mixtures of intermediate complexity ( in gel digestions ) can be lost to the surface of autosampler vials made from a variety of materials , respectively . tested a variety of carriers to determine which one improved recovery of peptides . as these two papers have shown , and as experience has taught us , the more samples are handled and exposed to surfaces , the greater the loss . this study compares sample losses for a shotgun proteomics experiment using three different methods of sample introduction using two different quantitation methods . hek293 cells ( crl-1573 ) purchased from atcc were seeded into a t25 flask in supplemented media ( dmem medium supplemented with 10% fetal bovine serum , 1% ( v / v ) penicillin / streptomycin , 2 mm l - glutamine , and 200 g / ml g418 ) from ( gibco , invitrogen)),and maintained with regular media changes for 3 weeks before they were considered to be stable cell lines . cultured cells were harvested at 80% confluency with 0.05% trypsin and edta , centrifuged for 5 min at 4000 g at 4 c , and washed twice with pbs . cells were suspended in 8 m urea , 500 mm tris - hcl ph 8.5 supplemented with complete ultra tablets , mini , easypack ( roche , mannheim ) for protein extraction . denaturated protein lysate was precipitated with acetone and assayed using modified bicinchoninic ( bca ) method ( pierce , rockford il ) . resuspended protein was reduced with 5 mm tris(2-carboxyethyl)phosphine ( tcep ) for 30 min . cysteine residues were alkylated with 10 mm iodoacetamide for 20 min in the dark . samples were diluted to a final concentration 2 m urea with 100 mm tris - hcl , ph 8.5 prior to digestion with trypsin . for endopeptidase digestion , modified trypsin ( promega , madison , wi ) was added at 50:1 ( protein / protease mass ratio ) along with 1 mm cacl2 and incubated overnight in a thermoshaker at 600 rpm at 37 c . digested peptide solution was acidified using 90% fa to a final ph of 3.0 . an analytical rplc column was prepared by pulling a 100 m id/360 m od capillary ( polymicro technologies , phoenix , az ) to 5 m i d tip . reversed - phase resin ( aqua c18 , 3 m dia . , 90 pores , phenomenex , torrance , ca ) was packed directly into the pulled column at 700 psi until 12 cm long . the column was washed and equilibrated at 100 bar with buffer b , followed by buffer a. a multiprotein identification technology ( mudpit ) trapping column was prepared by creating a kasil frit at one end of an undeactivated 250 m id/360 m od capillary ( agilent technologies , santa clara , ca ) . the frit was prepared by briefly dipping a 20 cm capillary in well - mixed 300 l of kasil 1624 ( pq corporation , malvern , pa ) and 100 l of formamide , curing at 100 c overnight , and cutting the frit to 1.5 mm in length . triphasic or biphasic columns were successively packed with 2.5 cm scx particles ( partisphere scx , 5 m dia . , 100 pores , phenomenex ) and 2.5 cm rp resin ( aqua c18 , 3 m dia . , 125 pores , phenomenex ) , as shown in figure 1 . peptide samples ( 100 g ) were loaded onto triphasic columns for online mudpit . for offline mudpit , samples were loaded onto a biphasic column and ten scx offline fractions were collected in 1.5 ml eppendorf tubes . fractions were then loaded into a 2.5 rp resin column ( offline mudpit ) or purified by stage tip and placed in autosampler vials ( offline mudpit with autosampler [ easy - nlc ii , thermo ] ) . both mudpit and analytical columns were assembled using a zero - dead volume union ( upchurch scientific , oak harbor , wa ) . ( a ) hek293 protein lysate was digested and processed in three replicate runs with different mudpit panels : online ( automated panel ) , offline ( manual collection of fractions ) , and offline - as ( offline with autosampler where fractions were collected manually then cleaned up with c18 stage tip columns before being placed into autosampler ) . ( b ) offline fractions were collected for 5 min after the volatile salt pulse phase using 10100% ammonium acetate . sixplex tandem mass tag ( tmt ) labeling was performed according to the manufacturer s instructions ( thermo fisher scientific , rockford , il ) . as illustrated in figure 2 , tmt reagents ( 0.8 mg ) were dissolved in 40 l of anhydrous acn ( sigma , milwaukee ) . trypsin - digested hek293 cells samples ( 25 g / tag ) were equilibrated to room temperature for 5 min with occasional vortexing . samples were then resuspended in 100 mm teab and derivatized with sixplex chemical tags : 126 , 127 for online mudpit ; 128 , 129 for offline mudpit ; and 130 and 131 th ( thomson ) for offline mudpit with autosampler . the reaction mixtures were incubated at room temperature for 1 h and quenched with 15 l of 5% hydroxylamine solution in water . equal ratios of tmt- tagged samples were mixed and analyzed prior to fractionation to ensure unbiased and impartial labeling . fractions were massed up to same volume with 100 mm teab and equally combined into one sample before vacuum drying . the lyophilized tmt - labeled peptides ( 25 g ) were reconstituted with 50 l of buffer a ( 0.1% formic acid ( fa ) , 5% acetonitrile ( acn ) in water ) , centrifuged at 12 000 g for 30 min prior to mass spectrometric analysis . protein lysate was labeled in a sixplex tmt format : 126 and 127 for online mudpit , 128 and 129 for the offline mudpit , and 130 and 131 for the offline with autosampler mudpit ( offline - as ) . directly after labeling , an equal volume of the tmt tags was mixed and analyzed to ensure successful labeling ( labeling efficiency averaged 97.2% ) . samples were then processed with different mudpit platforms , massed up to same volume with 100 mm teab , and equally combined into one sample before drying and mass analysis . peptides were separated by an eksigent nanolc-2d system ( eksigent , dublin ) with or without autosampler unit ( 10 l peek sample loop , six - port titanium injection valve , 50 mm sus sample needle , 50 m i d fused silica tubing ) . the hplc system was either connected online or offline to thermo ltq xl ( for label - free quantification ) or ltq - orbitrap velos ( for tmt quantification ) using an in - house built nanoelectrospray stage . electrospray was performed directly from the analytical column by applying the esi voltage at a tee ( 150 m i d , upchurch scientific ) directly downstream of a 1:1000 split flow used to reduce the flow rate to 250 nl / min through the columns . ten - step mudpit experiments were performed either online or offline , with steps corresponding to 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , and 100% buffer c being run for 5 min at the beginning of a 120 min gradient . a three mobile phase system consisting of buffer a ( 5% acn ; 0.1% fa ( sigma - aldrich , st . louis , mo ) ) , buffer b ( 80% acn , 0.1% fa ) , and buffer c ( 500 mm ammonium acetate , 5% acn , 0.1% fa ) was used in the current experiment . the lc system was coupled to 224 nm laser - induced native fluorescence ( linf ) detector with elliptical flow cell for real - time peptide detection . data - dependent acquisition of ms / ms spectra was performed by dynamically choosing up to 5 or 10 most intense precursor ions from the survey scan for ltq xl or ltq orbitrap velos , respectively . , charge 25 , full - scan ms resolution of 30.000 ( ltq xl ) and 60.000 ( ltq orbitrap velos ) with a target value of 1 10 , and the maximal injection time of 200 ms . the lower threshold for targeting a precursor ion in the ms scan was 5000 counts and 2.5 kv maximum injection time for higher - energy collisional dissociation ( hcd)ms / ms analysis in the orbitrap . the hcd dissociation mode enables simultaneous production of tmt reporter ions and fragment ions of the peptides . ms / ms scans were acquired in the orbitrap with a mass resolution of 17 000 . the target value was 30 000 ions with injection time of 150 ms . once analyzed , the selected peptide ions were dynamically excluded from further analysis for 120 s to allow for the selection of lower - abundance ions for subsequent fragmentation and detection using the setting for repeat count = 1 , repeat duration = 30 ms , and exclusion list size = 500 . ions with singly or unassigned charge states were rejected . the m / z isolation width for ms / ms fragmentation was set to 2 th . for tandem mass spectra were extracted from raw files using rawextract 1.9.9 and searched with the prolucid algorithm against homo sapiens uniprot / swiss - prot database with reversed sequences ( 176 708 entries ) . the search space included all fully and semitryptic peptide candidates ( at least six amino acids ) . carbamidomethylation of cysteine ( 57.02146 amu ) was considered as a static modification as well as static n - terminus and lysine modification ( 229.1629 amu ) for sixplex tmt labels analysis . the search parameters include 10 ppm precursor mass tolerance and 0.6 da peptide mass tolerance . exported prolucid files were assembled and filtered using the dtaselect2.0 which combines xcorr and deltacn values using a quadratic discriminate function to compute a confidence score . the false - positive rate ( fdr ) was kept at 1% at the protein level . for quantitative analysis , census was used to extract the relative intensities of reporter ions for each peptide from the identified tandem mass spectra for normalization . the mass tolerance and intensity threshold for the reporter ions in census prolucid xcorr , deltacn , and zscore values were used to generate a bayesian discriminator . outlier points in the two distributions that had a mahalanobis distance greater than four were discarded . for label - free quantification , normalized spectral abundance factor , protein , peptide expression alteration ( fold changes ) , log values , and confidence were calculated based on spectral peak intensities generated from the mass spectrometric analysis after extracting confident protein spectra with p < 0.01 . for tmt analysis , the relative quantification between any experimental groups in the sixplex experiment was derived from the average ratio of the reporter ions of duplicate tags of one group over the average reporter ions of duplicate tags of the corresponding group . statistical computing and graphics were performed in r software environment and graphpad prism 5 . although the use of capillary chromatography has provided tremendous gains in sensitivity , the use of capillary chromatography also introduces sample - loading challenges . kennedy and jorgenson introduced a loading procedure that used a high - pressure device ( bomb ) to transfer samples directly from an eppendorf tube into the capillary column . when the bomb is pressurized , liquid is forced into the column from the eppendorf tube . the capability to analyze small amounts of materials allows access to samples such as biopsy samples , small sections of tissue such as brain sections , or even single - cell analysis such as neurons . as demonstrated by masuda et al . and thakur et al , as sample size decreases , sample handling and the nature of the chromatographic interface become important for good detection limits on peptides . in this study , we tested three methods for introducing complex peptide mixtures into a tandem mass spectrometer . the three multidimensional lc methods consisted of a direct online method using an integrated triphasic capillary column for the introduction of samples and two off - line methods involving collection of samples in eppendorf tubes . one of the off - line methods used an autosampler to introduce the samples into the analytical rp column , and the other used the same direct pressure loading system used to load samples for the online mdlc analysis . , all approaches used the same type of scx column to perform the iex fractionation , which consisted of a biphasic column . in addition , 10 salt steps were used in the online system , and 10 fractions were collected in the two off - line methods using the same elution method . the same analytical rp method was employed . to perform the comparison , a trypsin - digested hek293 sample was used that should be representative of the type of sample commonly analyzed in proteomics . for each method , two different quantitation methods were used to measure differences in peptide recoveries between the different methods . even with the current advanced mass spectrometric capabilities , multiple dimensions of separation improve comprehensive analysis of peptide mixtures . although a variety of multidimensional combinations have emerged in the past few years , the scx - rp combination is an efficient and highly resolving separation method for shotgun proteome analysis . the main impetus of this work is to provide a systematic comparative study of different lc / lc strategies in terms of performance , sensitivity , and recovery that should be applicable to all lc / lc methods regardless of the phases employed proteins from a hek293 cell line were digested with trypsin and aliquots were subjected to analysis by the three different platforms to measure peptide and protein identification efficiency for each method . for this measurement , the goal was simply to report how many peptides and proteins would be detected for the same sample using the different methods ( figure 1 ) . because of the complexity of the sample , 10 salt steps were used to ensure elution of most peptides from the column . three replicate analyses of each platform showed an average of 187 366 8545 , 168 750 3113 , and 140 000 7950 ms2 scans from online , offline , and offline - as groups , respectively . a total of 3383 386 , 2159 243 , and 1877 413 proteins were identified from the 12937 1533 , 7351 1201 , and 7146 829 peptides identified for the online , offline , and offline - as groups , respectively ( figure 3a , b ) . the higher mean of the online triphasic column was statistically significant ( confidence of 95% ) compared with the other methods . given the identical gradients and ms methods used for each approach , the peptide identification and protein identification numbers were similar to an improvement by more than 1.7 to 1.8 fold in the online method compared with both offline methods ( figure 3b ) . additionally , merging and removing redundant proteins / peptides increased protein / peptide identifications most in the online triphasic column method , less in the offline method , and least in the offline - as method . this comparison showed that recovery of peptides was best in the online method by virtue of the most peptide and protein identifications and the poorest in the off - line fractionation method with the introduction of samples through an autosampler . this result makes sense , as the samples being fractionated offline and introduced through an autosampler are being exposed to more new surfaces and are being subjected to the most manipulations . box and whiskers plots of averaged identified proteins ( a ) and peptides ( b ) for different mudpit platforms . a key measure in proteomics is overlap in peptide and protein identifications as a function of technical replicates . reproducibility between protein identifications is expected to be greater because a protein can be identified by different peptides . a high level of reproducibility in peptide identification is harder to achieve because it requires the system be near saturation . a comparison of reproducibility and overlap between the different systems is shown in figure 4 . proportional venn diagram demonstrating the overlap between protein identification of different mudpit triplicate runs ( ( a ) online , ( b ) offline ; and ( c ) offline - as ) or between different mudpit platforms ( d ) . reproducibility between runs was greatest among online replicates ( 60% overlap ) and lowest in the offline analysis coupled to the autosampler ( 45% overlap ) . in addition , a comparison of the three experimental strategies shows online lc / lc identifies more distinct proteins at 1% fdr compared with the other platforms ( figure 4d ) . differences in identification rates among the samples suggest that improved identification rates are a result of minimizing manual handling of fractions . by using an online system sample , losses are decreased , and this leads to improved recovery of peptides through the system and acquisition of more ms / ms . if there is an observed difference in peptide identification between the different methods , it begs the question of whether there are any differences in physicochemical characteristics of the peptides or proteins observed in the different methods . we analyzed the proteome metrics relevant to each experimental platform to determine if there were any peculiar physicochemical characteristics of peptides observed in one platform and not the other . in general , online mudpit showed only a modest increase in protein sequence coverage ( figure 5a ) over the offline methods . spectral count rank , for example , abundance of proteins , however , was significantly lower in the offline - as platform compared with the other methods ( figure 5b ) . because the major difference in the offline - as group is that the final peptide mixture is placed in autosampler vials , it is likely that the lower spectral counts are due to adsorptive loses of analyte on the surface of the polypropylene sample vials or that sample is lost in the flow path of the sample loop . peptide loss in the offline groups did not significantly correlate with pi , salt fraction , or peptide charge ( figure 5c e ) . although we included organic modifier ( 5% v / v acn ) in the iex elution buffer that reportedly reduces surface adsorption , our data suggest a modest loss of hydrophilic proteins in the offline - as group when plotted using bull breese or kyte doolittle scores ( figure 5f , g ) . this finding is in accordance with recent reports , describing a higher adsorptive tendency of soluble peptides to solid surfaces that ultimately affects peptide amount and quantification parameters . ( d ) number of peptides eluted in each salt fraction of the mudpit platforms . ( f ) bull breese hydrophobicity index was calculated based on the free energy of transfer to surface in kcl / mol . ( g ) kyte dolittle hydrophathy scoring ( gravy score ) was calculated based on the average amino acids score for a given protein . positive score is hydrophilic and negative score is hydrophobic in bull breese and vice versa for kyte dolittle . red is online mudpit , blue is offline mudpit , and black is offline - as mudpit . to further illustrate the changes in protein abundance between groups , we performed a statistical comparison of the average spectral count of triplicate runs from each experimental platform . as illustrated in figure 6 , although several proteins were quantified in high abundance between all platforms , we noticed that online proteins were more abundant and statistically more significant ( p 0.01 ) , especially when compared with either of the offline groups ( offline or offline - as ) . this significance was less obvious when comparing between the offline groups ( figure 6c ) . specifically , we found that 635 and 542 proteins were significantly higher in abundance in the online platform compared with offline and offline - as groups by 2.2 0.44 and 1.8 0.52 fold , respectively . label - free quantification based on normalized spectral abundance counts of different mudpit panels . ( a c ) volcano plots depict relationship between the p value and the magnitude of difference ( log2 ) in expression value between average technical replicates of two compared groups . ( d f ) surface 3d plots of the identified proteins in each experimental group based on their relative spectral counts ( % ) , molecular weight ( kd ) , and protein length . to verify our findings with an alternate quantitation method , peptides were labeled with different amine - reactive isobaric tags ( figure 2 ) . the tmt experiment was designed as another way to quantitate the differences between the strategies for lc / lc . a digested hek293 sample was aliquoted into six aliquots of 25 g each . two tagged samples each were used for online separation : two for off - line with pressure bomb loading and two for off - line fractionation with autosampling . the experiments were performed as for the label - free experiments except that the outflow from the rp capillary column was collected into a single tube for each sample . the volumes were adjusted ; then , all samples were combined into a single tube . this method ensures exact comigration with simultaneous and accurate peptide quantification using the mass tags that appears in the tandem mass spectrum . we found that peptide abundance was lower ( p 0.05 ) in the offline groups ( figure 7a c ) . again , this was not restricted to certain peptides properties ; in contrast , the majority of them showed similar trend patterns , denoting stochastic nonspecific loss . in addition , peptide ratios between online and offline groups disclosed a modest skew toward the online platform with a 1318% increase after peptide grouping and normalization ( figure 7d ) . cumulatively , the elevated ion intensity signaling for peptides detected in the automated online method corresponded to an average increase of 18% in protein abundance for more than 1100 proteins ( figure 7e ) . we noticed that the same proteins were underestimated in the offline groups with a significant correlation coefficient factor ( r = 0.76 , p = 0.05 ) when compared with the online platform ( figure 7e ) . ( b ) peptides were plotted with trend line pattern ( dashed red line ) . ( c ) perpendicular 3d plot revealed that most of the identified reporter ions were relatively higher in online mudpit with respect to the other offline formats . ( d ) frequency histogram of mudpit panels showing distribution of log2 peptide ratio observed between compared groups . ( e ) log log correlation plot of protein expression ratio of the online panel over offline panels . black and red dots represents proteins with higher intensities in online module with 1.4 to 2 fold , respectively . ( f ) example spectrum for peptide labeled with tmt isobaric mass tag labeling reagent . the ms / ms fragmentations were used to sequence the peptide . on the basis of the amino acid ladder , the peptide was identified as vnptvffdiavdgeplgr with the n - terminus modified by tmt isobaric mass tag labeling reagent . mass tags ( 126131 ) observed in the lower m / z region ( inserted figure ) indicate the relative abundance of this peptide in each group . the samples were labeled in the following order : online mudpit ( 126 , 127 ) , offline mudpit ( 128 , 129 ) , and offline mudpit with autosampler ( 130 , 131 ) . to answer the question of whether the lowered peptide intensity in the offline methods is due to peptide loss during processing or ion suppression as a result of high background noise ( chemical contaminants , atmospheric sources , or electrical interference ) , we utilized the high sensitivity of the linf detector ( 100 fold than uv detectors ) together with peptide quantification using modified bca method . peptide mapping at 220 nm provided a flat baseline with better sensitivity in the current experiment ( figure 8a ) , and detection of scx eluted peptides before mudpit processing did not show any significant differences between experimental groups ( figure 8b ) even at 280 nm associated with absorption of the aromatic amino acids ( data not shown ) . nevertheless , downstream peptide quantification , directly before mass spectrometry analysis , revealed potential low peptide yield ( p 0.05 ) in the offline methods ( figure 8c ) . a previous report demonstrated that the adsorption of biomolecules such as peptides followed a langmuir isotherm equation and was influenced by both solvents and the nature of the solid surfaces . moreover , we monitored the possible impact of sparse ion noise background on the desired peptide signal peak by calculating signal - to - noise ratio ( s / n ; figure 8d ) based on gygi s method . although most peptides in our analysis were analyzed at low s / n , the consequences of low signal levels on quantitative accuracy remain to be tested . interestingly , online mudpit generated slightly higher background noise compared with offline and offline - as ; the s / n ratio , however , was not significantly different between all platforms ( figure 8d ) , and both ms and ms / ms quality was comparable . this observation could be attributed to the higher signal ion intensity of the online mudpit method that maintained a constant s / n ratio between tested groups . impact of peptide loss and ion suppression of background noise on mudpit platforms . ( b ) real - time monitoring of peptide elution using 224 nm laser - induced native fluorescence ( linf ) detector . ( c ) modified bicinchoninic ( bca ) method for peptide fractionated with different mudpit platforms just before mass spectrometry . ( d ) signal - to - noise ( s / n ) ratio represents the intensity of the signal ( peak height ) to the intensity of the background noise ( in root - mean - square rms ) for mudpit platforms . red is online mudpit , blue is offline mudpit , and black is offline - as mudpit . it has long been known that peptides and proteins can be readily lost to surface adsorption to the materials they come in contact with . this issue was particularly troublesome when trying to purify proteins or peptides to homogeneity for analysis because these methods often required much sample manipulation . ultimately the sample losses associated with gel purification and in - gel - digestion - limited detection of proteins made these methods less attractive . one advantage of shotgun proteomics is the preparation of proteins en masse for analysis by the mass spectrometer . by preparing samples as a complex mixture , the more abundant proteins can act as carriers of the less abundant proteins . after digestion , the complex mixture of peptides needs to be separated by hplc for introduction into the mass spectrometer . several strategies have evolved to fractionate peptide mixtures prior to entry into the mass spectrometer . we demonstrated that the use of automated online mudpit results in more comprehensive peptide separation and substantially more protein and peptide identification in a label - free quantification experiment , although results were less striking for tmt quantification , where the nature of the experimental design may have complicated the comparison . differences attributed to sample loading are alleviated by normalization correction in both experiments , together with comparable ms / ms spectra ( similar s / n ratio ) , so it is likely that stochastic peptide loss due to adsorption could be affecting offline sample collection ( such as tubes and vials ) . certainly , this conclusion does not discourage using offline platforms because each format has its inherent advantages and disadvantages ( i.e. , flexibility of offline fraction collection and reduced labor time in online separation ) . however , because adsorption is a concentration - dependent surface phenomenon , one should critically consider the potential sample loss due to surface adsorption when considering offline fractionation platforms , especially when processing minute quantities of valuable samples .

large - scale proteomics often employs two orthogonal separation methods to fractionate complex peptide mixtures . fractionation can involve ion exchange separation coupled to reversed - phase separation or , more recently , two reversed - phase separations performed at different ph values . when multidimensional separations are combined with tandem mass spectrometry for protein identification , the strategy is often referred to as multidimensional protein identification technology ( mudpit ) . mudpit has been used in either an automated ( online ) or manual ( offline ) format . in this study , we evaluated the performance of different mudpit strategies by both label - free and tandem mass tag ( tmt ) isobaric tagging . our findings revealed that online mudpit provided more peptide / protein identifications and higher sequence coverage than offline platforms . when employing an off - line fractionation method with direct loading of samples onto the column from an eppendorf tube via a high - pressure device , a 5.3% loss in protein identifications is observed . when off - line fractionated samples are loaded via an autosampler , a 44.5% loss in protein identifications is observed compared with direct loading of samples onto a triphasic capillary column . moreover , peptide recovery was significantly lower after offline fractionation than in online fractionation . signal - to - noise ( s / n ) ratio , however , was not significantly altered between experimental groups . it is likely that offline sample collection results in stochastic peptide loss due to noncovalent adsorption to solid surfaces . therefore , the use of the offline approaches should be considered carefully when processing minute quantities of valuable samples .

Introduction Materials and Methods Results and Discussion Conclusions

as genomes have been sequenced and sequence databases have been compiled , these databases have been used to identify proteins present in biological samples using tandem mass spectrometry data . in a process called shotgun proteomics , mixtures of proteins from biological samples are digested to peptides using proteases , and liquid chromatography coupled to tandem mass spectrometry is used to directly identify the peptides present . identification of a peptide is used to infer the presence of the protein it is derived from . as more complex samples , such as cellular components and whole cells , have become targets of analysis , more comprehensive separations have become necessary to resolve complex peptide mixtures and increase dynamic range . for the analysis of complex peptide mixtures , several combinations of separation media have been used . the last phase in multidimensional liquid chromatography ( mdlc ) separations used for mass spectrometry is typically reversed - phase ( rp ) , which separates peptides by hydrophobicity and is effective at removing salts or other small molecule contaminants prior to introduction of peptides into the mass spectrometer . in this arrangement , a multidimensional separation is created by running buffer containing a set concentration of salt across the column to elute peptides from the scx phase onto the rp column . once the salt pulse is completed , the rp buffer is applied to the column at 0% b to remove salt from the column prior to running the gradient . after a rp gradient is completed , a second salt pulse with a higher concentration of salt is applied to the column to move a new population of peptides to the rp material . the sample is loaded directly onto the column from an eppendorf tube using a pressurized device , and the column is placed in line with the ion source with the voltage placed on a waste line at the backend of the column . this method is commonly referred to as online mdlc and may employ a bi- or triphasic column . advantages to off - line fractionation include the ability to add a high organic phase to the salt buffer ( e.g. , 25% organic in the iex buffer ) to minimize mixed - mode interactions , the capability to collect many fractions , and the capacity to load large amounts of material onto the column . in a clever use of off - line fractionation , wang et al . showed improvement in peptide identifications by combining fractions from different parts of a high ph rp separation to produce collections of peptides with different physical characteristics like hydrophobicity for the second - dimension , low - ph rp separation . after rp separation , excess solvent in the collected fractions is removed , and each fraction is loaded into an autosampler for introduction into the mass spectrometer . this system represents a compromise between the direct online and the off - line approaches . the valving system is used to redirect flow to shunt salt solutions used to elute peptides from an iex column to waste rather than have it run through the rp analytical column , or in the case of a rp - rp lclc system , the valves are used to direct peptides to the enrichment column to alter the ph of the buffer before the analytical separation . the end of the column was placed directly into a slurry of packing material and when the device was pressurized the packing material was driven into the column . this method has been adopted by others as a means to load small quantities of samples . because losses can occur on active surfaces such a glass and metal , efforts two recent papers showed that peptides can be lost when analyzed from autosamplers , and peptide mixtures of intermediate complexity ( in gel digestions ) can be lost to the surface of autosampler vials made from a variety of materials , respectively . as these two papers have shown , and as experience has taught us , the more samples are handled and exposed to surfaces , the greater the loss . this study compares sample losses for a shotgun proteomics experiment using three different methods of sample introduction using two different quantitation methods . cells were suspended in 8 m urea , 500 mm tris - hcl ph 8.5 supplemented with complete ultra tablets , mini , easypack ( roche , mannheim ) for protein extraction . reversed - phase resin ( aqua c18 , 3 m dia . the column was washed and equilibrated at 100 bar with buffer b , followed by buffer a. a multiprotein identification technology ( mudpit ) trapping column was prepared by creating a kasil frit at one end of an undeactivated 250 m id/360 m od capillary ( agilent technologies , santa clara , ca ) . for offline mudpit , samples were loaded onto a biphasic column and ten scx offline fractions were collected in 1.5 ml eppendorf tubes . fractions were then loaded into a 2.5 rp resin column ( offline mudpit ) or purified by stage tip and placed in autosampler vials ( offline mudpit with autosampler [ easy - nlc ii , thermo ] ) . ( a ) hek293 protein lysate was digested and processed in three replicate runs with different mudpit panels : online ( automated panel ) , offline ( manual collection of fractions ) , and offline - as ( offline with autosampler where fractions were collected manually then cleaned up with c18 stage tip columns before being placed into autosampler ) . sixplex tandem mass tag ( tmt ) labeling was performed according to the manufacturer s instructions ( thermo fisher scientific , rockford , il ) . protein lysate was labeled in a sixplex tmt format : 126 and 127 for online mudpit , 128 and 129 for the offline mudpit , and 130 and 131 for the offline with autosampler mudpit ( offline - as ) . samples were then processed with different mudpit platforms , massed up to same volume with 100 mm teab , and equally combined into one sample before drying and mass analysis . the hplc system was either connected online or offline to thermo ltq xl ( for label - free quantification ) or ltq - orbitrap velos ( for tmt quantification ) using an in - house built nanoelectrospray stage . the hcd dissociation mode enables simultaneous production of tmt reporter ions and fragment ions of the peptides . for tandem mass spectra were extracted from raw files using rawextract 1.9.9 and searched with the prolucid algorithm against homo sapiens uniprot / swiss - prot database with reversed sequences ( 176 708 entries ) . for label - free quantification , normalized spectral abundance factor , protein , peptide expression alteration ( fold changes ) , log values , and confidence were calculated based on spectral peak intensities generated from the mass spectrometric analysis after extracting confident protein spectra with p < 0.01 . for tmt analysis , the relative quantification between any experimental groups in the sixplex experiment was derived from the average ratio of the reporter ions of duplicate tags of one group over the average reporter ions of duplicate tags of the corresponding group . although the use of capillary chromatography has provided tremendous gains in sensitivity , the use of capillary chromatography also introduces sample - loading challenges . kennedy and jorgenson introduced a loading procedure that used a high - pressure device ( bomb ) to transfer samples directly from an eppendorf tube into the capillary column . when the bomb is pressurized , liquid is forced into the column from the eppendorf tube . and thakur et al , as sample size decreases , sample handling and the nature of the chromatographic interface become important for good detection limits on peptides . in this study , we tested three methods for introducing complex peptide mixtures into a tandem mass spectrometer . the three multidimensional lc methods consisted of a direct online method using an integrated triphasic capillary column for the introduction of samples and two off - line methods involving collection of samples in eppendorf tubes . one of the off - line methods used an autosampler to introduce the samples into the analytical rp column , and the other used the same direct pressure loading system used to load samples for the online mdlc analysis . in addition , 10 salt steps were used in the online system , and 10 fractions were collected in the two off - line methods using the same elution method . to perform the comparison , a trypsin - digested hek293 sample was used that should be representative of the type of sample commonly analyzed in proteomics . although a variety of multidimensional combinations have emerged in the past few years , the scx - rp combination is an efficient and highly resolving separation method for shotgun proteome analysis . the main impetus of this work is to provide a systematic comparative study of different lc / lc strategies in terms of performance , sensitivity , and recovery that should be applicable to all lc / lc methods regardless of the phases employed proteins from a hek293 cell line were digested with trypsin and aliquots were subjected to analysis by the three different platforms to measure peptide and protein identification efficiency for each method . because of the complexity of the sample , 10 salt steps were used to ensure elution of most peptides from the column . the higher mean of the online triphasic column was statistically significant ( confidence of 95% ) compared with the other methods . given the identical gradients and ms methods used for each approach , the peptide identification and protein identification numbers were similar to an improvement by more than 1.7 to 1.8 fold in the online method compared with both offline methods ( figure 3b ) . this comparison showed that recovery of peptides was best in the online method by virtue of the most peptide and protein identifications and the poorest in the off - line fractionation method with the introduction of samples through an autosampler . reproducibility between protein identifications is expected to be greater because a protein can be identified by different peptides . proportional venn diagram demonstrating the overlap between protein identification of different mudpit triplicate runs ( ( a ) online , ( b ) offline ; and ( c ) offline - as ) or between different mudpit platforms ( d ) . reproducibility between runs was greatest among online replicates ( 60% overlap ) and lowest in the offline analysis coupled to the autosampler ( 45% overlap ) . in addition , a comparison of the three experimental strategies shows online lc / lc identifies more distinct proteins at 1% fdr compared with the other platforms ( figure 4d ) . in general , online mudpit showed only a modest increase in protein sequence coverage ( figure 5a ) over the offline methods . spectral count rank , for example , abundance of proteins , however , was significantly lower in the offline - as platform compared with the other methods ( figure 5b ) . because the major difference in the offline - as group is that the final peptide mixture is placed in autosampler vials , it is likely that the lower spectral counts are due to adsorptive loses of analyte on the surface of the polypropylene sample vials or that sample is lost in the flow path of the sample loop . peptide loss in the offline groups did not significantly correlate with pi , salt fraction , or peptide charge ( figure 5c e ) . although we included organic modifier ( 5% v / v acn ) in the iex elution buffer that reportedly reduces surface adsorption , our data suggest a modest loss of hydrophilic proteins in the offline - as group when plotted using bull breese or kyte doolittle scores ( figure 5f , g ) . this finding is in accordance with recent reports , describing a higher adsorptive tendency of soluble peptides to solid surfaces that ultimately affects peptide amount and quantification parameters . to further illustrate the changes in protein abundance between groups , we performed a statistical comparison of the average spectral count of triplicate runs from each experimental platform . as illustrated in figure 6 , although several proteins were quantified in high abundance between all platforms , we noticed that online proteins were more abundant and statistically more significant ( p 0.01 ) , especially when compared with either of the offline groups ( offline or offline - as ) . specifically , we found that 635 and 542 proteins were significantly higher in abundance in the online platform compared with offline and offline - as groups by 2.2 0.44 and 1.8 0.52 fold , respectively . label - free quantification based on normalized spectral abundance counts of different mudpit panels . ( d f ) surface 3d plots of the identified proteins in each experimental group based on their relative spectral counts ( % ) , molecular weight ( kd ) , and protein length . two tagged samples each were used for online separation : two for off - line with pressure bomb loading and two for off - line fractionation with autosampling . the experiments were performed as for the label - free experiments except that the outflow from the rp capillary column was collected into a single tube for each sample . this method ensures exact comigration with simultaneous and accurate peptide quantification using the mass tags that appears in the tandem mass spectrum . again , this was not restricted to certain peptides properties ; in contrast , the majority of them showed similar trend patterns , denoting stochastic nonspecific loss . cumulatively , the elevated ion intensity signaling for peptides detected in the automated online method corresponded to an average increase of 18% in protein abundance for more than 1100 proteins ( figure 7e ) . we noticed that the same proteins were underestimated in the offline groups with a significant correlation coefficient factor ( r = 0.76 , p = 0.05 ) when compared with the online platform ( figure 7e ) . ( c ) perpendicular 3d plot revealed that most of the identified reporter ions were relatively higher in online mudpit with respect to the other offline formats . on the basis of the amino acid ladder , the peptide was identified as vnptvffdiavdgeplgr with the n - terminus modified by tmt isobaric mass tag labeling reagent . to answer the question of whether the lowered peptide intensity in the offline methods is due to peptide loss during processing or ion suppression as a result of high background noise ( chemical contaminants , atmospheric sources , or electrical interference ) , we utilized the high sensitivity of the linf detector ( 100 fold than uv detectors ) together with peptide quantification using modified bca method . peptide mapping at 220 nm provided a flat baseline with better sensitivity in the current experiment ( figure 8a ) , and detection of scx eluted peptides before mudpit processing did not show any significant differences between experimental groups ( figure 8b ) even at 280 nm associated with absorption of the aromatic amino acids ( data not shown ) . nevertheless , downstream peptide quantification , directly before mass spectrometry analysis , revealed potential low peptide yield ( p 0.05 ) in the offline methods ( figure 8c ) . a previous report demonstrated that the adsorption of biomolecules such as peptides followed a langmuir isotherm equation and was influenced by both solvents and the nature of the solid surfaces . moreover , we monitored the possible impact of sparse ion noise background on the desired peptide signal peak by calculating signal - to - noise ratio ( s / n ; figure 8d ) based on gygi s method . although most peptides in our analysis were analyzed at low s / n , the consequences of low signal levels on quantitative accuracy remain to be tested . interestingly , online mudpit generated slightly higher background noise compared with offline and offline - as ; the s / n ratio , however , was not significantly different between all platforms ( figure 8d ) , and both ms and ms / ms quality was comparable . this observation could be attributed to the higher signal ion intensity of the online mudpit method that maintained a constant s / n ratio between tested groups . ( c ) modified bicinchoninic ( bca ) method for peptide fractionated with different mudpit platforms just before mass spectrometry . ( d ) signal - to - noise ( s / n ) ratio represents the intensity of the signal ( peak height ) to the intensity of the background noise ( in root - mean - square rms ) for mudpit platforms . by preparing samples as a complex mixture , the more abundant proteins can act as carriers of the less abundant proteins . several strategies have evolved to fractionate peptide mixtures prior to entry into the mass spectrometer . we demonstrated that the use of automated online mudpit results in more comprehensive peptide separation and substantially more protein and peptide identification in a label - free quantification experiment , although results were less striking for tmt quantification , where the nature of the experimental design may have complicated the comparison . differences attributed to sample loading are alleviated by normalization correction in both experiments , together with comparable ms / ms spectra ( similar s / n ratio ) , so it is likely that stochastic peptide loss due to adsorption could be affecting offline sample collection ( such as tubes and vials ) . certainly , this conclusion does not discourage using offline platforms because each format has its inherent advantages and disadvantages ( i.e. , flexibility of offline fraction collection and reduced labor time in online separation ) . however , because adsorption is a concentration - dependent surface phenomenon , one should critically consider the potential sample loss due to surface adsorption when considering offline fractionation platforms , especially when processing minute quantities of valuable samples .

as genomes have been sequenced and sequence databases have been compiled , these databases have been used to identify proteins present in biological samples using tandem mass spectrometry data . in a process called shotgun proteomics , mixtures of proteins from biological samples are digested to peptides using proteases , and liquid chromatography coupled to tandem mass spectrometry is used to directly identify the peptides present . as more complex samples , such as cellular components and whole cells , have become targets of analysis , more comprehensive separations have become necessary to resolve complex peptide mixtures and increase dynamic range . for the analysis of complex peptide mixtures , several combinations of separation media have been used . the last phase in multidimensional liquid chromatography ( mdlc ) separations used for mass spectrometry is typically reversed - phase ( rp ) , which separates peptides by hydrophobicity and is effective at removing salts or other small molecule contaminants prior to introduction of peptides into the mass spectrometer . many different forms of mdlc have appeared over the years , from combinations of ion exchange ( iex ) with rp separations to combinations of capillary electrophoresis and liquid chromatography . in any multidimensional separation , how material is transferred from one separation stage to another is critical for maximizing peak capacity and optimizing sample recovery . in this arrangement , a multidimensional separation is created by running buffer containing a set concentration of salt across the column to elute peptides from the scx phase onto the rp column . after a rp gradient is completed , a second salt pulse with a higher concentration of salt is applied to the column to move a new population of peptides to the rp material . the sample is loaded directly onto the column from an eppendorf tube using a pressurized device , and the column is placed in line with the ion source with the voltage placed on a waste line at the backend of the column . , 25% organic in the iex buffer ) to minimize mixed - mode interactions , the capability to collect many fractions , and the capacity to load large amounts of material onto the column . showed improvement in peptide identifications by combining fractions from different parts of a high ph rp separation to produce collections of peptides with different physical characteristics like hydrophobicity for the second - dimension , low - ph rp separation . the valving system is used to redirect flow to shunt salt solutions used to elute peptides from an iex column to waste rather than have it run through the rp analytical column , or in the case of a rp - rp lclc system , the valves are used to direct peptides to the enrichment column to alter the ph of the buffer before the analytical separation . the end of the column was placed directly into a slurry of packing material and when the device was pressurized the packing material was driven into the column . an advantage of shotgun proteomics is the manipulation of complex protein mixtures where the more abundant proteins may presumably act as carrier proteins to protect lower abundance proteins from loss . because losses can occur on active surfaces such a glass and metal , efforts two recent papers showed that peptides can be lost when analyzed from autosamplers , and peptide mixtures of intermediate complexity ( in gel digestions ) can be lost to the surface of autosampler vials made from a variety of materials , respectively . as these two papers have shown , and as experience has taught us , the more samples are handled and exposed to surfaces , the greater the loss . hek293 cells ( crl-1573 ) purchased from atcc were seeded into a t25 flask in supplemented media ( dmem medium supplemented with 10% fetal bovine serum , 1% ( v / v ) penicillin / streptomycin , 2 mm l - glutamine , and 200 g / ml g418 ) from ( gibco , invitrogen)),and maintained with regular media changes for 3 weeks before they were considered to be stable cell lines . the frit was prepared by briefly dipping a 20 cm capillary in well - mixed 300 l of kasil 1624 ( pq corporation , malvern , pa ) and 100 l of formamide , curing at 100 c overnight , and cutting the frit to 1.5 mm in length . the lyophilized tmt - labeled peptides ( 25 g ) were reconstituted with 50 l of buffer a ( 0.1% formic acid ( fa ) , 5% acetonitrile ( acn ) in water ) , centrifuged at 12 000 g for 30 min prior to mass spectrometric analysis . protein lysate was labeled in a sixplex tmt format : 126 and 127 for online mudpit , 128 and 129 for the offline mudpit , and 130 and 131 for the offline with autosampler mudpit ( offline - as ) . directly after labeling , an equal volume of the tmt tags was mixed and analyzed to ensure successful labeling ( labeling efficiency averaged 97.2% ) . peptides were separated by an eksigent nanolc-2d system ( eksigent , dublin ) with or without autosampler unit ( 10 l peek sample loop , six - port titanium injection valve , 50 mm sus sample needle , 50 m i d fused silica tubing ) . the hplc system was either connected online or offline to thermo ltq xl ( for label - free quantification ) or ltq - orbitrap velos ( for tmt quantification ) using an in - house built nanoelectrospray stage . electrospray was performed directly from the analytical column by applying the esi voltage at a tee ( 150 m i d , upchurch scientific ) directly downstream of a 1:1000 split flow used to reduce the flow rate to 250 nl / min through the columns . the lc system was coupled to 224 nm laser - induced native fluorescence ( linf ) detector with elliptical flow cell for real - time peptide detection . the lower threshold for targeting a precursor ion in the ms scan was 5000 counts and 2.5 kv maximum injection time for higher - energy collisional dissociation ( hcd)ms / ms analysis in the orbitrap . once analyzed , the selected peptide ions were dynamically excluded from further analysis for 120 s to allow for the selection of lower - abundance ions for subsequent fragmentation and detection using the setting for repeat count = 1 , repeat duration = 30 ms , and exclusion list size = 500 . for tandem mass spectra were extracted from raw files using rawextract 1.9.9 and searched with the prolucid algorithm against homo sapiens uniprot / swiss - prot database with reversed sequences ( 176 708 entries ) . carbamidomethylation of cysteine ( 57.02146 amu ) was considered as a static modification as well as static n - terminus and lysine modification ( 229.1629 amu ) for sixplex tmt labels analysis . for quantitative analysis , census was used to extract the relative intensities of reporter ions for each peptide from the identified tandem mass spectra for normalization . for label - free quantification , normalized spectral abundance factor , protein , peptide expression alteration ( fold changes ) , log values , and confidence were calculated based on spectral peak intensities generated from the mass spectrometric analysis after extracting confident protein spectra with p < 0.01 . for tmt analysis , the relative quantification between any experimental groups in the sixplex experiment was derived from the average ratio of the reporter ions of duplicate tags of one group over the average reporter ions of duplicate tags of the corresponding group . although the use of capillary chromatography has provided tremendous gains in sensitivity , the use of capillary chromatography also introduces sample - loading challenges . in this study , we tested three methods for introducing complex peptide mixtures into a tandem mass spectrometer . the three multidimensional lc methods consisted of a direct online method using an integrated triphasic capillary column for the introduction of samples and two off - line methods involving collection of samples in eppendorf tubes . one of the off - line methods used an autosampler to introduce the samples into the analytical rp column , and the other used the same direct pressure loading system used to load samples for the online mdlc analysis . in addition , 10 salt steps were used in the online system , and 10 fractions were collected in the two off - line methods using the same elution method . to perform the comparison , a trypsin - digested hek293 sample was used that should be representative of the type of sample commonly analyzed in proteomics . even with the current advanced mass spectrometric capabilities , multiple dimensions of separation improve comprehensive analysis of peptide mixtures . although a variety of multidimensional combinations have emerged in the past few years , the scx - rp combination is an efficient and highly resolving separation method for shotgun proteome analysis . the main impetus of this work is to provide a systematic comparative study of different lc / lc strategies in terms of performance , sensitivity , and recovery that should be applicable to all lc / lc methods regardless of the phases employed proteins from a hek293 cell line were digested with trypsin and aliquots were subjected to analysis by the three different platforms to measure peptide and protein identification efficiency for each method . for this measurement , the goal was simply to report how many peptides and proteins would be detected for the same sample using the different methods ( figure 1 ) . a total of 3383 386 , 2159 243 , and 1877 413 proteins were identified from the 12937 1533 , 7351 1201 , and 7146 829 peptides identified for the online , offline , and offline - as groups , respectively ( figure 3a , b ) . the higher mean of the online triphasic column was statistically significant ( confidence of 95% ) compared with the other methods . given the identical gradients and ms methods used for each approach , the peptide identification and protein identification numbers were similar to an improvement by more than 1.7 to 1.8 fold in the online method compared with both offline methods ( figure 3b ) . additionally , merging and removing redundant proteins / peptides increased protein / peptide identifications most in the online triphasic column method , less in the offline method , and least in the offline - as method . this comparison showed that recovery of peptides was best in the online method by virtue of the most peptide and protein identifications and the poorest in the off - line fractionation method with the introduction of samples through an autosampler . proportional venn diagram demonstrating the overlap between protein identification of different mudpit triplicate runs ( ( a ) online , ( b ) offline ; and ( c ) offline - as ) or between different mudpit platforms ( d ) . in addition , a comparison of the three experimental strategies shows online lc / lc identifies more distinct proteins at 1% fdr compared with the other platforms ( figure 4d ) . differences in identification rates among the samples suggest that improved identification rates are a result of minimizing manual handling of fractions . if there is an observed difference in peptide identification between the different methods , it begs the question of whether there are any differences in physicochemical characteristics of the peptides or proteins observed in the different methods . we analyzed the proteome metrics relevant to each experimental platform to determine if there were any peculiar physicochemical characteristics of peptides observed in one platform and not the other . spectral count rank , for example , abundance of proteins , however , was significantly lower in the offline - as platform compared with the other methods ( figure 5b ) . because the major difference in the offline - as group is that the final peptide mixture is placed in autosampler vials , it is likely that the lower spectral counts are due to adsorptive loses of analyte on the surface of the polypropylene sample vials or that sample is lost in the flow path of the sample loop . peptide loss in the offline groups did not significantly correlate with pi , salt fraction , or peptide charge ( figure 5c e ) . although we included organic modifier ( 5% v / v acn ) in the iex elution buffer that reportedly reduces surface adsorption , our data suggest a modest loss of hydrophilic proteins in the offline - as group when plotted using bull breese or kyte doolittle scores ( figure 5f , g ) . ( f ) bull breese hydrophobicity index was calculated based on the free energy of transfer to surface in kcl / mol . to further illustrate the changes in protein abundance between groups , we performed a statistical comparison of the average spectral count of triplicate runs from each experimental platform . as illustrated in figure 6 , although several proteins were quantified in high abundance between all platforms , we noticed that online proteins were more abundant and statistically more significant ( p 0.01 ) , especially when compared with either of the offline groups ( offline or offline - as ) . specifically , we found that 635 and 542 proteins were significantly higher in abundance in the online platform compared with offline and offline - as groups by 2.2 0.44 and 1.8 0.52 fold , respectively . ( a c ) volcano plots depict relationship between the p value and the magnitude of difference ( log2 ) in expression value between average technical replicates of two compared groups . ( d f ) surface 3d plots of the identified proteins in each experimental group based on their relative spectral counts ( % ) , molecular weight ( kd ) , and protein length . the experiments were performed as for the label - free experiments except that the outflow from the rp capillary column was collected into a single tube for each sample . we found that peptide abundance was lower ( p 0.05 ) in the offline groups ( figure 7a c ) . in addition , peptide ratios between online and offline groups disclosed a modest skew toward the online platform with a 1318% increase after peptide grouping and normalization ( figure 7d ) . cumulatively , the elevated ion intensity signaling for peptides detected in the automated online method corresponded to an average increase of 18% in protein abundance for more than 1100 proteins ( figure 7e ) . we noticed that the same proteins were underestimated in the offline groups with a significant correlation coefficient factor ( r = 0.76 , p = 0.05 ) when compared with the online platform ( figure 7e ) . ( c ) perpendicular 3d plot revealed that most of the identified reporter ions were relatively higher in online mudpit with respect to the other offline formats . on the basis of the amino acid ladder , the peptide was identified as vnptvffdiavdgeplgr with the n - terminus modified by tmt isobaric mass tag labeling reagent . mass tags ( 126131 ) observed in the lower m / z region ( inserted figure ) indicate the relative abundance of this peptide in each group . to answer the question of whether the lowered peptide intensity in the offline methods is due to peptide loss during processing or ion suppression as a result of high background noise ( chemical contaminants , atmospheric sources , or electrical interference ) , we utilized the high sensitivity of the linf detector ( 100 fold than uv detectors ) together with peptide quantification using modified bca method . peptide mapping at 220 nm provided a flat baseline with better sensitivity in the current experiment ( figure 8a ) , and detection of scx eluted peptides before mudpit processing did not show any significant differences between experimental groups ( figure 8b ) even at 280 nm associated with absorption of the aromatic amino acids ( data not shown ) . nevertheless , downstream peptide quantification , directly before mass spectrometry analysis , revealed potential low peptide yield ( p 0.05 ) in the offline methods ( figure 8c ) . a previous report demonstrated that the adsorption of biomolecules such as peptides followed a langmuir isotherm equation and was influenced by both solvents and the nature of the solid surfaces . moreover , we monitored the possible impact of sparse ion noise background on the desired peptide signal peak by calculating signal - to - noise ratio ( s / n ; figure 8d ) based on gygi s method . although most peptides in our analysis were analyzed at low s / n , the consequences of low signal levels on quantitative accuracy remain to be tested . interestingly , online mudpit generated slightly higher background noise compared with offline and offline - as ; the s / n ratio , however , was not significantly different between all platforms ( figure 8d ) , and both ms and ms / ms quality was comparable . ( d ) signal - to - noise ( s / n ) ratio represents the intensity of the signal ( peak height ) to the intensity of the background noise ( in root - mean - square rms ) for mudpit platforms . ultimately the sample losses associated with gel purification and in - gel - digestion - limited detection of proteins made these methods less attractive . we demonstrated that the use of automated online mudpit results in more comprehensive peptide separation and substantially more protein and peptide identification in a label - free quantification experiment , although results were less striking for tmt quantification , where the nature of the experimental design may have complicated the comparison . differences attributed to sample loading are alleviated by normalization correction in both experiments , together with comparable ms / ms spectra ( similar s / n ratio ) , so it is likely that stochastic peptide loss due to adsorption could be affecting offline sample collection ( such as tubes and vials ) . however , because adsorption is a concentration - dependent surface phenomenon , one should critically consider the potential sample loss due to surface adsorption when considering offline fractionation platforms , especially when processing minute quantities of valuable samples .

uric acid ( ua ) is the end product of the metabolic pathway for purines , the main constituents of nucleotides . briefly , inosine monophosphate ( imp ) is derived from de novo purine synthesis and from purine salvage . hypoxanthine from imp is catalyzed to xanthine and then to uric acid by xanthine oxidase ( xo ) . de novo nucleotide synthesis generates imp via ribose-5-phosphate , catalyzed to 5-phosphoribosyl-1-pyrophosphate ( prpp ) . in the salvage pathway , hypoxanthine - guanine phosphoribosyl transferase ( hgprt ) plays an important role in generating imp , thereby inhibiting ua generation . since humans are unable to catabolize ua to the more soluble compound allantoin due to lack of urate oxidase or uricase , the serum ua concentration is higher in humans than almost all other mammals . however , this high ua level in humans has been regarded as being beneficial in the presence of elevated oxidative stress . ua is oxidized to allantoin and other metabolites via nonenzymatic oxidation and , thus , ua can function to neutralize prooxidant molecules , such as hydroxyl radicals , hydrogen peroxide , and peroxynitrite . ua shows the highest scavenging rate constant against o2 , with constants being low against ch3 and t - buoo . ua directly ( nonenzymatically ) and preferentially deletes nitric oxide ( no ) and forms 6-aminouracil in physiological environments or in association with antioxidants . in vitro , ua has both an antioxidant effect on native ldl and a prooxidant effect on mildly oxidized ldl . it has been suggested that this antioxidant effect of the high ua concentrations in humans contributes to neuroprotection in several neurodegenerative and neuroinflammatory diseases [ 814 ] . however , despite the potential antioxidant effect of ua itself , numerous studies have revealed close associations of serum ua concentrations and various disorders , most of which are included in the metabolic syndrome category . thus , ua metabolism may be a so - called double - edged sword as regards the inflammatory and/or oxidative responses in many organs , though on the whole , its harmful effects appear to outweigh the benefits of ua in most cases . in this review , we first explain the two putative molecular mechanisms underlying inflammation occurrence in relation to ua metabolism ; one is inflammasome activation via ua crystallization and the other involves superoxide free radicals generated by xo . while the ua crystallization mechanism would be dependent on a high serum ua concentration , the latter may not necessarily reflect the serum ua concentration though xo activity does lead to the production of reactive oxygen species ( ros ) . subsequently , lines of research showing relationships between ua metabolism and the development of various disorders are introduced and discussed . importantly , recent studies have demonstrated beneficial effects of xo inhibitors against the occurrence and/or progression of several disorders , particularly atherosclerosis and nonalcoholic steatohepatitis ( nash ) , both of which are associated with insulin resistance , hyperlipidemia , and/or obesity . in this review , atherosclerosis and nash are discussed extensively , while studies of gout and chronic kidney diseases ( ckd ) are mentioned briefly . in conclusion , we propose that such xo inhibitors may be more useful for preventing a variety of disorders , such as atherosclerosis and nash , than previously believed , probably via an anti - inflammatory effect . among the disorders related to hyperuricemia , gout is the most representative and well known . features of gout include painful arthritis affecting the limbs , caused by reduced ua crystals in the joints . while symptoms of a gout attack are typical of an acute inflammatory response , as indicated by the presence of swelling , heat , rubescence , and pain , there are many disorders with mild but chronic inflammation which are very likely to be related to ua metabolism . in the latter case , superoxide free radicals generated by xo are key players leading to chronic inflammatory processes eventually resulting in impaired organ functions . thus , we introduce two independent mechanisms underlying ua metabolism - induced inflammation . in 2002 , the inflammasome concept was proposed to involve multiple proteins and to control the cleavage of prointerleukin 1 ( il-1 ) . initially , inflammasomes were considered to play a role in immune responses and serve as defense systems against pathogens [ 16 , 17 ] . however , a line of subsequent studies has elucidated that inflammasomes are key players in the onsets of a wide range of diseases as well as host defense . excessive metabolites , such as atp or monosodium urate crystals ( muc ) , were also confirmed to be involved in the activation of inflammasomes , and inflammatory responses occurring via inflammasomes have been demonstrated to be linked to the onset and progression of human diseases , including gout , atherosclerosis and nash , as described below in detail [ 1824 ] . inflammasomes are known to be divided into discernible patterns , depending on component proteins . among them , the nlrp3 inflammasome , comprised of three major components , nod - like receptor 3 ( nlrp3 ) , apoptosis - associated speck - like protein containing a card ( asc ) and caspase-1 , has been well investigated . first , the toll - like receptor ligands , such as lipopolysaccharide ( lps ) , activate the nf-b pathway and upregulate the expression levels of interleukins , including pro - il-1 and pro - il-18 . subsequently , the inflammasome complex activated by pathogen - associated molecular patterns ( pamps ) or damage - associated molecular patterns ( damps ) cleaves pro - il-1 or pro - il-18 , resulting in the production of mature interleukins [ 1517 ] . although the mechanism of inflammasome activation by muc has not been fully elucidated , the following mechanism was proposed . muc stimulate the toll - like receptor 2/4-myd88 pathway and raise transcriptional levels of pro - il-1 through the nf-b pathway . it is theorized that muc - induced inflammasome activation is driven by two key factors . the other is the generation of ros , because an antioxidant , n - acetyl - cysteine , abolished il-1 secretion by muc . however , the relationship between intracellular k level changes and ros generation remains unknown , and future studies are expected to resolve this issue [ 27 , 28 ] . elevation of intracellular ros mediates the detachment of thioredoxin - interacting protein ( txnip ) from thioredoxin and enables txnip to associate with nlrp3 , leading to nlrp3 inflammasome activation [ 29 , 30 ] . thus , muc accumulation promotes inflammatory responses through inflammasomes ( figure 2 ) and thereby promotes the onset of diseases , such as gout . when mammalian xanthine dehydrogenase ( xdh ) is converted to xo under stressed conditions such as tissue damage and ischemia , superoxide anion and hydrogen peroxide are produced during molybdenum hydroxylase - catalyzed reactions in a molar ratio of about 1 : 3 . in essence , xo oxidizes a variety of purines and pterins , classified as molybdenum iron - sulfur flavin hydroxylases . when xo reacts with xanthine , electrons are transferred from mo , fe - s , and fad . only fadh2 reacts with o2 . in the ua metabolic pathway , xo oxidizes hypoxanthine from nucleic acid metabolites into xanthine and xanthine into ua ( figure 1 ) . xo , as well as nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase and the mitochondrial electron - transport chain , generates ros . treatment during pregnancy with allopurinol alters maternal vascular function involving 1-adrenergic stimulation and impairs the fetal 1-adrenergic vasoreflex response involving no . fetal xo is activated in vivo during hypoxia and xo - derived ros contributes to fetal peripheral vasoconstriction , leading to fetal defense against hypoxia . xo depletion induces renal interstitial fibrosis , and renal epithelial cells from xor ( / ) mice are more readily transformed into myofibroblasts . the tissue and cellular distributions of xo in mammals are highest in the liver and intestines due to xo - rich parenchymal cells . xanthine oxidoreductase ( xor ) is present in hepatocytes , while xo is present in bile duct epithelial cells , concentrated toward the luminal surface . moreover , in human liver disease , proliferating bile ducts are also strongly positive for xo . xo activity is low in human serum , the brain , heart , and skeletal muscle , while being rich in microvascular endothelial cells and is also present in macrophages . the study using electron spin resonance measurements revealed the contribution of increased xo activity to endothelial dysfunction in patients with coronary artery diseases . xo activation is induced by lps , angiotensin ii , nadph oxidase , hypoxia , hypoxia - inducible factor 1 , and inflammatory cytokines such as il-1 [ 4649 ] . the release of xo is increased in hypercholesterolemia , chronic hyperammonemia , thermal trauma , beta - thalassemia , brain ischemia , and pulmonary artery hypertension [ 5054 ] . indeed , xo was significantly higher in the aortic walls and skeletal muscles of old rats than in those of their young counterparts . the correlation between plasma xo activity and age is observed in both humans and rats . it appears that hyperglycemia itself has no impact on liver xo activity , though cardiac , renal , and brain xo activities were shown to be increased in rats with advanced diabetes [ 56 , 57 ] . xo activity rises remarkably in ischemic congestive heart failure and xo localizes within cd68 positive macrophages . xo is one of the major superoxide sources in ischemia / reperfusion injuries of the heart , forebrain , skin , liver [ 61 , 62 ] , and gastric mucosa , as well as multiple system organ failure after hind limb reperfusion . xo activity , along with lipid peroxidation , myeloperoxidase activity and no levels , is increased in the liver in response to renal ischemia / reperfusion in diabetic rats . ischemia / reperfusion injury is attributable to elevated xo activity and atp depletion related to increasing hypoxanthine and xanthine levels during ischemia , and reperfusion provides o2 for oxidation of these compounds . superoxide production by xo may also be enhanced by increasing the amount of its substrate , purine bodies . excess fructose metabolism results in atp depletion which is associated with degradation of amp to hypoxanthine , followed by conversion to ua by xo . atp depletion , such as that characteristic of glycogen storage disease type 1 , hypoglycemia , exercise , and starvation , also increases ua production . conditions associated with dna turnover , such as tumor progression and tumor lysis , are also mediated by xo . superoxide produced by xo is an important messenger inducing inflammation and signal transduction , leading to tissue damage . we found inflammatory cytokines to be induced via xo when foam cells form with lipid accumulation . xo regulates cyclooxygenase-2 in the inflammatory system , and xo appears to be critical for innate immune function . xo increased egr-1 mrna and protein , as well as the phosphorylation of erk1/2 , while pretreatment with an erk1/2 inhibitor prevented induction of egr-1 by xo . ros from xo augment trb3 expression in podocytes . as noted above , superoxide from xo has been suggested to play roles in various forms of inflammatory or ischemic pathophysiology ( figure 3 ) , not necessarily involving hyperuricemia . while gout is a disorder well known to be caused by the precipitation of ua crystals , the involvement of hyperuricemia in ckd is also widely recognized . the major causes of ckd have been regarded as diabetes mellitus and hypertension , and thus , hyperuricemia was long viewed as a consequence of ckd . in fact , loss of kidney function reduces the excretion of ua into urine , resulting in hyperuricemia . in contrast , recent studies demonstrated a significant association between serum ua and the development of ckd . while each metabolic syndrome component , including hyperglycemia , hyperlipidemia , and hypertension , was associated with an increased ckd risk , hyperuricemia was apparently an independent risk factor not influenced by the others . therefore , hyperuricemia is both a cause and a consequence of ckd and is frequently associated with other metabolic syndrome features . in terms of ckd pathogenesis , serum ua is likely to activate the renin - angiotensin system resulting in vascular smooth muscle cell proliferation and to induce an epithelial - to - mesenchymal transition of renal tubular cells . xo inhibitor treatment reportedly reduced intercellular adhesion molecule-1 ( icam-1 ) expression in tubular epithelial cells of mice . we speculate that ua itself and superoxide free radical generation both play roles in the molecular mechanisms underlying hyperuricemia - related ckd development , but further research is required to elucidate the complex mechanistic interactions between serum ua and ckd . as mentioned in section 2 , both ua and superoxide free radicals are simultaneously produced by xo and might be the pathophysiological cause of these diseases . as shown in figure 3 , chronic inflammation is also involved in pathophysiological processes , generally exhibiting a close relationship with oxidative stress . ros from xo induces lps - induced jnk activation via inactivation of mapk phosphatase- ( mkp- ) 1 and xo regulates cyclooxygenase-2 , one of the master regulators of inflammation . therefore , damage from ua , ros , and ua - induced and/or ros - induced inflammation might together contribute to the progression of certain diseases , and distinguishing which mechanism acts first is often difficult in lifestyle - related diseases . although the relationships between serum ua levels and atherosclerotic diseases , including hypertension [ 85 , 86 ] , have been documented , whether or not serum ua itself is an independent cardiovascular risk factor remains controversial as most hyperuricemic patients with cardiovascular diseases ( cvd ) have other complications such as hypertension , dyslipidemia , diabetes , and ckd as well , which are generally regarded as more established risk factors for cvd than hyperuricemia . recently , however , a growing body of evidence from both clinical and basic research supports the hypothesis that hyperuricemia , partly via elevated xo activity , is an independent risk factor for hypertension and cvd . despite the association between hyperuricemia and hypertension having been recognized since the 19th century , it was not until recently that hyperuricemia was demonstrated to be an independent risk factor for hypertension development [ 8793 ] . a recently published meta - analysis showed that the adjusted relative risk of developing hypertension was 1.48 for hyperuricemic patients , and this association was apparently much stronger in younger , early - onset hypertensive patients [ 86 , 95 ] . several clinical trials have demonstrated the beneficial effects of ua lowering therapy for hypertension [ 9699 ] . in a trial targeting prehypertensive obese adolescents , administration of either allopurinol ( xo inhibitor ) or probenecid ( uricosuric agent ) lowered blood pressure . consistently , both allopurinol and benziodarone ( uricosuric agent ) reduced blood pressure in rats with hypertension induced by hyperuricemia [ 100 , 101 ] , suggesting that not only xo activity but also ua itself plays an important role in the pathogenesis of hypertension . besides the association with hypertension , hyperuricemia or gout has been confirmed to be related to the morbidity and the mortality of cvd [ 102106 ] . according to a recently published meta - analysis , the relative risks of morbidity and mortality for coronary heart diseases were 1.13 and 1.27 , respectively , in hyperuricemic patients as compared to controls . several clinical studies have indicated the benefits of xo inhibitors for reducing the incidence of myocardial infarction , improving exercise tolerance in patients with stable angina , and enhancing endothelial function [ 110 , 111 ] . however , interestingly , unlike the case of treating hypertension , uricosuric agents have failed to show any benefits in patients with hyperuricemia or gout [ 110 , 112 ] . as described above , xo produces ros when converting hypoxanthine into xanthine and then ua . xo is also expressed in endothelial cells and was shown to be increased in the aortic endothelial cells of apoe mice , an established model of atherosclerosis . since oxidative stress inactivates no and leads to endothelial dysfunction , endothelial xo , especially given its enhanced expression during the development of atherosclerosis , contributes to vascular damage via ros production . recently , we established that xo activity in macrophages also plays a key role in the development of atherosclerosis . during atherosclerosis development , monocytes migrate beneath the endothelium and transform into macrophages , which then turn into foam cells by incorporating modified low density lipoproteins ( ldl ) ( such as oxidized ldl and acetyl ldl ) or very low density lipoproteins ( vldl ) . foam cells contribute to the formation of unstable plaques by secreting inflammatory mediators and matrix - degrading proteases ( such as matrix metalloproteinases ( mmps ) ) and by generating a prothrombotic necrotic core by eventually undergoing necrotic or apoptotic death . we demonstrated that allopurinol treatment ameliorated aortic lipid accumulation and calcification of the vessels of apoe - ko mice and that allopurinol markedly suppressed the transformation of j774.1 murine macrophages or primary cultured human macrophages into foam cells in response to stimulation with acetyl ldl or vldl . the expressions of scavenger receptors ( sr - a1 , sr - b1 , and sr - b2 ) and vldl receptors in j774.1 cells were upregulated by xor overexpression and downregulated by sirna - mediated xor suppression , raising the possibility that xo activity in macrophages positively regulates foam cell formation by increasing the uptake of modified ldl or vldl . conversely , expressions of abca1 and abcg1 , which regulate cellular cholesterol efflux , were decreased by xor overexpression and increased by xor knockdown . furthermore , allopurinol suppressed the expressions of inflammatory cytokines such as il-1 , il-6 , il-12 , and tnf , and the expressions of vcam1 , mcp-1 , and mmp2 , which were upregulated in j774.1 cells transformed into foam cells by atherosclerogenic serum . subsequently , febuxostat , another xo inhibitor , was also demonstrated to attenuate the development of atherosclerotic lesions in apoe mice . that study showed xo expression to be increased in macrophages infiltrating atherosclerotic plaques and that febuxostat diminished the ros level in the aortic walls of apoe mice . the authors demonstrated that cholesterol crystals ( ccs ) increased endogenous xo activity and ros production in macrophages and that ccs enhanced not only il-1 release via nlrp3 inflammasome activation but also secretions of other inflammatory cytokines such as il-1 , il-6 , and mcp-1 from macrophages , processes which in turn were suppressed by febuxostat or ros inhibitors . the significance of nlrp3 inflammasome activation in macrophages by ccs was verified by the observation that atherosclerosis in high - cholesterol diet fed ldl receptor- ( ldlr- ) deficient mice was alleviated by transplanting bone marrow from nlrp3-deficient , asc - deficient , or il-1/-deficient mice . taking these observations together , we can reasonably speculate that xo in macrophages enhances foam cell formation , ros production , and nlrp3 inflammasome activation , all three of which exacerbate inflammation and plaque formation , thereby contributing to the development of atherosclerotic diseases [ 75 , 114116 ] . independently of xo , ua itself is widely recognized to exert direct effects on vascular functions . vascular endothelial cells express several ua transporters and incorporated ua impairs no production and leads to endothelial dysfunction [ 118 , 119 ] . in vascular smooth muscle cells , ua stimulates proliferation and ros production and inhibits no production via increased angiotensin ii expression [ 81 , 120 ] . as noted above , not only xo inhibitors but also uricosuric agents markedly lowered blood pressure , especially in studies targeting early - stage hypertensive patients and those using animal models [ 100 , 101 ] . the results obtained suggest that ua presumably contributes to early - stage hypertension by promoting renal vasoconstriction via reduced no production and activation of the renin - angiotensin system [ 86 , 98 ] . the number of nonalcoholic fatty liver disease ( nafld ) patients including those with nash has been increasing worldwide and a portion of nash patients will progress to hepatocarcinoma onset [ 121123 ] . therefore , numerous investigations have been performed in efforts to elucidate the causes of nash . nash is characterized by fat deposition , inflammation and fibrosis in the liver , and a two - hit mechanism of onset has been proposed [ 124126 ] . this hypothesis is that fatty liver formation and subsequent injuries , including inflammation and oxidative stress , cause nash pathology . interestingly , recent studies have raised the possibility that ua is among the risk factors for nash pathology . many clinical studies have been carried out to investigate the relationship between serum ua levels and nafld / nash progression . for example , a cohort study in korea found the serum ua level to be a useful marker for predicting nafld development because the serum ua concentration correlated positively with the 5-year incidence rate of nafld . their conclusion is supported by another study showing that serum ua levels of nafld patients are higher than those of control groups . in addition , there are also studies demonstrating that serum ua is a risk factor for the development and/or progression of nafld including nash [ 130132 ] . consistent with these observations , hepatic xo activities and serum ua levels are reportedly increased in murine nafld / nash models [ 133 , 134 ] . moreover , a fraction of nafld / nash patients also have obesity , and hypertrophic adipocytes were also reported to secrete ua . taken together , these results indicate serum ua to be a good parameter for predicting the development of nafld / nash , and that xo inhibitors or uricosuric agents might have potential as treatments for ameliorating the features of nafld . as described above , increasing serum ua or xo activity apparently plays important roles in nafld / nash onset and progression . fructose treatment of hepg2 cells reportedly increased both the intracellular ua concentration and triglyceride ( tg ) accumulation , while allopurinol , an xo inhibitor , suppressed this fructose - mediated tg deposition . moreover , the application of ua alone was demonstrated to increase intracellular tg contents as well as ros generation in mitochondria . as a mechanism of ua - induced tg accumulation , the authors asserted that the elevation of intracellular ros by ua raised both the citrate concentration and atp citrate lyase activity via enhanced phosphorylation at s455 , resulting in the induction of lipogenesis . these observations are supported by those of another study in which pretreatment with antioxidants inhibited the elevation of triglyceride contents by ua . the authors asserted that ros generation by ua evoked endoplasmic reticulum stress , leading to upregulation of lipogenic genes , such as acetyl coa carboxylase1 and fasn . ros generation by ua is considered to depend on nadph oxidase activation [ 136 , 138 , 139 ] . for example , ua reportedly promotes translocation of the nadph oxidase subunit nox4 into mitochondria . it was also reported that ua treatment raises nadph oxidase activity and alters its localization , leading to lipid oxidation . in addition , xo may also function as a source of ros generation because xo activity is upregulated in the livers of murine nash models . collectively , these observations indicate that ua enhances fatty acid synthesis by regulating lipogenesis and induces ros generation by regulating nadph oxidase activity and upregulating fatty acid synthesis , thereby contributing to nash development . an initial study revealed that inflammasome impairment exacerbated the nash progression induced by feeding a methionine - choline deficient diet for 4 weeks to asc or il-1 ko mice . for example , it was reported that nlrp3 deficiency prevents liver fibrosis in response to a choline diet deficient in amino acids . in addition , caspase-1 deficient mice were also resistant to developing steatosis or fibrosis while being fed a high - fat diet . moreover , other groups have demonstrated that diets which lead to nash also increase the expressions of inflammasome components [ 143145 ] . taking these lines of evidence together , in the initial stage of nash , inflammasomes appear to exert a protective effect , but continuous inflammasome activation appears to cause excessive productions of inflammatory cytokines , ultimately resulting in liver injury . although , to date , numerous factors playing important roles in nash progression have been identified , ua also appears to be a key participant in the onset of nafld / nash . hyperuricemia was reportedly found to be related to insulin resistance in several clinical analyses [ 146152 ] . in addition , several meta - analyses have suggested the ua level to be positively associated with the development of type 2 diabetes mellitus ( dm ) [ 153156 ] , although mendelian randomization studies did not support circulating ua as being among the causes of dm development [ 157 , 158 ] . in metabolic syndrome patients , an oxidative stress marker , the myeloperoxidase level , was decreased by allopurinol and endothelial function improved . on the other hand , rapid ua reduction achieved by rasburicase , a urate oxidase , in obese subjects with high ua resulted in increasing the markers of systemic and skeletal muscle oxidative stress while having no effect on insulin sensitivity . furthermore , excess fructose intake is one of the major causes of the development of obesity with hyperuricemia , fatty liver , and metabolic syndrome . fructose is metabolized by fructokinase to fructose-1-phosphate and results in a drop in both intracellular phosphate and atp levels . the intracellular phosphate decrease stimulates amp deaminase ( ampd ) , the enzyme catalyzing the degradation of amp to inosine monophosphate and eventually ua . activated ampd increases the expressions of gluconeogenesis genes , that is , pepck and g6pase , via inhibition of amp - activated protein kinase ( ampk ) . the ua increased fructose - induced tg accumulation and decreased -hydroxybutyrate levels , dose - dependently , while allopurinol , a xo inhibitor , blocked it . because ua is the downstream product of ampd and allopurinol abolished fructose - induced lipid accumulation , ampd effects on ampk appeared to depend on ua . ua activates the transcription factor chrebp , which triggers a vicious cycle of fructokinase transcription and accelerated fructose metabolism . via these mechanisms , ua is considered to be an antioxidant in human blood , though ua induces oxidative stress in cells . the stimulation of nadph oxidase - dependent ros by ua resulted in the activation of map kinase p38 and erk1/2 , a decrease in no bioavailability , and increases in both protein nitrosylation and lipid oxidation . increased ua production , in turn , generates mitochondrial oxidants . mitochondrial oxidative stress inhibits aconitase in the krebs cycle , resulting in citrate accumulation and the stimulation of atp citrate lyase and fatty acid synthase , ultimately leading to de novo lipogenesis . in hepatocytes treated with high ua , oxidative stress is increased , which activates serine ( rat ser307 and human ser312 ) phosphorylation of irs-1 . this activity impairs akt phosphorylation , thereby resulting in acute hepatic insulin resistance after exposure to high ua levels . therefore , ua - induced lipid accumulation and oxidative stress are responsible for the development of insulin resistance and diabetes . involvement of increased xo catalyst activity in pathophysiological processes ( figure 4 ) suggests applications of xo inhibitors to the treatment of various disorders . at present , xo inhibitors , including allopurinol , oxypurinol , febuxostat , and topiroxostat , are widely used for treating gout and hyperuricemia . furthermore , xo inhibitors have been experimentally or clinically shown to exert beneficial effects by lowering serum ua and oxidative stress . febuxostat preserved renal function in 5/6 nephrectomized rats with and without coexisting hyperuricemia and prevented diabetic renal injury in streptozotocin - treated rats [ 166 , 167 ] . febuxostat also ameliorated tubular damage , diminished macrophage interstitial infiltration , and suppressed both proinflammatory cytokine activities and oxidative stress . febuxostat also reduced the induction of endoplasmic reticulum stress , as assessed by grp-78 ( glucose - regulated protein-78 ) , atf4 ( activating transcription factor-4 ) , and chop ( c / ebp homologous protein-10 ) . the clinical significance of measuring the serum ua level and xo inhibition for renal protection has largely been established by the results of recent studies [ 170173 ] . on the other hand , beneficial effects of xo inhibitors on atherosclerosis and nash constitute an evolving concept that has yet to be proven . in rats with fructose - induced metabolic syndrome , febuxostat treatment reversed hyperuricemia , hypertension , dyslipidemia , and insulin resistance the beneficial effects of xo inhibitors on nash are rarely reported , except by our research group , because animal models of nash with obesity , inflammation , and fibrosis have been difficult to establish . nash in response to the mcd diet , as used in our studies , caused primarily inflammation and also made the mice lean , such that no benefit of xo inhibition was obtained . thus , we next used a high - fat diet containing trans - fatty acids and a high - fructose diet to induce nash development in our animal models . another report showed that inhibition of xo activity also significantly prevents hepatic steatosis induced by a high - fat diet in mice . tungsten , acting as an xo inhibitor , has an inhibitory effect on both atherosclerosis and oxidative stress . we reported for the first time that more specific xo inhibition , using allopurinol rather than tungsten on macrophages , resulted in the inhibition of foam cell formation and reduced atherosclerotic lesions in apoe - ko mice , independently of the serum lipid profile . we also identified phenotypic changes of macrophages in response to allopurinol , such as alterations of gene expressions involved in lipid accumulation . moreover , both xo overexpression and knockdown of xo expression revealed vldl receptors to be dramatically upregulated by xo . febuxostat was also proven to have similar effects in terms of reducing the atherosclerotic lesions in apoe - ko mice , and oxidative stress was reduced in macrophages from atherosclerotic lesions . as a strategy for suppressing atherosclerosis , xo inhibition is expected to act on either macrophages or inflammatory cells . oxypurinol reduces o2 radical dot production and improves endothelial function in blood vessels from hyperlipidemic experimental animals . xo inhibition can also provide protection from radiation - induced endothelial dysfunction and cardiovascular complications . however , controversy persists as to whether the effect of xo on endothelial function is clinically relevant as an interventional target . pretreatment with xo inhibitors has beneficial effects on ischemia / reperfusion injuries of the intestine , in the impaired liver [ 61 , 62 ] , the edematous brain , kidneys with contrast induced nephropathy , and coronary ischemia . inflammation related to ua metabolism is induced via either inflammasome activation by ua crystal precipitation or free radical production in response to xo activity . in addition to gout , many disorders are known to be related to ua metabolism and xo inhibitor treatments have been shown to be effective for preventing the onset and/or the progression of such disorders . in particular , atherosclerosis and nash are diseases for which relationships with ua metabolism were not immediately recognized , but rodent model studies revealed the importance of ua metabolism maintenance for managing these disorders . we believe the impact of ua metabolism on many diseases accompanying chronic inflammation to have been underestimated . future studies are anticipated to reveal the pathological contribution of serum ua and/or xo activity to the specific processes underlying various disorders .

uric acid ( ua ) is the end product of purine metabolism and can reportedly act as an antioxidant . however , recently , numerous clinical and basic research approaches have revealed close associations of hyperuricemia with several disorders , particularly those comprising the metabolic syndrome . in this review , we first outline the two molecular mechanisms underlying inflammation occurrence in relation to ua metabolism ; one is inflammasome activation by ua crystallization and the other involves superoxide free radicals generated by xanthine oxidase ( xo ) . importantly , recent studies have demonstrated the therapeutic or preventive effects of xo inhibitors against atherosclerosis and nonalcoholic steatohepatitis , which were not previously considered to be related , at least not directly , to hyperuricemia . such beneficial effects of xo inhibitors have been reported for other organs including the kidneys and the heart . thus , a major portion of this review focuses on the relationships between ua metabolism and the development of atherosclerosis , nonalcoholic steatohepatitis , and related disorders . although further studies are necessary , xo inhibitors are a potentially novel strategy for reducing the risk of many forms of organ failure characteristic of the metabolic syndrome .

1. Introduction 2. Inflammation Occurrence Related to UA Metabolism 3. UA Metabolism and Chronic Renal Disease, Atherosclerosis, Heart Failure, and NASH 4. Beneficial Effects of XO Inhibitors 5. Conclusion

uric acid ( ua ) is the end product of the metabolic pathway for purines , the main constituents of nucleotides . briefly , inosine monophosphate ( imp ) is derived from de novo purine synthesis and from purine salvage . hypoxanthine from imp is catalyzed to xanthine and then to uric acid by xanthine oxidase ( xo ) . however , this high ua level in humans has been regarded as being beneficial in the presence of elevated oxidative stress . ua is oxidized to allantoin and other metabolites via nonenzymatic oxidation and , thus , ua can function to neutralize prooxidant molecules , such as hydroxyl radicals , hydrogen peroxide , and peroxynitrite . however , despite the potential antioxidant effect of ua itself , numerous studies have revealed close associations of serum ua concentrations and various disorders , most of which are included in the metabolic syndrome category . thus , ua metabolism may be a so - called double - edged sword as regards the inflammatory and/or oxidative responses in many organs , though on the whole , its harmful effects appear to outweigh the benefits of ua in most cases . in this review , we first explain the two putative molecular mechanisms underlying inflammation occurrence in relation to ua metabolism ; one is inflammasome activation via ua crystallization and the other involves superoxide free radicals generated by xo . subsequently , lines of research showing relationships between ua metabolism and the development of various disorders are introduced and discussed . importantly , recent studies have demonstrated beneficial effects of xo inhibitors against the occurrence and/or progression of several disorders , particularly atherosclerosis and nonalcoholic steatohepatitis ( nash ) , both of which are associated with insulin resistance , hyperlipidemia , and/or obesity . in this review , atherosclerosis and nash are discussed extensively , while studies of gout and chronic kidney diseases ( ckd ) are mentioned briefly . in conclusion , we propose that such xo inhibitors may be more useful for preventing a variety of disorders , such as atherosclerosis and nash , than previously believed , probably via an anti - inflammatory effect . among the disorders related to hyperuricemia , gout is the most representative and well known . while symptoms of a gout attack are typical of an acute inflammatory response , as indicated by the presence of swelling , heat , rubescence , and pain , there are many disorders with mild but chronic inflammation which are very likely to be related to ua metabolism . in the latter case , superoxide free radicals generated by xo are key players leading to chronic inflammatory processes eventually resulting in impaired organ functions . thus , we introduce two independent mechanisms underlying ua metabolism - induced inflammation . however , a line of subsequent studies has elucidated that inflammasomes are key players in the onsets of a wide range of diseases as well as host defense . excessive metabolites , such as atp or monosodium urate crystals ( muc ) , were also confirmed to be involved in the activation of inflammasomes , and inflammatory responses occurring via inflammasomes have been demonstrated to be linked to the onset and progression of human diseases , including gout , atherosclerosis and nash , as described below in detail [ 1824 ] . inflammasomes are known to be divided into discernible patterns , depending on component proteins . although the mechanism of inflammasome activation by muc has not been fully elucidated , the following mechanism was proposed . the other is the generation of ros , because an antioxidant , n - acetyl - cysteine , abolished il-1 secretion by muc . however , the relationship between intracellular k level changes and ros generation remains unknown , and future studies are expected to resolve this issue [ 27 , 28 ] . xo , as well as nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase and the mitochondrial electron - transport chain , generates ros . xo depletion induces renal interstitial fibrosis , and renal epithelial cells from xor ( / ) mice are more readily transformed into myofibroblasts . the tissue and cellular distributions of xo in mammals are highest in the liver and intestines due to xo - rich parenchymal cells . xo activity is low in human serum , the brain , heart , and skeletal muscle , while being rich in microvascular endothelial cells and is also present in macrophages . xo activation is induced by lps , angiotensin ii , nadph oxidase , hypoxia , hypoxia - inducible factor 1 , and inflammatory cytokines such as il-1 [ 4649 ] . the release of xo is increased in hypercholesterolemia , chronic hyperammonemia , thermal trauma , beta - thalassemia , brain ischemia , and pulmonary artery hypertension [ 5054 ] . indeed , xo was significantly higher in the aortic walls and skeletal muscles of old rats than in those of their young counterparts . it appears that hyperglycemia itself has no impact on liver xo activity , though cardiac , renal , and brain xo activities were shown to be increased in rats with advanced diabetes [ 56 , 57 ] . xo is one of the major superoxide sources in ischemia / reperfusion injuries of the heart , forebrain , skin , liver [ 61 , 62 ] , and gastric mucosa , as well as multiple system organ failure after hind limb reperfusion . excess fructose metabolism results in atp depletion which is associated with degradation of amp to hypoxanthine , followed by conversion to ua by xo . atp depletion , such as that characteristic of glycogen storage disease type 1 , hypoglycemia , exercise , and starvation , also increases ua production . xo regulates cyclooxygenase-2 in the inflammatory system , and xo appears to be critical for innate immune function . as noted above , superoxide from xo has been suggested to play roles in various forms of inflammatory or ischemic pathophysiology ( figure 3 ) , not necessarily involving hyperuricemia . while gout is a disorder well known to be caused by the precipitation of ua crystals , the involvement of hyperuricemia in ckd is also widely recognized . the major causes of ckd have been regarded as diabetes mellitus and hypertension , and thus , hyperuricemia was long viewed as a consequence of ckd . in contrast , recent studies demonstrated a significant association between serum ua and the development of ckd . while each metabolic syndrome component , including hyperglycemia , hyperlipidemia , and hypertension , was associated with an increased ckd risk , hyperuricemia was apparently an independent risk factor not influenced by the others . we speculate that ua itself and superoxide free radical generation both play roles in the molecular mechanisms underlying hyperuricemia - related ckd development , but further research is required to elucidate the complex mechanistic interactions between serum ua and ckd . as mentioned in section 2 , both ua and superoxide free radicals are simultaneously produced by xo and might be the pathophysiological cause of these diseases . although the relationships between serum ua levels and atherosclerotic diseases , including hypertension [ 85 , 86 ] , have been documented , whether or not serum ua itself is an independent cardiovascular risk factor remains controversial as most hyperuricemic patients with cardiovascular diseases ( cvd ) have other complications such as hypertension , dyslipidemia , diabetes , and ckd as well , which are generally regarded as more established risk factors for cvd than hyperuricemia . recently , however , a growing body of evidence from both clinical and basic research supports the hypothesis that hyperuricemia , partly via elevated xo activity , is an independent risk factor for hypertension and cvd . a recently published meta - analysis showed that the adjusted relative risk of developing hypertension was 1.48 for hyperuricemic patients , and this association was apparently much stronger in younger , early - onset hypertensive patients [ 86 , 95 ] . several clinical trials have demonstrated the beneficial effects of ua lowering therapy for hypertension [ 9699 ] . besides the association with hypertension , hyperuricemia or gout has been confirmed to be related to the morbidity and the mortality of cvd [ 102106 ] . several clinical studies have indicated the benefits of xo inhibitors for reducing the incidence of myocardial infarction , improving exercise tolerance in patients with stable angina , and enhancing endothelial function [ 110 , 111 ] . xo is also expressed in endothelial cells and was shown to be increased in the aortic endothelial cells of apoe mice , an established model of atherosclerosis . since oxidative stress inactivates no and leads to endothelial dysfunction , endothelial xo , especially given its enhanced expression during the development of atherosclerosis , contributes to vascular damage via ros production . recently , we established that xo activity in macrophages also plays a key role in the development of atherosclerosis . we demonstrated that allopurinol treatment ameliorated aortic lipid accumulation and calcification of the vessels of apoe - ko mice and that allopurinol markedly suppressed the transformation of j774.1 murine macrophages or primary cultured human macrophages into foam cells in response to stimulation with acetyl ldl or vldl . the expressions of scavenger receptors ( sr - a1 , sr - b1 , and sr - b2 ) and vldl receptors in j774.1 cells were upregulated by xor overexpression and downregulated by sirna - mediated xor suppression , raising the possibility that xo activity in macrophages positively regulates foam cell formation by increasing the uptake of modified ldl or vldl . conversely , expressions of abca1 and abcg1 , which regulate cellular cholesterol efflux , were decreased by xor overexpression and increased by xor knockdown . furthermore , allopurinol suppressed the expressions of inflammatory cytokines such as il-1 , il-6 , il-12 , and tnf , and the expressions of vcam1 , mcp-1 , and mmp2 , which were upregulated in j774.1 cells transformed into foam cells by atherosclerogenic serum . subsequently , febuxostat , another xo inhibitor , was also demonstrated to attenuate the development of atherosclerotic lesions in apoe mice . that study showed xo expression to be increased in macrophages infiltrating atherosclerotic plaques and that febuxostat diminished the ros level in the aortic walls of apoe mice . the authors demonstrated that cholesterol crystals ( ccs ) increased endogenous xo activity and ros production in macrophages and that ccs enhanced not only il-1 release via nlrp3 inflammasome activation but also secretions of other inflammatory cytokines such as il-1 , il-6 , and mcp-1 from macrophages , processes which in turn were suppressed by febuxostat or ros inhibitors . the significance of nlrp3 inflammasome activation in macrophages by ccs was verified by the observation that atherosclerosis in high - cholesterol diet fed ldl receptor- ( ldlr- ) deficient mice was alleviated by transplanting bone marrow from nlrp3-deficient , asc - deficient , or il-1/-deficient mice . taking these observations together , we can reasonably speculate that xo in macrophages enhances foam cell formation , ros production , and nlrp3 inflammasome activation , all three of which exacerbate inflammation and plaque formation , thereby contributing to the development of atherosclerotic diseases [ 75 , 114116 ] . as noted above , not only xo inhibitors but also uricosuric agents markedly lowered blood pressure , especially in studies targeting early - stage hypertensive patients and those using animal models [ 100 , 101 ] . the number of nonalcoholic fatty liver disease ( nafld ) patients including those with nash has been increasing worldwide and a portion of nash patients will progress to hepatocarcinoma onset [ 121123 ] . therefore , numerous investigations have been performed in efforts to elucidate the causes of nash . nash is characterized by fat deposition , inflammation and fibrosis in the liver , and a two - hit mechanism of onset has been proposed [ 124126 ] . interestingly , recent studies have raised the possibility that ua is among the risk factors for nash pathology . many clinical studies have been carried out to investigate the relationship between serum ua levels and nafld / nash progression . for example , a cohort study in korea found the serum ua level to be a useful marker for predicting nafld development because the serum ua concentration correlated positively with the 5-year incidence rate of nafld . in addition , there are also studies demonstrating that serum ua is a risk factor for the development and/or progression of nafld including nash [ 130132 ] . moreover , a fraction of nafld / nash patients also have obesity , and hypertrophic adipocytes were also reported to secrete ua . taken together , these results indicate serum ua to be a good parameter for predicting the development of nafld / nash , and that xo inhibitors or uricosuric agents might have potential as treatments for ameliorating the features of nafld . the authors asserted that ros generation by ua evoked endoplasmic reticulum stress , leading to upregulation of lipogenic genes , such as acetyl coa carboxylase1 and fasn . ros generation by ua is considered to depend on nadph oxidase activation [ 136 , 138 , 139 ] . moreover , other groups have demonstrated that diets which lead to nash also increase the expressions of inflammasome components [ 143145 ] . taking these lines of evidence together , in the initial stage of nash , inflammasomes appear to exert a protective effect , but continuous inflammasome activation appears to cause excessive productions of inflammatory cytokines , ultimately resulting in liver injury . although , to date , numerous factors playing important roles in nash progression have been identified , ua also appears to be a key participant in the onset of nafld / nash . hyperuricemia was reportedly found to be related to insulin resistance in several clinical analyses [ 146152 ] . in addition , several meta - analyses have suggested the ua level to be positively associated with the development of type 2 diabetes mellitus ( dm ) [ 153156 ] , although mendelian randomization studies did not support circulating ua as being among the causes of dm development [ 157 , 158 ] . in metabolic syndrome patients , an oxidative stress marker , the myeloperoxidase level , was decreased by allopurinol and endothelial function improved . on the other hand , rapid ua reduction achieved by rasburicase , a urate oxidase , in obese subjects with high ua resulted in increasing the markers of systemic and skeletal muscle oxidative stress while having no effect on insulin sensitivity . furthermore , excess fructose intake is one of the major causes of the development of obesity with hyperuricemia , fatty liver , and metabolic syndrome . because ua is the downstream product of ampd and allopurinol abolished fructose - induced lipid accumulation , ampd effects on ampk appeared to depend on ua . via these mechanisms , ua is considered to be an antioxidant in human blood , though ua induces oxidative stress in cells . the stimulation of nadph oxidase - dependent ros by ua resulted in the activation of map kinase p38 and erk1/2 , a decrease in no bioavailability , and increases in both protein nitrosylation and lipid oxidation . mitochondrial oxidative stress inhibits aconitase in the krebs cycle , resulting in citrate accumulation and the stimulation of atp citrate lyase and fatty acid synthase , ultimately leading to de novo lipogenesis . in hepatocytes treated with high ua , oxidative stress is increased , which activates serine ( rat ser307 and human ser312 ) phosphorylation of irs-1 . therefore , ua - induced lipid accumulation and oxidative stress are responsible for the development of insulin resistance and diabetes . involvement of increased xo catalyst activity in pathophysiological processes ( figure 4 ) suggests applications of xo inhibitors to the treatment of various disorders . at present , xo inhibitors , including allopurinol , oxypurinol , febuxostat , and topiroxostat , are widely used for treating gout and hyperuricemia . furthermore , xo inhibitors have been experimentally or clinically shown to exert beneficial effects by lowering serum ua and oxidative stress . on the other hand , beneficial effects of xo inhibitors on atherosclerosis and nash constitute an evolving concept that has yet to be proven . in rats with fructose - induced metabolic syndrome , febuxostat treatment reversed hyperuricemia , hypertension , dyslipidemia , and insulin resistance the beneficial effects of xo inhibitors on nash are rarely reported , except by our research group , because animal models of nash with obesity , inflammation , and fibrosis have been difficult to establish . thus , we next used a high - fat diet containing trans - fatty acids and a high - fructose diet to induce nash development in our animal models . another report showed that inhibition of xo activity also significantly prevents hepatic steatosis induced by a high - fat diet in mice . tungsten , acting as an xo inhibitor , has an inhibitory effect on both atherosclerosis and oxidative stress . we reported for the first time that more specific xo inhibition , using allopurinol rather than tungsten on macrophages , resulted in the inhibition of foam cell formation and reduced atherosclerotic lesions in apoe - ko mice , independently of the serum lipid profile . moreover , both xo overexpression and knockdown of xo expression revealed vldl receptors to be dramatically upregulated by xo . febuxostat was also proven to have similar effects in terms of reducing the atherosclerotic lesions in apoe - ko mice , and oxidative stress was reduced in macrophages from atherosclerotic lesions . as a strategy for suppressing atherosclerosis , xo inhibition is expected to act on either macrophages or inflammatory cells . however , controversy persists as to whether the effect of xo on endothelial function is clinically relevant as an interventional target . pretreatment with xo inhibitors has beneficial effects on ischemia / reperfusion injuries of the intestine , in the impaired liver [ 61 , 62 ] , the edematous brain , kidneys with contrast induced nephropathy , and coronary ischemia . inflammation related to ua metabolism is induced via either inflammasome activation by ua crystal precipitation or free radical production in response to xo activity . in addition to gout , many disorders are known to be related to ua metabolism and xo inhibitor treatments have been shown to be effective for preventing the onset and/or the progression of such disorders . in particular , atherosclerosis and nash are diseases for which relationships with ua metabolism were not immediately recognized , but rodent model studies revealed the importance of ua metabolism maintenance for managing these disorders . we believe the impact of ua metabolism on many diseases accompanying chronic inflammation to have been underestimated . future studies are anticipated to reveal the pathological contribution of serum ua and/or xo activity to the specific processes underlying various disorders .

uric acid ( ua ) is the end product of the metabolic pathway for purines , the main constituents of nucleotides . in the salvage pathway , hypoxanthine - guanine phosphoribosyl transferase ( hgprt ) plays an important role in generating imp , thereby inhibiting ua generation . it has been suggested that this antioxidant effect of the high ua concentrations in humans contributes to neuroprotection in several neurodegenerative and neuroinflammatory diseases [ 814 ] . however , despite the potential antioxidant effect of ua itself , numerous studies have revealed close associations of serum ua concentrations and various disorders , most of which are included in the metabolic syndrome category . thus , ua metabolism may be a so - called double - edged sword as regards the inflammatory and/or oxidative responses in many organs , though on the whole , its harmful effects appear to outweigh the benefits of ua in most cases . in this review , we first explain the two putative molecular mechanisms underlying inflammation occurrence in relation to ua metabolism ; one is inflammasome activation via ua crystallization and the other involves superoxide free radicals generated by xo . while the ua crystallization mechanism would be dependent on a high serum ua concentration , the latter may not necessarily reflect the serum ua concentration though xo activity does lead to the production of reactive oxygen species ( ros ) . importantly , recent studies have demonstrated beneficial effects of xo inhibitors against the occurrence and/or progression of several disorders , particularly atherosclerosis and nonalcoholic steatohepatitis ( nash ) , both of which are associated with insulin resistance , hyperlipidemia , and/or obesity . in this review , atherosclerosis and nash are discussed extensively , while studies of gout and chronic kidney diseases ( ckd ) are mentioned briefly . in conclusion , we propose that such xo inhibitors may be more useful for preventing a variety of disorders , such as atherosclerosis and nash , than previously believed , probably via an anti - inflammatory effect . while symptoms of a gout attack are typical of an acute inflammatory response , as indicated by the presence of swelling , heat , rubescence , and pain , there are many disorders with mild but chronic inflammation which are very likely to be related to ua metabolism . in 2002 , the inflammasome concept was proposed to involve multiple proteins and to control the cleavage of prointerleukin 1 ( il-1 ) . initially , inflammasomes were considered to play a role in immune responses and serve as defense systems against pathogens [ 16 , 17 ] . however , a line of subsequent studies has elucidated that inflammasomes are key players in the onsets of a wide range of diseases as well as host defense . excessive metabolites , such as atp or monosodium urate crystals ( muc ) , were also confirmed to be involved in the activation of inflammasomes , and inflammatory responses occurring via inflammasomes have been demonstrated to be linked to the onset and progression of human diseases , including gout , atherosclerosis and nash , as described below in detail [ 1824 ] . among them , the nlrp3 inflammasome , comprised of three major components , nod - like receptor 3 ( nlrp3 ) , apoptosis - associated speck - like protein containing a card ( asc ) and caspase-1 , has been well investigated . first , the toll - like receptor ligands , such as lipopolysaccharide ( lps ) , activate the nf-b pathway and upregulate the expression levels of interleukins , including pro - il-1 and pro - il-18 . subsequently , the inflammasome complex activated by pathogen - associated molecular patterns ( pamps ) or damage - associated molecular patterns ( damps ) cleaves pro - il-1 or pro - il-18 , resulting in the production of mature interleukins [ 1517 ] . although the mechanism of inflammasome activation by muc has not been fully elucidated , the following mechanism was proposed . elevation of intracellular ros mediates the detachment of thioredoxin - interacting protein ( txnip ) from thioredoxin and enables txnip to associate with nlrp3 , leading to nlrp3 inflammasome activation [ 29 , 30 ] . the tissue and cellular distributions of xo in mammals are highest in the liver and intestines due to xo - rich parenchymal cells . xanthine oxidoreductase ( xor ) is present in hepatocytes , while xo is present in bile duct epithelial cells , concentrated toward the luminal surface . xo activity is low in human serum , the brain , heart , and skeletal muscle , while being rich in microvascular endothelial cells and is also present in macrophages . the study using electron spin resonance measurements revealed the contribution of increased xo activity to endothelial dysfunction in patients with coronary artery diseases . xo activation is induced by lps , angiotensin ii , nadph oxidase , hypoxia , hypoxia - inducible factor 1 , and inflammatory cytokines such as il-1 [ 4649 ] . the release of xo is increased in hypercholesterolemia , chronic hyperammonemia , thermal trauma , beta - thalassemia , brain ischemia , and pulmonary artery hypertension [ 5054 ] . indeed , xo was significantly higher in the aortic walls and skeletal muscles of old rats than in those of their young counterparts . it appears that hyperglycemia itself has no impact on liver xo activity , though cardiac , renal , and brain xo activities were shown to be increased in rats with advanced diabetes [ 56 , 57 ] . xo is one of the major superoxide sources in ischemia / reperfusion injuries of the heart , forebrain , skin , liver [ 61 , 62 ] , and gastric mucosa , as well as multiple system organ failure after hind limb reperfusion . xo activity , along with lipid peroxidation , myeloperoxidase activity and no levels , is increased in the liver in response to renal ischemia / reperfusion in diabetic rats . ischemia / reperfusion injury is attributable to elevated xo activity and atp depletion related to increasing hypoxanthine and xanthine levels during ischemia , and reperfusion provides o2 for oxidation of these compounds . excess fructose metabolism results in atp depletion which is associated with degradation of amp to hypoxanthine , followed by conversion to ua by xo . xo increased egr-1 mrna and protein , as well as the phosphorylation of erk1/2 , while pretreatment with an erk1/2 inhibitor prevented induction of egr-1 by xo . while gout is a disorder well known to be caused by the precipitation of ua crystals , the involvement of hyperuricemia in ckd is also widely recognized . the major causes of ckd have been regarded as diabetes mellitus and hypertension , and thus , hyperuricemia was long viewed as a consequence of ckd . while each metabolic syndrome component , including hyperglycemia , hyperlipidemia , and hypertension , was associated with an increased ckd risk , hyperuricemia was apparently an independent risk factor not influenced by the others . in terms of ckd pathogenesis , serum ua is likely to activate the renin - angiotensin system resulting in vascular smooth muscle cell proliferation and to induce an epithelial - to - mesenchymal transition of renal tubular cells . we speculate that ua itself and superoxide free radical generation both play roles in the molecular mechanisms underlying hyperuricemia - related ckd development , but further research is required to elucidate the complex mechanistic interactions between serum ua and ckd . therefore , damage from ua , ros , and ua - induced and/or ros - induced inflammation might together contribute to the progression of certain diseases , and distinguishing which mechanism acts first is often difficult in lifestyle - related diseases . although the relationships between serum ua levels and atherosclerotic diseases , including hypertension [ 85 , 86 ] , have been documented , whether or not serum ua itself is an independent cardiovascular risk factor remains controversial as most hyperuricemic patients with cardiovascular diseases ( cvd ) have other complications such as hypertension , dyslipidemia , diabetes , and ckd as well , which are generally regarded as more established risk factors for cvd than hyperuricemia . recently , however , a growing body of evidence from both clinical and basic research supports the hypothesis that hyperuricemia , partly via elevated xo activity , is an independent risk factor for hypertension and cvd . despite the association between hyperuricemia and hypertension having been recognized since the 19th century , it was not until recently that hyperuricemia was demonstrated to be an independent risk factor for hypertension development [ 8793 ] . a recently published meta - analysis showed that the adjusted relative risk of developing hypertension was 1.48 for hyperuricemic patients , and this association was apparently much stronger in younger , early - onset hypertensive patients [ 86 , 95 ] . consistently , both allopurinol and benziodarone ( uricosuric agent ) reduced blood pressure in rats with hypertension induced by hyperuricemia [ 100 , 101 ] , suggesting that not only xo activity but also ua itself plays an important role in the pathogenesis of hypertension . besides the association with hypertension , hyperuricemia or gout has been confirmed to be related to the morbidity and the mortality of cvd [ 102106 ] . according to a recently published meta - analysis , the relative risks of morbidity and mortality for coronary heart diseases were 1.13 and 1.27 , respectively , in hyperuricemic patients as compared to controls . several clinical studies have indicated the benefits of xo inhibitors for reducing the incidence of myocardial infarction , improving exercise tolerance in patients with stable angina , and enhancing endothelial function [ 110 , 111 ] . however , interestingly , unlike the case of treating hypertension , uricosuric agents have failed to show any benefits in patients with hyperuricemia or gout [ 110 , 112 ] . xo is also expressed in endothelial cells and was shown to be increased in the aortic endothelial cells of apoe mice , an established model of atherosclerosis . since oxidative stress inactivates no and leads to endothelial dysfunction , endothelial xo , especially given its enhanced expression during the development of atherosclerosis , contributes to vascular damage via ros production . during atherosclerosis development , monocytes migrate beneath the endothelium and transform into macrophages , which then turn into foam cells by incorporating modified low density lipoproteins ( ldl ) ( such as oxidized ldl and acetyl ldl ) or very low density lipoproteins ( vldl ) . foam cells contribute to the formation of unstable plaques by secreting inflammatory mediators and matrix - degrading proteases ( such as matrix metalloproteinases ( mmps ) ) and by generating a prothrombotic necrotic core by eventually undergoing necrotic or apoptotic death . we demonstrated that allopurinol treatment ameliorated aortic lipid accumulation and calcification of the vessels of apoe - ko mice and that allopurinol markedly suppressed the transformation of j774.1 murine macrophages or primary cultured human macrophages into foam cells in response to stimulation with acetyl ldl or vldl . the expressions of scavenger receptors ( sr - a1 , sr - b1 , and sr - b2 ) and vldl receptors in j774.1 cells were upregulated by xor overexpression and downregulated by sirna - mediated xor suppression , raising the possibility that xo activity in macrophages positively regulates foam cell formation by increasing the uptake of modified ldl or vldl . furthermore , allopurinol suppressed the expressions of inflammatory cytokines such as il-1 , il-6 , il-12 , and tnf , and the expressions of vcam1 , mcp-1 , and mmp2 , which were upregulated in j774.1 cells transformed into foam cells by atherosclerogenic serum . the authors demonstrated that cholesterol crystals ( ccs ) increased endogenous xo activity and ros production in macrophages and that ccs enhanced not only il-1 release via nlrp3 inflammasome activation but also secretions of other inflammatory cytokines such as il-1 , il-6 , and mcp-1 from macrophages , processes which in turn were suppressed by febuxostat or ros inhibitors . the significance of nlrp3 inflammasome activation in macrophages by ccs was verified by the observation that atherosclerosis in high - cholesterol diet fed ldl receptor- ( ldlr- ) deficient mice was alleviated by transplanting bone marrow from nlrp3-deficient , asc - deficient , or il-1/-deficient mice . taking these observations together , we can reasonably speculate that xo in macrophages enhances foam cell formation , ros production , and nlrp3 inflammasome activation , all three of which exacerbate inflammation and plaque formation , thereby contributing to the development of atherosclerotic diseases [ 75 , 114116 ] . the results obtained suggest that ua presumably contributes to early - stage hypertension by promoting renal vasoconstriction via reduced no production and activation of the renin - angiotensin system [ 86 , 98 ] . the number of nonalcoholic fatty liver disease ( nafld ) patients including those with nash has been increasing worldwide and a portion of nash patients will progress to hepatocarcinoma onset [ 121123 ] . nash is characterized by fat deposition , inflammation and fibrosis in the liver , and a two - hit mechanism of onset has been proposed [ 124126 ] . many clinical studies have been carried out to investigate the relationship between serum ua levels and nafld / nash progression . for example , a cohort study in korea found the serum ua level to be a useful marker for predicting nafld development because the serum ua concentration correlated positively with the 5-year incidence rate of nafld . in addition , there are also studies demonstrating that serum ua is a risk factor for the development and/or progression of nafld including nash [ 130132 ] . taken together , these results indicate serum ua to be a good parameter for predicting the development of nafld / nash , and that xo inhibitors or uricosuric agents might have potential as treatments for ameliorating the features of nafld . fructose treatment of hepg2 cells reportedly increased both the intracellular ua concentration and triglyceride ( tg ) accumulation , while allopurinol , an xo inhibitor , suppressed this fructose - mediated tg deposition . as a mechanism of ua - induced tg accumulation , the authors asserted that the elevation of intracellular ros by ua raised both the citrate concentration and atp citrate lyase activity via enhanced phosphorylation at s455 , resulting in the induction of lipogenesis . collectively , these observations indicate that ua enhances fatty acid synthesis by regulating lipogenesis and induces ros generation by regulating nadph oxidase activity and upregulating fatty acid synthesis , thereby contributing to nash development . taking these lines of evidence together , in the initial stage of nash , inflammasomes appear to exert a protective effect , but continuous inflammasome activation appears to cause excessive productions of inflammatory cytokines , ultimately resulting in liver injury . although , to date , numerous factors playing important roles in nash progression have been identified , ua also appears to be a key participant in the onset of nafld / nash . in addition , several meta - analyses have suggested the ua level to be positively associated with the development of type 2 diabetes mellitus ( dm ) [ 153156 ] , although mendelian randomization studies did not support circulating ua as being among the causes of dm development [ 157 , 158 ] . in metabolic syndrome patients , an oxidative stress marker , the myeloperoxidase level , was decreased by allopurinol and endothelial function improved . on the other hand , rapid ua reduction achieved by rasburicase , a urate oxidase , in obese subjects with high ua resulted in increasing the markers of systemic and skeletal muscle oxidative stress while having no effect on insulin sensitivity . furthermore , excess fructose intake is one of the major causes of the development of obesity with hyperuricemia , fatty liver , and metabolic syndrome . the ua increased fructose - induced tg accumulation and decreased -hydroxybutyrate levels , dose - dependently , while allopurinol , a xo inhibitor , blocked it . because ua is the downstream product of ampd and allopurinol abolished fructose - induced lipid accumulation , ampd effects on ampk appeared to depend on ua . the stimulation of nadph oxidase - dependent ros by ua resulted in the activation of map kinase p38 and erk1/2 , a decrease in no bioavailability , and increases in both protein nitrosylation and lipid oxidation . mitochondrial oxidative stress inhibits aconitase in the krebs cycle , resulting in citrate accumulation and the stimulation of atp citrate lyase and fatty acid synthase , ultimately leading to de novo lipogenesis . therefore , ua - induced lipid accumulation and oxidative stress are responsible for the development of insulin resistance and diabetes . febuxostat preserved renal function in 5/6 nephrectomized rats with and without coexisting hyperuricemia and prevented diabetic renal injury in streptozotocin - treated rats [ 166 , 167 ] . febuxostat also reduced the induction of endoplasmic reticulum stress , as assessed by grp-78 ( glucose - regulated protein-78 ) , atf4 ( activating transcription factor-4 ) , and chop ( c / ebp homologous protein-10 ) . in rats with fructose - induced metabolic syndrome , febuxostat treatment reversed hyperuricemia , hypertension , dyslipidemia , and insulin resistance the beneficial effects of xo inhibitors on nash are rarely reported , except by our research group , because animal models of nash with obesity , inflammation , and fibrosis have been difficult to establish . nash in response to the mcd diet , as used in our studies , caused primarily inflammation and also made the mice lean , such that no benefit of xo inhibition was obtained . thus , we next used a high - fat diet containing trans - fatty acids and a high - fructose diet to induce nash development in our animal models . we reported for the first time that more specific xo inhibition , using allopurinol rather than tungsten on macrophages , resulted in the inhibition of foam cell formation and reduced atherosclerotic lesions in apoe - ko mice , independently of the serum lipid profile . we also identified phenotypic changes of macrophages in response to allopurinol , such as alterations of gene expressions involved in lipid accumulation . pretreatment with xo inhibitors has beneficial effects on ischemia / reperfusion injuries of the intestine , in the impaired liver [ 61 , 62 ] , the edematous brain , kidneys with contrast induced nephropathy , and coronary ischemia .

pharmacovigilance ( pv ) is defined as the science and activities relating to the detection , assessment , understanding and prevention of adverse effects and all other problems related to medicines . the 16th world health assembly ( 1963 ) adopted a resolution ( wha 16.36 ) that reaffirmed the need for early detection and rapid dissemination of information on adverse reactions due to medicines , and subsequently led to the creation of the who programme for international drug monitoring . under this programme , systems have been created in member states for the collection and evaluation of individual case safety reports ( icsrs ) . in all countries ( low , middle or high income ) , national pv systems rely heavily on spontaneous ( or voluntary ) reporting in which suspected adverse drug reactions ( adrs ) are reported to a national coordinating centre by health professionals , manufacturers or directly by patients . of all the sources of data for drug safety monitoring , the spontaneous reporting systems provide the highest volume of information at the lowest maintenance cost , and have proven their value in the early detection of patient safety issues related either to the products themselves or to their use . the most important function of spontaneous reporting systems is the early identification of signals and formulation of hypotheses , leading to further confirmatory investigations or sometimes regulatory warnings and changes of product information leaflets . in some instances , for example , in the case of cerivastatin , an association ( a signal ) between cerivastatin , myopathy and rhabdomyolysis was published , based on icsrs , by the uppsala monitoring centre ( umc ) in 1999 ; various regulatory decisions were announced between 1999 and 2001 in different countries . in spontaneous reporting , the most common form of pv , no measures are taken to systematically follow - up patients to record adrs that they may have experienced . the absence of an effective pv system with clear protocols , tools and a pv mandate can also act as a barrier to adr reporting . under these circumstances it is difficult to determine the actual number of individuals experiencing an adverse reaction to the medicine . this , together with the uncertainties around the number of patients exposed to the medicine in question , makes it difficult to estimate rates and frequencies of adrs through spontaneous reporting . methods of greater scientific rigour are needed to establish quantitative aspects of medicine safety , to better identify specific risk factors and high - risk groups , and to provide valid clinical characteristics of problems associated with specific medicines . public health programmes may treat a large population , in an organized and structured fashion , and record the number of patients treated , drugs used , doses given , etc . these programmes thus provide good platforms for capturing the quantitative aspects ( rates and frequencies ) of adrs and adverse events with the medicines used . in the management of hiv and tuberculosis ( tb ) patients , short- and long - term toxicity data play a critical role in informing treatment choices [ 1113 ] . in adopting a public health approach to the delivery of hiv and tb services , it is important to quantify and characterize treatment - related risks , as early as possible , to minimize the harm to public health as well as to sustain public confidence in the programme . for example , it is important to know what adrs may be expected with certain treatment and how many will experience this adr . such quantitative information will have a direct impact on treatment guidelines , policies and practices . while spontaneous reporting remains the bedrock of safety monitoring , additional methods are needed to quantify the frequency and severity of expected and unanticipated adrs occurring in patients receiving long - term treatment . in the context of hiv programmes which provide life - long therapy , a simple , inexpensive but in addition , we need to monitor adverse events that , instead of being directly attributed to the active substance , could be attributed more to aspects such as inappropriate use , medication errors , poor quality products or drug - drug interactions . the global fund urges countries to invest in pv programmes that monitor the quality , usage and efficacy of the hiv , tb and malaria medicines that it helps countries to buy . an effective pv programme is one that monitors medicines used in all public health programmes , to improve patient safety , and to improve the credibility and public perception of these programmes . cohort event monitoring ( cem ) [ 16 , 17 ] is a prospective , observational , cohort study of adverse events associated with one or more medicines . an adverse event is any untoward medical occurrence that may present during treatment with a pharmaceutical product , but which does not necessarily have a causal relationship with this treatment . a cem programme is essentially an observation of a new medicine in routine clinical practice in the early postmarketing phase , but it can be used for older medicines . it is based on the principles of the new zealand intensive medicines monitoring programme and the uk prescription event monitoring except that in most resource - limited countries , treatment within public health programmes ( such as tb and hiv ) is not provided on a prescription basis . cem is thus an early warning system that interviews patients on a certain treatment ( the cohort ) , for capturing problems ( the events ) with new medicines in public health programmes ; the patients are interviewed before and after starting treatment . cem captures all medicine - related events , including medication errors , problems due to poor storage conditions , poor quality or counterfeit medicines , and drug interactions . patients may be recruited from all health facilities involved in providing the medicines , or patients may be recruited from selected health facilities that are representative of the whole country . cem involves recording all events , regardless of its severity or seriousness , and includes the following steps : a patient cohort is established for the medicine of interest.adverse events experienced by patients in the cohort are recorded by interviewing patients as part of routine patient / health professional encounters.patient i d and demographic data , together with details of medicines , events and other relevant information , are recorded on questionnaires . adverse events experienced by patients in the cohort are recorded by interviewing patients as part of routine patient / health professional encounters . patient i d and demographic data , together with details of medicines , events and other relevant information , are recorded on questionnaires . it is normally recommended that a cohort of 10,000 patients be enrolled [ 16 , 17 ] . this gives a 95 % chance of identifying a specific event that has an incidence of 1:3000 ( that is , an uncommon or rare event ) . a cohort of 3000 patients gives a 95 % chance of identifying a single incident with an incidence of 1:1000 . for a meaningful assessment , at least three events need to be identified , hence the higher objective of 10,000 patients [ 16 , 17 ] . it is determined by the length of treatment to be monitored in individual patients , and the time it takes to reach the desired cohort size . in tb therapy , treatment of individual patients is monitored for a period that is considered to be appropriate for the identification of both short- and long - term effects ( e.g. for drug - resistant tb this might be for 2 years or more after ending a treatment course lasting 2 years ) . if there is special interest in subgroups such as pregnant women or children , or more information is needed on a type of event of particular concern , the programme may need to continue for a longer period to accrue sufficient numbers in these subgroups to be able to achieve statistical significance when testing associations . the data are reviewed at regular intervals ( e.g. 3-monthly ) to detect early trends that may require further monitoring . adverse events are recorded using treatment initiation and treatment review forms , to record events happening to patients during a control period , prior to and after treatment initiation , respectively . the length of the control period could vary but should not extend beyond what the patient may reasonably remember . by collecting data on events happening to patients before treatment , cem might involve special ethical considerations since it is a methodology that requires the collection of detailed personal data , for patient follow - up . since cem aims to measure incidence , it is essential that duplicate entries are avoided ; this can be done only if patients can be identified correctly . as a prerequisite to collecting patient data , it is important to seek the approval of the highest appropriate authority in the country . some countries may choose to obtain informed consent from every patient ; however , this will be time consuming . an alternative to obtaining informed consent opt out principle where information about the cem programme is provided publicly , and patients can then decide whether or not they wish to have their data stored as part of cem . systems for data storage should be password protected , with controlled access to the premises , etc . the umc has , in collaboration with the who , developed a specific software , cemflow , for the secure management and analysis of cem data . it is web - based but data entry can be done offline with regular uploads to the main database . detailed handbooks describing these principles for cem in public health programmes have been developed by the who [ 16 , 17 ] and can be downloaded from http://www.who.int/medicines/areas/quality_safety/safety_efficacy/pharmpubhealth/en/index.html . cem is currently being used for antimalarials in kenya , nigeria , the united republic of tanzania , and zimbabwe . cem of antiretroviral ( arv ) drugs is planned in belarus and in the united republic of tanzania . the who proposes targeted spontaneous reporting ( tsr ) as a methodology that builds on the principles of spontaneous reporting but applied in a defined setting . in this method , health professionals managing a well - defined group of patients [ e.g. patients on treatment for drug - resistant tb or those switching from first - line to second - line antiretroviral therapy ( art ) ] are sensitized to report specific safety concerns suspected to be medicine related . tsr addresses a distinct set of questions and provides a comprehensive monitoring method that is affordable , feasible and sustainable in settings with limited financial and human resources . it also promotes the role of pv as a best practice that improves quality of care . tsr was developed by the who in 2010 and is being piloted in the hiv treatment programmes in three countries ( kenya , vietnam and uganda ) . tsr may be adapted either to report all suspected reactions in the defined population or to focus only on specific reactions of particular concern , for example treatment - threatening toxicity , etc . this serves to limit the reporting workload to those adverse events that are most significant to individuals and to the programmes . poor adherence to treatment due to adverse events such as nausea can be included as one of the targeted events in the tsr concept for priority reporting . it is particularly useful in the targeted follow - up of patients with additional complications , such as patients with drug - resistant tb and lifelong art . in hiv care , compared with routine spontaneous reporting , tsr aims to increase reporting rates by targeting , training and mentoring reporters at selected high caseload clinics , and by task shifting the reporting to non - physician cadres of healthcare workers such as nurses , pharmacists , pharmacy technicians and patients living with hiv ( plhiv ) . by monitoring populations such as those receiving extended arv prophylaxis to prevent breast milk transmission of hiv , and by focusing on particular adrs of interest , such as renal toxicity related to tenofovir the who is now aiming to introduce tsr in tb treatment programmes . for many years , national tb programmes monitor patient outcomes in well - circumscribed groups termed cohorts . tsr can integrate monitoring of adrs as a standard of care , alongside the routine practice of monitoring success , death , default or failure of treatment within these tb cohorts . one benefit of monitoring within a treatment cohort is that the number and profiles of the exposed patients will be known . the number of patients in the treatment cohorts represent a denominator ; if all suspected cases of the targeted adr within this cohort are recorded , then it is possible to calculate simple frequencies of the targeted adr . tsr thus seeks to overcome the main disadvantages of spontaneous reporting , that is underreporting and the inability to calculate rates , while maintaining simplicity of use , low cost and linkage to existing systems . in all public health programmes reporting of drug - related adverse events more than 100 countries are members of the who pv programme and have existing systems in place for reporting adrs but do not always integrate with public health programmes . one of the objectives of tsr is to address this lack of integration between phps and pv programmes by leveraging existing pv systems , providing simple tools to phps , training and task shifting to encourage reporting . the steps required to meet the objectives of tsr include the following : monitoring for suspected drug - related problems is included as part of normal patient care . during patient investigation the possibility of a medicine - related problem is always considered . at every encounter , the healthcare professional screens for any suspected adr.suspicion of the possibility of a causal relationship between drug treatment and the event triggers the completion of an adr reporting form . the suspected adr is noted in the patient records.all healthcare professionals involved in patient care are sensitized to ask about and investigate adverse effects at every encounter.the reporting forms ( the generic national adr form ) and route for transmission of information are the same as those used in spontaneous reporting , but the forms are supported by specific guidance ( case definitions and written procedures ) on when to complete them and with details on standardized reporting of drug names and adrs.reporting may primarily target serious or treatment threatening / limiting ( treatment discontinuation , change of regimen , life - threatening event , hospitalization , poor adherence because of toxicity or death ) adrs rather than the notification of any suspected reaction . if the total burden of drug - related problems in the exposed population is of interest , health professionals can be instructed to report any suspected drug - related problem . if , however , the frequency of a specific problem suspected to be associated with the therapy given is the important question , e.g. vision disorders , a case definition for reporting can be given in the instructions to healthcare professionals.reporting lasts the whole length of treatment . in the case of tb , this is between 6 months and 2 years , and in the case of art , lifelongunlike cem , there are no baseline measurements nor is there any active follow - up of members of the cohort and , thus , less resources are required . monitoring for suspected drug - related problems is included as part of normal patient care . during patient investigation the possibility of a medicine - related problem is always considered . at every encounter , the healthcare professional screens for any suspected adr . suspicion of the possibility of a causal relationship between drug treatment and the event triggers the completion of an adr reporting form . all healthcare professionals involved in patient care are sensitized to ask about and investigate adverse effects at every encounter . the reporting forms ( the generic national adr form ) and route for transmission of information are the same as those used in spontaneous reporting , but the forms are supported by specific guidance ( case definitions and written procedures ) on when to complete them and with details on standardized reporting of drug names and adrs . reporting may primarily target serious or treatment threatening / limiting ( treatment discontinuation , change of regimen , life - threatening event , hospitalization , poor adherence because of toxicity or death ) adrs rather than the notification of any suspected reaction . if the total burden of drug - related problems in the exposed population is of interest , health professionals can be instructed to report any suspected drug - related problem . if , however , the frequency of a specific problem suspected to be associated with the therapy given is the important question , e.g. vision disorders , a case definition for reporting can be given in the instructions to healthcare professionals . reporting lasts the whole length of treatment . in the case of tb , this is between 6 months and 2 years , and in the case of art , lifelong unlike cem , there are no baseline measurements nor is there any active follow - up of members of the cohort and , thus , less resources are required . in public health programmes that integrate tsr , the routine patient record will include the question suspected adverse drug reaction : yes or no ? , thereby ensuring that it is always considered . the extent to which this information is recorded will indicate whether adr monitoring has become a part of normal practice . if safety monitoring of each patient is truly part of best practice and recording of whether the patient has experienced a suspected medicine - related problem or not is complete , the calculated reaction frequencies may be close estimates of true incidence rates . tsr provides the opportunity to monitor every single patient on treatment , as part of treatment and care . however , its successful implementation depends on the willingness of healthcare providers to participate in this monitoring exercise and to report their observations . adr reporting can be improved through interventions such as education , advocacy , mentoring , etc [ 22 , 23 ] . although different from these approaches , tsr , with its focus on targeted adrs and/or medicines , is likely to have a similar ( positive ) impact on reporting , albeit only for the targeted adrs , by reducing the reporting burden on overworked health professionals . all medical incidents ( events ) that patients experienced while on treatment can be captured by cem . those events considered noxious and unintended and suspected to be caused by the medicine are reportable as adrs through spontaneous reporting . tsr focuses on the collection of information on specific adrs , with specific medicines , in defined patient groups . adrs adverse drug reactions , cem cohort event monitoring , tsr targeted spontaneous reportingtable 1advantages and disadvantages of different types of pv methodsspontaneous reportingadvantages administratively simpler and less labour - intensive than cem less costly than cem has the potential of identifying very rare problems related to medicine use in any healthcare setting pv centres and health professionals are more likely to be familiar with this method as it is the most common method of pv used provides safety surveillance throughout the marketed life of all medicinesdisadvantages the data collected by this method are incomplete both in terms of quality and quantity underreporting is significant and widespread reliable rates can not be calculated and so risk can not be measured and risk factors can not be established with confidence there are strong biases in reporting deaths due to adrs may be incompletely recorded / investigated in some countries , especially when they occur outside healthcare special studies will need to be set up to obtain accurate information on areas of particular interest , e.g. pregnancy , children and specific events of concern . these special studies add to the cost and in turn reduce the cost advantage of spontaneous reportingcemadvantages the ability to produce rates the ability to produce a near - complete profile of the adverse events and/or adrs for the medicines of interest very effective in identifying signals at an early stage the ability to associate reactions with risk factors the ability to make accurate comparisons between medicines can detect reduced or failed therapeutic effect and can raise suspicion of medication errors , interactions , emerging resistance or poor - quality or counterfeit medicines the ability to record and examine details of all deaths and provide rates of deathdisadvantages the method is more labour - intensive , needs dedicated staff to perform treatment initiation ( baseline ) and treatment follow - up interviews more costly than spontaneous reporting patients may not turn up for follow - up ; potential for loss to follow - up patients may opt - out and refuse to be part of the cem ; this might make it difficult to reach the required cohort size takes certain expertise in recording adverse events , training will be necessary can not detect very rare problems with medicinestsradvantages is simpler , less costly and less labour - intensive than cem tsr represents an add - on to the routine monitoring of outcomes of patients can be focused on priority adrs the forms and routes for reporting are similar to those for routine spontaneous reporting can provide some measure of rates and incidences uses existing pv systems within countries links public health programmes to pv centres less likely to identify unanticipated reactionsdisadvantages the method is subject to individual willingness to monitor and report ; thus numerator ( number of individuals with the suspected adr ) may not be accurate completeness of reporting is therefore crucial there is limited experience with tsr and the technique needs to be field - testedadrs adverse drug reactions , cem cohort event monitoring , pv pharmacovigilance , tsr targeted spontaneous reporting the relationship between spontaneous reporting , tsr and cem . all medical incidents ( events ) that patients experienced while on treatment can be captured by cem . those events considered noxious and unintended and suspected to be caused by the medicine are reportable as adrs through spontaneous reporting . tsr focuses on the collection of information on specific adrs , with specific medicines , in defined patient groups . adrs adverse drug reactions , cem cohort event monitoring , tsr targeted spontaneous reporting advantages and disadvantages of different types of pv methods adrs adverse drug reactions , cem cohort event monitoring , pv pharmacovigilance , tsr targeted spontaneous reporting cohort event monitoring ( cem ) [ 16 , 17 ] is a prospective , observational , cohort study of adverse events associated with one or more medicines . an adverse event is any untoward medical occurrence that may present during treatment with a pharmaceutical product , but which does not necessarily have a causal relationship with this treatment . a cem programme is essentially an observation of a new medicine in routine clinical practice in the early postmarketing phase , but it can be used for older medicines . it is based on the principles of the new zealand intensive medicines monitoring programme and the uk prescription event monitoring except that in most resource - limited countries , treatment within public health programmes ( such as tb and hiv ) is not provided on a prescription basis . cem is thus an early warning system that interviews patients on a certain treatment ( the cohort ) , for capturing problems ( the events ) with new medicines in public health programmes ; the patients are interviewed before and after starting treatment . cem captures all medicine - related events , including medication errors , problems due to poor storage conditions , poor quality or counterfeit medicines , and drug interactions . patients may be recruited from all health facilities involved in providing the medicines , or patients may be recruited from selected health facilities that are representative of the whole country . cem involves recording all events , regardless of its severity or seriousness , and includes the following steps : a patient cohort is established for the medicine of interest.adverse events experienced by patients in the cohort are recorded by interviewing patients as part of routine patient / health professional encounters.patient i d and demographic data , together with details of medicines , events and other relevant information , are recorded on questionnaires . adverse events experienced by patients in the cohort are recorded by interviewing patients as part of routine patient / health professional encounters . patient i d and demographic data , together with details of medicines , events and other relevant information , are recorded on questionnaires . it is normally recommended that a cohort of 10,000 patients be enrolled [ 16 , 17 ] . this gives a 95 % chance of identifying a specific event that has an incidence of 1:3000 ( that is , an uncommon or rare event ) . a cohort of 3000 patients gives a 95 % chance of identifying a single incident with an incidence of 1:1000 . for a meaningful assessment , at least three events need to be identified , hence the higher objective of 10,000 patients [ 16 , 17 ] . it is determined by the length of treatment to be monitored in individual patients , and the time it takes to reach the desired cohort size . in tb therapy , treatment of individual patients is monitored for a period that is considered to be appropriate for the identification of both short- and long - term effects ( e.g. for drug - resistant tb this might be for 2 years or more after ending a treatment course lasting 2 years ) . if there is special interest in subgroups such as pregnant women or children , or more information is needed on a type of event of particular concern , the programme may need to continue for a longer period to accrue sufficient numbers in these subgroups to be able to achieve statistical significance when testing associations . the data are reviewed at regular intervals ( e.g. 3-monthly ) to detect early trends that may require further monitoring . adverse events are recorded using treatment initiation and treatment review forms , to record events happening to patients during a control period , prior to and after treatment initiation , respectively . the length of the control period could vary but should not extend beyond what the patient may reasonably remember . by collecting data on events happening to patients before treatment , cem might involve special ethical considerations since it is a methodology that requires the collection of detailed personal data , for patient follow - up . since cem aims to measure incidence , it is essential that duplicate entries are avoided ; this can be done only if patients can be identified correctly . as a prerequisite to collecting patient data , it is important to seek the approval of the highest appropriate authority in the country . some countries may choose to obtain informed consent from every patient ; however , this will be time consuming . an alternative to obtaining informed consent opt out principle where information about the cem programme is provided publicly , and patients can then decide whether or not they wish to have their data stored as part of cem . systems for data storage should be password protected , with controlled access to the premises , etc . the umc has , in collaboration with the who , developed a specific software , cemflow , for the secure management and analysis of cem data . it is web - based but data entry can be done offline with regular uploads to the main database . detailed handbooks describing these principles for cem in public health programmes have been developed by the who [ 16 , 17 ] and can be downloaded from http://www.who.int/medicines/areas/quality_safety/safety_efficacy/pharmpubhealth/en/index.html . cem is currently being used for antimalarials in kenya , nigeria , the united republic of tanzania , and zimbabwe . cem of antiretroviral ( arv ) drugs is planned in belarus and in the united republic of tanzania . the who proposes targeted spontaneous reporting ( tsr ) as a methodology that builds on the principles of spontaneous reporting but applied in a defined setting . in this method , health professionals managing a well - defined group of patients [ e.g. patients on treatment for drug - resistant tb or those switching from first - line to second - line antiretroviral therapy ( art ) ] are sensitized to report specific safety concerns suspected to be medicine related . tsr addresses a distinct set of questions and provides a comprehensive monitoring method that is affordable , feasible and sustainable in settings with limited financial and human resources . it also promotes the role of pv as a best practice that improves quality of care . tsr was developed by the who in 2010 and is being piloted in the hiv treatment programmes in three countries ( kenya , vietnam and uganda ) . tsr may be adapted either to report all suspected reactions in the defined population or to focus only on specific reactions of particular concern , for example treatment - threatening toxicity , etc . this serves to limit the reporting workload to those adverse events that are most significant to individuals and to the programmes . poor adherence to treatment due to adverse events such as nausea can be included as one of the targeted events in the tsr concept for priority reporting . it is particularly useful in the targeted follow - up of patients with additional complications , such as patients with drug - resistant tb and lifelong art . in hiv care , compared with routine spontaneous reporting , tsr aims to increase reporting rates by targeting , training and mentoring reporters at selected high caseload clinics , and by task shifting the reporting to non - physician cadres of healthcare workers such as nurses , pharmacists , pharmacy technicians and patients living with hiv ( plhiv ) . by monitoring populations such as those receiving extended arv prophylaxis to prevent breast milk transmission of hiv , and by focusing on particular adrs of interest , such as renal toxicity related to tenofovir the who is now aiming to introduce tsr in tb treatment programmes . for many years , national tb programmes monitor patient outcomes in well - circumscribed groups termed cohorts . tsr can integrate monitoring of adrs as a standard of care , alongside the routine practice of monitoring success , death , default or failure of treatment within these tb cohorts . one benefit of monitoring within a treatment cohort is that the number and profiles of the exposed patients will be known . the number of patients in the treatment cohorts represent a denominator ; if all suspected cases of the targeted adr within this cohort are recorded , then it is possible to calculate simple frequencies of the targeted adr . tsr thus seeks to overcome the main disadvantages of spontaneous reporting , that is underreporting and the inability to calculate rates , while maintaining simplicity of use , low cost and linkage to existing systems . in all public health programmes reporting of drug - related adverse events more than 100 countries are members of the who pv programme and have existing systems in place for reporting adrs but do not always integrate with public health programmes . one of the objectives of tsr is to address this lack of integration between phps and pv programmes by leveraging existing pv systems , providing simple tools to phps , training and task shifting to encourage reporting . the steps required to meet the objectives of tsr include the following : monitoring for suspected drug - related problems is included as part of normal patient care . during patient investigation the possibility of a medicine - related problem is always considered . at every encounter , the healthcare professional screens for any suspected adr.suspicion of the possibility of a causal relationship between drug treatment and the event triggers the completion of an adr reporting form . the suspected adr is noted in the patient records.all healthcare professionals involved in patient care are sensitized to ask about and investigate adverse effects at every encounter.the reporting forms ( the generic national adr form ) and route for transmission of information are the same as those used in spontaneous reporting , but the forms are supported by specific guidance ( case definitions and written procedures ) on when to complete them and with details on standardized reporting of drug names and adrs.reporting may primarily target serious or treatment threatening / limiting ( treatment discontinuation , change of regimen , life - threatening event , hospitalization , poor adherence because of toxicity or death ) adrs rather than the notification of any suspected reaction . if the total burden of drug - related problems in the exposed population is of interest , health professionals can be instructed to report any suspected drug - related problem . if , however , the frequency of a specific problem suspected to be associated with the therapy given is the important question , e.g. vision disorders , a case definition for reporting can be given in the instructions to healthcare professionals.reporting lasts the whole length of treatment . in the case of tb , this is between 6 months and 2 years , and in the case of art , lifelongunlike cem , there are no baseline measurements nor is there any active follow - up of members of the cohort and , thus , less resources are required . monitoring for suspected drug - related problems is included as part of normal patient care . during patient investigation the possibility of a medicine - related problem is always considered . at every encounter , the healthcare professional screens for any suspected adr . suspicion of the possibility of a causal relationship between drug treatment and the event triggers the completion of an adr reporting form . all healthcare professionals involved in patient care are sensitized to ask about and investigate adverse effects at every encounter . the reporting forms ( the generic national adr form ) and route for transmission of information are the same as those used in spontaneous reporting , but the forms are supported by specific guidance ( case definitions and written procedures ) on when to complete them and with details on standardized reporting of drug names and adrs . reporting may primarily target serious or treatment threatening / limiting ( treatment discontinuation , change of regimen , life - threatening event , hospitalization , poor adherence because of toxicity or death ) adrs rather than the notification of any suspected reaction . if the total burden of drug - related problems in the exposed population is of interest , health professionals can be instructed to report any suspected drug - related problem . if , however , the frequency of a specific problem suspected to be associated with the therapy given is the important question , e.g. vision disorders , a case definition for reporting can be given in the instructions to healthcare professionals . , this is between 6 months and 2 years , and in the case of art , lifelong unlike cem , there are no baseline measurements nor is there any active follow - up of members of the cohort and , thus , less resources are required . in public health programmes that integrate tsr , the routine patient record will include the question suspected adverse drug reaction : yes or no ? , thereby ensuring that it is always considered . the extent to which this information is recorded will indicate whether adr monitoring has become a part of normal practice . if safety monitoring of each patient is truly part of best practice and recording of whether the patient has experienced a suspected medicine - related problem or not is complete , the calculated reaction frequencies may be close estimates of true incidence rates . tsr provides the opportunity to monitor every single patient on treatment , as part of treatment and care . however , its successful implementation depends on the willingness of healthcare providers to participate in this monitoring exercise and to report their observations . adr reporting can be improved through interventions such as education , advocacy , mentoring , etc [ 22 , 23 ] . although different from these approaches , tsr , with its focus on targeted adrs and/or medicines , is likely to have a similar ( positive ) impact on reporting , albeit only for the targeted adrs , by reducing the reporting burden on overworked health professionals . all medical incidents ( events ) that patients experienced while on treatment can be captured by cem . those events considered noxious and unintended and suspected to be caused by the medicine are reportable as adrs through spontaneous reporting . tsr focuses on the collection of information on specific adrs , with specific medicines , in defined patient groups . adrs adverse drug reactions , cem cohort event monitoring , tsr targeted spontaneous reportingtable 1advantages and disadvantages of different types of pv methodsspontaneous reportingadvantages administratively simpler and less labour - intensive than cem less costly than cem has the potential of identifying very rare problems related to medicine use in any healthcare setting pv centres and health professionals are more likely to be familiar with this method as it is the most common method of pv used provides safety surveillance throughout the marketed life of all medicinesdisadvantages the data collected by this method are incomplete both in terms of quality and quantity underreporting is significant and widespread reliable rates can not be calculated and so risk can not be measured and risk factors can not be established with confidence there are strong biases in reporting deaths due to adrs may be incompletely recorded / investigated in some countries , especially when they occur outside healthcare special studies will need to be set up to obtain accurate information on areas of particular interest , e.g. pregnancy , children and specific events of concern . these special studies add to the cost and in turn reduce the cost advantage of spontaneous reportingcemadvantages the ability to produce rates the ability to produce a near - complete profile of the adverse events and/or adrs for the medicines of interest very effective in identifying signals at an early stage the ability to associate reactions with risk factors the ability to make accurate comparisons between medicines can detect reduced or failed therapeutic effect and can raise suspicion of medication errors , interactions , emerging resistance or poor - quality or counterfeit medicines the ability to record and examine details of all deaths and provide rates of deathdisadvantages the method is more labour - intensive , needs dedicated staff to perform treatment initiation ( baseline ) and treatment follow - up interviews more costly than spontaneous reporting patients may not turn up for follow - up ; potential for loss to follow - up patients may opt - out and refuse to be part of the cem ; this might make it difficult to reach the required cohort size takes certain expertise in recording adverse events , training will be necessary can not detect very rare problems with medicinestsradvantages is simpler , less costly and less labour - intensive than cem tsr represents an add - on to the routine monitoring of outcomes of patients can be focused on priority adrs the forms and routes for reporting are similar to those for routine spontaneous reporting can provide some measure of rates and incidences uses existing pv systems within countries links public health programmes to pv centres less likely to identify unanticipated reactionsdisadvantages the method is subject to individual willingness to monitor and report ; thus numerator ( number of individuals with the suspected adr ) may not be accurate completeness of reporting is therefore crucial there is limited experience with tsr and the technique needs to be field - testedadrs adverse drug reactions , cem cohort event monitoring , pv pharmacovigilance , tsr targeted spontaneous reporting the relationship between spontaneous reporting , tsr and cem . all medical incidents ( events ) that patients experienced while on treatment can be captured by cem . those events considered noxious and unintended and suspected to be caused by the medicine are reportable as adrs through spontaneous reporting . tsr focuses on the collection of information on specific adrs , with specific medicines , in defined patient groups . adrs adverse drug reactions , cem cohort event monitoring , tsr targeted spontaneous reporting advantages and disadvantages of different types of pv methods adrs adverse drug reactions , cem cohort event monitoring , pv pharmacovigilance , tsr targeted spontaneous reporting the who has organized training courses in tsr ( botswana , kenya , uganda and zimbabwe ) and cem ( belarus , burkina faso , ethiopia , ghana , kenya , nigeria , republic of moldova , ukraine , uganda , united republic of tanzania , and zambia ) . as mentioned above , several of these countries are in the process of implementing projects , either in cem or tsr , with technical support from the who , and with funds from the european commission , the bill and melinda gates foundation and others . preliminary feedback and data from these efforts are encouraging and there is general enthusiasm and interest in adopting these methods in other countries . good pv will identify the risks within the shortest possible time after the medicine has been marketed and will help establish or identify risk factors . when communicated effectively , this information allows for rational , evidence - based prescribing with the potential for preventing many adrs . such information will ultimately help each patient receive optimum therapy at a lower cost to the health system . the organizers of a monitoring programme for the safety of medicines used in public health programmes must have a clear sense of the questions they want to answer before developing their plan . routine safety monitoring , as a lifecycle approach for the product , is best handled by a spontaneous reporting system . but if the aim is to better understand , with minimum resources , the occurrence of a specific adr in a specific population , tsr is an appropriate choice . if the aim is to actively follow patients to characterize the safety profile of new medicines , then cem is a relevant choice . it is only with clear goals in mind that one can design a suitable data collection instrument and an analytical plan . whatever the method , all stakeholders must be fully informed of the reasons for monitoring , the methodology as far as it involves them , the value of safety monitoring , the contribution it will make to the health of the population , the potential for increasing the effectiveness of public health programmes and reducing health costs , and the contribution pv can make to the knowledge of medicines in public health programmes and their safety at country and global levels .

globally , national pharmacovigilance systems rely on spontaneous reporting in which suspected adverse drug reactions ( adrs ) are reported to a national coordinating centre by health professionals , manufacturers or patients . spontaneous reporting systems are the easiest to establish and the cheapest to run but suffer from poor - quality reports and underreporting . it is difficult to estimate rates and frequencies of adrs through spontaneous reporting . public health programmes need to quantify and characterize risks to individuals and communities from their medicines , to minimize harm and improve use , to sustain public confidence in the programmes , and to track problems due to medication errors and poor quality medicines . additional methods are therefore needed to monitor the quantitative aspects of medicine safety , to better identify specific risk factors and high - risk groups , and to characterize adrs associated with specific medicines and in specific populations . the present paper introduces two methods , cohort event monitoring and targeted spontaneous reporting , that are being implemented by the who , in its public health programmes , to complement spontaneous reporting . the advantages and disadvantages of these methods and how each can be applied in clinical practice are discussed .

Introduction Spontaneous Reporting System Methods that Complement Spontaneous Reporting Systems Cohort Event Monitoring (CEM) Targeted Spontaneous Reporting (TSR) CEM and TSR Projects in Other Countries Conclusion

the 16th world health assembly ( 1963 ) adopted a resolution ( wha 16.36 ) that reaffirmed the need for early detection and rapid dissemination of information on adverse reactions due to medicines , and subsequently led to the creation of the who programme for international drug monitoring . in all countries ( low , middle or high income ) , national pv systems rely heavily on spontaneous ( or voluntary ) reporting in which suspected adverse drug reactions ( adrs ) are reported to a national coordinating centre by health professionals , manufacturers or directly by patients . of all the sources of data for drug safety monitoring , the spontaneous reporting systems provide the highest volume of information at the lowest maintenance cost , and have proven their value in the early detection of patient safety issues related either to the products themselves or to their use . in some instances , for example , in the case of cerivastatin , an association ( a signal ) between cerivastatin , myopathy and rhabdomyolysis was published , based on icsrs , by the uppsala monitoring centre ( umc ) in 1999 ; various regulatory decisions were announced between 1999 and 2001 in different countries . under these circumstances it is difficult to determine the actual number of individuals experiencing an adverse reaction to the medicine . this , together with the uncertainties around the number of patients exposed to the medicine in question , makes it difficult to estimate rates and frequencies of adrs through spontaneous reporting . methods of greater scientific rigour are needed to establish quantitative aspects of medicine safety , to better identify specific risk factors and high - risk groups , and to provide valid clinical characteristics of problems associated with specific medicines . public health programmes may treat a large population , in an organized and structured fashion , and record the number of patients treated , drugs used , doses given , etc . these programmes thus provide good platforms for capturing the quantitative aspects ( rates and frequencies ) of adrs and adverse events with the medicines used . in adopting a public health approach to the delivery of hiv and tb services , it is important to quantify and characterize treatment - related risks , as early as possible , to minimize the harm to public health as well as to sustain public confidence in the programme . while spontaneous reporting remains the bedrock of safety monitoring , additional methods are needed to quantify the frequency and severity of expected and unanticipated adrs occurring in patients receiving long - term treatment . in the context of hiv programmes which provide life - long therapy , a simple , inexpensive but in addition , we need to monitor adverse events that , instead of being directly attributed to the active substance , could be attributed more to aspects such as inappropriate use , medication errors , poor quality products or drug - drug interactions . an effective pv programme is one that monitors medicines used in all public health programmes , to improve patient safety , and to improve the credibility and public perception of these programmes . cohort event monitoring ( cem ) [ 16 , 17 ] is a prospective , observational , cohort study of adverse events associated with one or more medicines . a cem programme is essentially an observation of a new medicine in routine clinical practice in the early postmarketing phase , but it can be used for older medicines . it is based on the principles of the new zealand intensive medicines monitoring programme and the uk prescription event monitoring except that in most resource - limited countries , treatment within public health programmes ( such as tb and hiv ) is not provided on a prescription basis . cem captures all medicine - related events , including medication errors , problems due to poor storage conditions , poor quality or counterfeit medicines , and drug interactions . it is determined by the length of treatment to be monitored in individual patients , and the time it takes to reach the desired cohort size . detailed handbooks describing these principles for cem in public health programmes have been developed by the who [ 16 , 17 ] and can be downloaded from http://www.who.int/medicines/areas/quality_safety/safety_efficacy/pharmpubhealth/en/index.html . the who proposes targeted spontaneous reporting ( tsr ) as a methodology that builds on the principles of spontaneous reporting but applied in a defined setting . tsr was developed by the who in 2010 and is being piloted in the hiv treatment programmes in three countries ( kenya , vietnam and uganda ) . this serves to limit the reporting workload to those adverse events that are most significant to individuals and to the programmes . poor adherence to treatment due to adverse events such as nausea can be included as one of the targeted events in the tsr concept for priority reporting . tsr thus seeks to overcome the main disadvantages of spontaneous reporting , that is underreporting and the inability to calculate rates , while maintaining simplicity of use , low cost and linkage to existing systems . in all public health programmes reporting of drug - related adverse events more than 100 countries are members of the who pv programme and have existing systems in place for reporting adrs but do not always integrate with public health programmes . the suspected adr is noted in the patient records.all healthcare professionals involved in patient care are sensitized to ask about and investigate adverse effects at every encounter.the reporting forms ( the generic national adr form ) and route for transmission of information are the same as those used in spontaneous reporting , but the forms are supported by specific guidance ( case definitions and written procedures ) on when to complete them and with details on standardized reporting of drug names and adrs.reporting may primarily target serious or treatment threatening / limiting ( treatment discontinuation , change of regimen , life - threatening event , hospitalization , poor adherence because of toxicity or death ) adrs rather than the notification of any suspected reaction . if the total burden of drug - related problems in the exposed population is of interest , health professionals can be instructed to report any suspected drug - related problem . in the case of tb , this is between 6 months and 2 years , and in the case of art , lifelongunlike cem , there are no baseline measurements nor is there any active follow - up of members of the cohort and , thus , less resources are required . if the total burden of drug - related problems in the exposed population is of interest , health professionals can be instructed to report any suspected drug - related problem . in public health programmes that integrate tsr , the routine patient record will include the question suspected adverse drug reaction : yes or no ? those events considered noxious and unintended and suspected to be caused by the medicine are reportable as adrs through spontaneous reporting . tsr focuses on the collection of information on specific adrs , with specific medicines , in defined patient groups . adrs adverse drug reactions , cem cohort event monitoring , tsr targeted spontaneous reportingtable 1advantages and disadvantages of different types of pv methodsspontaneous reportingadvantages administratively simpler and less labour - intensive than cem less costly than cem has the potential of identifying very rare problems related to medicine use in any healthcare setting pv centres and health professionals are more likely to be familiar with this method as it is the most common method of pv used provides safety surveillance throughout the marketed life of all medicinesdisadvantages the data collected by this method are incomplete both in terms of quality and quantity underreporting is significant and widespread reliable rates can not be calculated and so risk can not be measured and risk factors can not be established with confidence there are strong biases in reporting deaths due to adrs may be incompletely recorded / investigated in some countries , especially when they occur outside healthcare special studies will need to be set up to obtain accurate information on areas of particular interest , e.g. these special studies add to the cost and in turn reduce the cost advantage of spontaneous reportingcemadvantages the ability to produce rates the ability to produce a near - complete profile of the adverse events and/or adrs for the medicines of interest very effective in identifying signals at an early stage the ability to associate reactions with risk factors the ability to make accurate comparisons between medicines can detect reduced or failed therapeutic effect and can raise suspicion of medication errors , interactions , emerging resistance or poor - quality or counterfeit medicines the ability to record and examine details of all deaths and provide rates of deathdisadvantages the method is more labour - intensive , needs dedicated staff to perform treatment initiation ( baseline ) and treatment follow - up interviews more costly than spontaneous reporting patients may not turn up for follow - up ; potential for loss to follow - up patients may opt - out and refuse to be part of the cem ; this might make it difficult to reach the required cohort size takes certain expertise in recording adverse events , training will be necessary can not detect very rare problems with medicinestsradvantages is simpler , less costly and less labour - intensive than cem tsr represents an add - on to the routine monitoring of outcomes of patients can be focused on priority adrs the forms and routes for reporting are similar to those for routine spontaneous reporting can provide some measure of rates and incidences uses existing pv systems within countries links public health programmes to pv centres less likely to identify unanticipated reactionsdisadvantages the method is subject to individual willingness to monitor and report ; thus numerator ( number of individuals with the suspected adr ) may not be accurate completeness of reporting is therefore crucial there is limited experience with tsr and the technique needs to be field - testedadrs adverse drug reactions , cem cohort event monitoring , pv pharmacovigilance , tsr targeted spontaneous reporting the relationship between spontaneous reporting , tsr and cem . those events considered noxious and unintended and suspected to be caused by the medicine are reportable as adrs through spontaneous reporting . tsr focuses on the collection of information on specific adrs , with specific medicines , in defined patient groups . adrs adverse drug reactions , cem cohort event monitoring , tsr targeted spontaneous reporting advantages and disadvantages of different types of pv methods adrs adverse drug reactions , cem cohort event monitoring , pv pharmacovigilance , tsr targeted spontaneous reporting cohort event monitoring ( cem ) [ 16 , 17 ] is a prospective , observational , cohort study of adverse events associated with one or more medicines . a cem programme is essentially an observation of a new medicine in routine clinical practice in the early postmarketing phase , but it can be used for older medicines . it is based on the principles of the new zealand intensive medicines monitoring programme and the uk prescription event monitoring except that in most resource - limited countries , treatment within public health programmes ( such as tb and hiv ) is not provided on a prescription basis . cem captures all medicine - related events , including medication errors , problems due to poor storage conditions , poor quality or counterfeit medicines , and drug interactions . patients may be recruited from all health facilities involved in providing the medicines , or patients may be recruited from selected health facilities that are representative of the whole country . cem involves recording all events , regardless of its severity or seriousness , and includes the following steps : a patient cohort is established for the medicine of interest.adverse events experienced by patients in the cohort are recorded by interviewing patients as part of routine patient / health professional encounters.patient i d and demographic data , together with details of medicines , events and other relevant information , are recorded on questionnaires . it is determined by the length of treatment to be monitored in individual patients , and the time it takes to reach the desired cohort size . the umc has , in collaboration with the who , developed a specific software , cemflow , for the secure management and analysis of cem data . detailed handbooks describing these principles for cem in public health programmes have been developed by the who [ 16 , 17 ] and can be downloaded from http://www.who.int/medicines/areas/quality_safety/safety_efficacy/pharmpubhealth/en/index.html . the who proposes targeted spontaneous reporting ( tsr ) as a methodology that builds on the principles of spontaneous reporting but applied in a defined setting . tsr was developed by the who in 2010 and is being piloted in the hiv treatment programmes in three countries ( kenya , vietnam and uganda ) . this serves to limit the reporting workload to those adverse events that are most significant to individuals and to the programmes . poor adherence to treatment due to adverse events such as nausea can be included as one of the targeted events in the tsr concept for priority reporting . in hiv care , compared with routine spontaneous reporting , tsr aims to increase reporting rates by targeting , training and mentoring reporters at selected high caseload clinics , and by task shifting the reporting to non - physician cadres of healthcare workers such as nurses , pharmacists , pharmacy technicians and patients living with hiv ( plhiv ) . the number of patients in the treatment cohorts represent a denominator ; if all suspected cases of the targeted adr within this cohort are recorded , then it is possible to calculate simple frequencies of the targeted adr . tsr thus seeks to overcome the main disadvantages of spontaneous reporting , that is underreporting and the inability to calculate rates , while maintaining simplicity of use , low cost and linkage to existing systems . in all public health programmes reporting of drug - related adverse events more than 100 countries are members of the who pv programme and have existing systems in place for reporting adrs but do not always integrate with public health programmes . the suspected adr is noted in the patient records.all healthcare professionals involved in patient care are sensitized to ask about and investigate adverse effects at every encounter.the reporting forms ( the generic national adr form ) and route for transmission of information are the same as those used in spontaneous reporting , but the forms are supported by specific guidance ( case definitions and written procedures ) on when to complete them and with details on standardized reporting of drug names and adrs.reporting may primarily target serious or treatment threatening / limiting ( treatment discontinuation , change of regimen , life - threatening event , hospitalization , poor adherence because of toxicity or death ) adrs rather than the notification of any suspected reaction . if the total burden of drug - related problems in the exposed population is of interest , health professionals can be instructed to report any suspected drug - related problem . in the case of tb , this is between 6 months and 2 years , and in the case of art , lifelongunlike cem , there are no baseline measurements nor is there any active follow - up of members of the cohort and , thus , less resources are required . the reporting forms ( the generic national adr form ) and route for transmission of information are the same as those used in spontaneous reporting , but the forms are supported by specific guidance ( case definitions and written procedures ) on when to complete them and with details on standardized reporting of drug names and adrs . if the total burden of drug - related problems in the exposed population is of interest , health professionals can be instructed to report any suspected drug - related problem . in public health programmes that integrate tsr , the routine patient record will include the question suspected adverse drug reaction : yes or no ? although different from these approaches , tsr , with its focus on targeted adrs and/or medicines , is likely to have a similar ( positive ) impact on reporting , albeit only for the targeted adrs , by reducing the reporting burden on overworked health professionals . those events considered noxious and unintended and suspected to be caused by the medicine are reportable as adrs through spontaneous reporting . tsr focuses on the collection of information on specific adrs , with specific medicines , in defined patient groups . adrs adverse drug reactions , cem cohort event monitoring , tsr targeted spontaneous reportingtable 1advantages and disadvantages of different types of pv methodsspontaneous reportingadvantages administratively simpler and less labour - intensive than cem less costly than cem has the potential of identifying very rare problems related to medicine use in any healthcare setting pv centres and health professionals are more likely to be familiar with this method as it is the most common method of pv used provides safety surveillance throughout the marketed life of all medicinesdisadvantages the data collected by this method are incomplete both in terms of quality and quantity underreporting is significant and widespread reliable rates can not be calculated and so risk can not be measured and risk factors can not be established with confidence there are strong biases in reporting deaths due to adrs may be incompletely recorded / investigated in some countries , especially when they occur outside healthcare special studies will need to be set up to obtain accurate information on areas of particular interest , e.g. these special studies add to the cost and in turn reduce the cost advantage of spontaneous reportingcemadvantages the ability to produce rates the ability to produce a near - complete profile of the adverse events and/or adrs for the medicines of interest very effective in identifying signals at an early stage the ability to associate reactions with risk factors the ability to make accurate comparisons between medicines can detect reduced or failed therapeutic effect and can raise suspicion of medication errors , interactions , emerging resistance or poor - quality or counterfeit medicines the ability to record and examine details of all deaths and provide rates of deathdisadvantages the method is more labour - intensive , needs dedicated staff to perform treatment initiation ( baseline ) and treatment follow - up interviews more costly than spontaneous reporting patients may not turn up for follow - up ; potential for loss to follow - up patients may opt - out and refuse to be part of the cem ; this might make it difficult to reach the required cohort size takes certain expertise in recording adverse events , training will be necessary can not detect very rare problems with medicinestsradvantages is simpler , less costly and less labour - intensive than cem tsr represents an add - on to the routine monitoring of outcomes of patients can be focused on priority adrs the forms and routes for reporting are similar to those for routine spontaneous reporting can provide some measure of rates and incidences uses existing pv systems within countries links public health programmes to pv centres less likely to identify unanticipated reactionsdisadvantages the method is subject to individual willingness to monitor and report ; thus numerator ( number of individuals with the suspected adr ) may not be accurate completeness of reporting is therefore crucial there is limited experience with tsr and the technique needs to be field - testedadrs adverse drug reactions , cem cohort event monitoring , pv pharmacovigilance , tsr targeted spontaneous reporting the relationship between spontaneous reporting , tsr and cem . those events considered noxious and unintended and suspected to be caused by the medicine are reportable as adrs through spontaneous reporting . tsr focuses on the collection of information on specific adrs , with specific medicines , in defined patient groups . adrs adverse drug reactions , cem cohort event monitoring , tsr targeted spontaneous reporting advantages and disadvantages of different types of pv methods adrs adverse drug reactions , cem cohort event monitoring , pv pharmacovigilance , tsr targeted spontaneous reporting the who has organized training courses in tsr ( botswana , kenya , uganda and zimbabwe ) and cem ( belarus , burkina faso , ethiopia , ghana , kenya , nigeria , republic of moldova , ukraine , uganda , united republic of tanzania , and zambia ) . as mentioned above , several of these countries are in the process of implementing projects , either in cem or tsr , with technical support from the who , and with funds from the european commission , the bill and melinda gates foundation and others . whatever the method , all stakeholders must be fully informed of the reasons for monitoring , the methodology as far as it involves them , the value of safety monitoring , the contribution it will make to the health of the population , the potential for increasing the effectiveness of public health programmes and reducing health costs , and the contribution pv can make to the knowledge of medicines in public health programmes and their safety at country and global levels .

the 16th world health assembly ( 1963 ) adopted a resolution ( wha 16.36 ) that reaffirmed the need for early detection and rapid dissemination of information on adverse reactions due to medicines , and subsequently led to the creation of the who programme for international drug monitoring . of all the sources of data for drug safety monitoring , the spontaneous reporting systems provide the highest volume of information at the lowest maintenance cost , and have proven their value in the early detection of patient safety issues related either to the products themselves or to their use . in some instances , for example , in the case of cerivastatin , an association ( a signal ) between cerivastatin , myopathy and rhabdomyolysis was published , based on icsrs , by the uppsala monitoring centre ( umc ) in 1999 ; various regulatory decisions were announced between 1999 and 2001 in different countries . in spontaneous reporting , the most common form of pv , no measures are taken to systematically follow - up patients to record adrs that they may have experienced . this , together with the uncertainties around the number of patients exposed to the medicine in question , makes it difficult to estimate rates and frequencies of adrs through spontaneous reporting . methods of greater scientific rigour are needed to establish quantitative aspects of medicine safety , to better identify specific risk factors and high - risk groups , and to provide valid clinical characteristics of problems associated with specific medicines . in the management of hiv and tuberculosis ( tb ) patients , short- and long - term toxicity data play a critical role in informing treatment choices [ 1113 ] . in adopting a public health approach to the delivery of hiv and tb services , it is important to quantify and characterize treatment - related risks , as early as possible , to minimize the harm to public health as well as to sustain public confidence in the programme . in the context of hiv programmes which provide life - long therapy , a simple , inexpensive but in addition , we need to monitor adverse events that , instead of being directly attributed to the active substance , could be attributed more to aspects such as inappropriate use , medication errors , poor quality products or drug - drug interactions . the global fund urges countries to invest in pv programmes that monitor the quality , usage and efficacy of the hiv , tb and malaria medicines that it helps countries to buy . it is based on the principles of the new zealand intensive medicines monitoring programme and the uk prescription event monitoring except that in most resource - limited countries , treatment within public health programmes ( such as tb and hiv ) is not provided on a prescription basis . cem is thus an early warning system that interviews patients on a certain treatment ( the cohort ) , for capturing problems ( the events ) with new medicines in public health programmes ; the patients are interviewed before and after starting treatment . cem involves recording all events , regardless of its severity or seriousness , and includes the following steps : a patient cohort is established for the medicine of interest.adverse events experienced by patients in the cohort are recorded by interviewing patients as part of routine patient / health professional encounters.patient i d and demographic data , together with details of medicines , events and other relevant information , are recorded on questionnaires . in tb therapy , treatment of individual patients is monitored for a period that is considered to be appropriate for the identification of both short- and long - term effects ( e.g. if there is special interest in subgroups such as pregnant women or children , or more information is needed on a type of event of particular concern , the programme may need to continue for a longer period to accrue sufficient numbers in these subgroups to be able to achieve statistical significance when testing associations . patients on treatment for drug - resistant tb or those switching from first - line to second - line antiretroviral therapy ( art ) ] are sensitized to report specific safety concerns suspected to be medicine related . in hiv care , compared with routine spontaneous reporting , tsr aims to increase reporting rates by targeting , training and mentoring reporters at selected high caseload clinics , and by task shifting the reporting to non - physician cadres of healthcare workers such as nurses , pharmacists , pharmacy technicians and patients living with hiv ( plhiv ) . by monitoring populations such as those receiving extended arv prophylaxis to prevent breast milk transmission of hiv , and by focusing on particular adrs of interest , such as renal toxicity related to tenofovir the who is now aiming to introduce tsr in tb treatment programmes . the number of patients in the treatment cohorts represent a denominator ; if all suspected cases of the targeted adr within this cohort are recorded , then it is possible to calculate simple frequencies of the targeted adr . in all public health programmes reporting of drug - related adverse events more than 100 countries are members of the who pv programme and have existing systems in place for reporting adrs but do not always integrate with public health programmes . one of the objectives of tsr is to address this lack of integration between phps and pv programmes by leveraging existing pv systems , providing simple tools to phps , training and task shifting to encourage reporting . at every encounter , the healthcare professional screens for any suspected adr.suspicion of the possibility of a causal relationship between drug treatment and the event triggers the completion of an adr reporting form . the suspected adr is noted in the patient records.all healthcare professionals involved in patient care are sensitized to ask about and investigate adverse effects at every encounter.the reporting forms ( the generic national adr form ) and route for transmission of information are the same as those used in spontaneous reporting , but the forms are supported by specific guidance ( case definitions and written procedures ) on when to complete them and with details on standardized reporting of drug names and adrs.reporting may primarily target serious or treatment threatening / limiting ( treatment discontinuation , change of regimen , life - threatening event , hospitalization , poor adherence because of toxicity or death ) adrs rather than the notification of any suspected reaction . if the total burden of drug - related problems in the exposed population is of interest , health professionals can be instructed to report any suspected drug - related problem . in the case of tb , this is between 6 months and 2 years , and in the case of art , lifelongunlike cem , there are no baseline measurements nor is there any active follow - up of members of the cohort and , thus , less resources are required . the reporting forms ( the generic national adr form ) and route for transmission of information are the same as those used in spontaneous reporting , but the forms are supported by specific guidance ( case definitions and written procedures ) on when to complete them and with details on standardized reporting of drug names and adrs . if the total burden of drug - related problems in the exposed population is of interest , health professionals can be instructed to report any suspected drug - related problem . in the case of tb , this is between 6 months and 2 years , and in the case of art , lifelong unlike cem , there are no baseline measurements nor is there any active follow - up of members of the cohort and , thus , less resources are required . although different from these approaches , tsr , with its focus on targeted adrs and/or medicines , is likely to have a similar ( positive ) impact on reporting , albeit only for the targeted adrs , by reducing the reporting burden on overworked health professionals . adrs adverse drug reactions , cem cohort event monitoring , tsr targeted spontaneous reportingtable 1advantages and disadvantages of different types of pv methodsspontaneous reportingadvantages administratively simpler and less labour - intensive than cem less costly than cem has the potential of identifying very rare problems related to medicine use in any healthcare setting pv centres and health professionals are more likely to be familiar with this method as it is the most common method of pv used provides safety surveillance throughout the marketed life of all medicinesdisadvantages the data collected by this method are incomplete both in terms of quality and quantity underreporting is significant and widespread reliable rates can not be calculated and so risk can not be measured and risk factors can not be established with confidence there are strong biases in reporting deaths due to adrs may be incompletely recorded / investigated in some countries , especially when they occur outside healthcare special studies will need to be set up to obtain accurate information on areas of particular interest , e.g. these special studies add to the cost and in turn reduce the cost advantage of spontaneous reportingcemadvantages the ability to produce rates the ability to produce a near - complete profile of the adverse events and/or adrs for the medicines of interest very effective in identifying signals at an early stage the ability to associate reactions with risk factors the ability to make accurate comparisons between medicines can detect reduced or failed therapeutic effect and can raise suspicion of medication errors , interactions , emerging resistance or poor - quality or counterfeit medicines the ability to record and examine details of all deaths and provide rates of deathdisadvantages the method is more labour - intensive , needs dedicated staff to perform treatment initiation ( baseline ) and treatment follow - up interviews more costly than spontaneous reporting patients may not turn up for follow - up ; potential for loss to follow - up patients may opt - out and refuse to be part of the cem ; this might make it difficult to reach the required cohort size takes certain expertise in recording adverse events , training will be necessary can not detect very rare problems with medicinestsradvantages is simpler , less costly and less labour - intensive than cem tsr represents an add - on to the routine monitoring of outcomes of patients can be focused on priority adrs the forms and routes for reporting are similar to those for routine spontaneous reporting can provide some measure of rates and incidences uses existing pv systems within countries links public health programmes to pv centres less likely to identify unanticipated reactionsdisadvantages the method is subject to individual willingness to monitor and report ; thus numerator ( number of individuals with the suspected adr ) may not be accurate completeness of reporting is therefore crucial there is limited experience with tsr and the technique needs to be field - testedadrs adverse drug reactions , cem cohort event monitoring , pv pharmacovigilance , tsr targeted spontaneous reporting the relationship between spontaneous reporting , tsr and cem . adrs adverse drug reactions , cem cohort event monitoring , tsr targeted spontaneous reporting advantages and disadvantages of different types of pv methods adrs adverse drug reactions , cem cohort event monitoring , pv pharmacovigilance , tsr targeted spontaneous reporting cohort event monitoring ( cem ) [ 16 , 17 ] is a prospective , observational , cohort study of adverse events associated with one or more medicines . it is based on the principles of the new zealand intensive medicines monitoring programme and the uk prescription event monitoring except that in most resource - limited countries , treatment within public health programmes ( such as tb and hiv ) is not provided on a prescription basis . cem is thus an early warning system that interviews patients on a certain treatment ( the cohort ) , for capturing problems ( the events ) with new medicines in public health programmes ; the patients are interviewed before and after starting treatment . cem involves recording all events , regardless of its severity or seriousness , and includes the following steps : a patient cohort is established for the medicine of interest.adverse events experienced by patients in the cohort are recorded by interviewing patients as part of routine patient / health professional encounters.patient i d and demographic data , together with details of medicines , events and other relevant information , are recorded on questionnaires . if there is special interest in subgroups such as pregnant women or children , or more information is needed on a type of event of particular concern , the programme may need to continue for a longer period to accrue sufficient numbers in these subgroups to be able to achieve statistical significance when testing associations . patients on treatment for drug - resistant tb or those switching from first - line to second - line antiretroviral therapy ( art ) ] are sensitized to report specific safety concerns suspected to be medicine related . in hiv care , compared with routine spontaneous reporting , tsr aims to increase reporting rates by targeting , training and mentoring reporters at selected high caseload clinics , and by task shifting the reporting to non - physician cadres of healthcare workers such as nurses , pharmacists , pharmacy technicians and patients living with hiv ( plhiv ) . by monitoring populations such as those receiving extended arv prophylaxis to prevent breast milk transmission of hiv , and by focusing on particular adrs of interest , such as renal toxicity related to tenofovir the who is now aiming to introduce tsr in tb treatment programmes . the number of patients in the treatment cohorts represent a denominator ; if all suspected cases of the targeted adr within this cohort are recorded , then it is possible to calculate simple frequencies of the targeted adr . in all public health programmes reporting of drug - related adverse events more than 100 countries are members of the who pv programme and have existing systems in place for reporting adrs but do not always integrate with public health programmes . one of the objectives of tsr is to address this lack of integration between phps and pv programmes by leveraging existing pv systems , providing simple tools to phps , training and task shifting to encourage reporting . the suspected adr is noted in the patient records.all healthcare professionals involved in patient care are sensitized to ask about and investigate adverse effects at every encounter.the reporting forms ( the generic national adr form ) and route for transmission of information are the same as those used in spontaneous reporting , but the forms are supported by specific guidance ( case definitions and written procedures ) on when to complete them and with details on standardized reporting of drug names and adrs.reporting may primarily target serious or treatment threatening / limiting ( treatment discontinuation , change of regimen , life - threatening event , hospitalization , poor adherence because of toxicity or death ) adrs rather than the notification of any suspected reaction . if the total burden of drug - related problems in the exposed population is of interest , health professionals can be instructed to report any suspected drug - related problem . in the case of tb , this is between 6 months and 2 years , and in the case of art , lifelongunlike cem , there are no baseline measurements nor is there any active follow - up of members of the cohort and , thus , less resources are required . the reporting forms ( the generic national adr form ) and route for transmission of information are the same as those used in spontaneous reporting , but the forms are supported by specific guidance ( case definitions and written procedures ) on when to complete them and with details on standardized reporting of drug names and adrs . if the total burden of drug - related problems in the exposed population is of interest , health professionals can be instructed to report any suspected drug - related problem . , this is between 6 months and 2 years , and in the case of art , lifelong unlike cem , there are no baseline measurements nor is there any active follow - up of members of the cohort and , thus , less resources are required . although different from these approaches , tsr , with its focus on targeted adrs and/or medicines , is likely to have a similar ( positive ) impact on reporting , albeit only for the targeted adrs , by reducing the reporting burden on overworked health professionals . adrs adverse drug reactions , cem cohort event monitoring , tsr targeted spontaneous reportingtable 1advantages and disadvantages of different types of pv methodsspontaneous reportingadvantages administratively simpler and less labour - intensive than cem less costly than cem has the potential of identifying very rare problems related to medicine use in any healthcare setting pv centres and health professionals are more likely to be familiar with this method as it is the most common method of pv used provides safety surveillance throughout the marketed life of all medicinesdisadvantages the data collected by this method are incomplete both in terms of quality and quantity underreporting is significant and widespread reliable rates can not be calculated and so risk can not be measured and risk factors can not be established with confidence there are strong biases in reporting deaths due to adrs may be incompletely recorded / investigated in some countries , especially when they occur outside healthcare special studies will need to be set up to obtain accurate information on areas of particular interest , e.g. these special studies add to the cost and in turn reduce the cost advantage of spontaneous reportingcemadvantages the ability to produce rates the ability to produce a near - complete profile of the adverse events and/or adrs for the medicines of interest very effective in identifying signals at an early stage the ability to associate reactions with risk factors the ability to make accurate comparisons between medicines can detect reduced or failed therapeutic effect and can raise suspicion of medication errors , interactions , emerging resistance or poor - quality or counterfeit medicines the ability to record and examine details of all deaths and provide rates of deathdisadvantages the method is more labour - intensive , needs dedicated staff to perform treatment initiation ( baseline ) and treatment follow - up interviews more costly than spontaneous reporting patients may not turn up for follow - up ; potential for loss to follow - up patients may opt - out and refuse to be part of the cem ; this might make it difficult to reach the required cohort size takes certain expertise in recording adverse events , training will be necessary can not detect very rare problems with medicinestsradvantages is simpler , less costly and less labour - intensive than cem tsr represents an add - on to the routine monitoring of outcomes of patients can be focused on priority adrs the forms and routes for reporting are similar to those for routine spontaneous reporting can provide some measure of rates and incidences uses existing pv systems within countries links public health programmes to pv centres less likely to identify unanticipated reactionsdisadvantages the method is subject to individual willingness to monitor and report ; thus numerator ( number of individuals with the suspected adr ) may not be accurate completeness of reporting is therefore crucial there is limited experience with tsr and the technique needs to be field - testedadrs adverse drug reactions , cem cohort event monitoring , pv pharmacovigilance , tsr targeted spontaneous reporting the relationship between spontaneous reporting , tsr and cem . adrs adverse drug reactions , cem cohort event monitoring , tsr targeted spontaneous reporting advantages and disadvantages of different types of pv methods adrs adverse drug reactions , cem cohort event monitoring , pv pharmacovigilance , tsr targeted spontaneous reporting the who has organized training courses in tsr ( botswana , kenya , uganda and zimbabwe ) and cem ( belarus , burkina faso , ethiopia , ghana , kenya , nigeria , republic of moldova , ukraine , uganda , united republic of tanzania , and zambia ) . as mentioned above , several of these countries are in the process of implementing projects , either in cem or tsr , with technical support from the who , and with funds from the european commission , the bill and melinda gates foundation and others . whatever the method , all stakeholders must be fully informed of the reasons for monitoring , the methodology as far as it involves them , the value of safety monitoring , the contribution it will make to the health of the population , the potential for increasing the effectiveness of public health programmes and reducing health costs , and the contribution pv can make to the knowledge of medicines in public health programmes and their safety at country and global levels .

parkinson s disease ( pd ) is a synucleinopathy with heterogeneous progression of motor and non - motor dysfunction . in fact , several well - designed studies provided evidence that pd patients with the postural instability and gait disturbance ( pigd ) phenotype of motor symptoms showed a rapid progression of functional disability and cognitive deficit compared with patients with the tremor - dominant ( td ) motor phenotype [ 1 - 5 ] . although the mechanisms of the inherent heterogeneous progression of pd are still largely unknown , the heterogeneity of the disease may be associated with alterations of neural connectivity between the motor and cognitive control areas of the brain . the early development of dementia in pd patients is a major risk factor for poor prognosis and high mortality . therefore , the early detection of patients who are likely to develop dementia rapidly is crucial . it has been established that the overall prevalence of cognitive dysfunction without functional deficit sufficient for the diagnosis of dementia ( i.e. , cognitive impairment ) in pd is approximately 30% , and ~80% of patients with pd will develop overt dementia over 20 years after disease onset . there are currently no proven underlying pathogenic mechanisms of cognitive decline in pd patients . furthermore , several well - designed clinical studies to identify biochemical , imaging and genetic biomarkers of pd are emerging . among the large multicenter longitudinal pd biomarker studies , the parkinson progression marker initiative ( ppmi ) is the largest study to homogeneously enroll drug - nave pd patients at very early stages of the disease . the median duration of disease ( from diagnosis to enrollment ) was only 4.2 months ( range , 0.0338.83 ) , and the hoehn and yahr stage ( h&y ) of 99.5% of ppmi patients is i or ii . the primary objective of the ppmi study was to identify the biomarkers that predict pd progression . this review briefly provides evidence of the heterogeneous progression in the cognitive decline of pd patients , discusses the pathophysiologic mechanisms related to heterogeneous cognitive decline , and summarizes recent advances in the development of cerebrospinal fluid ( csf ) biomarkers to predict disease progression in the ppmi study . the existence of subgroups within pd with distinct clinical patterns of motor and non - motor symptoms is widely accepted . the clinical heterogeneity of pd may be associated with a variability in pathogenic mechanisms that may be controlled by inherited and environmental factors ( figure 1 ) . based on the motor symptoms of pd patients , formulas were developed to define the motor phenotypes using movement disorder society unified parkinson s disease rating scale ( mds - updrs ) , a revision of the original updrs . the motor phenotypes are most often classified by calculating the ratio between the scores of tremor - related and balance / gait / posture - related items in mds - updrs . using this system , however , despite evidence that the early development of cognitive deficits is significantly associated with pigd or non - td motor phenotypes , the motor phenotype of pd , particularly at a very early stage , might not yet be fully differentiated into two defined phenotypes ( i.e. , indeterminate type not specified as td or pigd ) . therefore , whether the motor phenotype is useful in predicting the development of dementia in pd patients with very early stage disease and/or with pd medication should be further evaluated . among the clinical symptoms that progress heterogeneously , the predictability of cognitive decline in pd is currently evaluated by neuropsychological , neuropathologic , neuroimaging and biologic biomarkers . the neuropsychological assessment of pd patients will be a tool to help predict the development of dementia in pd patients . in fact , patients who meet the criteria for mild cognitive impairment are more likely to develop dementia . the most common cognitive symptoms are attention deficits , executive functioning and visuospatial processing , and these variable profiles of cognitive dysfunction may be caused by the heterogeneous nature of the underlying neuropathology [ 19 - 21 ] . several studies suggested that a specific domain of memory and cognitive function or the malfunctioning of a specific region were associated with a rapid progression of cognitive deficit , although it remains to be elucidated whether a specific profile reflects the heterogeneous progression of pd dementia . for example , frontal cortex dysfunction assessed by executive difficulties is an early phenomenon , while visuospatial and semantic memory dysfunction , which reflect temporal and parietal involvement , are risk factors for parkinson s disease dementia ( pdd ) . although it is not sufficient , the measurement of csf alpha - synuclein ( -syn ) , dj-1 , tau , phosphorylated tau at thr181 ( p - tau ) or fms - related tyrosine kinase 3 ligand can differentiate pd patients from healthy controls ( hc ) . indeed , the level of csf -syn and tau proteins in pd patients was significantly lower than those in age - matched hc , with a marked overlap between groups . the mechanisms associated with lower levels of csf -syn in pd remain to be elucidated . the accumulation of -syn in the brain similar to amyloid ( a ) in alzheimer s disease ( ad)may be a mechanism for the reduction of csf -syn . although it is necessary to characterize the immunoassay platform , the measurement of -syn oligomers in csf improves diagnostic utility . interestingly , the level of tau proteins [ total tau ( t - tau ) and p - tau ] in the csf of pd patients was also lower than controls , although not all studies replicated this finding . taken together , the level of -syn and other proteins in csf may be useful in diagnosing pd , although it is too early to make definitive conclusions . revealed that the progression of -syn pathology throughout the brain is not random but follows a stereotypical caudal - to - rostral ascending progression ; however , not all neuropathological studies agreed with that topology . however , the detailed topographic patterns of the spread of -syn among pd patients are not homogeneous . the specific topographical pattern of lewy pathology ( i.e. , global involvement of cortical and limbic areas with lewy bodies and lewy neurites ) is significantly associated with cognitive deficits in pd , which suggests the underlying dementia - prone pattern in the spread of lewy pathology . although it is not clear which factors regulate the topographic spread of lewy pathology , human post - mortem studies suggested the cortical and limbic involvement of -syn pathology as a neuropathologic substrate were strongly correlated with pdd . furthermore , recent advances in the understanding of the neuropathologic substrates of pdd suggested that the interaction between -syn and ad pathology ( i.e. , a and tau pathology ) may regulate disease severity and progression [ 32 - 34 ] . provided evidence that the alzheimer s signature in csf ( lower level of csf a1 - 42 ) was significantly associated with more rapid cognitive decline in pd , and this finding was replicated by other studies [ 36 - 38 ] . the association of the ad - like csf signature with the rapid progression of cognitive dysfunction supported the hypothetical model that cross - seeding -syn and a or tau accelerate neurodegenerative pathology and disease progression . in support of this assertion , transgenic mice overexpressing human -syn ( wild type or ala53thr mutant ) with mutant forms of tau or amyloid precursor protein show greater neurodegeneration and functional deficits than mice overexpressing ad - associated proteins alone . furthermore , in vitro observation of enhanced fibril formation via cross - seed [ 41 - 43 ] and human studies in pd patients with the ala53thr snca mutation supported the contribution of ad pathology to the disease progression of pd . the rapid progression of cognitive deficits in pd patients could not be exclusively determined by pathological substrates of ad ; however , these previous studies in vitro , in animal models and in clinical cohorts suggested the increasingly important role of ad pathology in the development of pdd . for the prediction of cognitive progression in pd , genetic factors associated with neuropathological substrates of ad and/or pd may be an important contributor to cognitive deficits , along with -syn pathology . for example , genetic factors ( e.g. , mutation in -glucocerebrosidase ) associated with the hereditary form of pd are a genetic predictor of pdd . the apolipoprotein e ( apoe ) e4 allele , which has been widely accepted as a risk factor for ad , may confer an increased risk of rapid cognitive decline in pd . in contrast , the association of the apoe 4 allele with pdd was lost after adjustment for csf a1 - 42 level . in a large - scale study with autopsied subjects , however , the apoe 4 allele frequency was significantly higher not only in the ad group but also in the dementia with lewy bodies and pdd groups compared to the control group . therefore , the apoe 4 allele in pd may be a genetic risk factor for the cognitive deficits associated with pathways that are shared with and diverge from a-related pathogenesis . the existence of subgroups within pd with distinct clinical patterns of motor and non - motor symptoms is widely accepted . the clinical heterogeneity of pd may be associated with a variability in pathogenic mechanisms that may be controlled by inherited and environmental factors ( figure 1 ) . based on the motor symptoms of pd patients , formulas were developed to define the motor phenotypes using movement disorder society unified parkinson s disease rating scale ( mds - updrs ) , a revision of the original updrs . the motor phenotypes are most often classified by calculating the ratio between the scores of tremor - related and balance / gait / posture - related items in mds - updrs . using this system , however , despite evidence that the early development of cognitive deficits is significantly associated with pigd or non - td motor phenotypes , the motor phenotype of pd , particularly at a very early stage , might not yet be fully differentiated into two defined phenotypes ( i.e. , indeterminate type not specified as td or pigd ) . therefore , whether the motor phenotype is useful in predicting the development of dementia in pd patients with very early stage disease and/or with pd medication should be further evaluated . among the clinical symptoms that progress heterogeneously , the predictability of cognitive decline in pd is currently evaluated by neuropsychological , neuropathologic , neuroimaging and biologic biomarkers . the neuropsychological assessment of pd patients will be a tool to help predict the development of dementia in pd patients . in fact , patients who meet the criteria for mild cognitive impairment are more likely to develop dementia . the most common cognitive symptoms are attention deficits , executive functioning and visuospatial processing , and these variable profiles of cognitive dysfunction may be caused by the heterogeneous nature of the underlying neuropathology [ 19 - 21 ] . several studies suggested that a specific domain of memory and cognitive function or the malfunctioning of a specific region were associated with a rapid progression of cognitive deficit , although it remains to be elucidated whether a specific profile reflects the heterogeneous progression of pd dementia . for example , frontal cortex dysfunction assessed by executive difficulties is an early phenomenon , while visuospatial and semantic memory dysfunction , which reflect temporal and parietal involvement , are risk factors for parkinson s disease dementia ( pdd ) . although it is not sufficient , the measurement of csf alpha - synuclein ( -syn ) , dj-1 , tau , phosphorylated tau at thr181 ( p - tau ) or fms - related tyrosine kinase 3 ligand can differentiate pd patients from healthy controls ( hc ) . indeed , the level of csf -syn and tau proteins in pd patients was significantly lower than those in age - matched hc , with a marked overlap between groups . the mechanisms associated with lower levels of csf -syn in pd remain to be elucidated . the accumulation of -syn in the brain similar to amyloid ( a ) in alzheimer s disease ( ad)may be a mechanism for the reduction of csf -syn . although it is necessary to characterize the immunoassay platform , the measurement of -syn oligomers in csf improves diagnostic utility . interestingly , the level of tau proteins [ total tau ( t - tau ) and p - tau ] in the csf of pd patients was also lower than controls , although not all studies replicated this finding . taken together , the level of -syn and other proteins in csf may be useful in diagnosing pd , although it is too early to make definitive conclusions . . revealed that the progression of -syn pathology throughout the brain is not random but follows a stereotypical caudal - to - rostral ascending progression ; however , not all neuropathological studies agreed with that topology . however , the detailed topographic patterns of the spread of -syn among pd patients are not homogeneous . the specific topographical pattern of lewy pathology ( i.e. , global involvement of cortical and limbic areas with lewy bodies and lewy neurites ) is significantly associated with cognitive deficits in pd , which suggests the underlying dementia - prone pattern in the spread of lewy pathology . although it is not clear which factors regulate the topographic spread of lewy pathology , human post - mortem studies suggested the cortical and limbic involvement of -syn pathology as a neuropathologic substrate were strongly correlated with pdd . furthermore , recent advances in the understanding of the neuropathologic substrates of pdd suggested that the interaction between -syn and ad pathology ( i.e. , a and tau pathology ) may regulate disease severity and progression [ 32 - 34 ] . using csf biomarkers , siderowf et al . provided evidence that the alzheimer s signature in csf ( lower level of csf a1 - 42 ) was significantly associated with more rapid cognitive decline in pd , and this finding was replicated by other studies [ 36 - 38 ] . the association of the ad - like csf signature with the rapid progression of cognitive dysfunction supported the hypothetical model that cross - seeding -syn and a or tau accelerate neurodegenerative pathology and disease progression . in support of this assertion , transgenic mice overexpressing human -syn ( wild type or ala53thr mutant ) with mutant forms of tau or amyloid precursor protein show greater neurodegeneration and functional deficits than mice overexpressing ad - associated proteins alone . furthermore , in vitro observation of enhanced fibril formation via cross - seed [ 41 - 43 ] and human studies in pd patients with the ala53thr snca mutation supported the contribution of ad pathology to the disease progression of pd . the rapid progression of cognitive deficits in pd patients could not be exclusively determined by pathological substrates of ad ; however , these previous studies in vitro , in animal models and in clinical cohorts suggested the increasingly important role of ad pathology in the development of pdd . for the prediction of cognitive progression in pd , genetic factors associated with neuropathological substrates of ad and/or pd may be an important contributor to cognitive deficits , along with -syn pathology . for example , genetic factors ( e.g. , mutation in -glucocerebrosidase ) associated with the hereditary form of pd are a genetic predictor of pdd . the apolipoprotein e ( apoe ) e4 allele , which has been widely accepted as a risk factor for ad , may confer an increased risk of rapid cognitive decline in pd . in contrast , the association of the apoe 4 allele with pdd was lost after adjustment for csf a1 - 42 level . in a large - scale study with autopsied subjects , however , the apoe 4 allele frequency was significantly higher not only in the ad group but also in the dementia with lewy bodies and pdd groups compared to the control group . therefore , the apoe 4 allele in pd may be a genetic risk factor for the cognitive deficits associated with pathways that are shared with and diverge from a-related pathogenesis . the development of reliable and validated biomarkers for heterogeneous pd progression is a critical unmet need . validated pd progression markers are essential to accelerate research into pd pathogenesis , and the development of disease - modifying therapeutics ( dmt ) and would dramatically improve patient care . there are several important prerequisites for the development of valid biochemical progression biomarkers : the establishment of standardized protocols for the acquisition , transfer and analysis of biospecimens ; the optimization and verification of bioassays ; a sufficient longitudinal follow - up period to track heterogeneous progression ; and the recruitment of drug - nave patients at baseline . ppmi is a five - year observational , international , longitudinal study that aimed to identify biomarkers of pd progression that involve the collaborative effort of pd researchers with expertise in biomarker development , the clinical study of pd , bioinformatics , statistics and data management . analogous to the alzheimer s disease neuroimaging initiative ( adni ) , the ppmi is a public - private partnership , sponsored by the michael j fox foundation with industry partnership . the overall objective of the ppmi study was to identify the clinical , imaging , and biologic markers of pd progression for use in clinical trials of dmt . approximately 400 drug - nave pd patients at the early stage , and 200 age - matched hc were planned to be enrolled from 24 clinical sites in the united states , europe and australia ( figure 2 ) . the number of subjects was calculated with the power to detect a difference in prevalence of 13% ( for a dichotomous endpoint ) and a standardized mean difference of 0.24 ( for a continuous end - point ) . all pd patients were at the early stage ( diagnosis within 2 years and h&y stage < 2 ) and untreated with pd medication , as described in detail elsewhere and on the ppmi website ( http://www.ppmi-info.org/study-design/ ) . the longitudinal collection of biospecimens , including blood , csf and urine , is an essential component to discovering biological markers that are able to track disease progression . in particular , the collection , processing , aliquoting and storage of csf were remarkably standardized in the adni study . this review discusses the baseline csf biomarkers ( a1 - 42 , t - tau , p - tau , and -syn ) data of the ppmi cohort . the partial baseline csf results ( n = 102 ; pd = 63 , hc = 39 ) were published in 2013 . the initial data showed several interesting findings ; the lower levels of csf a1 - 42 and p - tau were significantly associated with the pigd phenotype in multiple logistic regression analysis with adjustment for confounders ; the level of -syn was significantly correlated with the level of t - tau and p - tau , and the levels of -syn and t - tau were associated with motor severity . a recent analysis of the full baseline dataset showed consistent results ( n = 660 ; pd = 412 , hc = 189 , subjects without evidence of dopamine deficit = 59 ) , but some results could not replicate the pilot findings . for example , the level of csf a1 - 42 or p - tau was not associated with motor phenotype , but the csf -syn level in pd patients with the non - td phenotype was significantly lower than pd patients with the td phenotype . in addition , there were no csf biomarkers that were significantly associated with motor severity when multivariate regression analysis with the adjustment of confounding factors was applied , although a low level of p - tau was marginally associated with disease severity . however , the strong correlation between the level of -syn and t - tau or p - tau in both pd and hc was replicable . consistent with the pilot study , the levels of csf -syn , t - tau , and p - tau , but not a1 - 42 , were significantly lower in pd compared to hc , while the diagnostic utility of each biomarker was limited due to a large overlap . the lower level of csf -syn in pd relative to hc implicates the accumulation of -syn in the brain of pd patients , analogous to the finding of lower levels of csf a1 - 42 in ad patients compared to hc . the mechanism of a reduction in tau proteins in pd compared with hc is unclear ; however , a possible interpretation is that the interaction between tau proteins and -syn may limit the release of tau proteins into csf . in connection with this , previous studies using in vitro , animal models [ 49 - 51 ] or postmortem brains of pd reported that the -syn pathology in the brain is accompanied by increased levels of hyperphosphorylated tau proteins and tau - positive tangles , and -syn positive lewy bodies may co - localize in the same neuron . the genome - wide association study also supported this hypothesis that mapt and snca , which encode tau and -syn , respectively , showed a genetic association with pd . therefore , the extent of the direct or indirect interaction between tau phosphorylation and -syn accumulation or the pattern of topological distribution of these pathogenic proteins may contribute to the heterogeneous progression of pd . although future long - term longitudinal observations in the ppmi cohort will be required to test the predictive performance of the csf biomarkers , the baseline data in this large cohort suggest that csf biomarkers in early pd patients already reflect disease heterogeneity and may have predictive value for disease progression . our findings of the association of csf biomarkers with cognitive function in the ppmi cohort was not consistent with other studies [ 26,35,37,56 - 60 ] . for example , the association of a higher csf -syn level with worsening cognitive decline was observed in the deprenyl and tocopherol antioxidative therapy of parkinsonism study . in contrast , the lower csf -syn level was associated with more severe neuropsychological function , including semantic fluency , visuospatial cognition and executive functioning in the ppmi cohort , which indicated that -syn pathology contributes to early cognitive impairment in pd . in addition , multivariate regression analysis of the ppmi baseline data did not fully reproduce the previous findings that the lower level of csf a1 - 42 was associated with cognitive impairment in pd . the lower level of a1 - 42 was significantly associated with processing speed / attention assessed by the symbol digit modality test ( sdmt ) but not with other cognitive functions , including the global cognitive function test and montreal cognitive assessment , in the ppmi cohort . instead , when the clinical variables of the group with the highest quintile levels of csf biomarkers were compared with those of the group with the lowest quintile levels , the csf a1 - 42 level showed significant associations with semantic fluency and sdmt score , and the t - tau / a1 - 42 ratio showed significant associations with memory ( total recall and delayed recall measured by hopkins verbal learning test - revised score ) , semantic fluency , sdmt and wechsler memory scaleiii letter - number sequencing score . it should be noted that the ppmi cohort included patients with very early stage and drug - nave disease at baseline ; therefore , whether csf biomarkers in early pd are associated with the risk of future cognitive decline and pdd should be determined in longitudinal analyses . a recent study that observed a group of ppmi pd patients ( n = 341 ) for 2 years found a significant association of lower baseline csf a1 - 42 level with higher odds of cognitive impairment , even though the baseline csf biomarker data showed a slight association of csf a1 - 42 with cognitive dysfunction in multivariate analysis . the discrepancy among studies in the association of csf biomarkers with clinical variables may be due to several demographic , biological and analytical factors , including but not limited to the different ages among cohorts , the contamination of blood in csf , the mixed pathology or disease stage of studied patients , and different immunoassay platforms . therefore , we should carefully interpret the results for the association of csf biomarkers with clinical variables . the development of reliable and validated biomarkers for heterogeneous pd progression is a critical unmet need . validated pd progression markers are essential to accelerate research into pd pathogenesis , and the development of disease - modifying therapeutics ( dmt ) and would dramatically improve patient care . there are several important prerequisites for the development of valid biochemical progression biomarkers : the establishment of standardized protocols for the acquisition , transfer and analysis of biospecimens ; the optimization and verification of bioassays ; a sufficient longitudinal follow - up period to track heterogeneous progression ; and the recruitment of drug - nave patients at baseline . ppmi is a five - year observational , international , longitudinal study that aimed to identify biomarkers of pd progression that involve the collaborative effort of pd researchers with expertise in biomarker development , the clinical study of pd , bioinformatics , statistics and data management . analogous to the alzheimer s disease neuroimaging initiative ( adni ) , the ppmi is a public - private partnership , sponsored by the michael j fox foundation with industry partnership . the overall objective of the ppmi study was to identify the clinical , imaging , and biologic markers of pd progression for use in clinical trials of dmt . approximately 400 drug - nave pd patients at the early stage , and 200 age - matched hc were planned to be enrolled from 24 clinical sites in the united states , europe and australia ( figure 2 ) . the number of subjects was calculated with the power to detect a difference in prevalence of 13% ( for a dichotomous endpoint ) and a standardized mean difference of 0.24 ( for a continuous end - point ) . all pd patients were at the early stage ( diagnosis within 2 years and h&y stage < 2 ) and untreated with pd medication , as described in detail elsewhere and on the ppmi website ( http://www.ppmi-info.org/study-design/ ) . the longitudinal collection of biospecimens , including blood , csf and urine , is an essential component to discovering biological markers that are able to track disease progression . in particular , the collection , processing , aliquoting and storage of csf were remarkably standardized in the adni study . this review discusses the baseline csf biomarkers ( a1 - 42 , t - tau , p - tau , and -syn ) data of the ppmi cohort . the partial baseline csf results ( n = 102 ; pd = 63 , hc = 39 ) were published in 2013 . the initial data showed several interesting findings ; the lower levels of csf a1 - 42 and p - tau were significantly associated with the pigd phenotype in multiple logistic regression analysis with adjustment for confounders ; the level of -syn was significantly correlated with the level of t - tau and p - tau , and the levels of -syn and t - tau were associated with motor severity . a recent analysis of the full baseline dataset showed consistent results ( n = 660 ; pd = 412 , hc = 189 , subjects without evidence of dopamine deficit = 59 ) , but some results could not replicate the pilot findings . for example , the level of csf a1 - 42 or p - tau was not associated with motor phenotype , but the csf -syn level in pd patients with the non - td phenotype was significantly lower than pd patients with the td phenotype . in addition , there were no csf biomarkers that were significantly associated with motor severity when multivariate regression analysis with the adjustment of confounding factors was applied , although a low level of p - tau was marginally associated with disease severity . however , the strong correlation between the level of -syn and t - tau or p - tau in both pd and hc was replicable . consistent with the pilot study , the levels of csf -syn , t - tau , and p - tau , but not a1 - 42 , were significantly lower in pd compared to hc , while the diagnostic utility of each biomarker was limited due to a large overlap . the lower level of csf -syn in pd relative to hc implicates the accumulation of -syn in the brain of pd patients , analogous to the finding of lower levels of csf a1 - 42 in ad patients compared to hc . the mechanism of a reduction in tau proteins in pd compared with hc is unclear ; however , a possible interpretation is that the interaction between tau proteins and -syn may limit the release of tau proteins into csf . in connection with this , previous studies using in vitro , animal models [ 49 - 51 ] or postmortem brains of pd reported that the -syn pathology in the brain is accompanied by increased levels of hyperphosphorylated tau proteins and tau - positive tangles , and -syn positive lewy bodies may co - localize in the same neuron . the genome - wide association study also supported this hypothesis that mapt and snca , which encode tau and -syn , respectively , showed a genetic association with pd . therefore , the extent of the direct or indirect interaction between tau phosphorylation and -syn accumulation or the pattern of topological distribution of these pathogenic proteins may contribute to the heterogeneous progression of pd . although future long - term longitudinal observations in the ppmi cohort will be required to test the predictive performance of the csf biomarkers , the baseline data in this large cohort suggest that csf biomarkers in early pd patients already reflect disease heterogeneity and may have predictive value for disease progression . our findings of the association of csf biomarkers with cognitive function in the ppmi cohort was not consistent with other studies [ 26,35,37,56 - 60 ] . for example , the association of a higher csf -syn level with worsening cognitive decline was observed in the deprenyl and tocopherol antioxidative therapy of parkinsonism study . in contrast , the lower csf -syn level was associated with more severe neuropsychological function , including semantic fluency , visuospatial cognition and executive functioning in the ppmi cohort , which indicated that -syn pathology contributes to early cognitive impairment in pd . in addition , multivariate regression analysis of the ppmi baseline data did not fully reproduce the previous findings that the lower level of csf a1 - 42 was associated with cognitive impairment in pd . the lower level of a1 - 42 was significantly associated with processing speed / attention assessed by the symbol digit modality test ( sdmt ) but not with other cognitive functions , including the global cognitive function test and montreal cognitive assessment , in the ppmi cohort . instead , when the clinical variables of the group with the highest quintile levels of csf biomarkers were compared with those of the group with the lowest quintile levels , the csf a1 - 42 level showed significant associations with semantic fluency and sdmt score , and the t - tau / a1 - 42 ratio showed significant associations with memory ( total recall and delayed recall measured by hopkins verbal learning test - revised score ) , semantic fluency , sdmt and wechsler memory scaleiii letter - number sequencing score . it should be noted that the ppmi cohort included patients with very early stage and drug - nave disease at baseline ; therefore , whether csf biomarkers in early pd are associated with the risk of future cognitive decline and pdd should be determined in longitudinal analyses . a recent study that observed a group of ppmi pd patients ( n = 341 ) for 2 years found a significant association of lower baseline csf a1 - 42 level with higher odds of cognitive impairment , even though the baseline csf biomarker data showed a slight association of csf a1 - 42 with cognitive dysfunction in multivariate analysis . the discrepancy among studies in the association of csf biomarkers with clinical variables may be due to several demographic , biological and analytical factors , including but not limited to the different ages among cohorts , the contamination of blood in csf , the mixed pathology or disease stage of studied patients , and different immunoassay platforms . therefore , we should carefully interpret the results for the association of csf biomarkers with clinical variables . not all pd patients develop dementia ; however , dementia is a frequent non - motor complication in pd with heterogeneous features . genetic , demographic and environmental factors may be related to the heterogeneous progression of cognitive decline in pd patients . thus , understanding the heterogeneity would provide insight into the pathogenic mechanism of pdd development , which is important for developing therapeutics as well as patient care . there are few studies on the association of heterogeneous cognitive decline with specific molecular signatures or pathogenesis in pd patients with a large number of subjects and in early stages of the disease . the evidence of molecular interactions between -syn and a1 - 42 and/or tau in the development of pdd in post - mortem samples implicate the necessity of the longitudinal observation of csf and imaging biomarkers in pd . in addition , the development of biomarkers for the early diagnosis of pd is an unmet need . because pd is not a disease with homogeneous features , the development of biomarkers of the heterogeneity in motor and non - motor dysfunction will provide the molecular basis for individualized therapeutics . finally , the combination of biochemical , imaging and genetic biomarkers rather than individual biomarkers may better accomplish this aim . to this end , the development of novel , promising and valid biochemical biomarkers and -syn imaging technologies is necessary .

parkinson s disease ( pd ) is a neurodegenerative disease with heterogeneous pathological and clinical features . cognitive dysfunction , a frequent non - motor complication , is a risk factor for poor prognosis and shows inter - individual variation in its progression . of the clinical studies performed to identify biomarkers of pd progression , the parkinson s progression markers initiative ( ppmi ) study is the largest study that enrolled drug - nave and very early stage pd patients . the baseline characteristics of the ppmi cohort were recently published . the diagnostic utility of cerebrospinal fluid ( csf ) biomarkers , including alpha - synuclein ( -syn ) , total tau , phosphorylated tau at thr181 , and amyloid 1 - 42 , was not satisfactory . however , the baseline data on csf biomarkers in the ppmi study suggested that the measurement of the csf biomarkers enables the prediction of future cognitive decline in pd patients , which was consistent with previous studies . to prove the hypothesis that the interaction between alzheimer s pathology and -syn pathology is important to the progression of cognitive dysfunction in pd , longitudinal observational studies must be followed . in this review , the neuropathological nature of heterogeneous cognitive decline in pd is briefly discussed , followed by a summarized interpretation of baseline csf biomarkers derived from the data in the ppmi study . the combination of clinical , biochemical , genetic and imaging biomarkers of pd constitutes a feasible strategy to predict the heterogeneous progression of pd .

INTRODUCTION EVIDENCE OF COGNITIVE HETEROGENEITY IN PD Clinical characteristics associated with cognitive heterogeneity Neuropathologic substrates associated with cognitive heterogeneity PARKINSONS PROGRESSION MARKERS INITIATIVE Overview Association of CSF biomarkers with clinical features PERSPECTIVE

parkinson s disease ( pd ) is a synucleinopathy with heterogeneous progression of motor and non - motor dysfunction . although the mechanisms of the inherent heterogeneous progression of pd are still largely unknown , the heterogeneity of the disease may be associated with alterations of neural connectivity between the motor and cognitive control areas of the brain . the early development of dementia in pd patients is a major risk factor for poor prognosis and high mortality . there are currently no proven underlying pathogenic mechanisms of cognitive decline in pd patients . furthermore , several well - designed clinical studies to identify biochemical , imaging and genetic biomarkers of pd are emerging . among the large multicenter longitudinal pd biomarker studies , the parkinson progression marker initiative ( ppmi ) is the largest study to homogeneously enroll drug - nave pd patients at very early stages of the disease . the primary objective of the ppmi study was to identify the biomarkers that predict pd progression . this review briefly provides evidence of the heterogeneous progression in the cognitive decline of pd patients , discusses the pathophysiologic mechanisms related to heterogeneous cognitive decline , and summarizes recent advances in the development of cerebrospinal fluid ( csf ) biomarkers to predict disease progression in the ppmi study . based on the motor symptoms of pd patients , formulas were developed to define the motor phenotypes using movement disorder society unified parkinson s disease rating scale ( mds - updrs ) , a revision of the original updrs . using this system , however , despite evidence that the early development of cognitive deficits is significantly associated with pigd or non - td motor phenotypes , the motor phenotype of pd , particularly at a very early stage , might not yet be fully differentiated into two defined phenotypes ( i.e. among the clinical symptoms that progress heterogeneously , the predictability of cognitive decline in pd is currently evaluated by neuropsychological , neuropathologic , neuroimaging and biologic biomarkers . the most common cognitive symptoms are attention deficits , executive functioning and visuospatial processing , and these variable profiles of cognitive dysfunction may be caused by the heterogeneous nature of the underlying neuropathology [ 19 - 21 ] . several studies suggested that a specific domain of memory and cognitive function or the malfunctioning of a specific region were associated with a rapid progression of cognitive deficit , although it remains to be elucidated whether a specific profile reflects the heterogeneous progression of pd dementia . although it is not sufficient , the measurement of csf alpha - synuclein ( -syn ) , dj-1 , tau , phosphorylated tau at thr181 ( p - tau ) or fms - related tyrosine kinase 3 ligand can differentiate pd patients from healthy controls ( hc ) . interestingly , the level of tau proteins [ total tau ( t - tau ) and p - tau ] in the csf of pd patients was also lower than controls , although not all studies replicated this finding . revealed that the progression of -syn pathology throughout the brain is not random but follows a stereotypical caudal - to - rostral ascending progression ; however , not all neuropathological studies agreed with that topology . , global involvement of cortical and limbic areas with lewy bodies and lewy neurites ) is significantly associated with cognitive deficits in pd , which suggests the underlying dementia - prone pattern in the spread of lewy pathology . furthermore , recent advances in the understanding of the neuropathologic substrates of pdd suggested that the interaction between -syn and ad pathology ( i.e. provided evidence that the alzheimer s signature in csf ( lower level of csf a1 - 42 ) was significantly associated with more rapid cognitive decline in pd , and this finding was replicated by other studies [ 36 - 38 ] . the association of the ad - like csf signature with the rapid progression of cognitive dysfunction supported the hypothetical model that cross - seeding -syn and a or tau accelerate neurodegenerative pathology and disease progression . furthermore , in vitro observation of enhanced fibril formation via cross - seed [ 41 - 43 ] and human studies in pd patients with the ala53thr snca mutation supported the contribution of ad pathology to the disease progression of pd . the rapid progression of cognitive deficits in pd patients could not be exclusively determined by pathological substrates of ad ; however , these previous studies in vitro , in animal models and in clinical cohorts suggested the increasingly important role of ad pathology in the development of pdd . for the prediction of cognitive progression in pd , genetic factors associated with neuropathological substrates of ad and/or pd may be an important contributor to cognitive deficits , along with -syn pathology . the apolipoprotein e ( apoe ) e4 allele , which has been widely accepted as a risk factor for ad , may confer an increased risk of rapid cognitive decline in pd . based on the motor symptoms of pd patients , formulas were developed to define the motor phenotypes using movement disorder society unified parkinson s disease rating scale ( mds - updrs ) , a revision of the original updrs . using this system , however , despite evidence that the early development of cognitive deficits is significantly associated with pigd or non - td motor phenotypes , the motor phenotype of pd , particularly at a very early stage , might not yet be fully differentiated into two defined phenotypes ( i.e. among the clinical symptoms that progress heterogeneously , the predictability of cognitive decline in pd is currently evaluated by neuropsychological , neuropathologic , neuroimaging and biologic biomarkers . the most common cognitive symptoms are attention deficits , executive functioning and visuospatial processing , and these variable profiles of cognitive dysfunction may be caused by the heterogeneous nature of the underlying neuropathology [ 19 - 21 ] . several studies suggested that a specific domain of memory and cognitive function or the malfunctioning of a specific region were associated with a rapid progression of cognitive deficit , although it remains to be elucidated whether a specific profile reflects the heterogeneous progression of pd dementia . although it is not sufficient , the measurement of csf alpha - synuclein ( -syn ) , dj-1 , tau , phosphorylated tau at thr181 ( p - tau ) or fms - related tyrosine kinase 3 ligand can differentiate pd patients from healthy controls ( hc ) . interestingly , the level of tau proteins [ total tau ( t - tau ) and p - tau ] in the csf of pd patients was also lower than controls , although not all studies replicated this finding . revealed that the progression of -syn pathology throughout the brain is not random but follows a stereotypical caudal - to - rostral ascending progression ; however , not all neuropathological studies agreed with that topology . , global involvement of cortical and limbic areas with lewy bodies and lewy neurites ) is significantly associated with cognitive deficits in pd , which suggests the underlying dementia - prone pattern in the spread of lewy pathology . furthermore , recent advances in the understanding of the neuropathologic substrates of pdd suggested that the interaction between -syn and ad pathology ( i.e. provided evidence that the alzheimer s signature in csf ( lower level of csf a1 - 42 ) was significantly associated with more rapid cognitive decline in pd , and this finding was replicated by other studies [ 36 - 38 ] . the association of the ad - like csf signature with the rapid progression of cognitive dysfunction supported the hypothetical model that cross - seeding -syn and a or tau accelerate neurodegenerative pathology and disease progression . furthermore , in vitro observation of enhanced fibril formation via cross - seed [ 41 - 43 ] and human studies in pd patients with the ala53thr snca mutation supported the contribution of ad pathology to the disease progression of pd . the rapid progression of cognitive deficits in pd patients could not be exclusively determined by pathological substrates of ad ; however , these previous studies in vitro , in animal models and in clinical cohorts suggested the increasingly important role of ad pathology in the development of pdd . for the prediction of cognitive progression in pd , genetic factors associated with neuropathological substrates of ad and/or pd may be an important contributor to cognitive deficits , along with -syn pathology . the apolipoprotein e ( apoe ) e4 allele , which has been widely accepted as a risk factor for ad , may confer an increased risk of rapid cognitive decline in pd . ppmi is a five - year observational , international , longitudinal study that aimed to identify biomarkers of pd progression that involve the collaborative effort of pd researchers with expertise in biomarker development , the clinical study of pd , bioinformatics , statistics and data management . analogous to the alzheimer s disease neuroimaging initiative ( adni ) , the ppmi is a public - private partnership , sponsored by the michael j fox foundation with industry partnership . the overall objective of the ppmi study was to identify the clinical , imaging , and biologic markers of pd progression for use in clinical trials of dmt . approximately 400 drug - nave pd patients at the early stage , and 200 age - matched hc were planned to be enrolled from 24 clinical sites in the united states , europe and australia ( figure 2 ) . this review discusses the baseline csf biomarkers ( a1 - 42 , t - tau , p - tau , and -syn ) data of the ppmi cohort . for example , the level of csf a1 - 42 or p - tau was not associated with motor phenotype , but the csf -syn level in pd patients with the non - td phenotype was significantly lower than pd patients with the td phenotype . consistent with the pilot study , the levels of csf -syn , t - tau , and p - tau , but not a1 - 42 , were significantly lower in pd compared to hc , while the diagnostic utility of each biomarker was limited due to a large overlap . the lower level of csf -syn in pd relative to hc implicates the accumulation of -syn in the brain of pd patients , analogous to the finding of lower levels of csf a1 - 42 in ad patients compared to hc . the mechanism of a reduction in tau proteins in pd compared with hc is unclear ; however , a possible interpretation is that the interaction between tau proteins and -syn may limit the release of tau proteins into csf . in connection with this , previous studies using in vitro , animal models [ 49 - 51 ] or postmortem brains of pd reported that the -syn pathology in the brain is accompanied by increased levels of hyperphosphorylated tau proteins and tau - positive tangles , and -syn positive lewy bodies may co - localize in the same neuron . therefore , the extent of the direct or indirect interaction between tau phosphorylation and -syn accumulation or the pattern of topological distribution of these pathogenic proteins may contribute to the heterogeneous progression of pd . although future long - term longitudinal observations in the ppmi cohort will be required to test the predictive performance of the csf biomarkers , the baseline data in this large cohort suggest that csf biomarkers in early pd patients already reflect disease heterogeneity and may have predictive value for disease progression . our findings of the association of csf biomarkers with cognitive function in the ppmi cohort was not consistent with other studies [ 26,35,37,56 - 60 ] . in contrast , the lower csf -syn level was associated with more severe neuropsychological function , including semantic fluency , visuospatial cognition and executive functioning in the ppmi cohort , which indicated that -syn pathology contributes to early cognitive impairment in pd . in addition , multivariate regression analysis of the ppmi baseline data did not fully reproduce the previous findings that the lower level of csf a1 - 42 was associated with cognitive impairment in pd . the lower level of a1 - 42 was significantly associated with processing speed / attention assessed by the symbol digit modality test ( sdmt ) but not with other cognitive functions , including the global cognitive function test and montreal cognitive assessment , in the ppmi cohort . instead , when the clinical variables of the group with the highest quintile levels of csf biomarkers were compared with those of the group with the lowest quintile levels , the csf a1 - 42 level showed significant associations with semantic fluency and sdmt score , and the t - tau / a1 - 42 ratio showed significant associations with memory ( total recall and delayed recall measured by hopkins verbal learning test - revised score ) , semantic fluency , sdmt and wechsler memory scaleiii letter - number sequencing score . it should be noted that the ppmi cohort included patients with very early stage and drug - nave disease at baseline ; therefore , whether csf biomarkers in early pd are associated with the risk of future cognitive decline and pdd should be determined in longitudinal analyses . a recent study that observed a group of ppmi pd patients ( n = 341 ) for 2 years found a significant association of lower baseline csf a1 - 42 level with higher odds of cognitive impairment , even though the baseline csf biomarker data showed a slight association of csf a1 - 42 with cognitive dysfunction in multivariate analysis . the discrepancy among studies in the association of csf biomarkers with clinical variables may be due to several demographic , biological and analytical factors , including but not limited to the different ages among cohorts , the contamination of blood in csf , the mixed pathology or disease stage of studied patients , and different immunoassay platforms . ppmi is a five - year observational , international , longitudinal study that aimed to identify biomarkers of pd progression that involve the collaborative effort of pd researchers with expertise in biomarker development , the clinical study of pd , bioinformatics , statistics and data management . analogous to the alzheimer s disease neuroimaging initiative ( adni ) , the ppmi is a public - private partnership , sponsored by the michael j fox foundation with industry partnership . the overall objective of the ppmi study was to identify the clinical , imaging , and biologic markers of pd progression for use in clinical trials of dmt . approximately 400 drug - nave pd patients at the early stage , and 200 age - matched hc were planned to be enrolled from 24 clinical sites in the united states , europe and australia ( figure 2 ) . this review discusses the baseline csf biomarkers ( a1 - 42 , t - tau , p - tau , and -syn ) data of the ppmi cohort . for example , the level of csf a1 - 42 or p - tau was not associated with motor phenotype , but the csf -syn level in pd patients with the non - td phenotype was significantly lower than pd patients with the td phenotype . consistent with the pilot study , the levels of csf -syn , t - tau , and p - tau , but not a1 - 42 , were significantly lower in pd compared to hc , while the diagnostic utility of each biomarker was limited due to a large overlap . the lower level of csf -syn in pd relative to hc implicates the accumulation of -syn in the brain of pd patients , analogous to the finding of lower levels of csf a1 - 42 in ad patients compared to hc . the mechanism of a reduction in tau proteins in pd compared with hc is unclear ; however , a possible interpretation is that the interaction between tau proteins and -syn may limit the release of tau proteins into csf . in connection with this , previous studies using in vitro , animal models [ 49 - 51 ] or postmortem brains of pd reported that the -syn pathology in the brain is accompanied by increased levels of hyperphosphorylated tau proteins and tau - positive tangles , and -syn positive lewy bodies may co - localize in the same neuron . therefore , the extent of the direct or indirect interaction between tau phosphorylation and -syn accumulation or the pattern of topological distribution of these pathogenic proteins may contribute to the heterogeneous progression of pd . although future long - term longitudinal observations in the ppmi cohort will be required to test the predictive performance of the csf biomarkers , the baseline data in this large cohort suggest that csf biomarkers in early pd patients already reflect disease heterogeneity and may have predictive value for disease progression . our findings of the association of csf biomarkers with cognitive function in the ppmi cohort was not consistent with other studies [ 26,35,37,56 - 60 ] . in contrast , the lower csf -syn level was associated with more severe neuropsychological function , including semantic fluency , visuospatial cognition and executive functioning in the ppmi cohort , which indicated that -syn pathology contributes to early cognitive impairment in pd . in addition , multivariate regression analysis of the ppmi baseline data did not fully reproduce the previous findings that the lower level of csf a1 - 42 was associated with cognitive impairment in pd . the lower level of a1 - 42 was significantly associated with processing speed / attention assessed by the symbol digit modality test ( sdmt ) but not with other cognitive functions , including the global cognitive function test and montreal cognitive assessment , in the ppmi cohort . instead , when the clinical variables of the group with the highest quintile levels of csf biomarkers were compared with those of the group with the lowest quintile levels , the csf a1 - 42 level showed significant associations with semantic fluency and sdmt score , and the t - tau / a1 - 42 ratio showed significant associations with memory ( total recall and delayed recall measured by hopkins verbal learning test - revised score ) , semantic fluency , sdmt and wechsler memory scaleiii letter - number sequencing score . it should be noted that the ppmi cohort included patients with very early stage and drug - nave disease at baseline ; therefore , whether csf biomarkers in early pd are associated with the risk of future cognitive decline and pdd should be determined in longitudinal analyses . a recent study that observed a group of ppmi pd patients ( n = 341 ) for 2 years found a significant association of lower baseline csf a1 - 42 level with higher odds of cognitive impairment , even though the baseline csf biomarker data showed a slight association of csf a1 - 42 with cognitive dysfunction in multivariate analysis . the discrepancy among studies in the association of csf biomarkers with clinical variables may be due to several demographic , biological and analytical factors , including but not limited to the different ages among cohorts , the contamination of blood in csf , the mixed pathology or disease stage of studied patients , and different immunoassay platforms . not all pd patients develop dementia ; however , dementia is a frequent non - motor complication in pd with heterogeneous features . genetic , demographic and environmental factors may be related to the heterogeneous progression of cognitive decline in pd patients . there are few studies on the association of heterogeneous cognitive decline with specific molecular signatures or pathogenesis in pd patients with a large number of subjects and in early stages of the disease . the evidence of molecular interactions between -syn and a1 - 42 and/or tau in the development of pdd in post - mortem samples implicate the necessity of the longitudinal observation of csf and imaging biomarkers in pd . because pd is not a disease with homogeneous features , the development of biomarkers of the heterogeneity in motor and non - motor dysfunction will provide the molecular basis for individualized therapeutics .

in fact , several well - designed studies provided evidence that pd patients with the postural instability and gait disturbance ( pigd ) phenotype of motor symptoms showed a rapid progression of functional disability and cognitive deficit compared with patients with the tremor - dominant ( td ) motor phenotype [ 1 - 5 ] . although the mechanisms of the inherent heterogeneous progression of pd are still largely unknown , the heterogeneity of the disease may be associated with alterations of neural connectivity between the motor and cognitive control areas of the brain . the median duration of disease ( from diagnosis to enrollment ) was only 4.2 months ( range , 0.0338.83 ) , and the hoehn and yahr stage ( h&y ) of 99.5% of ppmi patients is i or ii . this review briefly provides evidence of the heterogeneous progression in the cognitive decline of pd patients , discusses the pathophysiologic mechanisms related to heterogeneous cognitive decline , and summarizes recent advances in the development of cerebrospinal fluid ( csf ) biomarkers to predict disease progression in the ppmi study . based on the motor symptoms of pd patients , formulas were developed to define the motor phenotypes using movement disorder society unified parkinson s disease rating scale ( mds - updrs ) , a revision of the original updrs . using this system , however , despite evidence that the early development of cognitive deficits is significantly associated with pigd or non - td motor phenotypes , the motor phenotype of pd , particularly at a very early stage , might not yet be fully differentiated into two defined phenotypes ( i.e. therefore , whether the motor phenotype is useful in predicting the development of dementia in pd patients with very early stage disease and/or with pd medication should be further evaluated . among the clinical symptoms that progress heterogeneously , the predictability of cognitive decline in pd is currently evaluated by neuropsychological , neuropathologic , neuroimaging and biologic biomarkers . several studies suggested that a specific domain of memory and cognitive function or the malfunctioning of a specific region were associated with a rapid progression of cognitive deficit , although it remains to be elucidated whether a specific profile reflects the heterogeneous progression of pd dementia . although it is not sufficient , the measurement of csf alpha - synuclein ( -syn ) , dj-1 , tau , phosphorylated tau at thr181 ( p - tau ) or fms - related tyrosine kinase 3 ligand can differentiate pd patients from healthy controls ( hc ) . interestingly , the level of tau proteins [ total tau ( t - tau ) and p - tau ] in the csf of pd patients was also lower than controls , although not all studies replicated this finding . , global involvement of cortical and limbic areas with lewy bodies and lewy neurites ) is significantly associated with cognitive deficits in pd , which suggests the underlying dementia - prone pattern in the spread of lewy pathology . provided evidence that the alzheimer s signature in csf ( lower level of csf a1 - 42 ) was significantly associated with more rapid cognitive decline in pd , and this finding was replicated by other studies [ 36 - 38 ] . the association of the ad - like csf signature with the rapid progression of cognitive dysfunction supported the hypothetical model that cross - seeding -syn and a or tau accelerate neurodegenerative pathology and disease progression . in support of this assertion , transgenic mice overexpressing human -syn ( wild type or ala53thr mutant ) with mutant forms of tau or amyloid precursor protein show greater neurodegeneration and functional deficits than mice overexpressing ad - associated proteins alone . furthermore , in vitro observation of enhanced fibril formation via cross - seed [ 41 - 43 ] and human studies in pd patients with the ala53thr snca mutation supported the contribution of ad pathology to the disease progression of pd . the rapid progression of cognitive deficits in pd patients could not be exclusively determined by pathological substrates of ad ; however , these previous studies in vitro , in animal models and in clinical cohorts suggested the increasingly important role of ad pathology in the development of pdd . for the prediction of cognitive progression in pd , genetic factors associated with neuropathological substrates of ad and/or pd may be an important contributor to cognitive deficits , along with -syn pathology . in a large - scale study with autopsied subjects , however , the apoe 4 allele frequency was significantly higher not only in the ad group but also in the dementia with lewy bodies and pdd groups compared to the control group . based on the motor symptoms of pd patients , formulas were developed to define the motor phenotypes using movement disorder society unified parkinson s disease rating scale ( mds - updrs ) , a revision of the original updrs . using this system , however , despite evidence that the early development of cognitive deficits is significantly associated with pigd or non - td motor phenotypes , the motor phenotype of pd , particularly at a very early stage , might not yet be fully differentiated into two defined phenotypes ( i.e. therefore , whether the motor phenotype is useful in predicting the development of dementia in pd patients with very early stage disease and/or with pd medication should be further evaluated . several studies suggested that a specific domain of memory and cognitive function or the malfunctioning of a specific region were associated with a rapid progression of cognitive deficit , although it remains to be elucidated whether a specific profile reflects the heterogeneous progression of pd dementia . although it is not sufficient , the measurement of csf alpha - synuclein ( -syn ) , dj-1 , tau , phosphorylated tau at thr181 ( p - tau ) or fms - related tyrosine kinase 3 ligand can differentiate pd patients from healthy controls ( hc ) . interestingly , the level of tau proteins [ total tau ( t - tau ) and p - tau ] in the csf of pd patients was also lower than controls , although not all studies replicated this finding . provided evidence that the alzheimer s signature in csf ( lower level of csf a1 - 42 ) was significantly associated with more rapid cognitive decline in pd , and this finding was replicated by other studies [ 36 - 38 ] . the association of the ad - like csf signature with the rapid progression of cognitive dysfunction supported the hypothetical model that cross - seeding -syn and a or tau accelerate neurodegenerative pathology and disease progression . in support of this assertion , transgenic mice overexpressing human -syn ( wild type or ala53thr mutant ) with mutant forms of tau or amyloid precursor protein show greater neurodegeneration and functional deficits than mice overexpressing ad - associated proteins alone . furthermore , in vitro observation of enhanced fibril formation via cross - seed [ 41 - 43 ] and human studies in pd patients with the ala53thr snca mutation supported the contribution of ad pathology to the disease progression of pd . the rapid progression of cognitive deficits in pd patients could not be exclusively determined by pathological substrates of ad ; however , these previous studies in vitro , in animal models and in clinical cohorts suggested the increasingly important role of ad pathology in the development of pdd . for the prediction of cognitive progression in pd , genetic factors associated with neuropathological substrates of ad and/or pd may be an important contributor to cognitive deficits , along with -syn pathology . in a large - scale study with autopsied subjects , however , the apoe 4 allele frequency was significantly higher not only in the ad group but also in the dementia with lewy bodies and pdd groups compared to the control group . there are several important prerequisites for the development of valid biochemical progression biomarkers : the establishment of standardized protocols for the acquisition , transfer and analysis of biospecimens ; the optimization and verification of bioassays ; a sufficient longitudinal follow - up period to track heterogeneous progression ; and the recruitment of drug - nave patients at baseline . ppmi is a five - year observational , international , longitudinal study that aimed to identify biomarkers of pd progression that involve the collaborative effort of pd researchers with expertise in biomarker development , the clinical study of pd , bioinformatics , statistics and data management . approximately 400 drug - nave pd patients at the early stage , and 200 age - matched hc were planned to be enrolled from 24 clinical sites in the united states , europe and australia ( figure 2 ) . all pd patients were at the early stage ( diagnosis within 2 years and h&y stage < 2 ) and untreated with pd medication , as described in detail elsewhere and on the ppmi website ( http://www.ppmi-info.org/study-design/ ) . this review discusses the baseline csf biomarkers ( a1 - 42 , t - tau , p - tau , and -syn ) data of the ppmi cohort . the initial data showed several interesting findings ; the lower levels of csf a1 - 42 and p - tau were significantly associated with the pigd phenotype in multiple logistic regression analysis with adjustment for confounders ; the level of -syn was significantly correlated with the level of t - tau and p - tau , and the levels of -syn and t - tau were associated with motor severity . for example , the level of csf a1 - 42 or p - tau was not associated with motor phenotype , but the csf -syn level in pd patients with the non - td phenotype was significantly lower than pd patients with the td phenotype . in addition , there were no csf biomarkers that were significantly associated with motor severity when multivariate regression analysis with the adjustment of confounding factors was applied , although a low level of p - tau was marginally associated with disease severity . consistent with the pilot study , the levels of csf -syn , t - tau , and p - tau , but not a1 - 42 , were significantly lower in pd compared to hc , while the diagnostic utility of each biomarker was limited due to a large overlap . the lower level of csf -syn in pd relative to hc implicates the accumulation of -syn in the brain of pd patients , analogous to the finding of lower levels of csf a1 - 42 in ad patients compared to hc . the mechanism of a reduction in tau proteins in pd compared with hc is unclear ; however , a possible interpretation is that the interaction between tau proteins and -syn may limit the release of tau proteins into csf . in connection with this , previous studies using in vitro , animal models [ 49 - 51 ] or postmortem brains of pd reported that the -syn pathology in the brain is accompanied by increased levels of hyperphosphorylated tau proteins and tau - positive tangles , and -syn positive lewy bodies may co - localize in the same neuron . therefore , the extent of the direct or indirect interaction between tau phosphorylation and -syn accumulation or the pattern of topological distribution of these pathogenic proteins may contribute to the heterogeneous progression of pd . although future long - term longitudinal observations in the ppmi cohort will be required to test the predictive performance of the csf biomarkers , the baseline data in this large cohort suggest that csf biomarkers in early pd patients already reflect disease heterogeneity and may have predictive value for disease progression . our findings of the association of csf biomarkers with cognitive function in the ppmi cohort was not consistent with other studies [ 26,35,37,56 - 60 ] . in contrast , the lower csf -syn level was associated with more severe neuropsychological function , including semantic fluency , visuospatial cognition and executive functioning in the ppmi cohort , which indicated that -syn pathology contributes to early cognitive impairment in pd . in addition , multivariate regression analysis of the ppmi baseline data did not fully reproduce the previous findings that the lower level of csf a1 - 42 was associated with cognitive impairment in pd . the lower level of a1 - 42 was significantly associated with processing speed / attention assessed by the symbol digit modality test ( sdmt ) but not with other cognitive functions , including the global cognitive function test and montreal cognitive assessment , in the ppmi cohort . instead , when the clinical variables of the group with the highest quintile levels of csf biomarkers were compared with those of the group with the lowest quintile levels , the csf a1 - 42 level showed significant associations with semantic fluency and sdmt score , and the t - tau / a1 - 42 ratio showed significant associations with memory ( total recall and delayed recall measured by hopkins verbal learning test - revised score ) , semantic fluency , sdmt and wechsler memory scaleiii letter - number sequencing score . it should be noted that the ppmi cohort included patients with very early stage and drug - nave disease at baseline ; therefore , whether csf biomarkers in early pd are associated with the risk of future cognitive decline and pdd should be determined in longitudinal analyses . a recent study that observed a group of ppmi pd patients ( n = 341 ) for 2 years found a significant association of lower baseline csf a1 - 42 level with higher odds of cognitive impairment , even though the baseline csf biomarker data showed a slight association of csf a1 - 42 with cognitive dysfunction in multivariate analysis . the discrepancy among studies in the association of csf biomarkers with clinical variables may be due to several demographic , biological and analytical factors , including but not limited to the different ages among cohorts , the contamination of blood in csf , the mixed pathology or disease stage of studied patients , and different immunoassay platforms . there are several important prerequisites for the development of valid biochemical progression biomarkers : the establishment of standardized protocols for the acquisition , transfer and analysis of biospecimens ; the optimization and verification of bioassays ; a sufficient longitudinal follow - up period to track heterogeneous progression ; and the recruitment of drug - nave patients at baseline . ppmi is a five - year observational , international , longitudinal study that aimed to identify biomarkers of pd progression that involve the collaborative effort of pd researchers with expertise in biomarker development , the clinical study of pd , bioinformatics , statistics and data management . approximately 400 drug - nave pd patients at the early stage , and 200 age - matched hc were planned to be enrolled from 24 clinical sites in the united states , europe and australia ( figure 2 ) . all pd patients were at the early stage ( diagnosis within 2 years and h&y stage < 2 ) and untreated with pd medication , as described in detail elsewhere and on the ppmi website ( http://www.ppmi-info.org/study-design/ ) . the initial data showed several interesting findings ; the lower levels of csf a1 - 42 and p - tau were significantly associated with the pigd phenotype in multiple logistic regression analysis with adjustment for confounders ; the level of -syn was significantly correlated with the level of t - tau and p - tau , and the levels of -syn and t - tau were associated with motor severity . for example , the level of csf a1 - 42 or p - tau was not associated with motor phenotype , but the csf -syn level in pd patients with the non - td phenotype was significantly lower than pd patients with the td phenotype . in addition , there were no csf biomarkers that were significantly associated with motor severity when multivariate regression analysis with the adjustment of confounding factors was applied , although a low level of p - tau was marginally associated with disease severity . consistent with the pilot study , the levels of csf -syn , t - tau , and p - tau , but not a1 - 42 , were significantly lower in pd compared to hc , while the diagnostic utility of each biomarker was limited due to a large overlap . the lower level of csf -syn in pd relative to hc implicates the accumulation of -syn in the brain of pd patients , analogous to the finding of lower levels of csf a1 - 42 in ad patients compared to hc . the mechanism of a reduction in tau proteins in pd compared with hc is unclear ; however , a possible interpretation is that the interaction between tau proteins and -syn may limit the release of tau proteins into csf . in connection with this , previous studies using in vitro , animal models [ 49 - 51 ] or postmortem brains of pd reported that the -syn pathology in the brain is accompanied by increased levels of hyperphosphorylated tau proteins and tau - positive tangles , and -syn positive lewy bodies may co - localize in the same neuron . therefore , the extent of the direct or indirect interaction between tau phosphorylation and -syn accumulation or the pattern of topological distribution of these pathogenic proteins may contribute to the heterogeneous progression of pd . although future long - term longitudinal observations in the ppmi cohort will be required to test the predictive performance of the csf biomarkers , the baseline data in this large cohort suggest that csf biomarkers in early pd patients already reflect disease heterogeneity and may have predictive value for disease progression . our findings of the association of csf biomarkers with cognitive function in the ppmi cohort was not consistent with other studies [ 26,35,37,56 - 60 ] . in contrast , the lower csf -syn level was associated with more severe neuropsychological function , including semantic fluency , visuospatial cognition and executive functioning in the ppmi cohort , which indicated that -syn pathology contributes to early cognitive impairment in pd . the lower level of a1 - 42 was significantly associated with processing speed / attention assessed by the symbol digit modality test ( sdmt ) but not with other cognitive functions , including the global cognitive function test and montreal cognitive assessment , in the ppmi cohort . instead , when the clinical variables of the group with the highest quintile levels of csf biomarkers were compared with those of the group with the lowest quintile levels , the csf a1 - 42 level showed significant associations with semantic fluency and sdmt score , and the t - tau / a1 - 42 ratio showed significant associations with memory ( total recall and delayed recall measured by hopkins verbal learning test - revised score ) , semantic fluency , sdmt and wechsler memory scaleiii letter - number sequencing score . it should be noted that the ppmi cohort included patients with very early stage and drug - nave disease at baseline ; therefore , whether csf biomarkers in early pd are associated with the risk of future cognitive decline and pdd should be determined in longitudinal analyses . a recent study that observed a group of ppmi pd patients ( n = 341 ) for 2 years found a significant association of lower baseline csf a1 - 42 level with higher odds of cognitive impairment , even though the baseline csf biomarker data showed a slight association of csf a1 - 42 with cognitive dysfunction in multivariate analysis . the discrepancy among studies in the association of csf biomarkers with clinical variables may be due to several demographic , biological and analytical factors , including but not limited to the different ages among cohorts , the contamination of blood in csf , the mixed pathology or disease stage of studied patients , and different immunoassay platforms . the evidence of molecular interactions between -syn and a1 - 42 and/or tau in the development of pdd in post - mortem samples implicate the necessity of the longitudinal observation of csf and imaging biomarkers in pd .

determinants intrinsic to the structural precursor polyprotein ( gag ) that is encoded by the human immunodeficiency virus - type 1 ( hiv-1 ) and other retroviruses direct targeting of gag to the plasma membrane , membrane and genome rna binding , gag multimerization , and budding of the assemblage into the extracellular space as virus particles ( reviewed in [ 14 ] ) . through a proteomic search aimed at identification of cellular factors that might participate with gag and escrt , we identified the inositol 1,4,5-triphosphate receptor ( ip3r ) as a protein enriched in an endosome- and plasma - membrane - enriched fraction only when gag was expressed ( unpublished observation ) . ip3r protein forms a transmembrane calcium ion ( ca ) channel that is mostly found on the membrane of the endoplasmic reticulum ( er ) , the major intracellular ca store in the cell . ip3r has also been detected on the plasma membrane , late endosome / multivesicular bodies ( le / mvbs ) , and the nucleus ( reviewed in [ 68 ] ) . efficient hiv-1 gag trafficking and viral particle release were shown to require activation of ip3r . ip3r activation requires phospholipase - c- ( plc- ) catalyzed hydrolysis of pi(4,5)p2 to generate inositol 1,4,5-triphosphate ( ip3 ) , the activating ligand for the receptor ( reviewed in [ 68 ] ) . binding of ip3 initiates conformational changes leading to channel opening and release of ca into the cytosol . earlier studies on hiv particle production had demonstrated that induction of a transient rise in the cytosolic ca concentration resulted in a dramatic rise in viral particle release , suggesting that ca is a limiting factor in late - stage replication [ 11 , 12 ] . taken together , these observations collectively suggested that ip3r is the physiological provider of the required ca . the proteomic search also identified several additional proteins that function in regulation of ca signaling , including sprouty2 ( spry2 ) , a modulator of ca signaling and other modes of signaling [ 14 , 15 ] . we demonstrated that spry2 is also required for productive hiv egress [ 16 , 17 ] . proteins such as ip3r and spry2 have been shown to function with the same elements of cytoskeletal and vesicular transport that are integral to escrt machinery [ 1820 ] . over the past few years , a number of other non - escrt host proteins have been shown to be required for gag assembly . some of these have been discussed in recent reviews [ 2 , 21 , 22 ] . we will discuss how these host proteins set the stage for escrt recruitment and escrt - mediated abscission events . we apologize to those investigators whose studies may be pertinent but were not explicitly cited . the segment of the plasma membrane that serves as assembly platform evaginates to form the budded particle and becomes the viral envelope . since the gag precursor is the viral gene product that plays the key role in recruiting other viral components to the assembly site [ 23 , 24 ] , the assembly process must necessarily include a mechanism for stable localization of gag at the plasma membrane ( pm ) . once on the pm , gag has intrinsic assembly capability that is attributed to functions of its four domains ( matrix - capsid - nucleocapsid - p6 ) . the n - terminal matrix ( ma ) domain mediates membrane binding ( [ 2529 ] and references in ) . the capsid ( ca ) domain provides gag with capability for self - assembly into higher - order multimers ( [ 3035 ] and references in ) . the nucleocapsid domain ( nc ) mediates binding to viral rna and nonspecific rnas as well as promoting gag association [ 3739 ] and references in . the c - terminally located p6 region mediates the untethering of the assembled gag particle from the host [ 41 , 42 ] . orderly cleavage of gag at interdomain junctions within the structural precursor polyprotein by a virus - encoded proteinase [ 4347 ] occurring concurrently with budding results in mature proteins whose rearrangement transforms the bud to a mature , infectious particle [ 48 , 49 ] . the final step of the virus assembly process , which results in the pinching off of the particle from the host cell , is mediated by escrt proteins that have been recruited to the bud neck by motifs in p6 that are designated as late or l domains ( reviewed in and references in [ 50 , 51 ] ) . as a cytosolic protein , the synthesis of gag takes place on soluble polysomes in the cell interior . a myristoylation reaction occurs cotranslationally during which gag acquires a myristoyl moiety on the n - terminal glycine which plays a role in assembly [ 28 , 54 , 55 ] . at the earliest experimentally feasible time points , gag has been demonstrated to have a cytosolic distribution when examined by confocal microscopy , biochemical fractionation , and immunogold electron microscopy . eventually , the entire gag population becomes membrane associated with the pm as the preferred site at steady state ( references in ) . this is consistent with the results of in vitro binding studies wherein ma , which is highly basic ( [ 2529 ] and references in ) , mediates binding to membranes reconstituted with acidic phospholipids ( [ 26 , 27 ] and references in ) . it is also consistent with observations that the cytoplasmic leaflet of the pm is unique among cell membranes in having a net negative charge due to high levels of acidic phospholipids . the targeting phospholipid was identified as the complex acidic phospholipid , phosphatidylinositol 4,5 bis - phosphate ( pi(4,5)p2 ) . depletion of pi(4,5)p2 , using plasmamembrane - targeted lipid phosphatases , caused gag to be localized to le / mvbs and prevented gag localization to the pm . pi(4,5)p2 is mostly found on the pm where it represents a minor plasma membrane lipid component . structural analysis of pi(4,5)p2 binding to hiv-1 ma shows contacts made by the head group ( i.e. , phosphates and inositol ring ) with basic residues and the nestling of adjacent acyl groups into a hydrophobic cleft while studies with full - length gag underscored the importance of the phosphoinositide acyl chain . studies with the matrix protein show that pi(4,5)p2 binding results in exposure of the n - terminal myristate . studies with gag in the presence of nucleic acid reveal an interplay between binding to pi(4,5)p2 , binding to nucleic acid , and capsid ( ca ) domain - mediated self - association . the model of gag membrane association founded on gag interaction with pi(4,5)p2 is supported by the inhibitory effect on gag particle release of depletion of plasma membrane pi(4,5)p2 [ 59 , 64 , 65 ] . it should be noted that as important as pi(4,5)p2 is to hiv-1 gag membrane targeting , the importance of pi(4,5)p2 to targeting and release of other retroviral gags varies . equine infectious anemia virus ( eiav ) budding is less impacted by depletion of pi(4,5)p2 due to preferential binding to pi(3,5)p2 . pi(3,5)p2 is a phospholipid that is predominantly associated with endosomal compartments at steady state implying endosomal targeting of eaiv gag in the cell . eiav gag trafficking requires such targeting as inactivation of the pi(3)p2 5-kinase , which is responsible for the endosomal placement of pi(3,5)p2 , inhibits eiav gag vlp production . asv budding appears to rely on electrostatic interaction with acidic phospholipids and exhibits no specific reliance on phosphoinositide components of the pm . thus , hiv-1 gag membrane association is mediated by a specific bipartite determinant in the ma domain comprised of myristate and basic amino acid clusters with gag - pi(4,5)p2 binding serving as the basis for targeted membrane association . gag 's preferential association with the plasma membrane is due to two inherent features of pi(4,5)p2 : ( i ) the pm is where most of cellular pi(4,5)p2 is located and ( ii ) pi(4,5)p2 molecules are products of in situ synthesis ( i.e. , pm - localized molecules are produced at the pm ; ) . thus , pi(4,5)p2 targeting provides a mechanism to direct gag from its site of synthesis in the cell interior to the plasma membrane . detection of assembled hiv-1 gag inside membrane compartments with the characteristics of le / mvbs has been documented [ 70 , 71 ] , and altered gag residency in le / mvbs following stimulatory or inhibitory effects on virus production has been demonstrated [ 11 , 72 , 73 ] . additionally , the virus particle has components that are typical exosome markers . however , for macrophages , at least , those apparently intracellular membrane compartments with le / mvb features were demonstrated to be actually extracellular space delineated by intracytoplasmic plasma membrane [ 75 , 76 ] . moreover , gag particle production has been shown to be insensitive to interference with le / mvb function . the role of the le / mvb in gag assembly and release thus remains controversial . we suspect that at the root of this controversy is the complex nature of the le / mvb itself . it can not be precluded that the endosomal machinery can interact with gag in the traditional manner , wherein escrt machinery facilitates sorting of cargo proteins into mvbs for ultimate delivery to degradative compartments . however , the handling of sorted proteins by the mvb is not always unidirectional . though targeted to the le / mvb in both hela and jurkat cells , the 29ke/31ke gag mutant is released at near wild - type levels from jurkat cells but is trapped inside hela cells which shows that trafficking within the mvb can be influenced by its environment ( i.e. , cell dependence ) . eiav gag is another interesting case since , despite its endosomal targeting , eiav gag vlps are released from cells such as cos-1 and hela . it would be interesting to know if eiav gag induces any alteration in the mvb and , if so , whether this facilitates productive infection . direct delivery of gag to the site of release on the plasma membrane circumvents the potentially nonproductive outcome of gag association with endosomal machinery . a gag assembly model that incorporates gag - pi(4,5)p2-based targeting of gag to assembly sites on the pm permits a more productive path from gag synthesis to release of an assembled gag particle . budding structures accumulate on the plasma membrane if the c - terminal p6 region is missing from gag [ 41 , 42 ] . the p6 region bearing the l domain has counterparts in other retroviruses and is functionally exchangeable with these within and outside the genera ; for example , the ptap motif from the p6 region of hiv-1 gag was shown to substitute for the py motif in the l domain - bearing region ( p2b ) of the avian sarcoma virus ( asv ) and vice versa [ 7983 ] and references in [ 50 , 51 , 84 , 85 ] . functional exchangeability demonstrates that there are multiple , though not necessarily equally effective , ways for gag to access the escrt machinery . tsg101 as binding partner of the hiv ptap motif and nedd4 family members as binding partner of the asv py motif facilitate release of hiv-1 and asv , respectively , through functionally exchangeable but independent routes ( i.e. , tsg101 can replace nedd4 function in facilitating asv budding [ 86 , 87 ] ) . members of the nedd4 family of ubiquitin ligases can also replace tsg101 in facilitating hiv-1 release under certain circumstances [ 8891 ] . the binding of the escrt adaptor , alix , to the secondary l domain in gag serves this purpose as well ( reviewed in ) . the escrt machinery is now known to comprise > 25 proteins , organized into four complexes ( escrt-0 , -i , -ii , and -iii ) that function sequentially along with several additional associated factors ( reviewed in [ 9395 ] ) . irrespective of how gag is linked to the escrt machinery , in all cases escrt - iii and vps4 must be recruited to the bud neck at the membrane site to execute the final bud scission event and to release the escrt factors from the assemblage for recycling back to the cytosolic pool for participation in future events [ 96 , 97 ] . a feature of retroviral utilization of the escrt machinery is the selective use of the escrt complexes . viral particle production requires escrt - i and escrt - iii but not escrt - ii while asv requires escrt - ii but not escrt-1 . these observations , along with recognition that escrts , which normally function in transport of some cellular proteins to degradative cellular compartments , are required for exit of assembled gag from the cell , suggests that non - escrt host proteins may play a key role in allowing the escrt machinery to be utilized differentially by the virus compared to the host . thus , non - escrt proteins may permit hiv to exploit escrt machinery by preventing the gag - escrt complex from participating in interactions with escrt partners that are nonproductive for the virus . all organisms do things the same way except that it is completely different in every detail j. haber the abscission event in virus budding results in separation of the enveloped virus from the host cell . another process where the abscission event results in separation of two membrane - enclosed cellular entities is cytokinesis . abscission of the intercellular bridge / midbody results in separation of the mitotic daughter cells . recruitment of escrt and mediation of the abscission event by escrt is the basis for the parallel between hiv-1 budding and cytokinesis [ 101 , 102 ] . the parallel may extend to events occurring before escrt recruitment and participation , ( i.e. , in a pre - escrt stage ) . paradigms that govern the pre - escrt stage of cytokinesis , which has been an active area of research long before discovery of hiv , may likewise apply to the pre - escrt stage of viral budding . a theme that is emerging as a cell prepares for cytokinesis is the reshaping of calcium signaling . local and global elevations in cytosolic ca level are achieved by ion release from the er ( the cell 's major intracellular ca store ) and by influx from the extracellular environment . decrease in ca content of the er triggers activation of ca influx channels on the plasma membrane and refilling of the er store in a process called store - operated - calcium - entry ( soce ) [ 105 , 106 ] . a major cellular change that occurs during cell division prior to cytokinesis is the uncoupling of ca store depletion and soce [ 107 , 108 ] . why this is necessary is presently not known but the effect is to render the pre - escrt events in cytokinesis independent of soce and reliant on the internal stores as the ca source . independence from soce and reshaping of calcium signaling as a pre - escrt stage paradigm also appear to be the case for hiv-1 budding . blockade of soce with 2-aminoethoxydiphenylborate ( 2-apb ) , a small molecule inhibitor of store refilling through soce , had no effect on release of the hiv-1 gag particle . blockade of a g protein - coupled receptor cascade triggered by ca entry through receptor - operated calcium entry ( roce ; ) also had no effect on gag particle release . additionally , cells where productive gag budding is occurring ( i.e. , expression of wild - type gag ) exhibit higher cytosolic ca compared to mock - transfected cells or cells expressing a budding - impaired ptap gag mutant . possibly , insulating the calcium machinery from external ca sources allows both virus budding and cytokinesis to proceed more efficiently . the first step in both processes is the targeting of the requisite components to the eventual scission site , that is , the plasma membrane . formation of the cleavage furrow is a visual marker of initiation of cytokinesis and aspects of this event that appear similar to the budding process are furrow ingression , that is , a progressive narrowing of the eventual scission region to form a bud neck . in cytokinesis , the separating bodies are of comparable volumes ; in viral budding , they are of unequal volumes . ip3r , intact pi(4,5)p2 , pi(4,5)p2 hydrolysis , and ca are all required for the normal progression of cytokinesis in cellular systems where cell division has been well studied , for example , spermatocyte and oocytes [ 113116 ] . there is a requirement for ca to maintain furrow or neck stability , necessitating constant plc - mediated hydrolysis of pi(4,5)p2 [ 117 , 118 ] . components involved in ca mobilization and cytoskeleton remodeling are recruited to the furrow [ 117119 ] . similarly , in addition to intact pi(4,5)p2 , hiv budding requires ip3r and plc activity [ 9 , 110 ] . analogous to ip3r recruitment to the furrow in cytokinesis , there is also recruitment of ip3r to gag budding sites on the plasma membrane . in cytokinesis , cep55 recruits tsg101 , a component of escrt - i , and alix , an escrt adaptor protein that binds both escrt-1 and escrt - iii , to the eventual scission site once furrow ingression is completed [ 101 , 102 , 122124 ] . these escrt factors , in turn , recruit the escrt - iii complex required to carry out the terminal step in cytokinesis , abscission , that is , the severing of the thin intercellular bridge that connects the two daughter cells [ 125127 ] . the counterpart of the cep55-escrt link in viral budding is the targeting of gag to the eventual scission site on the plasma membrane and recruitment of tsg101 and/or alix through the l domains and eventually escrt - iii . autophagy , the process involved in the breakdown of intracellular proteins and organelles , is now appreciated as a mechanism of great importance in both cell survival and cell death . it is the latest cellular process linked to escrt function . indeed , autophagy is a necessary postabscission step in cytokinesis . following cytokinesis , the dividing cells are connected by an intracellular bridge that contains the midbody . this structure persists long after division as a midbody derivative that is inherited asymmetrically by the daughter cell with the older centrosome . recent findings in mammalian cells and in drosophila melanogaster indicate that escrts are required for efficient trafficking through the endolysosomal system where the autophagic cargo is degraded [ 130132 ] . as with cytokinesis and viral budding , ip3r - mediated ca signaling is emerging as critical for the pre - escrt stage in autophagy . pairing phosphoinositides with ca ions in endolysosomes has been suggested to control the direction and specificity of membrane trafficking . all three processes , cytokinesis , viral budding , and autophagy [ 138 , 139 ] , require or involve snares to conduct some of the critical events . the participation of calcium machinery components in all three processes suggests that the requirement for and reshaping of calcium signaling is a common feature governing their pre - escrt stages . for a number of non - escrt host proteins shown to be important for release of the gag particle [ 2 , 4 , 22 , 140 ] , disruption of the protein function does not result in the canonical l domain phenotype ( i.e. , arrested budding structures at the periphery of cells examined by em ) . rather , gag is found in the cell interior . we and others interpret this to indicate participation of these proteins in assembly step(s ) preceding escrt - mediated budding . some of these proteins have regulatory links to each other . among these are the human vacuolar protein sorting ( hvps ) protein 18 ( vps18 ) , a class c vps complex component , and mon2 . both have been shown to be required for gag pm localization and virus production . in yeast , class c vps proteins have been shown to regulate pm localization of at least one protein and to assume roles antagonistic to escrt in the recycling of membrane proteins . the human orthologue of mon2 ( hmon2 ) can bind and regulate the subcellular localization of adaptor proteins such as ap-1 , ap-3 , and arf1 which have previously been shown to be required for gag pm localization and gag particle production [ 72 , 144 , 145 ] . the notion of non - escrt proteins regulating the activity of other non - escrt proteins in the pre - escrt stage has a parallel in cytokinesis as illustrated by the host protein , tex14 . this non - escrt protein binds cep55 at the same motif used to recruit tsg101 or alix and negatively regulates escrt recruitment . through protein - protein interactions , non - escrt proteins could thus impose temporal and spatial control of the recruitment of participating proteins , including gag itself , to assembly sites on the pm during the pre - escrt stage . another pre - escrt event is alteration of the lipid composition of the assembly site . quantitative analyses indicate that the viral envelope differs from the pm of its host cell in having higher levels of cholesterol and pi(4,5)p2 [ 58 , 147 ] . since the viral envelope is derived from the pm microdomain serving as the gag assembly site , reorganization of the lipid bilayer in this location may occur as part of the assembly process . a feature of pm pi(4,5)p2 is that the greater majority is sequestered by electrostatic interaction with basic proteins that are resident at the pm ( e.g. , myristylated alanine - rich c kinase substrate ( marcks ; growth - associated protein ( gap)43 ; n - methyl - d - aspartate ( nmda ) receptor , and the epidermal growth factor receptor ( egfr ) ) and is only released by a local rise in ca . another property of pi(4,5)p2 is that it does not have a natural inclination for clustering due to the energy barrier posed by repulsion of the large polar head groups when they are in proximity . it has been shown that ca can reduce this barrier and induce pi(4,5)p2 clustering in lipid monolayers . recruitment of ip3r machinery to the cell periphery and release of ca may function to increase the portion of pm pi(4,5)p2 available for interaction with gag and to permit the clustering of pi(4,5)p2 molecules upon gag multimerization . this model is summarized in figure 3 and may explain how the budding requirement for both intact and hydrolyzed pi(4,5)p2 could be simultaneously resolved . that budding structures are still formed by gag mutants with disrupted ptap motifs despite their impairment in recruitment of tsg101 or in cells where tsg101 has been depleted [ 50 , 51 ] indicates that assembly site membrane deformation is a pre - escrt stage event . although not required for initiation , ca is required for furrow ingression and for stability of the intercellular bridge in cytokinesis [ 113116 ] . furrow ingression in the presence of ca leads to a productive escrt recruitment stage as indicated by completion of cytokinesis . analogous to furrow ingression is the formation of the virus bud neck where the escrt scission complex is recruited . the fact that the budding structures of gag mutants with disrupted ptap motifs accumulate on the plasma membrane indicates a failure in escrt recruitment even though the mutant has been demonstrated to be capable of employing alternative modes of linking to escrt ( i.e. , via nedd4 or alix ) . our study shows that , in cells expressing hiv-1 gag , ip3r was translocated from the cell interior to the periphery and colocalized with gag on the plasma membrane . interestingly , ip3r redistribution is not induced in cells expressing the ptap gag mutant even though release of the mutant , albeit inefficient , also requires ip3r - regulated machinery . the lack of ca store recruitment , which ip3r recruitment signifies , to the cell periphery of cells expressing such mutants indicates that , as is the case for furrow ingression , competency in linking to escrt is a property of bud necks formed in the presence of ca . the ability of the endoplasmic reticulum to form tubules and small vesicles is what permits the stores to be recruited . movement of ip3r - contaning er vesicles along microtubules has been shown to be facilitated by a kinesin . kinesins are a large family of cellular protein motors that use the energy of atp hydrolysis to induce movement along the microtubule . kinesins have been identified as being involved in an intracellular process required for gag release : ( i ) kinesin kif4 was reported to bind gag directly through the ma domain and was later found to regulate intracellular trafficking and stability of gag ; ( ii ) kinesin kif3 , a binding partner of ap-3 shown to be required for release of the viral particles assembled by gag , has also been reported to be involved in gag release . which particular kinesin is involved in ip3r transport kinesin - mediated translocation of ip3r along microtubules would allow for directed delivery of ca stores to the budding site and , thereby , establish a localized region where ca would be elevated . thus , for ca provision , utilization of the internal ca stores may provide a major advantage over ca influx which is mediated by channels that are homogenously distributed on the plasma membrane . the notion that intact pi(4,5)p2 is required for targeting gag to the plasma membrane and that plc - hydrolyzed pi(4,5)p2 is required for escrt - recruitment - competent bud neck ingression suggests the need for regulatory mechanisms that would ensure availability of the right form of the phospholipid for the right event in the pre - escrt stage . hydrolysis stimulates synthesis model proposes that hydrolysis and synthesis of pi(4,5)p2 are tightly coupled events such that synthesis stimulates hydrolysis while pi(4,5)p2 hydrolysis signals its production . ca might be a key regulator : ca is an activator of the lipid kinase that is critical for pi(4,5)p2 synthesis and of the plc that catalyzes pi(4,5)p2 hydrolysis . although it has been clearly demonstrated that depletion of pi(4,5)p2 with plasmamembrane - targeted lipid phosphatases prevents gag localization to the pm , other experimental approaches give different results . for example , increased gag pm targeting and vlp release were not observed following a clear increase in pm pi(4,5)p2 in cells treated with a plc inhibitor . also , a loss of gag pm targeting was reported in cells that did not exhibit a detectable change in pi(4,5)p2 level or subcellular distribution . there is growing recognition that pm pi(4,5)p2 exists in multiple pools and that the dynamic nature of these pools is important for cellular processes mediated by pi(4,5)p2 [ 148 , 156 ] . perhaps this conundrum , that is , the lack of a clear correlation between gag pm targeting and the pi(4,5)p2 level , reflects a requirement for a pi(4,5)p2 pool that is specifically made available for gag . the non - escrt proteins , spry2 and adp - ribosylation factor-1 ( arf1 ) , have activities that make them potential participants in such regulatory mechanisms . spry2 is required for gag particle budding [ 16 , 17 ] and for production of infectious virus ( ehrlich , khan , powell and carter , unpublished observations ) . it has several activities that can affect pi(4,5)p2 metabolism ; namely , binding of phospholipase c and of pi(4,5)p2 [ 13 , 17 ] and it can inhibit receptor - mediated activation of plc . although this protein is best known for its role in post - golgi trafficking , arf1 is also a stimulator of pi(4,5)p2 synthesis by directly activating pi(4)p 5-kinase and by inducing formation of an enhancer of the kinase . thus , together with local ca , sprouty and arf1 proteins have the potential to ensure the dynamic existence of pi(4,5)p2 pools specifically made available for interaction with gag . several other non - escrt proteins whose dysfunction inhibited transport of gag from the cell interior to the plasma membrane may also be involved in gag assembly as pre - escrt stage participants . admittedly , further studies will be needed to elucidate their exact contribution ; however , interestingly , these proteins also have links to cytokinesis and autophagy . in addition to the aforementioned snares , these include citron kinase , a rho effector ; rab9 and other gtpases ; posh ; ap-1 ; npc-1 ; and filamin a . direct participation in cytokinesis is documented for citron kinase , ap-1 , and filamin a [ 164166 ] . npc-1 and posh both affect the metabolism of two important factors in cytokinesis , cholesterol , and calcium , respectively . rab9 and other small gtpases have been implicated in cytokinesis and autophagy [ 118 , 169 ] . the formation of a gag - tsg101 complex occurs as part of the gag assembly process as long as l domain-1 is intact . although the precise stage at which tsg101 docks on the ptap motif is not known , association after stable bud neck formation might be more favorable as it precludes nonproductive interactions with escrt - ii that would signal internalization of the gag assemblage or premature escrt - iii scission . thus , spry2 facilitates release driven by both the primary and the secondary hiv-1 gag l domains , possibly due to its ability to compete with escrt - i factors for interaction with escrt - ii components . this notion is consistent with the fact that hiv-1 budding does not require escrt - ii [ 98 , 99 ] . not surprisingly since the interaction of escrt - i with escrt - ii leads to cargo internalization , it has been suggested that association with tsg101 increases susceptibility to internalization . delaying the recruitment of escrt machinery to the budding site may provide a means of maximizing viral budding efficiency . a parallel to this as a regulation possibility in cytokinesis may be the aforementioned function of tex14 , a protein believed to control premature progression to the abscission stage by competing with tsg101 and alix for binding to cep55 . in this paper , we have focused on proteins involved in steps in hiv-1 trafficking and budding that take place prior to gag recruitment of escrt machinery . as described here , proteins that function in pi(4,5)p2 binding , synthesis or hydrolysis , ca store recruitment , ip3r - mediated ca store release , and vesicular biogenesis or transport appear to comprise the major classes of participants in the pre - escrt stages . cellular activities in almost all cells are regulated by common signaling systems and ca is a ubiquitous intracellular messenger that is known to control a diverse range of processes . the discovery of ca signaling as a cofactor in hiv-1 protein trafficking and release , its potential link to exploitation of the escrt machinery by the virus for viral particle production , and the general similarity of this coupling to other cellular activities in which escrts participate , that is , cytokinesis and autophagy , may provide new therapeutic avenues for hiv treatment strategies .

more than a decade has elapsed since the link between the endosomal sorting complex required for transport ( escrt ) machinery and hiv-1 protein trafficking and budding was first identified . l domains in hiv-1 gag mediate recruitment of escrt which function in bud abscission releasing the viral particle from the host cell . beyond virus budding , the escrt machinery is also involved in the endocytic pathway , cytokinesis , and autophagy . in the past few years , the number of non - escrt host proteins shown to be required in the assembly process has also grown . in this paper , we highlight the role of recently identified cellular factors that link escrt machinery to calcium signaling machinery and we suggest that this liaison contributes to setting the stage for productive escrt recruitment and mediation of abscission . parallel paradigms for non - escrt roles in virus budding and cytokinesis will be discussed .

1. Focus of This Paper 2. Introduction 3. Plasma Membrane Targeting: Role of PI(4,5)P 4. Late Domains in Gag Recruit ESCRT Machinery 5. Parallels between HIV-1 Budding, Cytokinesis, and Autophagy 6. Non-ESCRT Proteins and Other Factors Engaged in the Pre-ESCRT Stages of HIV-1 Assembly 7. Non-ESCRT Proteins in the ESCRT Recruitment Stage 8. Concluding Remarks

determinants intrinsic to the structural precursor polyprotein ( gag ) that is encoded by the human immunodeficiency virus - type 1 ( hiv-1 ) and other retroviruses direct targeting of gag to the plasma membrane , membrane and genome rna binding , gag multimerization , and budding of the assemblage into the extracellular space as virus particles ( reviewed in [ 14 ] ) . through a proteomic search aimed at identification of cellular factors that might participate with gag and escrt , we identified the inositol 1,4,5-triphosphate receptor ( ip3r ) as a protein enriched in an endosome- and plasma - membrane - enriched fraction only when gag was expressed ( unpublished observation ) . ip3r protein forms a transmembrane calcium ion ( ca ) channel that is mostly found on the membrane of the endoplasmic reticulum ( er ) , the major intracellular ca store in the cell . ip3r has also been detected on the plasma membrane , late endosome / multivesicular bodies ( le / mvbs ) , and the nucleus ( reviewed in [ 68 ] ) . efficient hiv-1 gag trafficking and viral particle release were shown to require activation of ip3r . earlier studies on hiv particle production had demonstrated that induction of a transient rise in the cytosolic ca concentration resulted in a dramatic rise in viral particle release , suggesting that ca is a limiting factor in late - stage replication [ 11 , 12 ] . the proteomic search also identified several additional proteins that function in regulation of ca signaling , including sprouty2 ( spry2 ) , a modulator of ca signaling and other modes of signaling [ 14 , 15 ] . we demonstrated that spry2 is also required for productive hiv egress [ 16 , 17 ] . proteins such as ip3r and spry2 have been shown to function with the same elements of cytoskeletal and vesicular transport that are integral to escrt machinery [ 1820 ] . over the past few years , a number of other non - escrt host proteins have been shown to be required for gag assembly . we will discuss how these host proteins set the stage for escrt recruitment and escrt - mediated abscission events . since the gag precursor is the viral gene product that plays the key role in recruiting other viral components to the assembly site [ 23 , 24 ] , the assembly process must necessarily include a mechanism for stable localization of gag at the plasma membrane ( pm ) . the c - terminally located p6 region mediates the untethering of the assembled gag particle from the host [ 41 , 42 ] . the final step of the virus assembly process , which results in the pinching off of the particle from the host cell , is mediated by escrt proteins that have been recruited to the bud neck by motifs in p6 that are designated as late or l domains ( reviewed in and references in [ 50 , 51 ] ) . as a cytosolic protein , the synthesis of gag takes place on soluble polysomes in the cell interior . at the earliest experimentally feasible time points , gag has been demonstrated to have a cytosolic distribution when examined by confocal microscopy , biochemical fractionation , and immunogold electron microscopy . eventually , the entire gag population becomes membrane associated with the pm as the preferred site at steady state ( references in ) . it is also consistent with observations that the cytoplasmic leaflet of the pm is unique among cell membranes in having a net negative charge due to high levels of acidic phospholipids . depletion of pi(4,5)p2 , using plasmamembrane - targeted lipid phosphatases , caused gag to be localized to le / mvbs and prevented gag localization to the pm . studies with gag in the presence of nucleic acid reveal an interplay between binding to pi(4,5)p2 , binding to nucleic acid , and capsid ( ca ) domain - mediated self - association . it should be noted that as important as pi(4,5)p2 is to hiv-1 gag membrane targeting , the importance of pi(4,5)p2 to targeting and release of other retroviral gags varies . pi(3,5)p2 is a phospholipid that is predominantly associated with endosomal compartments at steady state implying endosomal targeting of eaiv gag in the cell . thus , hiv-1 gag membrane association is mediated by a specific bipartite determinant in the ma domain comprised of myristate and basic amino acid clusters with gag - pi(4,5)p2 binding serving as the basis for targeted membrane association . thus , pi(4,5)p2 targeting provides a mechanism to direct gag from its site of synthesis in the cell interior to the plasma membrane . detection of assembled hiv-1 gag inside membrane compartments with the characteristics of le / mvbs has been documented [ 70 , 71 ] , and altered gag residency in le / mvbs following stimulatory or inhibitory effects on virus production has been demonstrated [ 11 , 72 , 73 ] . however , for macrophages , at least , those apparently intracellular membrane compartments with le / mvb features were demonstrated to be actually extracellular space delineated by intracytoplasmic plasma membrane [ 75 , 76 ] . moreover , gag particle production has been shown to be insensitive to interference with le / mvb function . the role of the le / mvb in gag assembly and release thus remains controversial . it can not be precluded that the endosomal machinery can interact with gag in the traditional manner , wherein escrt machinery facilitates sorting of cargo proteins into mvbs for ultimate delivery to degradative compartments . however , the handling of sorted proteins by the mvb is not always unidirectional . though targeted to the le / mvb in both hela and jurkat cells , the 29ke/31ke gag mutant is released at near wild - type levels from jurkat cells but is trapped inside hela cells which shows that trafficking within the mvb can be influenced by its environment ( i.e. it would be interesting to know if eiav gag induces any alteration in the mvb and , if so , whether this facilitates productive infection . the p6 region bearing the l domain has counterparts in other retroviruses and is functionally exchangeable with these within and outside the genera ; for example , the ptap motif from the p6 region of hiv-1 gag was shown to substitute for the py motif in the l domain - bearing region ( p2b ) of the avian sarcoma virus ( asv ) and vice versa [ 7983 ] and references in [ 50 , 51 , 84 , 85 ] . functional exchangeability demonstrates that there are multiple , though not necessarily equally effective , ways for gag to access the escrt machinery . , tsg101 can replace nedd4 function in facilitating asv budding [ 86 , 87 ] ) . the escrt machinery is now known to comprise > 25 proteins , organized into four complexes ( escrt-0 , -i , -ii , and -iii ) that function sequentially along with several additional associated factors ( reviewed in [ 9395 ] ) . irrespective of how gag is linked to the escrt machinery , in all cases escrt - iii and vps4 must be recruited to the bud neck at the membrane site to execute the final bud scission event and to release the escrt factors from the assemblage for recycling back to the cytosolic pool for participation in future events [ 96 , 97 ] . a feature of retroviral utilization of the escrt machinery is the selective use of the escrt complexes . these observations , along with recognition that escrts , which normally function in transport of some cellular proteins to degradative cellular compartments , are required for exit of assembled gag from the cell , suggests that non - escrt host proteins may play a key role in allowing the escrt machinery to be utilized differentially by the virus compared to the host . thus , non - escrt proteins may permit hiv to exploit escrt machinery by preventing the gag - escrt complex from participating in interactions with escrt partners that are nonproductive for the virus . all organisms do things the same way except that it is completely different in every detail j. haber the abscission event in virus budding results in separation of the enveloped virus from the host cell . recruitment of escrt and mediation of the abscission event by escrt is the basis for the parallel between hiv-1 budding and cytokinesis [ 101 , 102 ] . the parallel may extend to events occurring before escrt recruitment and participation , ( i.e. , in a pre - escrt stage ) . paradigms that govern the pre - escrt stage of cytokinesis , which has been an active area of research long before discovery of hiv , may likewise apply to the pre - escrt stage of viral budding . a theme that is emerging as a cell prepares for cytokinesis is the reshaping of calcium signaling . independence from soce and reshaping of calcium signaling as a pre - escrt stage paradigm also appear to be the case for hiv-1 budding . blockade of soce with 2-aminoethoxydiphenylborate ( 2-apb ) , a small molecule inhibitor of store refilling through soce , had no effect on release of the hiv-1 gag particle . possibly , insulating the calcium machinery from external ca sources allows both virus budding and cytokinesis to proceed more efficiently . in cytokinesis , the separating bodies are of comparable volumes ; in viral budding , they are of unequal volumes . ip3r , intact pi(4,5)p2 , pi(4,5)p2 hydrolysis , and ca are all required for the normal progression of cytokinesis in cellular systems where cell division has been well studied , for example , spermatocyte and oocytes [ 113116 ] . components involved in ca mobilization and cytoskeleton remodeling are recruited to the furrow [ 117119 ] . analogous to ip3r recruitment to the furrow in cytokinesis , there is also recruitment of ip3r to gag budding sites on the plasma membrane . in cytokinesis , cep55 recruits tsg101 , a component of escrt - i , and alix , an escrt adaptor protein that binds both escrt-1 and escrt - iii , to the eventual scission site once furrow ingression is completed [ 101 , 102 , 122124 ] . these escrt factors , in turn , recruit the escrt - iii complex required to carry out the terminal step in cytokinesis , abscission , that is , the severing of the thin intercellular bridge that connects the two daughter cells [ 125127 ] . the counterpart of the cep55-escrt link in viral budding is the targeting of gag to the eventual scission site on the plasma membrane and recruitment of tsg101 and/or alix through the l domains and eventually escrt - iii . autophagy , the process involved in the breakdown of intracellular proteins and organelles , is now appreciated as a mechanism of great importance in both cell survival and cell death . following cytokinesis , the dividing cells are connected by an intracellular bridge that contains the midbody . as with cytokinesis and viral budding , ip3r - mediated ca signaling is emerging as critical for the pre - escrt stage in autophagy . all three processes , cytokinesis , viral budding , and autophagy [ 138 , 139 ] , require or involve snares to conduct some of the critical events . the participation of calcium machinery components in all three processes suggests that the requirement for and reshaping of calcium signaling is a common feature governing their pre - escrt stages . for a number of non - escrt host proteins shown to be important for release of the gag particle [ 2 , 4 , 22 , 140 ] , disruption of the protein function does not result in the canonical l domain phenotype ( i.e. rather , gag is found in the cell interior . both have been shown to be required for gag pm localization and virus production . in yeast , class c vps proteins have been shown to regulate pm localization of at least one protein and to assume roles antagonistic to escrt in the recycling of membrane proteins . the human orthologue of mon2 ( hmon2 ) can bind and regulate the subcellular localization of adaptor proteins such as ap-1 , ap-3 , and arf1 which have previously been shown to be required for gag pm localization and gag particle production [ 72 , 144 , 145 ] . the notion of non - escrt proteins regulating the activity of other non - escrt proteins in the pre - escrt stage has a parallel in cytokinesis as illustrated by the host protein , tex14 . this non - escrt protein binds cep55 at the same motif used to recruit tsg101 or alix and negatively regulates escrt recruitment . through protein - protein interactions , non - escrt proteins could thus impose temporal and spatial control of the recruitment of participating proteins , including gag itself , to assembly sites on the pm during the pre - escrt stage . another pre - escrt event is alteration of the lipid composition of the assembly site . quantitative analyses indicate that the viral envelope differs from the pm of its host cell in having higher levels of cholesterol and pi(4,5)p2 [ 58 , 147 ] . since the viral envelope is derived from the pm microdomain serving as the gag assembly site , reorganization of the lipid bilayer in this location may occur as part of the assembly process . , myristylated alanine - rich c kinase substrate ( marcks ; growth - associated protein ( gap)43 ; n - methyl - d - aspartate ( nmda ) receptor , and the epidermal growth factor receptor ( egfr ) ) and is only released by a local rise in ca . recruitment of ip3r machinery to the cell periphery and release of ca may function to increase the portion of pm pi(4,5)p2 available for interaction with gag and to permit the clustering of pi(4,5)p2 molecules upon gag multimerization . that budding structures are still formed by gag mutants with disrupted ptap motifs despite their impairment in recruitment of tsg101 or in cells where tsg101 has been depleted [ 50 , 51 ] indicates that assembly site membrane deformation is a pre - escrt stage event . although not required for initiation , ca is required for furrow ingression and for stability of the intercellular bridge in cytokinesis [ 113116 ] . furrow ingression in the presence of ca leads to a productive escrt recruitment stage as indicated by completion of cytokinesis . analogous to furrow ingression is the formation of the virus bud neck where the escrt scission complex is recruited . the fact that the budding structures of gag mutants with disrupted ptap motifs accumulate on the plasma membrane indicates a failure in escrt recruitment even though the mutant has been demonstrated to be capable of employing alternative modes of linking to escrt ( i.e. our study shows that , in cells expressing hiv-1 gag , ip3r was translocated from the cell interior to the periphery and colocalized with gag on the plasma membrane . the lack of ca store recruitment , which ip3r recruitment signifies , to the cell periphery of cells expressing such mutants indicates that , as is the case for furrow ingression , competency in linking to escrt is a property of bud necks formed in the presence of ca . the ability of the endoplasmic reticulum to form tubules and small vesicles is what permits the stores to be recruited . movement of ip3r - contaning er vesicles along microtubules has been shown to be facilitated by a kinesin . kinesins have been identified as being involved in an intracellular process required for gag release : ( i ) kinesin kif4 was reported to bind gag directly through the ma domain and was later found to regulate intracellular trafficking and stability of gag ; ( ii ) kinesin kif3 , a binding partner of ap-3 shown to be required for release of the viral particles assembled by gag , has also been reported to be involved in gag release . which particular kinesin is involved in ip3r transport kinesin - mediated translocation of ip3r along microtubules would allow for directed delivery of ca stores to the budding site and , thereby , establish a localized region where ca would be elevated . the notion that intact pi(4,5)p2 is required for targeting gag to the plasma membrane and that plc - hydrolyzed pi(4,5)p2 is required for escrt - recruitment - competent bud neck ingression suggests the need for regulatory mechanisms that would ensure availability of the right form of the phospholipid for the right event in the pre - escrt stage . the non - escrt proteins , spry2 and adp - ribosylation factor-1 ( arf1 ) , have activities that make them potential participants in such regulatory mechanisms . although this protein is best known for its role in post - golgi trafficking , arf1 is also a stimulator of pi(4,5)p2 synthesis by directly activating pi(4)p 5-kinase and by inducing formation of an enhancer of the kinase . several other non - escrt proteins whose dysfunction inhibited transport of gag from the cell interior to the plasma membrane may also be involved in gag assembly as pre - escrt stage participants . admittedly , further studies will be needed to elucidate their exact contribution ; however , interestingly , these proteins also have links to cytokinesis and autophagy . direct participation in cytokinesis is documented for citron kinase , ap-1 , and filamin a [ 164166 ] . npc-1 and posh both affect the metabolism of two important factors in cytokinesis , cholesterol , and calcium , respectively . thus , spry2 facilitates release driven by both the primary and the secondary hiv-1 gag l domains , possibly due to its ability to compete with escrt - i factors for interaction with escrt - ii components . not surprisingly since the interaction of escrt - i with escrt - ii leads to cargo internalization , it has been suggested that association with tsg101 increases susceptibility to internalization . delaying the recruitment of escrt machinery to the budding site may provide a means of maximizing viral budding efficiency . in this paper , we have focused on proteins involved in steps in hiv-1 trafficking and budding that take place prior to gag recruitment of escrt machinery . as described here , proteins that function in pi(4,5)p2 binding , synthesis or hydrolysis , ca store recruitment , ip3r - mediated ca store release , and vesicular biogenesis or transport appear to comprise the major classes of participants in the pre - escrt stages . the discovery of ca signaling as a cofactor in hiv-1 protein trafficking and release , its potential link to exploitation of the escrt machinery by the virus for viral particle production , and the general similarity of this coupling to other cellular activities in which escrts participate , that is , cytokinesis and autophagy , may provide new therapeutic avenues for hiv treatment strategies .

determinants intrinsic to the structural precursor polyprotein ( gag ) that is encoded by the human immunodeficiency virus - type 1 ( hiv-1 ) and other retroviruses direct targeting of gag to the plasma membrane , membrane and genome rna binding , gag multimerization , and budding of the assemblage into the extracellular space as virus particles ( reviewed in [ 14 ] ) . through a proteomic search aimed at identification of cellular factors that might participate with gag and escrt , we identified the inositol 1,4,5-triphosphate receptor ( ip3r ) as a protein enriched in an endosome- and plasma - membrane - enriched fraction only when gag was expressed ( unpublished observation ) . ip3r protein forms a transmembrane calcium ion ( ca ) channel that is mostly found on the membrane of the endoplasmic reticulum ( er ) , the major intracellular ca store in the cell . ip3r has also been detected on the plasma membrane , late endosome / multivesicular bodies ( le / mvbs ) , and the nucleus ( reviewed in [ 68 ] ) . ip3r activation requires phospholipase - c- ( plc- ) catalyzed hydrolysis of pi(4,5)p2 to generate inositol 1,4,5-triphosphate ( ip3 ) , the activating ligand for the receptor ( reviewed in [ 68 ] ) . earlier studies on hiv particle production had demonstrated that induction of a transient rise in the cytosolic ca concentration resulted in a dramatic rise in viral particle release , suggesting that ca is a limiting factor in late - stage replication [ 11 , 12 ] . taken together , these observations collectively suggested that ip3r is the physiological provider of the required ca . the proteomic search also identified several additional proteins that function in regulation of ca signaling , including sprouty2 ( spry2 ) , a modulator of ca signaling and other modes of signaling [ 14 , 15 ] . over the past few years , a number of other non - escrt host proteins have been shown to be required for gag assembly . we will discuss how these host proteins set the stage for escrt recruitment and escrt - mediated abscission events . since the gag precursor is the viral gene product that plays the key role in recruiting other viral components to the assembly site [ 23 , 24 ] , the assembly process must necessarily include a mechanism for stable localization of gag at the plasma membrane ( pm ) . once on the pm , gag has intrinsic assembly capability that is attributed to functions of its four domains ( matrix - capsid - nucleocapsid - p6 ) . the capsid ( ca ) domain provides gag with capability for self - assembly into higher - order multimers ( [ 3035 ] and references in ) . orderly cleavage of gag at interdomain junctions within the structural precursor polyprotein by a virus - encoded proteinase [ 4347 ] occurring concurrently with budding results in mature proteins whose rearrangement transforms the bud to a mature , infectious particle [ 48 , 49 ] . the final step of the virus assembly process , which results in the pinching off of the particle from the host cell , is mediated by escrt proteins that have been recruited to the bud neck by motifs in p6 that are designated as late or l domains ( reviewed in and references in [ 50 , 51 ] ) . as a cytosolic protein , the synthesis of gag takes place on soluble polysomes in the cell interior . a myristoylation reaction occurs cotranslationally during which gag acquires a myristoyl moiety on the n - terminal glycine which plays a role in assembly [ 28 , 54 , 55 ] . at the earliest experimentally feasible time points , gag has been demonstrated to have a cytosolic distribution when examined by confocal microscopy , biochemical fractionation , and immunogold electron microscopy . eventually , the entire gag population becomes membrane associated with the pm as the preferred site at steady state ( references in ) . this is consistent with the results of in vitro binding studies wherein ma , which is highly basic ( [ 2529 ] and references in ) , mediates binding to membranes reconstituted with acidic phospholipids ( [ 26 , 27 ] and references in ) . it is also consistent with observations that the cytoplasmic leaflet of the pm is unique among cell membranes in having a net negative charge due to high levels of acidic phospholipids . , phosphates and inositol ring ) with basic residues and the nestling of adjacent acyl groups into a hydrophobic cleft while studies with full - length gag underscored the importance of the phosphoinositide acyl chain . studies with gag in the presence of nucleic acid reveal an interplay between binding to pi(4,5)p2 , binding to nucleic acid , and capsid ( ca ) domain - mediated self - association . the model of gag membrane association founded on gag interaction with pi(4,5)p2 is supported by the inhibitory effect on gag particle release of depletion of plasma membrane pi(4,5)p2 [ 59 , 64 , 65 ] . eiav gag trafficking requires such targeting as inactivation of the pi(3)p2 5-kinase , which is responsible for the endosomal placement of pi(3,5)p2 , inhibits eiav gag vlp production . thus , hiv-1 gag membrane association is mediated by a specific bipartite determinant in the ma domain comprised of myristate and basic amino acid clusters with gag - pi(4,5)p2 binding serving as the basis for targeted membrane association . gag 's preferential association with the plasma membrane is due to two inherent features of pi(4,5)p2 : ( i ) the pm is where most of cellular pi(4,5)p2 is located and ( ii ) pi(4,5)p2 molecules are products of in situ synthesis ( i.e. thus , pi(4,5)p2 targeting provides a mechanism to direct gag from its site of synthesis in the cell interior to the plasma membrane . detection of assembled hiv-1 gag inside membrane compartments with the characteristics of le / mvbs has been documented [ 70 , 71 ] , and altered gag residency in le / mvbs following stimulatory or inhibitory effects on virus production has been demonstrated [ 11 , 72 , 73 ] . however , for macrophages , at least , those apparently intracellular membrane compartments with le / mvb features were demonstrated to be actually extracellular space delineated by intracytoplasmic plasma membrane [ 75 , 76 ] . we suspect that at the root of this controversy is the complex nature of the le / mvb itself . it can not be precluded that the endosomal machinery can interact with gag in the traditional manner , wherein escrt machinery facilitates sorting of cargo proteins into mvbs for ultimate delivery to degradative compartments . though targeted to the le / mvb in both hela and jurkat cells , the 29ke/31ke gag mutant is released at near wild - type levels from jurkat cells but is trapped inside hela cells which shows that trafficking within the mvb can be influenced by its environment ( i.e. direct delivery of gag to the site of release on the plasma membrane circumvents the potentially nonproductive outcome of gag association with endosomal machinery . a gag assembly model that incorporates gag - pi(4,5)p2-based targeting of gag to assembly sites on the pm permits a more productive path from gag synthesis to release of an assembled gag particle . the p6 region bearing the l domain has counterparts in other retroviruses and is functionally exchangeable with these within and outside the genera ; for example , the ptap motif from the p6 region of hiv-1 gag was shown to substitute for the py motif in the l domain - bearing region ( p2b ) of the avian sarcoma virus ( asv ) and vice versa [ 7983 ] and references in [ 50 , 51 , 84 , 85 ] . tsg101 as binding partner of the hiv ptap motif and nedd4 family members as binding partner of the asv py motif facilitate release of hiv-1 and asv , respectively , through functionally exchangeable but independent routes ( i.e. the escrt machinery is now known to comprise > 25 proteins , organized into four complexes ( escrt-0 , -i , -ii , and -iii ) that function sequentially along with several additional associated factors ( reviewed in [ 9395 ] ) . irrespective of how gag is linked to the escrt machinery , in all cases escrt - iii and vps4 must be recruited to the bud neck at the membrane site to execute the final bud scission event and to release the escrt factors from the assemblage for recycling back to the cytosolic pool for participation in future events [ 96 , 97 ] . a feature of retroviral utilization of the escrt machinery is the selective use of the escrt complexes . viral particle production requires escrt - i and escrt - iii but not escrt - ii while asv requires escrt - ii but not escrt-1 . these observations , along with recognition that escrts , which normally function in transport of some cellular proteins to degradative cellular compartments , are required for exit of assembled gag from the cell , suggests that non - escrt host proteins may play a key role in allowing the escrt machinery to be utilized differentially by the virus compared to the host . thus , non - escrt proteins may permit hiv to exploit escrt machinery by preventing the gag - escrt complex from participating in interactions with escrt partners that are nonproductive for the virus . recruitment of escrt and mediation of the abscission event by escrt is the basis for the parallel between hiv-1 budding and cytokinesis [ 101 , 102 ] . paradigms that govern the pre - escrt stage of cytokinesis , which has been an active area of research long before discovery of hiv , may likewise apply to the pre - escrt stage of viral budding . local and global elevations in cytosolic ca level are achieved by ion release from the er ( the cell 's major intracellular ca store ) and by influx from the extracellular environment . decrease in ca content of the er triggers activation of ca influx channels on the plasma membrane and refilling of the er store in a process called store - operated - calcium - entry ( soce ) [ 105 , 106 ] . why this is necessary is presently not known but the effect is to render the pre - escrt events in cytokinesis independent of soce and reliant on the internal stores as the ca source . , expression of wild - type gag ) exhibit higher cytosolic ca compared to mock - transfected cells or cells expressing a budding - impaired ptap gag mutant . the first step in both processes is the targeting of the requisite components to the eventual scission site , that is , the plasma membrane . formation of the cleavage furrow is a visual marker of initiation of cytokinesis and aspects of this event that appear similar to the budding process are furrow ingression , that is , a progressive narrowing of the eventual scission region to form a bud neck . in cytokinesis , cep55 recruits tsg101 , a component of escrt - i , and alix , an escrt adaptor protein that binds both escrt-1 and escrt - iii , to the eventual scission site once furrow ingression is completed [ 101 , 102 , 122124 ] . these escrt factors , in turn , recruit the escrt - iii complex required to carry out the terminal step in cytokinesis , abscission , that is , the severing of the thin intercellular bridge that connects the two daughter cells [ 125127 ] . the counterpart of the cep55-escrt link in viral budding is the targeting of gag to the eventual scission site on the plasma membrane and recruitment of tsg101 and/or alix through the l domains and eventually escrt - iii . autophagy , the process involved in the breakdown of intracellular proteins and organelles , is now appreciated as a mechanism of great importance in both cell survival and cell death . recent findings in mammalian cells and in drosophila melanogaster indicate that escrts are required for efficient trafficking through the endolysosomal system where the autophagic cargo is degraded [ 130132 ] . as with cytokinesis and viral budding , ip3r - mediated ca signaling is emerging as critical for the pre - escrt stage in autophagy . the participation of calcium machinery components in all three processes suggests that the requirement for and reshaping of calcium signaling is a common feature governing their pre - escrt stages . for a number of non - escrt host proteins shown to be important for release of the gag particle [ 2 , 4 , 22 , 140 ] , disruption of the protein function does not result in the canonical l domain phenotype ( i.e. in yeast , class c vps proteins have been shown to regulate pm localization of at least one protein and to assume roles antagonistic to escrt in the recycling of membrane proteins . the human orthologue of mon2 ( hmon2 ) can bind and regulate the subcellular localization of adaptor proteins such as ap-1 , ap-3 , and arf1 which have previously been shown to be required for gag pm localization and gag particle production [ 72 , 144 , 145 ] . the notion of non - escrt proteins regulating the activity of other non - escrt proteins in the pre - escrt stage has a parallel in cytokinesis as illustrated by the host protein , tex14 . through protein - protein interactions , non - escrt proteins could thus impose temporal and spatial control of the recruitment of participating proteins , including gag itself , to assembly sites on the pm during the pre - escrt stage . quantitative analyses indicate that the viral envelope differs from the pm of its host cell in having higher levels of cholesterol and pi(4,5)p2 [ 58 , 147 ] . since the viral envelope is derived from the pm microdomain serving as the gag assembly site , reorganization of the lipid bilayer in this location may occur as part of the assembly process . , myristylated alanine - rich c kinase substrate ( marcks ; growth - associated protein ( gap)43 ; n - methyl - d - aspartate ( nmda ) receptor , and the epidermal growth factor receptor ( egfr ) ) and is only released by a local rise in ca . recruitment of ip3r machinery to the cell periphery and release of ca may function to increase the portion of pm pi(4,5)p2 available for interaction with gag and to permit the clustering of pi(4,5)p2 molecules upon gag multimerization . the fact that the budding structures of gag mutants with disrupted ptap motifs accumulate on the plasma membrane indicates a failure in escrt recruitment even though the mutant has been demonstrated to be capable of employing alternative modes of linking to escrt ( i.e. our study shows that , in cells expressing hiv-1 gag , ip3r was translocated from the cell interior to the periphery and colocalized with gag on the plasma membrane . the lack of ca store recruitment , which ip3r recruitment signifies , to the cell periphery of cells expressing such mutants indicates that , as is the case for furrow ingression , competency in linking to escrt is a property of bud necks formed in the presence of ca . kinesins have been identified as being involved in an intracellular process required for gag release : ( i ) kinesin kif4 was reported to bind gag directly through the ma domain and was later found to regulate intracellular trafficking and stability of gag ; ( ii ) kinesin kif3 , a binding partner of ap-3 shown to be required for release of the viral particles assembled by gag , has also been reported to be involved in gag release . which particular kinesin is involved in ip3r transport kinesin - mediated translocation of ip3r along microtubules would allow for directed delivery of ca stores to the budding site and , thereby , establish a localized region where ca would be elevated . the notion that intact pi(4,5)p2 is required for targeting gag to the plasma membrane and that plc - hydrolyzed pi(4,5)p2 is required for escrt - recruitment - competent bud neck ingression suggests the need for regulatory mechanisms that would ensure availability of the right form of the phospholipid for the right event in the pre - escrt stage . ca might be a key regulator : ca is an activator of the lipid kinase that is critical for pi(4,5)p2 synthesis and of the plc that catalyzes pi(4,5)p2 hydrolysis . perhaps this conundrum , that is , the lack of a clear correlation between gag pm targeting and the pi(4,5)p2 level , reflects a requirement for a pi(4,5)p2 pool that is specifically made available for gag . although this protein is best known for its role in post - golgi trafficking , arf1 is also a stimulator of pi(4,5)p2 synthesis by directly activating pi(4)p 5-kinase and by inducing formation of an enhancer of the kinase . several other non - escrt proteins whose dysfunction inhibited transport of gag from the cell interior to the plasma membrane may also be involved in gag assembly as pre - escrt stage participants . although the precise stage at which tsg101 docks on the ptap motif is not known , association after stable bud neck formation might be more favorable as it precludes nonproductive interactions with escrt - ii that would signal internalization of the gag assemblage or premature escrt - iii scission . thus , spry2 facilitates release driven by both the primary and the secondary hiv-1 gag l domains , possibly due to its ability to compete with escrt - i factors for interaction with escrt - ii components . not surprisingly since the interaction of escrt - i with escrt - ii leads to cargo internalization , it has been suggested that association with tsg101 increases susceptibility to internalization . a parallel to this as a regulation possibility in cytokinesis may be the aforementioned function of tex14 , a protein believed to control premature progression to the abscission stage by competing with tsg101 and alix for binding to cep55 . in this paper , we have focused on proteins involved in steps in hiv-1 trafficking and budding that take place prior to gag recruitment of escrt machinery . as described here , proteins that function in pi(4,5)p2 binding , synthesis or hydrolysis , ca store recruitment , ip3r - mediated ca store release , and vesicular biogenesis or transport appear to comprise the major classes of participants in the pre - escrt stages . the discovery of ca signaling as a cofactor in hiv-1 protein trafficking and release , its potential link to exploitation of the escrt machinery by the virus for viral particle production , and the general similarity of this coupling to other cellular activities in which escrts participate , that is , cytokinesis and autophagy , may provide new therapeutic avenues for hiv treatment strategies .

seventy - seven days before the outbreak of the second world war , the german - language neurological and psychiatric journal zentralblatt announced a new , revolutionary method in the shock therapy of schizophrenia coming from rome. a method , as well as suitable apparatus , had apparently been found which superseded the convulsive therapy of schizophrenia , which had been introduced by ladislas meduna ( 18961964 ) in 1934 and relied on epileptic seizures triggered with camphor or pentylenetetrazol ( cardiazol ) . that same year , the new treatment was indeed introduced in many countries in europe , and in japan , latin america and the usa . with regards to europe , however , it is assumed that national socialism played a decisive role in the introduction of this new shock therapy . the research literature not only emphasises the flight of doctors and scientists of jewish descent or faith from national socialism , but particularly supports the thesis that the introduction of the new technology in the third reich had drawn special interest from high - ranking officials of the nazi regime . electroshock therapy ( now known as electroconvulsive therapy ( ect ) ) was swiftly integrated into the dialectics of healing and killing typical of medicine in nazi germany. since szasz s article from the slaughterhouse to the madhouse , the invention of electroshock therapy has generally been regarded as modern therapeutic totalitarianism. social histories such as braslow s mental ills and bodily cures seem to have reinforced the view popularised by one flew over the cuckoo s nest of electroshock therapy as a furthermore , it is frequently assumed that if electroshock therapy was indeed a disciplinary medical therapy , then its introduction and application in the third reich must have been particularly cruel . indeed , such highly regarded historical research on the third reich and the euthanasia murders as henry friedlander s the origins of nazi genocide : from euthanasia to the final solution claims that during the nazi period , electroshock was no therapy , but rather a treatment used to inflict torture. within the third reich , it had finally been the bureaucratic apparatus for organising the mass murder of mentally disabled and mentally ill people which under the conditions of war economy provided german psychiatric hospitals with the apparatuses necessary for electroconvulsive therapy and which considered the establishment of institutes for investigating the new technology . although most histories of electroshock therapy failed to engage with the complexities of the existence of electroshock therapy in the third reich , preliminary investigations on the chronology of the introduction and implementation of the new therapy in europe and especially in germany raise doubts as to whether national socialist health policy favoured the introduction of the new shock therapy for the german reich , or whether it in fact delayed it . following this line of enquiry , our article proposes a chronological analysis of the invention and subsequent introduction of electroshock therapy in the third reich and occupied territories . therapeutic enthusiasm concerning electroshock within the third reich is queried , as well as the existence of state - subsidised research institutions with the explicit purpose of studying and promoting electroshock therapy . our analysis relies on an exhaustive review of contemporary international publications as well as on archival records , including medical files from several countries in europe and the usa . methodologically , a social construction of technology perspective is taken to show how medical science worked relative to its social context . the ways in which new scientific knowledge was acquired and put into practice were many - faceted , and dependent on the diverging interests of physicians , industrial companies , mental health politics and the exigencies of total war . our article describes how the changing medico - political and economic context during the third reich and the second world war not only shaped the design of the devices for electroshock therapy but actually reversed their function as tools of medical therapy . in the spring of 1936 , ugo cerletti ( 18771963 ) , director of the psychiatric neurological university hospital in rome since 1935 , sent his assistants to vienna to study a new therapy for schizophrenia which had been recently introduced there , meduna s convulsive therapy. its principle was the injection of a pharmacological substance able to provoke a generalised epileptic seizure , the hypothesis being that this had a healing effect on schizophrenia . cerletti , with his longstanding research on epilepsy , immediately responded to the report of his assistants on their vienna observations with the idea of modifying meduna s procedure with the use of electricity instead of a pharmacological substance . in his epilepsy experiments on dogs , cerletti had already used electrical provocation to study histopathological brain changes following multiple epileptic seizures . however , until then , he had been reluctant during these experiments to attach the electrodes to the head of the dogs in view of the fact that the passage of an electric current through the brain several times might also produce some histological alterations. yet the method with one electrode in the mouth and the other in the rectum was relatively dangerous as the current passed the heart and led to the death of several of cerletti s dogs . therefore , still in october 1936 , cerletti entrusted his technically experienced assistant , dr lucio bini ( 190864 ) , with further studies on examining the possibility of safely causing epileptic seizures by applying electric current to the head . the initial results of these tests in dogs , utilising equipment designed by bini , were presented in may 1937 at an international conference in switzerland on the therapy of schizophrenia : insulin shock , cardiazol , permanent sleep. in contrast to the intravenous pharmacologic induction of epileptic seizures , electricity promised to be simpler , more reliable and less terrifying for the patient , as the application of the electric current immediately resulted in a loss of consciousness . motivated by this therapeutic concern , cerletti wished to change a technique initially developed to study the neurohistopathology of epilepsy into a therapy of schizophrenia ; the technique constructed for medical research was reshaped to enable medical therapy . it is evident , therefore , that the electroshock idea was not born in the roman slaughterhouse and it was not primarily tested on pigs as szasz suggests with his from the slaughterhouse to the madhouse story . on the contrary , cerletti had been quite reluctant to proceed to a clinical application : this possibility [ of using electricity to induce convulsions in people as they were then produced by cardiazol ] was often discussed at our institute and particularly when observing , after cardiazol injections , the painful reactions which shook fully conscious patients for over a minute as a result most of them refused the subsequent treatments . however , the idea of submitting man to convulsant electric discharges was considered utopian , barbaric , and dangerous : in everyone s mind was the spectre of the electric chair. and i myself found in the literature on electrology reports on death in man caused by electric currents of lower voltage than that used on our experimental dogs . this possibility [ of using electricity to induce convulsions in people as they were then produced by cardiazol ] was often discussed at our institute and particularly when observing , after cardiazol injections , the painful reactions which shook fully conscious patients for over a minute as a result most of them refused the subsequent treatments . however , the idea of submitting man to convulsant electric discharges was considered utopian , barbaric , and dangerous : in everyone s mind was the spectre of the electric chair. and i myself found in the literature on electrology reports on death in man caused by electric currents of lower voltage than that used on our experimental dogs indeed , cerletti continued in his autobiographical notes to say that it was a chance occurrence involving some new electrical equipment at the slaughterhouse in rome that allowed him to make the next step . having been told at first that here hogs were killed by electric current , cerletti first saw this information confirm his doubts regarding the danger of electric applications in man. however , having gone to the slaughterhouse to observe this so called electric slaughtering , he realised that it was a technique for anaesthesia ; the pigs were killed by other means while unconscious . source : excerpt from a french brochure by lotterschmidt & weinberger , without any date , siemens medarchives , erlangen , germany . source : excerpt from a french brochure by lotterschmidt & weinberger , without any date , siemens medarchives , erlangen , germany . in fact , the roman slaughterhouse was equipped with a new innovative electrical anaesthesia for pigs that had been developed between 1927 and 1930 and which in 1934 was already being sold throughout europe , especially in italy and the nordic countries ( see figure 1 ) . realising the astonishing similarities between this procedure and his experiments with dogs , cerletti realised that the hogs of the slaughterhouse could furnish the most valuable material for his investigations , which he again put in bini s hands as soon as bini had returned from the schizophrenia conference in switzerland . the chief veterinarian , professor ettore torti , indeed allowed bini further experiments to study the safety margin of the applied current . there was a clear difference between the time necessary for a current to provoke a fit ( s ) and the time necessary to provoke the death of an animal ( 60150s) , a safety margin big enough to make cerletti dare to undertake the first clinical applications . the story of these first trials on sick people in the roman clinic has been written several times . remarkably , the first trial was conducted in secret for fear of a major incident . cerletti was already 60 years old and a renowned authority in the field of neurology and psychiatry , and he was cautious of losing his reputation . in any case , the maestro , as he was called , did not need to discover something the fact that cerletti did choose a newly admitted patient , instead of a well - known patient of his own , has been widely critiqued ; however , in the light of the history of human experimentation in medicine , it appears neither as a feature exclusively characteristic of psychiatry and neurology , nor even of medicine in the third reich or in europe . the new electro - convulsive method proved so convincing that cerletti dared to present the results as early as 28 may 1938 to the royal medical academy in rome ; they were published in italian professional magazines as electric shock ( lelettroshock ) . the method was geared initially to apply a weak calibration current to two electrodes fixed with a special supporting structure to the patient s head , to be able to measure the so - called head resistance . on the basis of this value , a suitable voltage could subsequently be selected so that the electricity flowing through the patient s head via the same electrodes for fractions of a second would cause an epileptic seizure . electroshock apparatuses taken over in exclusive licence by the milanese company officine elettrotecniche italiane ing . ( arcioni ) ( see figure 2 ) . figure 2:illustration : italian manual source : collection dolhuys , museum van de geest , haarlem , the netherlands . however , in the first half of 1939 , the new method hardly attract[ed ] any interest , as max mller ( 18941980 ) , who had organised the swiss conference on the pharmacological shock therapies , later remembered . the apparatus developed by the italians was a trade secret of the company arcioni and difficult to get so that nobody was really keen on this apparently risky and still little tested method. max mller was nevertheless to apply electroshock therapy for the first time outside italy and launch it in switzerland as early as november 1939 . what motivated him to do so was an advertising visit by a german emigrant of jewish origin , lothar kalinowsky ( 18991992 ) , and a follow - up letter written by him from the italian clinic . two months after mller , in french - speaking switzerland , oscar louis forel ( 18911982 ) started to administer regular treatments to patients in prangins at the psychiatric hospital which he had founded . after the first 262 sessions , within eight months , he highly recommended the therapy . consequently , with italy s entry into the war , mller wanted to avoid the possibility that the supply of the devices he required for this very promising therapy might be threatened , and so he gave his arcioni apparatus to purtschert & co. in luzern for reproduction . only a few days before the german invasion of poland , bini and cerletti presented their research results from already 3000 cases of artificial inducement of epileptic seizures in humans at the third international congress for neurology in copenhagen in august 1939 in a special section on epilepsy . young psychiatrists such as the german adolf bingel ( 190182 ) or the danish arild faurbye ( 190783 ) , who had taken part , were enthusiastic about this new area of research and the excellent opportunities it offered for a scientific career . immediately upon their return from copenhagen , they sought support from engineers to construct a device with which they could examine the promising italian work on electroshock. faurbye started his first treatments in november 1940 : bingel , who had been senior physician at the university psychiatric and mental hospital in erlangen since 1937 , began in december 1939 . this early german start in germany was made possible because erlangen had the head office of the electro - medical research laboratory of siemens reiniger werke ( srw ) , the leading company for medical technology in the german reich . its head , the physicist and mathematician johannes ptzold ( 190780 ) , had already developed an interest in the method and its apparatus by the time of the copenhagen congress , although he was himself more concerned with high - frequency therapy techniques . indeed , by the summer of 1939 , srw had requested a report about the medical usefulness of the new srw had asked oswald bumke ( 18771950 ) , the director of the psychiatric clinic at the ludwig maximilian university of munich , who was also editor of the reference work handbook of mental diseases , for an expert opinion , because munich had obtained an arcioni apparatus as early as july 1939 thanks to lothar kalinowsky . however , it was not until october 1941 that bumke stated that , despite his predictions , the new convulsive therapy did produce positive results with those suffering from schizophrenia and other mental illnesses and that the treatment was therefore to be recommended . bingel s boss , the medical director and chair of the erlangen clinic , friedrich meggendorfer ( 18801953 ) , had been less sceptical about the new therapy . on 3 february 1940 , bingel and meggendorfer could thus describe their treatments as the first german tests , started in december 1939 with an apparatus produced by srw . following this , meggendorfer , who had been appreciated in the early 1930s for his genetic studies on huntington s disease and his eugenic thinking on moral insanity , or personality failures , saw in the official congratulations on his sixtieth birthday that the success of having been the first german psychiatrist to embrace the new therapy was not mentioned at all . the congratulations were written by ernst rdin ( 18741952 ) , who at this time viewed electroshock therapy , like both insulin coma and cardiazol - convulsive therapies , as a contradiction of the genetic view of endogenous psychoses , especially with regards to schizophrenia . rdin had been the major proponent of the expert committee on questions of population and racial policy that had prepared the scientific justification for the law for the prevention of hereditarily diseased offspring passed in 1934 . the law enabled forced sterilisation in over 400 000 cases of schizophrenia , epilepsy , huntington s disease , hereditary blindness or deafness , congenital mental deficiency or manic depressive illness until the end of the third reich . in spite of this initial reluctance towards the new therapy due to the preponderance of genetic thinking in german psychiatry and nazi mental health politics , srw were able to present a genuinely german electroshock apparatus under the name konvulsator at the end of june 1940 ( see figure 3 ) . in the light of some patent queries because of bini s application for a german patent , the patent department of srw had consciously chosen the name konvulsator to distinguish their equipment from the italian lelettroshock . ptzold had succeeded in changing the construction in such a way that the elements of bini s device which were most relevant for the patent had in any case been avoided ; the circuit omitted the preceding resistant measurement and also enabled the direct adjustment of the current in ma . additionally , and in contrast to the italian electrode clip or forceps maximally compressing the electrodes together and pressing them strongly against the skin , srw s electrodes were to be electroshock patent , srw saw themselves confronted with another competitive construction built by the vienna - based company reiner & co. it was developed by a graduate technician and psychiatrist at the vienna university psychiatric clinic , wolfgang holzer ( 190680 ) , who had been given the order to scrutinise the arcioni device and to adapt it to the needs of the vienna clinic . elkra i , a large standalone unit with an additional electrode holder , was constructed , and then elkra ii , where the entire apparatus was directly attached to the forceps ( see figure 4 ) . holzer had regarded it as established that suitably sized electrode holders proved to be considerably better than electrode appliances attached by means of headbands because the latter frequently could not even be attached to unsettled patients. moreover , with holzer s elkra ii , the treating physician was able to position the electrodes on the patient s head and simultaneously activate the treatment current without requiring additional staff . this advantage to enable shocking even the most unsettled patients in a comfortable manner had quickly piqued the interest of psychiatric hospitals even if due to the war events it was difficult to get such an apparatus. for example , the heilanstalt fr kopfverletzte und nervenkranke , another viennese clinic , which since the 1930s had distinguished itself through a special commitment to new therapies including convulsion therapy , bought an elkra ii in spite of having been one of the first clinics to own a konvulsator produced by srw . within the elkra ii design , the electrode forceps was an integral part of the overall device . this made srw find a remarkable way to protect themselves against elkra ii as competitor in the sales area of electroshock therapy in the german reich , to which austria officially belonged since the anschluss of 1938 . srw claimed to have a patent on the electrode forceps : the deutsche reichspatent ( d.r.p . ) 578 532 for the forceps form of the anaesthesia device for the electrical anaesthesia of animals to be slaughtered precisely the technique that bini had studied in the roman slaughterhouse to test the safety margin of the application of electricity to the head . as srw turned to reiner & co. to assert claims from this patent on the holder electrode of elkra i and ii , holzer responded with indignation : the subordination of electroshock under the broader term anaesthesia and of patients under animals to be slaughtered [ ] shall represent a unique curiosity for a leading electromedicine company . the subordination of electroshock under the broader term anaesthesia and of patients under animals to be slaughtered [ ] shall represent a unique curiosity for a leading electromedicine company . it does not apply for the patients in my sphere of influence . in march 1941 , reiner & co. nevertheless began to consider a contractual agreement with srw for a simple non - transmittable licence regarding a device for anaesthetizing animals to be slaughtered . in turn , reiner & co. committed themselves to pay a licence fee to the amount of one per cent from the list price of the apparatus for all electroshock devices produced and sold by reiner & co. meanwhile , srw got help from anton edler von braunmhl ( 190157 ) , at the mental hospital eglfing - haar , to also develop more comfortable electrodes , which were pressed to the patient s head and which enabled the current release at the same time ( see figure 5 ) . braunmhl had been the main protagonist of insulin coma therapy and cardiazol - convulsive therapy in the third reich . as early as 1939 , he had urged srw to give him a konvulsator prototype for testing the new therapy which he had learnt about on a trip to italy . in 1944 , braunmhl gained his postdoctoral qualification ( habilitation ) in munich thanks to his clinical research into shock therapies . although his electroshock developments as well as his comprehensive book physikalische medizin in diagnostik und therapie would have met by far the requirements to qualify for a habilitation , this did not happen during the nazi period . still , in september 1944 , holzer tried to get support for his research in electroshock therapy ; however , his idea of a whole scientific institute for investigating electroshock was never realised . source : picture dated 1955 , used in an advertising brochure in 1957 , siemens medarchives , erlangen , germany . illustration : siemens new electrodes relaying on braunmhl s recommendations . source : picture dated 1955 , used in an advertising brochure in 1957 , siemens medarchives , erlangen , germany . by the end of june 1941 , srw had delivered only fifty konvulsatoren , most of them abroad , including twenty - three devices to the occupied netherlands . grodeutsches reich , of which only five had gone to mental asylums ( including eglfing - haar ) . remarkably , the srw referred all other interested parties to the beginning of the assembly - line production . this was the experience of , for example , gottfried ewald ( 18881963 ) , who had been desperately looking for a device for the university psychiatric hospital in gttingen and had finally been able only as a result of personal initiative to acquire a device from switzerland , from purtschert & co. , in the summer of 1941 . the low sales figures of the konvulsator were not least the consequence of a politically desired radical policy of austerity in psychiatry in the german reich , intended to lead to the starvation of patients with lives unworthy of living. what was financed was the so - called euthanasia t4 campaign which paul nitsche ( 18761948 ) organised on behalf of the reich working party for mental asylums . from its start in october 1939 , until its official discontinuation in august 1941 , the construction of gas chambers on former hospital grounds and a bureaucratic procedure of camouflaging was funded to murder more than 700 000 patients with diagnoses such as schizophrenia , mental deficiency , epilepsy , dementia or huntington s disease . crucially , the consequences of another budget cut in psychiatry contributed to an increase of interest in convulsive therapy and particularly electroshock therapy in the spring of 1942 . with the responsibility for mental hospitals passing to herbert linden ( 18991945 ) , the beginning of new insulin coma cures had generally been forbidden as insulin was scarce and insulin coma therapy could not be regarded as a specific therapy for schizophrenia as there were also other methods ( such as cardiazol and electroshock ) available. indeed , the cherished genetic theory of endogen psychoses and especially of schizophrenia implied that insulin coma , cardiazol or electroshock therapies could only have unspecific impacts similar to states of shock induced by means of cold showers or a swivelchair ; a real therapy of the core group of schizophrenia was only conceivable by prevention , namely sterilisation . these genetic views and the consequent nazi sterilisation policy had at first considerably delayed the introduction of insulin coma therapy in germany in the mid-1930s and also caused an initial restraint towards electroshock therapy in well - known clinics . the university mental hospital of the charit in berlin , run by maximinian de crinis ( 18891945 ) , a key actor in the preparation and implementation of the national socialist euthanasia murders , had not begun testing until winter 1941/42 . in contrast , the small evangelical private clinic waldhaus am nikolassee , which became famous for employing those physicians and carers who have not received any employment in state facilities for political and racist reasons , had started with the new therapy by the spring of 1941 . its medical director , heinrich schulte ( 18981983 ) , recommended electroconvulsive therapy as an alternative to the pharmacological convulsive therapy as early as june 1941 , including at the conference of the berliner gesellschaft fr psychiatrie und neurologie , since it was less stressful for the patients , simpler , cheaper and also suitable for ambulant treatment . however , the horrifying sight of his demonstration of the electroconvulsion on two patients caused several listeners [ to ] leave the room. that the circle of leading national socialist psychiatrists , based around , amongst others , ernst rdin , maximinian de crinis , paul nitsche and carl schneider , finally began to advertise convulsive therapies was the result of a shift in nazi mental health politics ; to counter the public opinion that psychiatry is becoming more and more superfluous since mentally ill people would soon become extinct due to the racial hygienic laws , the convulsive methods , including prominently electroshock therapy , promised to justify a future eugenic national socialist psychiatry of sterilisation , euthanasia and therapy . probably with a view to introducing this new way for psychiatry in the third reich , a number of lectures on electroshock therapy , among them lectures by meggendorfer and bingel , max mller and also wolfgang holzer , had appeared on the agenda of the sixth annual assembly of the gesellschaft deutscher neurologen und psychiater scheduled for 57 october 1941 . yet , the whole conference was cancelled due to the war . while deaths from medicine overdoses and through starvation continued after the termination of the official t4 campaign in august 1941 , financial means for equipping the mental hospitals with therapy techniques were thus granted upon linden s mediation and an educational film on electroshock therapy was produced . facing continuing complaints , which also spread into public discourse , that the difficulties of material procurement do not favour the spreading of electroconvulsive treatment , ninety - five devices were finally ordered from srw at the end of february 1943 with a collective order by the management of the reichsauschusses zur wissenschaftlichen erfassung von erb- und anlagebedingten schweren leiden ( state committee for the scientific assessment of severe hereditary disease ) , who had also organised the t4 programme . such a collective order had become necessary after srw , upon linden s request , had made clear that the production of electroshock apparatuses would only be worthwhile for the company if higher quantities were sold . because technology central to the war effort , such as x - ray diagnostic devices , took priority with regards to the use of materials and production processes , the collective order had to be a formal wehrmacht order . those clinics which missed this order , offered by linden , were informed by srw that the delivery period for the device would be at least one and a half years. yet , even the delivery of the devices of the wehrmacht order happened technical reason for the delays was the fact that many clinics did not have any alternating current available , since the in - house electricity grid was exclusively supplied with direct current . under such circumstances , a direct current converter was necessary for the konvulsator . although srw did not itself produce it , they had promised to include the accessory device in the delivery of the konvulsator , although special convertors could not be obtained in the foreseeable future. remarkably enough , however , a konvulsator produced by srw had been in constant use in france at the paris hpital des enfants malades as early as february 1942 . the psychiatrist who used it was georges heuyer ( 18841977 ) . heuyer , known as the founder of french child psychiatry , had been another participant in the copenhagen neurological congress of 1939 ; it was here that he had learned about the new therapy from cerletti s and bini s lectures . although heuyer had even announced an export of konvulsatoren to france , at the occasion of the presentation of the initial dog experiments for a french apparatus at the conference of the socit mdico - psychologique in april 1941 , most psychiatric hospitals in the occupied france , as , for example , the hpital du vinatier near lyon , known from the study by isabelle von bueltzingsloewen on starvation in french psychiatry during the german occupation , used equipment from purtschert & co. on german territory against widespread assumptions to the contrary the war - related production bottlenecks meant that many of the psychiatric hospitals without university affiliation did not receive an apparatus before 1944 , and some , only after the war . in the later french occupation zone , for example , during the third reich , none of the big psychiatric asylums in bad schussenried , zwiefalten or weissenau had an electroshock apparatus in use . the french psychiatric commissioner sent to inspect the situation in the psychiatric hospitals at the end of the war in 1945 noticed with astonishment : this method has never been practised in these three institutions and this fact appears monstrous to the psychiatrist that knows of the remarkable results of electroconvulsive therapy in the treatment of acute psychosis . this method has never been practised in these three institutions and this fact appears monstrous to the psychiatrist that knows of the remarkable results of electroconvulsive therapy in the treatment of acute psychosis . the minor diffusion of electroshock therapy was not solely the result of an inadequate supply or inadequate diffusion of electroshock devices ; most of the ordered reichsausschuss konvulsatoren had finally been delivered to hospitals and asylums . however , few of the hospital asylums which did receive one appear to have been particularly eager to use it , if one believes the report of curd runckel ( 1913- ? ) , who was working with nitsche at the t4 unit . runckel had been sent into the hospitals and asylums to learn the reasons for the reluctance to the new therapies and euthanasia. in the summer of 1944 he wrote : in many places the electroshock apparatus is still regarded with certain scepticism . especially in lower silesia the treatment method is frequently belittled ; otherwise it would barely be possible , i think , that a huge asylum like lben sells its newly acquired electroshock apparatus . i continuously try to convince the medical directors that the modern methods are , concerning nursing care , surely greatly relieving . in many places the electroshock apparatus is still regarded with certain scepticism . especially in lower silesia the treatment method is frequently belittled ; otherwise it would barely be possible , i think , that a huge asylum like lben sells its newly acquired electroshock apparatus . i continuously try to convince the medical directors that the modern methods are , concerning nursing care , surely greatly relieving . also braunmhl had claimed that electroshock combined with insulin was to be preferred to electroshock alone , and the use of insulin for psychiatric therapy outlawed in february 1942 due to the lack of insulin was finally again permitted only in 1944 . indeed , the notion of a quick and extensive introduction of electroshock therapy in the third reich seems to be nothing more than a myth ; a clich , as heinz faulstich has argued in his major work , starvation in psychiatry 19141949 : a historically mistaken , if widely cherished , idea that psychiatrists during the time of national socialism were widely enthusiastic about euthanasia and shock therapies . correspondingly , historical studies that have relied on patient files have found that electroshock was seldom used . in saxony , for example , in the psychiatric hospital altscherbitz , an analysis of 1400 patient files , including 497 with the diagnosis of schizophrenia , found a single case of electroshock treatment . surely disastrous shortage in financial means and staff due to the fascist war. for the saxon psychiatric hospital groschweidnitz , the finding was similar : only with may 1942 are there a handful of treatment trials of the quite cheap treatment documented in the patient files ; however , it never achieved greater significance. in contrast , the asylum eglfing - haar near munich had a high incidence of electroshock therapy , as here anton von braunmhl , the most ardent proponent and defender of the somatic therapies in the third reich , had done everything to convince srw to give him the second prototype for clinical use . but besides the university hospitals and asylums with psychiatrists specifically interested in the treatment , the diffusion of electroshock therapy remained minimal in the daily practice of the mental institutions in the german reich until the end of the war . in psychiatric hospitals that had a special focus on electroshock therapy , sometimes explicit consent for shock therapy was required from the family . for this , at the university psychiatric and mental hospital erlangen for example , a letter was sent with the following wording : insulin- or cardiazol- and electroconvulsive treatment gives the possibility to approach mental diseases in a healing manner today . therefore we think it our duty as physicians to propose this treatment and we ask that you give us your consent for this treatment at your earliest convenience . as the treatment is invasive and may result in complications , we are informing you of our intentions . if you agree with the treatment , we ask that you sign and return this letter . insulin- or cardiazol- and electroconvulsive treatment gives the possibility to approach mental diseases in a healing manner today . therefore we think it our duty as physicians to propose this treatment and we ask that you give us your consent for this treatment at your earliest convenience . as the treatment is invasive and may result in complications , we are informing you of our intentions . if you agree with the treatment , we ask that you sign and return this letter . on the other hand , psychiatric hospitals whose clinical directors completely subscribed to the ideology of schizophrenia as a genetic disease such as the wittenauer heilsttten in berlin changed the diagnosis in the case of a successful electroshock treatment , for example , to reactive psychosis. it fits that an increasing number of reactive disorders , especially in soldiers , were treated with electroshock . in conclusion , the dialectics of healing and killing , as promoted in the 1943 denkschrift letter thoughts and suggestions concerning the future development of psychiatry written by the elite of the nazi psychiatrists , remained a pure ideal , unattained until the end of the third reich . regarding the everyday practice in most of the mental institutions of the german reich , there was no dialectics of healing and killing but a prevailing policy of starvation , leaving people to die and murder . the munich research institute for psychiatry ( deutsche forschungsanstalt ) run by rdin had no interest in research into electroshock therapy ; its clinical unit had in any case been closed in 1942 because of the war . however , even srw reacted with restraint to hints that a model institution for research into electroshock treatment was to emerge in southern germany , in co - operation with the heidelberg university hospital and under the direction of carl schneider and the mental hospital at wiesloch . actually , the reich working party for mental asylums , represented by linden and nitsche , had been planning the establishment of a psychiatric research institution with its main pillar in heidelberg yet this was intended less as a model institution for the research of electroshock treatment , as the srw branch in mannheim had understood from the discussions in heidelberg with linden and schneider , but rather for enhancing schneider s own genetic , constitutional and neurobiological examinations of mentally deficient children. whatever the potential plans concerning research into electroshock , ptzold explicitly pointed out that because of its extensive involvement in the war economy , srw had practically been deprived of all possibilities for more extensive development work for the duration of the war so that currently collaboration can only be in exchanging experiences and discussing pending problems in this field. however , srw did not want to make the same mistake as its predecessor companies during the first world war , when work on developments for civilian and medical- technology use had been considerably limited for reasons of war economy , with the consequence that srw had , after the end of the war , been left behind compared to the international competition . srw were therefore striving to avoid a repeat of this situation and to maintain developmental activity for civil commodities as far as possible . the company needed , in fact , to justify their already existing scientific collaboration with anton von braunmhl via nested lines of argument as relevant to the war in order to avoid seriously jeopardising their continuity . in this way , it had been alleged that the help offered by srw in procuring a purtschert & co. apparatus for anton von braunmhl , who was planning some major research comparing all apparatuses available on the market , was about a question irrelevant to the war for which an enormous amount of work was not justified. on the part of srw , the response to this was that mainly sick soldiers were treated with electric convulsion at dr von braunmhl s and hence comparative experiments for determining the reasonable method are thus also part of looking after sick soldiers and must also be regarded as relevant to war. the scope which the technical development department of srw was trying to create with this argument was , without doubt , extremely narrow . however , they took the position that tests for improving the apparatuses , whose production is not forbidden , are also allowed , and , beyond the wording of any regulations , it must be left to the company to decide which of the works will be postponed and which will not. yet , the conditions of total war in 1944 made any further research impossible . as for the heidelberg efforts in wiesloch , schneider was in any case resigned to the fact that after two attempts the war did not [ allow ] the realisation of the extensive projects planned at that time and that the implementation of total warfare after the disaster of stalingrad brought about the dissolution of the newly started wiesloch department again in 1944. some research had nevertheless been done on electroshock at the heidelberg clinic , and then also at the partner institution in wiesloch . as bingel had already warned in 1940 that the electroconvulsive treatment is not as free of complications as it seemed according to first reports by the italian authors , schneider s chief heidelberg physician , fritz schmieder ( 191188 ) , had started to examine the frequency of vertebral fractures under electroconvulsive therapy since february 1942 . the question whether a medicine affecting the muscle apparatus would be able to decrease the danger of a fracture , had already been internationally discussed in relation to pharmacological convulsion therapy since 1939 ; from 1940 onwards , it was also considered in relation to electroconvulsive therapy . consequently , studies on preventing vertebral fractures with convulsive shock were also planned for the research department , especially for schmieder s position here from july 1942 until the end of march 1943 . ernst - adolf schmorl ( 190664 ) , a leading assistant to nitsche with extensive experience in pharmacological convulsion therapy , meanwhile carried out experimental studies on the differences of induced and spontaneous convulsion in humans. for the studies on the prevention of vertebral fractures , schmieder had asked srw in november 1942 for technical as well as material support . schmieder wanted to carry out a series of tests with electroshocks on cats or rabbits using the konvulsator for this purpose the facilities were unfortunately inadequate for the housing of dogs . although srw did not yet have any experiences available from their own animal studies , ptzold was able to advise schmieder on the electrodes to be used for cats and the necessary device settings . thanks to this scientific collaboration , heidelberg ranked among the first clinics besides eglfing - haar that were not only lent the konvulsator ii in may 1943 as a simplified and handy ( although not battery - driven ) device for testing , but whose criticisms were taken into consideration in the future technical design . meanwhile , the problem of vertebral fractures had not remained of purely scientific or medical significance , as it influenced decisively the choice for linden s collective device order for the clinics and mental hospitals of the german reich and thereby consolidated srw s position on the electroshock market . it was precisely because of the claim that vertebral fractures were few with the srw apparatus compared to the competing devices that the collective order went to srw in spite of the konvulsator being the most expensive device ( 955 rm against 390 rm for elkra ii , for example ) : a claim that was pure advertising , as its author , meggendorfer , in erlangen , did not posses any clinical experience with the other devices . in the third reich , the distinctive conditions of national socialism and total war meant a particular interest in electroshock therapy . to this end , an electroshock apparatus was constructed at the concentration camp auschwitz iii , the working camp of the ig farben - werk in monowitz , in 1944 . with the unofficial agreement of the ss camp physicians eduard wirths ( 190945 ) and horst fischer ( 191666 ) , the polish detainee physician zenon drohocki ( 190378 ) , helped by a dutch engineer detainee , wanted to treat sick detainees with the apparatus in the neuropsychiatric department which he had established inside the monowitz camp detainee hospital . as part of his neurological and psychiatric education , drohocki had done research in berne , brussels and paris on electro - encephalography , and had also gained practical experience in electroconvulsive therapy in grenoble . during his attempt to flee to switzerland from france , then occupied by the germans , the senseless luxury of such a state - of - the - art healing method in view of the gas chambers , as drohocki retrospectively put it , had not least been the most prestigious project of the ss physicians wirths and fischer . thus , when the leading physician of the concentration camp , enno lolling ( 18881945 ) , visited at the end of 1943 , fischer demonstrated the therapy on a prisoner to demonstrate that electroshock can make emotionally disturbed people fit for work again. in his position , lolling was directly under the control of the head of amtsgruppe d at the ss - wirtschafts - verwaltungshauptamt ( ss head office for economic and administrative affairs ) , who constantly emphasised the optimisation of work amongst those prisoners employed in the german war effort . in fact , it seemed that the ss head office for economic and administrative affairs developed an enduring interest in the introduction of the method after the presentation , because in the spring of 1944 electroshock therapy was addressed again within a discussion amongst all the ss physicians employed in the concentration camps in auschwitz . prior to this , drohocki had received the order to write a scientific lecture about electroshocks in general , with particular reference to research in monowitz , which was subsequently presented by fischer . yet again , it concerned healing the mentally ill in order to reincorporate them into the working processes. while drohocki , because of a widespread shortage of anaesthesia , was himself testing electroshock as an anaesthetic method for surgical means , the ss s interest was in utilising humans as resources in the service of the nation and warfare . during the criminal proceedings against fischer in 1966 , many of the other detainee physicians who had , like drohocki , been involved in the electroshock therapy emphasised that they would have been able to save many a human life thanks to the device , in the future , if only their own at the time . after the war , however , it became known that in austria , part of the german reich , an electroshock device had indeed been used for euthanasia murder . psychiatric inpatients had been personally killed by physician emil gelny ( 18901961 ) , who had been medical director for the lower - austrian mental hospitals gugging and mauer hling since october 1943 . in gugging and mauer hling , gelny had used an elkra ii , which he had himself converted , on a total of around three hundred occasions . even if gelny had not developed this method of murder on the orders of the t4 organisation , he had nevertheless turned to them , and specifically to nitsche , in february 1944 with his euthanasia suggestion . this was at a time when the t4 had long been looking for a new solution to the e - problem , as it had been labelled in the correspondence of karl brandt ( 190448 ) , hitler s general commissioner for the medical and health system , in the sense of a centrally planned but locally conducted euthanasia . as a result , a meeting of doctors and the heads of those institutions directly involved in euthanasia took place in vienna in the summer of 1944 , on which occasion gelny demonstrated the use of the apparatus and the method with the murder of a mental patient in gugging . with regards to this demonstration , it also seemed very valuable to nitsche to be present at the inspection of the electrical method by dr gelny planned by brandt . however , gelny might not have been the first to develop the idea of using electroshock therapy for the explicit purpose of electrocution. dr elisabeth v. , a young psychiatrist at eichberg between 1942 and 1945 , remembered discussions on the topic of killing mental patients at eichberg : when he acquired an electroshock apparatus , director mennecke raised the question of whether one could effect their deaths through high levels of electric current passed through their brains . when he acquired an electroshock apparatus , director mennecke raised the question of whether one could effect their deaths through high levels of electric current passed through their brains . friedrich mennecke ( 190447 ) had been director of the psychiatric asylum in eichberg since 1939 ; during the nuremburg trials , mennecke was especially condemned for the murder of children in eichberg as part of schneider s research project in heidelberg . gelny s criminal case remains the only officially known murder of patients using a converted electroshock therapy device . in 1946 , holzer was appointed as an expert in the connected criminal case before the national court in vienna against the employees of the hospitals in gugging and mauer - hling , tasked with explaining the changes made by gelny to the elkra ii in order to exploit the therapeutic device to discreetly continue murdering patients . as , in the late summer of 1944 , holzer had contacted nitsche , the medical leader of the t4 campaign , to advertise for a research institute dedicated to electroshock therapy , it has been questioned as to whether holzer had himself been involved in the deliberations over the use of his elkra apparatus for euthanasia murders . holzer had requested the foundation of a research institute , reasoning that his application was timely because of the legal issues surrounding the euthanasia question. these attempts , according to holzer , forced the psychiatrist to arrive at a decision. however , holzer did not make an allusion to a euthanasia , but stressed instead that efforts around the basic problems of psychiatry and the development of therapies must be increased a thousand - fold in order to approach the euthanasia problems around psychoses at all. myths , prejudices and clichs have prevailed in many accounts of the history of electroshock therapy in the third reich . however , the clinical introduction of the new shock therapy in the german reich and occupied territories between 1936 and 1945 was neither especially swift nor radical. at the beginning , high - ranking members of the nazi academic and state elite saw electroshock therapy , like the preceding shock therapies , insulin coma and cardiazol convulsive therapies , as a contradiction of the genetic dogma of schizophrenia . core schizophrenia was a recessively inherited disease whose only causal treatment was primary prevention by sterilisation . in 1942 , however , the war - related shortages in insulin forced the commander responsible for the mental hospitals , linden , to forbid insulin coma therapy and propose electroshock therapy as an alternative . only after the end of the official euthanasia murder operation t4 in august 1941 , and increasingly confronted with the unexpected clinical successes reported by bumke in munich or braunmhl in eglfing - haar , did the national socialist psychiatry officials start to promote electroshock therapy as a modern unspecific treatment . euthanasia. however , war - related shortages even hindered the now politically supported production of electroshock devices . moreover , clinics or asylums that received a device with the exception of scientific centres and psychiatric hospitals especially interested in the new therapies and electroshock ( eglfing - haar , erlangen , gttingen etc . ) often made very limited use of it , occasionally changing the diagnosis of schizophrenia after successful treatments to reactive psychosis. of course , further analysis of medical files from different clinics is necessary if we are to draw a definite picture . the psychiatrist that indeed proposed the implementation of such a specialised institute , holzer , was not one of the nazi officials ; in fact , he was not even given his medical post - doctoral qualification ( habilitation ) until after the war and the fall of nazism . finally , the research done under carl schneider in heidelberg focused on the problem of vertebral fractures , and thus the clinical safety of electroshock therapy , research which was shared internationally . industrial companies like srw were decisive participants in the development of the electroshock technology , including in the solution of the therapy s safety issues . ultimately , they pursued their own interests . while designing their own devices and electrode holders , srw used the patents of slaughterhouse devices to protect themselves against competitors in the new electroshock therapy market . indeed , the subordination of electroshock under the broader term anaesthesia and of patients under animals to be slaughtered shall represent a unique curiosity for a leading electromedicine company : thus holzer had deplored it in 1941 . with regards to the use of a self - made electroshock device in the monowitz detainee hospital auschwitz iii , the working camp of the ig farben , there is no evidence of dubious experimentation or the introduction of electroshock therapy in the third reich is not adequately conceptualised under the paradigm of a dialectics of healing and killing. it did admittedly have the potential to become a very special version of that dialectic , where the difference was in the dose , and where anaesthesia was paired with the possibility of a healing convulsion or killing , both caused by electric current . this was cerletti s insight from the slaughterhouse studies , an insight that gave him justification to proceed to a clinical application , since the security margin was significant . cerletti was , however , not the last person to realise this . while scientists and technicians made great efforts to make euthanasia murders reflected in 1944 on the possibility of utilising electroshock therapy devices as killing instruments . it was this horrifying scenario which was indeed realised in southern austria . on a theoretical level , the analysis of electroshock therapy in the third reich demonstrates mechanisms in the introduction of a new medical therapy that went far beyond questions of therapeutic efficacy . medical and clinical needs played only a subsidiary role when confronted with the commercial interest of the major industrial companies involved , who came to redefine electroshock therapy as a problem of patent law . under the peculiar conditions of national socialism , fascism and the second world war , the process was in fact so flexible that it not only made electroshock therapy a state - of - the - art healing method utilised in proximity to the gas chambers of auschwitz but also perverted it into an instrument of euthanasia murder .

the history of electroshock therapy ( now known as electroconvulsive therapy ( ect ) ) in europe in the third reich is still a neglected chapter in medical history . since thomas szasz s from the slaughterhouse to the madhouse , prejudices have hindered a thorough historical analysis of the introduction and early application of electroshock therapy during the period of national socialism and the second world war . contrary to the assumption of a dialectics of healing and killing , the introduction of electroshock therapy in the german reich and occupied territories was neither especially swift nor radical . electroshock therapy , much like the preceding shock therapies , insulin coma therapy and cardiazol convulsive therapy , contradicted the genetic dogma of schizophrenia , in which only one treatment was permissible : primary prevention by sterilisation . however , industrial companies such as siemens reiniger werke ag ( srw ) embraced the new development in medical technology . moreover , they knew how to use existing patents on the electrical anaesthesia used for slaughtering to maintain a leading position in the new electroshock therapy market . only after the end of the official euthanasia murder operation in august 1941 , entitled t4 , did the psychiatric elite begin to promote electroshock therapy as a modern unspecific treatment in order to reframe psychiatry as an honorable medical discipline . war - related shortages hindered even the then politically supported production of electroshock devices . research into electroshock therapy remained minimal and was mainly concerned with internationally shared safety concerns regarding its clinical application . however , within the third reich , electroshock therapy was not only introduced in psychiatric hospitals , asylums , and in the auschwitz concentration camp in order to get patients back to work , it was also modified for euthanasia murder .

Introduction The Italian Invention of Electroshock Therapy: From a Science of Epilepsy to a Therapy of Schizophrenia The Way to the First German Experiences and the Siemens Konvulsator National Socialist Austerity Plans: The Timeline of the Introduction of Electroshock Therapy Therapeutic Enthusiasm? Electroshock Therapys Sales Markets during theWar Doing Research in Wartime? Prevention of Vertebral Fractures with ConvulsiveShock A Therapy in Auschwitz or a Solution to the E-Problem? Summary

seventy - seven days before the outbreak of the second world war , the german - language neurological and psychiatric journal zentralblatt announced a new , revolutionary method in the shock therapy of schizophrenia coming from rome. that same year , the new treatment was indeed introduced in many countries in europe , and in japan , latin america and the usa . with regards to europe , however , it is assumed that national socialism played a decisive role in the introduction of this new shock therapy . the research literature not only emphasises the flight of doctors and scientists of jewish descent or faith from national socialism , but particularly supports the thesis that the introduction of the new technology in the third reich had drawn special interest from high - ranking officials of the nazi regime . electroshock therapy ( now known as electroconvulsive therapy ( ect ) ) was swiftly integrated into the dialectics of healing and killing typical of medicine in nazi germany. since szasz s article from the slaughterhouse to the madhouse , the invention of electroshock therapy has generally been regarded as modern therapeutic totalitarianism. social histories such as braslow s mental ills and bodily cures seem to have reinforced the view popularised by one flew over the cuckoo s nest of electroshock therapy as a furthermore , it is frequently assumed that if electroshock therapy was indeed a disciplinary medical therapy , then its introduction and application in the third reich must have been particularly cruel . indeed , such highly regarded historical research on the third reich and the euthanasia murders as henry friedlander s the origins of nazi genocide : from euthanasia to the final solution claims that during the nazi period , electroshock was no therapy , but rather a treatment used to inflict torture. within the third reich , it had finally been the bureaucratic apparatus for organising the mass murder of mentally disabled and mentally ill people which under the conditions of war economy provided german psychiatric hospitals with the apparatuses necessary for electroconvulsive therapy and which considered the establishment of institutes for investigating the new technology . although most histories of electroshock therapy failed to engage with the complexities of the existence of electroshock therapy in the third reich , preliminary investigations on the chronology of the introduction and implementation of the new therapy in europe and especially in germany raise doubts as to whether national socialist health policy favoured the introduction of the new shock therapy for the german reich , or whether it in fact delayed it . following this line of enquiry , our article proposes a chronological analysis of the invention and subsequent introduction of electroshock therapy in the third reich and occupied territories . therapeutic enthusiasm concerning electroshock within the third reich is queried , as well as the existence of state - subsidised research institutions with the explicit purpose of studying and promoting electroshock therapy . the ways in which new scientific knowledge was acquired and put into practice were many - faceted , and dependent on the diverging interests of physicians , industrial companies , mental health politics and the exigencies of total war . our article describes how the changing medico - political and economic context during the third reich and the second world war not only shaped the design of the devices for electroshock therapy but actually reversed their function as tools of medical therapy . it is evident , therefore , that the electroshock idea was not born in the roman slaughterhouse and it was not primarily tested on pigs as szasz suggests with his from the slaughterhouse to the madhouse story . in any case , the maestro , as he was called , did not need to discover something the fact that cerletti did choose a newly admitted patient , instead of a well - known patient of his own , has been widely critiqued ; however , in the light of the history of human experimentation in medicine , it appears neither as a feature exclusively characteristic of psychiatry and neurology , nor even of medicine in the third reich or in europe . however , in the first half of 1939 , the new method hardly attract[ed ] any interest , as max mller ( 18941980 ) , who had organised the swiss conference on the pharmacological shock therapies , later remembered . this early german start in germany was made possible because erlangen had the head office of the electro - medical research laboratory of siemens reiniger werke ( srw ) , the leading company for medical technology in the german reich . however , it was not until october 1941 that bumke stated that , despite his predictions , the new convulsive therapy did produce positive results with those suffering from schizophrenia and other mental illnesses and that the treatment was therefore to be recommended . following this , meggendorfer , who had been appreciated in the early 1930s for his genetic studies on huntington s disease and his eugenic thinking on moral insanity , or personality failures , saw in the official congratulations on his sixtieth birthday that the success of having been the first german psychiatrist to embrace the new therapy was not mentioned at all . the congratulations were written by ernst rdin ( 18741952 ) , who at this time viewed electroshock therapy , like both insulin coma and cardiazol - convulsive therapies , as a contradiction of the genetic view of endogenous psychoses , especially with regards to schizophrenia . the law enabled forced sterilisation in over 400 000 cases of schizophrenia , epilepsy , huntington s disease , hereditary blindness or deafness , congenital mental deficiency or manic depressive illness until the end of the third reich . additionally , and in contrast to the italian electrode clip or forceps maximally compressing the electrodes together and pressing them strongly against the skin , srw s electrodes were to be electroshock patent , srw saw themselves confronted with another competitive construction built by the vienna - based company reiner & co. it was developed by a graduate technician and psychiatrist at the vienna university psychiatric clinic , wolfgang holzer ( 190680 ) , who had been given the order to scrutinise the arcioni device and to adapt it to the needs of the vienna clinic . this advantage to enable shocking even the most unsettled patients in a comfortable manner had quickly piqued the interest of psychiatric hospitals even if due to the war events it was difficult to get such an apparatus. this made srw find a remarkable way to protect themselves against elkra ii as competitor in the sales area of electroshock therapy in the german reich , to which austria officially belonged since the anschluss of 1938 . 578 532 for the forceps form of the anaesthesia device for the electrical anaesthesia of animals to be slaughtered precisely the technique that bini had studied in the roman slaughterhouse to test the safety margin of the application of electricity to the head . braunmhl had been the main protagonist of insulin coma therapy and cardiazol - convulsive therapy in the third reich . still , in september 1944 , holzer tried to get support for his research in electroshock therapy ; however , his idea of a whole scientific institute for investigating electroshock was never realised . the low sales figures of the konvulsator were not least the consequence of a politically desired radical policy of austerity in psychiatry in the german reich , intended to lead to the starvation of patients with lives unworthy of living. from its start in october 1939 , until its official discontinuation in august 1941 , the construction of gas chambers on former hospital grounds and a bureaucratic procedure of camouflaging was funded to murder more than 700 000 patients with diagnoses such as schizophrenia , mental deficiency , epilepsy , dementia or huntington s disease . crucially , the consequences of another budget cut in psychiatry contributed to an increase of interest in convulsive therapy and particularly electroshock therapy in the spring of 1942 . these genetic views and the consequent nazi sterilisation policy had at first considerably delayed the introduction of insulin coma therapy in germany in the mid-1930s and also caused an initial restraint towards electroshock therapy in well - known clinics . its medical director , heinrich schulte ( 18981983 ) , recommended electroconvulsive therapy as an alternative to the pharmacological convulsive therapy as early as june 1941 , including at the conference of the berliner gesellschaft fr psychiatrie und neurologie , since it was less stressful for the patients , simpler , cheaper and also suitable for ambulant treatment . that the circle of leading national socialist psychiatrists , based around , amongst others , ernst rdin , maximinian de crinis , paul nitsche and carl schneider , finally began to advertise convulsive therapies was the result of a shift in nazi mental health politics ; to counter the public opinion that psychiatry is becoming more and more superfluous since mentally ill people would soon become extinct due to the racial hygienic laws , the convulsive methods , including prominently electroshock therapy , promised to justify a future eugenic national socialist psychiatry of sterilisation , euthanasia and therapy . probably with a view to introducing this new way for psychiatry in the third reich , a number of lectures on electroshock therapy , among them lectures by meggendorfer and bingel , max mller and also wolfgang holzer , had appeared on the agenda of the sixth annual assembly of the gesellschaft deutscher neurologen und psychiater scheduled for 57 october 1941 . while deaths from medicine overdoses and through starvation continued after the termination of the official t4 campaign in august 1941 , financial means for equipping the mental hospitals with therapy techniques were thus granted upon linden s mediation and an educational film on electroshock therapy was produced . although heuyer had even announced an export of konvulsatoren to france , at the occasion of the presentation of the initial dog experiments for a french apparatus at the conference of the socit mdico - psychologique in april 1941 , most psychiatric hospitals in the occupied france , as , for example , the hpital du vinatier near lyon , known from the study by isabelle von bueltzingsloewen on starvation in french psychiatry during the german occupation , used equipment from purtschert & co. on german territory against widespread assumptions to the contrary the war - related production bottlenecks meant that many of the psychiatric hospitals without university affiliation did not receive an apparatus before 1944 , and some , only after the war . in the later french occupation zone , for example , during the third reich , none of the big psychiatric asylums in bad schussenried , zwiefalten or weissenau had an electroshock apparatus in use . the french psychiatric commissioner sent to inspect the situation in the psychiatric hospitals at the end of the war in 1945 noticed with astonishment : this method has never been practised in these three institutions and this fact appears monstrous to the psychiatrist that knows of the remarkable results of electroconvulsive therapy in the treatment of acute psychosis . this method has never been practised in these three institutions and this fact appears monstrous to the psychiatrist that knows of the remarkable results of electroconvulsive therapy in the treatment of acute psychosis . the minor diffusion of electroshock therapy was not solely the result of an inadequate supply or inadequate diffusion of electroshock devices ; most of the ordered reichsausschuss konvulsatoren had finally been delivered to hospitals and asylums . indeed , the notion of a quick and extensive introduction of electroshock therapy in the third reich seems to be nothing more than a myth ; a clich , as heinz faulstich has argued in his major work , starvation in psychiatry 19141949 : a historically mistaken , if widely cherished , idea that psychiatrists during the time of national socialism were widely enthusiastic about euthanasia and shock therapies . in saxony , for example , in the psychiatric hospital altscherbitz , an analysis of 1400 patient files , including 497 with the diagnosis of schizophrenia , found a single case of electroshock treatment . for the saxon psychiatric hospital groschweidnitz , the finding was similar : only with may 1942 are there a handful of treatment trials of the quite cheap treatment documented in the patient files ; however , it never achieved greater significance. in contrast , the asylum eglfing - haar near munich had a high incidence of electroshock therapy , as here anton von braunmhl , the most ardent proponent and defender of the somatic therapies in the third reich , had done everything to convince srw to give him the second prototype for clinical use . but besides the university hospitals and asylums with psychiatrists specifically interested in the treatment , the diffusion of electroshock therapy remained minimal in the daily practice of the mental institutions in the german reich until the end of the war . in psychiatric hospitals that had a special focus on electroshock therapy , sometimes explicit consent for shock therapy was required from the family . on the other hand , psychiatric hospitals whose clinical directors completely subscribed to the ideology of schizophrenia as a genetic disease such as the wittenauer heilsttten in berlin changed the diagnosis in the case of a successful electroshock treatment , for example , to reactive psychosis. in conclusion , the dialectics of healing and killing , as promoted in the 1943 denkschrift letter thoughts and suggestions concerning the future development of psychiatry written by the elite of the nazi psychiatrists , remained a pure ideal , unattained until the end of the third reich . regarding the everyday practice in most of the mental institutions of the german reich , there was no dialectics of healing and killing but a prevailing policy of starvation , leaving people to die and murder . however , even srw reacted with restraint to hints that a model institution for research into electroshock treatment was to emerge in southern germany , in co - operation with the heidelberg university hospital and under the direction of carl schneider and the mental hospital at wiesloch . actually , the reich working party for mental asylums , represented by linden and nitsche , had been planning the establishment of a psychiatric research institution with its main pillar in heidelberg yet this was intended less as a model institution for the research of electroshock treatment , as the srw branch in mannheim had understood from the discussions in heidelberg with linden and schneider , but rather for enhancing schneider s own genetic , constitutional and neurobiological examinations of mentally deficient children. however , srw did not want to make the same mistake as its predecessor companies during the first world war , when work on developments for civilian and medical- technology use had been considerably limited for reasons of war economy , with the consequence that srw had , after the end of the war , been left behind compared to the international competition . however , they took the position that tests for improving the apparatuses , whose production is not forbidden , are also allowed , and , beyond the wording of any regulations , it must be left to the company to decide which of the works will be postponed and which will not. meanwhile , the problem of vertebral fractures had not remained of purely scientific or medical significance , as it influenced decisively the choice for linden s collective device order for the clinics and mental hospitals of the german reich and thereby consolidated srw s position on the electroshock market . in the third reich , the distinctive conditions of national socialism and total war meant a particular interest in electroshock therapy . in fact , it seemed that the ss head office for economic and administrative affairs developed an enduring interest in the introduction of the method after the presentation , because in the spring of 1944 electroshock therapy was addressed again within a discussion amongst all the ss physicians employed in the concentration camps in auschwitz . during the criminal proceedings against fischer in 1966 , many of the other detainee physicians who had , like drohocki , been involved in the electroshock therapy emphasised that they would have been able to save many a human life thanks to the device , in the future , if only their own at the time . after the war , however , it became known that in austria , part of the german reich , an electroshock device had indeed been used for euthanasia murder . in 1946 , holzer was appointed as an expert in the connected criminal case before the national court in vienna against the employees of the hospitals in gugging and mauer - hling , tasked with explaining the changes made by gelny to the elkra ii in order to exploit the therapeutic device to discreetly continue murdering patients . as , in the late summer of 1944 , holzer had contacted nitsche , the medical leader of the t4 campaign , to advertise for a research institute dedicated to electroshock therapy , it has been questioned as to whether holzer had himself been involved in the deliberations over the use of his elkra apparatus for euthanasia murders . myths , prejudices and clichs have prevailed in many accounts of the history of electroshock therapy in the third reich . however , the clinical introduction of the new shock therapy in the german reich and occupied territories between 1936 and 1945 was neither especially swift nor radical. at the beginning , high - ranking members of the nazi academic and state elite saw electroshock therapy , like the preceding shock therapies , insulin coma and cardiazol convulsive therapies , as a contradiction of the genetic dogma of schizophrenia . in 1942 , however , the war - related shortages in insulin forced the commander responsible for the mental hospitals , linden , to forbid insulin coma therapy and propose electroshock therapy as an alternative . only after the end of the official euthanasia murder operation t4 in august 1941 , and increasingly confronted with the unexpected clinical successes reported by bumke in munich or braunmhl in eglfing - haar , did the national socialist psychiatry officials start to promote electroshock therapy as a modern unspecific treatment . however , war - related shortages even hindered the now politically supported production of electroshock devices . finally , the research done under carl schneider in heidelberg focused on the problem of vertebral fractures , and thus the clinical safety of electroshock therapy , research which was shared internationally . while designing their own devices and electrode holders , srw used the patents of slaughterhouse devices to protect themselves against competitors in the new electroshock therapy market . with regards to the use of a self - made electroshock device in the monowitz detainee hospital auschwitz iii , the working camp of the ig farben , there is no evidence of dubious experimentation or the introduction of electroshock therapy in the third reich is not adequately conceptualised under the paradigm of a dialectics of healing and killing. on a theoretical level , the analysis of electroshock therapy in the third reich demonstrates mechanisms in the introduction of a new medical therapy that went far beyond questions of therapeutic efficacy . medical and clinical needs played only a subsidiary role when confronted with the commercial interest of the major industrial companies involved , who came to redefine electroshock therapy as a problem of patent law . under the peculiar conditions of national socialism , fascism and the second world war , the process was in fact so flexible that it not only made electroshock therapy a state - of - the - art healing method utilised in proximity to the gas chambers of auschwitz but also perverted it into an instrument of euthanasia murder .

seventy - seven days before the outbreak of the second world war , the german - language neurological and psychiatric journal zentralblatt announced a new , revolutionary method in the shock therapy of schizophrenia coming from rome. a method , as well as suitable apparatus , had apparently been found which superseded the convulsive therapy of schizophrenia , which had been introduced by ladislas meduna ( 18961964 ) in 1934 and relied on epileptic seizures triggered with camphor or pentylenetetrazol ( cardiazol ) . the research literature not only emphasises the flight of doctors and scientists of jewish descent or faith from national socialism , but particularly supports the thesis that the introduction of the new technology in the third reich had drawn special interest from high - ranking officials of the nazi regime . social histories such as braslow s mental ills and bodily cures seem to have reinforced the view popularised by one flew over the cuckoo s nest of electroshock therapy as a furthermore , it is frequently assumed that if electroshock therapy was indeed a disciplinary medical therapy , then its introduction and application in the third reich must have been particularly cruel . within the third reich , it had finally been the bureaucratic apparatus for organising the mass murder of mentally disabled and mentally ill people which under the conditions of war economy provided german psychiatric hospitals with the apparatuses necessary for electroconvulsive therapy and which considered the establishment of institutes for investigating the new technology . although most histories of electroshock therapy failed to engage with the complexities of the existence of electroshock therapy in the third reich , preliminary investigations on the chronology of the introduction and implementation of the new therapy in europe and especially in germany raise doubts as to whether national socialist health policy favoured the introduction of the new shock therapy for the german reich , or whether it in fact delayed it . our article describes how the changing medico - political and economic context during the third reich and the second world war not only shaped the design of the devices for electroshock therapy but actually reversed their function as tools of medical therapy . on the contrary , cerletti had been quite reluctant to proceed to a clinical application : this possibility [ of using electricity to induce convulsions in people as they were then produced by cardiazol ] was often discussed at our institute and particularly when observing , after cardiazol injections , the painful reactions which shook fully conscious patients for over a minute as a result most of them refused the subsequent treatments . and i myself found in the literature on electrology reports on death in man caused by electric currents of lower voltage than that used on our experimental dogs indeed , cerletti continued in his autobiographical notes to say that it was a chance occurrence involving some new electrical equipment at the slaughterhouse in rome that allowed him to make the next step . in any case , the maestro , as he was called , did not need to discover something the fact that cerletti did choose a newly admitted patient , instead of a well - known patient of his own , has been widely critiqued ; however , in the light of the history of human experimentation in medicine , it appears neither as a feature exclusively characteristic of psychiatry and neurology , nor even of medicine in the third reich or in europe . indeed , by the summer of 1939 , srw had requested a report about the medical usefulness of the new srw had asked oswald bumke ( 18771950 ) , the director of the psychiatric clinic at the ludwig maximilian university of munich , who was also editor of the reference work handbook of mental diseases , for an expert opinion , because munich had obtained an arcioni apparatus as early as july 1939 thanks to lothar kalinowsky . following this , meggendorfer , who had been appreciated in the early 1930s for his genetic studies on huntington s disease and his eugenic thinking on moral insanity , or personality failures , saw in the official congratulations on his sixtieth birthday that the success of having been the first german psychiatrist to embrace the new therapy was not mentioned at all . in spite of this initial reluctance towards the new therapy due to the preponderance of genetic thinking in german psychiatry and nazi mental health politics , srw were able to present a genuinely german electroshock apparatus under the name konvulsator at the end of june 1940 ( see figure 3 ) . additionally , and in contrast to the italian electrode clip or forceps maximally compressing the electrodes together and pressing them strongly against the skin , srw s electrodes were to be electroshock patent , srw saw themselves confronted with another competitive construction built by the vienna - based company reiner & co. it was developed by a graduate technician and psychiatrist at the vienna university psychiatric clinic , wolfgang holzer ( 190680 ) , who had been given the order to scrutinise the arcioni device and to adapt it to the needs of the vienna clinic . as srw turned to reiner & co. to assert claims from this patent on the holder electrode of elkra i and ii , holzer responded with indignation : the subordination of electroshock under the broader term anaesthesia and of patients under animals to be slaughtered [ ] shall represent a unique curiosity for a leading electromedicine company . in turn , reiner & co. committed themselves to pay a licence fee to the amount of one per cent from the list price of the apparatus for all electroshock devices produced and sold by reiner & co. meanwhile , srw got help from anton edler von braunmhl ( 190157 ) , at the mental hospital eglfing - haar , to also develop more comfortable electrodes , which were pressed to the patient s head and which enabled the current release at the same time ( see figure 5 ) . this was the experience of , for example , gottfried ewald ( 18881963 ) , who had been desperately looking for a device for the university psychiatric hospital in gttingen and had finally been able only as a result of personal initiative to acquire a device from switzerland , from purtschert & co. , in the summer of 1941 . from its start in october 1939 , until its official discontinuation in august 1941 , the construction of gas chambers on former hospital grounds and a bureaucratic procedure of camouflaging was funded to murder more than 700 000 patients with diagnoses such as schizophrenia , mental deficiency , epilepsy , dementia or huntington s disease . with the responsibility for mental hospitals passing to herbert linden ( 18991945 ) , the beginning of new insulin coma cures had generally been forbidden as insulin was scarce and insulin coma therapy could not be regarded as a specific therapy for schizophrenia as there were also other methods ( such as cardiazol and electroshock ) available. indeed , the cherished genetic theory of endogen psychoses and especially of schizophrenia implied that insulin coma , cardiazol or electroshock therapies could only have unspecific impacts similar to states of shock induced by means of cold showers or a swivelchair ; a real therapy of the core group of schizophrenia was only conceivable by prevention , namely sterilisation . these genetic views and the consequent nazi sterilisation policy had at first considerably delayed the introduction of insulin coma therapy in germany in the mid-1930s and also caused an initial restraint towards electroshock therapy in well - known clinics . its medical director , heinrich schulte ( 18981983 ) , recommended electroconvulsive therapy as an alternative to the pharmacological convulsive therapy as early as june 1941 , including at the conference of the berliner gesellschaft fr psychiatrie und neurologie , since it was less stressful for the patients , simpler , cheaper and also suitable for ambulant treatment . that the circle of leading national socialist psychiatrists , based around , amongst others , ernst rdin , maximinian de crinis , paul nitsche and carl schneider , finally began to advertise convulsive therapies was the result of a shift in nazi mental health politics ; to counter the public opinion that psychiatry is becoming more and more superfluous since mentally ill people would soon become extinct due to the racial hygienic laws , the convulsive methods , including prominently electroshock therapy , promised to justify a future eugenic national socialist psychiatry of sterilisation , euthanasia and therapy . probably with a view to introducing this new way for psychiatry in the third reich , a number of lectures on electroshock therapy , among them lectures by meggendorfer and bingel , max mller and also wolfgang holzer , had appeared on the agenda of the sixth annual assembly of the gesellschaft deutscher neurologen und psychiater scheduled for 57 october 1941 . while deaths from medicine overdoses and through starvation continued after the termination of the official t4 campaign in august 1941 , financial means for equipping the mental hospitals with therapy techniques were thus granted upon linden s mediation and an educational film on electroshock therapy was produced . facing continuing complaints , which also spread into public discourse , that the difficulties of material procurement do not favour the spreading of electroconvulsive treatment , ninety - five devices were finally ordered from srw at the end of february 1943 with a collective order by the management of the reichsauschusses zur wissenschaftlichen erfassung von erb- und anlagebedingten schweren leiden ( state committee for the scientific assessment of severe hereditary disease ) , who had also organised the t4 programme . yet , even the delivery of the devices of the wehrmacht order happened technical reason for the delays was the fact that many clinics did not have any alternating current available , since the in - house electricity grid was exclusively supplied with direct current . although heuyer had even announced an export of konvulsatoren to france , at the occasion of the presentation of the initial dog experiments for a french apparatus at the conference of the socit mdico - psychologique in april 1941 , most psychiatric hospitals in the occupied france , as , for example , the hpital du vinatier near lyon , known from the study by isabelle von bueltzingsloewen on starvation in french psychiatry during the german occupation , used equipment from purtschert & co. on german territory against widespread assumptions to the contrary the war - related production bottlenecks meant that many of the psychiatric hospitals without university affiliation did not receive an apparatus before 1944 , and some , only after the war . the french psychiatric commissioner sent to inspect the situation in the psychiatric hospitals at the end of the war in 1945 noticed with astonishment : this method has never been practised in these three institutions and this fact appears monstrous to the psychiatrist that knows of the remarkable results of electroconvulsive therapy in the treatment of acute psychosis . indeed , the notion of a quick and extensive introduction of electroshock therapy in the third reich seems to be nothing more than a myth ; a clich , as heinz faulstich has argued in his major work , starvation in psychiatry 19141949 : a historically mistaken , if widely cherished , idea that psychiatrists during the time of national socialism were widely enthusiastic about euthanasia and shock therapies . in contrast , the asylum eglfing - haar near munich had a high incidence of electroshock therapy , as here anton von braunmhl , the most ardent proponent and defender of the somatic therapies in the third reich , had done everything to convince srw to give him the second prototype for clinical use . on the other hand , psychiatric hospitals whose clinical directors completely subscribed to the ideology of schizophrenia as a genetic disease such as the wittenauer heilsttten in berlin changed the diagnosis in the case of a successful electroshock treatment , for example , to reactive psychosis. in conclusion , the dialectics of healing and killing , as promoted in the 1943 denkschrift letter thoughts and suggestions concerning the future development of psychiatry written by the elite of the nazi psychiatrists , remained a pure ideal , unattained until the end of the third reich . actually , the reich working party for mental asylums , represented by linden and nitsche , had been planning the establishment of a psychiatric research institution with its main pillar in heidelberg yet this was intended less as a model institution for the research of electroshock treatment , as the srw branch in mannheim had understood from the discussions in heidelberg with linden and schneider , but rather for enhancing schneider s own genetic , constitutional and neurobiological examinations of mentally deficient children. whatever the potential plans concerning research into electroshock , ptzold explicitly pointed out that because of its extensive involvement in the war economy , srw had practically been deprived of all possibilities for more extensive development work for the duration of the war so that currently collaboration can only be in exchanging experiences and discussing pending problems in this field. however , srw did not want to make the same mistake as its predecessor companies during the first world war , when work on developments for civilian and medical- technology use had been considerably limited for reasons of war economy , with the consequence that srw had , after the end of the war , been left behind compared to the international competition . on the part of srw , the response to this was that mainly sick soldiers were treated with electric convulsion at dr von braunmhl s and hence comparative experiments for determining the reasonable method are thus also part of looking after sick soldiers and must also be regarded as relevant to war. as for the heidelberg efforts in wiesloch , schneider was in any case resigned to the fact that after two attempts the war did not [ allow ] the realisation of the extensive projects planned at that time and that the implementation of total warfare after the disaster of stalingrad brought about the dissolution of the newly started wiesloch department again in 1944. some research had nevertheless been done on electroshock at the heidelberg clinic , and then also at the partner institution in wiesloch . as bingel had already warned in 1940 that the electroconvulsive treatment is not as free of complications as it seemed according to first reports by the italian authors , schneider s chief heidelberg physician , fritz schmieder ( 191188 ) , had started to examine the frequency of vertebral fractures under electroconvulsive therapy since february 1942 . it was precisely because of the claim that vertebral fractures were few with the srw apparatus compared to the competing devices that the collective order went to srw in spite of the konvulsator being the most expensive device ( 955 rm against 390 rm for elkra ii , for example ) : a claim that was pure advertising , as its author , meggendorfer , in erlangen , did not posses any clinical experience with the other devices . with the unofficial agreement of the ss camp physicians eduard wirths ( 190945 ) and horst fischer ( 191666 ) , the polish detainee physician zenon drohocki ( 190378 ) , helped by a dutch engineer detainee , wanted to treat sick detainees with the apparatus in the neuropsychiatric department which he had established inside the monowitz camp detainee hospital . during his attempt to flee to switzerland from france , then occupied by the germans , the senseless luxury of such a state - of - the - art healing method in view of the gas chambers , as drohocki retrospectively put it , had not least been the most prestigious project of the ss physicians wirths and fischer . thus , when the leading physician of the concentration camp , enno lolling ( 18881945 ) , visited at the end of 1943 , fischer demonstrated the therapy on a prisoner to demonstrate that electroshock can make emotionally disturbed people fit for work again. in his position , lolling was directly under the control of the head of amtsgruppe d at the ss - wirtschafts - verwaltungshauptamt ( ss head office for economic and administrative affairs ) , who constantly emphasised the optimisation of work amongst those prisoners employed in the german war effort . in fact , it seemed that the ss head office for economic and administrative affairs developed an enduring interest in the introduction of the method after the presentation , because in the spring of 1944 electroshock therapy was addressed again within a discussion amongst all the ss physicians employed in the concentration camps in auschwitz . during the criminal proceedings against fischer in 1966 , many of the other detainee physicians who had , like drohocki , been involved in the electroshock therapy emphasised that they would have been able to save many a human life thanks to the device , in the future , if only their own at the time . this was at a time when the t4 had long been looking for a new solution to the e - problem , as it had been labelled in the correspondence of karl brandt ( 190448 ) , hitler s general commissioner for the medical and health system , in the sense of a centrally planned but locally conducted euthanasia . as a result , a meeting of doctors and the heads of those institutions directly involved in euthanasia took place in vienna in the summer of 1944 , on which occasion gelny demonstrated the use of the apparatus and the method with the murder of a mental patient in gugging . in 1946 , holzer was appointed as an expert in the connected criminal case before the national court in vienna against the employees of the hospitals in gugging and mauer - hling , tasked with explaining the changes made by gelny to the elkra ii in order to exploit the therapeutic device to discreetly continue murdering patients . as , in the late summer of 1944 , holzer had contacted nitsche , the medical leader of the t4 campaign , to advertise for a research institute dedicated to electroshock therapy , it has been questioned as to whether holzer had himself been involved in the deliberations over the use of his elkra apparatus for euthanasia murders . at the beginning , high - ranking members of the nazi academic and state elite saw electroshock therapy , like the preceding shock therapies , insulin coma and cardiazol convulsive therapies , as a contradiction of the genetic dogma of schizophrenia . only after the end of the official euthanasia murder operation t4 in august 1941 , and increasingly confronted with the unexpected clinical successes reported by bumke in munich or braunmhl in eglfing - haar , did the national socialist psychiatry officials start to promote electroshock therapy as a modern unspecific treatment . with regards to the use of a self - made electroshock device in the monowitz detainee hospital auschwitz iii , the working camp of the ig farben , there is no evidence of dubious experimentation or the introduction of electroshock therapy in the third reich is not adequately conceptualised under the paradigm of a dialectics of healing and killing. under the peculiar conditions of national socialism , fascism and the second world war , the process was in fact so flexible that it not only made electroshock therapy a state - of - the - art healing method utilised in proximity to the gas chambers of auschwitz but also perverted it into an instrument of euthanasia murder .

age - related macular degeneration ( amd ) is an ocular disease that involves the posterior aspect of the retina called the macula . the macula facilitates central vision and permits high - resolution visual acuity due to its dense concentration of cone photoreceptors ( fine et al 2000 ) . , there is formation of large drusen and pigmentary abnormalities ( fine et al 2000 ) . the nonexudative form is characterized by atrophic changes in the macula and clinically , has a slower deterioration and better preservation of visual acuity than exudative amd ( fine et al 2000 ) . amd involves choroidal neovascularization , which is the formation of new abnormal blood vessels in the choriocapillaries through bruch s membrane ( figure 1 ) . these vessels have a greater tendency of leakage and bleeding into the macula , ultimately leading to irreversible damage to the photoreceptors if left untreated ( fine et al 2000 ) . hence , the exudative form accounts for most cases of significant visual loss from amd ( ciulla et al 2001 ) . amd is the leading cause of visual deterioration and legal blindness in patients over 60 years of age ( klein et al 1997 ) . the loss of central vision and high - resolution visual acuity from untreated amd can lead to an irreversible loss of reading , facial recognition , and driving ( fine et al 2000 ) . the prevalence of amd among 40- to 49-year - olds is 2.1% , which increases dramatically to 35% among those over 80 years of age ( national eye institute 2007 ) . additionally , the disease can progress with approximately 10%20% of patients with nonexudative amd progressing to the exudative form if untreated ( hyman and neborsky 2002 ) . furthermore , almost 40% of patients have bilateral disease ( topouzis et al 2006 ) . the risk factors for amd include ethnicity , gender , hypertension , genetics , diet , and sunlight exposure . studies have shown that the late stages of amd are more common in whites than in other ethnic groups and that women are affected more than men ( hyman and neborsky 2002 ) . nevertheless , the strongest , most consistent risk factors are smoking and age ( hyman and neborsky 2002 ) . although aging will not inevitably lead to amd development , there are changes with age that can predispose the eye to development of amd . primary senescence of retinal pigment epithelial cells and bruch s membrane can lead to accumulation of metabolic debris and drusen . furthermore , primary abnormalities in ocular perfusion worsen with age , secondarily causing dysfunction of the retinal pigment epithelial cells , predisposing eyes to amd . these anatomical changes together with an individual s genetic make - up and environmental risk factors set the stage for the development of amd ( ciulla et al 2001 ) . in this report , we focus on the role that aging plays in the pathogenesis of amd . we start from a structural approach with alterations in the retinal pigment epithelium - bruch s membrane - choriocapillaris complex . then we focus on the vascular changes with an attention to ocular blood flow alterations that correlate with amd lesion development . the outer retina is made up of the retinal pigment epithelium and photoreceptors . the retinal pigment epithelial cells form the outer blood - retinal barrier to facilitate selective transport between the choroidal blood vessels and the outer retina ( salvi et al 2006 ) . therefore , they play a major role in rod and cone photoreceptor integrity . the retinal pigment epithelium is a phagocytic system that is essential to the renewal of photoreceptors ( de jong 2006 ) . when the tips ( or outer segments ) of photoreceptors are shed , retinal pigment epithelial cells have been shown to engulf and to degrade its components ( de jong 2006 ) . burns and feeney - burns ( 1980 ) speculate that the retinal pigment epithelium then transports the engulfed cytoplasmic material through bruch s membrane into the choriocapillaries . age - related processes that occur in the retinal pigment epithelium - bruch s membrane - choriocapillaris complex can precede the development of amd ( liang and godley 2003 ) . with advancing age , retinal pigment epithelial cells undergo an increase in pleomorphism , a decrease in the concentration of cells in the posterior pole , and decreased melanin content ( salvi et al 2006 ) . senescent retinal pigment epithelial cells accumulate metabolic debris from remnants of incomplete degradation of phagocytized rod and cone membranes ( ciulla et al 2001 ) . fatty acids from the outer segment receptors are degraded by lipid peroxidation , a process that increases with age in the macula ( zarbin 2004 ) . susuki et al ( 2007 ) found oxidized phospholipids present in photoreceptors and retinal pigment epithelial cells in the macula of the normal eyes , with increased quantities with advanced age . metabolic debris includes lipofuscin , an undegradable byproduct of outer segment photoreceptor metabolism , which increases in concentration within retinal pigment epithelial cell cytoplasm over time ( feeney - burns et al 1984 ) . it is believed that lipofuscin and fatty acids are continuously exposed to light and high oxygen tension , which initiates the production of reactive oxygen species ( zarbin 2004 ) . it is further postulated that these cause oxidative damage to the cellular mitochondria and to mitochondrial dna , which is most susceptible to this oxidative stress ( liang and godley 2003 ) . injury to the mitochondria leads to a reduction in energy production , compromises cellular and physiologic functioning , and can subsequently signal apoptosis in retinal pigment epithelial cells ( liang and godley 2003 ; zarbin 2004 ) . increased apoptosis eventually causes a decreased number of retinal pigment epithelial cells ( cai et al 1999 ) . in order to counteract the formation of reactive oxygen species , retinal pigment epithelial cells contain anti - oxidants , which decrease with age ( liang and godley 2003 ) the age - related eye disease study ( areds ) has demonstrated that the administration of anti - oxidants , such as vitamins b , c , e , and zinc , significantly slows the rate of progression from high - risk nonexudative amd to exudative amd ( areds 2001 ) . retinal pigment epithelial cell damage also leads to the production of an abnormal extracellular matrix , which alters retinal pigment epithelium function , and causes choriocapillary loss associated with geographic atrophy ( zarbin 2004 ) . damaged retinal pigment epithelial cells can promote an inflammatory response that has been implicated in the development of amd and the development of neovascularization ( moshfeghi and blumenkranz 2007 ) . once damaged , these retinal pigment epithelial cells can secrete growth factors , such as vascular endothelial growth factor ( vegf ) , basic fibroblast growth factor ( bfgf ) , and transforming growth factor beta ( tgf- ) . these cytokines play a major role in the formation of choroidal neovascularization in amd ( lutty et al 1999 ) . recent evidence suggests that within the eye , there is a fine balance between endogenous angiogenic and anti - angiogenic growth factors . imbalances between these factors can lead to choroidal neovascularization ( bhutto et al 2006 ) . the family of vegf proteins , including vegf - a , vegf - b , vegf - c , vegf - d , and plgf , exert their function through tyrosine kinase receptors ( andreoli and miller 2007 ) . vegf has been shown to be present in choroidal neovascularization membranes ( otani et al 2002 ) . conversely , pigment epithelium derived factor ( pedf ) is a potent endogenous anti - angiogenic growth factor . pedf has been shown to significantly suppress vegf - induced proliferation and migration of vascular endothelial cells ( bhutto et al 2006 ) . decreased pedf levels have been reported in the vitreous samples of exudative amd patients ( holekamp et al 2002 ) . choroidal pedf immunoreactivity is also reduced in amd patients compared to age - matched controls ( bhutto et al 2006 ) . currently , medications targeting vegf , such as bevacizumab and ranibizumab , are widely used in the treatment of neovascular amd . one such molecule , ubiquitin , is an intracellular protein that regulates numerous cellular processes , such as cell - cycle progression , signal transduction , transcriptional regulation , and endocytosis ( hershko and ciechanover 1998 ) . it has been implicated in immune responses and apoptosis ( hershko and ciechanover 1998 ) . ubiquitin is prominent in ganglion cells , retinal pigment epithelium and age - related subretinal pigment epithelium deposits ( shang and taylor 2004 ) . the first phase is ubiquitination whereby a substance that needs to be degraded is tagged with specific amino acid sequences . this process allows damaged or obsolete proteins to be degraded before they aggregate within the cytoplasm . over time , this protective pathway becomes compromised ( shang and taylor 2004 ) . additionally , hydrogen peroxide ( h2o2 ) increases the level of oxidized glutathione , which further interferes with ubiquitination ( shang and taylor 2004 ) . ubiquitin , without the active ubiquitin processing enzymes , is found in subretinal pigment epithelial deposits ( shang and taylor 2004 ) . it is speculated that with age , ubiquitinated proteins in retinal pigment epithelial cells do not undergo degradation by the ubiquitin processing enzymes ( shang and taylor 2004 ) . therefore , changes in the ubiquitin pathway compromise protein degradation and contribute to subretinal pigment epithelial protein accumulation , which occurs in both advanced age and amd . drusen are subretinal pigment epithelium deposits that are the clinical hallmark of amd ( sivaprasad et al 2005 ) . there are two classifications of drusen , depending on where they are located : basal linear and basal laminar deposits . basal linear deposits accumulate between the basal lamina of the retinal pigment epithelium and the inner collagenous zone of bruch s membrane ( sivaprasad et al 2005 ) . basal laminar deposits are found between the basement membrane and plasma membrane of the retinal pigment epithelium and consist of basement membrane proteins and collagen . the thickness of drusen deposits have been shown to correlate with the degree of retinal pigment epithelium degeneration , photoreceptor fallout , and vision loss ( sarks et al 2007 ) . drusen size , number , and degree of confluence are significant risk factors for the development of amd ( bressler et al 1990 ) . interestingly , the molecular composition of drusen and the atherosclerotic deposits within blood vessels share many similarities ( sivaprasad et al 2005 ) . both contain vitronectin ( a multifunctional plasma and extracellular matrix protein ) , apolipoprotein e , complement components and lipids ( mullins et al 2000 ) . nevertheless , a proteonomic analysis comparing drusen in amd patients and age - matched controls found that although two - thirds of the proteins were similar between the two groups , one - third of the proteins detected in drusen from amd patients were not found in controls ( crabb et al 2002 ) . furthermore , it is postulated that the development of drusen is most likely a selective and active process than a passive accumulation of proteins and lipids ( crabb et al 2002 ) . therefore , drusen formation may be influenced by an individual s genetic predisposition or environmental stressors , which helps explain why all aged patients do not develop amd ( figure 2 ) . with age , drusen can further accumulate on either side of the elastin layer in bruch s membrane ( feeney - burns and ellersieck 1985 ) . bruch s membrane is a connective tissue layer that lies between the metabolically active retinal pigment epithelial cells and the choriocapillaris . it supplies nutrition to retinal pigment epithelial cells ( sivaprasad et al 2005 ) and regulates ionic and metabolic exchange between the retinal pigment epithelium and choriocapillaries ( ramrattan et al 1994 ) . with age , bruch s membrane increases in thickness from 2 m in the first decade of life to 4.7 m in the 10th decade ( ramrattan et al 1994 ) . thickened bruch s membrane can predispose itself to crack formation ( ciulla et al 2001 ) . in patients with exudative amd , bruch s membrane is more calcified and fragmented than in age - matched controls or patients with nonexudative amd . these cracks can facilitate the development of choroidal neovascular membranes thus transforming nonexudative to exudative amd ( spraul and grossniklaus 1997 ) . the increased thickness of bruch s membrane involves both the elastin and collagenous layers and results from decreased degradation and increased production of the extracellular matrix ( zarbin 2004 ) . matrix mellatoproteinases ( mmps ) , which play a role in the degradation of extracellular matrix , and the tissue inhibitors of metalloproteinase ( timps ) , which regulate the activity of mmps , have been found to change with age ( zarbin 2004 ) . increased content of the inactive form of mmp-2 , mmp-9 as well as timp-3 have been found in the bruch s membrane of aged patients ( zarbin 2004 ) . activated by vegf , mmps break down the extracellular matrix , allowing the growth of new vessels ( steen et al 1998 ) . mmp-2 and mmp-9 have been localized to areas of new vessel development and to the enveloping bruchs - like membrane suggesting that these enzymes may be cooperatively involved in the progressive growth of choroidal neovascular membranes in amd . nevertheless , the amounts of noncollagen proteins , lipid deposits , heparin sulfate , laminin , and fibronectin are also increased in bruch s membrane with age ( zarbin 2004 ) . additionally , glycosaminoglycans increase in size and advanced glycation end products accumulate within bruch s membrane over time ( zarbin 2004 ) . the accumulation of cholesterol esters in bruch s membrane with advancing age is similar to that observed in the arterial intima in atherosclerosis ( curcio et al 2001 ) . this increase in lipid content creates a hydrophobic environment , which changes the flux of solutes and fluids across bruch s membrane ( bird and marshall 1986 ) . therefore , changes in thickness , molecular composition and electric charge leads to an age - related loss of permeability in bruch s membrane and alters the movement of nutritional factors from the choriocapillaries toward the retinal pigment epithelial layer and subretinal space , thereby affecting the available nutrients for the outer retina ( zarbin 2004 ) . there is a progressive decrease in the thickness of the choroid from 193 m in the first decade of life to 84 m in the 10th decade ( ramrattan et al 1994 ) . there is also a 45% decrease in choriocapillary density and a 34% decrease in the lumen diameter of the choriocapillaries with advancing age ( ramrattan et al 1994 ) . reduced numbers of macular arterioles and decreased intensity of dye in the macular region have been demonstrated over time using indocyanine green angiography ( nishiyama et al 2001 ) . these age - related changes were noted only in the arteries , but not in veins . furthermore , choroidal blood flow changes have been reported with advancing age ( ramrattan et al 1994 ) . pulsatile ocular blood flow reflects the total pulsatile component of ocular blood flow and since most of the ocular blood volume is present in the choroid , it mainly represents the choroidal circulation ( sandhu et al 2007 ) . ravalico et al ( 1996 ) and lam et al ( 2003 ) both found a decline in the pulsatile ocular blood flow with age . animal models have shown a 35%42% reduction in ocular blood flow and a 57%93% increase in ocular vascular resistance in aged rats compared with juvenile or adult rats ( salter et al 1998 ) . age - related changes in the reflexive choroidal vasodilatation following hypoperfusion have also been shown in rat models ( fitzgerald et al 2005 ) . neurogenic choroidal blood flow control declines with age both in human and in animal studies ( fitzgerald et al 2005 ) . with the use of laser doppler flowmetry , grunwald et al ( 1998 ) reported age - related decreases in choroidal blood flow in the fovea , which was related to decreases in blood volume but not velocity . the use of color doppler imaging provides measurements of the retrobulbar vasculature , namely the ophthalmic , central retinal and short posterior ciliary arteries . age - related decreases in peak systolic velocity and end - diastolic velocity of the ophthalmic artery have been reported ( harris et al 2000 ; lam et al 2003 ) . additionally , pourcelot s resistive index was increased in the ophthalmic artery ( harris et al 2000 , lam et al 2003 ) . groh et al ( 1996 ) found a decrease in central retinal artery blood flow by approximately 6%11% per decade with an increase in the resistive index . on the other hand , harris et al ( 2000 ) found that central retinal artery flow velocity was unaffected by age in both sexes . nevertheless , they did find that end - diastolic velocity of the posterior ciliary arteries decreased and the resistive index increased with age in women , but not in men . boehm et al ( 2005 ) used laser doppler flowmetry to find that volume and flow in the optic nerve decreased with age , suggesting that blood supply is reduced in elderly subjects . neuroretinal rim and lamina cribrosa blood flow also decreases with advancing age ( embelton et al 2002 ) . additionally , rizzo et al ( 1991 ) used a laser doppler technique to demonstrate that advancing age is associated with a reduction in capillary red blood cell speed at the optic nerve head . atherosclerosis has been postulated to be associated with late stages of amd through its effect on the choroidal circulation and possible deposition of lipids in the bruch s membrane ( freidman 2000 ; curcio et al 2001 ) . a history of cardiovascular disease and increased levels of cholesterol and stage 2 hypertension are strongly associated with neovascular amd ( hogg et al 2008 ) . the rotterdam eye study demonstrated that persons with subclinical cardiovascular disease , as manifested by carotid artery plaques , were nearly five times more likely to have late amd compared to patients without such plaques ( vingerling et al 1995a ) . the multiethnic study of atherosclerosis ( mesa ) found an association between the severity of the intima - media thickness of the carotid artery and early amd in black participants ( klein et al 2007a ) . nevertheless , they did not find an association between severity of subclinical atherosclerosis and early amd in the entire mesa cohort ( klein et al 2007a ) . the women s health initiative sight exam ancillary study ( whise ) further reported a positive association between elevated systolic blood pressure and the presence of soft drusen , which correlated inversely with retinal pigment epithelial depigmentation and exudative amd ( klein et al 2007b ) . diastolic blood pressures and mean arterial blood pressures positively correlated with an increased risk of early amd even after controlling for age ( klein et al 2007b ) . although the relationship between blood pressure and amd is not always consistent , several studies have demonstrated an association . in the beaver dam eye study , persons with hypertension at baseline were approximately two to three times more likely to develop neovascular macular degeneration after 10 years of follow - up than age - matched controls ( klein et al 2003 ) . furthermore , the age - related macular degeneration risk factor study reported hypertension and high cholesterol to be associated with increased risk of neovascular amd ( hyman et al 2000 ) . since the choriocapillaris supplies the retinal pigment epithelial cells and the outer retina , a primary perfusion defect in the choriocapillaries could lead to loss or dysfunction of retinal pigment epithelial cells ( ciulla et al 2001 ) . spraul et al ( 1996 ) found increased total luminal area of peripheral choroidal vessels and decreased density of choroidal vessels in the macular area of eyes with amd . in his theory on the development of amd , friedman ( 1997 ) hypothesized that with age , lipoid infiltration occurs within the sclera , bruch s membrane and the wall of blood vessels , which leads to a decrease in the scleral and choroidal vessels compliance . this decrease in compliance can increase the resistance of the choroidal blood vessels and decrease choroidal perfusion ( friedman 1997 ) . furthermore , scleral rigidity has been shown to increase with age ( lam et al 2003 ; pallikaris et al 2005 ) and in patients with amd ( friedman et al 1989 ) . all of these changes in the choriocapillaris can impair the retinal pigment epithelial cell transport , promote increased debris accumulation and cause dysfunction of the retinal pigment epithelial cells ( friedman 1997 ) to set the stage for amd development . blood flow analysis in patients with amd shows decreased choroidal blood flow , velocity and volume in the fovea in amd patients ( grunwald et al 2005 ) . in addition , the severity of amd increases in patients with the lowest circulatory parameters , possibly contributing to the ischemia and preceding the development of choroidal neovascularization ( grunwald et al 2005 ) . with the use of the laser doppler flowmetry , metelitsina et al ( 2006 ) further investigated choroidal blood flow in the fovea of patients with early amd with or without systemic hypertension . amd patients with systemic hypertension had 16.7% lower blood flow compared to patients without hypertension ( metelitsina et al 2006 ) . these results further suggest that systemic hypertension may contribute to the progression of amd and the development of choroidal neovascularization ( metelitsina et al 2006 ) . sato et al ( 2006 ) also used the laser doppler flowmetry to find that the pulsatility index , pulsatility ratio and resistive index were higher in patients with amd and increased with disease severity . they found no difference between the mean blood velocity and flow rates and no differences in arterial diameter , suggesting that patients with amd suffer from decreased compliance in the arterial vasculature ( sato et al 2006 ) . pulsatile ocular blood flow has been found to be decreased in exudative amd compared to nonexudative amd or normal subjects ( mori et al 2001 ) . chen et al ( 2001 ) found that the pulsatile ocular blood flow in eyes with drusen was lower than in eyes with choroidal neovascularization and higher in eyes with disciform scar , implying that hemodynamic changes can be partly responsible for asymmetry in some amd patients . nevertheless , a recent study of caucasian patients by sandhu et al ( 2007 ) demonstrated no differences between the pulsatile ocular blood flow in asymmetric eyes of patients with amd . moreover , delayed choroidal filling time has been found in patients with dry amd ( ciulla et al 2002 ) . prolonged choroidal filling phase was observed in 26% of eyes with early amd using fluorescein angiography and in 32% of eyes using indocyanine green ( pauleikoff et al 1999 ) . this prolonged filling was also associated with confluent drusen and geographic atrophy in the fellow eye ( pauleikoff et al 1999 ) . presence of macular watershed zones was also correlated with amd and one study found that choroidal neovascularization arose from the macular watershed zone in 91% of cases ( ross et al 1998 ) . changes in the retrobulbar blood flow can provide a better understanding of the primary or secondary role of decreased blood flow in amd . in an animal model , selective damage to retinal pigment epithelial cells caused degenerative changes to the choriocapillaris , suggesting that degeneration of choriocapillaries in amd can be secondary to dysfunction of the retinal pigment epithelial cells ( henkind and gartner 1983 ; del priore et al 1996 ) . therefore , secondary damage by retinal pigment epithelium is more likely to affect local blood vessels like the short posterior ciliary arteries and choriocapillaris than the ophthalmic or central retinal arteries . changes in these vessels can support a more primary role of decrease in blood flow in the development of amd , although a secondary autoregulatory response to a primary change elsewhere can not be ruled out ( ciulla et al 1999 ) . friedman et al ( 1995 ) reported that there is elevated pulsatility within all vessels including the ophthalmic , central retinal , and short posterior ciliary arteries , which can indicate an increase in the resistive index . hosal et al ( 1998 ) reported that patients with amd have lower blood velocity and higher resistive index in all retrobulbar vessels including the ophthalmic artery . studies have found that blood flow velocity was lower in the ophthalmic artery and short posterior ciliary arteries of eyes with choroidal neovascularization compared with eyes without choroidal neovascularization ( uretmen et al 2003 ; ciulla et al 1999 ) . various studies have reported decreases in the velocity of the short posterior ciliary arteries in amd patients ( friedman et al 1995 ; dimitrova et al 2002 ) . ciulla et al ( 1999 ) also found a decrease in the end - diastolic velocity and an increase in the resistive index of the central retinal artery . there are a couple of hypotheses for this change in the central retinal artery blood flow velocity . first , reductions in central retinal artery blood flow may be due to global decreases in retrobulbar blood flow from the ophthalmic artery and its branches . yannuzzi et al ( 2001 ) described retinal angiomatous proliferation as a phenomenon which occurs in a subgroup of neovascular amd patients , where angiomatous proliferation originates from the retina and extends posteriorly into the subretinal space , occasionally communicating with choroidal new vessels . increased flow through new choroidal vessels could decrease flow in the retina and the central retinal artery . nevertheless , further studies are necessary to determine whether primary or secondary vascular changes play a role in amd . although the hemodynamic abnormalities do not directly indicate that the vascular abnormalities precede the development of amd , they certainly suggest that hemodynamic changes play an important role in amd pathogenesis . friedman ( 1997 , 2004 ) uses vascular theory to help explain what is known about the amd pathophysiology . with advancing age , there is vascular stiffening and increased resistance . as overall vascular resistance increases , cerebral vascular resistance rises and leads to decreased choroidal perfusion and increased osmotic gradients against which retinal pigment epithelial cells must pump . furthermore , high choriocapillary pressure in the presence of calcification and fracture of bruch s membrane and ischemic damage to retinal pigment epithelial cells can cause the secretion of vascular growth factors leading to the development of choroidal neovascularization ( friedman 1997 , 2004 ) . although this theory has not been confirmed and does not elucidate the differences between races , it provides a well - rounded description of vascular alterations and how they contribute to the pathogenesis of amd . numerous population - based studies have reported differences in prevalence and incidence of amd between various ethnic / racial groups ( klein et al 1999 , 2006 ; mitchell et al 1995 ; vingerling et al 1995b ) . several studies have demonstrated an increased incidence of amd within families to provide further support for the role of genetics in amd ( seddon et al 1997 ; klaver et al 1998 ; grizzard et al 2003 ) . therefore , it is important for investigators to continue to explore the role of genetics and environment on the development of amd . although genetic predisposition combined with environmental stressors seem to lead to disease development ( swaroop et al 2007 ) , various studies have identified chromosomal abnormalities associated with amd . some of the chromosomal regions associated with amd include 1q3132 , 6q21 and 10q26 ( gold et al 2006 ; swaroop et al 2007 ) . within chromosome segment 10q26 , specific gene regions include plekha1 , loc387715 and htra1 ( swaroop et al 2007 ) . complement factor h , within chromosomal segment 1q3132 , is a key regulator in the alternative complement cascade and prevents uncontrolled complement activation ( yates et al 2007 ) . thakkinstian et al ( 2006 ) performed a meta - analysis between complement factor h y402h polymorphism and amd to find that heterozygotes ( tc ) and homozygotes ( cc ) were , respectively , 2.5 and 6 times more likely to have amd than homozygote patients with the tt genotype . nevertheless , a japanese study found that this complement factor h gene did not appear to increase risk of amd within their population ( gotoh et al 2006 ) . furthermore , within chromosome 6q21 , polymorphisms of complement factor b and complement component 2 have been shown to be a risk predictor of amd ( gold et al 2006 ) . these correlations between complement - related genes and amd further support the role of the inflammatory system in the development of amd . in addition , a previous study by mullins et al ( 2000 ) found that drusen can contain activated complement factors , acute phase reactants , coagulation proteins , immunoglobulins , and other proteins involved in the induction and activation of the immune response . not only can an individual s genetic predisposition influence the development of amd , but it is also associated with the rate of disease progression . seddon et al ( 2007 ) analyzed patients from the age - related eye disease study ( areds ) to determine whether variations within the complement factor h gene and the loc387715 gene have prognostic importance in amd disease progression . although genetic polymorphisms in the complement factor h y402h and loc387715 a69s were associated with progression to more advanced amd , the loc387715 genotype was also correlated with neovascular disease more than geographic atrophy . patients who were homozygous for the risk alleles had a nearly 50% probability for amd progression compared to the 5% risk in patients who are homozygous for the nonrisk alleles ( seddon et al 2007 ) . additionally , presence of the homozygous risk alleles combined with smoking and high body mass indices were associated with 19-fold increase of disease progression ( seddon et al 2007 ) . conversely , some genetic variations have been found to play a protective role in amd development and progression . apolipoprotein e is involved in transport and metabolism of lipid and cholesterol and in response to neural injury ( swaroop et al 2007 ) . the 4 allele of apolipoprotein e has been associated with the reduced prevalence of amd . there is strong evidence for the role of genetics in amd , but further research is necessary to determine how genetic differences can ultimately lead to amd . although amd development shares many correlations with normal aging processes , other factors , such as genetic predisposition or environmental stressors , may account for disease development ( figure 2 ) . furthermore , the similarities between drusen and atherosclerosis demonstrate that a vascular component may play a role in amd development . apart from the structural changes , age - related alterations in ocular hemodynamics can precede and correlate with the amd pathogenesis , which further supports the vascular theory of amd . although aging alone is not sufficient to form amd , it is apparent that the age - related changes and genetics are very important to the pathophysiology of amd . understanding the processes that develop with age and in age - related diseases can help guide new early treatment modalities . further studies are warranted in order to learn how age - associated vascular and ocular structural changes lead to disease development . this will help generate the knowledge needed to identify target areas that may be useful to prevent disease progression through medical or surgical treatment .

age - related macular degeneration ( amd ) is an ocular disease that causes damage to the retinal macula , mostly in the elderly . normal aging processes can lead to structural and blood flow changes that can predispose patients to amd , although advanced age does not inevitably cause amd . in this review , we describe changes that occur in the macular structure , such as the retinal pigment epithelium and bruch s membrane , with advancing age and in amd . the role of genetics in amd and age - related changes in ocular blood flow that may play a role in the pathogenesis of amd are also discussed . understanding the pathophysiology of amd development can help guide future research to further comprehend this disease and to develop better treatments to prevent its irreversible central vision loss in the elderly .

Introduction Structural changes with age and in AMD Ocular blood flow changes with age Ocular blood flow changes in AMD Role of genetics in AMD Conclusion

age - related macular degeneration ( amd ) is an ocular disease that involves the posterior aspect of the retina called the macula . the nonexudative form is characterized by atrophic changes in the macula and clinically , has a slower deterioration and better preservation of visual acuity than exudative amd ( fine et al 2000 ) . amd involves choroidal neovascularization , which is the formation of new abnormal blood vessels in the choriocapillaries through bruch s membrane ( figure 1 ) . these vessels have a greater tendency of leakage and bleeding into the macula , ultimately leading to irreversible damage to the photoreceptors if left untreated ( fine et al 2000 ) . the loss of central vision and high - resolution visual acuity from untreated amd can lead to an irreversible loss of reading , facial recognition , and driving ( fine et al 2000 ) . studies have shown that the late stages of amd are more common in whites than in other ethnic groups and that women are affected more than men ( hyman and neborsky 2002 ) . although aging will not inevitably lead to amd development , there are changes with age that can predispose the eye to development of amd . primary senescence of retinal pigment epithelial cells and bruch s membrane can lead to accumulation of metabolic debris and drusen . furthermore , primary abnormalities in ocular perfusion worsen with age , secondarily causing dysfunction of the retinal pigment epithelial cells , predisposing eyes to amd . in this report , we focus on the role that aging plays in the pathogenesis of amd . we start from a structural approach with alterations in the retinal pigment epithelium - bruch s membrane - choriocapillaris complex . then we focus on the vascular changes with an attention to ocular blood flow alterations that correlate with amd lesion development . the outer retina is made up of the retinal pigment epithelium and photoreceptors . the retinal pigment epithelial cells form the outer blood - retinal barrier to facilitate selective transport between the choroidal blood vessels and the outer retina ( salvi et al 2006 ) . the retinal pigment epithelium is a phagocytic system that is essential to the renewal of photoreceptors ( de jong 2006 ) . when the tips ( or outer segments ) of photoreceptors are shed , retinal pigment epithelial cells have been shown to engulf and to degrade its components ( de jong 2006 ) . burns and feeney - burns ( 1980 ) speculate that the retinal pigment epithelium then transports the engulfed cytoplasmic material through bruch s membrane into the choriocapillaries . age - related processes that occur in the retinal pigment epithelium - bruch s membrane - choriocapillaris complex can precede the development of amd ( liang and godley 2003 ) . with advancing age , retinal pigment epithelial cells undergo an increase in pleomorphism , a decrease in the concentration of cells in the posterior pole , and decreased melanin content ( salvi et al 2006 ) . susuki et al ( 2007 ) found oxidized phospholipids present in photoreceptors and retinal pigment epithelial cells in the macula of the normal eyes , with increased quantities with advanced age . it is further postulated that these cause oxidative damage to the cellular mitochondria and to mitochondrial dna , which is most susceptible to this oxidative stress ( liang and godley 2003 ) . injury to the mitochondria leads to a reduction in energy production , compromises cellular and physiologic functioning , and can subsequently signal apoptosis in retinal pigment epithelial cells ( liang and godley 2003 ; zarbin 2004 ) . in order to counteract the formation of reactive oxygen species , retinal pigment epithelial cells contain anti - oxidants , which decrease with age ( liang and godley 2003 ) the age - related eye disease study ( areds ) has demonstrated that the administration of anti - oxidants , such as vitamins b , c , e , and zinc , significantly slows the rate of progression from high - risk nonexudative amd to exudative amd ( areds 2001 ) . retinal pigment epithelial cell damage also leads to the production of an abnormal extracellular matrix , which alters retinal pigment epithelium function , and causes choriocapillary loss associated with geographic atrophy ( zarbin 2004 ) . damaged retinal pigment epithelial cells can promote an inflammatory response that has been implicated in the development of amd and the development of neovascularization ( moshfeghi and blumenkranz 2007 ) . once damaged , these retinal pigment epithelial cells can secrete growth factors , such as vascular endothelial growth factor ( vegf ) , basic fibroblast growth factor ( bfgf ) , and transforming growth factor beta ( tgf- ) . these cytokines play a major role in the formation of choroidal neovascularization in amd ( lutty et al 1999 ) . imbalances between these factors can lead to choroidal neovascularization ( bhutto et al 2006 ) . conversely , pigment epithelium derived factor ( pedf ) is a potent endogenous anti - angiogenic growth factor . choroidal pedf immunoreactivity is also reduced in amd patients compared to age - matched controls ( bhutto et al 2006 ) . currently , medications targeting vegf , such as bevacizumab and ranibizumab , are widely used in the treatment of neovascular amd . one such molecule , ubiquitin , is an intracellular protein that regulates numerous cellular processes , such as cell - cycle progression , signal transduction , transcriptional regulation , and endocytosis ( hershko and ciechanover 1998 ) . ubiquitin is prominent in ganglion cells , retinal pigment epithelium and age - related subretinal pigment epithelium deposits ( shang and taylor 2004 ) . therefore , changes in the ubiquitin pathway compromise protein degradation and contribute to subretinal pigment epithelial protein accumulation , which occurs in both advanced age and amd . basal linear deposits accumulate between the basal lamina of the retinal pigment epithelium and the inner collagenous zone of bruch s membrane ( sivaprasad et al 2005 ) . basal laminar deposits are found between the basement membrane and plasma membrane of the retinal pigment epithelium and consist of basement membrane proteins and collagen . the thickness of drusen deposits have been shown to correlate with the degree of retinal pigment epithelium degeneration , photoreceptor fallout , and vision loss ( sarks et al 2007 ) . nevertheless , a proteonomic analysis comparing drusen in amd patients and age - matched controls found that although two - thirds of the proteins were similar between the two groups , one - third of the proteins detected in drusen from amd patients were not found in controls ( crabb et al 2002 ) . with age , drusen can further accumulate on either side of the elastin layer in bruch s membrane ( feeney - burns and ellersieck 1985 ) . bruch s membrane is a connective tissue layer that lies between the metabolically active retinal pigment epithelial cells and the choriocapillaris . it supplies nutrition to retinal pigment epithelial cells ( sivaprasad et al 2005 ) and regulates ionic and metabolic exchange between the retinal pigment epithelium and choriocapillaries ( ramrattan et al 1994 ) . with age , bruch s membrane increases in thickness from 2 m in the first decade of life to 4.7 m in the 10th decade ( ramrattan et al 1994 ) . thickened bruch s membrane can predispose itself to crack formation ( ciulla et al 2001 ) . in patients with exudative amd , bruch s membrane is more calcified and fragmented than in age - matched controls or patients with nonexudative amd . the increased thickness of bruch s membrane involves both the elastin and collagenous layers and results from decreased degradation and increased production of the extracellular matrix ( zarbin 2004 ) . matrix mellatoproteinases ( mmps ) , which play a role in the degradation of extracellular matrix , and the tissue inhibitors of metalloproteinase ( timps ) , which regulate the activity of mmps , have been found to change with age ( zarbin 2004 ) . increased content of the inactive form of mmp-2 , mmp-9 as well as timp-3 have been found in the bruch s membrane of aged patients ( zarbin 2004 ) . mmp-2 and mmp-9 have been localized to areas of new vessel development and to the enveloping bruchs - like membrane suggesting that these enzymes may be cooperatively involved in the progressive growth of choroidal neovascular membranes in amd . nevertheless , the amounts of noncollagen proteins , lipid deposits , heparin sulfate , laminin , and fibronectin are also increased in bruch s membrane with age ( zarbin 2004 ) . additionally , glycosaminoglycans increase in size and advanced glycation end products accumulate within bruch s membrane over time ( zarbin 2004 ) . the accumulation of cholesterol esters in bruch s membrane with advancing age is similar to that observed in the arterial intima in atherosclerosis ( curcio et al 2001 ) . this increase in lipid content creates a hydrophobic environment , which changes the flux of solutes and fluids across bruch s membrane ( bird and marshall 1986 ) . therefore , changes in thickness , molecular composition and electric charge leads to an age - related loss of permeability in bruch s membrane and alters the movement of nutritional factors from the choriocapillaries toward the retinal pigment epithelial layer and subretinal space , thereby affecting the available nutrients for the outer retina ( zarbin 2004 ) . there is also a 45% decrease in choriocapillary density and a 34% decrease in the lumen diameter of the choriocapillaries with advancing age ( ramrattan et al 1994 ) . reduced numbers of macular arterioles and decreased intensity of dye in the macular region have been demonstrated over time using indocyanine green angiography ( nishiyama et al 2001 ) . these age - related changes were noted only in the arteries , but not in veins . furthermore , choroidal blood flow changes have been reported with advancing age ( ramrattan et al 1994 ) . pulsatile ocular blood flow reflects the total pulsatile component of ocular blood flow and since most of the ocular blood volume is present in the choroid , it mainly represents the choroidal circulation ( sandhu et al 2007 ) . ravalico et al ( 1996 ) and lam et al ( 2003 ) both found a decline in the pulsatile ocular blood flow with age . animal models have shown a 35%42% reduction in ocular blood flow and a 57%93% increase in ocular vascular resistance in aged rats compared with juvenile or adult rats ( salter et al 1998 ) . age - related changes in the reflexive choroidal vasodilatation following hypoperfusion have also been shown in rat models ( fitzgerald et al 2005 ) . neurogenic choroidal blood flow control declines with age both in human and in animal studies ( fitzgerald et al 2005 ) . with the use of laser doppler flowmetry , grunwald et al ( 1998 ) reported age - related decreases in choroidal blood flow in the fovea , which was related to decreases in blood volume but not velocity . age - related decreases in peak systolic velocity and end - diastolic velocity of the ophthalmic artery have been reported ( harris et al 2000 ; lam et al 2003 ) . groh et al ( 1996 ) found a decrease in central retinal artery blood flow by approximately 6%11% per decade with an increase in the resistive index . neuroretinal rim and lamina cribrosa blood flow also decreases with advancing age ( embelton et al 2002 ) . atherosclerosis has been postulated to be associated with late stages of amd through its effect on the choroidal circulation and possible deposition of lipids in the bruch s membrane ( freidman 2000 ; curcio et al 2001 ) . nevertheless , they did not find an association between severity of subclinical atherosclerosis and early amd in the entire mesa cohort ( klein et al 2007a ) . in the beaver dam eye study , persons with hypertension at baseline were approximately two to three times more likely to develop neovascular macular degeneration after 10 years of follow - up than age - matched controls ( klein et al 2003 ) . furthermore , the age - related macular degeneration risk factor study reported hypertension and high cholesterol to be associated with increased risk of neovascular amd ( hyman et al 2000 ) . since the choriocapillaris supplies the retinal pigment epithelial cells and the outer retina , a primary perfusion defect in the choriocapillaries could lead to loss or dysfunction of retinal pigment epithelial cells ( ciulla et al 2001 ) . spraul et al ( 1996 ) found increased total luminal area of peripheral choroidal vessels and decreased density of choroidal vessels in the macular area of eyes with amd . in his theory on the development of amd , friedman ( 1997 ) hypothesized that with age , lipoid infiltration occurs within the sclera , bruch s membrane and the wall of blood vessels , which leads to a decrease in the scleral and choroidal vessels compliance . all of these changes in the choriocapillaris can impair the retinal pigment epithelial cell transport , promote increased debris accumulation and cause dysfunction of the retinal pigment epithelial cells ( friedman 1997 ) to set the stage for amd development . blood flow analysis in patients with amd shows decreased choroidal blood flow , velocity and volume in the fovea in amd patients ( grunwald et al 2005 ) . in addition , the severity of amd increases in patients with the lowest circulatory parameters , possibly contributing to the ischemia and preceding the development of choroidal neovascularization ( grunwald et al 2005 ) . with the use of the laser doppler flowmetry , metelitsina et al ( 2006 ) further investigated choroidal blood flow in the fovea of patients with early amd with or without systemic hypertension . these results further suggest that systemic hypertension may contribute to the progression of amd and the development of choroidal neovascularization ( metelitsina et al 2006 ) . pulsatile ocular blood flow has been found to be decreased in exudative amd compared to nonexudative amd or normal subjects ( mori et al 2001 ) . nevertheless , a recent study of caucasian patients by sandhu et al ( 2007 ) demonstrated no differences between the pulsatile ocular blood flow in asymmetric eyes of patients with amd . presence of macular watershed zones was also correlated with amd and one study found that choroidal neovascularization arose from the macular watershed zone in 91% of cases ( ross et al 1998 ) . changes in the retrobulbar blood flow can provide a better understanding of the primary or secondary role of decreased blood flow in amd . in an animal model , selective damage to retinal pigment epithelial cells caused degenerative changes to the choriocapillaris , suggesting that degeneration of choriocapillaries in amd can be secondary to dysfunction of the retinal pigment epithelial cells ( henkind and gartner 1983 ; del priore et al 1996 ) . therefore , secondary damage by retinal pigment epithelium is more likely to affect local blood vessels like the short posterior ciliary arteries and choriocapillaris than the ophthalmic or central retinal arteries . changes in these vessels can support a more primary role of decrease in blood flow in the development of amd , although a secondary autoregulatory response to a primary change elsewhere can not be ruled out ( ciulla et al 1999 ) . studies have found that blood flow velocity was lower in the ophthalmic artery and short posterior ciliary arteries of eyes with choroidal neovascularization compared with eyes without choroidal neovascularization ( uretmen et al 2003 ; ciulla et al 1999 ) . various studies have reported decreases in the velocity of the short posterior ciliary arteries in amd patients ( friedman et al 1995 ; dimitrova et al 2002 ) . nevertheless , further studies are necessary to determine whether primary or secondary vascular changes play a role in amd . although the hemodynamic abnormalities do not directly indicate that the vascular abnormalities precede the development of amd , they certainly suggest that hemodynamic changes play an important role in amd pathogenesis . furthermore , high choriocapillary pressure in the presence of calcification and fracture of bruch s membrane and ischemic damage to retinal pigment epithelial cells can cause the secretion of vascular growth factors leading to the development of choroidal neovascularization ( friedman 1997 , 2004 ) . although this theory has not been confirmed and does not elucidate the differences between races , it provides a well - rounded description of vascular alterations and how they contribute to the pathogenesis of amd . several studies have demonstrated an increased incidence of amd within families to provide further support for the role of genetics in amd ( seddon et al 1997 ; klaver et al 1998 ; grizzard et al 2003 ) . therefore , it is important for investigators to continue to explore the role of genetics and environment on the development of amd . these correlations between complement - related genes and amd further support the role of the inflammatory system in the development of amd . not only can an individual s genetic predisposition influence the development of amd , but it is also associated with the rate of disease progression . seddon et al ( 2007 ) analyzed patients from the age - related eye disease study ( areds ) to determine whether variations within the complement factor h gene and the loc387715 gene have prognostic importance in amd disease progression . although genetic polymorphisms in the complement factor h y402h and loc387715 a69s were associated with progression to more advanced amd , the loc387715 genotype was also correlated with neovascular disease more than geographic atrophy . conversely , some genetic variations have been found to play a protective role in amd development and progression . there is strong evidence for the role of genetics in amd , but further research is necessary to determine how genetic differences can ultimately lead to amd . although amd development shares many correlations with normal aging processes , other factors , such as genetic predisposition or environmental stressors , may account for disease development ( figure 2 ) . furthermore , the similarities between drusen and atherosclerosis demonstrate that a vascular component may play a role in amd development . apart from the structural changes , age - related alterations in ocular hemodynamics can precede and correlate with the amd pathogenesis , which further supports the vascular theory of amd . although aging alone is not sufficient to form amd , it is apparent that the age - related changes and genetics are very important to the pathophysiology of amd . understanding the processes that develop with age and in age - related diseases can help guide new early treatment modalities . further studies are warranted in order to learn how age - associated vascular and ocular structural changes lead to disease development . this will help generate the knowledge needed to identify target areas that may be useful to prevent disease progression through medical or surgical treatment .

age - related macular degeneration ( amd ) is an ocular disease that involves the posterior aspect of the retina called the macula . the nonexudative form is characterized by atrophic changes in the macula and clinically , has a slower deterioration and better preservation of visual acuity than exudative amd ( fine et al 2000 ) . hence , the exudative form accounts for most cases of significant visual loss from amd ( ciulla et al 2001 ) . the loss of central vision and high - resolution visual acuity from untreated amd can lead to an irreversible loss of reading , facial recognition , and driving ( fine et al 2000 ) . the prevalence of amd among 40- to 49-year - olds is 2.1% , which increases dramatically to 35% among those over 80 years of age ( national eye institute 2007 ) . additionally , the disease can progress with approximately 10%20% of patients with nonexudative amd progressing to the exudative form if untreated ( hyman and neborsky 2002 ) . studies have shown that the late stages of amd are more common in whites than in other ethnic groups and that women are affected more than men ( hyman and neborsky 2002 ) . although aging will not inevitably lead to amd development , there are changes with age that can predispose the eye to development of amd . primary senescence of retinal pigment epithelial cells and bruch s membrane can lead to accumulation of metabolic debris and drusen . furthermore , primary abnormalities in ocular perfusion worsen with age , secondarily causing dysfunction of the retinal pigment epithelial cells , predisposing eyes to amd . these anatomical changes together with an individual s genetic make - up and environmental risk factors set the stage for the development of amd ( ciulla et al 2001 ) . in this report , we focus on the role that aging plays in the pathogenesis of amd . we start from a structural approach with alterations in the retinal pigment epithelium - bruch s membrane - choriocapillaris complex . then we focus on the vascular changes with an attention to ocular blood flow alterations that correlate with amd lesion development . the retinal pigment epithelial cells form the outer blood - retinal barrier to facilitate selective transport between the choroidal blood vessels and the outer retina ( salvi et al 2006 ) . when the tips ( or outer segments ) of photoreceptors are shed , retinal pigment epithelial cells have been shown to engulf and to degrade its components ( de jong 2006 ) . age - related processes that occur in the retinal pigment epithelium - bruch s membrane - choriocapillaris complex can precede the development of amd ( liang and godley 2003 ) . with advancing age , retinal pigment epithelial cells undergo an increase in pleomorphism , a decrease in the concentration of cells in the posterior pole , and decreased melanin content ( salvi et al 2006 ) . susuki et al ( 2007 ) found oxidized phospholipids present in photoreceptors and retinal pigment epithelial cells in the macula of the normal eyes , with increased quantities with advanced age . it is further postulated that these cause oxidative damage to the cellular mitochondria and to mitochondrial dna , which is most susceptible to this oxidative stress ( liang and godley 2003 ) . injury to the mitochondria leads to a reduction in energy production , compromises cellular and physiologic functioning , and can subsequently signal apoptosis in retinal pigment epithelial cells ( liang and godley 2003 ; zarbin 2004 ) . in order to counteract the formation of reactive oxygen species , retinal pigment epithelial cells contain anti - oxidants , which decrease with age ( liang and godley 2003 ) the age - related eye disease study ( areds ) has demonstrated that the administration of anti - oxidants , such as vitamins b , c , e , and zinc , significantly slows the rate of progression from high - risk nonexudative amd to exudative amd ( areds 2001 ) . retinal pigment epithelial cell damage also leads to the production of an abnormal extracellular matrix , which alters retinal pigment epithelium function , and causes choriocapillary loss associated with geographic atrophy ( zarbin 2004 ) . damaged retinal pigment epithelial cells can promote an inflammatory response that has been implicated in the development of amd and the development of neovascularization ( moshfeghi and blumenkranz 2007 ) . once damaged , these retinal pigment epithelial cells can secrete growth factors , such as vascular endothelial growth factor ( vegf ) , basic fibroblast growth factor ( bfgf ) , and transforming growth factor beta ( tgf- ) . these cytokines play a major role in the formation of choroidal neovascularization in amd ( lutty et al 1999 ) . recent evidence suggests that within the eye , there is a fine balance between endogenous angiogenic and anti - angiogenic growth factors . the family of vegf proteins , including vegf - a , vegf - b , vegf - c , vegf - d , and plgf , exert their function through tyrosine kinase receptors ( andreoli and miller 2007 ) . pedf has been shown to significantly suppress vegf - induced proliferation and migration of vascular endothelial cells ( bhutto et al 2006 ) . currently , medications targeting vegf , such as bevacizumab and ranibizumab , are widely used in the treatment of neovascular amd . one such molecule , ubiquitin , is an intracellular protein that regulates numerous cellular processes , such as cell - cycle progression , signal transduction , transcriptional regulation , and endocytosis ( hershko and ciechanover 1998 ) . ubiquitin is prominent in ganglion cells , retinal pigment epithelium and age - related subretinal pigment epithelium deposits ( shang and taylor 2004 ) . therefore , changes in the ubiquitin pathway compromise protein degradation and contribute to subretinal pigment epithelial protein accumulation , which occurs in both advanced age and amd . basal linear deposits accumulate between the basal lamina of the retinal pigment epithelium and the inner collagenous zone of bruch s membrane ( sivaprasad et al 2005 ) . basal laminar deposits are found between the basement membrane and plasma membrane of the retinal pigment epithelium and consist of basement membrane proteins and collagen . the thickness of drusen deposits have been shown to correlate with the degree of retinal pigment epithelium degeneration , photoreceptor fallout , and vision loss ( sarks et al 2007 ) . nevertheless , a proteonomic analysis comparing drusen in amd patients and age - matched controls found that although two - thirds of the proteins were similar between the two groups , one - third of the proteins detected in drusen from amd patients were not found in controls ( crabb et al 2002 ) . furthermore , it is postulated that the development of drusen is most likely a selective and active process than a passive accumulation of proteins and lipids ( crabb et al 2002 ) . therefore , drusen formation may be influenced by an individual s genetic predisposition or environmental stressors , which helps explain why all aged patients do not develop amd ( figure 2 ) . with age , drusen can further accumulate on either side of the elastin layer in bruch s membrane ( feeney - burns and ellersieck 1985 ) . it supplies nutrition to retinal pigment epithelial cells ( sivaprasad et al 2005 ) and regulates ionic and metabolic exchange between the retinal pigment epithelium and choriocapillaries ( ramrattan et al 1994 ) . with age , bruch s membrane increases in thickness from 2 m in the first decade of life to 4.7 m in the 10th decade ( ramrattan et al 1994 ) . in patients with exudative amd , bruch s membrane is more calcified and fragmented than in age - matched controls or patients with nonexudative amd . the increased thickness of bruch s membrane involves both the elastin and collagenous layers and results from decreased degradation and increased production of the extracellular matrix ( zarbin 2004 ) . matrix mellatoproteinases ( mmps ) , which play a role in the degradation of extracellular matrix , and the tissue inhibitors of metalloproteinase ( timps ) , which regulate the activity of mmps , have been found to change with age ( zarbin 2004 ) . increased content of the inactive form of mmp-2 , mmp-9 as well as timp-3 have been found in the bruch s membrane of aged patients ( zarbin 2004 ) . mmp-2 and mmp-9 have been localized to areas of new vessel development and to the enveloping bruchs - like membrane suggesting that these enzymes may be cooperatively involved in the progressive growth of choroidal neovascular membranes in amd . nevertheless , the amounts of noncollagen proteins , lipid deposits , heparin sulfate , laminin , and fibronectin are also increased in bruch s membrane with age ( zarbin 2004 ) . the accumulation of cholesterol esters in bruch s membrane with advancing age is similar to that observed in the arterial intima in atherosclerosis ( curcio et al 2001 ) . therefore , changes in thickness , molecular composition and electric charge leads to an age - related loss of permeability in bruch s membrane and alters the movement of nutritional factors from the choriocapillaries toward the retinal pigment epithelial layer and subretinal space , thereby affecting the available nutrients for the outer retina ( zarbin 2004 ) . there is a progressive decrease in the thickness of the choroid from 193 m in the first decade of life to 84 m in the 10th decade ( ramrattan et al 1994 ) . there is also a 45% decrease in choriocapillary density and a 34% decrease in the lumen diameter of the choriocapillaries with advancing age ( ramrattan et al 1994 ) . pulsatile ocular blood flow reflects the total pulsatile component of ocular blood flow and since most of the ocular blood volume is present in the choroid , it mainly represents the choroidal circulation ( sandhu et al 2007 ) . animal models have shown a 35%42% reduction in ocular blood flow and a 57%93% increase in ocular vascular resistance in aged rats compared with juvenile or adult rats ( salter et al 1998 ) . age - related changes in the reflexive choroidal vasodilatation following hypoperfusion have also been shown in rat models ( fitzgerald et al 2005 ) . age - related decreases in peak systolic velocity and end - diastolic velocity of the ophthalmic artery have been reported ( harris et al 2000 ; lam et al 2003 ) . nevertheless , they did find that end - diastolic velocity of the posterior ciliary arteries decreased and the resistive index increased with age in women , but not in men . atherosclerosis has been postulated to be associated with late stages of amd through its effect on the choroidal circulation and possible deposition of lipids in the bruch s membrane ( freidman 2000 ; curcio et al 2001 ) . a history of cardiovascular disease and increased levels of cholesterol and stage 2 hypertension are strongly associated with neovascular amd ( hogg et al 2008 ) . the multiethnic study of atherosclerosis ( mesa ) found an association between the severity of the intima - media thickness of the carotid artery and early amd in black participants ( klein et al 2007a ) . nevertheless , they did not find an association between severity of subclinical atherosclerosis and early amd in the entire mesa cohort ( klein et al 2007a ) . the women s health initiative sight exam ancillary study ( whise ) further reported a positive association between elevated systolic blood pressure and the presence of soft drusen , which correlated inversely with retinal pigment epithelial depigmentation and exudative amd ( klein et al 2007b ) . diastolic blood pressures and mean arterial blood pressures positively correlated with an increased risk of early amd even after controlling for age ( klein et al 2007b ) . in the beaver dam eye study , persons with hypertension at baseline were approximately two to three times more likely to develop neovascular macular degeneration after 10 years of follow - up than age - matched controls ( klein et al 2003 ) . furthermore , the age - related macular degeneration risk factor study reported hypertension and high cholesterol to be associated with increased risk of neovascular amd ( hyman et al 2000 ) . since the choriocapillaris supplies the retinal pigment epithelial cells and the outer retina , a primary perfusion defect in the choriocapillaries could lead to loss or dysfunction of retinal pigment epithelial cells ( ciulla et al 2001 ) . in his theory on the development of amd , friedman ( 1997 ) hypothesized that with age , lipoid infiltration occurs within the sclera , bruch s membrane and the wall of blood vessels , which leads to a decrease in the scleral and choroidal vessels compliance . all of these changes in the choriocapillaris can impair the retinal pigment epithelial cell transport , promote increased debris accumulation and cause dysfunction of the retinal pigment epithelial cells ( friedman 1997 ) to set the stage for amd development . blood flow analysis in patients with amd shows decreased choroidal blood flow , velocity and volume in the fovea in amd patients ( grunwald et al 2005 ) . in addition , the severity of amd increases in patients with the lowest circulatory parameters , possibly contributing to the ischemia and preceding the development of choroidal neovascularization ( grunwald et al 2005 ) . with the use of the laser doppler flowmetry , metelitsina et al ( 2006 ) further investigated choroidal blood flow in the fovea of patients with early amd with or without systemic hypertension . these results further suggest that systemic hypertension may contribute to the progression of amd and the development of choroidal neovascularization ( metelitsina et al 2006 ) . sato et al ( 2006 ) also used the laser doppler flowmetry to find that the pulsatility index , pulsatility ratio and resistive index were higher in patients with amd and increased with disease severity . they found no difference between the mean blood velocity and flow rates and no differences in arterial diameter , suggesting that patients with amd suffer from decreased compliance in the arterial vasculature ( sato et al 2006 ) . chen et al ( 2001 ) found that the pulsatile ocular blood flow in eyes with drusen was lower than in eyes with choroidal neovascularization and higher in eyes with disciform scar , implying that hemodynamic changes can be partly responsible for asymmetry in some amd patients . nevertheless , a recent study of caucasian patients by sandhu et al ( 2007 ) demonstrated no differences between the pulsatile ocular blood flow in asymmetric eyes of patients with amd . presence of macular watershed zones was also correlated with amd and one study found that choroidal neovascularization arose from the macular watershed zone in 91% of cases ( ross et al 1998 ) . changes in the retrobulbar blood flow can provide a better understanding of the primary or secondary role of decreased blood flow in amd . in an animal model , selective damage to retinal pigment epithelial cells caused degenerative changes to the choriocapillaris , suggesting that degeneration of choriocapillaries in amd can be secondary to dysfunction of the retinal pigment epithelial cells ( henkind and gartner 1983 ; del priore et al 1996 ) . changes in these vessels can support a more primary role of decrease in blood flow in the development of amd , although a secondary autoregulatory response to a primary change elsewhere can not be ruled out ( ciulla et al 1999 ) . studies have found that blood flow velocity was lower in the ophthalmic artery and short posterior ciliary arteries of eyes with choroidal neovascularization compared with eyes without choroidal neovascularization ( uretmen et al 2003 ; ciulla et al 1999 ) . ciulla et al ( 1999 ) also found a decrease in the end - diastolic velocity and an increase in the resistive index of the central retinal artery . yannuzzi et al ( 2001 ) described retinal angiomatous proliferation as a phenomenon which occurs in a subgroup of neovascular amd patients , where angiomatous proliferation originates from the retina and extends posteriorly into the subretinal space , occasionally communicating with choroidal new vessels . although the hemodynamic abnormalities do not directly indicate that the vascular abnormalities precede the development of amd , they certainly suggest that hemodynamic changes play an important role in amd pathogenesis . furthermore , high choriocapillary pressure in the presence of calcification and fracture of bruch s membrane and ischemic damage to retinal pigment epithelial cells can cause the secretion of vascular growth factors leading to the development of choroidal neovascularization ( friedman 1997 , 2004 ) . although this theory has not been confirmed and does not elucidate the differences between races , it provides a well - rounded description of vascular alterations and how they contribute to the pathogenesis of amd . numerous population - based studies have reported differences in prevalence and incidence of amd between various ethnic / racial groups ( klein et al 1999 , 2006 ; mitchell et al 1995 ; vingerling et al 1995b ) . several studies have demonstrated an increased incidence of amd within families to provide further support for the role of genetics in amd ( seddon et al 1997 ; klaver et al 1998 ; grizzard et al 2003 ) . thakkinstian et al ( 2006 ) performed a meta - analysis between complement factor h y402h polymorphism and amd to find that heterozygotes ( tc ) and homozygotes ( cc ) were , respectively , 2.5 and 6 times more likely to have amd than homozygote patients with the tt genotype . these correlations between complement - related genes and amd further support the role of the inflammatory system in the development of amd . seddon et al ( 2007 ) analyzed patients from the age - related eye disease study ( areds ) to determine whether variations within the complement factor h gene and the loc387715 gene have prognostic importance in amd disease progression . although genetic polymorphisms in the complement factor h y402h and loc387715 a69s were associated with progression to more advanced amd , the loc387715 genotype was also correlated with neovascular disease more than geographic atrophy . patients who were homozygous for the risk alleles had a nearly 50% probability for amd progression compared to the 5% risk in patients who are homozygous for the nonrisk alleles ( seddon et al 2007 ) . additionally , presence of the homozygous risk alleles combined with smoking and high body mass indices were associated with 19-fold increase of disease progression ( seddon et al 2007 ) . there is strong evidence for the role of genetics in amd , but further research is necessary to determine how genetic differences can ultimately lead to amd . apart from the structural changes , age - related alterations in ocular hemodynamics can precede and correlate with the amd pathogenesis , which further supports the vascular theory of amd .

dementia is a clinical syndrome characterized by a global deterioration of mental functioning in its cognitive , emotional , and cognitive aspects . dementia typically involves a long period of progressive decline in memory and other cognitive abilities secondary to brain dysfunction and is a major cause of disability in elderly people . alzheimer disease is the most common dementing disorder , followed by vascular dementia , frontal lobe dementia , and dementia with lewy bodies . an expert panel estimated that the global prevalence of dementia is 3.9% in people over 60 years of age , and the estimated global annual incidence of dementia is approximately 7.5 per 1000 people . the incidence of dementia ranges from approximately 1 per 100 person - years in people aged 60 to 64 years to > 70 per 1000 person - years in people older than 90 years . because of an increased life expectancy in modern years , the number of people suffering from dementia has rapidly increased , potentially reaching up to 63 million people by 2030 . the risk factors for dementia include an apolipoprotein e4 genotype , cardiovascular comorbidities , diabetes mellitus , cerebrovascular diseases , alcohol consumption , and a lower education level . benzodiazepine ( bzd ) and other non - selective -aminobutyric acid ( gaba ) agonists with hypnotic effects similar to those of zolpidem have been shown to disrupt the memory in both human participants and animal subjects . the results of previous studies have suggested that bzds are associated with an increased risk of dementia in the elderly population , and these risks decreased when bzd use is discontinued . however , the possibility that zolpidem used independently of benzodiazepine derivatives , increases the risk for dementia has not been proposed . zolpidem and its derivatives ( the z drugs ) are non - bzd hypnotic agent belonging to the imidazopyridine family . zolpidem acts as an agonist of the benzodiazepine 1 receptor component of the gabaa receptor complex and is commonly used in patients with insomnia , including elderly patients . zolpidem is well known for having a rapid onset ( usually several minutes ) , short duration of action ( the peak time is 2 hours , half time is 1.55.5 hours ) , low tolerance , and a low incidence of adverse effects in treating insomnia . the most frequent adverse effects associated with zolpidem are nausea , headache , dizziness , drowsiness , hallucination , and short - term memory loss . a 3-week clinical trial revealed psychomotor retardation in 2% of patients receiving zolpidem and in 0% of patients in the placebo group . however , there are limited clinical data concerning the effects of long - term zolpidem use on psychomotor or cognitive functions . thus , the relationship between the use of zolpidem and the potential risk of developing dementia remains unknown . in the present study , we used a national population data bank to explore the associations between zolpidem and all dementia , non - alzheimer disease dementia , and alzheimer disease . a case control study was conducted using data from the taiwan national health insurance research database ( nhird ) . the taiwan national health insurance research database ( nhird ) contains reimbursement claims data from the taiwan national health insurance ( nhi ) system , which was established in 1996 and has provided coverage for approximately 99% of the population since 1998 . the national health research institute ( nhri ) manages the annual claims data in the nhird , and the longitudinal health insurance database ( lhid ) was established for use in medical research . the demographic data , medications , treatments ( including operations ) , and disease diagnoses ( based on the international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] ) of patients are recorded in the nhird . the health facilities enrolled in the taiwan nhi include local clinics , community hospitals , regional hospitals , and medical centers . with the exception of some local clinics , the taiwanese nhi includes almost all the primary , secondary , tertiary , and quaternary health care facilities in taiwan . the lhid comprises historical claims data for 1 million patients randomly selected from the nhird . the nhri encrypts the patients personal information for privacy protection and provides researchers with anonymous identification numbers associated with the relevant claim information , which includes the patient 's sex , date of birth , registry of medical services , and medication prescriptions . this study was approved by the institutional review board of china medical university in central taiwan ( cmu - rec-101012 ) . a population - based case patients 65 years and older diagnosed with dementia ( icd-9-cm 290 , 294 ) and alzheimer disease ( 331.0 ) in 2006 to 2010 were defined as a case group . the patients with the diagnosis with alzheimer disease were diagnosed by the board - certified neurologists and meet the following criteria : did not have history of severe trauma , stroke , or other neurology diseases that might interfere with the cognitive functions ; have decreased score in mini - mental state examination and cognitive ability screening instrument , complete blood sampling studies , and imaging studies . the control group was selected from the people without dementia diagnosis in lhid observation and was 2-fold frequency matched according to sex , age ( per 5 years ) , and index year . if the patients ever used zolpidem before index date , they were grouped into zolpidem used group . however , patients without zolpidem used before index date classified into non - zolpidem used group . the average zolpidem exposure dose was calculated by dividing the total zolpidem exposure ( milligrams ) according to the period between the first exposure and the index date ( years ) . other dementia - associated comorbidities were also considered potential confounding factors to determine associations between dementia and zolpidem . data for the underlying comorbidities , including hypertension ( icd-9-cm 401405 ) , diabetes ( icd-9-cm 250 ) , coronary artery disease ( cad ) ( icd-9-cm 410414 ) , stroke ( icd-9-cm 430438 ) , hyperlipidemia ( icd-9-cm 272 ) , depression ( icd-9-cm 296.2296.3 and 300.4 ) , and anxiety ( icd-9-cm 300.0 , 300.2 , 300.3 , 308.3 , and 309.81 ) , were collected before the index date . drugs potentially associated with the development of dementia , including anti - hypertension agents such as calcium channel blockers ( anatomical therapeutic chemical [ atc ] code : c08 ) , beta - blockers ( atc code : c07 ) , alpha - blockers ( atc code : c02 ) , diuretics ( atc code : c03 ) , angiotensin - converting enzyme antagonists ( acei ) , angiotensin receptor blockers ( arb ) ( atc code : c09 ) , and anticholesterol statin drugs ( atc code : c10 ) benzodiazepine ( atc code : n05 ) , and similar derivative drugs that were available such as zopiclone ( atc code : n05 ) were analyzed . we also considered effect of anti - psychotic drug used ( first generation antipsychotics and second generation antipsychotics ) and anti - depression drug used ( including selective serotonin reuptake inhibitors , monoamine oxidase inhibitors , heterocyclic antidepressants , and others [ bupropion , venlafaxine , mirtazapine , and duloxetine ] ) . all of these drug definitions were considered the drug use group as the patients at least once use before the index date . the distribution of the study population , which was based on the demographic characteristics and the disease history data , was analyzed . the test was used for categorical variables , and student t test was used for continuous variables to evaluate the differences among the study groups . the odds ratio ( or ) and 95% confidence interval ( 95% ci ) were measured for each comparison to estimate the associations between zolpidem use and dementia using logistic regression . adjusted odds ratios ( aors ) were also determined after adjusting for potential confounders . to evaluate the dose response of the association between the average zolpidem dose and dementia , logistic regression was used , and the average zolpidem dose was treated as a continuous variable across a range of average doses to evaluate trends in dementia diagnosis . we tested the multiplicative interaction of zolpidem use and each comorbidities ( or drugs ) by logistic regression and presented the effect of zolpidem for dementia under the different level of each comorbidities ( or drugs ) . all data management and statistical analyses were performed using the sas 9.1.3 software ( sas institute , cary , nc ) . all statistical tests were 2-sided , and p values < 0.05 were considered statistically significant . a case control study was conducted using data from the taiwan national health insurance research database ( nhird ) . the taiwan national health insurance research database ( nhird ) contains reimbursement claims data from the taiwan national health insurance ( nhi ) system , which was established in 1996 and has provided coverage for approximately 99% of the population since 1998 . the national health research institute ( nhri ) manages the annual claims data in the nhird , and the longitudinal health insurance database ( lhid ) was established for use in medical research . the demographic data , medications , treatments ( including operations ) , and disease diagnoses ( based on the international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] ) of patients are recorded in the nhird . the health facilities enrolled in the taiwan nhi include local clinics , community hospitals , regional hospitals , and medical centers . with the exception of some local clinics , the taiwanese nhi includes almost all the primary , secondary , tertiary , and quaternary health care facilities in taiwan . the lhid comprises historical claims data for 1 million patients randomly selected from the nhird . the nhri encrypts the patients personal information for privacy protection and provides researchers with anonymous identification numbers associated with the relevant claim information , which includes the patient 's sex , date of birth , registry of medical services , and medication prescriptions . this study was approved by the institutional review board of china medical university in central taiwan ( cmu - rec-101012 ) . patients 65 years and older diagnosed with dementia ( icd-9-cm 290 , 294 ) and alzheimer disease ( 331.0 ) in 2006 to 2010 were defined as a case group . the patients with the diagnosis with alzheimer disease were diagnosed by the board - certified neurologists and meet the following criteria : did not have history of severe trauma , stroke , or other neurology diseases that might interfere with the cognitive functions ; have decreased score in mini - mental state examination and cognitive ability screening instrument , complete blood sampling studies , and imaging studies . the control group was selected from the people without dementia diagnosis in lhid observation and was 2-fold frequency matched according to sex , age ( per 5 years ) , and index year . if the patients ever used zolpidem before index date , they were grouped into zolpidem used group . however , patients without zolpidem used before index date classified into non - zolpidem used group . the average zolpidem exposure dose was calculated by dividing the total zolpidem exposure ( milligrams ) according to the period between the first exposure and the index date ( years ) . other dementia - associated comorbidities were also considered potential confounding factors to determine associations between dementia and zolpidem . data for the underlying comorbidities , including hypertension ( icd-9-cm 401405 ) , diabetes ( icd-9-cm 250 ) , coronary artery disease ( cad ) ( icd-9-cm 410414 ) , stroke ( icd-9-cm 430438 ) , hyperlipidemia ( icd-9-cm 272 ) , depression ( icd-9-cm 296.2296.3 and 300.4 ) , and anxiety ( icd-9-cm 300.0 , 300.2 , 300.3 , 308.3 , and 309.81 ) , were collected before the index date . drugs potentially associated with the development of dementia , including anti - hypertension agents such as calcium channel blockers ( anatomical therapeutic chemical [ atc ] code : c08 ) , beta - blockers ( atc code : c07 ) , alpha - blockers ( atc code : c02 ) , diuretics ( atc code : c03 ) , angiotensin - converting enzyme antagonists ( acei ) , angiotensin receptor blockers ( arb ) ( atc code : c09 ) , and anticholesterol statin drugs ( atc code : c10 ) benzodiazepine ( atc code : n05 ) , and similar derivative drugs that were available such as zopiclone ( atc code : n05 ) were analyzed . we also considered effect of anti - psychotic drug used ( first generation antipsychotics and second generation antipsychotics ) and anti - depression drug used ( including selective serotonin reuptake inhibitors , monoamine oxidase inhibitors , heterocyclic antidepressants , and others [ bupropion , venlafaxine , mirtazapine , and duloxetine ] ) . all of these drug definitions were considered the drug use group as the patients at least once use before the index date . the distribution of the study population , which was based on the demographic characteristics and the disease history data , was analyzed . the test was used for categorical variables , and student t test was used for continuous variables to evaluate the differences among the study groups . the odds ratio ( or ) and 95% confidence interval ( 95% ci ) were measured for each comparison to estimate the associations between zolpidem use and dementia using logistic regression . adjusted odds ratios ( aors ) were also determined after adjusting for potential confounders . to evaluate the dose response of the association between the average zolpidem dose and dementia , logistic regression was used , and the average zolpidem dose was treated as a continuous variable across a range of average doses to evaluate trends in dementia diagnosis . we tested the multiplicative interaction of zolpidem use and each comorbidities ( or drugs ) by logistic regression and presented the effect of zolpidem for dementia under the different level of each comorbidities ( or drugs ) . all data management and statistical analyses were performed using the sas 9.1.3 software ( sas institute , cary , nc ) . all statistical tests were 2-sided , and p values < 0.05 were considered statistically significant . a total of 8406 dementia patient files were identified , and the control group consisted of 16,812 patients . the demographic characteristics of the study population are presented in table 1 . comparing with the control group , the dementia patients have longer zolpidem exposure time ( 302.8 days vs 345.1 days , p = 0.001 ) and higher exposure dose , were more likely to have been exposed to zolpidem ( 44.8% vs 30% , p < 0.0001 ) , zopiclone ( 20.3% vs 12.9% , p < 0.0001 ) , and bzds ( 91.3% vs 83.6% , p < 0.0001 ) , to have received simultaneous treatments with anti - hypertension agents ( 79.8% vs 70.3% , p < 0.0001 ) and anti - cholesterol statin agents ( 26.3% vs 22% , p < 0.0001 ) , and to have had underlying diseases , such as hypertension ( 81.6% vs 72.6% , p < 0.0001 ) , diabetes ( 32.2% vs 24.1% , p < 0.0001 ) , stroke ( 28.3% vs 12.2% , p < 0.0001 ) , cad ( 51% vs 42% , p < 0.0001 ) , hyperlipidemia ( 41.2% vs 36% , p < 0.0001 ) , depression ( 17.1% vs 6.3% , p < 0.0001 ) , and anxiety disorder ( 27.4% vs 17.9% , p < 0.0001 ) . demographic status and comorbidity compared between control group and dementia group the logistic regression models were adjusted for potential confounders , such as age , sex , cad , diabetes , stroke , hyperlipidemia , depression , anxiety , and the use of antihypertension agents , anticholesterol statin agents zopiclone and bzd drug uses , to evaluate the relationship between zolpidem use and dementia ( table 2 ) . zolpidem use and dementia remained significantly associated ( dementia : aor = 1.33 , 95% ci 1.241.41 ; p < 0.0001 ; non - alzheimer disease dementia : aor = 1.33 , 95% ci 1.251.42 ; p < 0.0001 ) , although the relationship effects between the zolpidem use and alzheimer disease were not significant ( alzheimer disease : aor = 1.17 , 95% ci 0.901.54 , p = 0.2410 ) . effects of zolpidem exposure on dementia in individuals in different average cumulative exposure doses the average cumulative zolpidem doses were analyzed to identify dose effects . response effects for most of types of dementia except alzheimer disease ( dementia : < 170 mg / y [ aor = 1.18 , 95% ci 1.111.28 ] , 170819 mg / y [ aor = 1.50 , 95% ci 1.361.65 ] , 820 mg / y [ aor = 1.52 , 95% ci 1.381.68 ] , p value for trend < 0.001 ; non - alzheimer disease dementia : < 170 mg / y [ aor = 1.20 , 95% ci 1.111.29 ] , 170819 mg / y [ aor = 1.49 , 95% ci 1.341.65 ] , 820 mg / y [ aor = 1.55 , 95% ci 1.391.72 ] , p value for trend < 0.001 ; and alzheimer disease : < 170 mg / y [ aor = 0.97 , 95% ci 0.681.37 ] , 170819 mg / y [ aor = 1.65 , 95% ci 1.082.51 ] , 820 mg / y [ aor = 1.15 , 95% ci 0.781.78 ] , p value for trend = 0.4413 ) . potential modifying effects for comorbidities that might interfere with the association between dementia and zolpidem were analyzed ( table 3 ) . although effect modifiers such as hypertension , diabetes , stroke , cad , hyperlipidemia , and anxiety , depression , anti - psychotic agent , and anti - depressant use had positive effects for dementia ( hypertension : aor = 1.72 , 95% ci 1.621.83 ; diabetes : aor = 1.64 , 95% ci 1.481.81 ; stroke : aor = 1.39 , 95% ci 1.231.57 ; cad : aor = 1.74 , 95% ci 1.611.88 ; hyperlipidemia : aor = 1.74 , 95% ci 1.601.90 ; anxiety : aor = 1.66 , 95% ci 1.481.86 ; depression : aor = 1.37 , 95% ci 1.151.63 ) , zolpidem exposure alone also has more positive effects on dementia in most circumstances ( non - hypertension : aor = 2.30 , 95% ci 2.012.62 ; non - diabetes : aor = 1.95 , 95% ci 1.832.08 ; non - stroke : aor = 1.88 , 95% ci 1.762.00 ; non - cad : aor = 1.88 , 95% ci 1.762.00 ; non - hyperlipidemia : aor = 1.95 , 95% ci 1.822.10 ; non - statin : aor = 1.92 , 95% ci 1.802.04 ; non - bzd : aor = 1.96 , 95% ci 1.492.59 ; non - anxiety : aor = 1.76 , 95% ci 1.651.88 ; non - depression : aor = 1.68 , 95% ci 1.581.78 ) . patients receiving zolpidem with anti - psychotic or anti - depressant agents at the same time had more positive effects on dementia risk ( anti - psychotic agents vs non - antipsychotic agents : aor = 1.75 [ 95% ci 1.611.90 ] vs. 1.57 [ 95% ci 1.461.70 ] ; anti - depressant vs non - antidepressant agents : aor = 1.66 [ 95% ci 1.541.80 ] vs 1.54 [ 95% ci 1.421.68 ] ) . although large numbers of patients were co - prescribed zolpidem and bzd derivatives , the effects of interactions between zolpidem and bzd derivatives were not significant . control study suggests that zolpidem use might increase the risk of developing dementia in the elderly population . the accumulative dose of zolpidem , alone , or with other underlying diseases , such as hypertension , diabetes , and stroke , was significantly associated with dementia after controlling for potential confounders , such as age , sex , cad , diabetes , antihypertension drugs , stroke , anticholesterol statin drugs , depression , anxiety , and bzd use ; however , the effects of zolpidem on patients with alzheimer disease remained obscure . the adjusted odds ratio for patients whose cumulative exposure doses were between 170 and 819 mg / year ( adjusted or : 1.65 , 95% ci 1.082.51 , p = 0.0199 ) was significant ; however , the effects for lower and higher cumulative dose were not significant . alzheimer disease is the leading subtype of dementia and has been identified as a protein degenerative disease that is primarily caused by the accumulation of abnormally folded amyloid beta and phosphorylated tau proteins in the brain . clinical presentations , histories , neurological examinations , images , and biomarkers are required to diagnose alzheimer disease . dementias other than alzheimer disease ( non - alzheimer dementias ) comprise heterogeneous diseases , including vascular dementia , mixed dementia , parkinson disease , dementia with lewy bodies , huntington disease , creutzfeldt jakob disease , and frontotemporal dementia / pick disease . interactions between other factors , such as hypercholesterolemia , hypertension , atherosclerosis , coronary heart disease , smoking , obesity , diabetes , and sleep deprivation or sleep fragmentation , were reported to be risk factors that might contribute to dementia . however , few studies have addressed the potential long - term psychoneurological effects of drugs on non - alzheimer dementia or alzheimer disease . zolpidem is an effective non - bzd drug that is primarily used for treating insomnia in the elderly population . previous clinical trials have demonstrated the effects of zolpidem on the central nervous system ( eg , confusion , impaired cognitive and motor function , postural sway , and ataxia ) , and an increased risk of falling has also been observed . comparable dose - related impairment was also observed in healthy participants asked to perform cognitive - related tests , such as picture recall , digit entry and recall , a digit symbol substitution test , repeated acquisition and circular lights tasks , and balance tests . the cognition - impairing effects in executive function tasks were characterized in animal models before . previous study results suggested that zolpidem displays a high affinity for 1 subunit of gabaa receptors ; therefore , the drug impairs the human cognitive domain in the prefrontal cortical areas , which are involved in processes such as goal forming , planning , initiation , preservation and alteration of goal - directed behaviors , problem - solving , response inhibition , and cognitive flexibility . because the aging brain has increased 1 subunit of gabaa receptor binding sites and 1 subunit of gabaa receptor mrna levels , the cognition - impairing effects of zolpidem might reflect the elderly population 's increased risk of developing non - alzheimer dementia . in contrast , the 1 subunit of gabaa receptor protein and mrna levels were reduced in the brain cortex regions of alzheimer disease patients . the different compositions indicate that increases or decreases in the proportion of receptors containing a particular subunit may reflect the affinity of the receptor for zolpidem , resulting in altered drug responses between patients of non - alzheimer dementias and alzheimer disease in the elderly population . whether cognitive effects on zolpidem could extend to other z drugs still remain obscure . previous studies suggested zaleplon had a rapid elimination and had fewer residual side effects after taking a single dose at bedtime . by comparison , the differences in potency based on plasma concentrations suggested that there are differences in binding to the gaba receptor complex . for example , zopiclone interacts with both benzodiazepine 1and 2 gabaa receptors , and zolpidem mainly acts on benzodiazepine 1 gabaa receptors . therefore , there may be a prolonged drug effect and result in residual sedation and side effects . the z drugs undergoes hepatic metabolism by cytochrome p450 . the delayed clearances of the z drugs were significantly decreased in patients with preexisting advanced liver diseases . furthermore , variations in genetic polymorphism of the cytochrome p450 in different population might result in different responses as well . a study has suggested that the cyp3a4 and cyp2c19 genetic polymorphisms are associated with the poor metabolism of zolpidem in the chinese han population . a limited number of human studies have explored the relationship between neuropsychological drugs and the development of dementia or alzheimer disease . a meta - analysis setting out to ascertain which domains of cognitive function were influenced among the z drugs indicated very few studies evaluate the individual cognitive effects of the z drugs . most of the studies focused on the next - day residual cognitive effects of the z drugs following nocturnal administration . the z drugs have been heralded as a new frontier with consistent increase in the prescription in the pharmacological treatments available for insomnia patients due to the adverse and deleterious effects associated with long - term benzodiazepine use . there needs to be more thorough investigations into the possible effects on the daily functioning of individuals who take these medications . the results of the present study indicate the potential associations regarding the cumulative dose and interactions among commonly prescribed tranquilizers , zolpidem , underlying comorbidities , and the development of dementia or alzheimer disease in the elderly population . although detailed observations and medical records were not obtained and effect modifiers and confounders were not completely controlled , correlations between zolpidem and dementia in the elderly population were observed . the limitations of the present study include the limitations of the clinical data collected from the nhird , the difficulty associated with controlling for confounding factors in a retrospective study design , and the incomplete verification of the data in the nhird . the nhird does not provide detailed information regarding smoking habits , alcohol consumption , substance use , body mass index , physical activity , socioeconomic status , family history , and detailed medical records such as sleep quality records , reasons for zolpidem withdrawal , or intermittent used , which are potential confounding factors for this analysis . furthermore , some patients in control group might present prodromal symptoms like nxiety , depression , or insomnia before dementia diagnosis was made and led to misclassification and selection bias . the registries in the nhi claims system were primarily designed for administrative billing , and the registry data are not subjected to the stringent levels of verification appropriate for many types of scientific research . there was also no method for directly contacting the patients to obtain additional information on the use of zolpidem because the participants remained anonymous . however , the data from the nhird regarding prescriptions and the diagnosis of major underlying diseases and dementia are highly reliable . qualified neurologists performed a series of neurological examinations . because of the limitations of the nhird , the prescription records for zolpidem before 1996 were not acquired for analysis ; therefore , shorter follow - up examinations and lower cumulative doses for zolpidem were calculated , and the risk associations between zolpidem and dementia might also be underestimated . future studies , such as population - based unbiased randomized observational trials , are warranted to confirm the causal relationships between zolpidem use and dementia . in conclusion , zolpidem use might be associated with an increased risk for dementia in the elderly population . an increased accumulative dose might result in a significantly higher risk to develop dementia in patients with underlying diseases , such as hypertension , diabetes , and stroke . the long - term effects of zolpidem on patients with alzheimer disease might not be significant in the elderly population . therefore , the careful evaluation of the indications of zolpidem use and close follow - up examinations of the cognitive status of elderly patients receiving zolpidem are essential .

abstractwe evaluate the effects of zolpidem use to develop dementia or alzheimer disease from the taiwan national health insurance research database ( nhird).a retrospective population - based nested case control study . newly diagnosed dementia patients 65 years and older and controls were sampled . a total of 8406 dementia and 16,812 control subjects were enrolled from taiwan nhird during 2006 to 2010 . the relationships between zolpidem use and dementia were measured using odds and adjusted odds ratios . the relationship between the average cumulative doses for zolpidem and dementia was also analyzed.zolpidem alone or with other underlying diseases , such as hypertension , diabetes , and stroke , was significantly associated with dementia after controlling for potential confounders , such as age , sex , coronary artery disease , diabetes , anti - hypertension drugs , stroke , anticholesterol statin drugs , depression , anxiety , benzodiazepine , anti - psychotic , and anti - depressant agents use ( adjusted or = 1.33 , 95% ci 1.241.41 ) . zolpidem use also has significant dose response effects for most of the types of dementia . in patient with alzheimer diseases , the effects of zolpidem among patients with alzheimer 's disease remained obscure . the adjusted or for patients whose cumulative exposure doses were between 170 and 819 mg / year ( adjusted or : 1.65 , 95% ci 1.082.51 , p = 0.0199 ) was significant ; however , the effects for lower and higher cumulative dose were not significant.zolpidem used might be associated with increased risk for dementia in elderly population . increased accumulative dose might have higher risk to develop dementia , especially in patients with underlying diseases such as hypertension , diabetes , and stroke .

INTRODUCTION MATERIALS AND METHODS Data Source Study Design Statistical Analysis RESULTS DISCUSSION LIMITATIONS

the risk factors for dementia include an apolipoprotein e4 genotype , cardiovascular comorbidities , diabetes mellitus , cerebrovascular diseases , alcohol consumption , and a lower education level . the results of previous studies have suggested that bzds are associated with an increased risk of dementia in the elderly population , and these risks decreased when bzd use is discontinued . however , the possibility that zolpidem used independently of benzodiazepine derivatives , increases the risk for dementia has not been proposed . thus , the relationship between the use of zolpidem and the potential risk of developing dementia remains unknown . in the present study , we used a national population data bank to explore the associations between zolpidem and all dementia , non - alzheimer disease dementia , and alzheimer disease . a case control study was conducted using data from the taiwan national health insurance research database ( nhird ) . the taiwan national health insurance research database ( nhird ) contains reimbursement claims data from the taiwan national health insurance ( nhi ) system , which was established in 1996 and has provided coverage for approximately 99% of the population since 1998 . the national health research institute ( nhri ) manages the annual claims data in the nhird , and the longitudinal health insurance database ( lhid ) was established for use in medical research . a population - based case patients 65 years and older diagnosed with dementia ( icd-9-cm 290 , 294 ) and alzheimer disease ( 331.0 ) in 2006 to 2010 were defined as a case group . the patients with the diagnosis with alzheimer disease were diagnosed by the board - certified neurologists and meet the following criteria : did not have history of severe trauma , stroke , or other neurology diseases that might interfere with the cognitive functions ; have decreased score in mini - mental state examination and cognitive ability screening instrument , complete blood sampling studies , and imaging studies . data for the underlying comorbidities , including hypertension ( icd-9-cm 401405 ) , diabetes ( icd-9-cm 250 ) , coronary artery disease ( cad ) ( icd-9-cm 410414 ) , stroke ( icd-9-cm 430438 ) , hyperlipidemia ( icd-9-cm 272 ) , depression ( icd-9-cm 296.2296.3 and 300.4 ) , and anxiety ( icd-9-cm 300.0 , 300.2 , 300.3 , 308.3 , and 309.81 ) , were collected before the index date . drugs potentially associated with the development of dementia , including anti - hypertension agents such as calcium channel blockers ( anatomical therapeutic chemical [ atc ] code : c08 ) , beta - blockers ( atc code : c07 ) , alpha - blockers ( atc code : c02 ) , diuretics ( atc code : c03 ) , angiotensin - converting enzyme antagonists ( acei ) , angiotensin receptor blockers ( arb ) ( atc code : c09 ) , and anticholesterol statin drugs ( atc code : c10 ) benzodiazepine ( atc code : n05 ) , and similar derivative drugs that were available such as zopiclone ( atc code : n05 ) were analyzed . we also considered effect of anti - psychotic drug used ( first generation antipsychotics and second generation antipsychotics ) and anti - depression drug used ( including selective serotonin reuptake inhibitors , monoamine oxidase inhibitors , heterocyclic antidepressants , and others [ bupropion , venlafaxine , mirtazapine , and duloxetine ] ) . the odds ratio ( or ) and 95% confidence interval ( 95% ci ) were measured for each comparison to estimate the associations between zolpidem use and dementia using logistic regression . adjusted odds ratios ( aors ) were also determined after adjusting for potential confounders . to evaluate the dose response of the association between the average zolpidem dose and dementia , logistic regression was used , and the average zolpidem dose was treated as a continuous variable across a range of average doses to evaluate trends in dementia diagnosis . we tested the multiplicative interaction of zolpidem use and each comorbidities ( or drugs ) by logistic regression and presented the effect of zolpidem for dementia under the different level of each comorbidities ( or drugs ) . a case control study was conducted using data from the taiwan national health insurance research database ( nhird ) . the taiwan national health insurance research database ( nhird ) contains reimbursement claims data from the taiwan national health insurance ( nhi ) system , which was established in 1996 and has provided coverage for approximately 99% of the population since 1998 . the national health research institute ( nhri ) manages the annual claims data in the nhird , and the longitudinal health insurance database ( lhid ) was established for use in medical research . patients 65 years and older diagnosed with dementia ( icd-9-cm 290 , 294 ) and alzheimer disease ( 331.0 ) in 2006 to 2010 were defined as a case group . the patients with the diagnosis with alzheimer disease were diagnosed by the board - certified neurologists and meet the following criteria : did not have history of severe trauma , stroke , or other neurology diseases that might interfere with the cognitive functions ; have decreased score in mini - mental state examination and cognitive ability screening instrument , complete blood sampling studies , and imaging studies . data for the underlying comorbidities , including hypertension ( icd-9-cm 401405 ) , diabetes ( icd-9-cm 250 ) , coronary artery disease ( cad ) ( icd-9-cm 410414 ) , stroke ( icd-9-cm 430438 ) , hyperlipidemia ( icd-9-cm 272 ) , depression ( icd-9-cm 296.2296.3 and 300.4 ) , and anxiety ( icd-9-cm 300.0 , 300.2 , 300.3 , 308.3 , and 309.81 ) , were collected before the index date . drugs potentially associated with the development of dementia , including anti - hypertension agents such as calcium channel blockers ( anatomical therapeutic chemical [ atc ] code : c08 ) , beta - blockers ( atc code : c07 ) , alpha - blockers ( atc code : c02 ) , diuretics ( atc code : c03 ) , angiotensin - converting enzyme antagonists ( acei ) , angiotensin receptor blockers ( arb ) ( atc code : c09 ) , and anticholesterol statin drugs ( atc code : c10 ) benzodiazepine ( atc code : n05 ) , and similar derivative drugs that were available such as zopiclone ( atc code : n05 ) were analyzed . we also considered effect of anti - psychotic drug used ( first generation antipsychotics and second generation antipsychotics ) and anti - depression drug used ( including selective serotonin reuptake inhibitors , monoamine oxidase inhibitors , heterocyclic antidepressants , and others [ bupropion , venlafaxine , mirtazapine , and duloxetine ] ) . the odds ratio ( or ) and 95% confidence interval ( 95% ci ) were measured for each comparison to estimate the associations between zolpidem use and dementia using logistic regression . adjusted odds ratios ( aors ) were also determined after adjusting for potential confounders . to evaluate the dose response of the association between the average zolpidem dose and dementia , logistic regression was used , and the average zolpidem dose was treated as a continuous variable across a range of average doses to evaluate trends in dementia diagnosis . we tested the multiplicative interaction of zolpidem use and each comorbidities ( or drugs ) by logistic regression and presented the effect of zolpidem for dementia under the different level of each comorbidities ( or drugs ) . a total of 8406 dementia patient files were identified , and the control group consisted of 16,812 patients . comparing with the control group , the dementia patients have longer zolpidem exposure time ( 302.8 days vs 345.1 days , p = 0.001 ) and higher exposure dose , were more likely to have been exposed to zolpidem ( 44.8% vs 30% , p < 0.0001 ) , zopiclone ( 20.3% vs 12.9% , p < 0.0001 ) , and bzds ( 91.3% vs 83.6% , p < 0.0001 ) , to have received simultaneous treatments with anti - hypertension agents ( 79.8% vs 70.3% , p < 0.0001 ) and anti - cholesterol statin agents ( 26.3% vs 22% , p < 0.0001 ) , and to have had underlying diseases , such as hypertension ( 81.6% vs 72.6% , p < 0.0001 ) , diabetes ( 32.2% vs 24.1% , p < 0.0001 ) , stroke ( 28.3% vs 12.2% , p < 0.0001 ) , cad ( 51% vs 42% , p < 0.0001 ) , hyperlipidemia ( 41.2% vs 36% , p < 0.0001 ) , depression ( 17.1% vs 6.3% , p < 0.0001 ) , and anxiety disorder ( 27.4% vs 17.9% , p < 0.0001 ) . demographic status and comorbidity compared between control group and dementia group the logistic regression models were adjusted for potential confounders , such as age , sex , cad , diabetes , stroke , hyperlipidemia , depression , anxiety , and the use of antihypertension agents , anticholesterol statin agents zopiclone and bzd drug uses , to evaluate the relationship between zolpidem use and dementia ( table 2 ) . zolpidem use and dementia remained significantly associated ( dementia : aor = 1.33 , 95% ci 1.241.41 ; p < 0.0001 ; non - alzheimer disease dementia : aor = 1.33 , 95% ci 1.251.42 ; p < 0.0001 ) , although the relationship effects between the zolpidem use and alzheimer disease were not significant ( alzheimer disease : aor = 1.17 , 95% ci 0.901.54 , p = 0.2410 ) . effects of zolpidem exposure on dementia in individuals in different average cumulative exposure doses the average cumulative zolpidem doses were analyzed to identify dose effects . response effects for most of types of dementia except alzheimer disease ( dementia : < 170 mg / y [ aor = 1.18 , 95% ci 1.111.28 ] , 170819 mg / y [ aor = 1.50 , 95% ci 1.361.65 ] , 820 mg / y [ aor = 1.52 , 95% ci 1.381.68 ] , p value for trend < 0.001 ; non - alzheimer disease dementia : < 170 mg / y [ aor = 1.20 , 95% ci 1.111.29 ] , 170819 mg / y [ aor = 1.49 , 95% ci 1.341.65 ] , 820 mg / y [ aor = 1.55 , 95% ci 1.391.72 ] , p value for trend < 0.001 ; and alzheimer disease : < 170 mg / y [ aor = 0.97 , 95% ci 0.681.37 ] , 170819 mg / y [ aor = 1.65 , 95% ci 1.082.51 ] , 820 mg / y [ aor = 1.15 , 95% ci 0.781.78 ] , p value for trend = 0.4413 ) . although effect modifiers such as hypertension , diabetes , stroke , cad , hyperlipidemia , and anxiety , depression , anti - psychotic agent , and anti - depressant use had positive effects for dementia ( hypertension : aor = 1.72 , 95% ci 1.621.83 ; diabetes : aor = 1.64 , 95% ci 1.481.81 ; stroke : aor = 1.39 , 95% ci 1.231.57 ; cad : aor = 1.74 , 95% ci 1.611.88 ; hyperlipidemia : aor = 1.74 , 95% ci 1.601.90 ; anxiety : aor = 1.66 , 95% ci 1.481.86 ; depression : aor = 1.37 , 95% ci 1.151.63 ) , zolpidem exposure alone also has more positive effects on dementia in most circumstances ( non - hypertension : aor = 2.30 , 95% ci 2.012.62 ; non - diabetes : aor = 1.95 , 95% ci 1.832.08 ; non - stroke : aor = 1.88 , 95% ci 1.762.00 ; non - cad : aor = 1.88 , 95% ci 1.762.00 ; non - hyperlipidemia : aor = 1.95 , 95% ci 1.822.10 ; non - statin : aor = 1.92 , 95% ci 1.802.04 ; non - bzd : aor = 1.96 , 95% ci 1.492.59 ; non - anxiety : aor = 1.76 , 95% ci 1.651.88 ; non - depression : aor = 1.68 , 95% ci 1.581.78 ) . patients receiving zolpidem with anti - psychotic or anti - depressant agents at the same time had more positive effects on dementia risk ( anti - psychotic agents vs non - antipsychotic agents : aor = 1.75 [ 95% ci 1.611.90 ] vs. 1.57 [ 95% ci 1.461.70 ] ; anti - depressant vs non - antidepressant agents : aor = 1.66 [ 95% ci 1.541.80 ] vs 1.54 [ 95% ci 1.421.68 ] ) . although large numbers of patients were co - prescribed zolpidem and bzd derivatives , the effects of interactions between zolpidem and bzd derivatives were not significant . control study suggests that zolpidem use might increase the risk of developing dementia in the elderly population . the accumulative dose of zolpidem , alone , or with other underlying diseases , such as hypertension , diabetes , and stroke , was significantly associated with dementia after controlling for potential confounders , such as age , sex , cad , diabetes , antihypertension drugs , stroke , anticholesterol statin drugs , depression , anxiety , and bzd use ; however , the effects of zolpidem on patients with alzheimer disease remained obscure . the adjusted odds ratio for patients whose cumulative exposure doses were between 170 and 819 mg / year ( adjusted or : 1.65 , 95% ci 1.082.51 , p = 0.0199 ) was significant ; however , the effects for lower and higher cumulative dose were not significant . dementias other than alzheimer disease ( non - alzheimer dementias ) comprise heterogeneous diseases , including vascular dementia , mixed dementia , parkinson disease , dementia with lewy bodies , huntington disease , creutzfeldt jakob disease , and frontotemporal dementia / pick disease . interactions between other factors , such as hypercholesterolemia , hypertension , atherosclerosis , coronary heart disease , smoking , obesity , diabetes , and sleep deprivation or sleep fragmentation , were reported to be risk factors that might contribute to dementia . however , few studies have addressed the potential long - term psychoneurological effects of drugs on non - alzheimer dementia or alzheimer disease . previous clinical trials have demonstrated the effects of zolpidem on the central nervous system ( eg , confusion , impaired cognitive and motor function , postural sway , and ataxia ) , and an increased risk of falling has also been observed . comparable dose - related impairment was also observed in healthy participants asked to perform cognitive - related tests , such as picture recall , digit entry and recall , a digit symbol substitution test , repeated acquisition and circular lights tasks , and balance tests . because the aging brain has increased 1 subunit of gabaa receptor binding sites and 1 subunit of gabaa receptor mrna levels , the cognition - impairing effects of zolpidem might reflect the elderly population 's increased risk of developing non - alzheimer dementia . the different compositions indicate that increases or decreases in the proportion of receptors containing a particular subunit may reflect the affinity of the receptor for zolpidem , resulting in altered drug responses between patients of non - alzheimer dementias and alzheimer disease in the elderly population . a limited number of human studies have explored the relationship between neuropsychological drugs and the development of dementia or alzheimer disease . the results of the present study indicate the potential associations regarding the cumulative dose and interactions among commonly prescribed tranquilizers , zolpidem , underlying comorbidities , and the development of dementia or alzheimer disease in the elderly population . although detailed observations and medical records were not obtained and effect modifiers and confounders were not completely controlled , correlations between zolpidem and dementia in the elderly population were observed . the limitations of the present study include the limitations of the clinical data collected from the nhird , the difficulty associated with controlling for confounding factors in a retrospective study design , and the incomplete verification of the data in the nhird . however , the data from the nhird regarding prescriptions and the diagnosis of major underlying diseases and dementia are highly reliable . because of the limitations of the nhird , the prescription records for zolpidem before 1996 were not acquired for analysis ; therefore , shorter follow - up examinations and lower cumulative doses for zolpidem were calculated , and the risk associations between zolpidem and dementia might also be underestimated . future studies , such as population - based unbiased randomized observational trials , are warranted to confirm the causal relationships between zolpidem use and dementia . in conclusion , zolpidem use might be associated with an increased risk for dementia in the elderly population . an increased accumulative dose might result in a significantly higher risk to develop dementia in patients with underlying diseases , such as hypertension , diabetes , and stroke . the long - term effects of zolpidem on patients with alzheimer disease might not be significant in the elderly population . therefore , the careful evaluation of the indications of zolpidem use and close follow - up examinations of the cognitive status of elderly patients receiving zolpidem are essential .

an expert panel estimated that the global prevalence of dementia is 3.9% in people over 60 years of age , and the estimated global annual incidence of dementia is approximately 7.5 per 1000 people . benzodiazepine ( bzd ) and other non - selective -aminobutyric acid ( gaba ) agonists with hypnotic effects similar to those of zolpidem have been shown to disrupt the memory in both human participants and animal subjects . the results of previous studies have suggested that bzds are associated with an increased risk of dementia in the elderly population , and these risks decreased when bzd use is discontinued . zolpidem is well known for having a rapid onset ( usually several minutes ) , short duration of action ( the peak time is 2 hours , half time is 1.55.5 hours ) , low tolerance , and a low incidence of adverse effects in treating insomnia . thus , the relationship between the use of zolpidem and the potential risk of developing dementia remains unknown . in the present study , we used a national population data bank to explore the associations between zolpidem and all dementia , non - alzheimer disease dementia , and alzheimer disease . the demographic data , medications , treatments ( including operations ) , and disease diagnoses ( based on the international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] ) of patients are recorded in the nhird . the nhri encrypts the patients personal information for privacy protection and provides researchers with anonymous identification numbers associated with the relevant claim information , which includes the patient 's sex , date of birth , registry of medical services , and medication prescriptions . a population - based case patients 65 years and older diagnosed with dementia ( icd-9-cm 290 , 294 ) and alzheimer disease ( 331.0 ) in 2006 to 2010 were defined as a case group . the patients with the diagnosis with alzheimer disease were diagnosed by the board - certified neurologists and meet the following criteria : did not have history of severe trauma , stroke , or other neurology diseases that might interfere with the cognitive functions ; have decreased score in mini - mental state examination and cognitive ability screening instrument , complete blood sampling studies , and imaging studies . the control group was selected from the people without dementia diagnosis in lhid observation and was 2-fold frequency matched according to sex , age ( per 5 years ) , and index year . data for the underlying comorbidities , including hypertension ( icd-9-cm 401405 ) , diabetes ( icd-9-cm 250 ) , coronary artery disease ( cad ) ( icd-9-cm 410414 ) , stroke ( icd-9-cm 430438 ) , hyperlipidemia ( icd-9-cm 272 ) , depression ( icd-9-cm 296.2296.3 and 300.4 ) , and anxiety ( icd-9-cm 300.0 , 300.2 , 300.3 , 308.3 , and 309.81 ) , were collected before the index date . drugs potentially associated with the development of dementia , including anti - hypertension agents such as calcium channel blockers ( anatomical therapeutic chemical [ atc ] code : c08 ) , beta - blockers ( atc code : c07 ) , alpha - blockers ( atc code : c02 ) , diuretics ( atc code : c03 ) , angiotensin - converting enzyme antagonists ( acei ) , angiotensin receptor blockers ( arb ) ( atc code : c09 ) , and anticholesterol statin drugs ( atc code : c10 ) benzodiazepine ( atc code : n05 ) , and similar derivative drugs that were available such as zopiclone ( atc code : n05 ) were analyzed . we also considered effect of anti - psychotic drug used ( first generation antipsychotics and second generation antipsychotics ) and anti - depression drug used ( including selective serotonin reuptake inhibitors , monoamine oxidase inhibitors , heterocyclic antidepressants , and others [ bupropion , venlafaxine , mirtazapine , and duloxetine ] ) . the odds ratio ( or ) and 95% confidence interval ( 95% ci ) were measured for each comparison to estimate the associations between zolpidem use and dementia using logistic regression . to evaluate the dose response of the association between the average zolpidem dose and dementia , logistic regression was used , and the average zolpidem dose was treated as a continuous variable across a range of average doses to evaluate trends in dementia diagnosis . we tested the multiplicative interaction of zolpidem use and each comorbidities ( or drugs ) by logistic regression and presented the effect of zolpidem for dementia under the different level of each comorbidities ( or drugs ) . the nhri encrypts the patients personal information for privacy protection and provides researchers with anonymous identification numbers associated with the relevant claim information , which includes the patient 's sex , date of birth , registry of medical services , and medication prescriptions . patients 65 years and older diagnosed with dementia ( icd-9-cm 290 , 294 ) and alzheimer disease ( 331.0 ) in 2006 to 2010 were defined as a case group . the patients with the diagnosis with alzheimer disease were diagnosed by the board - certified neurologists and meet the following criteria : did not have history of severe trauma , stroke , or other neurology diseases that might interfere with the cognitive functions ; have decreased score in mini - mental state examination and cognitive ability screening instrument , complete blood sampling studies , and imaging studies . the control group was selected from the people without dementia diagnosis in lhid observation and was 2-fold frequency matched according to sex , age ( per 5 years ) , and index year . data for the underlying comorbidities , including hypertension ( icd-9-cm 401405 ) , diabetes ( icd-9-cm 250 ) , coronary artery disease ( cad ) ( icd-9-cm 410414 ) , stroke ( icd-9-cm 430438 ) , hyperlipidemia ( icd-9-cm 272 ) , depression ( icd-9-cm 296.2296.3 and 300.4 ) , and anxiety ( icd-9-cm 300.0 , 300.2 , 300.3 , 308.3 , and 309.81 ) , were collected before the index date . drugs potentially associated with the development of dementia , including anti - hypertension agents such as calcium channel blockers ( anatomical therapeutic chemical [ atc ] code : c08 ) , beta - blockers ( atc code : c07 ) , alpha - blockers ( atc code : c02 ) , diuretics ( atc code : c03 ) , angiotensin - converting enzyme antagonists ( acei ) , angiotensin receptor blockers ( arb ) ( atc code : c09 ) , and anticholesterol statin drugs ( atc code : c10 ) benzodiazepine ( atc code : n05 ) , and similar derivative drugs that were available such as zopiclone ( atc code : n05 ) were analyzed . we also considered effect of anti - psychotic drug used ( first generation antipsychotics and second generation antipsychotics ) and anti - depression drug used ( including selective serotonin reuptake inhibitors , monoamine oxidase inhibitors , heterocyclic antidepressants , and others [ bupropion , venlafaxine , mirtazapine , and duloxetine ] ) . the odds ratio ( or ) and 95% confidence interval ( 95% ci ) were measured for each comparison to estimate the associations between zolpidem use and dementia using logistic regression . to evaluate the dose response of the association between the average zolpidem dose and dementia , logistic regression was used , and the average zolpidem dose was treated as a continuous variable across a range of average doses to evaluate trends in dementia diagnosis . we tested the multiplicative interaction of zolpidem use and each comorbidities ( or drugs ) by logistic regression and presented the effect of zolpidem for dementia under the different level of each comorbidities ( or drugs ) . comparing with the control group , the dementia patients have longer zolpidem exposure time ( 302.8 days vs 345.1 days , p = 0.001 ) and higher exposure dose , were more likely to have been exposed to zolpidem ( 44.8% vs 30% , p < 0.0001 ) , zopiclone ( 20.3% vs 12.9% , p < 0.0001 ) , and bzds ( 91.3% vs 83.6% , p < 0.0001 ) , to have received simultaneous treatments with anti - hypertension agents ( 79.8% vs 70.3% , p < 0.0001 ) and anti - cholesterol statin agents ( 26.3% vs 22% , p < 0.0001 ) , and to have had underlying diseases , such as hypertension ( 81.6% vs 72.6% , p < 0.0001 ) , diabetes ( 32.2% vs 24.1% , p < 0.0001 ) , stroke ( 28.3% vs 12.2% , p < 0.0001 ) , cad ( 51% vs 42% , p < 0.0001 ) , hyperlipidemia ( 41.2% vs 36% , p < 0.0001 ) , depression ( 17.1% vs 6.3% , p < 0.0001 ) , and anxiety disorder ( 27.4% vs 17.9% , p < 0.0001 ) . demographic status and comorbidity compared between control group and dementia group the logistic regression models were adjusted for potential confounders , such as age , sex , cad , diabetes , stroke , hyperlipidemia , depression , anxiety , and the use of antihypertension agents , anticholesterol statin agents zopiclone and bzd drug uses , to evaluate the relationship between zolpidem use and dementia ( table 2 ) . zolpidem use and dementia remained significantly associated ( dementia : aor = 1.33 , 95% ci 1.241.41 ; p < 0.0001 ; non - alzheimer disease dementia : aor = 1.33 , 95% ci 1.251.42 ; p < 0.0001 ) , although the relationship effects between the zolpidem use and alzheimer disease were not significant ( alzheimer disease : aor = 1.17 , 95% ci 0.901.54 , p = 0.2410 ) . effects of zolpidem exposure on dementia in individuals in different average cumulative exposure doses the average cumulative zolpidem doses were analyzed to identify dose effects . response effects for most of types of dementia except alzheimer disease ( dementia : < 170 mg / y [ aor = 1.18 , 95% ci 1.111.28 ] , 170819 mg / y [ aor = 1.50 , 95% ci 1.361.65 ] , 820 mg / y [ aor = 1.52 , 95% ci 1.381.68 ] , p value for trend < 0.001 ; non - alzheimer disease dementia : < 170 mg / y [ aor = 1.20 , 95% ci 1.111.29 ] , 170819 mg / y [ aor = 1.49 , 95% ci 1.341.65 ] , 820 mg / y [ aor = 1.55 , 95% ci 1.391.72 ] , p value for trend < 0.001 ; and alzheimer disease : < 170 mg / y [ aor = 0.97 , 95% ci 0.681.37 ] , 170819 mg / y [ aor = 1.65 , 95% ci 1.082.51 ] , 820 mg / y [ aor = 1.15 , 95% ci 0.781.78 ] , p value for trend = 0.4413 ) . although effect modifiers such as hypertension , diabetes , stroke , cad , hyperlipidemia , and anxiety , depression , anti - psychotic agent , and anti - depressant use had positive effects for dementia ( hypertension : aor = 1.72 , 95% ci 1.621.83 ; diabetes : aor = 1.64 , 95% ci 1.481.81 ; stroke : aor = 1.39 , 95% ci 1.231.57 ; cad : aor = 1.74 , 95% ci 1.611.88 ; hyperlipidemia : aor = 1.74 , 95% ci 1.601.90 ; anxiety : aor = 1.66 , 95% ci 1.481.86 ; depression : aor = 1.37 , 95% ci 1.151.63 ) , zolpidem exposure alone also has more positive effects on dementia in most circumstances ( non - hypertension : aor = 2.30 , 95% ci 2.012.62 ; non - diabetes : aor = 1.95 , 95% ci 1.832.08 ; non - stroke : aor = 1.88 , 95% ci 1.762.00 ; non - cad : aor = 1.88 , 95% ci 1.762.00 ; non - hyperlipidemia : aor = 1.95 , 95% ci 1.822.10 ; non - statin : aor = 1.92 , 95% ci 1.802.04 ; non - bzd : aor = 1.96 , 95% ci 1.492.59 ; non - anxiety : aor = 1.76 , 95% ci 1.651.88 ; non - depression : aor = 1.68 , 95% ci 1.581.78 ) . patients receiving zolpidem with anti - psychotic or anti - depressant agents at the same time had more positive effects on dementia risk ( anti - psychotic agents vs non - antipsychotic agents : aor = 1.75 [ 95% ci 1.611.90 ] vs. 1.57 [ 95% ci 1.461.70 ] ; anti - depressant vs non - antidepressant agents : aor = 1.66 [ 95% ci 1.541.80 ] vs 1.54 [ 95% ci 1.421.68 ] ) . the accumulative dose of zolpidem , alone , or with other underlying diseases , such as hypertension , diabetes , and stroke , was significantly associated with dementia after controlling for potential confounders , such as age , sex , cad , diabetes , antihypertension drugs , stroke , anticholesterol statin drugs , depression , anxiety , and bzd use ; however , the effects of zolpidem on patients with alzheimer disease remained obscure . the adjusted odds ratio for patients whose cumulative exposure doses were between 170 and 819 mg / year ( adjusted or : 1.65 , 95% ci 1.082.51 , p = 0.0199 ) was significant ; however , the effects for lower and higher cumulative dose were not significant . alzheimer disease is the leading subtype of dementia and has been identified as a protein degenerative disease that is primarily caused by the accumulation of abnormally folded amyloid beta and phosphorylated tau proteins in the brain . dementias other than alzheimer disease ( non - alzheimer dementias ) comprise heterogeneous diseases , including vascular dementia , mixed dementia , parkinson disease , dementia with lewy bodies , huntington disease , creutzfeldt jakob disease , and frontotemporal dementia / pick disease . interactions between other factors , such as hypercholesterolemia , hypertension , atherosclerosis , coronary heart disease , smoking , obesity , diabetes , and sleep deprivation or sleep fragmentation , were reported to be risk factors that might contribute to dementia . previous clinical trials have demonstrated the effects of zolpidem on the central nervous system ( eg , confusion , impaired cognitive and motor function , postural sway , and ataxia ) , and an increased risk of falling has also been observed . comparable dose - related impairment was also observed in healthy participants asked to perform cognitive - related tests , such as picture recall , digit entry and recall , a digit symbol substitution test , repeated acquisition and circular lights tasks , and balance tests . previous study results suggested that zolpidem displays a high affinity for 1 subunit of gabaa receptors ; therefore , the drug impairs the human cognitive domain in the prefrontal cortical areas , which are involved in processes such as goal forming , planning , initiation , preservation and alteration of goal - directed behaviors , problem - solving , response inhibition , and cognitive flexibility . because the aging brain has increased 1 subunit of gabaa receptor binding sites and 1 subunit of gabaa receptor mrna levels , the cognition - impairing effects of zolpidem might reflect the elderly population 's increased risk of developing non - alzheimer dementia . the different compositions indicate that increases or decreases in the proportion of receptors containing a particular subunit may reflect the affinity of the receptor for zolpidem , resulting in altered drug responses between patients of non - alzheimer dementias and alzheimer disease in the elderly population . a meta - analysis setting out to ascertain which domains of cognitive function were influenced among the z drugs indicated very few studies evaluate the individual cognitive effects of the z drugs . the z drugs have been heralded as a new frontier with consistent increase in the prescription in the pharmacological treatments available for insomnia patients due to the adverse and deleterious effects associated with long - term benzodiazepine use . the results of the present study indicate the potential associations regarding the cumulative dose and interactions among commonly prescribed tranquilizers , zolpidem , underlying comorbidities , and the development of dementia or alzheimer disease in the elderly population . the limitations of the present study include the limitations of the clinical data collected from the nhird , the difficulty associated with controlling for confounding factors in a retrospective study design , and the incomplete verification of the data in the nhird . the nhird does not provide detailed information regarding smoking habits , alcohol consumption , substance use , body mass index , physical activity , socioeconomic status , family history , and detailed medical records such as sleep quality records , reasons for zolpidem withdrawal , or intermittent used , which are potential confounding factors for this analysis . because of the limitations of the nhird , the prescription records for zolpidem before 1996 were not acquired for analysis ; therefore , shorter follow - up examinations and lower cumulative doses for zolpidem were calculated , and the risk associations between zolpidem and dementia might also be underestimated . therefore , the careful evaluation of the indications of zolpidem use and close follow - up examinations of the cognitive status of elderly patients receiving zolpidem are essential .

consider a one - dimensional series yn = ( y1 , , yn ) of length n. mapping this series into a sequence of lagged vectors with size l , x1 , , xk , with xi = ( y1 , , yi+l1 ) r provides the trajectory matrix x=(xi , j)i , j=1l , k , where l(2 l n/2 ) is the window length and k = n l + 1;x=[x1, ,xk]=(xi , j)i , j=1l , k = y1y2 yky2y3 yk+1ylyl+1 yn.the trajectory matrix x is a hankel matrix as has equal elements on the antidiagonals i + j = const . the importance of x and its corresponding singular values can be seen in different areas including time series analysis , , biomedical signal processing , , mathematics , econometrics and physics . however , the distribution of eigenvalues / singular values and their closed form has not been studied adequately . for recent work on the generalized eigenvalues of hankel random matrices the eigenvalue distributions of beta - wishart matrices which is a special case of random matrix see edelman and plamen study . furthermore , such hankel matrix x naturally appears in multivariate analysis and signal processing , particularly in singular spectrum analysis , where each of it column represents the l - lagged vector of observations in r , . accordingly , the aim was to determine the accurate dimension of the system , that is the smallest dimension with which the filtered series is reconstructed from a noisy signal . in this case , the main analysis is based on the study of the eigenvalues and corresponding eigenvectors . if the signal component dominates the noise component , then the eigenvalues of the random matrix x have a few large eigenvalues and many small ones , suggesting that the variations in the data takes place mainly in the eigenspace corresponding to these few large eigenvalues . note that the number of correct singular values , r , for filtering and noise reduction , is increased with the increased l which makes the comparison among different choices ( l , r ) more difficult . furthermore , despite the fact that several approaches have been proposed to identify the values of r , due to a lack of substantial theoretical results , none of them consider the distribution of singular values of x. here , we study the empirical distribution of singular values of x for different situations considering various criteria . accordingly , the theoretical results on the eigenvalues of xx divided by its trace with a new view is considered in main results . the empirical results using simulated data are presented in the empirical distribution of i . some conclusions and recommendations for future research the singular values of x are the square root of the eigenvalues of the l by l matrix xx , where x is the conjugate transpose . for a fixed value of l and a series with length n , the trace of matrix xx , tr(xxt)=xf2=i=1li , where f denotes the frobenius norm , and i(i=1, note that the increase of sample size n leads to the increase of i which makes the situation more complex . to overcome this issue , we divide xx by its trace ( xxt/i=1li ) , which provides the following properties.proposition 1let 1 , , l denote eigenvalues of the matrix ( xxt/i=1li ) , where x is a hankel trajectory matrix with l rows , and i(i=1, thus , we have the following properties:1.0 l 1 1,2.i=1li=1,3.1 1/l,4.l 1/l.proofthe first two properties are simply obtained from matrix algebra and thus not provided here . the outermost inequalities are attained as equalities when , for example , yi = 1 for all i. to prove the third property , the first two properties are used as follows . + l = 1 . thus , using the first property , 1 i ( i = 2 , , l ) , we obtain 1 + 1 + + 1 = l1 1 1 1/l . similarly , for the fourth property , it is straightforward to show that l + l + + l = ll 1 l 1/l , since l i(i = 1 , 2 , , l 1 ) , and i = 1 . note also that if yl = 1 and yi = 0 for i l then 1 = , l = 1/l . rational number theory can also aid us to provide more informative inequalities ( for more information see ) . let 1 , , l denote eigenvalues of the matrix ( xxt/i=1li ) , where x is a hankel trajectory matrix with l rows , and i(i=1, thus , we have the following properties : the first two properties are simply obtained from matrix algebra and thus not provided here . the outermost inequalities are attained as equalities when , for example , yi = 1 for all i. to prove the third property , the first two properties are used as follows . + l = 1 . thus , using the first property , 1 i ( i = 2 , , + 1 = l1 1 1 1/l . similarly , for the fourth property , it is straightforward to show that l + l + + l = ll 1 l 1/l , since l i(i = 1 , 2 , , l 1 ) , and i = 1 . note also that if yl = 1 and yi = 0 for i l then 1 = , l = 1/l . rational number theory can also aid us to provide more informative inequalities ( for more information see ) . a series of length n from different distributions , is generated m times . for consistency and comparability of the results , a fixed value of l , here 10 , is used for all examples and case studies throughout the paper . for point estimation and comparing the mean value of eigenvalues , the average of each eigenvalue in m runs is used ; i as defined before , i = 1 , , here we consider eight different cases that can be seen in real life examples:(a)white noise ; wn.(b)uniform distribution with mean zero ; u( , ).(c)uniform distribution ; u(0 , ).(d)exponential distribution ; exp().(e) + exp().(f) + t.(g)sine wave series ; sin().(h) + sin( ) + sin(),where = 1 , = 2 , = 2t/12 , = 2t/5 , and t is the time which is used to generate the linear trend series . 1 demonstrates i for different values of n for cases ( ( a)(c ) ) considered in this study . in fig . 1 , i has a decreasing pattern for different values of n. it can be seen that , for a large n , i 1/10 for cases ( a ) and ( b ) . thus , increasing n clearly affects the values of i for the white noise ( a ) and uniform distribution ( b ) . however , there is no obvious effect on i for other cases . for example , for case ( c ) , 1 is approximately equal to 0.8 for different values of n , and i1 is less than 1/10 ( see fig . although the pattern of i for the uniform distribution ( c ) is similar to exponential case ( d ) , but for case ( c ) , 1 is greater than 1 comparing to the case ( d ) , whilst other i are smaller . it has been observed that i has similar patterns for cases ( ( c ) , the values of i for cases ( a ) and ( b ) , where yn generated from a symmetric distribution , are approximately the same . the results clearly indicate that increasing n does not have a significant influence on the mean of i for all cases except ( a ) and ( b ) . as a result , n will affect the value of i significantly . let us now consider the patterns of i for n = 10 . for the white noise distribution ( a ) and trend series ( f ) , i has different pattern . it is obvious that , for the white noise series , i converges asymptotically to 1/10 , whilst for the trend series 1 is approximately equal to 1 , and i1 tends to zero . similar results were obtained for the uniform distributions , cases ( b ) and ( c ) , respectively . however , it is noticed that adding an intercept to the exponential distribution , increases the value of 1 and decreases other i . the results indicate that 10.6 and 20.4 , whilst , other izero for sine wave ( g ) . it also indicates that , for sine case ( h ) , i(i = 1 , , 5 ) are not zero , whereas other i tend to zero . it was noticed that the value of 1 for sine wave ( h ) is greater than its value for sine case ( g ) , whilst the value of 2 is less . the distribution of i was assessed for different values of l. it was observed that the histograms of i are similar for different values of l ( the results are not presented here ) . therefore , for graphical aspect , and visualization purpose , l = 10 is considered here . the results are provided only for 1 , 5 and 10 , for the cases ( ( a ) , , ( d ) ) , as similar results are observed for other i . fig . 2 shows histogram of i(i = 1 , 5 , 10 ) for l = 10 , and m = 5000 simulations . it appears that the histogram of 1 , is skewed to the right for samples taken from wn ( a ) and uniform distributions ( b ) , whilst for the data generated from the uniform ( c ) and exponential ( d ) distributions , might be symmetric . for the middle i , the histogram might be symmetric for the four cases ( the results only provided for 5 ) , whilst the distribution of 10 , is skewed to the left . for cases , exponential distribution ( e ) , trend series ( f ) , and sine wave series ( g ) and complex series ( h ) , we have standardized i to have conveying information about their distributions . 3 shows the density of i ( i = 1 , 2 , 3 , 5 , 6 , 10 ) for those cases . it is clear that 1 has different histogram for these cases , and also different from what was achieved for the white noise and uniform distributions with zero mean . remember that , if yn generated from a symmetric distribution , like case ( a ) and ( b ) , 1 has a right skewed distribution . moreover , it is interesting that 10 has a negative skewed distribution for all cases except the trend series and sine cases ( ( g ) and ( h ) ) . additionally , it should be noted that , for sine series ( g ) , both 1 and 2 have similar distributions , whereas other i have right skewed distributions . it is obvious that the distribution of i for sine series ( h ) becomes skewed to the right for i ( i = 6 , , remember that the sine wave ( h ) was generated from an intercept and two pure sine waves . this means that the components related to the first five eigenvalues create the sine series ( h ) . the results confirm that adding even an intercept alone will change the pattern of i . note that an intercept can be considered as a trend in time series analysis . generally , if we add more non stochastic components to the noise series , for instance trend , harmonic and cyclical components , then the first few eigenvalues are related to those components and as soon as we reach the noise level the pattern of eigenvalues will be similar to those found for the noise series . usually every harmonic component with a different frequency produces two close eigenvalues ( except for frequency 0.5 which provides one eigenvalues ) . it will be clearer if n , l , and k are sufficiently large . in practice , the eigenvalues of a harmonic series are often close to each other , and this fact simplifies the visual identification of the harmonic components . thus , the results obtained here are very important for signal processing and time series techniques where noise reduction and filtering matter . generally , it is not easy to judge visually if i has a symmetric distribution , thus it is necessarily to consider other criteria like statistical test . we calculate the coefficient of skewness which is a measure for the degree of symmetry in the distribution of a variable . bulmer suggests that ; if skewness is less than 1 or greater than + 1 , the distribution is highly skewed ; if skewness is between 1 and 1/2 or between + 1/2 and + 1 , the distribution is moderately skewed , and finally if skewness is between 1/2 and + 1/2 , the distributions approximately symmetric . therefore , we can say that , for instance , the distribution of 1 for cases ( ( c ) , , ( f ) ) , and 5 for all cases might be symmetric . it is also known as the omnibus test because it uses the test statistics for both the skewness and kurtosis to come up with a single p - value and quantify how far from gaussian the distribution is in terms of asymmetry and shape . the p - value of dagostin test was significant , greater than 0.05 for 1 , for cases ( ( c ) , , ( f ) ) , whereas , it is less than 0.05 for other cases ( ( a ) , ( b ) , ( g ) , ( h ) ) . therefore , we accept the null hypothesis that the data of 1 for cases ( ( c ) , , ( f ) ) are not skewed and as a result are symmetric . moreover , 5 has a symmetric distribution for all cases , except the trend series and sine waves . the distribution of i(i = 2 , 4 ) , for the exponential case ( d ) is symmetric , whereas skewed for the exponential case with intercept ( e ) . in terms of the distribution of i for the trend series and sine wave ( g ) , the distributions of i=1,2 are totally different to the distributions of other i , which becomes skewed distribution . note that the distribution of i ( i = 1 , 2 ) for the trend series is symmetric , whilst skewed for sine wave ( g ) . for sine series ( h ) , the distribution of i ( i = 1 , , 5 ) is different from the distribution of i ( i = 6 , , 10 ) . it is obvious from the figure that i ( i = 6 , , 10 ) has a right skewed distribution . in this section , we consider the effect of the sample size , n on i . 1 demonstrates i for different values of n for cases ( ( a)(c ) ) considered in this study . in fig . 1 , i has a decreasing pattern for different values of n. it can be seen that , for a large n , i 1/10 for cases ( a ) and ( b ) . thus , increasing n clearly affects the values of i for the white noise ( a ) and uniform distribution ( b ) . however , there is no obvious effect on i for other cases . for example , for case ( c ) , 1 is approximately equal to 0.8 for different values of n , and i1 is less than 1/10 ( see fig . although the pattern of i for the uniform distribution ( c ) is similar to exponential case ( d ) , but for case ( c ) , 1 is greater than 1 comparing to the case ( d ) , whilst other i are smaller . it has been observed that i has similar patterns for cases ( ( c ) , the values of i for cases ( a ) and ( b ) , where yn generated from a symmetric distribution , are approximately the same . the results clearly indicate that increasing n does not have a significant influence on the mean of i for all cases except ( a ) and ( b ) . as a result , if yn is generated from wn or u(1 , 1 ) , then increasing n will affect the value of i significantly . let us now consider the patterns of i for n = 10 . for the white noise distribution ( a ) and trend series ( f ) it is obvious that , for the white noise series , i converges asymptotically to 1/10 , whilst for the trend series 1 is approximately equal to 1 , and i1 tends to zero . similar results were obtained for the uniform distributions , cases ( b ) and ( c ) , respectively . however , it is noticed that adding an intercept to the exponential distribution , increases the value of 1 and decreases other i . the results indicate that 10.6 and 20.4 , whilst , other izero for sine wave ( g ) . it also indicates that , for sine case ( h ) , i(i = 1 , , 5 ) are not zero , whereas other i tend to zero . it was noticed that the value of 1 for sine wave ( h ) is greater than its value for sine case ( g ) , whilst the value of 2 is less . the distribution of i was assessed for different values of l. it was observed that the histograms of i are similar for different values of l ( the results are not presented here ) . therefore , for graphical aspect , and visualization purpose , l = 10 is considered here . the results are provided only for 1 , 5 and 10 , for the cases ( ( a ) , , ( d ) ) , as similar results are observed for other i . fig . 2 shows histogram of i(i = 1 , 5 , 10 ) for l = 10 , and m = 5000 simulations . it appears that the histogram of 1 , is skewed to the right for samples taken from wn ( a ) and uniform distributions ( b ) , whilst for the data generated from the uniform ( c ) and exponential ( d ) distributions , might be symmetric . for the middle i , the histogram might be symmetric for the four cases ( the results only provided for 5 ) , whilst the distribution of 10 , is skewed to the left . for cases , exponential distribution ( e ) , trend series ( f ) , and sine wave series ( g ) and complex series ( h ) , we have standardized i to have conveying information about their distributions . 3 shows the density of i ( i = 1 , 2 , 3 , 5 , 6 , 10 ) for those cases . it is clear that 1 has different histogram for these cases , and also different from what was achieved for the white noise and uniform distributions with zero mean . remember that , if yn generated from a symmetric distribution , like case ( a ) and ( b ) , 1 has a right skewed distribution . moreover , it is interesting that 10 has a negative skewed distribution for all cases except the trend series and sine cases ( ( g ) and ( h ) ) . additionally , it should be noted that , for sine series ( g ) , both 1 and 2 have similar distributions , whereas other i have right skewed distributions . it is obvious that the distribution of i for sine series ( h ) becomes skewed to the right for i ( i = 6 , , remember that the sine wave ( h ) was generated from an intercept and two pure sine waves . this means that the components related to the first five eigenvalues create the sine series ( h ) . the results confirm that adding even an intercept alone will change the pattern of i . generally , if we add more non stochastic components to the noise series , for instance trend , harmonic and cyclical components , then the first few eigenvalues are related to those components and as soon as we reach the noise level the pattern of eigenvalues will be similar to those found for the noise series . usually every harmonic component with a different frequency produces two close eigenvalues ( except for frequency 0.5 which provides one eigenvalues ) . it will be clearer if n , l , and k are sufficiently large . in practice , the eigenvalues of a harmonic series are often close to each other , and this fact simplifies the visual identification of the harmonic components . thus , the results obtained here are very important for signal processing and time series techniques where noise reduction and filtering matter . generally , it is not easy to judge visually if i has a symmetric distribution , thus it is necessarily to consider other criteria like statistical test . we calculate the coefficient of skewness which is a measure for the degree of symmetry in the distribution of a variable . bulmer suggests that ; if skewness is less than 1 or greater than + 1 , the distribution is highly skewed ; if skewness is between 1 and 1/2 or between + 1/2 and + 1 , the distribution is moderately skewed , and finally if skewness is between 1/2 and + 1/2 , the distributions approximately symmetric . therefore , we can say that , for instance , the distribution of 1 for cases ( ( c ) , , ( f ) ) , and 5 for all cases might be symmetric . dagostino it is also known as the omnibus test because it uses the test statistics for both the skewness and kurtosis to come up with a single p - value and quantify how far from gaussian the distribution is in terms of asymmetry and shape . the p - value of dagostin test was significant , greater than 0.05 for 1 , for cases ( ( c ) , , ( f ) ) , whereas , it is less than 0.05 for other cases ( ( a ) , ( b ) , ( g ) , ( h ) ) . therefore , we accept the null hypothesis that the data of 1 for cases ( ( c ) , , ( f ) ) are not skewed and as a result are symmetric . moreover , 5 has a symmetric distribution for all cases , except the trend series and sine waves . the distribution of i(i = 2 , 4 ) , for the exponential case ( d ) is symmetric , whereas skewed for the exponential case with intercept ( e ) . in terms of the distribution of i for the trend series and sine wave ( g ) , the distributions of i=1,2 are totally different to the distributions of other i , which becomes skewed distribution . note that the distribution of i ( i = 1 , 2 ) for the trend series is symmetric , whilst skewed for sine wave ( g ) . for sine series ( h ) , the distribution of i ( i = 1 , , 5 ) is different from the distribution of i ( i = 6 , , 10 ) . it is obvious from the figure that i ( i = 6 , , 10 ) has a right skewed distribution . the pattern of the eigenvalues of the matrix xxt/i=1li , generated from different distributions was studied , and several properties were introduced . the results indicate that for a large sample size n , i ; n 1/l for the symmetric distributions ( the white noise and the uniform distributions with zero mean ) , whilst this convergence has not been observed for other cases . the results also indicate that , for the symmetric cases , the pattern of the first eigenvalue is skewed , whilst it can be symmetric for the trend and nonsymmetrical distributions . furthermore , for all cases under this study , the distribution of the middle i , for l = 10 , can be symmetric except the pattern of 5 for the trend case and both sine series . it is found that the last eigenvalue has a positive skewed distribution , for all cases except the trend series and sine waves . for future research , the theoretical distribution of the matrix xxt/i=1li is of our interest . furthermore , we aim to evaluate the applicability of the results found here for noise reduction of the chaotic series . additionally , we are applying the properties obtained here as extra criteria for filtering series with complex structure . we may also consider a test to evaluate the k largest eigenvalues , to decide whether the distribution of the eigenvalues can resemble the particular distribution of the eigenvalues . in addition , the distribution of the smallest eigenvalue is as well of great interest , for example , because its behavior is used to prove its convergence to the circular law . accordingly , the study of the local properties of the spectrum as well as the related distribution is of interest .

the empirical distribution of the eigenvalues of the matrix xxt divided by its trace is evaluated , where x is a random hankel matrix . the distribution of eigenvalues for symmetric and nonsymmetric distributions is assessed with various criteria . this yields several important properties with broad application , particularly for noise reduction and filtering in signal processing and time series analysis .

Introduction Main results The effect of The patterns of The empirical distribution of Conclusions Conflict of Interest Compliance with Ethics Requirements

consider a one - dimensional series yn = ( y1 , , yn ) of length n. mapping this series into a sequence of lagged vectors with size l , x1 , , xk , with xi = ( y1 , , yi+l1 ) r provides the trajectory matrix x=(xi , j)i , j=1l , k , where l(2 l n/2 ) is the window length and k = n l + 1;x=[x1, ,xk]=(xi , j)i , j=1l , k = y1y2 yky2y3 yk+1ylyl+1 yn.the trajectory matrix x is a hankel matrix as has equal elements on the antidiagonals i + j = const . the importance of x and its corresponding singular values can be seen in different areas including time series analysis , , biomedical signal processing , , mathematics , econometrics and physics . however , the distribution of eigenvalues / singular values and their closed form has not been studied adequately . for recent work on the generalized eigenvalues of hankel random matrices the eigenvalue distributions of beta - wishart matrices which is a special case of random matrix see edelman and plamen study . furthermore , such hankel matrix x naturally appears in multivariate analysis and signal processing , particularly in singular spectrum analysis , where each of it column represents the l - lagged vector of observations in r , . accordingly , the aim was to determine the accurate dimension of the system , that is the smallest dimension with which the filtered series is reconstructed from a noisy signal . in this case , the main analysis is based on the study of the eigenvalues and corresponding eigenvectors . if the signal component dominates the noise component , then the eigenvalues of the random matrix x have a few large eigenvalues and many small ones , suggesting that the variations in the data takes place mainly in the eigenspace corresponding to these few large eigenvalues . note that the number of correct singular values , r , for filtering and noise reduction , is increased with the increased l which makes the comparison among different choices ( l , r ) more difficult . furthermore , despite the fact that several approaches have been proposed to identify the values of r , due to a lack of substantial theoretical results , none of them consider the distribution of singular values of x. here , we study the empirical distribution of singular values of x for different situations considering various criteria . accordingly , the theoretical results on the eigenvalues of xx divided by its trace with a new view is considered in main results . the empirical results using simulated data are presented in the empirical distribution of i . some conclusions and recommendations for future research the singular values of x are the square root of the eigenvalues of the l by l matrix xx , where x is the conjugate transpose . for a fixed value of l and a series with length n , the trace of matrix xx , tr(xxt)=xf2=i=1li , where f denotes the frobenius norm , and i(i=1, note that the increase of sample size n leads to the increase of i which makes the situation more complex . to overcome this issue , we divide xx by its trace ( xxt/i=1li ) , which provides the following properties.proposition 1let 1 , , l denote eigenvalues of the matrix ( xxt/i=1li ) , where x is a hankel trajectory matrix with l rows , and i(i=1, thus , we have the following properties:1.0 l 1 1,2.i=1li=1,3.1 1/l,4.l 1/l.proofthe first two properties are simply obtained from matrix algebra and thus not provided here . the outermost inequalities are attained as equalities when , for example , yi = 1 for all i. to prove the third property , the first two properties are used as follows . + l = 1 . thus , using the first property , 1 i ( i = 2 , , l ) , we obtain 1 + 1 + + 1 = l1 1 1 1/l . similarly , for the fourth property , it is straightforward to show that l + l + + l = ll 1 l 1/l , since l i(i = 1 , 2 , , l 1 ) , and i = 1 . note also that if yl = 1 and yi = 0 for i l then 1 = , l = 1/l . let 1 , , l denote eigenvalues of the matrix ( xxt/i=1li ) , where x is a hankel trajectory matrix with l rows , and i(i=1, thus , we have the following properties : the first two properties are simply obtained from matrix algebra and thus not provided here . the outermost inequalities are attained as equalities when , for example , yi = 1 for all i. to prove the third property , the first two properties are used as follows . + l = 1 . note also that if yl = 1 and yi = 0 for i l then 1 = , l = 1/l . rational number theory can also aid us to provide more informative inequalities ( for more information see ) . a series of length n from different distributions , is generated m times . for consistency and comparability of the results , a fixed value of l , here 10 , is used for all examples and case studies throughout the paper . for point estimation and comparing the mean value of eigenvalues , the average of each eigenvalue in m runs is used ; i as defined before , i = 1 , , here we consider eight different cases that can be seen in real life examples:(a)white noise ; wn. (c)uniform distribution ; u(0 , ). (d)exponential distribution ; exp(). (e) + exp(). in fig . however , there is no obvious effect on i for other cases . although the pattern of i for the uniform distribution ( c ) is similar to exponential case ( d ) , but for case ( c ) , 1 is greater than 1 comparing to the case ( d ) , whilst other i are smaller . it has been observed that i has similar patterns for cases ( ( c ) , the values of i for cases ( a ) and ( b ) , where yn generated from a symmetric distribution , are approximately the same . the results clearly indicate that increasing n does not have a significant influence on the mean of i for all cases except ( a ) and ( b ) . as a result , n will affect the value of i significantly . however , it is noticed that adding an intercept to the exponential distribution , increases the value of 1 and decreases other i . the results indicate that 10.6 and 20.4 , whilst , other izero for sine wave ( g ) . it also indicates that , for sine case ( h ) , i(i = 1 , , 5 ) are not zero , whereas other i tend to zero . the distribution of i was assessed for different values of l. it was observed that the histograms of i are similar for different values of l ( the results are not presented here ) . for the middle i , the histogram might be symmetric for the four cases ( the results only provided for 5 ) , whilst the distribution of 10 , is skewed to the left . for cases , exponential distribution ( e ) , trend series ( f ) , and sine wave series ( g ) and complex series ( h ) , we have standardized i to have conveying information about their distributions . it is clear that 1 has different histogram for these cases , and also different from what was achieved for the white noise and uniform distributions with zero mean . additionally , it should be noted that , for sine series ( g ) , both 1 and 2 have similar distributions , whereas other i have right skewed distributions . it is obvious that the distribution of i for sine series ( h ) becomes skewed to the right for i ( i = 6 , , remember that the sine wave ( h ) was generated from an intercept and two pure sine waves . this means that the components related to the first five eigenvalues create the sine series ( h ) . note that an intercept can be considered as a trend in time series analysis . generally , if we add more non stochastic components to the noise series , for instance trend , harmonic and cyclical components , then the first few eigenvalues are related to those components and as soon as we reach the noise level the pattern of eigenvalues will be similar to those found for the noise series . usually every harmonic component with a different frequency produces two close eigenvalues ( except for frequency 0.5 which provides one eigenvalues ) . in practice , the eigenvalues of a harmonic series are often close to each other , and this fact simplifies the visual identification of the harmonic components . thus , the results obtained here are very important for signal processing and time series techniques where noise reduction and filtering matter . we calculate the coefficient of skewness which is a measure for the degree of symmetry in the distribution of a variable . bulmer suggests that ; if skewness is less than 1 or greater than + 1 , the distribution is highly skewed ; if skewness is between 1 and 1/2 or between + 1/2 and + 1 , the distribution is moderately skewed , and finally if skewness is between 1/2 and + 1/2 , the distributions approximately symmetric . therefore , we can say that , for instance , the distribution of 1 for cases ( ( c ) , , ( f ) ) , and 5 for all cases might be symmetric . it is also known as the omnibus test because it uses the test statistics for both the skewness and kurtosis to come up with a single p - value and quantify how far from gaussian the distribution is in terms of asymmetry and shape . therefore , we accept the null hypothesis that the data of 1 for cases ( ( c ) , , ( f ) ) are not skewed and as a result are symmetric . the distribution of i(i = 2 , 4 ) , for the exponential case ( d ) is symmetric , whereas skewed for the exponential case with intercept ( e ) . in terms of the distribution of i for the trend series and sine wave ( g ) , the distributions of i=1,2 are totally different to the distributions of other i , which becomes skewed distribution . note that the distribution of i ( i = 1 , 2 ) for the trend series is symmetric , whilst skewed for sine wave ( g ) . for sine series ( h ) , the distribution of i ( i = 1 , , 5 ) is different from the distribution of i ( i = 6 , , 10 ) . in this section , we consider the effect of the sample size , n on i . 1 demonstrates i for different values of n for cases ( ( a)(c ) ) considered in this study . thus , increasing n clearly affects the values of i for the white noise ( a ) and uniform distribution ( b ) . however , there is no obvious effect on i for other cases . for example , for case ( c ) , 1 is approximately equal to 0.8 for different values of n , and i1 is less than 1/10 ( see fig . it has been observed that i has similar patterns for cases ( ( c ) , the values of i for cases ( a ) and ( b ) , where yn generated from a symmetric distribution , are approximately the same . the results clearly indicate that increasing n does not have a significant influence on the mean of i for all cases except ( a ) and ( b ) . let us now consider the patterns of i for n = 10 . for the white noise distribution ( a ) and trend series ( f ) it is obvious that , for the white noise series , i converges asymptotically to 1/10 , whilst for the trend series 1 is approximately equal to 1 , and i1 tends to zero . similar results were obtained for the uniform distributions , cases ( b ) and ( c ) , respectively . however , it is noticed that adding an intercept to the exponential distribution , increases the value of 1 and decreases other i . the results indicate that 10.6 and 20.4 , whilst , other izero for sine wave ( g ) . it was noticed that the value of 1 for sine wave ( h ) is greater than its value for sine case ( g ) , whilst the value of 2 is less . the distribution of i was assessed for different values of l. it was observed that the histograms of i are similar for different values of l ( the results are not presented here ) . therefore , for graphical aspect , and visualization purpose , l = 10 is considered here . the results are provided only for 1 , 5 and 10 , for the cases ( ( a ) , , ( d ) ) , as similar results are observed for other i . 2 shows histogram of i(i = 1 , 5 , 10 ) for l = 10 , and m = 5000 simulations . it appears that the histogram of 1 , is skewed to the right for samples taken from wn ( a ) and uniform distributions ( b ) , whilst for the data generated from the uniform ( c ) and exponential ( d ) distributions , might be symmetric . for the middle i , the histogram might be symmetric for the four cases ( the results only provided for 5 ) , whilst the distribution of 10 , is skewed to the left . additionally , it should be noted that , for sine series ( g ) , both 1 and 2 have similar distributions , whereas other i have right skewed distributions . it is obvious that the distribution of i for sine series ( h ) becomes skewed to the right for i ( i = 6 , , remember that the sine wave ( h ) was generated from an intercept and two pure sine waves . this means that the components related to the first five eigenvalues create the sine series ( h ) . generally , if we add more non stochastic components to the noise series , for instance trend , harmonic and cyclical components , then the first few eigenvalues are related to those components and as soon as we reach the noise level the pattern of eigenvalues will be similar to those found for the noise series . in practice , the eigenvalues of a harmonic series are often close to each other , and this fact simplifies the visual identification of the harmonic components . thus , the results obtained here are very important for signal processing and time series techniques where noise reduction and filtering matter . we calculate the coefficient of skewness which is a measure for the degree of symmetry in the distribution of a variable . bulmer suggests that ; if skewness is less than 1 or greater than + 1 , the distribution is highly skewed ; if skewness is between 1 and 1/2 or between + 1/2 and + 1 , the distribution is moderately skewed , and finally if skewness is between 1/2 and + 1/2 , the distributions approximately symmetric . therefore , we can say that , for instance , the distribution of 1 for cases ( ( c ) , , ( f ) ) , and 5 for all cases might be symmetric . dagostino it is also known as the omnibus test because it uses the test statistics for both the skewness and kurtosis to come up with a single p - value and quantify how far from gaussian the distribution is in terms of asymmetry and shape . the p - value of dagostin test was significant , greater than 0.05 for 1 , for cases ( ( c ) , , ( f ) ) , whereas , it is less than 0.05 for other cases ( ( a ) , ( b ) , ( g ) , ( h ) ) . therefore , we accept the null hypothesis that the data of 1 for cases ( ( c ) , , ( f ) ) are not skewed and as a result are symmetric . the distribution of i(i = 2 , 4 ) , for the exponential case ( d ) is symmetric , whereas skewed for the exponential case with intercept ( e ) . in terms of the distribution of i for the trend series and sine wave ( g ) , the distributions of i=1,2 are totally different to the distributions of other i , which becomes skewed distribution . note that the distribution of i ( i = 1 , 2 ) for the trend series is symmetric , whilst skewed for sine wave ( g ) . for sine series ( h ) , the distribution of i ( i = 1 , , 5 ) is different from the distribution of i ( i = 6 , , 10 ) . the pattern of the eigenvalues of the matrix xxt/i=1li , generated from different distributions was studied , and several properties were introduced . the results also indicate that , for the symmetric cases , the pattern of the first eigenvalue is skewed , whilst it can be symmetric for the trend and nonsymmetrical distributions . furthermore , for all cases under this study , the distribution of the middle i , for l = 10 , can be symmetric except the pattern of 5 for the trend case and both sine series . it is found that the last eigenvalue has a positive skewed distribution , for all cases except the trend series and sine waves . for future research , the theoretical distribution of the matrix xxt/i=1li is of our interest . furthermore , we aim to evaluate the applicability of the results found here for noise reduction of the chaotic series . additionally , we are applying the properties obtained here as extra criteria for filtering series with complex structure . we may also consider a test to evaluate the k largest eigenvalues , to decide whether the distribution of the eigenvalues can resemble the particular distribution of the eigenvalues . in addition , the distribution of the smallest eigenvalue is as well of great interest , for example , because its behavior is used to prove its convergence to the circular law . accordingly , the study of the local properties of the spectrum as well as the related distribution is of interest .

consider a one - dimensional series yn = ( y1 , , yn ) of length n. mapping this series into a sequence of lagged vectors with size l , x1 , , xk , with xi = ( y1 , , yi+l1 ) r provides the trajectory matrix x=(xi , j)i , j=1l , k , where l(2 l n/2 ) is the window length and k = n l + 1;x=[x1, ,xk]=(xi , j)i , j=1l , k = y1y2 yky2y3 yk+1ylyl+1 yn.the trajectory matrix x is a hankel matrix as has equal elements on the antidiagonals i + j = const . the importance of x and its corresponding singular values can be seen in different areas including time series analysis , , biomedical signal processing , , mathematics , econometrics and physics . for recent work on the generalized eigenvalues of hankel random matrices the eigenvalue distributions of beta - wishart matrices which is a special case of random matrix see edelman and plamen study . furthermore , such hankel matrix x naturally appears in multivariate analysis and signal processing , particularly in singular spectrum analysis , where each of it column represents the l - lagged vector of observations in r , . accordingly , the aim was to determine the accurate dimension of the system , that is the smallest dimension with which the filtered series is reconstructed from a noisy signal . in this case , the main analysis is based on the study of the eigenvalues and corresponding eigenvectors . if the signal component dominates the noise component , then the eigenvalues of the random matrix x have a few large eigenvalues and many small ones , suggesting that the variations in the data takes place mainly in the eigenspace corresponding to these few large eigenvalues . note that the number of correct singular values , r , for filtering and noise reduction , is increased with the increased l which makes the comparison among different choices ( l , r ) more difficult . furthermore , despite the fact that several approaches have been proposed to identify the values of r , due to a lack of substantial theoretical results , none of them consider the distribution of singular values of x. here , we study the empirical distribution of singular values of x for different situations considering various criteria . accordingly , the theoretical results on the eigenvalues of xx divided by its trace with a new view is considered in main results . some conclusions and recommendations for future research the singular values of x are the square root of the eigenvalues of the l by l matrix xx , where x is the conjugate transpose . for a fixed value of l and a series with length n , the trace of matrix xx , tr(xxt)=xf2=i=1li , where f denotes the frobenius norm , and i(i=1, note that the increase of sample size n leads to the increase of i which makes the situation more complex . to overcome this issue , we divide xx by its trace ( xxt/i=1li ) , which provides the following properties.proposition 1let 1 , , l denote eigenvalues of the matrix ( xxt/i=1li ) , where x is a hankel trajectory matrix with l rows , and i(i=1, thus , we have the following properties:1.0 l 1 1,2.i=1li=1,3.1 1/l,4.l 1/l.proofthe first two properties are simply obtained from matrix algebra and thus not provided here . the outermost inequalities are attained as equalities when , for example , yi = 1 for all i. to prove the third property , the first two properties are used as follows . thus , using the first property , 1 i ( i = 2 , , l ) , we obtain 1 + 1 + + 1 = l1 1 1 1/l . similarly , for the fourth property , it is straightforward to show that l + l + + l = ll 1 l 1/l , since l i(i = 1 , 2 , , l 1 ) , and i = 1 . let 1 , , l denote eigenvalues of the matrix ( xxt/i=1li ) , where x is a hankel trajectory matrix with l rows , and i(i=1, thus , we have the following properties : the first two properties are simply obtained from matrix algebra and thus not provided here . the outermost inequalities are attained as equalities when , for example , yi = 1 for all i. to prove the third property , the first two properties are used as follows . thus , using the first property , 1 i ( i = 2 , , + 1 = l1 1 1 1/l . similarly , for the fourth property , it is straightforward to show that l + l + + l = ll 1 l 1/l , since l i(i = 1 , 2 , , l 1 ) , and i = 1 . for consistency and comparability of the results , a fixed value of l , here 10 , is used for all examples and case studies throughout the paper . for point estimation and comparing the mean value of eigenvalues , the average of each eigenvalue in m runs is used ; i as defined before , i = 1 , , here we consider eight different cases that can be seen in real life examples:(a)white noise ; wn. (h) + sin( ) + sin(),where = 1 , = 2 , = 2t/12 , = 2t/5 , and t is the time which is used to generate the linear trend series . 1 demonstrates i for different values of n for cases ( ( a)(c ) ) considered in this study . 1 , i has a decreasing pattern for different values of n. it can be seen that , for a large n , i 1/10 for cases ( a ) and ( b ) . thus , increasing n clearly affects the values of i for the white noise ( a ) and uniform distribution ( b ) . for example , for case ( c ) , 1 is approximately equal to 0.8 for different values of n , and i1 is less than 1/10 ( see fig . although the pattern of i for the uniform distribution ( c ) is similar to exponential case ( d ) , but for case ( c ) , 1 is greater than 1 comparing to the case ( d ) , whilst other i are smaller . it has been observed that i has similar patterns for cases ( ( c ) , the values of i for cases ( a ) and ( b ) , where yn generated from a symmetric distribution , are approximately the same . the results clearly indicate that increasing n does not have a significant influence on the mean of i for all cases except ( a ) and ( b ) . for the white noise distribution ( a ) and trend series ( f ) , i has different pattern . it is obvious that , for the white noise series , i converges asymptotically to 1/10 , whilst for the trend series 1 is approximately equal to 1 , and i1 tends to zero . however , it is noticed that adding an intercept to the exponential distribution , increases the value of 1 and decreases other i . it was noticed that the value of 1 for sine wave ( h ) is greater than its value for sine case ( g ) , whilst the value of 2 is less . the distribution of i was assessed for different values of l. it was observed that the histograms of i are similar for different values of l ( the results are not presented here ) . therefore , for graphical aspect , and visualization purpose , l = 10 is considered here . the results are provided only for 1 , 5 and 10 , for the cases ( ( a ) , , ( d ) ) , as similar results are observed for other i . 2 shows histogram of i(i = 1 , 5 , 10 ) for l = 10 , and m = 5000 simulations . it appears that the histogram of 1 , is skewed to the right for samples taken from wn ( a ) and uniform distributions ( b ) , whilst for the data generated from the uniform ( c ) and exponential ( d ) distributions , might be symmetric . for the middle i , the histogram might be symmetric for the four cases ( the results only provided for 5 ) , whilst the distribution of 10 , is skewed to the left . for cases , exponential distribution ( e ) , trend series ( f ) , and sine wave series ( g ) and complex series ( h ) , we have standardized i to have conveying information about their distributions . 3 shows the density of i ( i = 1 , 2 , 3 , 5 , 6 , 10 ) for those cases . it is clear that 1 has different histogram for these cases , and also different from what was achieved for the white noise and uniform distributions with zero mean . moreover , it is interesting that 10 has a negative skewed distribution for all cases except the trend series and sine cases ( ( g ) and ( h ) ) . it is obvious that the distribution of i for sine series ( h ) becomes skewed to the right for i ( i = 6 , , remember that the sine wave ( h ) was generated from an intercept and two pure sine waves . this means that the components related to the first five eigenvalues create the sine series ( h ) . generally , if we add more non stochastic components to the noise series , for instance trend , harmonic and cyclical components , then the first few eigenvalues are related to those components and as soon as we reach the noise level the pattern of eigenvalues will be similar to those found for the noise series . in practice , the eigenvalues of a harmonic series are often close to each other , and this fact simplifies the visual identification of the harmonic components . thus , the results obtained here are very important for signal processing and time series techniques where noise reduction and filtering matter . we calculate the coefficient of skewness which is a measure for the degree of symmetry in the distribution of a variable . bulmer suggests that ; if skewness is less than 1 or greater than + 1 , the distribution is highly skewed ; if skewness is between 1 and 1/2 or between + 1/2 and + 1 , the distribution is moderately skewed , and finally if skewness is between 1/2 and + 1/2 , the distributions approximately symmetric . therefore , we can say that , for instance , the distribution of 1 for cases ( ( c ) , , ( f ) ) , and 5 for all cases might be symmetric . it is also known as the omnibus test because it uses the test statistics for both the skewness and kurtosis to come up with a single p - value and quantify how far from gaussian the distribution is in terms of asymmetry and shape . the p - value of dagostin test was significant , greater than 0.05 for 1 , for cases ( ( c ) , , ( f ) ) , whereas , it is less than 0.05 for other cases ( ( a ) , ( b ) , ( g ) , ( h ) ) . therefore , we accept the null hypothesis that the data of 1 for cases ( ( c ) , , ( f ) ) are not skewed and as a result are symmetric . the distribution of i(i = 2 , 4 ) , for the exponential case ( d ) is symmetric , whereas skewed for the exponential case with intercept ( e ) . in terms of the distribution of i for the trend series and sine wave ( g ) , the distributions of i=1,2 are totally different to the distributions of other i , which becomes skewed distribution . note that the distribution of i ( i = 1 , 2 ) for the trend series is symmetric , whilst skewed for sine wave ( g ) . for sine series ( h ) , the distribution of i ( i = 1 , , 5 ) is different from the distribution of i ( i = 6 , , 10 ) . in this section , we consider the effect of the sample size , n on i . 1 demonstrates i for different values of n for cases ( ( a)(c ) ) considered in this study . 1 , i has a decreasing pattern for different values of n. it can be seen that , for a large n , i 1/10 for cases ( a ) and ( b ) . thus , increasing n clearly affects the values of i for the white noise ( a ) and uniform distribution ( b ) . although the pattern of i for the uniform distribution ( c ) is similar to exponential case ( d ) , but for case ( c ) , 1 is greater than 1 comparing to the case ( d ) , whilst other i are smaller . it has been observed that i has similar patterns for cases ( ( c ) , the values of i for cases ( a ) and ( b ) , where yn generated from a symmetric distribution , are approximately the same . the results clearly indicate that increasing n does not have a significant influence on the mean of i for all cases except ( a ) and ( b ) . for the white noise distribution ( a ) and trend series ( f ) it is obvious that , for the white noise series , i converges asymptotically to 1/10 , whilst for the trend series 1 is approximately equal to 1 , and i1 tends to zero . it was noticed that the value of 1 for sine wave ( h ) is greater than its value for sine case ( g ) , whilst the value of 2 is less . the distribution of i was assessed for different values of l. it was observed that the histograms of i are similar for different values of l ( the results are not presented here ) . the results are provided only for 1 , 5 and 10 , for the cases ( ( a ) , , ( d ) ) , as similar results are observed for other i . it appears that the histogram of 1 , is skewed to the right for samples taken from wn ( a ) and uniform distributions ( b ) , whilst for the data generated from the uniform ( c ) and exponential ( d ) distributions , might be symmetric . for the middle i , the histogram might be symmetric for the four cases ( the results only provided for 5 ) , whilst the distribution of 10 , is skewed to the left . for cases , exponential distribution ( e ) , trend series ( f ) , and sine wave series ( g ) and complex series ( h ) , we have standardized i to have conveying information about their distributions . it is obvious that the distribution of i for sine series ( h ) becomes skewed to the right for i ( i = 6 , , remember that the sine wave ( h ) was generated from an intercept and two pure sine waves . this means that the components related to the first five eigenvalues create the sine series ( h ) . generally , if we add more non stochastic components to the noise series , for instance trend , harmonic and cyclical components , then the first few eigenvalues are related to those components and as soon as we reach the noise level the pattern of eigenvalues will be similar to those found for the noise series . thus , the results obtained here are very important for signal processing and time series techniques where noise reduction and filtering matter . we calculate the coefficient of skewness which is a measure for the degree of symmetry in the distribution of a variable . bulmer suggests that ; if skewness is less than 1 or greater than + 1 , the distribution is highly skewed ; if skewness is between 1 and 1/2 or between + 1/2 and + 1 , the distribution is moderately skewed , and finally if skewness is between 1/2 and + 1/2 , the distributions approximately symmetric . therefore , we can say that , for instance , the distribution of 1 for cases ( ( c ) , , ( f ) ) , and 5 for all cases might be symmetric . dagostino it is also known as the omnibus test because it uses the test statistics for both the skewness and kurtosis to come up with a single p - value and quantify how far from gaussian the distribution is in terms of asymmetry and shape . the p - value of dagostin test was significant , greater than 0.05 for 1 , for cases ( ( c ) , , ( f ) ) , whereas , it is less than 0.05 for other cases ( ( a ) , ( b ) , ( g ) , ( h ) ) . therefore , we accept the null hypothesis that the data of 1 for cases ( ( c ) , , ( f ) ) are not skewed and as a result are symmetric . in terms of the distribution of i for the trend series and sine wave ( g ) , the distributions of i=1,2 are totally different to the distributions of other i , which becomes skewed distribution . for sine series ( h ) , the distribution of i ( i = 1 , , 5 ) is different from the distribution of i ( i = 6 , , 10 ) . the results indicate that for a large sample size n , i ; n 1/l for the symmetric distributions ( the white noise and the uniform distributions with zero mean ) , whilst this convergence has not been observed for other cases . the results also indicate that , for the symmetric cases , the pattern of the first eigenvalue is skewed , whilst it can be symmetric for the trend and nonsymmetrical distributions . furthermore , for all cases under this study , the distribution of the middle i , for l = 10 , can be symmetric except the pattern of 5 for the trend case and both sine series . furthermore , we aim to evaluate the applicability of the results found here for noise reduction of the chaotic series . accordingly , the study of the local properties of the spectrum as well as the related distribution is of interest .

pupae of g. pallidipes from a viral infected colony were received and maintained at the insect pest control laboratory of the international atomic energy agency ( iaea ) in vienna , austria . pupae were maintained at 24c , 70% rh , and a photoperiod of 12:12 ( ld ) h , as previous described in feldmann ( 1994 ) , gooding et al . ( 2007 ) . both sexes were fed a sugar solution and , when designated , given heated and defibrinated bovine blood using the membrane - feeding technique of feldmann ( 1994 ) . samples of cns , which included the brain , cervical connective ( cc ) and thoracico - abdominal ganglion ( tag ) were taken for morphological observations . moreover , specimens of both sexes , nonblood , and blood feeders ( at 48 and 72 hr postfeeding ) , were dissected to obtain samples of normal sglds and hypertrophied sglds . samples of the cns from normal and hypertrophied sglds were dissected in phosphate buffered saline ( pbs ) and immediately observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . for whole mount fluorescence immunocytochemistry of the cns , normal and hypertrophied sglds were fixed in 4% paraformaldehyde in pbs , washed in pbst ( pbs with 0.5% triton x-100 ) ( 5 changes , 30 min each ) and left in the last wash overnight at 4c . tissues were then blocked with 10% nonimmune goat serum / pbst ( 10% normal goat serum in pbst ) for 1 hr with agitation before application of primary antiserum . tissues were probed with a polyclonal anti - serotonin antiserum ( sigma - aldrich ) diluted in 10% ngs / pbst ( anti - serotonin 1:1,000 ) for 72 hr at 4c . probed tissues were washed in pbst ( five changes , 30 min each ) and again blocked in 10% ngs / pbst for 1 hr with agitation . tissues were soaked in fluorescein conjugated secondary antiserum ( 1:200 ) for 1 hr in darkness with agitation . finally , all samples were observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . pupae of g. pallidipes from a viral infected colony were received and maintained at the insect pest control laboratory of the international atomic energy agency ( iaea ) in vienna , austria . pupae were maintained at 24c , 70% rh , and a photoperiod of 12:12 ( ld ) h , as previous described in feldmann ( 1994 ) , gooding et al . ( 2007 ) . both sexes were fed a sugar solution and , when designated , given heated and defibrinated bovine blood using the membrane - feeding technique of feldmann ( 1994 ) . samples of cns , which included the brain , cervical connective ( cc ) and thoracico - abdominal ganglion ( tag ) were taken for morphological observations . moreover , specimens of both sexes , nonblood , and blood feeders ( at 48 and 72 hr postfeeding ) , were dissected to obtain samples of normal sglds and hypertrophied sglds . samples of the cns from normal and hypertrophied sglds were dissected in phosphate buffered saline ( pbs ) and immediately observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . for whole mount fluorescence immunocytochemistry of the cns , normal and hypertrophied sglds were fixed in 4% paraformaldehyde in pbs , washed in pbst ( pbs with 0.5% triton x-100 ) ( 5 changes , 30 min each ) and left in the last wash overnight at 4c . tissues were then blocked with 10% nonimmune goat serum / pbst ( 10% normal goat serum in pbst ) for 1 hr with agitation before application of primary antiserum . tissues were probed with a polyclonal anti - serotonin antiserum ( sigma - aldrich ) diluted in 10% ngs / pbst ( anti - serotonin 1:1,000 ) for 72 hr at 4c . probed tissues were washed in pbst ( five changes , 30 min each ) and again blocked in 10% ngs / pbst for 1 hr with agitation . tissues were soaked in fluorescein conjugated secondary antiserum ( 1:200 ) for 1 hr in darkness with agitation . finally , all samples were observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . pupae of g. pallidipes from a viral infected colony were received and maintained at the insect pest control laboratory of the international atomic energy agency ( iaea ) in vienna , austria . pupae were maintained at 24c , 70% rh , and a photoperiod of 12:12 ( ld ) h , as previous described in feldmann ( 1994 ) , gooding et al . ( 2007 ) . both sexes were fed a sugar solution and , when designated , given heated and defibrinated bovine blood using the membrane - feeding technique of feldmann ( 1994 ) . samples of cns , which included the brain , cervical connective ( cc ) and thoracico - abdominal ganglion ( tag ) were taken for morphological observations . moreover , specimens of both sexes , nonblood , and blood feeders ( at 48 and 72 hr postfeeding ) , were dissected to obtain samples of normal sglds and hypertrophied sglds . samples of the cns from normal and hypertrophied sglds were dissected in phosphate buffered saline ( pbs ) and immediately observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . for whole mount fluorescence immunocytochemistry of the cns , normal and hypertrophied sglds were fixed in 4% paraformaldehyde in pbs , washed in pbst ( pbs with 0.5% triton x-100 ) ( 5 changes , 30 min each ) and left in the last wash overnight at 4c . tissues were then blocked with 10% nonimmune goat serum / pbst ( 10% normal goat serum in pbst ) for 1 hr with agitation before application of primary antiserum . tissues were probed with a polyclonal anti - serotonin antiserum ( sigma - aldrich ) diluted in 10% ngs / pbst ( anti - serotonin 1:1,000 ) for 72 hr at 4c . probed tissues were washed in pbst ( five changes , 30 min each ) and again blocked in 10% ngs / pbst for 1 hr with agitation . tissues were soaked in fluorescein conjugated secondary antiserum ( 1:200 ) for 1 hr in darkness with agitation . finally , all samples were observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . the general layout of the cns in g. pallidipes is described in figure 1a . a stratified staining with serotonin immunoreactive fibers several immunopositive cells are observed in the protocerebrum region , in particular in the lobula ( fig . immunoreactive cells are also present in the subesophageal ganglion ( sog ) , as highlighted by the high magnification in figure 1c . close examination of figure 1d shows the plexus of the cc with numerous varicosities , which are outside the blood brain barrier . in the ventral surface of the tag several serotonin immunopositive cell clusters are observed ( fig . controls ( micrographs not shown ) revealed that the immunoreactivity demonstrated in the treated specimens was serotonin . ( a ) brain shown for easier comparison of the serotonergic nerves presented in b and c of the sog . ( b and c ) cell bodies and nerve tracts are seen in the ol area ( arrow ) and in sog ( arrow ) . ( d ) a network or neural plexus within the cc is visible exiting the brain and going to the tag . the presence of small boutons or varicosities being outside the brain barrier , suggests putative serotonin release sites . ( a and b ) the dorsal area of the tag is crossed by a dense network of axons and varicosities ( i.e. suggesting serotonin release sites ) , while in the ventral area of the tag ( c and d ) several clusters of serotonin immunopositive cell bodies are present . bars = 10 m serotonin immunoreactivity in the brain and cc . ( a ) brain shown for easier comparison of the serotonergic nerves presented in b and c of the sog . ( b and c ) cell bodies and nerve tracts are seen in the ol area ( arrow ) and in sog ( arrow ) . ( d ) a network or neural plexus within the cc is visible exiting the brain and going to the tag . the presence of small boutons or varicosities being outside the brain barrier , suggests putative serotonin release sites . ( a and b ) the dorsal area of the tag is crossed by a dense network of axons and varicosities ( i.e. suggesting serotonin release sites ) , while in the ventral area of the tag ( c and d ) several clusters of serotonin immunopositive cell bodies are present . bars = 10 m serotonin immunoreactivity is analyzed in the normal and hypertrophied sglds from female and male nonblood feeders and from flies of both sexes at 48 and 72 hr postblood feeding ( hpf ) . figure 3 shows the comparison between the serotonin immunolabeling in normal and hypertrophied sglds from female flies . in healthy sglds from flies prior to blood feeding , the serotonin immunoreactivity is detected throughout the secretory region of the glands and immunoreactive axons are closely associated with the muscles covering the secretory portion of the glands ( fig . an external immunostained axon transmits the serotonergic signal to the nerves radiating on the surface of the sgld ( see the asterisk in fig . the muscle fibers are detached from one another and , consequently , the nerve fiber pattern is altered ( fig . 3b ) . now the neural fibers do nt run parallel with the muscle fibers , but appear scattered . a similar organization and a comparable alteration of the serotonergic nerve plexus is seen in hypertrophied sglds of female flies at 48 hpf ( fig . also , at 72 hpf , the serotonin innervation of normal sglds glands shows the same features as those analyzed for previous stages of flies ( fig . 3e ) . a similar alteration of serotonergic nerves affects hypertrophied sglds from females at 72 hpf ( fig . the serotonin immunostaining was performed also in normal and hypertrophied sglds from male flies , prior to the blood feeding and at 4872 hpf , but the same organization of serotonin innervation and a comparable effect are observed respective to the corresponding specimens of sglds from female flies ( data not shown ) . ( a ) serotonin immunoreactivity is shown on the surface of the muscles of the secretory region of the sglds of asymptomatic flies prior to blood feeding . the nerve tracts generally are in parallel with the muscle fibers with some side branches ( see arrows ) . ( b ) in the hypertrophied sglds of symptomatic flies prior to blood feeding , the muscle fibers are altered ( see arrows ) and the nerve tracts do not follow the parallel orientation of the muscles , which is found in asymptomatic flies . ( c and d ) a similar organization and a comparable alteration of the serotonergic nerve plexus is seen in normal and hypertrophied , respectively , sglds of females 48 hr postblood meal . ( e and f ) the same pattern is seen in the sglds of asymptomatic and symptomatic flies 72 hpf . ( a ) serotonin immunoreactivity is shown on the surface of the muscles of the secretory region of the sglds of asymptomatic flies prior to blood feeding . the nerve tracts generally are in parallel with the muscle fibers with some side branches ( see arrows ) . ( b ) in the hypertrophied sglds of symptomatic flies prior to blood feeding , the muscle fibers are altered ( see arrows ) and the nerve tracts do not follow the parallel orientation of the muscles , which is found in asymptomatic flies . ( c and d ) a similar organization and a comparable alteration of the serotonergic nerve plexus is seen in normal and hypertrophied , respectively , sglds of females 48 hr postblood meal . ( e and f ) the same pattern is seen in the sglds of asymptomatic and symptomatic flies 72 hpf . two groups of insects have been used as model systems for studying insect sglds ( i.e. cockroaches - walz et al . , we are obtaining a better understanding of the role of various amines ( i.e. dopamine and serotonin for cockroaches and serotonin for the dipterans ) and neural impact on saliva production . most studies , however , focus on secretory production and not salivation control or deployment . for these two model systems , rapid salivary delivery may not be as important as it is for blood feeding insects such as the biting flies . it is surprising when examining the literature on sglds that there is no model biting fly system ; and , the overall lack of information on these potentially important vectors is so scarce . especially since the sglds are directly involved in pathogen transmission . tsetse flies , like other dipterans , have tubular and not acinar sglds ( i.e. cockroaches ) while previous studies have shown serotonin to be the neurohormone activating saliva production ( rser et al . 2012 ; baumann and bauer 2013 ) . in a previous report ( guerra et al . 2013 ) , significant changes in the sglds of viral , symptomatic adults by the gpsghv , both at the transmission electron microscope ( tem ) and scanning electron microscope ( sem ) level showed many changes in the glands which could have compromised normal saliva production and salivation . in the same report , no mention was made about neural control of the muscle coat covering only the secretory region of the glands . however , a later publication ( guerra et al . 2015 ) showed , at the tem level , the presence of a neuron whose secretory vesicles were translucent and not opaque ( i.e. suggestive of a neurotransmitter and not a neuropeptide ) ( fig . 3e of guerra et al . 2015 ) between two muscle fibers of the sglds . no neurons were seen in any of the cells of the secretory , reabsorptive , or proximal regions . this stimulated the present research to demonstrate neuronal stimulation of the muscle coating of the adult sglds of tsetse and to get a better idea of what the sgld hypertrophy virus has on this system . to date , no studies have reported or demonstrated the presence of serotonin in the cns of tsetse . immunocytochemistry shows that clusters of serotonin immunoreactive cell bodies are present in the optic lobes ( ols ) , sog , and in the ventral region of the tag , with a neural plexus of axons running parallel to the orientation of the cc and also in the dorsal region of the tag of g. pallidipes . at one time , both glossina and stomoxys belonged to the muscidae , but now glossina has its own family , glossinidae . regardless of this change , the serotonergic patterns in the brain and tag reported here for g. pallidipes are similar to those reported for stomoxys calcitrans ( liu et al . serotonergic cell bodies have been reported in a few dipteran species belonging to families other than muscidae / glossinidae ( nssel 1988 ) , but no studies have been reported on adult tsetse . the presence of serotonergic fibers forming a plexus in the dorsal region of the tag have been reported for other flies ( nssel et al . 1994 ; haselton et al . 2006 ) ; it is suggested that the dorsal plexus region is a site of neurohormonal serotonin release ( nssel and elekes 1985 ) and it is this hemolymph borne serotonin that is involved in stimulating the secretory region to produce saliva . it is also suggested that the tag plexus is involved in the release of neurohormonal serotonin , which has been proposed to act on nonblood feeding dipteran sglds ( trimmer 1985 ; ali 1997 ) and in adult phormia regina meigen to inhibit protein feeding ( haselton et al . the presence of the dorsal plexus suggests that sgld secretion is under the control of hormonal serotonin , like in nonblood feeding dipterans , while the muscles , presumed to be involved in dispensing luminal saliva anteriorly , are controlled by the serotonergic neural plexus ( suggestion by d. r. nssel , personal communication ) as was proposed by novak et al . ( 1995 ) for mosquitoes . because this present study only briefly examined the presence of serotonergic neurons in cns , one is referred to more comprehensive studies in other flies for locations , numbers , and functions for these neurons ( nssel et al . 1985 ) . transmission electron microscopic evidence for the release of serotonin into the muscle area of g. pallidipes sglds was shown in figure 3e of guerra et al . this micrograph shows the terminal end of what is believed to be one of the endings of the serotonergic neurons containing translucent vesicles in the vicinity of two muscle fibers . comprehensive tem of all of the areas of the sglds in our previous research ( guerra et al . 2013 ) only showed a muscle layer surrounding the secretory region of the glands and no evidence of neural innervation of any other region was found . our original hypothesis for the altered neural plexus of the muscles in symptomatic flies was that it resulted in the neurons breaking . in discussion with dick nssel ( personal communication ) , he suggested , since the neurons appeared healthy , that what was happening was neural proliferation due to the increase in size of the sglds and not breakage ( i.e. neuronal compensation for organ increase ) . a similar response resulting in neural proliferation has been reported for various pathological conditions and is known as neuronal proliferation ( demir et al . the article by chen and condron ( 2008 ) and personal correspondence with condron , provide supporting evidence for the neuronal proliferation hypothesis . the serotonergic arbor structure of the larval vnc ( sic drosophila melanogaster ) grows and adds branches and varicosities as it expands from l1 to l3f ( personal communication). thus , this hypothesis using symptomatic flies must now be tested . neuronal proliferation may have established new connections on the hypertrophied sgld , but the organelle disruption shown in a previous study ( guerra et al . 2013 ) , the dissociation of the muscle fibers ( guerra et al . 2015 ) , and the loss of collagen may produce muscle myopathy , just as it has been demonstrated in drosophila mutants ( kelemen - valkony et al . all of these abnormalities induced by the virus may affect normal saliva production and salivation . this study is the first to demonstrate that the muscles of the secretory region of the sglds of adult tsetse flies , or any dipteran species , are innervated and by serotonergic neurons . ( 2010 ) , which provided information that suggested sgld stimulation in tsetse might involve the biogenic amine serotonin . the only other report on how the sglds are stimulated in hematophagous dipterans is the report of novak et al . we would like to emphasize here that the studies in the diptera have focused on control of saliva production ( baumann and bauer 2013 ) and not on salivation release . the presence of muscles surrounding the secretory regions of the sgld in tsetse might be a mechanism for rapidly dispensing the saliva into the host , thus avoiding a host response . even though muscles surrounding the sglds of mosquitoes have not been found ( ribeiro , personal correspondence ) suggests that control of salivation may be different between the nematoceran and brachyceran diptera . unlike in the mosquito where only the female blood feeds and the sglds are innervated ( novak et al . 1995 ) , the sglds of both sexes of tsetse are innervated and both sexes feed on blood . 5-ht - immunoreactive innervation is absent in male sglds , suggesting that 5-ht is involved in blood - feeding. even though we found serotonin positive neurons in both the brain and tag , we did not follow the neural connection from the sglds to their cns origin . the presence of numerous variscosities in the cervical connector and , being outside the blood brain barrier , suggests serotonin release sites along the connective . in ae . ( 1995 ) reported that , using -methyltryptophan ( amtp ) ( i.e. an established and effective serotonin depleter ) , females injected with amtp and induced to salivate into mineral oil , produced less saliva and significantly lower apyrase . this led the authors to suggest that serotonin was important for salivation ; and , it probably was the cause of impaired salivation . normal feeding in symptomatic tsetse flies infected by the sghv has been reported as being normal for g. pallidipes , but abnormal for glossina morsitans centralis ( lietze et al . ( 2011 ) , however , notes that viral symptomatic g. pallidipes flies , 1015 d posteclosion , have altered feeding . the difference in these reported effects on different glossina species could be due to when the flies were tested ( i.e. older flies being more severely impaired ) . in addition , further experimentation concerning the regulation of salivation by serotonin needs to be done ; and , we need to know the effects of the virus on saliva output . as ribeiro and francischetti ( 2003 ) reported for hematophagous dipterans to steal blood and avoid a host response , it is essential to probe , salivate and find a vessel rapidly to successfully deploy their pharmacological salivary secretion . in order to accomplish this , we suggest some type of mechanism ( i.e. for tsetse an innervated muscle coating of the sglds ) should have evolved such as direct innervation for rapid ejection of saliva , as suggested here for g. pallidipes ( i.e. serotonergic regulation ) . nonhematophagous dipterans , however , have salivation regulated by serotonin , but in the dipteran species studied to date , serotonin acts as a neurohormone ( berridge and patel 1968 ; bay 1978 ; trimmer 1985 ) and not a neurotransmitter . ribeiro ( personal communication ) , however , suggested that the hematophagous nematoceran , versus the brachyceran diptera , may differ as to whether the sglds are directly innervated . also , it remains to be shown whether other brachyceran diptera , such as tabanids , have muscles on the sglds and whether they are directly innervated as shown here for tsetse . ( 2011 ) on s. calcitrans , using antibodies to serotonin , did not mention anything about innervation of the sglds . this study demonstrates that the gpsghv alters the neural plexus of the muscles surrounding the secretory region of the adult sglds of symptomatic tsetse flies . in a previous study ( guerra et al . 2015 ) , it was shown that the disrupted muscles lose contact with one another because of what was suggested as the loss of collagen , which may result from an up - regulation of the mp - nnase gene involved in regulating interstitial collagenase production . using the antibody to serotonin , we showed that the cns of tsetse has serotonergic neurons and the axons from some of these probably innervate the muscle coating of the sglds . the fact that symptomatic g. pallidipes has altered feeding behavior suggests that a study similar to that of lefvre et al . ( 2007 ) , which examined the effect of the trypanosome parasite on the host , should be conducted . these authors used proteomic analysis of brain tissue to compare both trypanosome infected and uninfected adults . their results showed differences between both the levels of serotonin and also the signal transduction protein , which includes meprin ( metalloendopeptidase ) , that could explain failure to feed in symptomatic adult tsetse , 1015 d posteclosion ( kariithi et al . 2011 ) , and other behaviors , in symptomatic , viral infected g. pallidipes . developing a comprehensive model of both saliva production and saliva deployment ( into the host or associated with nectar feeding ) in hematophagous dipterans

using a serotonin antibody and confocal microscopy , this study reports for the first time direct serotonergic innervation of the muscle sheath covering the secretory region of the salivary glands of adult tsetse fly , glossina pallidipes austen . reports to date , however , note that up until this finding , dipteran species previously studied lack a muscle sheath covering of the secretory region of the salivary glands . direct innervation of the salivary gland muscle sheath of tsetse would facilitate rapid deployment of saliva into the host , thus delaying a host response . our results also suggest that the neuronal and abnormal pattern seen in viral infected glands by the glossina pallidipes salivary gland hypertrophy virus ( gpsghv ) is due to a compensatory increased branching of the neurons of the salivary glands , which is associated with the increased size of the salivary glands in viral infected flies . this study shows for the first time serotonin in the cell bodies of the brain and thoracico - abdominal ganglion in adult tsetse , g. pallidipes austen ( diptera : glossinidae ) . a hypothesis is proposed as to whether innervation of the muscle sheath covering of the secretory region of the salivary glands is present in brachyceran compared with nematoceran dipterans ; and , a plea is made that more research is needed to develop a blood feeding model , similar to that in the blow flies , for elucidating the various mechanisms involved in production and deployment of saliva .

Materials and Methods None Animals Light Microscopy Immunocytochemistry Results Discussion Concluding Remarks

pupae of g. pallidipes from a viral infected colony were received and maintained at the insect pest control laboratory of the international atomic energy agency ( iaea ) in vienna , austria . both sexes were fed a sugar solution and , when designated , given heated and defibrinated bovine blood using the membrane - feeding technique of feldmann ( 1994 ) . samples of cns , which included the brain , cervical connective ( cc ) and thoracico - abdominal ganglion ( tag ) were taken for morphological observations . samples of the cns from normal and hypertrophied sglds were dissected in phosphate buffered saline ( pbs ) and immediately observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . for whole mount fluorescence immunocytochemistry of the cns , normal and hypertrophied sglds were fixed in 4% paraformaldehyde in pbs , washed in pbst ( pbs with 0.5% triton x-100 ) ( 5 changes , 30 min each ) and left in the last wash overnight at 4c . finally , all samples were observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . pupae of g. pallidipes from a viral infected colony were received and maintained at the insect pest control laboratory of the international atomic energy agency ( iaea ) in vienna , austria . both sexes were fed a sugar solution and , when designated , given heated and defibrinated bovine blood using the membrane - feeding technique of feldmann ( 1994 ) . samples of cns , which included the brain , cervical connective ( cc ) and thoracico - abdominal ganglion ( tag ) were taken for morphological observations . samples of the cns from normal and hypertrophied sglds were dissected in phosphate buffered saline ( pbs ) and immediately observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . for whole mount fluorescence immunocytochemistry of the cns , normal and hypertrophied sglds were fixed in 4% paraformaldehyde in pbs , washed in pbst ( pbs with 0.5% triton x-100 ) ( 5 changes , 30 min each ) and left in the last wash overnight at 4c . finally , all samples were observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . pupae of g. pallidipes from a viral infected colony were received and maintained at the insect pest control laboratory of the international atomic energy agency ( iaea ) in vienna , austria . both sexes were fed a sugar solution and , when designated , given heated and defibrinated bovine blood using the membrane - feeding technique of feldmann ( 1994 ) . samples of cns , which included the brain , cervical connective ( cc ) and thoracico - abdominal ganglion ( tag ) were taken for morphological observations . samples of the cns from normal and hypertrophied sglds were dissected in phosphate buffered saline ( pbs ) and immediately observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . for whole mount fluorescence immunocytochemistry of the cns , normal and hypertrophied sglds were fixed in 4% paraformaldehyde in pbs , washed in pbst ( pbs with 0.5% triton x-100 ) ( 5 changes , 30 min each ) and left in the last wash overnight at 4c . finally , all samples were observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . the general layout of the cns in g. pallidipes is described in figure 1a . immunoreactive cells are also present in the subesophageal ganglion ( sog ) , as highlighted by the high magnification in figure 1c . close examination of figure 1d shows the plexus of the cc with numerous varicosities , which are outside the blood brain barrier . in the ventral surface of the tag several serotonin immunopositive cell clusters are observed ( fig . controls ( micrographs not shown ) revealed that the immunoreactivity demonstrated in the treated specimens was serotonin . ( a ) brain shown for easier comparison of the serotonergic nerves presented in b and c of the sog . ( b and c ) cell bodies and nerve tracts are seen in the ol area ( arrow ) and in sog ( arrow ) . ( d ) a network or neural plexus within the cc is visible exiting the brain and going to the tag . suggesting serotonin release sites ) , while in the ventral area of the tag ( c and d ) several clusters of serotonin immunopositive cell bodies are present . bars = 10 m serotonin immunoreactivity in the brain and cc . ( a ) brain shown for easier comparison of the serotonergic nerves presented in b and c of the sog . ( b and c ) cell bodies and nerve tracts are seen in the ol area ( arrow ) and in sog ( arrow ) . ( d ) a network or neural plexus within the cc is visible exiting the brain and going to the tag . the presence of small boutons or varicosities being outside the brain barrier , suggests putative serotonin release sites . ( a and b ) the dorsal area of the tag is crossed by a dense network of axons and varicosities ( i.e. suggesting serotonin release sites ) , while in the ventral area of the tag ( c and d ) several clusters of serotonin immunopositive cell bodies are present . bars = 10 m serotonin immunoreactivity is analyzed in the normal and hypertrophied sglds from female and male nonblood feeders and from flies of both sexes at 48 and 72 hr postblood feeding ( hpf ) . in healthy sglds from flies prior to blood feeding , the serotonin immunoreactivity is detected throughout the secretory region of the glands and immunoreactive axons are closely associated with the muscles covering the secretory portion of the glands ( fig . an external immunostained axon transmits the serotonergic signal to the nerves radiating on the surface of the sgld ( see the asterisk in fig . the muscle fibers are detached from one another and , consequently , the nerve fiber pattern is altered ( fig . now the neural fibers do nt run parallel with the muscle fibers , but appear scattered . a similar organization and a comparable alteration of the serotonergic nerve plexus is seen in hypertrophied sglds of female flies at 48 hpf ( fig . also , at 72 hpf , the serotonin innervation of normal sglds glands shows the same features as those analyzed for previous stages of flies ( fig . the serotonin immunostaining was performed also in normal and hypertrophied sglds from male flies , prior to the blood feeding and at 4872 hpf , but the same organization of serotonin innervation and a comparable effect are observed respective to the corresponding specimens of sglds from female flies ( data not shown ) . ( a ) serotonin immunoreactivity is shown on the surface of the muscles of the secretory region of the sglds of asymptomatic flies prior to blood feeding . the nerve tracts generally are in parallel with the muscle fibers with some side branches ( see arrows ) . ( b ) in the hypertrophied sglds of symptomatic flies prior to blood feeding , the muscle fibers are altered ( see arrows ) and the nerve tracts do not follow the parallel orientation of the muscles , which is found in asymptomatic flies . ( c and d ) a similar organization and a comparable alteration of the serotonergic nerve plexus is seen in normal and hypertrophied , respectively , sglds of females 48 hr postblood meal . ( e and f ) the same pattern is seen in the sglds of asymptomatic and symptomatic flies 72 hpf . ( a ) serotonin immunoreactivity is shown on the surface of the muscles of the secretory region of the sglds of asymptomatic flies prior to blood feeding . the nerve tracts generally are in parallel with the muscle fibers with some side branches ( see arrows ) . ( b ) in the hypertrophied sglds of symptomatic flies prior to blood feeding , the muscle fibers are altered ( see arrows ) and the nerve tracts do not follow the parallel orientation of the muscles , which is found in asymptomatic flies . ( c and d ) a similar organization and a comparable alteration of the serotonergic nerve plexus is seen in normal and hypertrophied , respectively , sglds of females 48 hr postblood meal . ( e and f ) the same pattern is seen in the sglds of asymptomatic and symptomatic flies 72 hpf . , we are obtaining a better understanding of the role of various amines ( i.e. dopamine and serotonin for cockroaches and serotonin for the dipterans ) and neural impact on saliva production . most studies , however , focus on secretory production and not salivation control or deployment . for these two model systems , rapid salivary delivery may not be as important as it is for blood feeding insects such as the biting flies . it is surprising when examining the literature on sglds that there is no model biting fly system ; and , the overall lack of information on these potentially important vectors is so scarce . especially since the sglds are directly involved in pathogen transmission . 2013 ) , significant changes in the sglds of viral , symptomatic adults by the gpsghv , both at the transmission electron microscope ( tem ) and scanning electron microscope ( sem ) level showed many changes in the glands which could have compromised normal saliva production and salivation . in the same report , no mention was made about neural control of the muscle coat covering only the secretory region of the glands . however , a later publication ( guerra et al . no neurons were seen in any of the cells of the secretory , reabsorptive , or proximal regions . this stimulated the present research to demonstrate neuronal stimulation of the muscle coating of the adult sglds of tsetse and to get a better idea of what the sgld hypertrophy virus has on this system . to date , no studies have reported or demonstrated the presence of serotonin in the cns of tsetse . immunocytochemistry shows that clusters of serotonin immunoreactive cell bodies are present in the optic lobes ( ols ) , sog , and in the ventral region of the tag , with a neural plexus of axons running parallel to the orientation of the cc and also in the dorsal region of the tag of g. pallidipes . regardless of this change , the serotonergic patterns in the brain and tag reported here for g. pallidipes are similar to those reported for stomoxys calcitrans ( liu et al . serotonergic cell bodies have been reported in a few dipteran species belonging to families other than muscidae / glossinidae ( nssel 1988 ) , but no studies have been reported on adult tsetse . the presence of serotonergic fibers forming a plexus in the dorsal region of the tag have been reported for other flies ( nssel et al . 2006 ) ; it is suggested that the dorsal plexus region is a site of neurohormonal serotonin release ( nssel and elekes 1985 ) and it is this hemolymph borne serotonin that is involved in stimulating the secretory region to produce saliva . it is also suggested that the tag plexus is involved in the release of neurohormonal serotonin , which has been proposed to act on nonblood feeding dipteran sglds ( trimmer 1985 ; ali 1997 ) and in adult phormia regina meigen to inhibit protein feeding ( haselton et al . the presence of the dorsal plexus suggests that sgld secretion is under the control of hormonal serotonin , like in nonblood feeding dipterans , while the muscles , presumed to be involved in dispensing luminal saliva anteriorly , are controlled by the serotonergic neural plexus ( suggestion by d. r. nssel , personal communication ) as was proposed by novak et al . transmission electron microscopic evidence for the release of serotonin into the muscle area of g. pallidipes sglds was shown in figure 3e of guerra et al . this micrograph shows the terminal end of what is believed to be one of the endings of the serotonergic neurons containing translucent vesicles in the vicinity of two muscle fibers . comprehensive tem of all of the areas of the sglds in our previous research ( guerra et al . 2013 ) only showed a muscle layer surrounding the secretory region of the glands and no evidence of neural innervation of any other region was found . our original hypothesis for the altered neural plexus of the muscles in symptomatic flies was that it resulted in the neurons breaking . in discussion with dick nssel ( personal communication ) , he suggested , since the neurons appeared healthy , that what was happening was neural proliferation due to the increase in size of the sglds and not breakage ( i.e. the article by chen and condron ( 2008 ) and personal correspondence with condron , provide supporting evidence for the neuronal proliferation hypothesis . the serotonergic arbor structure of the larval vnc ( sic drosophila melanogaster ) grows and adds branches and varicosities as it expands from l1 to l3f ( personal communication). thus , this hypothesis using symptomatic flies must now be tested . 2013 ) , the dissociation of the muscle fibers ( guerra et al . all of these abnormalities induced by the virus may affect normal saliva production and salivation . this study is the first to demonstrate that the muscles of the secretory region of the sglds of adult tsetse flies , or any dipteran species , are innervated and by serotonergic neurons . ( 2010 ) , which provided information that suggested sgld stimulation in tsetse might involve the biogenic amine serotonin . we would like to emphasize here that the studies in the diptera have focused on control of saliva production ( baumann and bauer 2013 ) and not on salivation release . the presence of muscles surrounding the secretory regions of the sgld in tsetse might be a mechanism for rapidly dispensing the saliva into the host , thus avoiding a host response . unlike in the mosquito where only the female blood feeds and the sglds are innervated ( novak et al . even though we found serotonin positive neurons in both the brain and tag , we did not follow the neural connection from the sglds to their cns origin . the presence of numerous variscosities in the cervical connector and , being outside the blood brain barrier , suggests serotonin release sites along the connective . this led the authors to suggest that serotonin was important for salivation ; and , it probably was the cause of impaired salivation . normal feeding in symptomatic tsetse flies infected by the sghv has been reported as being normal for g. pallidipes , but abnormal for glossina morsitans centralis ( lietze et al . ( 2011 ) , however , notes that viral symptomatic g. pallidipes flies , 1015 d posteclosion , have altered feeding . the difference in these reported effects on different glossina species could be due to when the flies were tested ( i.e. in addition , further experimentation concerning the regulation of salivation by serotonin needs to be done ; and , we need to know the effects of the virus on saliva output . as ribeiro and francischetti ( 2003 ) reported for hematophagous dipterans to steal blood and avoid a host response , it is essential to probe , salivate and find a vessel rapidly to successfully deploy their pharmacological salivary secretion . for tsetse an innervated muscle coating of the sglds ) should have evolved such as direct innervation for rapid ejection of saliva , as suggested here for g. pallidipes ( i.e. nonhematophagous dipterans , however , have salivation regulated by serotonin , but in the dipteran species studied to date , serotonin acts as a neurohormone ( berridge and patel 1968 ; bay 1978 ; trimmer 1985 ) and not a neurotransmitter . ribeiro ( personal communication ) , however , suggested that the hematophagous nematoceran , versus the brachyceran diptera , may differ as to whether the sglds are directly innervated . ( 2011 ) on s. calcitrans , using antibodies to serotonin , did not mention anything about innervation of the sglds . this study demonstrates that the gpsghv alters the neural plexus of the muscles surrounding the secretory region of the adult sglds of symptomatic tsetse flies . 2015 ) , it was shown that the disrupted muscles lose contact with one another because of what was suggested as the loss of collagen , which may result from an up - regulation of the mp - nnase gene involved in regulating interstitial collagenase production . using the antibody to serotonin , we showed that the cns of tsetse has serotonergic neurons and the axons from some of these probably innervate the muscle coating of the sglds . the fact that symptomatic g. pallidipes has altered feeding behavior suggests that a study similar to that of lefvre et al . ( 2007 ) , which examined the effect of the trypanosome parasite on the host , should be conducted . their results showed differences between both the levels of serotonin and also the signal transduction protein , which includes meprin ( metalloendopeptidase ) , that could explain failure to feed in symptomatic adult tsetse , 1015 d posteclosion ( kariithi et al . 2011 ) , and other behaviors , in symptomatic , viral infected g. pallidipes . developing a comprehensive model of both saliva production and saliva deployment ( into the host or associated with nectar feeding ) in hematophagous dipterans

pupae of g. pallidipes from a viral infected colony were received and maintained at the insect pest control laboratory of the international atomic energy agency ( iaea ) in vienna , austria . both sexes were fed a sugar solution and , when designated , given heated and defibrinated bovine blood using the membrane - feeding technique of feldmann ( 1994 ) . samples of cns , which included the brain , cervical connective ( cc ) and thoracico - abdominal ganglion ( tag ) were taken for morphological observations . moreover , specimens of both sexes , nonblood , and blood feeders ( at 48 and 72 hr postfeeding ) , were dissected to obtain samples of normal sglds and hypertrophied sglds . samples of the cns from normal and hypertrophied sglds were dissected in phosphate buffered saline ( pbs ) and immediately observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . for whole mount fluorescence immunocytochemistry of the cns , normal and hypertrophied sglds were fixed in 4% paraformaldehyde in pbs , washed in pbst ( pbs with 0.5% triton x-100 ) ( 5 changes , 30 min each ) and left in the last wash overnight at 4c . tissues were then blocked with 10% nonimmune goat serum / pbst ( 10% normal goat serum in pbst ) for 1 hr with agitation before application of primary antiserum . tissues were probed with a polyclonal anti - serotonin antiserum ( sigma - aldrich ) diluted in 10% ngs / pbst ( anti - serotonin 1:1,000 ) for 72 hr at 4c . pupae of g. pallidipes from a viral infected colony were received and maintained at the insect pest control laboratory of the international atomic energy agency ( iaea ) in vienna , austria . both sexes were fed a sugar solution and , when designated , given heated and defibrinated bovine blood using the membrane - feeding technique of feldmann ( 1994 ) . samples of cns , which included the brain , cervical connective ( cc ) and thoracico - abdominal ganglion ( tag ) were taken for morphological observations . moreover , specimens of both sexes , nonblood , and blood feeders ( at 48 and 72 hr postfeeding ) , were dissected to obtain samples of normal sglds and hypertrophied sglds . samples of the cns from normal and hypertrophied sglds were dissected in phosphate buffered saline ( pbs ) and immediately observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . for whole mount fluorescence immunocytochemistry of the cns , normal and hypertrophied sglds were fixed in 4% paraformaldehyde in pbs , washed in pbst ( pbs with 0.5% triton x-100 ) ( 5 changes , 30 min each ) and left in the last wash overnight at 4c . tissues were then blocked with 10% nonimmune goat serum / pbst ( 10% normal goat serum in pbst ) for 1 hr with agitation before application of primary antiserum . tissues were probed with a polyclonal anti - serotonin antiserum ( sigma - aldrich ) diluted in 10% ngs / pbst ( anti - serotonin 1:1,000 ) for 72 hr at 4c . pupae of g. pallidipes from a viral infected colony were received and maintained at the insect pest control laboratory of the international atomic energy agency ( iaea ) in vienna , austria . both sexes were fed a sugar solution and , when designated , given heated and defibrinated bovine blood using the membrane - feeding technique of feldmann ( 1994 ) . samples of cns , which included the brain , cervical connective ( cc ) and thoracico - abdominal ganglion ( tag ) were taken for morphological observations . moreover , specimens of both sexes , nonblood , and blood feeders ( at 48 and 72 hr postfeeding ) , were dissected to obtain samples of normal sglds and hypertrophied sglds . samples of the cns from normal and hypertrophied sglds were dissected in phosphate buffered saline ( pbs ) and immediately observed using a computerized image analysis system , which included a zeiss light microscope ( axiophot ) equipped with a video color camera ( axio cam mrc , arese , milano - italy ) and imaging software ( ks 300 and axiovision ) . for whole mount fluorescence immunocytochemistry of the cns , normal and hypertrophied sglds were fixed in 4% paraformaldehyde in pbs , washed in pbst ( pbs with 0.5% triton x-100 ) ( 5 changes , 30 min each ) and left in the last wash overnight at 4c . tissues were then blocked with 10% nonimmune goat serum / pbst ( 10% normal goat serum in pbst ) for 1 hr with agitation before application of primary antiserum . tissues were probed with a polyclonal anti - serotonin antiserum ( sigma - aldrich ) diluted in 10% ngs / pbst ( anti - serotonin 1:1,000 ) for 72 hr at 4c . the general layout of the cns in g. pallidipes is described in figure 1a . a stratified staining with serotonin immunoreactive fibers several immunopositive cells are observed in the protocerebrum region , in particular in the lobula ( fig . immunoreactive cells are also present in the subesophageal ganglion ( sog ) , as highlighted by the high magnification in figure 1c . close examination of figure 1d shows the plexus of the cc with numerous varicosities , which are outside the blood brain barrier . in the ventral surface of the tag several serotonin immunopositive cell clusters are observed ( fig . controls ( micrographs not shown ) revealed that the immunoreactivity demonstrated in the treated specimens was serotonin . ( a ) brain shown for easier comparison of the serotonergic nerves presented in b and c of the sog . ( b and c ) cell bodies and nerve tracts are seen in the ol area ( arrow ) and in sog ( arrow ) . ( d ) a network or neural plexus within the cc is visible exiting the brain and going to the tag . the presence of small boutons or varicosities being outside the brain barrier , suggests putative serotonin release sites . ( a and b ) the dorsal area of the tag is crossed by a dense network of axons and varicosities ( i.e. suggesting serotonin release sites ) , while in the ventral area of the tag ( c and d ) several clusters of serotonin immunopositive cell bodies are present . ( a ) brain shown for easier comparison of the serotonergic nerves presented in b and c of the sog . ( b and c ) cell bodies and nerve tracts are seen in the ol area ( arrow ) and in sog ( arrow ) . ( d ) a network or neural plexus within the cc is visible exiting the brain and going to the tag . the presence of small boutons or varicosities being outside the brain barrier , suggests putative serotonin release sites . ( a and b ) the dorsal area of the tag is crossed by a dense network of axons and varicosities ( i.e. suggesting serotonin release sites ) , while in the ventral area of the tag ( c and d ) several clusters of serotonin immunopositive cell bodies are present . bars = 10 m serotonin immunoreactivity is analyzed in the normal and hypertrophied sglds from female and male nonblood feeders and from flies of both sexes at 48 and 72 hr postblood feeding ( hpf ) . figure 3 shows the comparison between the serotonin immunolabeling in normal and hypertrophied sglds from female flies . in healthy sglds from flies prior to blood feeding , the serotonin immunoreactivity is detected throughout the secretory region of the glands and immunoreactive axons are closely associated with the muscles covering the secretory portion of the glands ( fig . an external immunostained axon transmits the serotonergic signal to the nerves radiating on the surface of the sgld ( see the asterisk in fig . the muscle fibers are detached from one another and , consequently , the nerve fiber pattern is altered ( fig . a similar organization and a comparable alteration of the serotonergic nerve plexus is seen in hypertrophied sglds of female flies at 48 hpf ( fig . also , at 72 hpf , the serotonin innervation of normal sglds glands shows the same features as those analyzed for previous stages of flies ( fig . the serotonin immunostaining was performed also in normal and hypertrophied sglds from male flies , prior to the blood feeding and at 4872 hpf , but the same organization of serotonin innervation and a comparable effect are observed respective to the corresponding specimens of sglds from female flies ( data not shown ) . ( a ) serotonin immunoreactivity is shown on the surface of the muscles of the secretory region of the sglds of asymptomatic flies prior to blood feeding . ( b ) in the hypertrophied sglds of symptomatic flies prior to blood feeding , the muscle fibers are altered ( see arrows ) and the nerve tracts do not follow the parallel orientation of the muscles , which is found in asymptomatic flies . ( c and d ) a similar organization and a comparable alteration of the serotonergic nerve plexus is seen in normal and hypertrophied , respectively , sglds of females 48 hr postblood meal . ( e and f ) the same pattern is seen in the sglds of asymptomatic and symptomatic flies 72 hpf . ( a ) serotonin immunoreactivity is shown on the surface of the muscles of the secretory region of the sglds of asymptomatic flies prior to blood feeding . ( b ) in the hypertrophied sglds of symptomatic flies prior to blood feeding , the muscle fibers are altered ( see arrows ) and the nerve tracts do not follow the parallel orientation of the muscles , which is found in asymptomatic flies . ( c and d ) a similar organization and a comparable alteration of the serotonergic nerve plexus is seen in normal and hypertrophied , respectively , sglds of females 48 hr postblood meal . ( e and f ) the same pattern is seen in the sglds of asymptomatic and symptomatic flies 72 hpf . , we are obtaining a better understanding of the role of various amines ( i.e. dopamine and serotonin for cockroaches and serotonin for the dipterans ) and neural impact on saliva production . it is surprising when examining the literature on sglds that there is no model biting fly system ; and , the overall lack of information on these potentially important vectors is so scarce . 2013 ) , significant changes in the sglds of viral , symptomatic adults by the gpsghv , both at the transmission electron microscope ( tem ) and scanning electron microscope ( sem ) level showed many changes in the glands which could have compromised normal saliva production and salivation . in the same report , no mention was made about neural control of the muscle coat covering only the secretory region of the glands . 2015 ) showed , at the tem level , the presence of a neuron whose secretory vesicles were translucent and not opaque ( i.e. no neurons were seen in any of the cells of the secretory , reabsorptive , or proximal regions . this stimulated the present research to demonstrate neuronal stimulation of the muscle coating of the adult sglds of tsetse and to get a better idea of what the sgld hypertrophy virus has on this system . to date , no studies have reported or demonstrated the presence of serotonin in the cns of tsetse . immunocytochemistry shows that clusters of serotonin immunoreactive cell bodies are present in the optic lobes ( ols ) , sog , and in the ventral region of the tag , with a neural plexus of axons running parallel to the orientation of the cc and also in the dorsal region of the tag of g. pallidipes . regardless of this change , the serotonergic patterns in the brain and tag reported here for g. pallidipes are similar to those reported for stomoxys calcitrans ( liu et al . serotonergic cell bodies have been reported in a few dipteran species belonging to families other than muscidae / glossinidae ( nssel 1988 ) , but no studies have been reported on adult tsetse . the presence of serotonergic fibers forming a plexus in the dorsal region of the tag have been reported for other flies ( nssel et al . 2006 ) ; it is suggested that the dorsal plexus region is a site of neurohormonal serotonin release ( nssel and elekes 1985 ) and it is this hemolymph borne serotonin that is involved in stimulating the secretory region to produce saliva . it is also suggested that the tag plexus is involved in the release of neurohormonal serotonin , which has been proposed to act on nonblood feeding dipteran sglds ( trimmer 1985 ; ali 1997 ) and in adult phormia regina meigen to inhibit protein feeding ( haselton et al . the presence of the dorsal plexus suggests that sgld secretion is under the control of hormonal serotonin , like in nonblood feeding dipterans , while the muscles , presumed to be involved in dispensing luminal saliva anteriorly , are controlled by the serotonergic neural plexus ( suggestion by d. r. nssel , personal communication ) as was proposed by novak et al . because this present study only briefly examined the presence of serotonergic neurons in cns , one is referred to more comprehensive studies in other flies for locations , numbers , and functions for these neurons ( nssel et al . transmission electron microscopic evidence for the release of serotonin into the muscle area of g. pallidipes sglds was shown in figure 3e of guerra et al . this micrograph shows the terminal end of what is believed to be one of the endings of the serotonergic neurons containing translucent vesicles in the vicinity of two muscle fibers . comprehensive tem of all of the areas of the sglds in our previous research ( guerra et al . 2013 ) only showed a muscle layer surrounding the secretory region of the glands and no evidence of neural innervation of any other region was found . our original hypothesis for the altered neural plexus of the muscles in symptomatic flies was that it resulted in the neurons breaking . in discussion with dick nssel ( personal communication ) , he suggested , since the neurons appeared healthy , that what was happening was neural proliferation due to the increase in size of the sglds and not breakage ( i.e. the serotonergic arbor structure of the larval vnc ( sic drosophila melanogaster ) grows and adds branches and varicosities as it expands from l1 to l3f ( personal communication). 2015 ) , and the loss of collagen may produce muscle myopathy , just as it has been demonstrated in drosophila mutants ( kelemen - valkony et al . this study is the first to demonstrate that the muscles of the secretory region of the sglds of adult tsetse flies , or any dipteran species , are innervated and by serotonergic neurons . we would like to emphasize here that the studies in the diptera have focused on control of saliva production ( baumann and bauer 2013 ) and not on salivation release . the presence of muscles surrounding the secretory regions of the sgld in tsetse might be a mechanism for rapidly dispensing the saliva into the host , thus avoiding a host response . even though muscles surrounding the sglds of mosquitoes have not been found ( ribeiro , personal correspondence ) suggests that control of salivation may be different between the nematoceran and brachyceran diptera . even though we found serotonin positive neurons in both the brain and tag , we did not follow the neural connection from the sglds to their cns origin . the presence of numerous variscosities in the cervical connector and , being outside the blood brain barrier , suggests serotonin release sites along the connective . an established and effective serotonin depleter ) , females injected with amtp and induced to salivate into mineral oil , produced less saliva and significantly lower apyrase . normal feeding in symptomatic tsetse flies infected by the sghv has been reported as being normal for g. pallidipes , but abnormal for glossina morsitans centralis ( lietze et al . in addition , further experimentation concerning the regulation of salivation by serotonin needs to be done ; and , we need to know the effects of the virus on saliva output . as ribeiro and francischetti ( 2003 ) reported for hematophagous dipterans to steal blood and avoid a host response , it is essential to probe , salivate and find a vessel rapidly to successfully deploy their pharmacological salivary secretion . for tsetse an innervated muscle coating of the sglds ) should have evolved such as direct innervation for rapid ejection of saliva , as suggested here for g. pallidipes ( i.e. nonhematophagous dipterans , however , have salivation regulated by serotonin , but in the dipteran species studied to date , serotonin acts as a neurohormone ( berridge and patel 1968 ; bay 1978 ; trimmer 1985 ) and not a neurotransmitter . this study demonstrates that the gpsghv alters the neural plexus of the muscles surrounding the secretory region of the adult sglds of symptomatic tsetse flies . 2015 ) , it was shown that the disrupted muscles lose contact with one another because of what was suggested as the loss of collagen , which may result from an up - regulation of the mp - nnase gene involved in regulating interstitial collagenase production . using the antibody to serotonin , we showed that the cns of tsetse has serotonergic neurons and the axons from some of these probably innervate the muscle coating of the sglds . their results showed differences between both the levels of serotonin and also the signal transduction protein , which includes meprin ( metalloendopeptidase ) , that could explain failure to feed in symptomatic adult tsetse , 1015 d posteclosion ( kariithi et al .

kaposi sarcoma ( ks ) is an endothelial cell lineage tumor that is caused by ks - associated herpesvirus ( kshv ) . it is unclear whether these different clinical forms have the same molecular make - up and whether they would respond to the same treatment . the clinical forms of ks are classic ks , endemic ks , transplant - associated ks , and aids - associated ks . in the era of effective antiretroviral therapy ( art ) , we now also encounter a new type of ks that is hiv associated , but no longer aids - defining as it appears in individuals with near - normal cd4 counts . he identified five patients exhibiting hemorrhagic sarcoma of the skin as well as sarcoma of internal organs upon postmortem examination ( kaposi , 1872 ; abada et al . , 2008 ) . note that kaposi described an aggressive tumor in older hiv - negative men , whereas to date the classic form of ks is considered to be an indolent disease restricted to the skin . one report suggests a hereditary component for classic ks ( guttman - yassky et al . , 2004 ) . endemic ks was described as occurring in sub - saharan africa before the emergence of hiv ( maclean , 1963 ) . it tends to be aggressive , and it is regularly seen in children ( slavin et al . , 1970 ) . endemic ks is the leading cancer of children in some african countries owing to near universal infection with kshv and other yet to be identified co - factors . it is as frequent , and as characteristic for the malaria belt as epstein barr - virus ( ebv)-associated endemic burkitt lymphoma ( bl ) . the only treatment , if available , is radiation and/or cytotoxic chemotherapy . in severe cases of ks that are localized to the limbs amputation transplant - associated ks is a significant cause of morbidity and mortality in italy , turkey , saudia - arabia , e.g. , in developed countries with substantial kshv seroprevalence ( 818% ) . ks seen in solid organ transplant recipients is called iatrogenic ks ( harwood et al . , 1979 ) this often leads to lesions resolution ( or immune - mediated tumor regression ) , though it risks organ rejection . lately , rapamycin has emerged as an effective therapy for transplant - associated ks ( stallone et al . , 2005 ) , either as second - line substitutive immune suppressive regimen or as addition to cyclosporine mediated immune maintenance regimens . transplant - associated ks tends to occur later ( after the first year ) than viremic herpesvirus - associated complications . either the donor organ or the recipient can be the source of kshv in this setting , as well as de novo exposure post transplantation when immunosuppressive therapies are administered ( barozzi et al . , 2003 ) . note that ks can become an epidemic disease only in those hiv - positive populations that have high prevalence rates of kshv . in the us and other developing countries , these are men who have sex with men , not individuals who acquired hiv through blood transfusion prior to the introduction of mandatory hiv screening of blood supplies ( gao et al . , 1996a , b ; kedes et al . , 1996 ) . in regions of endemic ks , where initial exposure happens during childhood , these are all individuals children , men , and women ( though even here ks is twice as frequent in men as in women ) . in the context of substantial immune suppression , i.e. , terminal aids , ks can be highly aggressive , and spreads quickly to internal organs ( gottlieb et al . , 1981 ) . the clinical presentation of aids ks has become more diverse with the advent of art . first , approximately 30% of hiv - associated ks cases in the us now occur in the setting of successfully art - suppressed hiv viral load . second , terminal aids ks - associated with art failure remains the leading malignancy of hiv - positive men . third , ks is often still the first indication of hiv infection , particularly in sub - saharan africa . like transplant - associated ks fourth , the opposite clinical scenario has also been seen : hiv - infected individuals who newly start art develop ks within the first months of therapy , a manifestation that defines immune reconstitution inflammatory syndrome ( iris ; connick et al . , 2004 ; bower et al . , 2005 ; is there a molecular mechanism or signature that may be utilized as the basis of rational therapy choice ? and how do we measure ks responses to compare different therapeutic modalities ? as mentioned above ks can be limited or can present as fulminant disease , with internal organ involvement . persons with severe ks may also develop primary effusion lymphoma ( pel ) or suffer from concurrent kshv - associated multicentric castleman disease ( mcd ; described in detail in later sections ; see below ) . cutaneous lesions tend to be most common at the lower extremities , but can appear anywhere ( cheung et al . , 2005 ) . oral ks , concurrent with cutaneous ks or as a single lesion absent cutaneous involvement , has been reported in the context of hiv infection . whereas cutaneous lesions are a cause of stigma as much as physical pain , the more insidious presentation is that of ks in internal organs ( lung , liver ) without overt extensive cutaneous involvement , as is seen , e.g. , in some patient populations in brazil ( desouza , personal communication ) . cutaneous ks lesions are typically dark and can present as patches , papules , plaques , or nodules . no formal studies have explicitly linked these broad descriptors to survival or to response to therapy . ks can affect all internal organs including but not limited to the lungs , liver , lymph nodes , and the gastrointestinal tract . , lymph node involvement does not always portend the same poor prognosis as other cancers ( myskowski et al . , 1988 ) . the current staging system of aids - associated ks is based on the aids clinical trials group ( actg ) oncology committee ( krown et al . , 1989 , 1997 ) . akin to the standard tnm staging for solid tumors ( sobin et al . , 2009 ) , it incorporates extent of tumor , where t0 signifies that the ks is restricted to the skin and/or lymph nodes and/or minimal oral disease ( i.e. , non - nodular ks confined to the palate ) , and t1 signifies tumor - associated edema or ulceration , gastrointestinal ks , extensive oral ks , or ks in other non - nodal internal organs . other criteria in this classification include the severity of immunodeficiency ( i0 or i1 ) as measured by cd4 count and various systemic symptoms / illnesses ( s0 or s1 ) . both poor immune response ( i ) , as well as the stage of the tumor ( t ) , most accurately predicted survival of aids ks in the pre - art era ( krown et al . , both tumor stage and systemic illness also predicts dismal prognosis in the post - art era ( nasti et al . , 2003 ) . of note . notably this classification does not consider kshv viral load , as this classification system predates the discovery of kshv . it does not consider molecular markers of the tumor , such as proliferative index of the lesions or circulating biomarkers , such as il-6 . regardless of clinical acumen , lesional biopsy is required to establish ks , as lesions can be mistaken for hemangiomas , dermatofibromas , hematomas , purpuras etc . , and in developing countries , tuberculosis ( skin and internal ) . other diseases that may be mistaken for ks due to similar presentations include bacillary angiomatosis , lymphangioma , angiosarcoma , and hemangioendothelioma to list just a few . most individuals with ks lesions will have detectable kshv in blood and ks viral load is prognostic for ks . a study of classic ks reported an average of < 100 copies / ml ( guttman - yassky et al . , 2007 ) , whereas aids ks is almost always associated with > 1000 copies / ml . compared to other herpesvirus infections ( epstein barr - virus or human cytomegalovirus ) in the setting of aids or transplant - associated immune suppression , the level of viremia is low , reducing the sensitivity of kshv viral load based assays . by contrast specificity of kshv viral load assays is high , as a high viral load is almost always associated with overt or imminent ks . seropositivity for kshv latent nuclear antigen ( lana ) , which at present is the most specific assay , can not be used to diagnose ks . it establishes exposure to the risk factor kshv , however exposure precedes disease by a long time . a rise in serum antibody titers may happen in some instances , but not others , particularly in heavily immune deficient individuals . early studies estimated a median latency of 7 years between exposure and disease for pre - art aids ks ( gao et al . , the latency may be even longer ; for endemic ks in children the latency can be months . as of late , immunohistochemistry for the kshv antigen , lana , has been added to the diagnostic repertoire . as another alternative , kshv detection by pcr for viral dna or rna may be informative . the problem here is that sensitivity of the pcr depends on the accuracy and location of the excisional biopsy ; for instance , to minimize bleeding the most aggressive lesion is typically not biopsied . a confounding factor for rna - based diagnosis has been the rnase - rich environment of the lesions , which prevented rna analyses by traditional methods ( e.g. , northern blot ) until the introduction of more sensitive methods such as quantitative real - time pcr ( renne , lagunoff , dittmer , unpublished ) . lesions are composed of vascular spaces comprised of large endothelial cells that protrude into the vessel lumen due to thinning of the blood vessel wall . these proliferating spindle cells are the ks tumor cells and the target for anti - ks therapy . the presence of extravascular erythrocytes and narrow irregular , angulated slits is a classic hallmark of ks . inflammatory mononuclear cell infiltrates are seen consistently and are generally comprised of infiltrating lymphocytes , macrophages , and plasma cells . exactly how they contribute to lesion development ( or if at all ) is unclear . as there are many forms of ks , one must separately consider treatment options for each form . clinical trial data in ks , especially phase iii studies , are limited by declining numbers of patients in developed countries in the post - art era . therefore , published treatment recommendations are based on both older trial data , limited ( phase i / ii ) trials , and consensus opinion . of note , this review by no means offers specific recommendations . these fall into the purview of the treating physician . rather , we will focus on those regimens for which a reasonable rational basis exists and these regimens are often arrived at post facto . with rare exceptions , kaposi sarcoma , as an aids - presenting manifestation in art nave patients , often responds to art and the ensuing immune reconstitution alone . however , typically no more than half of patients achieve lesion resolution with successful art ( nguyen et al . , 2008 ) . at present there are no clinical or molecular parameters that distinguish between responders and non - responders . a phase iii clinical trial comparing art alone , or with delayed chemotherapy to art , with immediate adjunctive chemotherapy for limited aids ks in resource - limited settings ( react - ks ) has recently started enrolling . whether some art regimens are better suited to treat aids some hiv protease inhibitors have direct anti - tumor activity ( monini et al . , 2004 ) , and some also exhibit anti - viral kshv activity ( gantt et al . , antiretroviral combinations with non - protease - inhibitors can also induce ks regression ( bower et al . ks if lesions do not regress with primary art therapy , and can be useful in other situations as well . ks as an indicator of art failure requires second and/or third line therapy for hiv , again with concurrent or delayed chemotherapy . early systemic chemotherapy may help to suppress iris - associated flares ( leidner and aboulafia , 2005 ) . ks that develops in the presence of successful art also requires ks - targeted chemotherapy . in addition to art , radiotherapy or surgery can be used to treat isolated lesions . cytotoxic chemotherapy includes vincristine , bleomycin , doxorubicin , and etoposide , as single agents or in combination . in particular the liposomal formulations of pegylated - doxorubicin ( doxil ) or daunorubicin have shown clinical efficacy and are often the first - line agent used . these chemotherapy options are part of the standard repertoire of cytotoxic therapy for solid tumors , including sarcomas , and were developed and clinically evaluated in the pre - art and pre - kshv era . while these agents are efficacious , they were not chosen because of unique insights into the biology of the disease and they do not take into account any novel targets that the tumor - associated virus may present . future improvements in ks treatment ( i.e. , higher efficacy and lower toxicity ) will depend on better exploitation of these disease - specific targets . in patients afflicted with transplant - associated ks , complete regression of cutaneous ks was seen when immunosuppressive therapy was switched from cyclosporin to rapamycin ( campistol et al . , 2004 ; stallone et al . , these results have been recapitulated in immune deficient animal models of ks ( roy , dittmer , unpublished observation ) , and similar encouraging response rates have been seen by others ( gutierrez - dalmau et al . a recent study showed rapamycin ( sirolimus ) to be well tolerated over a long period of time ( 10 months ) and to induce encouraging molecular responses and disease stabilization in a limited study of aids ks ( krown et al . , 2012 ) . fkb12 complex inhibits mammalian target of rapamycin ( mtor ) kinase activity ( sabers et al . , 1995 ) . rapamycin is established clinically since 1999 as a second - generation immunosuppressive agent for organ transplantation , because it inhibits il-2 translation and secretion and thus t - cell proliferation . in this context the cell autonomous g1 arrest phenotype is augmented by inhibition of il-2 , which is a paracrine and autocrine growth factor for t cells . this has been established for the b - cell survival factors hil-6 and hil-10 ( komanduri et al . , 1996 ; asou et al . , 1998 ; aoki and tosato , 1999 ; drexler et al . , 1999 il-6 , il-10 , ifngamma , and il12p40 secretion is inhibited by rapamycin ( sin et al . , 2007 ) . other cytokines [ il-1 , il-1 , il-2 , il-3 , il-4 , il-5 , il-7 , il-8 , il-12(p70 ) , il-13 , il-15 , ip-10 , eotaxin , ifn , gm - csf , mcp-1 , mip-1 , rantes , and tnf ] are not affected . clinically , both direct and indirect mechanisms may contribute to these responses . on one hand , tumor regression coincided with the recovery of t - cell memory responses against kshv latent ( orf73 ) and lytic ( k8.1 ) antigens ( barozzi et al . on the other hand , rapamycin is directly toxic to kshv - infected cells ( sin et al . , 2007 ) , and kshv - associated tumors , i.e. , ks and pel , depend on the mtor signaling pathway , which is directly targeted by rapamycin ( sodhi et al . , 2006 ; ks lesions almost universally exhibit phosphorylated akt , which activates mtor kinase and phosphorylated s6 , which is a biomarker for mtor kinase activity ( roy and dittmer , 2011 ) . this is expected since the pi3k / akt pathway is activated upon of vegf vegfr stimulation in endothelial cells and by the activation of the b - cell receptor ( bcr ) complex in b cells . active akt kinase promotes multiple cellular survival mechanisms : ( i ) akt enhances protein synthesis through increasing the phosphorylation of mtor ( gingras et al . , 1998 ) , ( ii ) akt counteracts apoptosis by directly phosphorylating and inactivating pro - apoptotic factors such as bad ( datta et al . , 1997 ; 1998 ) , ( iii ) akt phosphorylates a family of transcription factors known as the forkhead ( fkhr ) or foxo transcription factors ; brunet et al . , 1999 ; kops and burgering , 1999 ; tang et al . , 1999 ) . members of this family include fkhr ( foxo1a ) , fkhrl1 ( foxo3 ) , and afx ( foxo4 ) . the net result of phosphorylation of the downstream targets of akt is cell survival via inactivation of the fkhr family , gsk-3b , caspase-9 , and bad ( cross et al . , 1995 ; datta et al . , 1997 ; del peso et al . , 1997 ; cardone et al . , rapamycin blocks focus formation induced by oncogenic alleles of the upstream mtor regulators , pi3k , or akt ( aoki et al . , 2001 ) . prolonged rapamycin treatment has been shown to lead to increased akt phosphorylation by the mtor rictor complex ( sarbassov et al . , 2005 , 2006 ) , though this was not seen in ks . thus , the efficacy of rapamycin ( and more modern rapamycin analogs ) in ks may plausibly be the result of a slightly different wiring of the pi3k / akt / mtor pathway in kshv - associated cancers . the discovery of kshv in ks prompted a frantic search for other cancers that may be associated with this new virus . following the paradigm of ebv , which is seen in nasopharyngeal carcinoma , a solid organ cancer , as well as in lymphomas , kshv sequences were rapidly identified in an uncommon type of b - cell lymphoma ( cesarman et al . , 1995 ) : the first cell lines for this lymphoma were obtained from body cavity effusions of what was hitherto called aids - associated lymphohematopoietic neoplasms . pel occurs with increased frequency in hiv - infected individuals ; including those with concurrent ks . this may be a skewed estimate , since it is may be quite common that aids patients die of other complications , including ks , before a diagnosis of pel can be made ( nador et al . the recognition that kshv was always found in pel , and ebv in 5080% of pel , led to their classification as a new sub - type of non - hodgkin lymphoma ( nador et al . pel cell lines contain many copies of the kshv genome as nuclear plasmids , which greatly facilitates molecular studies for this virus . these variants also carry the kshv genome and express at least the kshv latent genes ( nador et al . , 1996 ; said et al . , 1996 ; engels et al . , 2003 ; chadburn et al , 2004 ; carbone et al . , 2005 ; deloose et al . , 2005 ) . the diagnostic criteria for pel ( nador et al 2008 ) include immunoblastic anaplastic large - cell morphology , null - cell phenotype with no b - cell - associated antigen and immunoglobulin expression , and b - cell genotype as ascertained by bcr rearrangement . high cd138/syndecan-1 ( gaidano et al . , 1997 ) expression and hypermutation of immunoglobulin genes ( matolcsy et al . , 1998 ) established that pels are post - germinal center ( gc ) tumors at a pre - terminal stage prior to plasma - cell differentiation ( this does not exclude the possibility that pel arrived at this stage through extra - gc maturation ) . , 2003 ; klein et al . , 2003 ; ohara et al . , 2008 , 2009 ) . survival on conventional chemotherapy is very poor ( nador et al . , 1996 ; simonelli et al . , 2003 ) , but of course pel patients tend to have multiple comorbidities at presentation . the effusions may be managed by repeated drainage but eventually solid nests of pel abolish vital organ functions . in pel fhit and wwox , two fragile site tumor suppressor genes , are deleted in many pel cell lines ( roy et al . , 2011 ) , however these and other genome - wide association studies are limited by the scarcity of cases . individual case reports document responses to anti - viral therapy , bortezomib , rapamycin ( sin et al . , 2007 ) , rituximab - containing chemotherapy regimens ( oksenhendler et al . , 1998 ; boulanger et al . , 2001 ; , 2005 ; siddiqi and joyce , 2008 ) , or pleurodesis with bleomycin ( yiakoumis et al . , 2011 ) ( velcade ) primary mechanism of action is inhibition of the 26s proteasome and nuclear factor b ( nfb ) activity . rapamycin ( sirolimus ) inhibits mtor signaling , rituximab ( rituxan ) is a humanized monoclonal antibody against cd20 , and bleomycin induces dna breaks and eventually apoptosis in rapidly growing cells . this is a sub - type of castleman disease , specifically the plasma - cell type ( keller et al . , 1972 ) . castleman disease can be localized to just one , or multiple lymph nodes can be involved . mcd has mostly a plasma - cell type morphology and is accompanied by systemic symptoms ( waterston and bower , 2004 ) . these systemic symptoms may be a result of cytokines , and in particular interleukin-6 ( il-6 ) . the viral homolog , vil-6 is expressed in scattered plasmablasts surrounding the lymphoid follicles in mcd ( berti et al . , 1997 ; cannon et al . , 1999 ; thus , both kshv - induced human il-6 and virus - encoded vil-6 are found at high levels in mcd . recently , cases of an inflammatory syndrome with clinical symptoms similar to mcd have been described in aids patients with ks , but no diagnosis of mcd ( uldrick et al . , 2010 ) . here too , high amounts of vil-6 in the serum have been noted . this suggests excess cytokine production is one of the systemic features of mcd ( hasson , 1985 ) . multicentric castleman disease patients may also develop concurrent frank cancer , such as ks and nhl . kshv is invariably present in aids - associated mcd ( soulier et al . , 1995 ; larroche et al . , 2002 ) and the presence of ks and mcd in the same lymph node is not rare . in some cases high angiosclerosis , gc , and perifollicular vascular proliferation is seen , while plasmacytosis was less pronounced ( suda et al . , 2001 ) . studies are ongoing to better understand this disease and to arrive at a more succinct clinical description . the kshv - positive cases may represent a distinct morphologic variant from kshv - negative mcd ( dupin et al . mcd lymph nodes can contain multiple kshv - infected b cells , which may form microlymphomas or even frank lymphomas . here , kshv - infected plasmablasts are b - monotypic but polyclonal . they almost invariably express igm - lambda ( du et al . , 2001 ) . median survival of mcd in hiv - positive individuals in the pre - art era was approximately 14 months ( oksenhendler et al . evidence of kshv lytic viral protein expression led to testing of ganciclovir , with mixed results in small case series ( corbellino et al . this is not unexpected if for instance vil-6 expression is independent , rather than coincident with viral lytic gene expression ( chatterjee et al . , 2002 ) . under such a scenario the viral kinases orf36 and tk , which are required for ganciclovir efficacy would not be expressed . alternatively , the drug regimen of just one anti - viral may be insufficient as a two - drug regimen combining valganciclovir and zidovudine ( azt ) , showed promising results ( uldrick et al . , 2011 ) . in mcd rational therapy treatment with tocilizumab , an anti - human interleukin-6 ( il-6 ) receptor monoclonal antibody , resulted in clinical responses in mcd patients that did not have aids ( nishimoto et al . , 2005 ; song et al . , 2010 ) ; and the anti - cd20 antibody , rituximab , has shown responses in up to 70% of patients ( corbellino et al . , 2001 ; gerard et al . , 2007 ; bestawros et al . , 2008 ; bower , 2010 ; bower et al . , 2011 ) . the kshv - positive b cells frequently lack expression of cd20 ( chadburn et al . , 2008 ; naresh et al . , 2009 ) , so the source of systemic symptoms may be virus - negative plasma cells or other immune cells that respond to infection . antiretroviral therapy has increased the life expectancy of the hiv - infected population . however , as these individuals age , there is likely to be a corresponding increase in the incidence of cancers in the hiv - positive population . most of the current therapies do not target the unique viral etiology of cancers linked to kshv infection . one exception are the anti - herpes viral drugs which target lytic virus but do are not effective against latent virus . the interactions of chemotherapy with hiv protease inhibitors is also another consideration that needs to be addressed . in the future , it will be important to determine whether traditional chemotherapies are safe in the context of currently prescribed hiv protease inhibitors , and to devise newer therapies that directly target the viral etiology of these cancers . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

kaposi sarcoma ( ks ) is the most frequent aids - defining cancer worldwide . ks - associated herpesvirus ( kshv ) is the etiological agent of ks , and the virus is also associated with two lymphoproliferative diseases . both ks and kshv - associated lymphomas , are cancers of unique molecular composition . they represent a challenge for cancer treatment and an opportunity to identify new mechanisms of transformation . here , we review the current clinical insights into kshv - associated cancers and discuss scientific insights into the pathobiology of ks , primary effusion lymphoma , and multicentric castleman s disease .

Kaposi sarcoma Treatment Primary Effusion Lymphoma Multicentric Castleman Disease Challenges Conflict of Interest Statement

kaposi sarcoma ( ks ) is an endothelial cell lineage tumor that is caused by ks - associated herpesvirus ( kshv ) . the clinical forms of ks are classic ks , endemic ks , transplant - associated ks , and aids - associated ks . in the era of effective antiretroviral therapy ( art ) , we now also encounter a new type of ks that is hiv associated , but no longer aids - defining as it appears in individuals with near - normal cd4 counts . he identified five patients exhibiting hemorrhagic sarcoma of the skin as well as sarcoma of internal organs upon postmortem examination ( kaposi , 1872 ; abada et al . , 2008 ) . note that kaposi described an aggressive tumor in older hiv - negative men , whereas to date the classic form of ks is considered to be an indolent disease restricted to the skin . , 2004 ) . it tends to be aggressive , and it is regularly seen in children ( slavin et al . endemic ks is the leading cancer of children in some african countries owing to near universal infection with kshv and other yet to be identified co - factors . it is as frequent , and as characteristic for the malaria belt as epstein barr - virus ( ebv)-associated endemic burkitt lymphoma ( bl ) . in severe cases of ks that are localized to the limbs amputation transplant - associated ks is a significant cause of morbidity and mortality in italy , turkey , saudia - arabia , e.g. ks seen in solid organ transplant recipients is called iatrogenic ks ( harwood et al . lately , rapamycin has emerged as an effective therapy for transplant - associated ks ( stallone et al . transplant - associated ks tends to occur later ( after the first year ) than viremic herpesvirus - associated complications . either the donor organ or the recipient can be the source of kshv in this setting , as well as de novo exposure post transplantation when immunosuppressive therapies are administered ( barozzi et al . note that ks can become an epidemic disease only in those hiv - positive populations that have high prevalence rates of kshv . in the us and other developing countries , these are men who have sex with men , not individuals who acquired hiv through blood transfusion prior to the introduction of mandatory hiv screening of blood supplies ( gao et al . , 1996a , b ; kedes et al . in regions of endemic ks , where initial exposure happens during childhood , these are all individuals children , men , and women ( though even here ks is twice as frequent in men as in women ) . in the context of substantial immune suppression , i.e. , terminal aids , ks can be highly aggressive , and spreads quickly to internal organs ( gottlieb et al . first , approximately 30% of hiv - associated ks cases in the us now occur in the setting of successfully art - suppressed hiv viral load . second , terminal aids ks - associated with art failure remains the leading malignancy of hiv - positive men . like transplant - associated ks fourth , the opposite clinical scenario has also been seen : hiv - infected individuals who newly start art develop ks within the first months of therapy , a manifestation that defines immune reconstitution inflammatory syndrome ( iris ; connick et al . , 2004 ; bower et al . , 2005 ; is there a molecular mechanism or signature that may be utilized as the basis of rational therapy choice ? as mentioned above ks can be limited or can present as fulminant disease , with internal organ involvement . persons with severe ks may also develop primary effusion lymphoma ( pel ) or suffer from concurrent kshv - associated multicentric castleman disease ( mcd ; described in detail in later sections ; see below ) . oral ks , concurrent with cutaneous ks or as a single lesion absent cutaneous involvement , has been reported in the context of hiv infection . whereas cutaneous lesions are a cause of stigma as much as physical pain , the more insidious presentation is that of ks in internal organs ( lung , liver ) without overt extensive cutaneous involvement , as is seen , e.g. , in some patient populations in brazil ( desouza , personal communication ) . no formal studies have explicitly linked these broad descriptors to survival or to response to therapy . ks can affect all internal organs including but not limited to the lungs , liver , lymph nodes , and the gastrointestinal tract . , lymph node involvement does not always portend the same poor prognosis as other cancers ( myskowski et al . the current staging system of aids - associated ks is based on the aids clinical trials group ( actg ) oncology committee ( krown et al . akin to the standard tnm staging for solid tumors ( sobin et al . , 2009 ) , it incorporates extent of tumor , where t0 signifies that the ks is restricted to the skin and/or lymph nodes and/or minimal oral disease ( i.e. , non - nodular ks confined to the palate ) , and t1 signifies tumor - associated edema or ulceration , gastrointestinal ks , extensive oral ks , or ks in other non - nodal internal organs . both poor immune response ( i ) , as well as the stage of the tumor ( t ) , most accurately predicted survival of aids ks in the pre - art era ( krown et al . of note . notably this classification does not consider kshv viral load , as this classification system predates the discovery of kshv . regardless of clinical acumen , lesional biopsy is required to establish ks , as lesions can be mistaken for hemangiomas , dermatofibromas , hematomas , purpuras etc . , and in developing countries , tuberculosis ( skin and internal ) . other diseases that may be mistaken for ks due to similar presentations include bacillary angiomatosis , lymphangioma , angiosarcoma , and hemangioendothelioma to list just a few . most individuals with ks lesions will have detectable kshv in blood and ks viral load is prognostic for ks . a study of classic ks reported an average of < 100 copies / ml ( guttman - yassky et al . , 2007 ) , whereas aids ks is almost always associated with > 1000 copies / ml . compared to other herpesvirus infections ( epstein barr - virus or human cytomegalovirus ) in the setting of aids or transplant - associated immune suppression , the level of viremia is low , reducing the sensitivity of kshv viral load based assays . by contrast specificity of kshv viral load assays is high , as a high viral load is almost always associated with overt or imminent ks . seropositivity for kshv latent nuclear antigen ( lana ) , which at present is the most specific assay , can not be used to diagnose ks . it establishes exposure to the risk factor kshv , however exposure precedes disease by a long time . a rise in serum antibody titers may happen in some instances , but not others , particularly in heavily immune deficient individuals . early studies estimated a median latency of 7 years between exposure and disease for pre - art aids ks ( gao et al . , the latency may be even longer ; for endemic ks in children the latency can be months . the problem here is that sensitivity of the pcr depends on the accuracy and location of the excisional biopsy ; for instance , to minimize bleeding the most aggressive lesion is typically not biopsied . a confounding factor for rna - based diagnosis has been the rnase - rich environment of the lesions , which prevented rna analyses by traditional methods ( e.g. , northern blot ) until the introduction of more sensitive methods such as quantitative real - time pcr ( renne , lagunoff , dittmer , unpublished ) . lesions are composed of vascular spaces comprised of large endothelial cells that protrude into the vessel lumen due to thinning of the blood vessel wall . these proliferating spindle cells are the ks tumor cells and the target for anti - ks therapy . the presence of extravascular erythrocytes and narrow irregular , angulated slits is a classic hallmark of ks . inflammatory mononuclear cell infiltrates are seen consistently and are generally comprised of infiltrating lymphocytes , macrophages , and plasma cells . exactly how they contribute to lesion development ( or if at all ) is unclear . as there are many forms of ks , one must separately consider treatment options for each form . clinical trial data in ks , especially phase iii studies , are limited by declining numbers of patients in developed countries in the post - art era . therefore , published treatment recommendations are based on both older trial data , limited ( phase i / ii ) trials , and consensus opinion . of note , this review by no means offers specific recommendations . these fall into the purview of the treating physician . rather , we will focus on those regimens for which a reasonable rational basis exists and these regimens are often arrived at post facto . with rare exceptions , kaposi sarcoma , as an aids - presenting manifestation in art nave patients , often responds to art and the ensuing immune reconstitution alone . however , typically no more than half of patients achieve lesion resolution with successful art ( nguyen et al . a phase iii clinical trial comparing art alone , or with delayed chemotherapy to art , with immediate adjunctive chemotherapy for limited aids ks in resource - limited settings ( react - ks ) has recently started enrolling . whether some art regimens are better suited to treat aids some hiv protease inhibitors have direct anti - tumor activity ( monini et al . , 2004 ) , and some also exhibit anti - viral kshv activity ( gantt et al . ks if lesions do not regress with primary art therapy , and can be useful in other situations as well . ks as an indicator of art failure requires second and/or third line therapy for hiv , again with concurrent or delayed chemotherapy . early systemic chemotherapy may help to suppress iris - associated flares ( leidner and aboulafia , 2005 ) . ks that develops in the presence of successful art also requires ks - targeted chemotherapy . cytotoxic chemotherapy includes vincristine , bleomycin , doxorubicin , and etoposide , as single agents or in combination . in particular the liposomal formulations of pegylated - doxorubicin ( doxil ) or daunorubicin have shown clinical efficacy and are often the first - line agent used . these chemotherapy options are part of the standard repertoire of cytotoxic therapy for solid tumors , including sarcomas , and were developed and clinically evaluated in the pre - art and pre - kshv era . while these agents are efficacious , they were not chosen because of unique insights into the biology of the disease and they do not take into account any novel targets that the tumor - associated virus may present . future improvements in ks treatment ( i.e. in patients afflicted with transplant - associated ks , complete regression of cutaneous ks was seen when immunosuppressive therapy was switched from cyclosporin to rapamycin ( campistol et al . , these results have been recapitulated in immune deficient animal models of ks ( roy , dittmer , unpublished observation ) , and similar encouraging response rates have been seen by others ( gutierrez - dalmau et al . , 2012 ) . , 1995 ) . , 1996 ; asou et al . , 1999 il-6 , il-10 , ifngamma , and il12p40 secretion is inhibited by rapamycin ( sin et al . other cytokines [ il-1 , il-1 , il-2 , il-3 , il-4 , il-5 , il-7 , il-8 , il-12(p70 ) , il-13 , il-15 , ip-10 , eotaxin , ifn , gm - csf , mcp-1 , mip-1 , rantes , and tnf ] are not affected . on the other hand , rapamycin is directly toxic to kshv - infected cells ( sin et al . , 2007 ) , and kshv - associated tumors , i.e. , ks and pel , depend on the mtor signaling pathway , which is directly targeted by rapamycin ( sodhi et al . active akt kinase promotes multiple cellular survival mechanisms : ( i ) akt enhances protein synthesis through increasing the phosphorylation of mtor ( gingras et al . , 1997 ; 1998 ) , ( iii ) akt phosphorylates a family of transcription factors known as the forkhead ( fkhr ) or foxo transcription factors ; brunet et al . , 1999 ; kops and burgering , 1999 ; tang et al . , 1999 ) . members of this family include fkhr ( foxo1a ) , fkhrl1 ( foxo3 ) , and afx ( foxo4 ) . the net result of phosphorylation of the downstream targets of akt is cell survival via inactivation of the fkhr family , gsk-3b , caspase-9 , and bad ( cross et al . , 1997 ; cardone et al . , rapamycin blocks focus formation induced by oncogenic alleles of the upstream mtor regulators , pi3k , or akt ( aoki et al . , 2001 ) . thus , the efficacy of rapamycin ( and more modern rapamycin analogs ) in ks may plausibly be the result of a slightly different wiring of the pi3k / akt / mtor pathway in kshv - associated cancers . the discovery of kshv in ks prompted a frantic search for other cancers that may be associated with this new virus . following the paradigm of ebv , which is seen in nasopharyngeal carcinoma , a solid organ cancer , as well as in lymphomas , kshv sequences were rapidly identified in an uncommon type of b - cell lymphoma ( cesarman et al . , 1995 ) : the first cell lines for this lymphoma were obtained from body cavity effusions of what was hitherto called aids - associated lymphohematopoietic neoplasms . this may be a skewed estimate , since it is may be quite common that aids patients die of other complications , including ks , before a diagnosis of pel can be made ( nador et al . the recognition that kshv was always found in pel , and ebv in 5080% of pel , led to their classification as a new sub - type of non - hodgkin lymphoma ( nador et al . , 2003 ; chadburn et al , 2004 ; carbone et al . , 2005 ; deloose et al . the diagnostic criteria for pel ( nador et al 2008 ) include immunoblastic anaplastic large - cell morphology , null - cell phenotype with no b - cell - associated antigen and immunoglobulin expression , and b - cell genotype as ascertained by bcr rearrangement . , 2003 ; ohara et al . survival on conventional chemotherapy is very poor ( nador et al . the effusions may be managed by repeated drainage but eventually solid nests of pel abolish vital organ functions . in pel fhit and wwox , two fragile site tumor suppressor genes , are deleted in many pel cell lines ( roy et al . individual case reports document responses to anti - viral therapy , bortezomib , rapamycin ( sin et al . , 2007 ) , rituximab - containing chemotherapy regimens ( oksenhendler et al . , 2011 ) ( velcade ) primary mechanism of action is inhibition of the 26s proteasome and nuclear factor b ( nfb ) activity . rapamycin ( sirolimus ) inhibits mtor signaling , rituximab ( rituxan ) is a humanized monoclonal antibody against cd20 , and bleomycin induces dna breaks and eventually apoptosis in rapidly growing cells . , 1972 ) . these systemic symptoms may be a result of cytokines , and in particular interleukin-6 ( il-6 ) . the viral homolog , vil-6 is expressed in scattered plasmablasts surrounding the lymphoid follicles in mcd ( berti et al . , 1999 ; thus , both kshv - induced human il-6 and virus - encoded vil-6 are found at high levels in mcd . recently , cases of an inflammatory syndrome with clinical symptoms similar to mcd have been described in aids patients with ks , but no diagnosis of mcd ( uldrick et al . multicentric castleman disease patients may also develop concurrent frank cancer , such as ks and nhl . kshv is invariably present in aids - associated mcd ( soulier et al . , 1995 ; larroche et al . , 2002 ) and the presence of ks and mcd in the same lymph node is not rare . in some cases high angiosclerosis , gc , and perifollicular vascular proliferation is seen , while plasmacytosis was less pronounced ( suda et al . the kshv - positive cases may represent a distinct morphologic variant from kshv - negative mcd ( dupin et al . mcd lymph nodes can contain multiple kshv - infected b cells , which may form microlymphomas or even frank lymphomas . here , kshv - infected plasmablasts are b - monotypic but polyclonal . they almost invariably express igm - lambda ( du et al . evidence of kshv lytic viral protein expression led to testing of ganciclovir , with mixed results in small case series ( corbellino et al . in mcd rational therapy treatment with tocilizumab , an anti - human interleukin-6 ( il-6 ) receptor monoclonal antibody , resulted in clinical responses in mcd patients that did not have aids ( nishimoto et al . , 2010 ) ; and the anti - cd20 antibody , rituximab , has shown responses in up to 70% of patients ( corbellino et al . , 2001 ; gerard et al . , 2008 ; bower , 2010 ; bower et al . the kshv - positive b cells frequently lack expression of cd20 ( chadburn et al . , 2008 ; naresh et al . , 2009 ) , so the source of systemic symptoms may be virus - negative plasma cells or other immune cells that respond to infection . however , as these individuals age , there is likely to be a corresponding increase in the incidence of cancers in the hiv - positive population . most of the current therapies do not target the unique viral etiology of cancers linked to kshv infection . the interactions of chemotherapy with hiv protease inhibitors is also another consideration that needs to be addressed . in the future , it will be important to determine whether traditional chemotherapies are safe in the context of currently prescribed hiv protease inhibitors , and to devise newer therapies that directly target the viral etiology of these cancers .

kaposi sarcoma ( ks ) is an endothelial cell lineage tumor that is caused by ks - associated herpesvirus ( kshv ) . the clinical forms of ks are classic ks , endemic ks , transplant - associated ks , and aids - associated ks . in the era of effective antiretroviral therapy ( art ) , we now also encounter a new type of ks that is hiv associated , but no longer aids - defining as it appears in individuals with near - normal cd4 counts . he identified five patients exhibiting hemorrhagic sarcoma of the skin as well as sarcoma of internal organs upon postmortem examination ( kaposi , 1872 ; abada et al . note that kaposi described an aggressive tumor in older hiv - negative men , whereas to date the classic form of ks is considered to be an indolent disease restricted to the skin . one report suggests a hereditary component for classic ks ( guttman - yassky et al . endemic ks was described as occurring in sub - saharan africa before the emergence of hiv ( maclean , 1963 ) . it tends to be aggressive , and it is regularly seen in children ( slavin et al . endemic ks is the leading cancer of children in some african countries owing to near universal infection with kshv and other yet to be identified co - factors . it is as frequent , and as characteristic for the malaria belt as epstein barr - virus ( ebv)-associated endemic burkitt lymphoma ( bl ) . in severe cases of ks that are localized to the limbs amputation transplant - associated ks is a significant cause of morbidity and mortality in italy , turkey , saudia - arabia , e.g. , in developed countries with substantial kshv seroprevalence ( 818% ) . , 1979 ) this often leads to lesions resolution ( or immune - mediated tumor regression ) , though it risks organ rejection . lately , rapamycin has emerged as an effective therapy for transplant - associated ks ( stallone et al . , 2005 ) , either as second - line substitutive immune suppressive regimen or as addition to cyclosporine mediated immune maintenance regimens . transplant - associated ks tends to occur later ( after the first year ) than viremic herpesvirus - associated complications . either the donor organ or the recipient can be the source of kshv in this setting , as well as de novo exposure post transplantation when immunosuppressive therapies are administered ( barozzi et al . note that ks can become an epidemic disease only in those hiv - positive populations that have high prevalence rates of kshv . in the us and other developing countries , these are men who have sex with men , not individuals who acquired hiv through blood transfusion prior to the introduction of mandatory hiv screening of blood supplies ( gao et al . in regions of endemic ks , where initial exposure happens during childhood , these are all individuals children , men , and women ( though even here ks is twice as frequent in men as in women ) . in the context of substantial immune suppression , i.e. , terminal aids , ks can be highly aggressive , and spreads quickly to internal organs ( gottlieb et al . the clinical presentation of aids ks has become more diverse with the advent of art . first , approximately 30% of hiv - associated ks cases in the us now occur in the setting of successfully art - suppressed hiv viral load . second , terminal aids ks - associated with art failure remains the leading malignancy of hiv - positive men . third , ks is often still the first indication of hiv infection , particularly in sub - saharan africa . like transplant - associated ks fourth , the opposite clinical scenario has also been seen : hiv - infected individuals who newly start art develop ks within the first months of therapy , a manifestation that defines immune reconstitution inflammatory syndrome ( iris ; connick et al . , 2005 ; is there a molecular mechanism or signature that may be utilized as the basis of rational therapy choice ? and how do we measure ks responses to compare different therapeutic modalities ? as mentioned above ks can be limited or can present as fulminant disease , with internal organ involvement . persons with severe ks may also develop primary effusion lymphoma ( pel ) or suffer from concurrent kshv - associated multicentric castleman disease ( mcd ; described in detail in later sections ; see below ) . cutaneous lesions tend to be most common at the lower extremities , but can appear anywhere ( cheung et al . oral ks , concurrent with cutaneous ks or as a single lesion absent cutaneous involvement , has been reported in the context of hiv infection . whereas cutaneous lesions are a cause of stigma as much as physical pain , the more insidious presentation is that of ks in internal organs ( lung , liver ) without overt extensive cutaneous involvement , as is seen , e.g. cutaneous ks lesions are typically dark and can present as patches , papules , plaques , or nodules . ks can affect all internal organs including but not limited to the lungs , liver , lymph nodes , and the gastrointestinal tract . the current staging system of aids - associated ks is based on the aids clinical trials group ( actg ) oncology committee ( krown et al . , 2009 ) , it incorporates extent of tumor , where t0 signifies that the ks is restricted to the skin and/or lymph nodes and/or minimal oral disease ( i.e. , non - nodular ks confined to the palate ) , and t1 signifies tumor - associated edema or ulceration , gastrointestinal ks , extensive oral ks , or ks in other non - nodal internal organs . other criteria in this classification include the severity of immunodeficiency ( i0 or i1 ) as measured by cd4 count and various systemic symptoms / illnesses ( s0 or s1 ) . both poor immune response ( i ) , as well as the stage of the tumor ( t ) , most accurately predicted survival of aids ks in the pre - art era ( krown et al . , both tumor stage and systemic illness also predicts dismal prognosis in the post - art era ( nasti et al . notably this classification does not consider kshv viral load , as this classification system predates the discovery of kshv . it does not consider molecular markers of the tumor , such as proliferative index of the lesions or circulating biomarkers , such as il-6 . regardless of clinical acumen , lesional biopsy is required to establish ks , as lesions can be mistaken for hemangiomas , dermatofibromas , hematomas , purpuras etc . other diseases that may be mistaken for ks due to similar presentations include bacillary angiomatosis , lymphangioma , angiosarcoma , and hemangioendothelioma to list just a few . most individuals with ks lesions will have detectable kshv in blood and ks viral load is prognostic for ks . a study of classic ks reported an average of < 100 copies / ml ( guttman - yassky et al . compared to other herpesvirus infections ( epstein barr - virus or human cytomegalovirus ) in the setting of aids or transplant - associated immune suppression , the level of viremia is low , reducing the sensitivity of kshv viral load based assays . by contrast specificity of kshv viral load assays is high , as a high viral load is almost always associated with overt or imminent ks . seropositivity for kshv latent nuclear antigen ( lana ) , which at present is the most specific assay , can not be used to diagnose ks . it establishes exposure to the risk factor kshv , however exposure precedes disease by a long time . early studies estimated a median latency of 7 years between exposure and disease for pre - art aids ks ( gao et al . , the latency may be even longer ; for endemic ks in children the latency can be months . as of late , immunohistochemistry for the kshv antigen , lana , has been added to the diagnostic repertoire . the problem here is that sensitivity of the pcr depends on the accuracy and location of the excisional biopsy ; for instance , to minimize bleeding the most aggressive lesion is typically not biopsied . a confounding factor for rna - based diagnosis has been the rnase - rich environment of the lesions , which prevented rna analyses by traditional methods ( e.g. , northern blot ) until the introduction of more sensitive methods such as quantitative real - time pcr ( renne , lagunoff , dittmer , unpublished ) . lesions are composed of vascular spaces comprised of large endothelial cells that protrude into the vessel lumen due to thinning of the blood vessel wall . these proliferating spindle cells are the ks tumor cells and the target for anti - ks therapy . the presence of extravascular erythrocytes and narrow irregular , angulated slits is a classic hallmark of ks . inflammatory mononuclear cell infiltrates are seen consistently and are generally comprised of infiltrating lymphocytes , macrophages , and plasma cells . clinical trial data in ks , especially phase iii studies , are limited by declining numbers of patients in developed countries in the post - art era . therefore , published treatment recommendations are based on both older trial data , limited ( phase i / ii ) trials , and consensus opinion . rather , we will focus on those regimens for which a reasonable rational basis exists and these regimens are often arrived at post facto . with rare exceptions , kaposi sarcoma , as an aids - presenting manifestation in art nave patients , often responds to art and the ensuing immune reconstitution alone . at present there are no clinical or molecular parameters that distinguish between responders and non - responders . a phase iii clinical trial comparing art alone , or with delayed chemotherapy to art , with immediate adjunctive chemotherapy for limited aids ks in resource - limited settings ( react - ks ) has recently started enrolling . whether some art regimens are better suited to treat aids some hiv protease inhibitors have direct anti - tumor activity ( monini et al . , antiretroviral combinations with non - protease - inhibitors can also induce ks regression ( bower et al . ks if lesions do not regress with primary art therapy , and can be useful in other situations as well . ks as an indicator of art failure requires second and/or third line therapy for hiv , again with concurrent or delayed chemotherapy . ks that develops in the presence of successful art also requires ks - targeted chemotherapy . in particular the liposomal formulations of pegylated - doxorubicin ( doxil ) or daunorubicin have shown clinical efficacy and are often the first - line agent used . these chemotherapy options are part of the standard repertoire of cytotoxic therapy for solid tumors , including sarcomas , and were developed and clinically evaluated in the pre - art and pre - kshv era . while these agents are efficacious , they were not chosen because of unique insights into the biology of the disease and they do not take into account any novel targets that the tumor - associated virus may present . , higher efficacy and lower toxicity ) will depend on better exploitation of these disease - specific targets . in patients afflicted with transplant - associated ks , complete regression of cutaneous ks was seen when immunosuppressive therapy was switched from cyclosporin to rapamycin ( campistol et al . , these results have been recapitulated in immune deficient animal models of ks ( roy , dittmer , unpublished observation ) , and similar encouraging response rates have been seen by others ( gutierrez - dalmau et al . a recent study showed rapamycin ( sirolimus ) to be well tolerated over a long period of time ( 10 months ) and to induce encouraging molecular responses and disease stabilization in a limited study of aids ks ( krown et al . rapamycin is established clinically since 1999 as a second - generation immunosuppressive agent for organ transplantation , because it inhibits il-2 translation and secretion and thus t - cell proliferation . in this context the cell autonomous g1 arrest phenotype is augmented by inhibition of il-2 , which is a paracrine and autocrine growth factor for t cells . this has been established for the b - cell survival factors hil-6 and hil-10 ( komanduri et al . other cytokines [ il-1 , il-1 , il-2 , il-3 , il-4 , il-5 , il-7 , il-8 , il-12(p70 ) , il-13 , il-15 , ip-10 , eotaxin , ifn , gm - csf , mcp-1 , mip-1 , rantes , and tnf ] are not affected . on one hand , tumor regression coincided with the recovery of t - cell memory responses against kshv latent ( orf73 ) and lytic ( k8.1 ) antigens ( barozzi et al . on the other hand , rapamycin is directly toxic to kshv - infected cells ( sin et al . , ks and pel , depend on the mtor signaling pathway , which is directly targeted by rapamycin ( sodhi et al . , 2006 ; ks lesions almost universally exhibit phosphorylated akt , which activates mtor kinase and phosphorylated s6 , which is a biomarker for mtor kinase activity ( roy and dittmer , 2011 ) . this is expected since the pi3k / akt pathway is activated upon of vegf vegfr stimulation in endothelial cells and by the activation of the b - cell receptor ( bcr ) complex in b cells . active akt kinase promotes multiple cellular survival mechanisms : ( i ) akt enhances protein synthesis through increasing the phosphorylation of mtor ( gingras et al . , 1997 ; 1998 ) , ( iii ) akt phosphorylates a family of transcription factors known as the forkhead ( fkhr ) or foxo transcription factors ; brunet et al . the net result of phosphorylation of the downstream targets of akt is cell survival via inactivation of the fkhr family , gsk-3b , caspase-9 , and bad ( cross et al . , rapamycin blocks focus formation induced by oncogenic alleles of the upstream mtor regulators , pi3k , or akt ( aoki et al . prolonged rapamycin treatment has been shown to lead to increased akt phosphorylation by the mtor rictor complex ( sarbassov et al . thus , the efficacy of rapamycin ( and more modern rapamycin analogs ) in ks may plausibly be the result of a slightly different wiring of the pi3k / akt / mtor pathway in kshv - associated cancers . the discovery of kshv in ks prompted a frantic search for other cancers that may be associated with this new virus . following the paradigm of ebv , which is seen in nasopharyngeal carcinoma , a solid organ cancer , as well as in lymphomas , kshv sequences were rapidly identified in an uncommon type of b - cell lymphoma ( cesarman et al . , 1995 ) : the first cell lines for this lymphoma were obtained from body cavity effusions of what was hitherto called aids - associated lymphohematopoietic neoplasms . the recognition that kshv was always found in pel , and ebv in 5080% of pel , led to their classification as a new sub - type of non - hodgkin lymphoma ( nador et al . pel cell lines contain many copies of the kshv genome as nuclear plasmids , which greatly facilitates molecular studies for this virus . these variants also carry the kshv genome and express at least the kshv latent genes ( nador et al . the diagnostic criteria for pel ( nador et al 2008 ) include immunoblastic anaplastic large - cell morphology , null - cell phenotype with no b - cell - associated antigen and immunoglobulin expression , and b - cell genotype as ascertained by bcr rearrangement . , 1998 ) established that pels are post - germinal center ( gc ) tumors at a pre - terminal stage prior to plasma - cell differentiation ( this does not exclude the possibility that pel arrived at this stage through extra - gc maturation ) . , 2011 ) , however these and other genome - wide association studies are limited by the scarcity of cases . , 2011 ) ( velcade ) primary mechanism of action is inhibition of the 26s proteasome and nuclear factor b ( nfb ) activity . rapamycin ( sirolimus ) inhibits mtor signaling , rituximab ( rituxan ) is a humanized monoclonal antibody against cd20 , and bleomycin induces dna breaks and eventually apoptosis in rapidly growing cells . this is a sub - type of castleman disease , specifically the plasma - cell type ( keller et al . mcd has mostly a plasma - cell type morphology and is accompanied by systemic symptoms ( waterston and bower , 2004 ) . , 1999 ; thus , both kshv - induced human il-6 and virus - encoded vil-6 are found at high levels in mcd . recently , cases of an inflammatory syndrome with clinical symptoms similar to mcd have been described in aids patients with ks , but no diagnosis of mcd ( uldrick et al . kshv is invariably present in aids - associated mcd ( soulier et al . , 2002 ) and the presence of ks and mcd in the same lymph node is not rare . here , kshv - infected plasmablasts are b - monotypic but polyclonal . median survival of mcd in hiv - positive individuals in the pre - art era was approximately 14 months ( oksenhendler et al . evidence of kshv lytic viral protein expression led to testing of ganciclovir , with mixed results in small case series ( corbellino et al . alternatively , the drug regimen of just one anti - viral may be insufficient as a two - drug regimen combining valganciclovir and zidovudine ( azt ) , showed promising results ( uldrick et al . in mcd rational therapy treatment with tocilizumab , an anti - human interleukin-6 ( il-6 ) receptor monoclonal antibody , resulted in clinical responses in mcd patients that did not have aids ( nishimoto et al . , 2010 ) ; and the anti - cd20 antibody , rituximab , has shown responses in up to 70% of patients ( corbellino et al . however , as these individuals age , there is likely to be a corresponding increase in the incidence of cancers in the hiv - positive population . in the future , it will be important to determine whether traditional chemotherapies are safe in the context of currently prescribed hiv protease inhibitors , and to devise newer therapies that directly target the viral etiology of these cancers .

medication effects on human cardiovascular regulation are difficult to predict based on preclinical and clinical investigations . drugs could interfere with cardiovascular regulation in brain or periphery directly or through secondary baroreflexmediated adjustments in heart rate ( hr ) and vascular tone . highfidelity phenotyping during infusion of vasoactive medications can be used to detect drug effects on human cardiovascular control that could otherwise go undetected . hcn4 generates funny pacemaker f currents ( if ) promoting slow diastolic depolarization and cardiac rhythm generation.1 intracellular cyclic adenosine monophosphate ( camp ) modulates if 2 , and adrenergic stimulation augments camp , thereby increasing hcn4 conductivity and hr . given hcn4 's important role in autonomic hr adjustments , pharmacological hcn4 inhibition could profoundly affect human cardiovascular regulation . in fact , chronotropic competence is reduced3 or absent4 in mutated hcn4 channels unresponsive to camp . conversely , blockade of hcn4 channels on baroreceptor sensory neurons increased their excitability,5 , 6 which would tend to facilitate afferent baroreflex signaling and baroreflex function . the issue is relevant because ivabradine7 , 8 is used in the treatment of heart failure9 and angina pectoris10 ; however , the influences of hcn4 on human cardiovascular autonomic regulation have not been studied , aside from noninvasive baroreflex assessment of profound sympathetic activation.11 ivabradine is an openchannel blocker12 producing inhibition,13 thus ivabradine may be more effective at increased hr.14 we tested the hypothesis that pharmacological hcn4 blockade with ivabradine affects baroreflex hr regulation in healthy participants such that hr is reduced at a given blood pressure ( bp ) . moreover , we assessed whether ivabradine would interfere with baroreflex regulation of hr and sympathetic efferent traffic to resistance vessels and whether or not baroreflex bp buffering is perturbed . finally , we evaluated whether or not in vivo responses mirror ivabradine 's usedependent pharmacology . healthy men ( aged 1840 years ) with a body mass index between 18 and 30 kg / m and a resting hr 60 beats per minute ( bpm ) were eligible for this study . preexisting diseases were ruled out through a detailed history , medical examination , 12lead ecg , bp recordings , and blood sampling for routine laboratory measurements . all procedures were in accordance with the helsinki declaration of 1975 ( as revised in 1983 ) . a national competent authority and the local internal review board approved the studies . this randomized , doubleblind , 2period , 2sequence , crossover study was conducted at 2 sites ( experimental clinical research center , charit medical university , berlin , germany , and institute of clinical pharmacology , hannover medical school , hannover , germany ) . randomization for study drug sequence ( randomization block size : 6 ) and manufacturing and labeling of blinded medications was done centrally by the pharmacy of the charit medical university . medications were prepared as identical capsules in neutral containers labeled with the randomization code and a visit identifier . adherence to randomized medication sequence was secured by following the predefined numeric sequence of the visits . consequently , participants and investigators remained fully blinded until the database had been locked , double checked , and transferred to the institute of biostatistics at hannover medical school . on 2 separate occasions , participants ingested maximally recommended doses of ivabradine ( 7.5 mg ) or matching placebo 13 hours and 1 hour before testing . testing was conducted between 8 and 11 am in a quiet laboratory at an ambient temperature of 22 to 23c . recumbent systolic bp ( sbp ) , mean bp , and diastolic arterial bp were measured with an automated oscillometric device ( dinamap ; ge / critikon ) . we obtained blood samples after at least 30 minutes of rest for plasma catecholamine determination with highpressure liquid chromatography and consecutive electrochemical detection.15 stroke volume and cardiac output were obtained using an inert gas rebreathing technique ( innocor ; innovision ) . muscle sympathetic nerve activity ( msna ) was recorded from the right peroneal nerve ( nerve traffic analyzer 662c3 ; biomedical engineering department , university of iowa , iowa city , ia ) , as described previously.16 data were analogtodigital converted and analyzed using a program written by one of the authors ( a.d . ) . we determined the following msna parameters from the integrated nerve signal : burst frequency , or the number of msna bursts per minute ; burst incidence , or the number of bursts per 100 heart beats ; and total activity , or the area under the bursts per minute as arbitrary units per minute . following a resting period of at least 30 minutes after taking blood samples and measuring cardiac output , we performed a pharmacological baroreflex assessment using the nitric oxide donor sodium nitroprusside ( vasodilator ) and the 1adrenoceptor agonist phenylephrine ( vasoconstrictor ) . dose incrementation was stopped after the maximum dose of 2.1 g / kg per minute had been reached , sbp had changed by > 25 mm hg , or hr had dropped < 40 bpm . main end points of the study were normalized hr ( ie , hr at a given bp ) and resting msna burst frequency during supine rest . exploratory end points served to further characterize hemodynamic and autonomic responses to ivabradine at rest and during pharmacologic baroreflex challenge : arterial bp , stroke volume , cardiac output , total peripheral resistance , plasma catecholamine levels , cardiac and sympathetic baroreflex characteristics , and vasoactor sensitivity . the latter was defined as sbp change per dose increment of sodium nitroprusside and phenylephrine , respectively . ivabradine is expected to change hr directly , but it may do so also indirectly via baroreflexes through changes in bp . to remove confounding baroreflex influences on resting hr , we normalized hr before data unblinding ( example in figure 1 ) : we transferred the relationship between rr intervals ( rris ) and corresponding sbp values during pharmacological baroreflex testing into a mathematical function by linear regression or baroreflex curve fitting using a boltzmann sigmoidal function separately for the placebo and ivabradine condition for each participant ( sigmoidal curves in the example ) . resting sbp of both visits ( vertical dotted lines in the figure 1 ) were averaged . the average served as shared common ( standard ) pressure ( vertical solid line in the figure ) . this standard pressure was used as input ( abscissa value ) to the baroreflex curve function to calculate normalized rri and hr for each visit separately ( ordinate values ) . the example assumes lower sbp on ivabradine than on placebo ( 118 vs 122 mm hg ) . the solid vertical line represents mean sbp ( 120 mm hg ) , which determines normalized rri values . without normalization , ivabradine 's effect on hr would have been underestimated , as demonstrated by the smaller difference between the 2 raw rri values compared with the normalized rri values . hr indicates heart rate ; rri , rr interval ; sbp , systolic blood pressure . baroreflexmediated changes in the dependent parameters ( msna , rri ) were collected in 10mm hg bins separately for placebo and ivabradine . related bins for all participants , a boltzmann sigmoidal function was fitted to these merged data , and 95% confidence bands were calculated and plotted . note that the bins for lowest and highest bp are least occupied by values , thus their weight in calculations of curvefitting parameters and confidence bands are small ( figure 6a and 6b ) . with sd of 6.7 bpm in the group 's resting hr paired differences , =0.05 , and 2sided testing , 15 participants would provide 80% statistical power to detect such a difference.17 we included additional participants to allow for the meaningful analysis of secondary and exploratory end points . according to a prospective data analysis plan , primary and secondary end points were analyzed using the hillsarmitage approach.18 the method allows periodadjusted therapy effect estimation , namely , calculation of periodadjusted p values for mean differences between placebo and ivabradine data . to appreciate consistency of the results , several sensitivity analyses were performed ( univariate 1sample t tests and mixedmodel analysis with participant as random effect ) . exploratory variables have been tested with t tests for paired data and correlation analysis without adjustments for multiple testing . healthy men ( aged 1840 years ) with a body mass index between 18 and 30 kg / m and a resting hr 60 beats per minute ( bpm ) were eligible for this study . preexisting diseases were ruled out through a detailed history , medical examination , 12lead ecg , bp recordings , and blood sampling for routine laboratory measurements . all procedures were in accordance with the helsinki declaration of 1975 ( as revised in 1983 ) . a national competent authority and the local internal review board approved the studies . this randomized , doubleblind , 2period , 2sequence , crossover study was conducted at 2 sites ( experimental clinical research center , charit medical university , berlin , germany , and institute of clinical pharmacology , hannover medical school , hannover , germany ) . randomization for study drug sequence ( randomization block size : 6 ) and manufacturing and labeling of blinded medications was done centrally by the pharmacy of the charit medical university . medications were prepared as identical capsules in neutral containers labeled with the randomization code and a visit identifier . adherence to randomized medication sequence was secured by following the predefined numeric sequence of the visits . consequently , participants and investigators remained fully blinded until the database had been locked , double checked , and transferred to the institute of biostatistics at hannover medical school . on 2 separate occasions , participants ingested maximally recommended doses of ivabradine ( 7.5 mg ) or matching placebo 13 hours and 1 hour before testing . the washout period between study days was at least 3 weeks . testing was conducted between 8 and 11 am in a quiet laboratory at an ambient temperature of 22 to 23c . recumbent systolic bp ( sbp ) , mean bp , and diastolic arterial bp were measured with an automated oscillometric device ( dinamap ; ge / critikon ) . we obtained blood samples after at least 30 minutes of rest for plasma catecholamine determination with highpressure liquid chromatography and consecutive electrochemical detection.15 stroke volume and cardiac output were obtained using an inert gas rebreathing technique ( innocor ; innovision ) . muscle sympathetic nerve activity ( msna ) was recorded from the right peroneal nerve ( nerve traffic analyzer 662c3 ; biomedical engineering department , university of iowa , iowa city , ia ) , as described previously.16 data were analogtodigital converted and analyzed using a program written by one of the authors ( a.d . ) . we determined the following msna parameters from the integrated nerve signal : burst frequency , or the number of msna bursts per minute ; burst incidence , or the number of bursts per 100 heart beats ; and total activity , or the area under the bursts per minute as arbitrary units per minute . following a resting period of at least 30 minutes , we obtained resting baseline recordings in the supine position . after taking blood samples and measuring cardiac output , we performed a pharmacological baroreflex assessment using the nitric oxide donor sodium nitroprusside ( vasodilator ) and the 1adrenoceptor agonist phenylephrine ( vasoconstrictor ) . dose incrementation was stopped after the maximum dose of 2.1 g / kg per minute had been reached , sbp had changed by > 25 mm hg , or hr had dropped < 40 bpm . main end points of the study were normalized hr ( ie , hr at a given bp ) and resting msna burst frequency during supine rest . exploratory end points served to further characterize hemodynamic and autonomic responses to ivabradine at rest and during pharmacologic baroreflex challenge : arterial bp , stroke volume , cardiac output , total peripheral resistance , plasma catecholamine levels , cardiac and sympathetic baroreflex characteristics , and vasoactor sensitivity . the latter was defined as sbp change per dose increment of sodium nitroprusside and phenylephrine , respectively . ivabradine is expected to change hr directly , but it may do so also indirectly via baroreflexes through changes in bp . to remove confounding baroreflex influences on resting hr , we normalized hr before data unblinding ( example in figure 1 ) : we transferred the relationship between rr intervals ( rris ) and corresponding sbp values during pharmacological baroreflex testing into a mathematical function by linear regression or baroreflex curve fitting using a boltzmann sigmoidal function separately for the placebo and ivabradine condition for each participant ( sigmoidal curves in the example ) . resting sbp of both visits ( vertical dotted lines in the figure 1 ) were averaged . the average served as shared common ( standard ) pressure ( vertical solid line in the figure ) . this standard pressure was used as input ( abscissa value ) to the baroreflex curve function to calculate normalized rri and hr for each visit separately ( ordinate values ) . the example assumes lower sbp on ivabradine than on placebo ( 118 vs 122 mm hg ) . the solid vertical line represents mean sbp ( 120 mm hg ) , which determines normalized rri values . without normalization , ivabradine 's effect on hr would have been underestimated , as demonstrated by the smaller difference between the 2 raw rri values compared with the normalized rri values . hr indicates heart rate ; rri , rr interval ; sbp , systolic blood pressure . baroreflexmediated changes in the dependent parameters ( msna , rri ) were collected in 10mm hg bins separately for placebo and ivabradine . related bins for all participants using graphpad prism 5 , a boltzmann sigmoidal function was fitted to these merged data , and 95% confidence bands were calculated and plotted . note that the bins for lowest and highest bp are least occupied by values , thus their weight in calculations of curvefitting parameters and confidence bands are small ( figure 6a and 6b ) . with sd of 6.7 bpm in the group 's resting hr paired differences , =0.05 , and 2sided testing , 15 participants would provide 80% statistical power to detect such a difference.17 we included additional participants to allow for the meaningful analysis of secondary and exploratory end points . according to a prospective data analysis plan , primary and secondary end points were analyzed using the hillsarmitage approach.18 the method allows periodadjusted therapy effect estimation , namely , calculation of periodadjusted p values for mean differences between placebo and ivabradine data . to appreciate consistency of the results , several sensitivity analyses were performed ( univariate 1sample t tests and mixedmodel analysis with participant as random effect ) . exploratory variables have been tested with t tests for paired data and correlation analysis without adjustments for multiple testing . we screened 26 men : 23 met inclusion and exclusion criteria and entered the study . twentyone men completed both study visits ( 2 dropped out ) ( figure 2 ) . microneurographic recordings were obtained in 18 participants on both days . in 3 participants , we failed to obtain a stable nerve recording position at the second experimental day . the characteristics of the participants were as follows : age 26.84.2 years , height 1.820.06 m , body mass 79.810.5 kg , body mass index 24.13.0 kg / m , bp 1308/728 mm hg , and hr 67.45.8 bpm . ic indicates informed consent ; iva , ivabradine ; plc , placebo experimental data are shown in table . if blockade reduced resting hr with and without normalization for prevailing bp ( figure 3a ) ( for hr normalization , see methods ) . bp and stroke volume remained unchanged with a trend toward reduced cardiac output ( 7.6% ) and increased total peripheral resistance ( 5.6% ) on ivabradine . sbp sensitivity to sodium nitroprusside infusion was similar on both study days with a trend toward reduced sensitivity ( improved bp buffering ) to the vasoconstrictor phenylephrine with if blockade ( figure 5 ) . ivabradine did not alter pharmacological baroreflex sensitivities , such as the cardiac parasympathetic and vasoconstrictor sympathetic baroreflex gains . if blockade dampens the increase in msna burst frequency during higher sodium nitroprusside infusion rates , namely , when high vasoconstrictor activity and elevated hr cooccur ( figure 6a ) ; however , hrindependent msna parameters ( ie , msna burst incidence and total activity ) were not altered by hcn4 blockade during pharmacological baroreflex testing ( data not shown ) . upward shift of the cardiac baroreflex curve to longer rris ( figure 6b ) . consequently , rri lengthening by ivabradine was virtually independent of the prevailing hr during pharmacological baroreflex testing . hemodynamic , sympathetic , hormonal , and baroreflex parameters au indicates arbitrary units ; brg , baroreflex gain ( ie , baroreflex sensitivity ) ; co , cardiac output ; dbp , diastolic blood pressure ; hr , normalized ( baroreflexcorrected ) heart rate ; hrp , normalized heart rate during parasympathetic activation ; mbp , mean blood pressure ; msna , muscle sympathetic nerve activity ; ntp , sodium nitroprusside ; phe , phenylephrine ; sbp , systolic blood pressure ; sv , stroke volume ; tpr , total peripheral resistance . b , ivabradine 's use dependence : hr lowering is more pronounced in participants with higher resting hr . with resting values of 56.5 bpm , ivabradine has no effect ( unresponsive hr ) . c , effects of hcn4 inhibition on hr during parasympathetic activation ( hrp ) through baroreflex loading with phenylephrine . hrp was assessed as hr at the largest individual phenylephrine dose that was reached on both study days . d , participants with lower hrp on placebo exhibited a smaller reduction in hrp with ivabradine . the observation is consistent with ivabradine 's usedependent pharmacology ; however , parasympathetic activation shifted the unresponsive hr to 44.2 bpm , which is 12 bpm lower compared with resting conditions ( see panel b ) . b , individual changes in msna burst frequency , burst incidence , and total msna by ivabradine compared with placebo . total msna has been rescaled ( 20 ) for visual range harmonization among the 3 msna measures ( also see table ) . au indicates arbitrary units ; hb , heart beats ; msna , muscle sympathetic nerve activity . effects of hcn4 inhibition on bp sensitivity to infusion of the vasoconstrictor phenylephrine ( upper panel ) and the vasodilator sodium nitroprusside ( lower panel ) during placebo ( open circles ) and ivabradine ( closed circles ) . note the trend toward improved baroreflex buffering of bp increases on ivabradine ( upper panel ) . a , sympathetic baroreflex : although ivabradine may dampen the increase in msna burst frequency during nitroprusside infusion ( ie , when high vasoconstrictor activity and elevated hr cooccur ) , baroreflex curves are virtually identical if hrindependent msna measures ( burst incidence and total activity ) are used ( curves not shown ) . b , cardiac baroreflex : ivabradine shifts ( offsets ) the baroreflex curve upward ( to longer rris ) with virtually no effect on slope or range . note that the bands do not overlap with high blood pressure , indicating that baroreflexmediated hr slowing is intensified by ivabradine . dbp indicates diastolic blood pressure ; hr , heart rate ; msna , muscle sympathetic nerve activity ; rri , rr interval ; sbp , systolic blood pressure . participants with higher resting hr on placebo exhibited more pronounced hr slowing on ivabradine , showing use dependence we observed a similar pattern during baroreflex loading with phenylephrine ( figure 3c and 3d ) ; however , the hr at which ivabradine did not slow hr further was only 44.2 bpm ( ie , 12 bpm lower during parasympathetic activation ) . together , figure 3b and 3d visualize ivabradine 's usedependent characteristics ; however , they also show , as does figure 6b , that ivabradine and parasympathetic activation are additive in terms of hr reduction . we observed that hcn4 blockade with ivabradine reduces hr , leaving baroreflex hr and msna regulation intact . although hcn4 is involved in transducing autonomic activity at the sinus node level , baroreflex hr regulation was preserved on ivabradine . the cardiac baroreflex curve was parallelshifted toward lower hr without reductions in baroreflex gain or range . similarly , baroreflex hr slopes determined noninvasively by crossspectral analysis or the sequence method were unchanged with ivabradine in a human model of cardiac sympathetic activation.11 because msna discharges occur during diastole when arterial baroreceptors are unloaded , we expected reduced msna frequencies with compensatory increases in sympathetic action potentials within bursts ; however , ivabradineinduced hr reduction was not associated with resting msna changes . moreover , sympathetic baroreflex regulation was virtually identical on placebo and on ivabradine considering hrindependent msna measurements . finally , bp responses to vasoactive medications did not increase on ivabradine , suggesting that baroreflex bp buffering remained fully functional.19 together with previous studies in genetic animal models and in patients with familial sinus bradycardia due to hcn4 mutations,3 , 20 , 21 , 22 , 23 our study provides new insight into hcn4 's role in hr regulation . in patients with the 573x mutation , hcn4 does not respond to camp.3 nevertheless , hr increased 50 bpm during exercise . likewise , hr acceleration to adrenoreceptor stimulation was intact in adult hcn4deleted mice24 and in a heterozygous knockin model rendering hcn4 camp insensitive.4 on ivabradine , chronotropic competence in terms of hr acceleration and deceleration over a wide hr range assessed through baroreflex loading and unloading was unchanged . consequently , if can not be the crucial mechanism transducing autonomic signals at the level of the sinus node . cardiac pacemaker models are composed of several clocks,25 , 26 raising questions about hcn4 's dominant role in sinoatrial node automatism and rate regulation . instead , hcn4 may defend the sinus node against excess hyperpolarization,27 preventing bradycardia . despite its usedependent pharmacology,12 indicated by larger hr reduction in persons with higher resting hr , ivabradine aggravated baroreflexmediated bradycardia in our study . moreover , in the signify study , almost 10 000 patients with stable coronary artery disease received ivabradine doses up to 10 mg twice daily . bradycardia and atrial fibrillation occurred more often in the ivabradine group.28 together with our mechanismoriented study , these observations translate the preclinical concept that if is a defense mechanism against excess bradycardia to humans . using a similar approach , we previously observed that 2adrenoreceptor stimulation with clonidine also disables physiological safeguards maintaining hr.29 our study has potential limitations . we did not investigate responses to ivabradine under steady state conditions ; however , we showed earlier that acute dosing 13 hours and 1 hour before measurements was sufficient to attain ivabradine plasma concentrations in a therapeutic range.11 furthermore , the drug 's bradycardic activity consists of an initial rapid and subsequent relatively long duration of action caused by the ndealkylated metabolite and the parent drug , respectively.30 on the other hand , the 3week washout period was more than twice the time required to avoid carryover , according to the kinetics of the bradycardic effect with repeated oral administration.30 ivabradine 's pharmacological interaction with hcn4 channels complicates the interpretation of our study . as an openchannel blocker , for example , ivabradine features use dependence with trapping of the drug in the closed hcn4 channel.8 furthermore , hcn4 is also expressed in the nervous system , particularly in the thalamus,31 in spinal interneurons,32 and in baroreflex afferents.5 , 6 msna is a sensitive readout for changes in central autonomic regulation.33 , 34 , 35 because msna did not change , confounding effects by neural hcn4 inhibition is unlikely ; however , diseases could alter the ivabradine response . blunted baroreflex function in type 1 diabetic rats , for example , was associated with increased hcn expression and camp sensitivity.6 our findings in healthy participants can not be simply extrapolated to patients . finally , given the potential influence of respiration on postganglionic sympathetic discharge , it would have been desirable to also record and analyze breathing , yet others did not observe effects of ivabradine on respiratory rate either in healthy men36 or in patients with chronic obstructive pulmonary disease.37 highfidelity physiological phenotyping provides pharmacological insight that could not be gained by routine hr and bp measurements . we observed that hcn4 blockade with ivabradine reduced hr but did not impair sympathetic or parasympathetic baroreflex function . this feature preserves baroreflexdependent buffering of bp changes and aggravates parasympathetically mediated hr reduction ; therefore , ivabradine 's use dependence is not protective against marked bradycardia . moreover , our findings provide a better understanding of hcn4 's physiology in humans , which could be applied to elucidate diseases associated with perturbed hr regulation . excess hcn4 conductivity , for example , could contribute to postural tachycardia syndrome , a condition characterized by sinus tachycardia with standing and a mismatch between cardiac and vascular sympathetic activation.38 , 39 indeed , hcn4 inhibition may improve symptoms of postural tachycardia syndrome.40 although gainoffunction mutations in the hcn4 gene have not been discovered , functional hcn4 gain of function has been described in right atrial samples from patients with chronic atrial fibrillation.41 patients with heart failure or coronary artery disease , who are currently being considered for ivabradine treatment , also show redistribution of sympathetic activity toward the heart with an associated increase in hr.42 , 43 this study was funded by a grant of the german research foundation ( deutsche forschungsgemeinschaft , dfg ) awarded to jordan ( jo 284/81 ) . tank , heusser , and may were funded by a grant of the german aerospace center ( deutsches zentrum fr luft und raumfahrt , dlr , 50 wb1117 ) .

backgroundsympathetic and parasympathetic influences on heart rate ( hr ) , which are governed by baroreflex mechanisms , are integrated at the cardiac sinus node through hyperpolarizationactivated cyclic nucleotide gated channels ( hcn4 ) . we hypothesized that hcn4 blockade with ivabradine selectively attenuates hr and baroreflex hr regulation , leaving baroreflex control of muscle sympathetic nerve activity intact.methods and resultswe treated 21 healthy men with 27.5 mg ivabradine or placebo in a randomized crossover fashion . we recorded electrocardiogram , blood pressure , and muscle sympathetic nerve activity at rest and during pharmacological baroreflex testing . ivabradine reduced normalized hr from 65.98.1 to 58.46.2 beats per minute ( p<0.001 ) with unaffected blood pressure and muscle sympathetic nerve activity . on ivabradine , cardiac and sympathetic baroreflex gains and blood pressure responses to vasoactive drugs were unchanged . ivabradine aggravated bradycardia during baroreflex loading.conclusionshcn4 blockade with ivabradine reduced hr , leaving physiological regulation of hr and muscle sympathetic nerve activity as well as baroreflex blood pressure buffering intact . ivabradine could aggravate bradycardia during parasympathetic activation.clinical trial registrationurl : http://www.clinicaltrials.gov . unique identifier : nct00865917 .

Clinical Trial Registration Introduction Methods Participants Study Design and Protocol End Points HR Normalization Merged Baroreflex Curves Statistics Results Discussion Conclusion Sources of Funding Disclosures

drugs could interfere with cardiovascular regulation in brain or periphery directly or through secondary baroreflexmediated adjustments in heart rate ( hr ) and vascular tone . hcn4 generates funny pacemaker f currents ( if ) promoting slow diastolic depolarization and cardiac rhythm generation.1 intracellular cyclic adenosine monophosphate ( camp ) modulates if 2 , and adrenergic stimulation augments camp , thereby increasing hcn4 conductivity and hr . conversely , blockade of hcn4 channels on baroreceptor sensory neurons increased their excitability,5 , 6 which would tend to facilitate afferent baroreflex signaling and baroreflex function . the issue is relevant because ivabradine7 , 8 is used in the treatment of heart failure9 and angina pectoris10 ; however , the influences of hcn4 on human cardiovascular autonomic regulation have not been studied , aside from noninvasive baroreflex assessment of profound sympathetic activation.11 ivabradine is an openchannel blocker12 producing inhibition,13 thus ivabradine may be more effective at increased hr.14 we tested the hypothesis that pharmacological hcn4 blockade with ivabradine affects baroreflex hr regulation in healthy participants such that hr is reduced at a given blood pressure ( bp ) . moreover , we assessed whether ivabradine would interfere with baroreflex regulation of hr and sympathetic efferent traffic to resistance vessels and whether or not baroreflex bp buffering is perturbed . healthy men ( aged 1840 years ) with a body mass index between 18 and 30 kg / m and a resting hr 60 beats per minute ( bpm ) were eligible for this study . preexisting diseases were ruled out through a detailed history , medical examination , 12lead ecg , bp recordings , and blood sampling for routine laboratory measurements . this randomized , doubleblind , 2period , 2sequence , crossover study was conducted at 2 sites ( experimental clinical research center , charit medical university , berlin , germany , and institute of clinical pharmacology , hannover medical school , hannover , germany ) . randomization for study drug sequence ( randomization block size : 6 ) and manufacturing and labeling of blinded medications was done centrally by the pharmacy of the charit medical university . consequently , participants and investigators remained fully blinded until the database had been locked , double checked , and transferred to the institute of biostatistics at hannover medical school . on 2 separate occasions , participants ingested maximally recommended doses of ivabradine ( 7.5 mg ) or matching placebo 13 hours and 1 hour before testing . testing was conducted between 8 and 11 am in a quiet laboratory at an ambient temperature of 22 to 23c . recumbent systolic bp ( sbp ) , mean bp , and diastolic arterial bp were measured with an automated oscillometric device ( dinamap ; ge / critikon ) . we obtained blood samples after at least 30 minutes of rest for plasma catecholamine determination with highpressure liquid chromatography and consecutive electrochemical detection.15 stroke volume and cardiac output were obtained using an inert gas rebreathing technique ( innocor ; innovision ) . muscle sympathetic nerve activity ( msna ) was recorded from the right peroneal nerve ( nerve traffic analyzer 662c3 ; biomedical engineering department , university of iowa , iowa city , ia ) , as described previously.16 data were analogtodigital converted and analyzed using a program written by one of the authors ( a.d . ) we determined the following msna parameters from the integrated nerve signal : burst frequency , or the number of msna bursts per minute ; burst incidence , or the number of bursts per 100 heart beats ; and total activity , or the area under the bursts per minute as arbitrary units per minute . following a resting period of at least 30 minutes after taking blood samples and measuring cardiac output , we performed a pharmacological baroreflex assessment using the nitric oxide donor sodium nitroprusside ( vasodilator ) and the 1adrenoceptor agonist phenylephrine ( vasoconstrictor ) . dose incrementation was stopped after the maximum dose of 2.1 g / kg per minute had been reached , sbp had changed by > 25 mm hg , or hr had dropped < 40 bpm . main end points of the study were normalized hr ( ie , hr at a given bp ) and resting msna burst frequency during supine rest . exploratory end points served to further characterize hemodynamic and autonomic responses to ivabradine at rest and during pharmacologic baroreflex challenge : arterial bp , stroke volume , cardiac output , total peripheral resistance , plasma catecholamine levels , cardiac and sympathetic baroreflex characteristics , and vasoactor sensitivity . to remove confounding baroreflex influences on resting hr , we normalized hr before data unblinding ( example in figure 1 ) : we transferred the relationship between rr intervals ( rris ) and corresponding sbp values during pharmacological baroreflex testing into a mathematical function by linear regression or baroreflex curve fitting using a boltzmann sigmoidal function separately for the placebo and ivabradine condition for each participant ( sigmoidal curves in the example ) . the example assumes lower sbp on ivabradine than on placebo ( 118 vs 122 mm hg ) . the solid vertical line represents mean sbp ( 120 mm hg ) , which determines normalized rri values . hr indicates heart rate ; rri , rr interval ; sbp , systolic blood pressure . related bins for all participants , a boltzmann sigmoidal function was fitted to these merged data , and 95% confidence bands were calculated and plotted . with sd of 6.7 bpm in the group 's resting hr paired differences , =0.05 , and 2sided testing , 15 participants would provide 80% statistical power to detect such a difference.17 we included additional participants to allow for the meaningful analysis of secondary and exploratory end points . healthy men ( aged 1840 years ) with a body mass index between 18 and 30 kg / m and a resting hr 60 beats per minute ( bpm ) were eligible for this study . preexisting diseases were ruled out through a detailed history , medical examination , 12lead ecg , bp recordings , and blood sampling for routine laboratory measurements . this randomized , doubleblind , 2period , 2sequence , crossover study was conducted at 2 sites ( experimental clinical research center , charit medical university , berlin , germany , and institute of clinical pharmacology , hannover medical school , hannover , germany ) . consequently , participants and investigators remained fully blinded until the database had been locked , double checked , and transferred to the institute of biostatistics at hannover medical school . on 2 separate occasions , participants ingested maximally recommended doses of ivabradine ( 7.5 mg ) or matching placebo 13 hours and 1 hour before testing . testing was conducted between 8 and 11 am in a quiet laboratory at an ambient temperature of 22 to 23c . recumbent systolic bp ( sbp ) , mean bp , and diastolic arterial bp were measured with an automated oscillometric device ( dinamap ; ge / critikon ) . muscle sympathetic nerve activity ( msna ) was recorded from the right peroneal nerve ( nerve traffic analyzer 662c3 ; biomedical engineering department , university of iowa , iowa city , ia ) , as described previously.16 data were analogtodigital converted and analyzed using a program written by one of the authors ( a.d . ) we determined the following msna parameters from the integrated nerve signal : burst frequency , or the number of msna bursts per minute ; burst incidence , or the number of bursts per 100 heart beats ; and total activity , or the area under the bursts per minute as arbitrary units per minute . after taking blood samples and measuring cardiac output , we performed a pharmacological baroreflex assessment using the nitric oxide donor sodium nitroprusside ( vasodilator ) and the 1adrenoceptor agonist phenylephrine ( vasoconstrictor ) . dose incrementation was stopped after the maximum dose of 2.1 g / kg per minute had been reached , sbp had changed by > 25 mm hg , or hr had dropped < 40 bpm . main end points of the study were normalized hr ( ie , hr at a given bp ) and resting msna burst frequency during supine rest . exploratory end points served to further characterize hemodynamic and autonomic responses to ivabradine at rest and during pharmacologic baroreflex challenge : arterial bp , stroke volume , cardiac output , total peripheral resistance , plasma catecholamine levels , cardiac and sympathetic baroreflex characteristics , and vasoactor sensitivity . to remove confounding baroreflex influences on resting hr , we normalized hr before data unblinding ( example in figure 1 ) : we transferred the relationship between rr intervals ( rris ) and corresponding sbp values during pharmacological baroreflex testing into a mathematical function by linear regression or baroreflex curve fitting using a boltzmann sigmoidal function separately for the placebo and ivabradine condition for each participant ( sigmoidal curves in the example ) . the example assumes lower sbp on ivabradine than on placebo ( 118 vs 122 mm hg ) . the solid vertical line represents mean sbp ( 120 mm hg ) , which determines normalized rri values . hr indicates heart rate ; rri , rr interval ; sbp , systolic blood pressure . related bins for all participants using graphpad prism 5 , a boltzmann sigmoidal function was fitted to these merged data , and 95% confidence bands were calculated and plotted . with sd of 6.7 bpm in the group 's resting hr paired differences , =0.05 , and 2sided testing , 15 participants would provide 80% statistical power to detect such a difference.17 we included additional participants to allow for the meaningful analysis of secondary and exploratory end points . in 3 participants , we failed to obtain a stable nerve recording position at the second experimental day . the characteristics of the participants were as follows : age 26.84.2 years , height 1.820.06 m , body mass 79.810.5 kg , body mass index 24.13.0 kg / m , bp 1308/728 mm hg , and hr 67.45.8 bpm . ic indicates informed consent ; iva , ivabradine ; plc , placebo experimental data are shown in table . bp and stroke volume remained unchanged with a trend toward reduced cardiac output ( 7.6% ) and increased total peripheral resistance ( 5.6% ) on ivabradine . ivabradine did not alter pharmacological baroreflex sensitivities , such as the cardiac parasympathetic and vasoconstrictor sympathetic baroreflex gains . if blockade dampens the increase in msna burst frequency during higher sodium nitroprusside infusion rates , namely , when high vasoconstrictor activity and elevated hr cooccur ( figure 6a ) ; however , hrindependent msna parameters ( ie , msna burst incidence and total activity ) were not altered by hcn4 blockade during pharmacological baroreflex testing ( data not shown ) . upward shift of the cardiac baroreflex curve to longer rris ( figure 6b ) . consequently , rri lengthening by ivabradine was virtually independent of the prevailing hr during pharmacological baroreflex testing . hemodynamic , sympathetic , hormonal , and baroreflex parameters au indicates arbitrary units ; brg , baroreflex gain ( ie , baroreflex sensitivity ) ; co , cardiac output ; dbp , diastolic blood pressure ; hr , normalized ( baroreflexcorrected ) heart rate ; hrp , normalized heart rate during parasympathetic activation ; mbp , mean blood pressure ; msna , muscle sympathetic nerve activity ; ntp , sodium nitroprusside ; phe , phenylephrine ; sbp , systolic blood pressure ; sv , stroke volume ; tpr , total peripheral resistance . with resting values of 56.5 bpm , ivabradine has no effect ( unresponsive hr ) . c , effects of hcn4 inhibition on hr during parasympathetic activation ( hrp ) through baroreflex loading with phenylephrine . hrp was assessed as hr at the largest individual phenylephrine dose that was reached on both study days . d , participants with lower hrp on placebo exhibited a smaller reduction in hrp with ivabradine . the observation is consistent with ivabradine 's usedependent pharmacology ; however , parasympathetic activation shifted the unresponsive hr to 44.2 bpm , which is 12 bpm lower compared with resting conditions ( see panel b ) . b , individual changes in msna burst frequency , burst incidence , and total msna by ivabradine compared with placebo . au indicates arbitrary units ; hb , heart beats ; msna , muscle sympathetic nerve activity . note the trend toward improved baroreflex buffering of bp increases on ivabradine ( upper panel ) . a , sympathetic baroreflex : although ivabradine may dampen the increase in msna burst frequency during nitroprusside infusion ( ie , when high vasoconstrictor activity and elevated hr cooccur ) , baroreflex curves are virtually identical if hrindependent msna measures ( burst incidence and total activity ) are used ( curves not shown ) . b , cardiac baroreflex : ivabradine shifts ( offsets ) the baroreflex curve upward ( to longer rris ) with virtually no effect on slope or range . note that the bands do not overlap with high blood pressure , indicating that baroreflexmediated hr slowing is intensified by ivabradine . dbp indicates diastolic blood pressure ; hr , heart rate ; msna , muscle sympathetic nerve activity ; rri , rr interval ; sbp , systolic blood pressure . participants with higher resting hr on placebo exhibited more pronounced hr slowing on ivabradine , showing use dependence we observed a similar pattern during baroreflex loading with phenylephrine ( figure 3c and 3d ) ; however , the hr at which ivabradine did not slow hr further was only 44.2 bpm ( ie , 12 bpm lower during parasympathetic activation ) . together , figure 3b and 3d visualize ivabradine 's usedependent characteristics ; however , they also show , as does figure 6b , that ivabradine and parasympathetic activation are additive in terms of hr reduction . we observed that hcn4 blockade with ivabradine reduces hr , leaving baroreflex hr and msna regulation intact . although hcn4 is involved in transducing autonomic activity at the sinus node level , baroreflex hr regulation was preserved on ivabradine . the cardiac baroreflex curve was parallelshifted toward lower hr without reductions in baroreflex gain or range . similarly , baroreflex hr slopes determined noninvasively by crossspectral analysis or the sequence method were unchanged with ivabradine in a human model of cardiac sympathetic activation.11 because msna discharges occur during diastole when arterial baroreceptors are unloaded , we expected reduced msna frequencies with compensatory increases in sympathetic action potentials within bursts ; however , ivabradineinduced hr reduction was not associated with resting msna changes . moreover , sympathetic baroreflex regulation was virtually identical on placebo and on ivabradine considering hrindependent msna measurements . finally , bp responses to vasoactive medications did not increase on ivabradine , suggesting that baroreflex bp buffering remained fully functional.19 together with previous studies in genetic animal models and in patients with familial sinus bradycardia due to hcn4 mutations,3 , 20 , 21 , 22 , 23 our study provides new insight into hcn4 's role in hr regulation . likewise , hr acceleration to adrenoreceptor stimulation was intact in adult hcn4deleted mice24 and in a heterozygous knockin model rendering hcn4 camp insensitive.4 on ivabradine , chronotropic competence in terms of hr acceleration and deceleration over a wide hr range assessed through baroreflex loading and unloading was unchanged . consequently , if can not be the crucial mechanism transducing autonomic signals at the level of the sinus node . instead , hcn4 may defend the sinus node against excess hyperpolarization,27 preventing bradycardia . despite its usedependent pharmacology,12 indicated by larger hr reduction in persons with higher resting hr , ivabradine aggravated baroreflexmediated bradycardia in our study . we did not investigate responses to ivabradine under steady state conditions ; however , we showed earlier that acute dosing 13 hours and 1 hour before measurements was sufficient to attain ivabradine plasma concentrations in a therapeutic range.11 furthermore , the drug 's bradycardic activity consists of an initial rapid and subsequent relatively long duration of action caused by the ndealkylated metabolite and the parent drug , respectively.30 on the other hand , the 3week washout period was more than twice the time required to avoid carryover , according to the kinetics of the bradycardic effect with repeated oral administration.30 ivabradine 's pharmacological interaction with hcn4 channels complicates the interpretation of our study . finally , given the potential influence of respiration on postganglionic sympathetic discharge , it would have been desirable to also record and analyze breathing , yet others did not observe effects of ivabradine on respiratory rate either in healthy men36 or in patients with chronic obstructive pulmonary disease.37 highfidelity physiological phenotyping provides pharmacological insight that could not be gained by routine hr and bp measurements . we observed that hcn4 blockade with ivabradine reduced hr but did not impair sympathetic or parasympathetic baroreflex function . moreover , our findings provide a better understanding of hcn4 's physiology in humans , which could be applied to elucidate diseases associated with perturbed hr regulation . excess hcn4 conductivity , for example , could contribute to postural tachycardia syndrome , a condition characterized by sinus tachycardia with standing and a mismatch between cardiac and vascular sympathetic activation.38 , 39 indeed , hcn4 inhibition may improve symptoms of postural tachycardia syndrome.40 although gainoffunction mutations in the hcn4 gene have not been discovered , functional hcn4 gain of function has been described in right atrial samples from patients with chronic atrial fibrillation.41 patients with heart failure or coronary artery disease , who are currently being considered for ivabradine treatment , also show redistribution of sympathetic activity toward the heart with an associated increase in hr.42 , 43 this study was funded by a grant of the german research foundation ( deutsche forschungsgemeinschaft , dfg ) awarded to jordan ( jo 284/81 ) . tank , heusser , and may were funded by a grant of the german aerospace center ( deutsches zentrum fr luft und raumfahrt , dlr , 50 wb1117 ) .

medication effects on human cardiovascular regulation are difficult to predict based on preclinical and clinical investigations . drugs could interfere with cardiovascular regulation in brain or periphery directly or through secondary baroreflexmediated adjustments in heart rate ( hr ) and vascular tone . hcn4 generates funny pacemaker f currents ( if ) promoting slow diastolic depolarization and cardiac rhythm generation.1 intracellular cyclic adenosine monophosphate ( camp ) modulates if 2 , and adrenergic stimulation augments camp , thereby increasing hcn4 conductivity and hr . given hcn4 's important role in autonomic hr adjustments , pharmacological hcn4 inhibition could profoundly affect human cardiovascular regulation . conversely , blockade of hcn4 channels on baroreceptor sensory neurons increased their excitability,5 , 6 which would tend to facilitate afferent baroreflex signaling and baroreflex function . the issue is relevant because ivabradine7 , 8 is used in the treatment of heart failure9 and angina pectoris10 ; however , the influences of hcn4 on human cardiovascular autonomic regulation have not been studied , aside from noninvasive baroreflex assessment of profound sympathetic activation.11 ivabradine is an openchannel blocker12 producing inhibition,13 thus ivabradine may be more effective at increased hr.14 we tested the hypothesis that pharmacological hcn4 blockade with ivabradine affects baroreflex hr regulation in healthy participants such that hr is reduced at a given blood pressure ( bp ) . moreover , we assessed whether ivabradine would interfere with baroreflex regulation of hr and sympathetic efferent traffic to resistance vessels and whether or not baroreflex bp buffering is perturbed . finally , we evaluated whether or not in vivo responses mirror ivabradine 's usedependent pharmacology . healthy men ( aged 1840 years ) with a body mass index between 18 and 30 kg / m and a resting hr 60 beats per minute ( bpm ) were eligible for this study . this randomized , doubleblind , 2period , 2sequence , crossover study was conducted at 2 sites ( experimental clinical research center , charit medical university , berlin , germany , and institute of clinical pharmacology , hannover medical school , hannover , germany ) . recumbent systolic bp ( sbp ) , mean bp , and diastolic arterial bp were measured with an automated oscillometric device ( dinamap ; ge / critikon ) . we obtained blood samples after at least 30 minutes of rest for plasma catecholamine determination with highpressure liquid chromatography and consecutive electrochemical detection.15 stroke volume and cardiac output were obtained using an inert gas rebreathing technique ( innocor ; innovision ) . muscle sympathetic nerve activity ( msna ) was recorded from the right peroneal nerve ( nerve traffic analyzer 662c3 ; biomedical engineering department , university of iowa , iowa city , ia ) , as described previously.16 data were analogtodigital converted and analyzed using a program written by one of the authors ( a.d . ) we determined the following msna parameters from the integrated nerve signal : burst frequency , or the number of msna bursts per minute ; burst incidence , or the number of bursts per 100 heart beats ; and total activity , or the area under the bursts per minute as arbitrary units per minute . following a resting period of at least 30 minutes after taking blood samples and measuring cardiac output , we performed a pharmacological baroreflex assessment using the nitric oxide donor sodium nitroprusside ( vasodilator ) and the 1adrenoceptor agonist phenylephrine ( vasoconstrictor ) . dose incrementation was stopped after the maximum dose of 2.1 g / kg per minute had been reached , sbp had changed by > 25 mm hg , or hr had dropped < 40 bpm . main end points of the study were normalized hr ( ie , hr at a given bp ) and resting msna burst frequency during supine rest . exploratory end points served to further characterize hemodynamic and autonomic responses to ivabradine at rest and during pharmacologic baroreflex challenge : arterial bp , stroke volume , cardiac output , total peripheral resistance , plasma catecholamine levels , cardiac and sympathetic baroreflex characteristics , and vasoactor sensitivity . the latter was defined as sbp change per dose increment of sodium nitroprusside and phenylephrine , respectively . to remove confounding baroreflex influences on resting hr , we normalized hr before data unblinding ( example in figure 1 ) : we transferred the relationship between rr intervals ( rris ) and corresponding sbp values during pharmacological baroreflex testing into a mathematical function by linear regression or baroreflex curve fitting using a boltzmann sigmoidal function separately for the placebo and ivabradine condition for each participant ( sigmoidal curves in the example ) . resting sbp of both visits ( vertical dotted lines in the figure 1 ) were averaged . this standard pressure was used as input ( abscissa value ) to the baroreflex curve function to calculate normalized rri and hr for each visit separately ( ordinate values ) . without normalization , ivabradine 's effect on hr would have been underestimated , as demonstrated by the smaller difference between the 2 raw rri values compared with the normalized rri values . baroreflexmediated changes in the dependent parameters ( msna , rri ) were collected in 10mm hg bins separately for placebo and ivabradine . with sd of 6.7 bpm in the group 's resting hr paired differences , =0.05 , and 2sided testing , 15 participants would provide 80% statistical power to detect such a difference.17 we included additional participants to allow for the meaningful analysis of secondary and exploratory end points . according to a prospective data analysis plan , primary and secondary end points were analyzed using the hillsarmitage approach.18 the method allows periodadjusted therapy effect estimation , namely , calculation of periodadjusted p values for mean differences between placebo and ivabradine data . to appreciate consistency of the results , several sensitivity analyses were performed ( univariate 1sample t tests and mixedmodel analysis with participant as random effect ) . healthy men ( aged 1840 years ) with a body mass index between 18 and 30 kg / m and a resting hr 60 beats per minute ( bpm ) were eligible for this study . this randomized , doubleblind , 2period , 2sequence , crossover study was conducted at 2 sites ( experimental clinical research center , charit medical university , berlin , germany , and institute of clinical pharmacology , hannover medical school , hannover , germany ) . recumbent systolic bp ( sbp ) , mean bp , and diastolic arterial bp were measured with an automated oscillometric device ( dinamap ; ge / critikon ) . we obtained blood samples after at least 30 minutes of rest for plasma catecholamine determination with highpressure liquid chromatography and consecutive electrochemical detection.15 stroke volume and cardiac output were obtained using an inert gas rebreathing technique ( innocor ; innovision ) . muscle sympathetic nerve activity ( msna ) was recorded from the right peroneal nerve ( nerve traffic analyzer 662c3 ; biomedical engineering department , university of iowa , iowa city , ia ) , as described previously.16 data were analogtodigital converted and analyzed using a program written by one of the authors ( a.d . ) we determined the following msna parameters from the integrated nerve signal : burst frequency , or the number of msna bursts per minute ; burst incidence , or the number of bursts per 100 heart beats ; and total activity , or the area under the bursts per minute as arbitrary units per minute . following a resting period of at least 30 minutes , we obtained resting baseline recordings in the supine position . after taking blood samples and measuring cardiac output , we performed a pharmacological baroreflex assessment using the nitric oxide donor sodium nitroprusside ( vasodilator ) and the 1adrenoceptor agonist phenylephrine ( vasoconstrictor ) . dose incrementation was stopped after the maximum dose of 2.1 g / kg per minute had been reached , sbp had changed by > 25 mm hg , or hr had dropped < 40 bpm . main end points of the study were normalized hr ( ie , hr at a given bp ) and resting msna burst frequency during supine rest . exploratory end points served to further characterize hemodynamic and autonomic responses to ivabradine at rest and during pharmacologic baroreflex challenge : arterial bp , stroke volume , cardiac output , total peripheral resistance , plasma catecholamine levels , cardiac and sympathetic baroreflex characteristics , and vasoactor sensitivity . to remove confounding baroreflex influences on resting hr , we normalized hr before data unblinding ( example in figure 1 ) : we transferred the relationship between rr intervals ( rris ) and corresponding sbp values during pharmacological baroreflex testing into a mathematical function by linear regression or baroreflex curve fitting using a boltzmann sigmoidal function separately for the placebo and ivabradine condition for each participant ( sigmoidal curves in the example ) . resting sbp of both visits ( vertical dotted lines in the figure 1 ) were averaged . without normalization , ivabradine 's effect on hr would have been underestimated , as demonstrated by the smaller difference between the 2 raw rri values compared with the normalized rri values . baroreflexmediated changes in the dependent parameters ( msna , rri ) were collected in 10mm hg bins separately for placebo and ivabradine . with sd of 6.7 bpm in the group 's resting hr paired differences , =0.05 , and 2sided testing , 15 participants would provide 80% statistical power to detect such a difference.17 we included additional participants to allow for the meaningful analysis of secondary and exploratory end points . according to a prospective data analysis plan , primary and secondary end points were analyzed using the hillsarmitage approach.18 the method allows periodadjusted therapy effect estimation , namely , calculation of periodadjusted p values for mean differences between placebo and ivabradine data . to appreciate consistency of the results , several sensitivity analyses were performed ( univariate 1sample t tests and mixedmodel analysis with participant as random effect ) . the characteristics of the participants were as follows : age 26.84.2 years , height 1.820.06 m , body mass 79.810.5 kg , body mass index 24.13.0 kg / m , bp 1308/728 mm hg , and hr 67.45.8 bpm . if blockade reduced resting hr with and without normalization for prevailing bp ( figure 3a ) ( for hr normalization , see methods ) . bp and stroke volume remained unchanged with a trend toward reduced cardiac output ( 7.6% ) and increased total peripheral resistance ( 5.6% ) on ivabradine . sbp sensitivity to sodium nitroprusside infusion was similar on both study days with a trend toward reduced sensitivity ( improved bp buffering ) to the vasoconstrictor phenylephrine with if blockade ( figure 5 ) . if blockade dampens the increase in msna burst frequency during higher sodium nitroprusside infusion rates , namely , when high vasoconstrictor activity and elevated hr cooccur ( figure 6a ) ; however , hrindependent msna parameters ( ie , msna burst incidence and total activity ) were not altered by hcn4 blockade during pharmacological baroreflex testing ( data not shown ) . upward shift of the cardiac baroreflex curve to longer rris ( figure 6b ) . consequently , rri lengthening by ivabradine was virtually independent of the prevailing hr during pharmacological baroreflex testing . hemodynamic , sympathetic , hormonal , and baroreflex parameters au indicates arbitrary units ; brg , baroreflex gain ( ie , baroreflex sensitivity ) ; co , cardiac output ; dbp , diastolic blood pressure ; hr , normalized ( baroreflexcorrected ) heart rate ; hrp , normalized heart rate during parasympathetic activation ; mbp , mean blood pressure ; msna , muscle sympathetic nerve activity ; ntp , sodium nitroprusside ; phe , phenylephrine ; sbp , systolic blood pressure ; sv , stroke volume ; tpr , total peripheral resistance . b , ivabradine 's use dependence : hr lowering is more pronounced in participants with higher resting hr . c , effects of hcn4 inhibition on hr during parasympathetic activation ( hrp ) through baroreflex loading with phenylephrine . the observation is consistent with ivabradine 's usedependent pharmacology ; however , parasympathetic activation shifted the unresponsive hr to 44.2 bpm , which is 12 bpm lower compared with resting conditions ( see panel b ) . b , individual changes in msna burst frequency , burst incidence , and total msna by ivabradine compared with placebo . effects of hcn4 inhibition on bp sensitivity to infusion of the vasoconstrictor phenylephrine ( upper panel ) and the vasodilator sodium nitroprusside ( lower panel ) during placebo ( open circles ) and ivabradine ( closed circles ) . a , sympathetic baroreflex : although ivabradine may dampen the increase in msna burst frequency during nitroprusside infusion ( ie , when high vasoconstrictor activity and elevated hr cooccur ) , baroreflex curves are virtually identical if hrindependent msna measures ( burst incidence and total activity ) are used ( curves not shown ) . b , cardiac baroreflex : ivabradine shifts ( offsets ) the baroreflex curve upward ( to longer rris ) with virtually no effect on slope or range . dbp indicates diastolic blood pressure ; hr , heart rate ; msna , muscle sympathetic nerve activity ; rri , rr interval ; sbp , systolic blood pressure . participants with higher resting hr on placebo exhibited more pronounced hr slowing on ivabradine , showing use dependence we observed a similar pattern during baroreflex loading with phenylephrine ( figure 3c and 3d ) ; however , the hr at which ivabradine did not slow hr further was only 44.2 bpm ( ie , 12 bpm lower during parasympathetic activation ) . together , figure 3b and 3d visualize ivabradine 's usedependent characteristics ; however , they also show , as does figure 6b , that ivabradine and parasympathetic activation are additive in terms of hr reduction . we observed that hcn4 blockade with ivabradine reduces hr , leaving baroreflex hr and msna regulation intact . although hcn4 is involved in transducing autonomic activity at the sinus node level , baroreflex hr regulation was preserved on ivabradine . similarly , baroreflex hr slopes determined noninvasively by crossspectral analysis or the sequence method were unchanged with ivabradine in a human model of cardiac sympathetic activation.11 because msna discharges occur during diastole when arterial baroreceptors are unloaded , we expected reduced msna frequencies with compensatory increases in sympathetic action potentials within bursts ; however , ivabradineinduced hr reduction was not associated with resting msna changes . finally , bp responses to vasoactive medications did not increase on ivabradine , suggesting that baroreflex bp buffering remained fully functional.19 together with previous studies in genetic animal models and in patients with familial sinus bradycardia due to hcn4 mutations,3 , 20 , 21 , 22 , 23 our study provides new insight into hcn4 's role in hr regulation . likewise , hr acceleration to adrenoreceptor stimulation was intact in adult hcn4deleted mice24 and in a heterozygous knockin model rendering hcn4 camp insensitive.4 on ivabradine , chronotropic competence in terms of hr acceleration and deceleration over a wide hr range assessed through baroreflex loading and unloading was unchanged . bradycardia and atrial fibrillation occurred more often in the ivabradine group.28 together with our mechanismoriented study , these observations translate the preclinical concept that if is a defense mechanism against excess bradycardia to humans . we did not investigate responses to ivabradine under steady state conditions ; however , we showed earlier that acute dosing 13 hours and 1 hour before measurements was sufficient to attain ivabradine plasma concentrations in a therapeutic range.11 furthermore , the drug 's bradycardic activity consists of an initial rapid and subsequent relatively long duration of action caused by the ndealkylated metabolite and the parent drug , respectively.30 on the other hand , the 3week washout period was more than twice the time required to avoid carryover , according to the kinetics of the bradycardic effect with repeated oral administration.30 ivabradine 's pharmacological interaction with hcn4 channels complicates the interpretation of our study . as an openchannel blocker , for example , ivabradine features use dependence with trapping of the drug in the closed hcn4 channel.8 furthermore , hcn4 is also expressed in the nervous system , particularly in the thalamus,31 in spinal interneurons,32 and in baroreflex afferents.5 , 6 msna is a sensitive readout for changes in central autonomic regulation.33 , 34 , 35 because msna did not change , confounding effects by neural hcn4 inhibition is unlikely ; however , diseases could alter the ivabradine response . blunted baroreflex function in type 1 diabetic rats , for example , was associated with increased hcn expression and camp sensitivity.6 our findings in healthy participants can not be simply extrapolated to patients . finally , given the potential influence of respiration on postganglionic sympathetic discharge , it would have been desirable to also record and analyze breathing , yet others did not observe effects of ivabradine on respiratory rate either in healthy men36 or in patients with chronic obstructive pulmonary disease.37 highfidelity physiological phenotyping provides pharmacological insight that could not be gained by routine hr and bp measurements . excess hcn4 conductivity , for example , could contribute to postural tachycardia syndrome , a condition characterized by sinus tachycardia with standing and a mismatch between cardiac and vascular sympathetic activation.38 , 39 indeed , hcn4 inhibition may improve symptoms of postural tachycardia syndrome.40 although gainoffunction mutations in the hcn4 gene have not been discovered , functional hcn4 gain of function has been described in right atrial samples from patients with chronic atrial fibrillation.41 patients with heart failure or coronary artery disease , who are currently being considered for ivabradine treatment , also show redistribution of sympathetic activity toward the heart with an associated increase in hr.42 , 43 this study was funded by a grant of the german research foundation ( deutsche forschungsgemeinschaft , dfg ) awarded to jordan ( jo 284/81 ) .

ncbi has maintained the molecular modeling database ( mmdb ) ( 1 ) since 1996 , as a collection of publicly accessible experimentally determined macromolecular structures that have been deposited with the protein data bank ( pdb ) ( 2 ) . it facilitates searching for macromolecular structure data in ncbi s entrez query and retrieval system ( 3 ) ; links and associates macromolecular structure data with a variety of other resources such as gene , sequence , sequence variation , chemistry and literature databases ; provides sequence data for ncbi s blast ( 4 ) services ; and supports other ncbi resources such as the conserved domain database ( cdd ) ( 5 ) and ibis ( 6 ) . recently , mmdb has updated the default presentation of macromolecular structure data so that the biologically relevant macromolecular complexes ( termed biological units or biological assemblies ) , as defined by the structures authors or computed by the pdb ( 7 ) , are shown by default , and so that interactions between macromolecules or macromolecules and smaller chemical ligands are emphasized ( 1 ) . this presentation makes it easier to identify pairs of molecules that come into direct contact with each other in a macromolecular complex something that can be difficult to determine , for example , when visualizing large biological assemblies using 3d graphics software . another feature of mmdb is the association of 3d structures , through sequence similarity searches , with protein sequences that do not yet have solved structures , facilitating inference of protein function . a blast search of all sequences in the entrez protein database , against the subset of protein sequences from experimentally determined structures , maps a large fraction of the publicly available proteins to three - dimensional structure information in mmdb . for example , of the 35 138 human protein sequences tracked in ncbi s bioproject 178 030 , a genomic sequence data set from a human hydatidiform mole cell line , 76% appear similar to known 3d structures using standard protein - blast . an even larger fraction can be mapped to 3d structure using approaches with higher sensitivity , such as the identification of conserved domain signatures . thus , macromolecular structure data as provided by mmdb can be used to postulate homology - inferred function for a large number of functionally uncharacterized protein sequences and genes . what may be less well - known and used are structure neighboring data and a structure neighboring service available as part of mmdb , which identify similarly - shaped structures based on geometric criteria , regardless of the extent of sequence similarity . the resulting 3d structure alignments are helpful in understanding the functional consequences of sequence variation , as well as in discovering distant homologous relationships and subtle functional similarities . the vector alignment search tool ( vast ) ( 8) algorithm , which computes these similarities , was developed around 1995 and has been applied ever since to compute and maintain comprehensive and up - to - date lists of statistically significant similarities between known protein 3d structures . pre - computed similarities and alignments derived from structure superposition are available for all protein structures that have been included in mmdb and are suitable to be processed by vast . an interactive search tool , vast - search , facilitates structure similarity searches for protein structure queries that are not ( yet ) part of mmdb s collection , enabling a user to enter 3d coordinate data for comparison against all publicly available structures . in the past 25 years , a variety of methods have been developed to computationally characterize or measure structural similarities between macromolecules , resulting in an even larger variety of published methods , too numerous to be listed here ( 9 ) . the protein data bank , for example , reports structure neighbors from a subset of representatives computed with the jfatcat algorithm ( 10 ) and points to a handful of external resources that provide structural classifications and structure comparisons : scop ( 11 ) , cath ( 12 ) , vast ( 8) , fatcat ( 13 ) , dali ( 14 ) and superfamily ( 15 ) . scop is a hierarchical classification of domain structures that has been maintained by manual intervention and does not rely on computationally determined 3d structure similarity . cath classifies domain structures hierarchically as well , but makes systematic use of the ssap ( 16 ) algorithm to compute similarities on a 3d level . fatcat , a more recent development , uses dynamic programming to string together locally aligned pairs of structural fragments while allowing for a number of twists around pivot points , decomposing the match between two structures into a series of segment pairs that can be superimposed as rigid bodies . the jfatcat implementation used to pre - compute data for the pdb site falls back to reporting a single - segment rigid body superimposition , though . dali was one of the first structure comparison methods that relied solely on geometric criteria and has been available since the mid 1990s . another method that had been incorporated in the protein data bank , ce ( 17 ) , computes rigid body superimpositions for alignments found via combinatorial extension of aligned fragment pairs , as opposed to dynamic programming or monte carlo optimizations . most , if not all of these computational resources and the associated data have been maintained and available / accessible since their inception , although updates of data sets such as pre - computed structure alignments may not have happened frequently , as most structure comparison methods are computationally intensive . the dali database of pre - computed structure alignments , for example , currently reports a most recent update in march 2011 . also , most of the pre - computed structure neighboring data sets and search databases available for live neighboring have been reduced in size to contain representative structures only . pre - computed structure neighbors as found on the pdb web site , for example , have been obtained for representative structures from clusters formed at a threshold of 40% sequence identity , meaning that a structural alignment between an arbitrary pair of similar or related protein structures may not be readily available , which somewhat limits the practical applicability of the data and search implementations . the vast search database and database of pre - computed structure alignments have been maintained as complete and redundant collections since their launch , with automated updates occurring on a weekly basis . this was made possible by implementing a fast heuristic that uses a model for the statistical significance of initial alignments of secondary structure vectors ( which can be computed quickly ) , so that the database searches can avoid costly alignment refinements for the large majority of insignificant and uninteresting similarities . the drawbacks are that a heuristic will miss some potentially interesting similarities . the vast algorithm will not , for example , report similarities between structures deemed to have <3 secondary structure elements . searches for structural similarity can and should be complemented with searches for sequence similarity , as flexibility of molecular structure and limitations of the structure comparison method may preclude the detection of matches between structures of homologous polypeptides . in general , though , structure comparison methods will pick up many subtle similarities that evade detection by sequence comparison strategies , and there is no natural cutoff point for a ranked list of similar structures , unlike in the sequence comparison scenario , where matches to non - homologous gene products are considered accidental and uninformative , for the most part . results computed by the vast algorithm have been compared against other approaches a number of times ( 1719 ) . although there are subtle differences in retrieval sensitivity and alignment accuracy ( 20 ) , it appears fair to state that the large majority of extensive structural similarities , which are indicative of common evolutionary descent and could be used to infer functional similarities , are reported by vast ( and by most if not all of the alternative approaches to detect common substructures ) . as structure similarity search strategies have been developed to also detect distant relationships that might not be evident from sequence analysis , most if not all of the current approaches have been implemented so that they use a single protein molecule or rather a single domain as the unit of comparison . however , the protein data bank is continuing to accumulate structures of larger macromolecular complexes and has started to provide data on what constitutes functionally or biologically relevant macromolecular complexes or biological assemblies ( 1 ) . such assemblies range from simple homo - oligomers to intricate arrangements of many different components , revealing details on specific molecular interactions and on how these might constrain sequence variation . a small number of approaches have been published in the past few years that examine structural similarity of macromolecular complexes ( 21,22 ) . here we present a simple strategy that builds on the existing database of pairwise structure alignments computed by vast and supports the first ( to our knowledge ) comprehensive and regularly updated collection of macromolecular complex similarities . as information characterizing biological assemblies in macromolecular structure data has become available , it seemed that the biological assembly would be a convenient and informative unit of comparison between individual entries in the structure database . if the goal is to list structures most similar to any particular query , one would have to consider that the query itself may contain a macromolecular complex with a given stoichiometry , and that matching complexes with matching stoichiometry might be more informative structure neighbors than , for example , the structures that happen to contain molecules with the strongest local similarity to the query , irrespective of the context . vast+ builds on the existing vast database to generate such a report of structure neighbors . its goal is to find the largest set of pairs of matching macromolecules between two biological assemblies and to characterize that match and compute instructions for a global superimposition that can be used to visualize the structural similarity . for each pair of structures in mmdb , vast+ examines pre - computed structure alignments stored in the vast database that were computed for the full - length protein molecule components of the default biological assemblies . if such pairwise alignments are found , the alignments between individual protein components of the biological assemblies are compared with each other for compatibility , and compatible / matching alignments are clustered into sets of alignments that together constitute a biological assembly match . pairwise alignments are compatible ( i ) if they do not share the same macromolecules , i.e. a protein molecule from one assembly can not be aligned to two molecules from the other assembly at the same time and ( ii ) if they generate similar instructions ( spatial transformation matrices ) for the superpositions of coordinate sets . a simple distance metric can be used to compare transformation matrices and it lends itself to cluster alignment sets efficiently . each set of compatible pairwise alignments can be characterized by ( i ) the number of pairwise matches , i.e. the total number of pairs of protein molecules from the query and subject biological assemblies , that are simultaneously aligned with each other ; ( ii ) the rmsd of the superposition obtained from considering all alignments in the set ; ( iii ) the total length of all pairwise alignments , i.e. the total number of amino acids that are aligned in 3d space ; and ( iv ) percentage of identical residues in the alignments . for each pairwise comparison of two biological assemblies , only the match with the highest number of aligned molecules and the highest number of aligned residues is recorded and reported . currently , 53% of polypeptide - containing structures in mmdb have > 1 polypeptide chain . the histogram plotted in figure 1 breaks down the numbers by oligomer size and indicates that large fractions of the oligomeric assemblies have , in general , structure neighbors that match the entire assemblies . it should be noted that the fractions might be somewhat exaggerated , as exact duplicates of a structure would be counted as biological assembly matches , and no attempt was made to remove redundant structures or classify biological assembly matches as informative versus uninformative . figure 1.this histogram displays the number of structures in mmdb ( blue ) , categorized by the size of the biological assembly . monomers , dimers and higher oligomers up to dodecamers are plotted as separate categories , the 13th category summarizes tridecamers and all higher oligomers . red columns indicate the number of structures in that category that have at least one complete biological assembly match according to vast+ . this histogram displays the number of structures in mmdb ( blue ) , categorized by the size of the biological assembly . monomers , dimers and higher oligomers up to dodecamers are plotted as separate categories , the 13th category summarizes tridecamers and all higher oligomers . red columns indicate the number of structures in that category that have at least one complete biological assembly match according to vast+ . structure neighbors as computed by the vast+ algorithm will be used in the future to provide links to similar structures on entrez / structure document summaries . lists of similar structures are then summarized via a new interactive web service , which can also be used independently of the entrez query and retrieval system and provides tools for sub - setting results , at http://www.ncbi.nlm.nih.gov/structure/vastplus/vastplus.cgi . for a query structure specified by the user , the service lists similar structures , matches that associate each polymer chain of the query with a corresponding polymer chain of some other structure are considered complete and are indicated with full circles in the search results table ; partial matches are indicated with partially filled circles . the default ranking puts matches with the most matched components at the top of the list . not all queries that have similar structures according to vast+ are guaranteed to also have complete matches ( although monomers usually do ) . the search results tables provided by the vast+ web service give a concise summary of the matches and the extent / quality of the similarity . a clickable + symbol opens a panel for a selected match that provides more details and functionality . the 3d structures of superimposed biological assemblies may be visualized using the 3d viewer cn3d ( 23 ) , which has been re - released as a new version 4.3.1 to support the visualization style . currently , cn3d is able to display the structure superposition of the matched biological assemblies and all the protein chains involved , but it can only display one sequence alignment at a time . therefore , the individual protein match table as shown in figure 2 provides separate cn3d launch points for each matched / aligned protein pair . all of these launch points will result in the same 3d image and rendering , but they will differ in the pair of aligned sequences that are chosen as the content of cn3d s sequence / alignment viewer window . pairs of matching molecules are rendered in the same color , with unaligned segments rendered in gray . the default rendering settings , as generated and provided by the vast+ service , can be examined and modified via cn3d s style|annotate menu . figure 2.the vast+ web service generates lists of structures that have 3d similarity to the query . matches are evaluated with biological assemblies as the unit of comparison ( referred to as biological units ) and may summarize simultaneous alignment of several protein molecule pairs . the query structure 3o6f ( 24 ) currently yields 2712 structure neighbors . only the 115 neighbors with a complete biological assembly match have been selected in this example ( via the display filters menu , shown as collapsed in this figure ) . the 115 complete matches have been sorted by rmsd , and the third ranking match has been selected to provide more detail . the tabulated matches are shown with their pdb accession , descriptive text , the number of proteins aligned in the match , the total number of aligned residues , the sequence identity and the rmsd resulting from the simultaneous superimposition of all aligned molecules . in this example , the query 3o6f matches the structure 1j8h with a total of four aligned protein molecules , totaling 768 residues and resulting in a superposition with 2.55 rmsd . 80% of the residues in 3o6f and 1j8h that were spatially aligned by vast are identical . the extended panel characterizing this selected match contains a table that lists pairs of matching / aligned proteins , and it provides schematic depictions of each biological assembly s composition and interactions . the user can mouse - over those schematics to identify individual molecules and their corresponding match in the other structure ( as shown in this example ) . the individual protein match table contains action buttons that provide access to the pairwise sequence alignments as derived from the vast superimposition and launch points for visualization of the structure superimposition with the protein structure viewer cn3d ( 23 ) . button will open a superposition of the complete biological assembly alignment with the 3d view centered on the selected protein molecule and its sequence data featured in the cn3d sequence viewer window . next to the aligned molecules table , an information box lists some stats that characterize the matched biological assembly . figure 3.visualization of structurally matching biological assemblies , as rendered by the visualization tool cn3d . cn3d is a helper application for the web browser , available for windows and os - x platforms . the query structure , pdb accession 3o6f , represents the complex of an autoreactive t - cell receptor ( ms2 - 3c8 , molecules rendered in green and brown ) complexed with a self - peptide derived from myelin basic protein and the multiple sclerosis - associated mhc molecule hla - dr4 ( molecules rendered in magenta and blue ) ( 24 ) . the self - peptide has been fused with the mhc molecule for the experiment , which explains why the query is represented as a biological assembly with only four components ( figure 2 ) , and is rendered in gray , as is the default for all unaligned segments in cn3d visualization sessions launched from vast+ results pages . the left panel shows 3o6f superimposed with the structure neighbor , pdb accession 1j8h ( 25 ) , which contains a complex between hla - dr3 , an influenza hemagglutinin peptide , and a human alpha / beta t - cell receptor . the structures of the two complexes match well , resulting in a superimposition of 768 amino acid residues at 2.6 rmsd . this demonstrates how well the autoreactive t - cell receptor complex mimics complexes that include foreign peptides , and it is thought that this binding mode is responsible for the autoimmune tcr escaping negative selection . the right panel shows the vast+ alignment between 3o6f and the structure of a t - cell receptor from a patient with multiple sclerosis , complexed with a myelin basic protein - derived peptide and an hla - dr2 mhc , pdb accession 1ymm ( 26 ) . the conformations of the two complexes are different although their components are similar , and vast+ does not consider the complete biological assemblies to match . instead , it reports the most extensive sub - structure match , which in this case involves both subunits of the mhc ( molecules rendered in magenta and blue ) . the molecules corresponding to the tcr are rendered in gray color and would not be displayed by default . the unusual conformation of the complex reported in 1ymm is thought to represent an alternative binding mode that helps autoimmune tcrs to escape negative selection . the vast+ web service generates lists of structures that have 3d similarity to the query . matches are evaluated with biological assemblies as the unit of comparison ( referred to as biological units ) and may summarize simultaneous alignment of several protein molecule pairs . the query structure 3o6f ( 24 ) currently yields 2712 structure neighbors . only the 115 neighbors with a complete biological assembly match have been selected in this example ( via the display filters menu , shown as collapsed in this figure ) . the 115 complete matches have been sorted by rmsd , and the third ranking match has been selected to provide more detail . the tabulated matches are shown with their pdb accession , descriptive text , the number of proteins aligned in the match , the total number of aligned residues , the sequence identity and the rmsd resulting from the simultaneous superimposition of all aligned molecules . in this example , the query 1j8h with a total of four aligned protein molecules , totaling 768 residues and resulting in a superposition with 2.55 rmsd . 80% of the residues in 3o6f and 1j8h that were spatially aligned by vast are identical . the extended panel characterizing this selected match contains a table that lists pairs of matching / aligned proteins , and it provides schematic depictions of each biological assembly s composition and interactions . the user can mouse - over those schematics to identify individual molecules and their corresponding match in the other structure ( as shown in this example ) . the individual protein match table contains action buttons that provide access to the pairwise sequence alignments as derived from the vast superimposition and launch points for visualization of the structure superimposition with the protein structure viewer cn3d ( 23 ) . button will open a superposition of the complete biological assembly alignment with the 3d view centered on the selected protein molecule and its sequence data featured in the cn3d sequence viewer window . next to the aligned molecules table , an information box lists some stats that characterize the matched biological assembly . cn3d is a helper application for the web browser , available for windows and os - x platforms . the query structure , pdb accession 3o6f , represents the complex of an autoreactive t - cell receptor ( ms2 - 3c8 , molecules rendered in green and brown ) complexed with a self - peptide derived from myelin basic protein and the multiple sclerosis - associated mhc molecule hla - dr4 ( molecules rendered in magenta and blue ) ( 24 ) . the self - peptide has been fused with the mhc molecule for the experiment , which explains why the query is represented as a biological assembly with only four components ( figure 2 ) , and is rendered in gray , as is the default for all unaligned segments in cn3d visualization sessions launched from vast+ results pages . the left panel shows 3o6f superimposed with the structure neighbor , pdb accession 1j8h ( 25 ) , which contains a complex between hla - dr3 , an influenza hemagglutinin peptide , and a human alpha / beta t - cell receptor . the structures of the two complexes match well , resulting in a superimposition of 768 amino acid residues at 2.6 rmsd . this demonstrates how well the autoreactive t - cell receptor complex mimics complexes that include foreign peptides , and it is thought that this binding mode is responsible for the autoimmune tcr escaping negative selection . the right panel shows the vast+ alignment between 3o6f and the structure of a t - cell receptor from a patient with multiple sclerosis , complexed with a myelin basic protein - derived peptide and an hla - dr2 mhc , pdb accession 1ymm ( 26 ) . the conformations of the two complexes are different although their components are similar , and vast+ does not consider the complete biological assemblies to match . instead , it reports the most extensive sub - structure match , which in this case involves both subunits of the mhc ( molecules rendered in magenta and blue ) . the molecules corresponding to the tcr are rendered in gray color and would not be displayed by default . the unusual conformation of the complex reported in 1ymm is thought to represent an alternative binding mode that helps autoimmune tcrs to escape negative selection . mmdb is updated weekly , following pdb s schedule . with each update , computation of new structure neighbors is completed within a few days , and they are available as structure neighbors computed for biological assemblies via the vast+ service , as well as structure neighbors computed for individual protein chains and domains , via the original vast service . original vast that can be found near the top of the vast+ results page . at this point , the vast - search service , which accepts 3d structure data uploaded in pdb - format , remains unchanged , and presents similar structures in the original vast format ( table 1 ) . table 1.urls for mmdb and vast resourcesmmdbdatabase home pagehttp://www.ncbi.nlm.nih.gov / structuremmdb ftpdata distributionftp://ftp.ncbi.nih.gov / mmdb / vastidentify structurally similar individual protein moleculeshttp://www.ncbi.nlm.nih.gov / structure / vast / vast.shtmlvast+identify structurally similar macromolecular complexeshttp://www.ncbi.nlm.nih.gov / structure / vastplus / vastplus.cgivast searchinput the 3d coordinates of a query structure to search for similar structureshttp://www.ncbi.nlm.nih.gov / structure / vast / vastsearch.htmlcn3dmolecular graphics viewerhttp://www.ncbi.nlm.nih.gov / structure / cn3d / cn3d.shtmlcblastfind 3d structures that are related to a query protein via sequence comparisonhttp://www.ncbi.nlm.nih.gov / structure / cblast / cblast.cgi urls for mmdb and vast resources the current implementation of vast+ , the associated database of pre - computed structure comparison results and the web service represent a first attempt at providing a comprehensive set of structure neighboring information for biological assemblies . several issues may limit the potential applications of the idea , and we intend to address them in future releases of the service and the associated data . currently , vast+ neighboring is restricted to the default biological assembly for each structure , as determined by the content of the structure data and mmdb parsing . although multiple biological assemblies , if present in a structure entry , tend to be closely similar copies of each other , there are exceptions that should be considered explicitly . also , vast+ currently draws on results of vast neighboring as computed for complete protein molecules and ignores a larger set of results obtained for individual domains . this was done intentionally , so as to speed up the computation , and as the first implementation was intended to focus on structural similarities that are both strong and global . we anticipated that most cases , where two entire biological assemblies can be superimposed globally , would break down into individual protein pairs that can also be aligned and superimposed globally , and not just at the level of individual domains . more importantly , vast+ makes no attempt at this point at refining the alignment and superpositions after detecting a match between two biological assemblies . it is conceivable that such refinement would , in many cases , results in somewhat shorter alignments and lower rmsd values and might be useful in emphasizing the conserved contact interface between the components of a molecular complex . furthermore , a strategy for detecting biological assembly matches that considers multiple molecules simultaneously might exhibit higher sensitivity and pick up similarities that can not be found via the 2-tiered approach we have presented here . currently , vast+ ignores non - polypeptide components of macromolecular complexes , but certainly both nucleic acids and chemical ligands , if present , could be matched as well as the protein components . it should be mentioned that no vast+ neighboring data are available for structure database entries that lack assignment of biological assemblies , and currently vast+ skips biological assemblies whose size exceeds a threshold number of protein components the systematic evaluation of all possible multimolecule matches becomes too time - consuming , and will need to be supplemented by a suitable heuristic . intramural research program of the national library of medicine at national institutes of health / dhhs . funding for open access charge : intramural research program of the national library of medicine at the national institutes of health / dhhs .

the computational detection of similarities between protein 3d structures has become an indispensable tool for the detection of homologous relationships , the classification of protein families and functional inference . consequently , numerous algorithms have been developed that facilitate structure comparison , including rapid searches against a steadily growing collection of protein structures . to this end , ncbi s molecular modeling database ( mmdb ) , which is based on the protein data bank ( pdb ) , maintains a comprehensive and up - to - date archive of protein structure similarities computed with the vector alignment search tool ( vast ) . these similarities have been recorded on the level of single proteins and protein domains , comprising in excess of 1.5 billion pairwise alignments . here we present vast+ , an extension to the existing vast service , which summarizes and presents structural similarity on the level of biological assemblies or macromolecular complexes . vast+ simplifies structure neighboring results and shows , for macromolecular complexes tracked in mmdb , lists of similar complexes ranked by the extent of similarity . vast+ replaces the previous vast service as the default presentation of structure neighboring data in ncbi s entrez query and retrieval system . mmdb and vast+ can be accessed via http://www.ncbi.nlm.nih.gov/structure .

INTRODUCTION VAST+ AS AN EXTENSION TO EXISTING PROTEIN STRUCTURE COMPARISON THE VAST+ WEB SERVICE USING CN3D TO VISUALIZE BIOLOGICAL ASSEMBLY ALIGNMENTS SIMILAR SUBSTRUCTURES: ORIGINAL VAST AND VAST-SEARCH LIMITATIONS AND FUTURE WORK FUNDING

ncbi has maintained the molecular modeling database ( mmdb ) ( 1 ) since 1996 , as a collection of publicly accessible experimentally determined macromolecular structures that have been deposited with the protein data bank ( pdb ) ( 2 ) . it facilitates searching for macromolecular structure data in ncbi s entrez query and retrieval system ( 3 ) ; links and associates macromolecular structure data with a variety of other resources such as gene , sequence , sequence variation , chemistry and literature databases ; provides sequence data for ncbi s blast ( 4 ) services ; and supports other ncbi resources such as the conserved domain database ( cdd ) ( 5 ) and ibis ( 6 ) . recently , mmdb has updated the default presentation of macromolecular structure data so that the biologically relevant macromolecular complexes ( termed biological units or biological assemblies ) , as defined by the structures authors or computed by the pdb ( 7 ) , are shown by default , and so that interactions between macromolecules or macromolecules and smaller chemical ligands are emphasized ( 1 ) . this presentation makes it easier to identify pairs of molecules that come into direct contact with each other in a macromolecular complex something that can be difficult to determine , for example , when visualizing large biological assemblies using 3d graphics software . another feature of mmdb is the association of 3d structures , through sequence similarity searches , with protein sequences that do not yet have solved structures , facilitating inference of protein function . a blast search of all sequences in the entrez protein database , against the subset of protein sequences from experimentally determined structures , maps a large fraction of the publicly available proteins to three - dimensional structure information in mmdb . for example , of the 35 138 human protein sequences tracked in ncbi s bioproject 178 030 , a genomic sequence data set from a human hydatidiform mole cell line , 76% appear similar to known 3d structures using standard protein - blast . an even larger fraction can be mapped to 3d structure using approaches with higher sensitivity , such as the identification of conserved domain signatures . thus , macromolecular structure data as provided by mmdb can be used to postulate homology - inferred function for a large number of functionally uncharacterized protein sequences and genes . what may be less well - known and used are structure neighboring data and a structure neighboring service available as part of mmdb , which identify similarly - shaped structures based on geometric criteria , regardless of the extent of sequence similarity . the resulting 3d structure alignments are helpful in understanding the functional consequences of sequence variation , as well as in discovering distant homologous relationships and subtle functional similarities . the vector alignment search tool ( vast ) ( 8) algorithm , which computes these similarities , was developed around 1995 and has been applied ever since to compute and maintain comprehensive and up - to - date lists of statistically significant similarities between known protein 3d structures . pre - computed similarities and alignments derived from structure superposition are available for all protein structures that have been included in mmdb and are suitable to be processed by vast . an interactive search tool , vast - search , facilitates structure similarity searches for protein structure queries that are not ( yet ) part of mmdb s collection , enabling a user to enter 3d coordinate data for comparison against all publicly available structures . in the past 25 years , a variety of methods have been developed to computationally characterize or measure structural similarities between macromolecules , resulting in an even larger variety of published methods , too numerous to be listed here ( 9 ) . the protein data bank , for example , reports structure neighbors from a subset of representatives computed with the jfatcat algorithm ( 10 ) and points to a handful of external resources that provide structural classifications and structure comparisons : scop ( 11 ) , cath ( 12 ) , vast ( 8) , fatcat ( 13 ) , dali ( 14 ) and superfamily ( 15 ) . scop is a hierarchical classification of domain structures that has been maintained by manual intervention and does not rely on computationally determined 3d structure similarity . fatcat , a more recent development , uses dynamic programming to string together locally aligned pairs of structural fragments while allowing for a number of twists around pivot points , decomposing the match between two structures into a series of segment pairs that can be superimposed as rigid bodies . the jfatcat implementation used to pre - compute data for the pdb site falls back to reporting a single - segment rigid body superimposition , though . another method that had been incorporated in the protein data bank , ce ( 17 ) , computes rigid body superimpositions for alignments found via combinatorial extension of aligned fragment pairs , as opposed to dynamic programming or monte carlo optimizations . most , if not all of these computational resources and the associated data have been maintained and available / accessible since their inception , although updates of data sets such as pre - computed structure alignments may not have happened frequently , as most structure comparison methods are computationally intensive . the dali database of pre - computed structure alignments , for example , currently reports a most recent update in march 2011 . also , most of the pre - computed structure neighboring data sets and search databases available for live neighboring have been reduced in size to contain representative structures only . pre - computed structure neighbors as found on the pdb web site , for example , have been obtained for representative structures from clusters formed at a threshold of 40% sequence identity , meaning that a structural alignment between an arbitrary pair of similar or related protein structures may not be readily available , which somewhat limits the practical applicability of the data and search implementations . this was made possible by implementing a fast heuristic that uses a model for the statistical significance of initial alignments of secondary structure vectors ( which can be computed quickly ) , so that the database searches can avoid costly alignment refinements for the large majority of insignificant and uninteresting similarities . the vast algorithm will not , for example , report similarities between structures deemed to have <3 secondary structure elements . searches for structural similarity can and should be complemented with searches for sequence similarity , as flexibility of molecular structure and limitations of the structure comparison method may preclude the detection of matches between structures of homologous polypeptides . in general , though , structure comparison methods will pick up many subtle similarities that evade detection by sequence comparison strategies , and there is no natural cutoff point for a ranked list of similar structures , unlike in the sequence comparison scenario , where matches to non - homologous gene products are considered accidental and uninformative , for the most part . results computed by the vast algorithm have been compared against other approaches a number of times ( 1719 ) . although there are subtle differences in retrieval sensitivity and alignment accuracy ( 20 ) , it appears fair to state that the large majority of extensive structural similarities , which are indicative of common evolutionary descent and could be used to infer functional similarities , are reported by vast ( and by most if not all of the alternative approaches to detect common substructures ) . as structure similarity search strategies have been developed to also detect distant relationships that might not be evident from sequence analysis , most if not all of the current approaches have been implemented so that they use a single protein molecule or rather a single domain as the unit of comparison . however , the protein data bank is continuing to accumulate structures of larger macromolecular complexes and has started to provide data on what constitutes functionally or biologically relevant macromolecular complexes or biological assemblies ( 1 ) . a small number of approaches have been published in the past few years that examine structural similarity of macromolecular complexes ( 21,22 ) . here we present a simple strategy that builds on the existing database of pairwise structure alignments computed by vast and supports the first ( to our knowledge ) comprehensive and regularly updated collection of macromolecular complex similarities . as information characterizing biological assemblies in macromolecular structure data has become available , it seemed that the biological assembly would be a convenient and informative unit of comparison between individual entries in the structure database . if the goal is to list structures most similar to any particular query , one would have to consider that the query itself may contain a macromolecular complex with a given stoichiometry , and that matching complexes with matching stoichiometry might be more informative structure neighbors than , for example , the structures that happen to contain molecules with the strongest local similarity to the query , irrespective of the context . vast+ builds on the existing vast database to generate such a report of structure neighbors . its goal is to find the largest set of pairs of matching macromolecules between two biological assemblies and to characterize that match and compute instructions for a global superimposition that can be used to visualize the structural similarity . for each pair of structures in mmdb , vast+ examines pre - computed structure alignments stored in the vast database that were computed for the full - length protein molecule components of the default biological assemblies . if such pairwise alignments are found , the alignments between individual protein components of the biological assemblies are compared with each other for compatibility , and compatible / matching alignments are clustered into sets of alignments that together constitute a biological assembly match . pairwise alignments are compatible ( i ) if they do not share the same macromolecules , i.e. a simple distance metric can be used to compare transformation matrices and it lends itself to cluster alignment sets efficiently . each set of compatible pairwise alignments can be characterized by ( i ) the number of pairwise matches , i.e. the total number of pairs of protein molecules from the query and subject biological assemblies , that are simultaneously aligned with each other ; ( ii ) the rmsd of the superposition obtained from considering all alignments in the set ; ( iii ) the total length of all pairwise alignments , i.e. for each pairwise comparison of two biological assemblies , only the match with the highest number of aligned molecules and the highest number of aligned residues is recorded and reported . figure 1.this histogram displays the number of structures in mmdb ( blue ) , categorized by the size of the biological assembly . monomers , dimers and higher oligomers up to dodecamers are plotted as separate categories , the 13th category summarizes tridecamers and all higher oligomers . this histogram displays the number of structures in mmdb ( blue ) , categorized by the size of the biological assembly . monomers , dimers and higher oligomers up to dodecamers are plotted as separate categories , the 13th category summarizes tridecamers and all higher oligomers . structure neighbors as computed by the vast+ algorithm will be used in the future to provide links to similar structures on entrez / structure document summaries . lists of similar structures are then summarized via a new interactive web service , which can also be used independently of the entrez query and retrieval system and provides tools for sub - setting results , at http://www.ncbi.nlm.nih.gov/structure/vastplus/vastplus.cgi . for a query structure specified by the user , the service lists similar structures , matches that associate each polymer chain of the query with a corresponding polymer chain of some other structure are considered complete and are indicated with full circles in the search results table ; partial matches are indicated with partially filled circles . the default ranking puts matches with the most matched components at the top of the list . the search results tables provided by the vast+ web service give a concise summary of the matches and the extent / quality of the similarity . the 3d structures of superimposed biological assemblies may be visualized using the 3d viewer cn3d ( 23 ) , which has been re - released as a new version 4.3.1 to support the visualization style . currently , cn3d is able to display the structure superposition of the matched biological assemblies and all the protein chains involved , but it can only display one sequence alignment at a time . therefore , the individual protein match table as shown in figure 2 provides separate cn3d launch points for each matched / aligned protein pair . all of these launch points will result in the same 3d image and rendering , but they will differ in the pair of aligned sequences that are chosen as the content of cn3d s sequence / alignment viewer window . the default rendering settings , as generated and provided by the vast+ service , can be examined and modified via cn3d s style|annotate menu . figure 2.the vast+ web service generates lists of structures that have 3d similarity to the query . matches are evaluated with biological assemblies as the unit of comparison ( referred to as biological units ) and may summarize simultaneous alignment of several protein molecule pairs . only the 115 neighbors with a complete biological assembly match have been selected in this example ( via the display filters menu , shown as collapsed in this figure ) . the 115 complete matches have been sorted by rmsd , and the third ranking match has been selected to provide more detail . in this example , the query 3o6f matches the structure 1j8h with a total of four aligned protein molecules , totaling 768 residues and resulting in a superposition with 2.55 rmsd . the individual protein match table contains action buttons that provide access to the pairwise sequence alignments as derived from the vast superimposition and launch points for visualization of the structure superimposition with the protein structure viewer cn3d ( 23 ) . button will open a superposition of the complete biological assembly alignment with the 3d view centered on the selected protein molecule and its sequence data featured in the cn3d sequence viewer window . next to the aligned molecules table , an information box lists some stats that characterize the matched biological assembly . figure 3.visualization of structurally matching biological assemblies , as rendered by the visualization tool cn3d . the self - peptide has been fused with the mhc molecule for the experiment , which explains why the query is represented as a biological assembly with only four components ( figure 2 ) , and is rendered in gray , as is the default for all unaligned segments in cn3d visualization sessions launched from vast+ results pages . the left panel shows 3o6f superimposed with the structure neighbor , pdb accession 1j8h ( 25 ) , which contains a complex between hla - dr3 , an influenza hemagglutinin peptide , and a human alpha / beta t - cell receptor . this demonstrates how well the autoreactive t - cell receptor complex mimics complexes that include foreign peptides , and it is thought that this binding mode is responsible for the autoimmune tcr escaping negative selection . the conformations of the two complexes are different although their components are similar , and vast+ does not consider the complete biological assemblies to match . the vast+ web service generates lists of structures that have 3d similarity to the query . matches are evaluated with biological assemblies as the unit of comparison ( referred to as biological units ) and may summarize simultaneous alignment of several protein molecule pairs . the 115 complete matches have been sorted by rmsd , and the third ranking match has been selected to provide more detail . the tabulated matches are shown with their pdb accession , descriptive text , the number of proteins aligned in the match , the total number of aligned residues , the sequence identity and the rmsd resulting from the simultaneous superimposition of all aligned molecules . the individual protein match table contains action buttons that provide access to the pairwise sequence alignments as derived from the vast superimposition and launch points for visualization of the structure superimposition with the protein structure viewer cn3d ( 23 ) . button will open a superposition of the complete biological assembly alignment with the 3d view centered on the selected protein molecule and its sequence data featured in the cn3d sequence viewer window . next to the aligned molecules table , an information box lists some stats that characterize the matched biological assembly . the self - peptide has been fused with the mhc molecule for the experiment , which explains why the query is represented as a biological assembly with only four components ( figure 2 ) , and is rendered in gray , as is the default for all unaligned segments in cn3d visualization sessions launched from vast+ results pages . the left panel shows 3o6f superimposed with the structure neighbor , pdb accession 1j8h ( 25 ) , which contains a complex between hla - dr3 , an influenza hemagglutinin peptide , and a human alpha / beta t - cell receptor . this demonstrates how well the autoreactive t - cell receptor complex mimics complexes that include foreign peptides , and it is thought that this binding mode is responsible for the autoimmune tcr escaping negative selection . the conformations of the two complexes are different although their components are similar , and vast+ does not consider the complete biological assemblies to match . with each update , computation of new structure neighbors is completed within a few days , and they are available as structure neighbors computed for biological assemblies via the vast+ service , as well as structure neighbors computed for individual protein chains and domains , via the original vast service . at this point , the vast - search service , which accepts 3d structure data uploaded in pdb - format , remains unchanged , and presents similar structures in the original vast format ( table 1 ) . table 1.urls for mmdb and vast resourcesmmdbdatabase home pagehttp://www.ncbi.nlm.nih.gov / structuremmdb ftpdata distributionftp://ftp.ncbi.nih.gov / mmdb / vastidentify structurally similar individual protein moleculeshttp://www.ncbi.nlm.nih.gov / structure / vast / vast.shtmlvast+identify structurally similar macromolecular complexeshttp://www.ncbi.nlm.nih.gov / structure / vastplus / vastplus.cgivast searchinput the 3d coordinates of a query structure to search for similar structureshttp://www.ncbi.nlm.nih.gov / structure / vast / vastsearch.htmlcn3dmolecular graphics viewerhttp://www.ncbi.nlm.nih.gov / structure / cn3d / cn3d.shtmlcblastfind 3d structures that are related to a query protein via sequence comparisonhttp://www.ncbi.nlm.nih.gov / structure / cblast / cblast.cgi urls for mmdb and vast resources the current implementation of vast+ , the associated database of pre - computed structure comparison results and the web service represent a first attempt at providing a comprehensive set of structure neighboring information for biological assemblies . currently , vast+ neighboring is restricted to the default biological assembly for each structure , as determined by the content of the structure data and mmdb parsing . although multiple biological assemblies , if present in a structure entry , tend to be closely similar copies of each other , there are exceptions that should be considered explicitly . this was done intentionally , so as to speed up the computation , and as the first implementation was intended to focus on structural similarities that are both strong and global . we anticipated that most cases , where two entire biological assemblies can be superimposed globally , would break down into individual protein pairs that can also be aligned and superimposed globally , and not just at the level of individual domains . currently , vast+ ignores non - polypeptide components of macromolecular complexes , but certainly both nucleic acids and chemical ligands , if present , could be matched as well as the protein components . it should be mentioned that no vast+ neighboring data are available for structure database entries that lack assignment of biological assemblies , and currently vast+ skips biological assemblies whose size exceeds a threshold number of protein components the systematic evaluation of all possible multimolecule matches becomes too time - consuming , and will need to be supplemented by a suitable heuristic .

ncbi has maintained the molecular modeling database ( mmdb ) ( 1 ) since 1996 , as a collection of publicly accessible experimentally determined macromolecular structures that have been deposited with the protein data bank ( pdb ) ( 2 ) . it facilitates searching for macromolecular structure data in ncbi s entrez query and retrieval system ( 3 ) ; links and associates macromolecular structure data with a variety of other resources such as gene , sequence , sequence variation , chemistry and literature databases ; provides sequence data for ncbi s blast ( 4 ) services ; and supports other ncbi resources such as the conserved domain database ( cdd ) ( 5 ) and ibis ( 6 ) . recently , mmdb has updated the default presentation of macromolecular structure data so that the biologically relevant macromolecular complexes ( termed biological units or biological assemblies ) , as defined by the structures authors or computed by the pdb ( 7 ) , are shown by default , and so that interactions between macromolecules or macromolecules and smaller chemical ligands are emphasized ( 1 ) . this presentation makes it easier to identify pairs of molecules that come into direct contact with each other in a macromolecular complex something that can be difficult to determine , for example , when visualizing large biological assemblies using 3d graphics software . another feature of mmdb is the association of 3d structures , through sequence similarity searches , with protein sequences that do not yet have solved structures , facilitating inference of protein function . a blast search of all sequences in the entrez protein database , against the subset of protein sequences from experimentally determined structures , maps a large fraction of the publicly available proteins to three - dimensional structure information in mmdb . for example , of the 35 138 human protein sequences tracked in ncbi s bioproject 178 030 , a genomic sequence data set from a human hydatidiform mole cell line , 76% appear similar to known 3d structures using standard protein - blast . an even larger fraction can be mapped to 3d structure using approaches with higher sensitivity , such as the identification of conserved domain signatures . thus , macromolecular structure data as provided by mmdb can be used to postulate homology - inferred function for a large number of functionally uncharacterized protein sequences and genes . what may be less well - known and used are structure neighboring data and a structure neighboring service available as part of mmdb , which identify similarly - shaped structures based on geometric criteria , regardless of the extent of sequence similarity . the resulting 3d structure alignments are helpful in understanding the functional consequences of sequence variation , as well as in discovering distant homologous relationships and subtle functional similarities . the vector alignment search tool ( vast ) ( 8) algorithm , which computes these similarities , was developed around 1995 and has been applied ever since to compute and maintain comprehensive and up - to - date lists of statistically significant similarities between known protein 3d structures . an interactive search tool , vast - search , facilitates structure similarity searches for protein structure queries that are not ( yet ) part of mmdb s collection , enabling a user to enter 3d coordinate data for comparison against all publicly available structures . in the past 25 years , a variety of methods have been developed to computationally characterize or measure structural similarities between macromolecules , resulting in an even larger variety of published methods , too numerous to be listed here ( 9 ) . the protein data bank , for example , reports structure neighbors from a subset of representatives computed with the jfatcat algorithm ( 10 ) and points to a handful of external resources that provide structural classifications and structure comparisons : scop ( 11 ) , cath ( 12 ) , vast ( 8) , fatcat ( 13 ) , dali ( 14 ) and superfamily ( 15 ) . scop is a hierarchical classification of domain structures that has been maintained by manual intervention and does not rely on computationally determined 3d structure similarity . cath classifies domain structures hierarchically as well , but makes systematic use of the ssap ( 16 ) algorithm to compute similarities on a 3d level . fatcat , a more recent development , uses dynamic programming to string together locally aligned pairs of structural fragments while allowing for a number of twists around pivot points , decomposing the match between two structures into a series of segment pairs that can be superimposed as rigid bodies . the jfatcat implementation used to pre - compute data for the pdb site falls back to reporting a single - segment rigid body superimposition , though . another method that had been incorporated in the protein data bank , ce ( 17 ) , computes rigid body superimpositions for alignments found via combinatorial extension of aligned fragment pairs , as opposed to dynamic programming or monte carlo optimizations . most , if not all of these computational resources and the associated data have been maintained and available / accessible since their inception , although updates of data sets such as pre - computed structure alignments may not have happened frequently , as most structure comparison methods are computationally intensive . also , most of the pre - computed structure neighboring data sets and search databases available for live neighboring have been reduced in size to contain representative structures only . pre - computed structure neighbors as found on the pdb web site , for example , have been obtained for representative structures from clusters formed at a threshold of 40% sequence identity , meaning that a structural alignment between an arbitrary pair of similar or related protein structures may not be readily available , which somewhat limits the practical applicability of the data and search implementations . this was made possible by implementing a fast heuristic that uses a model for the statistical significance of initial alignments of secondary structure vectors ( which can be computed quickly ) , so that the database searches can avoid costly alignment refinements for the large majority of insignificant and uninteresting similarities . searches for structural similarity can and should be complemented with searches for sequence similarity , as flexibility of molecular structure and limitations of the structure comparison method may preclude the detection of matches between structures of homologous polypeptides . in general , though , structure comparison methods will pick up many subtle similarities that evade detection by sequence comparison strategies , and there is no natural cutoff point for a ranked list of similar structures , unlike in the sequence comparison scenario , where matches to non - homologous gene products are considered accidental and uninformative , for the most part . results computed by the vast algorithm have been compared against other approaches a number of times ( 1719 ) . although there are subtle differences in retrieval sensitivity and alignment accuracy ( 20 ) , it appears fair to state that the large majority of extensive structural similarities , which are indicative of common evolutionary descent and could be used to infer functional similarities , are reported by vast ( and by most if not all of the alternative approaches to detect common substructures ) . as structure similarity search strategies have been developed to also detect distant relationships that might not be evident from sequence analysis , most if not all of the current approaches have been implemented so that they use a single protein molecule or rather a single domain as the unit of comparison . however , the protein data bank is continuing to accumulate structures of larger macromolecular complexes and has started to provide data on what constitutes functionally or biologically relevant macromolecular complexes or biological assemblies ( 1 ) . such assemblies range from simple homo - oligomers to intricate arrangements of many different components , revealing details on specific molecular interactions and on how these might constrain sequence variation . a small number of approaches have been published in the past few years that examine structural similarity of macromolecular complexes ( 21,22 ) . here we present a simple strategy that builds on the existing database of pairwise structure alignments computed by vast and supports the first ( to our knowledge ) comprehensive and regularly updated collection of macromolecular complex similarities . as information characterizing biological assemblies in macromolecular structure data has become available , it seemed that the biological assembly would be a convenient and informative unit of comparison between individual entries in the structure database . if the goal is to list structures most similar to any particular query , one would have to consider that the query itself may contain a macromolecular complex with a given stoichiometry , and that matching complexes with matching stoichiometry might be more informative structure neighbors than , for example , the structures that happen to contain molecules with the strongest local similarity to the query , irrespective of the context . its goal is to find the largest set of pairs of matching macromolecules between two biological assemblies and to characterize that match and compute instructions for a global superimposition that can be used to visualize the structural similarity . for each pair of structures in mmdb , vast+ examines pre - computed structure alignments stored in the vast database that were computed for the full - length protein molecule components of the default biological assemblies . if such pairwise alignments are found , the alignments between individual protein components of the biological assemblies are compared with each other for compatibility , and compatible / matching alignments are clustered into sets of alignments that together constitute a biological assembly match . a protein molecule from one assembly can not be aligned to two molecules from the other assembly at the same time and ( ii ) if they generate similar instructions ( spatial transformation matrices ) for the superpositions of coordinate sets . the total number of pairs of protein molecules from the query and subject biological assemblies , that are simultaneously aligned with each other ; ( ii ) the rmsd of the superposition obtained from considering all alignments in the set ; ( iii ) the total length of all pairwise alignments , i.e. the total number of amino acids that are aligned in 3d space ; and ( iv ) percentage of identical residues in the alignments . for each pairwise comparison of two biological assemblies , only the match with the highest number of aligned molecules and the highest number of aligned residues is recorded and reported . the histogram plotted in figure 1 breaks down the numbers by oligomer size and indicates that large fractions of the oligomeric assemblies have , in general , structure neighbors that match the entire assemblies . figure 1.this histogram displays the number of structures in mmdb ( blue ) , categorized by the size of the biological assembly . this histogram displays the number of structures in mmdb ( blue ) , categorized by the size of the biological assembly . monomers , dimers and higher oligomers up to dodecamers are plotted as separate categories , the 13th category summarizes tridecamers and all higher oligomers . structure neighbors as computed by the vast+ algorithm will be used in the future to provide links to similar structures on entrez / structure document summaries . lists of similar structures are then summarized via a new interactive web service , which can also be used independently of the entrez query and retrieval system and provides tools for sub - setting results , at http://www.ncbi.nlm.nih.gov/structure/vastplus/vastplus.cgi . for a query structure specified by the user , the service lists similar structures , matches that associate each polymer chain of the query with a corresponding polymer chain of some other structure are considered complete and are indicated with full circles in the search results table ; partial matches are indicated with partially filled circles . the search results tables provided by the vast+ web service give a concise summary of the matches and the extent / quality of the similarity . the 3d structures of superimposed biological assemblies may be visualized using the 3d viewer cn3d ( 23 ) , which has been re - released as a new version 4.3.1 to support the visualization style . currently , cn3d is able to display the structure superposition of the matched biological assemblies and all the protein chains involved , but it can only display one sequence alignment at a time . therefore , the individual protein match table as shown in figure 2 provides separate cn3d launch points for each matched / aligned protein pair . all of these launch points will result in the same 3d image and rendering , but they will differ in the pair of aligned sequences that are chosen as the content of cn3d s sequence / alignment viewer window . matches are evaluated with biological assemblies as the unit of comparison ( referred to as biological units ) and may summarize simultaneous alignment of several protein molecule pairs . the tabulated matches are shown with their pdb accession , descriptive text , the number of proteins aligned in the match , the total number of aligned residues , the sequence identity and the rmsd resulting from the simultaneous superimposition of all aligned molecules . in this example , the query 3o6f matches the structure 1j8h with a total of four aligned protein molecules , totaling 768 residues and resulting in a superposition with 2.55 rmsd . the extended panel characterizing this selected match contains a table that lists pairs of matching / aligned proteins , and it provides schematic depictions of each biological assembly s composition and interactions . the individual protein match table contains action buttons that provide access to the pairwise sequence alignments as derived from the vast superimposition and launch points for visualization of the structure superimposition with the protein structure viewer cn3d ( 23 ) . button will open a superposition of the complete biological assembly alignment with the 3d view centered on the selected protein molecule and its sequence data featured in the cn3d sequence viewer window . next to the aligned molecules table , an information box lists some stats that characterize the matched biological assembly . figure 3.visualization of structurally matching biological assemblies , as rendered by the visualization tool cn3d . the query structure , pdb accession 3o6f , represents the complex of an autoreactive t - cell receptor ( ms2 - 3c8 , molecules rendered in green and brown ) complexed with a self - peptide derived from myelin basic protein and the multiple sclerosis - associated mhc molecule hla - dr4 ( molecules rendered in magenta and blue ) ( 24 ) . the self - peptide has been fused with the mhc molecule for the experiment , which explains why the query is represented as a biological assembly with only four components ( figure 2 ) , and is rendered in gray , as is the default for all unaligned segments in cn3d visualization sessions launched from vast+ results pages . the left panel shows 3o6f superimposed with the structure neighbor , pdb accession 1j8h ( 25 ) , which contains a complex between hla - dr3 , an influenza hemagglutinin peptide , and a human alpha / beta t - cell receptor . this demonstrates how well the autoreactive t - cell receptor complex mimics complexes that include foreign peptides , and it is thought that this binding mode is responsible for the autoimmune tcr escaping negative selection . the right panel shows the vast+ alignment between 3o6f and the structure of a t - cell receptor from a patient with multiple sclerosis , complexed with a myelin basic protein - derived peptide and an hla - dr2 mhc , pdb accession 1ymm ( 26 ) . instead , it reports the most extensive sub - structure match , which in this case involves both subunits of the mhc ( molecules rendered in magenta and blue ) . the unusual conformation of the complex reported in 1ymm is thought to represent an alternative binding mode that helps autoimmune tcrs to escape negative selection . matches are evaluated with biological assemblies as the unit of comparison ( referred to as biological units ) and may summarize simultaneous alignment of several protein molecule pairs . the tabulated matches are shown with their pdb accession , descriptive text , the number of proteins aligned in the match , the total number of aligned residues , the sequence identity and the rmsd resulting from the simultaneous superimposition of all aligned molecules . the individual protein match table contains action buttons that provide access to the pairwise sequence alignments as derived from the vast superimposition and launch points for visualization of the structure superimposition with the protein structure viewer cn3d ( 23 ) . button will open a superposition of the complete biological assembly alignment with the 3d view centered on the selected protein molecule and its sequence data featured in the cn3d sequence viewer window . the query structure , pdb accession 3o6f , represents the complex of an autoreactive t - cell receptor ( ms2 - 3c8 , molecules rendered in green and brown ) complexed with a self - peptide derived from myelin basic protein and the multiple sclerosis - associated mhc molecule hla - dr4 ( molecules rendered in magenta and blue ) ( 24 ) . the self - peptide has been fused with the mhc molecule for the experiment , which explains why the query is represented as a biological assembly with only four components ( figure 2 ) , and is rendered in gray , as is the default for all unaligned segments in cn3d visualization sessions launched from vast+ results pages . the left panel shows 3o6f superimposed with the structure neighbor , pdb accession 1j8h ( 25 ) , which contains a complex between hla - dr3 , an influenza hemagglutinin peptide , and a human alpha / beta t - cell receptor . this demonstrates how well the autoreactive t - cell receptor complex mimics complexes that include foreign peptides , and it is thought that this binding mode is responsible for the autoimmune tcr escaping negative selection . the right panel shows the vast+ alignment between 3o6f and the structure of a t - cell receptor from a patient with multiple sclerosis , complexed with a myelin basic protein - derived peptide and an hla - dr2 mhc , pdb accession 1ymm ( 26 ) . at this point , the vast - search service , which accepts 3d structure data uploaded in pdb - format , remains unchanged , and presents similar structures in the original vast format ( table 1 ) . table 1.urls for mmdb and vast resourcesmmdbdatabase home pagehttp://www.ncbi.nlm.nih.gov / structuremmdb ftpdata distributionftp://ftp.ncbi.nih.gov / mmdb / vastidentify structurally similar individual protein moleculeshttp://www.ncbi.nlm.nih.gov / structure / vast / vast.shtmlvast+identify structurally similar macromolecular complexeshttp://www.ncbi.nlm.nih.gov / structure / vastplus / vastplus.cgivast searchinput the 3d coordinates of a query structure to search for similar structureshttp://www.ncbi.nlm.nih.gov / structure / vast / vastsearch.htmlcn3dmolecular graphics viewerhttp://www.ncbi.nlm.nih.gov / structure / cn3d / cn3d.shtmlcblastfind 3d structures that are related to a query protein via sequence comparisonhttp://www.ncbi.nlm.nih.gov / structure / cblast / cblast.cgi urls for mmdb and vast resources the current implementation of vast+ , the associated database of pre - computed structure comparison results and the web service represent a first attempt at providing a comprehensive set of structure neighboring information for biological assemblies . several issues may limit the potential applications of the idea , and we intend to address them in future releases of the service and the associated data . currently , vast+ ignores non - polypeptide components of macromolecular complexes , but certainly both nucleic acids and chemical ligands , if present , could be matched as well as the protein components . it should be mentioned that no vast+ neighboring data are available for structure database entries that lack assignment of biological assemblies , and currently vast+ skips biological assemblies whose size exceeds a threshold number of protein components the systematic evaluation of all possible multimolecule matches becomes too time - consuming , and will need to be supplemented by a suitable heuristic .

urothelial cancer of the bladder ( ucb ) is the fourth most common malignancy diagnosed in american men.1 the majority of these cancers are non - muscle - invasive lesions at the time of diagnosis . non - muscle - invasive urothelial cancer has a high incidence of recurrence ( up to 75% ) which requires close monitoring.2,3 the main goal of intravesical therapy is to prevent tumor recurrence and progression following initial transurethral resection of bladder tumor ( turbt ) . intravesical bacillus calmette - gurin ( bcg ) is so far the most effective and common form of adjuvant therapy for high risk bladder cancer.4 compared with controls , bcg immunotherapy has superior advantage in preventing tumor recurrence than intravesical chemotherapy.5,6 in contrast to intravesical chemotherapy , bcg has also been shown to reduce the risk of tumor progression.3 despite its success , significant proportions ( 30%40% ) of patients do not respond to bcg therapy and 30% to 50% of initial responders have relapse within the first five years.7 furthermore , side - effects are common , which may be related to bcg dose , especially in highly sensitive patients.8 bcg elicits a non - specific immune response within the bladder wall . this response is t - lymphocyte dependent and is mediated by both t - helper 1 ( th1 ) and t - helper 2 ( th2 ) cytokines.9,10 cd4 th1 cytokines [ interleukin 2 ( il-2 ) and interferons ( ifns ) ] lead to activation of lymphokine activated killer cells , macrophages , nk cells , and apoptotic pathways.11,12 the apoptotic pathways are thought to be related to the tumor necrosis factor - related apoptosis - inducing ligand ( trail).13,14 trail can trigger both the extrinsic and intrinsic apoptotic pathways.15 preliminary clinical studies of combination therapy with ifn- plus low dose bcg demonstrate 42% to 53% tumor - free at 2 years in bcg refractory patients , for which effective alternative therapy remains very limited.16,17 also , combining low - dose bcg plus cd4 cytokines has been suggested to reduce bcg side effects.18 in addition , il-2 has been shown in some clinical studies to have a direct antitumor effect.19 furthermore , there is strong experimental evidence for maximally activating the apoptotic pathways with combination treatment of gemcitabine and trail in urothelial cancer cells.20 gemcitabine ( 2-deoxy-2 , 2-difluorocytidine , gemzar ) , is a nucleoside analogue with a molecular weight of 299.66 that is metabolized intracellularly by nucleoside kinases to the active diphosphate and triphosphate nucleosides . studies have shown it can also promote tumor cell apoptosis,21 which may enhance the antitumor effect of bcg if used together as an intravesical agent.22 the ultimate goal of cancer therapy is to achieve maximal antitumor efficacy and minimal toxicity . to reach this goal , we investigated the antitumor efficacy of some alternative modalities including gemcitabine , ifn- , il-2 , alone or in combination with bcg , which was compared to bcg monotherapy and saline control . ideally new therapeutic strategies should be tested rigorously in relevant animal models . thus , a previously well characterized orthotopic , immune competent rat bladder cancer model2325 was used in this study . the ay-27 cell line was originally induced in the bladders of fischer f344 rats by using the fanft carcinogen . the cells were cultured as monolayers in rpmi-1640 medium ( gibco - brl ) supplemented with 10% heat inactivated fetal bovine serum in 37 c humidified 5% co2 . these cells were passaged using standard trypsinization protocols and were previously characterized to confirm their urothelial cell origin by immunohistochemical analysis.23 connaught strain bcg was initially provided by aventis pharmaceuticals ( formerly pasteur - merieux connaught , montreal , qc , canada ) . it was assayed for colony forming unit ( cfu ) quality by the manufacturer and our treatment aliquots were based on these functional calculations . our hospitals later switched over to use tice strain bcg ( oncotice , organon canada ltd . , accordingly , we used tice strain bcg in the second part of the animal study . the medium bcg dose ( 2 10 cfu / ml ) used for the animal studies was based on the clinically used dose ( 18 10 cfu / ml ) . bcg was used in aliquots and never stored after being reconstituted in sterile saline ( 0.9% nacl ) . the lyophilized powder of gemcitabine ( eli lilly canada inc . , toronto , on ) was first reconstituted with saline , aliquoted and stored at 20 c . these aliquots were further diluted to the desired concentrations with dulbecco s phosphate buffered saline ( pbs , ph 7.4 ) before each experiment . all animal procedures were carried out in accordance with guidelines regulated by the canadian council on animal care and approved by the university of alberta institutional animal care committee . female fischer f344 rats weighing 150 gm were used for tumor implantation and randomly assigned to experimental groups ( tables 1 and 2 ) . animals were initially purchased from charles river laboratories ( quebec , canada ) and bred locally in the cross cancer institute vivarium . tumor cell implantation procedures were previously reported by us.23,26 briefly , animals were anesthetized with inhalation of 2% isoflurane in oxygen . the bladder was catheterized with an 18 gauge angiocatheter ( bd insyte , utah , usa ) and the mucosa preconditioned with 0.1 m hcl , neutralized with 0.1 m koh , and then flushed with sterile pbs ( ph 7.4 ) 3 times . single cell suspensions of ay-27 cells ( 3 10 ) in 500 l of serum - free medium were then instilled via the catheter and left indwelling for 1 h. the rats position was changed from side to side to facilitate full bladder wall exposure . the catheter was removed after 1 h and the rats were allowed to void spontaneously . , nearly 100% tumor engraftment has been achieved in syngeneic fisher f344 rats if 2 10 or more tumor cells are inoculated.2325 based on our prior study using reovirus in the same tumor model,26 treatments with dose escalation of intravesical bcg ( connaught strain ) or bcg plus il-2 ( invitrogen ) commenced on day 10 after tumor cell inoculation ( table 1 ) . bcg doses ranged from 5 10 cfu / ml ( low - dose ) to 5 10 cfu / ml ( high - dose ) . low - dose bcg plus il-2 ( 5 10 units ) was used for combination treatment . a purse - string suture was placed in the skin around the urethral meatus to keep the solutions in the bladder while the animals were under anesthesia . where urine could not be collected directly ( ie , empty bladder ) 0.5 ml of normal saline was flushed into the bladder and collected for cytology . a 9.4 t rodent magnetic resonance imaging ( mri , magnex scientific , oxford , uk ) although the 9.4 t mri had better resolution ( 0.5 mm ) than the 1.5 t clinical mri we used previously,23 it was still difficult to visualize tiny or flat tumors ( fig . 1 ) . the mri did detect papillary tumors and some were fairly large after 10 days post - inoculation ( fig . therefore in the second part of the study , treatments with gemcitabine commenced at day 6 post - implantation ( table 2 ) . graded doses of gemcitabine ranging from 0.5 mg / ml to 20 mg / ml , or bcg ( 2 10 cfu / ml ) , or recombinant rat ifn- ( invitrogen , 18000 units ) were given intravesically twice weekly for 3 weeks . combination therapies of bcg plus gemcitabine ( 0.5 mg / ml ) or ifn- were administered . however , urine cytology and mri were abandoned due to their limitations in tumor detection . once the animals completed the treatment regimes , they were observed for more than 90 days for signs and symptoms of bladder cancer progression ( body weight loss , hematuria , and urinary retention ) . animals were then euthanized using pentobarbital ( euthanyl , bimeda - mtc animal health inc . ) the bladders and kidneys were excised for gross and histological examination ( standard formalin fixation , sectioning , and hematoxylin and eosin staining ) . survival curves from different groups of animals were plotted by the kaplan - meier method . log - rank ( mantel - cox ) test ( graphpad prism , version 5.01 , san diego , usa ) was used to compare survival time distributions . individual groups were compared relative to control groups to calculate p values and a p < 0.05 was considered significant . the ay-27 cell line was originally induced in the bladders of fischer f344 rats by using the fanft carcinogen . the cells were cultured as monolayers in rpmi-1640 medium ( gibco - brl ) supplemented with 10% heat inactivated fetal bovine serum in 37 c humidified 5% co2 . these cells were passaged using standard trypsinization protocols and were previously characterized to confirm their urothelial cell origin by immunohistochemical analysis.23 connaught strain bcg was initially provided by aventis pharmaceuticals ( formerly pasteur - merieux connaught , montreal , qc , canada ) . it was assayed for colony forming unit ( cfu ) quality by the manufacturer and our treatment aliquots were based on these functional calculations . our hospitals later switched over to use tice strain bcg ( oncotice , organon canada ltd . , accordingly , we used tice strain bcg in the second part of the animal study . the medium bcg dose ( 2 10 cfu / ml ) used for the animal studies was based on the clinically used dose ( 18 10 cfu / ml ) . bcg was used in aliquots and never stored after being reconstituted in sterile saline ( 0.9% nacl ) . the lyophilized powder of gemcitabine ( eli lilly canada inc . , toronto , on ) was first reconstituted with saline , aliquoted and stored at 20 c . these aliquots were further diluted to the desired concentrations with dulbecco s phosphate buffered saline ( pbs , ph 7.4 ) before each experiment . all animal procedures were carried out in accordance with guidelines regulated by the canadian council on animal care and approved by the university of alberta institutional animal care committee . female fischer f344 rats weighing 150 gm were used for tumor implantation and randomly assigned to experimental groups ( tables 1 and 2 ) . animals were initially purchased from charles river laboratories ( quebec , canada ) and bred locally in the cross cancer institute vivarium . tumor cell implantation procedures were previously reported by us.23,26 briefly , animals were anesthetized with inhalation of 2% isoflurane in oxygen . the bladder was catheterized with an 18 gauge angiocatheter ( bd insyte , utah , usa ) and the mucosa preconditioned with 0.1 m hcl , neutralized with 0.1 m koh , and then flushed with sterile pbs ( ph 7.4 ) 3 times . single cell suspensions of ay-27 cells ( 3 10 ) in 500 l of serum - free medium were then instilled via the catheter and left indwelling for 1 h. the rats position was changed from side to side to facilitate full bladder wall exposure . the catheter was removed after 1 h and the rats were allowed to void spontaneously . , nearly 100% tumor engraftment has been achieved in syngeneic fisher f344 rats if 2 10 or more tumor cells are inoculated.2325 based on our prior study using reovirus in the same tumor model,26 treatments with dose escalation of intravesical bcg ( connaught strain ) or bcg plus il-2 ( invitrogen ) commenced on day 10 after tumor cell inoculation ( table 1 ) . bcg doses ranged from 5 10 cfu / ml ( low - dose ) to 5 10 cfu / ml ( high - dose ) . low - dose bcg plus il-2 ( 5 10 units ) was used for combination treatment . a purse - string suture was placed in the skin around the urethral meatus to keep the solutions in the bladder while the animals were under anesthesia . where urine could not be collected directly ( ie , empty bladder ) 0.5 ml of normal saline was flushed into the bladder and collected for cytology . a 9.4 t rodent magnetic resonance imaging ( mri , magnex scientific , oxford , uk ) was used to monitor bladder tumor growth . although the 9.4 t mri had better resolution ( 0.5 mm ) than the 1.5 t clinical mri we used previously,23 it was still difficult to visualize tiny or flat tumors ( fig . 1 ) . the mri did detect papillary tumors and some were fairly large after 10 days post - inoculation ( fig . therefore in the second part of the study , treatments with gemcitabine commenced at day 6 post - implantation ( table 2 ) . graded doses of gemcitabine ranging from 0.5 mg / ml to 20 mg / ml , or bcg ( 2 10 cfu / ml ) , or recombinant rat ifn- ( invitrogen , 18000 units ) were given intravesically twice weekly for 3 weeks . combination therapies of bcg plus gemcitabine ( 0.5 mg / ml ) or ifn- were administered . however , urine cytology and mri were abandoned due to their limitations in tumor detection . once the animals completed the treatment regimes , they were observed for more than 90 days for signs and symptoms of bladder cancer progression ( body weight loss , hematuria , and urinary retention ) . animals were then euthanized using pentobarbital ( euthanyl , bimeda - mtc animal health inc . ) the bladders and kidneys were excised for gross and histological examination ( standard formalin fixation , sectioning , and hematoxylin and eosin staining ) . survival curves from different groups of animals were plotted by the kaplan - meier method . log - rank ( mantel - cox ) test ( graphpad prism , version 5.01 , san diego , usa ) was used to compare survival time distributions . individual groups were compared relative to control groups to calculate p values and a p < 0.05 was considered significant . table 1 and figure 2 summarize the data from tumor - bearing rats treated by graded doses of bcg alone or low - dose bcg plus il-2 starting at 10 days post tumor cell inoculation . animals surviving greater than 90 days post - implantation were considered long - term survivals ( lts ) . a dose - dependent tumor cure ( tumor - free animals in lts ) and survival benefit were observed from intravesical bcg monotherapies . high - dose bcg attained significant survival benefit compared to control ( p = 0.03 ) , while low - dose bcg did not ( p = 0.115 ) . interestingly , when combining low - dose bcg with il-2 similar efficacy to high - dose bcg was obtained ( p = 0.01 ) . surprisingly , the benefit from medium - dose bcg ( equivalent to dose used clinically ) over saline instillations just reached significance ( p = 0.05 ) . the tumor burden at the time of treatment could have been a potential cause of failure , since mri and histological examination at 10 days post tumor cell inoculation revealed some relatively large papillary tumors with early invasion ( fig . bcg has been used mainly for treatment of carcinoma in situ or for minimal residual tumor following surgery to eliminate recurrence and progression . histological slides of bladder tissue did not detect marked difference of inflammatory cell infiltration between bcg and bcg plus il-2 treated groups , suggesting that il-2 may act directly on tumor cells . urine cytology detected less than 50% of bladder tumors when compared with histological examination at necrospy . during and after bcg therapy , animals showed various degrees of distress ( ocular porphyrin staining , hematuria and poor grooming ) . the main cause for animals to be terminated earlier was urinary retention ( lower abdomen distention ) resulting from bladder tumor and/or dystrophic stone obstruction of bladder outlet . pyelonephritis was observed in 15% of animals , which might be related to tumor obstruction or reflux which is common in this model . in the second part of the study , we investigated the efficacy of alternative agents in this tumor model using bcg as a standard treatment . in an attempt to treat animals with less tumor burden , intravesical treatments in this part of the study commenced at 6 days post - inoculation when mostly flat ucb was present ( fig . data from the second part of the study are shown in table 2 and figure 3 . consistent to the first part , animals treated with bcg ( tice strain , 2 10 cfu / ml ) instillations showed a significant survival advantage ( 66.7% lts , p = 0.002 ) and tumor cure ( tumor - free in lts ) compared with saline controls . animals receiving gemcitabine instillations ( 0.5 , 1 , and 2 mg / ml ) also attained 7090% long - term survival , with 4060% tumor cure , which were significantly different from animals receiving saline instillations ( p = 0.001 , = 0.001 , = 0.005 , respectively ) . since no dose - response was observed between the lower - dose gemcitabine groups , they were pooled into one group for survival analysis to generate a concise and more readable figure ( fig . however , it should be noted that animals treated with higher doses of gemcitabine ( 10 and 20 mg / ml ) died from toxicity after 2 instillations ; as such , they were excluded from analysis as they did not finish the treatment schedule . necropsy of these high - dose treated animals revealed hemorrhagic cystitis and gastrointestinal bleeding which appeared to be the etiology of death . to test if gemcitabine could increase efficacy of bcg by targeting both the intrinsic and extrinsic apoptotic pathways , one group of animals received bcg ( 2 10 cfu / ml ) plus gemcitabine ( 0.5 mg / ml ) instillations . unfortunately , this combination did not further increase effectiveness , but it slightly reduced the animals tolerance to treatment ( 44% lts ) . interestingly , combining bcg ( 2 10 cfu / ml ) with ifn- did not enhance effectiveness over high - dose bcg monotherapy . unfortunately , these tumors recur in 40% to 80% of patients following turbt.27 to prevent tumor recurrence and progression , intravesical instillation of bcg is the most commonly used adjuvant therapy . to date , there are limited proven effective alternative intravesical therapies for patients with bcg - refractory bladder cancers . increasing the dose of bcg or enhancing the treatment schedules was suggested , but was associated with higher toxicities.8,18 the doses of bcg used clinically are largely empirical and there is limited data in literature to show that bcg efficacy is dose - dependent . studies in animals and preliminary clinical trials have shown that gemcitabine and ifn may be promising intravesical agents which could be used as monotherapy or in combination therapy with bcg.12,17,18,21,28 however , studies to date have been performed in various different clinical settings . to the best of our knowledge , there has been no side - by - side comparative studies performed using all these agents in a single preclinical animal model . in the present study , we used a well characterized syngeneic rat orthotopic bladder tumor model to examine gemcitabine as an alternative regional chemotherapeutic agent ( which could be clinically administered earlier post - turbt than the infectious bcg ) and compared it with bcg . we also explored combination therapies of bcg plus gemcitabine , or t lymphocyte cytokines . the rationale for combining gemcitabine with bcg was to maximize apoptosis in ucc , as demonstrated in our previous in - vitro studies , where gemcitabine reduced bcl-2 expression and enhanced trail mediated apoptosis.20 we first performed a bcg dose escalation study because such a study is difficult to do in patients . a dose dependent antitumor and survival effect was shown in animals receiving bcg instillations started 10 days post - inoculation of ucc cells ( table 1 , fig . high - dose bcg treatments significantly improved survival benefit and tumor cure versus saline control . the clinically equivalent medium - dose bcg treatment demonstrated a marginally significant anti - tumor and survival benefit ( p = 0.05 , mantel - cox test ) over saline . combination therapy of low - dose bcg with il-2 significantly enhanced bcg s anti - tumor efficacy , which concurs with clinical results of combining low - dose bcg with inf-.16 urinary il-2 seems a positive predictor of response to bcg therapy.9,10 histological examination has not found noticeable difference of inflammatory cell infiltration between bcg and bcg plus il-2 treated bladder tissues , which may suggest il-2 ( effector ) has direct antitumor effect and enhances bcg s indirect ( activator ) activity.9 the reason for the lack of efficacy of low - dose bcg alone is not clear . we thought a heavier tumor burden at 10 days ( rather than 6 days , fig . this was based on the criteria established by zbar , that successful bcg therapy requires close contact between bcg and tumor cells , a limited tumor burden , a host capable of mounting an immunological reaction to mycobacterial antigens and adequate numbers of viable bcg organisms.4 clinically , bcg is used to treat bladder flat lesions ( cis ) or residual tumor cells . we then examined the efficacy of gemcitabine and ifn- , as well as in combination with high - dose bcg . the rationale of this was two - fold : 1 , gemcitabine and inf- are not infectious agents and can be used earlier than bcg in the clinic , and 2,the tumors tended to have early invasion ( pt1 disease ) beyond 6 days . gemcitabine ( low dose ; 0.52 mg / ml ) , ifn- , and bcg all demonstrated significantly superior antitumor responses ( tumor - free long - term survival ) when compared with saline controls ( table 2 and fig . these data show that gemcitabine and inf- may be promising alternative agents to bcg , especially in bcg - refractory patients . also , we believe this is the first time to examine the two commonly used bcg strains in one study . although the timing of bcg initiation was slightly different , both strains of high - dose bcg showed highly effective antitumor activity . animals treated with high concentrations of gemcitabine ( 1020 mg / ml ) could not tolerate beyond 2 instillations and died from drug related toxicities . these toxicities may be attributable to an acidic chemical effect on the thin bladder wall at high drug concentration ( ph of 3.0 ) and systemic absorption of the drug . absorption of gemcitabine ( molecular weight , 299.66 ) may be facilitated by vesicoureteral reflux to kidneys in our rat model.29 systemic absorption in dogs after instillation has also been reported.30 fortunately , absorption of gemcitabine following instillation in humans appears to be limited,28,31 which has allowed for dose escalation to facilitate a concentration gradient driven tissue penetration in human bladders . contrary to our hypothesis , combining high - dose bcg with gemcitabine or ifn- did not further increase their antitumor efficacy over monotherapy of either agent . it seems that a maximal antitumor activity of the monotherapy has been reached and there is limited room for further improvement from combining them in our model system . if this is the case , then rational combination therapy is more suitable for low - dose bcg plus gemcitabine or th1 cytokines in the treatment of early stage bladder cancer . based on the study in an orthotopic rat bladder tumor model , the antitumor efficacy of bcg is dose - dependent . il-2 enhanced low - dose bcg s efficacy to the level of high - dose bcg treatment . gemcitabine and ifn- monotherapy showed similar efficacy to high - dose bcg and are promising intravesical agents for treatment of early stage ucb . these alternative and combinatory strategies warrant further exploration in patients with non - muscle invasive ucb , especially in those with bcg - refractory diseases . bacillus calmette - gurin ; tumor necrosis factor - related apoptosis - inducing ligand ; transurethral resection of bladder tumor ; urothelial cancer of the bladder ; urothelial cell carcinoma .

objective : to reduce adverse effects and improve efficacy of intravesical bcg for bladder cancer , alternative treatment options were investigated in an orthotopic rat tumor model.methods:superficial bladder cancer was established in syngeneic female rat bladders by instillation of ay-27 cells . animals were randomly assigned to treatment groups including dose escalation of intravesical bcg with or without interferon- ( ifn- ) or interleukin-2 ( il-2 ) ; or graded doses of gemcitabine alone ; or bcg plus gemcitabine . treatments were given twice weekly for 3 weeks . rats in control groups received saline instillations . treatment response was monitored by animals well - being , survival days , tumor growth inhibition , and histological examination at necropsy.results:rats receiving monotherapy with intravesical bcg , gemcitabine , or ifn- , attained significantly better survival and tumor reduction compared with control ( p = 0.002 ; 0.001 ; 0.002 , respectively , log - rank test ) . a dose - dependent treatment response was observed in animals with established bladder tumor receiving escalated bcg instillations . only high - dose bcg significantly improved animal survival . although high - dose bcg plus gemcitabine or ifn- did not increase benefit over monotherapies , low - dose bcg plus il-2 did show improved efficacy ( p = 0.01).conclusion : intravesical monotherapies with gemcitabine and ifn- were as effective as bcg for treatment of early non - muscle - invasive urothelial bladder cancer in this immune competent rat model . combining these agents with high - dose bcg did not further increase efficacy . however , combining low - dose bcg with il-2 enhanced bcg effectiveness .

Introduction Materials and Methods Tumor cells, BCG, and gemcitabine Animals Tumor cell implantation and intravesical therapy Follow-up endpoint Statistic analysis Results Discussion Conclusions Abbreviations

urothelial cancer of the bladder ( ucb ) is the fourth most common malignancy diagnosed in american men.1 the majority of these cancers are non - muscle - invasive lesions at the time of diagnosis . non - muscle - invasive urothelial cancer has a high incidence of recurrence ( up to 75% ) which requires close monitoring.2,3 the main goal of intravesical therapy is to prevent tumor recurrence and progression following initial transurethral resection of bladder tumor ( turbt ) . intravesical bacillus calmette - gurin ( bcg ) is so far the most effective and common form of adjuvant therapy for high risk bladder cancer.4 compared with controls , bcg immunotherapy has superior advantage in preventing tumor recurrence than intravesical chemotherapy.5,6 in contrast to intravesical chemotherapy , bcg has also been shown to reduce the risk of tumor progression.3 despite its success , significant proportions ( 30%40% ) of patients do not respond to bcg therapy and 30% to 50% of initial responders have relapse within the first five years.7 furthermore , side - effects are common , which may be related to bcg dose , especially in highly sensitive patients.8 bcg elicits a non - specific immune response within the bladder wall . this response is t - lymphocyte dependent and is mediated by both t - helper 1 ( th1 ) and t - helper 2 ( th2 ) cytokines.9,10 cd4 th1 cytokines [ interleukin 2 ( il-2 ) and interferons ( ifns ) ] lead to activation of lymphokine activated killer cells , macrophages , nk cells , and apoptotic pathways.11,12 the apoptotic pathways are thought to be related to the tumor necrosis factor - related apoptosis - inducing ligand ( trail).13,14 trail can trigger both the extrinsic and intrinsic apoptotic pathways.15 preliminary clinical studies of combination therapy with ifn- plus low dose bcg demonstrate 42% to 53% tumor - free at 2 years in bcg refractory patients , for which effective alternative therapy remains very limited.16,17 also , combining low - dose bcg plus cd4 cytokines has been suggested to reduce bcg side effects.18 in addition , il-2 has been shown in some clinical studies to have a direct antitumor effect.19 furthermore , there is strong experimental evidence for maximally activating the apoptotic pathways with combination treatment of gemcitabine and trail in urothelial cancer cells.20 gemcitabine ( 2-deoxy-2 , 2-difluorocytidine , gemzar ) , is a nucleoside analogue with a molecular weight of 299.66 that is metabolized intracellularly by nucleoside kinases to the active diphosphate and triphosphate nucleosides . to reach this goal , we investigated the antitumor efficacy of some alternative modalities including gemcitabine , ifn- , il-2 , alone or in combination with bcg , which was compared to bcg monotherapy and saline control . thus , a previously well characterized orthotopic , immune competent rat bladder cancer model2325 was used in this study . the lyophilized powder of gemcitabine ( eli lilly canada inc . female fischer f344 rats weighing 150 gm were used for tumor implantation and randomly assigned to experimental groups ( tables 1 and 2 ) . animals were initially purchased from charles river laboratories ( quebec , canada ) and bred locally in the cross cancer institute vivarium . tumor cell implantation procedures were previously reported by us.23,26 briefly , animals were anesthetized with inhalation of 2% isoflurane in oxygen . the bladder was catheterized with an 18 gauge angiocatheter ( bd insyte , utah , usa ) and the mucosa preconditioned with 0.1 m hcl , neutralized with 0.1 m koh , and then flushed with sterile pbs ( ph 7.4 ) 3 times . single cell suspensions of ay-27 cells ( 3 10 ) in 500 l of serum - free medium were then instilled via the catheter and left indwelling for 1 h. the rats position was changed from side to side to facilitate full bladder wall exposure . , nearly 100% tumor engraftment has been achieved in syngeneic fisher f344 rats if 2 10 or more tumor cells are inoculated.2325 based on our prior study using reovirus in the same tumor model,26 treatments with dose escalation of intravesical bcg ( connaught strain ) or bcg plus il-2 ( invitrogen ) commenced on day 10 after tumor cell inoculation ( table 1 ) . bcg doses ranged from 5 10 cfu / ml ( low - dose ) to 5 10 cfu / ml ( high - dose ) . low - dose bcg plus il-2 ( 5 10 units ) was used for combination treatment . a purse - string suture was placed in the skin around the urethral meatus to keep the solutions in the bladder while the animals were under anesthesia . therefore in the second part of the study , treatments with gemcitabine commenced at day 6 post - implantation ( table 2 ) . graded doses of gemcitabine ranging from 0.5 mg / ml to 20 mg / ml , or bcg ( 2 10 cfu / ml ) , or recombinant rat ifn- ( invitrogen , 18000 units ) were given intravesically twice weekly for 3 weeks . combination therapies of bcg plus gemcitabine ( 0.5 mg / ml ) or ifn- were administered . however , urine cytology and mri were abandoned due to their limitations in tumor detection . once the animals completed the treatment regimes , they were observed for more than 90 days for signs and symptoms of bladder cancer progression ( body weight loss , hematuria , and urinary retention ) . animals were then euthanized using pentobarbital ( euthanyl , bimeda - mtc animal health inc . ) the bladders and kidneys were excised for gross and histological examination ( standard formalin fixation , sectioning , and hematoxylin and eosin staining ) . survival curves from different groups of animals were plotted by the kaplan - meier method . log - rank ( mantel - cox ) test ( graphpad prism , version 5.01 , san diego , usa ) was used to compare survival time distributions . individual groups were compared relative to control groups to calculate p values and a p < 0.05 was considered significant . the lyophilized powder of gemcitabine ( eli lilly canada inc . female fischer f344 rats weighing 150 gm were used for tumor implantation and randomly assigned to experimental groups ( tables 1 and 2 ) . animals were initially purchased from charles river laboratories ( quebec , canada ) and bred locally in the cross cancer institute vivarium . the bladder was catheterized with an 18 gauge angiocatheter ( bd insyte , utah , usa ) and the mucosa preconditioned with 0.1 m hcl , neutralized with 0.1 m koh , and then flushed with sterile pbs ( ph 7.4 ) 3 times . single cell suspensions of ay-27 cells ( 3 10 ) in 500 l of serum - free medium were then instilled via the catheter and left indwelling for 1 h. the rats position was changed from side to side to facilitate full bladder wall exposure . , nearly 100% tumor engraftment has been achieved in syngeneic fisher f344 rats if 2 10 or more tumor cells are inoculated.2325 based on our prior study using reovirus in the same tumor model,26 treatments with dose escalation of intravesical bcg ( connaught strain ) or bcg plus il-2 ( invitrogen ) commenced on day 10 after tumor cell inoculation ( table 1 ) . bcg doses ranged from 5 10 cfu / ml ( low - dose ) to 5 10 cfu / ml ( high - dose ) . low - dose bcg plus il-2 ( 5 10 units ) was used for combination treatment . a purse - string suture was placed in the skin around the urethral meatus to keep the solutions in the bladder while the animals were under anesthesia . a 9.4 t rodent magnetic resonance imaging ( mri , magnex scientific , oxford , uk ) was used to monitor bladder tumor growth . therefore in the second part of the study , treatments with gemcitabine commenced at day 6 post - implantation ( table 2 ) . graded doses of gemcitabine ranging from 0.5 mg / ml to 20 mg / ml , or bcg ( 2 10 cfu / ml ) , or recombinant rat ifn- ( invitrogen , 18000 units ) were given intravesically twice weekly for 3 weeks . combination therapies of bcg plus gemcitabine ( 0.5 mg / ml ) or ifn- were administered . however , urine cytology and mri were abandoned due to their limitations in tumor detection . once the animals completed the treatment regimes , they were observed for more than 90 days for signs and symptoms of bladder cancer progression ( body weight loss , hematuria , and urinary retention ) . animals were then euthanized using pentobarbital ( euthanyl , bimeda - mtc animal health inc . ) the bladders and kidneys were excised for gross and histological examination ( standard formalin fixation , sectioning , and hematoxylin and eosin staining ) . survival curves from different groups of animals were plotted by the kaplan - meier method . log - rank ( mantel - cox ) test ( graphpad prism , version 5.01 , san diego , usa ) was used to compare survival time distributions . table 1 and figure 2 summarize the data from tumor - bearing rats treated by graded doses of bcg alone or low - dose bcg plus il-2 starting at 10 days post tumor cell inoculation . a dose - dependent tumor cure ( tumor - free animals in lts ) and survival benefit were observed from intravesical bcg monotherapies . high - dose bcg attained significant survival benefit compared to control ( p = 0.03 ) , while low - dose bcg did not ( p = 0.115 ) . interestingly , when combining low - dose bcg with il-2 similar efficacy to high - dose bcg was obtained ( p = 0.01 ) . surprisingly , the benefit from medium - dose bcg ( equivalent to dose used clinically ) over saline instillations just reached significance ( p = 0.05 ) . the tumor burden at the time of treatment could have been a potential cause of failure , since mri and histological examination at 10 days post tumor cell inoculation revealed some relatively large papillary tumors with early invasion ( fig . bcg has been used mainly for treatment of carcinoma in situ or for minimal residual tumor following surgery to eliminate recurrence and progression . histological slides of bladder tissue did not detect marked difference of inflammatory cell infiltration between bcg and bcg plus il-2 treated groups , suggesting that il-2 may act directly on tumor cells . urine cytology detected less than 50% of bladder tumors when compared with histological examination at necrospy . pyelonephritis was observed in 15% of animals , which might be related to tumor obstruction or reflux which is common in this model . in the second part of the study , we investigated the efficacy of alternative agents in this tumor model using bcg as a standard treatment . in an attempt to treat animals with less tumor burden , intravesical treatments in this part of the study commenced at 6 days post - inoculation when mostly flat ucb was present ( fig . consistent to the first part , animals treated with bcg ( tice strain , 2 10 cfu / ml ) instillations showed a significant survival advantage ( 66.7% lts , p = 0.002 ) and tumor cure ( tumor - free in lts ) compared with saline controls . animals receiving gemcitabine instillations ( 0.5 , 1 , and 2 mg / ml ) also attained 7090% long - term survival , with 4060% tumor cure , which were significantly different from animals receiving saline instillations ( p = 0.001 , = 0.001 , = 0.005 , respectively ) . since no dose - response was observed between the lower - dose gemcitabine groups , they were pooled into one group for survival analysis to generate a concise and more readable figure ( fig . however , it should be noted that animals treated with higher doses of gemcitabine ( 10 and 20 mg / ml ) died from toxicity after 2 instillations ; as such , they were excluded from analysis as they did not finish the treatment schedule . necropsy of these high - dose treated animals revealed hemorrhagic cystitis and gastrointestinal bleeding which appeared to be the etiology of death . to test if gemcitabine could increase efficacy of bcg by targeting both the intrinsic and extrinsic apoptotic pathways , one group of animals received bcg ( 2 10 cfu / ml ) plus gemcitabine ( 0.5 mg / ml ) instillations . unfortunately , this combination did not further increase effectiveness , but it slightly reduced the animals tolerance to treatment ( 44% lts ) . interestingly , combining bcg ( 2 10 cfu / ml ) with ifn- did not enhance effectiveness over high - dose bcg monotherapy . unfortunately , these tumors recur in 40% to 80% of patients following turbt.27 to prevent tumor recurrence and progression , intravesical instillation of bcg is the most commonly used adjuvant therapy . increasing the dose of bcg or enhancing the treatment schedules was suggested , but was associated with higher toxicities.8,18 the doses of bcg used clinically are largely empirical and there is limited data in literature to show that bcg efficacy is dose - dependent . studies in animals and preliminary clinical trials have shown that gemcitabine and ifn may be promising intravesical agents which could be used as monotherapy or in combination therapy with bcg.12,17,18,21,28 however , studies to date have been performed in various different clinical settings . to the best of our knowledge , there has been no side - by - side comparative studies performed using all these agents in a single preclinical animal model . in the present study , we used a well characterized syngeneic rat orthotopic bladder tumor model to examine gemcitabine as an alternative regional chemotherapeutic agent ( which could be clinically administered earlier post - turbt than the infectious bcg ) and compared it with bcg . we also explored combination therapies of bcg plus gemcitabine , or t lymphocyte cytokines . the rationale for combining gemcitabine with bcg was to maximize apoptosis in ucc , as demonstrated in our previous in - vitro studies , where gemcitabine reduced bcl-2 expression and enhanced trail mediated apoptosis.20 we first performed a bcg dose escalation study because such a study is difficult to do in patients . a dose dependent antitumor and survival effect was shown in animals receiving bcg instillations started 10 days post - inoculation of ucc cells ( table 1 , fig . high - dose bcg treatments significantly improved survival benefit and tumor cure versus saline control . the clinically equivalent medium - dose bcg treatment demonstrated a marginally significant anti - tumor and survival benefit ( p = 0.05 , mantel - cox test ) over saline . combination therapy of low - dose bcg with il-2 significantly enhanced bcg s anti - tumor efficacy , which concurs with clinical results of combining low - dose bcg with inf-.16 urinary il-2 seems a positive predictor of response to bcg therapy.9,10 histological examination has not found noticeable difference of inflammatory cell infiltration between bcg and bcg plus il-2 treated bladder tissues , which may suggest il-2 ( effector ) has direct antitumor effect and enhances bcg s indirect ( activator ) activity.9 the reason for the lack of efficacy of low - dose bcg alone is not clear . we thought a heavier tumor burden at 10 days ( rather than 6 days , fig . this was based on the criteria established by zbar , that successful bcg therapy requires close contact between bcg and tumor cells , a limited tumor burden , a host capable of mounting an immunological reaction to mycobacterial antigens and adequate numbers of viable bcg organisms.4 clinically , bcg is used to treat bladder flat lesions ( cis ) or residual tumor cells . we then examined the efficacy of gemcitabine and ifn- , as well as in combination with high - dose bcg . the rationale of this was two - fold : 1 , gemcitabine and inf- are not infectious agents and can be used earlier than bcg in the clinic , and 2,the tumors tended to have early invasion ( pt1 disease ) beyond 6 days . gemcitabine ( low dose ; 0.52 mg / ml ) , ifn- , and bcg all demonstrated significantly superior antitumor responses ( tumor - free long - term survival ) when compared with saline controls ( table 2 and fig . these data show that gemcitabine and inf- may be promising alternative agents to bcg , especially in bcg - refractory patients . although the timing of bcg initiation was slightly different , both strains of high - dose bcg showed highly effective antitumor activity . animals treated with high concentrations of gemcitabine ( 1020 mg / ml ) could not tolerate beyond 2 instillations and died from drug related toxicities . absorption of gemcitabine ( molecular weight , 299.66 ) may be facilitated by vesicoureteral reflux to kidneys in our rat model.29 systemic absorption in dogs after instillation has also been reported.30 fortunately , absorption of gemcitabine following instillation in humans appears to be limited,28,31 which has allowed for dose escalation to facilitate a concentration gradient driven tissue penetration in human bladders . contrary to our hypothesis , combining high - dose bcg with gemcitabine or ifn- did not further increase their antitumor efficacy over monotherapy of either agent . if this is the case , then rational combination therapy is more suitable for low - dose bcg plus gemcitabine or th1 cytokines in the treatment of early stage bladder cancer . based on the study in an orthotopic rat bladder tumor model , the antitumor efficacy of bcg is dose - dependent . il-2 enhanced low - dose bcg s efficacy to the level of high - dose bcg treatment . gemcitabine and ifn- monotherapy showed similar efficacy to high - dose bcg and are promising intravesical agents for treatment of early stage ucb . these alternative and combinatory strategies warrant further exploration in patients with non - muscle invasive ucb , especially in those with bcg - refractory diseases . bacillus calmette - gurin ; tumor necrosis factor - related apoptosis - inducing ligand ; transurethral resection of bladder tumor ; urothelial cancer of the bladder ; urothelial cell carcinoma .

urothelial cancer of the bladder ( ucb ) is the fourth most common malignancy diagnosed in american men.1 the majority of these cancers are non - muscle - invasive lesions at the time of diagnosis . non - muscle - invasive urothelial cancer has a high incidence of recurrence ( up to 75% ) which requires close monitoring.2,3 the main goal of intravesical therapy is to prevent tumor recurrence and progression following initial transurethral resection of bladder tumor ( turbt ) . intravesical bacillus calmette - gurin ( bcg ) is so far the most effective and common form of adjuvant therapy for high risk bladder cancer.4 compared with controls , bcg immunotherapy has superior advantage in preventing tumor recurrence than intravesical chemotherapy.5,6 in contrast to intravesical chemotherapy , bcg has also been shown to reduce the risk of tumor progression.3 despite its success , significant proportions ( 30%40% ) of patients do not respond to bcg therapy and 30% to 50% of initial responders have relapse within the first five years.7 furthermore , side - effects are common , which may be related to bcg dose , especially in highly sensitive patients.8 bcg elicits a non - specific immune response within the bladder wall . this response is t - lymphocyte dependent and is mediated by both t - helper 1 ( th1 ) and t - helper 2 ( th2 ) cytokines.9,10 cd4 th1 cytokines [ interleukin 2 ( il-2 ) and interferons ( ifns ) ] lead to activation of lymphokine activated killer cells , macrophages , nk cells , and apoptotic pathways.11,12 the apoptotic pathways are thought to be related to the tumor necrosis factor - related apoptosis - inducing ligand ( trail).13,14 trail can trigger both the extrinsic and intrinsic apoptotic pathways.15 preliminary clinical studies of combination therapy with ifn- plus low dose bcg demonstrate 42% to 53% tumor - free at 2 years in bcg refractory patients , for which effective alternative therapy remains very limited.16,17 also , combining low - dose bcg plus cd4 cytokines has been suggested to reduce bcg side effects.18 in addition , il-2 has been shown in some clinical studies to have a direct antitumor effect.19 furthermore , there is strong experimental evidence for maximally activating the apoptotic pathways with combination treatment of gemcitabine and trail in urothelial cancer cells.20 gemcitabine ( 2-deoxy-2 , 2-difluorocytidine , gemzar ) , is a nucleoside analogue with a molecular weight of 299.66 that is metabolized intracellularly by nucleoside kinases to the active diphosphate and triphosphate nucleosides . studies have shown it can also promote tumor cell apoptosis,21 which may enhance the antitumor effect of bcg if used together as an intravesical agent.22 the ultimate goal of cancer therapy is to achieve maximal antitumor efficacy and minimal toxicity . to reach this goal , we investigated the antitumor efficacy of some alternative modalities including gemcitabine , ifn- , il-2 , alone or in combination with bcg , which was compared to bcg monotherapy and saline control . ideally new therapeutic strategies should be tested rigorously in relevant animal models . thus , a previously well characterized orthotopic , immune competent rat bladder cancer model2325 was used in this study . the ay-27 cell line was originally induced in the bladders of fischer f344 rats by using the fanft carcinogen . these cells were passaged using standard trypsinization protocols and were previously characterized to confirm their urothelial cell origin by immunohistochemical analysis.23 connaught strain bcg was initially provided by aventis pharmaceuticals ( formerly pasteur - merieux connaught , montreal , qc , canada ) . our hospitals later switched over to use tice strain bcg ( oncotice , organon canada ltd . , accordingly , we used tice strain bcg in the second part of the animal study . the medium bcg dose ( 2 10 cfu / ml ) used for the animal studies was based on the clinically used dose ( 18 10 cfu / ml ) . female fischer f344 rats weighing 150 gm were used for tumor implantation and randomly assigned to experimental groups ( tables 1 and 2 ) . tumor cell implantation procedures were previously reported by us.23,26 briefly , animals were anesthetized with inhalation of 2% isoflurane in oxygen . the bladder was catheterized with an 18 gauge angiocatheter ( bd insyte , utah , usa ) and the mucosa preconditioned with 0.1 m hcl , neutralized with 0.1 m koh , and then flushed with sterile pbs ( ph 7.4 ) 3 times . single cell suspensions of ay-27 cells ( 3 10 ) in 500 l of serum - free medium were then instilled via the catheter and left indwelling for 1 h. the rats position was changed from side to side to facilitate full bladder wall exposure . , nearly 100% tumor engraftment has been achieved in syngeneic fisher f344 rats if 2 10 or more tumor cells are inoculated.2325 based on our prior study using reovirus in the same tumor model,26 treatments with dose escalation of intravesical bcg ( connaught strain ) or bcg plus il-2 ( invitrogen ) commenced on day 10 after tumor cell inoculation ( table 1 ) . bcg doses ranged from 5 10 cfu / ml ( low - dose ) to 5 10 cfu / ml ( high - dose ) . low - dose bcg plus il-2 ( 5 10 units ) was used for combination treatment . a purse - string suture was placed in the skin around the urethral meatus to keep the solutions in the bladder while the animals were under anesthesia . where urine could not be collected directly ( ie , empty bladder ) 0.5 ml of normal saline was flushed into the bladder and collected for cytology . a 9.4 t rodent magnetic resonance imaging ( mri , magnex scientific , oxford , uk ) although the 9.4 t mri had better resolution ( 0.5 mm ) than the 1.5 t clinical mri we used previously,23 it was still difficult to visualize tiny or flat tumors ( fig . the mri did detect papillary tumors and some were fairly large after 10 days post - inoculation ( fig . therefore in the second part of the study , treatments with gemcitabine commenced at day 6 post - implantation ( table 2 ) . graded doses of gemcitabine ranging from 0.5 mg / ml to 20 mg / ml , or bcg ( 2 10 cfu / ml ) , or recombinant rat ifn- ( invitrogen , 18000 units ) were given intravesically twice weekly for 3 weeks . once the animals completed the treatment regimes , they were observed for more than 90 days for signs and symptoms of bladder cancer progression ( body weight loss , hematuria , and urinary retention ) . the bladders and kidneys were excised for gross and histological examination ( standard formalin fixation , sectioning , and hematoxylin and eosin staining ) . individual groups were compared relative to control groups to calculate p values and a p < 0.05 was considered significant . the ay-27 cell line was originally induced in the bladders of fischer f344 rats by using the fanft carcinogen . these cells were passaged using standard trypsinization protocols and were previously characterized to confirm their urothelial cell origin by immunohistochemical analysis.23 connaught strain bcg was initially provided by aventis pharmaceuticals ( formerly pasteur - merieux connaught , montreal , qc , canada ) . our hospitals later switched over to use tice strain bcg ( oncotice , organon canada ltd . , accordingly , we used tice strain bcg in the second part of the animal study . the medium bcg dose ( 2 10 cfu / ml ) used for the animal studies was based on the clinically used dose ( 18 10 cfu / ml ) . female fischer f344 rats weighing 150 gm were used for tumor implantation and randomly assigned to experimental groups ( tables 1 and 2 ) . tumor cell implantation procedures were previously reported by us.23,26 briefly , animals were anesthetized with inhalation of 2% isoflurane in oxygen . the bladder was catheterized with an 18 gauge angiocatheter ( bd insyte , utah , usa ) and the mucosa preconditioned with 0.1 m hcl , neutralized with 0.1 m koh , and then flushed with sterile pbs ( ph 7.4 ) 3 times . single cell suspensions of ay-27 cells ( 3 10 ) in 500 l of serum - free medium were then instilled via the catheter and left indwelling for 1 h. the rats position was changed from side to side to facilitate full bladder wall exposure . , nearly 100% tumor engraftment has been achieved in syngeneic fisher f344 rats if 2 10 or more tumor cells are inoculated.2325 based on our prior study using reovirus in the same tumor model,26 treatments with dose escalation of intravesical bcg ( connaught strain ) or bcg plus il-2 ( invitrogen ) commenced on day 10 after tumor cell inoculation ( table 1 ) . bcg doses ranged from 5 10 cfu / ml ( low - dose ) to 5 10 cfu / ml ( high - dose ) . a purse - string suture was placed in the skin around the urethral meatus to keep the solutions in the bladder while the animals were under anesthesia . where urine could not be collected directly ( ie , empty bladder ) 0.5 ml of normal saline was flushed into the bladder and collected for cytology . a 9.4 t rodent magnetic resonance imaging ( mri , magnex scientific , oxford , uk ) was used to monitor bladder tumor growth . although the 9.4 t mri had better resolution ( 0.5 mm ) than the 1.5 t clinical mri we used previously,23 it was still difficult to visualize tiny or flat tumors ( fig . the mri did detect papillary tumors and some were fairly large after 10 days post - inoculation ( fig . therefore in the second part of the study , treatments with gemcitabine commenced at day 6 post - implantation ( table 2 ) . graded doses of gemcitabine ranging from 0.5 mg / ml to 20 mg / ml , or bcg ( 2 10 cfu / ml ) , or recombinant rat ifn- ( invitrogen , 18000 units ) were given intravesically twice weekly for 3 weeks . once the animals completed the treatment regimes , they were observed for more than 90 days for signs and symptoms of bladder cancer progression ( body weight loss , hematuria , and urinary retention ) . individual groups were compared relative to control groups to calculate p values and a p < 0.05 was considered significant . table 1 and figure 2 summarize the data from tumor - bearing rats treated by graded doses of bcg alone or low - dose bcg plus il-2 starting at 10 days post tumor cell inoculation . a dose - dependent tumor cure ( tumor - free animals in lts ) and survival benefit were observed from intravesical bcg monotherapies . high - dose bcg attained significant survival benefit compared to control ( p = 0.03 ) , while low - dose bcg did not ( p = 0.115 ) . interestingly , when combining low - dose bcg with il-2 similar efficacy to high - dose bcg was obtained ( p = 0.01 ) . surprisingly , the benefit from medium - dose bcg ( equivalent to dose used clinically ) over saline instillations just reached significance ( p = 0.05 ) . the tumor burden at the time of treatment could have been a potential cause of failure , since mri and histological examination at 10 days post tumor cell inoculation revealed some relatively large papillary tumors with early invasion ( fig . bcg has been used mainly for treatment of carcinoma in situ or for minimal residual tumor following surgery to eliminate recurrence and progression . histological slides of bladder tissue did not detect marked difference of inflammatory cell infiltration between bcg and bcg plus il-2 treated groups , suggesting that il-2 may act directly on tumor cells . during and after bcg therapy , animals showed various degrees of distress ( ocular porphyrin staining , hematuria and poor grooming ) . in the second part of the study , we investigated the efficacy of alternative agents in this tumor model using bcg as a standard treatment . in an attempt to treat animals with less tumor burden , intravesical treatments in this part of the study commenced at 6 days post - inoculation when mostly flat ucb was present ( fig . consistent to the first part , animals treated with bcg ( tice strain , 2 10 cfu / ml ) instillations showed a significant survival advantage ( 66.7% lts , p = 0.002 ) and tumor cure ( tumor - free in lts ) compared with saline controls . animals receiving gemcitabine instillations ( 0.5 , 1 , and 2 mg / ml ) also attained 7090% long - term survival , with 4060% tumor cure , which were significantly different from animals receiving saline instillations ( p = 0.001 , = 0.001 , = 0.005 , respectively ) . since no dose - response was observed between the lower - dose gemcitabine groups , they were pooled into one group for survival analysis to generate a concise and more readable figure ( fig . however , it should be noted that animals treated with higher doses of gemcitabine ( 10 and 20 mg / ml ) died from toxicity after 2 instillations ; as such , they were excluded from analysis as they did not finish the treatment schedule . to test if gemcitabine could increase efficacy of bcg by targeting both the intrinsic and extrinsic apoptotic pathways , one group of animals received bcg ( 2 10 cfu / ml ) plus gemcitabine ( 0.5 mg / ml ) instillations . unfortunately , this combination did not further increase effectiveness , but it slightly reduced the animals tolerance to treatment ( 44% lts ) . unfortunately , these tumors recur in 40% to 80% of patients following turbt.27 to prevent tumor recurrence and progression , intravesical instillation of bcg is the most commonly used adjuvant therapy . increasing the dose of bcg or enhancing the treatment schedules was suggested , but was associated with higher toxicities.8,18 the doses of bcg used clinically are largely empirical and there is limited data in literature to show that bcg efficacy is dose - dependent . studies in animals and preliminary clinical trials have shown that gemcitabine and ifn may be promising intravesical agents which could be used as monotherapy or in combination therapy with bcg.12,17,18,21,28 however , studies to date have been performed in various different clinical settings . to the best of our knowledge , there has been no side - by - side comparative studies performed using all these agents in a single preclinical animal model . in the present study , we used a well characterized syngeneic rat orthotopic bladder tumor model to examine gemcitabine as an alternative regional chemotherapeutic agent ( which could be clinically administered earlier post - turbt than the infectious bcg ) and compared it with bcg . the rationale for combining gemcitabine with bcg was to maximize apoptosis in ucc , as demonstrated in our previous in - vitro studies , where gemcitabine reduced bcl-2 expression and enhanced trail mediated apoptosis.20 we first performed a bcg dose escalation study because such a study is difficult to do in patients . a dose dependent antitumor and survival effect was shown in animals receiving bcg instillations started 10 days post - inoculation of ucc cells ( table 1 , fig . the clinically equivalent medium - dose bcg treatment demonstrated a marginally significant anti - tumor and survival benefit ( p = 0.05 , mantel - cox test ) over saline . combination therapy of low - dose bcg with il-2 significantly enhanced bcg s anti - tumor efficacy , which concurs with clinical results of combining low - dose bcg with inf-.16 urinary il-2 seems a positive predictor of response to bcg therapy.9,10 histological examination has not found noticeable difference of inflammatory cell infiltration between bcg and bcg plus il-2 treated bladder tissues , which may suggest il-2 ( effector ) has direct antitumor effect and enhances bcg s indirect ( activator ) activity.9 the reason for the lack of efficacy of low - dose bcg alone is not clear . this was based on the criteria established by zbar , that successful bcg therapy requires close contact between bcg and tumor cells , a limited tumor burden , a host capable of mounting an immunological reaction to mycobacterial antigens and adequate numbers of viable bcg organisms.4 clinically , bcg is used to treat bladder flat lesions ( cis ) or residual tumor cells . we then examined the efficacy of gemcitabine and ifn- , as well as in combination with high - dose bcg . the rationale of this was two - fold : 1 , gemcitabine and inf- are not infectious agents and can be used earlier than bcg in the clinic , and 2,the tumors tended to have early invasion ( pt1 disease ) beyond 6 days . gemcitabine ( low dose ; 0.52 mg / ml ) , ifn- , and bcg all demonstrated significantly superior antitumor responses ( tumor - free long - term survival ) when compared with saline controls ( table 2 and fig . although the timing of bcg initiation was slightly different , both strains of high - dose bcg showed highly effective antitumor activity . absorption of gemcitabine ( molecular weight , 299.66 ) may be facilitated by vesicoureteral reflux to kidneys in our rat model.29 systemic absorption in dogs after instillation has also been reported.30 fortunately , absorption of gemcitabine following instillation in humans appears to be limited,28,31 which has allowed for dose escalation to facilitate a concentration gradient driven tissue penetration in human bladders . if this is the case , then rational combination therapy is more suitable for low - dose bcg plus gemcitabine or th1 cytokines in the treatment of early stage bladder cancer . based on the study in an orthotopic rat bladder tumor model , the antitumor efficacy of bcg is dose - dependent . these alternative and combinatory strategies warrant further exploration in patients with non - muscle invasive ucb , especially in those with bcg - refractory diseases . bacillus calmette - gurin ; tumor necrosis factor - related apoptosis - inducing ligand ; transurethral resection of bladder tumor ; urothelial cancer of the bladder ; urothelial cell carcinoma .

end - stage renal disease ( esrd ) is the condition in which the kidneys permanently cease to function at a level that will support life . the two basic therapies for treating this condition , dialysis and transplantation , were effectively developed by the early 1960 's . however , the high cost of both treatments prevented the widespread expansion of these therapies to all persons who could benefit from them . the fact that thousands of persons were being denied access to these life - saving therapies because of the prohibitively high costs led congress to extend medicare benefits to those persons with end - stage renal disease . entitlement to medicare benefits was extended to persons with end - stage renal disease with the enactment of the social security amendments of 1972 ( public law 92 - 603 , section 299i ) . coverage began on july 1 , 1973 . since that time expenditures rose from $ 229 million in 1974 to an estimated $ 1.8 billion in 1982 . rettig and marks ( 1981 , p. 8 . ) have put this problem in perspective : the figures used in the 1972 senate debate authorizing the programing were unreasonably low and quite misleading and , in early 1973 , the subject of a well - publicized controversy . the shift from unrealistic to realistic estimates contributed to a perception of costs out of control . unexpected or not , there can be no question that the esrd program is expensive . esrd beneficiaries comprise about one - fourth of 1 percent of all medicare beneficiaries . in 1983 , they accounted for an estimated 3.7 percent of total medicare expenditures ( parts a and b ) and 8.5 percent of part b expenditures ( health care financing administration , 1984 ) . when an easily identifiable group such as this has extremely high health care costs , it inevitably becomes the focus of considerable attention for cost - savings measures . two subsequent legislative actions , the end - stage renal disease program amendment of 1978 ( public law 95 - 292 ) and the omnibus budget reconciliation act ( obra ) of 1981 ( public law 97 - 35 ) contained provisions designed to increase the cost - efficiency of the esrd program . reacting to a decrease in the percent of patients dialyzing at home ( generally considered to be less expensive than facility dialysis ) , congress wrote into the 1978 amendments a number of provisions designed to promote home dialysis . among these were a waiver of the usual 3-month waiting period for entitlement for persons undergoing a self - dialysis training program , full coverage for home dialysis supplies , 100 percent reimbursement for home dialysis equipment , and authorization for the establishment of target - rate reimbursements to encourage home dialysis . these target rates were paid to facilities that would be responsible for providing the necessary supplies , equipment , and back - up to home patients . these included extension of post - transplant medicare entitlement from 1 to 3 years , clarification of coverage for live related donor costs , and clarification of the reimbursement principles on cadaveric organ procurement . the first directed the health care financing administration ( hcfa ) to develop incentive reimbursement rates per dialysis treatment based on a composite of facility and home dialysis rates . because the rates would be lower than the facility rates alone , facilities would have an incentive to dialyze patients at home , where costs are lower . the composite rates that went into effect on august 1 , 1983 , are $ 131 per dialysis treatment for hospital - based facilities and $ 127 per dialysis treatment for free - standing facilities . these replaced the $ 138 screen , applicable to both types of facilities , which had been in effect since the program 's inception . a second provision in obra makes medicare the secondary payer to other insurance coverage for care for the first year following renal failure . rettig and marks ( 1981 ) showed that although average benefit payments rose from $ 14,895 per person in 1974 to $ 20,149 per person in 1978 , when the figures are adjusted for inflation in medical care , the per capita rates remain virtually unchanged . another analysis by lowrie and hampers ( 1981 ) made essentially the same point . using data for the years 1974 through 1979 , they showed that per capita costs for the esrd program rose by 30.8 percent . during the same time , national per capita health expenditures rose by 74.9 percent and the cost per patient day in community hospitals rose by 91.4 percent . thus , it would seem that within the context of rapidly expanding health care costs , the medicare esrd program has experienced relatively modest cost increases . medicare pays for inpatient care and physician costs as well as for dialysis and transplantation . what is the distribution of these costs and have they changed over time ? do costs vary by age , sex , and race categories ? are there differences in the costs of care for different causes of renal failure ( e.g. , glomerulonephritis , primary hypertensive disease , diabetic nephropathy ) ? how do costs of dialysis patients compare with transplant patients ? has this relationship changed over time ? this article is a sequel to a previous analysis of the incidence and prevalence of esrd ( eggers et al . , 1984 ) and attempts to clarify some of the issues outlined above by presenting per capita medicare program expenditures for the years 1974 through 1981 . longitudinal analyses determine the sources of changes in program expenditures and cross - sectional analyses of 1979 data explore demographic and therapy variations in program expenditures . data for this study were taken from two sources : the medicare statistical system ( mss ) and the esrd program management and medical information system ( mmis ) . the mss is a byproduct of the basic administrative data system used to determine beneficiary eligibility and to monitor program utilization and expenditures for the 30 million beneficiaries currently entitled to medicare . the master beneficiary record , a part of the mss , is used to maintain individual entitlement information and to provide the basic age , sex , race , residence , entitlement , and death information used in the analysis . from 1974 through 1979 , approximately 100,000 different people were identified as medicare beneficiaries with esrd for some length of time . the analyses in this study are based on the universe of esrd patients covered by medicare during these years . expenditure data were taken from medicare billing records , also maintained as part of the mss . the bill records include inpatient and skilled nursing facility billings ( hcfa 1453 ) , outpatient billings ( hcfa 1483 ) , physician and supplier billings ( hcfa 1490 ) , and home health billings ( hcfa 1487 ) . specific mmis data elements were taken from various hcfa medical reporting forms developed for the esrd program : these include primary diagnosis and date of first dialysis ( hcfa form 2742 ) , evidence of dialysis services ( hcfa form 2743 ) , and date of transplantation ( hcfa form 2745 ) . nonresponse rates on the mmis forms were in the range of 50 percent or more during the period covered by this analysis . therefore , it was not possible to completely differentiate between dialysis and transplant patients or to assign primary cause of renal failure to all patients . thus , many of the ensuing analyses will , of necessity , be based on subsets of the population for whom mmis data were available . mss and mmis data were linked for each beneficiary to form a single record summarizing entitlement , reimbursement , and medical information . persons with less than a full year of medicare entitlement in any year ( either their first year of entitlement or the year in which they died ) were given a person - year weight equal to the number of months of entitlement divided by 12 . table 1 presents end - stage renal disease ( esrd ) reimbursements , enrollment , and per capita reimbursements for 1974 through 1981 . in 1974 , the first full year of coverage of esrd , total medicare reimbursements for persons with esrd were $ 229 million . by 1981 , reimbursements had risen to $ 1,471 million , over six times more than 1974 , or an annual growth rate of 30.5 percent . however , the rate of growth has slowed considerably in recent years . between 1974 and 1975 , for the most recent year 's experience ( 1980 to 1981 ) the growth has slowed to 17.7 percent . this pattern of growth in reimbursements is largely the result of growth in the esrd population . there were 16,000 esrd enrollees in 1974 . by 1981 , this total was 64,100 , or four times greater . enrollment increases were greatest in the early years of the program . although the overall annual growth rate has been 21.9 percent , it was 41.9 percent from 1974 to 1975 and only 10.9 percent from 1980 to 1981 . per capita reimbursements rose from $ 14,300 in 1974 to $ 23,000 in 1981 , an annual growth rate of 7.0 percent . the growth in total esrd reimbursements can be disaggregated into the two components of enrollment and per capita reimbursements . this analysis shows that 76 percent of the growth is the result of the increase in enrolled population and only 24 percent is because of the increase in reimbursements per enrollee ( klarman et al . , 1970 ) . the distribution of reimbursements by type of service has changed little since the program 's inception . table 2 shows program reimbursement by type of service for the years 1974 through 1981 . outpatient billings , which account for the majority of dialysis reimbursements , accounted for $ 135.5 million in 1974 , and 59 percent of total program expenditures . by 1981 , outpatient billings had risen to $ 732.1 million ( a 27-percent annual rate of increase ) , but accounted for only 50 percent of total program costs . most of this shift in reimbursements was accounted for by an increase in physician / supplier billings . physician / supplier billings include payments for physicians ' services as well as payments to suppliers for furnishing the materials needed for home dialysis . these payments grew from 12 percent of program costs in 1974 ( $ 27.6 million ) to 21 percent of program costs in 1981 ( $ 303.0 million ) . however , most of this shift is an artifact of the manner in which physicians have been paid . in 1974 , all physicians were paid $ 9.60 which was added to the dialysis fee screen ( referred to as the initial method ) . , hcfa instituted an alternative method in which physicians were paid an amount per patient per month . these billings would appear in the physician / supplier records . by 1979 , 75 percent of all physicians were paid through the alternative method . therefore , much of this change in mix of payments is an artifact of billing procedures and not because of any underlying change in the provision of care . interestingly , inpatient reimbursements have remained a relatively constant portion of program expenditures despite the rapid rise in hospital costs in general . inpatient costs were 28 percent of program costs in 1974 ( $ 64.9 million ) and were 29 percent in 1981 ( $ 430.7 million ) . first , transplant patients , who have very high inpatient costs , have decreased as a percent of program enrollment . in 1974 , almost 20 percent of medicare beneficiaries received transplants . by 1981 , a second reason for the stability of inpatient costs is the decrease in hospitalization rates for dialysis patients ( table 3 ) . table 3 shows discharge rates , average length of stay , rates for total days of care , and per capita inpatient reimbursement for dialysis patients for the years 1974 through 1979 . discharge rates decreased by 17 percent and average length of stay decreased by 23 percent , rsulting in a 35 percent decrease in the rate of total days of care . these decreases in total hospital use offset the general increases in hospital per diem costs to the point that per capita inpatient reimbursements in 1979 ( $ 4,924 ) were slightly less than in 1974 ( $ 4,963 ) . patient costs for dialysis and transplants are compared in table 4 . in 1974 , reimbursements for dialysis patients averaged $ 16,558 per person . reimbursements for transplant patients during the same year were $ 21,159 , a difference of 28 percent . however , in the following years , transplant patient costs rose twice as fast ( 10.5 percent per year ) as did dialysis patient costs ( 5.2 percent per year ) . as a result , transplant patient costs in 1979 ( $ 34,914 ) were 64 percent higher than dialysis patient costs ( $ 21,325 ) . of the total cost of care for a transplant patient in 1979 , approximately 45 percent , or $ 15,629 , was attributable to the hospital stay in which the transplant occurred ( table 5 ) . this table also shows hospital charges for the transplant stay broken down by cost center . of the $ 21,951 in total charges , 19 percent was for accommodation ( the average length of stay was nearly 30 days ) . another 7 percent was for intensive care unit services ( about 5 days per transplant stay were spent in the icu ) . laboratory services accounted for $ 3,910 ( 18 percent of the total ) and other ancillary services which include kidney acquisition and dialysis treatments accounted for $ 7,662 ( 25 percent of total charges ) . this section examines variations in medicare payments for esrd patients in 1979 by therapy type , by demographic categories , and by primary cause of renal failure . table 6 shows medicare reimbursements for home and in - unit hemodialysis patients . in 1979 , home patient costs averaged $ 18,659 per person , or 21 percent lower than the average program costs of $ 23,591 for in - unit dialysis patients . it is not possible to determine how much of this differential is a result of lower dialysis costs because much of the home dialysis costs are included with the physician / supplier costs . however , it is interesting to note that home patients use fewer hospital resources than do in - unit patients ( $ 4,743 and $ 5,519 , respectively ) . this may be attributed to the fact that home patients are believed to be in better health than in - unit patients . still , leaving aside the question of case mix , it appears that in 1979 the cost savings to medicare of having patients dialyze at home was approximately $ 5,000 per person . dialysis patient costs are compared to transplant patient costs in table 7 . as discussed above , in 1979 , . nevertheless , almost one - fourth of dialysis patient costs ( $ 4,924 ) are for inpatient care episodes . on average , transplant patients are hospitalized three times for a total of about 50 days during the year in which the transplant occurs ( including the stay for the transplant ) . transplant patients also have higher physician costs than do dialysis patients ( $ 5,722 and $ 3,521 , respectively ) primarily because of transplant surgeons ' fees . according to the 1983 esrd annual report to congress given a more or less random distribution of transplant dates , the average patient will have had 6 months of dialysis prior to the transplant . second , many transplants fail and the beneficiary returns to a dialysis regimen . the kidney graft in about 44 percent of cadaveric transplants and 25 percent of live related donor transplants will fail in the first year following the transplant ( krakauer et al . , 1983 ) therefore , some of the dialysis costs are incurred post transplantation . medicare reimbursements for dialysis and transplant patients by age , sex , and race persons under 25 years of age received $ 3,252 more in reimbursements than persons 65 years of age or over . however , part of this difference could be because of the under - reporting of transplants . about one - half of the transplants occurring in 1979 were reported to the esrd - mmis . the impact of this nonresponse is an upward bias of the dialysis patient costs and this bias is much greater in the under 25 age group where transplants account for 25 percent of all patients than in the 65 or over age group where transplants are very rare . so , the real reimbursement differences by age for dialysis patients are probably less than suggested by this table . there is also a small difference in reimbursements by sex with female dialysis patients accounting for 7 percent higher per capita reimbursements than male dialysis patients ( $ 22,048 and $ 20,693 , respectively ) . differences by race were very slight with white persons receiving two percent less in reimbursements than did black persons . persons 25 - 44 years of age and 45 - 64 years received 5 percent and 15 percent more in reimbursements than did persons under 25 years . increasing costs by age can be attributed to higher rates of transplant failure in the older age groups . as will be shown in a subsequent analysis , transplants that fail are markedly more expensive than successful transplants . it is of interest to examine the extent to which the primary cause of renal failure affects reimbursement amounts . the primary cause of renal failure has a significant impact on patient survival ( eggers et al . , 1984 ) . persons whose renal failure is a result of polycystic kidney disease have the best 5-year survival ( 58 percent ) and persons with diabetic nephropathy experience the worst 5-year survival rate ( 21 percent ) . table 9 shows the per capita reimbursements for dialysis and transplant patients for the five most commonly reported primary diagnoses leading to renal failure . there is very little difference in per capita reimbursements by primary diagnosis for dialysis patients . four of the five diagnoses show reimbursements in the range of $ 20,000 to $ 21,000 . patients with diabetic nephropathy had reimbursement rates of $ 22,770 which was about $ 2,000 higher than the other major diagnostic categories . some of this difference can be attributed to the higher mortality rate among diabetic patients . as shown in table 10 , dying dialysis patients have higher costs than do surviving dialysis patients . transplant patients whose renal failure was a result of hypertensive nephropathy had the highest reimbursements ( $ 38,028 ) , suggesting that these patients have more complications than other patients receiving transplants . table 10 presents 1979 medicare reimbursements for esrd patients by type of therapy and patient outcome . although the average transplant patient accrued $ 34,914 in reimbursements in 1979 , there were large differences depending on graft and patient outcome . for those transplants in which the patient survived and the graft was functioning at the end of the year , the costs ( $ 29,860 ) were $ 5,054 less than the overall average . if the graft failed but the patient survived and returned to dialysis , reimbursements averaged $ 42 , 432 , or $ 12,572 more than the successful transplant . these additional costs result from rejection costs ( the average hospital stay cost for a rejection with nephrectomy was $ 8,941 in 1979 ) and costs of maintenance dialysis after rejection . if the patient received a transplant and died in the same year , the costs were $ 60,679 . for patients receiving a transplant in a year prior to 1979 but having a graft rejection in 1979 , the per capita costs were $ 30,189 . however , for those earlier transplant patients whose graft continued to function through 1979 , the costs to medicare were $ 4,074 per person . for those successful transplant patients who lose entitlement 36 months after the transplant , the costs to medicare drop to $ 0 . recent enrollment data show that about one - half of transplant patients with functioning grafts remain sufficiently disabled to require continued medicare coverage . the remaining patients continue to receive medicare coverage as disabled beneficiaries . as shown in table 4 earlier , in table 10 , dialysis patients are divided into surviving and dying patients . for surviving dialysis patients the per capita costs were $ 19,541 whereas for decedents the costs were $ 28,253 . the costs presented in table 10 have been used with patient and transplant graft survival rates to model the general cost effectiveness of kidney transplantation ( eggers , 1983 ) . the results of that analysis show that the higher initial costs of transplantation are paid back in about four years in terms of person life years and lower program costs when compared with the relatively constant costs of dialysis therapy . if one assumes that a functioning transplant provides a patient with a higher quality of life , then the relative cost - effectiveness of transplantation is increased . table 1 presents end - stage renal disease ( esrd ) reimbursements , enrollment , and per capita reimbursements for 1974 through 1981 . in 1974 , the first full year of coverage of esrd , total medicare reimbursements for persons with esrd were $ 229 million . by 1981 , reimbursements had risen to $ 1,471 million , over six times more than 1974 , or an annual growth rate of 30.5 percent . however , the rate of growth has slowed considerably in recent years . between 1974 and 1975 , for the most recent year 's experience ( 1980 to 1981 ) the growth has slowed to 17.7 percent . this pattern of growth in reimbursements is largely the result of growth in the esrd population . there were 16,000 esrd enrollees in 1974 . by 1981 , this total was 64,100 , or four times greater . enrollment increases were greatest in the early years of the program . although the overall annual growth rate has been 21.9 percent , it was 41.9 percent from 1974 to 1975 and only 10.9 percent from 1980 to 1981 . per capita reimbursements rose from $ 14,300 in 1974 to $ 23,000 in 1981 , an annual growth rate of 7.0 percent . the growth in total esrd reimbursements can be disaggregated into the two components of enrollment and per capita reimbursements . this analysis shows that 76 percent of the growth is the result of the increase in enrolled population and only 24 percent is because of the increase in reimbursements per enrollee ( klarman et al . , 1970 ) . the distribution of reimbursements by type of service has changed little since the program 's inception . table 2 shows program reimbursement by type of service for the years 1974 through 1981 . outpatient billings , which account for the majority of dialysis reimbursements , accounted for $ 135.5 million in 1974 , and 59 percent of total program expenditures . by 1981 , outpatient billings had risen to $ 732.1 million ( a 27-percent annual rate of increase ) , but accounted for only 50 percent of total program costs . most of this shift in reimbursements was accounted for by an increase in physician / supplier billings . physician / supplier billings include payments for physicians ' services as well as payments to suppliers for furnishing the materials needed for home dialysis . these payments grew from 12 percent of program costs in 1974 ( $ 27.6 million ) to 21 percent of program costs in 1981 ( $ 303.0 million ) . however , most of this shift is an artifact of the manner in which physicians have been paid . in 1974 , all physicians were paid $ 9.60 which was added to the dialysis fee screen ( referred to as the initial method ) . , hcfa instituted an alternative method in which physicians were paid an amount per patient per month . these billings would appear in the physician / supplier records . by 1979 , 75 percent of all physicians were paid through the alternative method . therefore , much of this change in mix of payments is an artifact of billing procedures and not because of any underlying change in the provision of care . interestingly , inpatient reimbursements have remained a relatively constant portion of program expenditures despite the rapid rise in hospital costs in general . inpatient costs were 28 percent of program costs in 1974 ( $ 64.9 million ) and were 29 percent in 1981 ( $ 430.7 million ) . first , transplant patients , who have very high inpatient costs , have decreased as a percent of program enrollment . in 1974 , almost 20 percent of medicare beneficiaries received transplants . by 1981 , a second reason for the stability of inpatient costs is the decrease in hospitalization rates for dialysis patients ( table 3 ) . table 3 shows discharge rates , average length of stay , rates for total days of care , and per capita inpatient reimbursement for dialysis patients for the years 1974 through 1979 . discharge rates decreased by 17 percent and average length of stay decreased by 23 percent , rsulting in a 35 percent decrease in the rate of total days of care . these decreases in total hospital use offset the general increases in hospital per diem costs to the point that per capita inpatient reimbursements in 1979 ( $ 4,924 ) were slightly less than in 1974 ( $ 4,963 ) . patient costs for dialysis and transplants are compared in table 4 . in 1974 , reimbursements for dialysis patients averaged $ 16,558 per person . reimbursements for transplant patients during the same year were $ 21,159 , a difference of 28 percent . however , in the following years , transplant patient costs rose twice as fast ( 10.5 percent per year ) as did dialysis patient costs ( 5.2 percent per year ) . as a result , transplant patient costs in 1979 ( $ 34,914 ) were 64 percent higher than dialysis patient costs ( $ 21,325 ) . of the total cost of care for a transplant patient in 1979 , approximately 45 percent , or $ 15,629 , was attributable to the hospital stay in which the transplant occurred ( table 5 ) . this table also shows hospital charges for the transplant stay broken down by cost center . of the $ 21,951 in total charges , 19 percent was for accommodation ( the average length of stay was nearly 30 days ) . another 7 percent was for intensive care unit services ( about 5 days per transplant stay were spent in the icu ) . laboratory services accounted for $ 3,910 ( 18 percent of the total ) and other ancillary services which include kidney acquisition and dialysis treatments accounted for $ 7,662 ( 25 percent of total charges ) . this section examines variations in medicare payments for esrd patients in 1979 by therapy type , by demographic categories , and by primary cause of renal failure . table 6 shows medicare reimbursements for home and in - unit hemodialysis patients . in 1979 , home patient costs averaged $ 18,659 per person , or 21 percent lower than the average program costs of $ 23,591 for in - unit dialysis patients . it is not possible to determine how much of this differential is a result of lower dialysis costs because much of the home dialysis costs are included with the physician / supplier costs . however , it is interesting to note that home patients use fewer hospital resources than do in - unit patients ( $ 4,743 and $ 5,519 , respectively ) . this may be attributed to the fact that home patients are believed to be in better health than in - unit patients . still , leaving aside the question of case mix , it appears that in 1979 the cost savings to medicare of having patients dialyze at home was approximately $ 5,000 per person . dialysis patient costs are compared to transplant patient costs in table 7 . as discussed above , in 1979 , . nevertheless , almost one - fourth of dialysis patient costs ( $ 4,924 ) are for inpatient care episodes . on average , transplant patients are hospitalized three times for a total of about 50 days during the year in which the transplant occurs ( including the stay for the transplant ) . transplant patients also have higher physician costs than do dialysis patients ( $ 5,722 and $ 3,521 , respectively ) primarily because of transplant surgeons ' fees . according to the 1983 esrd annual report to congress given a more or less random distribution of transplant dates , the average patient will have had 6 months of dialysis prior to the transplant . the kidney graft in about 44 percent of cadaveric transplants and 25 percent of live related donor transplants will fail in the first year following the transplant ( krakauer et al . , 1983 ) . therefore , some of the dialysis costs are incurred post transplantation . medicare reimbursements for dialysis and transplant patients by age , sex , and race . for dialysis patients there appears to be a slight inverse relationship of reimbursements with age . persons under 25 years of age received $ 3,252 more in reimbursements than persons 65 years of age or over . however , part of this difference could be because of the under - reporting of transplants . about one - half of the transplants occurring in 1979 were reported to the esrd - mmis . the impact of this nonresponse is an upward bias of the dialysis patient costs and this bias is much greater in the under 25 age group where transplants account for 25 percent of all patients than in the 65 or over age group where transplants are very rare . so , the real reimbursement differences by age for dialysis patients are probably less than suggested by this table . there is also a small difference in reimbursements by sex with female dialysis patients accounting for 7 percent higher per capita reimbursements than male dialysis patients ( $ 22,048 and $ 20,693 , respectively ) . differences by race were very slight with white persons receiving two percent less in reimbursements than did black persons . for transplant patients persons 25 - 44 years of age and 45 - 64 years received 5 percent and 15 percent more in reimbursements than did persons under 25 years . increasing costs by age can be attributed to higher rates of transplant failure in the older age groups . as will be shown in a subsequent analysis , transplants that fail it is of interest to examine the extent to which the primary cause of renal failure affects reimbursement amounts . the primary cause of renal failure has a significant impact on patient survival ( eggers et al . , 1984 ) . persons whose renal failure is a result of polycystic kidney disease have the best 5-year survival ( 58 percent ) and persons with diabetic nephropathy experience the worst 5-year survival rate ( 21 percent ) . table 9 shows the per capita reimbursements for dialysis and transplant patients for the five most commonly reported primary diagnoses leading to renal failure . there is very little difference in per capita reimbursements by primary diagnosis for dialysis patients . four of the five diagnoses show reimbursements in the range of $ 20,000 to $ 21,000 . patients with diabetic nephropathy had reimbursement rates of $ 22,770 which was about $ 2,000 higher than the other major diagnostic categories . some of this difference can be attributed to the higher mortality rate among diabetic patients . as shown in table 10 , transplant patients whose renal failure was a result of hypertensive nephropathy had the highest reimbursements ( $ 38,028 ) , suggesting that these patients have more complications than other patients receiving transplants . table 10 presents 1979 medicare reimbursements for esrd patients by type of therapy and patient outcome . the first five figures show reimbursement amounts for transplant patients by transplant outcome . although the average transplant patient accrued $ 34,914 in reimbursements in 1979 , there were large differences depending on graft and patient outcome . for those transplants in which the patient survived and the graft was functioning at the end of the year , the costs ( $ 29,860 ) were $ 5,054 less than the overall average . if the graft failed but the patient survived and returned to dialysis , reimbursements averaged $ 42 , 432 , or $ 12,572 more than the successful transplant . these additional costs result from rejection costs ( the average hospital stay cost for a rejection with nephrectomy was $ 8,941 in 1979 ) and costs of maintenance dialysis after rejection . if the patient received a transplant and died in the same year , the costs were $ 60,679 . for patients receiving a transplant in a year prior to 1979 but having a graft rejection in 1979 , the per capita costs were $ 30,189 . however , for those earlier transplant patients whose graft continued to function through 1979 , the costs to medicare were $ 4,074 per person . for those successful transplant patients who lose entitlement 36 months after the transplant , the costs to medicare drop to $ 0 . recent enrollment data show that about one - half of transplant patients with functioning grafts remain sufficiently disabled to require continued medicare coverage . the remaining patients continue to receive medicare coverage as disabled beneficiaries . as shown in table 4 earlier , the per capita costs were $ 19,541 whereas for decedents the costs were $ 28,253 . the costs presented in table 10 have been used with patient and transplant graft survival rates to model the general cost effectiveness of kidney transplantation ( eggers , 1983 ) . paid back in about four years in terms of person life years and lower program costs when compared with the relatively constant costs of dialysis therapy . if one assumes that a functioning transplant provides a patient with a higher quality of life , then the relative cost - effectiveness of transplantation is increased . the analyses presented in this article have shown some of the reasons for increases in program expenditures during the early years of the program as well as some variations in program costs in the most recent year for which patient specific data are available ( 1979 ) . the results show that although expenditures rose at a 30-percent annual rate in the first 8 years of the esrd program , most of that increase ( 76 percent ) can be attributed to a rapid growth in the beneficiary population . the per capita costs of the program have risen at a 7-percent rate , well below the general inflation in medical care prices . per capita costs have not risen uniformly . this is most likely because of the fact that providers have been reimbursed for the costs of dialysis only up to a predetermined fee screen . about 75 percent of total costs are attributable to dialysis treatments and physicians ' services . for transplant patients , about one - half of total costs are for the transplant stay itself with an additional $ 2,000 for the surgeon 's fee . however , cumulative transplant patient costs after about four years following a transplant are low enough to offset the higher initial costs . in 1979 , total program costs for home dialysis patients were about $ 5,000 less than for in - unit dialysis patients . although much of this difference is a result of lower dialysis costs , it is likely that home patients are in better health as evidenced by lower hospitalization costs . age appears to have a slight impact on dialysis costs and a more significant impact on transplant costs . the age impact on transplant costs is probably related to the more prevalent use of live related donor kidneys ( and consequent better success rates ) among younger patients . neither sex , race , nor primary diagnosis had much impact on patient costs . although transplant patient costs were rising faster than dialysis patient costs , the impact was partially offset by the fact that transplants are a decreasing percent of the patient population . the trends in program expenditures since 1979 need to be examined as there have been a number of changes that could impact on per capita costs . from 1978 through 1983 , hcfa granted exceptions to the $ 138 dialysis fee screens to 503 facilities . on the other hand , the composite rate that went into effect in august 1983 should have the immediate effect of reducing dialysis costs and should be noticeable in 1984 expenditures . in addition , the 1981 obra change which makes medicare the second payer for the first year after renal failure should reduce medicare expenditures somewhat . hcfa 's office of financial and actuarial analysis estimates that this provision will reduce program expenditures by 1.7 percent by the end of the decade ( hcfa , 1984 ) . two therapy changes following the period covered in this article could impact on program expenditures . one was the introduction and rapid proliferation of continuous ambulatory peritoneal dialysis ( capd ) . in 1979 , only a few hundred persons were on this therapy . by the end of 1983 , over 8,500 persons were on this therapy , almost twice as many as on home hemodialysis . although the dialysis costs may be low , there is some concern that problems with peritonitis may push up hospitalization costs , thus negating any dialysis savings . the other change in therapy is the introduction of cyclosporin as an immunosuppressant for transplant patients . this drug has the potential to reduce graft rejection rates and thus not only improve patient outcomes but decrease program costs as well . recent changes in medicare legislation ( public law 98 - 21 ) will impact on hospitalization through the implementation of the prospective payment system . the impact of this change should be to reduce the rate of increase in transplant patient costs but it will impact on dialysis patients as well . thus , there is reason to believe that medicare program expenditures for esrd will increase at a lower rate in the future . however , the full impact of specific regulatory and legislative changes have yet to be determined . as more current data become available , it will be possible to assess not only changes in reimbursement levels but changes in the population being served and utilization of available therapies .

this article presents detailed analyses of the trends in medicare expenditures for persons with end - stage renal disease . program expenditures increased at an annual rate of 30.5 percent from 1974 to 1981 . three - fourths of this increase was a result of increases in enrollment . per capita reimbursements for dialysis patients increased at a 5.2-percent annual rate and per capita reimbursements for transplant patients increased at a 10.5-percent annual rate . in 1979 , per capita reimbursements for home dialysis patients were $ 5,000 less than for in - unit dialysis patients . patient characteristics such as age , sex , race , and cause of renal failure were , for the most part , unrelated to the costs of dialysis and transplantation .

Introduction Data and methods Results Trends in reimbursements Program expenditures: 1979 Summary and conclusions

end - stage renal disease ( esrd ) is the condition in which the kidneys permanently cease to function at a level that will support life . the two basic therapies for treating this condition , dialysis and transplantation , were effectively developed by the early 1960 's . the fact that thousands of persons were being denied access to these life - saving therapies because of the prohibitively high costs led congress to extend medicare benefits to those persons with end - stage renal disease . entitlement to medicare benefits was extended to persons with end - stage renal disease with the enactment of the social security amendments of 1972 ( public law 92 - 603 , section 299i ) . in 1983 , they accounted for an estimated 3.7 percent of total medicare expenditures ( parts a and b ) and 8.5 percent of part b expenditures ( health care financing administration , 1984 ) . two subsequent legislative actions , the end - stage renal disease program amendment of 1978 ( public law 95 - 292 ) and the omnibus budget reconciliation act ( obra ) of 1981 ( public law 97 - 35 ) contained provisions designed to increase the cost - efficiency of the esrd program . among these were a waiver of the usual 3-month waiting period for entitlement for persons undergoing a self - dialysis training program , full coverage for home dialysis supplies , 100 percent reimbursement for home dialysis equipment , and authorization for the establishment of target - rate reimbursements to encourage home dialysis . a second provision in obra makes medicare the secondary payer to other insurance coverage for care for the first year following renal failure . rettig and marks ( 1981 ) showed that although average benefit payments rose from $ 14,895 per person in 1974 to $ 20,149 per person in 1978 , when the figures are adjusted for inflation in medical care , the per capita rates remain virtually unchanged . using data for the years 1974 through 1979 , they showed that per capita costs for the esrd program rose by 30.8 percent . medicare pays for inpatient care and physician costs as well as for dialysis and transplantation . do costs vary by age , sex , and race categories ? are there differences in the costs of care for different causes of renal failure ( e.g. how do costs of dialysis patients compare with transplant patients ? this article is a sequel to a previous analysis of the incidence and prevalence of esrd ( eggers et al . , 1984 ) and attempts to clarify some of the issues outlined above by presenting per capita medicare program expenditures for the years 1974 through 1981 . the mss is a byproduct of the basic administrative data system used to determine beneficiary eligibility and to monitor program utilization and expenditures for the 30 million beneficiaries currently entitled to medicare . the master beneficiary record , a part of the mss , is used to maintain individual entitlement information and to provide the basic age , sex , race , residence , entitlement , and death information used in the analysis . from 1974 through 1979 , approximately 100,000 different people were identified as medicare beneficiaries with esrd for some length of time . specific mmis data elements were taken from various hcfa medical reporting forms developed for the esrd program : these include primary diagnosis and date of first dialysis ( hcfa form 2742 ) , evidence of dialysis services ( hcfa form 2743 ) , and date of transplantation ( hcfa form 2745 ) . therefore , it was not possible to completely differentiate between dialysis and transplant patients or to assign primary cause of renal failure to all patients . persons with less than a full year of medicare entitlement in any year ( either their first year of entitlement or the year in which they died ) were given a person - year weight equal to the number of months of entitlement divided by 12 . table 1 presents end - stage renal disease ( esrd ) reimbursements , enrollment , and per capita reimbursements for 1974 through 1981 . in 1974 , the first full year of coverage of esrd , total medicare reimbursements for persons with esrd were $ 229 million . by 1981 , reimbursements had risen to $ 1,471 million , over six times more than 1974 , or an annual growth rate of 30.5 percent . between 1974 and 1975 , for the most recent year 's experience ( 1980 to 1981 ) the growth has slowed to 17.7 percent . although the overall annual growth rate has been 21.9 percent , it was 41.9 percent from 1974 to 1975 and only 10.9 percent from 1980 to 1981 . per capita reimbursements rose from $ 14,300 in 1974 to $ 23,000 in 1981 , an annual growth rate of 7.0 percent . the growth in total esrd reimbursements can be disaggregated into the two components of enrollment and per capita reimbursements . this analysis shows that 76 percent of the growth is the result of the increase in enrolled population and only 24 percent is because of the increase in reimbursements per enrollee ( klarman et al . outpatient billings , which account for the majority of dialysis reimbursements , accounted for $ 135.5 million in 1974 , and 59 percent of total program expenditures . physician / supplier billings include payments for physicians ' services as well as payments to suppliers for furnishing the materials needed for home dialysis . however , most of this shift is an artifact of the manner in which physicians have been paid . by 1981 , a second reason for the stability of inpatient costs is the decrease in hospitalization rates for dialysis patients ( table 3 ) . table 3 shows discharge rates , average length of stay , rates for total days of care , and per capita inpatient reimbursement for dialysis patients for the years 1974 through 1979 . these decreases in total hospital use offset the general increases in hospital per diem costs to the point that per capita inpatient reimbursements in 1979 ( $ 4,924 ) were slightly less than in 1974 ( $ 4,963 ) . patient costs for dialysis and transplants are compared in table 4 . in 1974 , reimbursements for dialysis patients averaged $ 16,558 per person . reimbursements for transplant patients during the same year were $ 21,159 , a difference of 28 percent . as a result , transplant patient costs in 1979 ( $ 34,914 ) were 64 percent higher than dialysis patient costs ( $ 21,325 ) . of the total cost of care for a transplant patient in 1979 , approximately 45 percent , or $ 15,629 , was attributable to the hospital stay in which the transplant occurred ( table 5 ) . this section examines variations in medicare payments for esrd patients in 1979 by therapy type , by demographic categories , and by primary cause of renal failure . table 6 shows medicare reimbursements for home and in - unit hemodialysis patients . in 1979 , home patient costs averaged $ 18,659 per person , or 21 percent lower than the average program costs of $ 23,591 for in - unit dialysis patients . it is not possible to determine how much of this differential is a result of lower dialysis costs because much of the home dialysis costs are included with the physician / supplier costs . however , it is interesting to note that home patients use fewer hospital resources than do in - unit patients ( $ 4,743 and $ 5,519 , respectively ) . this may be attributed to the fact that home patients are believed to be in better health than in - unit patients . as discussed above , in 1979 , . on average , transplant patients are hospitalized three times for a total of about 50 days during the year in which the transplant occurs ( including the stay for the transplant ) . transplant patients also have higher physician costs than do dialysis patients ( $ 5,722 and $ 3,521 , respectively ) primarily because of transplant surgeons ' fees . according to the 1983 esrd annual report to congress given a more or less random distribution of transplant dates , the average patient will have had 6 months of dialysis prior to the transplant . medicare reimbursements for dialysis and transplant patients by age , sex , and race persons under 25 years of age received $ 3,252 more in reimbursements than persons 65 years of age or over . about one - half of the transplants occurring in 1979 were reported to the esrd - mmis . so , the real reimbursement differences by age for dialysis patients are probably less than suggested by this table . there is also a small difference in reimbursements by sex with female dialysis patients accounting for 7 percent higher per capita reimbursements than male dialysis patients ( $ 22,048 and $ 20,693 , respectively ) . it is of interest to examine the extent to which the primary cause of renal failure affects reimbursement amounts . the primary cause of renal failure has a significant impact on patient survival ( eggers et al . persons whose renal failure is a result of polycystic kidney disease have the best 5-year survival ( 58 percent ) and persons with diabetic nephropathy experience the worst 5-year survival rate ( 21 percent ) . table 9 shows the per capita reimbursements for dialysis and transplant patients for the five most commonly reported primary diagnoses leading to renal failure . there is very little difference in per capita reimbursements by primary diagnosis for dialysis patients . some of this difference can be attributed to the higher mortality rate among diabetic patients . transplant patients whose renal failure was a result of hypertensive nephropathy had the highest reimbursements ( $ 38,028 ) , suggesting that these patients have more complications than other patients receiving transplants . for those transplants in which the patient survived and the graft was functioning at the end of the year , the costs ( $ 29,860 ) were $ 5,054 less than the overall average . for patients receiving a transplant in a year prior to 1979 but having a graft rejection in 1979 , the per capita costs were $ 30,189 . however , for those earlier transplant patients whose graft continued to function through 1979 , the costs to medicare were $ 4,074 per person . for those successful transplant patients who lose entitlement 36 months after the transplant , the costs to medicare drop to $ 0 . for surviving dialysis patients the per capita costs were $ 19,541 whereas for decedents the costs were $ 28,253 . the results of that analysis show that the higher initial costs of transplantation are paid back in about four years in terms of person life years and lower program costs when compared with the relatively constant costs of dialysis therapy . table 1 presents end - stage renal disease ( esrd ) reimbursements , enrollment , and per capita reimbursements for 1974 through 1981 . in 1974 , the first full year of coverage of esrd , total medicare reimbursements for persons with esrd were $ 229 million . by 1981 , reimbursements had risen to $ 1,471 million , over six times more than 1974 , or an annual growth rate of 30.5 percent . between 1974 and 1975 , for the most recent year 's experience ( 1980 to 1981 ) the growth has slowed to 17.7 percent . although the overall annual growth rate has been 21.9 percent , it was 41.9 percent from 1974 to 1975 and only 10.9 percent from 1980 to 1981 . per capita reimbursements rose from $ 14,300 in 1974 to $ 23,000 in 1981 , an annual growth rate of 7.0 percent . the growth in total esrd reimbursements can be disaggregated into the two components of enrollment and per capita reimbursements . outpatient billings , which account for the majority of dialysis reimbursements , accounted for $ 135.5 million in 1974 , and 59 percent of total program expenditures . by 1981 , outpatient billings had risen to $ 732.1 million ( a 27-percent annual rate of increase ) , but accounted for only 50 percent of total program costs . physician / supplier billings include payments for physicians ' services as well as payments to suppliers for furnishing the materials needed for home dialysis . by 1981 , a second reason for the stability of inpatient costs is the decrease in hospitalization rates for dialysis patients ( table 3 ) . table 3 shows discharge rates , average length of stay , rates for total days of care , and per capita inpatient reimbursement for dialysis patients for the years 1974 through 1979 . these decreases in total hospital use offset the general increases in hospital per diem costs to the point that per capita inpatient reimbursements in 1979 ( $ 4,924 ) were slightly less than in 1974 ( $ 4,963 ) . in 1974 , reimbursements for dialysis patients averaged $ 16,558 per person . reimbursements for transplant patients during the same year were $ 21,159 , a difference of 28 percent . as a result , transplant patient costs in 1979 ( $ 34,914 ) were 64 percent higher than dialysis patient costs ( $ 21,325 ) . of the total cost of care for a transplant patient in 1979 , approximately 45 percent , or $ 15,629 , was attributable to the hospital stay in which the transplant occurred ( table 5 ) . this section examines variations in medicare payments for esrd patients in 1979 by therapy type , by demographic categories , and by primary cause of renal failure . table 6 shows medicare reimbursements for home and in - unit hemodialysis patients . in 1979 , home patient costs averaged $ 18,659 per person , or 21 percent lower than the average program costs of $ 23,591 for in - unit dialysis patients . it is not possible to determine how much of this differential is a result of lower dialysis costs because much of the home dialysis costs are included with the physician / supplier costs . however , it is interesting to note that home patients use fewer hospital resources than do in - unit patients ( $ 4,743 and $ 5,519 , respectively ) . this may be attributed to the fact that home patients are believed to be in better health than in - unit patients . still , leaving aside the question of case mix , it appears that in 1979 the cost savings to medicare of having patients dialyze at home was approximately $ 5,000 per person . as discussed above , in 1979 , . transplant patients also have higher physician costs than do dialysis patients ( $ 5,722 and $ 3,521 , respectively ) primarily because of transplant surgeons ' fees . according to the 1983 esrd annual report to congress given a more or less random distribution of transplant dates , the average patient will have had 6 months of dialysis prior to the transplant . medicare reimbursements for dialysis and transplant patients by age , sex , and race . for dialysis patients there appears to be a slight inverse relationship of reimbursements with age . however , part of this difference could be because of the under - reporting of transplants . about one - half of the transplants occurring in 1979 were reported to the esrd - mmis . the impact of this nonresponse is an upward bias of the dialysis patient costs and this bias is much greater in the under 25 age group where transplants account for 25 percent of all patients than in the 65 or over age group where transplants are very rare . so , the real reimbursement differences by age for dialysis patients are probably less than suggested by this table . there is also a small difference in reimbursements by sex with female dialysis patients accounting for 7 percent higher per capita reimbursements than male dialysis patients ( $ 22,048 and $ 20,693 , respectively ) . for transplant patients persons 25 - 44 years of age and 45 - 64 years received 5 percent and 15 percent more in reimbursements than did persons under 25 years . as will be shown in a subsequent analysis , transplants that fail it is of interest to examine the extent to which the primary cause of renal failure affects reimbursement amounts . the primary cause of renal failure has a significant impact on patient survival ( eggers et al . persons whose renal failure is a result of polycystic kidney disease have the best 5-year survival ( 58 percent ) and persons with diabetic nephropathy experience the worst 5-year survival rate ( 21 percent ) . table 9 shows the per capita reimbursements for dialysis and transplant patients for the five most commonly reported primary diagnoses leading to renal failure . there is very little difference in per capita reimbursements by primary diagnosis for dialysis patients . some of this difference can be attributed to the higher mortality rate among diabetic patients . as shown in table 10 , transplant patients whose renal failure was a result of hypertensive nephropathy had the highest reimbursements ( $ 38,028 ) , suggesting that these patients have more complications than other patients receiving transplants . the first five figures show reimbursement amounts for transplant patients by transplant outcome . for those transplants in which the patient survived and the graft was functioning at the end of the year , the costs ( $ 29,860 ) were $ 5,054 less than the overall average . if the patient received a transplant and died in the same year , the costs were $ 60,679 . for patients receiving a transplant in a year prior to 1979 but having a graft rejection in 1979 , the per capita costs were $ 30,189 . however , for those earlier transplant patients whose graft continued to function through 1979 , the costs to medicare were $ 4,074 per person . for those successful transplant patients who lose entitlement 36 months after the transplant , the costs to medicare drop to $ 0 . as shown in table 4 earlier , the per capita costs were $ 19,541 whereas for decedents the costs were $ 28,253 . paid back in about four years in terms of person life years and lower program costs when compared with the relatively constant costs of dialysis therapy . the analyses presented in this article have shown some of the reasons for increases in program expenditures during the early years of the program as well as some variations in program costs in the most recent year for which patient specific data are available ( 1979 ) . the results show that although expenditures rose at a 30-percent annual rate in the first 8 years of the esrd program , most of that increase ( 76 percent ) can be attributed to a rapid growth in the beneficiary population . the per capita costs of the program have risen at a 7-percent rate , well below the general inflation in medical care prices . this is most likely because of the fact that providers have been reimbursed for the costs of dialysis only up to a predetermined fee screen . for transplant patients , about one - half of total costs are for the transplant stay itself with an additional $ 2,000 for the surgeon 's fee . in 1979 , total program costs for home dialysis patients were about $ 5,000 less than for in - unit dialysis patients . although much of this difference is a result of lower dialysis costs , it is likely that home patients are in better health as evidenced by lower hospitalization costs . neither sex , race , nor primary diagnosis had much impact on patient costs . the trends in program expenditures since 1979 need to be examined as there have been a number of changes that could impact on per capita costs . in addition , the 1981 obra change which makes medicare the second payer for the first year after renal failure should reduce medicare expenditures somewhat . hcfa 's office of financial and actuarial analysis estimates that this provision will reduce program expenditures by 1.7 percent by the end of the decade ( hcfa , 1984 ) . two therapy changes following the period covered in this article could impact on program expenditures . in 1979 , only a few hundred persons were on this therapy . the other change in therapy is the introduction of cyclosporin as an immunosuppressant for transplant patients . recent changes in medicare legislation ( public law 98 - 21 ) will impact on hospitalization through the implementation of the prospective payment system . the impact of this change should be to reduce the rate of increase in transplant patient costs but it will impact on dialysis patients as well . thus , there is reason to believe that medicare program expenditures for esrd will increase at a lower rate in the future .

end - stage renal disease ( esrd ) is the condition in which the kidneys permanently cease to function at a level that will support life . however , the high cost of both treatments prevented the widespread expansion of these therapies to all persons who could benefit from them . the fact that thousands of persons were being denied access to these life - saving therapies because of the prohibitively high costs led congress to extend medicare benefits to those persons with end - stage renal disease . entitlement to medicare benefits was extended to persons with end - stage renal disease with the enactment of the social security amendments of 1972 ( public law 92 - 603 , section 299i ) . have put this problem in perspective : the figures used in the 1972 senate debate authorizing the programing were unreasonably low and quite misleading and , in early 1973 , the subject of a well - publicized controversy . in 1983 , they accounted for an estimated 3.7 percent of total medicare expenditures ( parts a and b ) and 8.5 percent of part b expenditures ( health care financing administration , 1984 ) . when an easily identifiable group such as this has extremely high health care costs , it inevitably becomes the focus of considerable attention for cost - savings measures . two subsequent legislative actions , the end - stage renal disease program amendment of 1978 ( public law 95 - 292 ) and the omnibus budget reconciliation act ( obra ) of 1981 ( public law 97 - 35 ) contained provisions designed to increase the cost - efficiency of the esrd program . reacting to a decrease in the percent of patients dialyzing at home ( generally considered to be less expensive than facility dialysis ) , congress wrote into the 1978 amendments a number of provisions designed to promote home dialysis . among these were a waiver of the usual 3-month waiting period for entitlement for persons undergoing a self - dialysis training program , full coverage for home dialysis supplies , 100 percent reimbursement for home dialysis equipment , and authorization for the establishment of target - rate reimbursements to encourage home dialysis . these target rates were paid to facilities that would be responsible for providing the necessary supplies , equipment , and back - up to home patients . these included extension of post - transplant medicare entitlement from 1 to 3 years , clarification of coverage for live related donor costs , and clarification of the reimbursement principles on cadaveric organ procurement . the first directed the health care financing administration ( hcfa ) to develop incentive reimbursement rates per dialysis treatment based on a composite of facility and home dialysis rates . because the rates would be lower than the facility rates alone , facilities would have an incentive to dialyze patients at home , where costs are lower . the composite rates that went into effect on august 1 , 1983 , are $ 131 per dialysis treatment for hospital - based facilities and $ 127 per dialysis treatment for free - standing facilities . these replaced the $ 138 screen , applicable to both types of facilities , which had been in effect since the program 's inception . a second provision in obra makes medicare the secondary payer to other insurance coverage for care for the first year following renal failure . rettig and marks ( 1981 ) showed that although average benefit payments rose from $ 14,895 per person in 1974 to $ 20,149 per person in 1978 , when the figures are adjusted for inflation in medical care , the per capita rates remain virtually unchanged . during the same time , national per capita health expenditures rose by 74.9 percent and the cost per patient day in community hospitals rose by 91.4 percent . thus , it would seem that within the context of rapidly expanding health care costs , the medicare esrd program has experienced relatively modest cost increases . are there differences in the costs of care for different causes of renal failure ( e.g. this article is a sequel to a previous analysis of the incidence and prevalence of esrd ( eggers et al . , 1984 ) and attempts to clarify some of the issues outlined above by presenting per capita medicare program expenditures for the years 1974 through 1981 . longitudinal analyses determine the sources of changes in program expenditures and cross - sectional analyses of 1979 data explore demographic and therapy variations in program expenditures . data for this study were taken from two sources : the medicare statistical system ( mss ) and the esrd program management and medical information system ( mmis ) . the mss is a byproduct of the basic administrative data system used to determine beneficiary eligibility and to monitor program utilization and expenditures for the 30 million beneficiaries currently entitled to medicare . the master beneficiary record , a part of the mss , is used to maintain individual entitlement information and to provide the basic age , sex , race , residence , entitlement , and death information used in the analysis . the analyses in this study are based on the universe of esrd patients covered by medicare during these years . specific mmis data elements were taken from various hcfa medical reporting forms developed for the esrd program : these include primary diagnosis and date of first dialysis ( hcfa form 2742 ) , evidence of dialysis services ( hcfa form 2743 ) , and date of transplantation ( hcfa form 2745 ) . nonresponse rates on the mmis forms were in the range of 50 percent or more during the period covered by this analysis . therefore , it was not possible to completely differentiate between dialysis and transplant patients or to assign primary cause of renal failure to all patients . thus , many of the ensuing analyses will , of necessity , be based on subsets of the population for whom mmis data were available . persons with less than a full year of medicare entitlement in any year ( either their first year of entitlement or the year in which they died ) were given a person - year weight equal to the number of months of entitlement divided by 12 . table 1 presents end - stage renal disease ( esrd ) reimbursements , enrollment , and per capita reimbursements for 1974 through 1981 . in 1974 , the first full year of coverage of esrd , total medicare reimbursements for persons with esrd were $ 229 million . between 1974 and 1975 , for the most recent year 's experience ( 1980 to 1981 ) the growth has slowed to 17.7 percent . this pattern of growth in reimbursements is largely the result of growth in the esrd population . the growth in total esrd reimbursements can be disaggregated into the two components of enrollment and per capita reimbursements . this analysis shows that 76 percent of the growth is the result of the increase in enrolled population and only 24 percent is because of the increase in reimbursements per enrollee ( klarman et al . outpatient billings , which account for the majority of dialysis reimbursements , accounted for $ 135.5 million in 1974 , and 59 percent of total program expenditures . these payments grew from 12 percent of program costs in 1974 ( $ 27.6 million ) to 21 percent of program costs in 1981 ( $ 303.0 million ) . however , most of this shift is an artifact of the manner in which physicians have been paid . therefore , much of this change in mix of payments is an artifact of billing procedures and not because of any underlying change in the provision of care . interestingly , inpatient reimbursements have remained a relatively constant portion of program expenditures despite the rapid rise in hospital costs in general . first , transplant patients , who have very high inpatient costs , have decreased as a percent of program enrollment . by 1981 , a second reason for the stability of inpatient costs is the decrease in hospitalization rates for dialysis patients ( table 3 ) . table 3 shows discharge rates , average length of stay , rates for total days of care , and per capita inpatient reimbursement for dialysis patients for the years 1974 through 1979 . discharge rates decreased by 17 percent and average length of stay decreased by 23 percent , rsulting in a 35 percent decrease in the rate of total days of care . these decreases in total hospital use offset the general increases in hospital per diem costs to the point that per capita inpatient reimbursements in 1979 ( $ 4,924 ) were slightly less than in 1974 ( $ 4,963 ) . reimbursements for transplant patients during the same year were $ 21,159 , a difference of 28 percent . however , in the following years , transplant patient costs rose twice as fast ( 10.5 percent per year ) as did dialysis patient costs ( 5.2 percent per year ) . of the total cost of care for a transplant patient in 1979 , approximately 45 percent , or $ 15,629 , was attributable to the hospital stay in which the transplant occurred ( table 5 ) . of the $ 21,951 in total charges , 19 percent was for accommodation ( the average length of stay was nearly 30 days ) . laboratory services accounted for $ 3,910 ( 18 percent of the total ) and other ancillary services which include kidney acquisition and dialysis treatments accounted for $ 7,662 ( 25 percent of total charges ) . this section examines variations in medicare payments for esrd patients in 1979 by therapy type , by demographic categories , and by primary cause of renal failure . in 1979 , home patient costs averaged $ 18,659 per person , or 21 percent lower than the average program costs of $ 23,591 for in - unit dialysis patients . it is not possible to determine how much of this differential is a result of lower dialysis costs because much of the home dialysis costs are included with the physician / supplier costs . however , it is interesting to note that home patients use fewer hospital resources than do in - unit patients ( $ 4,743 and $ 5,519 , respectively ) . this may be attributed to the fact that home patients are believed to be in better health than in - unit patients . still , leaving aside the question of case mix , it appears that in 1979 the cost savings to medicare of having patients dialyze at home was approximately $ 5,000 per person . on average , transplant patients are hospitalized three times for a total of about 50 days during the year in which the transplant occurs ( including the stay for the transplant ) . according to the 1983 esrd annual report to congress given a more or less random distribution of transplant dates , the average patient will have had 6 months of dialysis prior to the transplant . the kidney graft in about 44 percent of cadaveric transplants and 25 percent of live related donor transplants will fail in the first year following the transplant ( krakauer et al . medicare reimbursements for dialysis and transplant patients by age , sex , and race persons under 25 years of age received $ 3,252 more in reimbursements than persons 65 years of age or over . the impact of this nonresponse is an upward bias of the dialysis patient costs and this bias is much greater in the under 25 age group where transplants account for 25 percent of all patients than in the 65 or over age group where transplants are very rare . there is also a small difference in reimbursements by sex with female dialysis patients accounting for 7 percent higher per capita reimbursements than male dialysis patients ( $ 22,048 and $ 20,693 , respectively ) . persons 25 - 44 years of age and 45 - 64 years received 5 percent and 15 percent more in reimbursements than did persons under 25 years . increasing costs by age can be attributed to higher rates of transplant failure in the older age groups . it is of interest to examine the extent to which the primary cause of renal failure affects reimbursement amounts . persons whose renal failure is a result of polycystic kidney disease have the best 5-year survival ( 58 percent ) and persons with diabetic nephropathy experience the worst 5-year survival rate ( 21 percent ) . table 9 shows the per capita reimbursements for dialysis and transplant patients for the five most commonly reported primary diagnoses leading to renal failure . four of the five diagnoses show reimbursements in the range of $ 20,000 to $ 21,000 . patients with diabetic nephropathy had reimbursement rates of $ 22,770 which was about $ 2,000 higher than the other major diagnostic categories . transplant patients whose renal failure was a result of hypertensive nephropathy had the highest reimbursements ( $ 38,028 ) , suggesting that these patients have more complications than other patients receiving transplants . table 10 presents 1979 medicare reimbursements for esrd patients by type of therapy and patient outcome . for those transplants in which the patient survived and the graft was functioning at the end of the year , the costs ( $ 29,860 ) were $ 5,054 less than the overall average . if the graft failed but the patient survived and returned to dialysis , reimbursements averaged $ 42 , 432 , or $ 12,572 more than the successful transplant . these additional costs result from rejection costs ( the average hospital stay cost for a rejection with nephrectomy was $ 8,941 in 1979 ) and costs of maintenance dialysis after rejection . however , for those earlier transplant patients whose graft continued to function through 1979 , the costs to medicare were $ 4,074 per person . for those successful transplant patients who lose entitlement 36 months after the transplant , the costs to medicare drop to $ 0 . recent enrollment data show that about one - half of transplant patients with functioning grafts remain sufficiently disabled to require continued medicare coverage . as shown in table 4 earlier , in table 10 , dialysis patients are divided into surviving and dying patients . the costs presented in table 10 have been used with patient and transplant graft survival rates to model the general cost effectiveness of kidney transplantation ( eggers , 1983 ) . the results of that analysis show that the higher initial costs of transplantation are paid back in about four years in terms of person life years and lower program costs when compared with the relatively constant costs of dialysis therapy . if one assumes that a functioning transplant provides a patient with a higher quality of life , then the relative cost - effectiveness of transplantation is increased . in 1974 , the first full year of coverage of esrd , total medicare reimbursements for persons with esrd were $ 229 million . this analysis shows that 76 percent of the growth is the result of the increase in enrolled population and only 24 percent is because of the increase in reimbursements per enrollee ( klarman et al . therefore , much of this change in mix of payments is an artifact of billing procedures and not because of any underlying change in the provision of care . by 1981 , a second reason for the stability of inpatient costs is the decrease in hospitalization rates for dialysis patients ( table 3 ) . table 3 shows discharge rates , average length of stay , rates for total days of care , and per capita inpatient reimbursement for dialysis patients for the years 1974 through 1979 . discharge rates decreased by 17 percent and average length of stay decreased by 23 percent , rsulting in a 35 percent decrease in the rate of total days of care . these decreases in total hospital use offset the general increases in hospital per diem costs to the point that per capita inpatient reimbursements in 1979 ( $ 4,924 ) were slightly less than in 1974 ( $ 4,963 ) . however , in the following years , transplant patient costs rose twice as fast ( 10.5 percent per year ) as did dialysis patient costs ( 5.2 percent per year ) . of the total cost of care for a transplant patient in 1979 , approximately 45 percent , or $ 15,629 , was attributable to the hospital stay in which the transplant occurred ( table 5 ) . of the $ 21,951 in total charges , 19 percent was for accommodation ( the average length of stay was nearly 30 days ) . laboratory services accounted for $ 3,910 ( 18 percent of the total ) and other ancillary services which include kidney acquisition and dialysis treatments accounted for $ 7,662 ( 25 percent of total charges ) . this section examines variations in medicare payments for esrd patients in 1979 by therapy type , by demographic categories , and by primary cause of renal failure . it is not possible to determine how much of this differential is a result of lower dialysis costs because much of the home dialysis costs are included with the physician / supplier costs . however , it is interesting to note that home patients use fewer hospital resources than do in - unit patients ( $ 4,743 and $ 5,519 , respectively ) . still , leaving aside the question of case mix , it appears that in 1979 the cost savings to medicare of having patients dialyze at home was approximately $ 5,000 per person . on average , transplant patients are hospitalized three times for a total of about 50 days during the year in which the transplant occurs ( including the stay for the transplant ) . the kidney graft in about 44 percent of cadaveric transplants and 25 percent of live related donor transplants will fail in the first year following the transplant ( krakauer et al . the impact of this nonresponse is an upward bias of the dialysis patient costs and this bias is much greater in the under 25 age group where transplants account for 25 percent of all patients than in the 65 or over age group where transplants are very rare . there is also a small difference in reimbursements by sex with female dialysis patients accounting for 7 percent higher per capita reimbursements than male dialysis patients ( $ 22,048 and $ 20,693 , respectively ) . for transplant patients persons 25 - 44 years of age and 45 - 64 years received 5 percent and 15 percent more in reimbursements than did persons under 25 years . as will be shown in a subsequent analysis , transplants that fail it is of interest to examine the extent to which the primary cause of renal failure affects reimbursement amounts . persons whose renal failure is a result of polycystic kidney disease have the best 5-year survival ( 58 percent ) and persons with diabetic nephropathy experience the worst 5-year survival rate ( 21 percent ) . table 9 shows the per capita reimbursements for dialysis and transplant patients for the five most commonly reported primary diagnoses leading to renal failure . patients with diabetic nephropathy had reimbursement rates of $ 22,770 which was about $ 2,000 higher than the other major diagnostic categories . as shown in table 10 , transplant patients whose renal failure was a result of hypertensive nephropathy had the highest reimbursements ( $ 38,028 ) , suggesting that these patients have more complications than other patients receiving transplants . for those transplants in which the patient survived and the graft was functioning at the end of the year , the costs ( $ 29,860 ) were $ 5,054 less than the overall average . these additional costs result from rejection costs ( the average hospital stay cost for a rejection with nephrectomy was $ 8,941 in 1979 ) and costs of maintenance dialysis after rejection . for those successful transplant patients who lose entitlement 36 months after the transplant , the costs to medicare drop to $ 0 . the costs presented in table 10 have been used with patient and transplant graft survival rates to model the general cost effectiveness of kidney transplantation ( eggers , 1983 ) . the analyses presented in this article have shown some of the reasons for increases in program expenditures during the early years of the program as well as some variations in program costs in the most recent year for which patient specific data are available ( 1979 ) . the results show that although expenditures rose at a 30-percent annual rate in the first 8 years of the esrd program , most of that increase ( 76 percent ) can be attributed to a rapid growth in the beneficiary population . for transplant patients , about one - half of total costs are for the transplant stay itself with an additional $ 2,000 for the surgeon 's fee . the age impact on transplant costs is probably related to the more prevalent use of live related donor kidneys ( and consequent better success rates ) among younger patients . on the other hand , the composite rate that went into effect in august 1983 should have the immediate effect of reducing dialysis costs and should be noticeable in 1984 expenditures .

chronic disease is a serious and expensive public health problem worldwide . in chronic illness care , heart failure ( hf ) is one of the few cardiovascular diagnoses where disease prevalence is rising rather than falling.1,2 owing to a rapidly aging population and improved survival from acute cardiac events , approximately 5.8 million americans are living with hf , with an estimated incidence of 660,000 new cases each year.1,2 in adults , hf has emerged as a common principal medicare hospital discharge diagnosis , and a leading cause of readmission within 30 days of an hf admission.2,36 although patients hospitalized with acute decompensated hf improve during the incident admission , the long - term all - cause mortality rate remains high and has improved little over time despite important therapeutic advances and national quality improvement efforts.2,3 today , whether engaging in a health - promoting activity such as exercise or managing a chronic disease such as hf , the individual is responsible for actively managing day - to - day activities , a concept commonly referred to as self - management . self - management strategies have become a core component and major theme of many disease management programs and chronic care models at organizational levels.7 the clinical trajectory of chronic hf is characterized by chronic symptoms interspersed with acute symptoms , which often result in adverse events and poor outcomes . one process for reducing the burden of hf symptoms in the everyday life of those living with chronic hf is to identify effective approaches to chronic illness care , which support successful self - management.8,9 furthermore , to maximize effective behavioral interventions , efforts must focus on understanding the challenges individuals face in managing the complex demands of their illness and the often multiple and competing conditions . gaps in our understanding of the characteristics that influence self - management behaviors , and the lack of evidence that self - management translates to better health outcomes in hf patients , suggest a need for further dialog among health care providers and clinicians interested in improving care for people living with chronic hf . the aim of this narrative literature synthesis is to contribute to the evidence base on self - management in hf by describing where we have been , what additional information we may need , and where we need to go with self - management in chronic hf care . over the past few decades , the united states ( us ) has witnessed increasing prevalence of chronic diseases and skyrocketing health care costs . because of the tremendous clinical and financial impact of chronic illness care , managing chronic disease has become a focal point for health care stakeholders , policy makers , and researchers . adding to the mounting evidence that chronic illness care is fragmented and poorly coordinated is the criticism that us health care systems provide health care on an episodic , acute care basis , and lack systematic approaches to managing chronic disease.7,8 to correct deficiencies in the organization and delivery of chronic care and to reduce the burden of escalating costs , disease management emerged as a comprehensive approach and strategic model for chronic illness care.10 self - management is the catalyst or backbone for many disease management approaches because it is patient - centered , focuses on helping people with chronic disease become more informed about their illnesses , and actively engages patients in their own health care . the institute of medicine ( iom ) identified self - management as a top priority for us health care . in addition , the centers for disease control and prevention ( cdc ) state of aging and health in america report also prioritized self - management , with six of its seven calls - to - action identifying self - management as a central pathway to improving outcomes in people with chronic disease.7,11 for this review , chronic disease self - management ( cdsm ) will be used as an umbrella term to cover the broad diversity of programs , approaches , models , and interventions used to improve the quality of care for people living with chronic disease . it is important to highlight that the terms disease management and self - management have been used in a variety of ways in the literature . a major challenge for dissemination of research outcomes for chronic illness care has been the absence of universally accepted definitions for disease management and self - management , or standardization for cdsm programs and strategies.10 despite the encumbrance imposed by the lack of standardization , both private and public interest in cdsm grew , and many of these strategies were adopted on a wider scale by the mid-1990s . once established , this interest spread rapidly , accompanied by comprehensive initiatives to improve chronic illness care and outcomes while reducing health care expenditures.12 in broad terms , cdsm programs are population - based approaches that engage in collaborative practice using multidisciplinary health care teams with specialized education and training . the widespread attraction of self - management coincided with a period of significant transition within the us health care delivery system from a paternalistic model of acute medical care , whereby patients were passive health care recipients , to one in which patients became active participants in their health care . over the past two decades , research has concluded that chronic hf care is a staggering and expensive public health problem . us health care systems are under pressure to slow or reverse the often poor outcomes , escalating health care costs , and high utilization rates associated with chronic illness care.8,10,13 adding to this burden are demographic projections that suggest dramatic increases in chronic illness care and resource utilization . this concern led to recommendations from policy makers for further research to identify optimal cdsm programs , to change patient behaviors , and to improve the health of the chronically ill.14 as with other chronic conditions , the major goals of cdsm in hf are to reduce symptoms and medical care costs while improving clinical outcomes . the interchangeable use of the terms self - care and self - management and the struggle to reach consensus or a gold standard definition has imposed limitations on understanding and promoting self - management in chronic hf care . to remain consistent with national goals advocated by the iom , the cdc , the agency for healthcare research and quality ( ahrq ) , and the acronym cdsm , the term self - management will be used . self - management is derived from definitions set out by lorig and holman15 and barlow et al16 based on the individual s ability ( problem solving , decision making , resource utilization , formation of patient - provider partnerships , action planning , and tailoring of daily activities ) to undertake and manage day - to - day tasks , inherent lifestyle changes , physical symptoms , and psychosocial consequences of health and well - being over the lifetime of an illness . throughout the remainder of this review , self - management will be considered as interchangeable with the terms self - care or self - care behaviors . despite differences over terminology and definitions and their conceptualization in the hf literature , there is wide acceptance of the american heart association s ( aha ) recommended behaviors for persons living with hf ( medication adherence , symptom management , dietary adherence , exercise , smoking cessation , and preventative behaviors).17 hf patients are strongly encouraged by clinicians to regularly take medications , monitor their condition and symptoms , keep appointments , and contact their health care providers when needed.17 strategies such as monitoring weight , remembering to take medications daily and on time , and following a low - salt diet are among the core recommendations in hf guidelines and have been shown to be beneficial for hf patients.1,17 regardless of how seemingly simple these recommendations may appear , making plans to adhere to , and apply , the prescribed behavior changes in daily activities requires decision making and problem solving skills for self - management.17 with or without the help of family members or caregivers , it is the patient s responsibility to integrate the vast majority of hf care into his or her daily life.17 the challenges associated with managing hf and adhering to self - management are proving to be difficult problems for health - care providers , policy makers , and patients . first , hf is a complex disease that requires substantial resources for chronic medical management ; despite significant advances in hf therapies , hospital readmission rates in hf patients remain high . secondly , because it is difficult for clinicians to keep abreast of the latest recommendations and research findings , adherence to published guidelines and life saving therapies remains less than ideal.18 many people living with hf are elderly , are symptomatic , lack social and financial support , and have more than one comorbid condition , making their hf care and management complicated.19 finally , research on specific behaviors or characteristics of patients with hf is scarce . further insight into the answers or reasons for the lack of patient adherence , or the ability to identify potential barriers to self - management for risk - stratification in hf patients is greatly needed.20 hf places a tremendous strain on the patient , family members , community , and health care system because there is no silver bullet or are longer life expectancies and increasing numbers of people with hf living with other conditions . common comorbid conditions among medicare - aged beneficiaries with hf include hypertension , diabetes mellitus , dyslipidemia , chronic lung disease , renal dysfunction , cognitive impairment , and osteoarthritis / osteoporosis.21,22 in recognition of the complexity of the problem and the substantial demand for health care resources associated with chronic hf care , the aha , the iom , the american college of cardiology ( acc ) , the joint commission on accreditation of hospital organizations ( tjc ) , and the centers for medicare and medicaid services ( cms ) established key quality hf indicators and national goals to standardize hospital - level performance and reduce the high rates of hospital readmission and cost associated with poor clinical outcomes as a focus area for quality improvement efforts.1,6,7,23 research studies confirm that chronic hf can be extremely debilitating , with symptomatic exacerbations that often lead to episodes of acute decompensation , frequent hospital admissions , and premature death.36 patient hf hospitalization and rehospitalization rates have attracted considerable attention from policymakers as an indicator of the quality and effectiveness of hf care . public efforts to improve the use of evidence - based therapeutic approaches and clinical outcomes have focused on hospital - level performance as a core measure of the quality of hf care and a key strategy for reducing subsequent poor outcomes , most notably hospital readmission rates.5,6,23,24 included in these efforts is the implementation of a number of large national hf registries , such as the acute decompensated heart failure national registry ( adhere ) , the organized program to initiate lifesaving treatment in hospitalized patients with heart failure ( optimize - hf ) , and the initiation management pre - discharge of carvedilol heart failure ( impact - hf ) . these registries aim to collect observational data that will help describe characteristics , management , and outcomes in a broad sample of patients hospitalized for hf.2527 early observations from registry data document variability in hospital - level performance , delays in diagnosis and initiation of hf therapies , and under - use of evidenced - based hf guidelines along with high event rates ( death or readmission ) at hospital discharge in this subset of hf patients.25,27 general characteristics and select outcomes of medicare beneficiaries enrolled in registries appear similar to the broader , nonregistry medicare hf population , suggesting that ongoing registry work will provide a valuable resource and insight into clinical characteristics and patterns of care to guide treatment strategies for hospitalized hf patients . however , event rates remain high in this population , a signal that additional work is needed to identify the root cause for hf hospitalizations in order to improve long - term clinical outcomes.3,2729 in a systematic review of literature examining hf readmission rates , ross and colleagues6 concluded that hf patient hospitalizations are useful as a clinical marker for disease progression and have value as a means of understanding limited patient and health care system capacity as well as missed opportunities to better coordinate hf care . the authors concluded that the evidence supporting hf hospitalization or readmission as a quality indicator of hf care and outcomes is insufficient . jha et al30 examined national performance data on hospital discharge planning and associated rates of rehospitalization in hf patients and concluded that current efforts ( including public reporting on hospital performance ) are unlikely to yield large reductions in unnecessary hf patient hospital admissions . trends in the hospitalized hf medicare population have shown some promise , with incremental survival benefits , only minor fluctuations in cost , and isolated reductions in hospital readmission rates ; however , nearly 25% of hf patients are readmitted to the hospital within 30 days of their hospital discharge , an indication that more work needs to be done to change clinical practice and chronic care delivery for this high - risk population to improve their long - term outcomes.1,2,6,18,23,31 experts recognize that persons living with hf may influence their own health through effective self - management , and poor outcomes have been attributed to insufficient and ineffective self - management.3,9,18,23 for those interested in improving outcomes for people living with hf , an important first step requires a better understanding of what makes self - management in chronic hf care so difficult . polypharmacy problem ( both pharmacologic and nonpharmacologic interventions ) resulting from evidence - based care , dietary restrictions , lifestyle modifications , and ongoing need for frequent contact and medical care follow - up.32 adherence to complex medical regimens , along with the competing demands of daily life and , in particular , multiple chronic conditions , can become overwhelming . it is no surprise that suboptimal adherence to hf self - management has been reported as a major contributor to hf exacerbations that frequently lead to hospitalization and readmission in this population.19 moser et al33 studied the prevalence of risk factors for rehospitalization in 202 recently discharged hf patients , examining risk factors of functional and symptom status , comorbidity , living situation , anxiety , depression , quality of life ( qol ) , and adherence ( medications , low - salt diet , and symptom monitoring ) . results showed substantially impaired qol and high symptom burden , and significant impaired functional status ( 70% nyha iii ) , with 48% having more than 2 comorbid conditions , 50% having high levels of anxiety , and 69% having depression.33 the authors concluded that newly discharged hf patients exhibit psychosocial and behavioral risk for rehospitalization . evidence shows that poor adherence to prescribed treatments ( eg , low salt diet and medications ) is a risk factor that can trigger an hf hospitalization or readmission . lemon et al34 analyzed repeated cross - sectional probability sample surveys using data from national health and nutrition examination surveys ( nhanes ) from 1999 to 2006 and reported poor quality diets in persons with hf . characteristics associated with high sodium diets included male gender , lower education , low economic status , and no reported diagnosis of hypertension.34 ambardeckar and colleagues35 investigated a cohort of hospitalized patients to evaluate characteristics and in - hospital outcomes for nonadherent ( with diet and/or medications ) hf patients . ambardeckar and colleagues35 confirmed that nonadherence with diet and/or medications were identified causes for hf admissions . common patient characteristics identified by the authors included lower income , younger ( < 65 years ) patients , uninsured , and ethnic minorities . clinical characteristics identified by the researchers included higher risk hf profiles ( lower ejection fraction / worse cardiac function ) and symptom severity ( evidence of higher volume overload and more symptoms).35 understanding select patient characteristics that influence self - management is critical to providing a coordinated system of chronic hf care . in a model of self - care in chronic illness ( mscci ) , connelly36 identified 7 variables ( age , gender , income , education , social support , symptom severity , and comorbidity ) that influence effective self - management in patients with chronic disease . a more comprehensive literature review than set out in the present paper shows that a number of descriptive studies replicating the mscci36 model in hf patients have found contradictory results on which specific characteristics ( eg , age , gender ) influence self - management in hf patients . many of these studies identified age , functional status , comorbidity , emotional , and economical status as common characteristics that affect self - management behaviors and hf outcomes in the populations studied.3744 research that examined potential predictors and characteristics of self - management in hf patients are described below and summarized in table 1 . although not exhaustive , this list represents a cross - section of original research focused on self - management in hf published in the last 10 years . experts highlight the dynamic relationship between individual patient characteristics and self - management behaviors and recommend further research in this area.38 artinian and his colleagues39 examined relationships between select characteristics ( personal and environmental factors ) and self - management behaviors in a sample of 110 hf patients . although no statistically significant relationships were found between select characteristics and total self - care scores , the researchers concluded that certain trends they observed influenced individual self - management behaviors.39 older age positively influenced medication taking , keeping appointments , and receiving a flu shot . hf patients who lived alone were less likely to ask for help during shortness of breath ( sob ) and less likely to contact the doctor when they noticed symptoms . low - income patients living alone were more likely to eat canned or packaged foods , and individuals reporting poor health were more apt to rest , limit activities , ask for help , and contact their doctor regarding symptoms . cameron and colleagues19 found that 4 of the 7 hypothesized variables taken from the mscci model contributed significantly ( p < 0.05 ) to variance in hf self - management : male gender , moderate - to - severe comorbidity , depression , and confidence . in a nonexperimental replication study , chriss and colleagues37 found that increased age , male gender , and fewer comorbid conditions contributed to better hf self - management . in a similar study , rockwell and riegel38 replicated the mscci model of 7 characteristics , using data collected from 209 hf subjects participating in a community outpatient cdsm program . educational level and symptom severity were identified as predictors of hf self - management , explaining 10.3% of the variance . the authors concluded that those with higher educational attainment and greater symptom severity were more likely to be knowledgeable about the importance of hf symptoms.38 many people living with hf are elderly and symptomatic , lack social and financial support , and have more than one comorbid condition.19 therefore , implementing effective self - management strategies will require understanding the interplay between select patient characteristics and self - management behaviors to identify those in greatest need of cdsm support.19 to better understand the manner in which life situations facilitate or impede hf self - management , riegel and carlson40 conducted structured interviews with 26 hf patients , during which patients described the impact of hf on their daily living experiences and self - management behaviors . the researchers concluded that physical limitations , debilitating symptoms , difficulties coping with treatment , lack of knowledge , distressed emotions , multiple comorbid conditions , personal struggles , and poor family support were common characteristics that contributed to poor self - management in this small sample of hf patients.40 a limited number of studies have shown that mastering hf self - management is challenging and that few patients develop sufficient expertise to avoid repeated hospitalizations.41 cameron et al42 examined 143 elderly hospitalized hf patients and described differences in self - management skills between the novice patient ( < 2 months of hf symptoms ) and experienced patient ( > 2 months of hf symptoms ) . the authors concluded that experience was a determinant of self - management skills , but experience did not predict the patient s confidence to engage in self - management . riegel et al41 conducted a qualitative study in 29 chronic hf patients using in - person interviews and questionnaires measuring characteristics anticipated to influence self - care . the investigator found that only 10% of the sample were expert in hf self - management , and less daytime sleepiness and more family support distinguished good - vs - expert self - managers . 41 the researchers concluded that less daytime sleepiness and more family support contributed to better self - management.41 according to the investigators , the results of this study support prior research findings showing that self - management in persons with hf is poor , but the fact that only 1 in 10 hf patients can be expected to master self - management illustrates the need for further research in this area.41 emerging directions in health care policy have transformed the patient s role from a passive recipient to an active consumer and an engaged member of the health care team.8 this approach puts the patient in a key role to influence health care quality and cost.45 critical to achieving the desired outcomes is the active participation of an informed patient.45 in our current health care environments , people are being encouraged to take a more active role in self - managing their own health ; the rationale for this approach includes the fact that much of the self - management takes place in the individual s everyday life . research indicates that people who actively self - manage their own care receive higher quality health care and have better health care outcomes.4648 central to both consumer - driven health care systems and the chronic illness care model is an engaged and active patient as a member of the health care process.8,45 hibbard and her colleagues from the university of oregon have described the theory and measurement of consumer and patient activation.45,48,49 the degree to which an individual understands the necessity of taking an active role in managing personal health and health care , and feels capable of self - management is described as patient or consumer activation.49 more specifically , the term activation is defined by hibbard , and colleagues45 as having the information , motivation , and behavioral skills necessary to self - manage chronic illness , collaborate with health care providers , maintain functioning , and access appropriate care . the theory of activation and its measurement using the patient activation measure ( pam ) have been widely disseminated ; pam has acceptable psychometric properties , as noted by the authors , in patients with chronic illness , and consumers.45,50 table 2 summarizes selected research studies that examine predictive relationships between activation using the pam ( scores ) and health outcomes in people with chronic disease . in a controlled trial of 479 chronic - disease patients randomized to either a cdsm intervention or usual care control , hibbard et al51 showed that changes in participants levels of activation were accompanied by changes in select self - management behaviors . individuals with higher activation levels were more likely to engage in health behaviors , such as exercise and following a low fat diet . they are also more likely to engage in disease - specific self - management behaviors , such as taking medications , obtaining preventive care , and requesting and using health information.51 hibbard et al51 conclude that if activation , as measured by the self - reported pam score , was increased , a change in self - management behavior followed . mosen et al52 in a study of 4108 adults with chronic conditions , found that patients with high pam scores were significantly more likely to perform self - management behaviors , use self - management services , and report better medication adherence , compared with patients with low pam scores . the authors concluded that patient activation is predictive of health outcomes and health care utilization.52 dixon et al53 extended this work using face - to - face semi - structured interviews in a convenience sample of 27 adults with at least one chronic condition . results showed that those low in activation tended to see successful self - management as compliance , whereas the more activated patients ( high pam scores ) saw it as being in control and working in partnerships with health care professionals . barriers in self - management identified by the researchers in people with lower activation included a lack of confidence and knowledge about their condition and fewer strategies for coping with their chronic condition and stress.53 according to hibbard et al,45 a valid and reliable instrument to measure activation is necessary , to understand patient activation and its role in health care quality and outcomes in chronic illness care . most cdsm approaches seek to engage patients to self - manage their own care , but finding effective ways to engage the patient and provide the necessary support has been challenging . few experimental studies have examined whether chronic hf outcomes can be improved by increasing patient engagement , described by hibbard et al45 as activation in care and capability for self - management . the science of self - management in hf care is still young and there is limited empirical evidence of characteristics that predict self - management behaviors or describe the relationship between hf self - management and health outcomes.17 efforts to improve chronic hf care have concentrated on physiologic and clinical variables , hospital and provider performance , and public reporting on quality hf indicators to identify and characterize patient risk . there has been , to date , far less emphasis on patient - centered characteristics as a means of improving chronic hf care . persistence of high event rates for death or readmission in people with hf indicate that there is substantial room to improve outcomes that might be achieved through enhancing the quality of outpatient hf care and identifying strategies to stratify patient risk.6,23,31 in addition , these high event rates underscore the need for innovative management strategies that will coordinate the transition of patient care from the acute hospital environment to outpatient settings.5456 emerging directions in health care policy have focused attention on discharge failures and negative outcomes in chronic hf care , making it imperative that health care stakeholders translate evidence - based research into practice . the transitional care model ( tcm ) is an evidence - based model of care designed to assist elderly adults with chronic illnesses such as hf to transition from acute care settings into the home or other less intensive health care environments . the tcm has been rigorously tested and refined by a team of researchers at the university of pennsylvania in both academic and community settings.57,58 its core components include both in - person contact and a nurse - led , interdisciplinary team approach to increase self - management and improve patient outcomes.57 current efforts are underway to bring tcm into mainstream clinical practice.59 health care reform in the us has focused national improvement efforts in chronic hf care , on reducing 30-day all - cause readmission rates among patients discharged with hf or acute myocardial infarction by 20% nationally by december 2012 . reducing avoidable hospital readmissions in these patients presents an opportunity not only to improve quality chronic illness care , but also to reduce cost and prevent the loss of medicare reimbursement for hf readmissions . the hospital to home ( h2h ) national quality improvement initiative , led by the american college of cardiology ( acc ) and the institute for healthcare improvement ( ihi ) , is one strategy that is currently underway to improve the transition from acute inpatient care to outpatient care for chronic hf.60 performance improvement efforts emphasize key areas such as medication reconciliation , early discharge follow up , and symptom management . in clinical practice ( links to the h2h and tcm websites can be found in the references.59,60 ) . although both models of care coordination have been tested and show promising effects in improving desired outcomes and lowering costs for the high - risk chronically ill patient populations , research should examine the contributions of self - management strategies toward achieving these goals . future efforts focused on the dissemination and the evaluation of program outcomes for hf patients , are greatly needed . research has shown that effective cdsm approaches can play a significant role in optimizing hf outcomes and that self - management is central to most chronic hf care.10,6164 while the specific structure , goals , and dimensions of cdsm programs for hf patients vary significantly , most comprehensive hf programs include a ) practice redesign ( use of an integrated multidisciplinary team to provide continuous , coordinated care to patients ) ; b ) patient education and support ( knowledge , self - management , and behavioral change strategies ) ; and c ) clinical expertise ( teams typically led by nurse specialists with expertise in hf management).8,17 evidence surrounding hf self - management has focused on delivering an intervention for a set period of time and intensity in varied populations and on describing measurable outcomes based on the specific aim of the research . results of many of these studies illustrate diversity among characteristics and risk factors associated with poor self - management for hf , and highlight potential barriers and challenges that may contribute to problems with adherence in hf patients.19,39,40 future recommendations for cdsm research and practice include a ) developing strategies to aggressively address barriers and risk factors;33 b ) implementing approaches to effective assessment of self - management deficits so that educational and behavioral strategies can be tailored to individual needs;42 c ) identifying the modifiable and nonmodifiable behaviors and risk factor(s ) and selectively directing self - management strategies toward the most modifiable behavior or risk factor(s ) that will net the greatest improvement;19 and d ) improving prerequisite knowledge of self - management and the characteristics of target populations before designing cdsm programs.39 although , cdsm programs share core strategies ( eg , multidisciplinary teams , coordination of care , patient education and support , clinical expertise ) , the individual program components , targeted outcomes , methods of evaluation , and measurement are highly variable and contribute to both challenges and limitations when comparing and contrasting effectiveness and outcomes.10 widespread adoption of cdsm programs have been plagued by methodological shortcomings , limiting the validation of their effectiveness and clinical application.61,65 future efforts must include uniform comparison , both within and across conditions ( eg , programs and interventions ) in order to identify effective program components , populations and settings , and provide sufficient details of program design and methodology to allow for easy replication for the greatest success.65,66 sorting out the most advantageous approaches to better self - management in hf patients is essential to improve chronic hf care in this complex population.40 although the patient s role has not been fully integrated into clinical practice , the advent of health information technology and health care reform have shifted priorities toward consumer driven health care , so that patients are in a primary position to influence health care quality and cost . to improve the effectiveness of chronic hf care , an understanding of the specific difficulties of self - management is necessary in order to apply targeted interventions and effective cdsm support . experts in this area highlight these gaps in our current cdsm knowledge base : a ) understanding characteristics that influence self - management from a multidisciplinary viewpoint , b ) integrating biological and psychological perspectives , and c ) building on existing research to answer new questions.67 self - management is widely accepted as a central pathway for multidisciplinary cdsm programs and chronic care models ; however , successful quality improvement efforts must also focus on helping individuals become more informed about their illness , actively engage in their own care ( activation ) , and improve their skills for self - management . 7 most chronic hf therapy and treatments rely on self - management strategies ( eg , telehealth technology , electronic patient records , patient education ) . thus , cdsm approaches that encourage patients to become active partners ( participants ) in their care are necessary to improve the impact of self - management on long - term hf outcomes . cdsm has been viewed as a viable strategy to bridge the gap between the capacity of the individual and the health care system to meet the needs of individuals with chronic disease and effect improved outcomes . understanding the relationship among patient characteristics , activation , self - management , and the desired outcomes in hf patients is an important next step . evidence that patient characteristics , activation , and self - management are of consequence to hf outcomes , opens a new path of inquiry for health services researchers and clinicians . future research in this area is needed to inform health care stakeholders about the patient s critical role in designing , tailoring , and implementing cdsm care plans for chronic hf care . achieving national goals and improving outcomes for people with hf will require the implementation of effective cdsm strategies to close identified gaps in chronic hf care . improving health care quality and mitigating negative hf outcomes will require identifying barriers to self - management so that targeted strategies for cdsm support can be implemented . to date , quality improvement efforts in chronic hf care have concentrated on discharge failures , hospital and provider level performance , and public reporting of core hf indicators to identify and characterize patient risk . to improve the continuum of chronic hf care will require that health care stakeholders place more emphasis on patient - centered characteristics as a means of understanding patient risk and identifying barriers to self - management , so that effective multidisciplinary strategies for cdsm support can be implemented . understanding the interplay between patient characteristics , self - management , and activation in chronic hf care is not only timely but necessary to achieve the desired long - term outcomes for hf patients and other complex disease populations .

chronic conditions such as heart failure ( hf ) place a tremendous strain on patients , their families , the community , and the health care system because there are no real cures . adding to the burden are longer life expectancies and increased numbers of people living with multiple chronic conditions . today , whether engaging in a health - promoting activity , such as exercise , or living with a chronic disease such as hf , the individual is responsible for actively managing day - to - day activities , a concept referred to as self - management . self - management emerged as the cornerstone for chronic care models and multidisciplinary disease - management strategies in chronic illness care . moreover , self - management has been prioritized as a central pathway for improving the quality and effectiveness of most chronic hf care . adherence to self - management is vital to optimize the treatment outcomes in hf patients , but implementing chronic disease self - management ( cdsm ) strategies and identifying the difficulties in self - management has proved to be a challenge . understanding both where we have been and the future direction of self - management in hf care is not only timely , but a crucial aspect of improving long - term outcomes for people with hf and other chronic diseases .

Chronic heart failure: a common but serious problem Chronic disease self-management: historical perspectives Self-management in heart failure: where have we been? Self-management in heart failure: future directions

in chronic illness care , heart failure ( hf ) is one of the few cardiovascular diagnoses where disease prevalence is rising rather than falling.1,2 owing to a rapidly aging population and improved survival from acute cardiac events , approximately 5.8 million americans are living with hf , with an estimated incidence of 660,000 new cases each year.1,2 in adults , hf has emerged as a common principal medicare hospital discharge diagnosis , and a leading cause of readmission within 30 days of an hf admission.2,36 although patients hospitalized with acute decompensated hf improve during the incident admission , the long - term all - cause mortality rate remains high and has improved little over time despite important therapeutic advances and national quality improvement efforts.2,3 today , whether engaging in a health - promoting activity such as exercise or managing a chronic disease such as hf , the individual is responsible for actively managing day - to - day activities , a concept commonly referred to as self - management . self - management strategies have become a core component and major theme of many disease management programs and chronic care models at organizational levels.7 the clinical trajectory of chronic hf is characterized by chronic symptoms interspersed with acute symptoms , which often result in adverse events and poor outcomes . one process for reducing the burden of hf symptoms in the everyday life of those living with chronic hf is to identify effective approaches to chronic illness care , which support successful self - management.8,9 furthermore , to maximize effective behavioral interventions , efforts must focus on understanding the challenges individuals face in managing the complex demands of their illness and the often multiple and competing conditions . gaps in our understanding of the characteristics that influence self - management behaviors , and the lack of evidence that self - management translates to better health outcomes in hf patients , suggest a need for further dialog among health care providers and clinicians interested in improving care for people living with chronic hf . the aim of this narrative literature synthesis is to contribute to the evidence base on self - management in hf by describing where we have been , what additional information we may need , and where we need to go with self - management in chronic hf care . adding to the mounting evidence that chronic illness care is fragmented and poorly coordinated is the criticism that us health care systems provide health care on an episodic , acute care basis , and lack systematic approaches to managing chronic disease.7,8 to correct deficiencies in the organization and delivery of chronic care and to reduce the burden of escalating costs , disease management emerged as a comprehensive approach and strategic model for chronic illness care.10 self - management is the catalyst or backbone for many disease management approaches because it is patient - centered , focuses on helping people with chronic disease become more informed about their illnesses , and actively engages patients in their own health care . in addition , the centers for disease control and prevention ( cdc ) state of aging and health in america report also prioritized self - management , with six of its seven calls - to - action identifying self - management as a central pathway to improving outcomes in people with chronic disease.7,11 for this review , chronic disease self - management ( cdsm ) will be used as an umbrella term to cover the broad diversity of programs , approaches , models , and interventions used to improve the quality of care for people living with chronic disease . a major challenge for dissemination of research outcomes for chronic illness care has been the absence of universally accepted definitions for disease management and self - management , or standardization for cdsm programs and strategies.10 despite the encumbrance imposed by the lack of standardization , both private and public interest in cdsm grew , and many of these strategies were adopted on a wider scale by the mid-1990s . us health care systems are under pressure to slow or reverse the often poor outcomes , escalating health care costs , and high utilization rates associated with chronic illness care.8,10,13 adding to this burden are demographic projections that suggest dramatic increases in chronic illness care and resource utilization . the interchangeable use of the terms self - care and self - management and the struggle to reach consensus or a gold standard definition has imposed limitations on understanding and promoting self - management in chronic hf care . self - management is derived from definitions set out by lorig and holman15 and barlow et al16 based on the individual s ability ( problem solving , decision making , resource utilization , formation of patient - provider partnerships , action planning , and tailoring of daily activities ) to undertake and manage day - to - day tasks , inherent lifestyle changes , physical symptoms , and psychosocial consequences of health and well - being over the lifetime of an illness . despite differences over terminology and definitions and their conceptualization in the hf literature , there is wide acceptance of the american heart association s ( aha ) recommended behaviors for persons living with hf ( medication adherence , symptom management , dietary adherence , exercise , smoking cessation , and preventative behaviors).17 hf patients are strongly encouraged by clinicians to regularly take medications , monitor their condition and symptoms , keep appointments , and contact their health care providers when needed.17 strategies such as monitoring weight , remembering to take medications daily and on time , and following a low - salt diet are among the core recommendations in hf guidelines and have been shown to be beneficial for hf patients.1,17 regardless of how seemingly simple these recommendations may appear , making plans to adhere to , and apply , the prescribed behavior changes in daily activities requires decision making and problem solving skills for self - management.17 with or without the help of family members or caregivers , it is the patient s responsibility to integrate the vast majority of hf care into his or her daily life.17 the challenges associated with managing hf and adhering to self - management are proving to be difficult problems for health - care providers , policy makers , and patients . further insight into the answers or reasons for the lack of patient adherence , or the ability to identify potential barriers to self - management for risk - stratification in hf patients is greatly needed.20 hf places a tremendous strain on the patient , family members , community , and health care system because there is no silver bullet or are longer life expectancies and increasing numbers of people with hf living with other conditions . common comorbid conditions among medicare - aged beneficiaries with hf include hypertension , diabetes mellitus , dyslipidemia , chronic lung disease , renal dysfunction , cognitive impairment , and osteoarthritis / osteoporosis.21,22 in recognition of the complexity of the problem and the substantial demand for health care resources associated with chronic hf care , the aha , the iom , the american college of cardiology ( acc ) , the joint commission on accreditation of hospital organizations ( tjc ) , and the centers for medicare and medicaid services ( cms ) established key quality hf indicators and national goals to standardize hospital - level performance and reduce the high rates of hospital readmission and cost associated with poor clinical outcomes as a focus area for quality improvement efforts.1,6,7,23 research studies confirm that chronic hf can be extremely debilitating , with symptomatic exacerbations that often lead to episodes of acute decompensation , frequent hospital admissions , and premature death.36 patient hf hospitalization and rehospitalization rates have attracted considerable attention from policymakers as an indicator of the quality and effectiveness of hf care . public efforts to improve the use of evidence - based therapeutic approaches and clinical outcomes have focused on hospital - level performance as a core measure of the quality of hf care and a key strategy for reducing subsequent poor outcomes , most notably hospital readmission rates.5,6,23,24 included in these efforts is the implementation of a number of large national hf registries , such as the acute decompensated heart failure national registry ( adhere ) , the organized program to initiate lifesaving treatment in hospitalized patients with heart failure ( optimize - hf ) , and the initiation management pre - discharge of carvedilol heart failure ( impact - hf ) . however , event rates remain high in this population , a signal that additional work is needed to identify the root cause for hf hospitalizations in order to improve long - term clinical outcomes.3,2729 in a systematic review of literature examining hf readmission rates , ross and colleagues6 concluded that hf patient hospitalizations are useful as a clinical marker for disease progression and have value as a means of understanding limited patient and health care system capacity as well as missed opportunities to better coordinate hf care . trends in the hospitalized hf medicare population have shown some promise , with incremental survival benefits , only minor fluctuations in cost , and isolated reductions in hospital readmission rates ; however , nearly 25% of hf patients are readmitted to the hospital within 30 days of their hospital discharge , an indication that more work needs to be done to change clinical practice and chronic care delivery for this high - risk population to improve their long - term outcomes.1,2,6,18,23,31 experts recognize that persons living with hf may influence their own health through effective self - management , and poor outcomes have been attributed to insufficient and ineffective self - management.3,9,18,23 for those interested in improving outcomes for people living with hf , an important first step requires a better understanding of what makes self - management in chronic hf care so difficult . it is no surprise that suboptimal adherence to hf self - management has been reported as a major contributor to hf exacerbations that frequently lead to hospitalization and readmission in this population.19 moser et al33 studied the prevalence of risk factors for rehospitalization in 202 recently discharged hf patients , examining risk factors of functional and symptom status , comorbidity , living situation , anxiety , depression , quality of life ( qol ) , and adherence ( medications , low - salt diet , and symptom monitoring ) . in a model of self - care in chronic illness ( mscci ) , connelly36 identified 7 variables ( age , gender , income , education , social support , symptom severity , and comorbidity ) that influence effective self - management in patients with chronic disease . many of these studies identified age , functional status , comorbidity , emotional , and economical status as common characteristics that affect self - management behaviors and hf outcomes in the populations studied.3744 research that examined potential predictors and characteristics of self - management in hf patients are described below and summarized in table 1 . the authors concluded that those with higher educational attainment and greater symptom severity were more likely to be knowledgeable about the importance of hf symptoms.38 many people living with hf are elderly and symptomatic , lack social and financial support , and have more than one comorbid condition.19 therefore , implementing effective self - management strategies will require understanding the interplay between select patient characteristics and self - management behaviors to identify those in greatest need of cdsm support.19 to better understand the manner in which life situations facilitate or impede hf self - management , riegel and carlson40 conducted structured interviews with 26 hf patients , during which patients described the impact of hf on their daily living experiences and self - management behaviors . the researchers concluded that physical limitations , debilitating symptoms , difficulties coping with treatment , lack of knowledge , distressed emotions , multiple comorbid conditions , personal struggles , and poor family support were common characteristics that contributed to poor self - management in this small sample of hf patients.40 a limited number of studies have shown that mastering hf self - management is challenging and that few patients develop sufficient expertise to avoid repeated hospitalizations.41 cameron et al42 examined 143 elderly hospitalized hf patients and described differences in self - management skills between the novice patient ( < 2 months of hf symptoms ) and experienced patient ( > 2 months of hf symptoms ) . 41 the researchers concluded that less daytime sleepiness and more family support contributed to better self - management.41 according to the investigators , the results of this study support prior research findings showing that self - management in persons with hf is poor , but the fact that only 1 in 10 hf patients can be expected to master self - management illustrates the need for further research in this area.41 emerging directions in health care policy have transformed the patient s role from a passive recipient to an active consumer and an engaged member of the health care team.8 this approach puts the patient in a key role to influence health care quality and cost.45 critical to achieving the desired outcomes is the active participation of an informed patient.45 in our current health care environments , people are being encouraged to take a more active role in self - managing their own health ; the rationale for this approach includes the fact that much of the self - management takes place in the individual s everyday life . research indicates that people who actively self - manage their own care receive higher quality health care and have better health care outcomes.4648 central to both consumer - driven health care systems and the chronic illness care model is an engaged and active patient as a member of the health care process.8,45 hibbard and her colleagues from the university of oregon have described the theory and measurement of consumer and patient activation.45,48,49 the degree to which an individual understands the necessity of taking an active role in managing personal health and health care , and feels capable of self - management is described as patient or consumer activation.49 more specifically , the term activation is defined by hibbard , and colleagues45 as having the information , motivation , and behavioral skills necessary to self - manage chronic illness , collaborate with health care providers , maintain functioning , and access appropriate care . they are also more likely to engage in disease - specific self - management behaviors , such as taking medications , obtaining preventive care , and requesting and using health information.51 hibbard et al51 conclude that if activation , as measured by the self - reported pam score , was increased , a change in self - management behavior followed . barriers in self - management identified by the researchers in people with lower activation included a lack of confidence and knowledge about their condition and fewer strategies for coping with their chronic condition and stress.53 according to hibbard et al,45 a valid and reliable instrument to measure activation is necessary , to understand patient activation and its role in health care quality and outcomes in chronic illness care . the science of self - management in hf care is still young and there is limited empirical evidence of characteristics that predict self - management behaviors or describe the relationship between hf self - management and health outcomes.17 efforts to improve chronic hf care have concentrated on physiologic and clinical variables , hospital and provider performance , and public reporting on quality hf indicators to identify and characterize patient risk . persistence of high event rates for death or readmission in people with hf indicate that there is substantial room to improve outcomes that might be achieved through enhancing the quality of outpatient hf care and identifying strategies to stratify patient risk.6,23,31 in addition , these high event rates underscore the need for innovative management strategies that will coordinate the transition of patient care from the acute hospital environment to outpatient settings.5456 emerging directions in health care policy have focused attention on discharge failures and negative outcomes in chronic hf care , making it imperative that health care stakeholders translate evidence - based research into practice . the tcm has been rigorously tested and refined by a team of researchers at the university of pennsylvania in both academic and community settings.57,58 its core components include both in - person contact and a nurse - led , interdisciplinary team approach to increase self - management and improve patient outcomes.57 current efforts are underway to bring tcm into mainstream clinical practice.59 health care reform in the us has focused national improvement efforts in chronic hf care , on reducing 30-day all - cause readmission rates among patients discharged with hf or acute myocardial infarction by 20% nationally by december 2012 . research has shown that effective cdsm approaches can play a significant role in optimizing hf outcomes and that self - management is central to most chronic hf care.10,6164 while the specific structure , goals , and dimensions of cdsm programs for hf patients vary significantly , most comprehensive hf programs include a ) practice redesign ( use of an integrated multidisciplinary team to provide continuous , coordinated care to patients ) ; b ) patient education and support ( knowledge , self - management , and behavioral change strategies ) ; and c ) clinical expertise ( teams typically led by nurse specialists with expertise in hf management).8,17 evidence surrounding hf self - management has focused on delivering an intervention for a set period of time and intensity in varied populations and on describing measurable outcomes based on the specific aim of the research . results of many of these studies illustrate diversity among characteristics and risk factors associated with poor self - management for hf , and highlight potential barriers and challenges that may contribute to problems with adherence in hf patients.19,39,40 future recommendations for cdsm research and practice include a ) developing strategies to aggressively address barriers and risk factors;33 b ) implementing approaches to effective assessment of self - management deficits so that educational and behavioral strategies can be tailored to individual needs;42 c ) identifying the modifiable and nonmodifiable behaviors and risk factor(s ) and selectively directing self - management strategies toward the most modifiable behavior or risk factor(s ) that will net the greatest improvement;19 and d ) improving prerequisite knowledge of self - management and the characteristics of target populations before designing cdsm programs.39 although , cdsm programs share core strategies ( eg , multidisciplinary teams , coordination of care , patient education and support , clinical expertise ) , the individual program components , targeted outcomes , methods of evaluation , and measurement are highly variable and contribute to both challenges and limitations when comparing and contrasting effectiveness and outcomes.10 widespread adoption of cdsm programs have been plagued by methodological shortcomings , limiting the validation of their effectiveness and clinical application.61,65 future efforts must include uniform comparison , both within and across conditions ( eg , programs and interventions ) in order to identify effective program components , populations and settings , and provide sufficient details of program design and methodology to allow for easy replication for the greatest success.65,66 sorting out the most advantageous approaches to better self - management in hf patients is essential to improve chronic hf care in this complex population.40 although the patient s role has not been fully integrated into clinical practice , the advent of health information technology and health care reform have shifted priorities toward consumer driven health care , so that patients are in a primary position to influence health care quality and cost . to improve the effectiveness of chronic hf care , an understanding of the specific difficulties of self - management is necessary in order to apply targeted interventions and effective cdsm support . experts in this area highlight these gaps in our current cdsm knowledge base : a ) understanding characteristics that influence self - management from a multidisciplinary viewpoint , b ) integrating biological and psychological perspectives , and c ) building on existing research to answer new questions.67 self - management is widely accepted as a central pathway for multidisciplinary cdsm programs and chronic care models ; however , successful quality improvement efforts must also focus on helping individuals become more informed about their illness , actively engage in their own care ( activation ) , and improve their skills for self - management . cdsm has been viewed as a viable strategy to bridge the gap between the capacity of the individual and the health care system to meet the needs of individuals with chronic disease and effect improved outcomes . understanding the relationship among patient characteristics , activation , self - management , and the desired outcomes in hf patients is an important next step . achieving national goals and improving outcomes for people with hf will require the implementation of effective cdsm strategies to close identified gaps in chronic hf care . to improve the continuum of chronic hf care will require that health care stakeholders place more emphasis on patient - centered characteristics as a means of understanding patient risk and identifying barriers to self - management , so that effective multidisciplinary strategies for cdsm support can be implemented . understanding the interplay between patient characteristics , self - management , and activation in chronic hf care is not only timely but necessary to achieve the desired long - term outcomes for hf patients and other complex disease populations .

in chronic illness care , heart failure ( hf ) is one of the few cardiovascular diagnoses where disease prevalence is rising rather than falling.1,2 owing to a rapidly aging population and improved survival from acute cardiac events , approximately 5.8 million americans are living with hf , with an estimated incidence of 660,000 new cases each year.1,2 in adults , hf has emerged as a common principal medicare hospital discharge diagnosis , and a leading cause of readmission within 30 days of an hf admission.2,36 although patients hospitalized with acute decompensated hf improve during the incident admission , the long - term all - cause mortality rate remains high and has improved little over time despite important therapeutic advances and national quality improvement efforts.2,3 today , whether engaging in a health - promoting activity such as exercise or managing a chronic disease such as hf , the individual is responsible for actively managing day - to - day activities , a concept commonly referred to as self - management . self - management strategies have become a core component and major theme of many disease management programs and chronic care models at organizational levels.7 the clinical trajectory of chronic hf is characterized by chronic symptoms interspersed with acute symptoms , which often result in adverse events and poor outcomes . one process for reducing the burden of hf symptoms in the everyday life of those living with chronic hf is to identify effective approaches to chronic illness care , which support successful self - management.8,9 furthermore , to maximize effective behavioral interventions , efforts must focus on understanding the challenges individuals face in managing the complex demands of their illness and the often multiple and competing conditions . gaps in our understanding of the characteristics that influence self - management behaviors , and the lack of evidence that self - management translates to better health outcomes in hf patients , suggest a need for further dialog among health care providers and clinicians interested in improving care for people living with chronic hf . the aim of this narrative literature synthesis is to contribute to the evidence base on self - management in hf by describing where we have been , what additional information we may need , and where we need to go with self - management in chronic hf care . adding to the mounting evidence that chronic illness care is fragmented and poorly coordinated is the criticism that us health care systems provide health care on an episodic , acute care basis , and lack systematic approaches to managing chronic disease.7,8 to correct deficiencies in the organization and delivery of chronic care and to reduce the burden of escalating costs , disease management emerged as a comprehensive approach and strategic model for chronic illness care.10 self - management is the catalyst or backbone for many disease management approaches because it is patient - centered , focuses on helping people with chronic disease become more informed about their illnesses , and actively engages patients in their own health care . in addition , the centers for disease control and prevention ( cdc ) state of aging and health in america report also prioritized self - management , with six of its seven calls - to - action identifying self - management as a central pathway to improving outcomes in people with chronic disease.7,11 for this review , chronic disease self - management ( cdsm ) will be used as an umbrella term to cover the broad diversity of programs , approaches , models , and interventions used to improve the quality of care for people living with chronic disease . a major challenge for dissemination of research outcomes for chronic illness care has been the absence of universally accepted definitions for disease management and self - management , or standardization for cdsm programs and strategies.10 despite the encumbrance imposed by the lack of standardization , both private and public interest in cdsm grew , and many of these strategies were adopted on a wider scale by the mid-1990s . this concern led to recommendations from policy makers for further research to identify optimal cdsm programs , to change patient behaviors , and to improve the health of the chronically ill.14 as with other chronic conditions , the major goals of cdsm in hf are to reduce symptoms and medical care costs while improving clinical outcomes . self - management is derived from definitions set out by lorig and holman15 and barlow et al16 based on the individual s ability ( problem solving , decision making , resource utilization , formation of patient - provider partnerships , action planning , and tailoring of daily activities ) to undertake and manage day - to - day tasks , inherent lifestyle changes , physical symptoms , and psychosocial consequences of health and well - being over the lifetime of an illness . despite differences over terminology and definitions and their conceptualization in the hf literature , there is wide acceptance of the american heart association s ( aha ) recommended behaviors for persons living with hf ( medication adherence , symptom management , dietary adherence , exercise , smoking cessation , and preventative behaviors).17 hf patients are strongly encouraged by clinicians to regularly take medications , monitor their condition and symptoms , keep appointments , and contact their health care providers when needed.17 strategies such as monitoring weight , remembering to take medications daily and on time , and following a low - salt diet are among the core recommendations in hf guidelines and have been shown to be beneficial for hf patients.1,17 regardless of how seemingly simple these recommendations may appear , making plans to adhere to , and apply , the prescribed behavior changes in daily activities requires decision making and problem solving skills for self - management.17 with or without the help of family members or caregivers , it is the patient s responsibility to integrate the vast majority of hf care into his or her daily life.17 the challenges associated with managing hf and adhering to self - management are proving to be difficult problems for health - care providers , policy makers , and patients . further insight into the answers or reasons for the lack of patient adherence , or the ability to identify potential barriers to self - management for risk - stratification in hf patients is greatly needed.20 hf places a tremendous strain on the patient , family members , community , and health care system because there is no silver bullet or are longer life expectancies and increasing numbers of people with hf living with other conditions . common comorbid conditions among medicare - aged beneficiaries with hf include hypertension , diabetes mellitus , dyslipidemia , chronic lung disease , renal dysfunction , cognitive impairment , and osteoarthritis / osteoporosis.21,22 in recognition of the complexity of the problem and the substantial demand for health care resources associated with chronic hf care , the aha , the iom , the american college of cardiology ( acc ) , the joint commission on accreditation of hospital organizations ( tjc ) , and the centers for medicare and medicaid services ( cms ) established key quality hf indicators and national goals to standardize hospital - level performance and reduce the high rates of hospital readmission and cost associated with poor clinical outcomes as a focus area for quality improvement efforts.1,6,7,23 research studies confirm that chronic hf can be extremely debilitating , with symptomatic exacerbations that often lead to episodes of acute decompensation , frequent hospital admissions , and premature death.36 patient hf hospitalization and rehospitalization rates have attracted considerable attention from policymakers as an indicator of the quality and effectiveness of hf care . public efforts to improve the use of evidence - based therapeutic approaches and clinical outcomes have focused on hospital - level performance as a core measure of the quality of hf care and a key strategy for reducing subsequent poor outcomes , most notably hospital readmission rates.5,6,23,24 included in these efforts is the implementation of a number of large national hf registries , such as the acute decompensated heart failure national registry ( adhere ) , the organized program to initiate lifesaving treatment in hospitalized patients with heart failure ( optimize - hf ) , and the initiation management pre - discharge of carvedilol heart failure ( impact - hf ) . these registries aim to collect observational data that will help describe characteristics , management , and outcomes in a broad sample of patients hospitalized for hf.2527 early observations from registry data document variability in hospital - level performance , delays in diagnosis and initiation of hf therapies , and under - use of evidenced - based hf guidelines along with high event rates ( death or readmission ) at hospital discharge in this subset of hf patients.25,27 general characteristics and select outcomes of medicare beneficiaries enrolled in registries appear similar to the broader , nonregistry medicare hf population , suggesting that ongoing registry work will provide a valuable resource and insight into clinical characteristics and patterns of care to guide treatment strategies for hospitalized hf patients . however , event rates remain high in this population , a signal that additional work is needed to identify the root cause for hf hospitalizations in order to improve long - term clinical outcomes.3,2729 in a systematic review of literature examining hf readmission rates , ross and colleagues6 concluded that hf patient hospitalizations are useful as a clinical marker for disease progression and have value as a means of understanding limited patient and health care system capacity as well as missed opportunities to better coordinate hf care . trends in the hospitalized hf medicare population have shown some promise , with incremental survival benefits , only minor fluctuations in cost , and isolated reductions in hospital readmission rates ; however , nearly 25% of hf patients are readmitted to the hospital within 30 days of their hospital discharge , an indication that more work needs to be done to change clinical practice and chronic care delivery for this high - risk population to improve their long - term outcomes.1,2,6,18,23,31 experts recognize that persons living with hf may influence their own health through effective self - management , and poor outcomes have been attributed to insufficient and ineffective self - management.3,9,18,23 for those interested in improving outcomes for people living with hf , an important first step requires a better understanding of what makes self - management in chronic hf care so difficult . it is no surprise that suboptimal adherence to hf self - management has been reported as a major contributor to hf exacerbations that frequently lead to hospitalization and readmission in this population.19 moser et al33 studied the prevalence of risk factors for rehospitalization in 202 recently discharged hf patients , examining risk factors of functional and symptom status , comorbidity , living situation , anxiety , depression , quality of life ( qol ) , and adherence ( medications , low - salt diet , and symptom monitoring ) . results showed substantially impaired qol and high symptom burden , and significant impaired functional status ( 70% nyha iii ) , with 48% having more than 2 comorbid conditions , 50% having high levels of anxiety , and 69% having depression.33 the authors concluded that newly discharged hf patients exhibit psychosocial and behavioral risk for rehospitalization . many of these studies identified age , functional status , comorbidity , emotional , and economical status as common characteristics that affect self - management behaviors and hf outcomes in the populations studied.3744 research that examined potential predictors and characteristics of self - management in hf patients are described below and summarized in table 1 . the authors concluded that those with higher educational attainment and greater symptom severity were more likely to be knowledgeable about the importance of hf symptoms.38 many people living with hf are elderly and symptomatic , lack social and financial support , and have more than one comorbid condition.19 therefore , implementing effective self - management strategies will require understanding the interplay between select patient characteristics and self - management behaviors to identify those in greatest need of cdsm support.19 to better understand the manner in which life situations facilitate or impede hf self - management , riegel and carlson40 conducted structured interviews with 26 hf patients , during which patients described the impact of hf on their daily living experiences and self - management behaviors . the researchers concluded that physical limitations , debilitating symptoms , difficulties coping with treatment , lack of knowledge , distressed emotions , multiple comorbid conditions , personal struggles , and poor family support were common characteristics that contributed to poor self - management in this small sample of hf patients.40 a limited number of studies have shown that mastering hf self - management is challenging and that few patients develop sufficient expertise to avoid repeated hospitalizations.41 cameron et al42 examined 143 elderly hospitalized hf patients and described differences in self - management skills between the novice patient ( < 2 months of hf symptoms ) and experienced patient ( > 2 months of hf symptoms ) . 41 the researchers concluded that less daytime sleepiness and more family support contributed to better self - management.41 according to the investigators , the results of this study support prior research findings showing that self - management in persons with hf is poor , but the fact that only 1 in 10 hf patients can be expected to master self - management illustrates the need for further research in this area.41 emerging directions in health care policy have transformed the patient s role from a passive recipient to an active consumer and an engaged member of the health care team.8 this approach puts the patient in a key role to influence health care quality and cost.45 critical to achieving the desired outcomes is the active participation of an informed patient.45 in our current health care environments , people are being encouraged to take a more active role in self - managing their own health ; the rationale for this approach includes the fact that much of the self - management takes place in the individual s everyday life . research indicates that people who actively self - manage their own care receive higher quality health care and have better health care outcomes.4648 central to both consumer - driven health care systems and the chronic illness care model is an engaged and active patient as a member of the health care process.8,45 hibbard and her colleagues from the university of oregon have described the theory and measurement of consumer and patient activation.45,48,49 the degree to which an individual understands the necessity of taking an active role in managing personal health and health care , and feels capable of self - management is described as patient or consumer activation.49 more specifically , the term activation is defined by hibbard , and colleagues45 as having the information , motivation , and behavioral skills necessary to self - manage chronic illness , collaborate with health care providers , maintain functioning , and access appropriate care . they are also more likely to engage in disease - specific self - management behaviors , such as taking medications , obtaining preventive care , and requesting and using health information.51 hibbard et al51 conclude that if activation , as measured by the self - reported pam score , was increased , a change in self - management behavior followed . barriers in self - management identified by the researchers in people with lower activation included a lack of confidence and knowledge about their condition and fewer strategies for coping with their chronic condition and stress.53 according to hibbard et al,45 a valid and reliable instrument to measure activation is necessary , to understand patient activation and its role in health care quality and outcomes in chronic illness care . the science of self - management in hf care is still young and there is limited empirical evidence of characteristics that predict self - management behaviors or describe the relationship between hf self - management and health outcomes.17 efforts to improve chronic hf care have concentrated on physiologic and clinical variables , hospital and provider performance , and public reporting on quality hf indicators to identify and characterize patient risk . persistence of high event rates for death or readmission in people with hf indicate that there is substantial room to improve outcomes that might be achieved through enhancing the quality of outpatient hf care and identifying strategies to stratify patient risk.6,23,31 in addition , these high event rates underscore the need for innovative management strategies that will coordinate the transition of patient care from the acute hospital environment to outpatient settings.5456 emerging directions in health care policy have focused attention on discharge failures and negative outcomes in chronic hf care , making it imperative that health care stakeholders translate evidence - based research into practice . the tcm has been rigorously tested and refined by a team of researchers at the university of pennsylvania in both academic and community settings.57,58 its core components include both in - person contact and a nurse - led , interdisciplinary team approach to increase self - management and improve patient outcomes.57 current efforts are underway to bring tcm into mainstream clinical practice.59 health care reform in the us has focused national improvement efforts in chronic hf care , on reducing 30-day all - cause readmission rates among patients discharged with hf or acute myocardial infarction by 20% nationally by december 2012 . research has shown that effective cdsm approaches can play a significant role in optimizing hf outcomes and that self - management is central to most chronic hf care.10,6164 while the specific structure , goals , and dimensions of cdsm programs for hf patients vary significantly , most comprehensive hf programs include a ) practice redesign ( use of an integrated multidisciplinary team to provide continuous , coordinated care to patients ) ; b ) patient education and support ( knowledge , self - management , and behavioral change strategies ) ; and c ) clinical expertise ( teams typically led by nurse specialists with expertise in hf management).8,17 evidence surrounding hf self - management has focused on delivering an intervention for a set period of time and intensity in varied populations and on describing measurable outcomes based on the specific aim of the research . results of many of these studies illustrate diversity among characteristics and risk factors associated with poor self - management for hf , and highlight potential barriers and challenges that may contribute to problems with adherence in hf patients.19,39,40 future recommendations for cdsm research and practice include a ) developing strategies to aggressively address barriers and risk factors;33 b ) implementing approaches to effective assessment of self - management deficits so that educational and behavioral strategies can be tailored to individual needs;42 c ) identifying the modifiable and nonmodifiable behaviors and risk factor(s ) and selectively directing self - management strategies toward the most modifiable behavior or risk factor(s ) that will net the greatest improvement;19 and d ) improving prerequisite knowledge of self - management and the characteristics of target populations before designing cdsm programs.39 although , cdsm programs share core strategies ( eg , multidisciplinary teams , coordination of care , patient education and support , clinical expertise ) , the individual program components , targeted outcomes , methods of evaluation , and measurement are highly variable and contribute to both challenges and limitations when comparing and contrasting effectiveness and outcomes.10 widespread adoption of cdsm programs have been plagued by methodological shortcomings , limiting the validation of their effectiveness and clinical application.61,65 future efforts must include uniform comparison , both within and across conditions ( eg , programs and interventions ) in order to identify effective program components , populations and settings , and provide sufficient details of program design and methodology to allow for easy replication for the greatest success.65,66 sorting out the most advantageous approaches to better self - management in hf patients is essential to improve chronic hf care in this complex population.40 although the patient s role has not been fully integrated into clinical practice , the advent of health information technology and health care reform have shifted priorities toward consumer driven health care , so that patients are in a primary position to influence health care quality and cost . experts in this area highlight these gaps in our current cdsm knowledge base : a ) understanding characteristics that influence self - management from a multidisciplinary viewpoint , b ) integrating biological and psychological perspectives , and c ) building on existing research to answer new questions.67 self - management is widely accepted as a central pathway for multidisciplinary cdsm programs and chronic care models ; however , successful quality improvement efforts must also focus on helping individuals become more informed about their illness , actively engage in their own care ( activation ) , and improve their skills for self - management . to improve the continuum of chronic hf care will require that health care stakeholders place more emphasis on patient - centered characteristics as a means of understanding patient risk and identifying barriers to self - management , so that effective multidisciplinary strategies for cdsm support can be implemented .

the early ( 90day ) risk of recurrence of stroke and other vascular events following index transient ischemic attack ( tia ) and minor ischemic stroke is very high , even in patients treated with aspirin , the current standard of care . treatment with the combination of clopidogrel and aspirin taken soon after a tia or minor stroke was found to decrease the 90day risk of stroke ( hazard ratio 0.68 , 95% ci 0.57 to 0.81 , p<0.001 ) but did not increase the risk of hemorrhage compared with aspirin alone in the chance stroke trial ( clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ) . although the clopidogrelaspirin regimen is a reasonable early management for tia and minor ischemic stroke , the extent of its adoption and use in clinical practice will dependto some extenton its economic practicality . however , the costeffectiveness , which is very important for patients , clinicians , and policymakers , has not been evaluated . in this analysis , we sought to determine the costeffectiveness of adding clopidogrel to aspirin in patients with acute tia or minor stroke . markov models are generally used to estimate the long term costs and outcomes associated with a disease and a particular healthcare intervention , in which the disease is divided into distinct states and transits between these states over a discrete time period under assigned transitions probabilities . a markov model was developed ( figure 1 ) to simulate the costeffectiveness of two antiplatelet treatment strategies for highrisk patients with acute minor stroke or tia : ( 1 ) clopidogrelaspirin strategy : a 300mg loading dose of clopidogrel followed by 75 mg clopidogrel per day on days 2 to 90 plus aspirin 75 to 300 mg on day 1 followed by 75 mg / day on days 2 to 21 or ( 2 ) aspirinalone strategy : aspirin 75 to 300 mg on day 1 followed by 75 mg / day on days 2 to 90 . the basecase was a cohort of 100 000 patients ( 33% female ) , with mean age of 63 years at the time of acute ischemic minor stroke or tia , which is the sex distribution and mean age of the patients who were enrolled in the chance trial ( clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ) . patients in the two treatment arms entered the model at the markov health state of minor or no disability and transited to other health states ( ischemic stroke or intracranial hemorrhage with minor or no disability , moderate disability , severe disability , myocardial infarction , extracranial hemorrhage , and dead ) in the next cycle . death ( by stroke or other causes ) was the only absorbing state after which the patient was excluded from the model . total direct medical costs and qualityadjusted lifeyears ( qalys ) gained with each alternative were estimated for each health state at 90 days from onset of tia or minor stroke and then estimated annually for the remaining 30 years . locations in the model where prescribers can make a decision ( squares ) , chance nodes that are under the control of transition probabilities ( circles ) , and terminal nodes ( triangles ) are presented . transitions to future health states leading from the asaalone branch are the same as for the clopidogrelasa branch . asa indicates aspirin ; ech , extracranial hemorrhage ; ich , intracerebral hemorrhage ; m , markov node ; mi , myocardial infarction ; tia , transient ischemic attack . model input parameters were drawn from the published literature and directly from the results of the chance trial ( tables 1 and 2 ) . the death rates and the distribution of functional outcomes of patients treated with clopidogrelaspirin or aspirin alone in the first 90 days were based on results from the chance trial , derived directly from the trial database . major extracranial hemorrhage risk of the two treatment arms at 90 days were estimated by excluding intracranial hemorrhage from total hemorrhage defined by gusto ( global utilization of streptokinase and tissue plasminogen activator for occluded coronary arteries ) criteria , because extracranial hemorrhage were not explicitly defined in chance trial . we assumed all patients stopped the clopidogrelaspirin regimen and used aspirin alone after 90 days since dualantiplatelet therapy is not recommended for routine secondary stroke prevention and aspirin alone is more commonly used than clopidogrel alone in the longterm setting after tia and minor stroke . recurrent rates of stroke and the proportion of intracranial hemorrhage ( ich ) and fatal cases in recurrent stroke in years after the first 90 days of antiplatelet therapy were estimated from the china national stroke registry ( cnsr ) , a nationwide registry for patients with acute cerebrovascular events in china between september 2007 and august 2008 , recruiting 21 902 consecutive patients from 132 hospitals that cover all 27 provinces and four municipalities in china , and the nanjing stroke registry program ( nsrp ) , a total of 1432 patients with firstever ischemic stroke registered from july 2002 . we further assumed an increase in stroke recurrence rates by 1.017fold per lifeyear , according to the relative risk estimated from cnsr . patients remaining alive after recurrent stroke events were assumed to be reallocated equally among disability categories of equal and greater disability . for example , patients with minor or no disability who had a recurrent stroke and lived were allocated equally among minor or no disability , moderate disability , and severe disability categories . agespecific mortality rates for nonstroke death were derived from the most recent published census of china and adjusted by the causes of death of 2010 reported in the china health statistics yearbook 2012 . efficacy of clopidogrelaspirin regimen for tia and minor stroke tia indicates transient ischemic attack ; chance , clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ; mrs , modified rankin score ; ich , intracerebral hemorrhage ; ech , extracranial hemorrhage ; mi , myocardial infarction . model parameters and the range of values tested in sensitivity analyses all costs were converted to 2011 cny by using the medical care component of consumer price index ; to convert cny to us dollars , divide by 6.5 . cny indicates chinese yuan renminbi ; mrs , modified rankin score ; cnsr , china national stroke registry ; mi , myocardial infarction ; ech , extracranial hemorrhage ; ich , intracerebral hemorrhage . agespecific nonstroke death rate for only the number of 63yearolds ( 0.0089 ) and 93yearolds ( 0.1654 ) is presented . in our model , myocardial infarction and major extracranial hemorrhages for nonfatal myocardial infarction and nonfatal major extracranial hemorrhage , we further assumed that all patients entering these health states would have a shortterm disutility of only 2 weeks for nonfatal major extracranial hemorrhage and 30 days for nonfatal myocardial infarction . all costs were total costs , including both outofpocket costs and reimbursement levels , and converted to 2011 chinese yuan renminbi ( cny ) by using the medical care component of consumer price index . onetime hospitalization costs for major events and annual posthospitalization costs were based on cnsr and the china health statistics yearbook 2012 . additional costs of the 90day clopidogrelaspirin regimen were based on the retail price of clopidogrel ( brand ) and aspirin according to beijing municipal commission of development and reform . indirect economic costs such as lost work productivity were not included in this analysis . all costs and utilities , patients could undergo transitions between four poststroke disability states based on the modified rankin scale ( mrs ) : minor or no disability ( mrs 0 to 2 ) , moderate disability ( mrs 3 to 4 ) , severe disability ( mrs 5 ) , or death ( mrs 6 ) . at the end of each annual cycle , patients could remain in their current health state , transition to a lower health state due to recurrent stroke , or die due to a recurrent stroke or a nonvascular cause ( see figure 1 ) . health outcomes were measured in qalys by multiplying years of life by utility scores derived from the literature . utility estimates for stroke survivors were based on published utility values stratified by mrs category . economic costs were measured as the difference of health care costs between the two treatment alternatives . the economic costs included the additional cost of the 90day clopidogrelaspirin regimen , hospitalization for stroke or tia , myocardial infarction , extracranial hemorrhage , and posthospitalization longterm care associated with each health state . the incremental costeffectiveness ratio ( icer ) was calculated by dividing the cost difference by the difference in qalys . using the threshold of 3 gdp per capita of china in 2011 as the willingnesstopay per qaly , a threshold recommended by the commission on macroeconomics and health of world health organization , the intervention was considered costeffective if the icer was < cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) per qaly gained . oneway sensitivity analyses were performed to test the robustness of model results on all variables across plausible ranges determined a priori . plausible ranges were obtained from the literature or by varying estimates up to 20% in each direction ( table 2 ) . to evaluate the impact of the uncertainty in all variables simultaneously , a probabilistic sensitivity analysis was performed using monte carlo simulation in ersatz v1.3 ( a bootstrap addin for microsoft excel for windows ; epigear international pty ltd ) . markov models are generally used to estimate the long term costs and outcomes associated with a disease and a particular healthcare intervention , in which the disease is divided into distinct states and transits between these states over a discrete time period under assigned transitions probabilities . a markov model was developed ( figure 1 ) to simulate the costeffectiveness of two antiplatelet treatment strategies for highrisk patients with acute minor stroke or tia : ( 1 ) clopidogrelaspirin strategy : a 300mg loading dose of clopidogrel followed by 75 mg clopidogrel per day on days 2 to 90 plus aspirin 75 to 300 mg on day 1 followed by 75 mg / day on days 2 to 21 or ( 2 ) aspirinalone strategy : aspirin 75 to 300 mg on day 1 followed by 75 mg / day on days 2 to 90 . the basecase was a cohort of 100 000 patients ( 33% female ) , with mean age of 63 years at the time of acute ischemic minor stroke or tia , which is the sex distribution and mean age of the patients who were enrolled in the chance trial ( clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ) . patients in the two treatment arms entered the model at the markov health state of minor or no disability and transited to other health states ( ischemic stroke or intracranial hemorrhage with minor or no disability , moderate disability , severe disability , myocardial infarction , extracranial hemorrhage , and dead ) in the next cycle . death ( by stroke or other causes ) was the only absorbing state after which the patient was excluded from the model . total direct medical costs and qualityadjusted lifeyears ( qalys ) gained with each alternative were estimated for each health state at 90 days from onset of tia or minor stroke and then estimated annually for the remaining 30 years . locations in the model where prescribers can make a decision ( squares ) , chance nodes that are under the control of transition probabilities ( circles ) , and terminal nodes ( triangles ) are presented . transitions to future health states leading from the asaalone branch are the same as for the clopidogrelasa branch . asa indicates aspirin ; ech , extracranial hemorrhage ; ich , intracerebral hemorrhage ; m , markov node ; mi , myocardial infarction ; tia , transient ischemic attack . model input parameters were drawn from the published literature and directly from the results of the chance trial ( tables 1 and 2 ) . the death rates and the distribution of functional outcomes of patients treated with clopidogrelaspirin or aspirin alone in the first 90 days were based on results from the chance trial , derived directly from the trial database . major extracranial hemorrhage risk of the two treatment arms at 90 days were estimated by excluding intracranial hemorrhage from total hemorrhage defined by gusto ( global utilization of streptokinase and tissue plasminogen activator for occluded coronary arteries ) criteria , because extracranial hemorrhage were not explicitly defined in chance trial . we assumed all patients stopped the clopidogrelaspirin regimen and used aspirin alone after 90 days since dualantiplatelet therapy is not recommended for routine secondary stroke prevention and aspirin alone is more commonly used than clopidogrel alone in the longterm setting after tia and minor stroke . recurrent rates of stroke and the proportion of intracranial hemorrhage ( ich ) and fatal cases in recurrent stroke in years after the first 90 days of antiplatelet therapy were estimated from the china national stroke registry ( cnsr ) , a nationwide registry for patients with acute cerebrovascular events in china between september 2007 and august 2008 , recruiting 21 902 consecutive patients from 132 hospitals that cover all 27 provinces and four municipalities in china , and the nanjing stroke registry program ( nsrp ) , a total of 1432 patients with firstever ischemic stroke registered from july 2002 . we further assumed an increase in stroke recurrence rates by 1.017fold per lifeyear , according to the relative risk estimated from cnsr . patients remaining alive after recurrent stroke events were assumed to be reallocated equally among disability categories of equal and greater disability . for example , patients with minor or no disability who had a recurrent stroke and lived were allocated equally among minor or no disability , moderate disability , and severe disability categories . agespecific mortality rates for nonstroke death were derived from the most recent published census of china and adjusted by the causes of death of 2010 reported in the china health statistics yearbook 2012 . efficacy of clopidogrelaspirin regimen for tia and minor stroke tia indicates transient ischemic attack ; chance , clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ; mrs , modified rankin score ; ich , intracerebral hemorrhage ; ech , extracranial hemorrhage ; mi , myocardial infarction . model parameters and the range of values tested in sensitivity analyses all costs were converted to 2011 cny by using the medical care component of consumer price index ; to convert cny to us dollars , divide by 6.5 . cny indicates chinese yuan renminbi ; mrs , modified rankin score ; cnsr , china national stroke registry ; mi , myocardial infarction ; ech , extracranial hemorrhage ; ich , intracerebral hemorrhage . agespecific nonstroke death rate for only the number of 63yearolds ( 0.0089 ) and 93yearolds ( 0.1654 ) is presented . in our model , myocardial infarction and major extracranial hemorrhages these probabilities were derived from the literature . for nonfatal myocardial infarction and nonfatal major extracranial hemorrhage , we further assumed that all patients entering these health states would have a shortterm disutility of only 2 weeks for nonfatal major extracranial hemorrhage and 30 days for nonfatal myocardial infarction . all costs were total costs , including both outofpocket costs and reimbursement levels , and converted to 2011 chinese yuan renminbi ( cny ) by using the medical care component of consumer price index . onetime hospitalization costs for major events and annual posthospitalization costs were based on cnsr and the china health statistics yearbook 2012 . additional costs of the 90day clopidogrelaspirin regimen were based on the retail price of clopidogrel ( brand ) and aspirin according to beijing municipal commission of development and reform . in the model , patients could undergo transitions between four poststroke disability states based on the modified rankin scale ( mrs ) : minor or no disability ( mrs 0 to 2 ) , moderate disability ( mrs 3 to 4 ) , severe disability ( mrs 5 ) , or death ( mrs 6 ) . at the end of each annual cycle , patients could remain in their current health state , transition to a lower health state due to recurrent stroke , or die due to a recurrent stroke or a nonvascular cause ( see figure 1 ) . health outcomes were measured in qalys by multiplying years of life by utility scores derived from the literature . utility estimates for stroke survivors were based on published utility values stratified by mrs category . economic costs were measured as the difference of health care costs between the two treatment alternatives . the economic costs included the additional cost of the 90day clopidogrelaspirin regimen , hospitalization for stroke or tia , myocardial infarction , extracranial hemorrhage , and posthospitalization longterm care associated with each health state . the incremental costeffectiveness ratio ( icer ) was calculated by dividing the cost difference by the difference in qalys . using the threshold of 3 gdp per capita of china in 2011 as the willingnesstopay per qaly , a threshold recommended by the commission on macroeconomics and health of world health organization , the intervention was considered costeffective if the icer was < cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) per qaly gained . oneway sensitivity analyses were performed to test the robustness of model results on all variables across plausible ranges determined a priori . plausible ranges were obtained from the literature or by varying estimates up to 20% in each direction ( table 2 ) . to evaluate the impact of the uncertainty in all variables simultaneously , a probabilistic sensitivity analysis was performed using monte carlo simulation in ersatz v1.3 ( a bootstrap addin for microsoft excel for windows ; epigear international pty ltd ) . in the basecase scenario , for a 63yearold patient with acute minor stroke or tia , early 90day clopidogrelaspirin regimen would result in a lifetime gain of 0.037 qaly at an additional cost of cny 1250 ( us$ 192 ) ( table 3).therefore , the icer of early 90day clopidogrelaspirin regimen would be approximately cny 33 800 ( us$ 5200 ) per qaly gained . using the threshold of cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) as the willingnesstopay per qaly , cost and qalys per capita in basecase analysis qaly indicates qualityadjusted lifeyear ; cny , chinese yuan renminbi ; icer , incremental costeffectiveness ratio . the tornado diagram in figure 2 illustrates the effect of varying input parameters on the icer . overall , results were most sensitive to additional cost of 90day clopidogrel regimen and annual posthospitalization cost of disabling stroke ( mrs 3 to 5 ) . when additional cost of 90day clopidogrel regimen was decreased to cny 210 ( based on the generic clopidogrel price in the united states ) , the icer of clopidogrelaspirin decreased to cny 14 300/qaly , which represents a cost saving with improved health . when annual posthospitalization cost of disabling stroke was varied from cny 14254.6 to cny 3239.7 , the dualantiplatelet therapy 's icer increased from cny 25 000/qaly to cny 53 500/qaly . the icer was relatively insensitive to varying parameters of proportion of ich in recurrent stroke , utility of major extracranial bleed , utility of myocardial infarction , and hospitalization cost of ich . oneway sensitivity analyses on incremental cost per qualityadjusted lifeyear ( icer ) gained by 90day clopidogrelaspirin regimen . all parameters were analyzed , and only those with highest influence on icer are displayed . the first number listed after the variable name is the basecase value . solid vertical lines represent icer of the clopidogrelaspirin regimen at the basecase scenario ( cny 33 800 ) . cny indicates chinese yuan renminbi ; mi , myocardial infarction ; qalys , qualityadjusted lifeyears . results of 10 000 iteration probabilistic sensitivity analysis are shown in figure 3 . in 10.6% of simulation runs , clopidogrelaspirin therapy was costeffective in 95.7% of the simulations at a willingnesstopay threshold of cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) per qaly . it remained costeffective in 49.0% of the simulations at a willingnesstopay threshold of cny 35 100 ( 1 gdp per capita of china in 2011 , us$ 5400 ) per qaly . a costeffectiveness acceptability curve shows the probability of costeffectiveness of the clopidogrelaspirin regimen as a function of the willingnesstopay threshold ( figure 4 ) . the dark square represents the basecase ( 0.037 qaly gained at an incremental cost of cny 1250 ) . the curve presents the probability that the clopidogrelaspirin regimen is costeffective as a function of willingnesstopay threshold . in the basecase scenario , for a 63yearold patient with acute minor stroke or tia , early 90day clopidogrelaspirin regimen would result in a lifetime gain of 0.037 qaly at an additional cost of cny 1250 ( us$ 192 ) ( table 3).therefore , the icer of early 90day clopidogrelaspirin regimen would be approximately cny 33 800 ( us$ 5200 ) per qaly gained . using the threshold of cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) as the willingnesstopay per qaly , cost and qalys per capita in basecase analysis qaly indicates qualityadjusted lifeyear ; cny , chinese yuan renminbi ; icer , incremental costeffectiveness ratio . the tornado diagram in figure 2 illustrates the effect of varying input parameters on the icer . overall , results were most sensitive to additional cost of 90day clopidogrel regimen and annual posthospitalization cost of disabling stroke ( mrs 3 to 5 ) . when additional cost of 90day clopidogrel regimen was decreased to cny 210 ( based on the generic clopidogrel price in the united states ) , the icer of clopidogrelaspirin decreased to cny 14 300/qaly , which represents a cost saving with improved health . when annual posthospitalization cost of disabling stroke was varied from cny 14254.6 to cny 3239.7 , the dualantiplatelet therapy 's icer increased from cny 25 000/qaly to cny 53 500/qaly . the icer was relatively insensitive to varying parameters of proportion of ich in recurrent stroke , utility of major extracranial bleed , utility of myocardial infarction , and hospitalization cost of ich . oneway sensitivity analyses on incremental cost per qualityadjusted lifeyear ( icer ) gained by 90day clopidogrelaspirin regimen . all parameters were analyzed , and only those with highest influence on icer are displayed . solid vertical lines represent icer of the clopidogrelaspirin regimen at the basecase scenario ( cny 33 800 ) . cny indicates chinese yuan renminbi ; mi , myocardial infarction ; qalys , qualityadjusted lifeyears . results of 10 000 iteration probabilistic sensitivity analysis are shown in figure 3 . in 10.6% of simulation runs , clopidogrelaspirin therapy was costeffective in 95.7% of the simulations at a willingnesstopay threshold of cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) per qaly . it remained costeffective in 49.0% of the simulations at a willingnesstopay threshold of cny 35 100 ( 1 gdp per capita of china in 2011 , us$ 5400 ) per qaly . a costeffectiveness acceptability curve shows the probability of costeffectiveness of the clopidogrelaspirin regimen as a function of the willingnesstopay threshold ( figure 4 ) . the dark square represents the basecase ( 0.037 qaly gained at an incremental cost of cny 1250 ) . the curve presents the probability that the clopidogrelaspirin regimen is costeffective as a function of willingnesstopay threshold . based largely on the results of the chance trial , this costeffectiveness analysis demonstrates that a 90day clopidogrelaspirin regimen increases costs but improves quality of life . a patient on dual antiplatelet therapy gained an additional 0.037 qaly over a lifetime but at an additional of cny 1250 ( us$ 192 ) , resulting in an icer of cny 33 800 ( us$ 5200 ) per qaly . the robustness of our overall conclusion that 90day clopidogrelaspirin regimen is costeffective is supported by the sensitivity analysis . the lifetime gain of 0.037 qaly ( 0.44 qualityadjusted months ) for early 90day clopidogrelaspirin regimen of tia and minor stroke is comparable to that of most stroke treatments . for example , the lifetime qaly gain is 0.56 for tissue plasminogen activator treatment for acute ischemic stroke in the 3hour time window , 0.17 for clopidogrel ( ccompared with aspirin ) for secondary prevention in stroke patients . the gain of qalys associated with early 90day clopidogrelaspirin regimen of tia and minor stroke is relatively smaller than that of other treatments mainly because , unlike other analyses did , our analysis referred to nondisabling cerebrovascular events ( tia and minor stroke ) other than stroke with high severity . costeffectiveness was sensitive to the additional cost of 90day clopidogrel regimen and the annual posthospitalization cost of disabled stroke . when clopidogrel price was decreased to cny 78 ( us$ 12 , the generic clopidogrel price in us ) for a 1month supply , the additional cost of 90day clopidogrel regimen was decreased , and dualantiplatelet therapy became cost saving . if the annual posthospitalization cost of disabling stroke was reduced to approximately onethird of the basecase level , cost saving from dual antiplatelet therapy was decreased , but the resultant icer was still substantially lower than the willingnesstopay threshold . in current guidelines for the early management of patients with acute ischemic stroke published by both the cerebrovascular disease group of chinese medical association and by the american heart association / american stroke association , clopidogrel and aspirin combination therapy is not recommended for patients with minor ischemic stroke and tia . however , guidelines have not incorporated results of the chance trial and patients with aspirin monotherapy still have 90day stroke risk of 10% to 20% after minor ischemic stroke or tia . our study suggested that clopidogrelaspirin combination therapy is a costeffective alternative over aspirin monotherapy when taken soon after a tia or minor stroke . first , the external generalizability of our findings may be affected as it is based on the efficacy findings from a single trial performed in china ( the chance trial ) , whose participants were restricted to chinese patients . it is not known whether combination regimen will be shown to be similarly effective in other populations or settings . additionally , we used a basecase cohort based on the chance trial patient characteristics ( mean age was 63 years and 33% were female ) , with a low proportion of female tia patients . this was different from typical tia population from populationbased cohorts , and may result in different events rates and medical resource use . second , we used most estimates from cnsr and previously published literature for our input parameters . for example , we used the costs data from cnsr and inflate the costs to 2011 chinese yuan renminbi to estimate the onetime hospitalization costs of stroke . however , hospitalization costs of stroke differed widely by location and level of hospital . these estimates are higher than the official ( mean hospital cost ) figures from the ministry of health of china ( cny 7325.3 , us$ 1127 for ischemic stroke ; cny 11 802.1 us$ 1816 for ich ) , but comparable with those reported by the chinaquest study ( cny 10 689 , us$ 1527 for ischemic stroke ; cny 13 089 , us$ 1870 for ich , in 2006 ) . this may have been because many of the patients in cnsr and chinaquest studies were from urban locations where costs are greater . additionally , several surveys explored poor rehabilitation and decreased adherence to secondary prevention of ischemic stroke in china after discharge , which may dramatically influence the annual posthospitalization costs but would do so similarly in both groups . third , in the markov model , we assumed future transitions of health status based on events related to stroke recurrence and total nonstroke death and functional status as a result of other cause were not considered in this model . some studies have shown that disability status also affects survival , and this was not included in the present analysis . fourth , we assumed that patients remaining alive after recurrent stroke events were reallocated equally among disability categories of equal and greater disability . if a societal perspective was taken , indirect costs such as lost work productivity should be included and it may probably influence the results of the model . these assumptions may deviate from the real world , but are unlikely to affect the overall results of our study because our findings were robust over reasonable variations in all model inputs . , the monthly cost of clopidogrel ( cny 650 , us$ 100 ) is substantially greater in china than in the united states , where generic clopidogrel is available for cny 78 ( us$ 12 ) for a month supply while the costs of hospitalization and posthospital care are much greater in the united states . if generic is introduced , the price will come down dramatically and will likely be cost saving . early treatment with a 90day clopidogrelaspirin regimen for acute tia or minor stroke is highly costeffective in china setting . yongjun wang ( beijingtiantan hospital , principal investigator ) ; s.claiborne johnston ( departments of neurology and epidemiology , university of california , san francisco , usa , coprincipal investigator ) ; yilong wang ( beijingtiantan hospital , executive committee ) ; xingquan zhao ( beijingtiantan hospital , site investigator ) ; zhimin wang ( taizhou first people 's hospital , site investigator ) ; haiqin xia ( taiyuan iron and steel [ group ] co. , ltd . , general hospital , site investigator ) ; ( dagang oilfield gengeal hospital , site investigator ) ; guiru zhang ( penglai people 's hospital , site investigator ) ; xudong ren ( the third people 's hospital of datong , site investigator ) ; chunling ji ( the fourth central hospital of tianjin , site investigator ) ; guohua zhang ( the second hospital of hebei medical university , site investigator ) ; jianhua li ( the first hospital of fangshan district , beijing , site investigator ) ; bohua lu ( beijing puren hospital , site investigator ) ; liping wang ( tianjin ninghe district hospital , site investigator ) ; shutao feng ( the people 's hospital of zhengzhou , site investigator ) ; dali wang ( affiliated hospital of north china coal medical college , site investigator ) ; weiguotang ( zhejiang zhoushan hospital , site investigator ) ; juntao li ( han dan central hospital , site investigator ) ; hongtian zhang ( zhecheng people 's hospital , site investigator ) ; guanglai li ( shanxi medical university second hospital , site investigator ) ; baojun wang ( baotou central hospital , site investigator ) ; yuhua chen ( the general hospital of changjiang river shipping , site investigator ) ; ying lian ( dalian economic and technological development zone hospital , site investigator ) ; bin liu ( first neurology department , affiliated hospital of north china coal medical college , site investigator ) ; junfang teng ( the first affiliated hospital of zhengzhou university , site investigator ) ; rubo sui ( first affiliated hospital of liaoning medical , site investigator ) ; lejun li ( lianyungang municipal hospital of tcm , site investigator ) ; zhiling yuan ( central hospital in qiu county , site investigator ) ; dawei zang ( tianjin first center hospital , site investigator ) ; zuneng lu ( renmin hospital of wuhan university , site investigator ) ; li sun ( qingdao central hospital , site investigator ) ; dong wang ( baogang hospital , site investigator ) ; liying hou ( changzhi city people 's hospital of shanxi province , site investigator ) ; dongcai yuan ( halixun international peace hospital , site investigator ) ; yongliang cao ( people 's hospital of linzi district , zibo , site investigator ) ; hui li ( yantai city yantai mountain hospital , site investigator ) ; xiuge tan ( beijing pinggu district hospital , site investigator ) ; huicong wang ( taiyuan central hospital , site investigator ) ; haisong du ( chengde central hospital , site investigator ) ; mingyi liu ( shijiazhuang central hospital , site investigator ) ; suping wang ( first neurology department , dalian municipal central hospital , site investigator ) ; qiuwu liu ( xian 141 hospital , site investigator ) ; zhong zhang ( chengdu third municipal people 's hospital , site investigator ) ; qifu cui ( affiliated hospital of chifeng university , site investigator ) ; runqing wang ( zhengzhou central hospital , site investigator ) ; jialin zhao ( ningbo city , zhejiang province lihuili hospital medical center , site investigator ) ; jiewen zhang ( henan provincial people 's hospital , site investigator ) ; jianping zhao ( jinzhong city second hospital , site investigator ) ; qi bi ( beijing anzhen hospital , capital medical university , site investigator ) ; xiyou qi ( beijing huairou district chinese medicine hospital , site investigator ) ; junyan liu ( hebei medical university third hospital , site investigator ) ; changxin li ( first affiliated hospital shanxi medical unversity , site investigator ) ; ling li ( hebei provincial people 's hospital , site investigator ) ; xiaoping pan ( guangzhou first municipal peoples hospital , site investigator ) ; junling zhang ( central hospital in cangzhou , site investigator ) ; derang jiao ( the chinese people 's armed police force medical school affiliated hospital , site investigator ) ; zhao han ( zhejiang wenzhou medical college first affiliated hospital , site investigator ) ; dawei qian ( jilin central hospital , site investigator ) ; jin xiao ( anhui maanshan central hospital , site investigator ) ; yan xing ( beijing aviation industry central hospital , site investigator ) ; huishan du ( luhe hospital , tongzhou district , beijing , site investigator ) ; guang huang ( beijing fuxing hospital , capital medical university , site investigator ) ; yongqiang cui ( the 306th hospital of p.l.a , site investigator ) ; yan li ( the first affiliated hospital of tianjin university of chinese medicine , site investigator ) ; lianyuan feng ( baiqiuen international peace hospital of people 's liberation army , site investigator ) ; lianbo gao ( fourth affiliated hospital of china medical university , site investigator ) ; bo xiao ( xiangya hospital centralsouth university , site investigator ) ; yibin cao ( tangshan worker 's hospital , site investigator ) ; yiping wu ( the 1st hospital in handan , site investigator ) ; jinfeng liu ( yangquan coal ( group ) co. , ltd . general hospital , site investigator ) ; zhiming zhang ( tianjin tianhe hospital , site investigator ) ; zhengxie dong ( nantong first people 's hospital , site investigator ) ; limin wang ( the 1st hospital of zhangjiakou city , site investigator ) ; li he ( west china hospital , sichuan university , site investigator ) ; xinchen wang ( the second affiliated hospital of shandong university of tcm , site investigator ) ; xueying guo ( fenyang hospital of shanxi province , site investigator ) ; ming wang ( zhejiang zhoushan putuo district people 's hospital , site investigator ) ; xiaosha wang ( xiyuan hospital of china academy of chinese traditional medicine , site investigator ) ; jiandong jiang ( no.2 people 's hospital east in lianyungang city , site investigator ) ; renliang zhao ( affiliated hospital of qingdao university medical college , site investigator ) ; shengnian zhou ( qilu hospital of shandong university , site investigator ) ; haohu ( zibo hospital of traditional chinese medicine , site investigator ) ; maolin he ( beijing shijitan hospital , site investigator ) ; fengchun yu ( beijing haidian hospital , site investigator ) ; quping ouyang ( beijing shunyi district hospital , site investigator ) ; jingbo zhang ( dalian third municipal hospital , site investigator ) ; anding xu ( the first affliated hospital of jinan university , site investigator ) ; xiaokun qi ( navy genaral hospital of p.l.a , site investigator ) ; lei wang ( beijing second artillery general hospital , site investigator ) ; fuming shi ( beijing daxing district hospital , site investigator ) ; fuqiang guo ( sichuan province people 's hospital , site investigator ) ; jianfeng wang ( dalian municipal central hospital , site investigator ) ; fengli zhao ( the second hospital in baoding , site investigator ) ; ronghua dou ( the hospital combine traditional chinese and western medicine in cang zhou , site investigator ) ; dongning wei ( the 309th hospital of p.l.a , site investigator ) ; qingwei meng ( liangxiang hospital of fangshan district , beijing , site investigator ) ; yilu xia ( huaxin hospital first hospital of tsinghua university , site investigator ) ; shiminwang ( tianjinhuanhu hospital , site investigator ) ; zhangcang xue ( shijiazhuang first hospital , site investigator ) ; yuming xu ( the first affiliated hospital of zhengzhou university , site investigator ) ; liping ma ( xinzhou city people 's hospital , site investigator ) ; chun wang ( sichuan province people 's hospital of deyang city , site investigator ) ; jiang wu ( first hospital , jilin university , site investigator ) ; yifeng du ( shandong provincial hospital , site investigator ) ; yinzhou wang ( fujian province hospital , site investigator ) ; lijun xiao ( liaoyang city third people 's hospital , site investigator ) ; fucong song ( handan city center hospital , site investigator ) ; wenli hu ( beijing chaoyang hospital , capital medical university , site investigator ) ; zhigang chen ( beijing university of chinese medicine east hospital , site investigator ) ; qingrui liu ( hebei medical university fourth hospital , site investigator ) ; jiemin zhang ( the fourth affiliated hospital of soochow university , site investigator ) ; mei chen ( zhejiang university of chinese medicine affiliated first hospital , site investigator ) ; xiaodong yuan ( affiliated hospital of kailuan company ltd , site investigator ) ; zhihui liu ( affiliated hospital of weifang medical university , site investigator ) ; guozhong li ( the first hospital of harbin medical university , site investigator ) ; xiaohong li ( dalian friendship hospital , site investigator ) ; tingchen tian ( tianjin dagang hospital , site investigator ) .

backgroundtreatment with the combination of clopidogrel and aspirin taken soon after a transient ischemic attack ( tia ) or minor stroke was shown to reduce the 90day risk of stroke in a large trial in china , but the costeffectiveness is unknown . this study sought to estimate the costeffectiveness of the clopidogrelaspirin regimen for acute tia or minor stroke.methods and resultsa markov model was created to determine the costeffectiveness of treatment of acute tia or minor stroke patients with clopidogrelaspirin compared with aspirin alone . inputs for the model were obtained from clinical trial data , claims databases , and the published literature . the main outcome measure was cost per qualityadjusted lifeyears ( qalys ) gained . oneway and multivariable probabilistic sensitivity analyses were performed to test the robustness of the findings . compared with aspirin alone , clopidogrelaspirin resulted in a lifetime gain of 0.037 qalys at an additional cost of cny 1250 ( us$ 192 ) , yielding an incremental costeffectiveness ratio of cny 33 800 ( us$ 5200 ) per qaly gained . probabilistic sensitivity analysis showed that clopidogrelaspirin therapy was more costeffective in 95.7% of the simulations at a willingnesstopay threshold recommended by the world health organization of cny 105 000 ( us$ 16 200 ) per qaly.conclusionsearly 90day clopidogrelaspirin regimen for acute tia or minor stroke is highly costeffective in china . although clopidogrel is generic , plavix is brand in china . if plavix were generic , treatment with clopidogrelaspirin would have been cost saving .

Introduction Methods Model Overview Input Parameters Costs Health States Outcome Assessment Sensitivity Analysis Results BaseCase Analysis Sensitivity Analysis Discussion Conclusions The CHANCE Investigators

the early ( 90day ) risk of recurrence of stroke and other vascular events following index transient ischemic attack ( tia ) and minor ischemic stroke is very high , even in patients treated with aspirin , the current standard of care . treatment with the combination of clopidogrel and aspirin taken soon after a tia or minor stroke was found to decrease the 90day risk of stroke ( hazard ratio 0.68 , 95% ci 0.57 to 0.81 , p<0.001 ) but did not increase the risk of hemorrhage compared with aspirin alone in the chance stroke trial ( clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ) . although the clopidogrelaspirin regimen is a reasonable early management for tia and minor ischemic stroke , the extent of its adoption and use in clinical practice will dependto some extenton its economic practicality . in this analysis , we sought to determine the costeffectiveness of adding clopidogrel to aspirin in patients with acute tia or minor stroke . a markov model was developed ( figure 1 ) to simulate the costeffectiveness of two antiplatelet treatment strategies for highrisk patients with acute minor stroke or tia : ( 1 ) clopidogrelaspirin strategy : a 300mg loading dose of clopidogrel followed by 75 mg clopidogrel per day on days 2 to 90 plus aspirin 75 to 300 mg on day 1 followed by 75 mg / day on days 2 to 21 or ( 2 ) aspirinalone strategy : aspirin 75 to 300 mg on day 1 followed by 75 mg / day on days 2 to 90 . the basecase was a cohort of 100 000 patients ( 33% female ) , with mean age of 63 years at the time of acute ischemic minor stroke or tia , which is the sex distribution and mean age of the patients who were enrolled in the chance trial ( clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ) . total direct medical costs and qualityadjusted lifeyears ( qalys ) gained with each alternative were estimated for each health state at 90 days from onset of tia or minor stroke and then estimated annually for the remaining 30 years . locations in the model where prescribers can make a decision ( squares ) , chance nodes that are under the control of transition probabilities ( circles ) , and terminal nodes ( triangles ) are presented . model input parameters were drawn from the published literature and directly from the results of the chance trial ( tables 1 and 2 ) . the death rates and the distribution of functional outcomes of patients treated with clopidogrelaspirin or aspirin alone in the first 90 days were based on results from the chance trial , derived directly from the trial database . major extracranial hemorrhage risk of the two treatment arms at 90 days were estimated by excluding intracranial hemorrhage from total hemorrhage defined by gusto ( global utilization of streptokinase and tissue plasminogen activator for occluded coronary arteries ) criteria , because extracranial hemorrhage were not explicitly defined in chance trial . we assumed all patients stopped the clopidogrelaspirin regimen and used aspirin alone after 90 days since dualantiplatelet therapy is not recommended for routine secondary stroke prevention and aspirin alone is more commonly used than clopidogrel alone in the longterm setting after tia and minor stroke . recurrent rates of stroke and the proportion of intracranial hemorrhage ( ich ) and fatal cases in recurrent stroke in years after the first 90 days of antiplatelet therapy were estimated from the china national stroke registry ( cnsr ) , a nationwide registry for patients with acute cerebrovascular events in china between september 2007 and august 2008 , recruiting 21 902 consecutive patients from 132 hospitals that cover all 27 provinces and four municipalities in china , and the nanjing stroke registry program ( nsrp ) , a total of 1432 patients with firstever ischemic stroke registered from july 2002 . efficacy of clopidogrelaspirin regimen for tia and minor stroke tia indicates transient ischemic attack ; chance , clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ; mrs , modified rankin score ; ich , intracerebral hemorrhage ; ech , extracranial hemorrhage ; mi , myocardial infarction . additional costs of the 90day clopidogrelaspirin regimen were based on the retail price of clopidogrel ( brand ) and aspirin according to beijing municipal commission of development and reform . the economic costs included the additional cost of the 90day clopidogrelaspirin regimen , hospitalization for stroke or tia , myocardial infarction , extracranial hemorrhage , and posthospitalization longterm care associated with each health state . the incremental costeffectiveness ratio ( icer ) was calculated by dividing the cost difference by the difference in qalys . using the threshold of 3 gdp per capita of china in 2011 as the willingnesstopay per qaly , a threshold recommended by the commission on macroeconomics and health of world health organization , the intervention was considered costeffective if the icer was < cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) per qaly gained . oneway sensitivity analyses were performed to test the robustness of model results on all variables across plausible ranges determined a priori . to evaluate the impact of the uncertainty in all variables simultaneously , a probabilistic sensitivity analysis was performed using monte carlo simulation in ersatz v1.3 ( a bootstrap addin for microsoft excel for windows ; epigear international pty ltd ) . a markov model was developed ( figure 1 ) to simulate the costeffectiveness of two antiplatelet treatment strategies for highrisk patients with acute minor stroke or tia : ( 1 ) clopidogrelaspirin strategy : a 300mg loading dose of clopidogrel followed by 75 mg clopidogrel per day on days 2 to 90 plus aspirin 75 to 300 mg on day 1 followed by 75 mg / day on days 2 to 21 or ( 2 ) aspirinalone strategy : aspirin 75 to 300 mg on day 1 followed by 75 mg / day on days 2 to 90 . the basecase was a cohort of 100 000 patients ( 33% female ) , with mean age of 63 years at the time of acute ischemic minor stroke or tia , which is the sex distribution and mean age of the patients who were enrolled in the chance trial ( clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ) . total direct medical costs and qualityadjusted lifeyears ( qalys ) gained with each alternative were estimated for each health state at 90 days from onset of tia or minor stroke and then estimated annually for the remaining 30 years . locations in the model where prescribers can make a decision ( squares ) , chance nodes that are under the control of transition probabilities ( circles ) , and terminal nodes ( triangles ) are presented . model input parameters were drawn from the published literature and directly from the results of the chance trial ( tables 1 and 2 ) . the death rates and the distribution of functional outcomes of patients treated with clopidogrelaspirin or aspirin alone in the first 90 days were based on results from the chance trial , derived directly from the trial database . major extracranial hemorrhage risk of the two treatment arms at 90 days were estimated by excluding intracranial hemorrhage from total hemorrhage defined by gusto ( global utilization of streptokinase and tissue plasminogen activator for occluded coronary arteries ) criteria , because extracranial hemorrhage were not explicitly defined in chance trial . we assumed all patients stopped the clopidogrelaspirin regimen and used aspirin alone after 90 days since dualantiplatelet therapy is not recommended for routine secondary stroke prevention and aspirin alone is more commonly used than clopidogrel alone in the longterm setting after tia and minor stroke . recurrent rates of stroke and the proportion of intracranial hemorrhage ( ich ) and fatal cases in recurrent stroke in years after the first 90 days of antiplatelet therapy were estimated from the china national stroke registry ( cnsr ) , a nationwide registry for patients with acute cerebrovascular events in china between september 2007 and august 2008 , recruiting 21 902 consecutive patients from 132 hospitals that cover all 27 provinces and four municipalities in china , and the nanjing stroke registry program ( nsrp ) , a total of 1432 patients with firstever ischemic stroke registered from july 2002 . efficacy of clopidogrelaspirin regimen for tia and minor stroke tia indicates transient ischemic attack ; chance , clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ; mrs , modified rankin score ; ich , intracerebral hemorrhage ; ech , extracranial hemorrhage ; mi , myocardial infarction . additional costs of the 90day clopidogrelaspirin regimen were based on the retail price of clopidogrel ( brand ) and aspirin according to beijing municipal commission of development and reform . the economic costs included the additional cost of the 90day clopidogrelaspirin regimen , hospitalization for stroke or tia , myocardial infarction , extracranial hemorrhage , and posthospitalization longterm care associated with each health state . the incremental costeffectiveness ratio ( icer ) was calculated by dividing the cost difference by the difference in qalys . using the threshold of 3 gdp per capita of china in 2011 as the willingnesstopay per qaly , a threshold recommended by the commission on macroeconomics and health of world health organization , the intervention was considered costeffective if the icer was < cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) per qaly gained . oneway sensitivity analyses were performed to test the robustness of model results on all variables across plausible ranges determined a priori . to evaluate the impact of the uncertainty in all variables simultaneously , a probabilistic sensitivity analysis was performed using monte carlo simulation in ersatz v1.3 ( a bootstrap addin for microsoft excel for windows ; epigear international pty ltd ) . in the basecase scenario , for a 63yearold patient with acute minor stroke or tia , early 90day clopidogrelaspirin regimen would result in a lifetime gain of 0.037 qaly at an additional cost of cny 1250 ( us$ 192 ) ( table 3).therefore , the icer of early 90day clopidogrelaspirin regimen would be approximately cny 33 800 ( us$ 5200 ) per qaly gained . using the threshold of cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) as the willingnesstopay per qaly , cost and qalys per capita in basecase analysis qaly indicates qualityadjusted lifeyear ; cny , chinese yuan renminbi ; icer , incremental costeffectiveness ratio . when additional cost of 90day clopidogrel regimen was decreased to cny 210 ( based on the generic clopidogrel price in the united states ) , the icer of clopidogrelaspirin decreased to cny 14 300/qaly , which represents a cost saving with improved health . oneway sensitivity analyses on incremental cost per qualityadjusted lifeyear ( icer ) gained by 90day clopidogrelaspirin regimen . solid vertical lines represent icer of the clopidogrelaspirin regimen at the basecase scenario ( cny 33 800 ) . in 10.6% of simulation runs , clopidogrelaspirin therapy was costeffective in 95.7% of the simulations at a willingnesstopay threshold of cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) per qaly . it remained costeffective in 49.0% of the simulations at a willingnesstopay threshold of cny 35 100 ( 1 gdp per capita of china in 2011 , us$ 5400 ) per qaly . a costeffectiveness acceptability curve shows the probability of costeffectiveness of the clopidogrelaspirin regimen as a function of the willingnesstopay threshold ( figure 4 ) . the dark square represents the basecase ( 0.037 qaly gained at an incremental cost of cny 1250 ) . the curve presents the probability that the clopidogrelaspirin regimen is costeffective as a function of willingnesstopay threshold . in the basecase scenario , for a 63yearold patient with acute minor stroke or tia , early 90day clopidogrelaspirin regimen would result in a lifetime gain of 0.037 qaly at an additional cost of cny 1250 ( us$ 192 ) ( table 3).therefore , the icer of early 90day clopidogrelaspirin regimen would be approximately cny 33 800 ( us$ 5200 ) per qaly gained . using the threshold of cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) as the willingnesstopay per qaly , cost and qalys per capita in basecase analysis qaly indicates qualityadjusted lifeyear ; cny , chinese yuan renminbi ; icer , incremental costeffectiveness ratio . when additional cost of 90day clopidogrel regimen was decreased to cny 210 ( based on the generic clopidogrel price in the united states ) , the icer of clopidogrelaspirin decreased to cny 14 300/qaly , which represents a cost saving with improved health . oneway sensitivity analyses on incremental cost per qualityadjusted lifeyear ( icer ) gained by 90day clopidogrelaspirin regimen . solid vertical lines represent icer of the clopidogrelaspirin regimen at the basecase scenario ( cny 33 800 ) . in 10.6% of simulation runs , clopidogrelaspirin therapy was costeffective in 95.7% of the simulations at a willingnesstopay threshold of cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) per qaly . it remained costeffective in 49.0% of the simulations at a willingnesstopay threshold of cny 35 100 ( 1 gdp per capita of china in 2011 , us$ 5400 ) per qaly . a costeffectiveness acceptability curve shows the probability of costeffectiveness of the clopidogrelaspirin regimen as a function of the willingnesstopay threshold ( figure 4 ) . the dark square represents the basecase ( 0.037 qaly gained at an incremental cost of cny 1250 ) . the curve presents the probability that the clopidogrelaspirin regimen is costeffective as a function of willingnesstopay threshold . based largely on the results of the chance trial , this costeffectiveness analysis demonstrates that a 90day clopidogrelaspirin regimen increases costs but improves quality of life . a patient on dual antiplatelet therapy gained an additional 0.037 qaly over a lifetime but at an additional of cny 1250 ( us$ 192 ) , resulting in an icer of cny 33 800 ( us$ 5200 ) per qaly . the robustness of our overall conclusion that 90day clopidogrelaspirin regimen is costeffective is supported by the sensitivity analysis . the lifetime gain of 0.037 qaly ( 0.44 qualityadjusted months ) for early 90day clopidogrelaspirin regimen of tia and minor stroke is comparable to that of most stroke treatments . for example , the lifetime qaly gain is 0.56 for tissue plasminogen activator treatment for acute ischemic stroke in the 3hour time window , 0.17 for clopidogrel ( ccompared with aspirin ) for secondary prevention in stroke patients . the gain of qalys associated with early 90day clopidogrelaspirin regimen of tia and minor stroke is relatively smaller than that of other treatments mainly because , unlike other analyses did , our analysis referred to nondisabling cerebrovascular events ( tia and minor stroke ) other than stroke with high severity . costeffectiveness was sensitive to the additional cost of 90day clopidogrel regimen and the annual posthospitalization cost of disabled stroke . when clopidogrel price was decreased to cny 78 ( us$ 12 , the generic clopidogrel price in us ) for a 1month supply , the additional cost of 90day clopidogrel regimen was decreased , and dualantiplatelet therapy became cost saving . if the annual posthospitalization cost of disabling stroke was reduced to approximately onethird of the basecase level , cost saving from dual antiplatelet therapy was decreased , but the resultant icer was still substantially lower than the willingnesstopay threshold . in current guidelines for the early management of patients with acute ischemic stroke published by both the cerebrovascular disease group of chinese medical association and by the american heart association / american stroke association , clopidogrel and aspirin combination therapy is not recommended for patients with minor ischemic stroke and tia . however , guidelines have not incorporated results of the chance trial and patients with aspirin monotherapy still have 90day stroke risk of 10% to 20% after minor ischemic stroke or tia . our study suggested that clopidogrelaspirin combination therapy is a costeffective alternative over aspirin monotherapy when taken soon after a tia or minor stroke . first , the external generalizability of our findings may be affected as it is based on the efficacy findings from a single trial performed in china ( the chance trial ) , whose participants were restricted to chinese patients . for example , we used the costs data from cnsr and inflate the costs to 2011 chinese yuan renminbi to estimate the onetime hospitalization costs of stroke . these estimates are higher than the official ( mean hospital cost ) figures from the ministry of health of china ( cny 7325.3 , us$ 1127 for ischemic stroke ; cny 11 802.1 us$ 1816 for ich ) , but comparable with those reported by the chinaquest study ( cny 10 689 , us$ 1527 for ischemic stroke ; cny 13 089 , us$ 1870 for ich , in 2006 ) . additionally , several surveys explored poor rehabilitation and decreased adherence to secondary prevention of ischemic stroke in china after discharge , which may dramatically influence the annual posthospitalization costs but would do so similarly in both groups . , the monthly cost of clopidogrel ( cny 650 , us$ 100 ) is substantially greater in china than in the united states , where generic clopidogrel is available for cny 78 ( us$ 12 ) for a month supply while the costs of hospitalization and posthospital care are much greater in the united states . early treatment with a 90day clopidogrelaspirin regimen for acute tia or minor stroke is highly costeffective in china setting .

the early ( 90day ) risk of recurrence of stroke and other vascular events following index transient ischemic attack ( tia ) and minor ischemic stroke is very high , even in patients treated with aspirin , the current standard of care . treatment with the combination of clopidogrel and aspirin taken soon after a tia or minor stroke was found to decrease the 90day risk of stroke ( hazard ratio 0.68 , 95% ci 0.57 to 0.81 , p<0.001 ) but did not increase the risk of hemorrhage compared with aspirin alone in the chance stroke trial ( clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ) . although the clopidogrelaspirin regimen is a reasonable early management for tia and minor ischemic stroke , the extent of its adoption and use in clinical practice will dependto some extenton its economic practicality . in this analysis , we sought to determine the costeffectiveness of adding clopidogrel to aspirin in patients with acute tia or minor stroke . markov models are generally used to estimate the long term costs and outcomes associated with a disease and a particular healthcare intervention , in which the disease is divided into distinct states and transits between these states over a discrete time period under assigned transitions probabilities . a markov model was developed ( figure 1 ) to simulate the costeffectiveness of two antiplatelet treatment strategies for highrisk patients with acute minor stroke or tia : ( 1 ) clopidogrelaspirin strategy : a 300mg loading dose of clopidogrel followed by 75 mg clopidogrel per day on days 2 to 90 plus aspirin 75 to 300 mg on day 1 followed by 75 mg / day on days 2 to 21 or ( 2 ) aspirinalone strategy : aspirin 75 to 300 mg on day 1 followed by 75 mg / day on days 2 to 90 . the basecase was a cohort of 100 000 patients ( 33% female ) , with mean age of 63 years at the time of acute ischemic minor stroke or tia , which is the sex distribution and mean age of the patients who were enrolled in the chance trial ( clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ) . patients in the two treatment arms entered the model at the markov health state of minor or no disability and transited to other health states ( ischemic stroke or intracranial hemorrhage with minor or no disability , moderate disability , severe disability , myocardial infarction , extracranial hemorrhage , and dead ) in the next cycle . total direct medical costs and qualityadjusted lifeyears ( qalys ) gained with each alternative were estimated for each health state at 90 days from onset of tia or minor stroke and then estimated annually for the remaining 30 years . the death rates and the distribution of functional outcomes of patients treated with clopidogrelaspirin or aspirin alone in the first 90 days were based on results from the chance trial , derived directly from the trial database . major extracranial hemorrhage risk of the two treatment arms at 90 days were estimated by excluding intracranial hemorrhage from total hemorrhage defined by gusto ( global utilization of streptokinase and tissue plasminogen activator for occluded coronary arteries ) criteria , because extracranial hemorrhage were not explicitly defined in chance trial . we assumed all patients stopped the clopidogrelaspirin regimen and used aspirin alone after 90 days since dualantiplatelet therapy is not recommended for routine secondary stroke prevention and aspirin alone is more commonly used than clopidogrel alone in the longterm setting after tia and minor stroke . recurrent rates of stroke and the proportion of intracranial hemorrhage ( ich ) and fatal cases in recurrent stroke in years after the first 90 days of antiplatelet therapy were estimated from the china national stroke registry ( cnsr ) , a nationwide registry for patients with acute cerebrovascular events in china between september 2007 and august 2008 , recruiting 21 902 consecutive patients from 132 hospitals that cover all 27 provinces and four municipalities in china , and the nanjing stroke registry program ( nsrp ) , a total of 1432 patients with firstever ischemic stroke registered from july 2002 . agespecific mortality rates for nonstroke death were derived from the most recent published census of china and adjusted by the causes of death of 2010 reported in the china health statistics yearbook 2012 . efficacy of clopidogrelaspirin regimen for tia and minor stroke tia indicates transient ischemic attack ; chance , clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ; mrs , modified rankin score ; ich , intracerebral hemorrhage ; ech , extracranial hemorrhage ; mi , myocardial infarction . in our model , myocardial infarction and major extracranial hemorrhages for nonfatal myocardial infarction and nonfatal major extracranial hemorrhage , we further assumed that all patients entering these health states would have a shortterm disutility of only 2 weeks for nonfatal major extracranial hemorrhage and 30 days for nonfatal myocardial infarction . additional costs of the 90day clopidogrelaspirin regimen were based on the retail price of clopidogrel ( brand ) and aspirin according to beijing municipal commission of development and reform . all costs and utilities , patients could undergo transitions between four poststroke disability states based on the modified rankin scale ( mrs ) : minor or no disability ( mrs 0 to 2 ) , moderate disability ( mrs 3 to 4 ) , severe disability ( mrs 5 ) , or death ( mrs 6 ) . at the end of each annual cycle , patients could remain in their current health state , transition to a lower health state due to recurrent stroke , or die due to a recurrent stroke or a nonvascular cause ( see figure 1 ) . the economic costs included the additional cost of the 90day clopidogrelaspirin regimen , hospitalization for stroke or tia , myocardial infarction , extracranial hemorrhage , and posthospitalization longterm care associated with each health state . using the threshold of 3 gdp per capita of china in 2011 as the willingnesstopay per qaly , a threshold recommended by the commission on macroeconomics and health of world health organization , the intervention was considered costeffective if the icer was < cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) per qaly gained . to evaluate the impact of the uncertainty in all variables simultaneously , a probabilistic sensitivity analysis was performed using monte carlo simulation in ersatz v1.3 ( a bootstrap addin for microsoft excel for windows ; epigear international pty ltd ) . markov models are generally used to estimate the long term costs and outcomes associated with a disease and a particular healthcare intervention , in which the disease is divided into distinct states and transits between these states over a discrete time period under assigned transitions probabilities . a markov model was developed ( figure 1 ) to simulate the costeffectiveness of two antiplatelet treatment strategies for highrisk patients with acute minor stroke or tia : ( 1 ) clopidogrelaspirin strategy : a 300mg loading dose of clopidogrel followed by 75 mg clopidogrel per day on days 2 to 90 plus aspirin 75 to 300 mg on day 1 followed by 75 mg / day on days 2 to 21 or ( 2 ) aspirinalone strategy : aspirin 75 to 300 mg on day 1 followed by 75 mg / day on days 2 to 90 . the basecase was a cohort of 100 000 patients ( 33% female ) , with mean age of 63 years at the time of acute ischemic minor stroke or tia , which is the sex distribution and mean age of the patients who were enrolled in the chance trial ( clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ) . patients in the two treatment arms entered the model at the markov health state of minor or no disability and transited to other health states ( ischemic stroke or intracranial hemorrhage with minor or no disability , moderate disability , severe disability , myocardial infarction , extracranial hemorrhage , and dead ) in the next cycle . total direct medical costs and qualityadjusted lifeyears ( qalys ) gained with each alternative were estimated for each health state at 90 days from onset of tia or minor stroke and then estimated annually for the remaining 30 years . the death rates and the distribution of functional outcomes of patients treated with clopidogrelaspirin or aspirin alone in the first 90 days were based on results from the chance trial , derived directly from the trial database . major extracranial hemorrhage risk of the two treatment arms at 90 days were estimated by excluding intracranial hemorrhage from total hemorrhage defined by gusto ( global utilization of streptokinase and tissue plasminogen activator for occluded coronary arteries ) criteria , because extracranial hemorrhage were not explicitly defined in chance trial . we assumed all patients stopped the clopidogrelaspirin regimen and used aspirin alone after 90 days since dualantiplatelet therapy is not recommended for routine secondary stroke prevention and aspirin alone is more commonly used than clopidogrel alone in the longterm setting after tia and minor stroke . recurrent rates of stroke and the proportion of intracranial hemorrhage ( ich ) and fatal cases in recurrent stroke in years after the first 90 days of antiplatelet therapy were estimated from the china national stroke registry ( cnsr ) , a nationwide registry for patients with acute cerebrovascular events in china between september 2007 and august 2008 , recruiting 21 902 consecutive patients from 132 hospitals that cover all 27 provinces and four municipalities in china , and the nanjing stroke registry program ( nsrp ) , a total of 1432 patients with firstever ischemic stroke registered from july 2002 . efficacy of clopidogrelaspirin regimen for tia and minor stroke tia indicates transient ischemic attack ; chance , clopidogrel in highrisk patients with acute nondisabling cerebrovascular events trial ; mrs , modified rankin score ; ich , intracerebral hemorrhage ; ech , extracranial hemorrhage ; mi , myocardial infarction . for nonfatal myocardial infarction and nonfatal major extracranial hemorrhage , we further assumed that all patients entering these health states would have a shortterm disutility of only 2 weeks for nonfatal major extracranial hemorrhage and 30 days for nonfatal myocardial infarction . in the model , patients could undergo transitions between four poststroke disability states based on the modified rankin scale ( mrs ) : minor or no disability ( mrs 0 to 2 ) , moderate disability ( mrs 3 to 4 ) , severe disability ( mrs 5 ) , or death ( mrs 6 ) . at the end of each annual cycle , patients could remain in their current health state , transition to a lower health state due to recurrent stroke , or die due to a recurrent stroke or a nonvascular cause ( see figure 1 ) . the economic costs included the additional cost of the 90day clopidogrelaspirin regimen , hospitalization for stroke or tia , myocardial infarction , extracranial hemorrhage , and posthospitalization longterm care associated with each health state . using the threshold of 3 gdp per capita of china in 2011 as the willingnesstopay per qaly , a threshold recommended by the commission on macroeconomics and health of world health organization , the intervention was considered costeffective if the icer was < cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) per qaly gained . to evaluate the impact of the uncertainty in all variables simultaneously , a probabilistic sensitivity analysis was performed using monte carlo simulation in ersatz v1.3 ( a bootstrap addin for microsoft excel for windows ; epigear international pty ltd ) . in the basecase scenario , for a 63yearold patient with acute minor stroke or tia , early 90day clopidogrelaspirin regimen would result in a lifetime gain of 0.037 qaly at an additional cost of cny 1250 ( us$ 192 ) ( table 3).therefore , the icer of early 90day clopidogrelaspirin regimen would be approximately cny 33 800 ( us$ 5200 ) per qaly gained . using the threshold of cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) as the willingnesstopay per qaly , cost and qalys per capita in basecase analysis qaly indicates qualityadjusted lifeyear ; cny , chinese yuan renminbi ; icer , incremental costeffectiveness ratio . when additional cost of 90day clopidogrel regimen was decreased to cny 210 ( based on the generic clopidogrel price in the united states ) , the icer of clopidogrelaspirin decreased to cny 14 300/qaly , which represents a cost saving with improved health . when annual posthospitalization cost of disabling stroke was varied from cny 14254.6 to cny 3239.7 , the dualantiplatelet therapy 's icer increased from cny 25 000/qaly to cny 53 500/qaly . the icer was relatively insensitive to varying parameters of proportion of ich in recurrent stroke , utility of major extracranial bleed , utility of myocardial infarction , and hospitalization cost of ich . in 10.6% of simulation runs , clopidogrelaspirin therapy was costeffective in 95.7% of the simulations at a willingnesstopay threshold of cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) per qaly . it remained costeffective in 49.0% of the simulations at a willingnesstopay threshold of cny 35 100 ( 1 gdp per capita of china in 2011 , us$ 5400 ) per qaly . a costeffectiveness acceptability curve shows the probability of costeffectiveness of the clopidogrelaspirin regimen as a function of the willingnesstopay threshold ( figure 4 ) . in the basecase scenario , for a 63yearold patient with acute minor stroke or tia , early 90day clopidogrelaspirin regimen would result in a lifetime gain of 0.037 qaly at an additional cost of cny 1250 ( us$ 192 ) ( table 3).therefore , the icer of early 90day clopidogrelaspirin regimen would be approximately cny 33 800 ( us$ 5200 ) per qaly gained . using the threshold of cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) as the willingnesstopay per qaly , cost and qalys per capita in basecase analysis qaly indicates qualityadjusted lifeyear ; cny , chinese yuan renminbi ; icer , incremental costeffectiveness ratio . when additional cost of 90day clopidogrel regimen was decreased to cny 210 ( based on the generic clopidogrel price in the united states ) , the icer of clopidogrelaspirin decreased to cny 14 300/qaly , which represents a cost saving with improved health . when annual posthospitalization cost of disabling stroke was varied from cny 14254.6 to cny 3239.7 , the dualantiplatelet therapy 's icer increased from cny 25 000/qaly to cny 53 500/qaly . the icer was relatively insensitive to varying parameters of proportion of ich in recurrent stroke , utility of major extracranial bleed , utility of myocardial infarction , and hospitalization cost of ich . in 10.6% of simulation runs , clopidogrelaspirin therapy was costeffective in 95.7% of the simulations at a willingnesstopay threshold of cny 105 000 ( 3 gdp per capita of china in 2011 , us$ 16 200 ) per qaly . a costeffectiveness acceptability curve shows the probability of costeffectiveness of the clopidogrelaspirin regimen as a function of the willingnesstopay threshold ( figure 4 ) . based largely on the results of the chance trial , this costeffectiveness analysis demonstrates that a 90day clopidogrelaspirin regimen increases costs but improves quality of life . the lifetime gain of 0.037 qaly ( 0.44 qualityadjusted months ) for early 90day clopidogrelaspirin regimen of tia and minor stroke is comparable to that of most stroke treatments . for example , the lifetime qaly gain is 0.56 for tissue plasminogen activator treatment for acute ischemic stroke in the 3hour time window , 0.17 for clopidogrel ( ccompared with aspirin ) for secondary prevention in stroke patients . the gain of qalys associated with early 90day clopidogrelaspirin regimen of tia and minor stroke is relatively smaller than that of other treatments mainly because , unlike other analyses did , our analysis referred to nondisabling cerebrovascular events ( tia and minor stroke ) other than stroke with high severity . when clopidogrel price was decreased to cny 78 ( us$ 12 , the generic clopidogrel price in us ) for a 1month supply , the additional cost of 90day clopidogrel regimen was decreased , and dualantiplatelet therapy became cost saving . if the annual posthospitalization cost of disabling stroke was reduced to approximately onethird of the basecase level , cost saving from dual antiplatelet therapy was decreased , but the resultant icer was still substantially lower than the willingnesstopay threshold . in current guidelines for the early management of patients with acute ischemic stroke published by both the cerebrovascular disease group of chinese medical association and by the american heart association / american stroke association , clopidogrel and aspirin combination therapy is not recommended for patients with minor ischemic stroke and tia . these estimates are higher than the official ( mean hospital cost ) figures from the ministry of health of china ( cny 7325.3 , us$ 1127 for ischemic stroke ; cny 11 802.1 us$ 1816 for ich ) , but comparable with those reported by the chinaquest study ( cny 10 689 , us$ 1527 for ischemic stroke ; cny 13 089 , us$ 1870 for ich , in 2006 ) . third , in the markov model , we assumed future transitions of health status based on events related to stroke recurrence and total nonstroke death and functional status as a result of other cause were not considered in this model . , the monthly cost of clopidogrel ( cny 650 , us$ 100 ) is substantially greater in china than in the united states , where generic clopidogrel is available for cny 78 ( us$ 12 ) for a month supply while the costs of hospitalization and posthospital care are much greater in the united states .

however , under certain situations of stress as viral infection , toxic injury , and partial hepatectomy , they can divide in reaction to the loss of liver mass . among these different situations , the regeneration of liver after partial hepatectomy ( pht ) provides an in vivo model to dissect the mechanisms of control of a highly differentiated normal cell growth . indeed , surgical removal of 70% of the liver synchronized most hepatocytes and the cell cycle is characterized by a fast g0/g1 phase transition of the cell cycle after pht , followed by a well - synchronized long g1 phase [ 13 ] . there is an initial step priming phase , in which the activation of il6 and tnf alpha pathways allows hepatocytes to undergo the transition from g0 to g1 in vivo leading to activation of nf - kb , ap-1 , and stat3 . then , hepatocytes proliferation is regulated by different mitogens including hgf , igf1 , ligands of the egf , and fgf receptors [ 4 , 5 ] . in vitro , hepatocytes can also proliferate after growth factor stimulations and in vitro rat hepatocyte cell cycle progression highly mimicked the kinetic of cell proliferation during liver regeneration after pht [ 6 , 7 ] . in response to mitogens ( i.e. , egf , hgf , pdgf , tgf alpha ) , hepatocytes maintained in short - term culture can undergo one or two rounds of replication ( for reviews see [ 4 , 8 , 9 ] ) . this model has been extensively used by many laboratories illustrating that primary culture of hepatocytes can be a powerful model to study the precise sequences of events which are necessary for hepatocyte cell cycle progression from a g0-like state to s phase . there are four mapk families categorized by sequence homology and functions : erk1/2 , p38 , jnk , and erk5 . mostly , jnk and p38 are more activated in response to cellular stress and cytokines . numerous studies have shown that growth factor could enhance cell proliferation and survival through the activation of the mek1/2-erk1/2 pathway , including hepatocytes in primary culture . indeed , the erk1/2 are activated in response to external and internal stimuli in numerous cell types and play a central role in many signal transduction pathways . the ras - raf - mek1/2-erk1/2 pathway couples signal from the cell surface receptors to cytoplasmic substrates and transcription factors , which regulate gene expression [ 1012 ] . following binding of growth factors , cytokines , or extracellular matrix proteins to their receptors , activation of the pathway involves the activation of the mek1/2 , by c - raf which in turn , activates erk1/2 . erk1/2 can directly phosphorylate many targets ( over 160 ) including transcription factors ( e.g. , ets-1 , c - jun , c - myc , p53 ) which leads to the induction of many cell cycle proteins ( e.g. , p21 , cyclin d1 , cdk1 ) . erk1/2 can also phosphorylate and activate cytoplasmic substrates like the 90 kda ribosomal s6 kinase ( p90 rsk ) which leads to the activation of the creb transcription factor , apoptotic factors ( e.g. , caspase 9 , bad , bim ) , and also contribute to a mechanism of retrocontrol of the cascade by phosphorylation of the egfr , sos , and raf . in addition to proliferation , the ras - raf - mek1/2-erk1/2 cascade can antagonize cell death and activate survival signals . the mek - erk pathway has been implicated in the regulation of both g1/s and g2/m transitions and mitosis in somatic cells . whereas the possible specificity of mek1 and 2 , erk1 and 2 isoforms are still in debate , and disruption of erk2 leads to embryonic lethality early in mouse development after the implantation stage . conversely , erk1 knockout mice are viable and fertile , arguing for possible different roles of each kinase or / and that erk gene dosage is essential and could drive their apparent biological differences . there is an agreement that during liver regeneration , jnk activation is an early event while activation of erk1/2 occurs in early and mid - late g1 . p38 is present in normal liver and rapidly inactivated after pht suggesting a permissive role in dna replication . these last ten years , our laboratory has studied the role of the mek1/2-erk1/2 pathway in the regulation of the cell cycle and survival of hepatocytes stimulated by the egf . we looked at long - term survival , control of multiple cell cycles , apoptosis engagement of normal rodent hepatocytes , and rat and human hepatocarcinoma cell lines , in vitro and in vivo . the mek1/2-erk1/2 cascade is activated at two points of the g1 progression in mature rat hepatocytes : the first one occurs in early g1 after pht ; the second one occurs in mid - late g1 phase and is associated with the induction of cyclin d1 , a cyclin associated to late g1 phase progression of many cells including hepatocytes [ 23 , 24 ] . in vitro , during tissue disruption by collagenase , hepatocytes can enter into the g1 phase and undergo , depending on the culture conditions in primary culture , at least one round of division [ 6 , 7 , 25 ] . in the absence of growth factor , rat hepatocytes are blocked at 2/3 of g1 phase and rapidly progress through apoptosis [ 26 , 27 ] . the growth factor ( i.e. , egf ) is a morphogen in early g1 phase by inducing controlled spreading of hepatocytes via a mek / erk - integrin 1 regulation , in vitro . during hepatocyte spreading , rac1 trough nadph oxidase is part of the signalling pathway constituted by fak - rac1-erk that regulates focal adhesion disassembly important for the turnover of adhesion sites that leads to cell spread . the growth factor - induced nuclear translocation of erk is an adhesion - dependent event and requires signalling from rac1 . cell spread and migration are dynamic processes involving the focal adhesion assembly / disassembly and erk1/2 are activated downstream of fak while erk1/2 can mediate its phosphorylation . a mitogenic effect occurs in mid - late g1 phase and allows hepatocytes to progress through a growth factor restriction point at two thirds of the g1 phase . mek signaling cascade is essential for progression to late g1 phase in vitro as well as in vivo after pht . indeed , a growth factor - mek dependency could be defined in mid - late g1 phase in regenerating liver between 9 and 12 h after pht . this activation controls expression of cyclin d1 and cdk1 which are upregulated in the prereplicative phase of liver regeneration and in proliferating hepatocytes in vitro . very interesting results from the hansen 's lab have demonstrated that adhesion to polymerized collagen could induce growth arrest by inhibiting the ras / erk pathway to cyclin d1 required in late g1 [ 33 , 34 ] . moreover , the involvement of the cell shape / motility via an erk - mlck - p70s6 k - dependent regulation of g1/s was specified in proliferating hepatocytes and in other cell types [ 31 , 36 , 37 ] . all these results highlight the mechanisms by which a growth factor can temporally control morphogenic and mitogenic signals during g1 phase progression ( see scheme 1 ) . a precise location in the cell cycle appears determinant for the regulation of erk1/2 pathway and sequential checkpoints in early g1 , mid - late g1 , and g1/s transition control hepatocyte cell cycle progression , making them permissive for dna replication . the signaling crosstalk is an important aspect of the regulation of liver regeneration and other pathways ( i.e. , hgf / c - met , igf1/igf - r , gh ) are activated and required for efficient liver regeneration . liver regeneration and erk pathway are also impaired in mice with liver - specific knockouts of igf-1r or igf binding protein 1 [ 39 , 40 ] . hgf and igf-1 strongly activated akt and erk1/2 in vitro . in vivo , egf and hgf have been implicated in liver regeneration , but specific deletion of egf receptor in hepatocyte led to liver regeneration deficiencies after 2/3 pht in mice without activation defect of erk1/2 while p38 mapk and nf - kb activation was reduced in regenerating mutant livers , indicating an impaired stress response after hepatectomy . indeed , p38 mapk could play a permissive role in dna replication during liver regeneration consistent with a role in the maintenance of hepatocyte cell cycle arrest in adult liver , while jnk could be involved in the g0/g1 transition . interestingly , hepatocyte deletion of c - met which led to liver regeneration defect was associated with mek - erk pathway inhibition highlighting that hgf contributes dominantly to erk1/2 activation in vivo [ 43 , 44 ] . a persistent egf supplementation in vitro only partially rescues the effect of erk1/2 downregulation in c - met depleted hepatocytes and restores to some extent dna synthesis and protein levels of cdk1 , aurora a and b , and mad2 . different in vitro models have previously described that hepatocytes can undergo several cell cycles in primary cultures and long - term survival when appropriate culture conditions are provided [ 4552 ] . indeed , removal of egf in long - term survival dmso culture conditions followed by readdition of the growth factor was accompanied with an increase in dna synthesis , and multiple round of replication could be observed by alternating addition / removal [ 5355 ] . in coculture with liver biliary cell [ 5660 ] , egf alone prolonged cell progression up to late g1 phase , whereas tnf mediated extracellular remodeling is required for multiple division cycles . interestingly , tnf promoted an extracellular matrix degradation required for initiating a new hepatocyte division wave . furthermore , a network of ecm or polymerized collagen type i gel induces a highly differentiated but growth - arrested phenotype in primary cultures , whereas a film of collagen promotes cell cycle progression and dedifferentiation [ 34 , 61 , 62 ] . hepatocytes dedifferentiation is reversible in consequence of a specific network triggered by the extracellular matrix , an active process driven by fak - mediated akt and erk1/2 signaling . as well , in hepatocellular carcinoma cells , increasing matrix stiffness promotes proliferation whereas soft environment induces cellular dormancy . all these experiments and others indicated that adult hepatocytes could undergo long - term survival and multiple cell divisions . in this context , our group have demonstrated that rat hepatocytes seeded in the presence of egf ( in the absence of fcs ) increased cell spreading and greatly enhanced cell survival . however , only one peak of brdu incorporation was obtained in egf - seeded cultured whereas nearly 100% of the hepatocytes accomplished a complete cell cycle . some reports have suggested that sustained activation of erk inhibits hepatocyte dna replication and that transient activations of this pathway could stimulate dna synthesis [ 67 , 68 ] . we therefore hypothesized that maintained mek1/2-erk1/2 stimulation of hepatocytes by egf could lead to a sustained activation of erk responsible for the negative control of the progression in a second cell cycle . indeed , when the mek / erk pathway is transiently inhibited with the mek inhibitor u0126 about 60% of hepatocytes did replicate their dna showing that primary hepatocytes are able to perform 2 cell cycles when a break of the mek / erk signalling pathway activity is done . in addition , cyclin d1 , e , a2 , cdk1 , p21 , and p27 were downregulated in mek - inhibited cells and induced after the u0126 removal . a third peak of dna synthesis in egf - seeded hepatocytes by performing another 2 days - break of mek1/2-erk1/2 activity could be obtained demonstrating that egf - seeded hepatocytes were able to perform multiple division waves after sequential mek1/2-erk1/2 pathway inhibitions ( see scheme 2 ) . in hepatocytes , in addition to its proliferating properties , egf could induce survival . in vitro , in the absence of serum and growth factor stimulation , hepatocytes in primary culture adhered to the plastic support but underwent spreading with a very low efficiency and die as observed by rapid caspase3/7 activations evaluated using a devd - amc assay . at the opposite , hepatocytes seeded with egf alone and cultured with the growth factor all along the culture time present a high level of differentiation . cell survival can be maintained at least 15 to 20 days in this culture condition . albumin expression reached a level closed to half of freshly isolated cells , and cyp450s can be induced by 3mc or pb showing that the detoxification machinery is still fully operative . in these cells , erk localization could be determinant for the cell phenotype since rosseland et al . showed that the cytoplasmic retention of transient peroxide - activated erk provides survival in primary cultures of rat hepatocytes . indeed , mek1 and mek2 could regulate distinct functions by sorting erk2 to different intracellular compartments in response to growth factor and erk2 intracellular localization could determine whether growth factors mediate hepatocyte proliferation or survival in an adhesion - dependent manner [ 7072 ] . surprisingly , an improvement of the survival of hepatocytes continuously treated with the mek inhibitor u0126 can be obtained . indeed , a permanent treatment with u0126 keeps hepatocytes for more than 2 weeks in survival . all the genes of detoxification analyzed ( cyp 1a1 , 1a2 , 2b2 , 3a23 , and gsta2 ) as well as the aldolase b gene are induced all along the period of treatment . u0126 removal from the culture medium is accompanied with a fast decrease of the expression of these markers related to the reentry of the cells in a new cycle . in summary , early and sustained egf stimulation , in the absence of serum , could be a good compromise between classical monolayers with limited survival / differentiation , and long - term sophisticated and labor intensive cultures . this model emphasizes that early egf stimulation of hepatocytes in the absence of fcs and transient or sustained inhibition of the mek / erk pathway represent serum - free models ( scheme 2 ) that will be very helpful for pharmacological studies on drug metabolism and toxicity . the strict specificity of action of the mapks is still debated and today , no one can affirm with certitude the full redundancy of erk1 and erk2 or at the contrary the specificity of action of each protein . on one hand , the simple observation of the phenotypes of knockout animals for erk1 and erk2 fuels the idea that each erk isoform could regulate specific and non overlapping functions . invalidating erk1 has no strong and lethal impact on animals : mice are viable , fertile , and of normal size . thereby , erk1 was associated with maturation of thymocytes , development of adipose tissue , or osteoclast formation and differentiation [ 74 , 75 ] . erk2 knockout is much more severe as embryos die very early during development [ 17 , 76 ] , because of major defects in the establishment of extraembryonic tissues [ 17 , 76 , 77 ] . to counteract this embryonic lethality and ascertain the roles of erk2 in embryo or adult tissues , conditional expressions erk2 also regulates multiple stages of t - cells development [ 79 , 80 ] . invalidation of erk2 in the central neural system ( cns ) leads to anomalies in multiple aspects of social behaviors , decreased anxiety - related attitude , and impaired long - term memory . erk2 also protects the myocardium from ischemia - reperfusion injury in vivo as erk2 gene - targeted mice showed enhanced infarction areas . based on the strict observation of these phenotypes , one could easily conclude that erk1 and erk2 regulate specific functions . indeed , studies performed on animal and which attributed to erk1 or erk2 unique functions did not really took into account the expression level of each isoform in the tissues or cell types analyzed . in other words , the lethality of erk2 embryos could reflect a specific role of the isoform in the establishment of extraembryonic tissues or could be due to a higher expression of the erk2 isoform ( compared to erk1 ) in these tissues . we must be careful when drawing some conclusions about specific roles for erk1 and erk2 . besides , on a purely biochemical point of view , it has been difficult to associate erk1 or erk2 to cellular specific functions . they share a 84% homology at the protein level , seem to be activated in response to similar stimuli and to date no specific substrate for each kinase has been characterized . actually , only a few papers have reported biochemical differences between both proteins . thus , preferential activation of erk1 versus erk2 was reported in nfb4 cells after lpa stimulation . at the contrary , another biochemical difference concerns the identification of a specific scaffolding protein of erk1 called mp1 ( mek partner 1 ) . this protein interacts exclusively with mek1 and erk1 at the surface of late endosome [ 85 , 86 ] . finally , despite the fact that both kinases are simultaneously expressed in all cell types and tissues analyzed , the erk1 : erk2 ratio is quite variable . one of the best examples that illustrates this is the quite heterogenous expression profile of erk1 and erk2 mrna in brain . these are essentially the more pronounced biochemical differences that have been reported until today and finally erk1 and erk2 appear as tightly close enzymes . interestingly , erk is highly activated in ectoplacental cone and extraembryonic ectoderm , which both give rise to these extraembryonic tissues . even if the elevated erk activity in these tissues has not been attributed to erk1 or erk2 , it is likely to be mainly carried by erk2 isoform , which would explain the phenotype of erk2 knockout . it is indeed assumed that erk2 is more expressed than erk1 in nearly all tissues examined so far and , as a consequence invalidation of erk2 , is supposed to have a stronger impact on the global erk activity compared to erk1 knockout . actually , the only one way to compare the expression levels of erk1 and erk2 in cells is the use of a phosphospecific antibody , which recognizes the phosphorylation of activation loop residues thr202/tyr204 and thr185/tyr187 of erk1 and erk2 , respectively . this motif is recognized with the same affinity by the antibody . in a recent report , lefloch et al . have established a clear correlation between the expression ratio of erk1 and erk2 and their activation ratio . in this work , the authors have demonstrated that erk1 and erk2 are fully redundant kinases regarding the regulation of cell proliferation in nih3t3 cells . a similar observation was done on embryonic fibroblast genetically deficient for erk1 and/or erk2 . in this study , a strong correlation was drawn between the quantity of erk proteins inside the cell and the intensity of proliferation . in addition to these works based on the use of genetically deficient animals and riding the wave following rnai discovery , a sustained number of studies have emerged in the literature in order to decipher the roles of erk1 and erk2 . in skeletal myoblasts , proliferation requires one of the two isoforms , whatever it is , but terminal differentiation is strictly dependent on erk2 . according to liu et al . , if erk1 and erk2 silencing would both affect cell proliferation , each kinase would be involved at different phases of the cell cycle : erk1 would regulate g2/m transition while erk2 could be essential in g1 phase . a recent study done by john blenis ' group has shown that the erk2 isoform induces epithelial - to - mesenchymal transformation when overexpressed . interestingly , erk1 is not able to reach the same effect despite high expression levels . what about the roles of erk1 and erk2 in the physiology of normal hepatocytes ? interestingly , contrary to other cell types in which erk2 is much more expressed compared to erk1 , normal hepatocytes harbor closely similar levels of erk1 and erk2 , according to p - erk1 : p - erk2 ratio . we have shown that erk1 hepatocytes proliferate with similar kinetics as wild - type hepatocytes after in vivo pht and in vitro . at the contrary , silencing erk2 has a strong impact on cell proliferation [ 66 , 94 ] . these results are in accordance with several papers listed above that place erk2 as a positive regulator of cell proliferation . we went further in the analysis of putative roles of erk1 and erk2 in hepatic processes and established a link between erk1 and the cell survival of hepatocytes . indeed , silencing erk1 using rnai decreases susceptibility to apoptosis as it is observed in erk1-deficient hepatocytes . this is in accordance with the report from bourcier et al . which has shown that erk1 keratinocytes are resistant to apoptosis induced by different agents or stress . but this is probably not so simple and associating one isoform to the regulation of one specific cellular function could be a dangerous shortcut . as an example , in ovarian cells , the silencing of erk1 triggers the opposite phenotype to that observed in hepatocytes since cells become more sensitive to apoptosis . how the inhibition of the same protein could have radically opposite effects ? the localization of the isoforms inside the cell could be a response element . in that sense , mek1 and mek2 were proposed to regulate distinct cellular functions in hepatocytes by localizing erk2 to different regions of the cell . following activation by mek1 , erk2 translocates to the nucleus where it would trigger a proliferative response . at the opposite , when activated by mek2 , erk2 retains a cytoplasmic localization to mediate survival . this could explain why one isoform would regulate distinct functions according to the cell type . finally and in order to reconcile all these data , one can easily imagine that erk1 and erk2 regulate overlapping fundamental functions with regard to the most fundamental processes such as proliferation in nonspecialized cells . results obtained on mefs cells and which explicit a dose - response effect of erk on the intensity of cell proliferation argue in that sense . interestingly , all the major differences that were observed between erk isoforms have been made on more differentiated cells , and notably hepatocytes . this could suggest a specialization of erk1 and erk2 in the regulation of unique biological functions in differentiated cells . erk1 and erk2 also share dozen of substrates so one could speculate that potential differences in substrate affinity could explain the predominant role of one isoform in a function due to the presence of a particular set of partners , in other words in a particular cellular context . the mek / erk signaling pathway plays a central role in the regulation of various physiological processes such as proliferation , survival or cell motility . thus , its disregulation has often been associated with the promotion or development of tumor cell growth . indeed , the chemical inhibition of mek1/2 kinase activities blocks in vitro as well as in vivo proliferation of a variety of tumor models , including hepatocellular carcinoma ( hcc ) [ 99103 ] . moreover , active mutant forms of raf or mek have been shown to transform different cell types [ 90 , 104106 ] . the best illustration of mapk pathway importance in oncogenesis lies in the observation of an erk1/2 overactivation in 50 out of 138 human tumor cell lines . indeed , an increased expression and activation of the mek1/2 , and erk1/2 kinases has been reported in human and mice primary liver tumors [ 108111 ] . active forms of the mek / erk pathway components including praf1 , pmek1/2 and perk1/2 were also associated with poor prognosis in patients with hcc [ 112 , 113 ] . however , in hcc , the overactivation of the mek / erk pathway did not result ( or rarely ) from an activating mutation of an upstream protein , namely , the gtpase ras or the raf protein kinase . generally , the protein ras is found mutated in about 30% of all human cancers with a high prevalence in the pancreas ( 90% ) and colon ( 50% ) adenocarcinomas . the braf v600e mutation is on the other hand found in about 20% of tumors and especially in melanoma ( ~ 50% ) . in hcc , ras and braf mutations are rare in humans or could be related to some etiologic factors or genetic backgrounds [ 116119 ] . overactivation of the mek / erk pathway is rather a consequence of a disinhibition , an upregulation of upstream activators , or an oncogenic stimulation . have shown from 80 surgical resections of hcc that 100% of tested malignant tissues had a constitutive activation of ras and this was linked to the loss or reduction of the ras inhibitory proteins nore1a and rassf1a . different inhibitors of the mek / erk pathway like rkip , sprouty-2 , spred-1 , or spred-2 are also frequently downregulated in human tissues of hcc . these decreases , or losses of activity , are supposed to have an important impact on hcc development and progression since ectopic expression of these different inhibitors is sufficient to inhibit the mapk pathway but , most of all , to suppress tumor cell proliferation both in vitro and in vivo [ 121124 ] . the downregulation of the previously cited inhibitors would provide an increased activation of the kinases erk1/2 . moreover , in poor prognosis hcc , erk1/2 activity should also be unrestrained given the weakened expression of the mapk phosphatase dusp-1 found in those patients . in addition , the erk1/2 kinases could also undergo a more intense stimulation through the overexpression of various components of the mek / erk pathway . for instance , the protein c - raf could be an important source of erk overactivation . indeed , hwang and colleagues have shown that the c - raf kinase is upregulated in almost all cirrhosis and tumor tissues analyzed . a significantly higher level of expression of c - raf was also reported in hepatocellular carcinomas when compared to cirrhosis . the signaling via these receptors will thus activate the mapk pathway cascade and lead to a sustained activation of erk1/2 . for example , the egf receptor is overexpressed in about 40% to 70% of hcc [ 128130 ] . importantly , the transforming growth factor- which is one of the egfr ligands presents in parallel an increased expression in cirrhosis and early hcc . since the gene encoding for tgf- is also a target of the ras pathway , an autoamplification loop could be established ensuring , by the way , a persistent activation of the mek / erk pathway [ 131133 ] . hepatocellular carcinoma is also one of the most vascularized solid tumors links to a strong angiogenesis . therefore , it is not surprising to find an upregulation of various proangiogenic factors such as vegf , its receptor vegfr , or the pdgf . all of these proteins will provide activator signals for the mek / erk pathway [ 134136 ] . in hcc , a significant deregulation of the igf this was mediated by the upregulation of igf2 via epigenetic mechanisms and by the downregulation of the igf2r receptor , which normally lead to the lysosomal degradation of igfs [ 79 , 137 , 138 ] . in addition , an overexpression of c - met is observed in approximately 50% of hcc and this was associated with poor prognosis . finally , erk1/2 overactivation in hcc could be due to hepatitis - b virus ( hbv ) or c virus ( hcv ) infections , the two major etiologic factors of primary liver cancers . thus , the core protein and the envelope protein e2 of the hcv and the hbx protein and pres2-activator large surface protein of hbv have been shown to directly activate the mek / erk pathway but by different mechanisms [ 140144 ] . for instance , the hbx protein could upregulate the egfr and interacts with the protein pin1 to facilitate the dephosphorylation of c - raf while activation by the pres2-activator large surface protein used pkc - dependent mechanisms [ 142 , 143 , 145 , 146 ] . the envelope protein e2 of hcv stimulates the mek / erk pathway by binding to the cd81 receptor or to the low density lipoprotein receptor ( ldlr ) . anyway , mek / erk activation by viruses is postulated to promote hepatocarcinogenesis by facilitating the proliferation and survival of infected cells . indeed , mek1/2 inhibition , conducted by the use of different chemical inhibitors , could abolish the in vitro proliferation of numerous human and rat hepatocarcinoma cell lines . growth of xenograft tumors in mice is also severely impaired in a context of mek / erk inhibition [ 94 , 99 , 104 , 111 , 148152 ] . we have shown that mek1 deficiency suppressed both in vitro and in vivo proliferation of huh7 cells . on the other side , mek2 silencing did not affect the proliferation capacity of transformed cells . similar to normal hepatocytes , tumor growth is also supported by the kinase erk2 but not by erk1 . indeed , erk2 targeting by stable chemically modified sirna altered the in vitro proliferation as well as the in vivo growth of the highly tumorigenic f1 cells . we have also demonstrated that erk2 was primordial for the in vivo proliferation of the huh-7 cell line [ 99 , 151 ] . interestingly , it is noteworthy that hepatoma cells exhibit a higher expression of erk2 than erk1 while normal hepatocytes have a more balanced erk1 : erk2 ratio . this could reflect the difference of functions carried by these kinases in the particular context of liver . indeed , we showed that erk2 favored the proliferation of normal and transformed hepatocytes as opposed to erk1 which could promote a death signal ( guegan , personal data ) . given the permanent and sustained mek / erk activation in hcc , one could speculate that the newly transformed hepatocyte should thus prime erk2 functions while diminishing erk1 prodeath activity . moreover and besides its role in cell proliferation , we have shown that erk2 but not erk1 was involved in hepatoma cell motility and invasiveness by an upar and p70s6 k dependent mechanism . rnai - mediated inhibition of erk2 or p70s6 k led to strongly reduced cell motility . however , this is not the unique mechanism by which the mapk pathway regulates hcc invasion . have shown that an active mutant form of mek1 could suppress the e - cadherin mediated homotypic adhesion and thus potentiate cell migration . erk1/2 activity was also involved in the migration of three metastatic hcc cell lines but in a pkc- dependent mechanism . interestingly , erk activation status was shown to increase following the metastatic potential of the cell lines analyzed . hence , the mek / erk overactivation found in tumor cell could support the hcc progression and metastasis . indeed it has been shown that treatment of hepg2 or hep3b cells by mek1/2 inhibitors led to an apoptosis engagement . inhibition of mek1/2 could also sensitize hepatoma cells to the death induced by er - stress . moreover , active form of mek1 prevented serum deprivation - induced death of hepatocarcinoma cells and in hepg2 , mek / erk activity has been reported to contribute to cisplatin induced death . the mek / erk pathway has also been shown to protect transformed hepatocytes from tgf--induced apoptosis , a natural inducer of apoptosis in hepatocytes , produced in the liver by hepatic stellate cells . liver tumor bearing late tgf- gene signature is indeed more aggressive than those expressing early gene signature . hence the mek / erk overactivation might play an important role in the initiation or development of hcc . the prosurvival effects of the mek / erk pathway in tumor cells have been shown to pass through the upregulation or stimulation of different antiapoptotic factors such as bcl-2 , bim , or bad ( for review see ) . for instance , it has been shown in hepatocarcinoma cells that erk1/2 could phosphorylate the antiapoptotic factor mcl-1 on thr163 in order to stabilize it and to thus enhance its prosurvival function . the critical involvement of the mek / erk pathway in hcc tumorigenesis strongly suggests that the kinases mek1/2 or erk1/2 could be promising therapeutic targets . sorafenib advent in therapy has also clearly demonstrated the potential of targeting signaling pathways in hcc . given the predominant role of erk2 in transformed hepatocyte proliferation , survival , and motility and given the prodeath role of erk1 , it could be preferential to specifically target erk2 without affecting erk1 activity . by this way , this might have different effects compared to a nonspecific chemical inhibition of both kinases , what could ultimately improve therapeutic benefits .

primary cultures of hepatocytes are powerful models in studying the sequence of events that are necessary for cell progression from a g0-like state to s phase . the models mimic the physiological process of hepatic regeneration after liver injury or partial hepatectomy . many reports suggest that the mitogen - activated protein kinase ( mapk ) erk1/2 can support hepatocyte proliferation in vitro and in vivo and the mek / erk cascade acts as an essential element in hepatocyte responses induced by the egf . moreover , its disregulation has been associated with the promotion of tumor cell growth of a variety of tumors , including hepatocellular carcinoma . whereas the strict specificity of action of erk1 and erk2 is still debated , the mapks may have specific biological functions under certain contexts and according to the differentiation status of the cells , notably hepatocytes . in this paper , we will focus on mek1/2-erk1/2 activations and roles in normal rodent hepatocytes in vitro and in vivo after partial hepatectomy and in human hepatocarcinoma cells . the possible specificity of erk1 and erk2 in normal and transformed hepatocyte will be discussed in regard to other differentiated and undifferentiated cellular models .

1. Introduction 2. Mechanisms in the Sequential Control of Cell Morphology and G1 Phase Progression Involve MEK-ERK Activations in Normal Hepatocytes 3. Transient Blockade of the MEK/ERK Pathway Using Allows Multiple Cell Cycles 4. Early Sustained EGF Stimulation and MEK Inhibition Maintain Hepatocytes in a Long-Term Survival and Differentiated States 5. Specificity of the MAPK ERK1 and ERK2 Signaling: ERK2 Controls S Phase Entry whereas ERK1 Regulates Survival in 6. Roles of the MEK1/2-ERK1/2 Pathway in

however , under certain situations of stress as viral infection , toxic injury , and partial hepatectomy , they can divide in reaction to the loss of liver mass . among these different situations , the regeneration of liver after partial hepatectomy ( pht ) provides an in vivo model to dissect the mechanisms of control of a highly differentiated normal cell growth . indeed , surgical removal of 70% of the liver synchronized most hepatocytes and the cell cycle is characterized by a fast g0/g1 phase transition of the cell cycle after pht , followed by a well - synchronized long g1 phase [ 13 ] . there is an initial step priming phase , in which the activation of il6 and tnf alpha pathways allows hepatocytes to undergo the transition from g0 to g1 in vivo leading to activation of nf - kb , ap-1 , and stat3 . then , hepatocytes proliferation is regulated by different mitogens including hgf , igf1 , ligands of the egf , and fgf receptors [ 4 , 5 ] . in vitro , hepatocytes can also proliferate after growth factor stimulations and in vitro rat hepatocyte cell cycle progression highly mimicked the kinetic of cell proliferation during liver regeneration after pht [ 6 , 7 ] . this model has been extensively used by many laboratories illustrating that primary culture of hepatocytes can be a powerful model to study the precise sequences of events which are necessary for hepatocyte cell cycle progression from a g0-like state to s phase . numerous studies have shown that growth factor could enhance cell proliferation and survival through the activation of the mek1/2-erk1/2 pathway , including hepatocytes in primary culture . , ets-1 , c - jun , c - myc , p53 ) which leads to the induction of many cell cycle proteins ( e.g. erk1/2 can also phosphorylate and activate cytoplasmic substrates like the 90 kda ribosomal s6 kinase ( p90 rsk ) which leads to the activation of the creb transcription factor , apoptotic factors ( e.g. the mek - erk pathway has been implicated in the regulation of both g1/s and g2/m transitions and mitosis in somatic cells . whereas the possible specificity of mek1 and 2 , erk1 and 2 isoforms are still in debate , and disruption of erk2 leads to embryonic lethality early in mouse development after the implantation stage . these last ten years , our laboratory has studied the role of the mek1/2-erk1/2 pathway in the regulation of the cell cycle and survival of hepatocytes stimulated by the egf . we looked at long - term survival , control of multiple cell cycles , apoptosis engagement of normal rodent hepatocytes , and rat and human hepatocarcinoma cell lines , in vitro and in vivo . the mek1/2-erk1/2 cascade is activated at two points of the g1 progression in mature rat hepatocytes : the first one occurs in early g1 after pht ; the second one occurs in mid - late g1 phase and is associated with the induction of cyclin d1 , a cyclin associated to late g1 phase progression of many cells including hepatocytes [ 23 , 24 ] . , egf ) is a morphogen in early g1 phase by inducing controlled spreading of hepatocytes via a mek / erk - integrin 1 regulation , in vitro . mek signaling cascade is essential for progression to late g1 phase in vitro as well as in vivo after pht . this activation controls expression of cyclin d1 and cdk1 which are upregulated in the prereplicative phase of liver regeneration and in proliferating hepatocytes in vitro . moreover , the involvement of the cell shape / motility via an erk - mlck - p70s6 k - dependent regulation of g1/s was specified in proliferating hepatocytes and in other cell types [ 31 , 36 , 37 ] . the signaling crosstalk is an important aspect of the regulation of liver regeneration and other pathways ( i.e. hgf and igf-1 strongly activated akt and erk1/2 in vitro . in vivo , egf and hgf have been implicated in liver regeneration , but specific deletion of egf receptor in hepatocyte led to liver regeneration deficiencies after 2/3 pht in mice without activation defect of erk1/2 while p38 mapk and nf - kb activation was reduced in regenerating mutant livers , indicating an impaired stress response after hepatectomy . interestingly , hepatocyte deletion of c - met which led to liver regeneration defect was associated with mek - erk pathway inhibition highlighting that hgf contributes dominantly to erk1/2 activation in vivo [ 43 , 44 ] . different in vitro models have previously described that hepatocytes can undergo several cell cycles in primary cultures and long - term survival when appropriate culture conditions are provided [ 4552 ] . hepatocytes dedifferentiation is reversible in consequence of a specific network triggered by the extracellular matrix , an active process driven by fak - mediated akt and erk1/2 signaling . as well , in hepatocellular carcinoma cells , increasing matrix stiffness promotes proliferation whereas soft environment induces cellular dormancy . however , only one peak of brdu incorporation was obtained in egf - seeded cultured whereas nearly 100% of the hepatocytes accomplished a complete cell cycle . we therefore hypothesized that maintained mek1/2-erk1/2 stimulation of hepatocytes by egf could lead to a sustained activation of erk responsible for the negative control of the progression in a second cell cycle . indeed , when the mek / erk pathway is transiently inhibited with the mek inhibitor u0126 about 60% of hepatocytes did replicate their dna showing that primary hepatocytes are able to perform 2 cell cycles when a break of the mek / erk signalling pathway activity is done . in vitro , in the absence of serum and growth factor stimulation , hepatocytes in primary culture adhered to the plastic support but underwent spreading with a very low efficiency and die as observed by rapid caspase3/7 activations evaluated using a devd - amc assay . cell survival can be maintained at least 15 to 20 days in this culture condition . albumin expression reached a level closed to half of freshly isolated cells , and cyp450s can be induced by 3mc or pb showing that the detoxification machinery is still fully operative . showed that the cytoplasmic retention of transient peroxide - activated erk provides survival in primary cultures of rat hepatocytes . indeed , mek1 and mek2 could regulate distinct functions by sorting erk2 to different intracellular compartments in response to growth factor and erk2 intracellular localization could determine whether growth factors mediate hepatocyte proliferation or survival in an adhesion - dependent manner [ 7072 ] . surprisingly , an improvement of the survival of hepatocytes continuously treated with the mek inhibitor u0126 can be obtained . u0126 removal from the culture medium is accompanied with a fast decrease of the expression of these markers related to the reentry of the cells in a new cycle . this model emphasizes that early egf stimulation of hepatocytes in the absence of fcs and transient or sustained inhibition of the mek / erk pathway represent serum - free models ( scheme 2 ) that will be very helpful for pharmacological studies on drug metabolism and toxicity . the strict specificity of action of the mapks is still debated and today , no one can affirm with certitude the full redundancy of erk1 and erk2 or at the contrary the specificity of action of each protein . on one hand , the simple observation of the phenotypes of knockout animals for erk1 and erk2 fuels the idea that each erk isoform could regulate specific and non overlapping functions . to counteract this embryonic lethality and ascertain the roles of erk2 in embryo or adult tissues , conditional expressions erk2 also regulates multiple stages of t - cells development [ 79 , 80 ] . based on the strict observation of these phenotypes , one could easily conclude that erk1 and erk2 regulate specific functions . in other words , the lethality of erk2 embryos could reflect a specific role of the isoform in the establishment of extraembryonic tissues or could be due to a higher expression of the erk2 isoform ( compared to erk1 ) in these tissues . we must be careful when drawing some conclusions about specific roles for erk1 and erk2 . they share a 84% homology at the protein level , seem to be activated in response to similar stimuli and to date no specific substrate for each kinase has been characterized . at the contrary , another biochemical difference concerns the identification of a specific scaffolding protein of erk1 called mp1 ( mek partner 1 ) . one of the best examples that illustrates this is the quite heterogenous expression profile of erk1 and erk2 mrna in brain . these are essentially the more pronounced biochemical differences that have been reported until today and finally erk1 and erk2 appear as tightly close enzymes . actually , the only one way to compare the expression levels of erk1 and erk2 in cells is the use of a phosphospecific antibody , which recognizes the phosphorylation of activation loop residues thr202/tyr204 and thr185/tyr187 of erk1 and erk2 , respectively . this motif is recognized with the same affinity by the antibody . have established a clear correlation between the expression ratio of erk1 and erk2 and their activation ratio . in this work , the authors have demonstrated that erk1 and erk2 are fully redundant kinases regarding the regulation of cell proliferation in nih3t3 cells . in this study , a strong correlation was drawn between the quantity of erk proteins inside the cell and the intensity of proliferation . in addition to these works based on the use of genetically deficient animals and riding the wave following rnai discovery , a sustained number of studies have emerged in the literature in order to decipher the roles of erk1 and erk2 . in skeletal myoblasts , proliferation requires one of the two isoforms , whatever it is , but terminal differentiation is strictly dependent on erk2 . , if erk1 and erk2 silencing would both affect cell proliferation , each kinase would be involved at different phases of the cell cycle : erk1 would regulate g2/m transition while erk2 could be essential in g1 phase . what about the roles of erk1 and erk2 in the physiology of normal hepatocytes ? interestingly , contrary to other cell types in which erk2 is much more expressed compared to erk1 , normal hepatocytes harbor closely similar levels of erk1 and erk2 , according to p - erk1 : p - erk2 ratio . we have shown that erk1 hepatocytes proliferate with similar kinetics as wild - type hepatocytes after in vivo pht and in vitro . we went further in the analysis of putative roles of erk1 and erk2 in hepatic processes and established a link between erk1 and the cell survival of hepatocytes . as an example , in ovarian cells , the silencing of erk1 triggers the opposite phenotype to that observed in hepatocytes since cells become more sensitive to apoptosis . this could explain why one isoform would regulate distinct functions according to the cell type . finally and in order to reconcile all these data , one can easily imagine that erk1 and erk2 regulate overlapping fundamental functions with regard to the most fundamental processes such as proliferation in nonspecialized cells . interestingly , all the major differences that were observed between erk isoforms have been made on more differentiated cells , and notably hepatocytes . this could suggest a specialization of erk1 and erk2 in the regulation of unique biological functions in differentiated cells . erk1 and erk2 also share dozen of substrates so one could speculate that potential differences in substrate affinity could explain the predominant role of one isoform in a function due to the presence of a particular set of partners , in other words in a particular cellular context . the mek / erk signaling pathway plays a central role in the regulation of various physiological processes such as proliferation , survival or cell motility . thus , its disregulation has often been associated with the promotion or development of tumor cell growth . indeed , the chemical inhibition of mek1/2 kinase activities blocks in vitro as well as in vivo proliferation of a variety of tumor models , including hepatocellular carcinoma ( hcc ) [ 99103 ] . indeed , an increased expression and activation of the mek1/2 , and erk1/2 kinases has been reported in human and mice primary liver tumors [ 108111 ] . active forms of the mek / erk pathway components including praf1 , pmek1/2 and perk1/2 were also associated with poor prognosis in patients with hcc [ 112 , 113 ] . however , in hcc , the overactivation of the mek / erk pathway did not result ( or rarely ) from an activating mutation of an upstream protein , namely , the gtpase ras or the raf protein kinase . overactivation of the mek / erk pathway is rather a consequence of a disinhibition , an upregulation of upstream activators , or an oncogenic stimulation . have shown from 80 surgical resections of hcc that 100% of tested malignant tissues had a constitutive activation of ras and this was linked to the loss or reduction of the ras inhibitory proteins nore1a and rassf1a . different inhibitors of the mek / erk pathway like rkip , sprouty-2 , spred-1 , or spred-2 are also frequently downregulated in human tissues of hcc . these decreases , or losses of activity , are supposed to have an important impact on hcc development and progression since ectopic expression of these different inhibitors is sufficient to inhibit the mapk pathway but , most of all , to suppress tumor cell proliferation both in vitro and in vivo [ 121124 ] . moreover , in poor prognosis hcc , erk1/2 activity should also be unrestrained given the weakened expression of the mapk phosphatase dusp-1 found in those patients . in addition , the erk1/2 kinases could also undergo a more intense stimulation through the overexpression of various components of the mek / erk pathway . for example , the egf receptor is overexpressed in about 40% to 70% of hcc [ 128130 ] . importantly , the transforming growth factor- which is one of the egfr ligands presents in parallel an increased expression in cirrhosis and early hcc . since the gene encoding for tgf- is also a target of the ras pathway , an autoamplification loop could be established ensuring , by the way , a persistent activation of the mek / erk pathway [ 131133 ] . hepatocellular carcinoma is also one of the most vascularized solid tumors links to a strong angiogenesis . all of these proteins will provide activator signals for the mek / erk pathway [ 134136 ] . in hcc , a significant deregulation of the igf this was mediated by the upregulation of igf2 via epigenetic mechanisms and by the downregulation of the igf2r receptor , which normally lead to the lysosomal degradation of igfs [ 79 , 137 , 138 ] . in addition , an overexpression of c - met is observed in approximately 50% of hcc and this was associated with poor prognosis . thus , the core protein and the envelope protein e2 of the hcv and the hbx protein and pres2-activator large surface protein of hbv have been shown to directly activate the mek / erk pathway but by different mechanisms [ 140144 ] . for instance , the hbx protein could upregulate the egfr and interacts with the protein pin1 to facilitate the dephosphorylation of c - raf while activation by the pres2-activator large surface protein used pkc - dependent mechanisms [ 142 , 143 , 145 , 146 ] . the envelope protein e2 of hcv stimulates the mek / erk pathway by binding to the cd81 receptor or to the low density lipoprotein receptor ( ldlr ) . anyway , mek / erk activation by viruses is postulated to promote hepatocarcinogenesis by facilitating the proliferation and survival of infected cells . indeed , mek1/2 inhibition , conducted by the use of different chemical inhibitors , could abolish the in vitro proliferation of numerous human and rat hepatocarcinoma cell lines . growth of xenograft tumors in mice is also severely impaired in a context of mek / erk inhibition [ 94 , 99 , 104 , 111 , 148152 ] . we have shown that mek1 deficiency suppressed both in vitro and in vivo proliferation of huh7 cells . indeed , erk2 targeting by stable chemically modified sirna altered the in vitro proliferation as well as the in vivo growth of the highly tumorigenic f1 cells . we have also demonstrated that erk2 was primordial for the in vivo proliferation of the huh-7 cell line [ 99 , 151 ] . indeed , we showed that erk2 favored the proliferation of normal and transformed hepatocytes as opposed to erk1 which could promote a death signal ( guegan , personal data ) . given the permanent and sustained mek / erk activation in hcc , one could speculate that the newly transformed hepatocyte should thus prime erk2 functions while diminishing erk1 prodeath activity . interestingly , erk activation status was shown to increase following the metastatic potential of the cell lines analyzed . hence , the mek / erk overactivation found in tumor cell could support the hcc progression and metastasis . inhibition of mek1/2 could also sensitize hepatoma cells to the death induced by er - stress . moreover , active form of mek1 prevented serum deprivation - induced death of hepatocarcinoma cells and in hepg2 , mek / erk activity has been reported to contribute to cisplatin induced death . the mek / erk pathway has also been shown to protect transformed hepatocytes from tgf--induced apoptosis , a natural inducer of apoptosis in hepatocytes , produced in the liver by hepatic stellate cells . hence the mek / erk overactivation might play an important role in the initiation or development of hcc . the prosurvival effects of the mek / erk pathway in tumor cells have been shown to pass through the upregulation or stimulation of different antiapoptotic factors such as bcl-2 , bim , or bad ( for review see ) . for instance , it has been shown in hepatocarcinoma cells that erk1/2 could phosphorylate the antiapoptotic factor mcl-1 on thr163 in order to stabilize it and to thus enhance its prosurvival function . the critical involvement of the mek / erk pathway in hcc tumorigenesis strongly suggests that the kinases mek1/2 or erk1/2 could be promising therapeutic targets . given the predominant role of erk2 in transformed hepatocyte proliferation , survival , and motility and given the prodeath role of erk1 , it could be preferential to specifically target erk2 without affecting erk1 activity .

among these different situations , the regeneration of liver after partial hepatectomy ( pht ) provides an in vivo model to dissect the mechanisms of control of a highly differentiated normal cell growth . indeed , surgical removal of 70% of the liver synchronized most hepatocytes and the cell cycle is characterized by a fast g0/g1 phase transition of the cell cycle after pht , followed by a well - synchronized long g1 phase [ 13 ] . there is an initial step priming phase , in which the activation of il6 and tnf alpha pathways allows hepatocytes to undergo the transition from g0 to g1 in vivo leading to activation of nf - kb , ap-1 , and stat3 . then , hepatocytes proliferation is regulated by different mitogens including hgf , igf1 , ligands of the egf , and fgf receptors [ 4 , 5 ] . in vitro , hepatocytes can also proliferate after growth factor stimulations and in vitro rat hepatocyte cell cycle progression highly mimicked the kinetic of cell proliferation during liver regeneration after pht [ 6 , 7 ] . , egf , hgf , pdgf , tgf alpha ) , hepatocytes maintained in short - term culture can undergo one or two rounds of replication ( for reviews see [ 4 , 8 , 9 ] ) . this model has been extensively used by many laboratories illustrating that primary culture of hepatocytes can be a powerful model to study the precise sequences of events which are necessary for hepatocyte cell cycle progression from a g0-like state to s phase . indeed , the erk1/2 are activated in response to external and internal stimuli in numerous cell types and play a central role in many signal transduction pathways . the ras - raf - mek1/2-erk1/2 pathway couples signal from the cell surface receptors to cytoplasmic substrates and transcription factors , which regulate gene expression [ 1012 ] . following binding of growth factors , cytokines , or extracellular matrix proteins to their receptors , activation of the pathway involves the activation of the mek1/2 , by c - raf which in turn , activates erk1/2 . , ets-1 , c - jun , c - myc , p53 ) which leads to the induction of many cell cycle proteins ( e.g. erk1/2 can also phosphorylate and activate cytoplasmic substrates like the 90 kda ribosomal s6 kinase ( p90 rsk ) which leads to the activation of the creb transcription factor , apoptotic factors ( e.g. , caspase 9 , bad , bim ) , and also contribute to a mechanism of retrocontrol of the cascade by phosphorylation of the egfr , sos , and raf . the mek - erk pathway has been implicated in the regulation of both g1/s and g2/m transitions and mitosis in somatic cells . whereas the possible specificity of mek1 and 2 , erk1 and 2 isoforms are still in debate , and disruption of erk2 leads to embryonic lethality early in mouse development after the implantation stage . conversely , erk1 knockout mice are viable and fertile , arguing for possible different roles of each kinase or / and that erk gene dosage is essential and could drive their apparent biological differences . these last ten years , our laboratory has studied the role of the mek1/2-erk1/2 pathway in the regulation of the cell cycle and survival of hepatocytes stimulated by the egf . we looked at long - term survival , control of multiple cell cycles , apoptosis engagement of normal rodent hepatocytes , and rat and human hepatocarcinoma cell lines , in vitro and in vivo . the mek1/2-erk1/2 cascade is activated at two points of the g1 progression in mature rat hepatocytes : the first one occurs in early g1 after pht ; the second one occurs in mid - late g1 phase and is associated with the induction of cyclin d1 , a cyclin associated to late g1 phase progression of many cells including hepatocytes [ 23 , 24 ] . in vitro , during tissue disruption by collagenase , hepatocytes can enter into the g1 phase and undergo , depending on the culture conditions in primary culture , at least one round of division [ 6 , 7 , 25 ] . during hepatocyte spreading , rac1 trough nadph oxidase is part of the signalling pathway constituted by fak - rac1-erk that regulates focal adhesion disassembly important for the turnover of adhesion sites that leads to cell spread . cell spread and migration are dynamic processes involving the focal adhesion assembly / disassembly and erk1/2 are activated downstream of fak while erk1/2 can mediate its phosphorylation . this activation controls expression of cyclin d1 and cdk1 which are upregulated in the prereplicative phase of liver regeneration and in proliferating hepatocytes in vitro . very interesting results from the hansen 's lab have demonstrated that adhesion to polymerized collagen could induce growth arrest by inhibiting the ras / erk pathway to cyclin d1 required in late g1 [ 33 , 34 ] . moreover , the involvement of the cell shape / motility via an erk - mlck - p70s6 k - dependent regulation of g1/s was specified in proliferating hepatocytes and in other cell types [ 31 , 36 , 37 ] . a precise location in the cell cycle appears determinant for the regulation of erk1/2 pathway and sequential checkpoints in early g1 , mid - late g1 , and g1/s transition control hepatocyte cell cycle progression , making them permissive for dna replication . the signaling crosstalk is an important aspect of the regulation of liver regeneration and other pathways ( i.e. , hgf / c - met , igf1/igf - r , gh ) are activated and required for efficient liver regeneration . in vivo , egf and hgf have been implicated in liver regeneration , but specific deletion of egf receptor in hepatocyte led to liver regeneration deficiencies after 2/3 pht in mice without activation defect of erk1/2 while p38 mapk and nf - kb activation was reduced in regenerating mutant livers , indicating an impaired stress response after hepatectomy . indeed , p38 mapk could play a permissive role in dna replication during liver regeneration consistent with a role in the maintenance of hepatocyte cell cycle arrest in adult liver , while jnk could be involved in the g0/g1 transition . interestingly , hepatocyte deletion of c - met which led to liver regeneration defect was associated with mek - erk pathway inhibition highlighting that hgf contributes dominantly to erk1/2 activation in vivo [ 43 , 44 ] . a persistent egf supplementation in vitro only partially rescues the effect of erk1/2 downregulation in c - met depleted hepatocytes and restores to some extent dna synthesis and protein levels of cdk1 , aurora a and b , and mad2 . indeed , removal of egf in long - term survival dmso culture conditions followed by readdition of the growth factor was accompanied with an increase in dna synthesis , and multiple round of replication could be observed by alternating addition / removal [ 5355 ] . in coculture with liver biliary cell [ 5660 ] , egf alone prolonged cell progression up to late g1 phase , whereas tnf mediated extracellular remodeling is required for multiple division cycles . furthermore , a network of ecm or polymerized collagen type i gel induces a highly differentiated but growth - arrested phenotype in primary cultures , whereas a film of collagen promotes cell cycle progression and dedifferentiation [ 34 , 61 , 62 ] . hepatocytes dedifferentiation is reversible in consequence of a specific network triggered by the extracellular matrix , an active process driven by fak - mediated akt and erk1/2 signaling . in this context , our group have demonstrated that rat hepatocytes seeded in the presence of egf ( in the absence of fcs ) increased cell spreading and greatly enhanced cell survival . we therefore hypothesized that maintained mek1/2-erk1/2 stimulation of hepatocytes by egf could lead to a sustained activation of erk responsible for the negative control of the progression in a second cell cycle . indeed , when the mek / erk pathway is transiently inhibited with the mek inhibitor u0126 about 60% of hepatocytes did replicate their dna showing that primary hepatocytes are able to perform 2 cell cycles when a break of the mek / erk signalling pathway activity is done . in addition , cyclin d1 , e , a2 , cdk1 , p21 , and p27 were downregulated in mek - inhibited cells and induced after the u0126 removal . a third peak of dna synthesis in egf - seeded hepatocytes by performing another 2 days - break of mek1/2-erk1/2 activity could be obtained demonstrating that egf - seeded hepatocytes were able to perform multiple division waves after sequential mek1/2-erk1/2 pathway inhibitions ( see scheme 2 ) . in vitro , in the absence of serum and growth factor stimulation , hepatocytes in primary culture adhered to the plastic support but underwent spreading with a very low efficiency and die as observed by rapid caspase3/7 activations evaluated using a devd - amc assay . indeed , mek1 and mek2 could regulate distinct functions by sorting erk2 to different intracellular compartments in response to growth factor and erk2 intracellular localization could determine whether growth factors mediate hepatocyte proliferation or survival in an adhesion - dependent manner [ 7072 ] . all the genes of detoxification analyzed ( cyp 1a1 , 1a2 , 2b2 , 3a23 , and gsta2 ) as well as the aldolase b gene are induced all along the period of treatment . u0126 removal from the culture medium is accompanied with a fast decrease of the expression of these markers related to the reentry of the cells in a new cycle . in summary , early and sustained egf stimulation , in the absence of serum , could be a good compromise between classical monolayers with limited survival / differentiation , and long - term sophisticated and labor intensive cultures . this model emphasizes that early egf stimulation of hepatocytes in the absence of fcs and transient or sustained inhibition of the mek / erk pathway represent serum - free models ( scheme 2 ) that will be very helpful for pharmacological studies on drug metabolism and toxicity . the strict specificity of action of the mapks is still debated and today , no one can affirm with certitude the full redundancy of erk1 and erk2 or at the contrary the specificity of action of each protein . on one hand , the simple observation of the phenotypes of knockout animals for erk1 and erk2 fuels the idea that each erk isoform could regulate specific and non overlapping functions . to counteract this embryonic lethality and ascertain the roles of erk2 in embryo or adult tissues , conditional expressions erk2 also regulates multiple stages of t - cells development [ 79 , 80 ] . invalidation of erk2 in the central neural system ( cns ) leads to anomalies in multiple aspects of social behaviors , decreased anxiety - related attitude , and impaired long - term memory . erk2 also protects the myocardium from ischemia - reperfusion injury in vivo as erk2 gene - targeted mice showed enhanced infarction areas . in other words , the lethality of erk2 embryos could reflect a specific role of the isoform in the establishment of extraembryonic tissues or could be due to a higher expression of the erk2 isoform ( compared to erk1 ) in these tissues . at the contrary , another biochemical difference concerns the identification of a specific scaffolding protein of erk1 called mp1 ( mek partner 1 ) . finally , despite the fact that both kinases are simultaneously expressed in all cell types and tissues analyzed , the erk1 : erk2 ratio is quite variable . one of the best examples that illustrates this is the quite heterogenous expression profile of erk1 and erk2 mrna in brain . even if the elevated erk activity in these tissues has not been attributed to erk1 or erk2 , it is likely to be mainly carried by erk2 isoform , which would explain the phenotype of erk2 knockout . it is indeed assumed that erk2 is more expressed than erk1 in nearly all tissues examined so far and , as a consequence invalidation of erk2 , is supposed to have a stronger impact on the global erk activity compared to erk1 knockout . actually , the only one way to compare the expression levels of erk1 and erk2 in cells is the use of a phosphospecific antibody , which recognizes the phosphorylation of activation loop residues thr202/tyr204 and thr185/tyr187 of erk1 and erk2 , respectively . in this work , the authors have demonstrated that erk1 and erk2 are fully redundant kinases regarding the regulation of cell proliferation in nih3t3 cells . in addition to these works based on the use of genetically deficient animals and riding the wave following rnai discovery , a sustained number of studies have emerged in the literature in order to decipher the roles of erk1 and erk2 . , if erk1 and erk2 silencing would both affect cell proliferation , each kinase would be involved at different phases of the cell cycle : erk1 would regulate g2/m transition while erk2 could be essential in g1 phase . interestingly , contrary to other cell types in which erk2 is much more expressed compared to erk1 , normal hepatocytes harbor closely similar levels of erk1 and erk2 , according to p - erk1 : p - erk2 ratio . we went further in the analysis of putative roles of erk1 and erk2 in hepatic processes and established a link between erk1 and the cell survival of hepatocytes . as an example , in ovarian cells , the silencing of erk1 triggers the opposite phenotype to that observed in hepatocytes since cells become more sensitive to apoptosis . finally and in order to reconcile all these data , one can easily imagine that erk1 and erk2 regulate overlapping fundamental functions with regard to the most fundamental processes such as proliferation in nonspecialized cells . results obtained on mefs cells and which explicit a dose - response effect of erk on the intensity of cell proliferation argue in that sense . erk1 and erk2 also share dozen of substrates so one could speculate that potential differences in substrate affinity could explain the predominant role of one isoform in a function due to the presence of a particular set of partners , in other words in a particular cellular context . the mek / erk signaling pathway plays a central role in the regulation of various physiological processes such as proliferation , survival or cell motility . indeed , the chemical inhibition of mek1/2 kinase activities blocks in vitro as well as in vivo proliferation of a variety of tumor models , including hepatocellular carcinoma ( hcc ) [ 99103 ] . the best illustration of mapk pathway importance in oncogenesis lies in the observation of an erk1/2 overactivation in 50 out of 138 human tumor cell lines . active forms of the mek / erk pathway components including praf1 , pmek1/2 and perk1/2 were also associated with poor prognosis in patients with hcc [ 112 , 113 ] . however , in hcc , the overactivation of the mek / erk pathway did not result ( or rarely ) from an activating mutation of an upstream protein , namely , the gtpase ras or the raf protein kinase . generally , the protein ras is found mutated in about 30% of all human cancers with a high prevalence in the pancreas ( 90% ) and colon ( 50% ) adenocarcinomas . have shown from 80 surgical resections of hcc that 100% of tested malignant tissues had a constitutive activation of ras and this was linked to the loss or reduction of the ras inhibitory proteins nore1a and rassf1a . these decreases , or losses of activity , are supposed to have an important impact on hcc development and progression since ectopic expression of these different inhibitors is sufficient to inhibit the mapk pathway but , most of all , to suppress tumor cell proliferation both in vitro and in vivo [ 121124 ] . moreover , in poor prognosis hcc , erk1/2 activity should also be unrestrained given the weakened expression of the mapk phosphatase dusp-1 found in those patients . a significantly higher level of expression of c - raf was also reported in hepatocellular carcinomas when compared to cirrhosis . since the gene encoding for tgf- is also a target of the ras pathway , an autoamplification loop could be established ensuring , by the way , a persistent activation of the mek / erk pathway [ 131133 ] . in hcc , a significant deregulation of the igf this was mediated by the upregulation of igf2 via epigenetic mechanisms and by the downregulation of the igf2r receptor , which normally lead to the lysosomal degradation of igfs [ 79 , 137 , 138 ] . thus , the core protein and the envelope protein e2 of the hcv and the hbx protein and pres2-activator large surface protein of hbv have been shown to directly activate the mek / erk pathway but by different mechanisms [ 140144 ] . for instance , the hbx protein could upregulate the egfr and interacts with the protein pin1 to facilitate the dephosphorylation of c - raf while activation by the pres2-activator large surface protein used pkc - dependent mechanisms [ 142 , 143 , 145 , 146 ] . the envelope protein e2 of hcv stimulates the mek / erk pathway by binding to the cd81 receptor or to the low density lipoprotein receptor ( ldlr ) . indeed , erk2 targeting by stable chemically modified sirna altered the in vitro proliferation as well as the in vivo growth of the highly tumorigenic f1 cells . moreover and besides its role in cell proliferation , we have shown that erk2 but not erk1 was involved in hepatoma cell motility and invasiveness by an upar and p70s6 k dependent mechanism . moreover , active form of mek1 prevented serum deprivation - induced death of hepatocarcinoma cells and in hepg2 , mek / erk activity has been reported to contribute to cisplatin induced death . the prosurvival effects of the mek / erk pathway in tumor cells have been shown to pass through the upregulation or stimulation of different antiapoptotic factors such as bcl-2 , bim , or bad ( for review see ) . the critical involvement of the mek / erk pathway in hcc tumorigenesis strongly suggests that the kinases mek1/2 or erk1/2 could be promising therapeutic targets . given the predominant role of erk2 in transformed hepatocyte proliferation , survival , and motility and given the prodeath role of erk1 , it could be preferential to specifically target erk2 without affecting erk1 activity .

equilibrium black - hole solutions to einstein s equations have been known since the advent of general relativity . the most obvious reason such solutions are of physical interest is the expectation that they arise as the end state of catastrophic gravitational collapse of some suitably localised matter distribution . a less obvious reason such solutions are important is that they have played a key role in guiding studies of quantum gravity . . however , the laws of black hole mechanics have a formal similarity with the laws of thermodynamics . by studying quantum fields in a black - hole background , hawking demonstrated that this is not a mere analogy and in fact quantum mechanically black holes are a thermodynamic system . the black hole radiates at small temperature 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t_h } = { { \hbar \kappa } \over { 2\pi}}$$\end{document } proportional to the surface gravity of the horizon , and possesses a large entropy 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\rm{bh } } } } = { { { a_h } } \over { 4\hbar}}$$\end{document } proportional to the area ah of spatial cross sections of the horizon.1 deriving these semi - classical thermodynamic formulae from statistical mechanics requires a microscopic understanding of the degrees of freedom of the black hole . this has been a major motivation and driving force for quantum gravity research over the last four decades , although it is fair to say this is still poorly understood . an equilibrium black hole is extremal ( or extreme or degenerate ) , if the surface gravity 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\kappa = 0.$$\end{document } it immediately follows that the hawking temperature vanishes extremal black holes do not radiate after all ! hence , even semi - classically , extremal black holes are inert objects2 and as such are expected to have a simpler quantum description . the main purpose of this review is to discuss the classification of the near - horizon geometries of extremal black holes . there are a number of different motivations for considering this , which we will briefly review . as alluded to above , the principle reasons stem from studies in quantum gravity . in section 1.1 and 1.2 we discuss the various ways extremal black holes and their near - horizon geometries have appeared in modern studies of quantum gravity . in section 1.3 we discuss the more general black hole classification problem ( which is also partly motivated by quantum gravity ) , and how near - horizon geometries provide a systematic tool for investigating certain aspects of this problem for extremal black holes . to date , the most promising candidate for a theory of quantum gravity is string theory . famously , this predicts the existence of extra spatial dimensions . as discussed above , an important test for any candidate theory of quantum gravity is that it is able to explain the semi - classical formulae ( 1 ) and ( 2 ) . a major breakthrough of strominger and vafa was to use string theory to supply a microscopic derivation of eq . the black holes in question are higher - dimensional counterparts of the extremal reissner - nordstrm ( rn ) black holes . these are supersymmetric solutions to a supergravity theory , which can be obtained as a consistent kaluza - klein ( kk ) reduction of the ten / eleven dimensional supergravity that describes string theory at low energies . supersymmetry was crucial for their calculation , since non - renormalisation results allowed them to perform a weak - coupling string calculation ( involving certain d - brane configurations ) to deduce the entropy of the semi - classical black holes that exist in the strong coupling regime ( see for a review ) . this was quickly generalised to supersymmetric black holes with angular momentum and supersymmetric four dimensional black holes . an important assumption in these string theory calculations is that a given black hole is uniquely specified by its conserved charges : its mass / energy , electric charge and angular momentum . for four dimensional einstein - maxwell theory this follows from the black - hole uniqueness theorem ( see for a review ) . however , emparan and reall demonstrated that black hole uniqueness is violated for five - dimensional asymptotically - flat vacuum spacetimes . this was via the construction of an explicit counterexample , the black ring , which is a black hole whose spatial horizon topology is s s. together with the higher - dimensional analogues of the kerr black hole ( which have spherical topology ) found by myers and perry , this established that the conserved charges are not sufficient to specify a black hole uniquely and also that other horizon topologies are possible . indeed , this remarkable result motivated the study of stationary black holes in higher dimensional spacetimes . subsequently , a supersymmetric black ring was constructed [ 64 , 66 ] that coexists with the spherical topology black holes used in the original entropy calculations . although microscopic descriptions for the black rings have been proposed [ 20 , 51 ] , it is fair to say that the description of black hole non - uniqueness within string theory is not properly understood ( see for a brief review ) . , a substantial amount of work has been directed at removing the assumption of supersymmetry and extremality , with the ultimate goal being a string theory derivation of the thermodynamics of realistic black holes such as the four dimensional schwarzschild or kerr black holes . although little progress has been made in the description of such non - extremal black holes , significant progress has been made for extremal non - supersymmetric black holes . the attractor mechanism is the phenomenon that the entropy of certain extremal black holes in string theory does not depend on the moduli of the theory ( typically scalar fields in the supergravity theory ) . this was first observed for supersymmetric static black holes [ 78 , 197 , 77 ] although later it was realised it is valid for generic extremal black holes [ 100 , 194 , 12 ] . the key idea is that extremal black holes have a well - defined near - horizon geometry that typically possesses an ads2 symmetry . assuming this symmetry , it was argued that the entropy must be independent of the moduli of the theory . motivated by this , it was then proved that the near - horizon geometry of any extremal black hole in this context must in fact possess an ads2 symmetry . this general attractor mechanism thus ensures the black hole entropy is independent of the string coupling , so it can be safely computed at weak coupling . this shows that in fact it is extremality , rather than supersymmetry , that is behind the success of the string theory microscopic calculations [ 52 , 13 ] . this also explains the success of the entropy calculations for extremal , non - supersymmetric black holes in four and five dimensions , e.g. , [ 192 , 71 , 140 ] . a significant breakthrough in the study of quantum gravity is the anti de sitter / conformal field theory ( ads / cft ) duality [ 169 , 206 , 207 , 109 ] . in principle , ads / cft asserts a fully non - perturbative equivalence of quantum gravity in asymptotically ads spacetimes with a conformally invariant quantum field theory in one lower spatial dimension . a crucial feature of the duality is that classical gravity in ads spacetimes is dual to the strongly - coupled regime in the cft . this provides a precise framework to analyse the microscopic description of black holes in terms of well - defined quantum field theories . the duality was originally proposed in the context of string theory on ads5 s , in which case the cft is the maximally supersymmetric four dimensional su(n ) yang - mills gauge theory . however , the original idea has subsequently been generalised to a number of dimensions and theories , and such gauge / gravity dualities are believed to hold more generally . classical non - extremal ads black holes represent high - energy thermal states in the dual theory at large n and strong coupling . strong coupling poses the main obstacle to providing a precise entropy counting for such black holes , although excellent qualitative agreement can be found via extrapolating weak coupling calculations [ 108 , 23 , 130 ] . precise agreement has been achieved for the asymptotically ads3 baados - teitelboim - zanelli ( btz ) black hole ( even for the non - extremal case ) . this is because generically any theory of quantum gravity in ads3 must be described by a two dimensional cft2 with a specific central charge . this allows one to compute the entropy from cardy s formula , without requiring an understanding of the microscopic degrees of freedom . in fact the string theory calculations described in section 1.1 can be thought of as applications of this method . this is because the black holes in question can be viewed ( from a higher - dimensional viewpoint ) as black strings with an ads3 factor in the near - horizon geometry , allowing ads3/cft2 to be applied . a major open problem is to successfully account for black - hole entropy using a higher dimensional cft . the best understood case is when the cft is four dimensional , in which case the black holes are asymptotically ads5 . as in the original string calculations , a strategy to overcome the strong - coupling problem is to focus on supersymmetric ads black holes . the dual cft states then belong to certain bogomolnyi - prasad - sommerfield ( bps ) representations , and so weak - coupling calculations may not receive quantum corrections . it turns out that such black - hole solutions must rotate and hence are difficult to construct . in fact the first examples of supersymmetric ads5 black holes [ 120 , 119 ] were found via a classification of near - horizon geometries . subsequently , a more general four - parameter family of black - hole solutions were found [ 38 , 160 ] . the problem of classifying all supersymmetric ads black holes motivated further classifications of near - horizon geometries , which have ruled out the possibility of other types of black hole such as supersymmetric ads black rings [ 154 , 161 , 106 ] . despite significant effort , a microscopic derivation of the entropy from the cft has not yet been achieved in this context . due to the low amount of supersymmetry preserved by the black holes , it appears that non - zero coupling effects must be taken into account [ 149 , 22 , 148 , 21 , 36 ] . the original ads / cft duality was established by arguing that there exist two complementary descriptions of the low energy physics of the string theory of a stack of n extremal d3 black branes . near the horizon of the d - brane only low energy excitations survive , which are thus described by string theory in the ads5 s near - horizon geometry . on the other hand , the massless degrees of freedom on a d - brane arrange themselves into ( super ) su(n ) yang - mills theory . since extremal black holes typically possess an ads2 factor in their near - horizon geometry , one may then hope that an ads2/cft1 duality could provide a microscopic description of such black holes . however , it appears that the black - hole entropy can be reproduced from the degeneracy of the ground states of the dual conformal mechanics [ 170 , 195 ] . another recently - developed approach is to generalise the ads3/cft2 derivation of the btz entropy to describe more general black holes . this involves finding an asymptotic symmetry group of a given near - horizon geometry that contains a virasoro algebra and then applying cardy s formula . this was applied to the extremal kerr black hole and led to the kerr / cft correspondence , which is a proposal that quantum gravity in the near - horizon geometry of extremal kerr is described by a chiral cft2 ( see the reviews [ 31 , 48 ] ) . this technique has provided a successful counting of the entropy of many black holes . however , as in the ads2/cft1 case , the duality is poorly understood and it appears that non - trivial excitations of the near - horizon geometry do not exist [ 60 , 4 ] . the relation between these various approaches has been investigated in the special case of btz . furthermore , a cft2 description has been proposed for a certain class of near - extremal black holes , which possess a local near - horizon ads3 factor but a vanishing horizon area in the extremal limit [ 196 , 146].3 recently , ideas from the gauge / gravity duality have been used to model certain phase transitions that occur in condensed - matter systems , such as superfluids or superconductors [ 107 , 125 ] . the key motivation to this line of research , in contrast to the above , is to use knowledge of the gravitational system to learn about strongly - coupled field theories . charged black holes in ads describe the finite temperature phases . the non - superconducting phase is dual to the standard planar rn - ads black hole of einstein - maxwell theory , which is stable at high enough temperature . however , at low enough temperatures this solution is unstable to the formation of a charged scalar condensate . the dominant phase at low temperatures is a charged black hole with scalar hair , which describes the superconducting phase . this instability of ( near)-extremal rn - ads can be understood as occurring due to the violation of the breitenlohner - freedmann bound in the ads2 factor of the near - horizon geometry . the near - horizon ads2 has also been used to provide holographic descriptions of quantum critical points and fermi surfaces . the classification of higher - dimensional stationary black - hole solutions to einstein s equations is a major open problem in higher dimensional general relativity ( see [ 75 , 136 ] for reviews ) . as explained above , the main physical motivation stems from studies of quantum gravity and high energy physics . however , its study is also of intrinsic value both physically and mathematically . on the physical side we gain insight into the behaviour of gravity in higher - dimensional spacetimes , which in turn often provides renewed perspective for the classic four - dimensional results . on the mathematical side , solutions to einstein s equation have also been of interest in differential geometry .4 in four dimensions the black - hole uniqueness theorem provides an answer to the classification problem for asymptotically - flat black - hole solutions of einstein - maxwell theory ( see for a review).5 however , in higher dimensions , uniqueness is violated even for asymptotically - flat vacuum black holes . to date , the explicit black - hole solutions known are the spherical horizon topology myers - perry black holes and the black rings [ 73 , 187 ] that have s s horizon topology ( see for a review ) . if one allows for more complicated boundary conditions , such as kk asymptotics , then uniqueness is violated even for static black holes ( see , e.g. , ) . although of less obvious physical relevance , the investigation of asymptotically - flat vacuum black holes is the fundamental starting case to consider in higher dimensions , since such solutions can be viewed as limits of black holes with more general asymptotics such as kk , ads and matter fields . general results have been derived that constrain the topology of black holes . by generalising hawking s horizon topology theorem to higher dimensions , galloway and schoen have shown that the spatial topology of the horizon must be such that it admits a positive scalar curvature metric ( i.e. , positive yamabe type ) . horizon topologies are further constrained by topological censorship [ 85 , 44 ] . for asymptotically - flat ( and globally ads ) black holes , this implies that there must be a simply connected ( oriented ) cobordism between cross sections of the horizon and the ( d 2)-dimensional sphere at spatial infinity.6 in d = 4 , this rules out toroidal black holes , although for d = 5 it imposes no constraint . for d > 5 this does provide a logically independent constraint in addition to the positive yamabe condition [ 191 , 156 ] . firstly , asymptotically - flat , static vacuum black holes must be spherically symmetric and hence are uniquely given by the higher dimensional schwarzschild black hole .7 by generalising hawking s rigidity theorem , it was shown that asymptotically - flat and ads stationary non - extremal rotating black holes must admit at least u(1 ) isometry [ 137 , 180 ] ( for partial results pertaining to extremal rotating black holes see ) . this additional isometry can be used to further refine the allowed set of d = 5 black hole horizon topologies . an important class of spacetimes , for which substantial progress towards classification has been made , are the generalised weyl solutions [ 72 , 124 ] . by definition these possess an u(1 ) symmetry group and generalise d = 4 stationary and axisymmetric spacetimes . as in the d = 4 case , it turns out that the vacuum einstein equations for spacetimes with these symmetries are integrable . for d = 5 this structure has allowed one to prove certain uniqueness theorems for asymptotically - flat black holes with u(1 ) symmetry , using the same methods as for d = 4 . furthermore , this has led to the explicit construction of several novel asymptotically - flat , stationary , multi - black - hole vacuum solutions , the first example being a ( non - linear ) superposition of a black ring and a spherical black hole . for d > 5 , the symmetry of these spacetimes is not compatible with asymptotic flatness , that would require the number of commuting rotational symmetries to not exceed \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\lfloor { { { d - 1 } \over 2 } } \right\rfloor$\end{document } , the rank of the rotation group so(d 1 ) . in this case , weyl solutions are compatible with kk asymptotics and this has been used to prove uniqueness theorems for ( uniform ) kk black holes / strings . the general topology and symmetry constraints discussed above become increasingly weak as one increases the number of dimensions . furthermore , there is evidence that black hole uniqueness will be violated much more severely as one increases the dimensions . for example , by an analysis of gravitational perturbations of the myers - perry black hole , evidence for a large new family of black holes was found . furthermore , the investigation of blackfolds , where the long - range effective dynamics of certain types of black holes can be analysed , suggests that many new types of black holes should exist , see for a review . in the absence of new ideas , it appears that the general classification problem for asymptotically - flat black holes is hopelessly out of reach . as discussed in section 1.2 , the black - hole - classification problem for asymptotically ads black holes is of interest in the context of gauge / gravity dualities . the presence of a ( negative ) cosmological constant renders the problem even more complicated . even in four dimensions one reason for this comes from the fact that einstein s equations with a cosmological constant for stationary and axisymmetric metrics are not integrable . this also means that constructing charged generalisations , in four and higher dimensions , from a neutral seed can not be accomplished using standard solution generating methods . in fact , perturbation analyses of known solutions ( e.g. , kerr - ads and higher dimensional generalisations ) , reveal that if the black holes rotate sufficiently fast , super - radiant instabilities exist [ 130 , 34 , 159 , 35 ] . it has been suggested that the endpoint of these instabilities are new types of non - axisymmetric black - hole solutions that are not stationary in the usual sense , but instead invariant under a single killing field co - rotating with the horizon . a further complication in ads comes from the choice of asymptotic boundary conditions . in ads there is the option of replacing the sphere on the conformal boundary with more general manifolds , in which case topological censorship permits more black - hole topologies . it is clear that supersymmetry provides a technically - simplifying assumption to classifying spacetimes , since it reduces the problem to solving first - order killing spinor equations rather than the full einstein equations . a great deal of work has been devoted to developing systematic techniques for constructing supersymmetric solutions , most notably in five - dimensional ungauged supergravity and gauged supergravity . these have been used to construct new five - dimensional supersymmetric black - hole solutions , which are asymptotically flat [ 66 , 64 ] and ads [ 120 , 119 , 160 ] , respectively . furthermore , the first uniqueness theorem for asymptotically - flat supersymmetric black holes was proved using these methods . less obviously , it turns out that the weaker assumption of extremality can also be used as a simplifying assumption , as follows . the event horizon of all known extremal black holes is a degenerate killing horizon with compact spatial cross sections h. it turns out that restricting einstein s equations for a d - dimensional spacetime to a degenerate horizon gives a set of geometric equations for the induced metric on such ( d 2)-dimensional cross sections h , that depend only on quantities intrinsic to h. by studying solutions to this problem of riemannian geometry on a compact manifold h , one can thus consider the possible horizon geometries ( and topologies ) independently of the full parent spacetime . this strategy often also works in cases where the standard black hole uniqueness / classification techniques do not apply ( e.g. , ads , higher dimensions etc . ) . one can understand this feature of degenerate horizons in terms of the near - horizon limit , which , as we explain in section 2 , exists for any spacetime containing a degenerate horizon . this allows one to define an associated near - horizon geometry , which must also satisfy the full einstein equations [ 191 , 162 ] , so classifying near - horizon geometries is then equivalent to classifying possible horizon geometries ( and topologies ) . indeed , the topic of this review is the classification of near - horizon geometries in diverse dimensions and theories . the classification of near - horizon geometries allows one to explore in a simplified setup the main issues that appear in the general black - hole classification problem , such as the horizon topology , spacetime symmetry and the number of solutions . the main drawback of this approach is that the existence of a near - horizon geometry solution does not guarantee the existence of a corresponding black - hole solution ( let alone its uniqueness).8 hence , one must keep this in mind when interpreting near - horizon classifications in the context of black holes , although definite statements can be learned . in particular , one can use this method to rule out possible black - hole horizon topologies , for if one can classify near - horizon geometries completely and a certain horizon topology does not appear , this implies there can be no extremal black hole with that horizon topology either . a notable example of this method has been a proof of the non - existence of supersymmetric ads black rings in d = 5 minimal gauged supergravity [ 161 , 106 ] . in this review we will consider near - horizon geometries of solutions to einstein s equations , in all dimensions d 3 , that contain smooth degenerate horizons . our aim is to provide a unified treatment of such near - horizon solutions in diverse theories with matter content ranging from vacuum gravity , to einstein - maxwell theories and various ( minimal ) supergravity theories . we do not assume the near - horizon geometry arises as a near - horizon limit of a black - hole solution . however , due to the application to extremal black holes we will mostly consider horizons that admit a spatial cross section that is compact . as we will see in various setups , compactness often allows one to avoid explicitly solving the full einstein equations and instead use global arguments to constrain the space of solutions . as a result , the classification of near - horizon geometries with non - compact horizon cross sections is a much more difficult problem about which less is known . this is relevant to the classification of extremal black branes and therefore lies outside the scope of this article . nevertheless , along the way , we will point out cases in which classification has been achieved without the assumption of compactness , and in section 7.5 we briefly discuss extremal branes in this context . although this is a review article , we streamline some of the known proofs and we also present several new results that fill in various gaps in the literature . most notably , we fully classify three dimensional near - horizon geometries in vacuum gravity and einstein - maxwell - chern - simons theories , in section 4.2 and 6.1 respectively , and classify homogeneous near - horizon geometries in five dimensional einstein - maxwell - chern - simons theories in section 6.3.2 . in section 2 we provide key definitions , introduce a suitably general notion of a near - horizon geometry and set up the einstein equations for such near - horizon geometries . in section 3 we review various general results that constrain the topology and symmetry of near - horizon geometries . we also discuss the physical charges one can calculate from a near - horizon geometry . in section 4 we discuss the classification of near - horizon geometries in vacuum gravity , including a cosmological constant , organised by dimension . in cases where classification results are not known , we discuss the classification of supersymmetric near - horizon geometries in various supergravity theories , organised by dimension . in section 6 this includes d = 3 , 4 , 5 einstein - maxwell theories , allowing for chern - simons terms where appropriate , and d = 4 einstein - yang - mills theory . in section 7 this includes uniqueness / classification theorems of the corresponding extremal black - hole solutions , stability of near - horizon geometries and extremal black holes , geometric inequalities , analytic continuation of near - horizon geometries , and extremal branes and their near - horizon geometries . in this section we will introduce a general notion of a near - horizon geometry . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } be a smooth9 codimension-1 null hypersurface in a d dimensional spacetime ( m , g ) . in a neighbourhood of any such hypersurface there exists an adapted coordinate chart called gaussian null coordinates that we now recall [ 179 , 86 ] . let n be a vector field normal to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } whose integral curves are future - directed null geodesic generators of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document}. in general these will be non - affinely parameterised so on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } we have nn = n for some function . now let h denote a smooth ( d 2)-dimensional spacelike submanifold of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } , such that each integral curve of n crosses h exactly once : we term h a cross section of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } and assume such submanifolds exist . on h , d 2 , containing some point p h. starting from p h , consider the point in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } a parameter value v along the integral curve of n. now assign coordinates ( v , x ) to such a point , i.e. , we extend the functions x into \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } by keeping them constant along such a curve . this defines a set of coordinates ( v , x ) in a tubular neighbourhood of the integral curves of n through p h , such that n = /v . since n is normal to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } we have n n = gvv = 0 and n /x = gva = 0 on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document}. we now extend these coordinates into a neighbourhood of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } in m as follows . for any point \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q \in { \mathcal n}$\end{document } contained in the above coordinates ( v , x ) , let l be the unique past - directed null vector satisfying l consider the point in m an affine parameter value r along the null geodesic with tangent vector l. define the coordinates of such a point in m by ( v , r , x ) , i.e. , the functions v , x are extended into m by requiring them to be constant along such null geodesics . this provides coordinates ( v , r , x ) defined in a neighbourhood of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } in m , as required . we extend the definitions of n and l into m by n = /v and l = /r . by construction the integral curves of l = /r are null geodesics and hence grr = 0 everywhere in the neighbourhood of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } in m in question . furthermore , using the fact that n and l commute ( they are coordinate vector fields ) , we have 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ l}(l \cdot n ) = l \cdot ( { \nabla _ l}n ) = l \cdot ( { \nabla _ n}l ) = { 1 \over 2}{\nabla _ n}(l \cdot l ) = 0$$\end{document } and therefore l n = gvr = 1 for all r. a similar argument shows l /x = gra = 0 for all r. these considerations show that , in a neighbourhood of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } in m , the spacetime metric g written in gaussian null coordinates ( v , r , x ) is of the form 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = 2\;{\rm{d}}v({\rm{d}}r + r{h_a}{\rm{d}}{x^a } + { 1 \over 2}rf{\rm{d}}v ) + { \gamma _ { ab}}{\rm{d}}{x^a}{\rm{d}}{x^b},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } is the hypersurface { r = 0 } , the metric components f , ha , ab are smooth functions of all the coordinates , and ab is an invertible ( d 2 ) ( d 2 ) matrix . this coordinate chart is unique up to choice of cross section h and choice of coordinates ( x ) on h. upon a change of coordinates on h the quantities f , ha , ab transform as a function , 1-form and non - degenerate metric , respectively . hence they may be thought of as components of a globally - defined function , 1-form and riemannian metric on h. the coordinates developed above are valid in the neighbourhood of any smooth null hypersurface \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document}. in this work we will in fact be concerned with smooth killing horizons . these are null hypersurfaces that possess a normal that is a killing field k in m. hence we may set n = k in the above construction . since k = /v we deduce that in the neighbourhood of a killing horizon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } , the metric can be written as eq . ( 5 ) where the functions f , ha , ab are all independent of the coordinate v. using the killing property one can rewrite k k = k as d(k k ) = 2k on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } , where is now the usual surface gravity of a killing horizon . we may now introduce the main objects we will study in this work : degenerate killing horizons . these are defined as killing horizons \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } such that the normal killing field k is tangent to affinely parameterised null geodesics on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } , i.e. , 0 . therefore , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{d}}(k \cdot k){\vert _ { \mathcal n } } = 0$\end{document } , which implies that in gaussian null coordinates ( rgvv)|r = o = 0 . it follows that gvv = rf for some smooth function f. therefore , in the neighbourhood of any smooth degenerate killing horizon the metric in gaussian null coordinates reads 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = 2\;{\rm{d}}v({\rm{d}}r + r{h_a}(r , x){\rm{d}}{x^a } + { 1 \over 2}{r^2}f(r , x){\rm{d}}v ) + { \gamma _ { ab}}(r , x){\rm{d}}{x^a}{\rm{d}}{x^b}{.}$$\end{document } we are now ready to define the near - horizon geometry of a d - dimensional spacetime ( m , g ) containing such a degenerate horizon . given any > 0 , consider the diffeomorphism defined by v v/ and r r . the metric in gaussian null coordinates transforms g g where g is given by eq . x ) f(r , x ) , ha(r , x ) ha(r , x ) and ab(r , x ) ab(r , x ) . the near - horizon limit is then defined as the 0 limit of g. it is clear this limit always exists since all metric functions are smooth at r = 0 . the resulting metric is called the near - horizon geometry and is given by 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{{\rm{nh } } } } = 2\;{\rm{d}}v({\rm{d}}r + r{h_a}(x){\rm{d}}{x^a } + { 1 \over 2}{r^2}f(x){\rm{d}}v ) + { \gamma _ { ab}}(x){\rm{d}}{x^a}{\rm{d}}{x^b},$$\end{document } where f(x ) = f(0 , x ) , ha(x ) = ha(0 , x ) and ab(x ) = ab(0 , x ) . in fact the near - horizon geometry is completely specified by the following geometric data on the ( d 2)-dimensional cross section h : a smooth function f , 1-form ha and riemannian metric ab . we will often refer to the triple of data ( f , ha , ab ) on h as the near - horizon data . intuitively , the near - horizon limit is a scaling limit that focuses on the spacetime near the horizon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document}. we emphasise that the degenerate assumption gvv = o(r ) is crucial for defining this limit and such a general notion of a near - horizon limit does not exist for a non - degenerate killing horizon . as we will see , geometric equations ( such as einstein s equations ) for a near - horizon geometry can be equivalently written as geometric equations defined purely on a ( d 2)-dimensional cross section manifold h of the horizon . in this section we will write down general formulae relating the curvature of a near - horizon geometry to the curvature of the horizon h. for convenience we will denote the dimension of h by n = d 2 . it is convenient to introduce a null - orthonormal frame for the near - horizon metric ( 7 ) , denoted by ( e ) , where a = ( + , , a ) , a = 1 , , n and 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^ + } = \;{\rm{d}}v,\quad { e^ -}{\rm{= d}}r + r{h_a}{\hat e^a } + { 1 \over 2}{r^2}f{\rm{d}}v,\quad { e^a } = { \hat e^a},$$\end{document } so that g = abee = 2ee + ee , where are vielbeins for the horizon metric = .10 the dual basis vectors are 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e _ + } = { \partial _ v } - { 1 \over 2}f{r^2}{\partial _ r},\quad { e _ - } = { \partial _ r},\quad { e_a } = { \hat \partial _ a } - r{h_a}{\partial _ r},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat \partial}_a}$\end{document } denote the dual vectors to . the connection 1-forms satisfy de = e and are given by 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \omega _ { + - } } = rf{e^ + } + { 1 \over 2}{h_a}{e^a},\quad { \omega _ { + a } } = { 1 \over 2}{r^2}({{\hat \partial}_a}f - f{h_a}){e^ + } - { 1 \over 2}{h_a}{e^ - } + r{{\hat \nabla}_{\left [ a \right.}}{h_{\left . b \right]}}{e^b } , } \\ { { \omega _ { - a } } = - { 1 \over 2}{h_a}{e^ + } , \quad \quad \quad { \omega _ { ab } } = { { \hat \omega}_{ab } } - r{{\hat \nabla}_{\left [ a \right.}}{h_{\left . b \right]}}{e^ + } , } \quad\quad\quad\quad\quad\quad\quad\quad\quad\quad\\ \end{array}$$\end{document } where ab and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat \nabla}_a}$\end{document } are the connection 1-forms and levi - civita connection of the metric ab on h respectively . the curvature two - forms defined by ab = dab + ac give the riemann tensor in this basis using ab = rabcde e. the curvature two forms are : 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \omega _ { ab } } = { { \hat \omega}_{ab } } + { e^ + } \wedge { e^ -}{{\hat \nabla}_{\left [ a \right.}}{h_{\left . b \right ] } } + r{e^ + } \wedge { e^d}\left({- { h_d}{{\hat \nabla}_{\left [ a \right.}}{h_{\left . b \right ] } } + { { \hat \nabla}_d}{{\hat \nabla}_{\left [ a \right.}}{h_{\left . b \right ] } } + { 1 \over 2}{h_a}{{\hat \nabla}_{\left [ b \right.}}{h_{\left . d \right ] } } - { 1 \over 2}{h_b}{{\hat \nabla}_{\left [ a \right.}}{h_{\left . d \right ] } } } \right ) , } \\{{\omega _ { + - } } = ( { 1 \over 4}{h_a}{h_a } - f){e^ + } \wedge { e^ - } + r{e^b } \wedge { e^ + } \left({{{\hat \partial}_b}f - f{h_b } + { 1 \over 2}{h_a}{{\hat \nabla}_{\left [ a \right.}}{h_{\left . b \right ] } } } \right ) + { 1 \over 2}{{\hat \nabla}_{\left [ a \right.}}{h_{\left . b \right]}}{e^a } \wedge { e^b},}\qquad \qquad \\{{\omega _ { + a } } = { r^2}{e^ + } \wedge { e^d}\left [ { \left({- { 1 \over 2}{{\hat \nabla}_d } + { h_d } } \right)({{\hat \partial}_a}f - f{h_a } ) + { 1 \over 2}f{{\hat \nabla}_{\left [ a \right.}}{h_{\left . d \right ] } } + { { \hat \nabla}_{\left [ c \right.}}{h_{\left . a \right]}}{{\hat \nabla}_{\left [ c \right.}}{h_{\left . d \right]}}f } \right]}\quad \\+ r{e^ + } \wedge { e^ -}\left({{h_a}f - { { \hat \partial}_a}f - { 1 \over 2}{h_b}{{\hat \nabla}_{\left [ b \right.}}{h_{\left . a \right ] } } } \right ) + { 1 \over 2}{e^ - } \wedge { e^b}\left({{{\hat \nabla}_a}{h_b } - { 1 \over 2}{h_a}{h_b } } \right)\qquad\qquad \quad\ \\{+ r{e^b } \wedge { e^d}\left({- { { \hat \nabla}_d}{{\hat \nabla}_{\left [ a \right.}}{h_{\left . b \right ] } } + { 1 \over 2}{h_a}{{\hat \nabla}_{\left [ d \right.}}{h_{\left . b \right ] } } - { 1 \over 2}{h_b}{{\hat \nabla}_{\left [ a \right.}}{h_{\left . d \right ] } } } \right),}\qquad\qquad\qquad\qquad \qquad \qquad\\{{\omega _ { - a } } = { 1 \over 2}{e^ + } \wedge { e^b}\left({{{\hat \nabla}_b}{h_a } - { 1 \over 2}{h_a}{h_b } } \right),}\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat \omega}_{ab}}$\end{document } is the curvature of ab on h. the non - vanishing components of the ricci tensor are thus given by : 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { r_{+ - } } = f - { 1 \over 2}{h_a}{h_a } + { 1 \over 2}{{\hat \nabla}_a}{h_a},\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad}\\ { { r_{ab } } = { { \hat r}_{ab } } + { { \hat \nabla}_{(a}}{h_{b ) } } - { 1 \over 2}{h_a}{h_b},\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad}\\ { { r_{+ + } } = { r^2}\left [ { - { 1 \over 2}{{\hat \nabla}^2}f + { 3 \over 2}{h^a}{{\hat \nabla}_a}f + { 1 \over 2}f{{\hat \nabla}^a}{h_a } - f{h_a}{h_a } + { { \hat \nabla}_{\left [ c \right.}}{h_{\left . a \right]}}{{\hat \nabla}_{\left [ c \right.}}{h_{\left . a \right ] } } } \right ] \equiv { r^2}{s_{+ + } } , } \\ { { r_{+ a } } = r\left [ { { { \hat \nabla}_a}f - f{h_a } - 2{h_b}{{\hat \nabla}_{\left [ a \right.}}{h_{\left . b \right ] } } } \right ] \equiv r{s_{+ a}},\quad \quad \quad \quad \quad \quad \quad \quad}\\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat r}_{ab}}$\end{document } is the ricci tensor of the metric ab on h. the spacetime contracted bianchi identity implies the following identities on h : 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{+ + } } = - { 1 \over 2}({\hat \nabla ^a } - 2{h^a}){s_{+ a}},$$\end{document } 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{+ a } } = - { \hat \nabla ^b}[{r_{ba } } - { 1 \over 2}{\gamma _ { ba}}({r^c}_c + 2{r_{+ - } } ) ] + { h^b}{r_{ba } } - { h_a}{r_{+ -}},$$\end{document } which may also be verified directly from the above expressions . it is worth noting that the following components of the weyl tensor automatically vanish : cab = 0 and cabc = 0 this means that e = r is a multiple weyl aligned null direction and hence any near - horizon geometry is at least algebraically special of type ii within the classification of . in fact , it can be checked that the null geodesic vector field r has vanishing expansion , shear and twist and therefore any near - horizon geometry is a kundt spacetime.11 indeed , by inspection of eq . ( 7 ) it is clear that near - horizon geometries are a subclass of the degenerate kundt spacetimes,12 which are all algebraically special of at least type ii . henceforth , we will drop the hats on all horizon quantities , so rab and a refer to the ricci tensor and levi - civita connection of ab on h. we will consider spacetimes that are solutions to einstein s equations : 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_{\mu \nu } } = \lambda { g_{\mu \nu } } + { t_{\mu \nu } } - { 1 \over n}{g^{\rho \sigma}}{t_{\rho \sigma}}{g_{\mu \nu}},$$\end{document } where t is the energy - momentum and is the cosmological constant of our spacetime . we will be interested in a variety of possible energy momentum tensors and thus in this section we will keep the discussion general . an important fact is that if a spacetime containing a degenerate horizon satisfies einstein s equations then so does its near - horizon geometry . ( 6 ) satisfies einstein s equations , then so will the 1-parameter family of diffeomorphic metrics g for any > 0 . hence the limiting metric 0 , which by definition is the near - horizon geometry , must also satisfy the einstein equations . the near - horizon limit of the energy momentum tensor thus must also exist and takes the form 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t = 2\;{\rm{d}}v({t_{+ -}}{\rm{d}}r + r({\beta _ a } + { t_{+ -}}{h_a}){\rm{d}}{x^a } + { 1 \over 2}{r^2}(\alpha + { t_{+ -}}f){\rm{d}}v ) + { t_{ab}}{\rm{d}}{x^a}{\rm{d}}{x^b},$$\end{document } where t+ , are functions on h and a is a 1-form on h. working in the vielbein frame ( 8) , it is then straightforward to verify that the ab and + components of the einstein equations for the near - horizon geometry give the following equations on the cross section h : 17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_{ab } } = { 1 \over 2}{h_a}{h_b } - { \nabla _ { ( a}}{h_{b ) } } + \lambda { \gamma _ { ab } } + { p_{ab}},$$\end{document } 18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f = { 1 \over 2}{h^2 } - { 1 \over 2}{\nabla _ a}{h^a } + \lambda - e,$$\end{document } where we have defined 19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_{ab } } \equiv { t_{ab } } - { 1 \over n}({\gamma ^{cd}}{t_{cd } } + 2{t_{+ -}}){\gamma _ { ab}},$$\end{document } 20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e \equiv - \left({{{n - 2 } \over n } } \right){t_{+ - } } + { 1 \over n}{\gamma ^{ab}}{t_{ab}}.$$\end{document } it may be shown that the rest of the einstein equations are automatically satisfied as a consequence of eqs . ( 17 ) , ( 18 ) and the matter field equations , as follows . this is equivalent to the following equations on h : 21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha = - { 1 \over 2}({\nabla ^a } - 2{h^a}){\beta _ a},\quad { \beta _ a } = - ( { \nabla ^b } - { h^b}){t_{ab } } - { h_a}{t_{+ -}},$$\end{document } which thus determine the components of the energy momentum tensor , a in terms of t+ , tab . the + + and + a components of the einstein equations are s++ = and s+a = a respectively , where s++ and s+a are defined in eq . the first equation in ( 21 ) and the identity ( 13 ) imply that the + + equation is satisfied as a consequence of the + a equation . finally , substituting eqs . ( 17 ) and ( 18 ) into the identity ( 14 ) , and using the second equation in ( 21 ) , implies the + a equation . although the energy momentum tensor must have a near - horizon limit , it is not obvious that the matter fields themselves must . thus , consider the full spacetime before taking the near - horizon limit . recall that for any killing horizon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_{\mu \nu}}{k^\mu}{k^\nu}\vert { _ \mathcal n } = 0$\end{document } and therefore \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${t_{\mu \nu}}{k^\mu}{k^\nu}{\vert _ we will illustrate this for einstein - maxwell theory whose energy - momentum tensor is 22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t_{\mu \nu } } = 2({{\mathcal f}_{\mu \rho}}{\mathcal f}_\nu ^\rho - { 1 \over 4}{{\mathcal f}^2}{g_{\mu \nu}}),$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } is the maxwell 2-form , which must satisfy the bianchi identity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{d\mathcal f = 0}}$\end{document}. it can be checked that in gaussian null coordinates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${t_{\mu \nu}}{k^\mu}{k^\nu}{\vert _ \mathcal n } = 2{\mathcal f_{\upsilon a}}{\mathcal f_{\upsilon b}}{\gamma ^{ab}}{\vert _ { r = 0}}$\end{document } and hence we deduce that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f_{\upsilon a}}{\vert _ { r = 0 } } = 0$\end{document}. thus , smoothness requires \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f_{\upsilon a } } = { \mathcal o(r)}$\end{document } , which implies the near - horizon limit of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } in fact exists . furthermore , imposing the bianchi identity to the near - horizon limit of the maxwell field relates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f_{\upsilon r}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f_{\upsilon a}}$\end{document } , allowing one to write 23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal f}_{{\rm{nh } } } } = { \rm{d}}(r\delta ( x){\rm{d}}v ) + { 1 \over 2}{b_{ab}}(x){\rm{d}}{x^a } \wedge { \rm{d}}{x^b},$$\end{document } where is a function on h and b is a closed 2-form on h. the 2-form b is the maxwell field induced on h and locally can be written as b = da for some 1-form potential a on h. it can be checked that for the near - horizon limit 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = { { 2(n - 1 ) } \over n}{\delta ^2 } + { 1 \over n}{b_{ab}}{b^{ab}},$$\end{document } 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_{ab } } = 2{b_{ac}}b_b^c + \left({{2 \over n}{\delta ^2 } - { { { b_{cd}}{b^{cd } } } \over n } } \right){\gamma _ { ab}}.$$\end{document } we will present the maxwell equations in a variety of dimensions in section 6 . it is worth remarking that the above naturally generalises to p - form electrodynamics , with p 2 , for which the energy momentum tensor is 26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t_{\mu \nu } } = { 2 \over { ( p - 1)!}}\left({{{\mathcal f}_{\mu { \rho _ 1 } \ldots { \rho _ { p - 1}}}}{\mathcal f}_\nu ^{{\rho _ 1 } \ldots { \rho _ { p - 1 } } } - { 1 \over { 2p}}{{\mathcal f}^2}{g_{\mu \nu } } } \right),$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } is a p - form field strength satisfying the bianchi identity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{d\mathcal f = 0}}$\end{document}. it is then easily checked that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${t_{\mu \nu}}{k^\mu}{k^\nu}{\vert _ \mathcal n } = 0$\end{document } implies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f_{\upsilon { a_{1 \cdots}}{a_{p - 1}}}}{\mathcal f_\upsilon}^{{a_{1 \cdots}}{a_{p - 1}}}{\vert _ { r = 0 } } = 0$\end{document } and hence \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f_{\upsilon { a_{1 \cdots}}{a_{p - 1}}}}{\vert _ { r = 0 } } = 0$\end{document}. thus , smoothness requires \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f_{\upsilon { a_{1 \cdots}}{a_{p - 1 } } } } = { \mathcal o(r)}$\end{document } , which implies that the near - horizon limit of the p - form exists . the bianchi identity then implies that the most general form for the near - horizon limit is 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal f}_{{\rm{nh } } } } = { \rm{d}}(y \wedge r{\rm{d}}v ) + x,$$\end{document } where y is a ( p 2)-form on h and x is a closed p - form on h. the einstein equations for a near - horizon geometry can also be interpreted as geometrical equations arising from the restriction of the einstein equations for the full spacetime to a degenerate horizon , without taking the near - horizon limit , as follows . the near - horizon limit can be thought of as the 0 limit of the boost transformation ( k , l ) ( k , l ) . this implies that restricting the boost - invariant components of the einstein equations for the full spacetime to a degenerate horizon is equivalent to the boost invariant components of the einstein equations for the near - horizon geometry . the boost - invariant components are + and ab and hence we see that eqs . ( 17 ) and ( 18 ) are also valid for the full spacetime quantities restricted to the horizon . we deduce that the restriction of these components of the einstein equations depends only on data intrinsic to h : this special feature only arises for degenerate horizons.13 it is worth noting that the horizon equations ( 17 ) and ( 18 ) remain valid in the more general context of extremal isolated horizons [ 163 , 209 , 28 ] and kundt metrics . the positivity of e and pab can be related to standard energy conditions . for a near - horizon geometry \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_{\mu \nu}}{(k - l)^\mu}{(k - l)^\nu}{\vert _ \mathcal n } = - 2{r_{\mu \nu}}{k^\mu}{l^\nu}{\vert _ \mathcal n}$\end{document}. since k l is timelike on the horizon , the strong energy condition implies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_{\mu \nu}}{k^\mu}{l^\nu}{\vert _ \mathcal n } \leq 0$\end{document}. hence , noting that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_{\mu \nu}}{k^\mu}{l^\nu}{\vert _ \mathcal n } = { r_{+ - } } = - e + \lambda$\end{document } we deduce that the strong energy condition implies 28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e \geq\lambda .$$\end{document } on the other hand the dominant energy condition implies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${t_{\mu \nu}}{k^\mu}{l^\nu}{\vert _ n } \leq 0$\end{document}. one can show \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${t_{\mu \nu}}{k^\mu}{l^\nu}{\vert _ \mathcal n } = - { 1 \over 2}{p_{ab}}{\gamma ^{ab}}$\end{document}. therefore , the dominant energy condition implies 29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_\gamma } \equiv { p_{ab}}{\gamma ^{ab } } \geq0.$$\end{document } since p = 2t+ , if n 2 the dominant energy condition implies e 0 : hence , if 0 the dominant energy condition implies both eqs . ( 28 ) and ( 29 ) . observe that einstein - maxwell theory with 0 satisfies both of these conditions . in this review , we describe the current understanding of the space of solutions to the basic horizon equation ( 17 ) , together with the appropriate horizon matter field equations , in a variety of dimensions and theories . so far we have considered near - horizon geometries independently of any extremal - black - hole solutions . in this section we will assume that the near - horizon geometry arises from a near - horizon limit of an extremal black hole . this limit discards the asymptotic data of the parent - black - hole solution . as a result , only a subset of the physical properties of a black hole can be calculated from the near - horizon geometry alone . in particular , information about the asymptotic stationary killing vector field is lost and hence one can not compute the mass from a komar integral , nor can one compute the angular velocity of the horizon with respect to infinity . below we discuss physical properties that can be computed purely from the near - horizon geometry [ 123 , 79 , 154 ] . area . the area of cross sections of the horizon h is defined by 30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_h } = \int\nolimits_h { { \epsilon _ \gamma},}$$\end{document } where is the volume form associated to the induced riemannian metric ab on h. for definiteness we now assume the parent black hole is asymptotically flat . angular momentum . the conserved angular momentum associated with a rotational symmetry , generated by a killing vector m , is given by a komar integral on a sphere at spacelike infinity s:14 31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j = { 1 \over { 16\pi}}\int\nolimits_{{s_\infty } } { \star{\rm{d}}m.}$$\end{document } this expression can be rewritten as an integral of the near - horizon data over h , by applying stokes theorem to a spacelike hypersurface with boundary s h. the field equations can be used to evaluate the volume integral that is of the form r(m ) , where r(m) = rm . in particular , for vacuum gravity one simply has : 32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j = { 1 \over { 16\pi}}\int\nolimits_h { ( h \cdot m){\epsilon _ \gamma}.}$$\end{document } for einstein - maxwell theories the integral r(m ) can also be written as an integral over h , giving extra terms that correspond to the contribution of the matter fields to the angular momentum . for example , consider pure einstein - maxwell theory in any dimension so the maxwell equation is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{d}}\star { \mathcal f } = 0$\end{document}. parameterising the near - horizon maxwell field by ( 23 ) one can show that , in the gauge ma = 0 , 33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j = { 1 \over { 16\pi}}\int\nolimits_h { ( h\cdot m + 4(m\cdot a)\delta){\epsilon_\gamma},}$$\end{document } so the angular momentum is indeed determined by the near - horizon data . in five spacetime dimensions it is natural to couple einstein - maxwell theory to a chern - simons ( cs ) term . while the einstein equations are unchanged , the maxwell equation now becomes 34\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{d } } \star { \mathcal f } + { { 2\xi } \over { \sqrt 3}}{\mathcal f } \wedge { \mathcal f } = 0,$$\end{document } where is the cs coupling constant . the angular momentum in this case can also be written purely as an integral over h : 35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j = { 1 \over { 16\pi}}\int\nolimits_h { ( h\cdot m + 4(m\cdot a)\delta){\epsilon_\gamma } + { { 16 } \over { 3\sqrt 3}}\xi ( m \cdot a)a \wedge b.}$$\end{document } of particular interest is the theory defined by cs coupling = 1 , since this corresponds to the bosonic sector of minimal supergravity . for example , in pure einstein - maxwell theory in any dimension , the electric charge is written as an integral over spatial infinity : 36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${q_e } = { 1 \over { 4\pi}}\int\nolimits_{{s_\infty } } { \star { \mathcal f}}.$$\end{document } by applying stokes theorem to a spacelike hypersurface as above , and using the maxwell equation , one easily finds 37\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${q_e } = { 1 \over { 4\pi}}\int\nolimits_h { \delta { \epsilon _ \gamma } } .$$\end{document } for d = 5 einstein - maxwell - cs theory one instead gets 38\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${q_e } = { 1 \over { 4\pi}}\int\nolimits_h { \delta { \epsilon_\gamma } + { 2 \over { \sqrt 3}}\xi a \wedge b } .$$\end{document } for d = 4 one also has a conserved magnetic charge \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${q_m } = { 1 \over { 4\pi}}\int\nolimits_{{s_\infty } } { \mathcal f}$\end{document}. using the bianchi identity this can be written as 39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${q_m } = { 1 \over { 4\pi}}\int\nolimits_h b .$$\end{document } for d > 4 asymptotically - flat black holes there is no conserved magnetic charge . however , for d = 5 black rings h s s , one can define a quasi - local dipole charge over the s 40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal d } = { 1 \over { 2\pi}}\int\nolimits_{{s^2 } } { { \mathcal f } = { 1 \over { 2\pi}}\int\nolimits_{{s^2 } } b } , $ $ \end{document } where in the second equality we have expressed it in terms of the horizon maxwell field . note that in general the gauge field a will not be globally defined on h , so care must be taken to evaluate expressions such as ( 35 ) and ( 38 ) , see [ 123 , 155 ] . in this section we describe a number of general results concerning the topology and symmetry of near - horizon geometries under various assumptions . hawking s horizon topology theorem is one of the fundamental ingredients of the classic four - dimensional black - hole uniqueness theorems . it states that cross sections of the event horizon of an asymptotically - flat , stationary , black - hole solution to einstein s equations , satisfying the dominant energy condition , must be homeomorphic to s. the proof is an elegant variational argument that shows that any cross section with negative euler characteristic can be deformed outside the event horizon such that its outward null geodesics converge . this means one has an outer trapped surface outside the event horizon , which is not allowed by general results on black holes.15 galloway and schoen have shown how to generalise hawking s horizon topology theorem to higher dimensional spacetimes . their theorem states if the dominant energy condition holds , a cross section h of the horizon of a black hole , or more generally a marginally outer trapped surface , must have positive yamabe invariant.16 the positivity of the yamabe invariant , which we define below , is equivalent to the existence of a positive scalar curvature metric and is well known to impose restrictions on the topology , see , e.g. , . for example , when h is three dimensional , the only possibilities are connected sums of s ( and their quotients ) and s s , consistent with the known examples of black - hole solutions . in the special case of degenerate horizons a simple proof of this topology theorem can be given directly from the near - horizon geometry . this is essentially a specialisation of the simplified proof of the galloway - schoen theorem given in . however , we note that since we only use properties of the near - horizon geometry , in particular only the horizon equation ( 17 ) , we do not require the existence of a black hole . for four dimensional spacetimes , so dim h = 2 , the proof is immediate , see , e.g. , [ 144 , 152 ] . consider a spacetime containing a degenerate horizon with a compact cross section h and assume the dominant energy condition holds . if 0 then h s , except for the special case where the near - horizon geometry is flat ( so = 0 ) and h t. if < 0 and (h ) < 0 the area of h satisfies ah 2(h ) with equality if and only if the near - horizon geometry is ads2 g , where g is a compact quotient of hyperbolic space of genus g. the proof is elementary . ( 17 ) over h to get 41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi ( h ) = { 1 \over { 4\pi}}\int\nolimits_h { { r_\gamma}{\epsilon _ \gamma } = { 1 \over { 8\pi } } } \int\nolimits_h { ( h \cdot h + 4\lambda + 2{p_\gamma}){\epsilon _ \gamma } } , $ $ \end{document } where is the volume form of the horizon metric . therefore , for 0 and matter satisfying the dominant energy condition p 0 ( see eq . ( 29 ) ) , it follows that (h ) 0 . equality can only occur if = 0 , p 0 , ha 0 : using eqs . ( 17 ) and ( 18 ) this implies rab = 0 and f = 0 , so the near - horizon geometry is the trivial flat solution t. for the < 0 case the above argument fails and one finds no restriction on the topology of h. instead , for (h ) < 0 , one can derive a lower bound for the area of h : 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_h } = { { 2\pi \vert \chi ( h)\vert } \over { \vert \lambda \vert } } + { 1 \over { 4\vert \lambda \vert}}\int\nolimits_h { ( h \cdot h + 2{p_\gamma}){\epsilon _ \gamma } \geq } { { 2\pi \vert \chi ( h)\vert } \over { \vert \lambda \vert}}.$$\end{document } this agrees with the lower bounds found in [ 95 , 208 ] in the more general context of apparent horizons . ( 42 ) is saturated if and only if ha 0 , p 0 , which implies rab = ||ab and f = , so the near - horizon geometry is ads2 g . it is of interest to generalise these results to higher dimensions along the lines of galloway and schoen . as is well known , the total integral of the scalar curvature in itself does not constrain the topology of h in this case . an analogue of this invariant for dim h = n 3 is given by the yamabe invariant (h ) . this is defined via the yamabe constant associated to a given conformal class of metrics [ ] on h. first consider the volume - normalised einstein - hilbert functional 43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e[{\gamma ^\prime } ] \equiv { { \int\nolimits_h { { r_{{\gamma ^\prime}}}{\epsilon_{{\gamma ^\prime } } } } } \over { { { ( \int\nolimits_h { { \epsilon_{{\gamma ^\prime}}}})}^{{{n - 2 } \over n}}}}},$$\end{document } where is a riemannian metric on h and is the associated volume form . as is well known , this functional is neither bounded from above or below . however , the restriction of e to any conformal class of metrics is always bounded from below : the yamabe constant y ( h , [ ] ) for a given conformal class is then defined as the infimum of this functional . parameterising the conformal class by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\gamma ^\prime } = { \phi ^{{4 \over { n - 2}}}}\gamma$\end{document } , for smooth positive functions , we have y ( h , [ ] ) inf > 0 e [ ] , where 44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_\gamma}[\phi ] \equiv { { \int\nolimits_h { \left({{{4(n - 1 ) } \over { n - 2}}\vert \nabla \phi\vert ^{2 } + { r_\gamma}{\phi ^2 } } \right){\epsilon _ \gamma } } } \over { { { \left({\int\nolimits_h { { \phi ^{{{2n } \over { n - 2 } } } } } { \epsilon _ \gamma } } \right)}^{{{n - 2 } \over n}}}}}.$$\end{document } the yamabe invariant (h ) is defined by (h ) = sup[ ] y ( h , [ ] ) , where the supremum is taken over all possible conformal classes . the solution to the yamabe problem states the following remarkable fact : for every conformal class [ ] on compact h , the functional e[ ] achieves its infimum and this occurs for a constant scalar curvature metric . consider a spacetime containing a degenerate horizon with a compact cross section h and assume the dominant energy condition holds . if 0 , then either (h ) > 0 or the induced metric on the horizon is ricci flat . if < 0 and (h ) < 0 the area of h satisfies 45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_h } \geq{\left({{{\sigma ( h ) } \over { n\lambda } } } \right)^{n/2}}.$$\end{document } a simple proof exploits the solution to the yamabe problem mentioned above [ 189 , 166 ] . first observe that if there exists a conformal class of metrics for which the yamabe constant y ( h , [ ] ) > 0 then it follows that (h ) > 0 . therefore , to establish that h has positive yamabe invariant , it is sufficient to show that for some ab the functional e [ ] > 0 for all > 0 , since the solution to the yamabe problem then tells us that y ( h , [ ] ) = e [ 0 ] > 0 for some 0 > 0 . for our riemannian manifolds ( h , ) it is easy to show , except for one exceptional circumstance , that e [ ] > 0 for all > 0 and thus y ( h , [ ] ) > 0 . the horizon equation ( 17 ) can be used to establish the identity 46\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2\vert \nabla \phi { \vert ^2 } + { r_\gamma}{\phi ^2 } = 2\vert { \rm{d}}\phi { \vert ^2 } - \nabla \cdot ( { \phi ^2}h ) + ( \lambda n + { p_\gamma}){\phi ^2}$$\end{document } for all , where we have defined the differential operator da a + ha . it is worth noting that this identity relies crucially on the precise constants appearing in eq . this implies the following integral identity over h : 47\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_h { \left [ { { { 4(n - 1 ) } \over { n - 2}}\vert \nabla \phi { \vert ^2 } + { r_\gamma}{\phi ^2 } } \right]{\epsilon _ \gamma } } = \int\nolimits_h { \left [ { 2\vert { \rm{d}}\phi { \vert ^2 } + { { 2n } \over { n - 2}}\vert \nabla \phi { \vert ^2 } + ( n\lambda + { p_\gamma}){\phi ^2 } } \right]{\epsilon _ \gamma}.}$$\end{document } if 0 , the dominant energy condition p 0 implies e [ ] 0 for all > 0 with equality only if ha 0 , p 0 and = 0 . the exceptional case ha 0 , p 0 and = 0 implies r = 0 , which allows one to infer that either rab = 0 or h admits a metric of positive scalar curvature ( and is thus positive yamabe after all ) . as in four spacetime dimensions the above argument fails for < 0 , and thus provides no restriction of the topology of h. instead , assuming the dominant energy condition , eq . ( 47 ) implies 48\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_\gamma}[\phi ] \geq - n\vert \lambda \vert { { \int\nolimits_h { { \phi ^2}{\epsilon _ \gamma } } } \over { { { ( \int\nolimits_h { { \phi ^{{{2n } \over { n - 2}}}}{\epsilon _ \gamma}})}^{{{n - 2 } \over n } } } } } \geq - n\vert \lambda \vert a_h^{2/n}$$\end{document } for any > 0 , where the second inequality follows from hlder s inequality . therefore , by the definition of y ( h , [ ] ) , we deduce that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$y(h,\left [ \gamma \right ] ) \geq - n\vert \lambda \vert a_h^{2/n}$\end{document}. it follows that if (h ) < 0 , we get the stated non - trivial lower bound on the area of h. we note that the lower bound can only be achieved if ha 0 and p 0 , which implies r = n|| , in which case necessarily minimises the functional e in the conformal class [ ] so that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$y(h,\left [ \gamma \right ] ) \geq - n\vert \lambda \vert a_h^{2/n}$\end{document}. however , since y(h , [ ] ) |(h)| , it need not be the case that the lower bound in eq . ( 45 ) is saturated by such horizon metrics ( in contrast to the n = 2 case above ) . we note that the above topology theorems in fact only employ the scalar curvature of the horizon metric and not the full horizon equation ( 17 ) . it would be interesting if one could use the non - trace part of the horizon equation to derive further topological restrictions . ( 7 ) , possesses enhanced symmetry : in addition to the translation symmetry v v + c one also has a dilation symmetry ( v , r ) ( v , r ) where 0 and together these form a two - dimensional non - abelian isometry group . in this section we will discuss various near - horizon symmetry theorems that guarantee further enhanced symmetry . a static near - horizon geometry is one for which the normal killing field k is hypersurface orthogonal , i.e. , k dk 0 everywhere . any static near - horizon geometry is locally a warped product of ads2 , ds2 or and h. if h is simply connected this statement is global . in this case if h is compact and the strong energy conditions holds it must be the ads2 case or the direct product h. proof : as a 1-form k = k dx = dr + rha dx + rf dv . a short calculation then reveals that k dk = 0 if and only if 49\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{d}}h = 0\quad { \rm{d}}f = hf,$$\end{document } which are the staticity conditions for a near - horizon geometry . locally they can be solved by 50\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h = { \rm{d}}\lambda \quad f = { a_0}{e^\lambda},$$\end{document } where (x ) is a function on h and a0 is a constant . substituting these into the near - horizon geometry and changing the affine parameter r er gives : 51\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = { e^{- \lambda ( x)}}[{a_0}{r^2}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r ] + { \gamma _ { ab}}(x){\rm{d}}{x^a}{\rm{d}}{x^b}.$$\end{document } the metric in the square bracket is a maximally symmetric space : ads2 for a0 < 0 , ds2 for a0 > 0 and for a0 = 0 . if h is simply connected then is globally defined on h. now consider eq . ( 18 ) , which in this case reduces to 52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_0 } = { 1 \over 2}{\nabla ^2}{e^{- \lambda } } - { e^{- \lambda}}(e - \lambda){.}$$\end{document } assume is a globally - defined function . integrating over h shows that if the strong energy condition ( 28 ) holds then a0 < 0 . the equality a0 = 0 occurs if and only if e = , in which case is harmonic and hence a constant . we begin by considering near - horizon geometries with a u(1 ) rotational symmetry , whose orbits are generically cohomogeneity-1 on cross sections of the horizon h. the orbit spaces h / u(1 ) have been classified and are homeomorphic to either the closed interval or a circle , see , e.g. , . the former corresponds to h of topology s , s , l(p , q ) times an appropriate dimensional torus , whereas the latter corresponds to h t. unless otherwise stated we will assume non - toroidal topology . it turns out to be convenient to work with a geometrically - defined set of coordinates as introduced in . let for i = 1 d 3 be the killing vector fields generating the isometry . define the 1-form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sigma = - { i_{{m_1 } } } \ldots { i_{{m_{d - 3}}}}{\epsilon _ \gamma}$\end{document } , where is the volume form associated with the metric on h. note that is closed and invariant under the killing fields mi and so defines a closed one - form on the orbit space . hence there exists a globally - defined invariant function x on h such that 53\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{d}}x = - { i_{{m_1 } } } \cdots { i_{{m_{d - 3}}}}{\epsilon _ \gamma}.$$\end{document } it follows that |dx| = det b , where bij (mi , mj ) , so dx vanishes precisely at the endpoints of the closed interval where the matrix bij has rank d 4 . as a function on h , x has precisely one minimum x1 and one maximum x2 , which must occur at the endpoints of the orbit space . hence h / u ( 1 ) d [ x1 , x2 ] . introducing coordinates adapted to the killing fields mi = / , we can use ( x , ) as a chart on h everywhere except the endpoints of the orbit space . the metric for x1 < x < x2 then reads 54\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { ab}}{\rm{d}}{x^a}{\rm{d}}{x^b } = { { { \rm{d}}{x^2 } } \over { \det \;b } } + { b_{ij}}(x){\rm{d}}{\phi ^i}{\rm{d}}{\phi ^j}.$$\end{document } by standard results , the one - form h may be decomposed globally on h as 55\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h = \beta + { \rm{d}}\lambda , $ $ \end{document } where is co - closed . since h is invariant under the m , it follows that and d are as well . further , periodicity of the orbits of the mi implies mi d = 0 , i.e. , = (x ) . it is convenient to define the globally - defined positive function 56\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma ( x ) \equiv { e^{- \lambda}}.$$\end{document } next , writing = x dx + i d and imposing that is co - closed , implies ( det b ) = c , where c is a constant . it follows i = c and since vanishes at the fixed points of the mi , this implies c must vanish . hence we may write 57\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h = { { { k_i}{\rm{d}}{\phi ^i } } \over \gamma } - { { \gamma ^{\prime}{\rm{d}}x } \over \gamma},$$\end{document } where we define ki(x ) hi . it is worth noting that in the toroidal case one can introduce coordinates ( x , ) so that the horizon metric takes the same form , with x now periodic and det b > 0 everywhere , although now the one - form h may have an extra term since the constant c need not vanish . we are now ready to state the simplest of the ads2 near - horizon symmetry enhancement theorems : theorem 3.4 ( ) . consider a d - dimensional spacetime containing a degenerate horizon , invariant under an u(1 ) isometry group , and satisfying the einstein equations r = g. then the near - horizon geometry has a global g u(1 ) symmetry , where g is either o(2 , 1 ) or the 2d poincar group . furthermore , if 0 and the near - horizon geometry is non - static the poincar case is excluded . proof : for the non - toroidal case we use the above coordinates . by examining the ( xv ) and ( xi ) components of the spacetime einstein equations and changing the affine parameter r (x)r , one can show 58\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = \gamma ( x)[{a_0}{r^2}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r ] + { { { \rm{d}}{x^2 } } \over { \det b } } + { b_{ij}}(x)({\rm{d}}{\phi ^i } + { k^i}r\;{\rm{d}}v)({\rm{d}}{\phi ^j } + { k^j}r\;{\rm{d}}v),$$\end{document } where a0 and k are constants . the metric in the square bracket is a maximally - symmetric space : ads2 for a0 < 0 , ds2 for a0 > 0 and for a0 = 0 . any isometry of these 2d base spaces transforms r dv r dv + d , for some function (v , r ) . k , the full near - horizon geometry inherits the full isometry group g of the 2d base , which for a0 = 0 is o(2 , 1 ) and for a0 = 0 is the 2d poincar group . the toroidal case can in fact be excluded , although as remarked above the coordinate system needs to be developed differently . in fact as we will see in section 4 one can completely solve for the near - horizon geometries of the above form in the = 0 case . the above result has a natural generalisation for d = 4 , 5 einstein - maxwell theories . for the sake of generality , consider a general 2-derivative theory describing einstein gravity coupled to abelian vectors a ( i = 1 n ) and uncharged scalars ( a = 1 m ) in d = 4 , 5 dimensions , with action 59\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s = \int { { { \rm{d}}^d } } x\sqrt { - g } \left({r - { 1 \over 2}{f_{ab}}(\phi){\partial _ \mu}{\phi ^a}{\partial ^\mu}{\phi ^b } - v(\phi ) - { 1 \over 4}{g_{ij}}(\phi)f_{\mu \nu}^i{f^{{j_{\mu \nu } } } } } \right ) + { s_{{\rm{top}}}},$$\end{document } where f da , v( ) is an arbitrary scalar potential ( which allows for a cosmological constant ) , and 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\rm{top } } } } = { 1 \over 2}\int { { h_{ij}}(\phi){f^i } \wedge { f^j}\quad { \rm{if}}\;d{\rm{= 4,}}}$$\end{document } or 61\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\rm{top } } } } = { 1 \over 6}\int { { c_{ijk}}{f^i } \wedge { f^j } \wedge { a^k}\quad { \rm{if}}\;d{\rm{= 5}},}$$\end{document } where cijk are constants . this encompasses many theories of interest , e.g. , vacuum gravity with a cosmological constant , einstein - maxwell theory , and various ( possibly gauged ) supergravity theories arising from compactification from ten or eleven dimensions . consider an extremal - black - hole solution of the above d = 4 , 5 theory with u(1 ) symmetry . the near - horizon limit of this solution has a global g u(1 ) symmetry , where g is either so(2 , 1 ) or ( the orientation - preserving subgroup of ) the 2d poincar group . the poincar - symmetric case is excluded if fab ( ) and gij( ) are positive definite , the scalar potential is non - positive , and the horizon topology is not t. proof : the maxwell fields f and the scalar fields are invariant under the killing fields mi , hence = (x ) . by examining the ( xv ) and ( xi ) components of einstein s equations for the above general theory , and changing the affine parameter r (x)r ( 58 ) and the maxwell fields are given by 62\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f^i } = { \rm{d}}[e^{i}r\;{\rm{d}}v + b_i^i(x)\;({\rm{d}}{\phi ^i } + { k^i}r\;{\rm{d}}v)],$$\end{document } where e are constants . invariant under g. generic orbits of the symmetry group have the structure of t fibred over a 2d maximally - symmetric space , i.e. , ads2 , ds2 or . ads2 and ds2 give so(2 , 1 ) symmetry , whereas gives poincar symmetry . the ds2 and cases are excluded subject to the additional assumptions mentioned , which ensure that the theory obeys the strong energy condition . remarks : in the original statement of this theorem asymptotically - flat or ads boundary conditions were assumed . these were only used at one point in the proof , where the property that the generator of each rotational symmetry must vanish somewhere in the asymptotic region ( on the axis of the symmetry ) was used to constrain the maxwell fields . in fact , using the general form for the near - horizon limit of a maxwell field ( 23 ) and the fact that for non - toroidal topology at least one of the rotational killing fields must vanish somewhere , allows one to remove any assumptions on the asymptotics of the black - hole spacetime.in the context of black holes , toroidal topology is excluded for = 0 when the dominant energy condition holds by the black - hole - topology theorems . an important corollary of the above theorems is : in the original statement of this theorem asymptotically - flat or ads boundary conditions were assumed . these were only used at one point in the proof , where the property that the generator of each rotational symmetry must vanish somewhere in the asymptotic region ( on the axis of the symmetry ) was used to constrain the maxwell fields . in fact , using the general form for the near - horizon limit of a maxwell field ( 23 ) and the fact that for non - toroidal topology at least one of the rotational killing fields must vanish somewhere , allows one to remove any assumptions on the asymptotics of the black - hole spacetime . in the context of black holes , toroidal topology is excluded for = 0 when the dominant energy condition holds by the black - hole - topology theorems . consider a d 5 spacetime with a degenerate horizon invariant under a u(1 ) symmetry as in theorem 3.4 and 3.5 . the near - horizon geometry is static if it is either a warped product of ads2 and h , or it is a warped product of locally ads3 and a ( d 3)-manifold . the static conditions ( 49 ) for eq . ( 58 ) occur if and only if : ( i ) k = 0 for all i = 1 , , d 3 , or ( ii ) ki = const and kki = f with a0 = < 0 . case ( i ) gives a warped product of a 2d maximally - symmetric space and h as in theorem ( 3.3 ) . for case ( ii ) one can introduce u(1 ) coordinates ( y , y ) where i = 2 , , d 3 , not necessarily periodic , so that k = /y and 63\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = \gamma ( x)\left [ { - { { { r^2 } } \over { { \ell ^2}}}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r + { { \left({{\rm{d}}{y^1 } + { r \over \ell}{\rm{d}}v } \right)}^2 } } \right ] + { { { \rm{d}}{x^2 } } \over { b(x ) } } + { b_{ij}}(x){\rm{d}}{y^i}{\rm{d}}{y^j}.$$\end{document } the metric in the square brackets is locally isometric to ads3 . if y is a periodic coordinate the horizon topology is s b , where b is some ( d 3)-dimensional manifold . theorem ( 3.5 ) can be extended to higher - derivative theories of gravity as follows . consider a general theory of gravity coupled to abelian vectors a and uncharged scalars with action 64\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s = { s_2 } + \sum\limits_{m \geq1 } { { \lambda ^m}\int { \sqrt { - g } { { \mathcal l}_m},}}$$\end{document } where s2 is the 2-derivative action above , is a coupling constant , and m is constructed by contracting ( derivatives of ) the riemann tensor , volume form , scalar fields and maxwell fields in such a way that the action is diffeomorphism and gauge - invariant . consider an extremal black - hole solution of the above higher - derivative theory , obeying the same assumptions as in theorem 3.5 . assume there is a regular horizon when = 0 with so(2 , 1 ) u(1 ) near - horizon symmetry , and the near - horizon solution is analytic in . then the near - horizon solution has so(2 , 1 ) u(1 ) symmetry to all orders in . hence theorem 3.5 is stable with respect to higher - derivative corrections . however , it does not apply to small black holes ( i.e. , if there is no regular black hole for = 0 ) . so far , the results described all assume d 3 commuting rotational killing fields . for d = 4 , 5 this is the same number as the rank of the rotation group so(d 1 ) , so the above results are applicable to asymptotically - flat or globally - ads black holes . for d > 5 the rank of this rotation group is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\lfloor { { { d - 1 } \over 2 } } \right\rfloor$\end{document } , which is smaller than d 3 , so the above theorems do not apply to asymptotically - flat or ads black holes . an important open question is whether the above theorems generalise when fewer than d 3 commuting rotational isometries are assumed , in particular the case with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\lfloor { { { d - 1 } \over 2 } } \right\rfloor$\end{document } commuting rotational symmetries . to this end , partial results have been obtained assuming a certain non - abelian cohomogeneity-1 rotational isometry . proposition 3.2 ( ) . consider a near - horizon geometry with a rotational isometry group u(1 ) k , whose generic orbit on h is a cohomogeneity-1 t - bundle over a k - invariant homogeneous space b. furthermore , assume b does not admit any k - invariant one - forms . if einstein s equations r = g hold , then the near - horizon geometry possesses a g u(1 ) k isometry group , where g is either 0(2 , 1 ) or the 2d poincar group . furthermore , if 0 and the near - horizon geometry is non - static then the poincar group is excluded . the assumptions in the above result reduce the einstein equations for the near - horizon geometry to odes , which can be solved in the same way as in theorem 3.4 . the special case k = su(q ) , b = with m = 1 and m = 2 , gives a near - horizon geometry of the type that occurs for a myers - perry black hole with all the angular momenta of set equal in 2q + 2 dimensions , or all but one set equal in 2q + 3 dimensions , respectively . theorem 3.6 ( ) . consider a spacetime containing a degenerate horizon invariant under orthogonally transitive17 isometry group u(1 ) , where 1 n d 3 , such that the surfaces orthogonal to the surfaces of transitivity are simply connected . then the near - horizon geometry has an isometry group g u(1 ) , where g is either so(2 , 1 ) or the 2d poincar group . furthermore , if the strong energy condition holds and the near - horizon geometry is non - static , the poincar case is excluded . the near - horizon geometry in this case can be written as 65\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = \gamma ( y)[{a_0}{r^2}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r ] + { \gamma _ { ij}}(y)({\rm{d}}{\phi ^i } + { k^i}r\;{\rm{d}}v)({\rm{d}}{\phi ^j } + { k^j}r\;{\rm{d}}v ) + { \gamma _ { mn}}(y){\rm{d}}{y^m}{\rm{d}}{y^n},$$\end{document } where as in the above cases we have rescaled the affine parameter r r . for the n = d 3 case , orthogonal transitivity follows from einstein s equations , which provides another proof of theorem 3.4 and 3.5 . for n < d 3 this result guarantees an ads2 symmetry for all known extremal - black - hole solutions , since all known explicit solutions possess orthogonally - transitive symmetry groups . in these higher cohomogeneity cases , the relation between einstein s equations and orthogonal transitivity is not understood . the einstein equations for a near - horizon geometry ( 7 ) in the absence of matter fields are equivalent to the following equation on h 66\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_{ab } } = { 1 \over 2}{h_a}{h_b } - { \nabla _ { ( a}}{h_{b ) } } + \lambda { \gamma _ { ab}},$$\end{document } with the function f determined by 67\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f = { 1 \over 2}{h^a}{h_a } - { 1 \over 2}{\nabla _ a}{h^a } + \lambda , $ $ \end{document } see eqs . it is useful to note that the contracted bianchi identity for the horizon metric is equivalent to 68\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ a}f - f{h_a } - 2{h^b}{\nabla _ { \left [ a \right.}}{h_{\left . b \right ] } } + { \nabla ^b}{\nabla _ { \left [ a \right.}}{h_{\left . b \right ] } } = 0.$$\end{document } a complete classification is possible for 0 . recall from section 3.2.1 , the staticity conditions for a near - horizon geometry are dh = 0 and df = hf . theorem 4.1 ( ) . the only vacuum static near - horizon geometry for 0 and compact h is given by ha 0 , f = and rab = ab . for d proof : a simple proof of the first statement is as follows . substituting the staticity conditions ( 50 ) into ( 67 ) gives 69\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla ^2}{\psi ^2 } + 2\lambda { \psi ^2 } = 2{a_0},$$\end{document } where e. irrespective of the topology of h one can argue that is a globally - defined function ( for simply connected h this is automatic , otherwise it can be shown by working in patches on h and exploiting the fact that in each patch is only defined up to an additive constant ) . for = 0 it is then clear that compactness implies must be a constant . for < 0 , if one assumes is non - constant one can easily derive a contradiction by evaluating the above equation at the maximum and minimum of ( which must exist by compactness ) . hence in either case ha 0 , which gives the claimed near - horizon data . in four dimensions ( 66 ) in general without assuming compactness of h. for non - constant and any one obtains the near - horizon geometry ( sending r r ) 70\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = { \psi ^2}[{a_0}{r^2}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r ] + { { { \rm{d}}{\psi ^2 } } \over { p(\psi ) } } + p(\psi){\rm{d}}{\chi ^2},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p(\psi ) = { a_0 } + \beta { \psi ^ { - 1 } } - { 1 \over 3}\lambda { \psi ^2}$\end{document}. ( this is an analytically - continued schwarzschild with a cosmological constant . ) this local form of the metric can be used to show that for > 0 and non - constant , there are also no smooth horizon metrics on a compact h. the classification of near - horizon geometries in d = 3 vacuum gravity with a cosmological constant can be completely solved . although very simple , to the best of our knowledge this has not been presented before , so for completeness we include it here . the main simplification comes from the fact that cross sections of the horizon h are one - dimensional , so the horizon equations are automatically odes . furthermore , there is no intrinsic geometry on h and so the only choice concerns its global topology , which must be either h s or h . theorem 4.2 . consider a near - horizon geometry with compact cross section h s , which satisfies the vacuum einstein equations including cosmological constant . if < 0 the near - horizon geometry is given by the quotient of ads3 in eq . = 0 the only solution is the trivial flat geometry s. there are no solutions for > 0 . proof : we may choose a periodic coordinate x on h so the horizon metric is simply = dx and the 1-form h = h(x)dx . observe that h(x ) must be a globally - defined function and hence must be a periodic function of x. since the curvature and metric connection trivially vanish , the horizon equations ( 66 ) and ( 67 ) simplify to 71\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h^{\prime } = { 1 \over 2}{h^2 } + \lambda , $ $ \end{document } 72\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f = { 1 \over 2}{h^2 } - { 1 \over 2}h^{\prime } + \lambda .$$\end{document } this system of odes can be explicitly integrated as we explain below . instead , we will avoid this and employ a global argument on h. if 0 , integrate eq . ( 71 ) over h to deduce that h 0 and = 0 , which gives the trivial flat near - horizon geometry s. for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\lambda = - { 2 \over \ell } < 0$\end{document } we argue as follows . ( 71 ) by h and integrate over h to find \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\int\nolimits_{{s^1 } } { { h^{\prime 2 } } } = 0$\end{document}. hence h must be a constant and substituting into the horizon equations gives \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h = { 2 \over \ell}$\end{document } and f = 0 ( without loss of generality we have chosen a sign for h ) . the near - horizon geometry is then 73\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = 2\;{\rm{d}}v\;{\rm{d}}r + { { 4r } \over \ell}{\rm{d}}v\;{\rm{d}}x + { \rm{d}}{x^2 } = - { { 4{r^2 } } \over { { \ell ^2}}}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r + { \left({{\rm{d}}x + { { 2r } \over \ell}{\rm{d}}v } \right)^2}.$$\end{document } this metric is locally ads3 and in the second equality we have written it as a fibration over ads2 . it is worth remarking that the ode ( 71 ) can be completely integrated without assuming compactness . for < 0 this reveals a second solution \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h = - { 2 \over \ell}\tanh \left({{x \over \ell } } \right)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f = - { 2 \over { { \ell ^2}}}{\rm{sec}}{{\rm{h}}^2}\left({{x \over \ell } } \right)$\end{document } , where we have set the integration constant to zero by translating the coordinate x. upon changing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r \rightarrow r\ { \cosh ^2}\left({{x \over \ell } } \right)$\end{document } the resulting near - horizon geometry is : 74\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = { \cosh ^2}\left({{x \over \ell } } \right)\left({- { { { r^2 } } \over { { \ell ^2}}}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r } \right ) + { \rm{d}}{x^2}.$$\end{document } again , this metric is locally ads3 . unlike the previous case though , h can not be taken to be compact and hence we must have h . for = 0 there is also a second solution given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h = - { 2 \over x}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f = - { 1 \over { { x^2}}}$\end{document } , although this is singular . for > 0 there is a unique solution given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h = { 2 \over \ell}\tan \left({{x \over \ell } } \right)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f = { 1 \over { { \ell ^2}}}{\sec ^2}\left({{x \over \ell } } \right)$\end{document } , although this is also singular.18 the general solution to eq . ( 66 ) is not known in this case . in view of the rigidity theorem if one assumes such a symmetry , the problem becomes of ode type and it is possible to completely solve it . the result is summarised by the following theorem , first proved in [ 122 , 163 ] for = 0 and in for < 0 . consider a spacetime containing a degenerate horizon , invariant under an u(1 ) isometry , satisfying the vacuum einstein equations including a cosmological constant . any non - static near - horizon geometry , with compact cross section , is given by the near - horizon limit of the extremal kerr or kerr-(a)ds black hole . as described in section 3.2.2 , axisymmetry implies one can introduce coordinates on h so that 75\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { ab}}{\rm{d}}{x^a}{\rm{d}}{x^b } = { { { \rm{d}}{x^2 } } \over { b(x ) } } + b(x){\rm{d}}{\phi ^2},\quad \quad h = { bk(x ) \over \gamma}{\rm{d}}\phi - { \gamma ^{\prime } \over \gamma}{\rm{d}}x.$$\end{document } the x component of eq . ( ( 68 ) then implies 76\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f = { { { a_0 } } \over \gamma } + { { b{k^2 } } \over { { \gamma ^2}}},$$\end{document } where a0 is a constant . substituting this into eq . ( 67 ) gives 77\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_0 } = { 1 \over 2}{\nabla ^2}\gamma - { { b{k^2 } } \over { 2\gamma } } + \lambda \gamma .$$\end{document } now subtracting the xx component from the component of eq . ( 66 ) gives 78\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2\gamma^{\prime\prime } - { { { \gamma ^{\prime 2 } } } \over \gamma } - { { { k^2 } } \over \gamma } = 0.$$\end{document } a non - static near - horizon geometry must have k 0 and therefore from the above equation is non - constant . ( 77 ) as 79\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left({{{b\gamma } \over { { \gamma ^\prime } } } } \right)^\prime } = { { 2({a_0 } - \lambda \gamma)\gamma } \over { { \gamma ^{\prime2}}}}.$$\end{document } the solution to the ode for is given by 80\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma = { { { k^2 } } \over \beta } + { { \beta { x^2 } } \over 4},$$\end{document } where is a positive constant , which can then be used to solve the ode for b : 81\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b = { { p(x ) } \over \gamma},$$\end{document } where 82\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(x ) = - { { \beta \lambda { x^4 } } \over { 12 } } + ( { a_0 } - 2\lambda { k^2}{\beta ^{- 1}}){x^2 } + { c_1}x - { { 4{k^2 } } \over { { \beta ^2}}}({a_0 } - \lambda { k^2}{\beta ^{- 1}})$$\end{document } and c1 is a constant . changing affine parameter r (x)r in the full near - horizon geometry finally gives 83\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = \gamma ( x)[{a_0}{r^2}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r ] + { { \gamma ( x ) } \over { p(x)}}{\rm{d}}{x^2 } + { { p(x ) } \over { \gamma ( x)}}{({\rm{d}}\phi { \rm{+}}kr\;{\rm{d}}v)^2},$$\end{document } with , p determined above . observe that this derivation is purely local and does not assume anything about the topology of h ( unlike the derivation in , which assumed compactness ) . if k = 0 we recover the general static solution ( 70 ) , hence let us now assume k 0 . now assume h is compact , so by axisymmetry one must have either s or t. integrating eq . ( 77 ) over h then shows that if 0 then a0 < 0 and so the metric in square brackets is ads2 . the horizon metric extends to a smooth metric on h s if and only if c1 = 0 . it can then be checked the near - horizon geometry is isometric to that of extremal kerr for = 0 or kerr - ads for < 0 . it is also easy to check that for > 0 it corresponds to extremal kerr - ds . in the non - static case , the horizon topology theorem excludes the possibility of h t for 0 . if < 0 the non - static possibility with h t can also be excluded . it would be interesting to remove the assumption of axisymmetry in the above theorem . in it ( 66 ) on h s must be axisymmetric and hence given by the above theorem . we may define a homogeneous near - horizon geometry to be one for which the riemannian manifold ( h , ab ) is a homogeneous space whose transitive isometry group k also leaves the rest of the near - horizon data ( f , ha ) invariant . since any near - horizon geometry ( 7 ) possesses the 2d symmetry generated by v v + c and ( v , r ) ( v , r ) where 0 , it is clear that this definition guarantees the near - horizon geometry itself is a homogeneous spacetime . conversely , if the near - horizon geometry is a homogeneous spacetime , then any cross section ( h , ab ) must be a homogeneous space under a subgroup k of the spacetime isometry group , which commutes with the 2d symmetry in the ( v , r ) plane ( since h is a constant ( v , r ) submanifold ) . it follows that ( f , ha ) must also be invariant under the isometry k , showing that our original definition is indeed equivalent to the near - horizon geometry being a homogeneous spacetime . any vacuum , homogeneous , non - static near - horizon geometry is locally isometric to 84\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = ( - { 1 \over 2}{k^2 } + \lambda){r^2}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r + { ( \hat \omega + kr\;{\rm{d}}v)^2 } + \hat g,$$\end{document } where is a u(1)-connection \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{ric}}(\hat g ) = \hat \lambda \hat g$\end{document } with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \lambda = { 1 \over 2}{k^2 } + 2\lambda$\end{document}. the curvature of the connection is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{d}}\hat \omega = \sqrt { { k^2 } + 2\lambda } \hat \epsilon$\end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat \epsilon}$\end{document } is the volume form of the 2d base , and k + 2 0 . the proof uses the fact that homogeneity implies h must be a killing field and then one reduces the problem onto the 2d orbit space . for k 0 and 0 we see that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \lambda > 0$\end{document } so that the 2d metric is a round s and the horizon metric is locally isometric to a homogeneously squashed s. hence we have : corollary 4.1 . any vacuum , homogeneous , non - static near - horizon geometry is locally isometric to the near - horizon limit of the extremal myers - perry black hole with su(2 ) u(1 ) rotational symmetry ( i.e. , equal angular momenta ) . for > 0 one gets the same result with the myers - perry black hole replaced by its generalisation with a cosmological constant . for < 0 we see that there are more possibilities depending on the sign of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat \lambda}$\end{document}. if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \lambda > 0$\end{document } we again have a horizon geometry locally isometric to a homogeneous s. if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \lambda > 0$\end{document } , we can write = dx + dy and the u(1)-connection \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \omega = \sqrt { 2\left\vert \lambda \right.\left\vert { ( x{\rm{d}}y - y{\rm{d}}x ) } \right.}$\end{document } is non - trivial , so the cross sections h are the nil group manifold with its standard homogeneous metric . for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \lambda > 0$\end{document } , we can write \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat g = { \rm{d}}{x^2 } + { \rm{d}}{y^2}$\end{document } and the connection \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\vert { \hat \lambda } \right.\left\vert { \hat \omega } \right . = \sqrt { { k^2 } + 2\lambda } \,{\rm{d}}x / y$\end{document } , so the cross sections h are the sl(2 , ) group manifold with its standard homogeneous metric , unless k = 2 , which gives h = . hence we have : corollary 4.2 . for < 0 any vacuum , homogeneous , non - static near - horizon geometry is locally isometric to either the near - horizon limit of the extremal rotating black hole with su(2 ) u(1 ) rotational symmetry , or a near - horizon geometry with : ( i ) h = nil and its standard homogenous metric , ( ii ) h = sl(2 , ) and its standard homogeneous metric or ( iii ) h = . this is analogous to a classification first obtained for supersymmetric near - horizon geometries in gauged supergravity , see proposition ( 5.3 ) . a u(1)-rotational isometry is natural in five dimensions and all known explicit black - hole solutions have this symmetry . consider a vacuum non - static near - horizon geometry with a u(1)-rotational isometry and a compact cross section h. it must be globally isometric to the near - horizon geometry of one of the following families of black - hole solutions : h s s : the 3-parameter boosted extremal kerr string.h s : the 2-parameter extremal myers - perry black hole or the 3-parameter l(p , q ) : the lens space quotients of the above h s solutions . h s : the 2-parameter extremal myers - perry black hole or the 3-parameter fast rotating extremal kk black hole . l(p , q ) : the lens space quotients of the above h s solutions . remarks : the near - horizon geometry of the vacuum extremal black ring is a 2-parameter subfamily of case 1 , corresponding to a kerr string with vanishing tension .the near - horizon geometry of the slowly rotating extremal kk black hole is identical to that of the 2-parameter extremal myers - perry in case 2.the h s cases can be written as a single 3-parameter family of near - horizon geometries .the h t case has been ruled out . for 0 , the analogous problem has not been solved . the only known solution in this case is the h s near - horizon geometry of the rotating black hole with a cosmological constant , which generalises the myers - perry black hole . it would be interesting to classify near - horizon geometries with h s s in this case since this would capture the near - horizon geometry of the yet - to - be - found asymptotically - ads5 black ring . the near - horizon geometry of the vacuum extremal black ring is a 2-parameter subfamily of case 1 , corresponding to a kerr string with vanishing tension . the near - horizon geometry of the slowly rotating extremal kk black hole is identical to that of the 2-parameter extremal myers - perry in case 2 . the h s cases can be written as a single 3-parameter family of near - horizon geometries . d 6 , so the horizon cross section dim h 4 , the horizon equation ( 66 ) is far less constraining than in lower dimensions . few general classification results are known , although several large families of vacuum near - horizon geometries have been constructed . the only known classification result for vacuum d > 5 near - horizon geometries is for = 0 solutions with u(1)-rotational symmetry . these generalise the d = 4 axisymmetric solutions and d = 5 solutions with u(1)-symmetry discussed in sections 4.3 and 4.4 respectively . by performing a detailed study of the orbit spaces h / u(1 ) it has been shown that the only possible topologies for h are : s t , s t , l(p , q ) t , and t . an explicit classification of the possible near - horizon geometries ( for the non - toroidal case ) was derived in , see their theorem 1 . using their theorem it is easy to show that the most general solution with h s t is in fact isometric to the near - horizon geometry of a boosted extremal kerr - membrane ( i.e. , perform a general boost of kerr along the { t } coordinates and then compactify t ) . non - static near - horizon geometries with h t have been ruled out ( including a cosmological constant ) . the myers - perry ( mp ) black - hole solutions generically have isometry groups u(1 ) where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$s = \left\lfloor { { { d - 1 } \over 2 } } \right\rfloor$\end{document}. observe that if d > 5 then s < d 3 and hence these solutions fall outside the classification discussed in section 4.5.1 . they are parameterised by their mass parameter and angular momentum parameters ai for i = 1 , s. the topology of the horizon cross section h s. a generalisation of these metrics with non - zero cosmological constant has been found . we will focus on the = 0 case , although analogous results hold for the 0 solutions . the location of the horizon is determined by the largest positive number r+ such that in odd and even dimensions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\pi ( { r _ + } ) - \mu r _ r+ = 0 , respectively , where 85\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi ( r ) = \prod\limits_{i = 1}^s { ( { r^2 } + a_i^2).}$$\end{document } the extremal limit of these black holes in odd and even dimensions is given by (r+ ) = 2 r+ and (r+ ) = , respectively . these conditions hold only when the black hole is spinning in all the two planes available , i.e. , we need ai 0 for all i = 1 , , s. without loss of generality we will henceforth assume ai > 0 and use the extremality condition to eliminate the mass parameter . the near - horizon geometry of the extremal mp black holes can be written in a unified form : 86\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { g_{{\rm{mp } } } } = { f _ + } \left({- { { \pi^{\prime\prime}({r _ + } ) } \over { 2\pi ( { r _ + } ) } } { r^2}\;{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;dr } \right ) + { \gamma _ { { \mu _ i}{\mu _ j}}}\;{\rm{d}}{\mu ^i}{\rm{d}}{\mu ^j}}\qquad\qquad\quad\\ { + { \gamma _ { ij}}\left({{\rm{d}}{\phi ^i } + { { 2r + { a_i } } \over { { { ( r _ + ^2 + a_i^2)}^2}}}r\;{\rm{d}}v } \right)\left({{\rm{d}}{\phi ^j } + { { 2r + { a_j } } \over { { { ( r _ + ^2 + a_j^2)}^2}}}r\;{\rm{d}}v } \right),}\\ \end{array}$$\end{document } where 87\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f _ + } + 1 - \sum\limits_{i = 1}^s { { { a_i^2\mu _ i^2 } \over { r _ + ^2 + a_i^2}},\quad \quad { \gamma _ { ij } } = ( r _ + ^2 + a_i^2 ) } \mu _ i^2{\delta _ { ij } } + { 1 \over { { f _ + } } } { a_i}\mu _ i^2{a_j}\mu _ j^2,$$\end{document } and in odd and even dimensions 88\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { { \mu _ i}{\mu _ j}}}{\rm{d}}{\mu ^i}{\rm{d}}{\mu ^j } = \sum\limits_{i = 1}^s { ( r _ + ^2 + a_i^2)d\mu _ i^2,\quad \quad { \gamma _ { { \mu _ i}{\mu _ j}}}{\rm{d}}{\mu ^i}{\rm{d}}{\mu ^j } = r _ + ^2{\rm{d}}{\alpha ^2 } + } \sum\limits_{i = 1}^s { ( r _ + ^2 + a_i^2)\;{\rm{d}}\mu _ i^2 } , $ $ \end{document } respectively . the direction cosines i and take values in the range 0 i 1 with 1 1 and in odd and even dimensions satisfy 89\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sum\limits_{i = 1}^s { \mu _ i^2 = 1 , } \quad \sum\limits_{i = 1}^s { \mu _ i^2 + { \alpha ^2 } = 1,}$$\end{document } respectively . the generalisation of these near - horizon geometries for 0 was given in . it is worth noting that if subsets of the angular momentum parameters ai are set equal , the rotational symmetry enhances to a non - abelian unitary group . since these are vacuum solutions one can trivially add flat directions to generate new solutions . for example , by adding one flat direction one can generate a boosted mp string , whose near - horizon geometries have h s s topology . interestingly , for odd dimensions d the resulting geometry has \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\lfloor { { { d - 1 } \over 2 } } \right\rfloor$\end{document } commuting rotational isometries . for this reason , it was conjectured that a special case of this is also the near - horizon geometry of yet - to - be - found asymptotically - flat black rings ( as is known to be the case in five dimensions ) . despite the absence of explicit d > 5 black - hole solutions , a number of solutions to eq . it is an open problem as to whether there are corresponding black - hole solutions to these near - horizon geometries . let k be a compact fano khlereinstein manifold19 of complex dimension q 1 and a h(k , ) is the indivisible class given by c1(k ) = ia with i ( the fano index i and satisfies i q with equality iff k = ) . the khler - einstein metric on k is normalised as ric( ) = 2q and we denote its isometry group by g. the simplest example occurs for q = 2 , in which case k = s with = ( d + sin d ) . in even dimensions greater than four , an infinite class of near - horizon geometries is revealed by the following result . let m and pm be the principal s - bundle over any fano khler - einstein manifold k , specified by the characteristic class ma . for each m ( 66 ) on the associated s - bundles \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h \cong { p_m}{\times _ { { s^1}}}{s^2}$\end{document}. the dim h = 2q ansatz used for the near - horizon data is the u(1 ) g invariant form 90\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { ab}}{\rm{d}}{x^a}{\rm{d}}{x^b } = { { { \rm{d}}{x^2 } } \over { b(x ) } } + b(x)\omega \otimes \omega + a{(x)^2}\bar g,$$\end{document } 91\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h = { { kb(x)\omega } \over \gamma } - { { \gamma ^{\prime}(x ) } \over \gamma}{\rm{d}}x,$$\end{document } where is a u ( 1)-connection over k with curvature 2-ma . the solutions depend on one continuous parameter l > 0 and the integer m > i. the continuous parameter corresponds to the angular momentum j[ ] where generates the u(1)-isometry in the s - fibre . the various functions are given by a(x ) = l(1 x ) , (x ) = + x , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$k = \pm 2\sqrt \xi$\end{document } and b(x ) = p(x)/(a(x)(x ) ) where p is a polynomial in x and smoothness fixes to be a function of m. the simplest example is the q = 2 , = 0 solution , for which the near - horizon data takes the explicit form 92\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \gamma _ { ab}}\;{\rm{d}}{x^a}\;{\rm{d}}{x^b } = { { { l^2}({\xi _ m } + { x^2})(1 - { x^2})d{x^2 } } \over { ( 4 - { m^2}{x^2})({\xi _ m } - { { 4{x^2 } } \over { 3{m^2 } } } ) } } + { { { l^2}(4 - { m^2}{x^2})\left({{\xi _ m } - { { 4{x^2 } } \over { 3{m^2 } } } } \right ) } \over { ( { \xi _ m } + { x^2})(1 - { x^2})}}{{({\rm{d}}\phi + { 1 \over 2}\cos \theta { \rm{d}}\chi)}^2}}\\ { + { 1 \over 4}{l^2}(1 - { x^2})({\rm{d}}{\theta ^2 } + { { \sin}^2}\theta { \rm{d}}{\chi ^2}),}\qquad\qquad\qquad\qquad\qquad\\ \end{array}$$\end{document } 93\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h_{a\;}}{\rm{d}}{x^a } = \pm { { 2\sqrt { { \xi _ m } } { l^2}(4 - { m^2}{x^2})\left({{\xi _ m } - { { 4{x^2 } } \over { 3{m^2 } } } } \right ) } \over { { { ( { \xi _ m } + { x^2})}^2}(1 - { x^2})}}({\rm{d}}\phi + { 1 \over 2}\cos \theta { \rm{d}}\chi { \rm { ) -}}{{2x } \over { { \xi _ m } + { x^2}}}{\rm{d}}x,$$\end{document } where 94\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi _ m } = { 4 \over 3}\left({{{3 - { 4 \over { { m^2 } } } } \over { 4 + { m^2 } } } } \right),$$\end{document } and m > 2 . the coordinate ranges are 2/m x 2/m , 0 , + 2/m , + 2. cross sections of the horizon h , are homeomorphic to s s if m is even , or the non - trivial bundle \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${s^2}\tilde \times { s^2 } \cong \mathbb c{\mathbb p^2}\ # { \overline { \mathbb c \mathbb p } ^2}$\end{document } if m is odd . for > 2 the fano base k is higher dimensional and there are more choices available . the topology of the total space is always a non - trivial s - bundle over k and in fact different m give different topologies , so there are an infinite number of horizon topologies allowed . furthermore , one can choose k to have no continuous isometries giving examples of near - horizon geometries with a single u(1)-rotational isometry . hence , if there are black holes corresponding to these horizon geometries they would saturate the lower bound in the rigidity theorem . it is worth noting that the local form of the above class of near - horizon metrics includes as a special case that of the extremal mp metrics h s with equal angular momenta ( for m = i ) . the above class of horizon geometries are of the same form as the einstein metrics on complex line bundles , which in four dimensions corresponds to the page metric , although we may of course set 0 . similar constructions of increasing complexity can be made in odd dimensions , again revealing an infinite class of near - horizon geometries . let m and be the principal s - bundle over k specified by the characteristic class ma . ( 66 ) on h pm . as a simple example consider k = . this leads to an explicit homogeneous near - horizon geometry with 95\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \gamma _ { ab}}\;{\rm{d}}{x^a}\;{\rm{d}}{x^b } = \left({{{{k^2 } + 2\lambda } \over { 2{\lambda ^2 } } } } \right){{({\rm{d}}\psi + \cos { \theta _ 1}\;{\rm{d}}{\phi _ 1 } + \cos { \theta _ 2}\;{\rm{d}}{\phi _ 2})}^2}}\qquad\qquad\qquad\qquad\qquad\qquad\qquad\\ { + { 1 \over \lambda}({\rm{d}}\theta _ 1 ^ 2 + { { \sin}^2}{\theta _ 1}\;{\rm{d}}\phi _ 1 ^ 2 + { \rm{d}}\theta _ 2 ^ 2 + { { \sin}^2}{\theta _ 2}{\rm{d}}\phi _ 2 ^ 2)}\\ { { h^a}{\partial _ a } = k\sqrt { { { 2{\lambda ^2 } } \over { { k^2 } + 2\lambda } } } { \partial \over { \partial \psi}},}\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\\ \end{array}$$\end{document } where for convenience we have written h is a vector field , is a constant and = ( k + 6)/4 . regularity requires that the chern number m of the u(1)-fibration over each s to be the same and the period = 2/m . the total space is a lens space s/m - bundle over s and is topologically h s s. for k = 0 and > 0 this corresponds to a sasaki - einstein metric on s s sometimes known as t. the above proposition proposition 4.3 ( ) . given any fano khler - einstein manifold k of complex dimension q 1 and coprime p1,p2 satisfying 1 < ip1/p2 < 2 , there exists a 1-parameter family of solutions to eq . these examples have u(1 ) g symmetry , although possess only one independent angular momentum along the t - fibres . there also exist a more general class of non - sasakian horizons in odd dimensions . let pm1,m2 be the principal t - bundle over any fano khler - einstein manifold k , specified by the characteristic classes ( m1a , m2a , ) where m1,m2 . for a countably infinite set of non - zero integers ( m1 , m2 , j , k ) , there exists a two - parameter family of smooth solutions to eq . the dim h = 2q + 1 form of the near - horizon data in the previous two propositions is the u(1 ) g invariant form 96\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \gamma _ { ab}}\;{\rm{d}}{x^a}\;{\rm{d}}{x^b } = { { { \rm{d}}{x^2 } } \over { \det b } } + { b_{ij}}(x){\omega ^i}{\omega ^j } + a{{(x)}^2}\bar g,}\\ { { h_a}{\rm{d}}{x^a } = { { { b_{ij}}{k^j}{\omega ^i } } \over \gamma } - { { \gamma ^{\prime}(x ) } \over \gamma}{\rm{d}}x,}\qquad\qquad\\ \end{array}$$\end{document } where is a principal t - connection over k whose curvature is 2mia . the explicit functions a(x ) , (x ) are linear in x and bij(x ) are ratios of various polynomials in x. generically these solutions possess two independent angular momenta along the t - fibres . the sasakian horizon geometries of proposition 4.3 arise as a special case with m1 + m2 = ij and possess only one independent angular momentum . the base k = gives horizon topologies h s s or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h \cong { s^3}\tilde \times { s^2}$\end{document } depending on whether m1 + m2 is even or odd respectively . it is worth noting that the local form of this class of near - horizon metrics includes as a special case that of the extremal mp metrics h s with all but one equal angular momenta . the above class of horizon geometries are of the same form as the einstein metrics found in [ 37 , 157 ] . by definition , a supersymmetric solution of a supergravity theory is a solution that also admits a killing spinor , i.e. , a spinor field that satisfies d = 0 , where d is a spinorial covariant derivative that depends on the matter fields of the theory . given such a killing spinor , the bilinear \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${k^\mu } = \bar \psi { \gamma ^\mu}\psi$\end{document } is a non - spacelike killing field . by definition , a supersymmetric horizon is invariant under the killing field k and thus k must be tangent to the horizon . hence k must be null on the horizon ; in other words the horizon is a killing horizon of k. furthermore , since k 0 both outside and inside the horizon , it follows that on the horizon dk = 0 , i.e. , it must be a degenerate horizon . the simplest supergravity theory in four dimensions admitting supersymmetric black holes is minimal \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 2$\end{document } supergravity , whose bosonic sector is simply standard einstein - maxwell theory . the general supersymmetric solution in this theory is given by the israel - wilson - perjs metrics . using this fact , the following near - horizon uniqueness theorem has been proved : theorem 5.1 ( ) . any supersymmetric near - horizon geometry in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 2$\end{document } minimal supergravity is one of the maximally supersymmetric solutions t or ads2 we will discuss the implications of this result for uniqueness of supersymmetric black holes in four dimensions . for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 2$\end{document } gauged supergravity , whose bosonic sector is einstein - maxwell theory with a negative cosmological constant , an analogous classification of supersymmetric near - horizon geometries has not been performed . nevertheless , one may deduce the following result , from a classification of all near - horizon geometries of this theory under the additional assumption of axisymmetry : proposition 5.1 ( ) . any supersymmetric , axisymmetric , near - horizon geometry in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 2$\end{document } gauged supergravity , is given by the near - horizon limit of the 1-parameter family of supersymmetric kerr - newman - ads4 black holes . this is related to the fact that supersymmetric ads black holes must carry angular momentum . supersymmetric black holes are not expected to exist in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 1$\end{document } supergravity . for the general \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 1$\end{document } supergravity the following result , supporting this expectation , has been established . a supersymmetric near - horizon geometry of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 1$\end{document } supergravity is either the trivial solution t , or s where s may be a non - round sphere . an example of a supersymmetric near - horizon geometry of the form s , with a round s , was given in . the simplest d = 5 supergravity theory admitting supersymmetric black holes is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } the bosonic sector is einstein - maxwell theory with a chern - simons term given by eq . this was used to obtain a complete classification of supersymmetric near - horizon geometries in this theory . any supersymmetric near - horizon geometry of minimal supergravity is locally isometric to one of the following maximally supersymmetric solutions : ads3 s , t , or the near - horizon geometry of the breckenridge - myers - peet - vafa ( bmpv ) black hole ( of which ads2 s is a special case ) . the ads3 s near - horizon geometry has cross sections h s s and arises as the near - horizon limit of supersymmetric black rings [ 64 , 65 ] and supersymmetric black strings [ 93 , 19 ] . analogous results have been obtained in d = 5 minimal supergravity coupled to an arbitrary number of vector multiplets . as discussed in section 7 , the above theorem can be used to prove a uniqueness theorem for topologically spherical supersymmetric black holes . the corresponding problem for minimal gauged supergravity the bosonic sector of this theory is einstein - maxwell - chern - simons theory with a negative cosmological constant . the theory admits asymptotically ads5 black - hole solutions that are relevant in the context of the ads / cft correspondence [ 120 , 38 ] . proposition 5.3 ( ) . consider a supersymmetric , homogeneous near - horizon geometry of minimal gauged supergravity . cross sections of the horizon must be one of the following : a homogeneously squashed s , nil or sl(2 , ) manifold . the near - horizon geometry of the s case was used to construct the first example of an asymptotically ads5 supersymmetric black hole . analogous results in gauged supergravity coupled to an arbitrary number of vector multiplets ( this includes u(1 ) gauged supergravity ) were obtained in . unlike the ungauged theory , homogeneity is not implied by supersymmetry , and indeed there are more general solutions . the most general supersymmetric near - horizon geometry in minimal gauged supergravity , admitting a u(1)-rotational symmetry and a compact horizon section , is the near - horizon limit of the topologically - spherical supersymmetric black holes of . the motivation for assuming this isometry group is that all known black - hole solutions in five dimensions possess this . interestingly , this result implies the non - existence of supersymmetric ads5 black rings with u(1 ) isometry . in fact , recent results allow one to remove all assumptions and obtain a complete classification . any -supersymmetric near - horizon geometry in minimal gauged supergravity must be invariant under a local u(1)-rotational isometry . any supersymmetric near - horizon geometry in minimal gauged supergravity with a compact horizon section must preserve -supersymmetry . this latter result is proved using a lichnerowicz type identity to establish a correspondence between killing spinors and solutions to a horizon dirac equation , and then applying an index theorem . therefore , combining the previous three propositions gives a complete classification theorem for near - horizon geometries in minimal gauged supergravity . a supersymmetric near - horizon geometry in minimal gauged supergravity , with a compact horizon section , must be locally isometric to the near - horizon limit of the topologically - spherical supersymmetric black holes , or the homogeneous near - horizon geometries with the nil or sl(2 , ) horizons . we emphasise that the absence of supersymmetric ads5 black rings is rather suprising , given asymptotically - flat counterparts are known to exist . parts of the above analysis have been generalised by u(1 ) gauged supergravity , although the results are slightly different . proposition 5.7 ( ) . consider a supersymmetric near - horizon geometry in u(1 ) minimal gauged supergravity with u(1)-rotational symmetry and a compact horizon section . it must be either : ( i ) the near - horizon limit of the topologically spherical black holes of ; or ( ii ) ads3 s with h s s or ( iii ) ads3 t with h t. these latter two cases have constant scalars and only exist in certain regions of the scalar moduli space ( not including the minimal theory ) . therefore , in this theory one can not rule out the existence of supersymmetric ads5 black rings ( although as argued in they would not be connected to the asymptotically - flat black rings ) . it would be interesting to complete the classification of near - horizon geometries in this more general theory , along the lines of the minimal theory . the bosonic field context of this theory is a metric and a 2-form potential with self - dual field strength . this was used to work out a complete classification of supersymmetric near - horizon geometries . any supersymmetric near - horizon geometry of d = 6 minimal supergravity , with a compact horizon cross section , is either t , k3 or locally ads3 s. the ads3 s solution has h s s and arises as the near - horizon limit of a supersymmetric rotating black string . analogous results have been obtained for minimal supergravity coupled to an arbitrary number of scalar and tensor multiplets . the bosonic field content of d = 10 heterotic supergravity consist of the metric , a 2-form gauge potential and a scalar field ( dilaton ) . one is the direct product h , with vanishing flux and constant dilaton , where h is spin(7 ) holonomy manifold , which generically preserves one supersymmetry ( there are solutions in this class which preserve more supersymmetry provided h has certain special holonomy ) . in the second class , the near - horizon geometry is a fibration of ads3 over a base b7 ( with a u(1)-connection ) with a g2 structure , which must preserve 2 supersymmetries . this class may preserve 4 , 6 , 8 supersymmetries if b7 is further restricted . any supersymmetric near - horizon geometry of heterotic supergravity invariant under 8 supersymmetries , with a compact horizon cross section , must be locally isometric to one of ads3 s t , ads3 s k3 or t k3 ( with constant dilaton ) . a large number of heterotic horizons preserving 4 supersymmetries have been constructed , including explicit examples where h is su(3 ) and s s t . the bosonic field content of type iib supergravity consists of a metric , a complex scalar , a complex 2-form potential and a self - dual 5-form field strength . a variety of results concerning the classification of supersymmetric near - horizon geometries in this theory have been derived [ 102 , 101 , 103 ] . certain explicit classification results are known for near - horizon geometries with just a 5-form flux preserving more than two supersymmetries , albeit under certain restrictive assumptions . more generally , the existence of one supersymmetry places rather weak geometric constraints on the horizon cross sections h : generically h may be any almost hermitian spinc manifold . there are also special cases for which h has a spin(7 ) structure and those for which it has an su(4 ) structure ( where the killing spinor is pure ) . more recently , it has been shown that any supersymmetric near - horizon geometry in type iib supergravity must preserve an even number of supersymmetries , and furthermore , if a certain horizon dirac operator has non - trivial kernel the bosonic symmetry group must contain so(22 , 1 ) . the bosonic field content of d = 11 supergravity consists of a metric and a 3-form potential c. the near - horizon limit of the 4-form field strength \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f } = { \rm{d}}c$\end{document } can be written as eq . ( 27 ) , where y and x are a 2-form and closed 4-form respectively on the 9-dimensional horizon cross sections h. the near - horizon einstein equations are eqs . ( 17 ) and ( 18 ) with = 0 and the matter field terms are given by 97\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_{ab } } = - { 1 \over 2}{y_{ac}}{y_b}^{\;c } + { 1 \over { 12}}{x_{a{c_1}{c_2}{c_3}}}x_b^{{c_1}{c_2}{c_3 } } + { \gamma _ { ab}}\left({{1 \over { 12}}{y^2 } - { 1 \over { 144}}{x^2 } } \right),$$\end{document } 98\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = { 1 \over 6}{y^2 } + { 1 \over { 144}}{x^2}.$$\end{document } we note that the dominant and strong energy conditions are satisfied : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${p_{ac}}{\gamma ^{ab } } = { 1 \over 4}{y^2 } + { 1 \over { 48}}{x^2 } \geq 0$\end{document } and e 0 . therefore , the general results established under these assumptions , discussed in section 3 , are all valid , including most notably the horizon topology theorem . various classification results have been derived for supersymmetric near - horizon geometry solutions under the assumption that cross sections of the horizon are compact . static supersymmetric near - horizon geometries are warped products of either or ads2 with m9 , where m9 admits a particular g - structure . we note that these warped product forms are guaranteed by the general analysis of static near - horizon geometries in section 3.2.1 . theorem 5.5 ( ) . any supersymmetric near - horizon geometry solution to eleven dimensional supergravity , with compact horizon the proof of this follows by first establishing a lichnerowicz type identity for certain horizon dirac operators and then application of an index theorem . as far as bosonic symmetry is concerned , the above result is a direct analogue of the various near - horizon symmetry theorems discussed in section 3.2 , which are instead established under various assumptions of rotational symmetry . in this section we will consider general near - horizon geometries coupled to non - trivial gauge fields . we will mostly focus on theories that are in the bosonic sector of minimal supergravity theories ( since these are the best understood cases ) . extremal , non - supersymmetric , near - horizon geometries may be thought of as interpolating between vacuum and supersymmetric solutions . they consist of a much larger class of solutions , which , at least in higher dimensions , are much more difficult to classify . in particular , we consider d = 3 , 4 , 5 einstein - maxwell theory , possibly coupled to a chern - simons term in odd dimensions , and d = 4 einstein - yang - mills theory . the classification of near - horizon geometries in d = 3 einstein - maxwell theory with a cosmological constant can be completely solved . to the best of our knowledge this has not been presented before , so for completeness we include it here . it should be noted though that partial results which capture the main result were previously shown in . since cross sections h of the horizon are one - dimensional , the maxwell 2-form induced on h must vanish identically . hence , the most general near - horizon maxwell field ( 23 ) in three dimensions is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${f_{{\rm{nh } } } } = { \rm{d}}(r\delta ( x){\rm{d}}\upsilon)$\end{document}. it is straightforward to show that the 3d maxwell equation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{d } } \star { \mathcal f } = 0$\end{document } , where is the hodge dual with respect to the spacetime metric , is equivalent to the following equation on h : 99\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ^{\prime } = h\delta .$$\end{document } the near - horizon einstein equations ( 17 ) and ( 18 ) are simply 100\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h^{\prime } = { 1 \over 2}{h^2 } + 2{\delta ^2 } + \lambda , $ $ \end{document } 101\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f = { 1 \over 2}{h^2 } - { 1 \over 2}h^{\prime } + \lambda .$$\end{document } theorem 6.1 . consider a near - horizon geometry with a compact horizon cross section h s in einstein - maxwell- theory . if < 0 the near - horizon geometry must be either ads2 s with a constant ads2 maxwell field , or the quotient of ads3 eq . if = 0 the only solution is the trivial flat near - horizon geometry s. if > 0 there are no solutions . proof : rather that solving the above odes we may use a global argument . compactness requires x to be a periodic coordinate on h s and since h , are globally defined they must be periodic functions of x. for 0 simply integrate eq . ( 100 ) over h to find that the only solution is the trivial flat one h 0 , 0 and = 0 . for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\lambda \equiv - { 2 \over { { \ell ^2 } } } < 0$\end{document } we may argue as follows . multiply eq . ( 100 ) by h and integrate over h to obtain 102\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 = \int\nolimits_{{s^1 } } { ( { h^{\prime 2 } } - 2{\delta ^2}{h^\prime}){\rm{d}}x } = \int\nolimits_{{s^1 } } { ( { h^{\prime2 } } + 4{\delta ^2}{h^2}){\rm{d}}x,}$$\end{document } where in the second equality we have integrated by parts and used eq . we deduce the only possible solutions are h = 0 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta = \pm { 1 \over \ell}$\end{document } , or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h = \pm { 2 \over \ell}$\end{document } , = 0 . the former gives a near - horizon geometry ads2 s and the latter is the vacuum solution locally isometric to ads3 . this result implies that the near - horizon limit of any charged rotating black - hole solution to 3d einstein - maxwell- theory either has vanishing charge or angular momentum . the ads2 s solution is the near - horizon limit of the non - rotating extremal charged btz black hole , whereas the ads3 solution is the near - horizon limit of the vacuum rotating extremal btz . charged rotating black holes were first obtained within a wide class of stationary and axisymmetric solutions to einstein - maxwell- theory , and later by applying a solution generating technique to the charged non - rotating black hole [ 46 , 174 ] . we have checked that in the extremal limit their near - horizon geometry is the ads2 s solution , so the angular momentum is lost in the near - horizon limit , in agreement with the above analysis . it would be interesting to investigate whether charged rotating black holes exist that instead possess a locally ads3 near - horizon geometry . in this case , charge would not be captured by the near - horizon geometry , a phenomenon that is known to occur for five - dimensional supersymmetric black rings whose near - horizon geometry is locally ads3 s. in 2 + 1 dimensions an abelian gauge field monopole is not isolated . electric charge can be screened by adding a mass term to the gauge field . a natural way to do this is to add a chern - simons term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mu \int { { \mathcal a } \wedge { \mathcal f}}$\end{document } to the spacetime action , resulting in a topologically - massive gauge theory . this only modifies the maxwell equation : 103\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{d}}\star { \mathcal f } + \mu { \mathcal f } = 0,$$\end{document } where is the mass parameter of the gauge field . for the near - horizon maxwell field it can be shown that this is equivalent to 104\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta^{\prime } = ( h + \mu)\delta .$$\end{document } as in the pure einstein - maxwell case , a complete classification of near - horizon geometries to this theory is possible . to the best of our knowledge this has not been presented before . consider a near - horizon geometry with a cross section h s in einstein - maxwell- theory with a chern - simons term and mass . if < 0 the functions and h are constant and the near - horizon geometry is the homogeneous s - bundle over ads2 ( 106 ) . if = 0 the only solution is the trivial flat near - horizon geometry s. if > 0 there are no solutions . for 0 the proof of this is identical to the einstein - maxwell case above . for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\lambda = - { 2 \over { { \ell ^2}}}$\end{document } one can also use the same argument as the einstein - maxwell case . using the horizon equation ( 100 ) and maxwell equation ( 104 ) one can show 105\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 = \int\nolimits_{{s^1 } } { ( { h^{\prime 2 } } - 2{\delta ^2}{h^\prime}){\rm{d}}x } = \int\nolimits_{{s^1 } } { ( { h^{\prime2 } } + 4{\delta ^2}{{(h + \mu)}^2}){\rm{d}}x,}$$\end{document } which implies h must be a constant and (h + ) 0 . if 0 then h = and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f = { 1 \over 2}{\mu ^2 } + \lambda$\end{document}. the rest of the near - horizon data is given by f = + and hence the near - horizon geometry is 106\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { g = - 2{\delta ^2}{r^2}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r - 2\mu r\;{\rm{d}}v\;{\rm{d}}x + { \rm{d}}{x^2}}\qquad\quad\\ { = - \left({1 \over 2}{\mu ^2 } + { 2 \over { { \ell ^2}}}\right){r^2}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r + { { ( { \rm{d}}x - \mu r\;{\rm{d}}v)}^2}}\\ \end{array}$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{a}}d{s_3 } \times { s^2}$\end{document}. note that if = 0 we recover the ads2 s solution , whereas if 0 we recover the vacuum ads3 solution . this implies that the near - horizon geometry of any charged rotating black - hole solution to this theory is either the vacuum ads3 solution , or the non - trivial solution ( 106 ) , which is sometimes referred to as warped ads3 . ( 15 ) , ( 22 ) with n = 2 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{d } } \star { \mathcal f } = 0$\end{document } , where is the hodge dual with respect to the spacetime metric , and the bianchi identity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{d}}{\mathcal f } = 0$\end{document}. the near - horizon maxwell field is given by ( 23 ) . ( 17 ) and ( 18 ) , where 107\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_{ab } } = 2{b_{ac}}{b_{bd}}{\gamma ^{cd } } + { \delta ^2}{\gamma _ { ab } } - { { { \gamma _ { ab } } } \over 2}{b^2},$$\end{document } 108\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = { \delta ^2 } + { { { b^2 } } \over 2},$$\end{document } 109\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{d}}{\star _ 2}\;b = { \star _ 2}{i_h}b + { \star _ 2}({\rm{d}}\delta - \delta h),$$\end{document } where 2 is the hodge dual with respect to the horizon metric ab . observe that 2b is a function on h. static near - horizon geometries have been completely classified . for = 0 this was first derived in , and generalised to 0 in . theorem 6.3 ( [ 43 , 154 ] ) . consider a static near - horizon geometry in d = 4 einstein - maxwell- theory , with compact horizon cross section h. for 0 it must be ads2 s. for < 0 it must be ads2 h where h is one of the constant curvature surfaces s , t , g . it is worth remarking that if one removes the assumption of compactness one can still completely classify near - horizon geometries . the extra solution one obtains can be written as a warped product 110\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = { \psi ^2}({a_0}{r^2}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r ) + { { { \rm{d}}{\psi ^2 } } \over { p(\psi ) } } + p(\psi){\rm{d}}{\phi ^2},$$\end{document } 111\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal f } = e\;{\rm{d}}r \wedge { \rm{d}}v + b{\psi ^{- 2}}{\rm{d}}\psi \wedge { \rm{d}}\phi , $ $ \end{document } where p( ) = a0 c(2 ) ( e + b) /3 , which is an analyticaly continued reissner - nordstrm- solution . non - static near - horizon geometries are not fully classified , except under the additional assumption of axisymmetry . theorem 6.4 ( [ 163 , 154 ] ) . any axisymmetric , non - static near - horizon geometry in d = 4 einstein - maxwell- theory , with a compact horizon cross section , must be given by the near - horizon geometry of an extremal kerr - newman- black hole . solved the = 0 in the context of isolated degenerate horizons , where the same equations on h arise . note that the horizon topology theorem excludes the possibility of toroidal horizon cross sections for 0 . also excluded the possibility of a toroidal horizon cross section if < 0 under the assumptions of the above theorem . it is worth noting that the results presented in this section , as well as the techniques used to establish them , are entirely analogous to the vacuum case presented in section 4.3 . the field equations of d = 5 einstein - maxwell theory coupled to a chern - simons term are given by eqs . ( 15 ) and ( 22 ) with n = 3 and 112\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{d}}\star{\mathcal f } + { { 2\xi } \over { \sqrt 3}}{\mathcal f } \wedge { \mathcal f } = 0$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f}$\end{document } is the maxwell two form and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{d}}{\mathcal f } = 0$\end{document}. the cases of most interest are = 0 and = 1 , which correspond to pure einstein - maxwell theory and the bosonic sector of minimal supergravity respectively . ( 17 ) and ( 18 ) and 113\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_{ab } } = 2{b_{ac}}{b_{bd}}{\gamma ^{cd } } + \left({{{2{\delta ^2 } } \over 3 } - { 1 \over 3}{b^2 } } \right){\gamma _ { ab}},$$\end{document } 114\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = { { 4{\delta ^2 } } \over 3 } + { { { b^2 } } \over 3},$$\end{document } 115\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{d}}{\star _ { \;3}}\;b = - { \star _ 3}{i_h}b - { \star _ 3}({\rm{d}}\delta - \delta h ) + { { 4\xi } \over { \sqrt 3}}\delta b,$$\end{document } where 3 is the hodge dual with respect to the 3d horizon metric ab . in this section we summarise what is known about solutions to these equations , which is mostly restricted to the = 0 case . hence , unless otherwise stated , we assume = 0 in this section . a number of new complications arise that render the classification problem more difficult , most obviously the lack of electro - magnetic duality . therefore , purely electric solutions , which correspond to 0 and b 0 , are qualitatively different to purely magnetic solutions , which correspond to 0 and b 0 . perhaps somewhat surprisingly a complete classification of static near - horizon geometries in this theory has not yet been achieved . all the results summarised in this section were proved in . as in other five dimensional near - horizon geometry classifications , the assumption of u(1 ) rotational symmetry proves to be useful . static near - horizon geometries in this class in general are either warped products of ads2 and h , or ads3 and a 2d manifold , see the corollary 3.1 . the ads3 near - horizon geometries are necessarily purely magnetic and can be classified for any . proposition 6.1 . any static ads3 near - horizon geometry with a u(1)-rotational symmetry , in einstein - maxwell - chern - simons theory , with a compact horizon cross section , is the direct product of a quotient of ads3 and a round s. this classifies a subset of purely magnetic geometries . by combining the results of together with proposition 6.4 of , it can be deduced that for = 1 there are no purely magnetic ads2 geometries ; therefore , with these symmetries , one has a complete classification of purely magnetic geometries . corollary 6.1 . any static , purely magnetic , near - horizon geometry in minimal supergravity , possessing u(1)-rotational symmetry and compact cross sections h , must be locally isometric to ads3 s with h = s s. we now turn to purely electric geometries . proposition 6.2 . consider a static , purely electric , near - horizon geometry in einstein - maxwell - chern - simons theory , with a u(1)-rotational symmetry and compact cross section . it must be given by either ads2 s , or a warped product of ads2 and an inhomogeneous s. the latter non - trivial solution is in fact the near - horizon limit of an extremal rn black hole immersed in a background electric field ( this can be generated via a harrison type transformation ) . finally , we turn to the case where the near - horizon geometry possess both electric and magnetic fields . in fact one can prove a general result in this case , i.e. , without the assumption of rotational symmetries . any static near - horizon geometry with compact cross sections h , in einstein - maxwell - chern - simons theory with coupling = 0 , with non - trivial electric and magnetic fields , is a direct product of ads2 h , where the metric on h is not einstein . < 1/4 were also found , which all have h = s with u(1)-rotational symmetry . however , we should emphasise that no examples are known for minimal supergravity ( = 1 ) . hence there is the possibility that in this case static near - horizon geometries with non - trivial electric and magnetic fields do not exist , although this has not yet been shown . if this is the case , then the above results fully classify static near - horizon geometries with u(1)-rotational symmetry . for = 0 the analysis of electro - magnetic geometries is analogous to the purely magnetic case above ; in fact there exists a dyonic ads2 geometry that is a direct product ads2 s s and it is conjectured there are no others . the classification of homogeneous near - horizon geometries can be completely solved , even including a cosmological constant . this does not appear to have been presented explicitly before , so for completeness we include it here with a brief derivation . the riemannian manifold ( h , ab ) is a homogeneous space , i.e. , admits a transitive isometry group k , such that the rest of the near - horizon data ( f , ha , , bab ) are invariant under k. as discussed in section ( 4.4 ) , this is equivalent to the near - horizon geometry being a homogeneous spacetime with a maxwell field invariant under its isometry group . an immediate consequence of homogeneity is that an invariant function must be a constant and any invariant 1-form must be a killing field . hence the 1-forms h and j 3b are killing and the functions f , , h , j must be constants . thus , the horizon einstein equations ( 17 ) , ( 18 ) , ( 113 ) , ( 114 ) and maxwell equation ( 115 ) simplify . firstly , note that if h and j vanish identically then h is einstein rab = ( + 2)ab , so h is a constant curvature space s , , ( the latter two can only occur if < 0 ) . h. now consider the case where at least one of h and j is non - vanishing . by contracting the maxwell equation ( 115 ) with jj ne can show that ( j h ) = jh and hence by the cauchy - schwarz inequality j and h must be parallel as long as they are both non - zero . thus , if one of h , j is non - vanishing , we can write ha = kua and ja = qua for some constants k , q where ua is a unit normalised killing vector field . the near - horizon equations now reduce to 116\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_{ab } } = { 1 \over 2}({k^2 } - 4{q^2}){u_a}{u_b } + \left({4 \over 3}{q^2 } + { 1 \over 2}{\delta ^2 } + \lambda\right){\gamma _ { ab}},$$\end{document } 117\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f = { 1 \over 2}{k^2 } - { 2 \over 3}{q^2 } - { \delta ^2 } + \lambda , $ $ \end{document } 118\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$qdu = \left({{{\sqrt 3 } \over 2}k + 2\xi q } \right)\delta\star u.$$\end{document } this allows one to prove : theorem 6.5 . any homogeneous near - horizon geometry in einstein - maxwell - cs- theory for which one of the constants k , q is non - zero , must be locally isometric to 119\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = \left(- { 1 \over 2}{k^2 } - { 2 \over 3}{q^2 } - { \delta ^2 } + \lambda\right){r^2}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r + { ( \hat \omega + kr\;{\rm{d}}v)^2 } + \hat g,$$\end{document } 120\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal f } = \delta { \rm{d}}r\ ; \wedge { \rm{d}}v + q\hat \epsilon , $ $ \end{document } where is a u(1)-connection over a 2d base with metric satisfying \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{ric}}(\hat g ) = \hat \lambda \hat g$\end{document } , with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \lambda = { 1 \over 2}{k^2 } + { 2 \over 3}{q^2 } + { \delta ^2 } + 2\lambda$\end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat \epsilon}$\end{document } is the volume form of the 2d base . the curvature of the connection is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{d}}\hat \omega = { [ { k^2 } - { 3 \over 4}{q^2 } + { \delta ^2}2\lambda ] ^{1/2}}\hat \epsilon$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${k^2 } - { 3 \over 4}{q^2 } + { \delta ^2}2\lambda \geq 0$\end{document}. if q 0 the constants must satisfy 121\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${k^2 } - { 4 \over 3}{q^2 } + { \delta ^2 } + 2\lambda = { 1 \over 4}{q^{- 2}}{\delta ^2}{\left({\sqrt 3 k + 4\xi q } \right)^2},$$\end{document } whereas if q = 0 one must have k = 0 . the proof of this proceeds as in the vacuum case , by reducing the horizon equations to the 2d orbit space defined by the killing field u. the above result contains a number of special cases of interest , which we now elaborate upon . firstly , note that q = = 0 reduces to the vacuum case , see theorem 4.4 . before discussing the general case consider k = 0 , so the near - horizon geometry is static , which connects to section 6.3.1 . the constraint on the parameters is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(1 - 4{\xi ^2}){\delta ^2 } = { 3 \over 4}{q^2 } + 2\lambda$\end{document } and hence , if 0 one must have 0 < . for = 0 the connection is trivial and hence the near - horizon geometry is locally isometric to the dyonic ads2 s s solution . for 0 < < we get examples of the geometries in proposition ( 6.3 ) , where h s with its standard homogeneous metric . now we consider = 0 in generality so at least one of k , q , is non - zero , in which case \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \lambda = 0$\end{document } and so is the round s. the horizon is then either h s with its homogeneous metric or h s s , depending on whether the connection is non - trivial or not , respectively . any homogeneous near - horizon geometry of minimal supergravity is locally isometric to ads3 s , or the near - horizon limit of either ( i ) the bmpv black hole ( including ads2 s ) , or ( ii ) an extremal nonsupersymmetric charged black hole with su(2 ) u(1 ) rotational symmetry . the proof of this follows immediately from theorem 6.5 with = 0 and = 1 . in this case the constraint on the parameters factorises to give two branches of possible solutions ( a ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$k = - 2q/\sqrt 3$\end{document } or ( b ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\delta ^2}(k + 2\sqrt { 3q } ) = 4{q^2}(k - 2q/\sqrt 3)/3$\end{document}. case ( a ) gives two solutions . if 0 it must have h s and corresponds to the bmpv solution ( i ) ( for q 0 this reduces to ads2 s ) , whereas if = 0 it is the ads3 s solution with h s s. case ( b ) also gives two solutions . if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$k = 2q/\sqrt 3$\end{document } then = 0 , which gives ads3 s with h s s , otherwise we get solution ( ii ) with h s. note that solution ( ii ) reduces to the vacuum case as ,q 0 . generically , the extremal limit depends on 3-parameters with two independent angular momenta j1 , j2 and u(1 ) symmetry . setting |j1| = |j2| gives two distinct branches of 2-parameter extremal black - hole solutions with enhanced su(2 ) u(1 ) rotational symmetry corresponding to the bmpv solution ( i ) ( which reduces to the rn solution if j = 0 ) or solution ( ii ) ( which reduces to the vacuum extremal mp black hole in the neutral limit ) . it is interesting to note the analogous result for pure einstein - maxwell theory : corollary 6.3 . any homogeneous near - horizon geometry of einstein - maxwell theory is locally isometric to either ( i ) 122\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { g = - \left({1 \over 2}{k^2 } + 2{q^2}\right){r^2}{\rm{d}}v^{2}+2{\rm{d}}v\;{\rm{d}}r + { { \left({d\psi + { { k\cos \theta \;{\rm{d}}\phi } \over { { 1 \over 2}{k^2 } + 2{q^2 } } } + kr\;{\rm{d}}v } \right)}^2 } + { { { \rm{d}}{\theta ^2 } + { { \sin}^2}\theta { \rm{d}}{\phi ^2 } } \over { { 1 \over 2}{k^2 } + 2{q^2}}},}\\ { { \mathcal f } = \pm { 2 \over { \sqrt 3}}q\;{\rm{d}}r \wedge { \rm{d}}v + { { q\sin \theta \;{\rm{d}}\theta \wedge { \rm{d}}\phi } \over { { 1 \over 2}{k^2 } + 2{q^2}}},}\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\\ \end{array}$$\end{document } or ( ii ) 123\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { g = - \left({\delta ^2 } + { 4 \over 3}{q^2}\right){r^2}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r + { { \left({d\psi + { { \delta \cos \theta \;{\rm{d}}\phi } \over { { \delta ^2 } + { 4 \over 3}{q^2 } } } \pm { 2 \over { \sqrt 3}}qr\;{\rm{d}}v } \right)}^2 } + { { { \rm{d}}{\theta ^2 } + { { \sin}^2}\theta { \rm{d}}{\phi ^2 } } \over { { \delta ^2 } + { 4 \over 3}{q^2}}},}\\ { { \mathcal f } = \delta \;{\rm{d}}r \wedge { \rm{d}}v + { { q\sin \theta \;{\rm{d}}\theta \wedge { \rm{d}}\phi } \over { { \delta ^2 } + { 4 \over 3}{q^2}}}.}\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\;\;\\ \end{array}$$\end{document } this also follows from application of theorem 6.5 with = 0 and = 0 . solution ( i ) for q 0 reduces to the vacuum solution of theorem 4.4 , whereas for k = 0 , it gives the static dyonic ads2 s s solution . solution ( ii ) for = 0 gives ads3 s , whereas for 0 it gives a near - horizon geometry with h s , which for q 0 is ads2 s. a charged rotating black - hole solution to einstein - maxwell theory generalising mp is not known explicitly . hence this corollary could be of use for constructing such an extremal charged rotating black hole with su(2 ) u(1 ) symmetry . for = 4/ < 0 there are even more possibilities , since \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat \lambda}$\end{document } may be positive , zero , or negative . one then gets near - horizon geometries that generically have h s , nil , sl(2 , ) , respectively , equipped with their standard homogeneous metrics . each of these may have a degenerate limit with h s s , t , s , as occurs in the vacuum and supersymmetric cases . the full space of solutions interpolates between the vacuum case given in corollary 4.2 , and the supersymmetric near - horizon geometries of gauged supergravity of proposition 5.3 . for example , the supersymmetric horizons correspond to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$k = - 2\sqrt 3 q$\end{document } and k = 9/ with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \lambda = { \delta ^2 } - 3{\ell ^{- 2}}$\end{document}. we will not investigate the full space of solutions in detail here . the classification of near - horizon geometries in d = 5 einstein - maxwell - cs theory , under the assumption of u(1 ) symmetry , turns out to be significantly more complicated than the vacuum case . this is unsurprising ; solutions may carry two independent angular momenta , electric charge and dipole / magnetic charge ( depending on the spacetime asymptotics ) . as a result , there are several ways for a black hole to achieve extremality . furthermore , such horizons may be deformed by background electric fields . in the special case of minimal supergravity any near - horizon geometry of minimal supergravity with u(1)-rotational symmetry takes the form of eqs . ( 58 ) and ( 62 ) , where the functional form of (x ) , bij(x ) , bi(x ) can be fully determined in terms of rational functions of x. in particular , (x ) is a quadratic function . although this solves the problem in principle , it turns out that in practice it is very complicated to disentangle the constraints on the constants that specify the solution . hence an explicit classification of all possible solutions has not yet been obtained , although in principle it is contained in the above result . we now summarise all known examples of five dimensional non - static near - horizon geometries with non - trivial gauge fields , which arise as near - horizon limits of known black hole or black string solutions . all these examples possess u(1 ) rotational symmetry and hence fall into the class of solutions covered by theorem 3.5 , so the near - horizon metric and field strength \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(g,{\mathcal f})$\end{document } take the form of eqs . charged myers - perry black holes : this asymptotically - flat solution is known explicitly only for minimal supergravity = 1 ( in particular it is not known in pure einstein - maxwell = 0 ) , since it can be constructed by a solution - generating procedure starting with the vacuum mp solution . it depends on four parameters m , j1,j2 , and q corresponding to the adm mass , two independent angular momenta and an electric charge . the extremal limit generically depends on three parameters and gives a near - horizon geometry with h s. charged kaluza - klein black holes : the most general known solution to date was found in ( see references therein for a list of previously known solutions ) and carries a mass , two independent angular momenta , a kk monopole charge , an electric charge and a magnetic charge20 . the extremal limit will generically depend on five - parameters , however , as for the vacuum case the extremal locus must have more than one connected component . these solutions give a large family of near - horizon geometries with h s. supersymmetric black rings and strings : the asymptotically - flat supersymmetric black ring and the supersymmetric taub - nut black ring both have a near - horizon geometry that is locally ads3 s. there are also supersymmetric black string solutions with such near - horizon geometries [ 93 , 19 ] . dipole black rings : the singly - spinning dipole black ring is a solution to einstein - maxwell - cs for all . it is a 3-parameter family with a single angular momentum and dipole charge possessing a 2-parameter extremal limit . the resulting near - horizon geometry with h s s is parameterised by 4-parameters ( q , , r1 , r2 ) with one constraint relating them . asymptotic flatness of the full black - hole solution imposes one further constraint , although from the viewpoint of the near - horizon geometry , this is strictly an external condition and we will deal with the general case here . the solution is explicitly given by 124\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { g = \gamma ( x)\left [ { - { { { r^2}{\rm{d}}{v^2 } } \over { { \ell ^2 } } } + 2\;{\rm{d}}v\;{\rm{d}}r } \right ] + { { { \ell ^2}\gamma ( x){\rm{d}}{x^2 } } \over { ( 1 - { x^2})}}}\qquad\qquad\qquad\qquad\qquad\qquad\qquad\\ { + { { r_1 ^ 2\lambda ( 1 + \lambda)h(x ) } \over { q(1 - \lambda)f(x)}}{{\left({{\rm{d}}{\phi ^1 } + { { ( 1 - \lambda ) } \over { \lambda { r_1}{r_2}}}\sqrt { { { 1 - \lambda } \over { 1 + \lambda } } } r\;{\rm{d}}v } \right)}^2 } + { { r_2 ^ 2{q^2}\omega _ 0 ^ 2(1 - { x^2 } ) } \over { h{{(x)}^2}}}{{({\rm{d}}{\phi ^2})}^2},}\\ { { \mathcal f } = { { \sqrt 3 } \over 2}{\rm{d}}\left [ { \sqrt { { { 1 - q } \over { 1 + q } } } { { { \omega _ 0}q{r_2}(1 + x ) } \over { h(x)}}{\rm{d}}{\phi ^2 } } \right],}\qquad\qquad\qquad\qquad\qquad\qquad\qquad\quad\\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma ( x ) = \sqrt { { { q(1 - \lambda ) } \over { \lambda ( 1 + \lambda ) } } } f(x)h(x)$\end{document } with f(x ) = 1 + x , h(x ) = 1 qx and we have also defined the length scale \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\ell ^2 } = r_2 ^ 2\sqrt { { { \lambda ( 1 + \lambda){q^3 } } \over { 1 - \lambda}}}$\end{document}. the parameters satisfy 0 < , q < 1 . the local metric induced on spatial cross sections h extends smoothly to a metric on s s provided \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ 0 } = \sqrt { f(1)h{{(1)}^3 } } = \sqrt { f(- 1)h{{(- 1)}^3}}$\end{document}. charged non - supersymmetric black rings : the dipole ring solution admits a three - parameter charged generalisation with one independent angular momentum and electric and dipole charges ( the removal of dirac - misner string singularities imposes an additional constraint , so this solution has the same number of parameters as that of ) . the charged black ring has a two - parameter extremal limit with a corresponding two - parameter near - horizon geometry . as in the above case , at the level of near - horizon geometries there is an additional independent parameter corresponding to the arbitrary size of the radius of the s. electro - magnetic kerr black strings : black string solutions have been constructed carrying five independent charges : mass m , linear momentum p along the s of the string , angular momentum j along the internal s , as well as electric qe and magnetic charge qm . these solutions admit a four parameter extremal limit , which in turn give rise to a five - parameter family of non - static near horizon geometries ( the additional parameter is the radius of the s at spatial infinity ) . for simplicity we will restrict our attention to the solutions with p = qe = 0 . the resulting near - horizon solution is parameterised by ( a , c , s ) with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$c_\beta ^2 - s_\beta ^2 = 1$\end{document } and corresponds to an extremal string with non - zero magnetic charge and internal angular momentum : 125\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { g = \gamma ( x)\left [ { - { { { r^2}{\rm{d}}{v^2 } } \over { { \ell ^2 } } } + 2\;{\rm{d}}v\;{\rm{d}}r } \right ] + { { { \ell ^2}\gamma ( x){\rm{d}}{x^2 } } \over { ( 1 - { x^2 } ) } } + { { 4{a^4}{{(c_\beta ^4 + s_\beta ^2)}^2}(1 - { x^2 } ) } \over { { \ell ^2}\gamma ( x)}}{\omega ^2}}\qquad\qquad\qquad\\ { + { a^2}{{\left [ { { { r{\rm{d}}{\phi ^1 } } \over a } - { { 8{a^2}c_\beta ^3s_\beta ^3 } \over { { \ell ^4}}}r\;{\rm{d}}v + { { 2{a^2}{c_\beta}{s_\beta}(c_\beta ^2 + s_\beta ^2)(1 - { x^2 } ) } \over { { \ell ^2}\gamma ( x)}}\omega } \right]}^2},}\qquad\qquad\qquad\qquad\\ { { \mathcal f } = { { 2\sqrt 3 { a^3}{s_\beta}{c_\beta}(c_\beta ^4 + s_\beta ^4 ) } \over { { \ell ^2}}}{\rm{d}}\left [ { { x \over { \gamma ( x)}}\omega } \right],}\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\qquad\\ \end{array}$$\end{document } where we have defined the one - form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega = { \rm{d}}{\phi ^2 } - { { ( c_\beta ^2 + s_\beta ^2 ) } \over { { \ell ^2}(c_\beta ^4 + s_\beta ^4)}}r{\rm{d}}\upsilon$\end{document } , the function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma ( x ) = { { { a^2 } } \over { { \ell ^2}}}(1 + { x^2 } + 4c_\beta ^2s_\beta ^2)$\end{document } and the length scale \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\ell ^2 } = 2{a^2}(c_\beta ^4 + s_\beta ^4)$\end{document}. the induced metric on cross sections of the horizon h extends smoothly to a cohomogeneity-1 metric on s s. although the two solutions ( 124 ) and ( 125 ) share many features , it is important to emphasise that only the former is known to correspond to the near - horizon geometry of an asymptotically - flat black ring . it is conjectured that there exists a general black - ring solution to minimal supergravity that carries mass , two angular momenta , electric and dipole charges all independently . discusses the possibility that the tensionless kerr - string solution is the near - horizon geometry of these yet - to - be - constructed black rings . one may represent such solutions by a three - dimensional metric h and a set of scalar fields ( potentials ) all defined on a three - dimensional manifold , see , e.g. , . equivalently , such solutions can be derived from the field equations of a three - dimensional theory of gravity coupled to a scalar harmonic map whose target manifold is parameterised by the with metric gmn ( ) determined by the specific theory . in certain theories of special interest , the scalar manifold is a symmetric space g / k equipped with the bi - invariant metric 126\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{mn}}(\phi){\rm{d}}{\phi ^m}{\rm{d}}{\phi ^n } = { 1 \over { 4m}}{\rm{tr}}({\phi ^{- 1}}{\rm{d}}\phi ) ^{\rm{2}},$$\end{document } where is a coset representative of g / k and m is a normalisation constant dependent on the theory . then the theory is equivalent to a three - dimensional theory of gravity coupled to a nonlinear sigma model with target space g / k . it can be shown [ 162 , 135 , 155 ] that the classification problem reduces to an ode on the orbit space h / u(1 ) for the , while h is completely determined . we will assume non - toroidal horizon topology so the orbit space is an interval , which without loss of generality we take to be [ 1 , 1 ] parameterised by the coordinate x. the ode is the equation of motion for a non - linear sigma model defined on this interval : 127\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{d } } \over { { \rm{d}}x}}\left [ { ( 1 - { x^2}){\phi ^{- 1}}{{{\rm{d}}\phi } \over { { \rm{d}}x } } } \right ] = 0,$$\end{document } where the a coset representative depends only on x. it is straightforward to integrate this matrix equation and completely solve for the scalar fields , which can then be used to reconstruct the full d - dimensional solution . however , in practice , reconstructing the near - horizon data is hindered by the non - linearity of the scalar metric . the most notable example which can be treated in the above formalism is vacuum d - dimensional gravity for which g / k = sl(d 2 , )/so(d 2 ) . the classification problem for near - horizon geometries has been completely solved using this approach , as discussed earlier in section 4.5.1 . four - dimensional einstein - maxwell also possesses such a structure where the coset is now g / k = su(2 , 1)/su(2 ) , although the near - horizon classification discussed earlier in section 6.2 does not exploit this fact . it turns out that d = 5 minimal supergravity also has a non - linear sigma model structure with g / k = g2,2/so(4 ) ( g2,2 refers to the split real form of the exceptional lie group g2 ) . the classification of near - horizon geometries in this case was analysed in using the hidden symmetry and some partial results were obtained , see proposition 6.4 . . it would be interesting to use it to classify near - horizon geometries in other theories possessing such hidden symmetry . we note that near - horizon geometries in this class extremise the energy functional of the harmonic map : h / u(1 ) g / k , with boundary conditions chosen such that the corresponding near - horizon data is smooth . explicitly , this functional is given by 128\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e[\phi ] = \int\nolimits_{- 1}^1 { \left [ { ( 1 - { x^2}){1 \over { 4m}}{\rm{tr}}({\phi ^{- 1}}{\partial _ x}\phi)^{2 } - { 2 \over { 1 - { x^2 } } } } \right]{\rm{d}}x}$$\end{document } and from it can be deduced this vanishes on such extrema . for vacuum gravity , for which m = 1 , it was proved that e[ ] 0 with equality if and only if corresponds to a near - horizon geometry , i.e. , near - horizon geometries are global minimisers of this functional [ 1 , 132 ] . this result has also been demonstrated for four dimensional einstein - maxwell theory and d = 4 , 5 einstein - maxwell - dilaton theory [ 210 , 211 ] . it would be interesting if this result could be generalised to other theories with hidden symmetry such as minimal supergravity . much less work has been done on classifying extremal black holes and their near - horizon geometries coupled to non - abelian gauge fields . the simplest setup for this is four - dimensional einstein - yang - mills theory . as is well known , the standard four dimensional black - hole uniqueness theorems fail in this case ( at least in the non - extremal case ) , for a review , see . nevertheless , near - horizon geometry uniqueness theorems analogous to the einstein - maxwell case have recently been established for this theory . theorem 6.6 ( ) . consider d = 4 einstein - yang - mills- theory with a compact semi - simple gauge group g. any static near - horizon geometry with compact horizon cross section is given by : ads2 s if > 0 ; ads2 h where h is one of s , t , g if < 0 . however , it should be noted that the yang - mills field need not be that of the abelian embedded solution . the horizon gauge field may be any yang - mills connection on s , or on the higher genus surface as appropriate , with a gauge group g ( if there is a non - zero electric field e the gauge group g is broken to the centraliser of e ) . the moduli space of such connections have been previously classified . hence , unless the gauge group is su(2 ) , one may have genuinely non - abelian solutions . it should be noted that static near - horizon geometries have been previously considered in einstein - yang - mills - higgs under certain restrictive assumptions . non - static near - horizon geometries have also been classified under the assumption of axisymmetry . any axisymmetric non - static near - horizon geometry with compact horizon cross section , in d = 4 einstein - yang - mills- with a compact semi - simple gauge group , must be given by the near - horizon geometry of an abelian embedded extreme kerr - newman- black hole . the proof of this actually requires new ingredients as compared to the einstein - maxwell theory . the ads2-symmetry enhancement theorems discussed in section 3.2 do not apply in the presence of a non - abelian gauge field . nevertheless , assuming the horizon cross sections are of s topology allows one to use a global argument to show the symmetry enhancement phenomenon still occurs . this implies the solution is effectively abelian and allows one to avoid finding the general solution to the odes that result from the reduction of the einstein - yang - mills equations . one can also rule out toroidal horizon cross sections , hence giving a complete classification of horizons with a u(1)-symmetry . interestingly , einstein - yang - mills theory with a negative cosmological constant is a consistent truncation of the bosonic sector of d = 11 supergravity on a squashed s . it would be interesting if near - horizon classification results could be obtained in more general theories with non - abelian yang - mills fields , such as the full \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 8$\end{document } , d = 4 , so(8)-gauged supergravity that arises as a truncation of d = 11 supergravity on s. one of the main motivations for classifying near - horizon geometries is to prove uniqueness theorems for the corresponding extremal black - hole solutions . this turns out to be a much harder problem and has only been achieved when extra structure is present that constrains certain global aspects of the spacetime . the role of the near - horizon geometry is to provide the correct boundary conditions near the horizon for the global - black - hole solution . uniqueness theorems for supersymmetric black holes have been proved in the simplest four and five - dimensional supergravity theories , by employing the associated near - horizon classifications described in section 5 . in four dimensions , the simplest supergravity theory that admits supersymmetric black holes is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 2$\end{document } minimal supergravity ; its bosonic sector is simply einstein - maxwell theory . theorem 7.1 ( ) . consider an asymptotically - flat , supersymmetric black - hole solution to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 2$\end{document } , d = 4 minimal supergravity . it would be interesting to remove the assumption on the supersymmetric killing vector field to provide a complete classification of supersymmetric black holes in this case . in five dimensions , the simplest supergravity theory that admits supersymmetric black holes is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 2$\end{document } minimal supergravity . using general properties of supersymmetric solutions in this theory , as well as the near - horizon classification discussed in section 5 , the following uniqueness theorem has been obtained . theorem 7.2 ( ) . consider an asymptotically - flat , supersymmetric black - hole solution to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 2$\end{document } , d = 5 minimal supergravity , with horizon cross section h s. assume that the supersymmetric killing vector field is timelike everywhere outside the horizon remarks : the bmpv solution is a stationary , non - static , non - rotating black hole with angular momentum j and electric charge q. for j = 0 it reduces to the extremal rn black hole.it would be interesting to investigate the classification of supersymmetric black holes without the assumption on the supersymmetric killing vector field.an analogous uniqueness theorem for supersymmetric black rings , i.e. , for h s s , remains an open problem.an analogous result has been obtained for minimal supergravity theory coupled to an arbitrary number of vector multiplets . the bmpv solution is a stationary , non - static , non - rotating black hole with angular momentum j and electric charge q. for j = 0 it reduces to the extremal rn black hole . it would be interesting to investigate the classification of supersymmetric black holes without the assumption on the supersymmetric killing vector field . an analogous uniqueness theorem for supersymmetric black rings , i.e. , for h s s , remains an open problem . an analogous result has been obtained for minimal supergravity theory coupled to an arbitrary number of vector multiplets . analogous results for asymptotically ads black holes in gauged supergravity have yet - to - be - obtained and this remains a very interesting open problem . this is particularly significant due to the lack of black - hole uniqueness theorems even for pure gravity in ads . however , it is worth mentioning that the analysis of used the homogeneous near - horizon geometry of theorem 5.3 with h s , together with supersymmetry , to explicitly integrate for the full cohomogeneity-1 ads5 black hole solution . it would be interesting to prove a uniqueness theorem for supersymmetric ads5 black holes assuming u(1 ) symmetry . the classic black - hole uniqueness theorem of general relativity roughly states that any stationary , asymptotically - flat black - hole solution to the vacuum einstein equations must be given by the kerr solution . traditionally this theorem assumed that the event horizon is non - degenerate , at a number of key steps . most notably , the rigidity theorem , which states that a stationary rotating black hole must be axisymmetric , is proved by first showing that the event horizon is a killing horizon . although the original arguments assumed non - degeneracy of the horizon , in four dimensions this assumption can be removed [ 179 , 180 , 134 ] . this allows one to reduce the problem to a boundary value problem on a two - dimensional domain ( the orbit space ) , just as in the case of a non - degenerate horizon . however , the boundary conditions near the boundary corresponding to the horizon depend on whether the surface gravity vanishes or not . unsurprisingly , the boundary conditions near a degenerate horizon can be deduced from the near - horizon geometry . this has allowed one to extend the uniqueness theorem for kerr to the degenerate case . the only four - dimensional , asymptotically - flat , stationary and axisymmetric , rotating , black - hole solution of the einstein vacuum equations , with a connected degenerate horizon with non - toroidal horizon sections , is the extremal kerr solution . the remaining proofs employ the near - horizon geometry classification theorem discussed in section 4.3 together with the standard method used to prove uniqueness of non - extremal kerr . the above uniqueness theorem has also been established for the extremal kerr - newman black hole in einstein - maxwell theory [ 5 , 40 , 177 ] . the assumption of a non - toroidal horizon is justified by the black - hole - horizon topology theorems . similarly , as discussed above , axisymmetry is justified by the rigidity theorem under the assumption of analyticity . together with these results , the above theorem provides a complete classification of rotating vacuum black holes with a single degenerate horizon , under the stated assumptions . the proof that a non - rotating black hole must be static has only been established for non - degenerate horizons , so the classification of non - rotating degenerate black holes remains an open problem . of course , in higher dimensions , there is no such simple general uniqueness theorem . for spacetimes with u(1 ) symmetry though , one has a mathematical structure analogous to d = 4 stationary and axisymmetric spacetimes . namely , one can reduce the problem to an integrable boundary - value problem on a 2d orbit space . it was first shown that non - degenerate black - hole solutions in this class are uniquely specified by certain topological data , which specifies the u(1)-action on the manifold , referred to as interval data ( i.e. , rod structure ) [ 138 , 139 ] . the proof is entirely analogous to that for uniqueness of kerr amongst stationary and axisymmetric black holes . this has been extended to cover the degenerate case , again by employing the near - horizon geometry to determine the correct boundary conditions . proposition 7.1 ( ) . consider a five - dimensional , asymptotically - flat , stationary black - hole solution of the vacuum einstein equations , with u(1 ) isometry group and a connected degenerate horizon ( with non - toroidal sections ) . there exists at most one such solution with given angular momenta and a given interval structure . it is worth emphasising that although there is no near - horizon uniqueness theorem in this case , see section 4.4 , this result actually only requires the general so(2 , 1 ) u(1 ) form for the near - horizon geometry and not its detailed functional form . it seems likely that the results of this section could be extended to u(1 ) invariant extremal black holes in einstein - maxwell type theories in higher dimensions . in d = 5 it is known that coupling a cs term in such a way to give the bosonic sector of minimal supergravity , implies such solutions are determined by a non - linear sigma model analogous to the pure vacuum case . hence it should be straightforward to generalise the vacuum uniqueness theorems to this theory . all known near - horizon geometries are fibrations of the horizon cross section h over an ads2 base ( see section 3.2 ) . by writing the ads2 in global explicitly , by converting the near - horizon geometry ( 65 ) to ads2 global coordinates , such spacetimes take the general form 129\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \rm{d}}{{\rm{s}}^2 } = { \ell ^2}\gamma ( y)\left [ - { { \cosh}^2}\rho { \rm{d}}{t^2 } + { \rm{d}}{\rho ^2}\right ] + { \gamma _ { mn}}(y){\rm{d}}{y^m}{\rm{d}}{y^n}}\qquad\qquad\qquad\quad\\ { + { \gamma _ { ij}}(y)({\rm{d}}{\phi ^i } + { \ell ^2}{k^i}\sinh \rho \;{\rm{d}}t)({\rm{d}}{\phi ^j } + { \ell ^2}{k^j}\sinh \rho \;{\rm{d}}t),}\\ \end{array}$$\end{document } where we have written the constant a0 = in terms of the radius of ads2 . these spacetimes possess two timelike boundaries , and of course do not contain a horizon . in fact , general arguments suggest that any near - horizon geometry must be unstable when backreaction is taken into account and the nearby solution must be singular . this follows from the fact that h is marginally trapped , so there exist perturbations that create a trapped surface and by the singularity theorems the resulting spacetime must be geodesically incomplete . if the perturbed solution is a black hole sitting inside the near - horizon geometry , then this need not be an issue.21 for ads2 s , heuristic arguments also indicating its instability have been obtained by dimensional reduction to an ads2 theory of gravity . in particular , this suggests that the backreaction of matter in ads2 s , is not consistent with a fall - off preserving both of the ads2 boundaries . so far we have been talking about the non - linear stability of near - horizon geometries . a massless scalar field in the near - horizon geometry of an extremal kerr black hole ( nhek ) reduces to a massive charged scalar field in ads2 with a homogeneous electric field . this turns out to capture the main features of the teukolsky equation for nhek , which describes linearised gravitational perturbations of nhek [ 4 , 60 ] . in contrast to the above instability , these works revealed the stability of nhek against linearised gravitational perturbations . in fact , one can prove a non - linear uniqueness theorem in this context : any stationary and axisymmetric solution that is asymptotic to nhek , possibly containing a smooth horizon , must in fact be nhek . so far we have discussed the stability of near - horizon geometries as spacetimes in their own right . a natural question is what information about the stability of an extremal black hole can be deduced from the stability properties of its near horizon geometry . clearly , stability of the near - horizon geometry is insufficient to establish stability of the black hole , but it has been argued that certain instabilities of the near - horizon geometry imply instability of the black hole . for higher dimensional vacuum near - horizon geometries , one can construct gauge - invariant quantities ( weyl scalars ) , whose perturbation equations decouple , generalising the teukolsky equation .22 one can then perform a kk reduction on h to find that linearised gravitational ( and electromagnetic ) perturbations reduce to an equation for a massive charged scalar field in ads2 with a homogeneous electric field ( as for the nhek case above ) . the authors of define the near - horizon geometry to be unstable if the effective breitenlohner - freedman bound for this charged scalar field is violated . they propose the following conjecture : an instability of the near - horizon geometry ( in the above sense ) , implies an instability of the associated extreme black hole , provided the unstable mode satisfies a certain symmetry requirement . this conjecture is supported by the linear stability of nhek and was verified for the known instabilities of odd dimensional cohomogeneity-1 mp black holes . it is also supported by the known stability results for the five - dimensional mp black hole and the kerr - ads4 black hole . this conjecture suggests that even - dimensional near - extremal mp black holes , which are more difficult to analyse directly , are also unstable . recently , it has been shown that extremal black holes exhibit linearised instabilities at the horizon . this was first observed for a massless scalar field in an extremal rn and extremal kerr - black - hole background [ 8 , 7 , 6 , 9 ] . while the scalar decays everywhere on and outside the horizon , the first transverse derivative of the scalar does not generically decay on the horizon , and furthermore the kth - transverse derivative blows up as v along the horizon . these results follow from the existence of a non - vanishing conserved quantity on the horizon linear in the scalar field . if the conserved quantity vanishes it has been shown that a similar , albeit milder , instability still occurs on the horizon [ 54 , 26 , 11 , 167 ] . it should be noted that this instability is not in contradiction with the above linear stability of the near - horizon geometry . from the point of view of the near - horizon geometry , it is merely a coordinate artefact corresponding to the fact that the poincar horizon of ads2 is not invariantly defined . the horizon instability has been generalised to a massless scalar in an arbitrary extremal black hole in any dimension , provided the near - horizon geometry satisfies a certain assumption . this assumption in fact follows from the ads2-symmetry theorems and hence is satisfied by all known extremal black holes . furthermore , by considering the teukolsky equation , it was shown that a similar horizon instability occurs for linearised gravitational perturbations of extremal kerr . using moncrief s perturbation formalism for rn , it was shown that coupled gravitational and electromagnetic perturbations of extremal rn within einstein - maxwell theory also exhibit such a horizon instability . an interesting open question is what is the fate of the non - linear evolution of such horizon instabilities . to this end interestingly , near - horizon geometries saturate certain geometric bounds relating the area and conserved angular momentum and charge of dynamical axisymmetric horizons , see for a review . in particular , for four - dimensional dynamical axially - symmetric spacetimes , the area of blackhole horizons with a given angular momentum is minimised by the extremal kerr black hole . the precise statement is : theorem 7.4 ( [ 55 , 143 ] ) . consider a spacetime satisfying the einstein equations with a nonnegative cosmological constant and matter obeying the dominant energy condition . the area of any axisymmetric closed ( stably outermost ) marginally - outer - trapped surface s satisfies 130\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a \geq8\pi \vert j\vert , $ $ \end{document } where j is the angular momentum of s. furthermore , this bound is saturated if and only if the metric induced on s is that of the ( near-)horizon geometry of an extreme kerr black hole . furthermore , it has been shown that if s is a section of an isolated horizon the above equality is saturated if and only if the surface gravity vanishes ( see also ) . an analogous area - angular momentum - charge inequality has been derived in einstein - maxwell theory , which is saturated by the extreme kerr - newman black hole . the above result can be generalised to higher dimensions , albeit under stronger symmetry assumptions . theorem 7.5 ( ) . consider a d - dimensional spacetime satisfying the vacuum einstein equations with non - negative cosmological constant that admits a u(1)-rotational isometry . then the area of any ( stably outer ) marginally - outer - trapped surface satisfies a 8|j+j| where j are distinguished components of the angular momenta associated to the rotational killing fields , which have fixed points on the horizon . further , if = 0 then equality is achieved if the spacetime is a near - horizon geometry and conversely , if the bound is saturated , the induced geometry on spatial cross sections of the horizon is that of a near - horizon geometry . in particular , for d = 4 , the horizon is topologically s and j+ = j and one recovers ( 130 ) . other generalisations of such inequalities have been obtained in d = 4 , 5 einstein - maxwell - dilaton theories [ 210 , 211 ] . the proof of the above results involve demonstrating that axisymmetric near - horizon geometries are global minimisers of a functional of the form ( 128 ) that is essentially the energy of a harmonic map , as discussed in section 6.4 . in this section we discuss analytic continuation of near - horizon geometries to obtain other lorentzian or riemannian metrics . as we will see , this uncovers a number of surprising connections between seemingly different spacetimes and geometries . as discussed in section 3.2 , generically , the orbits of this isometry are three - dimensional line or circle bundles over ads2 . one can often analytically continue these geometries so ads2 s and so(2 , 1 ) su(2 ) ( or so(3 ) ) with orbits that are circle bundles over s. it is most natural to work with the near - horizon geometry written in global ads2 coordinates ( 129 ) . such analytic continuations are then obtained by setting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\rho \rightarrow i(\theta - { \pi \over 2})$\end{document } and k ik . one can perform an analytic continuation of the near - horizon geometry of extremal kerr to obtain the zero mass lorentzian taub - nut solution . more generally , there is an analytic continuation of the near - horizon geometry of extremal kerr - newman- to the zero mass lorentzian rn - taub - nut- solution . in fact , page constructed a smooth compact riemannian metric on the non - trivial s - bundle over s with su(2 ) u(1 ) isometry , by taking a certain limit of the euclidean kerr - ds metric . he showed that locally his metric is the euclidean taub - nut- with zero mass . hence , it follows that there exists an analytic continuation of the near - horizon geometry of extremal kerr- to page s einstein manifold . ( also we deduce that page s limit is a riemannian version of a combined extremal and near - horizon limit ) . explicitly , the analytic continuation of the near - horizon extremal kerr- metric ( 83 ) to the page metric , can be obtained as follows . first write the near - horizon geometry in global coordinates ( 129 ) , then analytically continue \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\rho \rightarrow i(\theta - { \pi \over 2})$\end{document } and k = 2i , = 4 and redefine the coordinates ( t , ) ( , ) appropriately , to find 131\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{d}}{s^2 } = { { { \alpha ^2}(1 - { x^2}){\rm{d}}{x^2 } } \over { p(x ) } } + { { 4{\alpha ^2}p(x ) } \over { ( 1 - { x^2})}}{({\rm{d}}\psi + \cos \theta \;{\rm{d}}\phi)^2 } + { \alpha ^2}(1 - { x^2})({\rm{d}}{\theta ^2 } + { \sin ^2}\theta \;{\rm{d}}{\phi ^2})$$\end{document } with 132\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(x ) = 1 + { x^2 } - ( 1 + 2{x^2 } - { 1 \over 3}{x^4}){\alpha ^2}\lambda .$$\end{document } by an appropriate choice of parameters , this metric extends to a smooth global , inhomogeneous einstein metric on the non - trivial s bundle over s , as follows . compactness and positive definiteness requires one to take > 0 and x1 < x < x1 where x1 are two adjacent roots of p(x ) such that |x1| < 1 . the ( x , ) part of the metric has conical singularities at x = x1 , which are removed by imposing 133\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \psi = { { 2\pi { x_1 } } \over { 1 - \lambda { \alpha ^2}(1 - x_1 ^ 2)}}$$\end{document } resulting in an s fibre . this fibration is globally defined if m = 4 , where m , so combining these results implies 134\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m = { { 4{x_1}(3 + x_1 ^ 2 ) } \over { 3 + 6x_1 ^ 2 - x_1 ^ 4}}.$$\end{document } as page showed , the only solution is m = 1 , which implies the s - bundle is non - trivial . this manifold is diffeomorphic to the first del pezzo surface \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathbb c{\mathbb p^2}\ # { \overline { \mathbb c \mathbb p } ^2}$\end{document}. similarly , there is an analytic continuation of the near - horizon geometry of the extremal kerr - newman- that gives a family of smooth riemannian metrics on s - bundles over s that satisfy the riemannian einstein - maxwell equations ( and hence have constant scalar curvature ) . interestingly , this leads to an infinite class of metrics ( i.e. , there are an infinite number of possibilities for the integer m ) . the local solution can be derived directly by classifying solutions of the riemannian einstein - maxwell equations with su(2 ) u(1 ) isometry group acting on three - dimensional orbits ( see , e.g. , ) . one finds the geometry is given by ( 131 ) but with p(x ) now given by 135\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(x ) = 1 + { x^2 } + c - ( 1 + 2{x^2 } - { 1 \over 3}{x^4}){\alpha ^2}\lambda , $ $ \end{document } where c is a constant related to the electric and magnetic charges of the analytically continued extremal kerr - newmann black hole . the regularity condition now becomes 136\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m = { { 4{x_1}(3 + x_1 ^ 2 ) } \over { 3 + 6x_1 ^ 2 - x_1 ^ 4 } } + { { 8c(3 - x_1 ^ 2){x_1 } } \over { ( 1 - x_1 ^ 2)(3 + 6x_1 ^ 2 - x_1 ^ 4)}},$$\end{document } which for c = 0 reduces to eq . one can check the 2nd term above is monotonically increasing in the range 0 < x1 < 1 and unbounded as x1 1 . if c > 0 then there is a unique solution for every integer m 1 . for c < 0 the only allowed solution is m = 1 , and in fact there exist values of c < 0 such that there are two solutions with m = 1 . for m even , the metric extends to a global metric on the trivial s bundle over s , whereas for m odd , globally the space is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathbb c{\mathbb p^2}\ # { \overline { \mathbb c \mathbb p } ^2}$\end{document}. hence one obtains a generalisation of the page metric . curiously , in five dimensions there exist analytic continuations of near - horizon geometries to stationary black - hole solutions . for example , one can perform an analytic continuation of the near - horizon geometry of an extremal mp black hole with j1 j2 to obtain the full cohomogeneity-1 mp black hole with j1 = j2 ( which need not be extremal ) . in this case the s bundle over s that results after analytic continuation is the homogenous s of the resulting black hole . interestingly , this also works with the near - horizon geometries of extremal black rings . for example , there is an analytic continuation of the near - horizon geometry of the extremal dipole black ring that gives a static charged squashed kk black hole with s horizon topology . in these five - dimensional cases , the isometry of the near - horizon geometries is so(2 , 1 ) u(1 ) , which has 4d orbits ; hence one can arrange the new time coordinate to lie in these orbits in such a way it is not acted upon by the su(2 ) . this avoids nut charge , which is inevitable in the four - dimensional case discussed above . as in the four - dimensional case , there are analytic continuations which result in einstein metrics on compact manifolds . for example , there is an analytic continuation of the near - horizon geometry of extremal mp- that gives an infinite class of einstein metrics on s - bundles over s , which were first found by taking a page limit of the mp - ds black hole . in higher than five dimensions one can similarly perform analytic continuations of einstein near - horizon geometries to obtain examples of compact einstein manifolds . the near - horizon geometries of mp- give the einstein manifolds that have been constructed by a page limit of the mp - ds metrics . on the other hand , the new families of near - horizon geometries [ 156 , 157 ] , discussed in section 4.5.3 , analytically continue to new examples of einstein metrics on compact manifolds that have yet to be explored . so far we have discussed analytic continuations in which the ads2 is replaced by s. another possibility is to replace the ads2 with hyperbolic space . for simplicity let us focus on static near - horizon geometries . such an analytic continuation is then easily achieved by replacing the global ads2 time with imaginary time , i.e. , t it in eq . ( 129 ) . in this case , instead of a horizon , one gets a new asymptotic region corresponding to . general static riemannian manifolds possessing an end that is asymptotically extremal in this sense were introduced in . essentially , they are defined as static manifolds possessing an end in which the metric can be written as an euclideanised static spacetime containing a smooth degenerate horizon . it was shown that ricci flow preserves this class of manifolds , and furthermore asymptotically - extremal ricci solitons must be einstein spaces . these results were used to numerically simulate ricci flow to find a new einstein metric that has an interesting interpretation in the ads / cft correspondence , which we briefly discuss in section 7.5 . it would be interesting to investigate non - static near - horizon geometries in this context . due to the applications to black - hole solutions , we have mostly focused on the near - horizon geometries of degenerate horizons with cross sections h that are compact . however , as we discussed in section 2 , the concept of a near - horizon geometry exists for any spacetime containing a degenerate horizon , independent of the topology of h. in particular , extremal black branes possess horizons with non - compact cross sections h. hence the general techniques discussed in this review may be used to investigate the classification of the near - horizon geometries of extremal black branes . in general , this is a more difficult problem , since as we have seen , compactness of h can often be used to avoid solving for the general local metric by employing global arguments . since it is outside the scope of this review , we will not give a comprehensive overview of this topic ; instead we shall select a few noteworthy examples . first consider adsd space written in poincar coordinates 137\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{d}}{s^2 } = { y^2}(- { \rm{d}}{t^2 } + { \rm{d}}{x^i}{\rm{d}}{x^i } ) + { { { \rm{d}}{y^2 } } \over { { y^2}}},$$\end{document } where i = 1 , , d 2 . the surface y = 0 , often called the poincar horizon , is a degenerate killing horizon of the killing field k = /t . these coordinates are not valid at y = 0 ( the induced metric is singular ) and hence are unsuitable for extracting the geometry of the poincar horizon . one may introduce coordinates adapted to the poincar horizons by constructing gaussian null coordinates as described in section 2 . we need to find null geodesics ( ) that in particular satisfy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$k \cdot \dot \gamma = 1$\end{document}. explicitly , this condition is simply = y . now , since /x are killing fields , along any geodesic the quantities \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\partial/\partial { x^i } ) \cdot \dot \gamma$\end{document } must be constant ; this gives = ky , where k are constant along the geodesics . the null constraint now simplifies to = 1 kk and so kk < 1 . this latter equation is easily integrated to give y( ) and using the above we may now integrate for t( ) and x( ) . the result is 138\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$y = \sqrt { ( 1 - { k^i}{k^i } ) } \lambda , \quad t = v + { 1 \over { ( 1 - { k^i}{k^i})\lambda}},\quad { x^i } = { { { k^i } } \over { ( 1 - { k^i}{k^i})\lambda}},$$\end{document } where v is an integration constant and we have set the other integration constants to zero to ensure the horizon is at = 0 . this gives a family of null geodesics parameterised by ( v , k ) , which shoot out from every point on the horizon ; hence we may take the ( v , k ) as coordinates on the horizon . we can then change from poincar coordinates ( t , x , y ) to the desired gaussian null coordinates ( v , , k ) . indeed , one can check that in the coordinates ( v , , k ) , the killing field k = /v and the metric takes the form ( 6 ) ( with r = ) . in fact , as is often the case , it is convenient to use a different affine parameter r = (1 kk ) . also , since kk < 1 we may write k = tanh , where = 1 parameterise a unit ( d 3)-sphere . now the coordinate transformation becomes 139\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$y = r\cosh \eta , \quad t = v + { 1 \over r},\quad { x^i } = { { \tanh \eta \;{\mu ^i } } \over r},$$\end{document } and the adsd metric in these coordinates is 140\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{d}}{s^2 } = { \cosh ^2}\eta ( - { r^2}{\rm{d}}{v^2 } + 2{\rm{d}}v\;{\rm{d}}r ) + { \rm{d}}{\eta ^2 } + { \sinh ^2}\eta \;{\rm{d}}\omega _ { d - 3}^2.$$\end{document } it is now manifest that the surface r = 0 is a smooth degenerate killing horizon of /v , corresponding to the poincar horizon , which we may now extend onto and through by taking r 0 . cross sections of the poincar horizon are non - compact and of topology with a ( non - flat ) induced metric given by the standard einstein metric on hyperbolic space . observe that the above expresses adsd as a warped product of ads2 and hyperbolic space , i.e. , as a static near - horizon geometry ( with no need to take a near - horizon limit ! ) , as observed in . the bps extremal d3 , m2 , m5 black branes of 10,11-dimensional supergravity are well known to have a near - horizon geometry given by ads5 s , ads4 s and ads7 s respectively with their horizons corresponding to a poincar horizon in the ads factor . however , as discussed above , the standard poincar coordinates are not valid on the horizon and hence unsuitable if one wants to extend the brane geometry onto and beyond the horizon . to this end , it is straightforward to construct gaussian null coordinates adapted to the horizon of these black branes and check their near - horizon limit is indeed given by eq . ( 140 ) plus the appropriate sphere.23 of course , extremal branes occur in other contexts . for example , the randall - sundrum model posits that we live on a 3 + 1 dimensional brane in a 4 + 1-dimensional bulk spacetime with a negative cosmological constant . a longstanding open problem has been to construct solutions to the five - dimensional einstein equations with a black hole localised on such a brane , the brane - world black hole.24 in the five - dimensional spacetime , the horizons of such black holes extend out into the bulk . constructed the near - horizon geometry of an extremal charged black hole on a brane . this involved constructing ( numerically ) the most general five dimensional static near - horizon geometry with so(3 ) rotational symmetry , which turns out to be a 1-parameter family generalisation of eq . ( 140 ) ( this is the 5d analogue of the 4d general static near - horizon geometry with a non - compact horizon , see section 4.1 ) . notably , numerically constructed the first example of a brane - world black - hole solution . this corresponds to a schwarzschild - like black hole on a brane suspended above the poincar horizon in ads5 . an important step towards this solution was the construction of a novel asymptotically ads einstein metric with a schwarzschild conformal boundary metric and an extremal poincar horizon in the bulk ( sometimes called a black droplet ) . this was found by numerically simulating ricci flow on a suitable class of stationary and axisymmetric metrics . this solution is particularly interesting since by the ads / cft duality it is the gravity dual to a strongly coupled cft in the schwarzschild black hole , thus allowing one to investigate strongly coupled qft in black - hole backgrounds . recently , generalisations in which the boundary black hole is rotating have been constructed , in which case there is also the possibility of making the black hole on the boundary extremal [ 82 , 84 ] .

any spacetime containing a degenerate killing horizon , such as an extremal black hole , possesses a well - defined notion of a near - horizon geometry . we review such near - horizon geometry solutions in a variety of dimensions and theories in a unified manner . we discuss various general results including horizon topology and near - horizon symmetry enhancement . we also discuss the status of the classification of near - horizon geometries in theories ranging from vacuum gravity to einstein - maxwell theory and supergravity theories . finally , we discuss applications to the classification of extremal black holes and various related topics . several new results are presented and open problems are highlighted throughout .

Introduction Degenerate Horizons and Near-Horizon Geometry General Results Vacuum Solutions Supersymmetric Solutions Solutions with Gauge Fields Applications and Related Topics

the main purpose of this review is to discuss the classification of the near - horizon geometries of extremal black holes . in section 1.1 and 1.2 we discuss the various ways extremal black holes and their near - horizon geometries have appeared in modern studies of quantum gravity . in section 1.3 we discuss the more general black hole classification problem ( which is also partly motivated by quantum gravity ) , and how near - horizon geometries provide a systematic tool for investigating certain aspects of this problem for extremal black holes . the key idea is that extremal black holes have a well - defined near - horizon geometry that typically possesses an ads2 symmetry . furthermore , a cft2 description has been proposed for a certain class of near - extremal black holes , which possess a local near - horizon ads3 factor but a vanishing horizon area in the extremal limit [ 196 , 146].3 recently , ideas from the gauge / gravity duality have been used to model certain phase transitions that occur in condensed - matter systems , such as superfluids or superconductors [ 107 , 125 ] . the event horizon of all known extremal black holes is a degenerate killing horizon with compact spatial cross sections h. it turns out that restricting einstein s equations for a d - dimensional spacetime to a degenerate horizon gives a set of geometric equations for the induced metric on such ( d 2)-dimensional cross sections h , that depend only on quantities intrinsic to h. by studying solutions to this problem of riemannian geometry on a compact manifold h , one can thus consider the possible horizon geometries ( and topologies ) independently of the full parent spacetime . one can understand this feature of degenerate horizons in terms of the near - horizon limit , which , as we explain in section 2 , exists for any spacetime containing a degenerate horizon . indeed , the topic of this review is the classification of near - horizon geometries in diverse dimensions and theories . the classification of near - horizon geometries allows one to explore in a simplified setup the main issues that appear in the general black - hole classification problem , such as the horizon topology , spacetime symmetry and the number of solutions . our aim is to provide a unified treatment of such near - horizon solutions in diverse theories with matter content ranging from vacuum gravity , to einstein - maxwell theories and various ( minimal ) supergravity theories . most notably , we fully classify three dimensional near - horizon geometries in vacuum gravity and einstein - maxwell - chern - simons theories , in section 4.2 and 6.1 respectively , and classify homogeneous near - horizon geometries in five dimensional einstein - maxwell - chern - simons theories in section 6.3.2 . in section 2 we provide key definitions , introduce a suitably general notion of a near - horizon geometry and set up the einstein equations for such near - horizon geometries . in section 3 we review various general results that constrain the topology and symmetry of near - horizon geometries . in section 4 we discuss the classification of near - horizon geometries in vacuum gravity , including a cosmological constant , organised by dimension . in cases where classification results are not known , we discuss the classification of supersymmetric near - horizon geometries in various supergravity theories , organised by dimension . in section 7 this includes uniqueness / classification theorems of the corresponding extremal black - hole solutions , stability of near - horizon geometries and extremal black holes , geometric inequalities , analytic continuation of near - horizon geometries , and extremal branes and their near - horizon geometries . we emphasise that the degenerate assumption gvv = o(r ) is crucial for defining this limit and such a general notion of a near - horizon limit does not exist for a non - degenerate killing horizon . furthermore , imposing the bianchi identity to the near - horizon limit of the maxwell field relates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f_{\upsilon r}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal f_{\upsilon a}}$\end{document } , allowing one to write 23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal f}_{{\rm{nh } } } } = { \rm{d}}(r\delta ( x){\rm{d}}v ) + { 1 \over 2}{b_{ab}}(x){\rm{d}}{x^a } \wedge { \rm{d}}{x^b},$$\end{document } where is a function on h and b is a closed 2-form on h. the 2-form b is the maxwell field induced on h and locally can be written as b = da for some 1-form potential a on h. it can be checked that for the near - horizon limit 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = { { 2(n - 1 ) } \over n}{\delta ^2 } + { 1 \over n}{b_{ab}}{b^{ab}},$$\end{document } 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_{ab } } = 2{b_{ac}}b_b^c + \left({{2 \over n}{\delta ^2 } - { { { b_{cd}}{b^{cd } } } \over n } } \right){\gamma _ { ab}}.$$\end{document } we will present the maxwell equations in a variety of dimensions in section 6 . the bianchi identity then implies that the most general form for the near - horizon limit is 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal f}_{{\rm{nh } } } } = { \rm{d}}(y \wedge r{\rm{d}}v ) + x,$$\end{document } where y is a ( p 2)-form on h and x is a closed p - form on h. the einstein equations for a near - horizon geometry can also be interpreted as geometrical equations arising from the restriction of the einstein equations for the full spacetime to a degenerate horizon , without taking the near - horizon limit , as follows . in this review , we describe the current understanding of the space of solutions to the basic horizon equation ( 17 ) , together with the appropriate horizon matter field equations , in a variety of dimensions and theories . this local form of the metric can be used to show that for > 0 and non - constant , there are also no smooth horizon metrics on a compact h. the classification of near - horizon geometries in d = 3 vacuum gravity with a cosmological constant can be completely solved . remarks : the near - horizon geometry of the vacuum extremal black ring is a 2-parameter subfamily of case 1 , corresponding to a kerr string with vanishing tension .the near - horizon geometry of the slowly rotating extremal kk black hole is identical to that of the 2-parameter extremal myers - perry in case 2.the h s cases can be written as a single 3-parameter family of near - horizon geometries .the h t case has been ruled out . the near - horizon geometry of the extremal mp black holes can be written in a unified form : 86\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { g_{{\rm{mp } } } } = { f _ + } \left({- { { \pi^{\prime\prime}({r _ + } ) } \over { 2\pi ( { r _ + } ) } } { r^2}\;{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;dr } \right ) + { \gamma _ { { \mu _ i}{\mu _ j}}}\;{\rm{d}}{\mu ^i}{\rm{d}}{\mu ^j}}\qquad\qquad\quad\\ { + { \gamma _ { ij}}\left({{\rm{d}}{\phi ^i } + { { 2r + { a_i } } \over { { { ( r _ + ^2 + a_i^2)}^2}}}r\;{\rm{d}}v } \right)\left({{\rm{d}}{\phi ^j } + { { 2r + { a_j } } \over { { { ( r _ + ^2 + a_j^2)}^2}}}r\;{\rm{d}}v } \right),}\\ \end{array}$$\end{document } where 87\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f _ + } + 1 - \sum\limits_{i = 1}^s { { { a_i^2\mu _ i^2 } \over { r _ + ^2 + a_i^2}},\quad \quad { \gamma _ { ij } } = ( r _ + ^2 + a_i^2 ) } \mu _ i^2{\delta _ { ij } } + { 1 \over { { f _ + } } } { a_i}\mu _ i^2{a_j}\mu _ j^2,$$\end{document } and in odd and even dimensions 88\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { { \mu _ i}{\mu _ j}}}{\rm{d}}{\mu ^i}{\rm{d}}{\mu ^j } = \sum\limits_{i = 1}^s { ( r _ + ^2 + a_i^2)d\mu _ i^2,\quad \quad { \gamma _ { { \mu _ i}{\mu _ j}}}{\rm{d}}{\mu ^i}{\rm{d}}{\mu ^j } = r _ + ^2{\rm{d}}{\alpha ^2 } + } \sum\limits_{i = 1}^s { ( r _ + ^2 + a_i^2)\;{\rm{d}}\mu _ i^2 } , $ $ \end{document } respectively . a supersymmetric near - horizon geometry in minimal gauged supergravity , with a compact horizon section , must be locally isometric to the near - horizon limit of the topologically - spherical supersymmetric black holes , or the homogeneous near - horizon geometries with the nil or sl(2 , ) horizons . it would be interesting to complete the classification of near - horizon geometries in this more general theory , along the lines of the minimal theory . a variety of results concerning the classification of supersymmetric near - horizon geometries in this theory have been derived [ 102 , 101 , 103 ] . the classification of near - horizon geometries in d = 3 einstein - maxwell theory with a cosmological constant can be completely solved . for the near - horizon maxwell field it can be shown that this is equivalent to 104\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta^{\prime } = ( h + \mu)\delta .$$\end{document } as in the pure einstein - maxwell case , a complete classification of near - horizon geometries to this theory is possible . solution ( ii ) for = 0 gives ads3 s , whereas for 0 it gives a near - horizon geometry with h s , which for q 0 is ads2 s. a charged rotating black - hole solution to einstein - maxwell theory generalising mp is not known explicitly . the classification of near - horizon geometries in d = 5 einstein - maxwell - cs theory , under the assumption of u(1 ) symmetry , turns out to be significantly more complicated than the vacuum case . the classification of near - horizon geometries in this case was analysed in using the hidden symmetry and some partial results were obtained , see proposition 6.4 . it would be interesting if near - horizon classification results could be obtained in more general theories with non - abelian yang - mills fields , such as the full \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 8$\end{document } , d = 4 , so(8)-gauged supergravity that arises as a truncation of d = 11 supergravity on s. one of the main motivations for classifying near - horizon geometries is to prove uniqueness theorems for the corresponding extremal black - hole solutions . however , as we discussed in section 2 , the concept of a near - horizon geometry exists for any spacetime containing a degenerate horizon , independent of the topology of h. in particular , extremal black branes possess horizons with non - compact cross sections h. hence the general techniques discussed in this review may be used to investigate the classification of the near - horizon geometries of extremal black branes .

this was via the construction of an explicit counterexample , the black ring , which is a black hole whose spatial horizon topology is s s. together with the higher - dimensional analogues of the kerr black hole ( which have spherical topology ) found by myers and perry , this established that the conserved charges are not sufficient to specify a black hole uniquely and also that other horizon topologies are possible . this was applied to the extremal kerr black hole and led to the kerr / cft correspondence , which is a proposal that quantum gravity in the near - horizon geometry of extremal kerr is described by a chiral cft2 ( see the reviews [ 31 , 48 ] ) . furthermore , a cft2 description has been proposed for a certain class of near - extremal black holes , which possess a local near - horizon ads3 factor but a vanishing horizon area in the extremal limit [ 196 , 146].3 recently , ideas from the gauge / gravity duality have been used to model certain phase transitions that occur in condensed - matter systems , such as superfluids or superconductors [ 107 , 125 ] . on the mathematical side , solutions to einstein s equation have also been of interest in differential geometry .4 in four dimensions the black - hole uniqueness theorem provides an answer to the classification problem for asymptotically - flat black - hole solutions of einstein - maxwell theory ( see for a review).5 however , in higher dimensions , uniqueness is violated even for asymptotically - flat vacuum black holes . although of less obvious physical relevance , the investigation of asymptotically - flat vacuum black holes is the fundamental starting case to consider in higher dimensions , since such solutions can be viewed as limits of black holes with more general asymptotics such as kk , ads and matter fields . for asymptotically - flat ( and globally ads ) black holes , this implies that there must be a simply connected ( oriented ) cobordism between cross sections of the horizon and the ( d 2)-dimensional sphere at spatial infinity.6 in d = 4 , this rules out toroidal black holes , although for d = 5 it imposes no constraint . for d > 5 , the symmetry of these spacetimes is not compatible with asymptotic flatness , that would require the number of commuting rotational symmetries to not exceed \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\lfloor { { { d - 1 } \over 2 } } \right\rfloor$\end{document } , the rank of the rotation group so(d 1 ) . the event horizon of all known extremal black holes is a degenerate killing horizon with compact spatial cross sections h. it turns out that restricting einstein s equations for a d - dimensional spacetime to a degenerate horizon gives a set of geometric equations for the induced metric on such ( d 2)-dimensional cross sections h , that depend only on quantities intrinsic to h. by studying solutions to this problem of riemannian geometry on a compact manifold h , one can thus consider the possible horizon geometries ( and topologies ) independently of the full parent spacetime . the main drawback of this approach is that the existence of a near - horizon geometry solution does not guarantee the existence of a corresponding black - hole solution ( let alone its uniqueness).8 hence , one must keep this in mind when interpreting near - horizon classifications in the context of black holes , although definite statements can be learned . most notably , we fully classify three dimensional near - horizon geometries in vacuum gravity and einstein - maxwell - chern - simons theories , in section 4.2 and 6.1 respectively , and classify homogeneous near - horizon geometries in five dimensional einstein - maxwell - chern - simons theories in section 6.3.2 . in section 7 this includes uniqueness / classification theorems of the corresponding extremal black - hole solutions , stability of near - horizon geometries and extremal black holes , geometric inequalities , analytic continuation of near - horizon geometries , and extremal branes and their near - horizon geometries . by construction the integral curves of l = /r are null geodesics and hence grr = 0 everywhere in the neighbourhood of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } in m in question . hence they may be thought of as components of a globally - defined function , 1-form and riemannian metric on h. the coordinates developed above are valid in the neighbourhood of any smooth null hypersurface \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document}. since k = /v we deduce that in the neighbourhood of a killing horizon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } , the metric can be written as eq . ( 5 ) where the functions f , ha , ab are all independent of the coordinate v. using the killing property one can rewrite k k = k as d(k k ) = 2k on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document } , where is now the usual surface gravity of a killing horizon . it follows that gvv = rf for some smooth function f. therefore , in the neighbourhood of any smooth degenerate killing horizon the metric in gaussian null coordinates reads 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = 2\;{\rm{d}}v({\rm{d}}r + r{h_a}(r , x){\rm{d}}{x^a } + { 1 \over 2}{r^2}f(r , x){\rm{d}}v ) + { \gamma _ { ab}}(r , x){\rm{d}}{x^a}{\rm{d}}{x^b}{. intuitively , the near - horizon limit is a scaling limit that focuses on the spacetime near the horizon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n}$\end{document}. henceforth , we will drop the hats on all horizon quantities , so rab and a refer to the ricci tensor and levi - civita connection of ab on h. we will consider spacetimes that are solutions to einstein s equations : 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_{\mu \nu } } = \lambda { g_{\mu \nu } } + { t_{\mu \nu } } - { 1 \over n}{g^{\rho \sigma}}{t_{\rho \sigma}}{g_{\mu \nu}},$$\end{document } where t is the energy - momentum and is the cosmological constant of our spacetime . by examining the ( xv ) and ( xi ) components of the spacetime einstein equations and changing the affine parameter r (x)r , one can show 58\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g = \gamma ( x)[{a_0}{r^2}{\rm{d}}{v^2 } + 2\;{\rm{d}}v\;{\rm{d}}r ] + { { { \rm{d}}{x^2 } } \over { \det b } } + { b_{ij}}(x)({\rm{d}}{\phi ^i } + { k^i}r\;{\rm{d}}v)({\rm{d}}{\phi ^j } + { k^j}r\;{\rm{d}}v),$$\end{document } where a0 and k are constants . by examining the ( xv ) and ( xi ) components of einstein s equations for the above general theory , and changing the affine parameter r (x)r ( 58 ) and the maxwell fields are given by 62\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f^i } = { \rm{d}}[e^{i}r\;{\rm{d}}v + b_i^i(x)\;({\rm{d}}{\phi ^i } + { k^i}r\;{\rm{d}}v)],$$\end{document } where e are constants . we will assume non - toroidal horizon topology so the orbit space is an interval , which without loss of generality we take to be [ 1 , 1 ] parameterised by the coordinate x. the ode is the equation of motion for a non - linear sigma model defined on this interval : 127\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\rm{d } } \over { { \rm{d}}x}}\left [ { ( 1 - { x^2}){\phi ^{- 1}}{{{\rm{d}}\phi } \over { { \rm{d}}x } } } \right ] = 0,$$\end{document } where the a coset representative depends only on x. it is straightforward to integrate this matrix equation and completely solve for the scalar fields , which can then be used to reconstruct the full d - dimensional solution . it would be interesting if near - horizon classification results could be obtained in more general theories with non - abelian yang - mills fields , such as the full \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal n } = 8$\end{document } , d = 4 , so(8)-gauged supergravity that arises as a truncation of d = 11 supergravity on s. one of the main motivations for classifying near - horizon geometries is to prove uniqueness theorems for the corresponding extremal black - hole solutions . by writing the ads2 in global explicitly , by converting the near - horizon geometry ( 65 ) to ads2 global coordinates , such spacetimes take the general form 129\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \rm{d}}{{\rm{s}}^2 } = { \ell ^2}\gamma ( y)\left [ - { { \cosh}^2}\rho { \rm{d}}{t^2 } + { \rm{d}}{\rho ^2}\right ] + { \gamma _ { mn}}(y){\rm{d}}{y^m}{\rm{d}}{y^n}}\qquad\qquad\qquad\quad\\ { + { \gamma _ { ij}}(y)({\rm{d}}{\phi ^i } + { \ell ^2}{k^i}\sinh \rho \;{\rm{d}}t)({\rm{d}}{\phi ^j } + { \ell ^2}{k^j}\sinh \rho \;{\rm{d}}t),}\\ \end{array}$$\end{document } where we have written the constant a0 = in terms of the radius of ads2 . for m even , the metric extends to a global metric on the trivial s bundle over s , whereas for m odd , globally the space is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathbb c{\mathbb p^2}\ # { \overline { \mathbb c \mathbb p } ^2}$\end{document}.

participants enrolled in this study were part of a national institutes of health funded cohort study conducted at the university of alabama at birmingham : the african descent and glaucoma evaluation study ( adages ) . only subjects without evidence of ocular diseases that would affect the optic nerve , retina , or choroid , as defined below , were invited to participate . the university of alabama at birmingham institutional review board approved the study methods that adhered to the tenets of the declaration of helsinki . each participant underwent a complete ophthalmological exam that included medical history , snellen best - corrected visual acuity , early treatment diabetic retinopathy study ( etdrs ) visual acuity , color vision , slit - lamp biomicroscopy , applanation tonometry for iop , central corneal thickness ( cct ) measurement , axial length ( al ) measurement , dilated funduscopy , stereoscopic ophthalmoscopy of the optic disc , and stereoscopic fundus photography . standard swedish interactive thresholding algorithm 24 - 2 perimetry was performed to define normality for inclusion in this study ( see below ) . additionally , all subjects underwent testing with the ocular response analyzer ( ora ) to obtain measurements of corneal hysteresis ( ch ) and corneal resistance factor ( crf ) preceding baseline edi - sdoct imaging for the compliance testing . eligible participants had open iridocorneal angles , a best - corrected acuity of 20/40 or better , and refractive error 5.0 diopters sphere and 3.0 diopters cylinder . patients with diabetes and any retinal , corneal , or optic nerve disease were excluded . participants were excluded if they had a history of intraocular surgery ( except for uncomplicated cataract surgery ) , elevated iop ( > 22 mm hg ) at the time of the study , a history of elevated iop , prior use of glaucoma medication , other intraocular eye disease , or other diseases affecting visual field ( e.g. , pituitary lesions , demyelinating diseases , human immunodeficiency virus positive or acquired immune deficiency syndrome ) . further , patients were excluded if there was a diagnosis of alzheimer 's disease , parkinson 's disease , or previous brain injury including stroke , psychoses , or other neurological or psychiatric conditions that would prevent participation in a psychophysical test . we required two reliable tests ( less than 25% fixation losses , false negatives , and false positives ) on 24 - 2 standard automated perimetry ( carl zeiss meditec , dublin , ca , usa ) using the swedish interactive threshold algorithm in both eyes at baseline for inclusion in the study . reliability was defined as < 33% false positives , false negatives , and fixation losses . a field was considered normal if the pattern standard deviation was not triggered at 5% or less , the glaucoma hemifield test was within normal limits , and the field showed no sign of glaucomatous defect based on subjective evaluation from the enrolling clinician . the optic nerves were assessed by stereoscopic photos by a fellowship - trained glaucoma specialist . subjects with any structural finding suggestive of glaucoma ( i.e. , notching , rim thinning , disc hemorrhages , or retinal nerve fiber layer defects as judged ) were excluded . twenty - four eyes of 12 individuals of ad and 18 eyes of 9 individuals of ed underwent ocular mechanical compliance testing , as follows . baseline edi - sdoct imaging of the optic nerve was performed prior to iop elevation with the spectralis oct ( heidelberg engineering ; family acquisition module 5.4.8.0 ) with 24 radial scans centered on the center of the optic nerve head using enhanced depth imaging . scans were evaluated for imaging quality by the operator , and images were re - acquired if there was improper b - scan positioning in the imaging frame , a quality score < 20 , or poor centration of the optic nerve head . radial scans were acquired using a signal averaging of nine b - scans . a sample of the effect of the acute iop elevation is reported in figure 1 . for visualization purposes , the bruch 's membrane in the b - scan acquired at elevated iop ( fig . 1b ) is used as a reference line to align the scan at the reference iop ( fig . 1a ) ; from the superimposition of anterior lamina cribrosa surface ( alcs ) outline at reference- and high - pressure , it is noticeable the shift of the alcs caused by the iop elevation ( fig . it is important to note that the 3d quantification method used in this study and described in the next paragraph does not require an alignment to the b - scans to the bm plane , and that the alcs depth variation is estimated in the actual 3d space and not the 2d image plane . simplified representation of the morphological changes in alcs depth visible in a comparison of the same radial b - scan before ( a ) and after ( b ) the iop elevation . a variation of the lc is visible with the superimposition of the reference ( green ) and elevated ( red ) iop b - scans , respectively ( c ) . in order to induce a temporary elevation of the iop , a compressive force of approximately 35 pa on the inferior temporal globe was applied by means of an ophthalmodynamometer , in a similar manner described by agoumi et al . this compressive force caused increase in iop of approximately 14 mm hg and did not visibly disturb oct image quality . compression was maintained while the following steps were performed : 60 seconds waiting time allowing short - time mechanical effects of the tissue to be minimized ; an edi - sdoct scan was re - acquired in a similar manner to baseline testing ; iop measurement was re - performed with a tonopen to estimate the actual increase of pressure . after 5 minutes following initial iop elevation , an additional measurement of iop was performed . in order to enhance the visibility of the deeper tissue , all b - scans from the edi - sdoct volumes were postprocessed using adaptive light attenuation compensation , in order to increase a clearer visualization of the lamina cribrosa . light attenuation compensated scan volumes were then loaded in a custom software developed for 3d delineation of histologic and oct data that has been described in prior publications based on the visualization toolkit ( vtk , clifton park , ny , usa ) . a trained observer masked to subject characteristics manually delineated the internal limiting membrane ( ilm ) , bmo , inner and outer choroidal layers , and the anterior surface of the lc in 24 equally spaced radial sections of each oct scan . an average of 202 points ( range , 23534 ) were used to define the lc anterior surface . considering that former studies observed significant differences in the onh morphology of ed and ad for all the oct scans , differences in laminar visibility by race and iop elevation were assessed and reported in the results section . since the difference in alcs position seen with iop elevation was independent of the chosen reference plane , the commonly used bmo reference plane - based measures were used for the present analysis . this similarity between reference planes is possibly due to the lack of significant change in choroidal thickness with the acute increase of iop ( p = 0.94 ) . since the bmo is nearly planar , its best - fitting plane , computed through principal component analysis of the bmo samples , can serve as a reference plane . the depth of a laminar point is its distance from this bmo plane , positive only on the laminar side of the bmo plane . laminar depth is sampled across a reconstruction of the lamina , leading to a computation of mean laminar depth , as follows . to build a uniform sampling of the lc sections , each section is downsampled ( while preserving shape , using the douglas - peucker decimation algorithm ) , interpolated by a b - spline curve , then uniformly sampled . an optimal triangle mesh is built from this uniform sampling of the lc sections ( using the classic contour reconstruction algorithm of song et al . ) . the centroids of each triangle of this mesh are found , and the laminar depth at each centroid is measured ( its distance from the bmo plane ) . mean laminar depth is now a weighted linear combination of these centroid depths and weighted by triangle area ( weight = triangle area / mesh area ) in order to achieve a standardized quantification independent of the number of sampling points used for the definition of the laminar surface morphology . the prelaminar tissue depth of a point of the ilm is its distance from the bmo reference plane . as with laminar depth , a mesh is constructed and depths are sampled across this mesh to calculate the mean depth of the ilm . for prelaminar tissue depth , measurements are constrained to the cylinder above the best - fitting ellipse of the bmo . prelaminar tissue depth was quantified based on distance from the bmo reference plane to the surface of the manually delineated ilm . as with laminar depth , a mesh was constructed and depths were sampled uniformly across this mesh to calculate the depth of the ilm . measurements were constrained to the cylinder above the best - fitting ellipse of the bmo . prelaminar tissue depth is positive on the laminar side of the bmo plane , otherwise negative . the methods for computing alcs and plt depth are described in detail in a prior publication . in order to assess the dependency of the measurements from the interrater variability , we assessed the bland - altman limit - of - agreement and the intraclass correlation coefficient ( icc ) for the laminar depth measurement . for that purpose , 12 oct scans were manually delineated from both raters , and laminar depth was computed using the quantification method described in the previous paragraph . a bland - altam plot reporting the limit - of - agreement ( 18 m , 95% confidence interval [ ci ] ) for the mean laminar depth estimated by the two raters is shown in figure 3 ( left ) . in right plot of figure 3 , it can be noticed that the magnitude of disagreement was correlated ( pearson = 0.62 ) with the magnitude of the measurement that invalidates the interpretation of the limit - of - agreement as an estimate of the interrater variability . the lower agreement between raters observed for shallow laminae and a higher agreement for deeper laminae were plausibly imputable to the lamina visibility change as a function of mean depth ; a higher amount of plt present in shallower onhs would diminish the visibility of the anterior laminar surface . for the intraclass correlation coefficient , the i score at 95% ci was 0.932 < icc < 0.994 , showing a robust delineation and quantification procedure in the assessment of the laminar depth measurement . generalized estimating equations ( gees ) accounting for the correlation among fellow eyes from the same individual were used in multivariate models where the response variables were the change in depth of the lc and plt . only covariates that showed a significant association with the response variables were used in the multivariate model . jackknife resampling and bootstrapping were used to assess the dependency of the outcomes from high leverage data points and the sample size . statistical analyses were performed using the r software ( r foundation for statistical computing , vienna , austria ) . eligible participants had open iridocorneal angles , a best - corrected acuity of 20/40 or better , and refractive error 5.0 diopters sphere and 3.0 diopters cylinder . patients with diabetes and any retinal , corneal , or optic nerve disease were excluded . participants were excluded if they had a history of intraocular surgery ( except for uncomplicated cataract surgery ) , elevated iop ( > 22 mm hg ) at the time of the study , a history of elevated iop , prior use of glaucoma medication , other intraocular eye disease , or other diseases affecting visual field ( e.g. , pituitary lesions , demyelinating diseases , human immunodeficiency virus positive or acquired immune deficiency syndrome ) . further , patients were excluded if there was a diagnosis of alzheimer 's disease , parkinson 's disease , or previous brain injury including stroke , psychoses , or other neurological or psychiatric conditions that would prevent participation in a psychophysical test . we required two reliable tests ( less than 25% fixation losses , false negatives , and false positives ) on 24 - 2 standard automated perimetry ( carl zeiss meditec , dublin , ca , usa ) using the swedish interactive threshold algorithm in both eyes at baseline for inclusion in the study . reliability was defined as < 33% false positives , false negatives , and fixation losses . a field was considered normal if the pattern standard deviation was not triggered at 5% or less , the glaucoma hemifield test was within normal limits , and the field showed no sign of glaucomatous defect based on subjective evaluation from the enrolling clinician . the optic nerves were assessed by stereoscopic photos by a fellowship - trained glaucoma specialist . subjects with any structural finding suggestive of glaucoma ( i.e. , notching , rim thinning , disc hemorrhages , or retinal nerve fiber layer defects as judged ) were excluded . twenty - four eyes of 12 individuals of ad and 18 eyes of 9 individuals of ed underwent ocular mechanical compliance testing , as follows . baseline edi - sdoct imaging of the optic nerve was performed prior to iop elevation with the spectralis oct ( heidelberg engineering ; family acquisition module 5.4.8.0 ) with 24 radial scans centered on the center of the optic nerve head using enhanced depth imaging . scans were evaluated for imaging quality by the operator , and images were re - acquired if there was improper b - scan positioning in the imaging frame , a quality score < 20 , or poor centration of the optic nerve head . radial scans were acquired using a signal averaging of nine b - scans . a sample of the effect of the acute iop elevation is reported in figure 1 . for visualization purposes , the bruch 's membrane in the b - scan acquired at elevated iop ( fig . 1b ) is used as a reference line to align the scan at the reference iop ( fig . 1a ) ; from the superimposition of anterior lamina cribrosa surface ( alcs ) outline at reference- and high - pressure , it is noticeable the shift of the alcs caused by the iop elevation ( fig . it is important to note that the 3d quantification method used in this study and described in the next paragraph does not require an alignment to the b - scans to the bm plane , and that the alcs depth variation is estimated in the actual 3d space and not the 2d image plane . simplified representation of the morphological changes in alcs depth visible in a comparison of the same radial b - scan before ( a ) and after ( b ) the iop elevation . a variation of the lc is visible with the superimposition of the reference ( green ) and elevated ( red ) iop b - scans , respectively ( c ) . in order to induce a temporary elevation of the iop , a compressive force of approximately 35 pa on the inferior temporal globe was applied by means of an ophthalmodynamometer , in a similar manner described by agoumi et al . this compressive force caused increase in iop of approximately 14 mm hg and did not visibly disturb oct image quality . compression was maintained while the following steps were performed : 60 seconds waiting time allowing short - time mechanical effects of the tissue to be minimized ; an edi - sdoct scan was re - acquired in a similar manner to baseline testing ; iop measurement was re - performed with a tonopen to estimate the actual increase of pressure . after 5 minutes following initial iop elevation , an additional measurement of iop was performed . in order to enhance the visibility of the deeper tissue , all b - scans from the edi - sdoct volumes were postprocessed using adaptive light attenuation compensation , in order to increase a clearer visualization of the lamina cribrosa . light attenuation compensated scan volumes were then loaded in a custom software developed for 3d delineation of histologic and oct data that has been described in prior publications based on the visualization toolkit ( vtk , clifton park , ny , usa ) . a trained observer masked to subject characteristics manually delineated the internal limiting membrane ( ilm ) , bmo , inner and outer choroidal layers , and the anterior surface of the lc in 24 equally spaced radial sections of each oct scan . an average of 202 points ( range , 23534 ) were used to define the lc anterior surface . considering that former studies observed significant differences in the onh morphology of ed and ad for all the oct scans , the area of the 3d surface representing the delineated inner laminar surface was estimated . differences in laminar visibility by race and iop elevation were assessed and reported in the results section . since the difference in alcs position seen with iop elevation was independent of the chosen reference plane , the commonly used bmo reference plane - based measures were used for the present analysis . this similarity between reference planes is possibly due to the lack of significant change in choroidal thickness with the acute increase of iop ( p = 0.94 ) . since the bmo is nearly planar , its best - fitting plane , computed through principal component analysis of the bmo samples , can serve as a reference plane . the depth of a laminar point is its distance from this bmo plane , positive only on the laminar side of the bmo plane . laminar depth is sampled across a reconstruction of the lamina , leading to a computation of mean laminar depth , as follows . to build a uniform sampling of the lc sections , each section is downsampled ( while preserving shape , using the douglas - peucker decimation algorithm ) , interpolated by a b - spline curve , then uniformly sampled . an optimal triangle mesh is built from this uniform sampling of the lc sections ( using the classic contour reconstruction algorithm of song et al . ) . the centroids of each triangle of this mesh are found , and the laminar depth at each centroid is measured ( its distance from the bmo plane ) . mean laminar depth is now a weighted linear combination of these centroid depths and weighted by triangle area ( weight = triangle area / mesh area ) in order to achieve a standardized quantification independent of the number of sampling points used for the definition of the laminar surface morphology . the prelaminar tissue depth of a point of the ilm is its distance from the bmo reference plane . as with laminar depth , a mesh is constructed and depths are sampled across this mesh to calculate the mean depth of the ilm . for prelaminar tissue depth , measurements are constrained to the cylinder above the best - fitting ellipse of the bmo . prelaminar tissue depth was quantified based on distance from the bmo reference plane to the surface of the manually delineated ilm . as with laminar depth , a mesh was constructed and depths were sampled uniformly across this mesh to calculate the depth of the ilm . measurements were constrained to the cylinder above the best - fitting ellipse of the bmo . prelaminar tissue depth is positive on the laminar side of the bmo plane , otherwise negative . the methods for computing alcs and plt depth are described in detail in a prior publication . in order to assess the dependency of the measurements from the interrater variability , we assessed the bland - altman limit - of - agreement and the intraclass correlation coefficient ( icc ) for the laminar depth measurement . for that purpose , 12 oct scans were manually delineated from both raters , and laminar depth was computed using the quantification method described in the previous paragraph . a bland - altam plot reporting the limit - of - agreement ( 18 m , 95% confidence interval [ ci ] ) for the mean laminar depth estimated by the two raters in right plot of figure 3 , it can be noticed that the magnitude of disagreement was correlated ( pearson = 0.62 ) with the magnitude of the measurement that invalidates the interpretation of the limit - of - agreement as an estimate of the interrater variability . the lower agreement between raters observed for shallow laminae and a higher agreement for deeper laminae were plausibly imputable to the lamina visibility change as a function of mean depth ; a higher amount of plt present in shallower onhs would diminish the visibility of the anterior laminar surface . for the intraclass correlation coefficient , the i score at 95% ci was 0.932 < icc < 0.994 , showing a robust delineation and quantification procedure in the assessment of the laminar depth measurement . generalized estimating equations ( gees ) accounting for the correlation among fellow eyes from the same individual were used in multivariate models where the response variables were the change in depth of the lc and plt . only covariates that showed a significant association with the response variables were used in the multivariate model . jackknife resampling and bootstrapping were used to assess the dependency of the outcomes from high leverage data points and the sample size . statistical analyses were performed using the r software ( r foundation for statistical computing , vienna , austria ) . demographic and ocular characteristics from the enrolled patients in each racial group are illustrated in table 1 . the african descent group was significantly younger than the ed group ( table 1 ) . thus , age was accounted as a continuous variable when found significantly associated with the response variable . the african descent group had significantly thinner cct than the ed groups ( p = 0.0086 , table 1 ) ; al and ora parameters were similar across racial groups . the ophthalmodynamometry - induced iop elevation was on average 13.5 4.9 mm hg , but it varied with race ( fig . 2 ) ; the average increase in iop in the ed group was 11.5 mm hg , which was significantly lower than in the ad group 15 mm hg , p = 0.0235 . after 5 minutes from the ophthalmodynamometry compression , iop dropped from a baseline of 18.5 mm hg to 15.1 mm hg , p < 0.001 ; iop at 5 minutes was not significantly different between ed and ad , p = 0.526 ( table 1 ) . scatterplots of relevant covariates that showed significant correlations with change in laminar position in response to iop are shown in figure 4 . in agreement with published studies , bmo area was 22% larger in the ad group ( p < 0.001 ) , but despite this morphological difference the variation of the bmo area with iop was not significant for either groups , nor was correlated with laminar displacement . furthermore , laminar visibility was not significantly different ( p = 0.51 ) with race or as a consequence of the iop elevation ( p = 0.92 ) , as shown in figure 5 . demographic and ocular characteristics across ad and ed groups box - and - whisker plots of the iop distributions for the ed ( blue ) and ad ( red ) individuals . morphological parameters here reported correspond to the baseline iop and after 2 minutes ( min . ) of the ophthalmodynamometry . disagreement in the estimate of the mean laminar depth ( lc depth ) against the mean value estimated by the two raters is reported in the left plot . dashed line = 95% ci . the right plot reports the association between magnitude of the disagreement and measurement magnitude computed as suggested by the authors in altam et al . and scatterplots , regression line , and 95% cis of the following outcomes : ( a ) anterior lamina cribrosa surface displacement ( lc displ . ) resulted negatively correlated to an increase of iop increase ( iop ) in the ed group ( blue ) ; the ad group ( red ) showed a inverse response to iop compared to eds ( p = 0.0207 ) consisting in a posterior displacement of the lc with increasing iop . was positively associated to crf ; the association did not show significant differences with race ; ( c ) lc displ . regression with al ; in longer eyes the lc displaced significantly more posteriorly ( 0.0169 ) . ( d ) the prelaminar tissue ( plt ) tightly followed the direction of the lc displ . ( p < 0.001 ) ; the effect was not found different in the racial strata . difference in the visible anterior surface area before ( reference , ref . ) and after acute pressure elevation ( deformed , def . ) for the ed and ad groups . lamina visibility did not show a significant association across racial strata and iop increase , p = 0.51 . there was no significant association between age or bruch 's membrane opening and the alcs displacement . for each millimeter of al increase above the average , there was an associated increase in the alcs posterior displacement of 10.44 m ( p = 0.0169 , fig . 4c ) . increasing corneal resistance factor ( 7.58 m/m , p = 0.0078 ) , cct ( 0.26 m / mm hg , p = 0.0137 ) , and ch ( 5.47 m / mm hg , p = 0.020 ) were all corneal resistance factor , ch , and cct were highly correlated ( pearson correlation coefficient : cct / ch 0.617 ; cct / crf 0.534 ; ch / crf 0.768 ) ; hence in order to avoid multicollinearity effects only one of three corneal parameters was used as a covariate in the multivariable model . the corneal resistance factor was chosen based on an assessment of the goodness of fitting gaged by the akaike information criterion score . the results of the multivariable model for the amount of anterior laminar surface depth change and plt across racial groups are shown in table 2 . adjusted for age , al , crf , and the actual iop , the lc in the ed group showed a significant anterior displacement ( 9.75 m ; p = 0.0149 ) compared to ad ( 19 m ; p = 0.0092 ) . in the ed group , the alcs displaced anteriorly ( 3.34 m / mm hg ; p = 0.01 ) in response to increased iop . the african descent group showed a significantly different response than the ed group ( p = 0.0207 ; table 2 ) with a greater posterior displacement of the alcs , as shown in figure 4a . multivariable model of the average anterior laminar surface ( lc displ . ) and plt displacement ( plt displ . ) following the acute iop elevation while plt strongly correlated with changes in the underlying laminar surface ( p < 0.001 , se = 0.012 ; table 2 ; fig . 4d ) , the change in plt did not reach statistical significance across racial groups ( p = 0.064 ) or vary with al ( p = 0.99 ) . there were similar independent associations seen between plt with the change in iop and the iop at baseline ( table 2 ) . the results of this study suggest that the direction and magnitude of displacement of the lamina cribrosa following acute elevation of iop is dependent with the magnitude of the iop increase , differences in al , corneal thickness and ora parameters , and race ( ad and ed ) . specifically , after adjusting for the magnitude of the iop increase , greater posterior displacement of the lamina cribrosa was associated with longer al , thicker and stiffer ( higher ch , and crf ) corneas , and in individuals of ad . these results are consistent with published computational modeling studies , in addition to experimental in vivo and ex vivo studies of the human optic nerve head and peripapillary sclera as reviewed below . this finding of both anterior and posterior displacement of the lamina cribrosa following an increase in iop can be mechanistically understood by considering two counterbalancing forces that impact the optic nerve region , the out - of - plane compression caused by the iop , and in - plane stretching caused by the canal border displacement . using numerical simulations , sigal and coworkers estimated that the direction of the laminar surface displacement was tightly dependent of the mechanical rigidity of the sclera and lamina cribrosa , in addition to laminar thickness . in these computational models , it was shown that the direction of deformation of the lamina was primarily regulated by peripapillary sclera rigidity . indeed , for the same magnitude of iop increase , in an eye with a more rigid sclera it was associated with a posterior deformation of the lamina , while in an eye with a more compliant peripapillary sclera it was associated with an anteriorization ( shallowing ) of the lamina . this mechanism of deformation can be easily understood by looing at the lamina as a deformable membrane structure anchored to the onh canal borders and subject to two opposing hydrostatic forces that are the iop and the cerebrospinal fluid pressure . these forces create a direct translaminar pressure gradient that engenders posterior laminar displacement . along with these out - of - plane forces , the canal enlargement creates a third in - plane ( membrane ) force that will instead draw the lamina cribrosa taut and pull anteriorly . for example , in a compliant eye , an increase of iop will likely generate an enlargement of the canal that will increase the in - plane strain of the lamina , flattening it . on the opposite side , a more rigid peripapillary sclera will allow only a small enlargement of the onh canal , resulting in an in - plane tensile strain in the lamina that will not be enough to counterbalance the increase of hydrostatic pressure above the laminar surface ; consequentially , a posterior bowing of the lamina will be observed . the interplay between these in - plane and out - of - plane forces sets the biomechanical behavior of the laminar microenvironment . indeed , anterior migration of the lamina cribrosa has also been demonstrated in human glaucoma in an ed population . considering these complex interactions , the greater posterior displacement in the ad group observed in the current study is in agreement with previous works from our group showing increased scleral stiffness in normal eyes from ad donors discussed below . fazio et al . have published experimental results using laser electronic speckle pattern interferometry ( espi ) to quantify the displacement field in mechanical inflation testing of the posterior pole in normal human donor eyes . with this approach , we demonstrated that there is age - related stiffening of the peripapillary sclera , that these changes occur most dramatically in regions of the onh known to be susceptible to glaucoma , and that these age - related changes differ significantly across ad and ed groups . . showed that a stiffer ring of collagen fibrils around the scleral canal promotes posterior displacements of the lamina cribrosa while reducing scleral canal expansion . recently , we have shown that the peripapillary sclera is stiffer and stiffens more rapidly with iop in donors of ad compared to ed , due to a higher shear stiffness and a lower level of stretch at which the collagen fibrils uncrimp . consequently , the greater posterior displacement in patients of ad may be due to a stiffer peripapillary sclera in this racial subpopulation . the stiffer peripapillary sclera may be , in part , due to the higher percentage of occurrence of meridional fibers that yan et al . a stiffer peripapillary sclera would inhibit the canal expansion to increasing iop and promote the larger posterior displacement of the plt that was found associated with higher baseline iop , as peripapillary sclera stiffens nonlinearly with increasing iop . unfortunately , the currently poor visibility in oct images of the scleral tissue does not permit direct observation of the canal response to the iop changes . furthermore , coudrillier et al . estimated that a stiffer sclera is associated with glaucomatous eyes . the same authors also showed that biomechanics of the peripapillary sclera is directly linked to the deformability of the onh region . these age - related changes and racial differences in scleral material properties and mechanical behavior likely play a critical role in modulating acute strain within the lamina cribrosa that is thought to be important in strain - mediated laminar remodeling in glaucoma . greater posterior displacement of the lamina cribrosa in the ad group is also consistent with our previous findings in vivo that there is an anterior shift in the laminar surface associated with aging in ed , but not ad normals . these differential aging effects could reflect the impact of remodeling due to racial variation in acute strain within the lamina and peripapillary sclera suggested in the current in vivo study . in the current study , the laminar position also shifted anteriorly with acute iop elevation prominently in the ed group ( 9.8 m , p = 0.0149 ; table 2 ; fig . these data suggest that the ad group may experience a proportionally greater translaminar strain compared to membrane strain than the ed group , which would result in greater posterior rather than anterior displacement and may have an impact on long - term age - related remodeling of these load bearing tissues . the relationship between cct , ch , and crf with laminar displacement can also be understood within the context of this model . a thicker or stiffer cornea could correspond to a stiffer and thicker ( more rigid ) sclera ; hence for the same increase of iop the lamina would more likely be displaced posteriorly than stretched because of the reduced enlargement of the onh canal in stiffer sclera . however , the relationship between the morphology and material properties of the cornea and the posterior sclera and optic nerve , while hypothesized , has not been clearly demonstrated experimentally . it is very important to notice that many factors seem to affect the laminar displacement . interestingly , the three eyes in figure 4a that show a very large deepening of the lamina indeed have at the same time a very high crf ( stiff eye ) and long al ( thinner lamina ) . the positive correlation between al and posterior deformation for the lamina is less intuitive . a plausible explanation of this association could be suggested by the negative correlation between laminar thickness and al found in normal and glaucomatous individuals . a thinner lamina would be more compliant to the direct effect of increased iop than a thicker lamina , and this could explain why with an increase of al we observe a more posterior displacement of the lamina ( fig . agoumi et al . , using an approach similar to ours to evaluate how shifts in laminar position differed between a group of 12 patients with open angle glaucoma and 12 age - matched controls , found that while overall there was minimal difference in the means for laminar surface position at high and low iop , there was a range of anterior and posterior shifts in response to iop increase . these findings did not show a difference between glaucomatous and normal eyes , which was not evaluated in the current study ; however , the study participants were all of ed , only four best corresponding scans at baseline and during iop elevation were used for quantification of the morphological parameters , and edi method was not available at the time of the study . this is still consistent with our results in that overt posterior deformation was prominently seen in the ad group . furthermore , we employed a 3d delineation system that allows for visualization and quantification of the entire visualizable lamina cribrosa rather than using a few selected b - scans . also the 3d approach mitigates laminar depth quantification errors arising form the misalignments between the low- and high - pressure b - scans . indeed , it is important to consider that the scan - points that lie in each b - scan of the low - pressure oct acquisition do not necessarily lie in a plane after the iop - induced morphological deformations . the measurement of iop during pressure elevation , while immediately preceding imaging , is not simultaneous to the oct acquisition . since the force was manually applied , there could be some variation in iop between when iop is measured and the oct image is obtained . . however , any effect would likely be nondifferential across racial groups and consistent iop could be maintained easily for over 60 seconds in our preliminary testing using the ophthalmodynamometry . in order to minimize testing time for the enrolled individuals , in this study we set the in - between low- and high - pressure oct scans waiting time to 60 seconds , under the assumption that the viscoelastic response of the lamina and peripapillary sclera lay in the same temporal order of magnitude . to the best of our knowledge , there is no available data that allow quantifying the time - scale of viscosity effects on the onh deformation . empirically , we know that the deformations of the peripapillary sclera following an ex - vivo acute pressure elevation stabilizes after a range of 5 to 20 seconds . peripapillary sclera deformations are certainly linked to laminar deformation , but we do not know to what extent the viscoelastic properties of these tissues are alike . it is plausible that viscoelastic properties of the lamina differ across races and this could affect the magnitude of displacement ; despite that a racial difference of the viscoelastic properties of the onh region could not cause the opposite laminar displacement directions observed in the two groups . the anterior lamina cribrosa surface could be only delineated in areas where its superior surface was clearly visible . laminar visibility is affected by several factors ; the most influential factors are the presence of shadowing retinal vessels and other individual - specific morphological features ( thick plt ) that can rapidly weaken the oct signal . it is important to notice that laminar visibility can affect the sampling region and this can cause a bias in the estimate of the laminar depth , although , our custom resampling approach was developed to minimize this bias . that considered , laminar visibility was not different for the two racial groups , neither changed after iop elevation , as shown in figure 5 . furthermore , it is important to keep in mind that the objective of this study is not the assessment of the actual laminar depth but its variation following an iop increase , in the same eye . moreover , the main outcome of this study is the observation in the two racial groups of an inverse trend in laminar direction of deformation , and there is no plausible explanation of how laminar visibility could oppositely bias the direction of laminar displacement in the two groups . on the statistical model , we included all the measurable parameters that showed a significant association with laminar depth displacement , but we are certain that other influential factors that are not currently measured in this study have a significant influence on the laminar depth displacement . for example , the morphology of the canal is likely a very important biomechanical influential parameter for lamina deformation , but the current oct technology does not allow an accurate estimate of that region . interestingly , disk size ( bmo area ) did not show a significant association with the change in laminar depth , suggesting that this parameter is not a biomechanical surrogate for the canal size . the current linear regression model can not provide an estimate of the lamina cribrosa surface displacement to small iop increases . considering that at zero iop the lc displacement should be zero , the actual functional relation between lc displacement and increase of iop ( lc displ . versus iop ; fig . 4a ) is likely a high - degree nonmonotonic curve whose statistical modeling would require a much larger number of observations at lower iop than those available in this study . ideally , a larger set of data points associated with low iop values would allow an estimate of the regression shape close to the zero pressure increase point . it is very important to notice that while age , crf , and al vary cross - sectionally among individuals , the lamina cribrosa displacement is longitudinally measured in the same eye , so each laminar depth change is computed against its internal control . in summary , we demonstrated that there was greater posterior deformation in the lamina cribrosa in ad compared to ed normal individuals in response to an acute increase of iop . this significant variation in laminar compliance across racial groups may be related to described differences in the morphometric and material properties of the onh region and could explain some of the differential susceptibility to glaucomatous injury in the at - risk ad population . dedicated studies are needed to determine if longitudinal variations in compliance of the lamina cribrosa are associated with the development and progression of glaucoma and how compliance changes over time with the disease .

purposeto assess if the in vivo mechanical displacement of the anterior laminar cribrosa surface ( alcs ) as a response of an acute elevation in intraocular pressure ( iop ) differs in individuals of european ( ed ) and african descent ( ad).methodsspectral - domain optical coherence tomography ( sdoct ) scans were obtained from 24 eyes of 12 individuals of ad and 18 eyes of 9 individuals of ed at their normal baseline iop and after 60 seconds iop elevation using ophthalmodynamometry . change in depth ( displacement ) of the lc and to the prelaminar tissue ( plt ) were computed in association with the change ( delta ) in iop ( iop ) , race , age , corneal thickness , corneal rigidity ( ocular response analyzer [ ora ] ) , and axial.resultsin the ed group for small iop elevations ( iop < 12 mm hg ) , the alcs initially displaced posteriorly but for larger increase of iop an anterior displacement of the lamina followed . inversely , in the ad group the alcs did not show a significant posterior displacement for small iop , while for larger iop increases the alcs significantly displaced posteriorly . posterior displacement of the lamina cribrosa ( lc ) was also significantly correlated with longer axial length , higher corneal thickness , and ora parameters . prelaminar tissue posteriorly displaced for any magnitude of iop , in both groups.conclusionsthe african descent group demonstrated a greater acute posterior bowing of the lc after adjustment for age , axial length , bruch 's membrane opening ( bmo ) area , and ora parameters . greater plt posterior displacement was also seen in the ad group with increasing iop , which was tightly correlated with the displacement of the lc .

Materials and Methods Inclusion/Exclusion Criteria Compliance Testing: Acute IOP Elevation and SDOCT Image Acquisition Protocol EDI-SDOCT Image Processing and Quantification Computing Laminar and Prelaminar Tissue (PLT) Depth Statistical Analysis Results Discussion

each participant underwent a complete ophthalmological exam that included medical history , snellen best - corrected visual acuity , early treatment diabetic retinopathy study ( etdrs ) visual acuity , color vision , slit - lamp biomicroscopy , applanation tonometry for iop , central corneal thickness ( cct ) measurement , axial length ( al ) measurement , dilated funduscopy , stereoscopic ophthalmoscopy of the optic disc , and stereoscopic fundus photography . additionally , all subjects underwent testing with the ocular response analyzer ( ora ) to obtain measurements of corneal hysteresis ( ch ) and corneal resistance factor ( crf ) preceding baseline edi - sdoct imaging for the compliance testing . participants were excluded if they had a history of intraocular surgery ( except for uncomplicated cataract surgery ) , elevated iop ( > 22 mm hg ) at the time of the study , a history of elevated iop , prior use of glaucoma medication , other intraocular eye disease , or other diseases affecting visual field ( e.g. twenty - four eyes of 12 individuals of ad and 18 eyes of 9 individuals of ed underwent ocular mechanical compliance testing , as follows . scans were evaluated for imaging quality by the operator , and images were re - acquired if there was improper b - scan positioning in the imaging frame , a quality score < 20 , or poor centration of the optic nerve head . for visualization purposes , the bruch 's membrane in the b - scan acquired at elevated iop ( fig . 1a ) ; from the superimposition of anterior lamina cribrosa surface ( alcs ) outline at reference- and high - pressure , it is noticeable the shift of the alcs caused by the iop elevation ( fig . it is important to note that the 3d quantification method used in this study and described in the next paragraph does not require an alignment to the b - scans to the bm plane , and that the alcs depth variation is estimated in the actual 3d space and not the 2d image plane . simplified representation of the morphological changes in alcs depth visible in a comparison of the same radial b - scan before ( a ) and after ( b ) the iop elevation . a variation of the lc is visible with the superimposition of the reference ( green ) and elevated ( red ) iop b - scans , respectively ( c ) . in order to induce a temporary elevation of the iop , a compressive force of approximately 35 pa on the inferior temporal globe was applied by means of an ophthalmodynamometer , in a similar manner described by agoumi et al . in order to enhance the visibility of the deeper tissue , all b - scans from the edi - sdoct volumes were postprocessed using adaptive light attenuation compensation , in order to increase a clearer visualization of the lamina cribrosa . a trained observer masked to subject characteristics manually delineated the internal limiting membrane ( ilm ) , bmo , inner and outer choroidal layers , and the anterior surface of the lc in 24 equally spaced radial sections of each oct scan . this similarity between reference planes is possibly due to the lack of significant change in choroidal thickness with the acute increase of iop ( p = 0.94 ) . the prelaminar tissue depth of a point of the ilm is its distance from the bmo reference plane . for prelaminar tissue depth , measurements are constrained to the cylinder above the best - fitting ellipse of the bmo . the lower agreement between raters observed for shallow laminae and a higher agreement for deeper laminae were plausibly imputable to the lamina visibility change as a function of mean depth ; a higher amount of plt present in shallower onhs would diminish the visibility of the anterior laminar surface . generalized estimating equations ( gees ) accounting for the correlation among fellow eyes from the same individual were used in multivariate models where the response variables were the change in depth of the lc and plt . only covariates that showed a significant association with the response variables were used in the multivariate model . participants were excluded if they had a history of intraocular surgery ( except for uncomplicated cataract surgery ) , elevated iop ( > 22 mm hg ) at the time of the study , a history of elevated iop , prior use of glaucoma medication , other intraocular eye disease , or other diseases affecting visual field ( e.g. we required two reliable tests ( less than 25% fixation losses , false negatives , and false positives ) on 24 - 2 standard automated perimetry ( carl zeiss meditec , dublin , ca , usa ) using the swedish interactive threshold algorithm in both eyes at baseline for inclusion in the study . a field was considered normal if the pattern standard deviation was not triggered at 5% or less , the glaucoma hemifield test was within normal limits , and the field showed no sign of glaucomatous defect based on subjective evaluation from the enrolling clinician . twenty - four eyes of 12 individuals of ad and 18 eyes of 9 individuals of ed underwent ocular mechanical compliance testing , as follows . scans were evaluated for imaging quality by the operator , and images were re - acquired if there was improper b - scan positioning in the imaging frame , a quality score < 20 , or poor centration of the optic nerve head . for visualization purposes , the bruch 's membrane in the b - scan acquired at elevated iop ( fig . 1a ) ; from the superimposition of anterior lamina cribrosa surface ( alcs ) outline at reference- and high - pressure , it is noticeable the shift of the alcs caused by the iop elevation ( fig . it is important to note that the 3d quantification method used in this study and described in the next paragraph does not require an alignment to the b - scans to the bm plane , and that the alcs depth variation is estimated in the actual 3d space and not the 2d image plane . simplified representation of the morphological changes in alcs depth visible in a comparison of the same radial b - scan before ( a ) and after ( b ) the iop elevation . a variation of the lc is visible with the superimposition of the reference ( green ) and elevated ( red ) iop b - scans , respectively ( c ) . in order to induce a temporary elevation of the iop , a compressive force of approximately 35 pa on the inferior temporal globe was applied by means of an ophthalmodynamometer , in a similar manner described by agoumi et al . in order to enhance the visibility of the deeper tissue , all b - scans from the edi - sdoct volumes were postprocessed using adaptive light attenuation compensation , in order to increase a clearer visualization of the lamina cribrosa . a trained observer masked to subject characteristics manually delineated the internal limiting membrane ( ilm ) , bmo , inner and outer choroidal layers , and the anterior surface of the lc in 24 equally spaced radial sections of each oct scan . considering that former studies observed significant differences in the onh morphology of ed and ad for all the oct scans , the area of the 3d surface representing the delineated inner laminar surface was estimated . since the difference in alcs position seen with iop elevation was independent of the chosen reference plane , the commonly used bmo reference plane - based measures were used for the present analysis . this similarity between reference planes is possibly due to the lack of significant change in choroidal thickness with the acute increase of iop ( p = 0.94 ) . a bland - altam plot reporting the limit - of - agreement ( 18 m , 95% confidence interval [ ci ] ) for the mean laminar depth estimated by the two raters in right plot of figure 3 , it can be noticed that the magnitude of disagreement was correlated ( pearson = 0.62 ) with the magnitude of the measurement that invalidates the interpretation of the limit - of - agreement as an estimate of the interrater variability . the lower agreement between raters observed for shallow laminae and a higher agreement for deeper laminae were plausibly imputable to the lamina visibility change as a function of mean depth ; a higher amount of plt present in shallower onhs would diminish the visibility of the anterior laminar surface . generalized estimating equations ( gees ) accounting for the correlation among fellow eyes from the same individual were used in multivariate models where the response variables were the change in depth of the lc and plt . only covariates that showed a significant association with the response variables were used in the multivariate model . the african descent group was significantly younger than the ed group ( table 1 ) . the african descent group had significantly thinner cct than the ed groups ( p = 0.0086 , table 1 ) ; al and ora parameters were similar across racial groups . 2 ) ; the average increase in iop in the ed group was 11.5 mm hg , which was significantly lower than in the ad group 15 mm hg , p = 0.0235 . in agreement with published studies , bmo area was 22% larger in the ad group ( p < 0.001 ) , but despite this morphological difference the variation of the bmo area with iop was not significant for either groups , nor was correlated with laminar displacement . furthermore , laminar visibility was not significantly different ( p = 0.51 ) with race or as a consequence of the iop elevation ( p = 0.92 ) , as shown in figure 5 . demographic and ocular characteristics across ad and ed groups box - and - whisker plots of the iop distributions for the ed ( blue ) and ad ( red ) individuals . morphological parameters here reported correspond to the baseline iop and after 2 minutes ( min . ) disagreement in the estimate of the mean laminar depth ( lc depth ) against the mean value estimated by the two raters is reported in the left plot . and scatterplots , regression line , and 95% cis of the following outcomes : ( a ) anterior lamina cribrosa surface displacement ( lc displ . ) resulted negatively correlated to an increase of iop increase ( iop ) in the ed group ( blue ) ; the ad group ( red ) showed a inverse response to iop compared to eds ( p = 0.0207 ) consisting in a posterior displacement of the lc with increasing iop . ( d ) the prelaminar tissue ( plt ) tightly followed the direction of the lc displ . lamina visibility did not show a significant association across racial strata and iop increase , p = 0.51 . there was no significant association between age or bruch 's membrane opening and the alcs displacement . for each millimeter of al increase above the average , there was an associated increase in the alcs posterior displacement of 10.44 m ( p = 0.0169 , fig . increasing corneal resistance factor ( 7.58 m/m , p = 0.0078 ) , cct ( 0.26 m / mm hg , p = 0.0137 ) , and ch ( 5.47 m / mm hg , p = 0.020 ) were all corneal resistance factor , ch , and cct were highly correlated ( pearson correlation coefficient : cct / ch 0.617 ; cct / crf 0.534 ; ch / crf 0.768 ) ; hence in order to avoid multicollinearity effects only one of three corneal parameters was used as a covariate in the multivariable model . adjusted for age , al , crf , and the actual iop , the lc in the ed group showed a significant anterior displacement ( 9.75 m ; p = 0.0149 ) compared to ad ( 19 m ; p = 0.0092 ) . in the ed group , the alcs displaced anteriorly ( 3.34 m / mm hg ; p = 0.01 ) in response to increased iop . the african descent group showed a significantly different response than the ed group ( p = 0.0207 ; table 2 ) with a greater posterior displacement of the alcs , as shown in figure 4a . multivariable model of the average anterior laminar surface ( lc displ . ) following the acute iop elevation while plt strongly correlated with changes in the underlying laminar surface ( p < 0.001 , se = 0.012 ; table 2 ; fig . 4d ) , the change in plt did not reach statistical significance across racial groups ( p = 0.064 ) or vary with al ( p = 0.99 ) . there were similar independent associations seen between plt with the change in iop and the iop at baseline ( table 2 ) . the results of this study suggest that the direction and magnitude of displacement of the lamina cribrosa following acute elevation of iop is dependent with the magnitude of the iop increase , differences in al , corneal thickness and ora parameters , and race ( ad and ed ) . specifically , after adjusting for the magnitude of the iop increase , greater posterior displacement of the lamina cribrosa was associated with longer al , thicker and stiffer ( higher ch , and crf ) corneas , and in individuals of ad . this finding of both anterior and posterior displacement of the lamina cribrosa following an increase in iop can be mechanistically understood by considering two counterbalancing forces that impact the optic nerve region , the out - of - plane compression caused by the iop , and in - plane stretching caused by the canal border displacement . using numerical simulations , sigal and coworkers estimated that the direction of the laminar surface displacement was tightly dependent of the mechanical rigidity of the sclera and lamina cribrosa , in addition to laminar thickness . indeed , for the same magnitude of iop increase , in an eye with a more rigid sclera it was associated with a posterior deformation of the lamina , while in an eye with a more compliant peripapillary sclera it was associated with an anteriorization ( shallowing ) of the lamina . this mechanism of deformation can be easily understood by looing at the lamina as a deformable membrane structure anchored to the onh canal borders and subject to two opposing hydrostatic forces that are the iop and the cerebrospinal fluid pressure . for example , in a compliant eye , an increase of iop will likely generate an enlargement of the canal that will increase the in - plane strain of the lamina , flattening it . on the opposite side , a more rigid peripapillary sclera will allow only a small enlargement of the onh canal , resulting in an in - plane tensile strain in the lamina that will not be enough to counterbalance the increase of hydrostatic pressure above the laminar surface ; consequentially , a posterior bowing of the lamina will be observed . considering these complex interactions , the greater posterior displacement in the ad group observed in the current study is in agreement with previous works from our group showing increased scleral stiffness in normal eyes from ad donors discussed below . a stiffer peripapillary sclera would inhibit the canal expansion to increasing iop and promote the larger posterior displacement of the plt that was found associated with higher baseline iop , as peripapillary sclera stiffens nonlinearly with increasing iop . unfortunately , the currently poor visibility in oct images of the scleral tissue does not permit direct observation of the canal response to the iop changes . greater posterior displacement of the lamina cribrosa in the ad group is also consistent with our previous findings in vivo that there is an anterior shift in the laminar surface associated with aging in ed , but not ad normals . these differential aging effects could reflect the impact of remodeling due to racial variation in acute strain within the lamina and peripapillary sclera suggested in the current in vivo study . in the current study , the laminar position also shifted anteriorly with acute iop elevation prominently in the ed group ( 9.8 m , p = 0.0149 ; table 2 ; fig . these data suggest that the ad group may experience a proportionally greater translaminar strain compared to membrane strain than the ed group , which would result in greater posterior rather than anterior displacement and may have an impact on long - term age - related remodeling of these load bearing tissues . a thicker or stiffer cornea could correspond to a stiffer and thicker ( more rigid ) sclera ; hence for the same increase of iop the lamina would more likely be displaced posteriorly than stretched because of the reduced enlargement of the onh canal in stiffer sclera . interestingly , the three eyes in figure 4a that show a very large deepening of the lamina indeed have at the same time a very high crf ( stiff eye ) and long al ( thinner lamina ) . a thinner lamina would be more compliant to the direct effect of increased iop than a thicker lamina , and this could explain why with an increase of al we observe a more posterior displacement of the lamina ( fig . these findings did not show a difference between glaucomatous and normal eyes , which was not evaluated in the current study ; however , the study participants were all of ed , only four best corresponding scans at baseline and during iop elevation were used for quantification of the morphological parameters , and edi method was not available at the time of the study . this is still consistent with our results in that overt posterior deformation was prominently seen in the ad group . in order to minimize testing time for the enrolled individuals , in this study we set the in - between low- and high - pressure oct scans waiting time to 60 seconds , under the assumption that the viscoelastic response of the lamina and peripapillary sclera lay in the same temporal order of magnitude . it is plausible that viscoelastic properties of the lamina differ across races and this could affect the magnitude of displacement ; despite that a racial difference of the viscoelastic properties of the onh region could not cause the opposite laminar displacement directions observed in the two groups . moreover , the main outcome of this study is the observation in the two racial groups of an inverse trend in laminar direction of deformation , and there is no plausible explanation of how laminar visibility could oppositely bias the direction of laminar displacement in the two groups . interestingly , disk size ( bmo area ) did not show a significant association with the change in laminar depth , suggesting that this parameter is not a biomechanical surrogate for the canal size . the current linear regression model can not provide an estimate of the lamina cribrosa surface displacement to small iop increases . considering that at zero iop the lc displacement should be zero , the actual functional relation between lc displacement and increase of iop ( lc displ . it is very important to notice that while age , crf , and al vary cross - sectionally among individuals , the lamina cribrosa displacement is longitudinally measured in the same eye , so each laminar depth change is computed against its internal control . in summary , we demonstrated that there was greater posterior deformation in the lamina cribrosa in ad compared to ed normal individuals in response to an acute increase of iop . dedicated studies are needed to determine if longitudinal variations in compliance of the lamina cribrosa are associated with the development and progression of glaucoma and how compliance changes over time with the disease .

each participant underwent a complete ophthalmological exam that included medical history , snellen best - corrected visual acuity , early treatment diabetic retinopathy study ( etdrs ) visual acuity , color vision , slit - lamp biomicroscopy , applanation tonometry for iop , central corneal thickness ( cct ) measurement , axial length ( al ) measurement , dilated funduscopy , stereoscopic ophthalmoscopy of the optic disc , and stereoscopic fundus photography . participants were excluded if they had a history of intraocular surgery ( except for uncomplicated cataract surgery ) , elevated iop ( > 22 mm hg ) at the time of the study , a history of elevated iop , prior use of glaucoma medication , other intraocular eye disease , or other diseases affecting visual field ( e.g. baseline edi - sdoct imaging of the optic nerve was performed prior to iop elevation with the spectralis oct ( heidelberg engineering ; family acquisition module 5.4.8.0 ) with 24 radial scans centered on the center of the optic nerve head using enhanced depth imaging . scans were evaluated for imaging quality by the operator , and images were re - acquired if there was improper b - scan positioning in the imaging frame , a quality score < 20 , or poor centration of the optic nerve head . 1a ) ; from the superimposition of anterior lamina cribrosa surface ( alcs ) outline at reference- and high - pressure , it is noticeable the shift of the alcs caused by the iop elevation ( fig . it is important to note that the 3d quantification method used in this study and described in the next paragraph does not require an alignment to the b - scans to the bm plane , and that the alcs depth variation is estimated in the actual 3d space and not the 2d image plane . simplified representation of the morphological changes in alcs depth visible in a comparison of the same radial b - scan before ( a ) and after ( b ) the iop elevation . in order to induce a temporary elevation of the iop , a compressive force of approximately 35 pa on the inferior temporal globe was applied by means of an ophthalmodynamometer , in a similar manner described by agoumi et al . compression was maintained while the following steps were performed : 60 seconds waiting time allowing short - time mechanical effects of the tissue to be minimized ; an edi - sdoct scan was re - acquired in a similar manner to baseline testing ; iop measurement was re - performed with a tonopen to estimate the actual increase of pressure . in order to enhance the visibility of the deeper tissue , all b - scans from the edi - sdoct volumes were postprocessed using adaptive light attenuation compensation , in order to increase a clearer visualization of the lamina cribrosa . light attenuation compensated scan volumes were then loaded in a custom software developed for 3d delineation of histologic and oct data that has been described in prior publications based on the visualization toolkit ( vtk , clifton park , ny , usa ) . considering that former studies observed significant differences in the onh morphology of ed and ad for all the oct scans , differences in laminar visibility by race and iop elevation were assessed and reported in the results section . since the difference in alcs position seen with iop elevation was independent of the chosen reference plane , the commonly used bmo reference plane - based measures were used for the present analysis . mean laminar depth is now a weighted linear combination of these centroid depths and weighted by triangle area ( weight = triangle area / mesh area ) in order to achieve a standardized quantification independent of the number of sampling points used for the definition of the laminar surface morphology . in order to assess the dependency of the measurements from the interrater variability , we assessed the bland - altman limit - of - agreement and the intraclass correlation coefficient ( icc ) for the laminar depth measurement . a bland - altam plot reporting the limit - of - agreement ( 18 m , 95% confidence interval [ ci ] ) for the mean laminar depth estimated by the two raters is shown in figure 3 ( left ) . in right plot of figure 3 , it can be noticed that the magnitude of disagreement was correlated ( pearson = 0.62 ) with the magnitude of the measurement that invalidates the interpretation of the limit - of - agreement as an estimate of the interrater variability . the lower agreement between raters observed for shallow laminae and a higher agreement for deeper laminae were plausibly imputable to the lamina visibility change as a function of mean depth ; a higher amount of plt present in shallower onhs would diminish the visibility of the anterior laminar surface . for the intraclass correlation coefficient , the i score at 95% ci was 0.932 < icc < 0.994 , showing a robust delineation and quantification procedure in the assessment of the laminar depth measurement . generalized estimating equations ( gees ) accounting for the correlation among fellow eyes from the same individual were used in multivariate models where the response variables were the change in depth of the lc and plt . participants were excluded if they had a history of intraocular surgery ( except for uncomplicated cataract surgery ) , elevated iop ( > 22 mm hg ) at the time of the study , a history of elevated iop , prior use of glaucoma medication , other intraocular eye disease , or other diseases affecting visual field ( e.g. baseline edi - sdoct imaging of the optic nerve was performed prior to iop elevation with the spectralis oct ( heidelberg engineering ; family acquisition module 5.4.8.0 ) with 24 radial scans centered on the center of the optic nerve head using enhanced depth imaging . scans were evaluated for imaging quality by the operator , and images were re - acquired if there was improper b - scan positioning in the imaging frame , a quality score < 20 , or poor centration of the optic nerve head . it is important to note that the 3d quantification method used in this study and described in the next paragraph does not require an alignment to the b - scans to the bm plane , and that the alcs depth variation is estimated in the actual 3d space and not the 2d image plane . simplified representation of the morphological changes in alcs depth visible in a comparison of the same radial b - scan before ( a ) and after ( b ) the iop elevation . in order to induce a temporary elevation of the iop , a compressive force of approximately 35 pa on the inferior temporal globe was applied by means of an ophthalmodynamometer , in a similar manner described by agoumi et al . compression was maintained while the following steps were performed : 60 seconds waiting time allowing short - time mechanical effects of the tissue to be minimized ; an edi - sdoct scan was re - acquired in a similar manner to baseline testing ; iop measurement was re - performed with a tonopen to estimate the actual increase of pressure . in order to enhance the visibility of the deeper tissue , all b - scans from the edi - sdoct volumes were postprocessed using adaptive light attenuation compensation , in order to increase a clearer visualization of the lamina cribrosa . light attenuation compensated scan volumes were then loaded in a custom software developed for 3d delineation of histologic and oct data that has been described in prior publications based on the visualization toolkit ( vtk , clifton park , ny , usa ) . considering that former studies observed significant differences in the onh morphology of ed and ad for all the oct scans , the area of the 3d surface representing the delineated inner laminar surface was estimated . mean laminar depth is now a weighted linear combination of these centroid depths and weighted by triangle area ( weight = triangle area / mesh area ) in order to achieve a standardized quantification independent of the number of sampling points used for the definition of the laminar surface morphology . in order to assess the dependency of the measurements from the interrater variability , we assessed the bland - altman limit - of - agreement and the intraclass correlation coefficient ( icc ) for the laminar depth measurement . a bland - altam plot reporting the limit - of - agreement ( 18 m , 95% confidence interval [ ci ] ) for the mean laminar depth estimated by the two raters in right plot of figure 3 , it can be noticed that the magnitude of disagreement was correlated ( pearson = 0.62 ) with the magnitude of the measurement that invalidates the interpretation of the limit - of - agreement as an estimate of the interrater variability . the lower agreement between raters observed for shallow laminae and a higher agreement for deeper laminae were plausibly imputable to the lamina visibility change as a function of mean depth ; a higher amount of plt present in shallower onhs would diminish the visibility of the anterior laminar surface . for the intraclass correlation coefficient , the i score at 95% ci was 0.932 < icc < 0.994 , showing a robust delineation and quantification procedure in the assessment of the laminar depth measurement . generalized estimating equations ( gees ) accounting for the correlation among fellow eyes from the same individual were used in multivariate models where the response variables were the change in depth of the lc and plt . 2 ) ; the average increase in iop in the ed group was 11.5 mm hg , which was significantly lower than in the ad group 15 mm hg , p = 0.0235 . after 5 minutes from the ophthalmodynamometry compression , iop dropped from a baseline of 18.5 mm hg to 15.1 mm hg , p < 0.001 ; iop at 5 minutes was not significantly different between ed and ad , p = 0.526 ( table 1 ) . in agreement with published studies , bmo area was 22% larger in the ad group ( p < 0.001 ) , but despite this morphological difference the variation of the bmo area with iop was not significant for either groups , nor was correlated with laminar displacement . disagreement in the estimate of the mean laminar depth ( lc depth ) against the mean value estimated by the two raters is reported in the left plot . resulted negatively correlated to an increase of iop increase ( iop ) in the ed group ( blue ) ; the ad group ( red ) showed a inverse response to iop compared to eds ( p = 0.0207 ) consisting in a posterior displacement of the lc with increasing iop . increasing corneal resistance factor ( 7.58 m/m , p = 0.0078 ) , cct ( 0.26 m / mm hg , p = 0.0137 ) , and ch ( 5.47 m / mm hg , p = 0.020 ) were all corneal resistance factor , ch , and cct were highly correlated ( pearson correlation coefficient : cct / ch 0.617 ; cct / crf 0.534 ; ch / crf 0.768 ) ; hence in order to avoid multicollinearity effects only one of three corneal parameters was used as a covariate in the multivariable model . adjusted for age , al , crf , and the actual iop , the lc in the ed group showed a significant anterior displacement ( 9.75 m ; p = 0.0149 ) compared to ad ( 19 m ; p = 0.0092 ) . the african descent group showed a significantly different response than the ed group ( p = 0.0207 ; table 2 ) with a greater posterior displacement of the alcs , as shown in figure 4a . following the acute iop elevation while plt strongly correlated with changes in the underlying laminar surface ( p < 0.001 , se = 0.012 ; table 2 ; fig . the results of this study suggest that the direction and magnitude of displacement of the lamina cribrosa following acute elevation of iop is dependent with the magnitude of the iop increase , differences in al , corneal thickness and ora parameters , and race ( ad and ed ) . specifically , after adjusting for the magnitude of the iop increase , greater posterior displacement of the lamina cribrosa was associated with longer al , thicker and stiffer ( higher ch , and crf ) corneas , and in individuals of ad . these results are consistent with published computational modeling studies , in addition to experimental in vivo and ex vivo studies of the human optic nerve head and peripapillary sclera as reviewed below . this finding of both anterior and posterior displacement of the lamina cribrosa following an increase in iop can be mechanistically understood by considering two counterbalancing forces that impact the optic nerve region , the out - of - plane compression caused by the iop , and in - plane stretching caused by the canal border displacement . using numerical simulations , sigal and coworkers estimated that the direction of the laminar surface displacement was tightly dependent of the mechanical rigidity of the sclera and lamina cribrosa , in addition to laminar thickness . indeed , for the same magnitude of iop increase , in an eye with a more rigid sclera it was associated with a posterior deformation of the lamina , while in an eye with a more compliant peripapillary sclera it was associated with an anteriorization ( shallowing ) of the lamina . this mechanism of deformation can be easily understood by looing at the lamina as a deformable membrane structure anchored to the onh canal borders and subject to two opposing hydrostatic forces that are the iop and the cerebrospinal fluid pressure . on the opposite side , a more rigid peripapillary sclera will allow only a small enlargement of the onh canal , resulting in an in - plane tensile strain in the lamina that will not be enough to counterbalance the increase of hydrostatic pressure above the laminar surface ; consequentially , a posterior bowing of the lamina will be observed . the interplay between these in - plane and out - of - plane forces sets the biomechanical behavior of the laminar microenvironment . considering these complex interactions , the greater posterior displacement in the ad group observed in the current study is in agreement with previous works from our group showing increased scleral stiffness in normal eyes from ad donors discussed below . with this approach , we demonstrated that there is age - related stiffening of the peripapillary sclera , that these changes occur most dramatically in regions of the onh known to be susceptible to glaucoma , and that these age - related changes differ significantly across ad and ed groups . recently , we have shown that the peripapillary sclera is stiffer and stiffens more rapidly with iop in donors of ad compared to ed , due to a higher shear stiffness and a lower level of stretch at which the collagen fibrils uncrimp . a stiffer peripapillary sclera would inhibit the canal expansion to increasing iop and promote the larger posterior displacement of the plt that was found associated with higher baseline iop , as peripapillary sclera stiffens nonlinearly with increasing iop . these age - related changes and racial differences in scleral material properties and mechanical behavior likely play a critical role in modulating acute strain within the lamina cribrosa that is thought to be important in strain - mediated laminar remodeling in glaucoma . greater posterior displacement of the lamina cribrosa in the ad group is also consistent with our previous findings in vivo that there is an anterior shift in the laminar surface associated with aging in ed , but not ad normals . these differential aging effects could reflect the impact of remodeling due to racial variation in acute strain within the lamina and peripapillary sclera suggested in the current in vivo study . in the current study , the laminar position also shifted anteriorly with acute iop elevation prominently in the ed group ( 9.8 m , p = 0.0149 ; table 2 ; fig . these data suggest that the ad group may experience a proportionally greater translaminar strain compared to membrane strain than the ed group , which would result in greater posterior rather than anterior displacement and may have an impact on long - term age - related remodeling of these load bearing tissues . however , the relationship between the morphology and material properties of the cornea and the posterior sclera and optic nerve , while hypothesized , has not been clearly demonstrated experimentally . interestingly , the three eyes in figure 4a that show a very large deepening of the lamina indeed have at the same time a very high crf ( stiff eye ) and long al ( thinner lamina ) . a thinner lamina would be more compliant to the direct effect of increased iop than a thicker lamina , and this could explain why with an increase of al we observe a more posterior displacement of the lamina ( fig . , using an approach similar to ours to evaluate how shifts in laminar position differed between a group of 12 patients with open angle glaucoma and 12 age - matched controls , found that while overall there was minimal difference in the means for laminar surface position at high and low iop , there was a range of anterior and posterior shifts in response to iop increase . these findings did not show a difference between glaucomatous and normal eyes , which was not evaluated in the current study ; however , the study participants were all of ed , only four best corresponding scans at baseline and during iop elevation were used for quantification of the morphological parameters , and edi method was not available at the time of the study . in order to minimize testing time for the enrolled individuals , in this study we set the in - between low- and high - pressure oct scans waiting time to 60 seconds , under the assumption that the viscoelastic response of the lamina and peripapillary sclera lay in the same temporal order of magnitude . it is plausible that viscoelastic properties of the lamina differ across races and this could affect the magnitude of displacement ; despite that a racial difference of the viscoelastic properties of the onh region could not cause the opposite laminar displacement directions observed in the two groups . moreover , the main outcome of this study is the observation in the two racial groups of an inverse trend in laminar direction of deformation , and there is no plausible explanation of how laminar visibility could oppositely bias the direction of laminar displacement in the two groups . on the statistical model , we included all the measurable parameters that showed a significant association with laminar depth displacement , but we are certain that other influential factors that are not currently measured in this study have a significant influence on the laminar depth displacement . for example , the morphology of the canal is likely a very important biomechanical influential parameter for lamina deformation , but the current oct technology does not allow an accurate estimate of that region . in summary , we demonstrated that there was greater posterior deformation in the lamina cribrosa in ad compared to ed normal individuals in response to an acute increase of iop . this significant variation in laminar compliance across racial groups may be related to described differences in the morphometric and material properties of the onh region and could explain some of the differential susceptibility to glaucomatous injury in the at - risk ad population .

thirty percent of heart transplant ( htx ) recipients experience a rejection episode during the first year of htx.1 ) currently , periodic endomyocardial biopsy is the standard method for the surveillance of acute allograft rejection during the first 6 to 12 months after htx ( level of recommendation iia).2 ) according to the representative example of the biopsy schedule described in the guideline , a patient needs repeated endomyocardial biopsy for a total of 16 times during the first year after htx.2 ) however , endomyocardial biopsy is an invasive procedure that requires the patient 's hospitalization , and may also be accompanied by harmful complications such as traumatic tricuspid regurgitation . the patients , especially those with higher numbers of endomyocardial biopsy , have an increased risk of severe tricuspid regurgitation.3 ) the incidence of tricuspid regurgitation after htx was not rare ( 7 - 24%),4 ) and tricuspid regurgitation of more than moderate degree is associated with worse outcome after htx.5 ) furthermore , the recent advance of immunosuppressive therapy has lowered the incidence of rejection episodes requiring additional intensive immunosuppressive treatment . according to the 2012 registry of the international society for heart and lung transplantation ( ishlt ) data , more than 80 percent of endomyocardial biopsy episodes were enough to be observed without further intensive immunosuppressive therapy.1 ) there has been an effort to predict acute allograft rejection noninvasively using various biomarkers . repeated measurement of n - terminal pro - b - type natriuretic peptide ( nt - probnp ) or c - reactive protein ( crp ) has been reported to be ineffective in predicting acute cellular rejection.6 ) meanwhile , high - sensitivity cardiac troponin i ( hs - tni ) showed promising results with good correlation with acute cellular rejection.7)8 ) however , significant overlap of hs - tni between two groups with or without acute allograft rejection as well as conflicting data of cardiac troponin t ( tnt ) prevents clinicians from adopting biomarkers as a full substitute for endomyocardial biopsy.6)7)8 ) soluble st2 ( sst2 ) is a novel cardiovascular marker associated with cardiac hypertrophy , fibrosis , and ventricular dysfunction.9 ) in the patients with heart failure or acute coronary syndromes , sst2 is a well - known powerful predictor for cardiovascular events.9)10)11 ) even in subjects without structural heart disease , a higher level of sst2 was also associated with increased risk of death and heart failure events.12 ) furthermore , sst2 has been reported to be associated with the severity of inflammation of cardiomyocytes in acute lymphocytic myocarditis.13 ) therefore , in the present study , we investigated the possibility of using sst2 as a non - invasive marker to predict acute allograft rejection after htx . sixty - seven cases of htx in samsung medical center from september 2006 to august 2014 were included in this study . all of them agreed to provide their blood samples collected at the time of endomyocardial biopsy for the research with an informed consent form . the patients ' clinical and laboratory findings were investigated retrospectively by reviewing the medical charts . the study protocol was approved by the institutional review board of our institute ( 2014 - 12 - 111 ) . endomyocardial biopsy was repeated based on the predefined protocol after htx for the surveillance of acute allograft rejection in our center . in the early periods of the study , endomyocardial biopsy was performed at 2 , 4 , 8 , 12 , and 18 weeks , and 6 , 9 , 12 , 18 , and 24 months after htx . in the later periods of the study beginning from august 2011 , the routine surveillance beyond the first year of htx was omitted . since august 2011 , the patients without evidence of allograft rejection at one year after the transplant did not undergo further endomyocardial biopsy routinely . meanwhile , when htx recipients experienced new - onset symptoms suggesting heart failure ( dyspnea , fatigue , or syncope , etc . ) without any explainable causes , an additional endomyocardial biopsy schedule was arranged at the time of symptoms . when significant rejection ( grade 2r ) was detected pathologically in a biopsy specimen , follow - up biopsy was performed obligatorily 2 to 4 weeks after intensive immunosuppressive treatment . endomyocardial biopsy was performed via a right internal jugular vein approach , and 5 to 7 specimens were acquired from the right ventricle each time . the definition of acute allograft rejection follows the ishlt guideline ( 0r to 3r).14 ) in this study , " any rejection " was defined as grading 1r or higher , and " significant rejection " was defined as a rejection grade of 2r or 3r . additionally , sudden events of hemodynamic instability without any evidence of coronary ischemia were also included in the " significant rejection " grouping . all patients diagnosed with significant rejection were prescribed intensive immunosuppressive therapy including steroid pulse therapy . the protocol using steroid pulse in our center was comprised of 1000 mg of methylprednisolone every 24 hours for 3 days , and then administration of 1 mg / kg of prednisone per day for 3 days . after this was performed , a daily dose of prednisone was tapered by 5 mg every 3 days to reach a dose of 5 - 7.5 mg per day . in only one case , in which the patient had a body weight below 35 kg , was a lower dose of steroid used ( 500 mg of methylprednisolone once a day for 3 days ) instead of 1000 mg of methylprednisolone . if the patients were hemodynamically stable , a high dose of steroid was given only after the identification of pathologically significant rejection from a biopsy specimen . in those cases , the time interval between endomyocardial biopsy and intensive immunosuppressive therapy is 2 - 4 days . when htx recipients were hemodynamically unstable , steroid pulse therapy was begun immediately after endomyocardial biopsy before the confirmation of pathological findings . if the biopsy specimen revealed acute humoral rejection by c4d special staining , rituximab of 375 mg / m of body surface area was administered every week for a period of 2 - 4 weeks . in some ishlt 2r grade cases , steroid pulse therapy could be substituted by increasing the dose of mycophenolate from 1 - 1.5 g / day to 2 g / day . this kind of practice was allowed in the limited number of 2r cases that were stable without any symptoms . the patients taking higher mycophenolate as an intensive immunosuppressive therapy for the allograft rejection obligatorily had repeated endomyocardial biopsy after 2 - 4 weeks . if the repeated biopsy still showed significant rejection , steroid pulse therapy was performed right after the verification of significant rejection . blood samples were obtained by venipuncture at the time of endomyocardial biopsy and collected in a heparin tube . the sst2 levels were measured by personnel who were blinded to the clinical status of the patients , and were later matched with the medical information for each case . serum sst2 concentration was measured by enzymelinked immunosorbent assay ( elisa ) using presage st2 ( critical diagnostics , san diego , ca , usa ) . the presence of normal distribution in each variable was investigated with a kolmogorov - smirnov test . because the sst2 levels did not follow normal distribution , the correlation between sst2 levels and other continuous variables including age , white blood cell ( wbc ) counts , lymphocyte counts , the level of serum creatinine , c - reactive protein ( crp ) , ctni and nt - probnp were tested by spearman 's correlation test . to identify the independent correlates with sst2 , a multiple linear regression analysis was performed using statistically significant variables from a univariate spearman 's correlation test . consequently , wbc counts , lymphocyte counts , serum creatinine , ctni , nt - probnp , and time after htx were included in the multiple linear regression analysis . sst2 levels according to rejection grade were compared using a mann - whitney test or kruskal - wallis test where appropriate . the p value was corrected by multiplying by 6 after the kruskal - wallis test based on the bonferroni principle to control the problem brought by multiple comparisons . to evaluate changes of sst2 at the specific times in each patient , the association between sst2 and the presence of rejection was evaluated with repeated measures analysis using mixed modeling with sst2 as an outcome . when the patients had two episodes of significant rejection , first rejection episodes were chosen for within - subject analysis . because two patients had significant rejection twice , twenty cases with significant rejection and measured sst2 were used in within - subject analysis excluding the repeated episodes in the two patients . in the patients with significant rejection , sst2 and nt - probnp levels statistical analyses were performed with statistical package ibm spss statistics 20.0 software ( ibm inc . , sixty - seven cases of htx in samsung medical center from september 2006 to august 2014 were included in this study . all of them agreed to provide their blood samples collected at the time of endomyocardial biopsy for the research with an informed consent form . the patients ' clinical and laboratory findings were investigated retrospectively by reviewing the medical charts . the study protocol was approved by the institutional review board of our institute ( 2014 - 12 - 111 ) . endomyocardial biopsy was repeated based on the predefined protocol after htx for the surveillance of acute allograft rejection in our center . in the early periods of the study , endomyocardial biopsy was performed at 2 , 4 , 8 , 12 , and 18 weeks , and 6 , 9 , 12 , 18 , and 24 months after htx . in the later periods of the study beginning from august 2011 , the routine surveillance beyond the first year of htx was omitted . since august 2011 , the patients without evidence of allograft rejection at one year after the transplant did not undergo further endomyocardial biopsy routinely . meanwhile , when htx recipients experienced new - onset symptoms suggesting heart failure ( dyspnea , fatigue , or syncope , etc . ) without any explainable causes , an additional endomyocardial biopsy schedule was arranged at the time of symptoms . when significant rejection ( grade 2r ) was detected pathologically in a biopsy specimen , follow - up biopsy was performed obligatorily 2 to 4 weeks after intensive immunosuppressive treatment . endomyocardial biopsy was performed via a right internal jugular vein approach , and 5 to 7 specimens were acquired from the right ventricle each time . the definition of acute allograft rejection follows the ishlt guideline ( 0r to 3r).14 ) in this study , " any rejection " was defined as grading 1r or higher , and " significant rejection " was defined as a rejection grade of 2r or 3r . additionally , sudden events of hemodynamic instability without any evidence of coronary ischemia were also included in the " significant rejection " grouping . all patients diagnosed with significant rejection were prescribed intensive immunosuppressive therapy including steroid pulse therapy . the protocol using steroid pulse in our center was comprised of 1000 mg of methylprednisolone every 24 hours for 3 days , and then administration of 1 mg / kg of prednisone per day for 3 days . after this was performed , a daily dose of prednisone was tapered by 5 mg every 3 days to reach a dose of 5 - 7.5 mg per day . in only one case , in which the patient had a body weight below 35 kg , was a lower dose of steroid used ( 500 mg of methylprednisolone once a day for 3 days ) instead of 1000 mg of methylprednisolone . if the patients were hemodynamically stable , a high dose of steroid was given only after the identification of pathologically significant rejection from a biopsy specimen . in those cases , the time interval between endomyocardial biopsy and intensive immunosuppressive therapy is 2 - 4 days . when htx recipients were hemodynamically unstable , steroid pulse therapy was begun immediately after endomyocardial biopsy before the confirmation of pathological findings . if the biopsy specimen revealed acute humoral rejection by c4d special staining , rituximab of 375 mg / m of body surface area was administered every week for a period of 2 - 4 weeks . in some ishlt 2r grade cases , steroid pulse therapy could be substituted by increasing the dose of mycophenolate from 1 - 1.5 g / day to 2 g / day . this kind of practice was allowed in the limited number of 2r cases that were stable without any symptoms . the patients taking higher mycophenolate as an intensive immunosuppressive therapy for the allograft rejection obligatorily had repeated endomyocardial biopsy after 2 - 4 weeks . if the repeated biopsy still showed significant rejection , steroid pulse therapy was performed right after the verification of significant rejection . blood samples were obtained by venipuncture at the time of endomyocardial biopsy and collected in a heparin tube . the sst2 levels were measured by personnel who were blinded to the clinical status of the patients , and were later matched with the medical information for each case . serum sst2 concentration was measured by enzymelinked immunosorbent assay ( elisa ) using presage st2 ( critical diagnostics , san diego , ca , usa ) . the presence of normal distribution in each variable was investigated with a kolmogorov - smirnov test . because the sst2 levels did not follow normal distribution , the correlation between sst2 levels and other continuous variables including age , white blood cell ( wbc ) counts , lymphocyte counts , the level of serum creatinine , c - reactive protein ( crp ) , ctni and nt - probnp were tested by spearman 's correlation test . to identify the independent correlates with sst2 , a multiple linear regression analysis was performed using statistically significant variables from a univariate spearman 's correlation test . consequently , wbc counts , lymphocyte counts , serum creatinine , ctni , nt - probnp , and time after htx were included in the multiple linear regression analysis . sst2 levels according to rejection grade were compared using a mann - whitney test or kruskal - wallis test where appropriate . the p value was corrected by multiplying by 6 after the kruskal - wallis test based on the bonferroni principle to control the problem brought by multiple comparisons . to evaluate changes of sst2 at the specific times in each patient , one - way repeated measured analysis of variance was conducted . the association between sst2 and the presence of rejection was evaluated with repeated measures analysis using mixed modeling with sst2 as an outcome . when the patients had two episodes of significant rejection , first rejection episodes were chosen for within - subject analysis . because two patients had significant rejection twice , twenty cases with significant rejection and measured sst2 were used in within - subject analysis excluding the repeated episodes in the two patients . in the patients with significant rejection , sst2 and statistical analyses were performed with statistical package ibm spss statistics 20.0 software ( ibm inc . , sixty - seven htx cases from 65 patients and their 494 blood samples were examined in this study . among the 67 cases of htx , 4 ( 6.0% ) cases were retransplantation , and the other 63 ( 94.0% ) cases were de novo htx . their mean age at htx was 45.516.8 years , and 41 cases ( 61.2% ) were male . dilated cardiomyopathy was the most common etiology of heart failure ( 41 cases , 61.1% ) , followed by 7 cases of ischemic cardiomyopathy ( 10.4% ) , 6 cases of hypertrophic cardiomyopathy ( 9.0% ) , and 6 cases of valvular heart disease ( 9.0% ) . other causes of heart failure included 4 cases of allograft rejection of a previously transplanted heart ( 6.0% ) , 1 case of myocarditis , 1 case of refractory ventricular tachycardia and 1 case of chemotherapy - induced cardiomyopathy . initial immunosuppressive therapy included steroid in 67 cases ( 100% ) , cyclosporine in 21 cases ( 31.3% ) , tacrolimus in 46 cases ( 68.7% ) , and mycophenolate in 52 cases ( 77.6% ) . the median follow - up duration after htx was 25.4 ( 14.7 - 54.8 ) months , and the number of repetitive endomyocardial biopsies per case is 7.41.9 . the mean age of donors was 38.011.9 years , and 25 ( 73.5% ) cases were male . the median value of sst2 in all 494 blood samples was 32.0 ( 18.8 - 82.0 ) ng / ml . the level of sst2 was positively correlated with wbc counts ( r=0.50 , p<0.001 ) as well as the level of ctni ( r=0.53 , p<0.001 ) or nt - probnp ( r=0.61 , p<0.001 ) , and negatively correlated with time after htx ( r=-0.65 , p<0.001 ) and lymphocyte counts ( r=-0.44 , p<0.001 ) ( table 2 , fig . 1 and 2a ) . meanwhile , serum creatinine level showed only weak association with sst2 ( r=-0.09 , p=0.040 ) . sst2 showed no statistical correlation with patients ' age , crp level or left ventricular ejection fraction ( lvef ) measured using echocardiography . from multiple linear regression analysis , wbc counts , lymphocyte counts , ctni and nt - probnp remained as independent variables associated with sst2 ( all p<0.001 ) with r value of 0.54 of the model ( p<0.001 ) . when we selected sst2 at a specific time point after htx ( 0.5 , 1 , 2 , 3 , 6 , 9 , and 12 months ) in accordance with the predefined biopsy protocol , 381 samples in 67 htx cases were available . sst2 levels at each time point were 15512 , 11612 , 467 , 344 , 314 , 212 and 192 ng / ml , respectively ( p<0.001 ) ( fig . 2b ) . of the entire population of 494 samples , 166 ( 33.6% ) and 25 ( 5.1% ) samples were matched with pathologic results showing any rejection and significant rejection , respectively ; 328 ( 66.4% ) samples of 0r , 141 ( 28.5% ) samples of 1r , 19 ( 3.8% ) samples of 2r , 3 ( 0.6% ) samples of 3r and 3 ( 0.6% ) samples of hemodynamically unstable humoral rejection . the st2 level was statistically different according to the grades of acute rejection ( 36 [ 19 - 98 ] , 28 [ 18 - 62 ] , 15 [ 16 - 37 ] , and 191 [ 85 - 343 ] ng / ml , in rejection grades of 0r , 1r , 2r , and 3r+ [ 3r plus hemodynamically unstable humoral rejection ] , respectively p=0.003 ) ( fig . the median value of sst2 was found to be different only between the 2r and 3r+ groups ( corrected p=0.038 ) . when sst2 was dichotomously analyzed according to the presence of significant rejection , there was no significant difference ( 32 [ 19 - 81 ] and 27 [ 16 - 94 ] ng / ml , p=0.72 ) ( fig . when the same analysis was performed divided by pre - and post-90 days after htx , the sst2 level was not different statistically ; pre-90 days ( 93 [ 47 - 155 ] , 63 [ 30 - 132 ] , 96 [ 82 - 110 ] , and 105 [ 221 - 337 ] ng / ml , consecutively 0r to 3r+ , p=0.09 ) and post-90 days ( 23 [ 15 - 34 ] , 14 [ 20 - 28 ] , 19 [ 15 - 31 ] , and 191 [ 65 - 319 ] ng / ml , consecutively 0r to 3r+ , p=0.052 ) ( fig . 4 ) a total of 58 ( 86.7% ) htx patients experienced any acute allograft rejection first at 1.8 ( 0.4 - 3.4 ) months after htx . among them , first significant allograft rejection occurred in 20 ( 29.9% ) cases at 4.0 ( 2.0 - 9.0 ) months after htx . the baseline clinical characteristics of the two groups with or without significant rejection were not significantly different for recipient age , etiology of heart failure or initial immunosuppressive agents ( supplementary table 1 in the online - only data supplement ) . there was only a higher tendency of second htx in the group with significant rejection ( 3 [ 15% ] vs. 1 [ 2.1% ] , p=0.08 ) . when we studied within - subject effects of sst2 using a mixed model , the sst2 level according to the predefined time point was not different according to the presence of significant rejection ( p for interaction=0.94 ) . the result was the same when we selected cases only after 90 days of htx ( p for interaction=0.74 ) . the level of sst2 before , at the time of , and after treatment of acute significant rejection , were available in 16 , 18 and 17 patients among 20 cases experiencing significant rejection . overall , sst2 at the pre- and post - rejection periods were not different statistically ( 32 [ 19 - 57 ] , 24 [ 12 - 45 ] , and 18 [ 15 - 50 ] ng / ml , before , at the time of , and after treatment of acute significant rejection , respectively , p=0.87 ) ( fig . 5a ) . the change in sst2 levels were also analyzed after categorization according to postoperative days ; 90 days ( fig . 5c ) . there were no statistical difference in sst2 levels at the pre- and post - rejection periods in the early postoperative period ( 109 [ 74 - 173 ] , 74 [ 30 - 173 ] , and 31 [ 17 - 213 ] ng / ml , p=0.31 ) or late postoperative period ( 19 [ 14 - 31 ] , 29 [ 12 - 32 ] , and 50 [ 17 - 107 ] ng / ml , p=0.16 ) . when we drew lines connecting the dots belonging to the same individual , the level of sst2 was not consistently decreased after the treatment of rejection , regardless of pathological improvement . similarly , nt - probnp showed no statistical difference according to time frame of significant rejection ( 770 [ 405 - 6435 ] , 1683 [ 268 - 6882 ] , and 827 [ 246 - 1195 ] detailed information for 20 cases with significant rejection are presented in table 3 . in a large portion of cases ( 11/20 , 55.0% ) , the patients were treated with steroid pulse therapy . among them , only one case ( case # 10 ) failed to recover and needed retransplantation . among 6 cases treated with increasing doses of mycophenolate , one case ( case # 17 ) sustained significant rejection on follow - up biopsy and had subsequent steroid pulse therapy . among 20 cases with significant rejection , 5 ( 29.4% ) cases at pre - rejection , 10 ( 55.6% ) cases at rejection , and 8 ( 40.0% ) cases at the postrejection period showed sst2 > two standard deviations at the same time point after htx , the values of which were elicited from all 381 samples . sixty - seven htx cases from 65 patients and their 494 blood samples were examined in this study . among the 67 cases of htx , 4 ( 6.0% ) cases were retransplantation , and the other 63 ( 94.0% ) cases were de novo htx . their mean age at htx was 45.516.8 years , and 41 cases ( 61.2% ) were male . dilated cardiomyopathy was the most common etiology of heart failure ( 41 cases , 61.1% ) , followed by 7 cases of ischemic cardiomyopathy ( 10.4% ) , 6 cases of hypertrophic cardiomyopathy ( 9.0% ) , and 6 cases of valvular heart disease ( 9.0% ) . other causes of heart failure included 4 cases of allograft rejection of a previously transplanted heart ( 6.0% ) , 1 case of myocarditis , 1 case of refractory ventricular tachycardia and 1 case of chemotherapy - induced cardiomyopathy . initial immunosuppressive therapy included steroid in 67 cases ( 100% ) , cyclosporine in 21 cases ( 31.3% ) , tacrolimus in 46 cases ( 68.7% ) , and mycophenolate in 52 cases ( 77.6% ) . the median follow - up duration after htx was 25.4 ( 14.7 - 54.8 ) months , and the number of repetitive endomyocardial biopsies per case is 7.41.9 . the mean age of donors was 38.011.9 years , and 25 ( 73.5% ) cases were male . the median value of sst2 in all 494 blood samples was 32.0 ( 18.8 - 82.0 ) ng / ml . the level of sst2 was positively correlated with wbc counts ( r=0.50 , p<0.001 ) as well as the level of ctni ( r=0.53 , p<0.001 ) or nt - probnp ( r=0.61 , p<0.001 ) , and negatively correlated with time after htx ( r=-0.65 , p<0.001 ) and lymphocyte counts ( r=-0.44 , p<0.001 ) ( table 2 , fig . 1 and 2a ) . meanwhile , serum creatinine level showed only weak association with sst2 ( r=-0.09 , p=0.040 ) . sst2 showed no statistical correlation with patients ' age , crp level or left ventricular ejection fraction ( lvef ) measured using echocardiography . from multiple linear regression analysis , wbc counts , lymphocyte counts , ctni and nt - probnp remained as independent variables associated with sst2 ( all p<0.001 ) with r value of 0.54 of the model ( p<0.001 ) . when we selected sst2 at a specific time point after htx ( 0.5 , 1 , 2 , 3 , 6 , 9 , and 12 months ) in accordance with the predefined biopsy protocol , 381 samples in 67 htx cases were available . sst2 levels at each time point were 15512 , 11612 , 467 , 344 , 314 , 212 and 192 ng / ml , respectively ( p<0.001 ) ( fig . of the entire population of 494 samples , 166 ( 33.6% ) and 25 ( 5.1% ) samples were matched with pathologic results showing any rejection and significant rejection , respectively ; 328 ( 66.4% ) samples of 0r , 141 ( 28.5% ) samples of 1r , 19 ( 3.8% ) samples of 2r , 3 ( 0.6% ) samples of 3r and 3 ( 0.6% ) samples of hemodynamically unstable humoral rejection . the st2 level was statistically different according to the grades of acute rejection ( 36 [ 19 - 98 ] , 28 [ 18 - 62 ] , 15 [ 16 - 37 ] , and 191 [ 85 - 343 ] ng / ml , in rejection grades of 0r , 1r , 2r , and 3r+ [ 3r plus hemodynamically unstable humoral rejection ] , respectively p=0.003 ) ( fig . the median value of sst2 was found to be different only between the 2r and 3r+ groups ( corrected p=0.038 ) . when sst2 was dichotomously analyzed according to the presence of significant rejection , there was no significant difference ( 32 [ 19 - 81 ] and 27 [ 16 - 94 ] ng / ml , p=0.72 ) ( fig . when the same analysis was performed divided by pre - and post-90 days after htx , the sst2 level was not different statistically ; pre-90 days ( 93 [ 47 - 155 ] , 63 [ 30 - 132 ] , 96 [ 82 - 110 ] , and 105 [ 221 - 337 ] ng / ml , consecutively 0r to 3r+ , p=0.09 ) and post-90 days ( 23 [ 15 - 34 ] , 14 [ 20 - 28 ] , 19 [ 15 - 31 ] , and 191 [ 65 - 319 ] ng / ml , consecutively 0r to 3r+ , p=0.052 ) ( fig . 4 ) a total of 58 ( 86.7% ) htx patients experienced any acute allograft rejection first at 1.8 ( 0.4 - 3.4 ) months after htx . among them , first significant allograft rejection occurred in 20 ( 29.9% ) cases at 4.0 ( 2.0 - 9.0 ) months after htx . the baseline clinical characteristics of the two groups with or without significant rejection were not significantly different for recipient age , etiology of heart failure or initial immunosuppressive agents ( supplementary table 1 in the online - only data supplement ) . there was only a higher tendency of second htx in the group with significant rejection ( 3 [ 15% ] vs. 1 [ 2.1% ] , p=0.08 ) . when we studied within - subject effects of sst2 using a mixed model , the sst2 level according to the predefined time point was not different according to the presence of significant rejection ( p for interaction=0.94 ) . the result was the same when we selected cases only after 90 days of htx ( p for interaction=0.74 ) . the level of sst2 before , at the time of , and after treatment of acute significant rejection , were available in 16 , 18 and 17 patients among 20 cases experiencing significant rejection . overall , sst2 at the pre- and post - rejection periods were not different statistically ( 32 [ 19 - 57 ] , 24 [ 12 - 45 ] , and 18 [ 15 - 50 ] ng / ml , before , at the time of , and after treatment of acute significant rejection , respectively , p=0.87 ) ( fig . 5a ) . the change in sst2 levels were also analyzed after categorization according to postoperative days ; 90 days ( fig . 5c ) . there were no statistical difference in sst2 levels at the pre- and post - rejection periods in the early postoperative period ( 109 [ 74 - 173 ] , 74 [ 30 - 173 ] , and 31 [ 17 - 213 ] ng / ml , p=0.31 ) or late postoperative period ( 19 [ 14 - 31 ] , 29 [ 12 - 32 ] , and 50 [ 17 - 107 ] ng / ml , p=0.16 ) . when we drew lines connecting the dots belonging to the same individual , the level of sst2 was not consistently decreased after the treatment of rejection , regardless of pathological improvement . similarly , nt - probnp showed no statistical difference according to time frame of significant rejection ( 770 [ 405 - 6435 ] , 1683 [ 268 - 6882 ] , and 827 [ 246 - 1195 ] pg / ml , p=0.51 , respectively ) . detailed information for 20 cases with significant rejection are presented in table 3 . in a large portion of cases ( 11/20 , 55.0% ) , the patients were treated with steroid pulse therapy . among them , only one case ( case # 10 ) failed to recover and needed retransplantation . among 6 cases treated with increasing doses of mycophenolate , one case ( case # 17 ) sustained significant rejection on follow - up biopsy and had subsequent steroid pulse therapy . among 20 cases with significant rejection , 5 ( 29.4% ) cases at pre - rejection , 10 ( 55.6% ) cases at rejection , and 8 ( 40.0% ) cases at the postrejection period showed sst2 > two standard deviations at the same time point after htx , the values of which were elicited from all 381 samples . the sst2 level after htx was correlated with time after transplant , wbc count , lymphocytes count , ctni and nt - pro bnp . however , correlations with pathologically confirmed ishlt rejection score were not observed after control of the time after htx . many cases showing significant rejection were not accompanied by significant elevation in sst2 above two standard deviations of the same time point after htx . noninvasive methods to detect allograft rejection after htx using biomarkers have been studied several times . measurement of nt - probnp , tnt , crp , or combinations of nt - probnp and crp failed to prove clinical usefulness in predicting acute allograft rejection after htx.6)15 ) battes et al . studied nt - probnp or crp using 1136 blood samples taken at the time of endomyocardial biopsies in 77 htx cases.6 ) the level of nt - pro bnp and crp were high immediately after the surgery and decreased to reach steady status at 12 weeks post - transplantation . however , repeated measurement of nt - pro bnp or crp was ineffective in predicting acute cellular rejection both in the early periods and late periods after htx . meanwhile , high - sensitivity cardiac hs - tni has been reported to have good correlation with acute cellular rejection.7)8 ) patel et al.7 ) analyzed 418 serum samples of 35 htx recipients and reported that the median value of hs - tni was significantly high in the group with acute rejection , and that a cutoff point 0.015 ng / ml of ctni could discriminate acute rejection with 94% sensitivity and a 99% negative predictive value . however , significant overlap was noted when hs - tni was re - categorized by ishlt grade . similarly , in the recent study using 252 samples in 59 htx cases , the absolute value of hs - tni was not found to be different according to the presence of acute rejection.8 ) although the median value of ctni in the samples with acute cellular rejection was higher than the samples without acute rejection , a statistical difference was not observed . reflecting all these clinical data , current guidelines recommend that routine noninvasive testing modalities including cardiac troponin i or t can not substitute endomyocardial biopsy in adult htx recipients ( level of recommendation iii).2 ) the role of sst2 as a biomarker to predict acute rejection after htx has been investigated previously.16)17 ) pascual - figal et al.16 ) reported that sst2 levels were increased as compared with the pre - rejection period , and decreased again after treatment of acute rejection . instead , the samples before and after rejection were utilized as a control group . in our study , both groups with or without rejection were included , and the data of the patients with rejection were compared with those of the patients without rejection . in the present study , a statistical difference was observed in the sst2 level according to the grades of the allograft rejection . however , correcting for confounding factors of the time after htx , a statistical difference was not found . more recently , mathews et al . reported that a higher level of sst2 ( > 600 pg / ml ) could discriminate graft rejection with an area under the curve of 0.720.05 in pediatric htx patients.17 ) however , they had higher rates of rejection events , and included a large proportion of acute humoral rejection among the significant rejection group . similarly in our study , the level of antibody - mediated rejection was higher than others ( # 1 , 2 , and 7 in table 3 ) . over 40% of patients ( especially cases with acute cellular rejection ) maintained sst2 levels in the range of 2 standard deviations calculated by the patients without acute rejection . on the other hand , sst2 can be affected by other cardiovascular and inflammatory conditions . as the il-33/st2 signaling pathway also involves t helper type 2 cell differentiation,18 ) it is not unusual that sst2 was higher in the cases with systemic infection or inflammation.19 ) the correlation of sst2 level and wbc / lymphocyte counts that appeared in our study also supports the possible correlation between inflammation and sst2 levels . accordingly , sst2 was reported to be increased in chronic obstructive lung disease , asthma , pulmonary embolism , pulmonary hypertension , and alcohol abuse in other previous studies.19 ) some extreme cases of sst2 levels in our data ( arrows in fig . 3b ) also reflected critical events that occurred within several days ; 1 ) generalized seizure suspected to have resulted from posterior reversible encephalopathy syndrome , 2 ) ischemic injury that appeared in endomyocardial biopsy , and 3 ) septic shock two days after endomyocardial biopsy . because htx recipients are susceptible to complications by multiple comorbidities in the early periods of htx , sst2 is expected to have a limited role in predicating acute rejection specifically . first , as a retrospective study , not all of the included patients have consecutive samples to present sequential change in sst2 after htx , and there were missing values at certain time points after htx . nonetheless , we included 67 htx cases with 494 samples ; these numbers are quite comparable to those of previous retrospective studies.6)7)8)15)16 ) second , because the presence of acute rejection was detected by endomyocardial biopsy surveillance , the follow - up duration of the included patients had limited time duration of 1 - 2 years after htx . hence , development of chronic vasculopathy or overall long - term cardiovascular mortality after htx can not be investigated . like the general population without cardiovascular disease,12 ) sst2 may be associated with long - term cardiovascular outcome rather than acute allograft rejection developed in the early periods after htx . additionally , the number of acute rejection cases was too small to understand the role of certain biomarkers fully ; 20 patients with 22 blood samples ( 29.9% of total patients and 4.5% in total blood samples ) , even though the incidence rate was quite comparable with the rates of previous reports ( 37% of the patients).7 ) a larger number of subjects would definitely be required to obtain an adequate number of acute rejection episodes . although sst2 is known as a promising predictor for cardiovascular events , its role in htx patients in predicting acute allograft rejection seems to be limited . further prospective studies including larger populations with longer follow - up duration are needed in order to figure out the further clinical implications of sst2 in htx recipients . although sst2 is known as a promising predictor for cardiovascular events , its role in htx patients in predicting acute allograft rejection seems to be limited . further prospective studies including larger populations with longer follow - up duration are needed in order to figure out the further clinical implications of sst2 in htx recipients .

background and objectivesendomyocardial biopsy is obligatory during the first year after heart transplant ( htx ) for the surveillance of acute rejection . previous attempts using cardiac biomarkers for the detection of rejection failed to show enough evidence to substitute endomyocardial biopsy . therefore , this study sought the possibility of using soluble st2 ( sst2 ) , a novel cardiovascular marker , as a surrogate marker for acute allograft rejection after htx.subjects and methodsa total of 494 blood samples acquired at the time of endomyocardial biopsy were analyzed in 67 htx cases from september 2006 to august 2014 . significant rejection was defined as international society of heart and lung transplant ( ishlt ) score 2r and humoral rejection accompanied by hemodynamic instability.resultstwenty cases of htx with 22 blood samples showed significant rejection in endomyocardial biopsy at 4.0 ( 2.0 - 9.0 ) months after htx . the level of sst2 showed positive correlation with cardiac troponin i , and n - terminal pro - b - type natriuretic peptide ( all p<0.001 ) , and negative correlation with post - htx months ( p<0.001 ) . the levels of sst2 according to the ishlt scores were 36 ( 19 - 98 ) , 28 ( 18 - 62 ) , 15 ( 16 - 37 ) , and 191 ( 85 - 343 ) ng / ml , consecutively 0r , 1r , 2r , and 3r+ ( 3r plus hemodynamically - unstable humoral rejection ) ( p=0.003 ) . however , when we studied within - subject effects of sst2 using a mixed model , the sst2 level according to the predefined time point was not different according to the presence of significant rejection ( p for interaction=0.94).conclusionalthough sst2 is known as a promising predictor for cardiovascular events , its role in htx patients to predict acute allograft rejection seems to be limited .

Introduction Subjects and Methods Patients The protocol of endomyocardial biopsy The definition and treatment of acute allograft rejection Measurement of soluble ST2 Statistical analysis Results Subjects' characteristics Clinical variables associated with sST2 after heart transplant sST2 according to the grades of acute allograft rejection Discussion Conclusion Supplementary Materials

thirty percent of heart transplant ( htx ) recipients experience a rejection episode during the first year of htx.1 ) currently , periodic endomyocardial biopsy is the standard method for the surveillance of acute allograft rejection during the first 6 to 12 months after htx ( level of recommendation iia).2 ) according to the representative example of the biopsy schedule described in the guideline , a patient needs repeated endomyocardial biopsy for a total of 16 times during the first year after htx.2 ) however , endomyocardial biopsy is an invasive procedure that requires the patient 's hospitalization , and may also be accompanied by harmful complications such as traumatic tricuspid regurgitation . the patients , especially those with higher numbers of endomyocardial biopsy , have an increased risk of severe tricuspid regurgitation.3 ) the incidence of tricuspid regurgitation after htx was not rare ( 7 - 24%),4 ) and tricuspid regurgitation of more than moderate degree is associated with worse outcome after htx.5 ) furthermore , the recent advance of immunosuppressive therapy has lowered the incidence of rejection episodes requiring additional intensive immunosuppressive treatment . according to the 2012 registry of the international society for heart and lung transplantation ( ishlt ) data , more than 80 percent of endomyocardial biopsy episodes were enough to be observed without further intensive immunosuppressive therapy.1 ) there has been an effort to predict acute allograft rejection noninvasively using various biomarkers . repeated measurement of n - terminal pro - b - type natriuretic peptide ( nt - probnp ) or c - reactive protein ( crp ) has been reported to be ineffective in predicting acute cellular rejection.6 ) meanwhile , high - sensitivity cardiac troponin i ( hs - tni ) showed promising results with good correlation with acute cellular rejection.7)8 ) however , significant overlap of hs - tni between two groups with or without acute allograft rejection as well as conflicting data of cardiac troponin t ( tnt ) prevents clinicians from adopting biomarkers as a full substitute for endomyocardial biopsy.6)7)8 ) soluble st2 ( sst2 ) is a novel cardiovascular marker associated with cardiac hypertrophy , fibrosis , and ventricular dysfunction.9 ) in the patients with heart failure or acute coronary syndromes , sst2 is a well - known powerful predictor for cardiovascular events.9)10)11 ) even in subjects without structural heart disease , a higher level of sst2 was also associated with increased risk of death and heart failure events.12 ) furthermore , sst2 has been reported to be associated with the severity of inflammation of cardiomyocytes in acute lymphocytic myocarditis.13 ) therefore , in the present study , we investigated the possibility of using sst2 as a non - invasive marker to predict acute allograft rejection after htx . sixty - seven cases of htx in samsung medical center from september 2006 to august 2014 were included in this study . all of them agreed to provide their blood samples collected at the time of endomyocardial biopsy for the research with an informed consent form . endomyocardial biopsy was repeated based on the predefined protocol after htx for the surveillance of acute allograft rejection in our center . the definition of acute allograft rejection follows the ishlt guideline ( 0r to 3r).14 ) in this study , " any rejection " was defined as grading 1r or higher , and " significant rejection " was defined as a rejection grade of 2r or 3r . blood samples were obtained by venipuncture at the time of endomyocardial biopsy and collected in a heparin tube . because the sst2 levels did not follow normal distribution , the correlation between sst2 levels and other continuous variables including age , white blood cell ( wbc ) counts , lymphocyte counts , the level of serum creatinine , c - reactive protein ( crp ) , ctni and nt - probnp were tested by spearman 's correlation test . to evaluate changes of sst2 at the specific times in each patient , the association between sst2 and the presence of rejection was evaluated with repeated measures analysis using mixed modeling with sst2 as an outcome . , sixty - seven cases of htx in samsung medical center from september 2006 to august 2014 were included in this study . all of them agreed to provide their blood samples collected at the time of endomyocardial biopsy for the research with an informed consent form . endomyocardial biopsy was repeated based on the predefined protocol after htx for the surveillance of acute allograft rejection in our center . the definition of acute allograft rejection follows the ishlt guideline ( 0r to 3r).14 ) in this study , " any rejection " was defined as grading 1r or higher , and " significant rejection " was defined as a rejection grade of 2r or 3r . blood samples were obtained by venipuncture at the time of endomyocardial biopsy and collected in a heparin tube . because the sst2 levels did not follow normal distribution , the correlation between sst2 levels and other continuous variables including age , white blood cell ( wbc ) counts , lymphocyte counts , the level of serum creatinine , c - reactive protein ( crp ) , ctni and nt - probnp were tested by spearman 's correlation test . , sixty - seven htx cases from 65 patients and their 494 blood samples were examined in this study . the median value of sst2 in all 494 blood samples was 32.0 ( 18.8 - 82.0 ) ng / ml . the level of sst2 was positively correlated with wbc counts ( r=0.50 , p<0.001 ) as well as the level of ctni ( r=0.53 , p<0.001 ) or nt - probnp ( r=0.61 , p<0.001 ) , and negatively correlated with time after htx ( r=-0.65 , p<0.001 ) and lymphocyte counts ( r=-0.44 , p<0.001 ) ( table 2 , fig . from multiple linear regression analysis , wbc counts , lymphocyte counts , ctni and nt - probnp remained as independent variables associated with sst2 ( all p<0.001 ) with r value of 0.54 of the model ( p<0.001 ) . when we selected sst2 at a specific time point after htx ( 0.5 , 1 , 2 , 3 , 6 , 9 , and 12 months ) in accordance with the predefined biopsy protocol , 381 samples in 67 htx cases were available . sst2 levels at each time point were 15512 , 11612 , 467 , 344 , 314 , 212 and 192 ng / ml , respectively ( p<0.001 ) ( fig . of the entire population of 494 samples , 166 ( 33.6% ) and 25 ( 5.1% ) samples were matched with pathologic results showing any rejection and significant rejection , respectively ; 328 ( 66.4% ) samples of 0r , 141 ( 28.5% ) samples of 1r , 19 ( 3.8% ) samples of 2r , 3 ( 0.6% ) samples of 3r and 3 ( 0.6% ) samples of hemodynamically unstable humoral rejection . the st2 level was statistically different according to the grades of acute rejection ( 36 [ 19 - 98 ] , 28 [ 18 - 62 ] , 15 [ 16 - 37 ] , and 191 [ 85 - 343 ] ng / ml , in rejection grades of 0r , 1r , 2r , and 3r+ [ 3r plus hemodynamically unstable humoral rejection ] , respectively p=0.003 ) ( fig . when sst2 was dichotomously analyzed according to the presence of significant rejection , there was no significant difference ( 32 [ 19 - 81 ] and 27 [ 16 - 94 ] ng / ml , p=0.72 ) ( fig . when the same analysis was performed divided by pre - and post-90 days after htx , the sst2 level was not different statistically ; pre-90 days ( 93 [ 47 - 155 ] , 63 [ 30 - 132 ] , 96 [ 82 - 110 ] , and 105 [ 221 - 337 ] ng / ml , consecutively 0r to 3r+ , p=0.09 ) and post-90 days ( 23 [ 15 - 34 ] , 14 [ 20 - 28 ] , 19 [ 15 - 31 ] , and 191 [ 65 - 319 ] ng / ml , consecutively 0r to 3r+ , p=0.052 ) ( fig . 4 ) a total of 58 ( 86.7% ) htx patients experienced any acute allograft rejection first at 1.8 ( 0.4 - 3.4 ) months after htx . among them , first significant allograft rejection occurred in 20 ( 29.9% ) cases at 4.0 ( 2.0 - 9.0 ) months after htx . when we studied within - subject effects of sst2 using a mixed model , the sst2 level according to the predefined time point was not different according to the presence of significant rejection ( p for interaction=0.94 ) . the level of sst2 before , at the time of , and after treatment of acute significant rejection , were available in 16 , 18 and 17 patients among 20 cases experiencing significant rejection . overall , sst2 at the pre- and post - rejection periods were not different statistically ( 32 [ 19 - 57 ] , 24 [ 12 - 45 ] , and 18 [ 15 - 50 ] ng / ml , before , at the time of , and after treatment of acute significant rejection , respectively , p=0.87 ) ( fig . there were no statistical difference in sst2 levels at the pre- and post - rejection periods in the early postoperative period ( 109 [ 74 - 173 ] , 74 [ 30 - 173 ] , and 31 [ 17 - 213 ] ng / ml , p=0.31 ) or late postoperative period ( 19 [ 14 - 31 ] , 29 [ 12 - 32 ] , and 50 [ 17 - 107 ] ng / ml , p=0.16 ) . when we drew lines connecting the dots belonging to the same individual , the level of sst2 was not consistently decreased after the treatment of rejection , regardless of pathological improvement . among 20 cases with significant rejection , 5 ( 29.4% ) cases at pre - rejection , 10 ( 55.6% ) cases at rejection , and 8 ( 40.0% ) cases at the postrejection period showed sst2 > two standard deviations at the same time point after htx , the values of which were elicited from all 381 samples . sixty - seven htx cases from 65 patients and their 494 blood samples were examined in this study . the median value of sst2 in all 494 blood samples was 32.0 ( 18.8 - 82.0 ) ng / ml . the level of sst2 was positively correlated with wbc counts ( r=0.50 , p<0.001 ) as well as the level of ctni ( r=0.53 , p<0.001 ) or nt - probnp ( r=0.61 , p<0.001 ) , and negatively correlated with time after htx ( r=-0.65 , p<0.001 ) and lymphocyte counts ( r=-0.44 , p<0.001 ) ( table 2 , fig . when we selected sst2 at a specific time point after htx ( 0.5 , 1 , 2 , 3 , 6 , 9 , and 12 months ) in accordance with the predefined biopsy protocol , 381 samples in 67 htx cases were available . sst2 levels at each time point were 15512 , 11612 , 467 , 344 , 314 , 212 and 192 ng / ml , respectively ( p<0.001 ) ( fig . of the entire population of 494 samples , 166 ( 33.6% ) and 25 ( 5.1% ) samples were matched with pathologic results showing any rejection and significant rejection , respectively ; 328 ( 66.4% ) samples of 0r , 141 ( 28.5% ) samples of 1r , 19 ( 3.8% ) samples of 2r , 3 ( 0.6% ) samples of 3r and 3 ( 0.6% ) samples of hemodynamically unstable humoral rejection . the st2 level was statistically different according to the grades of acute rejection ( 36 [ 19 - 98 ] , 28 [ 18 - 62 ] , 15 [ 16 - 37 ] , and 191 [ 85 - 343 ] ng / ml , in rejection grades of 0r , 1r , 2r , and 3r+ [ 3r plus hemodynamically unstable humoral rejection ] , respectively p=0.003 ) ( fig . when sst2 was dichotomously analyzed according to the presence of significant rejection , there was no significant difference ( 32 [ 19 - 81 ] and 27 [ 16 - 94 ] ng / ml , p=0.72 ) ( fig . when the same analysis was performed divided by pre - and post-90 days after htx , the sst2 level was not different statistically ; pre-90 days ( 93 [ 47 - 155 ] , 63 [ 30 - 132 ] , 96 [ 82 - 110 ] , and 105 [ 221 - 337 ] ng / ml , consecutively 0r to 3r+ , p=0.09 ) and post-90 days ( 23 [ 15 - 34 ] , 14 [ 20 - 28 ] , 19 [ 15 - 31 ] , and 191 [ 65 - 319 ] ng / ml , consecutively 0r to 3r+ , p=0.052 ) ( fig . 4 ) a total of 58 ( 86.7% ) htx patients experienced any acute allograft rejection first at 1.8 ( 0.4 - 3.4 ) months after htx . among them , first significant allograft rejection occurred in 20 ( 29.9% ) cases at 4.0 ( 2.0 - 9.0 ) months after htx . when we studied within - subject effects of sst2 using a mixed model , the sst2 level according to the predefined time point was not different according to the presence of significant rejection ( p for interaction=0.94 ) . the level of sst2 before , at the time of , and after treatment of acute significant rejection , were available in 16 , 18 and 17 patients among 20 cases experiencing significant rejection . overall , sst2 at the pre- and post - rejection periods were not different statistically ( 32 [ 19 - 57 ] , 24 [ 12 - 45 ] , and 18 [ 15 - 50 ] ng / ml , before , at the time of , and after treatment of acute significant rejection , respectively , p=0.87 ) ( fig . there were no statistical difference in sst2 levels at the pre- and post - rejection periods in the early postoperative period ( 109 [ 74 - 173 ] , 74 [ 30 - 173 ] , and 31 [ 17 - 213 ] ng / ml , p=0.31 ) or late postoperative period ( 19 [ 14 - 31 ] , 29 [ 12 - 32 ] , and 50 [ 17 - 107 ] ng / ml , p=0.16 ) . when we drew lines connecting the dots belonging to the same individual , the level of sst2 was not consistently decreased after the treatment of rejection , regardless of pathological improvement . similarly , nt - probnp showed no statistical difference according to time frame of significant rejection ( 770 [ 405 - 6435 ] , 1683 [ 268 - 6882 ] , and 827 [ 246 - 1195 ] pg / ml , p=0.51 , respectively ) . among 20 cases with significant rejection , 5 ( 29.4% ) cases at pre - rejection , 10 ( 55.6% ) cases at rejection , and 8 ( 40.0% ) cases at the postrejection period showed sst2 > two standard deviations at the same time point after htx , the values of which were elicited from all 381 samples . measurement of nt - probnp , tnt , crp , or combinations of nt - probnp and crp failed to prove clinical usefulness in predicting acute allograft rejection after htx.6)15 ) battes et al . studied nt - probnp or crp using 1136 blood samples taken at the time of endomyocardial biopsies in 77 htx cases.6 ) the level of nt - pro bnp and crp were high immediately after the surgery and decreased to reach steady status at 12 weeks post - transplantation . similarly , in the recent study using 252 samples in 59 htx cases , the absolute value of hs - tni was not found to be different according to the presence of acute rejection.8 ) although the median value of ctni in the samples with acute cellular rejection was higher than the samples without acute rejection , a statistical difference was not observed . reflecting all these clinical data , current guidelines recommend that routine noninvasive testing modalities including cardiac troponin i or t can not substitute endomyocardial biopsy in adult htx recipients ( level of recommendation iii).2 ) the role of sst2 as a biomarker to predict acute rejection after htx has been investigated previously.16)17 ) pascual - figal et al.16 ) reported that sst2 levels were increased as compared with the pre - rejection period , and decreased again after treatment of acute rejection . in the present study , a statistical difference was observed in the sst2 level according to the grades of the allograft rejection . however , correcting for confounding factors of the time after htx , a statistical difference was not found . reported that a higher level of sst2 ( > 600 pg / ml ) could discriminate graft rejection with an area under the curve of 0.720.05 in pediatric htx patients.17 ) however , they had higher rates of rejection events , and included a large proportion of acute humoral rejection among the significant rejection group . similarly in our study , the level of antibody - mediated rejection was higher than others ( # 1 , 2 , and 7 in table 3 ) . nonetheless , we included 67 htx cases with 494 samples ; these numbers are quite comparable to those of previous retrospective studies.6)7)8)15)16 ) second , because the presence of acute rejection was detected by endomyocardial biopsy surveillance , the follow - up duration of the included patients had limited time duration of 1 - 2 years after htx . additionally , the number of acute rejection cases was too small to understand the role of certain biomarkers fully ; 20 patients with 22 blood samples ( 29.9% of total patients and 4.5% in total blood samples ) , even though the incidence rate was quite comparable with the rates of previous reports ( 37% of the patients).7 ) a larger number of subjects would definitely be required to obtain an adequate number of acute rejection episodes . although sst2 is known as a promising predictor for cardiovascular events , its role in htx patients in predicting acute allograft rejection seems to be limited . although sst2 is known as a promising predictor for cardiovascular events , its role in htx patients in predicting acute allograft rejection seems to be limited .

thirty percent of heart transplant ( htx ) recipients experience a rejection episode during the first year of htx.1 ) currently , periodic endomyocardial biopsy is the standard method for the surveillance of acute allograft rejection during the first 6 to 12 months after htx ( level of recommendation iia).2 ) according to the representative example of the biopsy schedule described in the guideline , a patient needs repeated endomyocardial biopsy for a total of 16 times during the first year after htx.2 ) however , endomyocardial biopsy is an invasive procedure that requires the patient 's hospitalization , and may also be accompanied by harmful complications such as traumatic tricuspid regurgitation . the patients , especially those with higher numbers of endomyocardial biopsy , have an increased risk of severe tricuspid regurgitation.3 ) the incidence of tricuspid regurgitation after htx was not rare ( 7 - 24%),4 ) and tricuspid regurgitation of more than moderate degree is associated with worse outcome after htx.5 ) furthermore , the recent advance of immunosuppressive therapy has lowered the incidence of rejection episodes requiring additional intensive immunosuppressive treatment . according to the 2012 registry of the international society for heart and lung transplantation ( ishlt ) data , more than 80 percent of endomyocardial biopsy episodes were enough to be observed without further intensive immunosuppressive therapy.1 ) there has been an effort to predict acute allograft rejection noninvasively using various biomarkers . repeated measurement of n - terminal pro - b - type natriuretic peptide ( nt - probnp ) or c - reactive protein ( crp ) has been reported to be ineffective in predicting acute cellular rejection.6 ) meanwhile , high - sensitivity cardiac troponin i ( hs - tni ) showed promising results with good correlation with acute cellular rejection.7)8 ) however , significant overlap of hs - tni between two groups with or without acute allograft rejection as well as conflicting data of cardiac troponin t ( tnt ) prevents clinicians from adopting biomarkers as a full substitute for endomyocardial biopsy.6)7)8 ) soluble st2 ( sst2 ) is a novel cardiovascular marker associated with cardiac hypertrophy , fibrosis , and ventricular dysfunction.9 ) in the patients with heart failure or acute coronary syndromes , sst2 is a well - known powerful predictor for cardiovascular events.9)10)11 ) even in subjects without structural heart disease , a higher level of sst2 was also associated with increased risk of death and heart failure events.12 ) furthermore , sst2 has been reported to be associated with the severity of inflammation of cardiomyocytes in acute lymphocytic myocarditis.13 ) therefore , in the present study , we investigated the possibility of using sst2 as a non - invasive marker to predict acute allograft rejection after htx . in the early periods of the study , endomyocardial biopsy was performed at 2 , 4 , 8 , 12 , and 18 weeks , and 6 , 9 , 12 , 18 , and 24 months after htx . in only one case , in which the patient had a body weight below 35 kg , was a lower dose of steroid used ( 500 mg of methylprednisolone once a day for 3 days ) instead of 1000 mg of methylprednisolone . because the sst2 levels did not follow normal distribution , the correlation between sst2 levels and other continuous variables including age , white blood cell ( wbc ) counts , lymphocyte counts , the level of serum creatinine , c - reactive protein ( crp ) , ctni and nt - probnp were tested by spearman 's correlation test . to evaluate changes of sst2 at the specific times in each patient , the association between sst2 and the presence of rejection was evaluated with repeated measures analysis using mixed modeling with sst2 as an outcome . in the early periods of the study , endomyocardial biopsy was performed at 2 , 4 , 8 , 12 , and 18 weeks , and 6 , 9 , 12 , 18 , and 24 months after htx . in only one case , in which the patient had a body weight below 35 kg , was a lower dose of steroid used ( 500 mg of methylprednisolone once a day for 3 days ) instead of 1000 mg of methylprednisolone . because the sst2 levels did not follow normal distribution , the correlation between sst2 levels and other continuous variables including age , white blood cell ( wbc ) counts , lymphocyte counts , the level of serum creatinine , c - reactive protein ( crp ) , ctni and nt - probnp were tested by spearman 's correlation test . dilated cardiomyopathy was the most common etiology of heart failure ( 41 cases , 61.1% ) , followed by 7 cases of ischemic cardiomyopathy ( 10.4% ) , 6 cases of hypertrophic cardiomyopathy ( 9.0% ) , and 6 cases of valvular heart disease ( 9.0% ) . other causes of heart failure included 4 cases of allograft rejection of a previously transplanted heart ( 6.0% ) , 1 case of myocarditis , 1 case of refractory ventricular tachycardia and 1 case of chemotherapy - induced cardiomyopathy . initial immunosuppressive therapy included steroid in 67 cases ( 100% ) , cyclosporine in 21 cases ( 31.3% ) , tacrolimus in 46 cases ( 68.7% ) , and mycophenolate in 52 cases ( 77.6% ) . the median follow - up duration after htx was 25.4 ( 14.7 - 54.8 ) months , and the number of repetitive endomyocardial biopsies per case is 7.41.9 . the level of sst2 was positively correlated with wbc counts ( r=0.50 , p<0.001 ) as well as the level of ctni ( r=0.53 , p<0.001 ) or nt - probnp ( r=0.61 , p<0.001 ) , and negatively correlated with time after htx ( r=-0.65 , p<0.001 ) and lymphocyte counts ( r=-0.44 , p<0.001 ) ( table 2 , fig . from multiple linear regression analysis , wbc counts , lymphocyte counts , ctni and nt - probnp remained as independent variables associated with sst2 ( all p<0.001 ) with r value of 0.54 of the model ( p<0.001 ) . when we selected sst2 at a specific time point after htx ( 0.5 , 1 , 2 , 3 , 6 , 9 , and 12 months ) in accordance with the predefined biopsy protocol , 381 samples in 67 htx cases were available . of the entire population of 494 samples , 166 ( 33.6% ) and 25 ( 5.1% ) samples were matched with pathologic results showing any rejection and significant rejection , respectively ; 328 ( 66.4% ) samples of 0r , 141 ( 28.5% ) samples of 1r , 19 ( 3.8% ) samples of 2r , 3 ( 0.6% ) samples of 3r and 3 ( 0.6% ) samples of hemodynamically unstable humoral rejection . the st2 level was statistically different according to the grades of acute rejection ( 36 [ 19 - 98 ] , 28 [ 18 - 62 ] , 15 [ 16 - 37 ] , and 191 [ 85 - 343 ] ng / ml , in rejection grades of 0r , 1r , 2r , and 3r+ [ 3r plus hemodynamically unstable humoral rejection ] , respectively p=0.003 ) ( fig . when sst2 was dichotomously analyzed according to the presence of significant rejection , there was no significant difference ( 32 [ 19 - 81 ] and 27 [ 16 - 94 ] ng / ml , p=0.72 ) ( fig . when the same analysis was performed divided by pre - and post-90 days after htx , the sst2 level was not different statistically ; pre-90 days ( 93 [ 47 - 155 ] , 63 [ 30 - 132 ] , 96 [ 82 - 110 ] , and 105 [ 221 - 337 ] ng / ml , consecutively 0r to 3r+ , p=0.09 ) and post-90 days ( 23 [ 15 - 34 ] , 14 [ 20 - 28 ] , 19 [ 15 - 31 ] , and 191 [ 65 - 319 ] ng / ml , consecutively 0r to 3r+ , p=0.052 ) ( fig . the baseline clinical characteristics of the two groups with or without significant rejection were not significantly different for recipient age , etiology of heart failure or initial immunosuppressive agents ( supplementary table 1 in the online - only data supplement ) . when we studied within - subject effects of sst2 using a mixed model , the sst2 level according to the predefined time point was not different according to the presence of significant rejection ( p for interaction=0.94 ) . the level of sst2 before , at the time of , and after treatment of acute significant rejection , were available in 16 , 18 and 17 patients among 20 cases experiencing significant rejection . overall , sst2 at the pre- and post - rejection periods were not different statistically ( 32 [ 19 - 57 ] , 24 [ 12 - 45 ] , and 18 [ 15 - 50 ] ng / ml , before , at the time of , and after treatment of acute significant rejection , respectively , p=0.87 ) ( fig . there were no statistical difference in sst2 levels at the pre- and post - rejection periods in the early postoperative period ( 109 [ 74 - 173 ] , 74 [ 30 - 173 ] , and 31 [ 17 - 213 ] ng / ml , p=0.31 ) or late postoperative period ( 19 [ 14 - 31 ] , 29 [ 12 - 32 ] , and 50 [ 17 - 107 ] ng / ml , p=0.16 ) . when we drew lines connecting the dots belonging to the same individual , the level of sst2 was not consistently decreased after the treatment of rejection , regardless of pathological improvement . among 20 cases with significant rejection , 5 ( 29.4% ) cases at pre - rejection , 10 ( 55.6% ) cases at rejection , and 8 ( 40.0% ) cases at the postrejection period showed sst2 > two standard deviations at the same time point after htx , the values of which were elicited from all 381 samples . dilated cardiomyopathy was the most common etiology of heart failure ( 41 cases , 61.1% ) , followed by 7 cases of ischemic cardiomyopathy ( 10.4% ) , 6 cases of hypertrophic cardiomyopathy ( 9.0% ) , and 6 cases of valvular heart disease ( 9.0% ) . other causes of heart failure included 4 cases of allograft rejection of a previously transplanted heart ( 6.0% ) , 1 case of myocarditis , 1 case of refractory ventricular tachycardia and 1 case of chemotherapy - induced cardiomyopathy . the median follow - up duration after htx was 25.4 ( 14.7 - 54.8 ) months , and the number of repetitive endomyocardial biopsies per case is 7.41.9 . the level of sst2 was positively correlated with wbc counts ( r=0.50 , p<0.001 ) as well as the level of ctni ( r=0.53 , p<0.001 ) or nt - probnp ( r=0.61 , p<0.001 ) , and negatively correlated with time after htx ( r=-0.65 , p<0.001 ) and lymphocyte counts ( r=-0.44 , p<0.001 ) ( table 2 , fig . from multiple linear regression analysis , wbc counts , lymphocyte counts , ctni and nt - probnp remained as independent variables associated with sst2 ( all p<0.001 ) with r value of 0.54 of the model ( p<0.001 ) . when we selected sst2 at a specific time point after htx ( 0.5 , 1 , 2 , 3 , 6 , 9 , and 12 months ) in accordance with the predefined biopsy protocol , 381 samples in 67 htx cases were available . of the entire population of 494 samples , 166 ( 33.6% ) and 25 ( 5.1% ) samples were matched with pathologic results showing any rejection and significant rejection , respectively ; 328 ( 66.4% ) samples of 0r , 141 ( 28.5% ) samples of 1r , 19 ( 3.8% ) samples of 2r , 3 ( 0.6% ) samples of 3r and 3 ( 0.6% ) samples of hemodynamically unstable humoral rejection . the st2 level was statistically different according to the grades of acute rejection ( 36 [ 19 - 98 ] , 28 [ 18 - 62 ] , 15 [ 16 - 37 ] , and 191 [ 85 - 343 ] ng / ml , in rejection grades of 0r , 1r , 2r , and 3r+ [ 3r plus hemodynamically unstable humoral rejection ] , respectively p=0.003 ) ( fig . when the same analysis was performed divided by pre - and post-90 days after htx , the sst2 level was not different statistically ; pre-90 days ( 93 [ 47 - 155 ] , 63 [ 30 - 132 ] , 96 [ 82 - 110 ] , and 105 [ 221 - 337 ] ng / ml , consecutively 0r to 3r+ , p=0.09 ) and post-90 days ( 23 [ 15 - 34 ] , 14 [ 20 - 28 ] , 19 [ 15 - 31 ] , and 191 [ 65 - 319 ] ng / ml , consecutively 0r to 3r+ , p=0.052 ) ( fig . the baseline clinical characteristics of the two groups with or without significant rejection were not significantly different for recipient age , etiology of heart failure or initial immunosuppressive agents ( supplementary table 1 in the online - only data supplement ) . when we studied within - subject effects of sst2 using a mixed model , the sst2 level according to the predefined time point was not different according to the presence of significant rejection ( p for interaction=0.94 ) . overall , sst2 at the pre- and post - rejection periods were not different statistically ( 32 [ 19 - 57 ] , 24 [ 12 - 45 ] , and 18 [ 15 - 50 ] ng / ml , before , at the time of , and after treatment of acute significant rejection , respectively , p=0.87 ) ( fig . there were no statistical difference in sst2 levels at the pre- and post - rejection periods in the early postoperative period ( 109 [ 74 - 173 ] , 74 [ 30 - 173 ] , and 31 [ 17 - 213 ] ng / ml , p=0.31 ) or late postoperative period ( 19 [ 14 - 31 ] , 29 [ 12 - 32 ] , and 50 [ 17 - 107 ] ng / ml , p=0.16 ) . among 20 cases with significant rejection , 5 ( 29.4% ) cases at pre - rejection , 10 ( 55.6% ) cases at rejection , and 8 ( 40.0% ) cases at the postrejection period showed sst2 > two standard deviations at the same time point after htx , the values of which were elicited from all 381 samples . measurement of nt - probnp , tnt , crp , or combinations of nt - probnp and crp failed to prove clinical usefulness in predicting acute allograft rejection after htx.6)15 ) battes et al . studied nt - probnp or crp using 1136 blood samples taken at the time of endomyocardial biopsies in 77 htx cases.6 ) the level of nt - pro bnp and crp were high immediately after the surgery and decreased to reach steady status at 12 weeks post - transplantation . meanwhile , high - sensitivity cardiac hs - tni has been reported to have good correlation with acute cellular rejection.7)8 ) patel et al.7 ) analyzed 418 serum samples of 35 htx recipients and reported that the median value of hs - tni was significantly high in the group with acute rejection , and that a cutoff point 0.015 ng / ml of ctni could discriminate acute rejection with 94% sensitivity and a 99% negative predictive value . similarly , in the recent study using 252 samples in 59 htx cases , the absolute value of hs - tni was not found to be different according to the presence of acute rejection.8 ) although the median value of ctni in the samples with acute cellular rejection was higher than the samples without acute rejection , a statistical difference was not observed . reflecting all these clinical data , current guidelines recommend that routine noninvasive testing modalities including cardiac troponin i or t can not substitute endomyocardial biopsy in adult htx recipients ( level of recommendation iii).2 ) the role of sst2 as a biomarker to predict acute rejection after htx has been investigated previously.16)17 ) pascual - figal et al.16 ) reported that sst2 levels were increased as compared with the pre - rejection period , and decreased again after treatment of acute rejection . reported that a higher level of sst2 ( > 600 pg / ml ) could discriminate graft rejection with an area under the curve of 0.720.05 in pediatric htx patients.17 ) however , they had higher rates of rejection events , and included a large proportion of acute humoral rejection among the significant rejection group . as the il-33/st2 signaling pathway also involves t helper type 2 cell differentiation,18 ) it is not unusual that sst2 was higher in the cases with systemic infection or inflammation.19 ) the correlation of sst2 level and wbc / lymphocyte counts that appeared in our study also supports the possible correlation between inflammation and sst2 levels . accordingly , sst2 was reported to be increased in chronic obstructive lung disease , asthma , pulmonary embolism , pulmonary hypertension , and alcohol abuse in other previous studies.19 ) some extreme cases of sst2 levels in our data ( arrows in fig . 3b ) also reflected critical events that occurred within several days ; 1 ) generalized seizure suspected to have resulted from posterior reversible encephalopathy syndrome , 2 ) ischemic injury that appeared in endomyocardial biopsy , and 3 ) septic shock two days after endomyocardial biopsy . nonetheless , we included 67 htx cases with 494 samples ; these numbers are quite comparable to those of previous retrospective studies.6)7)8)15)16 ) second , because the presence of acute rejection was detected by endomyocardial biopsy surveillance , the follow - up duration of the included patients had limited time duration of 1 - 2 years after htx . additionally , the number of acute rejection cases was too small to understand the role of certain biomarkers fully ; 20 patients with 22 blood samples ( 29.9% of total patients and 4.5% in total blood samples ) , even though the incidence rate was quite comparable with the rates of previous reports ( 37% of the patients).7 ) a larger number of subjects would definitely be required to obtain an adequate number of acute rejection episodes .

several biochemical and physiological studies have demonstrated the importance of calcium in regulating vascular and myocardial contraction , activating membrane receptors during cellular signal transduction , releasing many hormones by exocytosis , controlling several transport processes and promoting thrombus formation as co - factor iv ( 1 - 4 ) . total serum calcium exists in three forms : 1 ) ionized , normally 50% of the total ; 2 ) bound to plasma proteins such as albumin , usually 40% of the total ; and 3 ) complexed to anions such as lactate and phosphate , usually 10% of the total . initial ionized calcium ( ica ) , the physiologically active form of calcium found in the blood is regulated by homeostasis ( 5 ) . hypocalcemia has been reported in critically ill patients , most commonly in association with septic condition ( 6 ) . it may vary from an asymptomatic biochemical abnormality to a severe life - threatening condition depending on the duration , severity , and rapidity of development . the causes of hypocalcemia arise either from increased loss of calcium from the circulation or from insufficient entry of calcium into the circulation . it is well recognized that all pathophysiologic changes in shock and trauma have their basis at the cellular and molecular levels . a recent study observed hypocalcemia in 88% of critically ill patients , and a correlation between decreased calcium levels and increased mortality ( 6 ) . however , the relationship between hypocalcemia and mortality risk in trauma patients has not been well defined . base deficit , systemic inflammatory response syndrome ( sirs ) score , and triage - revised trauma score ( t - rts ) are three well - known predictors for the mortality in trauma patients as well as triage tools . therefore , the purpose of the present study was to assess the usefulness of initial ica in predicting mortality , and evaluate its superiority over these three triage tools in the trauma population . arterial blood gas analysis ( abga ) was performed on all trauma patients satisfying the following inclusion criteria in our emergency medical center ( emc ) : 1 ) altered mental status ( glasgow coma scale [ gcs ] score < 13 ) ; 2 ) hemodynamic instability ( initial systolic blood pressure [ sbp ] < 90 mmhg or heart rate > 100 beats per minute ) ; 3 ) respiratory compromise ( < 10 or > 29 breaths per minute ) ; 4 ) severe craniofacial fractures with extensive hemorrhage and/or airway compromise ; 5 ) flail chest ; 6 ) any penetrating injuries to the head , neck , torso , or extremities proximal to the elbow and knee ; 7 ) limb paralysis ; 8) amputation proximal to wrist and ankle ; 9 ) two or more proximal long bone fractures ; 10 ) pelvic bone fractures ; 11 ) falls of > 6 meters ; or 12 ) high speed auto crash , roll - over , or pedestrian run over . the following patients were excluded in this study : 1 ) more than 24 hr of time interval from injury onset to arrival on emc ; 2 ) known underlying liver cirrhosis ; 3 ) known underlying chronic renal failure ; 4 ) known parathyroid disease ; 5 ) current treatment for malignancy ; and 6 ) age younger than 16 years . of 278 consecutive trauma patients admitted to our emc from january to december , 2005 who underwent abga , 23 lost to follow - up ( 18 were transferred to other hospitals and 5 had omitted data ) . arterial blood gas , complete blood cell count , gcs score , and vital signs were measured to obtain ica , base deficit , sirs score , and t - rts . abga was performed by an emergency physician within 10 min after arrival at the emergency department ( blood gas analyzer gem premier 3000 , lexington , ma , u.s.a . ) . serum ph , ica , arterial oxygen pressure ( pao2 ) , arterial carbon dioxide pressure ( paco2 ) , serum bicarbonate , base deficit , and arterial oxygen saturation ( sao2 ) levels were automatically measured by abga . of the two values of ica , i.e. , uncorrected and corrected to ph=7.40 , presented in the abga results , the former was used because it seemed to reflect the biochemical and physiological status of patient more closely . the latter was automatically calculated by following equation : corrected ica = uncorrected ica 10 . the measurement 's coefficient of variation was 2.5% and the ica normal range was 1.15 - 1.35 sirs score was calculated by using body temperature , pulse rate , respiratory rate or paco2 , and white blood cell count , and ranged from 0 to 4 . the t - rts is a compound indicator consisting of gcs score , sbp , and respiratory rate , and ranges from 0 to 12 ( 8) . in addition , the following data were obtained in this study : age , sex , mechanism of injury , amount of intravenous fluid ( crystalloid or colloid ) administered in the prehospital phase of care , transfusion amount ( before and after arrival in the emc ) , injury severity score ( iss ) , revised trauma score ( rts ) , emergency operation , infection , and survival . most of the above data had been measured before discharge from the emc and collected prospectively , but the following data had been done retrospectively : transfusion amount after arrival in the emc , iss and infection . furthermore , the abilities of ica and the other three indicators to predict mortality were compared in the following aspects . first , the areas under the curves ( aucs ) on receiver operating characteristic ( roc ) curve analysis of these indicators were obtained to assess their discriminative power for mortality prediction . second , the sensitivities , specificities , and accuracies of these indicators were determined by constructing a contingency table according to survival after these indicators were transformed into dichotomous variables based on severity . sirs score was easily converted into a dichotomous variable via the above definition ( 2 ) . however , having no definite criteria on major trauma , the other indicators were assessed by obtaining the cut - off point with the best separation between survivors and non - survivors on roc curve analysis . , chicago , il , u.s.a . ) and medcalc ( medcalc 9.3 version , medcalc inc . , univariate analysis was performed by using the student 's t test for continuous variables or the chi - square test for categorical variables . all variables found to be significant by univariate analysis then underwent multivariate logistic regression analysis . in addition , the amounts of intravenous fluid ( crystalloid or colloid ) and transfusion administered before arrival in emc were adjusted for multivariate analysis because of the possible effect on ica ( 9 - 11 ) . the differences between the aucs of ica , base deficit , sirs score , and t - rts were determined by using the nonparametric method proposed by hanley and mcneil ( 12 ) . the differences between the sensitivities , specificities , and accuracies of these indicators were determined by using the mcnemar test . all tests for significance were two - tailed with an alpha level of 0.05 . type i error was corrected by the bonferroni 's method for comparisons of 3 or more variables . arterial blood gas analysis ( abga ) was performed on all trauma patients satisfying the following inclusion criteria in our emergency medical center ( emc ) : 1 ) altered mental status ( glasgow coma scale [ gcs ] score < 13 ) ; 2 ) hemodynamic instability ( initial systolic blood pressure [ sbp ] < 90 mmhg or heart rate > 100 beats per minute ) ; 3 ) respiratory compromise ( < 10 or > 29 breaths per minute ) ; 4 ) severe craniofacial fractures with extensive hemorrhage and/or airway compromise ; 5 ) flail chest ; 6 ) any penetrating injuries to the head , neck , torso , or extremities proximal to the elbow and knee ; 7 ) limb paralysis ; 8) amputation proximal to wrist and ankle ; 9 ) two or more proximal long bone fractures ; 10 ) pelvic bone fractures ; 11 ) falls of > 6 meters ; or 12 ) high speed auto crash , roll - over , or pedestrian run over . the following patients were excluded in this study : 1 ) more than 24 hr of time interval from injury onset to arrival on emc ; 2 ) known underlying liver cirrhosis ; 3 ) known underlying chronic renal failure ; 4 ) known parathyroid disease ; 5 ) current treatment for malignancy ; and 6 ) age younger than 16 years . of 278 consecutive trauma patients admitted to our emc from january to december , 2005 who underwent abga , 23 lost to follow - up ( 18 were transferred to other hospitals and 5 had omitted data ) . arterial blood gas , complete blood cell count , gcs score , and vital signs were measured to obtain ica , base deficit , sirs score , and t - rts . abga was performed by an emergency physician within 10 min after arrival at the emergency department ( blood gas analyzer gem premier 3000 , lexington , ma , u.s.a . ) . serum ph , ica , arterial oxygen pressure ( pao2 ) , arterial carbon dioxide pressure ( paco2 ) , serum bicarbonate , base deficit , and arterial oxygen saturation ( sao2 ) levels were automatically measured by abga . of the two values of ica , i.e. , uncorrected and corrected to ph=7.40 , presented in the abga results , the former was used because it seemed to reflect the biochemical and physiological status of patient more closely . the latter was automatically calculated by following equation : corrected ica = uncorrected ica 10 . the measurement 's coefficient of variation was 2.5% and the ica normal range was 1.15 - 1.35 sirs score was calculated by using body temperature , pulse rate , respiratory rate or paco2 , and white blood cell count , and ranged from 0 to 4 . the t - rts is a compound indicator consisting of gcs score , sbp , and respiratory rate , and ranges from 0 to 12 ( 8) . in addition , the following data were obtained in this study : age , sex , mechanism of injury , amount of intravenous fluid ( crystalloid or colloid ) administered in the prehospital phase of care , transfusion amount ( before and after arrival in the emc ) , injury severity score ( iss ) , revised trauma score ( rts ) , emergency operation , infection , and survival . most of the above data had been measured before discharge from the emc and collected prospectively , but the following data had been done retrospectively : transfusion amount after arrival in the emc , iss and infection . furthermore , the abilities of ica and the other three indicators to predict mortality were compared in the following aspects . first , the areas under the curves ( aucs ) on receiver operating characteristic ( roc ) curve analysis of these indicators were obtained to assess their discriminative power for mortality prediction . second , the sensitivities , specificities , and accuracies of these indicators were determined by constructing a contingency table according to survival after these indicators were transformed into dichotomous variables based on severity . sirs score was easily converted into a dichotomous variable via the above definition ( 2 ) . however , having no definite criteria on major trauma , the other indicators were assessed by obtaining the cut - off point with the best separation between survivors and non - survivors on roc curve analysis . chicago , il , u.s.a . ) and medcalc ( medcalc 9.3 version , medcalc inc . , univariate analysis was performed by using the student 's t test for continuous variables or the chi - square test for categorical variables . all variables found to be significant by univariate analysis then underwent multivariate logistic regression analysis . in addition , the amounts of intravenous fluid ( crystalloid or colloid ) and transfusion administered before arrival in emc were adjusted for multivariate analysis because of the possible effect on ica ( 9 - 11 ) . the differences between the aucs of ica , base deficit , sirs score , and t - rts were determined by using the nonparametric method proposed by hanley and mcneil ( 12 ) . the differences between the sensitivities , specificities , and accuracies of these indicators were determined by using the mcnemar test . all tests for significance were two - tailed with an alpha level of 0.05 . type i error was corrected by the bonferroni 's method for comparisons of 3 or more variables . the mean age of the study population was 47.216.0 yr with a range from 16 to 90 yr . the mean iss was 18.59.2 ( range 4 - 75 ) and mean rts was 7.081.39 ( range 0 - 7.84 ) . blunt trauma was the predominant mechanism of injury at 95.3% ( 243 patients ) , with penetrating injury accounting for only 4.7% . traffic accident injuries were the most common at 60.0% , followed by injuries due to falls at 25.5% , other blunt traumas at 9.8% , and stab injuries 4.7% . age , sex , and mechanism of injury were not associated with mortality ( table 1 ) . univariate analysis confirmed the following to be correlated with mortality : ica , gcs score , serum bicarbonate , base deficit , sao2 , initial sbp , pulse rate , body temperature , sirs score , t - rts , emergency operation , transfusion amount , iss , and rts ( p<0.05 , table 2 ) . on roc curve analysis , the ica cut - off point for mortality prediction was 0.88 mm / l ( fig . the patients were divided into three groups according to this ica cut - off point and the recognized cut - off point for normal condition : 1 ) normal ica concentration ( 1.15 the frequencies of these groups were 7 ( 2.7% ) , 195 ( 76.5% ) , and 53 ( 20.8% ) , respectively . the severity of ionized hypocalcemia was significantly associated with mortality ( p=0.003 , table 3 ) . the cut - off points of the base deficit and t - rts were -6.3 mm / l and 11 , respectively . the cut - off point of sirs score was determined by the inherent sirs score definition of 2 or more . based on these cut - off points , the four indicators were transformed into dichotomous variables , after which the other significant factors as well as amounts of intravenous fluid and blood transfusion administered in the pre - hospital phase were adjusted for multivariate analysis . multivariate logistic regression analysis confirmed ica ( 0.88 mm / l ) , gcs score , and transfusion amount after arrival in the emc to be associated with mortality ( p<0.05 , table 4 ) . the auc of t - rts for mortality prediction was 0.8750.043 ( 95% confidence interval [ ci ] ) and it exhibited excellent discrimination . those of ica and sirs score were 0.6070.062 ( 95% ci ) and 0.6940.059 ( 95% ci ) , respectively , with poor discrimination ( fig . the auc of ica was not different from that of sirs , but smaller than those of the other predictors ( p<0.05 ) . the sensitivities of ica , base deficit , sirs score , and t - rts were 82.9% , 76.4% , 67.1% , and 74.5% , their specificities were 41.0% , 64.1% , 64.1% , and 87.2% , and their accuracies were 76.5% , 74.5% , 66.7% , and 76.5% , respectively ( table 5 ) . the sensitivity of ica was higher than that of sirs score , but the specificity of ica was lower than that of t - rts ( p<0.01 ) . ionized hypocalcemia is a common finding in the intensive care unit ( icu ) , particularly in patients with sepsis , major trauma or pancreatitis ( 4 , 6 , 13 ) . it has been shown to be of prognostic value due to its relation with mortality in critically ill patients ( 6 , 10 , 13 - 15 ) . insufficient secretion or inhibited action of parathyroid hormone , decreased vitamin d3 production , and increased calcium deposition , both intra- and extra - cellularly , have all been suggested to be involved in the pathogenesis of hypocalcemia ( 4 ) . , calcium plays a critical role , but few studies have attempted to clearly define the consequences of low ica concentrations such as compromised hemodynamics . the contractile function of the heart is compromised during hypocalcemia but rapid and reliable recovery is achieved by calcium administration ( 16 ) . frank cardiac failure and a prolonged qt interval have been observed at ica levels of approximately 0.5 mm / l ( 17 ) . in addition , blood coagulation may be compromised due to hypocalcemia at ica levels of < 0.6 - 0.7 mm / l ( 18 ) . hastbacka and pettila ( 19 ) measured ica in a cohort of 941 consecutive , critically ill patients . mm / l ) in 85% of patients at icu admission . in their study , they first defined the cut - off point for severe ionized hypocalcemia as 0.9 mm / l , but did not present clear evidence for this definition . our study results supported an ica cut - off point for mortality prediction of 0.88 mm / l , with a lower level indicating severe ionized hypocalcemia . as expected , our criterion of severe ionized hypocalcemia was similar to theirs . vivien et al . ( 10 ) measured ica in a cohort of 212 consecutive , severe trauma patients . they found mild ( 0.90 - 1.14 mm / l ) and severe ( < 0.90 mm / l ) ionized hypocalcemia in 64% and 10% of patients at hospital admission , respectively . their study results indicated a progressive increase in mortality with decreasing ica levels , in agreement with our own results ( table 3 ) . in the present study , severe ionized hypocalcemia , low gcs score , and large transfusion amount received after arrival in the emc were significantly associated with mortality on multivariate logistic regression analysis . such a correlation of severe ionized hypocalcemia to mortality was also shown by previous studies using multivariate analysis ( 19 , 20 ) . several studies have reported that ica concentration might be influenced by intravenous fluid ( crystalloid or colloid ) or blood transfusion . fulgenico et al . ( 9 ) reported that the amount of crystalloids administrated to the patient after brain death was significantly correlated with plasma ionized calcium concentration . vivien et al . ( 10 ) demonstrated that the volume of colloids administered was significantly and negatively correlated with ica concentration . another study found that elevations in circulating citrate levels and speed of transfusion were correlated with decreases in ica concentration during blood transfusion ( 11 ) . based on these reported findings , we included the amounts of these factors administrated in the prehospital phase of care in the multivariate regression , even though they were not associated with mortality on univariate analysis . however , the result was same as that of multivariate regression analysis with these factors excluded . this unexpected result was thought to be due to the lack of any significant association of ica concentration with these factors on correlation analysis and/or to the small patient population underwent blood transfusion ( eight cases ) and colloid infusion ( only one case ) in the prehospital phase ( data not shown ) . as aforementioned however , this may not be reflected in a sufficient discriminative power of ica for mortality prediction ( 21 , 22 ) . we therefore performed two discriminative analyses : 1 ) corresponding statistics of classification tabulations , i.e. , sensitivities , specificities and accuracies ; and 2 ) auc by roc analysis . only one previous study by hastbacka and pettila performed such discriminative analyses for ica mortality prediction ( 19 ) . the discrimination of roc analysis in their study was an auc of 0.636 ( 95% ci 0.591 - 0.681 ) , while the sensitivity and specificity of severe ionized hypocalcemia were 98% and 7% for mortality , respectively . the discrepancies of the sensitivities and specificities between the two studies seem to have been due to differences in patient selection : all subjects in our study were trauma patients , whereas only 3% in their study were . we recently reported that base deficit , t - rts , and sirs score were associated with mortality and that the first two were significant mortality predictors , despite their different discriminative powers in the following order : t - rts > base deficit > sirs score ( 23 ) . however , no study has compared the discriminative powers of ionized hypocalcemia and these predictors before the present study . in our study , using corresponding statistics of classification tabulations for mortality , the discriminative power of ica was the same as or inferior to that of base deficit and t - rts , except for sirs score . moreover , discrimination by roc analysis for mortality prediction revealed that auc of ica was 0.607 , which was considered a poor discrimination in general . on the other hand , the aucs of base deficit ( 0.736 ) and t - rts ( 0.875 ) represented acceptable and excellent discrimination , respectively , as in our previous study ( 23 ) . abga requires only one puncture for blood sampling and can be performed within a few minutes . all the obtained results including ica and base deficit , can be easily utilized without any modification . however , sirs score and t - rts are not single factors , but are compound factors consisting of several factors that require certain modification by recoding . in addition , the former shows little variation because they comprise instrument - measured data , whereas the latter may suffer from greater variation due to partial or complete human measurement , i.e. , doctors , nurses , or emergency medical technicians . in a recent study , gcs score and its components , one of the three parts of the t - rts compound indicator , were found to have only a moderate degree of interrater agreement ( 24 ) . unfortunately , despite its several advantages , ica suffered from the fatal disadvantage of a low discriminative power , which is the most important property for mortality predictor . the limitation of this study was a possible selection bias in selecting the patient population , which may have resulted from the small sample size and the single center approach . therefore , we suggest that a multi - center or randomized trial be conducted in the future to avoid these biases and confirm our results . in conclusion , ica was a significant risk factor associated with mortality , but with a poorer discriminative power for mortality prediction and triage than previous mortality predictors , especially t - rts . the ica predictor is a single factor , whereas t - rts is a compound indicator consisting of three factors ( gcs score , sbp , and respiratory rate ) which have been recognized as mortality predictors . considering the difficulty and complexity in measuring t - rts , its superiority to ica as a mortality predictor is understandable . if a new predictor combining ica with the other significant factors known to be associated with mortality can be developed , we expect that it may be a superior mortality predictor than t - rts .

ionized hypocalcemia is a common finding in critically ill patients , but the relationship between ionized hypocalcemia and mortality risk in trauma patients has not been well established . the aim of this study was to assess the usefulness of initial ionized calcium ( ica ) in predicting mortality in the trauma population , and evaluate its superiority over the three other triage tools : base deficit , systemic inflammatory response syndrome ( sirs ) score , and triage - revised trauma score ( t - rts ) . a proand retrospective study was performed on 255 consecutive trauma patients admitted to our emergency medical center from january to december , 2005 , who underwent arterial blood gas analysis . multivariate logistic regression analysis confirmed ica ( 0.88 mm / l ) , low glasgow coma scale score , and a large transfusion amount to be significant risk factors associated with mortality ( p<0.05 ) . the sensitivities of ica , base deficit , sirs score , and t - rts were 82.9% , 76.4% , 67.1% , and 74.5% , and their specificities were 41.0% , 64.1% , 64.1% , and 87.2% , respectively . receiver operating characteristic curve analysis determined the areas under the curves of these parameters to be 0.6070.062 , 0.7360.056 , 0.6940.059 , and 0.875 0.043 , respectively ( 95% confidence interval ) . although initial ica ( 0.88 mm / l ) was confirmed as a significant risk factor associated with mortality , it exhibited a poorer discriminative power for mortality prediction than other predictors , especially t - rts .

INTRODUCTION MATERIALS AND METHODS Patient population Data collection and assessment Statistical analysis RESULTS DISCUSSION

initial ionized calcium ( ica ) , the physiologically active form of calcium found in the blood is regulated by homeostasis ( 5 ) . hypocalcemia has been reported in critically ill patients , most commonly in association with septic condition ( 6 ) . it may vary from an asymptomatic biochemical abnormality to a severe life - threatening condition depending on the duration , severity , and rapidity of development . a recent study observed hypocalcemia in 88% of critically ill patients , and a correlation between decreased calcium levels and increased mortality ( 6 ) . however , the relationship between hypocalcemia and mortality risk in trauma patients has not been well defined . base deficit , systemic inflammatory response syndrome ( sirs ) score , and triage - revised trauma score ( t - rts ) are three well - known predictors for the mortality in trauma patients as well as triage tools . therefore , the purpose of the present study was to assess the usefulness of initial ica in predicting mortality , and evaluate its superiority over these three triage tools in the trauma population . arterial blood gas analysis ( abga ) was performed on all trauma patients satisfying the following inclusion criteria in our emergency medical center ( emc ) : 1 ) altered mental status ( glasgow coma scale [ gcs ] score < 13 ) ; 2 ) hemodynamic instability ( initial systolic blood pressure [ sbp ] < 90 mmhg or heart rate > 100 beats per minute ) ; 3 ) respiratory compromise ( < 10 or > 29 breaths per minute ) ; 4 ) severe craniofacial fractures with extensive hemorrhage and/or airway compromise ; 5 ) flail chest ; 6 ) any penetrating injuries to the head , neck , torso , or extremities proximal to the elbow and knee ; 7 ) limb paralysis ; 8) amputation proximal to wrist and ankle ; 9 ) two or more proximal long bone fractures ; 10 ) pelvic bone fractures ; 11 ) falls of > 6 meters ; or 12 ) high speed auto crash , roll - over , or pedestrian run over . the following patients were excluded in this study : 1 ) more than 24 hr of time interval from injury onset to arrival on emc ; 2 ) known underlying liver cirrhosis ; 3 ) known underlying chronic renal failure ; 4 ) known parathyroid disease ; 5 ) current treatment for malignancy ; and 6 ) age younger than 16 years . of 278 consecutive trauma patients admitted to our emc from january to december , 2005 who underwent abga , 23 lost to follow - up ( 18 were transferred to other hospitals and 5 had omitted data ) . arterial blood gas , complete blood cell count , gcs score , and vital signs were measured to obtain ica , base deficit , sirs score , and t - rts . abga was performed by an emergency physician within 10 min after arrival at the emergency department ( blood gas analyzer gem premier 3000 , lexington , ma , u.s.a . ) serum ph , ica , arterial oxygen pressure ( pao2 ) , arterial carbon dioxide pressure ( paco2 ) , serum bicarbonate , base deficit , and arterial oxygen saturation ( sao2 ) levels were automatically measured by abga . of the two values of ica , i.e. , uncorrected and corrected to ph=7.40 , presented in the abga results , the former was used because it seemed to reflect the biochemical and physiological status of patient more closely . the measurement 's coefficient of variation was 2.5% and the ica normal range was 1.15 - 1.35 sirs score was calculated by using body temperature , pulse rate , respiratory rate or paco2 , and white blood cell count , and ranged from 0 to 4 . the t - rts is a compound indicator consisting of gcs score , sbp , and respiratory rate , and ranges from 0 to 12 ( 8) . in addition , the following data were obtained in this study : age , sex , mechanism of injury , amount of intravenous fluid ( crystalloid or colloid ) administered in the prehospital phase of care , transfusion amount ( before and after arrival in the emc ) , injury severity score ( iss ) , revised trauma score ( rts ) , emergency operation , infection , and survival . most of the above data had been measured before discharge from the emc and collected prospectively , but the following data had been done retrospectively : transfusion amount after arrival in the emc , iss and infection . furthermore , the abilities of ica and the other three indicators to predict mortality were compared in the following aspects . first , the areas under the curves ( aucs ) on receiver operating characteristic ( roc ) curve analysis of these indicators were obtained to assess their discriminative power for mortality prediction . second , the sensitivities , specificities , and accuracies of these indicators were determined by constructing a contingency table according to survival after these indicators were transformed into dichotomous variables based on severity . sirs score was easily converted into a dichotomous variable via the above definition ( 2 ) . however , having no definite criteria on major trauma , the other indicators were assessed by obtaining the cut - off point with the best separation between survivors and non - survivors on roc curve analysis . , univariate analysis was performed by using the student 's t test for continuous variables or the chi - square test for categorical variables . all variables found to be significant by univariate analysis then underwent multivariate logistic regression analysis . in addition , the amounts of intravenous fluid ( crystalloid or colloid ) and transfusion administered before arrival in emc were adjusted for multivariate analysis because of the possible effect on ica ( 9 - 11 ) . the differences between the aucs of ica , base deficit , sirs score , and t - rts were determined by using the nonparametric method proposed by hanley and mcneil ( 12 ) . the differences between the sensitivities , specificities , and accuracies of these indicators were determined by using the mcnemar test . arterial blood gas analysis ( abga ) was performed on all trauma patients satisfying the following inclusion criteria in our emergency medical center ( emc ) : 1 ) altered mental status ( glasgow coma scale [ gcs ] score < 13 ) ; 2 ) hemodynamic instability ( initial systolic blood pressure [ sbp ] < 90 mmhg or heart rate > 100 beats per minute ) ; 3 ) respiratory compromise ( < 10 or > 29 breaths per minute ) ; 4 ) severe craniofacial fractures with extensive hemorrhage and/or airway compromise ; 5 ) flail chest ; 6 ) any penetrating injuries to the head , neck , torso , or extremities proximal to the elbow and knee ; 7 ) limb paralysis ; 8) amputation proximal to wrist and ankle ; 9 ) two or more proximal long bone fractures ; 10 ) pelvic bone fractures ; 11 ) falls of > 6 meters ; or 12 ) high speed auto crash , roll - over , or pedestrian run over . the following patients were excluded in this study : 1 ) more than 24 hr of time interval from injury onset to arrival on emc ; 2 ) known underlying liver cirrhosis ; 3 ) known underlying chronic renal failure ; 4 ) known parathyroid disease ; 5 ) current treatment for malignancy ; and 6 ) age younger than 16 years . of 278 consecutive trauma patients admitted to our emc from january to december , 2005 who underwent abga , 23 lost to follow - up ( 18 were transferred to other hospitals and 5 had omitted data ) . arterial blood gas , complete blood cell count , gcs score , and vital signs were measured to obtain ica , base deficit , sirs score , and t - rts . abga was performed by an emergency physician within 10 min after arrival at the emergency department ( blood gas analyzer gem premier 3000 , lexington , ma , u.s.a . ) serum ph , ica , arterial oxygen pressure ( pao2 ) , arterial carbon dioxide pressure ( paco2 ) , serum bicarbonate , base deficit , and arterial oxygen saturation ( sao2 ) levels were automatically measured by abga . of the two values of ica , i.e. the measurement 's coefficient of variation was 2.5% and the ica normal range was 1.15 - 1.35 sirs score was calculated by using body temperature , pulse rate , respiratory rate or paco2 , and white blood cell count , and ranged from 0 to 4 . the t - rts is a compound indicator consisting of gcs score , sbp , and respiratory rate , and ranges from 0 to 12 ( 8) . in addition , the following data were obtained in this study : age , sex , mechanism of injury , amount of intravenous fluid ( crystalloid or colloid ) administered in the prehospital phase of care , transfusion amount ( before and after arrival in the emc ) , injury severity score ( iss ) , revised trauma score ( rts ) , emergency operation , infection , and survival . most of the above data had been measured before discharge from the emc and collected prospectively , but the following data had been done retrospectively : transfusion amount after arrival in the emc , iss and infection . furthermore , the abilities of ica and the other three indicators to predict mortality were compared in the following aspects . first , the areas under the curves ( aucs ) on receiver operating characteristic ( roc ) curve analysis of these indicators were obtained to assess their discriminative power for mortality prediction . second , the sensitivities , specificities , and accuracies of these indicators were determined by constructing a contingency table according to survival after these indicators were transformed into dichotomous variables based on severity . sirs score was easily converted into a dichotomous variable via the above definition ( 2 ) . however , having no definite criteria on major trauma , the other indicators were assessed by obtaining the cut - off point with the best separation between survivors and non - survivors on roc curve analysis . , univariate analysis was performed by using the student 's t test for continuous variables or the chi - square test for categorical variables . all variables found to be significant by univariate analysis then underwent multivariate logistic regression analysis . in addition , the amounts of intravenous fluid ( crystalloid or colloid ) and transfusion administered before arrival in emc were adjusted for multivariate analysis because of the possible effect on ica ( 9 - 11 ) . the differences between the aucs of ica , base deficit , sirs score , and t - rts were determined by using the nonparametric method proposed by hanley and mcneil ( 12 ) . the differences between the sensitivities , specificities , and accuracies of these indicators were determined by using the mcnemar test . traffic accident injuries were the most common at 60.0% , followed by injuries due to falls at 25.5% , other blunt traumas at 9.8% , and stab injuries 4.7% . age , sex , and mechanism of injury were not associated with mortality ( table 1 ) . univariate analysis confirmed the following to be correlated with mortality : ica , gcs score , serum bicarbonate , base deficit , sao2 , initial sbp , pulse rate , body temperature , sirs score , t - rts , emergency operation , transfusion amount , iss , and rts ( p<0.05 , table 2 ) . on roc curve analysis , the ica cut - off point for mortality prediction was 0.88 mm / l ( fig . the patients were divided into three groups according to this ica cut - off point and the recognized cut - off point for normal condition : 1 ) normal ica concentration ( 1.15 the frequencies of these groups were 7 ( 2.7% ) , 195 ( 76.5% ) , and 53 ( 20.8% ) , respectively . the severity of ionized hypocalcemia was significantly associated with mortality ( p=0.003 , table 3 ) . the cut - off points of the base deficit and t - rts were -6.3 mm / l and 11 , respectively . the cut - off point of sirs score was determined by the inherent sirs score definition of 2 or more . multivariate logistic regression analysis confirmed ica ( 0.88 mm / l ) , gcs score , and transfusion amount after arrival in the emc to be associated with mortality ( p<0.05 , table 4 ) . the auc of t - rts for mortality prediction was 0.8750.043 ( 95% confidence interval [ ci ] ) and it exhibited excellent discrimination . those of ica and sirs score were 0.6070.062 ( 95% ci ) and 0.6940.059 ( 95% ci ) , respectively , with poor discrimination ( fig . the auc of ica was not different from that of sirs , but smaller than those of the other predictors ( p<0.05 ) . the sensitivities of ica , base deficit , sirs score , and t - rts were 82.9% , 76.4% , 67.1% , and 74.5% , their specificities were 41.0% , 64.1% , 64.1% , and 87.2% , and their accuracies were 76.5% , 74.5% , 66.7% , and 76.5% , respectively ( table 5 ) . the sensitivity of ica was higher than that of sirs score , but the specificity of ica was lower than that of t - rts ( p<0.01 ) . ionized hypocalcemia is a common finding in the intensive care unit ( icu ) , particularly in patients with sepsis , major trauma or pancreatitis ( 4 , 6 , 13 ) . it has been shown to be of prognostic value due to its relation with mortality in critically ill patients ( 6 , 10 , 13 - 15 ) . insufficient secretion or inhibited action of parathyroid hormone , decreased vitamin d3 production , and increased calcium deposition , both intra- and extra - cellularly , have all been suggested to be involved in the pathogenesis of hypocalcemia ( 4 ) . , calcium plays a critical role , but few studies have attempted to clearly define the consequences of low ica concentrations such as compromised hemodynamics . frank cardiac failure and a prolonged qt interval have been observed at ica levels of approximately 0.5 mm / l ( 17 ) . in addition , blood coagulation may be compromised due to hypocalcemia at ica levels of < 0.6 - 0.7 mm / l ( 18 ) . hastbacka and pettila ( 19 ) measured ica in a cohort of 941 consecutive , critically ill patients . mm / l ) in 85% of patients at icu admission . in their study , they first defined the cut - off point for severe ionized hypocalcemia as 0.9 mm / l , but did not present clear evidence for this definition . our study results supported an ica cut - off point for mortality prediction of 0.88 mm / l , with a lower level indicating severe ionized hypocalcemia . as expected , our criterion of severe ionized hypocalcemia was similar to theirs . ( 10 ) measured ica in a cohort of 212 consecutive , severe trauma patients . they found mild ( 0.90 - 1.14 mm / l ) and severe ( < 0.90 mm / l ) ionized hypocalcemia in 64% and 10% of patients at hospital admission , respectively . in the present study , severe ionized hypocalcemia , low gcs score , and large transfusion amount received after arrival in the emc were significantly associated with mortality on multivariate logistic regression analysis . such a correlation of severe ionized hypocalcemia to mortality was also shown by previous studies using multivariate analysis ( 19 , 20 ) . based on these reported findings , we included the amounts of these factors administrated in the prehospital phase of care in the multivariate regression , even though they were not associated with mortality on univariate analysis . however , the result was same as that of multivariate regression analysis with these factors excluded . this unexpected result was thought to be due to the lack of any significant association of ica concentration with these factors on correlation analysis and/or to the small patient population underwent blood transfusion ( eight cases ) and colloid infusion ( only one case ) in the prehospital phase ( data not shown ) . as aforementioned however , this may not be reflected in a sufficient discriminative power of ica for mortality prediction ( 21 , 22 ) . only one previous study by hastbacka and pettila performed such discriminative analyses for ica mortality prediction ( 19 ) . the discrimination of roc analysis in their study was an auc of 0.636 ( 95% ci 0.591 - 0.681 ) , while the sensitivity and specificity of severe ionized hypocalcemia were 98% and 7% for mortality , respectively . the discrepancies of the sensitivities and specificities between the two studies seem to have been due to differences in patient selection : all subjects in our study were trauma patients , whereas only 3% in their study were . we recently reported that base deficit , t - rts , and sirs score were associated with mortality and that the first two were significant mortality predictors , despite their different discriminative powers in the following order : t - rts > base deficit > sirs score ( 23 ) . however , no study has compared the discriminative powers of ionized hypocalcemia and these predictors before the present study . in our study , using corresponding statistics of classification tabulations for mortality , the discriminative power of ica was the same as or inferior to that of base deficit and t - rts , except for sirs score . moreover , discrimination by roc analysis for mortality prediction revealed that auc of ica was 0.607 , which was considered a poor discrimination in general . on the other hand , the aucs of base deficit ( 0.736 ) and t - rts ( 0.875 ) represented acceptable and excellent discrimination , respectively , as in our previous study ( 23 ) . all the obtained results including ica and base deficit , can be easily utilized without any modification . however , sirs score and t - rts are not single factors , but are compound factors consisting of several factors that require certain modification by recoding . , doctors , nurses , or emergency medical technicians . in a recent study , gcs score and its components , one of the three parts of the t - rts compound indicator , were found to have only a moderate degree of interrater agreement ( 24 ) . unfortunately , despite its several advantages , ica suffered from the fatal disadvantage of a low discriminative power , which is the most important property for mortality predictor . the limitation of this study was a possible selection bias in selecting the patient population , which may have resulted from the small sample size and the single center approach . in conclusion , ica was a significant risk factor associated with mortality , but with a poorer discriminative power for mortality prediction and triage than previous mortality predictors , especially t - rts . the ica predictor is a single factor , whereas t - rts is a compound indicator consisting of three factors ( gcs score , sbp , and respiratory rate ) which have been recognized as mortality predictors . considering the difficulty and complexity in measuring t - rts , its superiority to ica as a mortality predictor is understandable . if a new predictor combining ica with the other significant factors known to be associated with mortality can be developed , we expect that it may be a superior mortality predictor than t - rts .

several biochemical and physiological studies have demonstrated the importance of calcium in regulating vascular and myocardial contraction , activating membrane receptors during cellular signal transduction , releasing many hormones by exocytosis , controlling several transport processes and promoting thrombus formation as co - factor iv ( 1 - 4 ) . total serum calcium exists in three forms : 1 ) ionized , normally 50% of the total ; 2 ) bound to plasma proteins such as albumin , usually 40% of the total ; and 3 ) complexed to anions such as lactate and phosphate , usually 10% of the total . initial ionized calcium ( ica ) , the physiologically active form of calcium found in the blood is regulated by homeostasis ( 5 ) . hypocalcemia has been reported in critically ill patients , most commonly in association with septic condition ( 6 ) . it may vary from an asymptomatic biochemical abnormality to a severe life - threatening condition depending on the duration , severity , and rapidity of development . the causes of hypocalcemia arise either from increased loss of calcium from the circulation or from insufficient entry of calcium into the circulation . it is well recognized that all pathophysiologic changes in shock and trauma have their basis at the cellular and molecular levels . a recent study observed hypocalcemia in 88% of critically ill patients , and a correlation between decreased calcium levels and increased mortality ( 6 ) . however , the relationship between hypocalcemia and mortality risk in trauma patients has not been well defined . base deficit , systemic inflammatory response syndrome ( sirs ) score , and triage - revised trauma score ( t - rts ) are three well - known predictors for the mortality in trauma patients as well as triage tools . therefore , the purpose of the present study was to assess the usefulness of initial ica in predicting mortality , and evaluate its superiority over these three triage tools in the trauma population . arterial blood gas analysis ( abga ) was performed on all trauma patients satisfying the following inclusion criteria in our emergency medical center ( emc ) : 1 ) altered mental status ( glasgow coma scale [ gcs ] score < 13 ) ; 2 ) hemodynamic instability ( initial systolic blood pressure [ sbp ] < 90 mmhg or heart rate > 100 beats per minute ) ; 3 ) respiratory compromise ( < 10 or > 29 breaths per minute ) ; 4 ) severe craniofacial fractures with extensive hemorrhage and/or airway compromise ; 5 ) flail chest ; 6 ) any penetrating injuries to the head , neck , torso , or extremities proximal to the elbow and knee ; 7 ) limb paralysis ; 8) amputation proximal to wrist and ankle ; 9 ) two or more proximal long bone fractures ; 10 ) pelvic bone fractures ; 11 ) falls of > 6 meters ; or 12 ) high speed auto crash , roll - over , or pedestrian run over . the following patients were excluded in this study : 1 ) more than 24 hr of time interval from injury onset to arrival on emc ; 2 ) known underlying liver cirrhosis ; 3 ) known underlying chronic renal failure ; 4 ) known parathyroid disease ; 5 ) current treatment for malignancy ; and 6 ) age younger than 16 years . of 278 consecutive trauma patients admitted to our emc from january to december , 2005 who underwent abga , 23 lost to follow - up ( 18 were transferred to other hospitals and 5 had omitted data ) . arterial blood gas , complete blood cell count , gcs score , and vital signs were measured to obtain ica , base deficit , sirs score , and t - rts . serum ph , ica , arterial oxygen pressure ( pao2 ) , arterial carbon dioxide pressure ( paco2 ) , serum bicarbonate , base deficit , and arterial oxygen saturation ( sao2 ) levels were automatically measured by abga . , uncorrected and corrected to ph=7.40 , presented in the abga results , the former was used because it seemed to reflect the biochemical and physiological status of patient more closely . the measurement 's coefficient of variation was 2.5% and the ica normal range was 1.15 - 1.35 sirs score was calculated by using body temperature , pulse rate , respiratory rate or paco2 , and white blood cell count , and ranged from 0 to 4 . the t - rts is a compound indicator consisting of gcs score , sbp , and respiratory rate , and ranges from 0 to 12 ( 8) . in addition , the following data were obtained in this study : age , sex , mechanism of injury , amount of intravenous fluid ( crystalloid or colloid ) administered in the prehospital phase of care , transfusion amount ( before and after arrival in the emc ) , injury severity score ( iss ) , revised trauma score ( rts ) , emergency operation , infection , and survival . most of the above data had been measured before discharge from the emc and collected prospectively , but the following data had been done retrospectively : transfusion amount after arrival in the emc , iss and infection . furthermore , the abilities of ica and the other three indicators to predict mortality were compared in the following aspects . first , the areas under the curves ( aucs ) on receiver operating characteristic ( roc ) curve analysis of these indicators were obtained to assess their discriminative power for mortality prediction . second , the sensitivities , specificities , and accuracies of these indicators were determined by constructing a contingency table according to survival after these indicators were transformed into dichotomous variables based on severity . however , having no definite criteria on major trauma , the other indicators were assessed by obtaining the cut - off point with the best separation between survivors and non - survivors on roc curve analysis . in addition , the amounts of intravenous fluid ( crystalloid or colloid ) and transfusion administered before arrival in emc were adjusted for multivariate analysis because of the possible effect on ica ( 9 - 11 ) . the differences between the aucs of ica , base deficit , sirs score , and t - rts were determined by using the nonparametric method proposed by hanley and mcneil ( 12 ) . the differences between the sensitivities , specificities , and accuracies of these indicators were determined by using the mcnemar test . arterial blood gas analysis ( abga ) was performed on all trauma patients satisfying the following inclusion criteria in our emergency medical center ( emc ) : 1 ) altered mental status ( glasgow coma scale [ gcs ] score < 13 ) ; 2 ) hemodynamic instability ( initial systolic blood pressure [ sbp ] < 90 mmhg or heart rate > 100 beats per minute ) ; 3 ) respiratory compromise ( < 10 or > 29 breaths per minute ) ; 4 ) severe craniofacial fractures with extensive hemorrhage and/or airway compromise ; 5 ) flail chest ; 6 ) any penetrating injuries to the head , neck , torso , or extremities proximal to the elbow and knee ; 7 ) limb paralysis ; 8) amputation proximal to wrist and ankle ; 9 ) two or more proximal long bone fractures ; 10 ) pelvic bone fractures ; 11 ) falls of > 6 meters ; or 12 ) high speed auto crash , roll - over , or pedestrian run over . the following patients were excluded in this study : 1 ) more than 24 hr of time interval from injury onset to arrival on emc ; 2 ) known underlying liver cirrhosis ; 3 ) known underlying chronic renal failure ; 4 ) known parathyroid disease ; 5 ) current treatment for malignancy ; and 6 ) age younger than 16 years . of 278 consecutive trauma patients admitted to our emc from january to december , 2005 who underwent abga , 23 lost to follow - up ( 18 were transferred to other hospitals and 5 had omitted data ) . arterial blood gas , complete blood cell count , gcs score , and vital signs were measured to obtain ica , base deficit , sirs score , and t - rts . serum ph , ica , arterial oxygen pressure ( pao2 ) , arterial carbon dioxide pressure ( paco2 ) , serum bicarbonate , base deficit , and arterial oxygen saturation ( sao2 ) levels were automatically measured by abga . , uncorrected and corrected to ph=7.40 , presented in the abga results , the former was used because it seemed to reflect the biochemical and physiological status of patient more closely . the measurement 's coefficient of variation was 2.5% and the ica normal range was 1.15 - 1.35 sirs score was calculated by using body temperature , pulse rate , respiratory rate or paco2 , and white blood cell count , and ranged from 0 to 4 . the t - rts is a compound indicator consisting of gcs score , sbp , and respiratory rate , and ranges from 0 to 12 ( 8) . in addition , the following data were obtained in this study : age , sex , mechanism of injury , amount of intravenous fluid ( crystalloid or colloid ) administered in the prehospital phase of care , transfusion amount ( before and after arrival in the emc ) , injury severity score ( iss ) , revised trauma score ( rts ) , emergency operation , infection , and survival . most of the above data had been measured before discharge from the emc and collected prospectively , but the following data had been done retrospectively : transfusion amount after arrival in the emc , iss and infection . furthermore , the abilities of ica and the other three indicators to predict mortality were compared in the following aspects . first , the areas under the curves ( aucs ) on receiver operating characteristic ( roc ) curve analysis of these indicators were obtained to assess their discriminative power for mortality prediction . second , the sensitivities , specificities , and accuracies of these indicators were determined by constructing a contingency table according to survival after these indicators were transformed into dichotomous variables based on severity . however , having no definite criteria on major trauma , the other indicators were assessed by obtaining the cut - off point with the best separation between survivors and non - survivors on roc curve analysis . in addition , the amounts of intravenous fluid ( crystalloid or colloid ) and transfusion administered before arrival in emc were adjusted for multivariate analysis because of the possible effect on ica ( 9 - 11 ) . the differences between the aucs of ica , base deficit , sirs score , and t - rts were determined by using the nonparametric method proposed by hanley and mcneil ( 12 ) . the differences between the sensitivities , specificities , and accuracies of these indicators were determined by using the mcnemar test . the mean age of the study population was 47.216.0 yr with a range from 16 to 90 yr . the mean iss was 18.59.2 ( range 4 - 75 ) and mean rts was 7.081.39 ( range 0 - 7.84 ) . blunt trauma was the predominant mechanism of injury at 95.3% ( 243 patients ) , with penetrating injury accounting for only 4.7% . traffic accident injuries were the most common at 60.0% , followed by injuries due to falls at 25.5% , other blunt traumas at 9.8% , and stab injuries 4.7% . age , sex , and mechanism of injury were not associated with mortality ( table 1 ) . univariate analysis confirmed the following to be correlated with mortality : ica , gcs score , serum bicarbonate , base deficit , sao2 , initial sbp , pulse rate , body temperature , sirs score , t - rts , emergency operation , transfusion amount , iss , and rts ( p<0.05 , table 2 ) . on roc curve analysis , the ica cut - off point for mortality prediction was 0.88 mm / l ( fig . the patients were divided into three groups according to this ica cut - off point and the recognized cut - off point for normal condition : 1 ) normal ica concentration ( 1.15 the frequencies of these groups were 7 ( 2.7% ) , 195 ( 76.5% ) , and 53 ( 20.8% ) , respectively . the severity of ionized hypocalcemia was significantly associated with mortality ( p=0.003 , table 3 ) . the cut - off points of the base deficit and t - rts were -6.3 mm / l and 11 , respectively . based on these cut - off points , the four indicators were transformed into dichotomous variables , after which the other significant factors as well as amounts of intravenous fluid and blood transfusion administered in the pre - hospital phase were adjusted for multivariate analysis . multivariate logistic regression analysis confirmed ica ( 0.88 mm / l ) , gcs score , and transfusion amount after arrival in the emc to be associated with mortality ( p<0.05 , table 4 ) . the auc of t - rts for mortality prediction was 0.8750.043 ( 95% confidence interval [ ci ] ) and it exhibited excellent discrimination . those of ica and sirs score were 0.6070.062 ( 95% ci ) and 0.6940.059 ( 95% ci ) , respectively , with poor discrimination ( fig . the auc of ica was not different from that of sirs , but smaller than those of the other predictors ( p<0.05 ) . the sensitivities of ica , base deficit , sirs score , and t - rts were 82.9% , 76.4% , 67.1% , and 74.5% , their specificities were 41.0% , 64.1% , 64.1% , and 87.2% , and their accuracies were 76.5% , 74.5% , 66.7% , and 76.5% , respectively ( table 5 ) . the sensitivity of ica was higher than that of sirs score , but the specificity of ica was lower than that of t - rts ( p<0.01 ) . ionized hypocalcemia is a common finding in the intensive care unit ( icu ) , particularly in patients with sepsis , major trauma or pancreatitis ( 4 , 6 , 13 ) . it has been shown to be of prognostic value due to its relation with mortality in critically ill patients ( 6 , 10 , 13 - 15 ) . insufficient secretion or inhibited action of parathyroid hormone , decreased vitamin d3 production , and increased calcium deposition , both intra- and extra - cellularly , have all been suggested to be involved in the pathogenesis of hypocalcemia ( 4 ) . , calcium plays a critical role , but few studies have attempted to clearly define the consequences of low ica concentrations such as compromised hemodynamics . frank cardiac failure and a prolonged qt interval have been observed at ica levels of approximately 0.5 mm / l ( 17 ) . in addition , blood coagulation may be compromised due to hypocalcemia at ica levels of < 0.6 - 0.7 mm / l ( 18 ) . hastbacka and pettila ( 19 ) measured ica in a cohort of 941 consecutive , critically ill patients . in their study , they first defined the cut - off point for severe ionized hypocalcemia as 0.9 mm / l , but did not present clear evidence for this definition . our study results supported an ica cut - off point for mortality prediction of 0.88 mm / l , with a lower level indicating severe ionized hypocalcemia . they found mild ( 0.90 - 1.14 mm / l ) and severe ( < 0.90 mm / l ) ionized hypocalcemia in 64% and 10% of patients at hospital admission , respectively . their study results indicated a progressive increase in mortality with decreasing ica levels , in agreement with our own results ( table 3 ) . in the present study , severe ionized hypocalcemia , low gcs score , and large transfusion amount received after arrival in the emc were significantly associated with mortality on multivariate logistic regression analysis . ( 9 ) reported that the amount of crystalloids administrated to the patient after brain death was significantly correlated with plasma ionized calcium concentration . ( 10 ) demonstrated that the volume of colloids administered was significantly and negatively correlated with ica concentration . another study found that elevations in circulating citrate levels and speed of transfusion were correlated with decreases in ica concentration during blood transfusion ( 11 ) . based on these reported findings , we included the amounts of these factors administrated in the prehospital phase of care in the multivariate regression , even though they were not associated with mortality on univariate analysis . this unexpected result was thought to be due to the lack of any significant association of ica concentration with these factors on correlation analysis and/or to the small patient population underwent blood transfusion ( eight cases ) and colloid infusion ( only one case ) in the prehospital phase ( data not shown ) . as aforementioned however , this may not be reflected in a sufficient discriminative power of ica for mortality prediction ( 21 , 22 ) . the discrimination of roc analysis in their study was an auc of 0.636 ( 95% ci 0.591 - 0.681 ) , while the sensitivity and specificity of severe ionized hypocalcemia were 98% and 7% for mortality , respectively . the discrepancies of the sensitivities and specificities between the two studies seem to have been due to differences in patient selection : all subjects in our study were trauma patients , whereas only 3% in their study were . we recently reported that base deficit , t - rts , and sirs score were associated with mortality and that the first two were significant mortality predictors , despite their different discriminative powers in the following order : t - rts > base deficit > sirs score ( 23 ) . however , no study has compared the discriminative powers of ionized hypocalcemia and these predictors before the present study . in our study , using corresponding statistics of classification tabulations for mortality , the discriminative power of ica was the same as or inferior to that of base deficit and t - rts , except for sirs score . moreover , discrimination by roc analysis for mortality prediction revealed that auc of ica was 0.607 , which was considered a poor discrimination in general . on the other hand , the aucs of base deficit ( 0.736 ) and t - rts ( 0.875 ) represented acceptable and excellent discrimination , respectively , as in our previous study ( 23 ) . however , sirs score and t - rts are not single factors , but are compound factors consisting of several factors that require certain modification by recoding . in a recent study , gcs score and its components , one of the three parts of the t - rts compound indicator , were found to have only a moderate degree of interrater agreement ( 24 ) . therefore , we suggest that a multi - center or randomized trial be conducted in the future to avoid these biases and confirm our results . in conclusion , ica was a significant risk factor associated with mortality , but with a poorer discriminative power for mortality prediction and triage than previous mortality predictors , especially t - rts . the ica predictor is a single factor , whereas t - rts is a compound indicator consisting of three factors ( gcs score , sbp , and respiratory rate ) which have been recognized as mortality predictors . if a new predictor combining ica with the other significant factors known to be associated with mortality can be developed , we expect that it may be a superior mortality predictor than t - rts .

the hydrated excess proton is in fact a unit of net positive charge due to a missing electron that can be passed between and among biomolecules in a seemingly simple dance of charge neutralization . however , the molecular nature of this dance is complicated , involving a dynamic charge delocalization between molecules and a constant restructuring of covalent and hydrogen bond topology . understanding and being able to characterize the migration of excess protons is important given the many roles that proton transport ( pt ) plays throughout biology . virtually all biomolecules are sensitive to ph , and many incorporate pt into their functional mechanisms , including transporters , proton pumps , proton channels , and enzymes . for example , the bacterial h / cl antiporter clc - ec1 , a homologue of mammalian clc antiporters , energetically couples the transmembrane transport of two cl ions and one proton . the mammalian clcs have many physiological functions , including maintenance of the membrane potential , regulation of transepithelial cl transport , and control of ph in the cytoplasm and intracellular organelles . cytochrome c oxidase ( cco ) , a proton pump in the respiratory chain of mitochondria and bacteria , reduces oxygen to water and utilizes the released free energy to pump protons across the membrane , contributing to the transmembrane electrochemical potential gradient that is necessary for atp synthesis . the influenza a m2 proton channel protein transports the protons across the influenza virus membrane and triggers the dissociation of the viral matrix proteins , which is an essential step in the influenza virus replication cycle this is a short list of the many systems in which pt plays a role in a functional mechanism . for such systems , the ionizable amino acids that influence for example in the clc - ec1 antiporter , an intracellular - facing glutamic acid , e203 , shuttles an excess proton to an extracellular - facing glutamic acid , e148 , during transmembrane pt . in cco , the highly conserved glutamic acid e286 is the key for both transmembrane proton pumping and pt for the chemical reaction . in the influenza a m2 channel , a highly conserved tetrad of histidine residues ( h37 ) is responsible for the ph - dependent channel activation and proton selectivity . computational approaches can play an important role in the investigation of pt mechanisms in proteins , adding molecular level insight as well as increased temporal and spatial resolution to experimental data . however , it is very challenging to explicitly model the pt process , even in simple bulk water solution , because it involves charge defect delocalization , grotthuss shuttling ( proton hopping ) , and solvent reorganization . moreover , the migration of an excess proton in proteins and other confined spaces can be nontrivially coupled with changes in the hydration along the pt pathway . as will be discussed later , water molecules move in / out of internal protein cavities in response to the excess positive charge as it moves between water molecules and ionizable amino acids . therefore , to more completely describe pt in biological systems , a computational method should ( 1 ) explicitly treat charge defect delocalization and grotthuss shuttling of the excess proton(s ) between water molecules and ionizable amino acids undergoing protonation / deprotonation ; ( 2 ) allow exchange of water molecules between different protein internal cavities as well as between those cavities and bulk solution ; and ( 3 ) be computationally efficient enough to achieve sufficient sampling of the charge translocation and protein and solvent configuration space for large - scale biomolecular systems , including protein , solvent , ions , and ( where needed ) the membrane . quantum mechanical approaches , such as ab initio molecular dynamics ( aimd ) or quantum mechanics molecular mechanics ( qm / mm ) md methods , can in principle explicitly treat the reactive nature of amino acid protonation / deprotonation and the grotthuss hopping phenomenon . however , their computational expense limits the application of the former to small systems much smaller than proteins and restricts the free energy sampling of the latter when applied to large biomolecular systems . a lack of sufficient free energy sampling generally leads to results with large systematic errors that depend on the initial state of the system . in addition , qm / mm methods without adaptive partitioning prohibit the exchange of mm and qm atoms across the qm / mm boundary , which is unphysical . although adaptive qm / mm partitioning methods enable the exchange of mm and qm atoms across the boundary , they can suffer from both inaccuracy of forces on atoms near the boundary and a lack of sufficient energy conservation . the qm / mm boundary may be especially problematic when the hydration along the pt pathway changes in response to the migration of the excess proton charge defect since this necessitates the exchange of water molecules between protein cavities and bulk solution , which often occurs beyond the nanosecond time scale . an alternative approach that explicitly accounts for the reactive nature of the hydrated excess proton is the multiscale reactive molecular dynamics ( ms - rmd ) method . this approach describes the migration of an excess proton including explicit grotthuss shuttling and charge defect delocalization by evolving the system on a reactive potential energy surface defined as a dynamical linear combination of diabatic basis states , as in the earlier multistate empirical valence bond ( ms - evb ) method . as will be shown in detail later , the newer ms - rmd approach differs from the older ms - evb approach primarily because the underlying reactive force field of ms - rmd is largely derived from aimd or qm / mm data via force - matching and other means , rendering it far less empirical than the original ms - evb approach . ms - rmd is also multiscale in the sense that quantum information on the electronic states is variationally bridged upward in scale to describe the forces felt by the system nuclei in the rmd model . as shown later as well , ms - rmd is several orders of magnitude more computationally efficient than md from qm / mm , while still describing the charge delocalization and reactive nature of the pt . the ms - rmd method is also not complicated by the qm / mm boundary issues because the molecules that participate in the proton charge defect charge delocalization are dynamically redefined at each time step in such a way that the forces on the atoms are more continuous . thus , as water molecules or residues move away from the excess proton , the forces acting on the atoms gradually transition to those described by the classical force field . these advantages make the ms - rmd method ( and ms - evb before it ) a powerful tool for investigating pt in a variety of biological systems . however , the primary challenge of the ms - rmd method is that it needs to be properly parametrized in order to faithfully simulate pt . the parametrization of ms - rmd models is the focus of this work . in ref ( 31 ) , an approach for parametrizing ms - rmd models for ionizable amino acids in bulk water was developed . in this fitting approach , forces from qm / mm calculations are bridged via an iterative variational framework into the reactive ms - rmd model . in particular , the ms - rmd model parameters are obtained by sampling configurations with a guess ms - rmd model and then fitting the model parameters to best reproduce forces from qm / mm calculations that are as accurate as possible . our fitting procedure was partially motivated by the work of wang and co - workers , in which force fields for liquid water were developed with an adaptive force matching method . it also bears similarity to the work of zhou and pu on reactive path force matching . because of the use of configurations from condensed phase ms - rmd trajectories , the resulting model takes into account the condensed phase environment , which is not captured in gas phase fitting . for a given ms - rmd potential energy functional form and with restrictions on the ranges of parameter values , this fitting approach was shown to work well for investigating glutamic acid ( glu ) and aspartic acid ( asp ) deprotonation in bulk water . however , one can not simply use these models to study the deprotonation of amino acids such as asp and glu within proteins . the electrostatic and hydration environment affecting ionizable amino acid protonation / deprotonation inside biomolecules for example , deprotonated asp and glu residues often form salt bridges with positively charged residues in proteins , and the surrounding hydration structure is rarely bulk - like . as a consequence , the pka values of amino acids inside proteins are often largely shifted from those measured in bulk solution . these differences require reparameterization of the ms - rmd models to fit the protein environment . in this work , we demonstrated the use of fitrmd parametrization to generate ms - rmd models for ionizable amino acid in proteins . we first outline a framework for fitting ms - rmd models for amino acid residues in biomolecular systems . we then give the computational details for following this framework in two example systems : the clc - ec1 antiporter and cco . the ability of the generated ms - rmd models to faithfully reproduce the free energy profiles ( potentials of mean force , pmfs ) of the reference qm / mm hamiltonian is demonstrated for pt inside the clc - ec1 channel in two different states . the importance of sampling and related qm / mm limitations are also demonstrated in clc - ec1 . finally , the advantages of developing efficient ms - rmd models are demonstrated with the calculation of 2-dimensional free energy profiles ( 2d - pmfs ) of pt coupled to hydration changes in the central hydrophobic region of cco . the ms - rmd ( and ms - evb ) method describes electronic delocalization of the excess proton as a linear combination of distinct states with different chemical bonding topologies . in the central region of the clc - ec1 antiporter ( see figure 1 for the images of the whole protein structure and its central region ) , figure 2 shows examples of a set of states in certain configurations , where the excess proton is delocalized into a glutamate residue and surrounding water molecules . the hamiltonian is defined as1where r are the system nuclear coordinates , hii is the potential energy for state i described by a classical force field , and hij is the coupling between states |i and |j. the diagonalization of the hamiltonian matrix on the fly gives the energy and eigenvector of the ground state for every configuration of nuclei r:2 ( a ) overview of the structure of the clc - ec1 antiporter and transport pathways for cl ( green dashed ) and h ( red dashed ) . ( b ) representative configurations of the central region with ( left ) clcen absent and ( right ) present for the p state in the pmfs in figure 3 . the protein and water molecules ( in sticks ) and clcen ( in a sphere ) are included in the qm region in the qm / mm calculation of the reference force . representative configurations of a set of ms - rmd states with different bonding topologies for the same atomic coordinates in the clc - ec1 antiporter . the configurations represent the three mostly populated diabatic states ( with the highest values of ci2(r ) ) , captured at the windows for r = 0.8 , when clcen is either absent ( a c ) or present ( d f ) . the excess proton is attached to e148 ( a and d ) , the first water from e148 ( b and e ) , or the second water ( c and f ) . the protonated species in each diabatic state is shown inside the blue dashed line . pmfs of pt through the central region of the clc - ec1 antiporter with ( a ) clcen absent and ( b ) present , as calculated with ms - rmd ( blue ) and qm / mm ( red ) . labels r , t , and p indicate the reactant state , the center of the cv range , and the product state , respectively . the cv r varies from 0 to 1 as the excess proton cec moves from e203 to e148 . t in the ms - rmd systems with ( c ) clcen absent or ( d ) present . the solvent positions and proton center of the excess charge ( cec , yellow sphere ) are taken from the last snapshot . it is important to note that the method explicitly treats the excess proton charge defect delocalization , grotthuss shuttling , and the polarization effect associated with the excess proton complex . the diagonal elements hii of the ms - rmd hamiltonian are given by the potential energy function of each basis state i. note that there is a single excess proton in the system and that either glutamate or water is protonated at each state . the hii corresponding to the state with protonated glutamate ( gluh ) is described as3where the first three terms are the inter- and the intramolecular potentials of protonated glutamate and all other surrounding molecules , such as waters , other protein residues , lipids , and ions in the system . they are computed with the charmm22 force field , with the exception of the o h bond in the carboxyl ( cooh ) group of glutamate . to properly represent its dissociation as a proton transfers from the carboxyl oxygen to the water oxygen , the harmonic bond stretch potential h bond length , and d , , and r0 are parameters , which are taken from our previous work . since the classical force fields between two protonated forms of glutamate and water do not share a common energy origin , vii is introduced to compensate the constant energy shift between the two states . in order to correct overestimated electrostatic interaction at a short distance between opposite charges on hydronium and deprotonated glutamate , two repulsive terms , vookrep and vhjokrep , are introduced in hii corresponding to the state with deprotonated glutamate:56where roo is the distance between the hydronium oxygen , o , and the carboxyl oxygen of glutamate , o ( oe1 and oe2 in the pdb ) , and rhjo is the distance between each of the three hydronium hydrogen atoms , hj , and the carboxyl oxygen of glutamate . the functional forms for the repulsive terms are the same as those used in the ms - evb3.1 model.b , b , b , c , and c are fitted parameters , and doo0 and doh0 are fixed with the same value used in ms - evb3.1 , which are 2.4 and 1.0 , respectively . the off - diagonal element hij for the coupling between protonated glutamate and water is given by7where roh is the distance between the donor oxygen of the carboxyl group of glutamate and the acceptor hydrogen of the adjacent hydronium molecule . hij for the coupling between hydronium and water is the same as the one used in the ms - evb3.1 model . the ms - rmd ( and ms - evb ) formalism also provides a convenient and physically intuitive description of the excess proton center of the excess charge ( cec ) , defined as8where the nevb is the total number of evb states , ci2(r ) is the population of state i contributing to the ms - rmd ground state , and ricoc is the geometric center - of - charge for state i. this cec definition allows the use of a continuous reaction coordinate ( further discussed below ) for the pmf calculation of pt in biological systems . the protonated moiety in the state with the largest coefficient , c1 ( eq 8) , possesses the majority of the excess positive charge , when in bulk water this state is the most hydronium - like species ( or the so - called pivot hydronium , a technical term used below ) . one possible fitting procedure of an ms - rmd potential energy function to qm / mm data was first described in ref ( 31 ) . in such an approach , configurations along the pt reaction coordinate are sampled by ms - rmd umbrella sampling simulations with the initial guess ms - rmd model parameter set . in the present article , the initial guess amino acid models were taken from the previous work done in bulk water , except that the constant energy shift between protonated / deprotonated states in the model ( vii ) was determined by the difference in the coulomb energy of the rmd ( evb ) complex between the most favorable hydronium state and the protonated state . the range of the pt reaction coordinate was set to sample configurations for both protonated / deprotonated states ( further defined below ) . next , for each configuration a qm / mm calculation was performed to collect the reference forces on each atom in the ms - rmd reactive complex . then , the ms - rmd model parameter set was optimized by minimizing the variational residual:9where nc and na are the number of configurations and the number atoms in each configuration , respectively . the weight of each atom w(rij ) is set to unity here , but it should be noted that other choices are possible . the atomic force fij is the one obtained from the current ms - rmd model parameter set , and fijqm / mm is the reference force from the qm / mm calculations . the whole set of the model parameters were then divided into two groups : ( 1 ) the diagonal terms vii and ( 2 ) the off - diagonal and repulsive terms . the model optimization was done individually for each group . first , the off - diagonal and repulsive terms were fit with the value of vii fixed , and then vii was refit with new values of the off - diagonal and repulsive terms held fixed . ms - rmd simulations of the pt process in the bacterial clc homologue , clc - ec1 , are extensively described in ref ( 39 ) . the fitrmd method was used to parametrize the ms - rmd models from qm / mm data for the two glutamate residues in the central region , namely , the e148 ( gluex ) and the e203 ( gluin ) , which have been identified as intermediate sites for proton binding along the transport pathway ( see figure 1 ) . depending on the presence of cl at the central site ( clcen ) , two different systems were setup . for each system setup , two sets of umbrella sampling simulations were performed ( deprotonating either e148 or e203 ) to sample configurations for the generation of the qm / mm force data . in order to obtain the initial configurations for the umbrella sampling , 50 ns of unconstrained classical md was run both with clcen present and absent . in this time , water penetrated the central region from the intracellular side of the membrane , forming a continuous hydrogen bonded network between the carboxyl oxygens of e148 and e203 . a harmonic potential with a force constant of 20 kcalmol was applied to a collective variable ( cv ) , defined as |rcec rx| , where x is the carboxyl center of mass for either e148 or e203 . the centers of the windows ranged from 2.0 to 4.0 and were separated by 0.25 . at cv = 2.0 , all configurations have the amino acid fully protonated , while at cv = 4.0 all were deprotonated with protonated state contributing less than 0.01% ( ci2(r ) 10 ) . the configurations were selected with a 2 ps interval from each window for 500 configurations for each protonation site . for each configuration , a single point qm / mm calculation was performed to evaluate the forces acting on the qm atoms . as shown in figure 1b , the qm region included clcen , if present , the water molecules in the central region , and the side chain of the pore - lining residues , including e113 , e148 , e202 , e203 , y445 , and s107 . the waters in the central region within the third solvation shell from e148 and e203 the qm box size was set to be 2030 in each dimension to ensure that it was 68 larger than the size of the qm atoms in each dimension . the gaussian expansion of the electrostatic potential ( geep ) scheme was used to treat the qm / mm electrostatic coupling with periodic boundary conditions ( pbcs ) , and the spurious qm / qm periodic image interactions were decoupled as described in ref ( 42 ) . the cc chemical bonds that crossed the qm / mm boundary were capped with hydrogen atoms , the forces on which were calculated following the imomm scheme with a scaling factor of 1.50 . the qm atoms were treated with density functional theory ( dft ) using the blyp functional with empirical dispersion corrections , under the gaussian and plane wave ( gpw ) scheme . . after the qm / mm forces were obtained , the ms - rmd model parameters were optimized by minimizing the residual given in eq 9 using a genetic algorithm . all atoms in the reactive complex , as defined by the ms - rmd state - selection algorithm , were included in the fitting ( i.e. , the glutamic acid side chains and solvent atoms in the central region ) . the vii and the off - diagonal terms were iteratively optimized for 36 rounds , depending on the system , until they changed less than 1% . the models for e203 and e148 were developed independently because they are separated by 1014 , depending on the presence of clcen , and therefore never participate in coupled delocalization of the excess charge . the ms - rmd simulations were performed with the raptor software interfaced with the lammps md package ( http://lammps.sandia.gov ) , and the umbrella potentials were controlled by the plumed package . all single point qm / mm calculations are performed by the cp2k package . parameters for e148 and e203 for clc - ec1 with clcen either absent or present are given in table 1 . in order to directly compare ms - rmd and qm / mm free energy profiles , umbrella sampling was performed with a cv defined as r , which is a function of the excess proton cec ( rcec ) and the center of mass of the carboxyl groups of the e203 ( r203 ) and the e148 ( r148 ) residues:10 the excess proton cec coordinate , however , was defined as11where the r is the position of the jth heavy atom in the qm region , which is either the water oxygen atoms or the carboxyl oxygen atoms in the e203 or the e148 , and the r is the position of the ith hydrogen atom bound to those heavy atoms . ( it should be noted that this cec definition was used for both the qm / mm and ms - rmd simulations , as opposed to eq 8 for the latter , in order to have a common definition of the cv in the two pmfs for comparison . ) the weighting factor , w , was set to be two for all the water oxygen atoms and zero for carboxyl oxygen atoms in the e203 and e148 , which reflect the hydrogen coordination number in the deprotonated state of the heavy atom . the term dxjhi denotes the distance between xj and hi atoms , and the fsw(dxjhi ) = 1/(1 + exp[(dxjhi rsw)/dsw ] ) is the switching function describing the coordination number of hi to xj , with the parameters chosen as dsw = 0.04 , rsw = 1.25 . the cv r varies from 0 to 1 as the excess proton cec moves from e203 to e148 . the centers of the harmonic umbrella potentials were separated by 0.10.2 between adjacent windows . the umbrella sampling force constant for r was chosen to be in the range of 30007000 kcalmol , depending on the sampling overlap between umbrella windows . r203| + |rcec r148| , is 15 , the effective force constant acting on |rcec because the central region is accessible by the water molecules from the intracellular bulk , the cec can locate outside the central region at the windows near e203 when a restraint is only placed on r . to avoid sampling irrelevant positions ( leading to the intracellular bulk phase ) , an additional restraint defining the upper limit for |rcec r148| was applied such that the cec was always situated between e148 and e203 . the details of the preparation of the initial configuration and the classical md equilibration for the clc - ec1 antiporter are described in ref ( 39 ) . the initial configurations used for the fitrmd umbrella sampling were used to initiate the ms - rmd umbrella sampling simulations . near the middle of the reaction path , when e203 and e148 were both deprotonated , a water molecule close to the center of each umbrella window was replaced with a hydronium cation . all windows were first equilibrated for 100 ps , followed by production runs of 2 ns . the integration time step was 1 fs . the cv r was collected every 10 time steps ( 10 fs ) , and the pmf was constructed by the weighted histogram analysis method ( wham ) . statistical error bars in the pmfs were estimated using the block averaging method by dividing each trajectory into four consecutive blocks . the initial configurations used in the ms - rmd simulations were also used for the qm / mm umbrella sampling simulations . the simulation details for the qm / mm md were kept consistent with the single point force calculations used in fitrmd . the window spacing and force constants for the umbrella windows were similar to those used in the ms - rmd simulations . all windows were equilibrated with qm / mm md for another 5 ps , followed by production runs of 20 ps . ( it should be noted that in our experience if a fitrmd model differs significantly from the underlying qm / mm forces due to a bad fit , then subsequent qm / mm md configurations taken starting with the ms - rmd initial conditions will diverge quickly from the ms - rmd ones . ) the integration time step was 0.5 fs , and the cv r was collected every time step . all qm / mm md simulations were performed by the cp2k package . upon finding that the pmfs ( see figure 3 ) calculated with qm / mm and ms - rmd md disagreed for the clcen absent system in the cv range 0.18 < r < 0.26 , a second set of qm / mm simulations was run in order to investigate the origin of the pmf discrepancy . the second set of qm / mm umbrella sampling simulations were initiated from the last md snapshots of the ms - rmd umbrella sampling simulations , which had an increased hydration level compared to that of the original qm / mm simulations . all windows in the cv range 0.18 < r < 0.26 were then sampled for 5 ps . all other simulation details were unchanged from the original qm / mm umbrella sampling . structural elements of the clc - ec1 protein are shown in figures 16 . to obtain these structures , the production run first , configurations were aligned based on carbon atoms at least 15 away from the central region . second , the positions of the side chains of the four residues in the central region ( s107 , e148 , e203 , and y445 ) and clcen , if present , were averaged , and full moieties representative of these averages were depicted . third , the positions of the water molecules and the excess proton were taken from the last md frame . in the system with clcen absent , rdfs from e148 carboxyl carbon ( cd in the pdb ) to water oxygens are shown in a1 to a3 , for the r , t , and p states ( figure 3 ) , respectively . the rdfs from the e203 carboxyl carbon to water oxygens are shown in b1 to b3 in the same order . the averaged positions of the four side chains are shown in c1 to c3 in the same order , with the qm / mm structures in red and the ms - rmd structures in blue . in the clc system as in figure 4 but with clcen present . the second qm / mm pmf ( green ) was initiated from the final ms - rmd configuration in each window . ( b ) the average and the standard error of c12(r ) , reflecting the delocalization of the hydrated excess proton ( see text ) , from the original qm / mm , the ms - rmd , and the second qm / mm umbrella sampling at the window r = 0.24 . panels c e : the averaged structure of the four residues and the water structure in the last snapshot from the ( c ) the qm / mm , ( d ) the ms - rmd , and ( e ) the second qm / mm umbrella sampling at the window r = 0.24 . the region circled by a gray line represents the position of the second water . panel f : the coordination number of the pivot hydronium to oxygen atoms of surrounding water molecules or the carboxyl group of e203 in the ms - rmd simulation , initiated from the configuration at low hydration level , which mimics the configuration of the qm / mm in c. to calculate the time scale for the transition from a low to high hydration state in the central region of clc - ec1 , the low hydration state first needed to be created in the ms - rmd structure . to do this , a high hydration configuration was taken from the last ms - rmd snapshot at umbrella window r = 0.24 ( figure 6d ) . an additional harmonic potential was applied to the water density cv ( nw ) in the predefined box ( see refs ( 23 ) and ( 39 ) and for definition ) . the force constant of the harmonic potential was set to be 10 kcalmol , and the center of the potential was at nw = 0 . the center of the box was chosen to be the midpoint between the center of mass of each carboxyl group of e148 and e203 , and the box size was set to be 2 2 2 , which covered the position of the second water . the second water present in the initial configuration was expelled from the central region by the harmonic potential centered at nw = 0 . the ms - rmd simulation was run for 200 ps , until the system was equilibrated in the absence of the second water . ten independent trajectories were initiated from the last md frame of the simulation above , after the velocities of all atoms were randomized and the harmonic potential centered at nw = 0 was released . the coordination number of the pivot hydronium was defined as the number of oxygen atoms ( from water or the e203 carboxyl group ) within 3 from the pivot hydronium oxygen . the time for the second water entering the central region was estimated by averaging the 10 simulations time to when there was a productive ( not transient ) transition of the coordination number from 2 to 3 . the fitrmd method was also used to parametrize the ms - rmd models from qm / mm data for three protonatable sites in the hydrophobic cavity of cco , including the e286 , prda3 , and praa3 ( figure 7 ) . umbrella sampling simulations were first carried out along the pt pathway identified by the ms - rmd metadynamics ( mtd ) simulation . then for each protonatable site , 100 configurations were selected from the trajectories from windows within 4 of the transition state of proton dissociation ( defined as the windows with 50% of the configurations having the largest amplitude on the amino acid and the other 50% on the first water molecule ) for each protonatable site . single point qm / mm calculations were then performed for each configuration using the b3lyp level density functional theory . the mm models were the charmm22 and charmm36 force fields for the protein and lipids , respectively . the qm region included the side chain of each protonatable amino acid , the hydrated excess proton , and water molecules within 3 solvation shells of the carboxyl group ( figure 7b ) . in all calculations , the qm box size was chosen to be 68 larger than the actual size of the qm atoms in each dimension . the geep scheme was used to treat the qm / mm electrostatic coupling with periodic boundary conditions ( pbcs ) , and the spurious qm / qm periodic image interactions were decoupled as described in ref ( 42 ) . the cc chemical bonds that cross the qm / mm boundary were capped with hydrogen atoms , the forces on which were calculated following the imomm scheme with a scaling factor of 1.50 . the forces generated by the qm / mm calculations were used to parametrize the ms - rmd parameters for the protonatable sites using fitrmd approach . the ms - rmd simulations were performed with the raptor software interfaced with the lammps md package , as described earlier . the qm / mm calculation was performed with the cp2k package , and fitrmd was carried out with in - house software , again as described earlier . the subunits i , ii , iii , and iv are colored in blue , red , gray , and orange , respectively . the yellow rectangular box highlights the e286 residue and the heme - copper groups that are essential for proton pumping and reaction . ( b ) enlargement of the region where e286 , heme a , heme a3 , and bnc are located . e286 is shown in green , the propionate groups of the pls in yellow , the hydrated excess proton in purple , the iron atom of the heme groups in gray , and the copper atom in the bnc in orange . , the qm atoms include the e286 side chain , the hydronium , and the 3 solvation shells of water molecules around the e286 residue ( shown in vdw representation ) . the qm / mm calculations for cco were performed at the b3lyp dft level and as such were much more expensive than the blyp level dft qm / mm calculations carried out for clc - ec1 . as a result , fewer configurations were used for cco than for clc - ec1 in the fitrmd , and for the same reason , no explicit pmf was calculated with the b3lyp level qm / mm for cco ( see below ) . in addition , the cco system is too complex to converge any reasonable accurate qm / mm pmf , and this fact serves to highlight the strength of the ms - rmd approach . full details of the cco simulations and pmf calculations are presented in ref ( 60 ) . some of the relevant details are given here , including the fact that the ms - rmd umbrella sampling simulations in the hydrophobic cavity ( hc ) were carried out by restraining the excess proton cec position ( eq 8) along the pt pathway defined from the mtd simulations and the water density in a predefined box that encompasses the hc ( see ref ( 23 ) for the definition ) . the force constant for the harmonic umbrella sampling restraint potential was 10 kcal / mol / on the proton migration cv and 20 kcal / mol on the water density . the window spacing was 0.5 for the cec and 0.5 for the water density . for each umbrella window , the ms - rmd simulation length was 500 ps . the integration time step was 1 fs . the cv r was collected every 10 time steps ( 10 fs ) . the 2d pmf was constructed by the wham . the computational speed for the ms - rmd , self - consistent density functional tight binding ( scc - dftb)-based qm / mm , and blyp - based qm / mm methods were compared for the md simulation of the cco system . ( the qm / mm with b3lyp is much too slow for viable qm / mm md in these systems . ) for ms - rmd , the setup was the same as described in the above section . for the blyp qm / mm method , the qm / mm setup was the same as described in the above section for fitrmd , except that the qm atoms were treated by the blyp functional with empirical dispersion corrections . more details on the blyp - based qm / mm md simulation setup are presented in ref ( 60 ) . for the scc - dftb - based qm / mm setup , the qm atoms were the same as those in the blyp - based qm / mm setup . the point charge based ewald summation was used to treat qm / mm electrostatic coupling under pbcs . more details on the scc - dftb - based qm / mm setup are described in ref ( 64 ) . the ms - rmd simulation and blyp - based qm / mm simulations were performed as described previously . the scc - dftb - based qm / mm simulation was performed with the charmm package . an important measure of success of the fitrmd method is its ability to reproduce results of the reference hamiltonian for properties other than those that were fit ( atomistic forces ) , and there is arguably no property more important than the pt free energy profile ( pmf ) . unlike our previous work demonstrating the fitrmd method for amino acid deprotonation in bulk water , calculating a pmf with qm / mm is possible in a protein environment because the water molecules are more confined . ( in the bulk environment , the qm / mm boundary issues introduce such large errors that a direct comparison between ms - rmd and qm / mm pmfs is highly problematic at best . ) however , for many protein cavities the waters involved in pt between two residues are largely surrounded by protein , and they all fit into the qm region , making exchange across the qm / mm boundary less of a complication . although many protein systems will still require extensive sampling ( beyond the limits of qm / mm ) as well as exhibit water exchange across the qm / mm boundary on longer time scales , the level of confinement in the clc - ec1 antiporter system enables a direct comparison of the qm / mm and ms - rmd pmfs . pt through the central region of clc antiporters is one of the essential intermediate steps in cl / h exchange . the migration of an excess proton from the internal e203 to the external e148 through water molecules in the central region is coupled with migration of approximately 2 cl ions in the opposite direction ( figure 1 ) . therefore , the pmfs for pt in the central region of the clc - ec1 antiporter were calculated both in the presence and absence of cl at the central binding site , clcen . the ms - rmd and qm / mm pmfs ( figures 3a and b ) show excellent agreement for most parts of the reaction coordinate and for both states of the system ( with and without clcen ) , although there are some differences ( discussed below ) . the free energy barrier of the pt process is significantly decreased when clcen is present , mainly due to the electrostatic interaction between the excess proton and the clcen ion . these results demonstrate that the fitrmd approach is capable of generating ms - rmd models that reproduce the free energy profile of the reference qm / mm hamiltonian , using only forces on the atomic nuclei from a relatively small set of configurations as input for the fitting . moreover , the fitrmd approach is robust enough to quantitatively capture the effect of the clcen ion on the free energy surface of the reference qm / mm hamiltonian . this suggests that the fitrmd approach is also capable of describing the shift in proton affinity in different protein environments , which has significant value for simulating pt in different protein systems with the ms - rmd method . the radial distribution functions ( rdfs ) from the carboxyl carbon of either e148 or e203 to the water and hydronium oxygens were calculated with the excess proton cec restrained in the reactant , transition , and product states of the pmf ( the positions are labeled as r , t , and p in figure 3a and b ) . the rdfs ( figures 4 and 5 with and without clcen , respectively ) demonstrate that the solvation structure around e203 and e148 is quite similar between the qm / mm and ms - rmd methods . this result provides additional evidence that the model generated by the fitrmd approach faithfully reproduces the underlying free energy landscape of the reference qm / mm hamiltonian , even though only qm / mm forces on the atom nuclei were used as input for the fitting . the discrepancies in the rdfs shown in figures 4 and 5 are likely due to the different water behavior of qm versus mm water as well as short qm / mm sampling where the water dynamics are slow in the confined space of the central region , which will be discussed below . we note , however , that this level of agreement may not be expected for more bulk - like water environments since the qm and mm water will have such different structural properties . although the pmfs in figure 3a and b show that the ms - rmd and qm / mm pmfs generally agree well , some discrepancies appear . focusing on the most significant , figure 6a highlights the region 0.18 < r < 0.26 from figure 3a , where the ms - rmd pmf ( blue ) dips to a modest metastable minimum , but the qm / mm pmf ( red ) shows an uphill rise with only a slight dip at r 0.21 . this discrepancy between the two pmfs is caused by the change of the local environment for the excess proton . the structures of the central region captured from the r = 0.24 umbrella windows , where the two pmfs disagree most , are shown in figure 6c ( qm / mm ) and d ( ms - rmd ) . the ms - rmd structure shows one additional water ( gray circle ) compared to the qm / mm structure . the center of excess charge ( yellow sphere ) is close to the first water coordinated to e203 in this window for both the qm / mm and ms - rmd configurations , meaning that the first water is the most hydronium - like species ( the pivot hydronium , which has the largest ms - rmd state coefficient , c1 , in eq 8) . to better understand the difference in solvation structures and charge delocalization , we calculated the average value of c12 for all configurations in the r = 0.24 umbrella windows from the qm / mm and ms - rmd simulations ( figure 6b ) . it should be kept in mind that the c12 value for an eigen cation ( h9o4 ) is 0.65 , which indicates that the pivot hydronium holds 65% of the positive charge ( hydronium - like ) defect , while that for a zundel cation ( h5o2 ) is 0.5 since in that case , excess charge is more equally shared between two water molecules . the ms - rmd simulations yield larger c12(r) values ( 0.7 ) because the pivot hydronium is coordinated to two waters in the central region , in addition to the carboxyl group , leading to a stable eigen - like ( co - h7o3 ) complex . however , one of these waters is missing in the qm / mm simulations ( figure 6c , gray circle ) , shifting c12(r) to a lower value ( 0.55 ) and the delocalization to more of a zundel - like complex between the carboxyl group and first water molecule ( co - h3o ) . the ms - rmd eigen - like complex forms a contact ion pair ( cip ) with the carboxyl group of deprotonated e203 and the stabilizing electrostatic attraction between oppositely charged ions causes a small energy well ( 1 kcal / mol ) in the ms - rmd pmf . however , in the qm / mm configurations , no hydrogen bond acceptor is found near one of the hydrogen atoms of the pivot hydronium , causing the pmf to continue its uphill climb . to see if the pmf discrepancy was caused by sampling different conformational phase space , another set of qm / mm umbrella sampling simulations was run for 5 ps ( as described earlier ) , initiated from the last snapshots from the ms - rmd umbrella sampling simulations in the cv range 0.18 < r < 0.26 . the resulting qm / mm pmf ( green in figure 6a ) shows much better agreement with the ms - rmd pmf . figure 6b and e show that for this second qm / mm simulation the value of c12(r) is close to the ms - rmd value , and that the missing second water is present . thus , the pmf discrepancy is indeed caused by sampling different conformational phase space with that sampled by ms - rmd having one additional water molecule to stabilize the excess proton . to estimate the time scale required for the additional water molecule ( missing in the original qm / mm simulations ) to enter the central region , an ms - rmd configuration was prepared ( see methods ) at r = 0.24 in which this water was removed ( mimicking the qm / mm solvation structure shown in figure 6c ) . the system was equilibrated in this low solvation state , and then 10 independent ms - rmd simulations were run for 500 ps . figure 6f shows the coordination number of the pivot hydronium to the oxygen atoms of the surrounding water molecules or the carboxyl group of e203 in one of the trajectories . the coordination number at t = 0 ps is 2 , when the pivot hydronium is coordinated to one water oxygen and e203 s carboxyl oxygen . although there are some transient transitions to a coordination of 3 around t 3070 ps , due to an additional water oxygen , the second water is not stable for another 100 ps . the coordination number remains 3 for most of the last 400 ps of the trajectory ( 89% of the time ) . this trajectory is representative of the other nine trajectories , in which the second water did not enter the central region until t = 60185 ps ( t = 99 ps ) , after which the coordination number remained 3 for 88 5% of the rest of the trajectories . these results suggest that the entrance of the second water is energetically favored in this umbrella window but that the time scale of this event exceeds the sampling time possible in the qm / mm simulations . although the discrepancy that motivated this detailed investigation was small , it highlights another important limitation of qm / mm md pmfs . in addition to the errors and artificial dynamics introduced by qm / mm boundary issues , limited sampling , especially of slow degrees of freedom such as changing hydration , can hide insidious pmf errors . this reinforces the importance of being able to map accurate ab initio forces onto an efficient method that is capable of extensive sampling . free energy profiles in complicated condensed phase environments are generally a balance of enthalpic and entropic contributions , requiring both accurate potential energy representations and extensive sampling . cco offers another example of pt through an interior protein region being coupled to hydration changes . during the reaction cycle of aa3-type cco , as found in mitochondria , protons from the intracellular side of the membrane are transported through the so - called d - channel to the glutamic acid e286 in the middle of the membrane . the protons are then transported through water molecules in a hydrophobic cavity ( the hc ) either to the pump loading site ( pls ) to be further released to the periplasmic side of the membrane or to the binuclear center ( bnc ) to react with oxygen and form water . a particularly interesting aspect of the proton pumping mechanism in cco is the role of water molecules in the hc during pt from amino acid e286 to the pls or bnc ( figure 7b ) . the number of water molecules in the hc and their role in pt has been the focus of much debate . to further complicate this issue , the water molecules can move in and out of the hc in cco in response to the migration of the excess proton , and the two processes can be intrinsically coupled to each other . capturing this type of cooperativity , we show that the computationally efficient ms - rmd method parametrized by the fitrmd approach allowed us to address this challenge . we calculated 2d pmfs for the pt from e286 to the pls in different redox states during the a f transition of cco . the collective variables used to define these 2d pmfs are ( 1 ) the progress of the excess proton cec through the hc ( horizontal axis ) and ( 2 ) the degree of hydration of the hc ( vertical axis ) ( figure 8 ; see methods for more discussion ) . the 2d pmf shown in figure 8 clearly reveals the cooperativity between the pt and dynamic hydration in the hc . the minimum free energy pathway ( black line ) along the 2d pmf ( figure 8) verifies that as the excess proton migrates from e286 to the prda3 , the hydration level of hc gradually increases . the nonhorizontal feature of the minimum free energy pathway indicates that the hc hydration is intrinsically coupled to the proton charge defect translocation in this activated process . two - dimensional free energy profiles ( 2d - pmfs ) for pt from the e286 to the pls in the pm state , as a function of the cec coordinate through the hydrophobic cavity ( hc ) as the horizontal axis and the water hydration in the hc as the vertical axis . the minimum free energy pathways ( black lines ) are diagonal in nature , indicating that the two processes are coupled . it is also important to emphasize how the computational efficiency of the ms - rmd method makes it feasible to calculate such 2d pmfs . figure 9 shows a relative speed and scaling over the processor plot of the ms - rmd , scc - dftb - based qm / mm , and blyp - based qm / mm md methods for cco . it is seen that ms - rmd is at least 3 orders of magnitude faster than the blyp - based qm / mm simulation and 2 orders of magnitude faster than the scc - dftb - based qm / mm simulation . ms - rmd also scales better over processors than the other two methods ( the scc - dftb charmm code is not scalable at all . ) scaling plot for the ms - rmd , scc - dftb - based qm / mm , and blyp - based qm / mm simulations of cco . the blue curve is for ms - rmd , the green is for scc - dftb , and the red curve is for blyp - based qm / mm . the ms - rmd method is at least 3 orders of magnitude faster than the blyp - based qm / mm method and 2 orders of magnitude faster than scc - dftb - based qm / mm , while showing more favorable scaling properties over processors ( the scc - dftb charmm code is not scalable ) . describing reactive processes in biomolecular systems remains a challenging domain for molecular simulations . the ab initio methods that are capable of describing chemical reactions generally do not scale to the size of most biomolecules or to the time scales needed to converge multidimensional free energy profiles of rare events in condensed phase environments . thus , bridging a quantum description of the reactive processes with more computationally efficient approaches ( e.g. , classical dynamics and enhanced sampling methods ) that are capable of extensive phase space sampling is of great utility and fundamental importance . the work presented herein contributes a multiscale framework aimed at accomplishing this multiscale bridging , specifically for the purpose of simulating the protonation / deprotonation of ionizable moieties such as amino acids in biomolecules . the core of our framework is the ms - rmd method , which describes reactive processes in classical md and explicitly treats the charge defect delocalization and grotthuss shuttling of the hydrated excess proton in the pt process . however , ms - rmd must be carefully parametrized in order to simulate pt faithfully , and the approach is not yet a black box . considerable effort has gone into the parametrization of the ms - rmd model for pt in water and amino acids . herein , we have extended to proteins the parametrization scheme from qm / mm data to ms - rmd models ( fitrmd ) and demonstrated its use on protonatable amino acids in biomolecular systems . as presented here , the fitrmd approach variationally maps quantum data ( dft - level qm / mm forces in this case ) onto the ms - rmd nonlinear reactive force field . we have demonstrated how fitrmd can be used to parametrize ms - rmd models for amino acids in proteins with two example systems , clc - ec1 and cco . in clc - ec1 , the ms - rmd models were shown to faithfully reproduce the pt pmfs of the reference qm / mm md , both in the presence and absence of a cl ion in the central region . thus , the fitrmd parametrization was robust enough to capture quite different free energy profiles due to the presence or absence of a single ion . moreover , the local structure of the protein and confined water was shown to be quite similar between the ms - rmd and qm / mm simulations . in cco , the developed ms - rmd models were shown to be efficient enough to capture the coupling between pt and hydration changes in the hc region . two - dimensional free energy surfaces in which both pt and hydration levels were explicitly sampled are required for this analysis . given that individual umbrella windows often required over 500 ps to converge , these calculations would not be possible with a qm / mm md approach . moreover , qm / mm boundary issues , such as the lack of exchange of water molecules , can lead to large systematic errors in such pt pmfs . even for the clc - ec1 system where exchange of water across the qm / mm boundary is minimal due to protein confinement , lack of water exchange was argued to introduce errors in the calculated qm / mm free energy profile . it is worth noting that in this article the original parametrization method of nelson et al . for example , in clc - ec1 the ms - rmd models produce a pmf that is consistent with the qm / mm hamiltonian even though the cv used for the configuration sampling ( absolute distance ) is different from the cv used for the pmf calculation ( ratio of distances ) ( see methods for a full description ) . moreover , different models for the same glu residues were parametrized in different states , with and without the central cl ion , resulting in quite different pmfs that were independently consistent with the qm / mm pmfs . it should also be emphasized that although a low - level qm method ( blyp - d ) was used here in the qm / mm calculations , our goal was to demonstrate that a reference ab initio hamiltonian ( and free energy surface ) can be reproduced . thus , an ms - rmd model obtained from fitrmd using a higher level reference ab initio method can be expected to also faithfully reproduce the free energy surface from the latter , if only the latter could be calculated , which it currently can not be . in principle , therefore , our multiscale fitrmd with ms - rmd framework can be used in the future to estimate high - level ( e.g. , mp2 ) free energy profiles for reactive processes , although the accuracy of the surrounding environment will remain dependent on the chosen classical force field . it also seems clear that the ms - rmd model parameters will depend on the given system under study and hence not likely to be transferable to some other system . however , this is precisely the point of the ms - rmd approach ( and also the qm / mm approach which it is fitting ) . it is very unlikely that it is generally possible to have transferable potential parameters for reactive processes . however , we note that the ms - rmd fitting methodology is itself transferable to different systems and that its use is practical because the total computational cost for both the model fit and the md sampling with ms - rmd will be still much cheaper than directly performing qm / mm md simulations on the same system . our future efforts will focus on improving the fitrmd protocol to make it more robust to the choice of the trial ms - rmd model , as free as possible from the coevolution of parameters , and insensitive to discrepancies between the qm and mm descriptions of nonreactive atoms in the system . we anticipate that reactive processes in many biomolecular and other systems can eventually be studied with the ms - rmd approach .

an important challenge in the simulation of biomolecular systems is a quantitative description of the protonation and deprotonation process of amino acid residues . despite the seeming simplicity of adding or removing a positively charged hydrogen nucleus , simulating the actual protonation / deprotonation process is inherently difficult . it requires both the explicit treatment of the excess proton , including its charge defect delocalization and grotthuss shuttling through inhomogeneous moieties ( water and amino residues ) , and extensive sampling of coupled condensed phase motions . in a recent paper ( j. chem . theory comput.2014 , 10 , 2729273725061442 ) , a multiscale approach was developed to map high - level quantum mechanics / molecular mechanics ( qm / mm ) data into a multiscale reactive molecular dynamics ( ms - rmd ) model in order to describe amino acid deprotonation in bulk water . in this article , we extend the fitting approach ( called fitrmd ) to create ms - rmd models for ionizable amino acids within proteins . the resulting models are shown to faithfully reproduce the free energy profiles of the reference qm / mm hamiltonian for pt inside an example protein , the clc - ec1 h+/cl antiporter . moreover , we show that the resulting ms - rmd models are computationally efficient enough to then characterize more complex 2-dimensional free energy surfaces due to slow degrees of freedom such as water hydration of internal protein cavities that can be inherently coupled to the excess proton charge translocation . the fitrmd method is thus shown to be an effective way to map ab initio level accuracy into a much more computationally efficient reactive md method in order to explicitly simulate and quantitatively describe amino acid protonation / deprotonation in proteins .

Introduction Methods Results and Discussion Conclusions

for such systems , the ionizable amino acids that influence for example in the clc - ec1 antiporter , an intracellular - facing glutamic acid , e203 , shuttles an excess proton to an extracellular - facing glutamic acid , e148 , during transmembrane pt . however , it is very challenging to explicitly model the pt process , even in simple bulk water solution , because it involves charge defect delocalization , grotthuss shuttling ( proton hopping ) , and solvent reorganization . as will be discussed later , water molecules move in / out of internal protein cavities in response to the excess positive charge as it moves between water molecules and ionizable amino acids . therefore , to more completely describe pt in biological systems , a computational method should ( 1 ) explicitly treat charge defect delocalization and grotthuss shuttling of the excess proton(s ) between water molecules and ionizable amino acids undergoing protonation / deprotonation ; ( 2 ) allow exchange of water molecules between different protein internal cavities as well as between those cavities and bulk solution ; and ( 3 ) be computationally efficient enough to achieve sufficient sampling of the charge translocation and protein and solvent configuration space for large - scale biomolecular systems , including protein , solvent , ions , and ( where needed ) the membrane . quantum mechanical approaches , such as ab initio molecular dynamics ( aimd ) or quantum mechanics molecular mechanics ( qm / mm ) md methods , can in principle explicitly treat the reactive nature of amino acid protonation / deprotonation and the grotthuss hopping phenomenon . the qm / mm boundary may be especially problematic when the hydration along the pt pathway changes in response to the migration of the excess proton charge defect since this necessitates the exchange of water molecules between protein cavities and bulk solution , which often occurs beyond the nanosecond time scale . an alternative approach that explicitly accounts for the reactive nature of the hydrated excess proton is the multiscale reactive molecular dynamics ( ms - rmd ) method . this approach describes the migration of an excess proton including explicit grotthuss shuttling and charge defect delocalization by evolving the system on a reactive potential energy surface defined as a dynamical linear combination of diabatic basis states , as in the earlier multistate empirical valence bond ( ms - evb ) method . as shown later as well , ms - rmd is several orders of magnitude more computationally efficient than md from qm / mm , while still describing the charge delocalization and reactive nature of the pt . the ms - rmd method is also not complicated by the qm / mm boundary issues because the molecules that participate in the proton charge defect charge delocalization are dynamically redefined at each time step in such a way that the forces on the atoms are more continuous . however , the primary challenge of the ms - rmd method is that it needs to be properly parametrized in order to faithfully simulate pt . in ref ( 31 ) , an approach for parametrizing ms - rmd models for ionizable amino acids in bulk water was developed . for a given ms - rmd potential energy functional form and with restrictions on the ranges of parameter values , this fitting approach was shown to work well for investigating glutamic acid ( glu ) and aspartic acid ( asp ) deprotonation in bulk water . the electrostatic and hydration environment affecting ionizable amino acid protonation / deprotonation inside biomolecules for example , deprotonated asp and glu residues often form salt bridges with positively charged residues in proteins , and the surrounding hydration structure is rarely bulk - like . in this work , we demonstrated the use of fitrmd parametrization to generate ms - rmd models for ionizable amino acid in proteins . we first outline a framework for fitting ms - rmd models for amino acid residues in biomolecular systems . the ability of the generated ms - rmd models to faithfully reproduce the free energy profiles ( potentials of mean force , pmfs ) of the reference qm / mm hamiltonian is demonstrated for pt inside the clc - ec1 channel in two different states . finally , the advantages of developing efficient ms - rmd models are demonstrated with the calculation of 2-dimensional free energy profiles ( 2d - pmfs ) of pt coupled to hydration changes in the central hydrophobic region of cco . in the central region of the clc - ec1 antiporter ( see figure 1 for the images of the whole protein structure and its central region ) , figure 2 shows examples of a set of states in certain configurations , where the excess proton is delocalized into a glutamate residue and surrounding water molecules . pmfs of pt through the central region of the clc - ec1 antiporter with ( a ) clcen absent and ( b ) present , as calculated with ms - rmd ( blue ) and qm / mm ( red ) . it is important to note that the method explicitly treats the excess proton charge defect delocalization , grotthuss shuttling , and the polarization effect associated with the excess proton complex . in order to correct overestimated electrostatic interaction at a short distance between opposite charges on hydronium and deprotonated glutamate , two repulsive terms , vookrep and vhjokrep , are introduced in hii corresponding to the state with deprotonated glutamate:56where roo is the distance between the hydronium oxygen , o , and the carboxyl oxygen of glutamate , o ( oe1 and oe2 in the pdb ) , and rhjo is the distance between each of the three hydronium hydrogen atoms , hj , and the carboxyl oxygen of glutamate . the ms - rmd ( and ms - evb ) formalism also provides a convenient and physically intuitive description of the excess proton center of the excess charge ( cec ) , defined as8where the nevb is the total number of evb states , ci2(r ) is the population of state i contributing to the ms - rmd ground state , and ricoc is the geometric center - of - charge for state i. this cec definition allows the use of a continuous reaction coordinate ( further discussed below ) for the pmf calculation of pt in biological systems . in the present article , the initial guess amino acid models were taken from the previous work done in bulk water , except that the constant energy shift between protonated / deprotonated states in the model ( vii ) was determined by the difference in the coulomb energy of the rmd ( evb ) complex between the most favorable hydronium state and the protonated state . the fitrmd method was used to parametrize the ms - rmd models from qm / mm data for the two glutamate residues in the central region , namely , the e148 ( gluex ) and the e203 ( gluin ) , which have been identified as intermediate sites for proton binding along the transport pathway ( see figure 1 ) . in order to directly compare ms - rmd and qm / mm free energy profiles , umbrella sampling was performed with a cv defined as r , which is a function of the excess proton cec ( rcec ) and the center of mass of the carboxyl groups of the e203 ( r203 ) and the e148 ( r148 ) residues:10 the excess proton cec coordinate , however , was defined as11where the r is the position of the jth heavy atom in the qm region , which is either the water oxygen atoms or the carboxyl oxygen atoms in the e203 or the e148 , and the r is the position of the ith hydrogen atom bound to those heavy atoms . ( it should be noted that this cec definition was used for both the qm / mm and ms - rmd simulations , as opposed to eq 8 for the latter , in order to have a common definition of the cv in the two pmfs for comparison . ) upon finding that the pmfs ( see figure 3 ) calculated with qm / mm and ms - rmd md disagreed for the clcen absent system in the cv range 0.18 < r < 0.26 , a second set of qm / mm simulations was run in order to investigate the origin of the pmf discrepancy . the averaged positions of the four side chains are shown in c1 to c3 in the same order , with the qm / mm structures in red and the ms - rmd structures in blue . ( b ) the average and the standard error of c12(r ) , reflecting the delocalization of the hydrated excess proton ( see text ) , from the original qm / mm , the ms - rmd , and the second qm / mm umbrella sampling at the window r = 0.24 . panels c e : the averaged structure of the four residues and the water structure in the last snapshot from the ( c ) the qm / mm , ( d ) the ms - rmd , and ( e ) the second qm / mm umbrella sampling at the window r = 0.24 . panel f : the coordination number of the pivot hydronium to oxygen atoms of surrounding water molecules or the carboxyl group of e203 in the ms - rmd simulation , initiated from the configuration at low hydration level , which mimics the configuration of the qm / mm in c. to calculate the time scale for the transition from a low to high hydration state in the central region of clc - ec1 , the low hydration state first needed to be created in the ms - rmd structure . the fitrmd method was also used to parametrize the ms - rmd models from qm / mm data for three protonatable sites in the hydrophobic cavity of cco , including the e286 , prda3 , and praa3 ( figure 7 ) . in addition , the cco system is too complex to converge any reasonable accurate qm / mm pmf , and this fact serves to highlight the strength of the ms - rmd approach . some of the relevant details are given here , including the fact that the ms - rmd umbrella sampling simulations in the hydrophobic cavity ( hc ) were carried out by restraining the excess proton cec position ( eq 8) along the pt pathway defined from the mtd simulations and the water density in a predefined box that encompasses the hc ( see ref ( 23 ) for the definition ) . the computational speed for the ms - rmd , self - consistent density functional tight binding ( scc - dftb)-based qm / mm , and blyp - based qm / mm methods were compared for the md simulation of the cco system . an important measure of success of the fitrmd method is its ability to reproduce results of the reference hamiltonian for properties other than those that were fit ( atomistic forces ) , and there is arguably no property more important than the pt free energy profile ( pmf ) . unlike our previous work demonstrating the fitrmd method for amino acid deprotonation in bulk water , calculating a pmf with qm / mm is possible in a protein environment because the water molecules are more confined . although many protein systems will still require extensive sampling ( beyond the limits of qm / mm ) as well as exhibit water exchange across the qm / mm boundary on longer time scales , the level of confinement in the clc - ec1 antiporter system enables a direct comparison of the qm / mm and ms - rmd pmfs . the free energy barrier of the pt process is significantly decreased when clcen is present , mainly due to the electrostatic interaction between the excess proton and the clcen ion . these results demonstrate that the fitrmd approach is capable of generating ms - rmd models that reproduce the free energy profile of the reference qm / mm hamiltonian , using only forces on the atomic nuclei from a relatively small set of configurations as input for the fitting . moreover , the fitrmd approach is robust enough to quantitatively capture the effect of the clcen ion on the free energy surface of the reference qm / mm hamiltonian . this result provides additional evidence that the model generated by the fitrmd approach faithfully reproduces the underlying free energy landscape of the reference qm / mm hamiltonian , even though only qm / mm forces on the atom nuclei were used as input for the fitting . the structures of the central region captured from the r = 0.24 umbrella windows , where the two pmfs disagree most , are shown in figure 6c ( qm / mm ) and d ( ms - rmd ) . the center of excess charge ( yellow sphere ) is close to the first water coordinated to e203 in this window for both the qm / mm and ms - rmd configurations , meaning that the first water is the most hydronium - like species ( the pivot hydronium , which has the largest ms - rmd state coefficient , c1 , in eq 8) . figure 6b and e show that for this second qm / mm simulation the value of c12(r) is close to the ms - rmd value , and that the missing second water is present . in addition to the errors and artificial dynamics introduced by qm / mm boundary issues , limited sampling , especially of slow degrees of freedom such as changing hydration , can hide insidious pmf errors . to further complicate this issue , the water molecules can move in and out of the hc in cco in response to the migration of the excess proton , and the two processes can be intrinsically coupled to each other . capturing this type of cooperativity , we show that the computationally efficient ms - rmd method parametrized by the fitrmd approach allowed us to address this challenge . the nonhorizontal feature of the minimum free energy pathway indicates that the hc hydration is intrinsically coupled to the proton charge defect translocation in this activated process . two - dimensional free energy profiles ( 2d - pmfs ) for pt from the e286 to the pls in the pm state , as a function of the cec coordinate through the hydrophobic cavity ( hc ) as the horizontal axis and the water hydration in the hc as the vertical axis . the work presented herein contributes a multiscale framework aimed at accomplishing this multiscale bridging , specifically for the purpose of simulating the protonation / deprotonation of ionizable moieties such as amino acids in biomolecules . the core of our framework is the ms - rmd method , which describes reactive processes in classical md and explicitly treats the charge defect delocalization and grotthuss shuttling of the hydrated excess proton in the pt process . considerable effort has gone into the parametrization of the ms - rmd model for pt in water and amino acids . herein , we have extended to proteins the parametrization scheme from qm / mm data to ms - rmd models ( fitrmd ) and demonstrated its use on protonatable amino acids in biomolecular systems . as presented here , the fitrmd approach variationally maps quantum data ( dft - level qm / mm forces in this case ) onto the ms - rmd nonlinear reactive force field . we have demonstrated how fitrmd can be used to parametrize ms - rmd models for amino acids in proteins with two example systems , clc - ec1 and cco . in clc - ec1 , the ms - rmd models were shown to faithfully reproduce the pt pmfs of the reference qm / mm md , both in the presence and absence of a cl ion in the central region . thus , the fitrmd parametrization was robust enough to capture quite different free energy profiles due to the presence or absence of a single ion . moreover , the local structure of the protein and confined water was shown to be quite similar between the ms - rmd and qm / mm simulations . in cco , the developed ms - rmd models were shown to be efficient enough to capture the coupling between pt and hydration changes in the hc region . even for the clc - ec1 system where exchange of water across the qm / mm boundary is minimal due to protein confinement , lack of water exchange was argued to introduce errors in the calculated qm / mm free energy profile . for example , in clc - ec1 the ms - rmd models produce a pmf that is consistent with the qm / mm hamiltonian even though the cv used for the configuration sampling ( absolute distance ) is different from the cv used for the pmf calculation ( ratio of distances ) ( see methods for a full description ) . it should also be emphasized that although a low - level qm method ( blyp - d ) was used here in the qm / mm calculations , our goal was to demonstrate that a reference ab initio hamiltonian ( and free energy surface ) can be reproduced . thus , an ms - rmd model obtained from fitrmd using a higher level reference ab initio method can be expected to also faithfully reproduce the free energy surface from the latter , if only the latter could be calculated , which it currently can not be . our future efforts will focus on improving the fitrmd protocol to make it more robust to the choice of the trial ms - rmd model , as free as possible from the coevolution of parameters , and insensitive to discrepancies between the qm and mm descriptions of nonreactive atoms in the system .

the mammalian clcs have many physiological functions , including maintenance of the membrane potential , regulation of transepithelial cl transport , and control of ph in the cytoplasm and intracellular organelles . cytochrome c oxidase ( cco ) , a proton pump in the respiratory chain of mitochondria and bacteria , reduces oxygen to water and utilizes the released free energy to pump protons across the membrane , contributing to the transmembrane electrochemical potential gradient that is necessary for atp synthesis . the influenza a m2 proton channel protein transports the protons across the influenza virus membrane and triggers the dissociation of the viral matrix proteins , which is an essential step in the influenza virus replication cycle this is a short list of the many systems in which pt plays a role in a functional mechanism . for such systems , the ionizable amino acids that influence for example in the clc - ec1 antiporter , an intracellular - facing glutamic acid , e203 , shuttles an excess proton to an extracellular - facing glutamic acid , e148 , during transmembrane pt . therefore , to more completely describe pt in biological systems , a computational method should ( 1 ) explicitly treat charge defect delocalization and grotthuss shuttling of the excess proton(s ) between water molecules and ionizable amino acids undergoing protonation / deprotonation ; ( 2 ) allow exchange of water molecules between different protein internal cavities as well as between those cavities and bulk solution ; and ( 3 ) be computationally efficient enough to achieve sufficient sampling of the charge translocation and protein and solvent configuration space for large - scale biomolecular systems , including protein , solvent , ions , and ( where needed ) the membrane . quantum mechanical approaches , such as ab initio molecular dynamics ( aimd ) or quantum mechanics molecular mechanics ( qm / mm ) md methods , can in principle explicitly treat the reactive nature of amino acid protonation / deprotonation and the grotthuss hopping phenomenon . the qm / mm boundary may be especially problematic when the hydration along the pt pathway changes in response to the migration of the excess proton charge defect since this necessitates the exchange of water molecules between protein cavities and bulk solution , which often occurs beyond the nanosecond time scale . this approach describes the migration of an excess proton including explicit grotthuss shuttling and charge defect delocalization by evolving the system on a reactive potential energy surface defined as a dynamical linear combination of diabatic basis states , as in the earlier multistate empirical valence bond ( ms - evb ) method . as will be shown in detail later , the newer ms - rmd approach differs from the older ms - evb approach primarily because the underlying reactive force field of ms - rmd is largely derived from aimd or qm / mm data via force - matching and other means , rendering it far less empirical than the original ms - evb approach . the ability of the generated ms - rmd models to faithfully reproduce the free energy profiles ( potentials of mean force , pmfs ) of the reference qm / mm hamiltonian is demonstrated for pt inside the clc - ec1 channel in two different states . finally , the advantages of developing efficient ms - rmd models are demonstrated with the calculation of 2-dimensional free energy profiles ( 2d - pmfs ) of pt coupled to hydration changes in the central hydrophobic region of cco . in the central region of the clc - ec1 antiporter ( see figure 1 for the images of the whole protein structure and its central region ) , figure 2 shows examples of a set of states in certain configurations , where the excess proton is delocalized into a glutamate residue and surrounding water molecules . the diagonalization of the hamiltonian matrix on the fly gives the energy and eigenvector of the ground state for every configuration of nuclei r:2 ( a ) overview of the structure of the clc - ec1 antiporter and transport pathways for cl ( green dashed ) and h ( red dashed ) . the hii corresponding to the state with protonated glutamate ( gluh ) is described as3where the first three terms are the inter- and the intramolecular potentials of protonated glutamate and all other surrounding molecules , such as waters , other protein residues , lipids , and ions in the system . in order to correct overestimated electrostatic interaction at a short distance between opposite charges on hydronium and deprotonated glutamate , two repulsive terms , vookrep and vhjokrep , are introduced in hii corresponding to the state with deprotonated glutamate:56where roo is the distance between the hydronium oxygen , o , and the carboxyl oxygen of glutamate , o ( oe1 and oe2 in the pdb ) , and rhjo is the distance between each of the three hydronium hydrogen atoms , hj , and the carboxyl oxygen of glutamate . the functional forms for the repulsive terms are the same as those used in the ms - evb3.1 model.b , b , b , c , and c are fitted parameters , and doo0 and doh0 are fixed with the same value used in ms - evb3.1 , which are 2.4 and 1.0 , respectively . the ms - rmd ( and ms - evb ) formalism also provides a convenient and physically intuitive description of the excess proton center of the excess charge ( cec ) , defined as8where the nevb is the total number of evb states , ci2(r ) is the population of state i contributing to the ms - rmd ground state , and ricoc is the geometric center - of - charge for state i. this cec definition allows the use of a continuous reaction coordinate ( further discussed below ) for the pmf calculation of pt in biological systems . the protonated moiety in the state with the largest coefficient , c1 ( eq 8) , possesses the majority of the excess positive charge , when in bulk water this state is the most hydronium - like species ( or the so - called pivot hydronium , a technical term used below ) . in the present article , the initial guess amino acid models were taken from the previous work done in bulk water , except that the constant energy shift between protonated / deprotonated states in the model ( vii ) was determined by the difference in the coulomb energy of the rmd ( evb ) complex between the most favorable hydronium state and the protonated state . the fitrmd method was used to parametrize the ms - rmd models from qm / mm data for the two glutamate residues in the central region , namely , the e148 ( gluex ) and the e203 ( gluin ) , which have been identified as intermediate sites for proton binding along the transport pathway ( see figure 1 ) . in order to directly compare ms - rmd and qm / mm free energy profiles , umbrella sampling was performed with a cv defined as r , which is a function of the excess proton cec ( rcec ) and the center of mass of the carboxyl groups of the e203 ( r203 ) and the e148 ( r148 ) residues:10 the excess proton cec coordinate , however , was defined as11where the r is the position of the jth heavy atom in the qm region , which is either the water oxygen atoms or the carboxyl oxygen atoms in the e203 or the e148 , and the r is the position of the ith hydrogen atom bound to those heavy atoms . second , the positions of the side chains of the four residues in the central region ( s107 , e148 , e203 , and y445 ) and clcen , if present , were averaged , and full moieties representative of these averages were depicted . in the system with clcen absent , rdfs from e148 carboxyl carbon ( cd in the pdb ) to water oxygens are shown in a1 to a3 , for the r , t , and p states ( figure 3 ) , respectively . panels c e : the averaged structure of the four residues and the water structure in the last snapshot from the ( c ) the qm / mm , ( d ) the ms - rmd , and ( e ) the second qm / mm umbrella sampling at the window r = 0.24 . panel f : the coordination number of the pivot hydronium to oxygen atoms of surrounding water molecules or the carboxyl group of e203 in the ms - rmd simulation , initiated from the configuration at low hydration level , which mimics the configuration of the qm / mm in c. to calculate the time scale for the transition from a low to high hydration state in the central region of clc - ec1 , the low hydration state first needed to be created in the ms - rmd structure . some of the relevant details are given here , including the fact that the ms - rmd umbrella sampling simulations in the hydrophobic cavity ( hc ) were carried out by restraining the excess proton cec position ( eq 8) along the pt pathway defined from the mtd simulations and the water density in a predefined box that encompasses the hc ( see ref ( 23 ) for the definition ) . the computational speed for the ms - rmd , self - consistent density functional tight binding ( scc - dftb)-based qm / mm , and blyp - based qm / mm methods were compared for the md simulation of the cco system . an important measure of success of the fitrmd method is its ability to reproduce results of the reference hamiltonian for properties other than those that were fit ( atomistic forces ) , and there is arguably no property more important than the pt free energy profile ( pmf ) . although many protein systems will still require extensive sampling ( beyond the limits of qm / mm ) as well as exhibit water exchange across the qm / mm boundary on longer time scales , the level of confinement in the clc - ec1 antiporter system enables a direct comparison of the qm / mm and ms - rmd pmfs . the ms - rmd and qm / mm pmfs ( figures 3a and b ) show excellent agreement for most parts of the reaction coordinate and for both states of the system ( with and without clcen ) , although there are some differences ( discussed below ) . these results demonstrate that the fitrmd approach is capable of generating ms - rmd models that reproduce the free energy profile of the reference qm / mm hamiltonian , using only forces on the atomic nuclei from a relatively small set of configurations as input for the fitting . the radial distribution functions ( rdfs ) from the carboxyl carbon of either e148 or e203 to the water and hydronium oxygens were calculated with the excess proton cec restrained in the reactant , transition , and product states of the pmf ( the positions are labeled as r , t , and p in figure 3a and b ) . this result provides additional evidence that the model generated by the fitrmd approach faithfully reproduces the underlying free energy landscape of the reference qm / mm hamiltonian , even though only qm / mm forces on the atom nuclei were used as input for the fitting . the discrepancies in the rdfs shown in figures 4 and 5 are likely due to the different water behavior of qm versus mm water as well as short qm / mm sampling where the water dynamics are slow in the confined space of the central region , which will be discussed below . focusing on the most significant , figure 6a highlights the region 0.18 < r < 0.26 from figure 3a , where the ms - rmd pmf ( blue ) dips to a modest metastable minimum , but the qm / mm pmf ( red ) shows an uphill rise with only a slight dip at r 0.21 . the center of excess charge ( yellow sphere ) is close to the first water coordinated to e203 in this window for both the qm / mm and ms - rmd configurations , meaning that the first water is the most hydronium - like species ( the pivot hydronium , which has the largest ms - rmd state coefficient , c1 , in eq 8) . to better understand the difference in solvation structures and charge delocalization , we calculated the average value of c12 for all configurations in the r = 0.24 umbrella windows from the qm / mm and ms - rmd simulations ( figure 6b ) . it should be kept in mind that the c12 value for an eigen cation ( h9o4 ) is 0.65 , which indicates that the pivot hydronium holds 65% of the positive charge ( hydronium - like ) defect , while that for a zundel cation ( h5o2 ) is 0.5 since in that case , excess charge is more equally shared between two water molecules . however , one of these waters is missing in the qm / mm simulations ( figure 6c , gray circle ) , shifting c12(r) to a lower value ( 0.55 ) and the delocalization to more of a zundel - like complex between the carboxyl group and first water molecule ( co - h3o ) . the ms - rmd eigen - like complex forms a contact ion pair ( cip ) with the carboxyl group of deprotonated e203 and the stabilizing electrostatic attraction between oppositely charged ions causes a small energy well ( 1 kcal / mol ) in the ms - rmd pmf . to see if the pmf discrepancy was caused by sampling different conformational phase space , another set of qm / mm umbrella sampling simulations was run for 5 ps ( as described earlier ) , initiated from the last snapshots from the ms - rmd umbrella sampling simulations in the cv range 0.18 < r < 0.26 . to estimate the time scale required for the additional water molecule ( missing in the original qm / mm simulations ) to enter the central region , an ms - rmd configuration was prepared ( see methods ) at r = 0.24 in which this water was removed ( mimicking the qm / mm solvation structure shown in figure 6c ) . this trajectory is representative of the other nine trajectories , in which the second water did not enter the central region until t = 60185 ps ( t = 99 ps ) , after which the coordination number remained 3 for 88 5% of the rest of the trajectories . the protons are then transported through water molecules in a hydrophobic cavity ( the hc ) either to the pump loading site ( pls ) to be further released to the periplasmic side of the membrane or to the binuclear center ( bnc ) to react with oxygen and form water . the collective variables used to define these 2d pmfs are ( 1 ) the progress of the excess proton cec through the hc ( horizontal axis ) and ( 2 ) the degree of hydration of the hc ( vertical axis ) ( figure 8 ; see methods for more discussion ) . two - dimensional free energy profiles ( 2d - pmfs ) for pt from the e286 to the pls in the pm state , as a function of the cec coordinate through the hydrophobic cavity ( hc ) as the horizontal axis and the water hydration in the hc as the vertical axis . the ms - rmd method is at least 3 orders of magnitude faster than the blyp - based qm / mm method and 2 orders of magnitude faster than scc - dftb - based qm / mm , while showing more favorable scaling properties over processors ( the scc - dftb charmm code is not scalable ) . the work presented herein contributes a multiscale framework aimed at accomplishing this multiscale bridging , specifically for the purpose of simulating the protonation / deprotonation of ionizable moieties such as amino acids in biomolecules . the core of our framework is the ms - rmd method , which describes reactive processes in classical md and explicitly treats the charge defect delocalization and grotthuss shuttling of the hydrated excess proton in the pt process . herein , we have extended to proteins the parametrization scheme from qm / mm data to ms - rmd models ( fitrmd ) and demonstrated its use on protonatable amino acids in biomolecular systems . for example , in clc - ec1 the ms - rmd models produce a pmf that is consistent with the qm / mm hamiltonian even though the cv used for the configuration sampling ( absolute distance ) is different from the cv used for the pmf calculation ( ratio of distances ) ( see methods for a full description ) . however , we note that the ms - rmd fitting methodology is itself transferable to different systems and that its use is practical because the total computational cost for both the model fit and the md sampling with ms - rmd will be still much cheaper than directly performing qm / mm md simulations on the same system . our future efforts will focus on improving the fitrmd protocol to make it more robust to the choice of the trial ms - rmd model , as free as possible from the coevolution of parameters , and insensitive to discrepancies between the qm and mm descriptions of nonreactive atoms in the system .

diabetes mellitus , a complex disorder , is a combination of metabolic disorders associated with hyperglycemia due to inadequate insulin production or insulin action . type 1 is characterized by beta - cell destruction leading to insulin deficiency due to autoantibody formation . type 2 is characterized by insulin resistance and eventual reduced insulin secretion due to pancreatic scaring and loss of beta - cells . type 2 diabetes , comprising 90 - 95% of all cases of diabetes mellitus , is one of the most lethal diseases in the world . the main cause of death in type 2 diabetes is cardiovascular disease , specifically atherosclerosis . atherosclerosis is initiated by sequences of alterations in the structure and function of the vascular endothelium . however , experimental evidences suggest that imbalance between oxidative stress and host antioxidant defense along with pro - inflammatory and anti - inflammatory factors play critical roles in early vascular endothelial dysfunction . some ( but not all ) previous exercise interventions have resulted in enhanced vascular endothelial function in type 2 diabetics . however , limited information is available on the most effective type of exercise training program or mechanisms responsible for the improvements seen in vascular endothelial function . thus , this review has the following three aims : 1 ) to introduce presumed diabetes - specific mechanisms responsible for dysfunctional vascular endothelium ; 2 ) to summarize and investigate current evidence of the effect of exercise training on conduit vessel endothelial function in type 2 diabetic patients ; and 3 ) to present possible future directions to what should be further explored to expand our knowledge on this research topic . human vasculature is composed of three layers : the endothelium ( intima ) , smooth muscle cells ( media ) , and surrounding elastic and connective tissues ( adventitia ) . the vascular endothelium comprises the innermost layer of the vasculature , which directly senses changes in blood flow and interacts with hormones and neurotransmitters through various receptor - ligand complexes at its membrane , producing vasoactive agents such as nitric oxide ( no ) , prostacyclin ( pgi ) , endothelium - derived hyperpolarizing factors ( edhf ) , and endothelin-1 . these agents control vascular tone at the vascular smooth muscle level either through vasoconstriction or vasodilatation . the vasculature is relaxed or dilated if the effect of dilatory agents overrides that of constricting agents , such as the basal sympathetic tone and endothelin-1 , whereas vasoconstriction occurs if the dilatory signals are overpowered . nitric oxide or no , the most prominent vasodilatory agent , is produced by the l - arginine - endothelial nitric oxide synthase ( enos ) pathway as a byproduct . the l - citrulline in the vascular endothelium then diffuses into vascular smooth muscle cells and facilitates soluble guanylyl cyclase to convert guanosine triphosphate ( gtp ) to cyclic guanosine monophosphaste ( cgmp ) , which in turn leads to calcium ion movement into the sarcoplasmic reticulum , decreasing calcium ion concentration within the cytoplasm of smooth muscle cells , thus causing the vascular smooth muscle cells lose their tonicity . similarly , pgis are produced when cyclooxygenase ( cox ) uses arachidonic acid ( aa ) as a substrate in vascular endothelium to move into neighboring vascular smooth muscle cells , which then convert adenosine triphosphate ( atp ) into cyclic adenosine monophosphate ( camp ) through activated adenylyl cyclase ( ac ) , inducing vasodilation as a result of decreased calcium ion concentration in vascular smooth muscle cells . the endothelin derived hyperpolarizing factor ( edhf ) signaling pathway still remains to be elucidated although there have been many studies . however , it is known that edhfs are generated by the enzymatic activity of cytochrome p-450 within the golgi apparaturs in endothelial cells . like cox , aa used as a substrate for the enzymatic activity of cytochrome p-450 eventually leads to edhf stimulation or opening of potassium ion channels on the vascular smooth muscle cell membrane , resulting in hyperpolarization of vascular smooth muscle cells . this potassium mediated hyperpolarization reduces the duration of calcium ion channel opening , resulting in decreased calcium ion influx into vascular smooth muscle cells which leads to vascular smooth muscle relaxation . endothelin-1 ( et-1 ) is an endogenous constricting agent produced in vascular endothelial cells that acts as a vascular smooth muscle constricting agent after binding to et-1 receptors ( et - a or b receptors ) on the vascular smooth muscle cell membrane . as previously mentioned , the modulation of human vasculatures is very complicated since there are numerous physiological mediators involved neural ( release of norepinephrine ) and humoral ( angiotensin ii and epinephrine ) regulators which systemically influence and interact with more local regulators , such as the myogenic response and endothelium - derived relaxing factors ( edrf : no , pgi , and edhf ) . however , edrfs are considered to be the most important vasoregulators because they override resting vascular tone controlled by systemic regulators and fine tuning other local mediators . nitric oxide ( no ) also prevents abnormal growth of vascular smooth muscle cells and maintains optimal , healthy vascular wall structure . functionally , no is the major vasodilator modulating systemic vascular resistance , whereas pgi and edhf are less influential by providing supplementary means for vasodilatation . improved no production along with no bioavailability depend on the enos substrate l - arginine s availability , enos phosphorylation and dimeric coupling , availability of enos cofactors ( tetrahydrobipterin : bh4 , heat shock protein-90 : hsp-90 , and calmodulin ) , and a balance between oxidants and antioxidants . thus , it is important to assess systemic biomarkers with no - mediated vasodilation in order to understand the mechanisms involved in the structural and functional changes that occur in diabetes patients . unlike other tissues and cells not affected by abnormal systemic glucose concentration , vascular endothelium is very sensitive to alterations of blood glucose . oxidative stress has emerged as a major player in the cause of vascular endothelial dysfunction in other chronic diseases such as diabetes as well as in normal aging . however , in diabetes , it has been recognized that hyperglycemia - induced generation of reactive oxygen species ( ros ) in the vascular endothelium is the foundation of micro- and macro - vascular complications . four putative mechanisms of diabetes - mediated vascular complications have been indicated by well - designed molecular studies . firstly , the altered role of aldose reductase through sorbitol aldose reductase pathway increases oxidative stress , thus inducing the formation of uncoupled enos . unlike its normal function to convert aldehyde to inactive alcohol , aldose reductase converts glucose into sorbitol which abnormally increases intracellular glucose concentration . during this signaling process , aldose reductase uses nicotinamide adenine dinucleotide phosphate ( nadph ) as a cofactor . thus , the absolute quantity of nadph , a major cofactor in regenerating reduced glutathione , is decreased , further augmenting intracellular oxidative stress . secondly , hyperglycemia - induced advanced glycation end products ( ages ) in vascular endothelium cause increased production of ros and inflammatory cytokines as well as increased receptor ( rage ) activity and pro - atherogenic lipoprotein modification . thirdly , an overall increase in diacylglycerol ( dag ) production leads to protein kinase c ( pkc ) activation . dag , a mediator in the second messenger system , is recruited for intracellular lipid metabolism . after dag activation , pkc is involved not only in enos activity , but also in a lot of gene expression and cytokine production in the vascular endothelium . activated pkc increases ros generation by nadph oxidase , vascular adhesion molecules , pro - inflammatory cytokines , and growth factors , but decreases enos activity , resulting in a decrease of overall no bioavailability , a major characteristic of vascular endothelial dysfunction . fourthly , fructose 6-phosphate can be converted into glucosamine 6-phosphate by glucosamine 6-phosphate transferase through the hexosamine pathway in the hyperglycemic cellular environment , instead of being converted to glyceraldehyde 3-phosphate in the glycolytic pathway . uridine diphosphate ( udp ) n - acetyl glucosamine , the cytosolic end product of the hexosamine pathway , decreases enos phosphorylation but increases transforming growth factor-1 and plasminogen activator inhibitor-1 production , which in turn facilitates the pathological processes of diabetic vascular complications . however , they appear to have a common upstream inducer hyperglycemia and abnormally augmented ros production along the electron transport chains ( etc ) within vascular endothelial cell mitochondria . according to brownlee s concept on the role of oxidative stress in diabetes - mediated vascular complications , hyperglycemia facilitates the rate of glycolysis and the krebs cycle , which aberrantly generates more nicotinamide adenine dinucleotide ( nadh ) and flavin adenine dinucleotide ( fadh2 ) and abnormally over - activates etc . once the etc is activated , more hydrogen ions come out of the mitochondrial inner membrane which increase membrane potential . to bring the membrane potential back down to normal , complex iii in the etc is blocked , electrons from coenzyme q combined with oxygen molecules then generate more superoxide anions . however , hyperglycemia reduces both enzymatic ( superoxide dismutase , catalase , and glutathione peroxidase ) and non - enzymatic ( vitamin e , coenzyme q , lipoic acid , and glutathione ) antioxidants through the over production of ros . thus , the balance between oxidants and antioxidants is disrupted which becomes a main trigger of diabetic vascular complications . nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase , a major ros source in the cytosol , leads to augmented superoxide production in vascular endothelium in the presence of abnormal glucose concentrations . hyperglycemia and increased ros also results in structural and functional abnormality of the enos system . insulin resistance , another major symptom of diabetes , inhibits enos phosphorylation by down - regulating phosphoinositide 3-kinase and pi3k / akt pathway in vascular endothelium , decreasing no production and thus its bioavailability . the increased generation of reactive nitrogen species ( i.e. , peroxynitrite ) within the vascular endothelium in diabetics could be also due to hyperglycemia . these reactive nitrogen species help facilitate lipid peroxidation , nitrotyrosine production , and dna modification , leading to cardiovascular complications such as atherosclerosis . activated vascular adhesion molecules pooled into vascular endothelium also contribute to atherosclerotic plaque development in diabetics . putative pathways related to hyperglycemia - induced vascular endothelial dysfunction are illustrated in figure 2 . flow mediated dilation or fmd is the non - invasive gold - standard way to assess no - mediated vascular endothelial function in response to a physiological stimulus as seen in reactive hyperemia . since this methodological approach was introduced by dr . celermajer in the lancet journal in 1992 , many cardiovascular physiologists who work on human subjects have employed and further developed this methodology . unlike other methods such as venous occlusion plethysmography which indirectly assesses changes in local smaller vessel blood flow as segmental volume changes of a limb in response to local invasive pharmacological stimulus ( i.e. , acetylcholine ) , fmd directly measures changes in larger conduit vessel diameter in response to reactive hyperemia , which occurs after 5 minutes of local limb blood flow occlusion . fmd determines the dilatory capacity of larger vessels by determining the difference between the resting diameter and the diameter during peak reactive hyperemia . fmd also has a strong correlation with invasive coronary epicardial vasoreactivity when evaluating conduit artery function . it has benefits in assessing other important functional parameters such as blood flow , velocity and vascular conductance . in addition , it needs unified guidelines and standardizations in order to have validity when comparing results to other studies . recently , dr . dan green s group provided a methodological guideline to assess fmd . although there are differences in operators , equipment , devices , analysis tools , and software between laboratories , unified technical guidelines will help researchers to accurately measure and evaluate fmd , and to compare the outcomes found in different laboratories . most importantly , vascular smooth muscle responsiveness to a nitric oxide donor ( i.e. , nitroglycerin ) needs to be tested if fmd method is used in order to ensure that results are coming specifically in an endothelium - dependent manner . in many studies , the outcomes of fmd have been supported by assays of numerous systemic biomarkers . since dr . feng and dr . colombo introduced and developed a state - of - the - art methodological approach which collects vascular endothelial cells by inserting guide wires into artery or vein via catheterization and evaluates local endothelial cell protein expressions using immunofluorescence , such method has provided an abundant amount of scientific evidence to understand the mechanisms associated with vascular endothelial function [ 9,22,23,28,32 , 39,62,63,73,74 ] . thus , assessing both systemic and local biomarkers with fmd would be a great methodological approach to understand the mechanisms involved in any functional changes . although there has been a substantial effort to promote vascular health in type 2 diabetic patients , only a few randomized controlled trials have tested the effect of aerobic exercise training on vascular endothelial function in type 2 diabetic patients with inconclusive and controversial results . some experimental evidence demonstrated a positive influence of regular physical activity and aerobic exercise on the improvement of the vascular endothelial function through systemic inflammatory biomarkers in diabetics . however , other studies indicated that regular aerobic exercise did not enhance the impaired endothelial function or systemic biomarkers in type 2 diabetic patients . although there has been a lot of attention on the effect of aerobic exercise on large conduit artery health mainly assessed by fmd , only a couple of studies have evaluated the effect of aerobic exercise training specifically on conduit artery endothelial function in type 2 diabetics . according to the recent collaborative statement by the american college of sports medicine ( acsm ) and the american diabetes association ( ada ) , either aerobic or combined exercise ( aerobic & resistance training ) is recommended to prevent diabetes and the associated complications specifically to improve diabetic vascular health . however , the recommended guidelines for aerobic exercise training are still nonspecific without specific aerobic exercise regimen aimed specifically towards helping improve vascular endothelial function in type 2 diabetic patients . this is partly due to the fact the mechanisms of the beneficial effects that aerobic exercise training has on diabetic vascular complications have not yet been fully elucidated . recently , dr . wisloff and his colleagues have demonstrated that 16 weeks of high - intensive interval training was greatly superior to moderate continuous exercise on the improvement of the vascular endothelial function , insulin signaling , and overall blood glucose in metabolic syndrome patients , including patients with prediabetes . the proposed effects of high - intensity interval training has caused patients and health care providers alike to become skeptical on its benefits when considering its potential risks and secondary detrimental effects it might have on individuals who are not physically fit . however , it has been recently demonstrated that high - intensity interval training can be safely employed in patients of high risk groups such as those with heart failure , metabolic syndrome , coronary artery disease , or hypertension . high - intensity interval training has been tested to over 2000 hours of intervention without any negative events . accordingly , future studies should demonstrate the benefit of high - intensity interval training on vascular endothelial function in type 2 diabetic patients . furthermore , the optimal training modality , intensity , frequency , and duration to improve impaired and dysfunctional diabetic vasculature should be emphasized . human vasculature is composed of three layers : the endothelium ( intima ) , smooth muscle cells ( media ) , and surrounding elastic and connective tissues ( adventitia ) . the vascular endothelium comprises the innermost layer of the vasculature , which directly senses changes in blood flow and interacts with hormones and neurotransmitters through various receptor - ligand complexes at its membrane , producing vasoactive agents such as nitric oxide ( no ) , prostacyclin ( pgi ) , endothelium - derived hyperpolarizing factors ( edhf ) , and endothelin-1 . these agents control vascular tone at the vascular smooth muscle level either through vasoconstriction or vasodilatation . the vasculature is relaxed or dilated if the effect of dilatory agents overrides that of constricting agents , such as the basal sympathetic tone and endothelin-1 , whereas vasoconstriction occurs if the dilatory signals are overpowered . nitric oxide or no , the most prominent vasodilatory agent , is produced by the l - arginine - endothelial nitric oxide synthase ( enos ) pathway as a byproduct . the l - citrulline in the vascular endothelium then diffuses into vascular smooth muscle cells and facilitates soluble guanylyl cyclase to convert guanosine triphosphate ( gtp ) to cyclic guanosine monophosphaste ( cgmp ) , which in turn leads to calcium ion movement into the sarcoplasmic reticulum , decreasing calcium ion concentration within the cytoplasm of smooth muscle cells , thus causing the vascular smooth muscle cells lose their tonicity . similarly , pgis are produced when cyclooxygenase ( cox ) uses arachidonic acid ( aa ) as a substrate in vascular endothelium to move into neighboring vascular smooth muscle cells , which then convert adenosine triphosphate ( atp ) into cyclic adenosine monophosphate ( camp ) through activated adenylyl cyclase ( ac ) , inducing vasodilation as a result of decreased calcium ion concentration in vascular smooth muscle cells . the endothelin derived hyperpolarizing factor ( edhf ) signaling pathway still remains to be elucidated although there have been many studies . however , it is known that edhfs are generated by the enzymatic activity of cytochrome p-450 within the golgi apparaturs in endothelial cells . like cox , aa used as a substrate for the enzymatic activity of cytochrome p-450 eventually leads to edhf stimulation or opening of potassium ion channels on the vascular smooth muscle cell membrane , resulting in hyperpolarization of vascular smooth muscle cells . this potassium mediated hyperpolarization reduces the duration of calcium ion channel opening , resulting in decreased calcium ion influx into vascular smooth muscle cells which leads to vascular smooth muscle relaxation . endothelin-1 ( et-1 ) is an endogenous constricting agent produced in vascular endothelial cells that acts as a vascular smooth muscle constricting agent after binding to et-1 receptors ( et - a or b receptors ) on the vascular smooth muscle cell membrane . as previously mentioned , the modulation of human vasculatures is very complicated since there are numerous physiological mediators involved neural ( release of norepinephrine ) and humoral ( angiotensin ii and epinephrine ) regulators which systemically influence and interact with more local regulators , such as the myogenic response and endothelium - derived relaxing factors ( edrf : no , pgi , and edhf ) . however , edrfs are considered to be the most important vasoregulators because they override resting vascular tone controlled by systemic regulators and fine tuning other local mediators . nitric oxide ( no ) also prevents abnormal growth of vascular smooth muscle cells and maintains optimal , healthy vascular wall structure . functionally , no is the major vasodilator modulating systemic vascular resistance , whereas pgi and edhf are less influential by providing supplementary means for vasodilatation . improved no production along with no bioavailability depend on the enos substrate l - arginine s availability , enos phosphorylation and dimeric coupling , availability of enos cofactors ( tetrahydrobipterin : bh4 , heat shock protein-90 : hsp-90 , and calmodulin ) , and a balance between oxidants and antioxidants . thus , it is important to assess systemic biomarkers with no - mediated vasodilation in order to understand the mechanisms involved in the structural and functional changes that occur in diabetes patients . unlike other tissues and cells not affected by abnormal systemic glucose concentration , vascular endothelium is very sensitive to alterations of blood glucose . oxidative stress has emerged as a major player in the cause of vascular endothelial dysfunction in other chronic diseases such as diabetes as well as in normal aging . however , in diabetes , it has been recognized that hyperglycemia - induced generation of reactive oxygen species ( ros ) in the vascular endothelium is the foundation of micro- and macro - vascular complications . four putative mechanisms of diabetes - mediated vascular complications have been indicated by well - designed molecular studies . firstly , the altered role of aldose reductase through sorbitol aldose reductase pathway increases oxidative stress , thus inducing the formation of uncoupled enos . unlike its normal function to convert aldehyde to inactive alcohol , aldose reductase converts glucose into sorbitol which abnormally increases intracellular glucose concentration . during this signaling process , thus , the absolute quantity of nadph , a major cofactor in regenerating reduced glutathione , is decreased , further augmenting intracellular oxidative stress . secondly , hyperglycemia - induced advanced glycation end products ( ages ) in vascular endothelium cause increased production of ros and inflammatory cytokines as well as increased receptor ( rage ) activity and pro - atherogenic lipoprotein modification . thirdly , an overall increase in diacylglycerol ( dag ) production leads to protein kinase c ( pkc ) activation . dag , a mediator in the second messenger system , is recruited for intracellular lipid metabolism . after dag activation , pkc is involved not only in enos activity , but also in a lot of gene expression and cytokine production in the vascular endothelium . activated pkc increases ros generation by nadph oxidase , vascular adhesion molecules , pro - inflammatory cytokines , and growth factors , but decreases enos activity , resulting in a decrease of overall no bioavailability , a major characteristic of vascular endothelial dysfunction . fourthly , fructose 6-phosphate can be converted into glucosamine 6-phosphate by glucosamine 6-phosphate transferase through the hexosamine pathway in the hyperglycemic cellular environment , instead of being converted to glyceraldehyde 3-phosphate in the glycolytic pathway . uridine diphosphate ( udp ) n - acetyl glucosamine , the cytosolic end product of the hexosamine pathway , decreases enos phosphorylation but increases transforming growth factor-1 and plasminogen activator inhibitor-1 production , which in turn facilitates the pathological processes of diabetic vascular complications . however , they appear to have a common upstream inducer hyperglycemia and abnormally augmented ros production along the electron transport chains ( etc ) within vascular endothelial cell mitochondria . according to brownlee s concept on the role of oxidative stress in diabetes - mediated vascular complications , hyperglycemia facilitates the rate of glycolysis and the krebs cycle , which aberrantly generates more nicotinamide adenine dinucleotide ( nadh ) and flavin adenine dinucleotide ( fadh2 ) and abnormally over - activates etc . once the etc is activated , more hydrogen ions come out of the mitochondrial inner membrane which increase membrane potential . to bring the membrane potential back down to normal , complex iii in the etc is blocked , electrons from coenzyme q combined with oxygen molecules then generate more superoxide anions . however , hyperglycemia reduces both enzymatic ( superoxide dismutase , catalase , and glutathione peroxidase ) and non - enzymatic ( vitamin e , coenzyme q , lipoic acid , and glutathione ) antioxidants through the over production of ros . thus , the balance between oxidants and antioxidants is disrupted which becomes a main trigger of diabetic vascular complications . nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase , a major ros source in the cytosol , leads to augmented superoxide production in vascular endothelium in the presence of abnormal glucose concentrations . hyperglycemia and increased ros also results in structural and functional abnormality of the enos system . insulin resistance , another major symptom of diabetes , inhibits enos phosphorylation by down - regulating phosphoinositide 3-kinase and pi3k / akt pathway in vascular endothelium , decreasing no production and thus its bioavailability . the increased generation of reactive nitrogen species ( i.e. , peroxynitrite ) within the vascular endothelium in diabetics could be also due to hyperglycemia . these reactive nitrogen species help facilitate lipid peroxidation , nitrotyrosine production , and dna modification , leading to cardiovascular complications such as atherosclerosis . activated vascular adhesion molecules pooled into vascular endothelium also contribute to atherosclerotic plaque development in diabetics . putative pathways related to hyperglycemia - induced vascular endothelial dysfunction are illustrated in figure 2 . flow mediated dilation or fmd is the non - invasive gold - standard way to assess no - mediated vascular endothelial function in response to a physiological stimulus as seen in reactive hyperemia . since this methodological approach was introduced by dr . celermajer in the lancet journal in 1992 , many cardiovascular physiologists who work on human subjects have employed and further developed this methodology . unlike other methods such as venous occlusion plethysmography which indirectly assesses changes in local smaller vessel blood flow as segmental volume changes of a limb in response to local invasive pharmacological stimulus ( i.e. , acetylcholine ) , fmd directly measures changes in larger conduit vessel diameter in response to reactive hyperemia , which occurs after 5 minutes of local limb blood flow occlusion . fmd determines the dilatory capacity of larger vessels by determining the difference between the resting diameter and the diameter during peak reactive hyperemia . fmd also has a strong correlation with invasive coronary epicardial vasoreactivity when evaluating conduit artery function . it has benefits in assessing other important functional parameters such as blood flow , velocity and vascular conductance . in addition , it needs unified guidelines and standardizations in order to have validity when comparing results to other studies . recently , dr . dan green s group provided a methodological guideline to assess fmd . although there are differences in operators , equipment , devices , analysis tools , and software between laboratories , unified technical guidelines will help researchers to accurately measure and evaluate fmd , and to compare the outcomes found in different laboratories . most importantly , vascular smooth muscle responsiveness to a nitric oxide donor ( i.e. , nitroglycerin ) needs to be tested if fmd method is used in order to ensure that results are coming specifically in an endothelium - dependent manner . in many studies , the outcomes of fmd have been supported by assays of numerous systemic biomarkers . since dr . feng and dr . colombo introduced and developed a state - of - the - art methodological approach which collects vascular endothelial cells by inserting guide wires into artery or vein via catheterization and evaluates local endothelial cell protein expressions using immunofluorescence , such method has provided an abundant amount of scientific evidence to understand the mechanisms associated with vascular endothelial function [ 9,22,23,28,32 , 39,62,63,73,74 ] . thus , assessing both systemic and local biomarkers with fmd would be a great methodological approach to understand the mechanisms involved in any functional changes . although there has been a substantial effort to promote vascular health in type 2 diabetic patients , only a few randomized controlled trials have tested the effect of aerobic exercise training on vascular endothelial function in type 2 diabetic patients with inconclusive and controversial results . some experimental evidence demonstrated a positive influence of regular physical activity and aerobic exercise on the improvement of the vascular endothelial function through systemic inflammatory biomarkers in diabetics . however , other studies indicated that regular aerobic exercise did not enhance the impaired endothelial function or systemic biomarkers in type 2 diabetic patients . although there has been a lot of attention on the effect of aerobic exercise on large conduit artery health mainly assessed by fmd , only a couple of studies have evaluated the effect of aerobic exercise training specifically on conduit artery endothelial function in type 2 diabetics . according to the recent collaborative statement by the american college of sports medicine ( acsm ) and the american diabetes association ( ada ) , either aerobic or combined exercise ( aerobic & resistance training ) is recommended to prevent diabetes and the associated complications specifically to improve diabetic vascular health . however , the recommended guidelines for aerobic exercise training are still nonspecific without specific aerobic exercise regimen aimed specifically towards helping improve vascular endothelial function in type 2 diabetic patients . this is partly due to the fact the mechanisms of the beneficial effects that aerobic exercise training has on diabetic vascular complications have not yet been fully elucidated . recently , dr . wisloff and his colleagues have demonstrated that 16 weeks of high - intensive interval training was greatly superior to moderate continuous exercise on the improvement of the vascular endothelial function , insulin signaling , and overall blood glucose in metabolic syndrome patients , including patients with prediabetes . the proposed effects of high - intensity interval training has caused patients and health care providers alike to become skeptical on its benefits when considering its potential risks and secondary detrimental effects it might have on individuals who are not physically fit . however , it has been recently demonstrated that high - intensity interval training can be safely employed in patients of high risk groups such as those with heart failure , metabolic syndrome , coronary artery disease , or hypertension . high - intensity interval training has been tested to over 2000 hours of intervention without any negative events . accordingly , future studies should demonstrate the benefit of high - intensity interval training on vascular endothelial function in type 2 diabetic patients . furthermore , the optimal training modality , intensity , frequency , and duration to improve impaired and dysfunctional diabetic vasculature should be emphasized . nitric oxide ( no ) is a major constituent necessary to maintain vascular integrity and dilatory capacity . increased ros generation of endothelial mitochondria has been implicated as a common precursor mechanism in the formation of diabetic vascular complications . even though some studies have explored the effects of exercise on the human vascular endothelial function and related mechanisms , thus , future studies need to demonstrate the optimal exercise regimen for impaired diabetic vasculature and to directly investigate related molecular and biochemical mechanisms involved in the induction and prevention of endothelial injury within collected vascular endothelial cells in addition to traditional systemic biomarkers .

[ purpose]vascular endothelial dysfunction is an early marker of atherosclerosis characterized by decreased nitric oxide bioavailability in the vascular endothelium and smooth muscle cells . recently , some animal models and in vitro trials demonstrated that excessive superoxide production from mitochondria within vascular endothelial cells played a role in the pathogenesis of atherosclerosis in type 2 diabetes . this review provides a systematic assessment of the effectiveness of exercise to identify effective approaches to recognize diabetes risk and prevent progression to heart disease.[methods]a systematic literature search was conducted to retrieve articles from 1979 to 2013 using the following databases : the medline , pubmed . articles had to describe an intervention that physical activity and exercise to identify effective approaches to heart and vascular endothelium.[results]currently , physical activity and exercise guidelines aimed to improve cardiovascular health in patients with type 2 diabetes are nonspecific . benefit of aerobic exercise training on vascular endothelial function in type 2 diabetic patients is still controversial.[conclusion]it is necessary to demonstrate the mechanism of endothelial dysfunction from live human tissues so that we can provide more specific exercise training regimens to enhance cardiovascular health in type 2 diabetic patients .

INTRODUCTION Relaxation of vascular endothelium: a major determinant for vascular integrity Potential mechanisms of diabetes-mediated vascular endothelial dysfunction Assessment of conduit artery endothelial function: Flow-mediated dilation (FMD) Aerobic exercise training and vascular endothelial function in type 2 diabetic patients CONCLUSION

type 1 is characterized by beta - cell destruction leading to insulin deficiency due to autoantibody formation . type 2 is characterized by insulin resistance and eventual reduced insulin secretion due to pancreatic scaring and loss of beta - cells . type 2 diabetes , comprising 90 - 95% of all cases of diabetes mellitus , is one of the most lethal diseases in the world . the main cause of death in type 2 diabetes is cardiovascular disease , specifically atherosclerosis . atherosclerosis is initiated by sequences of alterations in the structure and function of the vascular endothelium . however , experimental evidences suggest that imbalance between oxidative stress and host antioxidant defense along with pro - inflammatory and anti - inflammatory factors play critical roles in early vascular endothelial dysfunction . some ( but not all ) previous exercise interventions have resulted in enhanced vascular endothelial function in type 2 diabetics . however , limited information is available on the most effective type of exercise training program or mechanisms responsible for the improvements seen in vascular endothelial function . thus , this review has the following three aims : 1 ) to introduce presumed diabetes - specific mechanisms responsible for dysfunctional vascular endothelium ; 2 ) to summarize and investigate current evidence of the effect of exercise training on conduit vessel endothelial function in type 2 diabetic patients ; and 3 ) to present possible future directions to what should be further explored to expand our knowledge on this research topic . human vasculature is composed of three layers : the endothelium ( intima ) , smooth muscle cells ( media ) , and surrounding elastic and connective tissues ( adventitia ) . the vascular endothelium comprises the innermost layer of the vasculature , which directly senses changes in blood flow and interacts with hormones and neurotransmitters through various receptor - ligand complexes at its membrane , producing vasoactive agents such as nitric oxide ( no ) , prostacyclin ( pgi ) , endothelium - derived hyperpolarizing factors ( edhf ) , and endothelin-1 . these agents control vascular tone at the vascular smooth muscle level either through vasoconstriction or vasodilatation . the l - citrulline in the vascular endothelium then diffuses into vascular smooth muscle cells and facilitates soluble guanylyl cyclase to convert guanosine triphosphate ( gtp ) to cyclic guanosine monophosphaste ( cgmp ) , which in turn leads to calcium ion movement into the sarcoplasmic reticulum , decreasing calcium ion concentration within the cytoplasm of smooth muscle cells , thus causing the vascular smooth muscle cells lose their tonicity . similarly , pgis are produced when cyclooxygenase ( cox ) uses arachidonic acid ( aa ) as a substrate in vascular endothelium to move into neighboring vascular smooth muscle cells , which then convert adenosine triphosphate ( atp ) into cyclic adenosine monophosphate ( camp ) through activated adenylyl cyclase ( ac ) , inducing vasodilation as a result of decreased calcium ion concentration in vascular smooth muscle cells . however , it is known that edhfs are generated by the enzymatic activity of cytochrome p-450 within the golgi apparaturs in endothelial cells . like cox , aa used as a substrate for the enzymatic activity of cytochrome p-450 eventually leads to edhf stimulation or opening of potassium ion channels on the vascular smooth muscle cell membrane , resulting in hyperpolarization of vascular smooth muscle cells . this potassium mediated hyperpolarization reduces the duration of calcium ion channel opening , resulting in decreased calcium ion influx into vascular smooth muscle cells which leads to vascular smooth muscle relaxation . endothelin-1 ( et-1 ) is an endogenous constricting agent produced in vascular endothelial cells that acts as a vascular smooth muscle constricting agent after binding to et-1 receptors ( et - a or b receptors ) on the vascular smooth muscle cell membrane . nitric oxide ( no ) also prevents abnormal growth of vascular smooth muscle cells and maintains optimal , healthy vascular wall structure . thus , it is important to assess systemic biomarkers with no - mediated vasodilation in order to understand the mechanisms involved in the structural and functional changes that occur in diabetes patients . unlike other tissues and cells not affected by abnormal systemic glucose concentration , vascular endothelium is very sensitive to alterations of blood glucose . oxidative stress has emerged as a major player in the cause of vascular endothelial dysfunction in other chronic diseases such as diabetes as well as in normal aging . however , in diabetes , it has been recognized that hyperglycemia - induced generation of reactive oxygen species ( ros ) in the vascular endothelium is the foundation of micro- and macro - vascular complications . secondly , hyperglycemia - induced advanced glycation end products ( ages ) in vascular endothelium cause increased production of ros and inflammatory cytokines as well as increased receptor ( rage ) activity and pro - atherogenic lipoprotein modification . dag , a mediator in the second messenger system , is recruited for intracellular lipid metabolism . after dag activation , pkc is involved not only in enos activity , but also in a lot of gene expression and cytokine production in the vascular endothelium . activated pkc increases ros generation by nadph oxidase , vascular adhesion molecules , pro - inflammatory cytokines , and growth factors , but decreases enos activity , resulting in a decrease of overall no bioavailability , a major characteristic of vascular endothelial dysfunction . fourthly , fructose 6-phosphate can be converted into glucosamine 6-phosphate by glucosamine 6-phosphate transferase through the hexosamine pathway in the hyperglycemic cellular environment , instead of being converted to glyceraldehyde 3-phosphate in the glycolytic pathway . uridine diphosphate ( udp ) n - acetyl glucosamine , the cytosolic end product of the hexosamine pathway , decreases enos phosphorylation but increases transforming growth factor-1 and plasminogen activator inhibitor-1 production , which in turn facilitates the pathological processes of diabetic vascular complications . however , they appear to have a common upstream inducer hyperglycemia and abnormally augmented ros production along the electron transport chains ( etc ) within vascular endothelial cell mitochondria . once the etc is activated , more hydrogen ions come out of the mitochondrial inner membrane which increase membrane potential . to bring the membrane potential back down to normal , complex iii in the etc is blocked , electrons from coenzyme q combined with oxygen molecules then generate more superoxide anions . nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase , a major ros source in the cytosol , leads to augmented superoxide production in vascular endothelium in the presence of abnormal glucose concentrations . hyperglycemia and increased ros also results in structural and functional abnormality of the enos system . insulin resistance , another major symptom of diabetes , inhibits enos phosphorylation by down - regulating phosphoinositide 3-kinase and pi3k / akt pathway in vascular endothelium , decreasing no production and thus its bioavailability . , peroxynitrite ) within the vascular endothelium in diabetics could be also due to hyperglycemia . activated vascular adhesion molecules pooled into vascular endothelium also contribute to atherosclerotic plaque development in diabetics . putative pathways related to hyperglycemia - induced vascular endothelial dysfunction are illustrated in figure 2 . flow mediated dilation or fmd is the non - invasive gold - standard way to assess no - mediated vascular endothelial function in response to a physiological stimulus as seen in reactive hyperemia . celermajer in the lancet journal in 1992 , many cardiovascular physiologists who work on human subjects have employed and further developed this methodology . it has benefits in assessing other important functional parameters such as blood flow , velocity and vascular conductance . recently , dr . most importantly , vascular smooth muscle responsiveness to a nitric oxide donor ( i.e. colombo introduced and developed a state - of - the - art methodological approach which collects vascular endothelial cells by inserting guide wires into artery or vein via catheterization and evaluates local endothelial cell protein expressions using immunofluorescence , such method has provided an abundant amount of scientific evidence to understand the mechanisms associated with vascular endothelial function [ 9,22,23,28,32 , 39,62,63,73,74 ] . although there has been a substantial effort to promote vascular health in type 2 diabetic patients , only a few randomized controlled trials have tested the effect of aerobic exercise training on vascular endothelial function in type 2 diabetic patients with inconclusive and controversial results . some experimental evidence demonstrated a positive influence of regular physical activity and aerobic exercise on the improvement of the vascular endothelial function through systemic inflammatory biomarkers in diabetics . however , other studies indicated that regular aerobic exercise did not enhance the impaired endothelial function or systemic biomarkers in type 2 diabetic patients . although there has been a lot of attention on the effect of aerobic exercise on large conduit artery health mainly assessed by fmd , only a couple of studies have evaluated the effect of aerobic exercise training specifically on conduit artery endothelial function in type 2 diabetics . according to the recent collaborative statement by the american college of sports medicine ( acsm ) and the american diabetes association ( ada ) , either aerobic or combined exercise ( aerobic & resistance training ) is recommended to prevent diabetes and the associated complications specifically to improve diabetic vascular health . however , the recommended guidelines for aerobic exercise training are still nonspecific without specific aerobic exercise regimen aimed specifically towards helping improve vascular endothelial function in type 2 diabetic patients . this is partly due to the fact the mechanisms of the beneficial effects that aerobic exercise training has on diabetic vascular complications have not yet been fully elucidated . recently , dr . wisloff and his colleagues have demonstrated that 16 weeks of high - intensive interval training was greatly superior to moderate continuous exercise on the improvement of the vascular endothelial function , insulin signaling , and overall blood glucose in metabolic syndrome patients , including patients with prediabetes . however , it has been recently demonstrated that high - intensity interval training can be safely employed in patients of high risk groups such as those with heart failure , metabolic syndrome , coronary artery disease , or hypertension . accordingly , future studies should demonstrate the benefit of high - intensity interval training on vascular endothelial function in type 2 diabetic patients . furthermore , the optimal training modality , intensity , frequency , and duration to improve impaired and dysfunctional diabetic vasculature should be emphasized . human vasculature is composed of three layers : the endothelium ( intima ) , smooth muscle cells ( media ) , and surrounding elastic and connective tissues ( adventitia ) . the vascular endothelium comprises the innermost layer of the vasculature , which directly senses changes in blood flow and interacts with hormones and neurotransmitters through various receptor - ligand complexes at its membrane , producing vasoactive agents such as nitric oxide ( no ) , prostacyclin ( pgi ) , endothelium - derived hyperpolarizing factors ( edhf ) , and endothelin-1 . these agents control vascular tone at the vascular smooth muscle level either through vasoconstriction or vasodilatation . nitric oxide or no , the most prominent vasodilatory agent , is produced by the l - arginine - endothelial nitric oxide synthase ( enos ) pathway as a byproduct . the l - citrulline in the vascular endothelium then diffuses into vascular smooth muscle cells and facilitates soluble guanylyl cyclase to convert guanosine triphosphate ( gtp ) to cyclic guanosine monophosphaste ( cgmp ) , which in turn leads to calcium ion movement into the sarcoplasmic reticulum , decreasing calcium ion concentration within the cytoplasm of smooth muscle cells , thus causing the vascular smooth muscle cells lose their tonicity . similarly , pgis are produced when cyclooxygenase ( cox ) uses arachidonic acid ( aa ) as a substrate in vascular endothelium to move into neighboring vascular smooth muscle cells , which then convert adenosine triphosphate ( atp ) into cyclic adenosine monophosphate ( camp ) through activated adenylyl cyclase ( ac ) , inducing vasodilation as a result of decreased calcium ion concentration in vascular smooth muscle cells . like cox , aa used as a substrate for the enzymatic activity of cytochrome p-450 eventually leads to edhf stimulation or opening of potassium ion channels on the vascular smooth muscle cell membrane , resulting in hyperpolarization of vascular smooth muscle cells . this potassium mediated hyperpolarization reduces the duration of calcium ion channel opening , resulting in decreased calcium ion influx into vascular smooth muscle cells which leads to vascular smooth muscle relaxation . endothelin-1 ( et-1 ) is an endogenous constricting agent produced in vascular endothelial cells that acts as a vascular smooth muscle constricting agent after binding to et-1 receptors ( et - a or b receptors ) on the vascular smooth muscle cell membrane . nitric oxide ( no ) also prevents abnormal growth of vascular smooth muscle cells and maintains optimal , healthy vascular wall structure . thus , it is important to assess systemic biomarkers with no - mediated vasodilation in order to understand the mechanisms involved in the structural and functional changes that occur in diabetes patients . oxidative stress has emerged as a major player in the cause of vascular endothelial dysfunction in other chronic diseases such as diabetes as well as in normal aging . however , in diabetes , it has been recognized that hyperglycemia - induced generation of reactive oxygen species ( ros ) in the vascular endothelium is the foundation of micro- and macro - vascular complications . secondly , hyperglycemia - induced advanced glycation end products ( ages ) in vascular endothelium cause increased production of ros and inflammatory cytokines as well as increased receptor ( rage ) activity and pro - atherogenic lipoprotein modification . dag , a mediator in the second messenger system , is recruited for intracellular lipid metabolism . after dag activation , pkc is involved not only in enos activity , but also in a lot of gene expression and cytokine production in the vascular endothelium . activated pkc increases ros generation by nadph oxidase , vascular adhesion molecules , pro - inflammatory cytokines , and growth factors , but decreases enos activity , resulting in a decrease of overall no bioavailability , a major characteristic of vascular endothelial dysfunction . fourthly , fructose 6-phosphate can be converted into glucosamine 6-phosphate by glucosamine 6-phosphate transferase through the hexosamine pathway in the hyperglycemic cellular environment , instead of being converted to glyceraldehyde 3-phosphate in the glycolytic pathway . uridine diphosphate ( udp ) n - acetyl glucosamine , the cytosolic end product of the hexosamine pathway , decreases enos phosphorylation but increases transforming growth factor-1 and plasminogen activator inhibitor-1 production , which in turn facilitates the pathological processes of diabetic vascular complications . however , they appear to have a common upstream inducer hyperglycemia and abnormally augmented ros production along the electron transport chains ( etc ) within vascular endothelial cell mitochondria . once the etc is activated , more hydrogen ions come out of the mitochondrial inner membrane which increase membrane potential . to bring the membrane potential back down to normal , complex iii in the etc is blocked , electrons from coenzyme q combined with oxygen molecules then generate more superoxide anions . nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase , a major ros source in the cytosol , leads to augmented superoxide production in vascular endothelium in the presence of abnormal glucose concentrations . hyperglycemia and increased ros also results in structural and functional abnormality of the enos system . , peroxynitrite ) within the vascular endothelium in diabetics could be also due to hyperglycemia . activated vascular adhesion molecules pooled into vascular endothelium also contribute to atherosclerotic plaque development in diabetics . putative pathways related to hyperglycemia - induced vascular endothelial dysfunction are illustrated in figure 2 . flow mediated dilation or fmd is the non - invasive gold - standard way to assess no - mediated vascular endothelial function in response to a physiological stimulus as seen in reactive hyperemia . celermajer in the lancet journal in 1992 , many cardiovascular physiologists who work on human subjects have employed and further developed this methodology . it has benefits in assessing other important functional parameters such as blood flow , velocity and vascular conductance . recently , dr . most importantly , vascular smooth muscle responsiveness to a nitric oxide donor ( i.e. colombo introduced and developed a state - of - the - art methodological approach which collects vascular endothelial cells by inserting guide wires into artery or vein via catheterization and evaluates local endothelial cell protein expressions using immunofluorescence , such method has provided an abundant amount of scientific evidence to understand the mechanisms associated with vascular endothelial function [ 9,22,23,28,32 , 39,62,63,73,74 ] . although there has been a substantial effort to promote vascular health in type 2 diabetic patients , only a few randomized controlled trials have tested the effect of aerobic exercise training on vascular endothelial function in type 2 diabetic patients with inconclusive and controversial results . some experimental evidence demonstrated a positive influence of regular physical activity and aerobic exercise on the improvement of the vascular endothelial function through systemic inflammatory biomarkers in diabetics . however , other studies indicated that regular aerobic exercise did not enhance the impaired endothelial function or systemic biomarkers in type 2 diabetic patients . although there has been a lot of attention on the effect of aerobic exercise on large conduit artery health mainly assessed by fmd , only a couple of studies have evaluated the effect of aerobic exercise training specifically on conduit artery endothelial function in type 2 diabetics . however , the recommended guidelines for aerobic exercise training are still nonspecific without specific aerobic exercise regimen aimed specifically towards helping improve vascular endothelial function in type 2 diabetic patients . this is partly due to the fact the mechanisms of the beneficial effects that aerobic exercise training has on diabetic vascular complications have not yet been fully elucidated . recently , dr . wisloff and his colleagues have demonstrated that 16 weeks of high - intensive interval training was greatly superior to moderate continuous exercise on the improvement of the vascular endothelial function , insulin signaling , and overall blood glucose in metabolic syndrome patients , including patients with prediabetes . however , it has been recently demonstrated that high - intensity interval training can be safely employed in patients of high risk groups such as those with heart failure , metabolic syndrome , coronary artery disease , or hypertension . accordingly , future studies should demonstrate the benefit of high - intensity interval training on vascular endothelial function in type 2 diabetic patients . nitric oxide ( no ) is a major constituent necessary to maintain vascular integrity and dilatory capacity . increased ros generation of endothelial mitochondria has been implicated as a common precursor mechanism in the formation of diabetic vascular complications . even though some studies have explored the effects of exercise on the human vascular endothelial function and related mechanisms , thus , future studies need to demonstrate the optimal exercise regimen for impaired diabetic vasculature and to directly investigate related molecular and biochemical mechanisms involved in the induction and prevention of endothelial injury within collected vascular endothelial cells in addition to traditional systemic biomarkers .

type 2 is characterized by insulin resistance and eventual reduced insulin secretion due to pancreatic scaring and loss of beta - cells . type 2 diabetes , comprising 90 - 95% of all cases of diabetes mellitus , is one of the most lethal diseases in the world . atherosclerosis is initiated by sequences of alterations in the structure and function of the vascular endothelium . however , experimental evidences suggest that imbalance between oxidative stress and host antioxidant defense along with pro - inflammatory and anti - inflammatory factors play critical roles in early vascular endothelial dysfunction . however , limited information is available on the most effective type of exercise training program or mechanisms responsible for the improvements seen in vascular endothelial function . thus , this review has the following three aims : 1 ) to introduce presumed diabetes - specific mechanisms responsible for dysfunctional vascular endothelium ; 2 ) to summarize and investigate current evidence of the effect of exercise training on conduit vessel endothelial function in type 2 diabetic patients ; and 3 ) to present possible future directions to what should be further explored to expand our knowledge on this research topic . human vasculature is composed of three layers : the endothelium ( intima ) , smooth muscle cells ( media ) , and surrounding elastic and connective tissues ( adventitia ) . the vascular endothelium comprises the innermost layer of the vasculature , which directly senses changes in blood flow and interacts with hormones and neurotransmitters through various receptor - ligand complexes at its membrane , producing vasoactive agents such as nitric oxide ( no ) , prostacyclin ( pgi ) , endothelium - derived hyperpolarizing factors ( edhf ) , and endothelin-1 . nitric oxide or no , the most prominent vasodilatory agent , is produced by the l - arginine - endothelial nitric oxide synthase ( enos ) pathway as a byproduct . the l - citrulline in the vascular endothelium then diffuses into vascular smooth muscle cells and facilitates soluble guanylyl cyclase to convert guanosine triphosphate ( gtp ) to cyclic guanosine monophosphaste ( cgmp ) , which in turn leads to calcium ion movement into the sarcoplasmic reticulum , decreasing calcium ion concentration within the cytoplasm of smooth muscle cells , thus causing the vascular smooth muscle cells lose their tonicity . similarly , pgis are produced when cyclooxygenase ( cox ) uses arachidonic acid ( aa ) as a substrate in vascular endothelium to move into neighboring vascular smooth muscle cells , which then convert adenosine triphosphate ( atp ) into cyclic adenosine monophosphate ( camp ) through activated adenylyl cyclase ( ac ) , inducing vasodilation as a result of decreased calcium ion concentration in vascular smooth muscle cells . like cox , aa used as a substrate for the enzymatic activity of cytochrome p-450 eventually leads to edhf stimulation or opening of potassium ion channels on the vascular smooth muscle cell membrane , resulting in hyperpolarization of vascular smooth muscle cells . this potassium mediated hyperpolarization reduces the duration of calcium ion channel opening , resulting in decreased calcium ion influx into vascular smooth muscle cells which leads to vascular smooth muscle relaxation . endothelin-1 ( et-1 ) is an endogenous constricting agent produced in vascular endothelial cells that acts as a vascular smooth muscle constricting agent after binding to et-1 receptors ( et - a or b receptors ) on the vascular smooth muscle cell membrane . as previously mentioned , the modulation of human vasculatures is very complicated since there are numerous physiological mediators involved neural ( release of norepinephrine ) and humoral ( angiotensin ii and epinephrine ) regulators which systemically influence and interact with more local regulators , such as the myogenic response and endothelium - derived relaxing factors ( edrf : no , pgi , and edhf ) . nitric oxide ( no ) also prevents abnormal growth of vascular smooth muscle cells and maintains optimal , healthy vascular wall structure . improved no production along with no bioavailability depend on the enos substrate l - arginine s availability , enos phosphorylation and dimeric coupling , availability of enos cofactors ( tetrahydrobipterin : bh4 , heat shock protein-90 : hsp-90 , and calmodulin ) , and a balance between oxidants and antioxidants . thus , it is important to assess systemic biomarkers with no - mediated vasodilation in order to understand the mechanisms involved in the structural and functional changes that occur in diabetes patients . oxidative stress has emerged as a major player in the cause of vascular endothelial dysfunction in other chronic diseases such as diabetes as well as in normal aging . however , in diabetes , it has been recognized that hyperglycemia - induced generation of reactive oxygen species ( ros ) in the vascular endothelium is the foundation of micro- and macro - vascular complications . four putative mechanisms of diabetes - mediated vascular complications have been indicated by well - designed molecular studies . firstly , the altered role of aldose reductase through sorbitol aldose reductase pathway increases oxidative stress , thus inducing the formation of uncoupled enos . thus , the absolute quantity of nadph , a major cofactor in regenerating reduced glutathione , is decreased , further augmenting intracellular oxidative stress . secondly , hyperglycemia - induced advanced glycation end products ( ages ) in vascular endothelium cause increased production of ros and inflammatory cytokines as well as increased receptor ( rage ) activity and pro - atherogenic lipoprotein modification . after dag activation , pkc is involved not only in enos activity , but also in a lot of gene expression and cytokine production in the vascular endothelium . activated pkc increases ros generation by nadph oxidase , vascular adhesion molecules , pro - inflammatory cytokines , and growth factors , but decreases enos activity , resulting in a decrease of overall no bioavailability , a major characteristic of vascular endothelial dysfunction . uridine diphosphate ( udp ) n - acetyl glucosamine , the cytosolic end product of the hexosamine pathway , decreases enos phosphorylation but increases transforming growth factor-1 and plasminogen activator inhibitor-1 production , which in turn facilitates the pathological processes of diabetic vascular complications . according to brownlee s concept on the role of oxidative stress in diabetes - mediated vascular complications , hyperglycemia facilitates the rate of glycolysis and the krebs cycle , which aberrantly generates more nicotinamide adenine dinucleotide ( nadh ) and flavin adenine dinucleotide ( fadh2 ) and abnormally over - activates etc . however , hyperglycemia reduces both enzymatic ( superoxide dismutase , catalase , and glutathione peroxidase ) and non - enzymatic ( vitamin e , coenzyme q , lipoic acid , and glutathione ) antioxidants through the over production of ros . nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase , a major ros source in the cytosol , leads to augmented superoxide production in vascular endothelium in the presence of abnormal glucose concentrations . insulin resistance , another major symptom of diabetes , inhibits enos phosphorylation by down - regulating phosphoinositide 3-kinase and pi3k / akt pathway in vascular endothelium , decreasing no production and thus its bioavailability . flow mediated dilation or fmd is the non - invasive gold - standard way to assess no - mediated vascular endothelial function in response to a physiological stimulus as seen in reactive hyperemia . unlike other methods such as venous occlusion plethysmography which indirectly assesses changes in local smaller vessel blood flow as segmental volume changes of a limb in response to local invasive pharmacological stimulus ( i.e. fmd determines the dilatory capacity of larger vessels by determining the difference between the resting diameter and the diameter during peak reactive hyperemia . although there are differences in operators , equipment , devices , analysis tools , and software between laboratories , unified technical guidelines will help researchers to accurately measure and evaluate fmd , and to compare the outcomes found in different laboratories . colombo introduced and developed a state - of - the - art methodological approach which collects vascular endothelial cells by inserting guide wires into artery or vein via catheterization and evaluates local endothelial cell protein expressions using immunofluorescence , such method has provided an abundant amount of scientific evidence to understand the mechanisms associated with vascular endothelial function [ 9,22,23,28,32 , 39,62,63,73,74 ] . thus , assessing both systemic and local biomarkers with fmd would be a great methodological approach to understand the mechanisms involved in any functional changes . although there has been a substantial effort to promote vascular health in type 2 diabetic patients , only a few randomized controlled trials have tested the effect of aerobic exercise training on vascular endothelial function in type 2 diabetic patients with inconclusive and controversial results . some experimental evidence demonstrated a positive influence of regular physical activity and aerobic exercise on the improvement of the vascular endothelial function through systemic inflammatory biomarkers in diabetics . although there has been a lot of attention on the effect of aerobic exercise on large conduit artery health mainly assessed by fmd , only a couple of studies have evaluated the effect of aerobic exercise training specifically on conduit artery endothelial function in type 2 diabetics . according to the recent collaborative statement by the american college of sports medicine ( acsm ) and the american diabetes association ( ada ) , either aerobic or combined exercise ( aerobic & resistance training ) is recommended to prevent diabetes and the associated complications specifically to improve diabetic vascular health . however , the recommended guidelines for aerobic exercise training are still nonspecific without specific aerobic exercise regimen aimed specifically towards helping improve vascular endothelial function in type 2 diabetic patients . this is partly due to the fact the mechanisms of the beneficial effects that aerobic exercise training has on diabetic vascular complications have not yet been fully elucidated . wisloff and his colleagues have demonstrated that 16 weeks of high - intensive interval training was greatly superior to moderate continuous exercise on the improvement of the vascular endothelial function , insulin signaling , and overall blood glucose in metabolic syndrome patients , including patients with prediabetes . the proposed effects of high - intensity interval training has caused patients and health care providers alike to become skeptical on its benefits when considering its potential risks and secondary detrimental effects it might have on individuals who are not physically fit . however , it has been recently demonstrated that high - intensity interval training can be safely employed in patients of high risk groups such as those with heart failure , metabolic syndrome , coronary artery disease , or hypertension . accordingly , future studies should demonstrate the benefit of high - intensity interval training on vascular endothelial function in type 2 diabetic patients . the vascular endothelium comprises the innermost layer of the vasculature , which directly senses changes in blood flow and interacts with hormones and neurotransmitters through various receptor - ligand complexes at its membrane , producing vasoactive agents such as nitric oxide ( no ) , prostacyclin ( pgi ) , endothelium - derived hyperpolarizing factors ( edhf ) , and endothelin-1 . nitric oxide or no , the most prominent vasodilatory agent , is produced by the l - arginine - endothelial nitric oxide synthase ( enos ) pathway as a byproduct . the l - citrulline in the vascular endothelium then diffuses into vascular smooth muscle cells and facilitates soluble guanylyl cyclase to convert guanosine triphosphate ( gtp ) to cyclic guanosine monophosphaste ( cgmp ) , which in turn leads to calcium ion movement into the sarcoplasmic reticulum , decreasing calcium ion concentration within the cytoplasm of smooth muscle cells , thus causing the vascular smooth muscle cells lose their tonicity . similarly , pgis are produced when cyclooxygenase ( cox ) uses arachidonic acid ( aa ) as a substrate in vascular endothelium to move into neighboring vascular smooth muscle cells , which then convert adenosine triphosphate ( atp ) into cyclic adenosine monophosphate ( camp ) through activated adenylyl cyclase ( ac ) , inducing vasodilation as a result of decreased calcium ion concentration in vascular smooth muscle cells . like cox , aa used as a substrate for the enzymatic activity of cytochrome p-450 eventually leads to edhf stimulation or opening of potassium ion channels on the vascular smooth muscle cell membrane , resulting in hyperpolarization of vascular smooth muscle cells . as previously mentioned , the modulation of human vasculatures is very complicated since there are numerous physiological mediators involved neural ( release of norepinephrine ) and humoral ( angiotensin ii and epinephrine ) regulators which systemically influence and interact with more local regulators , such as the myogenic response and endothelium - derived relaxing factors ( edrf : no , pgi , and edhf ) . improved no production along with no bioavailability depend on the enos substrate l - arginine s availability , enos phosphorylation and dimeric coupling , availability of enos cofactors ( tetrahydrobipterin : bh4 , heat shock protein-90 : hsp-90 , and calmodulin ) , and a balance between oxidants and antioxidants . thus , it is important to assess systemic biomarkers with no - mediated vasodilation in order to understand the mechanisms involved in the structural and functional changes that occur in diabetes patients . oxidative stress has emerged as a major player in the cause of vascular endothelial dysfunction in other chronic diseases such as diabetes as well as in normal aging . however , in diabetes , it has been recognized that hyperglycemia - induced generation of reactive oxygen species ( ros ) in the vascular endothelium is the foundation of micro- and macro - vascular complications . firstly , the altered role of aldose reductase through sorbitol aldose reductase pathway increases oxidative stress , thus inducing the formation of uncoupled enos . during this signaling process , thus , the absolute quantity of nadph , a major cofactor in regenerating reduced glutathione , is decreased , further augmenting intracellular oxidative stress . secondly , hyperglycemia - induced advanced glycation end products ( ages ) in vascular endothelium cause increased production of ros and inflammatory cytokines as well as increased receptor ( rage ) activity and pro - atherogenic lipoprotein modification . after dag activation , pkc is involved not only in enos activity , but also in a lot of gene expression and cytokine production in the vascular endothelium . activated pkc increases ros generation by nadph oxidase , vascular adhesion molecules , pro - inflammatory cytokines , and growth factors , but decreases enos activity , resulting in a decrease of overall no bioavailability , a major characteristic of vascular endothelial dysfunction . uridine diphosphate ( udp ) n - acetyl glucosamine , the cytosolic end product of the hexosamine pathway , decreases enos phosphorylation but increases transforming growth factor-1 and plasminogen activator inhibitor-1 production , which in turn facilitates the pathological processes of diabetic vascular complications . according to brownlee s concept on the role of oxidative stress in diabetes - mediated vascular complications , hyperglycemia facilitates the rate of glycolysis and the krebs cycle , which aberrantly generates more nicotinamide adenine dinucleotide ( nadh ) and flavin adenine dinucleotide ( fadh2 ) and abnormally over - activates etc . however , hyperglycemia reduces both enzymatic ( superoxide dismutase , catalase , and glutathione peroxidase ) and non - enzymatic ( vitamin e , coenzyme q , lipoic acid , and glutathione ) antioxidants through the over production of ros . nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase , a major ros source in the cytosol , leads to augmented superoxide production in vascular endothelium in the presence of abnormal glucose concentrations . insulin resistance , another major symptom of diabetes , inhibits enos phosphorylation by down - regulating phosphoinositide 3-kinase and pi3k / akt pathway in vascular endothelium , decreasing no production and thus its bioavailability . flow mediated dilation or fmd is the non - invasive gold - standard way to assess no - mediated vascular endothelial function in response to a physiological stimulus as seen in reactive hyperemia . colombo introduced and developed a state - of - the - art methodological approach which collects vascular endothelial cells by inserting guide wires into artery or vein via catheterization and evaluates local endothelial cell protein expressions using immunofluorescence , such method has provided an abundant amount of scientific evidence to understand the mechanisms associated with vascular endothelial function [ 9,22,23,28,32 , 39,62,63,73,74 ] . thus , assessing both systemic and local biomarkers with fmd would be a great methodological approach to understand the mechanisms involved in any functional changes . although there has been a substantial effort to promote vascular health in type 2 diabetic patients , only a few randomized controlled trials have tested the effect of aerobic exercise training on vascular endothelial function in type 2 diabetic patients with inconclusive and controversial results . some experimental evidence demonstrated a positive influence of regular physical activity and aerobic exercise on the improvement of the vascular endothelial function through systemic inflammatory biomarkers in diabetics . although there has been a lot of attention on the effect of aerobic exercise on large conduit artery health mainly assessed by fmd , only a couple of studies have evaluated the effect of aerobic exercise training specifically on conduit artery endothelial function in type 2 diabetics . according to the recent collaborative statement by the american college of sports medicine ( acsm ) and the american diabetes association ( ada ) , either aerobic or combined exercise ( aerobic & resistance training ) is recommended to prevent diabetes and the associated complications specifically to improve diabetic vascular health . however , the recommended guidelines for aerobic exercise training are still nonspecific without specific aerobic exercise regimen aimed specifically towards helping improve vascular endothelial function in type 2 diabetic patients . this is partly due to the fact the mechanisms of the beneficial effects that aerobic exercise training has on diabetic vascular complications have not yet been fully elucidated . wisloff and his colleagues have demonstrated that 16 weeks of high - intensive interval training was greatly superior to moderate continuous exercise on the improvement of the vascular endothelial function , insulin signaling , and overall blood glucose in metabolic syndrome patients , including patients with prediabetes . the proposed effects of high - intensity interval training has caused patients and health care providers alike to become skeptical on its benefits when considering its potential risks and secondary detrimental effects it might have on individuals who are not physically fit . however , it has been recently demonstrated that high - intensity interval training can be safely employed in patients of high risk groups such as those with heart failure , metabolic syndrome , coronary artery disease , or hypertension . even though some studies have explored the effects of exercise on the human vascular endothelial function and related mechanisms , thus , future studies need to demonstrate the optimal exercise regimen for impaired diabetic vasculature and to directly investigate related molecular and biochemical mechanisms involved in the induction and prevention of endothelial injury within collected vascular endothelial cells in addition to traditional systemic biomarkers .

this study provides class ii evidence that teriflunomide generally does not adversely impact the ability of patients with rms to mount immune responses to influenza vaccination . the teriflunomide and vaccination ( teriva ) study ( nct01403376 ) was a multicenter , multinational , parallel - group study involving 128 patients in 3 treatment groups . groups 1 and 2 included patients with rms treated with either teriflunomide 7 mg or 14 mg once daily for at least 6 months over the course of 2 long - term extension studies ( nct00228163 : open - label extension of a phase ii study , which started in 2001 ; nct00803049 : blinded extension of the phase iii temso study , which started in 2004 ) . group 3 included patients with rms who had received a stable dose of ifn--1 for at least 6 months , and represents a reference population of patients with rms who have previously been reported to mount normal immune responses to seasonal influenza vaccination . the study comprised a screening period of up to 21 days , followed by administration of seasonal influenza vaccine on day 1 and antibody assessments at day 28 ( 2 days ) postimmunization ( figure e-1 on the neurology web site at www.neurology.org ) . patients were immunized with a single i m or intradermal administration of the 2011/2012 inactivated seasonal influenza vaccines , vaxigrip or mutagrip ( both sanofi pasteur , lyon , france ) . both vaccines contained the following influenza strains : a / california/7/2009 ( h1n1 ) , a / perth/16/2009 ( h3n2 ) , and b / brisbane/60/2008 ( b ) . choice of vaccine was performed according to country standards ; mutagrip was administered only in germany to 20 patients ( 12 in the ifn--1 group , and 3 and 5 in the 7-mg and 14-mg teriflunomide groups , respectively ) . the teriva study protocol and participation consents were submitted to independent ethics committees or institutional review boards , and subsequently reviewed and approved . the study was performed at sites approved to participate in either of the 2 long - term teriflunomide extension studies . subjects participating in these extension studies were asked to participate voluntarily in the teriva study . male or female subjects aged between 18 and 60 years with rms treated for at least 6 months with once - daily teriflunomide ( 7 mg or 14 mg ) or ifn--1 , and who were expected to remain on treatment for the duration of the study and who signed a specific informed consent form , were eligible for inclusion in the teriva study . subjects with concomitant infectious pathology at the time of vaccination , ms relapse within 1 month of vaccination , or administered systemic corticosteroids within 1 month of vaccination were excluded from the study . additional exclusion criteria included contraindication to influenza vaccine or in receipt of any vaccination within the last 6 months ; prior use of investigational drugs or participation in a clinical trial within 1 year before screening ( for patients in the ifn--1 group only ) ; prior / concomitant use ( within 1 year of study entry ) of cladribine , mitoxantrone , or other immunosuppressant agents ; prior / concomitant use of glatiramer acetate ( within 1 year of study entry ) ; or iv immunoglobulins ( within 3 months of study entry ) . pregnant or breastfeeding women were also excluded from the study . the hemagglutination inhibition assay ( hia ) hias were performed using serial 2-fold dilutions of serum and results were presented as titers ( i.e. , the highest dilution of serum that achieved complete inhibition of hemagglutination ) . evaluation of vaccine effectiveness in this study was performed in accordance with european guidelines , which apply 3 criteria to evaluate the immune response to influenza vaccine . first , the seroprotection rate ( i.e. , the proportion of subjects achieving a postvaccination titer 40 with the hia ) should be achieved in 70% of vaccinees postvaccination for individuals aged 1860 years and in 60% for those over 60 years . second , the mean geometric increase ( i.e. , the quotient of postvaccination and prevaccination geometric mean titers [ gmts ] ) should be 2.5 in individuals aged 1860 years and 2.0 in individuals over 60 years . third , the seroresponse rate ( i.e. , the proportion of previously seronegative subjects exceeding a postvaccination titer of 40 , and the proportion of previously seropositive subjects with a 4-fold increase in prevaccination and postvaccination sera ) should be achieved in 40% of vaccinees aged 1860 years and in 30% in those over 60 years . for each virus strain and each age class , at least one of the 3 criteria should be met . in this study , the primary efficacy endpoint was the proportion of patients who achieved seroprotection to the influenza vaccine strains , defined as an influenza antibody titer 40 for each strain 28 days postvaccination . the following secondary endpoints were also assessed : the proportion of patients with seroconversion ( i.e. , a prevaccination antibody titer 10 and a postvaccination antibody titer 40 ) ; the proportion of patients with a prevaccination antibody titer < 40 achieving seroprotection ; the proportion of patients with either a 2- or 4-fold increase in strain - specific antibody titers compared with prevaccination titers at 28 days postvaccination ; gmts at baseline and day 28 for each strain , and the corresponding gmt ratio ( postvaccination / prevaccination ) . assuming a response rate of around 70% ( measured as the proportion of patients with antibody titers 40 ) , sample size was calculated as 37 evaluable subjects per group based on the required number of subjects needed to obtain a precision of 12.5% ( corresponding to confidence interval [ ci ] widths of 25% ) around the point estimate of the true response rate under a 90% ci for the true response rate . efficacy analyses were conducted in the per - protocol population , which comprised all patients consenting to enter the study who received influenza vaccination and had an antibody titer at day 28 . the safety population comprised all patients who consented and were enrolled into the study . for the primary efficacy analysis , the proportion of patients with influenza antibody titers 40 at 28 days differences between each teriflunomide treatment group and the ifn--1 reference group were estimated along with 90% cis for each vaccine strain . although there were 2 teriflunomide treatments and 3 viral strains , no adjustments for multiplicity were made . for secondary efficacy analyses , the evaluation of 2- or 4-fold increases in influenza antibody titers compared with prevaccination titers , proportions of patients , and corresponding 90% cis using normal approximation were calculated for each strain and each treatment group . gmts at baseline and day 28 and the respective ratios ( postvaccination / prevaccination ) were summarized by strain and treatment group . for analysis of safety , incidences of treatment - emergent adverse events ( teaes ) , treatment - emergent serious adverse events ( saes ) , and adverse events leading to permanent treatment discontinuation occurring from time of vaccination to day 28 were documented within each group . the teriflunomide and vaccination ( teriva ) study ( nct01403376 ) was a multicenter , multinational , parallel - group study involving 128 patients in 3 treatment groups . groups 1 and 2 included patients with rms treated with either teriflunomide 7 mg or 14 mg once daily for at least 6 months over the course of 2 long - term extension studies ( nct00228163 : open - label extension of a phase ii study , which started in 2001 ; nct00803049 : blinded extension of the phase iii temso study , which started in 2004 ) . group 3 included patients with rms who had received a stable dose of ifn--1 for at least 6 months , and represents a reference population of patients with rms who have previously been reported to mount normal immune responses to seasonal influenza vaccination . the study comprised a screening period of up to 21 days , followed by administration of seasonal influenza vaccine on day 1 and antibody assessments at day 28 ( 2 days ) postimmunization ( figure e-1 on the neurology web site at www.neurology.org ) . patients were immunized with a single i m or intradermal administration of the 2011/2012 inactivated seasonal influenza vaccines , vaxigrip or mutagrip ( both sanofi pasteur , lyon , france ) . both vaccines contained the following influenza strains : a / california/7/2009 ( h1n1 ) , a / perth/16/2009 ( h3n2 ) , and b / brisbane/60/2008 ( b ) . choice of vaccine was performed according to country standards ; mutagrip was administered only in germany to 20 patients ( 12 in the ifn--1 group , and 3 and 5 in the 7-mg and 14-mg teriflunomide groups , respectively ) . the teriva study protocol and participation consents were submitted to independent ethics committees or institutional review boards , and subsequently reviewed and approved . the study was performed at sites approved to participate in either of the 2 long - term teriflunomide extension studies . subjects participating in these extension studies were asked to participate voluntarily in the teriva study . male or female subjects aged between 18 and 60 years with rms treated for at least 6 months with once - daily teriflunomide ( 7 mg or 14 mg ) or ifn--1 , and who were expected to remain on treatment for the duration of the study and who signed a specific informed consent form , were eligible for inclusion in the teriva study . subjects with concomitant infectious pathology at the time of vaccination , ms relapse within 1 month of vaccination , or administered systemic corticosteroids within 1 month of vaccination were excluded from the study . additional exclusion criteria included contraindication to influenza vaccine or in receipt of any vaccination within the last 6 months ; prior use of investigational drugs or participation in a clinical trial within 1 year before screening ( for patients in the ifn--1 group only ) ; prior / concomitant use ( within 1 year of study entry ) of cladribine , mitoxantrone , or other immunosuppressant agents ; prior / concomitant use of glatiramer acetate ( within 1 year of study entry ) ; or iv immunoglobulins ( within 3 months of study entry ) . the hemagglutination inhibition assay ( hia ) was used to detect strain - specific anti - influenza antibodies 28 days postimmunization . hias were performed using serial 2-fold dilutions of serum and results were presented as titers ( i.e. , the highest dilution of serum that achieved complete inhibition of hemagglutination ) . evaluation of vaccine effectiveness in this study was performed in accordance with european guidelines , which apply 3 criteria to evaluate the immune response to influenza vaccine . first , the seroprotection rate ( i.e. , the proportion of subjects achieving a postvaccination titer 40 with the hia ) should be achieved in 70% of vaccinees postvaccination for individuals aged 1860 years and in 60% for those over 60 years . second , the mean geometric increase ( i.e. , the quotient of postvaccination and prevaccination geometric mean titers [ gmts ] ) should be 2.5 in individuals aged 1860 years and 2.0 in individuals over 60 years . third , the seroresponse rate ( i.e. , the proportion of previously seronegative subjects exceeding a postvaccination titer of 40 , and the proportion of previously seropositive subjects with a 4-fold increase in prevaccination and postvaccination sera ) should be achieved in 40% of vaccinees aged 1860 years and in 30% in those over 60 years . for each virus strain and each age class , the primary efficacy endpoint was the proportion of patients who achieved seroprotection to the influenza vaccine strains , defined as an influenza antibody titer 40 for each strain 28 days postvaccination . the following secondary endpoints were also assessed : the proportion of patients with seroconversion ( i.e. , a prevaccination antibody titer 10 and a postvaccination antibody titer 40 ) ; the proportion of patients with a prevaccination antibody titer < 40 achieving seroprotection ; the proportion of patients with either a 2- or 4-fold increase in strain - specific antibody titers compared with prevaccination titers at 28 days postvaccination ; gmts at baseline and day 28 for each strain , and the corresponding gmt ratio ( postvaccination / prevaccination ) . assuming a response rate of around 70% ( measured as the proportion of patients with antibody titers 40 ) , sample size was calculated as 37 evaluable subjects per group based on the required number of subjects needed to obtain a precision of 12.5% ( corresponding to confidence interval [ ci ] widths of 25% ) around the point estimate of the true response rate under a 90% ci for the true response rate . efficacy analyses were conducted in the per - protocol population , which comprised all patients consenting to enter the study who received influenza vaccination and had an antibody titer at day 28 . the safety population comprised all patients who consented and were enrolled into the study . for the primary efficacy analysis , the proportion of patients with influenza antibody titers 40 at 28 days differences between each teriflunomide treatment group and the ifn--1 reference group were estimated along with 90% cis for each vaccine strain . although there were 2 teriflunomide treatments and 3 viral strains , no adjustments for multiplicity were made . for secondary efficacy analyses , the evaluation of 2- or 4-fold increases in influenza antibody titers compared with prevaccination titers , proportions of patients , and corresponding 90% cis using normal approximation were calculated for each strain and each treatment group . gmts at baseline and day 28 and the respective ratios ( postvaccination / prevaccination ) were summarized by strain and treatment group . for analysis of safety , incidences of treatment - emergent adverse events ( teaes ) , treatment - emergent serious adverse events ( saes ) , and adverse events leading to permanent treatment discontinuation occurring from time of vaccination to day 28 were documented within each group . the teriva study ran from september 2011 to january 2012 and involved 14 different study sites in 5 countries ( austria , canada , germany , russian federation , and ukraine ) . a total of 128 patients were enrolled into the study : 41 , 41 , and 46 patients in the teriflunomide 7 mg , teriflunomide 14 mg , and ifn--1 groups , respectively . the majority of patients in the ifn--1 group were receiving avonex ( ifn--1a ; 30 g once - weekly i m ; 34.8% ) , rebif ( ifn--1a ; 44 g 3 times per week sc ; 28.3% ) , or betaseron ( ifn--1b ; 0.25 mg every other day sc ; 21.7% ) ; the remaining patients received either genfaxone ( ifn--1a ; 44 g 3 times per week i m [ russia only ] ; 8.7% ) or rebif ( ifn--1a ; 22 g , 3 times per week sc ; 6.5% ) . of the 128 patients enrolled , 122 were included in the per - protocol population ( 40 , 39 , and 43 in the 7-mg , 14-mg , and ifn--1 groups , respectively ) . five patients were excluded from the per - protocol population because of recent or concomitant exposure to a systemic or topical corticosteroid , and one patient in the ifn--1 group was excluded because of poor compliance with study treatment . patients had received teriflunomide treatment for a median of 5.7 years ( range 1.610.4 years ) and the reference population received ifn--1 for a median duration of 5.7 years ( range 1.017.9 years ; tables e-1 and e-2 ) . more than half of the per - protocol population ( 57% ) had received influenza vaccination previously , more so for patients in the ifn--1 group ( 69.6% ) than either of the teriflunomide groups ( 43.9% and 56.1% for 7 mg and 14 mg , respectively ) . baseline demographics and characteristics ( per - protocol population ) more than 90% of patients achieved postvaccination antibody titers 40 for h1n1 and b in all groups . the proportion of patients achieving seroprotection to h3n2 was lower in the teriflunomide 14-mg group ( 76.9% ) compared with the teriflunomide 7-mg ( 90.0% ) and ifn--1 ( 90.7% ) groups ( table 2 ) . rates of seroprotection in the teriflunomide groups were consistent regardless of the total duration of treatment exposure prior to vaccination or the nature of the extension study from which patients were enrolled ( tables e-3 and e-4 ) . thus , more than 70% of patients achieved an hi titer of 40 for all influenza strains across all treatment groups ( table 2 and figure 1 ) . primary and secondary efficacy endpoints european criteria for efficacy of influenza vaccination in an 18- to 60-year - old population require achievement of a hemagglutination inhibition titer 40 by 70% of patients , as indicated by the dashed line . a high proportion of patients across all treatment groups had high hi titers ( 40 ) for each vaccine strain at baseline ( table 1 ) . this reflects the fact that between 44% and 70% of the study population had received influenza vaccination previously and also because the influenza vaccines used in this study included the same strains as those used in the 2010/2011 vaccine . of the limited number of patients with a prevaccination titer 10 , approximately 50% or more achieved seroconversion ( day 28 titer 40 ) , with similar proportions across each of the treatment groups . the lowest seroconversion rates were observed for the h3n2 strain in all 3 groups ( table 2 ) . the proportion of patients with a prevaccination titer < 40 achieving seroprotection was 61% across the 3 treatment groups and 3 influenza strains . however , fewer patients in the teriflunomide 14-mg group than the teriflunomide 7-mg or ifn--1 groups exhibited seroprotection to the h3n2 strain ( table 2 ) . the proportion of patients with a 2- or 4-fold increase in influenza strain - specific antibody titers from baseline to day 28 was slightly higher in the ifn--1 group than either of the teriflunomide groups ( table 2 ) . across all influenza strains , the gmt ratio postvaccination / prevaccination ranged from 2.3 to 3.1 in the teriflunomide groups and from 3.4 to 4.7 in the ifn--1 group . the ratio exceeded 2.5 for all treatment groups and for all influenza strains , with the exception of h1n1 in the 14-mg teriflunomide group ( table 2 ) . the study demonstrated no new safety concerns with teriflunomide administration , and influenza vaccination was generally well tolerated by the entire study population ( table e-5 ) . the first was a case of thoracic herpes zoster in a female patient in the teriflunomide 14-mg group , which occurred 9 days postvaccination , persisted for 14 days , was mild in severity , and responded to antiviral treatment . the second was a case of infective cholecystitis , also in the teriflunomide 14-mg group , which was associated with an increase in white blood count and facial rash , which had recovered by the end of the study . these 2 cases were not considered as saes . indeed , no saes were reported in this study , and the overall incidence of teaes was higher in the ifn--1 group ( 45.7% ) than the 2 teriflunomide groups ( 26.8% and 36.6% for 7 mg and 14 mg , respectively ) . the teriva study ran from september 2011 to january 2012 and involved 14 different study sites in 5 countries ( austria , canada , germany , russian federation , and ukraine ) . a total of 128 patients were enrolled into the study : 41 , 41 , and 46 patients in the teriflunomide 7 mg , teriflunomide 14 mg , and ifn--1 groups , respectively . the majority of patients in the ifn--1 group were receiving avonex ( ifn--1a ; 30 g once - weekly i m ; 34.8% ) , rebif ( ifn--1a ; 44 g 3 times per week sc ; 28.3% ) , or betaseron ( ifn--1b ; 0.25 mg every other day sc ; 21.7% ) ; the remaining patients received either genfaxone ( ifn--1a ; 44 g 3 times per week i m [ russia only ] ; 8.7% ) or rebif ( ifn--1a ; 22 g , 3 times per week sc ; 6.5% ) . of the 128 patients enrolled , 122 were included in the per - protocol population ( 40 , 39 , and 43 in the 7-mg , 14-mg , and ifn--1 groups , respectively ) . five patients were excluded from the per - protocol population because of recent or concomitant exposure to a systemic or topical corticosteroid , and one patient in the ifn--1 group was excluded because of poor compliance with study treatment . patients had received teriflunomide treatment for a median of 5.7 years ( range 1.610.4 years ) and the reference population received ifn--1 for a median duration of 5.7 years ( range 1.017.9 years ; tables e-1 and e-2 ) . more than half of the per - protocol population ( 57% ) had received influenza vaccination previously , more so for patients in the ifn--1 group ( 69.6% ) than either of the teriflunomide groups ( 43.9% and 56.1% for 7 mg and 14 mg , respectively ) . more than 90% of patients achieved postvaccination antibody titers 40 for h1n1 and b in all groups . the proportion of patients achieving seroprotection to h3n2 was lower in the teriflunomide 14-mg group ( 76.9% ) compared with the teriflunomide 7-mg ( 90.0% ) and ifn--1 ( 90.7% ) groups ( table 2 ) . rates of seroprotection in the teriflunomide groups were consistent regardless of the total duration of treatment exposure prior to vaccination or the nature of the extension study from which patients were enrolled ( tables e-3 and e-4 ) . thus , more than 70% of patients achieved an hi titer of 40 for all influenza strains across all treatment groups ( table 2 and figure 1 ) . primary and secondary efficacy endpoints european criteria for efficacy of influenza vaccination in an 18- to 60-year - old population require achievement of a hemagglutination inhibition titer 40 by 70% of patients , as indicated by the dashed line . a high proportion of patients across all treatment groups had high hi titers ( 40 ) for each vaccine strain at baseline ( table 1 ) . this reflects the fact that between 44% and 70% of the study population had received influenza vaccination previously and also because the influenza vaccines used in this study included the same strains as those used in the 2010/2011 vaccine . of the limited number of patients with a prevaccination titer 10 , approximately 50% or more achieved seroconversion ( day 28 titer 40 ) , with similar proportions across each of the treatment groups . the lowest seroconversion rates were observed for the h3n2 strain in all 3 groups ( table 2 ) . the proportion of patients with a prevaccination titer < 40 achieving seroprotection was 61% across the 3 treatment groups and 3 influenza strains . however , fewer patients in the teriflunomide 14-mg group than the teriflunomide 7-mg or ifn--1 groups exhibited seroprotection to the h3n2 strain ( table 2 ) . the proportion of patients with a 2- or 4-fold increase in influenza strain - specific antibody titers from baseline to day 28 was slightly higher in the ifn--1 group than either of the teriflunomide groups ( table 2 ) . across all influenza strains , the gmt ratio postvaccination / prevaccination ranged from 2.3 to 3.1 in the teriflunomide groups and from 3.4 to 4.7 in the ifn--1 group . the ratio exceeded 2.5 for all treatment groups and for all influenza strains , with the exception of h1n1 in the 14-mg teriflunomide group ( table 2 ) . the study demonstrated no new safety concerns with teriflunomide administration , and influenza vaccination was generally well tolerated by the entire study population ( table e-5 ) . the first was a case of thoracic herpes zoster in a female patient in the teriflunomide 14-mg group , which occurred 9 days postvaccination , persisted for 14 days , was mild in severity , and responded to antiviral treatment . the second was a case of infective cholecystitis , also in the teriflunomide 14-mg group , which was associated with an increase in white blood count and facial rash , which had recovered by the end of the study . these 2 cases were not considered as saes . indeed , no saes were reported in this study , and the overall incidence of teaes was higher in the ifn--1 group ( 45.7% ) than the 2 teriflunomide groups ( 26.8% and 36.6% for 7 mg and 14 mg , respectively ) . the teriva study demonstrates that patients with rms treated with teriflunomide are generally able to mount effective immune responses to seasonal influenza vaccination . specifically , the proportion of patients with postvaccination antibody titers 40 to all influenza strains was greater than 70% in both teriflunomide treatment groups ( range 76.997.5% ) , thereby meeting the european criterion for the efficacy of influenza vaccine in adult subjects aged 1860 years , which was a priori identified as the primary criterion . neither the duration of teriflunomide treatment prior to vaccination nor the origin of the teriflunomide extension study in which patients were enrolled had a significant impact on overall seroprotection rates . thus , vaccine responses were preserved in patients treated with teriflunomide across a broad range of treatment exposure , ranging from 1.6 to 10.4 years . although the response to all vaccine strains in all treatment groups was sufficient for vaccination to be considered protective , it should be noted that there was a slightly diminished response in the teriflunomide 14-mg group with respect to some evaluations for the h3n2 and h1n1 strains . nevertheless , the development of adequate protective immune responses confirms the ability of teriflunomide to inhibit the activity of pathogenic autoreactive lymphocytes without causing global immunosuppression . no death , sae , or teae leading to treatment discontinuation was reported in the study , and no new or unexpected safety observations arose in either of the teriflunomide treatment groups . these safety data are in accordance with those from the temso trial , in which teriflunomide was generally well tolerated with a well - characterized safety profile . furthermore , the low incidence of serious infections and the absence of serious opportunistic infections seen in temso lends further support to the observations made in this study that immune surveillance is preserved under teriflunomide treatment . immune responses to influenza vaccine in patients with ms treated with ifn--1a were similar to untreated patients with respect to the proportion of patients achieving hi titers 40 . our study was consistent with the literature showing that effective immune responses were observed in a reference rms population treated with ifn--1 for at least 6 months , with good safety and tolerability . it should be noted that this study was not designed or powered to make direct comparisons between immune responses in the teriflunomide groups and the ifn--1 reference population . effective influenza vaccination remains important in patients with ms because influenza can cause serious complications and has been shown to be associated with a higher occurrence of exacerbations in patients with ms . the observations made in this study are therefore highly reassuring for patients administered teriflunomide treatment . we conclude that influenza vaccine was found to be safe and effective in patients with ms receiving teriflunomide . our findings , combined with efficacy and safety data from the temso study , support the view that while teriflunomide appears to effectively limit abnormal activation of pathogenic lymphocyte responses implicated in ms relapses , teriflunomide therapy does not significantly interfere with adaptive activation of immune responses and generally appears to spare the serologic response to influenza antigens . amit bar - or , franoise menguy - vacheron , deborah bauer , stefan jodl , philippe truffinet , and myriam benamor contributed to the development of the study design , data analysis , and interpretation . they were all involved in the development , reviewing , and revising of the manuscript . mark s. freedman , marcelo kremenchutzky , and paul w. oconnor all contributed to the development of the study design . deborah bauer was responsible for all statistical analyses of the data , as well as development and revising of the manuscript . scott chambers is a medical writer who provided editorial support in the preparation and editing of the manuscript text , figures , and tables , addressing author , reviewer , and editorial comments , and assisting with the submission process . a. bar - or : speaker , consulting fees , and/or research support : amplimmune , aventis , bayhill therapeutics , berlex / bayer , biogen idec , bioms , diogenix , eli - lilly , emd serono , genentech , genzyme - sanofi , gsk , guthy jackson greater good foundation , medimmune , mitsubishi pharma , novartis , ono pharmacia , receptos , roche , teva neuroscience , wyeth . freedman : research / educational grant support : genzyme ; consulting fees : actelion , bayer , biogen idec , celgene , genzyme , glycominds , teva , merck serono , novartis , opexa , and sanofi - aventis . m. kremenchutzky : consulting fees and/or research support from bayer , biogen idec , emd serono , genzyme , novartis , sanofi - aventis , and teva scott chambers : employee of fishawack communications ltd , contracted to provide editorial services to sanofi , funded by genzyme , a sanofi company . p.w . oconnor : consulting fees and/or research support : actelion , bayer , biogen idec , bioms , cognosci , daiichi sankyo , emd serono , genentech , genmab , novartis , roche , sanofi - aventis , teva , and warburg pincus .

objective : to investigate the effect of teriflunomide on the efficacy and safety of seasonal influenza vaccine.methods:the 2011/2012 seasonal influenza vaccine ( containing h1n1 , h3n2 , and b strains ) was administered to patients with relapsing forms of multiple sclerosis ( rms ) treated for 6 months with teriflunomide 7 mg ( n = 41 ) or 14 mg ( n = 41 ) , or interferon--1 ( ifn--1 ; n = 46 ) . the primary endpoint was the proportion of patients with influenza strain specific antibody titers 40 , 28 days postvaccination.results:more than 90% of patients achieved postvaccination antibody titers 40 for h1n1 and b in all groups . for h3n2 , titers 40 were achieved in 90% of patients in the 7 mg and ifn--1 groups , and in 77% of the 14-mg group , respectively . a high proportion of patients already had detectable antibodies for each influenza strain at baseline . geometric mean titer ratios ( post / prevaccination ) were 2.5 for all groups and strains , except for h1n1 in the 14-mg group ( 2.3 ) . the proportion of patients with a prevaccination titer < 40 achieving seroprotection was 61% across the 3 treatment groups and 3 influenza strains . however , fewer patients in the 14-mg than the 7-mg or ifn--1 groups exhibited seroprotection to h3n2 ( 61% vs 78% and 82% , respectively).conclusion : teriflunomide - treated patients generally mounted effective immune responses to seasonal influenza vaccination , consistent with preservation of protective immune responses.classification of evidence : this study provides class ii evidence that teriflunomide generally does not adversely impact the ability of patients with rms to mount immune responses to influenza vaccination .

Classification of evidence: METHODS Study design. Standard protocol approvals, registrations, and patient consents. Study population. Study endpoints. Statistical analysis. RESULTS Study disposition and baseline demographics. Efficacy. Safety and tolerability. DISCUSSION Supplementary Material AUTHOR CONTRIBUTIONS STUDY FUNDING DISCLOSURE

this study provides class ii evidence that teriflunomide generally does not adversely impact the ability of patients with rms to mount immune responses to influenza vaccination . the teriflunomide and vaccination ( teriva ) study ( nct01403376 ) was a multicenter , multinational , parallel - group study involving 128 patients in 3 treatment groups . groups 1 and 2 included patients with rms treated with either teriflunomide 7 mg or 14 mg once daily for at least 6 months over the course of 2 long - term extension studies ( nct00228163 : open - label extension of a phase ii study , which started in 2001 ; nct00803049 : blinded extension of the phase iii temso study , which started in 2004 ) . group 3 included patients with rms who had received a stable dose of ifn--1 for at least 6 months , and represents a reference population of patients with rms who have previously been reported to mount normal immune responses to seasonal influenza vaccination . the study comprised a screening period of up to 21 days , followed by administration of seasonal influenza vaccine on day 1 and antibody assessments at day 28 ( 2 days ) postimmunization ( figure e-1 on the neurology web site at www.neurology.org ) . choice of vaccine was performed according to country standards ; mutagrip was administered only in germany to 20 patients ( 12 in the ifn--1 group , and 3 and 5 in the 7-mg and 14-mg teriflunomide groups , respectively ) . male or female subjects aged between 18 and 60 years with rms treated for at least 6 months with once - daily teriflunomide ( 7 mg or 14 mg ) or ifn--1 , and who were expected to remain on treatment for the duration of the study and who signed a specific informed consent form , were eligible for inclusion in the teriva study . additional exclusion criteria included contraindication to influenza vaccine or in receipt of any vaccination within the last 6 months ; prior use of investigational drugs or participation in a clinical trial within 1 year before screening ( for patients in the ifn--1 group only ) ; prior / concomitant use ( within 1 year of study entry ) of cladribine , mitoxantrone , or other immunosuppressant agents ; prior / concomitant use of glatiramer acetate ( within 1 year of study entry ) ; or iv immunoglobulins ( within 3 months of study entry ) . , the proportion of subjects achieving a postvaccination titer 40 with the hia ) should be achieved in 70% of vaccinees postvaccination for individuals aged 1860 years and in 60% for those over 60 years . , the proportion of previously seronegative subjects exceeding a postvaccination titer of 40 , and the proportion of previously seropositive subjects with a 4-fold increase in prevaccination and postvaccination sera ) should be achieved in 40% of vaccinees aged 1860 years and in 30% in those over 60 years . in this study , the primary efficacy endpoint was the proportion of patients who achieved seroprotection to the influenza vaccine strains , defined as an influenza antibody titer 40 for each strain 28 days postvaccination . the following secondary endpoints were also assessed : the proportion of patients with seroconversion ( i.e. , a prevaccination antibody titer 10 and a postvaccination antibody titer 40 ) ; the proportion of patients with a prevaccination antibody titer < 40 achieving seroprotection ; the proportion of patients with either a 2- or 4-fold increase in strain - specific antibody titers compared with prevaccination titers at 28 days postvaccination ; gmts at baseline and day 28 for each strain , and the corresponding gmt ratio ( postvaccination / prevaccination ) . assuming a response rate of around 70% ( measured as the proportion of patients with antibody titers 40 ) , sample size was calculated as 37 evaluable subjects per group based on the required number of subjects needed to obtain a precision of 12.5% ( corresponding to confidence interval [ ci ] widths of 25% ) around the point estimate of the true response rate under a 90% ci for the true response rate . for the primary efficacy analysis , the proportion of patients with influenza antibody titers 40 at 28 days differences between each teriflunomide treatment group and the ifn--1 reference group were estimated along with 90% cis for each vaccine strain . for secondary efficacy analyses , the evaluation of 2- or 4-fold increases in influenza antibody titers compared with prevaccination titers , proportions of patients , and corresponding 90% cis using normal approximation were calculated for each strain and each treatment group . gmts at baseline and day 28 and the respective ratios ( postvaccination / prevaccination ) were summarized by strain and treatment group . the teriflunomide and vaccination ( teriva ) study ( nct01403376 ) was a multicenter , multinational , parallel - group study involving 128 patients in 3 treatment groups . groups 1 and 2 included patients with rms treated with either teriflunomide 7 mg or 14 mg once daily for at least 6 months over the course of 2 long - term extension studies ( nct00228163 : open - label extension of a phase ii study , which started in 2001 ; nct00803049 : blinded extension of the phase iii temso study , which started in 2004 ) . group 3 included patients with rms who had received a stable dose of ifn--1 for at least 6 months , and represents a reference population of patients with rms who have previously been reported to mount normal immune responses to seasonal influenza vaccination . the study comprised a screening period of up to 21 days , followed by administration of seasonal influenza vaccine on day 1 and antibody assessments at day 28 ( 2 days ) postimmunization ( figure e-1 on the neurology web site at www.neurology.org ) . choice of vaccine was performed according to country standards ; mutagrip was administered only in germany to 20 patients ( 12 in the ifn--1 group , and 3 and 5 in the 7-mg and 14-mg teriflunomide groups , respectively ) . male or female subjects aged between 18 and 60 years with rms treated for at least 6 months with once - daily teriflunomide ( 7 mg or 14 mg ) or ifn--1 , and who were expected to remain on treatment for the duration of the study and who signed a specific informed consent form , were eligible for inclusion in the teriva study . additional exclusion criteria included contraindication to influenza vaccine or in receipt of any vaccination within the last 6 months ; prior use of investigational drugs or participation in a clinical trial within 1 year before screening ( for patients in the ifn--1 group only ) ; prior / concomitant use ( within 1 year of study entry ) of cladribine , mitoxantrone , or other immunosuppressant agents ; prior / concomitant use of glatiramer acetate ( within 1 year of study entry ) ; or iv immunoglobulins ( within 3 months of study entry ) . , the proportion of subjects achieving a postvaccination titer 40 with the hia ) should be achieved in 70% of vaccinees postvaccination for individuals aged 1860 years and in 60% for those over 60 years . , the proportion of previously seronegative subjects exceeding a postvaccination titer of 40 , and the proportion of previously seropositive subjects with a 4-fold increase in prevaccination and postvaccination sera ) should be achieved in 40% of vaccinees aged 1860 years and in 30% in those over 60 years . for each virus strain and each age class , the primary efficacy endpoint was the proportion of patients who achieved seroprotection to the influenza vaccine strains , defined as an influenza antibody titer 40 for each strain 28 days postvaccination . the following secondary endpoints were also assessed : the proportion of patients with seroconversion ( i.e. , a prevaccination antibody titer 10 and a postvaccination antibody titer 40 ) ; the proportion of patients with a prevaccination antibody titer < 40 achieving seroprotection ; the proportion of patients with either a 2- or 4-fold increase in strain - specific antibody titers compared with prevaccination titers at 28 days postvaccination ; gmts at baseline and day 28 for each strain , and the corresponding gmt ratio ( postvaccination / prevaccination ) . assuming a response rate of around 70% ( measured as the proportion of patients with antibody titers 40 ) , sample size was calculated as 37 evaluable subjects per group based on the required number of subjects needed to obtain a precision of 12.5% ( corresponding to confidence interval [ ci ] widths of 25% ) around the point estimate of the true response rate under a 90% ci for the true response rate . for the primary efficacy analysis , the proportion of patients with influenza antibody titers 40 at 28 days differences between each teriflunomide treatment group and the ifn--1 reference group were estimated along with 90% cis for each vaccine strain . gmts at baseline and day 28 and the respective ratios ( postvaccination / prevaccination ) were summarized by strain and treatment group . a total of 128 patients were enrolled into the study : 41 , 41 , and 46 patients in the teriflunomide 7 mg , teriflunomide 14 mg , and ifn--1 groups , respectively . the majority of patients in the ifn--1 group were receiving avonex ( ifn--1a ; 30 g once - weekly i m ; 34.8% ) , rebif ( ifn--1a ; 44 g 3 times per week sc ; 28.3% ) , or betaseron ( ifn--1b ; 0.25 mg every other day sc ; 21.7% ) ; the remaining patients received either genfaxone ( ifn--1a ; 44 g 3 times per week i m [ russia only ] ; 8.7% ) or rebif ( ifn--1a ; 22 g , 3 times per week sc ; 6.5% ) . of the 128 patients enrolled , 122 were included in the per - protocol population ( 40 , 39 , and 43 in the 7-mg , 14-mg , and ifn--1 groups , respectively ) . more than half of the per - protocol population ( 57% ) had received influenza vaccination previously , more so for patients in the ifn--1 group ( 69.6% ) than either of the teriflunomide groups ( 43.9% and 56.1% for 7 mg and 14 mg , respectively ) . baseline demographics and characteristics ( per - protocol population ) more than 90% of patients achieved postvaccination antibody titers 40 for h1n1 and b in all groups . the proportion of patients achieving seroprotection to h3n2 was lower in the teriflunomide 14-mg group ( 76.9% ) compared with the teriflunomide 7-mg ( 90.0% ) and ifn--1 ( 90.7% ) groups ( table 2 ) . thus , more than 70% of patients achieved an hi titer of 40 for all influenza strains across all treatment groups ( table 2 and figure 1 ) . a high proportion of patients across all treatment groups had high hi titers ( 40 ) for each vaccine strain at baseline ( table 1 ) . of the limited number of patients with a prevaccination titer 10 , approximately 50% or more achieved seroconversion ( day 28 titer 40 ) , with similar proportions across each of the treatment groups . the proportion of patients with a prevaccination titer < 40 achieving seroprotection was 61% across the 3 treatment groups and 3 influenza strains . however , fewer patients in the teriflunomide 14-mg group than the teriflunomide 7-mg or ifn--1 groups exhibited seroprotection to the h3n2 strain ( table 2 ) . the proportion of patients with a 2- or 4-fold increase in influenza strain - specific antibody titers from baseline to day 28 was slightly higher in the ifn--1 group than either of the teriflunomide groups ( table 2 ) . across all influenza strains , the gmt ratio postvaccination / prevaccination ranged from 2.3 to 3.1 in the teriflunomide groups and from 3.4 to 4.7 in the ifn--1 group . the ratio exceeded 2.5 for all treatment groups and for all influenza strains , with the exception of h1n1 in the 14-mg teriflunomide group ( table 2 ) . the first was a case of thoracic herpes zoster in a female patient in the teriflunomide 14-mg group , which occurred 9 days postvaccination , persisted for 14 days , was mild in severity , and responded to antiviral treatment . the second was a case of infective cholecystitis , also in the teriflunomide 14-mg group , which was associated with an increase in white blood count and facial rash , which had recovered by the end of the study . indeed , no saes were reported in this study , and the overall incidence of teaes was higher in the ifn--1 group ( 45.7% ) than the 2 teriflunomide groups ( 26.8% and 36.6% for 7 mg and 14 mg , respectively ) . a total of 128 patients were enrolled into the study : 41 , 41 , and 46 patients in the teriflunomide 7 mg , teriflunomide 14 mg , and ifn--1 groups , respectively . the majority of patients in the ifn--1 group were receiving avonex ( ifn--1a ; 30 g once - weekly i m ; 34.8% ) , rebif ( ifn--1a ; 44 g 3 times per week sc ; 28.3% ) , or betaseron ( ifn--1b ; 0.25 mg every other day sc ; 21.7% ) ; the remaining patients received either genfaxone ( ifn--1a ; 44 g 3 times per week i m [ russia only ] ; 8.7% ) or rebif ( ifn--1a ; 22 g , 3 times per week sc ; 6.5% ) . of the 128 patients enrolled , 122 were included in the per - protocol population ( 40 , 39 , and 43 in the 7-mg , 14-mg , and ifn--1 groups , respectively ) . more than half of the per - protocol population ( 57% ) had received influenza vaccination previously , more so for patients in the ifn--1 group ( 69.6% ) than either of the teriflunomide groups ( 43.9% and 56.1% for 7 mg and 14 mg , respectively ) . more than 90% of patients achieved postvaccination antibody titers 40 for h1n1 and b in all groups . the proportion of patients achieving seroprotection to h3n2 was lower in the teriflunomide 14-mg group ( 76.9% ) compared with the teriflunomide 7-mg ( 90.0% ) and ifn--1 ( 90.7% ) groups ( table 2 ) . thus , more than 70% of patients achieved an hi titer of 40 for all influenza strains across all treatment groups ( table 2 and figure 1 ) . a high proportion of patients across all treatment groups had high hi titers ( 40 ) for each vaccine strain at baseline ( table 1 ) . of the limited number of patients with a prevaccination titer 10 , approximately 50% or more achieved seroconversion ( day 28 titer 40 ) , with similar proportions across each of the treatment groups . the proportion of patients with a prevaccination titer < 40 achieving seroprotection was 61% across the 3 treatment groups and 3 influenza strains . however , fewer patients in the teriflunomide 14-mg group than the teriflunomide 7-mg or ifn--1 groups exhibited seroprotection to the h3n2 strain ( table 2 ) . the proportion of patients with a 2- or 4-fold increase in influenza strain - specific antibody titers from baseline to day 28 was slightly higher in the ifn--1 group than either of the teriflunomide groups ( table 2 ) . across all influenza strains , the gmt ratio postvaccination / prevaccination ranged from 2.3 to 3.1 in the teriflunomide groups and from 3.4 to 4.7 in the ifn--1 group . the ratio exceeded 2.5 for all treatment groups and for all influenza strains , with the exception of h1n1 in the 14-mg teriflunomide group ( table 2 ) . the first was a case of thoracic herpes zoster in a female patient in the teriflunomide 14-mg group , which occurred 9 days postvaccination , persisted for 14 days , was mild in severity , and responded to antiviral treatment . indeed , no saes were reported in this study , and the overall incidence of teaes was higher in the ifn--1 group ( 45.7% ) than the 2 teriflunomide groups ( 26.8% and 36.6% for 7 mg and 14 mg , respectively ) . the teriva study demonstrates that patients with rms treated with teriflunomide are generally able to mount effective immune responses to seasonal influenza vaccination . specifically , the proportion of patients with postvaccination antibody titers 40 to all influenza strains was greater than 70% in both teriflunomide treatment groups ( range 76.997.5% ) , thereby meeting the european criterion for the efficacy of influenza vaccine in adult subjects aged 1860 years , which was a priori identified as the primary criterion . although the response to all vaccine strains in all treatment groups was sufficient for vaccination to be considered protective , it should be noted that there was a slightly diminished response in the teriflunomide 14-mg group with respect to some evaluations for the h3n2 and h1n1 strains . nevertheless , the development of adequate protective immune responses confirms the ability of teriflunomide to inhibit the activity of pathogenic autoreactive lymphocytes without causing global immunosuppression . no death , sae , or teae leading to treatment discontinuation was reported in the study , and no new or unexpected safety observations arose in either of the teriflunomide treatment groups . immune responses to influenza vaccine in patients with ms treated with ifn--1a were similar to untreated patients with respect to the proportion of patients achieving hi titers 40 . it should be noted that this study was not designed or powered to make direct comparisons between immune responses in the teriflunomide groups and the ifn--1 reference population . our findings , combined with efficacy and safety data from the temso study , support the view that while teriflunomide appears to effectively limit abnormal activation of pathogenic lymphocyte responses implicated in ms relapses , teriflunomide therapy does not significantly interfere with adaptive activation of immune responses and generally appears to spare the serologic response to influenza antigens .

groups 1 and 2 included patients with rms treated with either teriflunomide 7 mg or 14 mg once daily for at least 6 months over the course of 2 long - term extension studies ( nct00228163 : open - label extension of a phase ii study , which started in 2001 ; nct00803049 : blinded extension of the phase iii temso study , which started in 2004 ) . group 3 included patients with rms who had received a stable dose of ifn--1 for at least 6 months , and represents a reference population of patients with rms who have previously been reported to mount normal immune responses to seasonal influenza vaccination . the study comprised a screening period of up to 21 days , followed by administration of seasonal influenza vaccine on day 1 and antibody assessments at day 28 ( 2 days ) postimmunization ( figure e-1 on the neurology web site at www.neurology.org ) . choice of vaccine was performed according to country standards ; mutagrip was administered only in germany to 20 patients ( 12 in the ifn--1 group , and 3 and 5 in the 7-mg and 14-mg teriflunomide groups , respectively ) . male or female subjects aged between 18 and 60 years with rms treated for at least 6 months with once - daily teriflunomide ( 7 mg or 14 mg ) or ifn--1 , and who were expected to remain on treatment for the duration of the study and who signed a specific informed consent form , were eligible for inclusion in the teriva study . additional exclusion criteria included contraindication to influenza vaccine or in receipt of any vaccination within the last 6 months ; prior use of investigational drugs or participation in a clinical trial within 1 year before screening ( for patients in the ifn--1 group only ) ; prior / concomitant use ( within 1 year of study entry ) of cladribine , mitoxantrone , or other immunosuppressant agents ; prior / concomitant use of glatiramer acetate ( within 1 year of study entry ) ; or iv immunoglobulins ( within 3 months of study entry ) . , the proportion of subjects achieving a postvaccination titer 40 with the hia ) should be achieved in 70% of vaccinees postvaccination for individuals aged 1860 years and in 60% for those over 60 years . , the proportion of previously seronegative subjects exceeding a postvaccination titer of 40 , and the proportion of previously seropositive subjects with a 4-fold increase in prevaccination and postvaccination sera ) should be achieved in 40% of vaccinees aged 1860 years and in 30% in those over 60 years . in this study , the primary efficacy endpoint was the proportion of patients who achieved seroprotection to the influenza vaccine strains , defined as an influenza antibody titer 40 for each strain 28 days postvaccination . , a prevaccination antibody titer 10 and a postvaccination antibody titer 40 ) ; the proportion of patients with a prevaccination antibody titer < 40 achieving seroprotection ; the proportion of patients with either a 2- or 4-fold increase in strain - specific antibody titers compared with prevaccination titers at 28 days postvaccination ; gmts at baseline and day 28 for each strain , and the corresponding gmt ratio ( postvaccination / prevaccination ) . assuming a response rate of around 70% ( measured as the proportion of patients with antibody titers 40 ) , sample size was calculated as 37 evaluable subjects per group based on the required number of subjects needed to obtain a precision of 12.5% ( corresponding to confidence interval [ ci ] widths of 25% ) around the point estimate of the true response rate under a 90% ci for the true response rate . for the primary efficacy analysis , the proportion of patients with influenza antibody titers 40 at 28 days differences between each teriflunomide treatment group and the ifn--1 reference group were estimated along with 90% cis for each vaccine strain . for secondary efficacy analyses , the evaluation of 2- or 4-fold increases in influenza antibody titers compared with prevaccination titers , proportions of patients , and corresponding 90% cis using normal approximation were calculated for each strain and each treatment group . for analysis of safety , incidences of treatment - emergent adverse events ( teaes ) , treatment - emergent serious adverse events ( saes ) , and adverse events leading to permanent treatment discontinuation occurring from time of vaccination to day 28 were documented within each group . groups 1 and 2 included patients with rms treated with either teriflunomide 7 mg or 14 mg once daily for at least 6 months over the course of 2 long - term extension studies ( nct00228163 : open - label extension of a phase ii study , which started in 2001 ; nct00803049 : blinded extension of the phase iii temso study , which started in 2004 ) . the study comprised a screening period of up to 21 days , followed by administration of seasonal influenza vaccine on day 1 and antibody assessments at day 28 ( 2 days ) postimmunization ( figure e-1 on the neurology web site at www.neurology.org ) . choice of vaccine was performed according to country standards ; mutagrip was administered only in germany to 20 patients ( 12 in the ifn--1 group , and 3 and 5 in the 7-mg and 14-mg teriflunomide groups , respectively ) . male or female subjects aged between 18 and 60 years with rms treated for at least 6 months with once - daily teriflunomide ( 7 mg or 14 mg ) or ifn--1 , and who were expected to remain on treatment for the duration of the study and who signed a specific informed consent form , were eligible for inclusion in the teriva study . additional exclusion criteria included contraindication to influenza vaccine or in receipt of any vaccination within the last 6 months ; prior use of investigational drugs or participation in a clinical trial within 1 year before screening ( for patients in the ifn--1 group only ) ; prior / concomitant use ( within 1 year of study entry ) of cladribine , mitoxantrone , or other immunosuppressant agents ; prior / concomitant use of glatiramer acetate ( within 1 year of study entry ) ; or iv immunoglobulins ( within 3 months of study entry ) . , the proportion of subjects achieving a postvaccination titer 40 with the hia ) should be achieved in 70% of vaccinees postvaccination for individuals aged 1860 years and in 60% for those over 60 years . , the proportion of previously seronegative subjects exceeding a postvaccination titer of 40 , and the proportion of previously seropositive subjects with a 4-fold increase in prevaccination and postvaccination sera ) should be achieved in 40% of vaccinees aged 1860 years and in 30% in those over 60 years . for each virus strain and each age class , the primary efficacy endpoint was the proportion of patients who achieved seroprotection to the influenza vaccine strains , defined as an influenza antibody titer 40 for each strain 28 days postvaccination . , a prevaccination antibody titer 10 and a postvaccination antibody titer 40 ) ; the proportion of patients with a prevaccination antibody titer < 40 achieving seroprotection ; the proportion of patients with either a 2- or 4-fold increase in strain - specific antibody titers compared with prevaccination titers at 28 days postvaccination ; gmts at baseline and day 28 for each strain , and the corresponding gmt ratio ( postvaccination / prevaccination ) . assuming a response rate of around 70% ( measured as the proportion of patients with antibody titers 40 ) , sample size was calculated as 37 evaluable subjects per group based on the required number of subjects needed to obtain a precision of 12.5% ( corresponding to confidence interval [ ci ] widths of 25% ) around the point estimate of the true response rate under a 90% ci for the true response rate . for the primary efficacy analysis , the proportion of patients with influenza antibody titers 40 at 28 days differences between each teriflunomide treatment group and the ifn--1 reference group were estimated along with 90% cis for each vaccine strain . for secondary efficacy analyses , the evaluation of 2- or 4-fold increases in influenza antibody titers compared with prevaccination titers , proportions of patients , and corresponding 90% cis using normal approximation were calculated for each strain and each treatment group . for analysis of safety , incidences of treatment - emergent adverse events ( teaes ) , treatment - emergent serious adverse events ( saes ) , and adverse events leading to permanent treatment discontinuation occurring from time of vaccination to day 28 were documented within each group . a total of 128 patients were enrolled into the study : 41 , 41 , and 46 patients in the teriflunomide 7 mg , teriflunomide 14 mg , and ifn--1 groups , respectively . the majority of patients in the ifn--1 group were receiving avonex ( ifn--1a ; 30 g once - weekly i m ; 34.8% ) , rebif ( ifn--1a ; 44 g 3 times per week sc ; 28.3% ) , or betaseron ( ifn--1b ; 0.25 mg every other day sc ; 21.7% ) ; the remaining patients received either genfaxone ( ifn--1a ; 44 g 3 times per week i m [ russia only ] ; 8.7% ) or rebif ( ifn--1a ; 22 g , 3 times per week sc ; 6.5% ) . patients had received teriflunomide treatment for a median of 5.7 years ( range 1.610.4 years ) and the reference population received ifn--1 for a median duration of 5.7 years ( range 1.017.9 years ; tables e-1 and e-2 ) . more than half of the per - protocol population ( 57% ) had received influenza vaccination previously , more so for patients in the ifn--1 group ( 69.6% ) than either of the teriflunomide groups ( 43.9% and 56.1% for 7 mg and 14 mg , respectively ) . the proportion of patients achieving seroprotection to h3n2 was lower in the teriflunomide 14-mg group ( 76.9% ) compared with the teriflunomide 7-mg ( 90.0% ) and ifn--1 ( 90.7% ) groups ( table 2 ) . rates of seroprotection in the teriflunomide groups were consistent regardless of the total duration of treatment exposure prior to vaccination or the nature of the extension study from which patients were enrolled ( tables e-3 and e-4 ) . primary and secondary efficacy endpoints european criteria for efficacy of influenza vaccination in an 18- to 60-year - old population require achievement of a hemagglutination inhibition titer 40 by 70% of patients , as indicated by the dashed line . however , fewer patients in the teriflunomide 14-mg group than the teriflunomide 7-mg or ifn--1 groups exhibited seroprotection to the h3n2 strain ( table 2 ) . the proportion of patients with a 2- or 4-fold increase in influenza strain - specific antibody titers from baseline to day 28 was slightly higher in the ifn--1 group than either of the teriflunomide groups ( table 2 ) . across all influenza strains , the gmt ratio postvaccination / prevaccination ranged from 2.3 to 3.1 in the teriflunomide groups and from 3.4 to 4.7 in the ifn--1 group . the ratio exceeded 2.5 for all treatment groups and for all influenza strains , with the exception of h1n1 in the 14-mg teriflunomide group ( table 2 ) . the first was a case of thoracic herpes zoster in a female patient in the teriflunomide 14-mg group , which occurred 9 days postvaccination , persisted for 14 days , was mild in severity , and responded to antiviral treatment . the second was a case of infective cholecystitis , also in the teriflunomide 14-mg group , which was associated with an increase in white blood count and facial rash , which had recovered by the end of the study . indeed , no saes were reported in this study , and the overall incidence of teaes was higher in the ifn--1 group ( 45.7% ) than the 2 teriflunomide groups ( 26.8% and 36.6% for 7 mg and 14 mg , respectively ) . a total of 128 patients were enrolled into the study : 41 , 41 , and 46 patients in the teriflunomide 7 mg , teriflunomide 14 mg , and ifn--1 groups , respectively . the majority of patients in the ifn--1 group were receiving avonex ( ifn--1a ; 30 g once - weekly i m ; 34.8% ) , rebif ( ifn--1a ; 44 g 3 times per week sc ; 28.3% ) , or betaseron ( ifn--1b ; 0.25 mg every other day sc ; 21.7% ) ; the remaining patients received either genfaxone ( ifn--1a ; 44 g 3 times per week i m [ russia only ] ; 8.7% ) or rebif ( ifn--1a ; 22 g , 3 times per week sc ; 6.5% ) . patients had received teriflunomide treatment for a median of 5.7 years ( range 1.610.4 years ) and the reference population received ifn--1 for a median duration of 5.7 years ( range 1.017.9 years ; tables e-1 and e-2 ) . more than half of the per - protocol population ( 57% ) had received influenza vaccination previously , more so for patients in the ifn--1 group ( 69.6% ) than either of the teriflunomide groups ( 43.9% and 56.1% for 7 mg and 14 mg , respectively ) . the proportion of patients achieving seroprotection to h3n2 was lower in the teriflunomide 14-mg group ( 76.9% ) compared with the teriflunomide 7-mg ( 90.0% ) and ifn--1 ( 90.7% ) groups ( table 2 ) . rates of seroprotection in the teriflunomide groups were consistent regardless of the total duration of treatment exposure prior to vaccination or the nature of the extension study from which patients were enrolled ( tables e-3 and e-4 ) . primary and secondary efficacy endpoints european criteria for efficacy of influenza vaccination in an 18- to 60-year - old population require achievement of a hemagglutination inhibition titer 40 by 70% of patients , as indicated by the dashed line . however , fewer patients in the teriflunomide 14-mg group than the teriflunomide 7-mg or ifn--1 groups exhibited seroprotection to the h3n2 strain ( table 2 ) . the proportion of patients with a 2- or 4-fold increase in influenza strain - specific antibody titers from baseline to day 28 was slightly higher in the ifn--1 group than either of the teriflunomide groups ( table 2 ) . across all influenza strains , the gmt ratio postvaccination / prevaccination ranged from 2.3 to 3.1 in the teriflunomide groups and from 3.4 to 4.7 in the ifn--1 group . the first was a case of thoracic herpes zoster in a female patient in the teriflunomide 14-mg group , which occurred 9 days postvaccination , persisted for 14 days , was mild in severity , and responded to antiviral treatment . the second was a case of infective cholecystitis , also in the teriflunomide 14-mg group , which was associated with an increase in white blood count and facial rash , which had recovered by the end of the study . indeed , no saes were reported in this study , and the overall incidence of teaes was higher in the ifn--1 group ( 45.7% ) than the 2 teriflunomide groups ( 26.8% and 36.6% for 7 mg and 14 mg , respectively ) . specifically , the proportion of patients with postvaccination antibody titers 40 to all influenza strains was greater than 70% in both teriflunomide treatment groups ( range 76.997.5% ) , thereby meeting the european criterion for the efficacy of influenza vaccine in adult subjects aged 1860 years , which was a priori identified as the primary criterion . neither the duration of teriflunomide treatment prior to vaccination nor the origin of the teriflunomide extension study in which patients were enrolled had a significant impact on overall seroprotection rates . although the response to all vaccine strains in all treatment groups was sufficient for vaccination to be considered protective , it should be noted that there was a slightly diminished response in the teriflunomide 14-mg group with respect to some evaluations for the h3n2 and h1n1 strains . nevertheless , the development of adequate protective immune responses confirms the ability of teriflunomide to inhibit the activity of pathogenic autoreactive lymphocytes without causing global immunosuppression . furthermore , the low incidence of serious infections and the absence of serious opportunistic infections seen in temso lends further support to the observations made in this study that immune surveillance is preserved under teriflunomide treatment . our study was consistent with the literature showing that effective immune responses were observed in a reference rms population treated with ifn--1 for at least 6 months , with good safety and tolerability . our findings , combined with efficacy and safety data from the temso study , support the view that while teriflunomide appears to effectively limit abnormal activation of pathogenic lymphocyte responses implicated in ms relapses , teriflunomide therapy does not significantly interfere with adaptive activation of immune responses and generally appears to spare the serologic response to influenza antigens . a. bar - or : speaker , consulting fees , and/or research support : amplimmune , aventis , bayhill therapeutics , berlex / bayer , biogen idec , bioms , diogenix , eli - lilly , emd serono , genentech , genzyme - sanofi , gsk , guthy jackson greater good foundation , medimmune , mitsubishi pharma , novartis , ono pharmacia , receptos , roche , teva neuroscience , wyeth . m. kremenchutzky : consulting fees and/or research support from bayer , biogen idec , emd serono , genzyme , novartis , sanofi - aventis , and teva scott chambers : employee of fishawack communications ltd , contracted to provide editorial services to sanofi , funded by genzyme , a sanofi company .

adoptive therapy using t cells for cancer therapy is a promising strategy that has curative potential and broad applicability . cytokine - induced killer ( cik ) cells are generated by in vitro expansion of peripheral blood lymphocytes ( pbl ) using anti - cd3 antibodies , ifn- , and il-2 . cik cells are a heterogeneous population of effector t cells , some of which possessing non - mhc - restricted cytolytic activities against cancer cells . although clinical studies have confirmed the benefit and safety of cik cell - based therapy for patients with hematopoietic and solid tumors , there are many factors impacting the clinical response due to the characteristics of ex vivo expanded lymphocyte cells . it was shown that the use of minimally cultured young less - differentiated tumor infiltrating lymphocytes ( tils ) , with stronger proliferative ability and higher levels of the costimulatory molecules cd27 and cd28 , is an important factor for success . retronectin is a chimeric peptide of recombinant e. coli - expressed human fibronectin fragments , with two functional domains which can interact with integrin molecules : very late antigen-4 ( vla-4 , 41 ) and very late antigen-5 ( vla-5 , 51 ) of the target cells . retronectin is always used to enhance retroviral mediated gene transduction by colocalizing target cells and virions on the retronectin molecules . it was also found that t lymphocytes could be efficiently expanded by stimulation with a combination of immobilized retronectin and anti - cd3 mab , resulting in a higher expansion fold and more naive t cells than other methods . there were reports that retronectin activated lymphocytes could be used to treat solid tumors , proving that these cells were safe for clinical use [ 6 , 7 ] . the detailed mechanism of how retronectin affects the biologic character of cik cells , however , has not been systemically researched . in order to find an effective way to increase the number of high quality cik cells for clinical use , we investigated the impact of using retronectin on proliferation , cytotoxic activity , phenotype alternation , and cytokine secretion of cik cells the cik cells we used in this study were generated from the peripheral blood mononuclear cells of pancreatic cancer patients . we found that retronectin could increase the proliferative ability of the resultant cik cells , increase growth time when il-2 was withdrawn from the culture system , and make the cells secrete higher levels of il-2 and lower levels of il-4 and il-5 . moreover , these cik cells could be safely used to treat pancreatic cancer , as some of the patients get significant therapeutic efficacy . all patients were approved by the ethics committee of affiliated cancer hospital zhengzhou university and signed an informed consent prior to initiation of lymphocyte cultures . peripheral blood mononuclear cells ( pbmcs ) were separated from the blood by density gradient centrifugation . the pbmcs of group i were seeded into a 75 cm flask precoated with cd3 mab ( okt3 ) ( cuba cimab sa , cuba ) , while the pbmcs of group ii were seeded into another 75 cm flask precoated with retronectin ( takara , japan ) and okt3 . both groups of pbmcs were cultured with gt - t551 serum - free culture medium ( takara , japan ) supplemented with 2% autologous plasma , 1000 u / ml ifn- ( shanghai kai mao biotechnology co. ltd . , china ) , and 1000 u / ml il-2 ( shandong quangang pharmaceutical co. ltd . , china ) . after 4 days in culture , the two group cells in the 75 cm flasks were pipetted up completely to gt - t610 culture bags ( takara , japan ) , with fresh medium containing 1000 u / ml il-2 to 3 times the volume of the original medium added in the flask . fresh culture medium containing 1000 u / ml il-2 was added in the culture bags every 3 days . the cell product in the flask precoated with retronectin and okt3 was named r - cik cells , while the cell product in the flask precoated with okt3 only was named okt - cik cells . k562 human immortalized myelogenous leukemia cells ( atcc ) were cultured with rpmi-1640 medium ( gibico , usa ) containing 10% fetal calf serum ( gibico , usa ) at 37c and 5% co2 incubator . the daily growth conditions of the cells were observed . logarithmic growth phase of the k562 cells were used for cytotoxicity assays . after 4 days in culture , 5 ml medium containing okt - cik or r - cik cells was extracted with a syringe from the 75 cm flasks and then cultured in a 25 cm flask in gt - t551 medium supplemented with 1000 u / ml of il-2 . the cell number was counted once every 3 days , and the expansion multiple was calculated by comparison with the original seeded cell number . we checked the continuing proliferative ability of the resultant okt - cik and r - cik cells in the medium without il-2 by performing il-2 withdrawal tests . after 12 days in culture , parts of the okt - cik and r - cik cells cultured in the culture bag were extracted and continued to be cultured in 24-well plates without il-2 , each sample in triplicate , with 1 10 cells per well containing 1 ml medium . cell numbers in the 24-well plate were counted every 2 days ; the expansion multiple was calculated and the growth curve was drawn according to the multiple . apoptosis of the okt - cik and r - cik cells was measured by annexin v and propidium iodide ( pi ) staining using an annexin v - fitc apoptosis detection kit ( keygen , china ) . the cells were harvested and washed in cold pbs , then resuspended in 500 l binding buffer supplied by the kit , and stained with annexin v - fitc 5 l , pi 5 l for 10 minutes at room temperature . the cells were analyzed by flow cytometry ( bd biosciences , san jose , ca , usa ) within 1 hour . okt - cik and r - cik cells were measured on the 10th and 16th days after retronectin and okt3 stimulating . the cells were harvested and washed in cold pbs and resuspended at 1 10 in 100 l cold pbs consisting of 5% serum . the cells were stained for anti - cd3-apc , anti - cd4-percp , anti - cd8-percp , anti - cd27-fitc , anti - cd28-pe , anti - cd56-pe , and anti - pd-1-pe purchased from bd biosciences ( san jose , ca , usa ) for 20 minutes on ice . facs analysis was performed by using a facscalibur and cellquest software ( bd biosciences , san jose , ca , usa ) . these cells were suspended at 1 10 cells / ml and labeled with 1.25 mol / l cfse ( invitrogen , usa ) for 5 minutes at 37c . the reaction was stopped by addition of an equal volume of rpmi1640 containing 10% fetal calf serum . then , cfse - labeled cells were centrifuged and resuspended in 10 ml rpmi1640 containing 10% fetal calf serum and incubated at 37c for 30 minutes . before use , cfse - labeled k562 cells were washed two times with pbs and resuspended in gt - t551 medium at the cell concentration of 5 10 cells / ml . adjusting the concentration of okt - cik and r - cik cells at 5 10 cells / ml as effector cells , the effector and target cells were added at different effector - target ratios at 40 : 1 ( 400 l/100 l ) , 20/1 ( 330 l/165 l ) , and 10/1 ( 250 l/250 l ) and were mixed to a final volume of 500 l in 48-well plates . the plates were then incubated in a humidified atmosphere of 5% co2 and 37c for 4 hours . to stain for dead cells , propidium iodide ( final concentration 1 g / ml ) was added 5 minutes before analysis . when using flow cytometry analysis , the cfsepi cells were alive target cells and the cfsepi cells were dead target cells . spontaneous dead targets were obtained from targets incubated with medium alone and with pi staining which is positive . the percentage of cfsepi or pi k562 cells was performed by using a facscalibur and cellquest software ( bd biosciences , san jose , ca , usa ) . the percentage of specific target cell death ( cytotoxicity ) was then calculated as(1)dead targets in the sample % spontaneously dead targets % 100%spontaneously dead targets % 100% . after 15 days in culture , okt - cik and r - cik cells ( 1 10 ) were plated at 1 10 cells per well in a 96-well flat - bottom plate , with 1 10 k562 tumor cells and 200 l gt - t551 medium without il-2 . after 24-hour coculture , supernatants were harvested and cytokine secretion was quantified by bd cytometric bead array ( cba ) human th1/th2 cytokine kit ii ( bd biosciences , san jose , ca , usa ) according to the protocol of the kit . from september 2010 to december 2013 , 13 patients with advanced pancreatic cancer from affiliated cancer hospital of zhengzhou university were treated by autologous r - cik cells for at least 2 cycles , with or without chemotherapy according to the patients eastern cooperative oncology group performance status ( ecog ps ) ( table 1 ) . eleven patients were diagnosed with metastatic pancreatic cancer ; the other two were diagnosed with local advanced cancer . from 10 patients who were treated as first - line the median age of these patients was 70 ( 4979 ) years old , seven males and six females . eight patients among them had histologically or cytologically confirmed pancreatic adenocarcinoma and the other five patients were diagnosed with computed tomography / magnetic resonance imaging ( ct / mri ) combined with abnormal increase of cancer and tissue - specific marker carbohydrate antigen-199 ( ca-199 ) . patients whose expected survival time was less than 2 months were excluded from the trial . the protocol of r - cik cells culture from the patients was similar to the protocol described above . briefly , 50 ml of heparinized peripheral blood was obtained from the patients , after 11~16 days in culture , and the r - cik cells were harvested and infused to those patients who were fit to clinical use through quality control . all patients who were entered into the clinical protocols signed informed consent forms that were approved by the ethics committee of affiliated cancer hospital of zhengzhou university prior to initiation of lymphocyte cultures . china ) based chemoimmunotherapy ; the dose of s-1 used in each cycle was 80 , 100 , or 120 mg / d according to body - surface area on days 1 through 14 of a 21-day cycle . after 24~48 hours of the last day 's s-1 oral administration , 3~5 10 autologous r - cik cells were intravenous drop infused to each patient , followed by 2 million units of interleukin-2 intravenous drop infusion each day , for 3 days . three patients received gemcitabine ( haosenh pharmaceutical co. , china ) based chemoimmunotherapy ; the dose of gemcitabine used in each cycle was 1000 mg / m on days 1 and 8 of a 21-day cycle . after 24~72 hours of the second gemcitabine dose infusion , 3~5 10 autologous r - cik cells were intravenous drop infused to each patient , followed by 2 million units of interleukin-2 intravenous drop infusion each day , for 3 days . these patients received 1~6 cycles chemoimmunotherapy altogether , according to the patients eastern cooperative oncology group performance status ( ecog ps ) . after those chemoimmunotherapy cycles were completed , 1~4 doses of more r - cik cells were infused after the last chemotherapy cycle ; each dose contained about 3~5 10 r - cik cells . four patients received only r - cik cells infusion without treatment of chemotherapy due to age or refusal to use chemotherapy drugs . these patients received 2~10 doses of r - cik cells infusion , respectively , twice a month ; each dose contained about 3~5 10 r - cik cells . after r - cik cells infusion , patients received 2 million units of interleukin-2 intravenous drop infusion each day , for 5 days . tumor response was determined according to the national cancer institute ( bethesda , md ) response evaluation criteria in solid tumors ( recist1.1 ) . patients were assessed serially using ct / mri of chest , abdomen , pelvis , and brain and technetium bone scan . overall survival was calculated from the date of therapy initiation until the date of death or december 31 , 2013 . overall survival ( os ) was calculated from initiation of therapy to death , and patients alive were censored at the time of last contact . distributions of survival time and rate were estimated by using the kaplan - meier method . three days after activation by okt3 alone or okt3 combined with retronectin , both okt - cik and r - cik cells began a logarithmic growth stage , but the growth speed of r - cik cells was much higher than that of okt - cik cells ( figure 1(a ) ) . both okt - cik and r - cik cells achieved growth platform after 15 days in culture : the okt - cik cells displayed 100 times amplification at this time , while the r - cik cells displayed 200 times amplification ( figure 1(a ) ) . coincident with the growth speed , the spontaneous apoptosis of r - cik cells was lower than that of okt - cik cells ( figure 1(b ) ) . after il-2 was withdrawn from the medium , r - cik cells could continue growing to 6 times amplification , while okt - cik cells could only grow to 3 times amplification ( figure 1(c ) ) . these results indicate that r - cik cells activated by retronectin have much stronger proliferative ability than only cd3 activated cik cells . there were also some shape differences between okt - cik and r - cik cells ; okt - cik cells displayed easily formed cells aggregates , while r - cik cells displayed dispersed growth ( figure 1(d ) ) . we analyzed the cell subpopulations in okt - cik and r - cik cells cultured on the 10th and 16th days , including cd3cd4 , cd3cd8 , cd3cd56 , cd3cd27 , cd3cd28 , and cd3pd-1 cells . as shown in table 2 and figure 2 , the percentage of cd3cd4 cells and the percentage of cd3cd28 cells were higher in r - cik cells on the 10th day ( p < 0.05 ) , but they became equal on the 16th day . conversely , the percentage of cd3cd56 cells was lower in r - cik cells on the 10th day ( p < 0.05 ) ; it also became equal on the 16th day . there was no difference seen between the okt - cik and r - cik cells when compared to other subpopulation cells ( p > 0.05 ) . as the culture time prolonged , cd3cd8 cell counts were raised in both okt - cik and r - cik cells ; their percentages were higher on the 16th day than on the 10th day . correspondingly , cd3cd4 cells decreased in both okt - cik and r - cik cells at the same time . we checked the cytokines secreted by okt - cik and r - cik cells when they were cocultured with k562 cells using the cytometric bead array ( cba ) human th1/th2 cytokine kit . the cytokines checked included il-2 , il-4 , il-5 , il-10 , tnf- , and ifn-. as shown in figure 3 , compared with okt - cik cells , r - cik cells secreted higher il-2 , while they secreted lower level il-4 and il-5 ( p < 0.05 ) . there was no difference between the okt - cik and r - cik cells when compared to il-10 , tnf- , and ifn- ( p > 0.05 ) . the okt - cik and r - cik cells were tested for cytotoxicity against the k562 tumor cells , measured by cfse / pi double staining and flow cytometry analysis . as shown in figure 4 , both okt - cik and r - cik cells could kill k562 cells at different effector / target ratio , but there was no statistical difference between the two group cells ( p > 0.05 ) . of the 13 pancreatic cancer patients studied , one patient ( 7.7% ) achieved a complete remission ( cr ) : this patient had local relapsed pancreatic adenocarcinoma after surgery and preventive radiotherapy . two patients ( 15.4% ) who received chemotherapy combined r - cik cells infusion achieved a partial remission ( pr ) . the overall objective response rate ( cr+pr ) was 23.1% and the disease control rate ( cr+pr+sd ) was 84.6% . the median survival time ( mos ) after first r - cik cells infusion was 10.57 months ( 95% ci , 6.6 to 14.6 months ; figure 5 ) . common toxicities consisting of bone marrow suppression , anorexia , and fatigue were mainly in the chemotherapy group . the toxicities in the chemotherapy group were more frequent and serious than those in the group without chemotherapy . cik cells are generated by culturing peripheral blood lymphocytes ( pbl ) with interferon- ( inf- ) , monoclonal antibody against cd3 ( anti - cd3 ) , and il-2 in a particular time schedule . the cytokines inf- and il-2 are crucial for the cytotoxicity of the cells and anti - cd3 provides mitogenic signals to t cells for proliferation . cd3cd8cd56 cells are referred to as natural killer t ( nk - t ) cells and represent the cell type with the greatest cytotoxicity in the cik cell population . among different adoptive lymphocyte therapies , cik cells have a particularly advantageous profile as these cells are easily available , have a high proliferative rate , own non - mhc - restricted cytotoxicity ability , and exhibit a high antitumor activity . there are reports that cik cells could even kill cancer stem cells [ 11 , 12 ] . both autologous and allogeneic cik cells have been used in phases 1 and 2 of clinical trials for the treatment of different tumor types . in these trials , they displayed limited toxicity , whereas evidence has been obtained that they exert antitumor activity . although clinical studies have confirmed benefit and safety of cik cell - based therapy for patients with malignancies , the efficacy was usually low , so enhancement of the potency of cik cells is very important . in order to find an easy way to improve the efficacy of cik cells for clinical use , we added retronectin to the traditional cik cell culture system . we found that , after using retronectin , most of the biologic characters of the r - cik cells were similar to unaltered cik cells . for example , cik and r - cik cells had similar t cell subpopulations after 16 days in culture ; their cytotoxicity abilities against lymphoma cell line k562 cells were also equal . retronectin , however , could make r - cik cells secrete more th1 cell - associated cytokine il-2 and secrete less th2 cell - associated cytokine il-4 and il-5 , indicating that retronectin could enhance the activity of th1 cells , while suppressing the activity of th2 cells . this is a clinically significant advantage for r - cik cells , since cancer immunotherapy needs the help of th1 cells , while the th2 cells always inhibit antitumor activity of immune system . another advantage of retronectin is that it could promote the proliferative ability of cik cells ; the r - cik cells ' growth speed was faster , with a lower ratio of spontaneous apoptosis . this characteristic of retronectin could shorten the culturing time in vitro to get a certain amount of cells for clinical use ; it could also improve the quality of resultant lymphocytes , as it has been reported that the use of less - differentiated tils with stronger proliferative ability was an important factor for success . more importantly , after il-2 was withdrawn from the medium , r - cik cells continue growing for a longer time and expanded double numbers of okt - cik cells which were not excited by retronectin . this characteristic of r - cik cells made them have two merits for clinical use . first , traditional cik cells should stop growing soon after they were infused into the patient blood , because the concentration of il-2 in the patient 's body is very low , while retronectin activated r - cik cells may continue to grow for a longer time . second , the patient 's blood may be the most favorable culture medium for r - cik cells ' growth : if the patient is given il-2 several days after infusion , the cells may continue to grow for more days in the patient body , so the therapeutic number of r - cik cells needed for cancer patients could be less than that of traditional cik cells . indeed , we used average 4 10 r - cik cells in each dose to treat pancreatic cancer in our study ; it seems that r - cik cells could work at this order of magnitude , while other researchers usually use more than 1 10 cik cells to treat cancer patients [ 15 , 16 ] . pancreatic cancer is a relatively common malignancy , with around 43,000 new cases diagnosed in the u.s . in 2012 . advanced disease is associated with a dismal outcome , with a median survival of 36 months . although advanced pancreatic cancer shows a modest response to chemotherapy , different studies suggest that pancreatic cancer can elicit antitumor immune responses [ 1821 ] ; thus , immunotherapy could be of great importance in treatment . recently , immunotherapy has been shown to be a novel approach with the potential to function alone or in concert with traditional therapies : results in many clinical trials have shown improved survival without added toxicity . in a large retrospective cohort analysis , 138 palliative treatment pancreatic cancer patients had added treatment with dendritic cells . median survival was significantly higher in the group of patients who started immunotherapy within 2 months following diagnosis or repeated immunotherapy . cik cells have been used to treat many types of malignant tumors in clinical trials , where they displayed limited toxicity and some efficacy . and , in our previous study , we found that r - cik cells combined with conventional therapies could improve the prognosis of metastatic brain tumor patients , especially of those with adenocarcinoma of the lung . in the present study , we use r - cik cells to treat 13 pancreatic cancer patients alone or combined with chemotherapy . eleven patients were diagnosed with metastatic pancreatic cancer ; the other two were local advanced . the overall objective response rate was 23.1% , including one patient who achieved a complete remission ( cr ) after receiving r - cik cells infusion alone ; two patients achieved a partial remission ( pr ) after receiving chemotherapy combined with r - cik cells infusion . the overall objective response rate ( cr+pr ) was 23.1% and disease control rate ( cr+pr+sd ) was 84.6% . the median survival time ( mos ) after first r - cik cell infusion was 10.57 months , and the 1-year survival rate was 38.5% . there was no serious toxicity associated with r - cik cells infusion ; one patient had a mild fever . although the patients ' number was low , it seems that r - cik greatly improved the survival time of the metastatic pancreatic cancer . in conclusion , our results showed that after activation by retronectin , cik cells displayed stronger and faster proliferative ability , even in an environment without extraneous il-2 , and enhanced the activity of th1 cells . the r - cik cells can be safely used to treat pancreatic cancer patients , and some patients can experience significant results from the treatment . due to the limited number of cases in our study , however , additional studies should be performed .

adoptive cell therapy ( act ) using autologous cytokine - induced killer ( cik ) cells is a promising treatment for metastatic carcinomas . in this study , we investigated the impact of retronectin on the proliferation , phenotype alternation , cytokine secretion , and cytotoxic activity of cik cells from pancreatic cancer patients . furthermore , we treated 13 patients with metastatic or locally advanced pancreatic cancer using autologous retronectin - activated cik cells ( r - cik cells ) alone or in combination with chemotherapy . compared with only cd3 activated cik cells ( okt - cik cells ) , r - cik cells showed stronger and faster proliferative ability , with a lower ratio of spontaneous apoptosis . moreover , this ability continued after il-2 was withdrawn from the culture system . r - cik cells could also secrete higher levels of il-2 and lower levels of il-4 and il-5 versus okt - cik cells . there was no difference between okt - cik and r - cik cells in cytotoxic ability against lymphoma cell line k562 . in patients who received auto - r - cik cell infusion therapy , the overall objective response rate was 23.1% . median survival time ( mos ) after first r - cik cell infusion was 10.57 months ; the 1-year survival rate was 38.5% . no serious toxicity was associated with r - cik cell infusion . in conclusion , retronectin may enhance antitumor activity of cik cells : it is safe for use in treating pancreatic cancer .

1. Introduction 2. Material and Methods 3. Results 4. Discussion

adoptive therapy using t cells for cancer therapy is a promising strategy that has curative potential and broad applicability . cytokine - induced killer ( cik ) cells are generated by in vitro expansion of peripheral blood lymphocytes ( pbl ) using anti - cd3 antibodies , ifn- , and il-2 . although clinical studies have confirmed the benefit and safety of cik cell - based therapy for patients with hematopoietic and solid tumors , there are many factors impacting the clinical response due to the characteristics of ex vivo expanded lymphocyte cells . it was shown that the use of minimally cultured young less - differentiated tumor infiltrating lymphocytes ( tils ) , with stronger proliferative ability and higher levels of the costimulatory molecules cd27 and cd28 , is an important factor for success . retronectin is a chimeric peptide of recombinant e. coli - expressed human fibronectin fragments , with two functional domains which can interact with integrin molecules : very late antigen-4 ( vla-4 , 41 ) and very late antigen-5 ( vla-5 , 51 ) of the target cells . it was also found that t lymphocytes could be efficiently expanded by stimulation with a combination of immobilized retronectin and anti - cd3 mab , resulting in a higher expansion fold and more naive t cells than other methods . there were reports that retronectin activated lymphocytes could be used to treat solid tumors , proving that these cells were safe for clinical use [ 6 , 7 ] . the detailed mechanism of how retronectin affects the biologic character of cik cells , however , has not been systemically researched . in order to find an effective way to increase the number of high quality cik cells for clinical use , we investigated the impact of using retronectin on proliferation , cytotoxic activity , phenotype alternation , and cytokine secretion of cik cells the cik cells we used in this study were generated from the peripheral blood mononuclear cells of pancreatic cancer patients . we found that retronectin could increase the proliferative ability of the resultant cik cells , increase growth time when il-2 was withdrawn from the culture system , and make the cells secrete higher levels of il-2 and lower levels of il-4 and il-5 . moreover , these cik cells could be safely used to treat pancreatic cancer , as some of the patients get significant therapeutic efficacy . peripheral blood mononuclear cells ( pbmcs ) were separated from the blood by density gradient centrifugation . , china ) , and 1000 u / ml il-2 ( shandong quangang pharmaceutical co. ltd . after 4 days in culture , the two group cells in the 75 cm flasks were pipetted up completely to gt - t610 culture bags ( takara , japan ) , with fresh medium containing 1000 u / ml il-2 to 3 times the volume of the original medium added in the flask . fresh culture medium containing 1000 u / ml il-2 was added in the culture bags every 3 days . the cell product in the flask precoated with retronectin and okt3 was named r - cik cells , while the cell product in the flask precoated with okt3 only was named okt - cik cells . k562 human immortalized myelogenous leukemia cells ( atcc ) were cultured with rpmi-1640 medium ( gibico , usa ) containing 10% fetal calf serum ( gibico , usa ) at 37c and 5% co2 incubator . after 4 days in culture , 5 ml medium containing okt - cik or r - cik cells was extracted with a syringe from the 75 cm flasks and then cultured in a 25 cm flask in gt - t551 medium supplemented with 1000 u / ml of il-2 . the cell number was counted once every 3 days , and the expansion multiple was calculated by comparison with the original seeded cell number . we checked the continuing proliferative ability of the resultant okt - cik and r - cik cells in the medium without il-2 by performing il-2 withdrawal tests . after 12 days in culture , parts of the okt - cik and r - cik cells cultured in the culture bag were extracted and continued to be cultured in 24-well plates without il-2 , each sample in triplicate , with 1 10 cells per well containing 1 ml medium . cell numbers in the 24-well plate were counted every 2 days ; the expansion multiple was calculated and the growth curve was drawn according to the multiple . apoptosis of the okt - cik and r - cik cells was measured by annexin v and propidium iodide ( pi ) staining using an annexin v - fitc apoptosis detection kit ( keygen , china ) . okt - cik and r - cik cells were measured on the 10th and 16th days after retronectin and okt3 stimulating . the cells were stained for anti - cd3-apc , anti - cd4-percp , anti - cd8-percp , anti - cd27-fitc , anti - cd28-pe , anti - cd56-pe , and anti - pd-1-pe purchased from bd biosciences ( san jose , ca , usa ) for 20 minutes on ice . adjusting the concentration of okt - cik and r - cik cells at 5 10 cells / ml as effector cells , the effector and target cells were added at different effector - target ratios at 40 : 1 ( 400 l/100 l ) , 20/1 ( 330 l/165 l ) , and 10/1 ( 250 l/250 l ) and were mixed to a final volume of 500 l in 48-well plates . when using flow cytometry analysis , the cfsepi cells were alive target cells and the cfsepi cells were dead target cells . after 15 days in culture , okt - cik and r - cik cells ( 1 10 ) were plated at 1 10 cells per well in a 96-well flat - bottom plate , with 1 10 k562 tumor cells and 200 l gt - t551 medium without il-2 . from september 2010 to december 2013 , 13 patients with advanced pancreatic cancer from affiliated cancer hospital of zhengzhou university were treated by autologous r - cik cells for at least 2 cycles , with or without chemotherapy according to the patients eastern cooperative oncology group performance status ( ecog ps ) ( table 1 ) . eleven patients were diagnosed with metastatic pancreatic cancer ; the other two were diagnosed with local advanced cancer . patients whose expected survival time was less than 2 months were excluded from the trial . the protocol of r - cik cells culture from the patients was similar to the protocol described above . briefly , 50 ml of heparinized peripheral blood was obtained from the patients , after 11~16 days in culture , and the r - cik cells were harvested and infused to those patients who were fit to clinical use through quality control . all patients who were entered into the clinical protocols signed informed consent forms that were approved by the ethics committee of affiliated cancer hospital of zhengzhou university prior to initiation of lymphocyte cultures . after 24~48 hours of the last day 's s-1 oral administration , 3~5 10 autologous r - cik cells were intravenous drop infused to each patient , followed by 2 million units of interleukin-2 intravenous drop infusion each day , for 3 days . after 24~72 hours of the second gemcitabine dose infusion , 3~5 10 autologous r - cik cells were intravenous drop infused to each patient , followed by 2 million units of interleukin-2 intravenous drop infusion each day , for 3 days . after those chemoimmunotherapy cycles were completed , 1~4 doses of more r - cik cells were infused after the last chemotherapy cycle ; each dose contained about 3~5 10 r - cik cells . four patients received only r - cik cells infusion without treatment of chemotherapy due to age or refusal to use chemotherapy drugs . these patients received 2~10 doses of r - cik cells infusion , respectively , twice a month ; each dose contained about 3~5 10 r - cik cells . after r - cik cells infusion , patients received 2 million units of interleukin-2 intravenous drop infusion each day , for 5 days . three days after activation by okt3 alone or okt3 combined with retronectin , both okt - cik and r - cik cells began a logarithmic growth stage , but the growth speed of r - cik cells was much higher than that of okt - cik cells ( figure 1(a ) ) . both okt - cik and r - cik cells achieved growth platform after 15 days in culture : the okt - cik cells displayed 100 times amplification at this time , while the r - cik cells displayed 200 times amplification ( figure 1(a ) ) . coincident with the growth speed , the spontaneous apoptosis of r - cik cells was lower than that of okt - cik cells ( figure 1(b ) ) . after il-2 was withdrawn from the medium , r - cik cells could continue growing to 6 times amplification , while okt - cik cells could only grow to 3 times amplification ( figure 1(c ) ) . these results indicate that r - cik cells activated by retronectin have much stronger proliferative ability than only cd3 activated cik cells . there were also some shape differences between okt - cik and r - cik cells ; okt - cik cells displayed easily formed cells aggregates , while r - cik cells displayed dispersed growth ( figure 1(d ) ) . we analyzed the cell subpopulations in okt - cik and r - cik cells cultured on the 10th and 16th days , including cd3cd4 , cd3cd8 , cd3cd56 , cd3cd27 , cd3cd28 , and cd3pd-1 cells . as shown in table 2 and figure 2 , the percentage of cd3cd4 cells and the percentage of cd3cd28 cells were higher in r - cik cells on the 10th day ( p < 0.05 ) , but they became equal on the 16th day . conversely , the percentage of cd3cd56 cells was lower in r - cik cells on the 10th day ( p < 0.05 ) ; it also became equal on the 16th day . there was no difference seen between the okt - cik and r - cik cells when compared to other subpopulation cells ( p > 0.05 ) . as the culture time prolonged , cd3cd8 cell counts were raised in both okt - cik and r - cik cells ; their percentages were higher on the 16th day than on the 10th day . correspondingly , cd3cd4 cells decreased in both okt - cik and r - cik cells at the same time . we checked the cytokines secreted by okt - cik and r - cik cells when they were cocultured with k562 cells using the cytometric bead array ( cba ) human th1/th2 cytokine kit . as shown in figure 3 , compared with okt - cik cells , r - cik cells secreted higher il-2 , while they secreted lower level il-4 and il-5 ( p < 0.05 ) . there was no difference between the okt - cik and r - cik cells when compared to il-10 , tnf- , and ifn- ( p > 0.05 ) . the okt - cik and r - cik cells were tested for cytotoxicity against the k562 tumor cells , measured by cfse / pi double staining and flow cytometry analysis . as shown in figure 4 , both okt - cik and r - cik cells could kill k562 cells at different effector / target ratio , but there was no statistical difference between the two group cells ( p > 0.05 ) . of the 13 pancreatic cancer patients studied , one patient ( 7.7% ) achieved a complete remission ( cr ) : this patient had local relapsed pancreatic adenocarcinoma after surgery and preventive radiotherapy . two patients ( 15.4% ) who received chemotherapy combined r - cik cells infusion achieved a partial remission ( pr ) . the overall objective response rate ( cr+pr ) was 23.1% and the disease control rate ( cr+pr+sd ) was 84.6% . the median survival time ( mos ) after first r - cik cells infusion was 10.57 months ( 95% ci , 6.6 to 14.6 months ; figure 5 ) . common toxicities consisting of bone marrow suppression , anorexia , and fatigue were mainly in the chemotherapy group . cik cells are generated by culturing peripheral blood lymphocytes ( pbl ) with interferon- ( inf- ) , monoclonal antibody against cd3 ( anti - cd3 ) , and il-2 in a particular time schedule . cd3cd8cd56 cells are referred to as natural killer t ( nk - t ) cells and represent the cell type with the greatest cytotoxicity in the cik cell population . among different adoptive lymphocyte therapies , cik cells have a particularly advantageous profile as these cells are easily available , have a high proliferative rate , own non - mhc - restricted cytotoxicity ability , and exhibit a high antitumor activity . there are reports that cik cells could even kill cancer stem cells [ 11 , 12 ] . in these trials , they displayed limited toxicity , whereas evidence has been obtained that they exert antitumor activity . although clinical studies have confirmed benefit and safety of cik cell - based therapy for patients with malignancies , the efficacy was usually low , so enhancement of the potency of cik cells is very important . in order to find an easy way to improve the efficacy of cik cells for clinical use , we added retronectin to the traditional cik cell culture system . we found that , after using retronectin , most of the biologic characters of the r - cik cells were similar to unaltered cik cells . for example , cik and r - cik cells had similar t cell subpopulations after 16 days in culture ; their cytotoxicity abilities against lymphoma cell line k562 cells were also equal . retronectin , however , could make r - cik cells secrete more th1 cell - associated cytokine il-2 and secrete less th2 cell - associated cytokine il-4 and il-5 , indicating that retronectin could enhance the activity of th1 cells , while suppressing the activity of th2 cells . this is a clinically significant advantage for r - cik cells , since cancer immunotherapy needs the help of th1 cells , while the th2 cells always inhibit antitumor activity of immune system . another advantage of retronectin is that it could promote the proliferative ability of cik cells ; the r - cik cells ' growth speed was faster , with a lower ratio of spontaneous apoptosis . this characteristic of retronectin could shorten the culturing time in vitro to get a certain amount of cells for clinical use ; it could also improve the quality of resultant lymphocytes , as it has been reported that the use of less - differentiated tils with stronger proliferative ability was an important factor for success . more importantly , after il-2 was withdrawn from the medium , r - cik cells continue growing for a longer time and expanded double numbers of okt - cik cells which were not excited by retronectin . this characteristic of r - cik cells made them have two merits for clinical use . first , traditional cik cells should stop growing soon after they were infused into the patient blood , because the concentration of il-2 in the patient 's body is very low , while retronectin activated r - cik cells may continue to grow for a longer time . second , the patient 's blood may be the most favorable culture medium for r - cik cells ' growth : if the patient is given il-2 several days after infusion , the cells may continue to grow for more days in the patient body , so the therapeutic number of r - cik cells needed for cancer patients could be less than that of traditional cik cells . indeed , we used average 4 10 r - cik cells in each dose to treat pancreatic cancer in our study ; it seems that r - cik cells could work at this order of magnitude , while other researchers usually use more than 1 10 cik cells to treat cancer patients [ 15 , 16 ] . pancreatic cancer is a relatively common malignancy , with around 43,000 new cases diagnosed in the u.s . advanced disease is associated with a dismal outcome , with a median survival of 36 months . although advanced pancreatic cancer shows a modest response to chemotherapy , different studies suggest that pancreatic cancer can elicit antitumor immune responses [ 1821 ] ; thus , immunotherapy could be of great importance in treatment . recently , immunotherapy has been shown to be a novel approach with the potential to function alone or in concert with traditional therapies : results in many clinical trials have shown improved survival without added toxicity . in a large retrospective cohort analysis , 138 palliative treatment pancreatic cancer patients had added treatment with dendritic cells . median survival was significantly higher in the group of patients who started immunotherapy within 2 months following diagnosis or repeated immunotherapy . cik cells have been used to treat many types of malignant tumors in clinical trials , where they displayed limited toxicity and some efficacy . and , in our previous study , we found that r - cik cells combined with conventional therapies could improve the prognosis of metastatic brain tumor patients , especially of those with adenocarcinoma of the lung . in the present study , we use r - cik cells to treat 13 pancreatic cancer patients alone or combined with chemotherapy . eleven patients were diagnosed with metastatic pancreatic cancer ; the other two were local advanced . the overall objective response rate was 23.1% , including one patient who achieved a complete remission ( cr ) after receiving r - cik cells infusion alone ; two patients achieved a partial remission ( pr ) after receiving chemotherapy combined with r - cik cells infusion . the overall objective response rate ( cr+pr ) was 23.1% and disease control rate ( cr+pr+sd ) was 84.6% . the median survival time ( mos ) after first r - cik cell infusion was 10.57 months , and the 1-year survival rate was 38.5% . there was no serious toxicity associated with r - cik cells infusion ; one patient had a mild fever . although the patients ' number was low , it seems that r - cik greatly improved the survival time of the metastatic pancreatic cancer . in conclusion , our results showed that after activation by retronectin , cik cells displayed stronger and faster proliferative ability , even in an environment without extraneous il-2 , and enhanced the activity of th1 cells . the r - cik cells can be safely used to treat pancreatic cancer patients , and some patients can experience significant results from the treatment .

adoptive therapy using t cells for cancer therapy is a promising strategy that has curative potential and broad applicability . cytokine - induced killer ( cik ) cells are generated by in vitro expansion of peripheral blood lymphocytes ( pbl ) using anti - cd3 antibodies , ifn- , and il-2 . cik cells are a heterogeneous population of effector t cells , some of which possessing non - mhc - restricted cytolytic activities against cancer cells . although clinical studies have confirmed the benefit and safety of cik cell - based therapy for patients with hematopoietic and solid tumors , there are many factors impacting the clinical response due to the characteristics of ex vivo expanded lymphocyte cells . it was shown that the use of minimally cultured young less - differentiated tumor infiltrating lymphocytes ( tils ) , with stronger proliferative ability and higher levels of the costimulatory molecules cd27 and cd28 , is an important factor for success . retronectin is a chimeric peptide of recombinant e. coli - expressed human fibronectin fragments , with two functional domains which can interact with integrin molecules : very late antigen-4 ( vla-4 , 41 ) and very late antigen-5 ( vla-5 , 51 ) of the target cells . retronectin is always used to enhance retroviral mediated gene transduction by colocalizing target cells and virions on the retronectin molecules . it was also found that t lymphocytes could be efficiently expanded by stimulation with a combination of immobilized retronectin and anti - cd3 mab , resulting in a higher expansion fold and more naive t cells than other methods . there were reports that retronectin activated lymphocytes could be used to treat solid tumors , proving that these cells were safe for clinical use [ 6 , 7 ] . the detailed mechanism of how retronectin affects the biologic character of cik cells , however , has not been systemically researched . in order to find an effective way to increase the number of high quality cik cells for clinical use , we investigated the impact of using retronectin on proliferation , cytotoxic activity , phenotype alternation , and cytokine secretion of cik cells the cik cells we used in this study were generated from the peripheral blood mononuclear cells of pancreatic cancer patients . we found that retronectin could increase the proliferative ability of the resultant cik cells , increase growth time when il-2 was withdrawn from the culture system , and make the cells secrete higher levels of il-2 and lower levels of il-4 and il-5 . moreover , these cik cells could be safely used to treat pancreatic cancer , as some of the patients get significant therapeutic efficacy . the pbmcs of group i were seeded into a 75 cm flask precoated with cd3 mab ( okt3 ) ( cuba cimab sa , cuba ) , while the pbmcs of group ii were seeded into another 75 cm flask precoated with retronectin ( takara , japan ) and okt3 . both groups of pbmcs were cultured with gt - t551 serum - free culture medium ( takara , japan ) supplemented with 2% autologous plasma , 1000 u / ml ifn- ( shanghai kai mao biotechnology co. ltd . after 4 days in culture , the two group cells in the 75 cm flasks were pipetted up completely to gt - t610 culture bags ( takara , japan ) , with fresh medium containing 1000 u / ml il-2 to 3 times the volume of the original medium added in the flask . the cell product in the flask precoated with retronectin and okt3 was named r - cik cells , while the cell product in the flask precoated with okt3 only was named okt - cik cells . after 4 days in culture , 5 ml medium containing okt - cik or r - cik cells was extracted with a syringe from the 75 cm flasks and then cultured in a 25 cm flask in gt - t551 medium supplemented with 1000 u / ml of il-2 . the cell number was counted once every 3 days , and the expansion multiple was calculated by comparison with the original seeded cell number . we checked the continuing proliferative ability of the resultant okt - cik and r - cik cells in the medium without il-2 by performing il-2 withdrawal tests . after 12 days in culture , parts of the okt - cik and r - cik cells cultured in the culture bag were extracted and continued to be cultured in 24-well plates without il-2 , each sample in triplicate , with 1 10 cells per well containing 1 ml medium . cell numbers in the 24-well plate were counted every 2 days ; the expansion multiple was calculated and the growth curve was drawn according to the multiple . apoptosis of the okt - cik and r - cik cells was measured by annexin v and propidium iodide ( pi ) staining using an annexin v - fitc apoptosis detection kit ( keygen , china ) . okt - cik and r - cik cells were measured on the 10th and 16th days after retronectin and okt3 stimulating . the cells were stained for anti - cd3-apc , anti - cd4-percp , anti - cd8-percp , anti - cd27-fitc , anti - cd28-pe , anti - cd56-pe , and anti - pd-1-pe purchased from bd biosciences ( san jose , ca , usa ) for 20 minutes on ice . before use , cfse - labeled k562 cells were washed two times with pbs and resuspended in gt - t551 medium at the cell concentration of 5 10 cells / ml . adjusting the concentration of okt - cik and r - cik cells at 5 10 cells / ml as effector cells , the effector and target cells were added at different effector - target ratios at 40 : 1 ( 400 l/100 l ) , 20/1 ( 330 l/165 l ) , and 10/1 ( 250 l/250 l ) and were mixed to a final volume of 500 l in 48-well plates . when using flow cytometry analysis , the cfsepi cells were alive target cells and the cfsepi cells were dead target cells . the percentage of specific target cell death ( cytotoxicity ) was then calculated as(1)dead targets in the sample % spontaneously dead targets % 100%spontaneously dead targets % 100% . after 15 days in culture , okt - cik and r - cik cells ( 1 10 ) were plated at 1 10 cells per well in a 96-well flat - bottom plate , with 1 10 k562 tumor cells and 200 l gt - t551 medium without il-2 . after 24-hour coculture , supernatants were harvested and cytokine secretion was quantified by bd cytometric bead array ( cba ) human th1/th2 cytokine kit ii ( bd biosciences , san jose , ca , usa ) according to the protocol of the kit . from september 2010 to december 2013 , 13 patients with advanced pancreatic cancer from affiliated cancer hospital of zhengzhou university were treated by autologous r - cik cells for at least 2 cycles , with or without chemotherapy according to the patients eastern cooperative oncology group performance status ( ecog ps ) ( table 1 ) . eleven patients were diagnosed with metastatic pancreatic cancer ; the other two were diagnosed with local advanced cancer . from 10 patients who were treated as first - line the median age of these patients was 70 ( 4979 ) years old , seven males and six females . eight patients among them had histologically or cytologically confirmed pancreatic adenocarcinoma and the other five patients were diagnosed with computed tomography / magnetic resonance imaging ( ct / mri ) combined with abnormal increase of cancer and tissue - specific marker carbohydrate antigen-199 ( ca-199 ) . the protocol of r - cik cells culture from the patients was similar to the protocol described above . briefly , 50 ml of heparinized peripheral blood was obtained from the patients , after 11~16 days in culture , and the r - cik cells were harvested and infused to those patients who were fit to clinical use through quality control . china ) based chemoimmunotherapy ; the dose of s-1 used in each cycle was 80 , 100 , or 120 mg / d according to body - surface area on days 1 through 14 of a 21-day cycle . after 24~48 hours of the last day 's s-1 oral administration , 3~5 10 autologous r - cik cells were intravenous drop infused to each patient , followed by 2 million units of interleukin-2 intravenous drop infusion each day , for 3 days . three patients received gemcitabine ( haosenh pharmaceutical co. , china ) based chemoimmunotherapy ; the dose of gemcitabine used in each cycle was 1000 mg / m on days 1 and 8 of a 21-day cycle . these patients received 1~6 cycles chemoimmunotherapy altogether , according to the patients eastern cooperative oncology group performance status ( ecog ps ) . after those chemoimmunotherapy cycles were completed , 1~4 doses of more r - cik cells were infused after the last chemotherapy cycle ; each dose contained about 3~5 10 r - cik cells . four patients received only r - cik cells infusion without treatment of chemotherapy due to age or refusal to use chemotherapy drugs . these patients received 2~10 doses of r - cik cells infusion , respectively , twice a month ; each dose contained about 3~5 10 r - cik cells . patients were assessed serially using ct / mri of chest , abdomen , pelvis , and brain and technetium bone scan . overall survival was calculated from the date of therapy initiation until the date of death or december 31 , 2013 . overall survival ( os ) was calculated from initiation of therapy to death , and patients alive were censored at the time of last contact . three days after activation by okt3 alone or okt3 combined with retronectin , both okt - cik and r - cik cells began a logarithmic growth stage , but the growth speed of r - cik cells was much higher than that of okt - cik cells ( figure 1(a ) ) . both okt - cik and r - cik cells achieved growth platform after 15 days in culture : the okt - cik cells displayed 100 times amplification at this time , while the r - cik cells displayed 200 times amplification ( figure 1(a ) ) . coincident with the growth speed , the spontaneous apoptosis of r - cik cells was lower than that of okt - cik cells ( figure 1(b ) ) . these results indicate that r - cik cells activated by retronectin have much stronger proliferative ability than only cd3 activated cik cells . there were also some shape differences between okt - cik and r - cik cells ; okt - cik cells displayed easily formed cells aggregates , while r - cik cells displayed dispersed growth ( figure 1(d ) ) . we analyzed the cell subpopulations in okt - cik and r - cik cells cultured on the 10th and 16th days , including cd3cd4 , cd3cd8 , cd3cd56 , cd3cd27 , cd3cd28 , and cd3pd-1 cells . as shown in table 2 and figure 2 , the percentage of cd3cd4 cells and the percentage of cd3cd28 cells were higher in r - cik cells on the 10th day ( p < 0.05 ) , but they became equal on the 16th day . conversely , the percentage of cd3cd56 cells was lower in r - cik cells on the 10th day ( p < 0.05 ) ; it also became equal on the 16th day . there was no difference seen between the okt - cik and r - cik cells when compared to other subpopulation cells ( p > 0.05 ) . as the culture time prolonged , cd3cd8 cell counts were raised in both okt - cik and r - cik cells ; their percentages were higher on the 16th day than on the 10th day . correspondingly , cd3cd4 cells decreased in both okt - cik and r - cik cells at the same time . we checked the cytokines secreted by okt - cik and r - cik cells when they were cocultured with k562 cells using the cytometric bead array ( cba ) human th1/th2 cytokine kit . as shown in figure 3 , compared with okt - cik cells , r - cik cells secreted higher il-2 , while they secreted lower level il-4 and il-5 ( p < 0.05 ) . there was no difference between the okt - cik and r - cik cells when compared to il-10 , tnf- , and ifn- ( p > 0.05 ) . the okt - cik and r - cik cells were tested for cytotoxicity against the k562 tumor cells , measured by cfse / pi double staining and flow cytometry analysis . as shown in figure 4 , both okt - cik and r - cik cells could kill k562 cells at different effector / target ratio , but there was no statistical difference between the two group cells ( p > 0.05 ) . of the 13 pancreatic cancer patients studied , one patient ( 7.7% ) achieved a complete remission ( cr ) : this patient had local relapsed pancreatic adenocarcinoma after surgery and preventive radiotherapy . the overall objective response rate ( cr+pr ) was 23.1% and the disease control rate ( cr+pr+sd ) was 84.6% . the median survival time ( mos ) after first r - cik cells infusion was 10.57 months ( 95% ci , 6.6 to 14.6 months ; figure 5 ) . the toxicities in the chemotherapy group were more frequent and serious than those in the group without chemotherapy . the cytokines inf- and il-2 are crucial for the cytotoxicity of the cells and anti - cd3 provides mitogenic signals to t cells for proliferation . cd3cd8cd56 cells are referred to as natural killer t ( nk - t ) cells and represent the cell type with the greatest cytotoxicity in the cik cell population . among different adoptive lymphocyte therapies , cik cells have a particularly advantageous profile as these cells are easily available , have a high proliferative rate , own non - mhc - restricted cytotoxicity ability , and exhibit a high antitumor activity . both autologous and allogeneic cik cells have been used in phases 1 and 2 of clinical trials for the treatment of different tumor types . although clinical studies have confirmed benefit and safety of cik cell - based therapy for patients with malignancies , the efficacy was usually low , so enhancement of the potency of cik cells is very important . in order to find an easy way to improve the efficacy of cik cells for clinical use , we added retronectin to the traditional cik cell culture system . we found that , after using retronectin , most of the biologic characters of the r - cik cells were similar to unaltered cik cells . retronectin , however , could make r - cik cells secrete more th1 cell - associated cytokine il-2 and secrete less th2 cell - associated cytokine il-4 and il-5 , indicating that retronectin could enhance the activity of th1 cells , while suppressing the activity of th2 cells . this is a clinically significant advantage for r - cik cells , since cancer immunotherapy needs the help of th1 cells , while the th2 cells always inhibit antitumor activity of immune system . another advantage of retronectin is that it could promote the proliferative ability of cik cells ; the r - cik cells ' growth speed was faster , with a lower ratio of spontaneous apoptosis . this characteristic of retronectin could shorten the culturing time in vitro to get a certain amount of cells for clinical use ; it could also improve the quality of resultant lymphocytes , as it has been reported that the use of less - differentiated tils with stronger proliferative ability was an important factor for success . more importantly , after il-2 was withdrawn from the medium , r - cik cells continue growing for a longer time and expanded double numbers of okt - cik cells which were not excited by retronectin . first , traditional cik cells should stop growing soon after they were infused into the patient blood , because the concentration of il-2 in the patient 's body is very low , while retronectin activated r - cik cells may continue to grow for a longer time . second , the patient 's blood may be the most favorable culture medium for r - cik cells ' growth : if the patient is given il-2 several days after infusion , the cells may continue to grow for more days in the patient body , so the therapeutic number of r - cik cells needed for cancer patients could be less than that of traditional cik cells . indeed , we used average 4 10 r - cik cells in each dose to treat pancreatic cancer in our study ; it seems that r - cik cells could work at this order of magnitude , while other researchers usually use more than 1 10 cik cells to treat cancer patients [ 15 , 16 ] . although advanced pancreatic cancer shows a modest response to chemotherapy , different studies suggest that pancreatic cancer can elicit antitumor immune responses [ 1821 ] ; thus , immunotherapy could be of great importance in treatment . recently , immunotherapy has been shown to be a novel approach with the potential to function alone or in concert with traditional therapies : results in many clinical trials have shown improved survival without added toxicity . median survival was significantly higher in the group of patients who started immunotherapy within 2 months following diagnosis or repeated immunotherapy . and , in our previous study , we found that r - cik cells combined with conventional therapies could improve the prognosis of metastatic brain tumor patients , especially of those with adenocarcinoma of the lung . in the present study , we use r - cik cells to treat 13 pancreatic cancer patients alone or combined with chemotherapy . the overall objective response rate was 23.1% , including one patient who achieved a complete remission ( cr ) after receiving r - cik cells infusion alone ; two patients achieved a partial remission ( pr ) after receiving chemotherapy combined with r - cik cells infusion . in conclusion , our results showed that after activation by retronectin , cik cells displayed stronger and faster proliferative ability , even in an environment without extraneous il-2 , and enhanced the activity of th1 cells . the r - cik cells can be safely used to treat pancreatic cancer patients , and some patients can experience significant results from the treatment .

cooperative catalysis between thioureas and brnsted acids , whereby the enhanced catalytic activity of the thiourea acts as an acid amplifier , has successfully been applied to asymmetric catalysis , and more recently , reports have emerged from the schmidt group applying this type of synergistic catalysis to glycosylation reactions involving o - glycosyl trichloroacetimidate donors to yield -selective glycosides . deoxy - hexoses are an important class of glycosides that occur widely in many natural products ranging from antibiotics to anticancer agents . the stereoselective formation of glycosidic linkages employing 2-deoxyglycosides , where a substituent at c-2 that can direct the coupling reaction is lacking , is a very challenging endeavor and many efforts have been devoted to achieve their stereoselective synthesis . acid - catalyzed direct nucleophilic substitution on a glycal is one of the most widely used and efficient methods for their synthesis , however these reactions often give anomeric mixtures and side products ( e.g. , ferrier rearrangement side - products ) . as part of our ongoing interest in developing stereoselective glycosylation methods , we decided to focus our attention on the synthesis of deoxyglycosides . recently , our team reported a mild organocatalytic method for the preparation of 2-deoxygalactosides employing schreiner s thiourea with excellent yields and -selectivity . although the method worked well with galactals , reactions needed to be refluxed over 24 h to reach completion , and in general , the thiourea catalyst was unable to activate glucal or rhamnal substrates . we postulated that synergistic acid / thiourea activation could provide a more efficient and practical glycosylation strategy for the preparation of deoxyglycosides than current methods that use hydrogen - bonding organocatalysts or acids as the sole promoters ( scheme 1 ) . to evaluate our hypothesis , thiourea 1 was chosen as the organocatalyst based on previous successful work from our group and others . moreover , binol - derived phosphoric acids have been shown to be particularly effective in many asymmetric transformations , including some success in glycosylations involving trichloroacetimidate glycoside donors . more recently , chiral phosphoric acids have also been shown to catalyze the spiroketalization of cyclic enol ethers bearing a pendant alcohol nucleophile in a highly diastereoselective syn - selective concerted mechanism whereby the phosphoric acid acts as a bifunctional catalyst activating both the alkene and alcohol nucleophile . initial experiments began with the screening of a series of commercial binol - derived phosphoric acids for their ability to promote the stereoselective glycosylation of perbenzylated galactal 2a with glucoside acceptor 3(12 ) in the absence and presence of 1 in ch2cl2 at room temperature . as summarized in table 1 , coupling reactions employing 10 mol % of chiral phosphoric acids ( s)-4a and ( r)-4b ( pka = 2.63 in dmso ) proceeded cleanly to give product 5(7 ) in 55 and 70% yields , albeit after 20 h ( entries 2 and 4 ) and with different degrees of : stereocontrol 9:1 and > 30:1 , respectively . when 10 mol % of 1 was added as the cocatalyst ( entries 3 and 5 ) , reactions were complete within 3 h , and glycoside 5 was obtained in improved yields of 82% and 89% , respectively , and with an : ratio of 7:1 for the ( s)-acid and > 30:1 for the ( r)-acid catalyst . these results demonstrate the strong influence of thiourea 1 , as a cocatalyst , on reaction rate and yield , while the stereochemistry of the acid catalyst has the biggest effect on the : selectivity of the glycosylation reaction . this observation is not completely surprising , as bennett and co - workers had already reported that the stereo - outcome of acid - catalyzed glycosylation reactions involving trichloroacetimidate donors was dependent on the chirality of the catalyst , the configuration of the leaving group in the glycoside donor , and the nature of the nucleophile acceptor . it is important to note that glycosylation reactions with 10 mol % of thiourea 1 in ch2cl2 ( in the absence of acid ) yielded only starting material ( entry 1 ) as previously observed by our team , likely due to catalyst inactivation via self - association at high catalyst loading , further demonstrating the importance of the synergy between both cocatalysts . 5 mol % , as reactions at 10 mol % lead to rapid hydrolysis of 2a . from h nmr ; n / a = not applicable the use of achiral p - toluenesulfonic acid ( ptsoh , 5 mol % ) 4e or bis(4-nirophenyl)hydrogen phosphate 4f or less acidic phosphoric catalysts 4c and 4d ( pka = 3.86 ) in combination with 1 proved to be detrimental to reaction rate , yield , and stereocontrol , with reactions needing 620 h for completion ( entries 69 ) . next , we decided to explore the reaction conditions using 4b and 1 as the cocatalyst . increasing the acid loading to 20 mol % in the presence of 10 mol % of 1 had no effect on the outcome of the model reaction ( entry 10 ) , while lowering the reaction temperature significantly slowed product formation ( entry 11 ) . changing the reaction solvent to either mecn or diethyl ether ( table 1 , entries 12 and 13 ) had a detrimental effect on the rate of the reaction . in the case of mecn , which is known to participate during the glycosylation reaction involving oxocarbenium ions favoring -glycoside products , significant erosion of the -selectivity was observed with 5 being isolated as a 3:1 : mixture ( entry 11 vs entry 5 ) . having established the optimum reaction conditions , our attention was then turned to exploring the scope of the cooperative catalytic system on coupling reactions between 2a and a range of oh nucleophiles 6a6j ( table 2 ) . in all cases , reactions proceeded smoothly within 26 h and in excellent yields and -selectivity , as determined by the characteristic anomeric signals in the h- and c nmr spectra , demonstrating that the catalytic system tolerates the presence of common alcohol and amine protecting groups such as acetals , ethers , esters , and carbamates . glycosylations with primary alcohols 6a6d and boc - protected serine 6h afforded the corresponding glycoside products in 8086% yield within 23 h and with a > 30:1 : ratio ( entries 14 and 8) . reactions with secondary alcohols such as phenols 6e , glycosides 6f and 6 g , boc - protected threonine 6i , or cholesterol 6j prove to be more challenging and required higher reaction temperatures ( 45 c ) and the use of 20 mol % of 1 in combination with 10 mol % of 4b in ch2cl2 , to drive reactions to completion within 56 h. under these optimized conditions , the desired products were thus isolated with similar high -selectivity ( > 30:1 : ratio ) and yields of 7485% ( table 2 , entries 57 , 9 , and 10 ) . reaction performed at 45 c , 1.5 equiv of donor and thiourea 1 ( 20 mol % ) ( 56 h ) . product was isolated as the desilylated disaccharide for purification purposes . our attention then turned to exploring the scope of the glycal donor . to that end , a series of differentially protected galactals 2b2f , glucals 8a8c , and l - rhamnal 9 bearing methyl , acetate , benzyl , allyl , silyl ether , and siloxane protecting groups were prepared and subjected to the reaction conditions with 3 or 6k , which bears a secondary oh , as the acceptor ( table 3 ) . pleasingly , high yields and excellent selectivities for -linked glycosides were obtained in most examples ( entries 1 , 35 , and 7 ) . although substrate 2d , which bears an acetate group at c-6 , shows that ester groups can be tolerated ( entry 3 ) , in the case of acetyl - protected galactal 2c ( entry 2 ) , an inseparable mixture of glycosides including ferrier side - products was observed after 20 h under the more forcing conditions ( 20 mol % of 4b and reflux in ch2cl2 ) . this lack of reactivity has been previously noted for glycals bearing a deactivating group ( e.g. , ester ) at c-3 , which is in close proximity to the reacting double bond . encouragingly , the reaction was also amenable to glycosylations with glucal substrates , and both benzyl - derivative 8a and 3,4-o - siloxane protected 8b(6b ) or 8c(6b ) afforded the corresponding glycosides with high -stereocontrol within 2 h , albeit 11a was isolated in a lower yield ( 40% ) than 11b ( 81% yield ) or 11c ( 82% ) . this is not completely unexpected , as 8b has been shown to be a more effective glycosylation donor than its benzylated counterpart ( table 3 , entries 68 ) . reaction performed at 45 c , 1.5 equiv of donor , and 0.2 equiv of thiourea . 2,6-dideoxyglycosides are also an important class of compounds , and their stereoselective synthesis is further complicated by the lack of oxygen substituents at both c-2 and c-6 . excitingly , activation of 3,4-o - siloxane protected l - rhamnal 9 afforded 12 in 74% yield within 3 h and with an 8:1 : ratio ( entry 9 ) . it is important to highlight that thiourea 1 alone was not able to activate any of the glucal and rhamnal substrates . these results further emphasize that the synergistic catalytic system works well across a range of reactivity profiles for both glycal donors and nucleophile acceptors . having found that thioglycosides are inert under the cooperative organocatalytic glycosylation conditions ( table 2 , entry 4 ) , we decided to evaluate the methodology in a three - component , one - pot synthesis of trisaccharide 13 ( scheme 2 ) . following disaccharide formation , acceptor 6c was added to the same pot along with nis and catalytic tmsotf and trisaccharide 13 was obtained in 58% yield with complete stereoselectivity . to probe the mechanism of our reaction , a 3:1 /-anomeric mixture of 5 was subjected to the reaction conditions and gave no change in the anomeric ratio , indicating that the high -selectivity is not the result of anomerization . moreover , reaction with deuterated galactal 14 yielded disaccharide 15 with the newly formed bonds cis to each other , as evidenced by the h nmr shifts associated with h-1 ( 4.62 ppm , d , 1h , j = 2.7 hz ) and h-2 ( 2.031.99 , m , 1h , h-2 ) of the deuterated 2-deoxygalactoside ( scheme 3 ) , which suggest that both the c h and the c o bond formation steps are syn - diastereoselective . h nmr spectroscopy studies in cd2cl2 of mixtures of thiourea 1 and acid 4b showed proton shifts associated with the aromatic signals from both acid and thiourea , indicating that an interaction between both catalysts occurs in solution . furthermore , nmr mixtures of thiourea 1 , acid 4b , and glycoside donor 2a showed additional downfield h - shifts associated with the anomeric protons in 2a ( 6.33 ppm ) , while shifts for the oh signal from acceptor 3 ( from 1.84 ppm to 2.54 ppm ) were detected in mixtures of 1 , 4b , and 3 ( see supporting information for details ) . thiourea 1 and chiral phosphoric acid 4b have been shown to individually hydrogen bond with oh nucleophiles . as expected , mixtures of 3 and 10 mol % of thiourea 1 or 3 mixed with 10 mol % of acid 4b also showed shifts associated with the oh protons of glycosyl acceptor 3 , although the magnitude of those shifts was different to that of mixtures of 3 and both cocatalysts ( 1 and 4b ) . these results further demonstrate that the combination of 1 and chiral acid ( r)-4b leads to a cooperative catalytic system that can activate both enol ether and oh nucleophiles in a synergistic fashion , leading to an enhancement in reaction rate and -stereocontrol of the glycosylation reaction . as proposed in scheme 3 , our findings suggest that a hydrogen - bond - mediated complex between thiourea 1 and acid 4b leads to a urea - induced acid amplification , and proton addition from this complex to the less hindered face of the enol ether ( i ) forms a short - lived oxocarbenium intermediate ( ii ) that is rapidly trapped by the oh nucleophile which is in turn activated by the phosphate intermediate that is generated in situ , to give 15 . this might help to explain the effect the chirality of the phosphoric acid has on the stereo outcome of the glycosylation reaction . in summary , we have described a practical , highly stereoselective , and efficient direct glycosylation method for glycals using a cooperative brnsted acid - organocatalytic promoter system . the reaction is widely applicable to a range of glycosyl donors and nucleophile acceptors , proceeds with excellent yields and high selectivity for the -anomer , and is tolerant of most common protecting groups . we note that the stereoselectivity of the reaction is highly dependent on the chirality of the acid , with ( r)-4b leading to the formation of -glycosides preferentially , while both yield and rate of reaction are greatly enhanced by the synergistic interaction between thiourea and acid . moreover , we exemplify the generality of the approach in the stereoselective synthesis of a series of disaccharides , glycosyl - amino acids , and other glycoconjugates and also in the one - pot chemo- and stereoselective synthesis of a trisaccharide . further work from our lab is underway to exploit the cooperative catalysis between organocatalysts and chiral acids for the stereoselective synthesis of other important glycosides and chiral acetals . dry dcm was obtained by distillation using standard procedures or by passage through a column of anhydrous alumina . glassware and needles were either flame - dried immediately prior to use or placed in an oven ( 150 c ) for at least 2 h and allowed to cool either in a desiccators or under reduced pressure . liquid reagents , solutions , or solvents were added via syringe through rubber septa . detection was by examination under uv light ( 254 nm ) and by charring with 10% sulfuric acid in ethanol . flash column chromatography was performed using silica gel [ merck , 230400 mesh ( 4063 m ) ] . extracts were concentrated in vacuo using both a bchi rotary evaporator ( bath temperatures up to 40 c ) at a pressure of either 15 mmhg ( diaphragm pump ) or 0.1 mmhg ( oil pump ) , as appropriate , and a high - vacuum line at room temperature . h nmr and c nmr spectra were measured in the solvent stated at 400 or 500 mhz . chemical shifts are quoted in parts per million from residual solvent peak ( cdcl3 : h , 7.26 ppm and c , 77.16 ppm ) and coupling constants ( j ) given in hertz . multiplicities are abbreviated as bs ( broad ) , s ( singlet ) , d ( doublet ) , t ( triplet ) , q ( quartet ) , m ( multiplet ) , or combinations thereof . positive ion matrix - assisted laser desorption ionization time - of - flight ( maldi - tof ) mass spectra were recorded using an hp - maldi instrument using gentisic acid matrix . electrospray ionization ( esi ) mass spectra were recorded on a micromass lct mass spectrometer or a vg quattro mass spectrometer . monosaccharide donor ( 1 equiv ) and acceptor ( 0.83 equiv ( 0.1 mmol ) ) were weighed into a microwave vial and placed under vacuum for 1 h , after which time the microwave vial was filled with n2 . a mixture of chiral acid ( 0.1 equiv ) and thiourea ( 0.1 equiv for primary acceptors and 0.2 equiv for secondary acceptors ) in anhydrous dcm ( 1 ml ) was stirred for 30 min and then added to the microwave vial containing the donor and acceptor . the reaction mixture was stirred at rt ( primary acceptors ) or heated at reflux ( secondary acceptors ) until the reaction was determined to be complete by either tlc or nmr analysis of the crude material . the reaction mixture was purified by silica gel flash column chromatography . following the general glycosylation procedure , donor 2a ( 50 mg , 0.12 mmol ) and acceptor 3 ( 46 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 6:1 to 3:1 ) : 5 as a yellow oil ( 68 mg , 89% ) . h nmr ( 400 mhz ; cdcl3 ) 7.397.19 ( 30h , m , ph ) , 5.02 ( 1h , app d , j = 2.4 hz , h-1 ) , 4.98 ( 1h , d , j = 10.8 hz , och hph ) , 4.91 ( 1h , d , j = 11.6 hz , och hph ) , 4.84 ( 1h , d , j = 10.9 hz , och hph ) , 4.80 ( 1h , d , j = 10.8 hz , ochh ph ) , 4.79 ( 1h , d , j = 12.2 hz , och hph ) , 4.68 ( 1h , d , j = 12.2 hz , ochh ph ) , 4.60 ( 1h , d , j = 3.6 hz , h-1 ) , 4.60 ( 1h , d , j = 11.6 hz , ochh ph ) , 4.57 ( 2h , s , och2 ph ) , 4.52 ( 1h , d , j = 10.9 hz , ochh ph ) , 4.41 ( 1h , d , j = 12.0 hz , och hph ) , 4.34 ( 1h , d , j = 12.0 hz , ochh ph ) , 3.98 ( 1h , t , j = 9.2 hz , h-3 ) , 3.903.84 ( 3h , m , h-3 , h-4 , h-5 ) , 3.81 ( 1h , dd , j = 11.2 , 4.6 hz , h-6a ) , 3.72 ( 1h , ddd , j = 10.0 , 4.5 , 1.6 hz , h-5 ) , 3.61 ( 1h , dd , j = 11.4 , 1.7 hz , h-6b ) , 3.54 ( 1h , dd , j = 9.5 , 7.3 hz , h-6a ) , 3.51 ( 1h , dd , j = 9.5 , 3.5 hz , h-2 ) , 3.50 ( 1h , dd , j = 9.5 , 5.9 hz , h-6b ) , 3.46 ( 1h , dd , j = 9.9 , 9.0 hz , h-4 ) , 3.31(3h , s , och3 ) , 2.20 ( 1h , td , j = 12.4 , 3.7 hz , h-2a ) , 2.01 ( 1h , app dd , j = 12.7 , 4.5 hz , h-2b ) ; c nmr ( 126 mhz ; cdcl3 ) 139.0 ( 4 c ) , 138.9 ( 4 c ) , 138.5 ( 4 c ) , 138.4 ( 4 c ) , 138.30 ( 4 c ) , 138.27 ( 4 c ) , 128.6 ( ch ) , 128.54 ( ch ) , 128.45 ( ch ) , 128.33 ( ch ) , 128.32 ( ch ) , 128.18 ( ch ) , 128.16 ( ch ) , 128.0 ( ch ) , 127.82 ( ch ) , 127.81 ( ch),127.78 ( ch ) , 127.7 ( ch ) , 127.6 ( ch ) , 127.5 ( ch ) , 98.4 ( c-1 ) , 98.0 ( c-1 ) , 82.3 ( c-3 ) , 80.1 ( c-2 ) , 78.1 ( c-4 ) , 76.0 ( c h2 ph ) , 75.1 ( c h2 ph ) , 74.41 , 74.35 ( c-3 , c h2 ph ) , 73.5 ( c h2 ph ) , 73.4 ( c h2 ph ) , 73.1 ( c-4 ) , 70.4 , 70.2 ( c-5 , ch2ph ) , 70.0 ( c-5 ) , 69.5 ( c-6 ) , 66.2 ( c-6 ) , 55.2 ( och3 ) , 31.2 ( c-2 ) . spectroscopic data in agreement with literature . following the general glycosylation procedure , donor 2a ( 50 mg , 0.12 mmol ) and acceptor 6a ( 11 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 10:1 to 8:1 ) : 7a as a colorless oil ( 42 mg , 80%).h nmr ( 500 mhz , cdcl3 ) 7.377.22 ( 20h , m , ph ) , 5.08 ( 1h , d , j = 3.5 hz , h-1 ) , 4.93 ( 1h , d , j = 11.6 hz , ochhph ) , 4.67 ( 1h , d , j = 11.9 hz , ochhph ) , 4.62 ( 1h , d , j = 11.7 hz , ochhph ) , 4.59 ( 1h , m , och2ph ) , 4.50 ( 1h , d , j = 11.8 hz , ochhph ) , 4.47 ( 2h , d , j = 11.9 hz , ochhph ) , 4.43 ( 1h , d , j = 11.8 hz , ochhph ) , 3.99 ( 1h , ddd , j = 12.0 , 4.6 , 2.5 hz , h-3 ) , 3.96 ( 1h , t , j = 6.7 hz , h-5 ) , 3.94 ( 1h , bs , h-4 ) , 3.61 ( 1h , dd , j = 9.4 , 6.8 hz , h-6a ) , 3.56 ( 1h , dd , j = 9.4 , 6.0 hz , h-6b ) , 2.25 ( 1h , td , j = 12.4 , 3.8 hz , h-2a ) , 2.072.01 ( 1h , m , h-2b ) ; c nmr ( 126 mhz , cdcl3 ) 139.1 ( 4 c ) , 138.7 ( 4 c ) , 138.3 ( 4 c ) , 138.0 ( 4 c ) , 128.54 ( ch ) , 128.52 ( ch ) , 128.51 ( ch ) , 128.37 ( ch ) , 128.35 ( ch ) , 128.1 ( ch ) , 127.9 ( ch ) , 127.81 ( ch ) , 127.77 ( ch ) , 127.7 ( ch ) , 127.6 ( ch ) , 127.5 ( ch ) , 97.3 ( h-1 ) , 75.0 ( c-3 ) , 74.4 ( och2ph ) , 73.6 ( och2ph ) , 73.2 ( c-4 ) , 70.7 ( och2ph ) , 70.3 ( c-5 ) , 69.7 ( c-6 ) , 69.1 ( och2ph ) , 31.3 ( c-2 ) . esi - hrms for c34h36nao5 ( mna ) calcd : 547.2460 ; found : 547.2459 . [ ]d = + 24 ( c = 1 , chcl3 ) . following the general glycosylation procedure , donor 2a ( 50 mg , 0.12 mmol ) and acceptor 6b ( 50 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 6:1 to 4:1 ) : 7b as a white solid ( 79 mg , 86% ) donor 2a ( 50 mg , 0.12 mmol ) and acceptor 6x ( 26 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 6:1 to 3:1 ) 7c as a colorless oil ( 56 mg , 83% ) . donor 2a ( 50 mg , 0.12 mmol ) and acceptor 6d ( 58 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 7:1 to 4:1 ) : 7d as a white solid ( 82 mg 82% ) . spectroscopic data in agreement with literature . following the general glycosylation procedure , donor 2a ( 50 mg , 0.12 mmol ) and acceptor 6e ( 9 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac 20:1 to 12:1 ) : 7e as a colorless oil ( 43 mg 84% ) : h nmr ( 400 mhz ; cdcl3 ) 7.426.98 ( 20h , m , ph ) , 5.59 ( 1h , d , j = 3.4 hz , h-1 ) , 4.85 ( 1h , d , j = 11.5 hz , ochhph ) , 4.72 ( 2h , bs , och2ph ) , 4.67 ( 1h , d , j = 11.5 hz , ochhph ) , 4.28 ( 1h , d , j = 11.6 hz , ochhph ) , 4.24 ( 1h , d , j = 11.6 hz , ochhph ) , 4.02 ( 1h , ddd , h-3 ) , 3.93 ( 1h , t , j = 7.3 hz , h-5 ) , 3.80 ( 1h , bs , h-4 ) , 3.53 ( 1h , dd , j = 9.3 , 7.3 hz , h-6a ) , 3.40 ( 1h , dd , j = 9.3 , 5.7 hz , h-6b ) , 2.28 ( 1h , td , j = 12.4 , 3.6 hz , h-2a ) , 2.08 ( 1h , dd , j = 12.4 , 4.4 hz , h-2b ) ; c nmr ( 101 mhz ; cdcl3 ) 156.8 ( 4 c ) , 138.8 ( 4 c ) , 138.4 ( 4 c ) , 138.0 ( 4 c ) , 128.4 ( ch ) , 128.3 ( ch ) , 128.2 ( ch ) , 128.2 ( ch ) , 128.1 ( ch ) , 128.2 ( ch ) , 127.7 ( ch ) , 127.6 ( ch ) , 127.5 ( ch ) , 127.3 ( ch ) , 121.9 ( ch ) , 116.6 ( ch ) , 96.5 ( c-1 ) , 74.5 ( ch2ph ) , 74.4 ( c-3 ) , 73.3 ( ch2ph ) , 72.8 ( c-4 ) , 70.6 ( ch2ph ) , 70.5 ( c-5 ) , 69.1 ( c-6 ) , 31.2 ( c-2 ) . esi - hrms for c33h34nao5 ( mna ) calcd : 533.2304 ; found : 533.2296 . the general glycosylation procedure was followed using donor 2a ( 50 mg , 0.12 mmol ) and acceptor 6f ( 53 mg , 0.10 mmol ) . the crude material was then dissolved in a 1 m thf solution of tbaf ( 0.5 ml , 0.5 mmol ) and stirred for 2 h. the solution was then concentrated in vacuo and purified by column chromatography ( hexane : etoac , 6:1 to 4:1 ) , affording 7f as a colorless oil ( 58 mg , 74% ) . donor 2a ( 50 mg , 0.12 mmol ) and acceptor 6 g ( 26 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 6:1 to 3:1 ) : 7 g as a white solid ( 53 mg 78% ) . spectroscopic data in agreement with literature . following the general glycosylation procedure , donor 2a ( 50 mg , 0.12 mmol ) and acceptor 6h ( 22 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 10:1 to 4:1 ) : 7h as a pale yellow oil ( 53 mg 83% ) . donor 2a ( 50 mg , 0.12 mmol ) and acceptor 6i ( 23 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 10:1 to 4:1 ) : 7i as a pale yellow oil ( 56 mg 85% ) . h nmr ( 500 mhz ; cdcl3 ) 7.377.23 ( 15h , m , ph ) , 5.14 ( 1h , j = 9.8 hz , nh ) , 4.944.89 ( 2h , m , h-1 , ochhph ) , 4.624.53 ( 3h , m , och2ph , h-6a ) , 4.51 ( 1h , d , j = 11.8 hz , ochhph ) , 4.43 ( 1h , d , j = 11.8 hz , ochhph ) , 4.304.25 ( 2h , m , ochhph , och(ch3)ch(nh ) ) , 3.943.89 ( 2h , m , h-6b , och(ch3)ch(nh ) ) , 3.84 ( 1h , m , h-3 ) , 3.72 ( 3h , s , och3 ) , 3.603.51 ( 2h , m , 2h-4 , h-5 ) , 2.15 ( 1h , td , j = 12.3 , 3.8 hz , h-2a ) , 1.86 ( 1h , dd , j = 12.4 , 4.4 hz , h-2b ) , 1.48 ( 9h , s , 3 ch3 ) , 1.25 ( 3h , d , j = 6.5 hz , och(ch3)ch(nh ) ) ; c nmr ( 126 mhz ; cdcl3 ) 171.6 ( co ) , 156.0 ( co ) , 138.7 ( 4 c ) , 138.3 ( 4 c ) , 138.0 ( 4 c ) , 128.4 ( ch ) , 128.4 ( ch ) , 128.3 ( ch ) , 128.2 ( ch ) , 128.1 ( ch ) , 127.6 ( ch ) , 127.6 ( ch ) , 127.5 ( ch ) , 127.4 ( ch ) , 99.3 ( c-1 ) , 80.0 ( 4 c ) , 75.3 ( ch2ph ) , 74.2 ( ch2ph ) , 74.2 ( c-3 ) , 73.5 ( ch2ph ) , 72.9 ( c-4 ) , 70.3 [ och(ch3)ch(nh ) ] , 70.3 ( c-6 ) , 58.2 [ och(ch3)ch(nh ) ] , 52.2 ( och3 ) , 31.2 ( c-2 ) , 28.3 ( 3 x och3 ) , 18.3 [ och(ch3)ch(nh ) ] . esi - hrms for c37h47no9na ( mna ) calcd : 672.3149 ; found : 672.3144 . [ ]d = + 20 ( c = 1 , chcl3 ) . following the general glycosylation procedure , donor 2a ( 50 mg , 0.12 mmol ) and acceptor 6j ( 39 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 10:1 to 6:1 ) : 7j as a colorless oil ( 57 mg 74% ) . spectroscopic data in agreement with literature . following the general glycosylation procedure , donor 2b ( 23 mg , 0.12 mmol ) and acceptor 3 ( 47 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 4:1 to 2:1 ) : 5b as a colorless oil ( 52 mg , 80% ) : h nmr ( 400 mhz ; cdcl3 ) 7.417.22 ( 15h , m , ph ) , 5.024.96 ( 2h , m , h-1 , ochhph ) , 4.92 ( 1h , d , j = 10.9 hz , ochhph ) , 4.834.77 ( 2h , m , 2 ochhph ) , 4.68 ( 1h , d , j = 12.2 hz , ochhph ) , 4.62 ( 1h , d , j = 4.6 hz , ochhph ) , 4.60 ( 1h , d , j = 2.9 hz , h-1 ) , 4.01 ( 1h , t , j = 9.2 hz , h-3 ) , 3.883.79 ( 2h , m , h-4 , 6a ) , 3.74 ( 1h , ddd , j = 9.9 , 4.4 , 1.8 hz , h-5 ) , 3.63 ( 1h , dd , j = 11.4 , 2.0 hz , h-6b ) , 3.613.56 ( 2h , m , 3,5 ) , 3.563.51 ( 5h , m , h-4 , och3 , h-2 ) , 3.513.40 ( 2h , m , h-6a , h-6b ) , 3.39 , 3.37 ( 6h , 2s , 2 och3 ) , 3.30 ( 3h , s , och3 ) , 2.041.90 ( 2h , m , h-2a,h-2b ) . c nmr ( 101 mhz , cdcl3 ) 138.6 ( 4 c ) , 138.2 ( 4 c ) , 138.1 ( 4 c ) , 128.5 ( ch ) , 128.4 ( ch ) , 128.4 ( ch ) , 128.1 ( ch ) , 128.1 ( ch ) , 127.9 ( ch ) , 127.7 ( ch ) , 98.2 ( c-1 ) , 97.9 ( c-1 ) , 82.1 ( c-3 ) , 80.0 ( c-2 ) , 77.8 ( c-1 ) , 75.9 , 75.8 , 74.9 ( c-3 , c-4 , c-5 ) , 74.4 ( ch2ph ) , 73.3 ( ch2ph ) , 71.5 ( ch2ph ) , 69.7 ( c-6 ) , 69.7 ( c-5 ) , 66.1 ( c-4 ) , 60.9 ( och3 ) , 59.1 ( och3 ) , 56.1 ( och3 ) , 55.1 ( och3 ) , 30.7 ( c-2 ) . esi - hrms for c37h48nao10 ( mna ) calcd : 675.3145 ; found : 675.3144 . following the general glycosylation procedure , donor 2d ( 50 mg , 0.12 mmol ) and acceptor 3 ( 47 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac 6:1 to 3:1 ) : 10d as a colorless oil ( 73 mg , 86% ) : h nmr ( 400 mhz ; cdcl3 ) 7.417.19 ( 25h , m , ph ) , 5.03 ( 1h , d , j = 3.3 hz , h-1 ) , 4.99 ( 1h , d , j = 10.7 hz , ochhph ) , 4.94 ( 1h , d , j = 11.7 hz , ochhph ) , 4.87 ( 1h , d , j = 11.0 hz , ochhph ) , 4.834.77 ( 2h , m , ochhph ) , 4.724.63 ( 2h , m , ochhph ) , 4.634.55 ( 3h , m , h-1 , ochhph ) , 4.48 ( 1h , d , j = 11.1 hz , ochhph ) , 4.113.95 ( 3h , m , h-3 , h-6a,h-6b ) , 3.84 ( 1h , ddd , j = 11.9 , 4.5 , 2.3 hz , h-3 ) , 3.74 ( 4h , m , h-4 , h-6a , h-2 , h-5 ) , 3.60 ( 1h , d , j = 10.5 hz , h-6b ) , 3.51 ( 1h , dd , j = 9.6 , 3.6 hz , h-2 ) , 3.43 ( 1h , t , j = 9.3 hz , h-4 ) , 3.31 ( 3h , s , och3 ) , 2.20 ( 1h , td , j = 12.3 , 3.6 hz , h-2a ) , 2.03 ( 1h , dd , j = 12.5 , 4.4 hz , h-2b ) , 1.85 ( 3h , s , ch3co ) . c nmr ( 101 mhz , cdcl3 ) 170.5 ( ch3co ) 138.6 ( 4 c ) , 138.4 ( 4 c ) , 138.2 ( 4 c ) , 128.5 ( ch ) , 128.4 ( ch ) , 128.4 ( ch ) , 128.4 ( ch ) , 128.3 ( ch ) , 128.1 ( ch ) , 127.9 ( ch ) , 127.7 ( ch ) , 127.7 ( ch ) , 127.5 ( ch ) , 127.4 ( ch ) , 98.0 ( c-1 ) , 97.8 ( c-1 ) , 82.1 ( c-3 ) , 79.9 ( c-2 ) , 77.9 ( c-4 ) , 75.8 ( ch2ph ) , 74.9 ( ch2ph ) , 74.2 ( c-3 ) , 74.0 ( ch2ph ) , 73.2 ( ch2ph ) , 72.5 ( c-5 ) , 70.3 ( ch2ph ) , 69.7 ( h-4 ) , 69.1(h-5 ) , 65.9 ( c-6 ) , 64.0 ( c-6 ) , 55.0 ( och3 ) , 30.7 ( c-2 ) , 20.8 ( coch3 ) . esi - hrms for c50h56nao11 ( mna ) calcd : 855.3720 ; found : 855.3711 . [ ]d = + 8 ( c = 1 , chcl3 ) . following the general glycosylation procedure , donor 2e ( 32 mg , 0.12 mmol ) and acceptor 3 ( 46 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 5:1 to 4:1 ) : 10e as a pale yellow oil ( 58 mg , 80% ) : h nmr ( 500 mhz ; cdcl3 ) 7.417.22 ( 15h , m , ph ) , 6.005.79 ( 3h , m , ch = ch2 ) , 5.30 ( 1h , dt , j = 3.4 , 1.6 hz , ch = ch2 ) , 5.26 ( 1h , q , j = 1.8 hz , ch = ch2 ) , 5.23 ( 1h , tt , j = 2.3 , 1.1 hz , ch = ch2 ) , 5.17 ( 1h , dt , j = 3.4 , 1.6 hz , ch = ch2 ) , 5.155.12 ( 2h , m , ch = ch2 ) , 5.01 ( 1h , d , j = 2.6 hz , h-1 ) , 4.99 ( 1h , d , j = 10.8 hz , ochhph ) , 4.91 ( 1h , d , j = 10.8 hz , ochhph ) , 4.82 ( 1h , d , j = 5.3 hz , ochhph ) , 4.80 ( 1h , d , j = 6.6 hz , ochhph ) , 4.69 ( 1h , d , j = 12.2 hz , ochhph ) , 4.62 ( 1h , d , j = 2.5 hz , h-1 ) , 4.61 ( 1h , d , j = 4.8 hz , ochhph ) , 4.35 ( 1h , ddt , j = 12.7 , 5.6 , 1.4 hz , ochhch = ch2 ) , 4.134.06 ( 1h , m , ochhch = ch2 ) , 4.054.02 ( 2h , m , ochhch = ch2 , h-3 ) , 4.013.97 ( 1h , m , ochhch = ch2 ) , 3.943.91 ( 2h , ochhch = ch2 ) , 3.883.81 ( 2h , m , h-4 , h-6a ) , 3.783.73 ( 3h , m , h-4 , h-3 , h-5 ) , 3.64 ( 1h , dd , j = 11.4 , 2.0 hz , h-6b ) , 3.58 ( 1h , dd , j = 9.3 , 7.3 hz , h-5 ) , 3.563.52 ( 1h , m , h-2 ) , 3.513.46 ( 2h , m , h-6a , h-6b ) , 3.37 ( 3h , s , och3 ) , 2.142.07 ( 1h , m , h-2a ) , 1.951.90 ( 1h , m , h-2b).c nmr ( 126 mhz , cdcl3 ) 138.7 ( 4 c ) , 138.2 ( 4 c ) , 138.1 ( 4 c ) , 135.6 ( ch = ch2 ) , 134.8 ( ch = ch2 ) , 134.6 ( ch = ch2 ) , 128.5 ( ch ) , 128.4 ( ch ) , 128.04 ( ch ) , 127.9 ( ch ) , 127.7 ( ch ) , 127.7 ( ch ) , 116.9 ( ch = ch2 ) , 116.8 ( ch = ch2 ) , 116.6 ( ch = ch2 ) , 98.2 ( c-1 ) , 97.9 ( c-1 ) , 82.1 ( c-3 ) , 80.0 ( c-5 ) , 77.9 ( c-2 ) , 75.8 ( ch2ph ) , 74.9 ( ch2ph ) , 73.9 ( c-4 ) , 73.5 ( och2ch ) , 73.3 ( ch2ph ) , 72.4 ( h-5 ) , 72.2 ( och2ch ) , 69.8 ( 2c , c-4 , c-3 ) , 69.2 ( och2ch ) , 69.1 ( c-6 ) , 66.0 ( c-6 ) , 55.0 ( och3 ) , 31.1 ( c-2 ) . esi - hrms for c43h54nao10 ( mna ) calcd : 753.3615 ; found : 753.3610 . following the general glycosylation procedure , donor 2f ( 50 mg , 0.10 mmol ) and acceptor 3 ( 39 mg , 0.09 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 5:1 to 4:1 ) : 10f as a colorless oil ( 64 mg , 79% ) : h nmr ( 400 mhz , cdcl3 ) : 7.387.24 ( 15h , m , ph ) , 4.994.95 ( 2h , m , h-1 , ochhph ) , 4.87 ( 1h , d , j = 10.8 hz , ochhph ) , 4.83 ( 1h , d , j = 10.7 hz , ochhph ) , 4.80 ( 1h , d , j = 12.2 hz , ochhph ) , 4.67 ( 1h , d , j = 12.2 hz , ochhph ) , 4.61 ( 1h , d , j = 10.8 hz , ochhph ) , 4.57 ( 1h , d , j = 3.6 hz , h-1 ) , 4.05 ( 1h , m , h-3 ) , 3.99 ( 1h , t , j = 9.2 hz , h-2 ) , 3.80 ( 1h , bs , h-4 ) , 3.773.73 ( 2h , m , h-3 , h-6a ) , 3.703.62 ( 4h , m , h-5 , h-6a , h-6b , h-6b ) , 3.49 ( 1h , dd , j = 9.6 , 3.6 hz , h-4 ) , 3.42 ( 1h , t , j = 9.6 h-5 ) , 3.36 ( 3h , s , och3 ) , 2.08 ( 1h , td , j = 12.1 , 3.5 , h-2a ) , 1.65 ( 1h , dd , j = 12.7 , 4.5 h-2b ) , 0.940.86 ( 27h , m , 3 sic(ch3)3 ) , 0.120.00 ( 18h , m , 6 x sich3 ) ; c nmr ( 126 mhz ; cdcl3 ) 138.7 ( 4 c ) , 138.2 ( 4 c ) , 138.1 ( 4 c ) , 128.4127.7 ( ph ) , 97.9 ( c-1 ) , 97.6 ( c-1 ) , 82.1 ( c-2 ) , 79.9 ( c-4 ) , 78.5 ( c-5 ) , 75.8 ( och2ph ) , 75.1 ( och2ph ) , 73.3 ( och2ph ) , 72.7 ( c-5 ) , 70.3 ( c-4 ) , 68.3 ( c-3 ) , 65.2 ( c-6 ) , 62.6 ( c-6 ) , 54.7 ( och3 ) , 33.5 ( c-2 ) , 26.2 ( sic(ch3)3 ) , 26.1 ( sic(ch3)3 ) , 25.8 ( sic(ch3)3 ) , 18.6 ( sic(ch3)3 ) , 18.5 ( sic(ch3)3 ) , 18.1 ( sic(ch3)3 ) , 3.9 ( sich3 ) , 4.4 ( sich3 ) , 4.8 ( sich3 ) , 5.0 ( sich3 ) , 5.3 ( sich3 ) , 5.4 ( sich3 ) . esi - hrms for c52h84nasi3o10 ( mna ) calcd : 975.5270 ; found : 975.5277 . [ ]d = + 16 ( c = 1 , chcl3 ) . following the general glycosylation procedure , donor 8a ( 50 mg , 0.10 mmol ) and acceptor 3 ( 50 mg , 0.08 mmol ) afforded the following purification by column chromatography ( hexane : etoac 5:1 to 4:1 ) : 11a as colorless oil ( 28 mg , 40% ) . donor 8b ( 50 mg , 0.10 mmol ) and acceptor 3 ( 40 mg , 0.08 mmol ) afforded the following purification by column chromatography ( hexane : etoac 5:1 to 4:1 ) : 11b as colorless oil ( 66 mg , 81% ) . donor 8c ( 50 mg , 0.10 mmol ) and acceptor 6k ( 30 mg , 0.08 mmol ) afforded the following purification by column chromatography ( hexane : etoac 5:1 to 4:1 ) : 11c as colorless oil ( 60 mg , 82% ) : h nmr ( 400 mhz , cdcl3 ) : 7.507.44 ( 2h , m , ph ) , 7.417.31 ( 5h , m , ph ) , 7.257.20 ( 3h , m , ph ) , 5.53 ( 1h , s , phcho2 ) , 5.09 ( 1h , d , j = 3.3 hz , h-1 ) , 4.99 ( 1h , d , j = 3.3 hz , h-1 ) , 4.88 ( 1h , d , j = 11.7 hz , ochhph ) , 4.78 ( 1h , d , j = 11.7 hz , ochhph ) , 4.29 ( 1h , dd , j = 9.9 , 4.5 hz , chh ) , 4.13 ( 1h , ddd , j = 11.3 , 8.3 , 5.3 hz , h-3 ) , 4.074.01 ( 1h , m , chh ) , 3.94 ( 1h , dd , j = 9.4 , 3.4 hz , h-2 ) , 3.92 ( 1h , dd , j = 9.4 , 8.9 hz , h-3 ) , 3.873.75 ( 3h , m , h-5 , h-5 , chh ) , 3.71 ( 1h , t , j = 10.1 hz , chh ) , 3.61 ( 1h , t , j = 9.1 hz , h-4 ) , 3.51 ( 1h , dd , j = 9.2 , 8.4 hz , h-4 ) , 3.43 ( 3h , s , och3 ) , 2.25 ( 1h , app dd , j = 13.2 , 5.1 hz , h-2a ) , 1.73 ( 1h , ddd , j = 13.5 , 11.4 , 3.7 hz , h-2b ) , 1.170.86 ( 49h , m , 7 x sich(ch3)2 ) ; c nmr ( 101 mhz , cdcl3 ) : 139.0 ( 4 c ) , 137.6 ( 4 c ) , 129.0 ( ch ) , 128.28 ( ch ) , 128.25 ( ch ) , 127.7 ( ch ) , 127.4 ( ch ) , 126.2 ( ch ) , 101.5 ( phcho2 ) , 97.4 ( c-1 ) , 92.9 ( c-1 ) , 81.5 ( h-4 ) , 77.3 ( c-3 ) , 75.2 ( phch2o ) , 74.7 ( c-4 ) , 73.7 ( ch ) , 73.5 ( c-2 ) , 71.9 ( c-3 ) , 69.2 , 63.5 ( c-6 , c-6 ) , 62.6 ( ch ) , 55.3 ( och3 ) , 38.0 ( c-2 ) , 18.24 ( si(ch(ch3)2 ) ) , 18.20 ( si(ch(ch3)2 ) ) , 18.15 ( si(ch(ch3)2 ) ) , 17.7 ( si(ch(ch3)2 ) ) , 17.6 ( si(ch(ch3)2))0 , 17.59 ( si(ch(ch3)2 ) ) , 17.53 ( si(ch(ch3)2 ) ) , 17.48 ( si(ch(ch3)2 ) ) , 17.45 ( si(ch(ch3)2 ) ) , 17.4 ( si(ch(ch3)2 ) ) , 13.1 ( si(ch(ch3)2 ) ) , 12.5 ( si(ch(ch3)2 ) ) , 12.4 ( si(ch(ch3)2 ) ) , 12.2 ( si(ch(ch3)2 ) ) , 12.2 ( si(ch(ch3)2 ) ) . maldi - hrms for c48h80nao11si3 ( mna ) calcd : 939.4869 ; found : 939.4896 . [ ]d22 = + 51 ( c = 0.0030 , chcl3 ) . following the general glycosylation procedure , donor 9 ( 50 mg , 0.13 mmol ) and acceptor 3 ( 52 mg , 0.11 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 8:1 to 4:1 ) 12 as a colorless oil ( 78 mg , 74% , : = 9:1 ) . spectroscopic data in agreement with literature . following the general procedure , donor 9 ( 50 mg , 1.3 mmol ) and acceptor 6k ( 42 mg , 1.1 mmol ) afforded the following purification by column chromatography ( hexane : etoac 20:1 to 10:1 ) : 12b as colorless oil ( 72 mg , 87% , : = 10:1 ) : h nmr ( 400 mhz , cdcl3 ) : 7.527.48 ( 2h , m , ph ) , 7.407.25 ( 8h , m , ph ) , 5.58 ( 1h , s , phcho2 -anomer ) , 5.56 ( 1h , s , phcho2 -anomer ) , 5.07 ( 1h , d , j = 3.9 hz , h-1 ) , 4.884.84 ( 2h , m , h-1 , ochhph ) , 4.78 ( 1h , d , j = 11.4 hz , ochhph ) , 4.30 ( 1h , dd , j = 9.9 , 4.5 hz , h-6a ) , 4.07 ( 1h , ddd , j = 11.3 , 8.3 , 5.3 hz , h-3 ) , 3.97 ( 1h , t , j = 9.3 , h-5 ) , 3.893.72 ( 3h , m , h-4 , h-5 , h-6b ) , 3.693.60 ( 2h , m , h-2 , h-3 ) , 3.45 ( 1h , s , och3 -anomer ) , 3.45 ( 1h , s , och3 -anomer ) , 3.27 ( 1h , j = 8.7 hz , h-4 ) ( 2.24 ( 1h , app dd , j = 13.2 , 5.1 hz , h-2a ) , 1.74 ( 1h , ddd , j = 13.5 , 11.4 , 3.7 hz , h-2b ) , 1.29 ( 3h , d , j = 6.3 hz , ch3 ) 1.141.02 ( 28h , m , 4 sich(ch3)2 ) ; c nmr ( 101 mhz , cdcl3 ) : 138.66 ( 4 c ) , 137.42 ( 4 c ) , 128.89 ( ch ) , 128.28 ( ch ) , 128.19 ( ch ) , 127.7 ( ch ) , 127.57 ( ch ) , 126.0 ( ch ) , 101.25 ( phcho2 ) , 100.6 ( c-1 ) , 100.25 ( c-1 ) , 82.0 ( c-2 ) , 80.37 ( c-3 ) , 79.99 ( c-4 ) , 77.94 ( c-5 ) , 75.25 ( phch2o ) , 71.22 ( c-3 ) , 69.07 ( c-6 ) , 68.3(c-5 ) , 62.23 ( c-4 ) , 55.2 ( och3 ) , 38.67 ( c-2 ) , 29.7 ( ch3 ) , 18.1412.27 ( 4 si(ch(ch3)2 ) ) . esi - hrms for c39h60nao10 ( mna ) calcd : 767.3623 ; found : 767.4002 . [ ]d = + 25 ( c = 1 , chcl3 ) . [ ]d22 = + 32 ( c = 1 , chcl3 ) . following general procedure , donor 2a ( 50 mg , 0.12 mmol ) , acceptor 6d ( 58 mg , 0.0998 mmol ) , and a solution mixture , prepared containing chiral acid ( 0.012 mmol ) and thiourea ( 0.012 mmol ) in anhydrous dcm ( 1 ml ) , were mixed together . after 2 h , the disaccharide formation was determined to be complete by tlc . the reaction mixture was then cooled to 0 c , and molecular sieves 4 were added . once cooled , a solution of 1,2:3,4-di - o - isopropylidene--d - galactopyranoside 6c ( 26 mg , 0.0998 mmol ) in anhydrous dcm ( 0.5 ml ) was added followed by addition of nis ( 45 mg , 0.2 mmol ) and finally the dropwise addition of a solution of tmsotf ( 95 l , 40 l in 20 ml anhydrous dcm , 0.001 mmol ) . after 1 h , the reaction was determined to be complete and was quenched with et3n ( 0.1 ml ) , diluted with dcm ( 25 ml ) , washed with na2s2o3 ( sat . ( 25 ml ) , brine ( 25 ml ) , dried over mgso4 , and concentrated in vacuo . purification by column chromatography ( hexane : etoac , 10:1 to 3:1 ) afforded product 13 as a white solid ( 59 mg , 51% ) . spectroscopic data in agreement with literature . following the general glycosylation procedure , donor 14 ( 50 mg , 0.12 mmol ) and acceptor 3 ( 46 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac 20:1 to 12:1 ) : 15 as a yellow oil ( 72 mg 83% ) : h nmr ( 500 mhz ; cdcl3 ) 7.407.21 ( m , 30h , ar h ) , 5.04 ( 1h , d , j = 1.3 hz , h-1 ) , 5.00 ( 1h , d , j = 10.8 hz , ochhph ) , 4.93 ( 1h , d , j = 11.6 hz , ochhph ) , 4.86 ( 1h , d , j = 10.9 hz , ochhph ) , 4.834.78 ( 2h , m , ochhph ) , 4.69 ( 1h , d , j = 12.2 hz , ochhph ) , 4.62 ( 1h , d , j = 2.7 hz , h-1 ) , 4.61 ( 1h , d , j = 5.4 hz , ochhph ) , 4.58 ( 2h , s , ochhph ) , 4.54 ( 1h , d , j = 10.8 hz , ochhph ) , 4.42 ( 1h , d , j = 11.8 hz , ochhph ) , 4.35 ( 1h , d , j = 11.8 hz , ochhph ) , 4.043.96 ( 1h , t , j = 9.3 hz , h-3 ) , 3.89 ( 3h , t , j = 3.1 hz , h-3 , h-4 , h-5 ) , 3.83 ( 1h , dd , j = 11.4 , 4.7 hz , h-6a ) , 3.74 ( 1h , ddd , j = 10.0 , 4.6 , 1.8 hz , h-5 ) , 3.63 ( 1h , dd , j = 11.4 , 1.9 hz , h-6b ) , 3.583.54 ( 2h , m , h-6a , h-2 ) , 3.543.51 ( 1h , m , h-6b ) , 3.48 ( 1h , dd , j = 10.1 , 8.9 hz , h-4 ) , 3.33 ( 3h , s , och3 ) , 2.031.99 ( 1h , m , h-2 ) . c nmr ( 126 mhz , cdcl3 ) 138.9 ( 4 c ) , 138.7 ( 4 c ) , 138.4 ( 4 c ) , 138.3 ( 4 c ) , 138.16 ( 4 c ) , 138.1 ( 4 c ) , 128.41 ( ch ) , 128.2 ( ch ) , 127.7 ( ch ) , 98.3 ( c-1 ) , 97.9 ( c-1 ) , 82.1 ( c-3 ) , 80.0 ( c-2 ) , 77.9 ( c-4 ) , 75.8 ( ch2ph ) , 74.9 ( ch2ph ) , 74.3 ( ch2ph ) , 74.16 ( c-3 ) , 73.3 ( ch2ph ) , 73.3 ( ch2ph ) , 72.9 ( c-4 ) , 70.2 ( ch2ph ) , 70.1 ( ch2ph ) , ( c-5 ) , 69.8 ( c-5 ) , 69.4 ( c-6 ) , 66.0 ( c-6 ) , 55.0 ( och3 ) , 29.7 ( c-2 ) . esi - hrms for c55h59dnao10 ( mna ) calcd : 904.4147 ; found : 904.4142 .

a practical approach for the -stereoselective synthesis of deoxyglycosides using cooperative brnsted acid - type organocatalysis has been developed . the method is tolerant of a wide range of glycoside donors and acceptors , and its versatility is exemplified in the one - pot synthesis of a trisaccharide . mechanistic studies suggest that thiourea - induced acid amplification of the chiral acid via h - bonding is key for the enhancement in reaction rate and yield , while stereocontrol is dependent on the chirality of the acid .

Introduction Results and Discussion Conclusions Experimental Section

cooperative catalysis between thioureas and brnsted acids , whereby the enhanced catalytic activity of the thiourea acts as an acid amplifier , has successfully been applied to asymmetric catalysis , and more recently , reports have emerged from the schmidt group applying this type of synergistic catalysis to glycosylation reactions involving o - glycosyl trichloroacetimidate donors to yield -selective glycosides . acid - catalyzed direct nucleophilic substitution on a glycal is one of the most widely used and efficient methods for their synthesis , however these reactions often give anomeric mixtures and side products ( e.g. as part of our ongoing interest in developing stereoselective glycosylation methods , we decided to focus our attention on the synthesis of deoxyglycosides . recently , our team reported a mild organocatalytic method for the preparation of 2-deoxygalactosides employing schreiner s thiourea with excellent yields and -selectivity . although the method worked well with galactals , reactions needed to be refluxed over 24 h to reach completion , and in general , the thiourea catalyst was unable to activate glucal or rhamnal substrates . we postulated that synergistic acid / thiourea activation could provide a more efficient and practical glycosylation strategy for the preparation of deoxyglycosides than current methods that use hydrogen - bonding organocatalysts or acids as the sole promoters ( scheme 1 ) . moreover , binol - derived phosphoric acids have been shown to be particularly effective in many asymmetric transformations , including some success in glycosylations involving trichloroacetimidate glycoside donors . initial experiments began with the screening of a series of commercial binol - derived phosphoric acids for their ability to promote the stereoselective glycosylation of perbenzylated galactal 2a with glucoside acceptor 3(12 ) in the absence and presence of 1 in ch2cl2 at room temperature . when 10 mol % of 1 was added as the cocatalyst ( entries 3 and 5 ) , reactions were complete within 3 h , and glycoside 5 was obtained in improved yields of 82% and 89% , respectively , and with an : ratio of 7:1 for the ( s)-acid and > 30:1 for the ( r)-acid catalyst . these results demonstrate the strong influence of thiourea 1 , as a cocatalyst , on reaction rate and yield , while the stereochemistry of the acid catalyst has the biggest effect on the : selectivity of the glycosylation reaction . this observation is not completely surprising , as bennett and co - workers had already reported that the stereo - outcome of acid - catalyzed glycosylation reactions involving trichloroacetimidate donors was dependent on the chirality of the catalyst , the configuration of the leaving group in the glycoside donor , and the nature of the nucleophile acceptor . it is important to note that glycosylation reactions with 10 mol % of thiourea 1 in ch2cl2 ( in the absence of acid ) yielded only starting material ( entry 1 ) as previously observed by our team , likely due to catalyst inactivation via self - association at high catalyst loading , further demonstrating the importance of the synergy between both cocatalysts . from h nmr ; n / a = not applicable the use of achiral p - toluenesulfonic acid ( ptsoh , 5 mol % ) 4e or bis(4-nirophenyl)hydrogen phosphate 4f or less acidic phosphoric catalysts 4c and 4d ( pka = 3.86 ) in combination with 1 proved to be detrimental to reaction rate , yield , and stereocontrol , with reactions needing 620 h for completion ( entries 69 ) . increasing the acid loading to 20 mol % in the presence of 10 mol % of 1 had no effect on the outcome of the model reaction ( entry 10 ) , while lowering the reaction temperature significantly slowed product formation ( entry 11 ) . changing the reaction solvent to either mecn or diethyl ether ( table 1 , entries 12 and 13 ) had a detrimental effect on the rate of the reaction . in the case of mecn , which is known to participate during the glycosylation reaction involving oxocarbenium ions favoring -glycoside products , significant erosion of the -selectivity was observed with 5 being isolated as a 3:1 : mixture ( entry 11 vs entry 5 ) . having established the optimum reaction conditions , our attention was then turned to exploring the scope of the cooperative catalytic system on coupling reactions between 2a and a range of oh nucleophiles 6a6j ( table 2 ) . in all cases , reactions proceeded smoothly within 26 h and in excellent yields and -selectivity , as determined by the characteristic anomeric signals in the h- and c nmr spectra , demonstrating that the catalytic system tolerates the presence of common alcohol and amine protecting groups such as acetals , ethers , esters , and carbamates . reactions with secondary alcohols such as phenols 6e , glycosides 6f and 6 g , boc - protected threonine 6i , or cholesterol 6j prove to be more challenging and required higher reaction temperatures ( 45 c ) and the use of 20 mol % of 1 in combination with 10 mol % of 4b in ch2cl2 , to drive reactions to completion within 56 h. under these optimized conditions , the desired products were thus isolated with similar high -selectivity ( > 30:1 : ratio ) and yields of 7485% ( table 2 , entries 57 , 9 , and 10 ) . our attention then turned to exploring the scope of the glycal donor . to that end , a series of differentially protected galactals 2b2f , glucals 8a8c , and l - rhamnal 9 bearing methyl , acetate , benzyl , allyl , silyl ether , and siloxane protecting groups were prepared and subjected to the reaction conditions with 3 or 6k , which bears a secondary oh , as the acceptor ( table 3 ) . pleasingly , high yields and excellent selectivities for -linked glycosides were obtained in most examples ( entries 1 , 35 , and 7 ) . although substrate 2d , which bears an acetate group at c-6 , shows that ester groups can be tolerated ( entry 3 ) , in the case of acetyl - protected galactal 2c ( entry 2 ) , an inseparable mixture of glycosides including ferrier side - products was observed after 20 h under the more forcing conditions ( 20 mol % of 4b and reflux in ch2cl2 ) . this lack of reactivity has been previously noted for glycals bearing a deactivating group ( e.g. encouragingly , the reaction was also amenable to glycosylations with glucal substrates , and both benzyl - derivative 8a and 3,4-o - siloxane protected 8b(6b ) or 8c(6b ) afforded the corresponding glycosides with high -stereocontrol within 2 h , albeit 11a was isolated in a lower yield ( 40% ) than 11b ( 81% yield ) or 11c ( 82% ) . this is not completely unexpected , as 8b has been shown to be a more effective glycosylation donor than its benzylated counterpart ( table 3 , entries 68 ) . reaction performed at 45 c , 1.5 equiv of donor , and 0.2 equiv of thiourea . 2,6-dideoxyglycosides are also an important class of compounds , and their stereoselective synthesis is further complicated by the lack of oxygen substituents at both c-2 and c-6 . it is important to highlight that thiourea 1 alone was not able to activate any of the glucal and rhamnal substrates . these results further emphasize that the synergistic catalytic system works well across a range of reactivity profiles for both glycal donors and nucleophile acceptors . having found that thioglycosides are inert under the cooperative organocatalytic glycosylation conditions ( table 2 , entry 4 ) , we decided to evaluate the methodology in a three - component , one - pot synthesis of trisaccharide 13 ( scheme 2 ) . to probe the mechanism of our reaction , a 3:1 /-anomeric mixture of 5 was subjected to the reaction conditions and gave no change in the anomeric ratio , indicating that the high -selectivity is not the result of anomerization . moreover , reaction with deuterated galactal 14 yielded disaccharide 15 with the newly formed bonds cis to each other , as evidenced by the h nmr shifts associated with h-1 ( 4.62 ppm , d , 1h , j = 2.7 hz ) and h-2 ( 2.031.99 , m , 1h , h-2 ) of the deuterated 2-deoxygalactoside ( scheme 3 ) , which suggest that both the c h and the c o bond formation steps are syn - diastereoselective . furthermore , nmr mixtures of thiourea 1 , acid 4b , and glycoside donor 2a showed additional downfield h - shifts associated with the anomeric protons in 2a ( 6.33 ppm ) , while shifts for the oh signal from acceptor 3 ( from 1.84 ppm to 2.54 ppm ) were detected in mixtures of 1 , 4b , and 3 ( see supporting information for details ) . these results further demonstrate that the combination of 1 and chiral acid ( r)-4b leads to a cooperative catalytic system that can activate both enol ether and oh nucleophiles in a synergistic fashion , leading to an enhancement in reaction rate and -stereocontrol of the glycosylation reaction . as proposed in scheme 3 , our findings suggest that a hydrogen - bond - mediated complex between thiourea 1 and acid 4b leads to a urea - induced acid amplification , and proton addition from this complex to the less hindered face of the enol ether ( i ) forms a short - lived oxocarbenium intermediate ( ii ) that is rapidly trapped by the oh nucleophile which is in turn activated by the phosphate intermediate that is generated in situ , to give 15 . this might help to explain the effect the chirality of the phosphoric acid has on the stereo outcome of the glycosylation reaction . in summary , we have described a practical , highly stereoselective , and efficient direct glycosylation method for glycals using a cooperative brnsted acid - organocatalytic promoter system . the reaction is widely applicable to a range of glycosyl donors and nucleophile acceptors , proceeds with excellent yields and high selectivity for the -anomer , and is tolerant of most common protecting groups . we note that the stereoselectivity of the reaction is highly dependent on the chirality of the acid , with ( r)-4b leading to the formation of -glycosides preferentially , while both yield and rate of reaction are greatly enhanced by the synergistic interaction between thiourea and acid . moreover , we exemplify the generality of the approach in the stereoselective synthesis of a series of disaccharides , glycosyl - amino acids , and other glycoconjugates and also in the one - pot chemo- and stereoselective synthesis of a trisaccharide . further work from our lab is underway to exploit the cooperative catalysis between organocatalysts and chiral acids for the stereoselective synthesis of other important glycosides and chiral acetals . extracts were concentrated in vacuo using both a bchi rotary evaporator ( bath temperatures up to 40 c ) at a pressure of either 15 mmhg ( diaphragm pump ) or 0.1 mmhg ( oil pump ) , as appropriate , and a high - vacuum line at room temperature . h nmr and c nmr spectra were measured in the solvent stated at 400 or 500 mhz . chemical shifts are quoted in parts per million from residual solvent peak ( cdcl3 : h , 7.26 ppm and c , 77.16 ppm ) and coupling constants ( j ) given in hertz . a mixture of chiral acid ( 0.1 equiv ) and thiourea ( 0.1 equiv for primary acceptors and 0.2 equiv for secondary acceptors ) in anhydrous dcm ( 1 ml ) was stirred for 30 min and then added to the microwave vial containing the donor and acceptor . the reaction mixture was stirred at rt ( primary acceptors ) or heated at reflux ( secondary acceptors ) until the reaction was determined to be complete by either tlc or nmr analysis of the crude material . h nmr ( 400 mhz ; cdcl3 ) 7.397.19 ( 30h , m , ph ) , 5.02 ( 1h , app d , j = 2.4 hz , h-1 ) , 4.98 ( 1h , d , j = 10.8 hz , och hph ) , 4.91 ( 1h , d , j = 11.6 hz , och hph ) , 4.84 ( 1h , d , j = 10.9 hz , och hph ) , 4.80 ( 1h , d , j = 10.8 hz , ochh ph ) , 4.79 ( 1h , d , j = 12.2 hz , och hph ) , 4.68 ( 1h , d , j = 12.2 hz , ochh ph ) , 4.60 ( 1h , d , j = 3.6 hz , h-1 ) , 4.60 ( 1h , d , j = 11.6 hz , ochh ph ) , 4.57 ( 2h , s , och2 ph ) , 4.52 ( 1h , d , j = 10.9 hz , ochh ph ) , 4.41 ( 1h , d , j = 12.0 hz , och hph ) , 4.34 ( 1h , d , j = 12.0 hz , ochh ph ) , 3.98 ( 1h , t , j = 9.2 hz , h-3 ) , 3.903.84 ( 3h , m , h-3 , h-4 , h-5 ) , 3.81 ( 1h , dd , j = 11.2 , 4.6 hz , h-6a ) , 3.72 ( 1h , ddd , j = 10.0 , 4.5 , 1.6 hz , h-5 ) , 3.61 ( 1h , dd , j = 11.4 , 1.7 hz , h-6b ) , 3.54 ( 1h , dd , j = 9.5 , 7.3 hz , h-6a ) , 3.51 ( 1h , dd , j = 9.5 , 3.5 hz , h-2 ) , 3.50 ( 1h , dd , j = 9.5 , 5.9 hz , h-6b ) , 3.46 ( 1h , dd , j = 9.9 , 9.0 hz , h-4 ) , 3.31(3h , s , och3 ) , 2.20 ( 1h , td , j = 12.4 , 3.7 hz , h-2a ) , 2.01 ( 1h , app dd , j = 12.7 , 4.5 hz , h-2b ) ; c nmr ( 126 mhz ; cdcl3 ) 139.0 ( 4 c ) , 138.9 ( 4 c ) , 138.5 ( 4 c ) , 138.4 ( 4 c ) , 138.30 ( 4 c ) , 138.27 ( 4 c ) , 128.6 ( ch ) , 128.54 ( ch ) , 128.45 ( ch ) , 128.33 ( ch ) , 128.32 ( ch ) , 128.18 ( ch ) , 128.16 ( ch ) , 128.0 ( ch ) , 127.82 ( ch ) , 127.81 ( ch),127.78 ( ch ) , 127.7 ( ch ) , 127.6 ( ch ) , 127.5 ( ch ) , 98.4 ( c-1 ) , 98.0 ( c-1 ) , 82.3 ( c-3 ) , 80.1 ( c-2 ) , 78.1 ( c-4 ) , 76.0 ( c h2 ph ) , 75.1 ( c h2 ph ) , 74.41 , 74.35 ( c-3 , c h2 ph ) , 73.5 ( c h2 ph ) , 73.4 ( c h2 ph ) , 73.1 ( c-4 ) , 70.4 , 70.2 ( c-5 , ch2ph ) , 70.0 ( c-5 ) , 69.5 ( c-6 ) , 66.2 ( c-6 ) , 55.2 ( och3 ) , 31.2 ( c-2 ) . spectroscopic data in agreement with literature . [ ]d = + 20 ( c = 1 , chcl3 ) . spectroscopic data in agreement with literature . [ ]d22 = + 32 ( c = 1 , chcl3 ) . following general procedure , donor 2a ( 50 mg , 0.12 mmol ) , acceptor 6d ( 58 mg , 0.0998 mmol ) , and a solution mixture , prepared containing chiral acid ( 0.012 mmol ) and thiourea ( 0.012 mmol ) in anhydrous dcm ( 1 ml ) , were mixed together . the reaction mixture was then cooled to 0 c , and molecular sieves 4 were added . once cooled , a solution of 1,2:3,4-di - o - isopropylidene--d - galactopyranoside 6c ( 26 mg , 0.0998 mmol ) in anhydrous dcm ( 0.5 ml ) was added followed by addition of nis ( 45 mg , 0.2 mmol ) and finally the dropwise addition of a solution of tmsotf ( 95 l , 40 l in 20 ml anhydrous dcm , 0.001 mmol ) . ( 25 ml ) , brine ( 25 ml ) , dried over mgso4 , and concentrated in vacuo . purification by column chromatography ( hexane : etoac , 10:1 to 3:1 ) afforded product 13 as a white solid ( 59 mg , 51% ) . spectroscopic data in agreement with literature . esi - hrms for c55h59dnao10 ( mna ) calcd : 904.4147 ; found : 904.4142 .

cooperative catalysis between thioureas and brnsted acids , whereby the enhanced catalytic activity of the thiourea acts as an acid amplifier , has successfully been applied to asymmetric catalysis , and more recently , reports have emerged from the schmidt group applying this type of synergistic catalysis to glycosylation reactions involving o - glycosyl trichloroacetimidate donors to yield -selective glycosides . the stereoselective formation of glycosidic linkages employing 2-deoxyglycosides , where a substituent at c-2 that can direct the coupling reaction is lacking , is a very challenging endeavor and many efforts have been devoted to achieve their stereoselective synthesis . we postulated that synergistic acid / thiourea activation could provide a more efficient and practical glycosylation strategy for the preparation of deoxyglycosides than current methods that use hydrogen - bonding organocatalysts or acids as the sole promoters ( scheme 1 ) . more recently , chiral phosphoric acids have also been shown to catalyze the spiroketalization of cyclic enol ethers bearing a pendant alcohol nucleophile in a highly diastereoselective syn - selective concerted mechanism whereby the phosphoric acid acts as a bifunctional catalyst activating both the alkene and alcohol nucleophile . initial experiments began with the screening of a series of commercial binol - derived phosphoric acids for their ability to promote the stereoselective glycosylation of perbenzylated galactal 2a with glucoside acceptor 3(12 ) in the absence and presence of 1 in ch2cl2 at room temperature . as summarized in table 1 , coupling reactions employing 10 mol % of chiral phosphoric acids ( s)-4a and ( r)-4b ( pka = 2.63 in dmso ) proceeded cleanly to give product 5(7 ) in 55 and 70% yields , albeit after 20 h ( entries 2 and 4 ) and with different degrees of : stereocontrol 9:1 and > 30:1 , respectively . when 10 mol % of 1 was added as the cocatalyst ( entries 3 and 5 ) , reactions were complete within 3 h , and glycoside 5 was obtained in improved yields of 82% and 89% , respectively , and with an : ratio of 7:1 for the ( s)-acid and > 30:1 for the ( r)-acid catalyst . these results demonstrate the strong influence of thiourea 1 , as a cocatalyst , on reaction rate and yield , while the stereochemistry of the acid catalyst has the biggest effect on the : selectivity of the glycosylation reaction . this observation is not completely surprising , as bennett and co - workers had already reported that the stereo - outcome of acid - catalyzed glycosylation reactions involving trichloroacetimidate donors was dependent on the chirality of the catalyst , the configuration of the leaving group in the glycoside donor , and the nature of the nucleophile acceptor . it is important to note that glycosylation reactions with 10 mol % of thiourea 1 in ch2cl2 ( in the absence of acid ) yielded only starting material ( entry 1 ) as previously observed by our team , likely due to catalyst inactivation via self - association at high catalyst loading , further demonstrating the importance of the synergy between both cocatalysts . from h nmr ; n / a = not applicable the use of achiral p - toluenesulfonic acid ( ptsoh , 5 mol % ) 4e or bis(4-nirophenyl)hydrogen phosphate 4f or less acidic phosphoric catalysts 4c and 4d ( pka = 3.86 ) in combination with 1 proved to be detrimental to reaction rate , yield , and stereocontrol , with reactions needing 620 h for completion ( entries 69 ) . increasing the acid loading to 20 mol % in the presence of 10 mol % of 1 had no effect on the outcome of the model reaction ( entry 10 ) , while lowering the reaction temperature significantly slowed product formation ( entry 11 ) . in the case of mecn , which is known to participate during the glycosylation reaction involving oxocarbenium ions favoring -glycoside products , significant erosion of the -selectivity was observed with 5 being isolated as a 3:1 : mixture ( entry 11 vs entry 5 ) . having established the optimum reaction conditions , our attention was then turned to exploring the scope of the cooperative catalytic system on coupling reactions between 2a and a range of oh nucleophiles 6a6j ( table 2 ) . in all cases , reactions proceeded smoothly within 26 h and in excellent yields and -selectivity , as determined by the characteristic anomeric signals in the h- and c nmr spectra , demonstrating that the catalytic system tolerates the presence of common alcohol and amine protecting groups such as acetals , ethers , esters , and carbamates . reactions with secondary alcohols such as phenols 6e , glycosides 6f and 6 g , boc - protected threonine 6i , or cholesterol 6j prove to be more challenging and required higher reaction temperatures ( 45 c ) and the use of 20 mol % of 1 in combination with 10 mol % of 4b in ch2cl2 , to drive reactions to completion within 56 h. under these optimized conditions , the desired products were thus isolated with similar high -selectivity ( > 30:1 : ratio ) and yields of 7485% ( table 2 , entries 57 , 9 , and 10 ) . to that end , a series of differentially protected galactals 2b2f , glucals 8a8c , and l - rhamnal 9 bearing methyl , acetate , benzyl , allyl , silyl ether , and siloxane protecting groups were prepared and subjected to the reaction conditions with 3 or 6k , which bears a secondary oh , as the acceptor ( table 3 ) . although substrate 2d , which bears an acetate group at c-6 , shows that ester groups can be tolerated ( entry 3 ) , in the case of acetyl - protected galactal 2c ( entry 2 ) , an inseparable mixture of glycosides including ferrier side - products was observed after 20 h under the more forcing conditions ( 20 mol % of 4b and reflux in ch2cl2 ) . encouragingly , the reaction was also amenable to glycosylations with glucal substrates , and both benzyl - derivative 8a and 3,4-o - siloxane protected 8b(6b ) or 8c(6b ) afforded the corresponding glycosides with high -stereocontrol within 2 h , albeit 11a was isolated in a lower yield ( 40% ) than 11b ( 81% yield ) or 11c ( 82% ) . having found that thioglycosides are inert under the cooperative organocatalytic glycosylation conditions ( table 2 , entry 4 ) , we decided to evaluate the methodology in a three - component , one - pot synthesis of trisaccharide 13 ( scheme 2 ) . to probe the mechanism of our reaction , a 3:1 /-anomeric mixture of 5 was subjected to the reaction conditions and gave no change in the anomeric ratio , indicating that the high -selectivity is not the result of anomerization . moreover , reaction with deuterated galactal 14 yielded disaccharide 15 with the newly formed bonds cis to each other , as evidenced by the h nmr shifts associated with h-1 ( 4.62 ppm , d , 1h , j = 2.7 hz ) and h-2 ( 2.031.99 , m , 1h , h-2 ) of the deuterated 2-deoxygalactoside ( scheme 3 ) , which suggest that both the c h and the c o bond formation steps are syn - diastereoselective . h nmr spectroscopy studies in cd2cl2 of mixtures of thiourea 1 and acid 4b showed proton shifts associated with the aromatic signals from both acid and thiourea , indicating that an interaction between both catalysts occurs in solution . furthermore , nmr mixtures of thiourea 1 , acid 4b , and glycoside donor 2a showed additional downfield h - shifts associated with the anomeric protons in 2a ( 6.33 ppm ) , while shifts for the oh signal from acceptor 3 ( from 1.84 ppm to 2.54 ppm ) were detected in mixtures of 1 , 4b , and 3 ( see supporting information for details ) . as expected , mixtures of 3 and 10 mol % of thiourea 1 or 3 mixed with 10 mol % of acid 4b also showed shifts associated with the oh protons of glycosyl acceptor 3 , although the magnitude of those shifts was different to that of mixtures of 3 and both cocatalysts ( 1 and 4b ) . these results further demonstrate that the combination of 1 and chiral acid ( r)-4b leads to a cooperative catalytic system that can activate both enol ether and oh nucleophiles in a synergistic fashion , leading to an enhancement in reaction rate and -stereocontrol of the glycosylation reaction . as proposed in scheme 3 , our findings suggest that a hydrogen - bond - mediated complex between thiourea 1 and acid 4b leads to a urea - induced acid amplification , and proton addition from this complex to the less hindered face of the enol ether ( i ) forms a short - lived oxocarbenium intermediate ( ii ) that is rapidly trapped by the oh nucleophile which is in turn activated by the phosphate intermediate that is generated in situ , to give 15 . in summary , we have described a practical , highly stereoselective , and efficient direct glycosylation method for glycals using a cooperative brnsted acid - organocatalytic promoter system . we note that the stereoselectivity of the reaction is highly dependent on the chirality of the acid , with ( r)-4b leading to the formation of -glycosides preferentially , while both yield and rate of reaction are greatly enhanced by the synergistic interaction between thiourea and acid . moreover , we exemplify the generality of the approach in the stereoselective synthesis of a series of disaccharides , glycosyl - amino acids , and other glycoconjugates and also in the one - pot chemo- and stereoselective synthesis of a trisaccharide . further work from our lab is underway to exploit the cooperative catalysis between organocatalysts and chiral acids for the stereoselective synthesis of other important glycosides and chiral acetals . a mixture of chiral acid ( 0.1 equiv ) and thiourea ( 0.1 equiv for primary acceptors and 0.2 equiv for secondary acceptors ) in anhydrous dcm ( 1 ml ) was stirred for 30 min and then added to the microwave vial containing the donor and acceptor . the crude material was then dissolved in a 1 m thf solution of tbaf ( 0.5 ml , 0.5 mmol ) and stirred for 2 h. the solution was then concentrated in vacuo and purified by column chromatography ( hexane : etoac , 6:1 to 4:1 ) , affording 7f as a colorless oil ( 58 mg , 74% ) . following the general glycosylation procedure , donor 2e ( 32 mg , 0.12 mmol ) and acceptor 3 ( 46 mg , 0.10 mmol ) afforded the following purification by column chromatography ( hexane : etoac , 5:1 to 4:1 ) : 10e as a pale yellow oil ( 58 mg , 80% ) : h nmr ( 500 mhz ; cdcl3 ) 7.417.22 ( 15h , m , ph ) , 6.005.79 ( 3h , m , ch = ch2 ) , 5.30 ( 1h , dt , j = 3.4 , 1.6 hz , ch = ch2 ) , 5.26 ( 1h , q , j = 1.8 hz , ch = ch2 ) , 5.23 ( 1h , tt , j = 2.3 , 1.1 hz , ch = ch2 ) , 5.17 ( 1h , dt , j = 3.4 , 1.6 hz , ch = ch2 ) , 5.155.12 ( 2h , m , ch = ch2 ) , 5.01 ( 1h , d , j = 2.6 hz , h-1 ) , 4.99 ( 1h , d , j = 10.8 hz , ochhph ) , 4.91 ( 1h , d , j = 10.8 hz , ochhph ) , 4.82 ( 1h , d , j = 5.3 hz , ochhph ) , 4.80 ( 1h , d , j = 6.6 hz , ochhph ) , 4.69 ( 1h , d , j = 12.2 hz , ochhph ) , 4.62 ( 1h , d , j = 2.5 hz , h-1 ) , 4.61 ( 1h , d , j = 4.8 hz , ochhph ) , 4.35 ( 1h , ddt , j = 12.7 , 5.6 , 1.4 hz , ochhch = ch2 ) , 4.134.06 ( 1h , m , ochhch = ch2 ) , 4.054.02 ( 2h , m , ochhch = ch2 , h-3 ) , 4.013.97 ( 1h , m , ochhch = ch2 ) , 3.943.91 ( 2h , ochhch = ch2 ) , 3.883.81 ( 2h , m , h-4 , h-6a ) , 3.783.73 ( 3h , m , h-4 , h-3 , h-5 ) , 3.64 ( 1h , dd , j = 11.4 , 2.0 hz , h-6b ) , 3.58 ( 1h , dd , j = 9.3 , 7.3 hz , h-5 ) , 3.563.52 ( 1h , m , h-2 ) , 3.513.46 ( 2h , m , h-6a , h-6b ) , 3.37 ( 3h , s , och3 ) , 2.142.07 ( 1h , m , h-2a ) , 1.951.90 ( 1h , m , h-2b).c nmr ( 126 mhz , cdcl3 ) 138.7 ( 4 c ) , 138.2 ( 4 c ) , 138.1 ( 4 c ) , 135.6 ( ch = ch2 ) , 134.8 ( ch = ch2 ) , 134.6 ( ch = ch2 ) , 128.5 ( ch ) , 128.4 ( ch ) , 128.04 ( ch ) , 127.9 ( ch ) , 127.7 ( ch ) , 127.7 ( ch ) , 116.9 ( ch = ch2 ) , 116.8 ( ch = ch2 ) , 116.6 ( ch = ch2 ) , 98.2 ( c-1 ) , 97.9 ( c-1 ) , 82.1 ( c-3 ) , 80.0 ( c-5 ) , 77.9 ( c-2 ) , 75.8 ( ch2ph ) , 74.9 ( ch2ph ) , 73.9 ( c-4 ) , 73.5 ( och2ch ) , 73.3 ( ch2ph ) , 72.4 ( h-5 ) , 72.2 ( och2ch ) , 69.8 ( 2c , c-4 , c-3 ) , 69.2 ( och2ch ) , 69.1 ( c-6 ) , 66.0 ( c-6 ) , 55.0 ( och3 ) , 31.1 ( c-2 ) . once cooled , a solution of 1,2:3,4-di - o - isopropylidene--d - galactopyranoside 6c ( 26 mg , 0.0998 mmol ) in anhydrous dcm ( 0.5 ml ) was added followed by addition of nis ( 45 mg , 0.2 mmol ) and finally the dropwise addition of a solution of tmsotf ( 95 l , 40 l in 20 ml anhydrous dcm , 0.001 mmol ) . c nmr ( 126 mhz , cdcl3 ) 138.9 ( 4 c ) , 138.7 ( 4 c ) , 138.4 ( 4 c ) , 138.3 ( 4 c ) , 138.16 ( 4 c ) , 138.1 ( 4 c ) , 128.41 ( ch ) , 128.2 ( ch ) , 127.7 ( ch ) , 98.3 ( c-1 ) , 97.9 ( c-1 ) , 82.1 ( c-3 ) , 80.0 ( c-2 ) , 77.9 ( c-4 ) , 75.8 ( ch2ph ) , 74.9 ( ch2ph ) , 74.3 ( ch2ph ) , 74.16 ( c-3 ) , 73.3 ( ch2ph ) , 73.3 ( ch2ph ) , 72.9 ( c-4 ) , 70.2 ( ch2ph ) , 70.1 ( ch2ph ) , ( c-5 ) , 69.8 ( c-5 ) , 69.4 ( c-6 ) , 66.0 ( c-6 ) , 55.0 ( och3 ) , 29.7 ( c-2 ) .

qx-314 ( n-[2,6-dimethylphenyl carbamoylmethyl ] triethylammonium bromide ) , a quaternary of lidocaine ( lido ) , is difficult to permeate through cell membranes because it has a permanent positive charge ( figure 1a ) . nevertheless , some studies have shown that qx-314 can produce long - lasting local anesthesia with a slow onset.1,2 therefore , the key is to improve the onset time of qx-314 . the transient receptor potential ( trp ) was found to be the primary pathway of qx-314 to elicit local anesthesia.3 an agonist of trp , capsaicin , can promote the effects of qx-314.4 however , capsaicin is limited for painful irritation and toxicity in local tissue.5 qx-314 produces long - lasting nerve blockade after a delayed onset when administered extracellularly as a peripheral local anesthetic . to improve its penetrability and accelerate the drug entry into cells , a derivative of qx-314 , qx - oh ( 2-(2,6-dimethylphenylamino)-n , n - diethyl - n-(2-hydroxyethyl)-2-oxoethanaminium bromide ; molecular weight , 359.3 previously , we showed in animal models that qx - oh alone can ( like qx-314 ) not only produce long - lasting local anesthesia but also moderate toxicity in local tissue compared with the same concentration of qx-314 . however , the onset time of qx - oh alone was still not improved significantly . bupivacaine ( bup ) , a local anesthetic with fast onset , can activate trp channels in the same way as capsaicin , inducing cellular entry of qx-314.6 therefore , we expect co - application of qx - oh and bup to induce a prolonged blockade with rapid onset . upon consideration of the severe system toxicity of bup , we choose the single levorotatory isomer of bup , levobupivacaine ( levo - bup ) , which is safer than bup . therefore , we conducted research on qx - oh , as well as a mixture of qx - oh and levo - bup , to meet the need for long - acting local anesthetics . previously , we found that 35 mm qx - oh in combination with 10 levobupivacaine , which we referred to as ll-1 , produced long - lasting sciatic nerve blockade in rats , which can be as long as 12 h , almost 3 times longer than 0.75% ( 23 mm ) bup alone , with minimal to mild local histological changes that were similar to those seen with 0.75% bup . moreover , the onset time of ll-1 was acceptable ( < 10 min ) . however , cardiotoxicity may be induced by qx - oh or ll-1 ( just as with other local anesthetics ) . severe cardiotoxicity after administration of local anesthetics always is lethal.7 therefore , it is crucial to evaluate the cardiotoxicity of qx - oh and ll-1 . the lipid solubility of qx - oh is less than that of qx-314 due to the changes in chemical structure . therefore , we hypothesized that the cardiotoxicity of qx - oh was less than that of qx-314 due to the improvements in physicochemical properties . in addition , whether qx - oh combined with levo - bup can increase cardiotoxicity is not known . therefore , we compared the cardiotoxicity of levo - bup , qx-314 , and qx - oh in anesthetized rats and assessed the interaction between levo - bup and qx - oh . we also evaluated the ability to resuscitate rats upon asystole after administration of these local anesthetics . male and female sprague dawley rats ( 230350 g ) were provided by the experimental animal centre of sichuan province . rats were housed in cages with free access to food and water and were maintained on a 12-h light dark cycle . the study was approved by the committee of animal care of west china hospital , sichuan university , people s republic of china ( approval number : 2015014a ) . the study was carried out in accordance with the guide for the care and use of laboratory animals ( us national institutes of health ; nih publication number 8023 [ revised 1996 ] ) . levo - bup was obtained from jiangsu hengrui medicine co. , ltd ( jiangsu , people s republic of china ) . qx-314 ( 35 mm ) and qx - oh ( 35 mm ) were dissolved in physiologic saline . ll-1 was prepared by mixing of qx - oh and levo - bup at a proportion of 35:10 mm . cd50c of levo - bup , qx-314 , qx - oh , and ll-1 was measured using the up - and - down method.8 this method , known as the staircase bioassay , is a procedure that has been proved to be effective in reducing the number of animals needed to determine a median dose . this method is appropriate for a variety of experimental end points , including toxicity studies . in the course of the experiment , if a positive result is observed , the subsequent dose of the study drug will be decreased by the dosing increment . this up - and - down procedure is repeated until 5 crossovers ( negative to positive or positive to negative effect ) are obtained . rats were anesthetized in a bell jar with 2% isoflurane in 100% oxygen . upon no aversive response to a tail - clamping stimulus , rats were then placed on a board under a warming lamp to maintain body temperature at 37c38c . three - lead electrocardiogram ( ecg ) was initiated by subcutaneous placement of needle electrodes at the left upper limb , right upper limb , and left lower limb.2 electrocardiographic data were acquired and displayed using a portable biological function experimental system ( bl-420f ; taimeng science and technology ltd . , chengdu , sichuan , people s republic of china ) equipped with alternating current and 50 hz filters and connected to a computer running the recording software . then , venipuncture was undertaken on the distal third of the tail with a 22 g intravenous cannula ( terumo medical corporation , tokyo , japan ) . after injection , we observed electrocardiographic evidence of the toxic effects on cardiac automaticity , conductivity , and rhythmicity for 15 min . positive result was defined as a change in the following end points : a decrease in baseline heart rate 30% , any evidence of atrioventricular conduction blockade , widening of the qrs complex , or other arrhythmias.9 for levo - bup , the starting dose was 1 mg / kg with a log dosing increment of 0.181 . for qx-314 , the starting dose was 5 mg / kg with a log dosing increment of 0.095 . for qx - oh , the starting dose was 5 mg / kg with a log dosing increment of 0.106 . for ll-1 , the starting dose ( qx - oh ) was 4 mg / kg with a log dosing increment of 0.095 . to evaluate si , we need to consider the tested drugs potency for local anesthesia . in rat sciatic nerve block model , we used a hot plate experiment to determine the median effective dose required to produce sensory blockade ( ed50 ) . rats were placed a left lateral position . an injection ( 23 g , 1 inch hypodermic needle ) was vertically inserted in the center of the line linking trochanter major and tuber ischiadicum of left leg . sensory block was measured in the same anatomic area using a modified hot plate ( model rb-200 hot plate analgesia meter ; chengdu technology & market co. , ltd . ) at a temperature of 56c . positive result was defined as the time that the animal left its paw was beyond 6 s.4 all rats were euthanized with isoflurane after each experiment . the method employed to measure the cd50a of levo - bup , qx-314 , qx - oh , first , the trachea was cannulated via tracheostomy , and then lungs were ventilated using a piston ventilator with a tidal volume of 1012 ml / kg at 5055 breaths / min to maintain the normal pressure of end - tidal carbon dioxide pressure . second , a positive result was defined as a lack of a recognizable beat on ecg for 1 min after appearance of the last systole or occurrence of ventricular fibrillation.10,11 third , based on our preliminary experiment , we needed to observe 60 min . the starting dose was 6 mg / kg with a log dosing increment of 0.119 for levo - bup . for qx-314 , the starting dose was 30 mg / kg with a log dosing increment of 0.084 . for qx - oh , the starting dose was 40 mg / kg with a log dosing increment of 0.059 . for ll-1 , the starting dose ( qx - oh ) was 20 mg / kg with a log dosing increment of 0.083 . the trachea was cannulated via tracheostomy , and lungs were ventilated mechanically as described in experiment 2 . ecg was recorded , and body temperature was maintained at 37c38c using a heating lamp . the left femoral artery was cannulated for blood sampling and monitoring of arterial pressure . through the left femoral vein after surgical preparation , rats were allowed to stabilize for 5 min in an atmosphere of 1% isoflurane and 100% oxygen . arterial blood gas was measured to confirm a ph between 7.30 and 7.45 and a serum lactate < 2.0 mmol / l before administration of local anesthetics . according to our preliminary experiment , rats were assigned randomly to receive 1.2-fold cd50a in the 4 groups a dose that reliably produces asystole from which hemodynamic recovery will occur with cardiopulmonary resuscitation . external cardiac compressions were applied immediately at a rate of ~300 beats / min to produce a systolic arterial pressure > 40 mmhg until spontaneous rhythm returned and were interrupted for 5 s every 1 min to evaluate circulatory function . simultaneously , epinephrine 10 g / kg was administered at 1-min intervals until return of spontaneous circulation ( rosc ) , which was defined as rate pressure product ( rpp ; systolic pressure heart rate ) 30% of the baseline value for 5 min . samples of arterial blood ( 0.3 ml ) were drawn in heparinized syringes before administration of local anesthetics ( baseline ) as well as 15 , 30 , 45 , and 60 min after rosc for the measurement of arterial blood gases . the following data were also collected in all 4 groups : time from initiation of injection of local anesthetics to asystole ( tasystole ) ; time from the start of asystole to rosc ( trecovery ) ; dose of epinephrine required for successful resuscitation ; duration of arrhythmia ( % ) after rosc ( if abnormal ecg was accompanied by abnormal arterial pulsation for 1 screen , this period was included in the duration of arrhythmia ) . data were analyzed using spss v19.0 ( ibm corporation , armonk , ny , usa ) . differences in the cd50c and cd50a among groups were considered significant if 95% confidence intervals did not overlap.12 the si was calculated by using the equation : cd50c2ed50=si(1)isobolographic analysis was used to evaluate the interaction between levo - bup and qx - oh.13,14 the interaction index ( ) indicates the changed potency of a combination and is defined using the equation : aa+bb=(2)where a and b are the doses of drug a ( alone ) and drug b ( alone ) , respectively , that give the specified effect and ( a , b ) is the combination dose that produces the same effect . if = 1 , the interaction is additive ; if < 1 , it is synergistic ; if > 1 , it is antagonistic . for experiment 3 , sample size calculation was based on the results of previous experiments comparing survival rate at 10 min after rosc among various groups . in a cochran armitage test for trend in proportions , sample sizes of 8 , 8 , 8 , and 8 are obtained from 4 groups with equally spaced x values and proportions equal to 0.80 , 0.80 , 0.40 , and 0.20 ( survival rate obtained from previous studies ) , respectively . the total sample of 32 subjects achieves 80% power to detect a linear trend using a 2-sided z test with continuity correction and a significance level of 0.05 . baseline parameters of cardiac function , weight , tasystole , trecovery , dose of epinephrine required for successful resuscitation , and duration of arrhythmia ( % ) after rosc among groups were analyzed by 1-way analysis of variance and scheffe post hoc tests . variables in arterial blood gas among groups after rosc were analyzed across time by analysis of variance with repeated - measures and scheffe post hoc tests . male and female sprague dawley rats ( 230350 g ) were provided by the experimental animal centre of sichuan province . rats were housed in cages with free access to food and water and were maintained on a 12-h light dark cycle . the study was approved by the committee of animal care of west china hospital , sichuan university , people s republic of china ( approval number : 2015014a ) . the study was carried out in accordance with the guide for the care and use of laboratory animals ( us national institutes of health ; nih publication number 8023 [ revised 1996 ] ) . levo - bup was obtained from jiangsu hengrui medicine co. , ltd ( jiangsu , people s republic of china ) . qx-314 ( 35 mm ) and qx - oh ( 35 mm ) were dissolved in physiologic saline . ll-1 was prepared by mixing of qx - oh and levo - bup at a proportion of 35:10 mm . cd50c of levo - bup , qx-314 , qx - oh , and ll-1 was measured using the up - and - down method.8 this method , known as the staircase bioassay , is a procedure that has been proved to be effective in reducing the number of animals needed to determine a median dose . this method is appropriate for a variety of experimental end points , including toxicity studies . in the course of the experiment , if a positive result is observed , the subsequent dose of the study drug will be decreased by the dosing increment . this up - and - down procedure is repeated until 5 crossovers ( negative to positive or positive to negative effect ) are obtained . rats were anesthetized in a bell jar with 2% isoflurane in 100% oxygen . upon no aversive response to a tail - clamping stimulus , rats were then placed on a board under a warming lamp to maintain body temperature at 37c38c . three - lead electrocardiogram ( ecg ) was initiated by subcutaneous placement of needle electrodes at the left upper limb , right upper limb , and left lower limb.2 electrocardiographic data were acquired and displayed using a portable biological function experimental system ( bl-420f ; taimeng science and technology ltd . , chengdu , sichuan , people s republic of china ) equipped with alternating current and 50 hz filters and connected to a computer running the recording software . then , venipuncture was undertaken on the distal third of the tail with a 22 g intravenous cannula ( terumo medical corporation , tokyo , japan ) . after injection , we observed electrocardiographic evidence of the toxic effects on cardiac automaticity , conductivity , and rhythmicity for 15 min . a positive result was defined as a change in the following end points : a decrease in baseline heart rate 30% , any evidence of atrioventricular conduction blockade , widening of the qrs complex , or other arrhythmias.9 for levo - bup , the starting dose was 1 mg / kg with a log dosing increment of 0.181 . for qx-314 , the starting dose was 5 mg / kg with a log dosing increment of 0.095 . for qx - oh , the starting dose was 5 mg / kg with a log dosing increment of 0.106 . for ll-1 , the starting dose ( qx - oh ) was 4 mg / kg with a log dosing increment of 0.095 . to evaluate si , we need to consider the tested drugs potency for local anesthesia . in rat sciatic nerve block model , we used a hot plate experiment to determine the median effective dose required to produce sensory blockade ( ed50 ) . rats were placed a left lateral position . an injection ( 23 g , 1 inch hypodermic needle ) was vertically inserted in the center of the line linking trochanter major and tuber ischiadicum of left leg . sensory block was measured in the same anatomic area using a modified hot plate ( model rb-200 hot plate analgesia meter ; chengdu technology & market co. , ltd . ) at a temperature of 56c . a positive result was defined as the time that the animal left its paw was beyond 6 s.4 all rats were euthanized with isoflurane after each experiment . the method employed to measure the cd50a of levo - bup , qx-314 , qx - oh , and ll-1 was the same as that for experiment 1 except for 3 differences . first , the trachea was cannulated via tracheostomy , and then lungs were ventilated using a piston ventilator with a tidal volume of 1012 ml / kg at 5055 breaths / min to maintain the normal pressure of end - tidal carbon dioxide pressure . second , a positive result was defined as a lack of a recognizable beat on ecg for 1 min after appearance of the last systole or occurrence of ventricular fibrillation.10,11 third , based on our preliminary experiment , we needed to observe 60 min . the starting dose was 6 mg / kg with a log dosing increment of 0.119 for levo - bup . for qx-314 , the starting dose was 30 mg / kg with a log dosing increment of 0.084 . for qx - oh , the starting dose was 40 mg / kg with a log dosing increment of 0.059 . for ll-1 , the starting dose ( qx - oh ) was 20 mg / kg with a log dosing increment of 0.083 . the trachea was cannulated via tracheostomy , and lungs were ventilated mechanically as described in experiment 2 . ecg was recorded , and body temperature was maintained at 37c38c using a heating lamp . the left femoral artery was cannulated for blood sampling and monitoring of arterial pressure . through the left femoral vein , after surgical preparation , rats were allowed to stabilize for 5 min in an atmosphere of 1% isoflurane and 100% oxygen . arterial blood gas was measured to confirm a ph between 7.30 and 7.45 and a serum lactate < 2.0 , rats were assigned randomly to receive 1.2-fold cd50a in the 4 groups a dose that reliably produces asystole from which hemodynamic recovery will occur with cardiopulmonary resuscitation . external cardiac compressions were applied immediately at a rate of ~300 beats / min to produce a systolic arterial pressure > 40 mmhg until spontaneous rhythm returned and were interrupted for 5 s every 1 min to evaluate circulatory function . simultaneously , epinephrine 10 g / kg was administered at 1-min intervals until return of spontaneous circulation ( rosc ) , which was defined as rate pressure product ( rpp ; systolic pressure heart rate ) 30% of the baseline value for 5 min . samples of arterial blood ( 0.3 ml ) were drawn in heparinized syringes before administration of local anesthetics ( baseline ) as well as 15 , 30 , 45 , and 60 min after rosc for the measurement of arterial blood gases . the following data were also collected in all 4 groups : time from initiation of injection of local anesthetics to asystole ( tasystole ) ; time from the start of asystole to rosc ( trecovery ) ; dose of epinephrine required for successful resuscitation ; duration of arrhythmia ( % ) after rosc ( if abnormal ecg was accompanied by abnormal arterial pulsation for 1 screen , this period was included in the duration of arrhythmia ) . data were analyzed using spss v19.0 ( ibm corporation , armonk , ny , usa ) . differences in the cd50c and cd50a among groups were considered significant if 95% confidence intervals did not overlap.12 the si was calculated by using the equation : cd50c2ed50=si(1)isobolographic analysis was used to evaluate the interaction between levo - bup and qx - oh.13,14 the interaction index ( ) indicates the changed potency of a combination and is defined using the equation : aa+bb=(2)where a and b are the doses of drug a ( alone ) and drug b ( alone ) , respectively , that give the specified effect and ( a , b ) is the combination dose that produces the same effect . if = 1 , the interaction is additive ; if < 1 , it is synergistic ; if > 1 , it is antagonistic . for experiment 3 , sample size calculation was based on the results of previous experiments comparing survival rate at 10 min after rosc among various groups . in a cochran armitage test for trend in proportions , sample sizes of 8 , 8 , 8 , and 8 are obtained from 4 groups with equally spaced x values and proportions equal to 0.80 , 0.80 , 0.40 , and 0.20 ( survival rate obtained from previous studies ) , respectively . the total sample of 32 subjects achieves 80% power to detect a linear trend using a 2-sided z test with continuity correction and a significance level of 0.05 . baseline parameters of cardiac function , weight , tasystole , trecovery , dose of epinephrine required for successful resuscitation , and duration of arrhythmia ( % ) after rosc among groups were analyzed by 1-way analysis of variance and scheffe post hoc tests . variables in arterial blood gas among groups after rosc were analyzed across time by analysis of variance with repeated - measures and scheffe post hoc tests . the baseline heart rate of rats was 367 28 , 367 28 , 355 46 , and 358 38 beats / min in levo - bup , qx-314 , qx - oh , and ll-1 groups , respectively ( p > 0.05 ) . in all groups , a decrease in heart rate and widening of the qrs complex after administration of study drugs was observed . the cd50c was significantly lower for levo - bup than for qx-314 or qx - oh . rats with a positive result developed asystole quickly after administration of levo - bup or ll-1 . however , all rats that tested positive developed ventricular fibrillation a few minutes after administration of qx-314 or qx - oh . the cd50a for levo - bup was significantly lower than that for qx-314 or qx - oh . however , the cd50a of qx - oh was significantly higher than that of qx-314 ( table 1 ) . the ed50 was 0.10 , 4.17 , and 3.60 mg / kg in levo - bup , qx-314 , and qx - oh groups . for ll-1 , the ed50 was 0.1 mg / kg for levo - bup and 0.43 mg / kg for qx - oh . compared with qx-314 , the margin of safety for qx - oh broadened to some extent . however , the si of qx-314 and qx - oh was significantly lower than that of levo - bup and ll-1 . that is , the dose needed to elicit cardiotoxicity by levo - bup or ll-1 was significantly higher than the dose resulting in sensory blockade ( table 1 ) . for cd50c , with addition of levo - bup , the cd50c of qx - oh was above the additive line , suggesting an antagonistic effect between levo - bup and qx - oh ( figure 2a ) . for cd50a , the between levo - bup and qx - oh was 1.202 . with addition of levo - bup , the cd50a of qx - oh was above or on the additive line , indicating a nonsynergistic effect between levo - bup and qx - oh ( figure 2b ) . thirty - four rats were studied in experiment 3 , but 2 rats in the qx-314 group were excluded because they did not develop asystole . groups were similar in terms of weight , heart rates , systolic blood pressure , rpp , and acid - base status at baseline ( table 2 ) . rosc was attained initially by 8 of 8 rats in the levo - bup group , 3 of 8 in the qx-314 group , 6 of 8 in the qx - oh group , and 8 of 8 in the ll-1 group ( table 3 ) . there were no significant differences among levo - bup , qx - oh , and ll-1 groups or between qx-314 vs. qx - oh groups ( p > 0.05 ) . compared with the qx-314 group , the number of rats achieving successful resuscitation increased significantly in levo - bup and ll-1 groups ( p < 0.05 ) . however , no rats could maintain rpp 30% baseline in qx-314 , qx - oh , and ll-1 groups at 10 , 20 , and 50 min after rosc , respectively . moreover , asystole occurred again in some rats that had attained spontaneous circulation : 2 of 3 in the qx-314 group , 4 of 6 in the qx - oh group , and 2 of 8 in the ll-1 group . tasystole of the qx - oh group was significantly longer than that of other groups ( p < 0.05 ) ( figure 3a ) . trecovery of the ll-1 group was significantly longer than that of qx-314 and qx - oh groups ( p < 0.05 ) . however , no significant differences were seen between levo - bup vs. other groups ( figure 3b ) . the dose of epinephrine required for successful resuscitation was 38.75 11.26 , 23.33 5.77 , 28.33 11.69 , and 65.00 32.51 mg / kg in levo - bup , qx-314 , qx - oh , and ll-1 groups , respectively ( figure 3c ) . compared with qx - oh group , more epinephrine was needed to resuscitate rats in the ll-1 group ( p < 0.05 ) . percent arrhythmia duration was significantly less than in the levo - bup group than in the other groups ( p < 0.05 ) . however , there were no significant differences among qx-314 , qx - oh , or ll-1 groups . there were no significant differences in ph , partial pressure of carbon dioxide , or partial pressure of oxygen among all groups ( p > 0.05 ) except that ph in the ll-1 group and partial pressure of carbon dioxide in the qx - oh group at 60 min after rosc were lower than those in the levo - bup group ( p < 0.05 ) . compared with the levo - bup group , lactate levels showed significant increases in the qx - oh group at 15 and 60 min and in the ll-1 group from 30 to 60 min after resuscitation ( p < 0.05 ) . the baseline heart rate of rats was 367 28 , 367 28 , 355 46 , and 358 38 beats / min in levo - bup , qx-314 , qx - oh , and ll-1 groups , respectively ( p > 0.05 ) . in all groups , a decrease in heart rate and widening of the qrs complex after administration of study drugs was observed . the cd50c was significantly lower for levo - bup than for qx-314 or qx - oh . rats with a positive result developed asystole quickly after administration of levo - bup or ll-1 . however , all rats that tested positive developed ventricular fibrillation a few minutes after administration of qx-314 or qx - oh . the cd50a for levo - bup was significantly lower than that for qx-314 or qx - oh . however , the cd50a of qx - oh was significantly higher than that of qx-314 ( table 1 ) . the ed50 was 0.10 , 4.17 , and 3.60 mg / kg in levo - bup , qx-314 , and qx - oh groups . for ll-1 , the ed50 was 0.1 mg / kg for levo - bup and 0.43 mg / kg for qx - oh . compared with qx-314 , the margin of safety for qx - oh broadened to some extent . however , the si of qx-314 and qx - oh was significantly lower than that of levo - bup and ll-1 . that is , the dose needed to elicit cardiotoxicity by levo - bup or ll-1 was significantly higher than the dose resulting in sensory blockade ( table 1 ) . with addition of levo - bup , the cd50c of qx - oh was above the additive line , suggesting an antagonistic effect between levo - bup and qx - oh ( figure 2a ) . for cd50a , the between levo - bup and qx - oh was 1.202 . with addition of levo - bup , the cd50a of qx - oh was above or on the additive line , indicating a nonsynergistic effect between levo - bup and qx - oh ( figure 2b ) . thirty - four rats were studied in experiment 3 , but 2 rats in the qx-314 group were excluded because they did not develop asystole . groups were similar in terms of weight , heart rates , systolic blood pressure , rpp , and acid - base status at baseline ( table 2 ) . rosc was attained initially by 8 of 8 rats in the levo - bup group , 3 of 8 in the qx-314 group , 6 of 8 in the qx - oh group , and 8 of 8 in the ll-1 group ( table 3 ) . there were no significant differences among levo - bup , qx - oh , and ll-1 groups or between qx-314 vs. qx - oh groups ( p > 0.05 ) . compared with the qx-314 group , the number of rats achieving successful resuscitation increased significantly in levo - bup and ll-1 groups ( p < 0.05 ) . however , no rats could maintain rpp 30% baseline in qx-314 , qx - oh , and ll-1 groups at 10 , 20 , and 50 min after rosc , respectively . moreover , asystole occurred again in some rats that had attained spontaneous circulation : 2 of 3 in the qx-314 group , 4 of 6 in the qx - oh group , and 2 of 8 in the ll-1 group . tasystole of the qx - oh group was significantly longer than that of other groups ( p < 0.05 ) ( figure 3a ) . trecovery of the ll-1 group was significantly longer than that of qx-314 and qx - oh groups ( p < 0.05 ) . however , no significant differences were seen between levo - bup vs. other groups ( figure 3b ) . the dose of epinephrine required for successful resuscitation was 38.75 11.26 , 23.33 5.77 , 28.33 11.69 , and 65.00 32.51 mg / kg in levo - bup , qx-314 , qx - oh , and ll-1 groups , respectively ( figure 3c ) . compared with qx - oh group , more epinephrine was needed to resuscitate rats in the ll-1 group ( p < 0.05 ) . percent arrhythmia duration was significantly less than in the levo - bup group than in the other groups ( p < 0.05 ) . however , there were no significant differences among qx-314 , qx - oh , or ll-1 groups . there were no significant differences in ph , partial pressure of carbon dioxide , or partial pressure of oxygen among all groups ( p > 0.05 ) except that ph in the ll-1 group and partial pressure of carbon dioxide in the qx - oh group at 60 min after rosc were lower than those in the levo - bup group ( p < 0.05 ) . compared with the levo - bup group , lactate levels showed significant increases in the qx - oh group at 15 and 60 min and in the ll-1 group from 30 to 60 min after resuscitation ( p < 0.05 ) . second , the interaction between qx - oh and levo - bup for cardiac effects was nonsynergistic . third , the initial ability to resuscitate rats in which asystole had been induced by levo - bup , qx - oh , or ll-1 was similar . however , it was more difficult to resuscitate rats after induction of systole by qx-314 than by levo - bup or ll-1 . moreover , advanced life support might be needed after rosc in qx-314- , qx - oh- , and ll-1-induced asystole . cd50c was measured by observing the ecg changes to identify the appearance of cardiotoxicity after administration of different local anesthetics . these changes occurred at a lower concentration range.15 cheung et al9 reported that qx-314 elicits cardiotoxicity in mice with a cd50 of 10.6 mg / kg , a value that is in accordance with our results . however , they observed cardiotoxicity only 30 s after injection of qx-314 , which was considerably shorter than our observing time ( the time to peak of qx-314 was ~13 min ) . according to the cd50c and cd50a , the potency for cardiac effects was levo - bup > qx-314 qx - oh ( table 1 ) . the potency for cardiotoxicity of local anesthetics is closely associated with the lipid solubility of drugs.16 compared with qx-314 and qx - oh , levo - bup is more lipid soluble , which could be why the cardiotoxicity of levo - bup was more severe than that of the other drugs we tested . the lipid solubility of qx - oh is less than that of qx-314 , which reduces the cardiotoxicity of qx - oh . it is not sufficient to evaluate safety based merely on the absolute dose required to elicit cardiotoxicity . the margin of safety also needs to be considered . in our study , even though the absolute dose of levo - bup resulting in cardiotoxicity was significantly lower than that of qx-314 or qx - oh ( 2.97 vs. 10.00 or 2.97 vs. 10.40 mg / kg ) , the si of levo - bup was significantly greater than that of qx-314 ( 10.60 vs. 1.20 ) or qx - oh ( 10.60 vs. 1.44 ) . however , the si of ll-1 was similar to that of levo - bup , suggesting that the safety of co - application of qx - oh and levo - bup was increased significantly compared with administration of qx - oh alone . interestingly , nonsynergistic interaction was found between levo - bup and qx - oh with regard to cardiac effects . systemic toxic effects of local anesthetic combinations appear to have additive effects.17 however , a mixture of bup and lido can impair the intraventricular conduction parameters to a lesser extent than that for bup itself.18 these different observations might have been resulted ( at least in part ) because of differences in experimental design and ethnicities of participants . from an electrophysiological viewpoint , local anesthetics elicit cardiotoxicity primarily by blocking sodium channels.19 lido blocks sodium channels in a fast - in , fast - out fashion , whereas bup blocks these channels in a slow - in , slow - out manner in low concentrations or a fast - in , slow - out manner at high concentrations.20 therefore , competitive blockade is present between bup and lido . qx - oh , a derivative of lido , may compete with levo - bup for blockade of sodium channels . meanwhile , the decreasing lipid solubility of qx - oh resulted in an extension of onset time on blocking the sodium channels of cardiac cells , which was longer than that of levo - bup . qx - oh might block sodium channels in a more slow - in , more slow - out fashion . thus , the moment when qx - oh produced its peak effect was later than that of levo - bup or was even in the recovery stage of levo - bup - induced cardiotoxicity . these phenomena could explain , to a certain extent , the antagonism between qx - oh and levo - bup . the interaction index ( ) between qx - oh and levo - bup for asystole was less than that for the appearance of cardiotoxicity ( 1.202 vs. 1.537 ) , suggesting that the degree of antagonism decreased with increasing dose of qx - oh and levo - bup . moreover , the cd50a of qx - oh was above or on the additivity line , indicating a nonsynergistic effect between levo - bup and qx - oh it has been reported that qx-314 alone at relatively high concentrations can activate trp channels without any adjuvant drug and may penetrate slowly through lipid membranes.1,21 thus , the time to peak effects of qx - oh was shortened due to activation of trp channels induced by a relatively massive dose of qx - oh . thus , the time to peak effects of qx - oh might overlap with that of levo - bup when ll-1 was administrated , inducing a nonsynergistic effect . the nonsynergistic effect was of great importance for the safety of the drug co - application . compared with qx - oh / levo - bup alone , the co - application of qx - oh and levo - bup produced the same or the lighter effects on cardiac functions under the same condition without affecting the potency of local anesthesia ( according to ed50 , the between qx - oh and levo - bup for sensory blockade was 0.817 , indicating a synergistic effect on local anesthesia ) . we therefore supposed that the compound preparation was reasonable in terms of effectiveness and safety . however , the precise mechanism of this interaction affecting the cardiac conductivity or contractility was still unknown in this study . with regard to the mechanism of death , asystole occurred rapidly in a fashion of disappearance of cardiac electrical activity after administration of levo - bup or ll-1 , whereas qx-314 and qx - oh brought about ventricular fibrillation a few minutes after injection of these drugs . ventricular fibrillation did not occur in the ll-1 group , suggesting that levo - bup in ll-1 had a very important role in eliciting asystole . we found that the ranking of tasystole was qx - oh > qx-314 > ll-1 levo - bup . local anesthetics need access to intracellular domains to block voltage - gated sodium channels.22 however , qx-314 can not block sodium channels if applied extracellularly.4 this difference is due to the physicochemical properties of drugs at least in part , especially lipid solubility and charge.23 tasystole in the ll-1 group was shorter than that in the qx - oh group , but it is not known whether this effect was due to the levo - bup in ll-1 or levo - bup - induced cellular entry of qx - oh or both . our results suggested that the ability to resuscitate initially rats was similar among levo - bup , ll-1 , and qx - oh groups . qx-314-induced asystole was relatively more difficult to resuscitate . however , sustainable arrhythmia occurred in qx-314 , qx - oh , and ll-1 groups , but not in the levo - bup group , after rosc , which led to circulatory collapse again . according to the manner of asystole or duration of arrhythmia ( % ) after rosc in qx-314 , qx - oh , and ll-1 groups , arrhythmia appeared to be the primary mechanism of cardiotoxicity induced by qx-314 and qx - oh . even though epinephrine can increase the number of ecg abnormalities after rosc,24 we think that arrhythmogenicity is the internal characteristic of qx-314 and qx - oh because ventricular fibrillation occurred before use of epinephrine . similarly , groban et al25 reported that continued therapy was required in 86% of dogs receiving lido compared with only 10%30% of dogs receiving bup , levo - bup , or ropivacaine ( rop ) . however , we think that arrhythmia is the primary mechanism of cardiotoxicity induced by qx-314 and qx - oh . local anesthetics decrease ventricular conduction in a stereospecific manner and decrease contractility in a non - stereospecific manner based on stereospecificity for blockade of sodium and potassium channels.2628 therefore , it is not known whether variation of the molecular structure of lido leads to the stereospecificity of the blockade of ion channels with lido . in a word , arrhythmogenicity of qx-314 and qx - oh was the reason why sustainable circulation could not be maintained after rosc in qx-314 , qx - oh , and ll-1 groups . it tells us that anti - arrhythmia and other continued therapy may be important for the treatment of severe cardiotoxicity induced by qx-314 , qx - oh , and ll-1 . also , it will be beneficial to monitor the dynamic change of vital signs for a relatively longer time after rosc . even though circulatory collapse occurred again in the ll-1 group due to the arrhythmogenicity of qx - oh , it can not deny absolutely the use of ll-1 because the dose required to elicit asystole was much larger than the dose produced local anesthesia . second , we did not monitor blood pressure of rats in measuring cd50c and cd50a . as we know , ecg changes induced by local anesthetics occurred in a low concentration range asystole occurred in a fashion of the disappearance of cardiac electrical activity or ventricular fibrillation in a preliminary experiment , which was confirmed by ecg . third , we did not use lipid emulsions in our resuscitation sequence . even though lipid emulsions have been approved by many anesthetists , epinephrine remains the first - line drug for cardiopulmonary resuscitation because of its positive chronotropy , inotropy , and vasopressor effects . however , epinephrine over a threshold dose near 10 mg / kg impairs lipid resuscitation from bup overdose.29 moreover , the lipid sink theory is the mechanism of lipid on the reversal of local anesthetic cardiotoxicity.30 some scholars have found no beneficial effects of lipid infusion on the less lipophilic mepivacaine- or rop - induced asystole , concluding that lipophilicity of local anesthetics has a marked impact on the efficacy of lipid infusion to treat asystole induced by these drugs.10,31 qx - oh is a less lipophilic drug . the cardiac effects of qx - oh and levo - bup were nonsynergistic . initial successful resuscitation could be achieved in qx - oh- and ll-1-induced asystole . though circulatory collapse occurred again , that did not deny the use of qx - oh or ll-1 because the dose required to elicit asystole was much larger than the dose used to elicit local anesthesia . further studies are needed to define what kind of advanced life support is needed after rosc to maintain stable circular function and the precise mechanism of cardiotoxicity with respect to cellular / molecular actions and pharmacokinetics .

objectivethis study was designed to evaluate the cardiotoxicity of a qx-314 analog ( qx - oh ) and a mixture of qx - oh and levobupivacaine ( ll-1 ) and to compare the ability to resuscitate rats after asystole induced by levobupivacaine ( levo - bup ) , qx-314 , qx - oh , and ll-1.methodsfirst , we used the up - and - down method to determine median dose resulting in appearance of cardiotoxicity ( cd50c ) and asystole ( cd50a ) of levo - bup , qx-314 , qx - oh , and ll-1 in rats . safety index ( si ; ratio of cd50c compared with 2-fold median effective dose needed to produce sensory blockade ) of the 4 drugs was calculated . isobolograms were used for drug interaction analysis . second , rats received 1.2-fold cd50a in the 4 groups . when asystole occurred , standard cardiopulmonary resuscitation was started and continued for 30 min or until return of spontaneous circulation ( rosc ) with native rate pressure product 30% baseline for 5 min.resultsranking of cd50c was levo - bup < qx-314 qx - oh . ranking of cd50a was levo - bup < qx-314 < qx - oh . however , the si of levo - bup was significantly higher than that of qx-314 ( 10.60 vs. 1.20 ) or qx - oh ( 10.60 vs. 1.44 ) . the si of ll-1 was similar to that of levo - bup . nonsynergistic interaction was observed for cardiac effects between qx - oh and levo - bup . rosc was attained initially by 8 of 8 rats in the levo - bup group , 3 of 8 in the qx-314 group , 6 of 8 in the qx - oh group , and 8 of 8 in the ll-1 group . sustained recovery was achieved in the levo - bup group but not in the other groups.conclusionlevo-bup and ll-1 are safer than qx-314 or qx - oh . cardiac effects between qx - oh and levo - bup were nonsynergistic . initial successful resuscitation could be achieved in the qx - oh- and ll-1-induced asystole , but advanced life support might be needed .

Introduction Methods Animals Drugs Experiment 1: Determination of median dose resulting in appearance of cardiotoxicity (CD Experiment 2: Determination of median dose resulting in asystole (CD Experiment 3: Resuscitation after induction of asystole with Levo-BUP, QX-314, QX-OH, or LL-1 Statistical analysis Results The CD The CD The SI of LL-1 was similar to Levo-BUP Nonsynergistic effect was revealed between QX-OH and Levo-BUP The ability to resuscitate rats Discussion Conclusion

to improve its penetrability and accelerate the drug entry into cells , a derivative of qx-314 , qx - oh ( 2-(2,6-dimethylphenylamino)-n , n - diethyl - n-(2-hydroxyethyl)-2-oxoethanaminium bromide ; molecular weight , 359.3 previously , we showed in animal models that qx - oh alone can ( like qx-314 ) not only produce long - lasting local anesthesia but also moderate toxicity in local tissue compared with the same concentration of qx-314 . bupivacaine ( bup ) , a local anesthetic with fast onset , can activate trp channels in the same way as capsaicin , inducing cellular entry of qx-314.6 therefore , we expect co - application of qx - oh and bup to induce a prolonged blockade with rapid onset . upon consideration of the severe system toxicity of bup , we choose the single levorotatory isomer of bup , levobupivacaine ( levo - bup ) , which is safer than bup . therefore , we conducted research on qx - oh , as well as a mixture of qx - oh and levo - bup , to meet the need for long - acting local anesthetics . severe cardiotoxicity after administration of local anesthetics always is lethal.7 therefore , it is crucial to evaluate the cardiotoxicity of qx - oh and ll-1 . therefore , we hypothesized that the cardiotoxicity of qx - oh was less than that of qx-314 due to the improvements in physicochemical properties . therefore , we compared the cardiotoxicity of levo - bup , qx-314 , and qx - oh in anesthetized rats and assessed the interaction between levo - bup and qx - oh . cd50c of levo - bup , qx-314 , qx - oh , and ll-1 was measured using the up - and - down method.8 this method , known as the staircase bioassay , is a procedure that has been proved to be effective in reducing the number of animals needed to determine a median dose . in rat sciatic nerve block model , we used a hot plate experiment to determine the median effective dose required to produce sensory blockade ( ed50 ) . the method employed to measure the cd50a of levo - bup , qx-314 , qx - oh , first , the trachea was cannulated via tracheostomy , and then lungs were ventilated using a piston ventilator with a tidal volume of 1012 ml / kg at 5055 breaths / min to maintain the normal pressure of end - tidal carbon dioxide pressure . according to our preliminary experiment , rats were assigned randomly to receive 1.2-fold cd50a in the 4 groups a dose that reliably produces asystole from which hemodynamic recovery will occur with cardiopulmonary resuscitation . simultaneously , epinephrine 10 g / kg was administered at 1-min intervals until return of spontaneous circulation ( rosc ) , which was defined as rate pressure product ( rpp ; systolic pressure heart rate ) 30% of the baseline value for 5 min . differences in the cd50c and cd50a among groups were considered significant if 95% confidence intervals did not overlap.12 the si was calculated by using the equation : cd50c2ed50=si(1)isobolographic analysis was used to evaluate the interaction between levo - bup and qx - oh.13,14 the interaction index ( ) indicates the changed potency of a combination and is defined using the equation : aa+bb=(2)where a and b are the doses of drug a ( alone ) and drug b ( alone ) , respectively , that give the specified effect and ( a , b ) is the combination dose that produces the same effect . ll-1 was prepared by mixing of qx - oh and levo - bup at a proportion of 35:10 mm . cd50c of levo - bup , qx-314 , qx - oh , and ll-1 was measured using the up - and - down method.8 this method , known as the staircase bioassay , is a procedure that has been proved to be effective in reducing the number of animals needed to determine a median dose . the method employed to measure the cd50a of levo - bup , qx-314 , qx - oh , and ll-1 was the same as that for experiment 1 except for 3 differences . arterial blood gas was measured to confirm a ph between 7.30 and 7.45 and a serum lactate < 2.0 , rats were assigned randomly to receive 1.2-fold cd50a in the 4 groups a dose that reliably produces asystole from which hemodynamic recovery will occur with cardiopulmonary resuscitation . simultaneously , epinephrine 10 g / kg was administered at 1-min intervals until return of spontaneous circulation ( rosc ) , which was defined as rate pressure product ( rpp ; systolic pressure heart rate ) 30% of the baseline value for 5 min . differences in the cd50c and cd50a among groups were considered significant if 95% confidence intervals did not overlap.12 the si was calculated by using the equation : cd50c2ed50=si(1)isobolographic analysis was used to evaluate the interaction between levo - bup and qx - oh.13,14 the interaction index ( ) indicates the changed potency of a combination and is defined using the equation : aa+bb=(2)where a and b are the doses of drug a ( alone ) and drug b ( alone ) , respectively , that give the specified effect and ( a , b ) is the combination dose that produces the same effect . the baseline heart rate of rats was 367 28 , 367 28 , 355 46 , and 358 38 beats / min in levo - bup , qx-314 , qx - oh , and ll-1 groups , respectively ( p > 0.05 ) . the cd50c was significantly lower for levo - bup than for qx-314 or qx - oh . the cd50a for levo - bup was significantly lower than that for qx-314 or qx - oh . however , the cd50a of qx - oh was significantly higher than that of qx-314 ( table 1 ) . however , the si of qx-314 and qx - oh was significantly lower than that of levo - bup and ll-1 . that is , the dose needed to elicit cardiotoxicity by levo - bup or ll-1 was significantly higher than the dose resulting in sensory blockade ( table 1 ) . for cd50c , with addition of levo - bup , the cd50c of qx - oh was above the additive line , suggesting an antagonistic effect between levo - bup and qx - oh ( figure 2a ) . with addition of levo - bup , the cd50a of qx - oh was above or on the additive line , indicating a nonsynergistic effect between levo - bup and qx - oh ( figure 2b ) . rosc was attained initially by 8 of 8 rats in the levo - bup group , 3 of 8 in the qx-314 group , 6 of 8 in the qx - oh group , and 8 of 8 in the ll-1 group ( table 3 ) . there were no significant differences among levo - bup , qx - oh , and ll-1 groups or between qx-314 vs. qx - oh groups ( p > 0.05 ) . compared with the qx-314 group , the number of rats achieving successful resuscitation increased significantly in levo - bup and ll-1 groups ( p < 0.05 ) . however , no rats could maintain rpp 30% baseline in qx-314 , qx - oh , and ll-1 groups at 10 , 20 , and 50 min after rosc , respectively . moreover , asystole occurred again in some rats that had attained spontaneous circulation : 2 of 3 in the qx-314 group , 4 of 6 in the qx - oh group , and 2 of 8 in the ll-1 group . tasystole of the qx - oh group was significantly longer than that of other groups ( p < 0.05 ) ( figure 3a ) . trecovery of the ll-1 group was significantly longer than that of qx-314 and qx - oh groups ( p < 0.05 ) . the dose of epinephrine required for successful resuscitation was 38.75 11.26 , 23.33 5.77 , 28.33 11.69 , and 65.00 32.51 mg / kg in levo - bup , qx-314 , qx - oh , and ll-1 groups , respectively ( figure 3c ) . compared with qx - oh group , more epinephrine was needed to resuscitate rats in the ll-1 group ( p < 0.05 ) . there were no significant differences in ph , partial pressure of carbon dioxide , or partial pressure of oxygen among all groups ( p > 0.05 ) except that ph in the ll-1 group and partial pressure of carbon dioxide in the qx - oh group at 60 min after rosc were lower than those in the levo - bup group ( p < 0.05 ) . compared with the levo - bup group , lactate levels showed significant increases in the qx - oh group at 15 and 60 min and in the ll-1 group from 30 to 60 min after resuscitation ( p < 0.05 ) . the baseline heart rate of rats was 367 28 , 367 28 , 355 46 , and 358 38 beats / min in levo - bup , qx-314 , qx - oh , and ll-1 groups , respectively ( p > 0.05 ) . the cd50c was significantly lower for levo - bup than for qx-314 or qx - oh . the cd50a for levo - bup was significantly lower than that for qx-314 or qx - oh . however , the cd50a of qx - oh was significantly higher than that of qx-314 ( table 1 ) . the ed50 was 0.10 , 4.17 , and 3.60 mg / kg in levo - bup , qx-314 , and qx - oh groups . however , the si of qx-314 and qx - oh was significantly lower than that of levo - bup and ll-1 . that is , the dose needed to elicit cardiotoxicity by levo - bup or ll-1 was significantly higher than the dose resulting in sensory blockade ( table 1 ) . with addition of levo - bup , the cd50c of qx - oh was above the additive line , suggesting an antagonistic effect between levo - bup and qx - oh ( figure 2a ) . with addition of levo - bup , the cd50a of qx - oh was above or on the additive line , indicating a nonsynergistic effect between levo - bup and qx - oh ( figure 2b ) . rosc was attained initially by 8 of 8 rats in the levo - bup group , 3 of 8 in the qx-314 group , 6 of 8 in the qx - oh group , and 8 of 8 in the ll-1 group ( table 3 ) . there were no significant differences among levo - bup , qx - oh , and ll-1 groups or between qx-314 vs. qx - oh groups ( p > 0.05 ) . compared with the qx-314 group , the number of rats achieving successful resuscitation increased significantly in levo - bup and ll-1 groups ( p < 0.05 ) . however , no rats could maintain rpp 30% baseline in qx-314 , qx - oh , and ll-1 groups at 10 , 20 , and 50 min after rosc , respectively . moreover , asystole occurred again in some rats that had attained spontaneous circulation : 2 of 3 in the qx-314 group , 4 of 6 in the qx - oh group , and 2 of 8 in the ll-1 group . tasystole of the qx - oh group was significantly longer than that of other groups ( p < 0.05 ) ( figure 3a ) . trecovery of the ll-1 group was significantly longer than that of qx-314 and qx - oh groups ( p < 0.05 ) . the dose of epinephrine required for successful resuscitation was 38.75 11.26 , 23.33 5.77 , 28.33 11.69 , and 65.00 32.51 mg / kg in levo - bup , qx-314 , qx - oh , and ll-1 groups , respectively ( figure 3c ) . compared with qx - oh group , more epinephrine was needed to resuscitate rats in the ll-1 group ( p < 0.05 ) . percent arrhythmia duration was significantly less than in the levo - bup group than in the other groups ( p < 0.05 ) . there were no significant differences in ph , partial pressure of carbon dioxide , or partial pressure of oxygen among all groups ( p > 0.05 ) except that ph in the ll-1 group and partial pressure of carbon dioxide in the qx - oh group at 60 min after rosc were lower than those in the levo - bup group ( p < 0.05 ) . compared with the levo - bup group , lactate levels showed significant increases in the qx - oh group at 15 and 60 min and in the ll-1 group from 30 to 60 min after resuscitation ( p < 0.05 ) . second , the interaction between qx - oh and levo - bup for cardiac effects was nonsynergistic . third , the initial ability to resuscitate rats in which asystole had been induced by levo - bup , qx - oh , or ll-1 was similar . moreover , advanced life support might be needed after rosc in qx-314- , qx - oh- , and ll-1-induced asystole . according to the cd50c and cd50a , the potency for cardiac effects was levo - bup > qx-314 qx - oh ( table 1 ) . the potency for cardiotoxicity of local anesthetics is closely associated with the lipid solubility of drugs.16 compared with qx-314 and qx - oh , levo - bup is more lipid soluble , which could be why the cardiotoxicity of levo - bup was more severe than that of the other drugs we tested . the lipid solubility of qx - oh is less than that of qx-314 , which reduces the cardiotoxicity of qx - oh . in our study , even though the absolute dose of levo - bup resulting in cardiotoxicity was significantly lower than that of qx-314 or qx - oh ( 2.97 vs. 10.00 or 2.97 vs. 10.40 mg / kg ) , the si of levo - bup was significantly greater than that of qx-314 ( 10.60 vs. 1.20 ) or qx - oh ( 10.60 vs. 1.44 ) . however , the si of ll-1 was similar to that of levo - bup , suggesting that the safety of co - application of qx - oh and levo - bup was increased significantly compared with administration of qx - oh alone . meanwhile , the decreasing lipid solubility of qx - oh resulted in an extension of onset time on blocking the sodium channels of cardiac cells , which was longer than that of levo - bup . thus , the moment when qx - oh produced its peak effect was later than that of levo - bup or was even in the recovery stage of levo - bup - induced cardiotoxicity . these phenomena could explain , to a certain extent , the antagonism between qx - oh and levo - bup . the interaction index ( ) between qx - oh and levo - bup for asystole was less than that for the appearance of cardiotoxicity ( 1.202 vs. 1.537 ) , suggesting that the degree of antagonism decreased with increasing dose of qx - oh and levo - bup . thus , the time to peak effects of qx - oh might overlap with that of levo - bup when ll-1 was administrated , inducing a nonsynergistic effect . compared with qx - oh / levo - bup alone , the co - application of qx - oh and levo - bup produced the same or the lighter effects on cardiac functions under the same condition without affecting the potency of local anesthesia ( according to ed50 , the between qx - oh and levo - bup for sensory blockade was 0.817 , indicating a synergistic effect on local anesthesia ) . local anesthetics need access to intracellular domains to block voltage - gated sodium channels.22 however , qx-314 can not block sodium channels if applied extracellularly.4 this difference is due to the physicochemical properties of drugs at least in part , especially lipid solubility and charge.23 tasystole in the ll-1 group was shorter than that in the qx - oh group , but it is not known whether this effect was due to the levo - bup in ll-1 or levo - bup - induced cellular entry of qx - oh or both . our results suggested that the ability to resuscitate initially rats was similar among levo - bup , ll-1 , and qx - oh groups . however , sustainable arrhythmia occurred in qx-314 , qx - oh , and ll-1 groups , but not in the levo - bup group , after rosc , which led to circulatory collapse again . according to the manner of asystole or duration of arrhythmia ( % ) after rosc in qx-314 , qx - oh , and ll-1 groups , arrhythmia appeared to be the primary mechanism of cardiotoxicity induced by qx-314 and qx - oh . in a word , arrhythmogenicity of qx-314 and qx - oh was the reason why sustainable circulation could not be maintained after rosc in qx-314 , qx - oh , and ll-1 groups . even though circulatory collapse occurred again in the ll-1 group due to the arrhythmogenicity of qx - oh , it can not deny absolutely the use of ll-1 because the dose required to elicit asystole was much larger than the dose produced local anesthesia . the cardiac effects of qx - oh and levo - bup were nonsynergistic . initial successful resuscitation could be achieved in qx - oh- and ll-1-induced asystole .

the transient receptor potential ( trp ) was found to be the primary pathway of qx-314 to elicit local anesthesia.3 an agonist of trp , capsaicin , can promote the effects of qx-314.4 however , capsaicin is limited for painful irritation and toxicity in local tissue.5 qx-314 produces long - lasting nerve blockade after a delayed onset when administered extracellularly as a peripheral local anesthetic . to improve its penetrability and accelerate the drug entry into cells , a derivative of qx-314 , qx - oh ( 2-(2,6-dimethylphenylamino)-n , n - diethyl - n-(2-hydroxyethyl)-2-oxoethanaminium bromide ; molecular weight , 359.3 previously , we showed in animal models that qx - oh alone can ( like qx-314 ) not only produce long - lasting local anesthesia but also moderate toxicity in local tissue compared with the same concentration of qx-314 . bupivacaine ( bup ) , a local anesthetic with fast onset , can activate trp channels in the same way as capsaicin , inducing cellular entry of qx-314.6 therefore , we expect co - application of qx - oh and bup to induce a prolonged blockade with rapid onset . upon consideration of the severe system toxicity of bup , we choose the single levorotatory isomer of bup , levobupivacaine ( levo - bup ) , which is safer than bup . therefore , we conducted research on qx - oh , as well as a mixture of qx - oh and levo - bup , to meet the need for long - acting local anesthetics . previously , we found that 35 mm qx - oh in combination with 10 levobupivacaine , which we referred to as ll-1 , produced long - lasting sciatic nerve blockade in rats , which can be as long as 12 h , almost 3 times longer than 0.75% ( 23 mm ) bup alone , with minimal to mild local histological changes that were similar to those seen with 0.75% bup . therefore , we hypothesized that the cardiotoxicity of qx - oh was less than that of qx-314 due to the improvements in physicochemical properties . therefore , we compared the cardiotoxicity of levo - bup , qx-314 , and qx - oh in anesthetized rats and assessed the interaction between levo - bup and qx - oh . cd50c of levo - bup , qx-314 , qx - oh , and ll-1 was measured using the up - and - down method.8 this method , known as the staircase bioassay , is a procedure that has been proved to be effective in reducing the number of animals needed to determine a median dose . positive result was defined as a change in the following end points : a decrease in baseline heart rate 30% , any evidence of atrioventricular conduction blockade , widening of the qrs complex , or other arrhythmias.9 for levo - bup , the starting dose was 1 mg / kg with a log dosing increment of 0.181 . the method employed to measure the cd50a of levo - bup , qx-314 , qx - oh , first , the trachea was cannulated via tracheostomy , and then lungs were ventilated using a piston ventilator with a tidal volume of 1012 ml / kg at 5055 breaths / min to maintain the normal pressure of end - tidal carbon dioxide pressure . the following data were also collected in all 4 groups : time from initiation of injection of local anesthetics to asystole ( tasystole ) ; time from the start of asystole to rosc ( trecovery ) ; dose of epinephrine required for successful resuscitation ; duration of arrhythmia ( % ) after rosc ( if abnormal ecg was accompanied by abnormal arterial pulsation for 1 screen , this period was included in the duration of arrhythmia ) . differences in the cd50c and cd50a among groups were considered significant if 95% confidence intervals did not overlap.12 the si was calculated by using the equation : cd50c2ed50=si(1)isobolographic analysis was used to evaluate the interaction between levo - bup and qx - oh.13,14 the interaction index ( ) indicates the changed potency of a combination and is defined using the equation : aa+bb=(2)where a and b are the doses of drug a ( alone ) and drug b ( alone ) , respectively , that give the specified effect and ( a , b ) is the combination dose that produces the same effect . in a cochran armitage test for trend in proportions , sample sizes of 8 , 8 , 8 , and 8 are obtained from 4 groups with equally spaced x values and proportions equal to 0.80 , 0.80 , 0.40 , and 0.20 ( survival rate obtained from previous studies ) , respectively . baseline parameters of cardiac function , weight , tasystole , trecovery , dose of epinephrine required for successful resuscitation , and duration of arrhythmia ( % ) after rosc among groups were analyzed by 1-way analysis of variance and scheffe post hoc tests . cd50c of levo - bup , qx-314 , qx - oh , and ll-1 was measured using the up - and - down method.8 this method , known as the staircase bioassay , is a procedure that has been proved to be effective in reducing the number of animals needed to determine a median dose . a positive result was defined as a change in the following end points : a decrease in baseline heart rate 30% , any evidence of atrioventricular conduction blockade , widening of the qrs complex , or other arrhythmias.9 for levo - bup , the starting dose was 1 mg / kg with a log dosing increment of 0.181 . arterial blood gas was measured to confirm a ph between 7.30 and 7.45 and a serum lactate < 2.0 , rats were assigned randomly to receive 1.2-fold cd50a in the 4 groups a dose that reliably produces asystole from which hemodynamic recovery will occur with cardiopulmonary resuscitation . the following data were also collected in all 4 groups : time from initiation of injection of local anesthetics to asystole ( tasystole ) ; time from the start of asystole to rosc ( trecovery ) ; dose of epinephrine required for successful resuscitation ; duration of arrhythmia ( % ) after rosc ( if abnormal ecg was accompanied by abnormal arterial pulsation for 1 screen , this period was included in the duration of arrhythmia ) . differences in the cd50c and cd50a among groups were considered significant if 95% confidence intervals did not overlap.12 the si was calculated by using the equation : cd50c2ed50=si(1)isobolographic analysis was used to evaluate the interaction between levo - bup and qx - oh.13,14 the interaction index ( ) indicates the changed potency of a combination and is defined using the equation : aa+bb=(2)where a and b are the doses of drug a ( alone ) and drug b ( alone ) , respectively , that give the specified effect and ( a , b ) is the combination dose that produces the same effect . in a cochran armitage test for trend in proportions , sample sizes of 8 , 8 , 8 , and 8 are obtained from 4 groups with equally spaced x values and proportions equal to 0.80 , 0.80 , 0.40 , and 0.20 ( survival rate obtained from previous studies ) , respectively . baseline parameters of cardiac function , weight , tasystole , trecovery , dose of epinephrine required for successful resuscitation , and duration of arrhythmia ( % ) after rosc among groups were analyzed by 1-way analysis of variance and scheffe post hoc tests . the baseline heart rate of rats was 367 28 , 367 28 , 355 46 , and 358 38 beats / min in levo - bup , qx-314 , qx - oh , and ll-1 groups , respectively ( p > 0.05 ) . for cd50c , with addition of levo - bup , the cd50c of qx - oh was above the additive line , suggesting an antagonistic effect between levo - bup and qx - oh ( figure 2a ) . with addition of levo - bup , the cd50a of qx - oh was above or on the additive line , indicating a nonsynergistic effect between levo - bup and qx - oh ( figure 2b ) . rosc was attained initially by 8 of 8 rats in the levo - bup group , 3 of 8 in the qx-314 group , 6 of 8 in the qx - oh group , and 8 of 8 in the ll-1 group ( table 3 ) . compared with the qx-314 group , the number of rats achieving successful resuscitation increased significantly in levo - bup and ll-1 groups ( p < 0.05 ) . moreover , asystole occurred again in some rats that had attained spontaneous circulation : 2 of 3 in the qx-314 group , 4 of 6 in the qx - oh group , and 2 of 8 in the ll-1 group . the dose of epinephrine required for successful resuscitation was 38.75 11.26 , 23.33 5.77 , 28.33 11.69 , and 65.00 32.51 mg / kg in levo - bup , qx-314 , qx - oh , and ll-1 groups , respectively ( figure 3c ) . there were no significant differences in ph , partial pressure of carbon dioxide , or partial pressure of oxygen among all groups ( p > 0.05 ) except that ph in the ll-1 group and partial pressure of carbon dioxide in the qx - oh group at 60 min after rosc were lower than those in the levo - bup group ( p < 0.05 ) . compared with the levo - bup group , lactate levels showed significant increases in the qx - oh group at 15 and 60 min and in the ll-1 group from 30 to 60 min after resuscitation ( p < 0.05 ) . the baseline heart rate of rats was 367 28 , 367 28 , 355 46 , and 358 38 beats / min in levo - bup , qx-314 , qx - oh , and ll-1 groups , respectively ( p > 0.05 ) . with addition of levo - bup , the cd50c of qx - oh was above the additive line , suggesting an antagonistic effect between levo - bup and qx - oh ( figure 2a ) . with addition of levo - bup , the cd50a of qx - oh was above or on the additive line , indicating a nonsynergistic effect between levo - bup and qx - oh ( figure 2b ) . rosc was attained initially by 8 of 8 rats in the levo - bup group , 3 of 8 in the qx-314 group , 6 of 8 in the qx - oh group , and 8 of 8 in the ll-1 group ( table 3 ) . compared with the qx-314 group , the number of rats achieving successful resuscitation increased significantly in levo - bup and ll-1 groups ( p < 0.05 ) . moreover , asystole occurred again in some rats that had attained spontaneous circulation : 2 of 3 in the qx-314 group , 4 of 6 in the qx - oh group , and 2 of 8 in the ll-1 group . the dose of epinephrine required for successful resuscitation was 38.75 11.26 , 23.33 5.77 , 28.33 11.69 , and 65.00 32.51 mg / kg in levo - bup , qx-314 , qx - oh , and ll-1 groups , respectively ( figure 3c ) . there were no significant differences in ph , partial pressure of carbon dioxide , or partial pressure of oxygen among all groups ( p > 0.05 ) except that ph in the ll-1 group and partial pressure of carbon dioxide in the qx - oh group at 60 min after rosc were lower than those in the levo - bup group ( p < 0.05 ) . compared with the levo - bup group , lactate levels showed significant increases in the qx - oh group at 15 and 60 min and in the ll-1 group from 30 to 60 min after resuscitation ( p < 0.05 ) . the potency for cardiotoxicity of local anesthetics is closely associated with the lipid solubility of drugs.16 compared with qx-314 and qx - oh , levo - bup is more lipid soluble , which could be why the cardiotoxicity of levo - bup was more severe than that of the other drugs we tested . in our study , even though the absolute dose of levo - bup resulting in cardiotoxicity was significantly lower than that of qx-314 or qx - oh ( 2.97 vs. 10.00 or 2.97 vs. 10.40 mg / kg ) , the si of levo - bup was significantly greater than that of qx-314 ( 10.60 vs. 1.20 ) or qx - oh ( 10.60 vs. 1.44 ) . however , the si of ll-1 was similar to that of levo - bup , suggesting that the safety of co - application of qx - oh and levo - bup was increased significantly compared with administration of qx - oh alone . systemic toxic effects of local anesthetic combinations appear to have additive effects.17 however , a mixture of bup and lido can impair the intraventricular conduction parameters to a lesser extent than that for bup itself.18 these different observations might have been resulted ( at least in part ) because of differences in experimental design and ethnicities of participants . from an electrophysiological viewpoint , local anesthetics elicit cardiotoxicity primarily by blocking sodium channels.19 lido blocks sodium channels in a fast - in , fast - out fashion , whereas bup blocks these channels in a slow - in , slow - out manner in low concentrations or a fast - in , slow - out manner at high concentrations.20 therefore , competitive blockade is present between bup and lido . meanwhile , the decreasing lipid solubility of qx - oh resulted in an extension of onset time on blocking the sodium channels of cardiac cells , which was longer than that of levo - bup . the interaction index ( ) between qx - oh and levo - bup for asystole was less than that for the appearance of cardiotoxicity ( 1.202 vs. 1.537 ) , suggesting that the degree of antagonism decreased with increasing dose of qx - oh and levo - bup . moreover , the cd50a of qx - oh was above or on the additivity line , indicating a nonsynergistic effect between levo - bup and qx - oh it has been reported that qx-314 alone at relatively high concentrations can activate trp channels without any adjuvant drug and may penetrate slowly through lipid membranes.1,21 thus , the time to peak effects of qx - oh was shortened due to activation of trp channels induced by a relatively massive dose of qx - oh . compared with qx - oh / levo - bup alone , the co - application of qx - oh and levo - bup produced the same or the lighter effects on cardiac functions under the same condition without affecting the potency of local anesthesia ( according to ed50 , the between qx - oh and levo - bup for sensory blockade was 0.817 , indicating a synergistic effect on local anesthesia ) . with regard to the mechanism of death , asystole occurred rapidly in a fashion of disappearance of cardiac electrical activity after administration of levo - bup or ll-1 , whereas qx-314 and qx - oh brought about ventricular fibrillation a few minutes after injection of these drugs . local anesthetics need access to intracellular domains to block voltage - gated sodium channels.22 however , qx-314 can not block sodium channels if applied extracellularly.4 this difference is due to the physicochemical properties of drugs at least in part , especially lipid solubility and charge.23 tasystole in the ll-1 group was shorter than that in the qx - oh group , but it is not known whether this effect was due to the levo - bup in ll-1 or levo - bup - induced cellular entry of qx - oh or both . according to the manner of asystole or duration of arrhythmia ( % ) after rosc in qx-314 , qx - oh , and ll-1 groups , arrhythmia appeared to be the primary mechanism of cardiotoxicity induced by qx-314 and qx - oh . even though epinephrine can increase the number of ecg abnormalities after rosc,24 we think that arrhythmogenicity is the internal characteristic of qx-314 and qx - oh because ventricular fibrillation occurred before use of epinephrine . local anesthetics decrease ventricular conduction in a stereospecific manner and decrease contractility in a non - stereospecific manner based on stereospecificity for blockade of sodium and potassium channels.2628 therefore , it is not known whether variation of the molecular structure of lido leads to the stereospecificity of the blockade of ion channels with lido . even though circulatory collapse occurred again in the ll-1 group due to the arrhythmogenicity of qx - oh , it can not deny absolutely the use of ll-1 because the dose required to elicit asystole was much larger than the dose produced local anesthesia . as we know , ecg changes induced by local anesthetics occurred in a low concentration range asystole occurred in a fashion of the disappearance of cardiac electrical activity or ventricular fibrillation in a preliminary experiment , which was confirmed by ecg . however , epinephrine over a threshold dose near 10 mg / kg impairs lipid resuscitation from bup overdose.29 moreover , the lipid sink theory is the mechanism of lipid on the reversal of local anesthetic cardiotoxicity.30 some scholars have found no beneficial effects of lipid infusion on the less lipophilic mepivacaine- or rop - induced asystole , concluding that lipophilicity of local anesthetics has a marked impact on the efficacy of lipid infusion to treat asystole induced by these drugs.10,31 qx - oh is a less lipophilic drug .

escherichia coli is a common inhabitant of the gastrointestinal tract of humans and animals . usually , e. coli forms a beneficial symbiotic relationship with its host and plays important roles in promoting the stability of the luminal microbial flora and in maintaining normal intestinal homeostasis . as a commensal , e. coli rather remains harmlessly confined to the intestinal lumen and rarely causes a disease . however , in the debilitated or immunosuppressed host , or when the gastrointestinal barriers are violated , even nonpathogenic - commensal strains of e. coli can cause infection . some strains of e. coli can diverge from their commensal cohorts , taking on a more pathogenic nature . these strains acquire specific virulence factors ( via dna horizontal transfer of transposons , plasmids , bacteriophages , and pathogenicity islands ) , which confer an increased ability to adapt to new niches and allow the bacteria to increase the ability to cause a broad spectrum of diseases . the pathogenic e. coli strains are broadly classified as either enteric / diarrheagenic e. coli or extraintestinal e. coli ( expec ) . pathotypes , including enteropathogenic e. coli ( epec ) , enterohemorrhagic e. coli ( ehec ) , enterotoxigenic e. coli ( etec ) , enteroaggregative e. coli ( eaec ) , enteroinvasive e. coli ( eiec ) , and diffusely adherent e. coli ( daec ) , are the enteric / diarrheagenic e. coli , and two pathotypes , neonatal meningitis e. coli ( nmec ) and uropathogenic e. coli ( upec ) , are the most common expec [ 2 , 3 ] . several pathotypes of enteric / diarrheagenic e. coli give rise to gastroenteritis but rarely cause disease outside of the intestinal tract . on the other hand , the expec strains maintain the ability to exist in the gut without consequence but have the capacity to disseminate and colonize other host niches including the blood , the central nervous system , and the urinary tract , resulting in disease . urinary tract infections ( utis ) are considered to be the most common infections in humans . the development of utis depends on anatomical factors , the integrity of host defense mechanisms , and the virulence of the infecting organisms . utis are classified into disease categories according to the site of infection : cystitis ( the bladder ) , pyelonephritis ( the kidney ) and bacteriuria ( the urine ) . successful establishment of infection by bacterial pathogens requires adhesion to host cells , colonization of tissues , and , in certain cases , cellular invasion , followed by intracellular multiplication , dissemination to other tissues , or persistence . colonization of the urine in the absence of the clinical symptoms is called asymptomatic bacteriuria ( abu ) . most patients with abu do not need treatment , and in many cases the colonizing by the abu strains may help to prevent infection by other more virulent bacteria [ 79 ] . the primary causative agents responsible for more than 80% of all utis , including both abu and symptomatic utis , are strains of uropathogenic e. coli [ 1012 ] . upec is the cause of community - acquired utis and a large portion of nosocomial utis , accounting for substantial medical costs and morbidity and mortality worldwide [ 6 , 1315 ] . the ability of upec to cause symptomatic utis is associated with expression of a broad spectrum of virulence factors , with adhesive molecules being arguably the most important determinants of pathogenicity . in contrast to symptomatic upec , the reason why abu patients do not develop symptoms is not properly understood . however , it has been explained by a number of observations that many abu strains are nonadherent and nonhaemolytic [ 1820 ] . the strain e. coli 83972 , which was isolated from a patient with abu who had carried it for 3 years , has lost the ability to express functional p and type 1 fimbriae and has , therefore , been able to persist without triggering the host immune response . in contrast to the microorganisms that have acquired genes encoding adhesins for pathogenesis , e. coli 83972 is adapted to commensalism through gene loss and mutation [ 21 , 22 ] . the symptomatic strains of upec , which colonize the urinary tract , may ascend towards bladder to cause cystitis , which is usually associated with the classic symptoms of utis , that is , pain ( painful urination ) , frequency ( frequent urination ) , and urgency ( sudden compelling desire to urinate ) . however , utis can proceed from the bladder , via the ureters to the kidney , to cause pyelonephritis with the possibility of causing irreversible kidney damage and death . among gram - negative bacteria , moreover , urological complications , for example after renal transplantation , are associated with utis and e. coli is the most common clinical isolate [ 2426 ] . acute allograft injury in the renal transplant population is also associated with both upec and clinical diagnosis of upper utis . in response to the breach by upec into normally sterile urinary tract , the robust host innate response the production of inflammatory mediators results in the rapid recruitment of neutrophils into the bladder lumen and in bacterial clearance [ 28 , 29 ] . moreover , the host inflammatory response leads to the exfoliation of infected bladder epithelial cells and generation of reactive nitrogen and oxygen species along with other antimicrobial compounds [ 30 , 31 ] . these bacteria might also possess multitude of strategies to delay or suppress innate immune response , which facilitate bacterial growth and persistence within the adverse settings of the urinary tract . this paper summarizes our knowledge about virulence factors of upec , the inflammatory responses , and development of tissue damage caused by utis . the mechanisms by which epec stimulates proinflammatory response , including the role of ca , and influence of this response to development of cystitis and pyelonephritis will also be discussed . upec strains encode a number of virulence factors , which enable the bacteria to colonize the urinary tract and persist in face of highly effective host defense . upec isolates exhibit a high degree of genetic diversity due to the possession of specialized virulence genes located on mobile genetic elements called pathogenicity islands [ 4 , 32 ] . virulence factors of e. coli that have been potentially implicated as important to establish utis can be divided into two groups : ( i ) virulence factors associated with the surface of bacterial cell and ( ii ) virulence factors , which are secreted and exported to the site of action . surface virulence factors of upec include a number of different types of adhesive organelles ( fimbriae ) , which promote bacterial attachment to host tissues within the urinary tract . the presentation of adhesive molecules ( adhesins ) by upec is the most important determinant of pathogenicity . upec adhesins can contribute to virulence in different ways : ( i ) directly triggering host and bacterial cell signaling pathways , ( ii ) facilitating the delivery of other bacterial products to host tissues , and ( iii ) promoting bacterial invasion . type 1 fimbriae are implicated as virulence factors in animal models of urinary tract infection , but their function in human pathology remains unclear [ 3539 ] . role of the type 1 fimbriae in human disease is difficult to reconcile because they are expressed in both pathogenic and commensal strains [ 40 , 41 ] . specifically , there is no significant difference in the fim gene frequency between more or less virulent strains in the urinary tract . in the murine utis model , the type 1 fimbriae have been shown to enhance bacterial survival , to stimulate mucosal inflammation , and to promote invasion and growth as a biofilm [ 17 , 37 , 4346 ] . the type 1 fimbriae bind to the urothelial mannosylated glycoproteins uroplakin ia and iiia ( upiiia ) via the adhesin subunit fimh , located at the fimbrial tip . this interaction leads to molecular phosphorylation events , which are required for stimulation of signaling pathways involved in invasion and apoptosis and may also contribute to elevation of the intracellular ca level in urothelial cells [ 4 , 17 , 47 ] . furthermore , tamm - horsfall protein ( thp ) is produced by kidney cells into human urine and can act as a soluble fimh receptor , obstructing bacterial - host cell interaction and limiting the ability of upec to colonize the urinary tract [ 48 , 49 ] . p fimbriae are the second common virulence factor of upec , which plays an important role in the pathogenesis of ascending utis and pyelonephritis in humans [ 5052 ] . they are responsible for adhesion to mucosal and tissue matrix and for the production of cytokines [ 5355 ] . p fimbriae consist of heteropolymeric fibres composed of different protein subunits , encoded by the papa - k gene operon . these fimbriae recognize kidney glycosphingolipids carrying the gal ( 14 ) gal determinant on renal epithelia via its papg adhesion [ 2 , 57 ] . attachment of p fimbriae to this receptor leads to the release of ceramide , which acts as an agonist of toll - like receptor 4 ( tlr4 ) , a receptor involved in activation of the immune cell response . this , in turn , leads to the development of the local inflammation and pain associated with utis . recently , melican and coworkers have defined previously unknown synergistic functions of the both types of fimbriae , which facilitate bacterial colonization under dynamic in vivo conditions . p fimbriae have been shown to enhance early colonization of the tubular epithelium , while the type 1 fimbriae mediate colonization of the center of the tubule via a mechanism that involves inter - bacterial binding and biofilm formation . the heterogeneous bacterial community within the tubule subsequently affects renal filtration leading to total obstruction of the nephron . the p fimbriae are also the most common virulence factor and papg class ii allele is predominant class of the p fimbriae isolated from the patients with acute renal dysfunction . additionally , in the renal transplant patients with upper utis , a majority have an acute allograft injury due to upec , which express the p fimbriae . both these types of fimbriae show binding to epithelial and endothelial cell lines derived from the lower human urinary tract and kidney [ 34 , 61 , 62 ] . the s fimbriae may facilitate bacterial dissemination within host tissues and are often associated with e. coli strains that cause sepsis , meningitis , and ascending utis . both the fimbrial dr and afimbrial afa adhesins of e. coli are associated with utis , in particular , with gestational pyelonephritis and recurring cystitis [ 6366 ] . dr adhesins bind to type iv collagen and decay - accelerating factor ( daf ) in the kidney . dr adhesins have been demonstrated to display a tropism to the basement membrane of the renal interstitium in a mouse model and , therefore , are critical for development of the chronic pyelonephritis . mutation within the dra region encoding for dr fimbriae prevents development of the tubulointerstitial nephritis . adhesins of the afa family are involved in utis , and upec strains expressing these adhesins have a unique renal tissue tropism [ 70 , 71 ] . clinical and experimental findings suggest that e. coli strains with afa adhesins have properties potentially favoring the establishment of chronic and/or recurrent infection [ 65 , 69 ] . virulence factors located on the bacterial surface include also the capsule and the lipopolysaccharide ( lps ) . the capsule is mainly a polysaccharide structure covering and protecting the bacterium from the host immune system . the capsule provides protection against phagocytic engulfment and complement - mediated bactericidal effect in the host . certain capsular types , for example , k1 and k5 , show a molecular mimicry to tissue components , preventing a proper humoral immune response of the infected host . the lps is an integral component of the cell wall of gram - negative bacteria . lps is known to activate host response and to induce nitric oxide and cytokine production . although lps of upec is important in activation of proinflammatory response in uncomplicated utis , it is not clear whether lps plays a role in mediating a renal failure and acute allograft injury in patients with ascending utis . it has been demonstrated in an animal model that the acute renal failure due to lps depends on the systemic response to lps and does not depend on expression of functional lps receptor , tlr4 , in the kidney . however , tlr4 is expressed in renal epithelia and in the renal pelvis , and these findings suggest that the ascending infection due to e. coli may stimulate the innate immune response associated with the acute allograft injury in patients with utis [ 76 , 77 ] . flagella , an organelle responsible for bacterial motility , is involved in the interaction of various pathogenic e. coli strains with epithelial cells . flagellated upec cause 7090% of all urinary tract infections , and their pathogenesis involves contact between the bacteria and epithelial cell surface of the urinary tract . the pyelonephritis - associated e. coli strains may invade renal collecting duct ( cd ) cells through flagellin , and the flagellin acts as an invasin in this process . other studies have suggested that e. coli flagella may be of importance in allowing the bacteria to ascend from the bladder and to initiate kidney infection in humans . the use of an antibody against the flagella could prevent the spread of upec into the kidneys . production of toxins by colonizing e. coli may cause an inflammatory response , a possible pathway for utis symptoms . the most important secreted virulence factor of uropathogenic e. coli is a lipoprotein called -haemolysin ( hlya ) , which is associated with upper utis such as pyelonephritis . the hlya is a pore - forming toxin , which belongs to the family of rtx ( repeats in toxin ) toxins that are widespread among the gram - negative pathogens [ 80 , 81 ] . this toxin has been shown to exert dual concentration - dependent activities on primary epithelial cells originating from renal proximal tubules . at high concentrations , hlya is able to lyse erythrocytes and nucleated host cells , a process that may enable extraintestinal pathogens like upec to better cross mucosal barriers , damage effector immune cells , and gain enhanced access to host nutrients and iron stores [ 73 , 83 , 84 ] . at low concentrations , hlya can induce the apoptosis of target host cells , including neutrophils , t lymphocytes , and renal cells , and promote the exfoliation of bladder epithelial cells [ 8588 ] . hlya has also been shown to induce ca oscillations in renal epithelial cells , resulting in increased production of il-6 and il-8 . approximately 50% of all cases of pyelonephritis , which leads to renal complications , are caused by hlya . the e. coli - expressed -hemolysin may induce endothelial damage and renal vasoconstriction , for example , by the intrarenal release of endothelin . moreover , permanent renal scarring is a common complication following hlya e. coli infection [ 91 , 92 ] and may be independent of bacterial adherence properties . moreover , it has been shown that hlya and other e. coli toxins cause inducible nitric - oxide - synthase-(inos- ) mediated cell membrane injury and apoptosis , a process that is regulated by extracellular signal regulated kinase ( erk ) independently of the p53 pathway . the cytotoxic necrotising factor 1 ( cnf1 ) [ 95 , 96 ] is produced by one - third of all pyelonephritis strains and may also be involved in kidney invasion . in vitro , this protein is secreted by e. coli and stimulates actin stress fibers formation and membrane ruffle formation in a rho gtpase - dependent manner , resulting in the entry of e. coli into the cells . however , the detailed role of cnf1 in invasion processes during pyelonephritis remains unclear and is a matter of debate . in vitro studies have also showed that cnf1 interferes with polymorphonuclear phagocytosis and evokes apoptotic death of bladder epithelial cells [ 97 , 98 ] . in vivo , cnf1 may lead to bladder cell exfoliation and to enhanced bacterial access to underlying tissue . secreted autotransporter toxin ( sat ) is a virulence factor of pyelonephritis e. coli strains . sat has a toxic activity against cell lines of bladder or kidney origin and , thus , may be important for pathogenesis of utis [ 99 , 100 ] . moreover , the cytolethal distending toxin ( cdt ) could be also considered as a virulence factor in utis caused by e. coli [ 101 , 102 ] . toll / interleukin ( il-1 ) receptor ( tir ) domain - containing protein ( tcp ) represents a novel class of virulence factors , which are able to subvert tlr signaling to gain a survival advantage during utis . the urinary tract is a typically sterile environment , which is maintained by a variety of host mechanisms to prevent bacterial colonization and survival . most of the pathogenic bacteria , which cause utis , are from the host own bowel flora and enter the bladder via the urethra . upec strains possess an impressive repertoire of adhesins that enable the bacteria to aggregate and adhere to the cellular surfaces [ 34 , 57 , 62 , 69 , 104 ] . consequently , the first line of host defense against utis is concentrated on preventing upec adherence to the bladder mucosa . the urinary tract has a number of specialized defenses against bacterial colonization , keeping the urine sterile . the bulk flow of the urine through the bladder and micturition can work to rinse away nonattached or weakly adherent microbes from bladder surface . secretion of glucosamines by bladder transitional cells prevents bacterial adherence by forming a mucin layer . the low ph and presence of salts , urea , and organic acids in urine can reduce bacterial survival within the urinary tract . the tamm - horsfall glycoprotein acts as an antiadhesive urinary factor by binding to upec expressing type 1 fimbriae and forming the complex , which is then cleared by voiding [ 106109 ] . defensins ( a group of small , highly cationic antimicrobial peptides ) are also produced in the urinary tract after exposure to the pathogens . these peptides attach to the anionic phospholipids on the cell wall of pathogens and disrupt their cell membrane function , increasing cell permeability and causing cell death . if a microbe manages to sidestep these constitutive host defenses and makes contact with the urothelium , its continued presence can trigger the activation of additional host defense mechanisms , leading to exfoliation of infected bladder epithelial cells and inflammation . a key feature of inflammation during utis is a disruption of the urothelial integrity due to the exfoliation and subsequent excretion of infected eukaryotic cells [ 111 , 112 ] . this fimh - dependent exfoliation process occurs via an apoptosis - like pathway that involves the activation of caspases , cysteine proteases implicated in the execution of apoptosis , and dna fragmentation [ 16 , 113 ] . the importance of this response as a host defense is illustrated by the fact that the inhibition of exfoliation with a pan - caspase inhibitor dramatically reduces bacterial secretion from the bladder early in the infection . the bacteria , which manage to escape from dying superficial cells before the exfoliation process is completed , can go on to infect surrounding and underlying tissue . this may not only promote bacterial dissemination within the urinary tract but could also allow upec to enter a sheltered environment within the bladder , where bacteria can persist for long time . thus , exfoliation is a powerful mechanism of eradication of both attached and internalized bacteria from the bladder epithelium . upon successful adherence to the uroepithelium , the presence of bacteria and bacteria factors / products within the urinary tract can trigger rapid and robust responses from the host . infection with upec elicits both innate and adaptive immune responses , although an efficient host defense against urinary tract infection is reliant upon an early activation of the innate immune response [ 115 , 116 ] . this response is characterized by the production of a number of proinflammatory mediators , including cytokines and chemokines [ 117119 ] . bladder and kidney epithelial cells appear to be a major source of interleukin-6 ( il-6 ) and interleukin-8 ( il-8 ) after infection with upec , which are important in the development of local tissue damage [ 119123 ] . il-6 possesses a variety of proinflammatory functions , including activation of signals involved in neutrophils recruitment and production of acute phase proteins . a high urinary concentration of il-6 during acute phase of pyelonephritis has been shown to correlate with an increased risk of permanent renal scars . il-6 gene polymorphisms have been connected to susceptibility to utis , but not scar formation in children . il-8 is a potent neutrophil chemotactic molecule . in humans , the induction of il-8 after infection with upec correlates with appearance of neutrophils in the urine . neutrophil recruitment to the site of infection has been shown to be critical for bacterial clearance from both the bladder and kidney , and the presence of neutrophils in the urine is a hallmark of utis . interactions between the neutrophil receptor cd11/cd18 and the adhesion molecule icam-1 on bladder epithelial cells seem to be critical for neutrophil migration into the urothelium . recently , it has been shown that il-17a , an immunomodulatory cytokine , is involved in the innate immune response to utis . il-17a importance for the innate immune response has been demonstrated by a defect in acute clearance of upec in il-17a mice . this clearance defect is likely a result of deficient cytokine and chemokine transcription and of impaired macrophage and neutrophil influx during infection . in conclusion , explanation of the signaling pathways involved in the neutrophil recruitment into the bladder and kidneys highlights the importance of cytokines and chemokines . importantly , activation of the innate immune response has a dual effect : it is necessary for eradication of pathogenic bacteria but may also lead to tissue damage and permanent scarring . ( 1 ) host signaling in response to upec recognitionthe activation of the innate immune response in the urinary tract is dependent on recognition of bacterial components / products by tlrs [ 128131 ] . in recent years , it has become clear that the immune activation of bladder and kidney epithelial cells depends on tlrs , including tlr4 , tlr5 , and tlr11 [ 74 , 77 , 128 , 129 , 132 ] . recognition of virulence factors by tlrs stimulates signaling pathways resulting in activation and translocation of nf-b . in the nucleus , nf-b activates the transcription of proinflammatory genes , such as those encoding il-6 and il-8 . however , in response to bacterial products , the bladder epithelial cells can activate nf-b - independent signaling pathway ( see below ) . more recently , the involvement of interferon regulatory transcriptional factor irf3 in antibacterial defense and immunoregulation by tlrs has received more attention [ 134137 ] . possession of multiple signaling pathways for production of cytokines is advantageous in case when bacteria have an ability to suppress certain signaling events important for cytokine and chemokine production ( figure 1).tlr4 attracts most of the attention in context of the mechanisms of immune defense in the urinary tract . tlr4 is expressed on the epithelial cells throughout the urinary tract and is required to mount an effective inflammatory response after infection with upec . however , both the role of upec lps and an existence of another bacterial trigger of tlr4 signaling are an area of debate [ 54 , 74 , 117 ] . several reports have claimed that uroepithelial cells are refractory to lps stimulation and have argued that , instead , bacterial fimbriae such as the type 1 fimbriae , p fimbriae drive the induction of cytokines in these cells [ 130 , 138 ] . studies using the human kidney cell line indicate that tlr4-mediated signaling pathway in response to upec is dependent on p fimbriae and can be initiated independently of lps / cd14 [ 54 , 58 , 130 , 139 ] . recently , the new irf3-dependent signaling pathway , which leads to induction of the innate immune response in tlr4- and p - fimbriae - dependent manner , has been described . mechanistic details regarding this phenomenon include p fimbriae binding to surface glycosphingolipids ( gsls ) and subsequent release of the gsl membrane - anchoring domain , ceramide [ 58 , 140 ] . this molecule appears to act as a tlr4 agonist and the putative intermediate for tlr4 signaling initiated by p fimbriae . ceramide - induced tlr4 signaling causes rapid phosphorylation of proteins , including tram , plc , fyn , pka , p38 map kinase , erk1/2 , and creb , which are implicated in nuclear translocation of ifr3 and activation of irf3/ifn-dependent antibacterial effector mechanisms . the irf3-dependent signaling pathway is essential for the host defense and is critical for distinguishing pathogens from normal flora at the mucosal barrier . in the absence of irf3 ( in the irf3-knockout mice ) the acute mortality , bacterial burden , abscess formation , and renal damage have been observed , consistent with the need for this pathway to maintain a functional antimicrobial defense . relevance of irf3 pathway for human disease was supported by data concerning polymorphic irf3 promoter sequences , which differ between children with severe , symptomatic kidney infection and children who were asymptomatic bacterial carriers . recently , the fimh tip adhesin of type 1 fimbriae has been shown to interact directly with tlr4an additional means for lps - independent stimulation of tlr4 [ 142 , 143].however , other results underline the important role for lps and tlr4 in the stimulation of bladder epithelial cells by type 1 fimbriae e. coli [ 74 , 118 ] . stimulation of tlr4 by lps and type 1 fimbriae correlates with the level of cd14 expression on bladder cells [ 117 , 133 , 144 ] . interestingly , cd14 expression is localized to the submucosa , and this may suggest that uroepithelial cells exposed to the lumen have little to no cd14 expression and , therefore , can not respond efficiently to lps alone . these results support a role for both lps - dependent and cooperative tlr4 stimulation by upec fimbriae . the inflammatory response to lps is mediated via specific lps - binding protein , accessory molecules cd14 and md2 , and tir domain . the tir domain of tlr4 interacts with the adaptors myd88 and tir domain containing adaptor protein ( tirap ) . activation of tlr4 is known to lead finally to p38 mapk activation and nuclear translocation of nf-b , and transcription of inflammatory response genes ( figure 1).tlr5 plays a crucial role in host defense to upec infection by mediating flagellin - induced inflammatory responses in the bladder [ 128 , 146 ] . hence , a study in tlr5 mice challenged with upec demonstrated a decreased inflammatory response early after urethral bacterial infection and , subsequently , a concomitantly increased bacterial burden in both the bladder and kidney in these mice .recently , a new tlr11 expressed in the kidney , bladder , and liver of mice was discovered . mice lacking tlr11 are highly susceptible to infection of the kidneys by upec indicating a potentially important role for tlr11 in preventing infection of internal organs of the urogenital system . in humans tlr11 might not play a significant role due to the abundance of stop codons occurring in the human tlr11 gene . the activation of the innate immune response in the urinary tract is dependent on recognition of bacterial components / products by tlrs [ 128131 ] . in recent years , it has become clear that the immune activation of bladder and kidney epithelial cells depends on tlrs , including tlr4 , tlr5 , and tlr11 [ 74 , 77 , 128 , 129 , 132 ] . recognition of virulence factors by tlrs stimulates signaling pathways resulting in activation and translocation of nf-b . in the nucleus , nf-b activates the transcription of proinflammatory genes , such as those encoding il-6 and il-8 . however , in response to bacterial products , the bladder epithelial cells can activate nf-b - independent signaling pathway ( see below ) . more recently , the involvement of interferon regulatory transcriptional factor irf3 in antibacterial defense and immunoregulation by tlrs has received more attention [ 134137 ] . possession of multiple signaling pathways for production of cytokines is advantageous in case when bacteria have an ability to suppress certain signaling events important for cytokine and chemokine production ( figure 1 ) . tlr4 attracts most of the attention in context of the mechanisms of immune defense in the urinary tract . tlr4 is expressed on the epithelial cells throughout the urinary tract and is required to mount an effective inflammatory response after infection with upec . however , both the role of upec lps and an existence of another bacterial trigger of tlr4 signaling are an area of debate [ 54 , 74 , 117 ] . several reports have claimed that uroepithelial cells are refractory to lps stimulation and have argued that , instead , bacterial fimbriae such as the type 1 fimbriae , p fimbriae drive the induction of cytokines in these cells [ 130 , 138 ] . studies using the human kidney cell line indicate that tlr4-mediated signaling pathway in response to upec is dependent on p fimbriae and can be initiated independently of lps / cd14 [ 54 , 58 , 130 , 139 ] . recently , the new irf3-dependent signaling pathway , which leads to induction of the innate immune response in tlr4- and p - fimbriae - dependent manner , has been described . mechanistic details regarding this phenomenon include p fimbriae binding to surface glycosphingolipids ( gsls ) and subsequent release of the gsl membrane - anchoring domain , ceramide [ 58 , 140 ] . this molecule appears to act as a tlr4 agonist and the putative intermediate for tlr4 signaling initiated by p fimbriae . ceramide - induced tlr4 signaling causes rapid phosphorylation of proteins , including tram , plc , fyn , pka , p38 map kinase , erk1/2 , and creb , which are implicated in nuclear translocation of ifr3 and activation of irf3/ifn-dependent antibacterial effector mechanisms . the irf3-dependent signaling pathway is essential for the host defense and is critical for distinguishing pathogens from normal flora at the mucosal barrier . in the absence of irf3 ( in the irf3-knockout mice ) the acute mortality , bacterial burden , abscess formation , and renal damage have been observed , consistent with the need for this pathway to maintain a functional antimicrobial defense . relevance of irf3 pathway for human disease was supported by data concerning polymorphic irf3 promoter sequences , which differ between children with severe , symptomatic kidney infection and children who were asymptomatic bacterial carriers . recently , the fimh tip adhesin of type 1 fimbriae has been shown to interact directly with tlr4an additional means for lps - independent stimulation of tlr4 [ 142 , 143 ] . however , other results underline the important role for lps and tlr4 in the stimulation of bladder epithelial cells by type 1 fimbriae e. coli [ 74 , 118 ] . stimulation of tlr4 by lps and type 1 fimbriae correlates with the level of cd14 expression on bladder cells [ 117 , 133 , 144 ] . interestingly , cd14 expression is localized to the submucosa , and this may suggest that uroepithelial cells exposed to the lumen have little to no cd14 expression and , therefore , can not respond efficiently to lps alone . these results support a role for both lps - dependent and cooperative tlr4 stimulation by upec fimbriae . the inflammatory response to lps is mediated via specific lps - binding protein , accessory molecules cd14 and md2 , and tir domain . the tir domain of tlr4 interacts with the adaptors myd88 and tir domain containing adaptor protein ( tirap ) . activation of tlr4 is known to lead finally to p38 mapk activation and nuclear translocation of nf-b , and transcription of inflammatory response genes ( figure 1 ) . tlr5 plays a crucial role in host defense to upec infection by mediating flagellin - induced inflammatory responses in the bladder [ 128 , 146 ] . hence , a study in tlr5 mice challenged with upec demonstrated a decreased inflammatory response early after urethral bacterial infection and , subsequently , a concomitantly increased bacterial burden in both the bladder and kidney in these mice . recently , a new tlr11 expressed in the kidney , bladder , and liver of mice was discovered . mice lacking tlr11 are highly susceptible to infection of the kidneys by upec indicating a potentially important role for tlr11 in preventing infection of internal organs of the urogenital system . in humans tlr11 might not play a significant role due to the abundance of stop codons occurring in the human tlr11 gene . ( 2 ) genetic polymorphismgene polymorphisms have been shown to have an influence on the inflammatory response of uroepithelium to bacteria as well as on susceptibility to kidney damage . polymorphism of the tlr4 gene is associated with hyporesponsiveness to lps , absence of neutrophil recruitment , and delayed clearance of bacteria from the urinary tract in mice . polymorphisms in the tlr4 gene may also have a role in the inflammatory response in humans . lorenz et al . have suggested that the tlr4 asp299gly allele predisposes individuals to septic shock and a higher prevalence of gram - negative bacteremia . recently , ragnarsdttir et al . have described a new concept for human tlr variation , based on tlr4 promoter polymorphisms that influence gene expression dynamics in vitro and the innate immune response dynamics in patients with asymptomatic bacteriuria . they suggest that reduced tlr4 expression attenuates the innate mucosal response , thus promoting an asymptomatic carrier state rather than severe disease . have shown that trl5 stop codon polymorphism abolishes flagellin signaling and is associated with increased susceptibility to legionnaire 's disease . these murine studies support a hypothesis that individuals , who possess this tlr5 variant , will also be more susceptible to uti . polymorphisms in cytokine genes have also been connected to frequency and severity of urinary tract infections , probably as a result of variability in immune responses in such patients [ 150 , 151 ] . for example , the gene polymorphism associated with tnf has been observed in patients with reflux nephropathy . similarly , cotton et al . have shown that variability in the transforming growth factor-1 ( tgf-1 ) gene predisposes individuals to postinfectious renal scarring . as previously described , tgf-1 , the production of which is activated by other cytokines ( il-6 , il-8 , il-1 , and tnf- ) , leads to the extracellular matrix deposition through an increased synthesis of matrix proteins , and at the same time by decreasing matrix protein degradation following a suppression of protease expression and an increased production of tissue inhibitor of matrix metalloproteinases ( timps ) . all of these facts support the hypothesis that tgf-1 is an important factor in the pathogenesis of renal parenchymal scarring following uti . variable constitutive or induced expression of tgf-1 protein could occur as a consequence of variability in the tgf-1 gene , which in turn could be associated with differential effects on cellular growth or extracellular matrix deposition leading to renal parenchymal scarring . gene polymorphisms have been shown to have an influence on the inflammatory response of uroepithelium to bacteria as well as on susceptibility to kidney damage . polymorphism of the tlr4 gene is associated with hyporesponsiveness to lps , absence of neutrophil recruitment , and delayed clearance of bacteria from the urinary tract in mice . polymorphisms in the tlr4 gene may also have a role in the inflammatory response in humans . lorenz et al . have suggested that the tlr4 asp299gly allele predisposes individuals to septic shock and a higher prevalence of gram - negative bacteremia . recently , ragnarsdttir et al . have described a new concept for human tlr variation , based on tlr4 promoter polymorphisms that influence gene expression dynamics in vitro and the innate immune response dynamics in patients with asymptomatic bacteriuria . they suggest that reduced tlr4 expression attenuates the innate mucosal response , thus promoting an asymptomatic carrier state rather than severe disease . have shown that trl5 stop codon polymorphism abolishes flagellin signaling and is associated with increased susceptibility to legionnaire 's disease . these murine studies support a hypothesis that individuals , who possess this tlr5 variant , will also be more susceptible to uti . polymorphisms in cytokine genes have also been connected to frequency and severity of urinary tract infections , probably as a result of variability in immune responses in such patients [ 150 , 151 ] . for example , the gene polymorphism associated with tnf has been observed in patients with reflux nephropathy . similarly , cotton et al . have shown that variability in the transforming growth factor-1 ( tgf-1 ) gene predisposes individuals to postinfectious renal scarring . as previously described , tgf-1 , the production of which is activated by other cytokines ( il-6 , il-8 , il-1 , and tnf- ) , leads to the extracellular matrix deposition through an increased synthesis of matrix proteins , and at the same time by decreasing matrix protein degradation following a suppression of protease expression and an increased production of tissue inhibitor of matrix metalloproteinases ( timps ) . all of these facts support the hypothesis that tgf-1 is an important factor in the pathogenesis of renal parenchymal scarring following uti . variable constitutive or induced expression of tgf-1 protein could occur as a consequence of variability in the tgf-1 gene , which in turn could be associated with differential effects on cellular growth or extracellular matrix deposition leading to renal parenchymal scarring . several lines of evidence indicate that ca - dependent signaling pathway interacts with signal transduction pathways implicated to the innate immune response . it supposes that any effect on ca regulation is likely to have some influence on the innate immune response . interestingly , expression of il-8 has been shown to be under control of artificially induced ca oscillations due to frequency - modulated expression of the transcription factor nf- b. moreover , il-6 is responsible for activation of response that involves not only nf- b pathway , but also other signaling , which implicates two secondary messengers , ca and camp , and mobilizes a transcriptional element known as camp response element - binding protein ( creb ) . different bacterial virulence factors can induce an increase of ca concentrations in the host cells [ 154156 ] . -haemolysin of upec elicits oscillatory fluctuations of intracellular ca when present in the sublytic concentrations [ 89 , 157 ] . it has been shown that ca oscillations , corresponding to a periodicity of 12 min , specifically stimulate production of il-6 and il-8 in human renal epithelial cell . experiments designed to identify components of the intracellular signaling pathway leading to ca oscillations revealed that specific activity of hlya is required together with an activation of l - type ca channels to obtain ca oscillations . ip3-receptor - gated ca stores of the endoplasmic reticulum are involved [ 157 , 158 ] . hlya has long been known to physically interact with lps , which plays an indirect role in the cytolytic activity of hlya in erythrocytes and in epithelial cells [ 159161 ] . moreover , the ability of hly to induce ca oscillations has been suggested to require lps as a cofactor . mnsson et al . have shown that the lps and hlya complex exploits the cd14/lps - binding protein ( lbp ) recognition system to bring hlya to the cell membrane , where intracellular ca signaling is initiated via specific activation of the small gtpase rhoa . additionally , hlya - induced ca signaling has been found to occur independently of the lps receptor tlr4 . also , the cytolytic effect triggered by exposure of cell to high hlya concentrations occurs independently of the cd14/lps - lbp complex , suggesting that cytolysis is induced through mechanism different from that used for induction of ca oscillations . ( 1 ) tlr4-mediated nf - b - independent signalingstudies on tlr4 signaling have revealed the existence of a distinct tlr4-mediated signaling pathway leading to il-6 secretion . this pathway is present in the bladder epithelial cells and is activated upon exposure to lps . interestingly , this signaling is independent of the pathway involving the nf-b and contains two well - known secondary messengers , ca and camp , which mobilize transcription factor creb . the creb binds to camp - response element ( cre ) promoter sites to regulate the transcription of numerous genes in response to a diverse stimuli [ 133 , 162 ] . intracellular camp is an important second messenger in several signaling pathways , including il-6 response . the increase in camp following bacterial exposure depends on both bacterial - associated lps and increase of intracellular ca . this e. coli - induced ca - dependent camp production strictly correlates with activation of adenyl cyclases ( acs ) . because there are currently ten known isoforms of mammalian acs , it is noteworthy that of the four acs isoforms expressed in bladder epithelial cells only adenyl cyclase 3 ( ac3 ) is known to be activated by increase in intracellular ca [ 163 , 164 ] . e. coli - induced ca spike leads to ac3-mediated increase in camp , protein kinase a ( pka ) activation , and phosphorylation of the creb . upon phosphorylation , creb promotes transcription of a number of genes , including il-6 and il-8 ( figure 1 ) [ 162 , 165 , 166 ] . using selective blockade of different signaling pathways , it has been determined that the activation of cytokine secretion by upec e. coli might even be faster via the creb than via the nf-b pathway . the capacity of the bladder epithelial cells to mobilize the secondary messengers and to evoke the rapid il-6 response could be critical in their role as the first responders to microbial challenge in the urinary tract . studies on tlr4 signaling have revealed the existence of a distinct tlr4-mediated signaling pathway leading to il-6 secretion . this pathway is present in the bladder epithelial cells and is activated upon exposure to lps . interestingly , this signaling is independent of the pathway involving the nf-b and contains two well - known secondary messengers , ca and camp , which mobilize transcription factor creb . the creb binds to camp - response element ( cre ) promoter sites to regulate the transcription of numerous genes in response to a diverse stimuli [ 133 , 162 ] . intracellular camp is an important second messenger in several signaling pathways , including il-6 response . the increase in camp following bacterial exposure depends on both bacterial - associated lps and increase of intracellular ca . this e. coli - induced ca - dependent camp production strictly correlates with activation of adenyl cyclases ( acs ) . because there are currently ten known isoforms of mammalian acs , it is noteworthy that of the four acs isoforms expressed in bladder epithelial cells only adenyl cyclase 3 ( ac3 ) is known to be activated by increase in intracellular ca [ 163 , 164 ] . e. coli - induced ca spike leads to ac3-mediated increase in camp , protein kinase a ( pka ) activation , and phosphorylation of the creb . upon phosphorylation , creb promotes transcription of a number of genes , including il-6 and il-8 ( figure 1 ) [ 162 , 165 , 166 ] . using selective blockade of different signaling pathways , it has been determined that the activation of cytokine secretion by upec e. coli might even be faster via the creb than via the nf-b pathway . the capacity of the bladder epithelial cells to mobilize the secondary messengers and to evoke the rapid il-6 response could be critical in their role as the first responders to microbial challenge in the urinary tract . ( 2 ) upec fimh - induced elevation in urothelial caactivation of the host signal transduction cascades by bacterial attachment is a well - recognized consequence of the host - pathogen interactions , and urothelial signaling events associate with upec invasion and urothelial cell apoptosis . fimh is involved in adhesion , invasion , and apoptosis of urothelial cells and initiates bladder pathology by binding to the uroplakin receptor complex . recently , hitherto undiscovered signaling role for the upiiia in bacterial invasion and apoptosis has been presented . the upiiia is the only major uroplakin with a potential cytoplasmic signaling domain . in response to fimh adhesin binding , the upiiia cytoplasmic tail undergoes phosphorylation on a specific threonine residue by casein kinase ii , followed by an increase in intracellular ca concentration . fimh - mediated ca elevation occurs as a result of ca release from intracellular stores and by influx from extracellular sources . ca elevation promotes global responses critical to upec pathogenesis , including cytokine stimulation , membrane trafficking , and apoptosis [ 133 , 156].upec - induced upiiia signaling is a critical mediator of the pathogenic cascade induced in the host cell and is a novel therapeutic target . activation of the host signal transduction cascades by bacterial attachment is a well - recognized consequence of the host - pathogen interactions , and urothelial signaling events associate with upec invasion and urothelial cell apoptosis . fimh is involved in adhesion , invasion , and apoptosis of urothelial cells and initiates bladder pathology by binding to the uroplakin receptor complex . recently , hitherto undiscovered signaling role for the upiiia in bacterial invasion and apoptosis has been presented . the upiiia is the only major uroplakin with a potential cytoplasmic signaling domain . in response to fimh adhesin binding , the upiiia cytoplasmic tail undergoes phosphorylation on a specific threonine residue by casein kinase ii , followed by an increase in intracellular ca concentration . fimh - mediated ca elevation occurs as a result of ca release from intracellular stores and by influx from extracellular sources . ca elevation promotes global responses critical to upec pathogenesis , including cytokine stimulation , membrane trafficking , and apoptosis [ 133 , 156].upec - induced upiiia signaling is a critical mediator of the pathogenic cascade induced in the host cell and is a novel therapeutic target . several lines of evidence suggest that upec might possess strategies to delay , attenuate , suppress , or subvert the activity of components of the innate immune response in the urinary tract , especially early in infection [ 16 , 113 , 169172 ] . suppression of nf-b activation by upec results in enhanced type-1-fimbriae - mediated apoptosis of urothelial cells and in decreased levels of inflammatory cytokines production as well as neutrophil recruitment [ 113 , 170 , 172 ] . by hindering host cytokine expression and ensuing inflammatory responses , upec may be better able to establish itself and multiply within the cells and tissues of the urinary tract . upec infection in the murine bladder upregulates expression of the suppressor of cytokine signaling 3 gene . this phenomenon might represent a conserved strategy to subvert host defenses , allowing upec to survive in the bladder . additionally , several genes involved in lps biosynthesis ( e.g. , rfa and rfb ) and sura gene implicated in biogenesis of outer membrane proteins are important for the phenotype , suggesting that alternation in lps structure may underline the notstimulatory properties of upec [ 169 , 172 ] . hilbert et al . have shown that the bladder epithelial cells secrete il-6 and il-8 in response to nonpathogenic e. coli but are unable to mount the same cytokine response following exposure to upec , revealing dominant suppression of the innate immune response through a pathway partially independent of lps and tlr4 . recently it has been shown that tir homologous protein tcpc inhibits myd88-dependent gene expression in infected cf073 human uroepithelial cells [ 103 , 174 ] . the effects of tcpc on bacterial persistence were attenuated in trif or il-1 mice , and innate immune responses were increased , confirming that trif and il-1-dependent targets might be involved in vivo , in addition to myd88 . loss of tcpc led to decreased bacterial burden in kidneys and to reduced renal damage , showing the importance of these proteins in pathogenesis of the urinary tract infection . additionally , it has been shown that upec downregulates neutrophil activity , a phenotype , which is important during initiation and progression of infection , or for subsequent establishment of upec reservoir in the bladder . the ability of upec to suppress the innate immune response plays a role in persistence of pathogens within urinary tract . however , identifying the gene(s ) and factors that are involved in this process will contribute to understanding of upec pathogenesis and provide potential novel diagnostic and therapeutic targets . renal bacterial infections are common infectious diseases that can impair renal function and/or lead to the renal tubulointerstitial nephritis . they can cause severe renal dysfunction and are associated with various kidney diseases , such as iga nephropathy , renal vasculitis , and lupus nephropathy in postinfectious glomerulonephritis [ 176178 ] . in the previous sections we have described mechanisms involved in induction of proinflammatory response to upec . the innate immune system recognizes the virulence determinants of pathogens via receptors and activates the line of defense against pathogens . however , if functionality of the proinflammatory response is delayed or suppressed , bacteria after colonization the bladder can ascend the ureters and the kidneys . at this juncture , a risk of permanent renal scarring exists , and bacteria can access the bloodstream . in response to upec , the renal cells activate proinflammatory mediators , which play essential roles in the first line of defense against pathogens in the kidney . however , when this response is excessive , acute , or chronic pyelonephritis may occur , leading to severe damage and renal failure [ 180182 ] . the acute pyelonephritis is an acute inflammation of the renal parenchyma and pelvis associated with bacterial infection . clinically , the acute pyelonephritis is a severe form of urinary tract infection with symptoms that range from mild discomfort to life - threatening illness or death . complications may result in chronic renal scarring ( atrophic pyelonephritis or reflux nephropathy ) and impairment of renal function [ 184 , 185 ] . the chronic pyelonephritis has been defined as a destructive inflammatory process involving both the pyelocaliceal system and renal parenchyma . utis caused by upec are also the most frequent infectious complications in renal transplant patients and can impair long - term renal graft function . in conclusion , further studies are required to determine the mechanisms by which virulence factors of upec interact with the kidneys and lead to the renal failure as well as to the deterioration of renal allograft function . concurrently , better understanding of functions of virulence factors implicated in renal damage could open the way to control the immune response in the kidney and may be helpful for the development of effective therapies for e. coli - caused kidney diseases . among the gram - negative bacteria , upec is the pathogen most frequently associated with utis . upec , which colonizes the urinary tract , may ascend towards bladder to cause cystitis . left untreated , bacteria ascend the ureters to the kidney and establish a secondary infection , acute pyelonephritis with the possibility of causing irreversible kidney damage leading to kidney failure and death . the specific host - pathogen interactions are required to activate inflammation based on production of cytokines and chemokines by epithelial cells of the urinary tract . expression of adhesive organelles allows upec to bind and to invade the host cells and tissues within the urinary tract . moreover , deployment of an array of toxins provides upec with the means to inflict an extensive tissue damage , facilitating bacterial dissemination as well as releasing host nutrients and disabling the immune effector cells . recognition of bacterial products by tlrs activates nf-b - dependent signaling pathway , leading to translocation of nf-b into the nucleus and to expression of proinflammatory mediators , such as il-6 and il-8 . additionally , the bladder epithelial cells activate other nf-b - independent signaling pathways , which results in ca , camp , pka , and creb activation . altered urothelial ca signaling can modulate gene transcription , can stimulate cytokine expression in response to lps and tlr4 , and can be initiated by the interaction of upec adhesin fimh with the integral membrane protein upiiia to cause urothelial invasion and apoptosis in the bladder . these findings suggest more complicated sequence of early events in the pathogenesis of utis that may enhance the potential for recurrent utis . moreover , recently described new irf3 signaling indicates that the genetic variation in irf3 influences individual susceptibility to the kidney infection and might serve as a new tool for future risk assessment in this patient group .

uropathogenic escherichia coli ( upec ) is a causative agent in the vast majority of urinary tract infections ( utis ) , including cystitis and pyelonephritis , and infectious complications , which may result in acute renal failure in healthy individuals as well as in renal transplant patients . upec expresses a multitude of virulence factors to break the inertia of the mucosal barrier . in response to the breach by upec into the normally sterile urinary tract , host inflammatory responses are triggered leading to cytokine production , neutrophil influx , and the exfoliation of infected bladder epithelial cells . several signaling pathways activated during upec infection , including the pathways known to activate the innate immune response , interact with calcium - dependent signaling pathways . some upec isolates , however , might possess strategies to delay or suppress the activation of components of the innate host response in the urinary tract . studies published in the recent past provide new information regarding how virulence factors of uropathogenic e. coli are involved in activation of the innate host response . despite numerous host defense mechanisms , upec can persist within the urinary tract and may serve as a reservoir for recurrent infections and serious complications . presentation of the molecular details of these events is essential for development of successful strategies for prevention of human utis and urological complications associated with utis .

1. Introduction 2. Virulence Factors of Uropathogenic 3. Host Defenses against UPEC Colonization of the Urinary Tract 4. Conclusion

pathotypes , including enteropathogenic e. coli ( epec ) , enterohemorrhagic e. coli ( ehec ) , enterotoxigenic e. coli ( etec ) , enteroaggregative e. coli ( eaec ) , enteroinvasive e. coli ( eiec ) , and diffusely adherent e. coli ( daec ) , are the enteric / diarrheagenic e. coli , and two pathotypes , neonatal meningitis e. coli ( nmec ) and uropathogenic e. coli ( upec ) , are the most common expec [ 2 , 3 ] . on the other hand , the expec strains maintain the ability to exist in the gut without consequence but have the capacity to disseminate and colonize other host niches including the blood , the central nervous system , and the urinary tract , resulting in disease . urinary tract infections ( utis ) are considered to be the most common infections in humans . the development of utis depends on anatomical factors , the integrity of host defense mechanisms , and the virulence of the infecting organisms . the primary causative agents responsible for more than 80% of all utis , including both abu and symptomatic utis , are strains of uropathogenic e. coli [ 1012 ] . the symptomatic strains of upec , which colonize the urinary tract , may ascend towards bladder to cause cystitis , which is usually associated with the classic symptoms of utis , that is , pain ( painful urination ) , frequency ( frequent urination ) , and urgency ( sudden compelling desire to urinate ) . among gram - negative bacteria , moreover , urological complications , for example after renal transplantation , are associated with utis and e. coli is the most common clinical isolate [ 2426 ] . in response to the breach by upec into normally sterile urinary tract , the robust host innate response the production of inflammatory mediators results in the rapid recruitment of neutrophils into the bladder lumen and in bacterial clearance [ 28 , 29 ] . moreover , the host inflammatory response leads to the exfoliation of infected bladder epithelial cells and generation of reactive nitrogen and oxygen species along with other antimicrobial compounds [ 30 , 31 ] . these bacteria might also possess multitude of strategies to delay or suppress innate immune response , which facilitate bacterial growth and persistence within the adverse settings of the urinary tract . this paper summarizes our knowledge about virulence factors of upec , the inflammatory responses , and development of tissue damage caused by utis . the mechanisms by which epec stimulates proinflammatory response , including the role of ca , and influence of this response to development of cystitis and pyelonephritis will also be discussed . upec strains encode a number of virulence factors , which enable the bacteria to colonize the urinary tract and persist in face of highly effective host defense . virulence factors of e. coli that have been potentially implicated as important to establish utis can be divided into two groups : ( i ) virulence factors associated with the surface of bacterial cell and ( ii ) virulence factors , which are secreted and exported to the site of action . surface virulence factors of upec include a number of different types of adhesive organelles ( fimbriae ) , which promote bacterial attachment to host tissues within the urinary tract . type 1 fimbriae are implicated as virulence factors in animal models of urinary tract infection , but their function in human pathology remains unclear [ 3539 ] . this interaction leads to molecular phosphorylation events , which are required for stimulation of signaling pathways involved in invasion and apoptosis and may also contribute to elevation of the intracellular ca level in urothelial cells [ 4 , 17 , 47 ] . furthermore , tamm - horsfall protein ( thp ) is produced by kidney cells into human urine and can act as a soluble fimh receptor , obstructing bacterial - host cell interaction and limiting the ability of upec to colonize the urinary tract [ 48 , 49 ] . p fimbriae are the second common virulence factor of upec , which plays an important role in the pathogenesis of ascending utis and pyelonephritis in humans [ 5052 ] . attachment of p fimbriae to this receptor leads to the release of ceramide , which acts as an agonist of toll - like receptor 4 ( tlr4 ) , a receptor involved in activation of the immune cell response . this , in turn , leads to the development of the local inflammation and pain associated with utis . additionally , in the renal transplant patients with upper utis , a majority have an acute allograft injury due to upec , which express the p fimbriae . dr adhesins have been demonstrated to display a tropism to the basement membrane of the renal interstitium in a mouse model and , therefore , are critical for development of the chronic pyelonephritis . adhesins of the afa family are involved in utis , and upec strains expressing these adhesins have a unique renal tissue tropism [ 70 , 71 ] . lps is known to activate host response and to induce nitric oxide and cytokine production . although lps of upec is important in activation of proinflammatory response in uncomplicated utis , it is not clear whether lps plays a role in mediating a renal failure and acute allograft injury in patients with ascending utis . it has been demonstrated in an animal model that the acute renal failure due to lps depends on the systemic response to lps and does not depend on expression of functional lps receptor , tlr4 , in the kidney . however , tlr4 is expressed in renal epithelia and in the renal pelvis , and these findings suggest that the ascending infection due to e. coli may stimulate the innate immune response associated with the acute allograft injury in patients with utis [ 76 , 77 ] . flagella , an organelle responsible for bacterial motility , is involved in the interaction of various pathogenic e. coli strains with epithelial cells . flagellated upec cause 7090% of all urinary tract infections , and their pathogenesis involves contact between the bacteria and epithelial cell surface of the urinary tract . the most important secreted virulence factor of uropathogenic e. coli is a lipoprotein called -haemolysin ( hlya ) , which is associated with upper utis such as pyelonephritis . at low concentrations , hlya can induce the apoptosis of target host cells , including neutrophils , t lymphocytes , and renal cells , and promote the exfoliation of bladder epithelial cells [ 8588 ] . in vitro , this protein is secreted by e. coli and stimulates actin stress fibers formation and membrane ruffle formation in a rho gtpase - dependent manner , resulting in the entry of e. coli into the cells . the urinary tract is a typically sterile environment , which is maintained by a variety of host mechanisms to prevent bacterial colonization and survival . the low ph and presence of salts , urea , and organic acids in urine can reduce bacterial survival within the urinary tract . defensins ( a group of small , highly cationic antimicrobial peptides ) are also produced in the urinary tract after exposure to the pathogens . if a microbe manages to sidestep these constitutive host defenses and makes contact with the urothelium , its continued presence can trigger the activation of additional host defense mechanisms , leading to exfoliation of infected bladder epithelial cells and inflammation . a key feature of inflammation during utis is a disruption of the urothelial integrity due to the exfoliation and subsequent excretion of infected eukaryotic cells [ 111 , 112 ] . this fimh - dependent exfoliation process occurs via an apoptosis - like pathway that involves the activation of caspases , cysteine proteases implicated in the execution of apoptosis , and dna fragmentation [ 16 , 113 ] . this may not only promote bacterial dissemination within the urinary tract but could also allow upec to enter a sheltered environment within the bladder , where bacteria can persist for long time . upon successful adherence to the uroepithelium , the presence of bacteria and bacteria factors / products within the urinary tract can trigger rapid and robust responses from the host . infection with upec elicits both innate and adaptive immune responses , although an efficient host defense against urinary tract infection is reliant upon an early activation of the innate immune response [ 115 , 116 ] . bladder and kidney epithelial cells appear to be a major source of interleukin-6 ( il-6 ) and interleukin-8 ( il-8 ) after infection with upec , which are important in the development of local tissue damage [ 119123 ] . neutrophil recruitment to the site of infection has been shown to be critical for bacterial clearance from both the bladder and kidney , and the presence of neutrophils in the urine is a hallmark of utis . interactions between the neutrophil receptor cd11/cd18 and the adhesion molecule icam-1 on bladder epithelial cells seem to be critical for neutrophil migration into the urothelium . recently , it has been shown that il-17a , an immunomodulatory cytokine , is involved in the innate immune response to utis . in conclusion , explanation of the signaling pathways involved in the neutrophil recruitment into the bladder and kidneys highlights the importance of cytokines and chemokines . importantly , activation of the innate immune response has a dual effect : it is necessary for eradication of pathogenic bacteria but may also lead to tissue damage and permanent scarring . ( 1 ) host signaling in response to upec recognitionthe activation of the innate immune response in the urinary tract is dependent on recognition of bacterial components / products by tlrs [ 128131 ] . however , in response to bacterial products , the bladder epithelial cells can activate nf-b - independent signaling pathway ( see below ) . possession of multiple signaling pathways for production of cytokines is advantageous in case when bacteria have an ability to suppress certain signaling events important for cytokine and chemokine production ( figure 1).tlr4 attracts most of the attention in context of the mechanisms of immune defense in the urinary tract . tlr4 is expressed on the epithelial cells throughout the urinary tract and is required to mount an effective inflammatory response after infection with upec . recently , the new irf3-dependent signaling pathway , which leads to induction of the innate immune response in tlr4- and p - fimbriae - dependent manner , has been described . the irf3-dependent signaling pathway is essential for the host defense and is critical for distinguishing pathogens from normal flora at the mucosal barrier . recently , the fimh tip adhesin of type 1 fimbriae has been shown to interact directly with tlr4an additional means for lps - independent stimulation of tlr4 [ 142 , 143].however , other results underline the important role for lps and tlr4 in the stimulation of bladder epithelial cells by type 1 fimbriae e. coli [ 74 , 118 ] . activation of tlr4 is known to lead finally to p38 mapk activation and nuclear translocation of nf-b , and transcription of inflammatory response genes ( figure 1).tlr5 plays a crucial role in host defense to upec infection by mediating flagellin - induced inflammatory responses in the bladder [ 128 , 146 ] . the activation of the innate immune response in the urinary tract is dependent on recognition of bacterial components / products by tlrs [ 128131 ] . in recent years , it has become clear that the immune activation of bladder and kidney epithelial cells depends on tlrs , including tlr4 , tlr5 , and tlr11 [ 74 , 77 , 128 , 129 , 132 ] . however , in response to bacterial products , the bladder epithelial cells can activate nf-b - independent signaling pathway ( see below ) . tlr4 is expressed on the epithelial cells throughout the urinary tract and is required to mount an effective inflammatory response after infection with upec . recently , the new irf3-dependent signaling pathway , which leads to induction of the innate immune response in tlr4- and p - fimbriae - dependent manner , has been described . the irf3-dependent signaling pathway is essential for the host defense and is critical for distinguishing pathogens from normal flora at the mucosal barrier . however , other results underline the important role for lps and tlr4 in the stimulation of bladder epithelial cells by type 1 fimbriae e. coli [ 74 , 118 ] . polymorphism of the tlr4 gene is associated with hyporesponsiveness to lps , absence of neutrophil recruitment , and delayed clearance of bacteria from the urinary tract in mice . variable constitutive or induced expression of tgf-1 protein could occur as a consequence of variability in the tgf-1 gene , which in turn could be associated with differential effects on cellular growth or extracellular matrix deposition leading to renal parenchymal scarring . polymorphism of the tlr4 gene is associated with hyporesponsiveness to lps , absence of neutrophil recruitment , and delayed clearance of bacteria from the urinary tract in mice . have described a new concept for human tlr variation , based on tlr4 promoter polymorphisms that influence gene expression dynamics in vitro and the innate immune response dynamics in patients with asymptomatic bacteriuria . polymorphisms in cytokine genes have also been connected to frequency and severity of urinary tract infections , probably as a result of variability in immune responses in such patients [ 150 , 151 ] . variable constitutive or induced expression of tgf-1 protein could occur as a consequence of variability in the tgf-1 gene , which in turn could be associated with differential effects on cellular growth or extracellular matrix deposition leading to renal parenchymal scarring . several lines of evidence indicate that ca - dependent signaling pathway interacts with signal transduction pathways implicated to the innate immune response . interestingly , expression of il-8 has been shown to be under control of artificially induced ca oscillations due to frequency - modulated expression of the transcription factor nf- b. moreover , il-6 is responsible for activation of response that involves not only nf- b pathway , but also other signaling , which implicates two secondary messengers , ca and camp , and mobilizes a transcriptional element known as camp response element - binding protein ( creb ) . experiments designed to identify components of the intracellular signaling pathway leading to ca oscillations revealed that specific activity of hlya is required together with an activation of l - type ca channels to obtain ca oscillations . hlya has long been known to physically interact with lps , which plays an indirect role in the cytolytic activity of hlya in erythrocytes and in epithelial cells [ 159161 ] . because there are currently ten known isoforms of mammalian acs , it is noteworthy that of the four acs isoforms expressed in bladder epithelial cells only adenyl cyclase 3 ( ac3 ) is known to be activated by increase in intracellular ca [ 163 , 164 ] . using selective blockade of different signaling pathways , it has been determined that the activation of cytokine secretion by upec e. coli might even be faster via the creb than via the nf-b pathway . the capacity of the bladder epithelial cells to mobilize the secondary messengers and to evoke the rapid il-6 response could be critical in their role as the first responders to microbial challenge in the urinary tract . because there are currently ten known isoforms of mammalian acs , it is noteworthy that of the four acs isoforms expressed in bladder epithelial cells only adenyl cyclase 3 ( ac3 ) is known to be activated by increase in intracellular ca [ 163 , 164 ] . using selective blockade of different signaling pathways , it has been determined that the activation of cytokine secretion by upec e. coli might even be faster via the creb than via the nf-b pathway . the capacity of the bladder epithelial cells to mobilize the secondary messengers and to evoke the rapid il-6 response could be critical in their role as the first responders to microbial challenge in the urinary tract . ca elevation promotes global responses critical to upec pathogenesis , including cytokine stimulation , membrane trafficking , and apoptosis [ 133 , 156].upec - induced upiiia signaling is a critical mediator of the pathogenic cascade induced in the host cell and is a novel therapeutic target . activation of the host signal transduction cascades by bacterial attachment is a well - recognized consequence of the host - pathogen interactions , and urothelial signaling events associate with upec invasion and urothelial cell apoptosis . ca elevation promotes global responses critical to upec pathogenesis , including cytokine stimulation , membrane trafficking , and apoptosis [ 133 , 156].upec - induced upiiia signaling is a critical mediator of the pathogenic cascade induced in the host cell and is a novel therapeutic target . several lines of evidence suggest that upec might possess strategies to delay , attenuate , suppress , or subvert the activity of components of the innate immune response in the urinary tract , especially early in infection [ 16 , 113 , 169172 ] . by hindering host cytokine expression and ensuing inflammatory responses , upec may be better able to establish itself and multiply within the cells and tissues of the urinary tract . have shown that the bladder epithelial cells secrete il-6 and il-8 in response to nonpathogenic e. coli but are unable to mount the same cytokine response following exposure to upec , revealing dominant suppression of the innate immune response through a pathway partially independent of lps and tlr4 . loss of tcpc led to decreased bacterial burden in kidneys and to reduced renal damage , showing the importance of these proteins in pathogenesis of the urinary tract infection . the ability of upec to suppress the innate immune response plays a role in persistence of pathogens within urinary tract . in response to upec , the renal cells activate proinflammatory mediators , which play essential roles in the first line of defense against pathogens in the kidney . utis caused by upec are also the most frequent infectious complications in renal transplant patients and can impair long - term renal graft function . in conclusion , further studies are required to determine the mechanisms by which virulence factors of upec interact with the kidneys and lead to the renal failure as well as to the deterioration of renal allograft function . concurrently , better understanding of functions of virulence factors implicated in renal damage could open the way to control the immune response in the kidney and may be helpful for the development of effective therapies for e. coli - caused kidney diseases . the specific host - pathogen interactions are required to activate inflammation based on production of cytokines and chemokines by epithelial cells of the urinary tract . recognition of bacterial products by tlrs activates nf-b - dependent signaling pathway , leading to translocation of nf-b into the nucleus and to expression of proinflammatory mediators , such as il-6 and il-8 . additionally , the bladder epithelial cells activate other nf-b - independent signaling pathways , which results in ca , camp , pka , and creb activation . altered urothelial ca signaling can modulate gene transcription , can stimulate cytokine expression in response to lps and tlr4 , and can be initiated by the interaction of upec adhesin fimh with the integral membrane protein upiiia to cause urothelial invasion and apoptosis in the bladder .

usually , e. coli forms a beneficial symbiotic relationship with its host and plays important roles in promoting the stability of the luminal microbial flora and in maintaining normal intestinal homeostasis . these strains acquire specific virulence factors ( via dna horizontal transfer of transposons , plasmids , bacteriophages , and pathogenicity islands ) , which confer an increased ability to adapt to new niches and allow the bacteria to increase the ability to cause a broad spectrum of diseases . pathotypes , including enteropathogenic e. coli ( epec ) , enterohemorrhagic e. coli ( ehec ) , enterotoxigenic e. coli ( etec ) , enteroaggregative e. coli ( eaec ) , enteroinvasive e. coli ( eiec ) , and diffusely adherent e. coli ( daec ) , are the enteric / diarrheagenic e. coli , and two pathotypes , neonatal meningitis e. coli ( nmec ) and uropathogenic e. coli ( upec ) , are the most common expec [ 2 , 3 ] . on the other hand , the expec strains maintain the ability to exist in the gut without consequence but have the capacity to disseminate and colonize other host niches including the blood , the central nervous system , and the urinary tract , resulting in disease . successful establishment of infection by bacterial pathogens requires adhesion to host cells , colonization of tissues , and , in certain cases , cellular invasion , followed by intracellular multiplication , dissemination to other tissues , or persistence . the ability of upec to cause symptomatic utis is associated with expression of a broad spectrum of virulence factors , with adhesive molecules being arguably the most important determinants of pathogenicity . the strain e. coli 83972 , which was isolated from a patient with abu who had carried it for 3 years , has lost the ability to express functional p and type 1 fimbriae and has , therefore , been able to persist without triggering the host immune response . the symptomatic strains of upec , which colonize the urinary tract , may ascend towards bladder to cause cystitis , which is usually associated with the classic symptoms of utis , that is , pain ( painful urination ) , frequency ( frequent urination ) , and urgency ( sudden compelling desire to urinate ) . among gram - negative bacteria , moreover , urological complications , for example after renal transplantation , are associated with utis and e. coli is the most common clinical isolate [ 2426 ] . in response to the breach by upec into normally sterile urinary tract , the robust host innate response the production of inflammatory mediators results in the rapid recruitment of neutrophils into the bladder lumen and in bacterial clearance [ 28 , 29 ] . moreover , the host inflammatory response leads to the exfoliation of infected bladder epithelial cells and generation of reactive nitrogen and oxygen species along with other antimicrobial compounds [ 30 , 31 ] . virulence factors of e. coli that have been potentially implicated as important to establish utis can be divided into two groups : ( i ) virulence factors associated with the surface of bacterial cell and ( ii ) virulence factors , which are secreted and exported to the site of action . upec adhesins can contribute to virulence in different ways : ( i ) directly triggering host and bacterial cell signaling pathways , ( ii ) facilitating the delivery of other bacterial products to host tissues , and ( iii ) promoting bacterial invasion . in the murine utis model , the type 1 fimbriae have been shown to enhance bacterial survival , to stimulate mucosal inflammation , and to promote invasion and growth as a biofilm [ 17 , 37 , 4346 ] . this interaction leads to molecular phosphorylation events , which are required for stimulation of signaling pathways involved in invasion and apoptosis and may also contribute to elevation of the intracellular ca level in urothelial cells [ 4 , 17 , 47 ] . furthermore , tamm - horsfall protein ( thp ) is produced by kidney cells into human urine and can act as a soluble fimh receptor , obstructing bacterial - host cell interaction and limiting the ability of upec to colonize the urinary tract [ 48 , 49 ] . p fimbriae have been shown to enhance early colonization of the tubular epithelium , while the type 1 fimbriae mediate colonization of the center of the tubule via a mechanism that involves inter - bacterial binding and biofilm formation . both the fimbrial dr and afimbrial afa adhesins of e. coli are associated with utis , in particular , with gestational pyelonephritis and recurring cystitis [ 6366 ] . dr adhesins have been demonstrated to display a tropism to the basement membrane of the renal interstitium in a mouse model and , therefore , are critical for development of the chronic pyelonephritis . although lps of upec is important in activation of proinflammatory response in uncomplicated utis , it is not clear whether lps plays a role in mediating a renal failure and acute allograft injury in patients with ascending utis . it has been demonstrated in an animal model that the acute renal failure due to lps depends on the systemic response to lps and does not depend on expression of functional lps receptor , tlr4 , in the kidney . however , tlr4 is expressed in renal epithelia and in the renal pelvis , and these findings suggest that the ascending infection due to e. coli may stimulate the innate immune response associated with the acute allograft injury in patients with utis [ 76 , 77 ] . at high concentrations , hlya is able to lyse erythrocytes and nucleated host cells , a process that may enable extraintestinal pathogens like upec to better cross mucosal barriers , damage effector immune cells , and gain enhanced access to host nutrients and iron stores [ 73 , 83 , 84 ] . moreover , it has been shown that hlya and other e. coli toxins cause inducible nitric - oxide - synthase-(inos- ) mediated cell membrane injury and apoptosis , a process that is regulated by extracellular signal regulated kinase ( erk ) independently of the p53 pathway . bladder and kidney epithelial cells appear to be a major source of interleukin-6 ( il-6 ) and interleukin-8 ( il-8 ) after infection with upec , which are important in the development of local tissue damage [ 119123 ] . in conclusion , explanation of the signaling pathways involved in the neutrophil recruitment into the bladder and kidneys highlights the importance of cytokines and chemokines . ( 1 ) host signaling in response to upec recognitionthe activation of the innate immune response in the urinary tract is dependent on recognition of bacterial components / products by tlrs [ 128131 ] . in recent years , it has become clear that the immune activation of bladder and kidney epithelial cells depends on tlrs , including tlr4 , tlr5 , and tlr11 [ 74 , 77 , 128 , 129 , 132 ] . possession of multiple signaling pathways for production of cytokines is advantageous in case when bacteria have an ability to suppress certain signaling events important for cytokine and chemokine production ( figure 1).tlr4 attracts most of the attention in context of the mechanisms of immune defense in the urinary tract . studies using the human kidney cell line indicate that tlr4-mediated signaling pathway in response to upec is dependent on p fimbriae and can be initiated independently of lps / cd14 [ 54 , 58 , 130 , 139 ] . recently , the new irf3-dependent signaling pathway , which leads to induction of the innate immune response in tlr4- and p - fimbriae - dependent manner , has been described . ceramide - induced tlr4 signaling causes rapid phosphorylation of proteins , including tram , plc , fyn , pka , p38 map kinase , erk1/2 , and creb , which are implicated in nuclear translocation of ifr3 and activation of irf3/ifn-dependent antibacterial effector mechanisms . in the absence of irf3 ( in the irf3-knockout mice ) the acute mortality , bacterial burden , abscess formation , and renal damage have been observed , consistent with the need for this pathway to maintain a functional antimicrobial defense . recently , the fimh tip adhesin of type 1 fimbriae has been shown to interact directly with tlr4an additional means for lps - independent stimulation of tlr4 [ 142 , 143].however , other results underline the important role for lps and tlr4 in the stimulation of bladder epithelial cells by type 1 fimbriae e. coli [ 74 , 118 ] . interestingly , cd14 expression is localized to the submucosa , and this may suggest that uroepithelial cells exposed to the lumen have little to no cd14 expression and , therefore , can not respond efficiently to lps alone . activation of tlr4 is known to lead finally to p38 mapk activation and nuclear translocation of nf-b , and transcription of inflammatory response genes ( figure 1).tlr5 plays a crucial role in host defense to upec infection by mediating flagellin - induced inflammatory responses in the bladder [ 128 , 146 ] . hence , a study in tlr5 mice challenged with upec demonstrated a decreased inflammatory response early after urethral bacterial infection and , subsequently , a concomitantly increased bacterial burden in both the bladder and kidney in these mice .recently , a new tlr11 expressed in the kidney , bladder , and liver of mice was discovered . in recent years , it has become clear that the immune activation of bladder and kidney epithelial cells depends on tlrs , including tlr4 , tlr5 , and tlr11 [ 74 , 77 , 128 , 129 , 132 ] . studies using the human kidney cell line indicate that tlr4-mediated signaling pathway in response to upec is dependent on p fimbriae and can be initiated independently of lps / cd14 [ 54 , 58 , 130 , 139 ] . recently , the new irf3-dependent signaling pathway , which leads to induction of the innate immune response in tlr4- and p - fimbriae - dependent manner , has been described . ceramide - induced tlr4 signaling causes rapid phosphorylation of proteins , including tram , plc , fyn , pka , p38 map kinase , erk1/2 , and creb , which are implicated in nuclear translocation of ifr3 and activation of irf3/ifn-dependent antibacterial effector mechanisms . in the absence of irf3 ( in the irf3-knockout mice ) the acute mortality , bacterial burden , abscess formation , and renal damage have been observed , consistent with the need for this pathway to maintain a functional antimicrobial defense . however , other results underline the important role for lps and tlr4 in the stimulation of bladder epithelial cells by type 1 fimbriae e. coli [ 74 , 118 ] . interestingly , cd14 expression is localized to the submucosa , and this may suggest that uroepithelial cells exposed to the lumen have little to no cd14 expression and , therefore , can not respond efficiently to lps alone . polymorphism of the tlr4 gene is associated with hyporesponsiveness to lps , absence of neutrophil recruitment , and delayed clearance of bacteria from the urinary tract in mice . have described a new concept for human tlr variation , based on tlr4 promoter polymorphisms that influence gene expression dynamics in vitro and the innate immune response dynamics in patients with asymptomatic bacteriuria . as previously described , tgf-1 , the production of which is activated by other cytokines ( il-6 , il-8 , il-1 , and tnf- ) , leads to the extracellular matrix deposition through an increased synthesis of matrix proteins , and at the same time by decreasing matrix protein degradation following a suppression of protease expression and an increased production of tissue inhibitor of matrix metalloproteinases ( timps ) . variable constitutive or induced expression of tgf-1 protein could occur as a consequence of variability in the tgf-1 gene , which in turn could be associated with differential effects on cellular growth or extracellular matrix deposition leading to renal parenchymal scarring . polymorphism of the tlr4 gene is associated with hyporesponsiveness to lps , absence of neutrophil recruitment , and delayed clearance of bacteria from the urinary tract in mice . have described a new concept for human tlr variation , based on tlr4 promoter polymorphisms that influence gene expression dynamics in vitro and the innate immune response dynamics in patients with asymptomatic bacteriuria . as previously described , tgf-1 , the production of which is activated by other cytokines ( il-6 , il-8 , il-1 , and tnf- ) , leads to the extracellular matrix deposition through an increased synthesis of matrix proteins , and at the same time by decreasing matrix protein degradation following a suppression of protease expression and an increased production of tissue inhibitor of matrix metalloproteinases ( timps ) . variable constitutive or induced expression of tgf-1 protein could occur as a consequence of variability in the tgf-1 gene , which in turn could be associated with differential effects on cellular growth or extracellular matrix deposition leading to renal parenchymal scarring . interestingly , expression of il-8 has been shown to be under control of artificially induced ca oscillations due to frequency - modulated expression of the transcription factor nf- b. moreover , il-6 is responsible for activation of response that involves not only nf- b pathway , but also other signaling , which implicates two secondary messengers , ca and camp , and mobilizes a transcriptional element known as camp response element - binding protein ( creb ) . experiments designed to identify components of the intracellular signaling pathway leading to ca oscillations revealed that specific activity of hlya is required together with an activation of l - type ca channels to obtain ca oscillations . have shown that the lps and hlya complex exploits the cd14/lps - binding protein ( lbp ) recognition system to bring hlya to the cell membrane , where intracellular ca signaling is initiated via specific activation of the small gtpase rhoa . also , the cytolytic effect triggered by exposure of cell to high hlya concentrations occurs independently of the cd14/lps - lbp complex , suggesting that cytolysis is induced through mechanism different from that used for induction of ca oscillations . because there are currently ten known isoforms of mammalian acs , it is noteworthy that of the four acs isoforms expressed in bladder epithelial cells only adenyl cyclase 3 ( ac3 ) is known to be activated by increase in intracellular ca [ 163 , 164 ] . using selective blockade of different signaling pathways , it has been determined that the activation of cytokine secretion by upec e. coli might even be faster via the creb than via the nf-b pathway . the capacity of the bladder epithelial cells to mobilize the secondary messengers and to evoke the rapid il-6 response could be critical in their role as the first responders to microbial challenge in the urinary tract . because there are currently ten known isoforms of mammalian acs , it is noteworthy that of the four acs isoforms expressed in bladder epithelial cells only adenyl cyclase 3 ( ac3 ) is known to be activated by increase in intracellular ca [ 163 , 164 ] . using selective blockade of different signaling pathways , it has been determined that the activation of cytokine secretion by upec e. coli might even be faster via the creb than via the nf-b pathway . the capacity of the bladder epithelial cells to mobilize the secondary messengers and to evoke the rapid il-6 response could be critical in their role as the first responders to microbial challenge in the urinary tract . ( 2 ) upec fimh - induced elevation in urothelial caactivation of the host signal transduction cascades by bacterial attachment is a well - recognized consequence of the host - pathogen interactions , and urothelial signaling events associate with upec invasion and urothelial cell apoptosis . ca elevation promotes global responses critical to upec pathogenesis , including cytokine stimulation , membrane trafficking , and apoptosis [ 133 , 156].upec - induced upiiia signaling is a critical mediator of the pathogenic cascade induced in the host cell and is a novel therapeutic target . activation of the host signal transduction cascades by bacterial attachment is a well - recognized consequence of the host - pathogen interactions , and urothelial signaling events associate with upec invasion and urothelial cell apoptosis . ca elevation promotes global responses critical to upec pathogenesis , including cytokine stimulation , membrane trafficking , and apoptosis [ 133 , 156].upec - induced upiiia signaling is a critical mediator of the pathogenic cascade induced in the host cell and is a novel therapeutic target . several lines of evidence suggest that upec might possess strategies to delay , attenuate , suppress , or subvert the activity of components of the innate immune response in the urinary tract , especially early in infection [ 16 , 113 , 169172 ] . suppression of nf-b activation by upec results in enhanced type-1-fimbriae - mediated apoptosis of urothelial cells and in decreased levels of inflammatory cytokines production as well as neutrophil recruitment [ 113 , 170 , 172 ] . , rfa and rfb ) and sura gene implicated in biogenesis of outer membrane proteins are important for the phenotype , suggesting that alternation in lps structure may underline the notstimulatory properties of upec [ 169 , 172 ] . have shown that the bladder epithelial cells secrete il-6 and il-8 in response to nonpathogenic e. coli but are unable to mount the same cytokine response following exposure to upec , revealing dominant suppression of the innate immune response through a pathway partially independent of lps and tlr4 . the effects of tcpc on bacterial persistence were attenuated in trif or il-1 mice , and innate immune responses were increased , confirming that trif and il-1-dependent targets might be involved in vivo , in addition to myd88 . in conclusion , further studies are required to determine the mechanisms by which virulence factors of upec interact with the kidneys and lead to the renal failure as well as to the deterioration of renal allograft function . concurrently , better understanding of functions of virulence factors implicated in renal damage could open the way to control the immune response in the kidney and may be helpful for the development of effective therapies for e. coli - caused kidney diseases . recognition of bacterial products by tlrs activates nf-b - dependent signaling pathway , leading to translocation of nf-b into the nucleus and to expression of proinflammatory mediators , such as il-6 and il-8 . altered urothelial ca signaling can modulate gene transcription , can stimulate cytokine expression in response to lps and tlr4 , and can be initiated by the interaction of upec adhesin fimh with the integral membrane protein upiiia to cause urothelial invasion and apoptosis in the bladder .

regular and widespread use of pharmaceuticals , which are frequently excreted as non- metabolized parent compounds , has led to growing concerns for the safety of drinking water . the vast range of pharmaceutical products that have been detected in sewage , surface , ground and drinking waters include bronchodilators , oral contraceptives , antidepressants , beta - blockers , anti - biotics , anti - inflammatories and analgesics [ 2 - 7 ] . even modern sewage treatment works are not constructed to specifically eliminate pharmaceuticals from potable water supplies . stanozolol , an anabolic steroid is a synthetic derivative of the endogenously - produced male - sex hormone testosterone . it is commonly misused as a performance enhancement drug because of its ability to enhance muscular strength . the world anti - doping agency ( wada ) has banned its use in- and out - of - competition . despite the restriction , stanozolol is one of the most commonly misused synthetic , anabolic steroids in sport and in veterinary practice , where it is used for growth promoting purposes . in humans , stanozolol is mainly metabolized by undergoing hydroxylation to form mono- and di - hydroxylated metabolites . less than 5% are excreted as non - conjugated fractions . according to wada , doping with stanozolol is confirmed if the urinary concentration of its major metabolite , 3-hydroxystanozolol exceeds 2 ng / ml . unlike testosterone , the synthetic stanozolol and/or its main metabolite should only appear in environmental waters if the former is used for veterinary purposes , taken under medical supervision or illegally by athletes for performance enhancement or if either one or both of these compounds are accidently discharged into environmental waters . in previous studies , we reported the detection of stanozolol in hair samples collected from subjects living in budapest . for this reason , we initiated this study to explore possible environmental sources of steroid contamination . the aim of this study was to develop a methodology for the detection of stanozolol in aqueous matrices . to achieve this , liquid - liquid extraction ( lle ) was employed for purification and concentration followed by direct determination using lc - ms / ms . extraction recovery was evaluated for aqueous matrices spiked with stanozolol at pg / ml levels . stanozolol and stanozolol d3 ( internal standard ) were obtained from lgc standards ( teddington , london , uk ) . pentane , deionised water , formic acid , and acetonitrile were obtained from sigma aldrich ( poole , dorset , uk ) . environmental water samples were obtained from budapest ( hungary ) and collected from the river danube and an urban tap ( drinking water ) in clean , amber bottles . samples from lake balaton and spring water ( rzsika forrs , solymr , near budapest ) were also collected for comparison . some commonly - consumed , bottled non - carbonated , natural mineral water samples were purchased from local supermarkets . the majority of the analysed , commercially available , bottled natural mineral waters are recognised by the european union . liquid - liquid extraction ( lle ) using pentane was employed for the extraction of stanozolol from water samples . suspended particles were not filtered from the river water so that the drug adsorbed on them could also be extracted efficiently . a 5 ml aliquot of each water sample was spiked with stanozolol d3 ( internal standard , 50 l of 10 ng / ml ) followed by the addition of 3 ml pentane . the contents were vortex mixed vigorously for 10 seconds followed by centrifugation at 3500 g at ambient temperature for 5 minutes . the pentane layer was separated and collected in a silanized glass tube . to ensure good recovery , both organic fractions were pooled and dried by evaporation at 45 c under a gentle stream of nitrogen gas . a 5 l aliquot of the reconstituted solution was injected into the lc - ms / ms system for analysis . the lc - ms / ms system consisted of an accela lc system ( thermo scientific , uk ) coupled to a tsq quantum triple quadrupole mass spectrometer ( thermo electron , uk ) without a flow splitter . the lc system was comprised of a quaternary pump , automatic solvent degasser , column heater and an auto - sampler equipped with tray chiller . chromatographic separation was obtained on an agilent zorbax sb - c18 column ( 2.1 mm 50 mm , 1.8 m ) maintained at 60 c . lc mobile phase gradient composition the mass spectrometer was equipped with an electrospray ionization ( esi ) source operated in positive ion mode . an ion spray voltage of 4000 v was essential for optimum ionization of stanozolol and stanozolol d3 ( internal standard ) . the protonated molecules , [ m+h ] , of stanozolol ( m / z 329.2 ) and stanozolol d3 ( m / z 332.2 ) , were used as precursor ions for collision induced dissociation ( cid ) for ms - ms analysis . selective reaction monitoring ( srm ) was used to monitor the precursor ions and diagnostic product ions for unambiguous quantification of stanozolol . the collision energies and srm , m / z transitions for stanozolol and internal standard ( i.s . ) are shown in table 2 . retention times , srm transitions and collision energies of stanozolol and stanozolol d3 ( internal standard ) the thermo scientific xcalibur software ( version 2.1 ) was used to control the lc system and mass spectrometer . the performance of the analytical method was validated for the following set of parameters : linearity , specificity , accuracy , lower limit of detection ( llod ) , lower limit of quantification ( lloq ) , inter - day precision and intra - day precision . calibration samples and quality control samples at low , medium and high concentration levels were prepared by fortifying 5 ml hplc grade water with known concentrations of stanozolol and i.s . the analyte - to - internal standard ratio was calculated by dividing the area of analyte peak by the area of the i.s . a calibration curve was constructed by plotting the analyte - to - internal standard ratio versus the known concentration of stanozolol in each sample . linear regression analysis using the least squares method was employed to evaluate the calibration curve of analyte as a function of its concentrations in water samples . the lloq or lowest point on the calibration curve was defined as the lowest concentration of analyte which could be quantified with a precision < 20% ( cv ) . to determine the lower limit of detection ( llod ) , a number of serial 1:2 dilutions were made from the low standard ( lloq ) . the lowest concentration which gave a response equivalent to three times the background noise was considered as the llod . the accuracy and intra - day precision was assessed by injecting qc samples in replicates at 3 different concentrations . this was repeated on three consecutive days to evaluate the inter - day precision of the assay . the average extraction recovery for the analyte was determined by comparing the analyte to internal standard peak area ratio obtained after extracting negative control water samples fortified with stanozolol at a final concentration of 2 pg / ml in presence of i.s with the un - extracted standard working solutions at the same concentrations . the matrix effects were assessed by comparing the responses of analyte and i.s . obtained from the extracted blank water samples ( hplc grade , tap water and river water ) spiked with known concentrations of stanozolol and stanozolol d3 after extraction to those obtained from neat standard solution at the same final concentrations . stanozolol and stanozolol d3 ( internal standard ) were obtained from lgc standards ( teddington , london , uk ) . pentane , deionised water , formic acid , and acetonitrile were obtained from sigma aldrich ( poole , dorset , uk ) . environmental water samples were obtained from budapest ( hungary ) and collected from the river danube and an urban tap ( drinking water ) in clean , amber bottles . samples from lake balaton and spring water ( rzsika forrs , solymr , near budapest ) were also collected for comparison . some commonly - consumed , bottled non - carbonated , natural mineral water samples were purchased from local supermarkets . the majority of the analysed , commercially available , bottled natural mineral waters are recognised by the european union . liquid - liquid extraction ( lle ) using pentane was employed for the extraction of stanozolol from water samples . suspended particles were not filtered from the river water so that the drug adsorbed on them could also be extracted efficiently . a 5 ml aliquot of each water sample was spiked with stanozolol d3 ( internal standard , 50 l of 10 ng / ml ) followed by the addition of 3 ml pentane . the contents were vortex mixed vigorously for 10 seconds followed by centrifugation at 3500 g at ambient temperature for 5 minutes . the pentane layer was separated and collected in a silanized glass tube . to ensure good recovery , both organic fractions were pooled and dried by evaporation at 45 c under a gentle stream of nitrogen gas . a 5 l aliquot of the reconstituted solution was injected into the lc - ms / ms system for analysis . the lc - ms / ms system consisted of an accela lc system ( thermo scientific , uk ) coupled to a tsq quantum triple quadrupole mass spectrometer ( thermo electron , uk ) without a flow splitter . the lc system was comprised of a quaternary pump , automatic solvent degasser , column heater and an auto - sampler equipped with tray chiller . chromatographic separation was obtained on an agilent zorbax sb - c18 column ( 2.1 mm 50 mm , 1.8 m ) maintained at 60 c . lc mobile phase gradient composition the mass spectrometer was equipped with an electrospray ionization ( esi ) source operated in positive ion mode . an ion spray voltage of 4000 v was essential for optimum ionization of stanozolol and stanozolol d3 ( internal standard ) . the protonated molecules , [ m+h ] , of stanozolol ( m / z 329.2 ) and stanozolol d3 ( m / z 332.2 ) , were used as precursor ions for collision induced dissociation ( cid ) for ms - ms analysis . selective reaction monitoring ( srm ) was used to monitor the precursor ions and diagnostic product ions for unambiguous quantification of stanozolol . the collision energies and srm , m / z transitions for stanozolol and internal standard ( i.s . ) are shown in table 2 . retention times , srm transitions and collision energies of stanozolol and stanozolol d3 ( internal standard ) the thermo scientific xcalibur software ( version 2.1 ) was used to control the lc system and mass spectrometer . the performance of the analytical method was validated for the following set of parameters : linearity , specificity , accuracy , lower limit of detection ( llod ) , lower limit of quantification ( lloq ) , inter - day precision and intra - day precision . calibration samples and quality control samples at low , medium and high concentration levels were prepared by fortifying 5 ml hplc grade water with known concentrations of stanozolol and i.s . the analyte - to - internal standard ratio was calculated by dividing the area of analyte peak by the area of the i.s . a calibration curve was constructed by plotting the analyte - to - internal standard ratio versus the known concentration of stanozolol in each sample . linear regression analysis using the least squares method was employed to evaluate the calibration curve of analyte as a function of its concentrations in water samples . the lloq or lowest point on the calibration curve was defined as the lowest concentration of analyte which could be quantified with a precision < 20% ( cv ) . to determine the lower limit of detection ( llod ) , a number of serial 1:2 dilutions were made from the low standard ( lloq ) . the lowest concentration which gave a response equivalent to three times the accuracy and intra - day precision was assessed by injecting qc samples in replicates at 3 different concentrations . this was repeated on three consecutive days to evaluate the inter - day precision of the assay . the average extraction recovery for the analyte was determined by comparing the analyte to internal standard peak area ratio obtained after extracting negative control water samples fortified with stanozolol at a final concentration of 2 pg / ml in presence of i.s with the un - extracted standard working solutions at the same concentrations . the matrix effects were assessed by comparing the responses of analyte and i.s . obtained from the extracted blank water samples ( hplc grade , tap water and river water ) spiked with known concentrations of stanozolol and stanozolol d3 after extraction to those obtained from neat standard solution at the same final concentrations . stanozolol was unambiguously analysed on the basis of its srm transition and retention time ( figures 1 and 2 ) via the method proposed and validated herein . regression analysis indicated that the assay showed excellent linearity within the quantification range of 0.5 to 200 pg / ml water for stanozolol . the lloq for stanozolol was found to be 0.5 pg / ml . the correlation coefficients were found to be greater than 0.996 during the method validation procedure . under the optimized lc - ms / ms conditions , the assay was capable of detecting ( llod ) stanozolol , without any interference , at a concentration as low as 0.25 pg / ml water when 5 ml water was processed . the analytical characteristics of this method including accuracy , linearity , llod , lloq , inter - day precision , intra - day precision and extraction recoveries from hplc grade , river and tap water are summarized in table 3 . the relative standard deviation ( rsd ) chromatogram and mass spectrum of stanozolol spiked to danube river water at a final concentration of 0.5 pg / ml . chromatogram and mass spectrum of stanozolol spiked to tap water at a final concentration of 0.5 pg / ml . matrix effects in river water led to a reduction in peak areas of stanozolol and stanozolol d3 by 22.3% and 18.4% , respectively . comparatively , tap water and hplc water showed lesser matrix effects . the reduction in peak areas was possibly attributed to ion suppression in the esi source . however , after internal standard correction , the matrix effects in all three types of water samples were comparable and in the range 95.4 - 97.3% as shown in table 4 . thus , stanozolol d3 was used as an internal standard to : i ) compensate for matrix induced changes in ionization of analyte , ii ) correct any loss of analyte during sample preparation , iii ) compensate for any variations in the instrument response from injection to injection . correction ) in three water types ; namely hplc grade water , danube river water and tap water were in the range 95.3% to 98.4% . the relative extraction recoveries ( with i.s . correction ) in all three water types were found to be in the range 94.2 to 95.5% for stanozolol as shown in table 5 . this indicated that the method is capable of detecting stanozolol in different types of aqueous matrix when only 5 ml water was processed . the analytical prerequisites for efficient detection of stanozolol at low levels in aqueous matrix were , purification of water samples using liquid - liquid extraction in presence of a deuterated internal standard followed by injecting only 5 l aliquot through the column combined with the optimized lc - ms / ms conditions employed for analysis . matrix effect results for stanozolol and stanozolol d3 in hplc water , tap water and river water meis matrix effect expressed as the ratio of mean peak area of analyte spiked postextraction to the mean peak area of the same analyte standard multiplied by 100 . corrected ) at 2 pg / ml gas chromatography mass spectrometry ( gc - ms ) and high performance liquid chromatography ( hplc ) coupled to fluorescence and uv detectors have been commonly employed to analyse steroids . however , hplc coupled to fluorescence detection involves laborious sample preparation steps and gc - ms requires a complicated sample derivatization step which makes the method more time consuming and expensive . hence , use of lc - ms / ms for analyzing steroids is a feasible approach as the sample preparation step involved is facile , economical and does not require any additional derivatization step . compared to previous methods for detecting steroids in environmental waters [ 15,17 - 19 ] , the major advantage of our method is that less volume ( only 5 ml , opposed to up to 1000 ml ) of water sample is required for analysis . another advantage includes the use of liquid - liquid extraction for purification of water samples , which is less time consuming and more economical in comparison to the solid phase extraction processes employed in previous studies [ 15,17 - 20 ] . in three out of six samples from the danube river , collected since december ' 09 , stanozolol was detected with levels up to 1.82 pg / ml . in contrast , only one sample of urban tap drinking water from budapest city was found to contain stanozolol at a concentration of 1.19 pg / ml . the results for stanozolol analysis in different water samples are shown in table 6 . determination of stanozolol in environmental and domestic water samples blq means below limit of quantification nd means not detectable the possible sources of stanozolol entering the river are unknown , but may be from human or animal consumption and excretion of un - metabolized drug or due to accidental discharge of the parent compound . it should be noted that stanozolol was only found once in tap water and that this level does not present a threat to health based on recommended intake levels . water samples from river and tap were collected periodically until november and stanozolol concentrations were found to be reducing over time as shown in table 5 . the possible reasons for a gradual reduction in concentration could be due to : i ) variations in rates of contamination , ii ) dilution of river water due to rise in water levels , ( see additional file 1 ) , iii ) degradation of the steroid in the river water due to other constituents in the river or photolysis , or deposition in the sediment . the ph values of all the river and tap water samples collected were found to be in the neutral range . stanozolol being basic in nature due to the presence of a pyrozole ring is found to be stable in neutral to slightly basic ph . further investigation needs to be carried out for determining the source of stanozolol and reasons for gradual decrease in its concentration . in recent years stanozolol has been detected ( qualitatively ) in sludge samples collected from huiyang and meihu waste water treatment plants . chang et al . have also reported the presence of stanozolol in beijing influent waste water at a concentration of ca . 0.54 pg / ml . recently , tlgyesi et al . have reported the presence of the steroids cortisol , dexamethasone , flumethasone , prednisolone and epitestosterone in danube river water , but their selection of analytes did not include stanozolol . our results indicate that stanozolol was present in the river danube and budapest tap water in the month of december 2009 , when the water level in the river was low ( additional file 2 ) . the national health service ( nhs ) recommends a minimum water intake of 1.2 litres every day . hence , individuals drinking stanozolol contaminated urban tap water ( 1.19 pg / ml ) will involuntarily consume approximately 1.43 ng stanozolol per day . since the effective doses of stanozolol for men and women are 50 - 100 mgs / day and 2.5 - 10 mgs / day respectively , such low levels detected in drinking water may not cause significant harm to the general public , especially as they were found only at one time point . in addition , a new biological sewage treatment plant opened in july 2010 in budapest in order to treat most of the water supplied to the city ( in contrast to only 30 - 40% water being treated in the past ) . this major environmental protection investment will potentially contribute to a further decrease in levels of stanozolol compared to those we previously observed . future studies , sampling from various river and tap water sites should , in due course , be able to provide evidence for this . stanozolol was unambiguously analysed on the basis of its srm transition and retention time ( figures 1 and 2 ) via the method proposed and validated herein . regression analysis indicated that the assay showed excellent linearity within the quantification range of 0.5 to 200 pg / ml water for stanozolol . the lloq for stanozolol was found to be 0.5 pg / ml . the correlation coefficients were found to be greater than 0.996 during the method validation procedure . under the optimized lc - ms / ms conditions , the assay was capable of detecting ( llod ) stanozolol , without any interference , at a concentration as low as 0.25 pg / ml water when 5 ml water was processed . the analytical characteristics of this method including accuracy , linearity , llod , lloq , inter - day precision , intra - day precision and extraction recoveries from hplc grade , river and tap water are summarized in table 3 . the relative standard deviation ( rsd ) chromatogram and mass spectrum of stanozolol spiked to danube river water at a final concentration of 0.5 pg / ml . chromatogram and mass spectrum of stanozolol spiked to tap water at a final concentration of 0.5 pg / ml . matrix effects in river water led to a reduction in peak areas of stanozolol and stanozolol d3 by 22.3% and 18.4% , respectively . comparatively , tap water and hplc water showed lesser matrix effects . the reduction in peak areas was possibly attributed to ion suppression in the esi source . however , after internal standard correction , the matrix effects in all three types of water samples were comparable and in the range 95.4 - 97.3% as shown in table 4 . thus , stanozolol d3 was used as an internal standard to : i ) compensate for matrix induced changes in ionization of analyte , ii ) correct any loss of analyte during sample preparation , iii ) compensate for any variations in the instrument response from injection to injection . correction ) in three water types ; namely hplc grade water , danube river water and tap water were in the range 95.3% to 98.4% . the relative extraction recoveries ( with i.s . correction ) in all three water types were found to be in the range 94.2 to 95.5% for stanozolol as shown in table 5 . this indicated that the method is capable of detecting stanozolol in different types of aqueous matrix when only 5 ml water was processed . the analytical prerequisites for efficient detection of stanozolol at low levels in aqueous matrix were , purification of water samples using liquid - liquid extraction in presence of a deuterated internal standard followed by injecting only 5 l aliquot through the column combined with the optimized lc - ms / ms conditions employed for analysis . matrix effect results for stanozolol and stanozolol d3 in hplc water , tap water and river water meis matrix effect expressed as the ratio of mean peak area of analyte spiked postextraction to the mean peak area of the same analyte standard multiplied by 100 . corrected ) at 2 pg / ml gas chromatography mass spectrometry ( gc - ms ) and high performance liquid chromatography ( hplc ) coupled to fluorescence and uv detectors have been commonly employed to analyse steroids . however , hplc coupled to fluorescence detection involves laborious sample preparation steps and gc - ms requires a complicated sample derivatization step which makes the method more time consuming and expensive . hence , use of lc - ms / ms for analyzing steroids is a feasible approach as the sample preparation step involved is facile , economical and does not require any additional derivatization step . compared to previous methods for detecting steroids in environmental waters [ 15,17 - 19 ] , the major advantage of our method is that less volume ( only 5 ml , opposed to up to 1000 ml ) of water sample is required for analysis . another advantage includes the use of liquid - liquid extraction for purification of water samples , which is less time consuming and more economical in comparison to the solid phase extraction processes employed in previous studies [ 15,17 - 20 ] . no stanozolol was detected in any of the fifteen bottled waters investigated . in three out of six samples from the danube river , collected since december ' 09 , in contrast , only one sample of urban tap drinking water from budapest city was found to contain stanozolol at a concentration of 1.19 pg / ml . the results for stanozolol analysis in different water samples are shown in table 6 . determination of stanozolol in environmental and domestic water samples blq means below limit of quantification nd means not detectable the possible sources of stanozolol entering the river are unknown , but may be from human or animal consumption and excretion of un - metabolized drug or due to accidental discharge of the parent compound . it should be noted that stanozolol was only found once in tap water and that this level does not present a threat to health based on recommended intake levels . water samples from river and tap were collected periodically until november and stanozolol concentrations were found to be reducing over time as shown in table 5 . the possible reasons for a gradual reduction in concentration could be due to : i ) variations in rates of contamination , ii ) dilution of river water due to rise in water levels , ( see additional file 1 ) , iii ) degradation of the steroid in the river water due to other constituents in the river or photolysis , or deposition in the sediment . the ph values of all the river and tap water samples collected were found to be in the neutral range . stanozolol being basic in nature due to the presence of a pyrozole ring is found to be stable in neutral to slightly basic ph . further investigation needs to be carried out for determining the source of stanozolol and reasons for gradual decrease in its concentration . in recent years stanozolol has been detected ( qualitatively ) in sludge samples collected from huiyang and meihu waste water treatment plants . chang et al . have also reported the presence of stanozolol in beijing influent waste water at a concentration of ca . 0.54 pg / ml . recently , tlgyesi et al . have reported the presence of the steroids cortisol , dexamethasone , flumethasone , prednisolone and epitestosterone in danube river water , but their selection of analytes did not include stanozolol . our results indicate that stanozolol was present in the river danube and budapest tap water in the month of december 2009 , when the water level in the river was low ( additional file 2 ) . the national health service ( nhs ) hence , individuals drinking stanozolol contaminated urban tap water ( 1.19 pg / ml ) will involuntarily consume approximately 1.43 ng stanozolol per day . since the effective doses of stanozolol for men and women are 50 - 100 mgs / day and 2.5 - 10 mgs / day respectively , such low levels detected in drinking water may not cause significant harm to the general public , especially as they were found only at one time point . in addition , a new biological sewage treatment plant opened in july 2010 in budapest in order to treat most of the water supplied to the city ( in contrast to only 30 - 40% water being treated in the past ) . this major environmental protection investment will potentially contribute to a further decrease in levels of stanozolol compared to those we previously observed . future studies , sampling from various river and tap water sites should , in due course , be able to provide evidence for this . in conclusion , a rapid , highly sensitive , robust and reproducible method has been developed to detect stanozolol in different types of water samples . the assay is capable of detecting stanozolol at a concentration as low as 0.25 pg / ml water when only 5 ml water is processed . the performance of this method gives acceptable relative recoveries for stanozolol river and tap water samples . the method can be extended to detect other chemicals and pharmaceutical drugs which may be hazardous to human health and environment . the supporting document reports the water level , volume , and temperature of river danube from december 2009 to november 2010 . details of bottled drinking water analysed . the supporting document reports the ph , eu recognition and origin of the bottled natural mineral water analysed . the authors thank julian swinden and iltaf shah for technical advice and help ; ip and fp for sample collection . the photos were taken by fp and zsb .

backgroundowing to frequent administration of a wide range of pharmaceutical products , various environmental waters have been found to be contaminated with pharmacologically active substances . for example , stanozolol , a synthetic anabolic steroid , is frequently misused for performance enhancement as well as for illegal growth promoting purposes in veterinary practice . previously we reported stanozolol in hair samples collected from subjects living in budapest . for this reason we initiated this study to explore possible environmental sources of steroid contamination . the aim of this study was to develop a method to monitor stanozolol in aqueous matrices using liquid chromatography tandem mass spectrometry ( lc - ms / ms).resultsliquid - liquid extraction using pentane was found to be an efficient method for the extraction of stanozolol from water samples . this was followed by direct detection using lc - ms / ms . the method was capable of detecting 0.25 pg / ml stanozolol when only 5 ml water was processed in the presence of stanozolol d3 as internal standard . fifteen bottled waters analysed were found to be negative for stanozolol . however , three out of six samples from the danube river , collected from december ' 09 to november ' 10 , were found to contain stanozolol at concentrations up to 1.82 pg / ml . in contrast , only one sample ( out of six ) of urban tap water from budapest city was found to contain stanozolol , at a concentration of 1.19 pg / ml.conclusionthe method developed is efficient , rapid , reproducible , sensitive and robust for the detection of stanozolol in aqueous matrices .

Background Experimental Reagents and chemicals Extraction procedure Instrumentation Results and discussion Method validation Water analyses results Conclusions Competing interests Authors' contributions Supplementary Material Acknowledgements

the vast range of pharmaceutical products that have been detected in sewage , surface , ground and drinking waters include bronchodilators , oral contraceptives , antidepressants , beta - blockers , anti - biotics , anti - inflammatories and analgesics [ 2 - 7 ] . stanozolol , an anabolic steroid is a synthetic derivative of the endogenously - produced male - sex hormone testosterone . despite the restriction , stanozolol is one of the most commonly misused synthetic , anabolic steroids in sport and in veterinary practice , where it is used for growth promoting purposes . according to wada , doping with stanozolol is confirmed if the urinary concentration of its major metabolite , 3-hydroxystanozolol exceeds 2 ng / ml . unlike testosterone , the synthetic stanozolol and/or its main metabolite should only appear in environmental waters if the former is used for veterinary purposes , taken under medical supervision or illegally by athletes for performance enhancement or if either one or both of these compounds are accidently discharged into environmental waters . in previous studies , we reported the detection of stanozolol in hair samples collected from subjects living in budapest . for this reason , we initiated this study to explore possible environmental sources of steroid contamination . the aim of this study was to develop a methodology for the detection of stanozolol in aqueous matrices . to achieve this , liquid - liquid extraction ( lle ) was employed for purification and concentration followed by direct determination using lc - ms / ms . extraction recovery was evaluated for aqueous matrices spiked with stanozolol at pg / ml levels . environmental water samples were obtained from budapest ( hungary ) and collected from the river danube and an urban tap ( drinking water ) in clean , amber bottles . liquid - liquid extraction ( lle ) using pentane was employed for the extraction of stanozolol from water samples . a 5 ml aliquot of each water sample was spiked with stanozolol d3 ( internal standard , 50 l of 10 ng / ml ) followed by the addition of 3 ml pentane . a 5 l aliquot of the reconstituted solution was injected into the lc - ms / ms system for analysis . the lc - ms / ms system consisted of an accela lc system ( thermo scientific , uk ) coupled to a tsq quantum triple quadrupole mass spectrometer ( thermo electron , uk ) without a flow splitter . an ion spray voltage of 4000 v was essential for optimum ionization of stanozolol and stanozolol d3 ( internal standard ) . the protonated molecules , [ m+h ] , of stanozolol ( m / z 329.2 ) and stanozolol d3 ( m / z 332.2 ) , were used as precursor ions for collision induced dissociation ( cid ) for ms - ms analysis . retention times , srm transitions and collision energies of stanozolol and stanozolol d3 ( internal standard ) the thermo scientific xcalibur software ( version 2.1 ) was used to control the lc system and mass spectrometer . calibration samples and quality control samples at low , medium and high concentration levels were prepared by fortifying 5 ml hplc grade water with known concentrations of stanozolol and i.s . a calibration curve was constructed by plotting the analyte - to - internal standard ratio versus the known concentration of stanozolol in each sample . linear regression analysis using the least squares method was employed to evaluate the calibration curve of analyte as a function of its concentrations in water samples . to determine the lower limit of detection ( llod ) , a number of serial 1:2 dilutions were made from the low standard ( lloq ) . the average extraction recovery for the analyte was determined by comparing the analyte to internal standard peak area ratio obtained after extracting negative control water samples fortified with stanozolol at a final concentration of 2 pg / ml in presence of i.s with the un - extracted standard working solutions at the same concentrations . obtained from the extracted blank water samples ( hplc grade , tap water and river water ) spiked with known concentrations of stanozolol and stanozolol d3 after extraction to those obtained from neat standard solution at the same final concentrations . stanozolol and stanozolol d3 ( internal standard ) were obtained from lgc standards ( teddington , london , uk ) . environmental water samples were obtained from budapest ( hungary ) and collected from the river danube and an urban tap ( drinking water ) in clean , amber bottles . liquid - liquid extraction ( lle ) using pentane was employed for the extraction of stanozolol from water samples . a 5 ml aliquot of each water sample was spiked with stanozolol d3 ( internal standard , 50 l of 10 ng / ml ) followed by the addition of 3 ml pentane . a 5 l aliquot of the reconstituted solution was injected into the lc - ms / ms system for analysis . the lc - ms / ms system consisted of an accela lc system ( thermo scientific , uk ) coupled to a tsq quantum triple quadrupole mass spectrometer ( thermo electron , uk ) without a flow splitter . an ion spray voltage of 4000 v was essential for optimum ionization of stanozolol and stanozolol d3 ( internal standard ) . the protonated molecules , [ m+h ] , of stanozolol ( m / z 329.2 ) and stanozolol d3 ( m / z 332.2 ) , were used as precursor ions for collision induced dissociation ( cid ) for ms - ms analysis . the collision energies and srm , m / z transitions for stanozolol and internal standard ( i.s . ) retention times , srm transitions and collision energies of stanozolol and stanozolol d3 ( internal standard ) the thermo scientific xcalibur software ( version 2.1 ) was used to control the lc system and mass spectrometer . a calibration curve was constructed by plotting the analyte - to - internal standard ratio versus the known concentration of stanozolol in each sample . the average extraction recovery for the analyte was determined by comparing the analyte to internal standard peak area ratio obtained after extracting negative control water samples fortified with stanozolol at a final concentration of 2 pg / ml in presence of i.s with the un - extracted standard working solutions at the same concentrations . obtained from the extracted blank water samples ( hplc grade , tap water and river water ) spiked with known concentrations of stanozolol and stanozolol d3 after extraction to those obtained from neat standard solution at the same final concentrations . regression analysis indicated that the assay showed excellent linearity within the quantification range of 0.5 to 200 pg / ml water for stanozolol . the lloq for stanozolol was found to be 0.5 pg / ml . the correlation coefficients were found to be greater than 0.996 during the method validation procedure . under the optimized lc - ms / ms conditions , the assay was capable of detecting ( llod ) stanozolol , without any interference , at a concentration as low as 0.25 pg / ml water when 5 ml water was processed . the analytical characteristics of this method including accuracy , linearity , llod , lloq , inter - day precision , intra - day precision and extraction recoveries from hplc grade , river and tap water are summarized in table 3 . the relative standard deviation ( rsd ) chromatogram and mass spectrum of stanozolol spiked to danube river water at a final concentration of 0.5 pg / ml . chromatogram and mass spectrum of stanozolol spiked to tap water at a final concentration of 0.5 pg / ml . however , after internal standard correction , the matrix effects in all three types of water samples were comparable and in the range 95.4 - 97.3% as shown in table 4 . thus , stanozolol d3 was used as an internal standard to : i ) compensate for matrix induced changes in ionization of analyte , ii ) correct any loss of analyte during sample preparation , iii ) compensate for any variations in the instrument response from injection to injection . correction ) in three water types ; namely hplc grade water , danube river water and tap water were in the range 95.3% to 98.4% . correction ) in all three water types were found to be in the range 94.2 to 95.5% for stanozolol as shown in table 5 . this indicated that the method is capable of detecting stanozolol in different types of aqueous matrix when only 5 ml water was processed . the analytical prerequisites for efficient detection of stanozolol at low levels in aqueous matrix were , purification of water samples using liquid - liquid extraction in presence of a deuterated internal standard followed by injecting only 5 l aliquot through the column combined with the optimized lc - ms / ms conditions employed for analysis . matrix effect results for stanozolol and stanozolol d3 in hplc water , tap water and river water meis matrix effect expressed as the ratio of mean peak area of analyte spiked postextraction to the mean peak area of the same analyte standard multiplied by 100 . corrected ) at 2 pg / ml gas chromatography mass spectrometry ( gc - ms ) and high performance liquid chromatography ( hplc ) coupled to fluorescence and uv detectors have been commonly employed to analyse steroids . however , hplc coupled to fluorescence detection involves laborious sample preparation steps and gc - ms requires a complicated sample derivatization step which makes the method more time consuming and expensive . hence , use of lc - ms / ms for analyzing steroids is a feasible approach as the sample preparation step involved is facile , economical and does not require any additional derivatization step . compared to previous methods for detecting steroids in environmental waters [ 15,17 - 19 ] , the major advantage of our method is that less volume ( only 5 ml , opposed to up to 1000 ml ) of water sample is required for analysis . another advantage includes the use of liquid - liquid extraction for purification of water samples , which is less time consuming and more economical in comparison to the solid phase extraction processes employed in previous studies [ 15,17 - 20 ] . in three out of six samples from the danube river , collected since december ' 09 , stanozolol was detected with levels up to 1.82 pg / ml . in contrast , only one sample of urban tap drinking water from budapest city was found to contain stanozolol at a concentration of 1.19 pg / ml . determination of stanozolol in environmental and domestic water samples blq means below limit of quantification nd means not detectable the possible sources of stanozolol entering the river are unknown , but may be from human or animal consumption and excretion of un - metabolized drug or due to accidental discharge of the parent compound . water samples from river and tap were collected periodically until november and stanozolol concentrations were found to be reducing over time as shown in table 5 . the ph values of all the river and tap water samples collected were found to be in the neutral range . stanozolol being basic in nature due to the presence of a pyrozole ring is found to be stable in neutral to slightly basic ph . further investigation needs to be carried out for determining the source of stanozolol and reasons for gradual decrease in its concentration . have also reported the presence of stanozolol in beijing influent waste water at a concentration of ca . have reported the presence of the steroids cortisol , dexamethasone , flumethasone , prednisolone and epitestosterone in danube river water , but their selection of analytes did not include stanozolol . our results indicate that stanozolol was present in the river danube and budapest tap water in the month of december 2009 , when the water level in the river was low ( additional file 2 ) . hence , individuals drinking stanozolol contaminated urban tap water ( 1.19 pg / ml ) will involuntarily consume approximately 1.43 ng stanozolol per day . since the effective doses of stanozolol for men and women are 50 - 100 mgs / day and 2.5 - 10 mgs / day respectively , such low levels detected in drinking water may not cause significant harm to the general public , especially as they were found only at one time point . in addition , a new biological sewage treatment plant opened in july 2010 in budapest in order to treat most of the water supplied to the city ( in contrast to only 30 - 40% water being treated in the past ) . regression analysis indicated that the assay showed excellent linearity within the quantification range of 0.5 to 200 pg / ml water for stanozolol . the lloq for stanozolol was found to be 0.5 pg / ml . the correlation coefficients were found to be greater than 0.996 during the method validation procedure . under the optimized lc - ms / ms conditions , the assay was capable of detecting ( llod ) stanozolol , without any interference , at a concentration as low as 0.25 pg / ml water when 5 ml water was processed . the relative standard deviation ( rsd ) chromatogram and mass spectrum of stanozolol spiked to danube river water at a final concentration of 0.5 pg / ml . chromatogram and mass spectrum of stanozolol spiked to tap water at a final concentration of 0.5 pg / ml . matrix effects in river water led to a reduction in peak areas of stanozolol and stanozolol d3 by 22.3% and 18.4% , respectively . however , after internal standard correction , the matrix effects in all three types of water samples were comparable and in the range 95.4 - 97.3% as shown in table 4 . thus , stanozolol d3 was used as an internal standard to : i ) compensate for matrix induced changes in ionization of analyte , ii ) correct any loss of analyte during sample preparation , iii ) compensate for any variations in the instrument response from injection to injection . correction ) in three water types ; namely hplc grade water , danube river water and tap water were in the range 95.3% to 98.4% . correction ) in all three water types were found to be in the range 94.2 to 95.5% for stanozolol as shown in table 5 . this indicated that the method is capable of detecting stanozolol in different types of aqueous matrix when only 5 ml water was processed . the analytical prerequisites for efficient detection of stanozolol at low levels in aqueous matrix were , purification of water samples using liquid - liquid extraction in presence of a deuterated internal standard followed by injecting only 5 l aliquot through the column combined with the optimized lc - ms / ms conditions employed for analysis . matrix effect results for stanozolol and stanozolol d3 in hplc water , tap water and river water meis matrix effect expressed as the ratio of mean peak area of analyte spiked postextraction to the mean peak area of the same analyte standard multiplied by 100 . corrected ) at 2 pg / ml gas chromatography mass spectrometry ( gc - ms ) and high performance liquid chromatography ( hplc ) coupled to fluorescence and uv detectors have been commonly employed to analyse steroids . however , hplc coupled to fluorescence detection involves laborious sample preparation steps and gc - ms requires a complicated sample derivatization step which makes the method more time consuming and expensive . hence , use of lc - ms / ms for analyzing steroids is a feasible approach as the sample preparation step involved is facile , economical and does not require any additional derivatization step . compared to previous methods for detecting steroids in environmental waters [ 15,17 - 19 ] , the major advantage of our method is that less volume ( only 5 ml , opposed to up to 1000 ml ) of water sample is required for analysis . another advantage includes the use of liquid - liquid extraction for purification of water samples , which is less time consuming and more economical in comparison to the solid phase extraction processes employed in previous studies [ 15,17 - 20 ] . in three out of six samples from the danube river , collected since december ' 09 , in contrast , only one sample of urban tap drinking water from budapest city was found to contain stanozolol at a concentration of 1.19 pg / ml . determination of stanozolol in environmental and domestic water samples blq means below limit of quantification nd means not detectable the possible sources of stanozolol entering the river are unknown , but may be from human or animal consumption and excretion of un - metabolized drug or due to accidental discharge of the parent compound . water samples from river and tap were collected periodically until november and stanozolol concentrations were found to be reducing over time as shown in table 5 . the ph values of all the river and tap water samples collected were found to be in the neutral range . stanozolol being basic in nature due to the presence of a pyrozole ring is found to be stable in neutral to slightly basic ph . in recent years stanozolol has been detected ( qualitatively ) in sludge samples collected from huiyang and meihu waste water treatment plants . have also reported the presence of stanozolol in beijing influent waste water at a concentration of ca . have reported the presence of the steroids cortisol , dexamethasone , flumethasone , prednisolone and epitestosterone in danube river water , but their selection of analytes did not include stanozolol . the national health service ( nhs ) hence , individuals drinking stanozolol contaminated urban tap water ( 1.19 pg / ml ) will involuntarily consume approximately 1.43 ng stanozolol per day . in addition , a new biological sewage treatment plant opened in july 2010 in budapest in order to treat most of the water supplied to the city ( in contrast to only 30 - 40% water being treated in the past ) . future studies , sampling from various river and tap water sites should , in due course , be able to provide evidence for this . in conclusion , a rapid , highly sensitive , robust and reproducible method has been developed to detect stanozolol in different types of water samples . the assay is capable of detecting stanozolol at a concentration as low as 0.25 pg / ml water when only 5 ml water is processed . the performance of this method gives acceptable relative recoveries for stanozolol river and tap water samples . the supporting document reports the water level , volume , and temperature of river danube from december 2009 to november 2010 .

regular and widespread use of pharmaceuticals , which are frequently excreted as non- metabolized parent compounds , has led to growing concerns for the safety of drinking water . the vast range of pharmaceutical products that have been detected in sewage , surface , ground and drinking waters include bronchodilators , oral contraceptives , antidepressants , beta - blockers , anti - biotics , anti - inflammatories and analgesics [ 2 - 7 ] . stanozolol , an anabolic steroid is a synthetic derivative of the endogenously - produced male - sex hormone testosterone . the world anti - doping agency ( wada ) has banned its use in- and out - of - competition . despite the restriction , stanozolol is one of the most commonly misused synthetic , anabolic steroids in sport and in veterinary practice , where it is used for growth promoting purposes . according to wada , doping with stanozolol is confirmed if the urinary concentration of its major metabolite , 3-hydroxystanozolol exceeds 2 ng / ml . unlike testosterone , the synthetic stanozolol and/or its main metabolite should only appear in environmental waters if the former is used for veterinary purposes , taken under medical supervision or illegally by athletes for performance enhancement or if either one or both of these compounds are accidently discharged into environmental waters . in previous studies , we reported the detection of stanozolol in hair samples collected from subjects living in budapest . the aim of this study was to develop a methodology for the detection of stanozolol in aqueous matrices . to achieve this , liquid - liquid extraction ( lle ) was employed for purification and concentration followed by direct determination using lc - ms / ms . extraction recovery was evaluated for aqueous matrices spiked with stanozolol at pg / ml levels . environmental water samples were obtained from budapest ( hungary ) and collected from the river danube and an urban tap ( drinking water ) in clean , amber bottles . samples from lake balaton and spring water ( rzsika forrs , solymr , near budapest ) were also collected for comparison . some commonly - consumed , bottled non - carbonated , natural mineral water samples were purchased from local supermarkets . the majority of the analysed , commercially available , bottled natural mineral waters are recognised by the european union . liquid - liquid extraction ( lle ) using pentane was employed for the extraction of stanozolol from water samples . a 5 l aliquot of the reconstituted solution was injected into the lc - ms / ms system for analysis . the lc - ms / ms system consisted of an accela lc system ( thermo scientific , uk ) coupled to a tsq quantum triple quadrupole mass spectrometer ( thermo electron , uk ) without a flow splitter . an ion spray voltage of 4000 v was essential for optimum ionization of stanozolol and stanozolol d3 ( internal standard ) . the protonated molecules , [ m+h ] , of stanozolol ( m / z 329.2 ) and stanozolol d3 ( m / z 332.2 ) , were used as precursor ions for collision induced dissociation ( cid ) for ms - ms analysis . selective reaction monitoring ( srm ) was used to monitor the precursor ions and diagnostic product ions for unambiguous quantification of stanozolol . retention times , srm transitions and collision energies of stanozolol and stanozolol d3 ( internal standard ) the thermo scientific xcalibur software ( version 2.1 ) was used to control the lc system and mass spectrometer . the performance of the analytical method was validated for the following set of parameters : linearity , specificity , accuracy , lower limit of detection ( llod ) , lower limit of quantification ( lloq ) , inter - day precision and intra - day precision . calibration samples and quality control samples at low , medium and high concentration levels were prepared by fortifying 5 ml hplc grade water with known concentrations of stanozolol and i.s . the analyte - to - internal standard ratio was calculated by dividing the area of analyte peak by the area of the i.s . a calibration curve was constructed by plotting the analyte - to - internal standard ratio versus the known concentration of stanozolol in each sample . linear regression analysis using the least squares method was employed to evaluate the calibration curve of analyte as a function of its concentrations in water samples . the lloq or lowest point on the calibration curve was defined as the lowest concentration of analyte which could be quantified with a precision < 20% ( cv ) . to determine the lower limit of detection ( llod ) , a number of serial 1:2 dilutions were made from the low standard ( lloq ) . this was repeated on three consecutive days to evaluate the inter - day precision of the assay . the average extraction recovery for the analyte was determined by comparing the analyte to internal standard peak area ratio obtained after extracting negative control water samples fortified with stanozolol at a final concentration of 2 pg / ml in presence of i.s with the un - extracted standard working solutions at the same concentrations . obtained from the extracted blank water samples ( hplc grade , tap water and river water ) spiked with known concentrations of stanozolol and stanozolol d3 after extraction to those obtained from neat standard solution at the same final concentrations . environmental water samples were obtained from budapest ( hungary ) and collected from the river danube and an urban tap ( drinking water ) in clean , amber bottles . some commonly - consumed , bottled non - carbonated , natural mineral water samples were purchased from local supermarkets . the majority of the analysed , commercially available , bottled natural mineral waters are recognised by the european union . liquid - liquid extraction ( lle ) using pentane was employed for the extraction of stanozolol from water samples . a 5 l aliquot of the reconstituted solution was injected into the lc - ms / ms system for analysis . the lc - ms / ms system consisted of an accela lc system ( thermo scientific , uk ) coupled to a tsq quantum triple quadrupole mass spectrometer ( thermo electron , uk ) without a flow splitter . an ion spray voltage of 4000 v was essential for optimum ionization of stanozolol and stanozolol d3 ( internal standard ) . the protonated molecules , [ m+h ] , of stanozolol ( m / z 329.2 ) and stanozolol d3 ( m / z 332.2 ) , were used as precursor ions for collision induced dissociation ( cid ) for ms - ms analysis . selective reaction monitoring ( srm ) was used to monitor the precursor ions and diagnostic product ions for unambiguous quantification of stanozolol . retention times , srm transitions and collision energies of stanozolol and stanozolol d3 ( internal standard ) the thermo scientific xcalibur software ( version 2.1 ) was used to control the lc system and mass spectrometer . the performance of the analytical method was validated for the following set of parameters : linearity , specificity , accuracy , lower limit of detection ( llod ) , lower limit of quantification ( lloq ) , inter - day precision and intra - day precision . calibration samples and quality control samples at low , medium and high concentration levels were prepared by fortifying 5 ml hplc grade water with known concentrations of stanozolol and i.s . the analyte - to - internal standard ratio was calculated by dividing the area of analyte peak by the area of the i.s . a calibration curve was constructed by plotting the analyte - to - internal standard ratio versus the known concentration of stanozolol in each sample . linear regression analysis using the least squares method was employed to evaluate the calibration curve of analyte as a function of its concentrations in water samples . to determine the lower limit of detection ( llod ) , a number of serial 1:2 dilutions were made from the low standard ( lloq ) . this was repeated on three consecutive days to evaluate the inter - day precision of the assay . the average extraction recovery for the analyte was determined by comparing the analyte to internal standard peak area ratio obtained after extracting negative control water samples fortified with stanozolol at a final concentration of 2 pg / ml in presence of i.s with the un - extracted standard working solutions at the same concentrations . obtained from the extracted blank water samples ( hplc grade , tap water and river water ) spiked with known concentrations of stanozolol and stanozolol d3 after extraction to those obtained from neat standard solution at the same final concentrations . stanozolol was unambiguously analysed on the basis of its srm transition and retention time ( figures 1 and 2 ) via the method proposed and validated herein . regression analysis indicated that the assay showed excellent linearity within the quantification range of 0.5 to 200 pg / ml water for stanozolol . under the optimized lc - ms / ms conditions , the assay was capable of detecting ( llod ) stanozolol , without any interference , at a concentration as low as 0.25 pg / ml water when 5 ml water was processed . the analytical characteristics of this method including accuracy , linearity , llod , lloq , inter - day precision , intra - day precision and extraction recoveries from hplc grade , river and tap water are summarized in table 3 . matrix effects in river water led to a reduction in peak areas of stanozolol and stanozolol d3 by 22.3% and 18.4% , respectively . however , after internal standard correction , the matrix effects in all three types of water samples were comparable and in the range 95.4 - 97.3% as shown in table 4 . thus , stanozolol d3 was used as an internal standard to : i ) compensate for matrix induced changes in ionization of analyte , ii ) correct any loss of analyte during sample preparation , iii ) compensate for any variations in the instrument response from injection to injection . correction ) in three water types ; namely hplc grade water , danube river water and tap water were in the range 95.3% to 98.4% . correction ) in all three water types were found to be in the range 94.2 to 95.5% for stanozolol as shown in table 5 . the analytical prerequisites for efficient detection of stanozolol at low levels in aqueous matrix were , purification of water samples using liquid - liquid extraction in presence of a deuterated internal standard followed by injecting only 5 l aliquot through the column combined with the optimized lc - ms / ms conditions employed for analysis . matrix effect results for stanozolol and stanozolol d3 in hplc water , tap water and river water meis matrix effect expressed as the ratio of mean peak area of analyte spiked postextraction to the mean peak area of the same analyte standard multiplied by 100 . corrected ) at 2 pg / ml gas chromatography mass spectrometry ( gc - ms ) and high performance liquid chromatography ( hplc ) coupled to fluorescence and uv detectors have been commonly employed to analyse steroids . hence , use of lc - ms / ms for analyzing steroids is a feasible approach as the sample preparation step involved is facile , economical and does not require any additional derivatization step . compared to previous methods for detecting steroids in environmental waters [ 15,17 - 19 ] , the major advantage of our method is that less volume ( only 5 ml , opposed to up to 1000 ml ) of water sample is required for analysis . another advantage includes the use of liquid - liquid extraction for purification of water samples , which is less time consuming and more economical in comparison to the solid phase extraction processes employed in previous studies [ 15,17 - 20 ] . determination of stanozolol in environmental and domestic water samples blq means below limit of quantification nd means not detectable the possible sources of stanozolol entering the river are unknown , but may be from human or animal consumption and excretion of un - metabolized drug or due to accidental discharge of the parent compound . it should be noted that stanozolol was only found once in tap water and that this level does not present a threat to health based on recommended intake levels . the possible reasons for a gradual reduction in concentration could be due to : i ) variations in rates of contamination , ii ) dilution of river water due to rise in water levels , ( see additional file 1 ) , iii ) degradation of the steroid in the river water due to other constituents in the river or photolysis , or deposition in the sediment . the ph values of all the river and tap water samples collected were found to be in the neutral range . stanozolol being basic in nature due to the presence of a pyrozole ring is found to be stable in neutral to slightly basic ph . further investigation needs to be carried out for determining the source of stanozolol and reasons for gradual decrease in its concentration . in recent years stanozolol has been detected ( qualitatively ) in sludge samples collected from huiyang and meihu waste water treatment plants . have reported the presence of the steroids cortisol , dexamethasone , flumethasone , prednisolone and epitestosterone in danube river water , but their selection of analytes did not include stanozolol . our results indicate that stanozolol was present in the river danube and budapest tap water in the month of december 2009 , when the water level in the river was low ( additional file 2 ) . since the effective doses of stanozolol for men and women are 50 - 100 mgs / day and 2.5 - 10 mgs / day respectively , such low levels detected in drinking water may not cause significant harm to the general public , especially as they were found only at one time point . in addition , a new biological sewage treatment plant opened in july 2010 in budapest in order to treat most of the water supplied to the city ( in contrast to only 30 - 40% water being treated in the past ) . this major environmental protection investment will potentially contribute to a further decrease in levels of stanozolol compared to those we previously observed . stanozolol was unambiguously analysed on the basis of its srm transition and retention time ( figures 1 and 2 ) via the method proposed and validated herein . regression analysis indicated that the assay showed excellent linearity within the quantification range of 0.5 to 200 pg / ml water for stanozolol . under the optimized lc - ms / ms conditions , the assay was capable of detecting ( llod ) stanozolol , without any interference , at a concentration as low as 0.25 pg / ml water when 5 ml water was processed . the analytical characteristics of this method including accuracy , linearity , llod , lloq , inter - day precision , intra - day precision and extraction recoveries from hplc grade , river and tap water are summarized in table 3 . however , after internal standard correction , the matrix effects in all three types of water samples were comparable and in the range 95.4 - 97.3% as shown in table 4 . thus , stanozolol d3 was used as an internal standard to : i ) compensate for matrix induced changes in ionization of analyte , ii ) correct any loss of analyte during sample preparation , iii ) compensate for any variations in the instrument response from injection to injection . correction ) in three water types ; namely hplc grade water , danube river water and tap water were in the range 95.3% to 98.4% . correction ) in all three water types were found to be in the range 94.2 to 95.5% for stanozolol as shown in table 5 . the analytical prerequisites for efficient detection of stanozolol at low levels in aqueous matrix were , purification of water samples using liquid - liquid extraction in presence of a deuterated internal standard followed by injecting only 5 l aliquot through the column combined with the optimized lc - ms / ms conditions employed for analysis . matrix effect results for stanozolol and stanozolol d3 in hplc water , tap water and river water meis matrix effect expressed as the ratio of mean peak area of analyte spiked postextraction to the mean peak area of the same analyte standard multiplied by 100 . corrected ) at 2 pg / ml gas chromatography mass spectrometry ( gc - ms ) and high performance liquid chromatography ( hplc ) coupled to fluorescence and uv detectors have been commonly employed to analyse steroids . hence , use of lc - ms / ms for analyzing steroids is a feasible approach as the sample preparation step involved is facile , economical and does not require any additional derivatization step . compared to previous methods for detecting steroids in environmental waters [ 15,17 - 19 ] , the major advantage of our method is that less volume ( only 5 ml , opposed to up to 1000 ml ) of water sample is required for analysis . another advantage includes the use of liquid - liquid extraction for purification of water samples , which is less time consuming and more economical in comparison to the solid phase extraction processes employed in previous studies [ 15,17 - 20 ] . in three out of six samples from the danube river , collected since december ' 09 , in contrast , only one sample of urban tap drinking water from budapest city was found to contain stanozolol at a concentration of 1.19 pg / ml . determination of stanozolol in environmental and domestic water samples blq means below limit of quantification nd means not detectable the possible sources of stanozolol entering the river are unknown , but may be from human or animal consumption and excretion of un - metabolized drug or due to accidental discharge of the parent compound . it should be noted that stanozolol was only found once in tap water and that this level does not present a threat to health based on recommended intake levels . the possible reasons for a gradual reduction in concentration could be due to : i ) variations in rates of contamination , ii ) dilution of river water due to rise in water levels , ( see additional file 1 ) , iii ) degradation of the steroid in the river water due to other constituents in the river or photolysis , or deposition in the sediment . the ph values of all the river and tap water samples collected were found to be in the neutral range . our results indicate that stanozolol was present in the river danube and budapest tap water in the month of december 2009 , when the water level in the river was low ( additional file 2 ) . since the effective doses of stanozolol for men and women are 50 - 100 mgs / day and 2.5 - 10 mgs / day respectively , such low levels detected in drinking water may not cause significant harm to the general public , especially as they were found only at one time point . in addition , a new biological sewage treatment plant opened in july 2010 in budapest in order to treat most of the water supplied to the city ( in contrast to only 30 - 40% water being treated in the past ) .

iron - sulfur ( fe - s ) clusters are important prosthetic groups with unusual chemical properties that enable the proteins that contain them ( fe - s proteins ) to function in pathways ranging from metabolism to dna repair . they are evolutionarily ancient and are present in essentially all organisms , including archaea , bacteria , plants and animals . the high level of evolutionary conservation is consistent with the possibility that fe - s clusters contributed to the success of early life forms . in the anaerobic atmosphere of ancient earth , fe - s inorganic metal compounds were probably already present in hydrothermal vents ( martin et al . , 2008 ) . evolving primitive organisms might have used these versatile fe - s compounds and incorporated them into early metabolic pathways ; in fact , fe - s cluster activities might have contributed to the origin of life itself . once primitive organisms evolved , they could use the reducing power of their metabolic pathways to generate organic molecules and to spread to less protected environments ( russell and martin , 2004 ) . however , as plants oxygenated the earth s atmosphere , fe - s clusters were no longer as readily assembled or as stable owing to the oxidation of iron and of fe - s clusters themselves ( imlay , 2006 ) . despite their inherent vulnerability to oxidation and degradation , fe - s clusters are crucial for facilitating enzyme activities in all kingdoms of life because they can bind electron - rich enzymatic substrates , accept or donate single electrons and stabilize specific protein conformations that are important to the activities of numerous proteins . their importance for normal mammalian physiology is illustrated by the fact that mutations in proteins involved in fe - s cluster biogenesis cause at least five distinctive human diseases ( table 1 ) , including friedreich s ataxia ( frda ) , iscu myopathy and a specific sideroblastic anemia , all of which seem to be caused by mutations in proteins in the main fe - s cluster biogenesis pathway ( reviewed by ye and rouault , 2010 ) . in addition , mutations in nubpl ( calvo et al . , 2010 ) , also known as ind1 , a protein involved in transferring fe - s clusters to complex i ( bych et al . , 2008 ; sheftel et al . , 2009 ) , were recently discovered to cause deficiency of respiratory chain complex i , resulting in mitochondrial encephalomyopathy . finally , two forms of a disease called multiple mitochondrial dysfunctions syndrome , caused by mutations in either nfu1 or bola3 , indicate that some fe - s proteins might acquire their clusters from a specific and essential subset of donor proteins ( cameron et al . careful phenotyping of humans with problems caused by defects in fe - s cluster biogenesis should help to clarify aspects of the pathway , particularly regarding how target fe - s proteins are identified , that have remained elusive in studies of bacteria and yeast model systems . determining why these diseases affect some tissues , but not others , is an important challenge that will probably be resolved by detailed studies of molecular pathophysiology . diseases caused by defects in fe - s cluster biogenesis insights into the pathophysiology of these diseases depends on gaining a better understanding of fe - s cluster biogenesis and its regulation in eukaryotic cells , particularly in the mitochondria , which must synthesize fe - s clusters for incorporation into the citric acid cycle enzymes aconitase and succinate dehydrogenase , and into respiratory chain complexes i iii . many studies have been performed to investigate fe - s cluster biogenesis in the model organism saccharomyces cerevisiae ; however , there are apparently differences between aspects of mammalian and yeast fe - s cluster biogenesis , including multicellularity , the role of frataxin ( an important component of the initial fe - s biogenesis complex ; see below ) ( bridwell - rabb et al . , 2011 ; schmucker et al . , 2011 ) and differences in the cytosolic fe - s cluster biogenesis machinery . notably , in mammalian cells , accumulating evidence suggests that several key fe - s cluster biogenesis proteins are present not only in mitochondria but also in cytosolic and/or nuclear compartments , whereas their yeast counterparts are alleged to reside only in mitochondria [ with the exception of cysteine desulfurase ( biederbick et al . , 2006 ) ] . thus , there is a need to clarify the situation in the mammalian system to obtain a better understanding of the role of the fe - s synthetic machinery in normal and diseased physiology . this commentary focuses on how advances in understanding the steps of basic fe - s cluster biogenesis can enhance our understanding of the pathogenesis of a relatively new class of human diseases . in addition , it highlights important features of the compartmentalization of the fe - s biogenesis machinery in mammalian cells , which seem to differ from those of the lower eukaryote s. cerevisiae . fe - s clusters are cofactors that are generally ligated to cysteine residues of proteins , where they facilitate numerous types of reactions . composed of iron and inorganic sulfur , they are most frequently found in a cubane form that contains four iron and four inorganic sulfur atoms ( meyer , 2008 ) . fe - s clusters are highly chemically versatile because both iron and sulfur can readily donate or accept multiple electrons ( beinert , 2000 ) , and these chemical features can synergize so that the affinity of an fe - s cluster for electrons can be fine - tuned across an extremely broad electrochemical range by its surrounding protein residues . for example , in mitochondrial complex i , seven fe - s clusters with gradually increasing reduction potentials align to form a wire - like pathway along which electrons ascend . thus , the ability of fe - s clusters to maintain low reduction potentials ( i.e. low affinity for electrons ) facilitates efficient capture of chemical energy from nadh as electrons move progressively through respiratory chain complexes ( hirst , 2010 ) . fe - s clusters are versatile in other ways , because they can directly facilitate chemical reactions by binding to an fe - s protein s substrate , as in the enzyme aconitase , which binds and interconverts citrate and isocitrate in the citric acid cycle . in addition , fe - s proteins have been found to function as sensors , as in the bacterial fnr and iscr proteins ( kiley and beinert , 2003 ) , and in mammalian irp1 , which regulates cytosolic iron metabolism in mammalian cells ( rouault , 2006 ; muckenthaler et al . , 2008 ) . the highly conserved general fe - s cluster biogenesis pathway has been the subject of intense study in numerous species of bacteria , plants , yeast and mammals since it was first described in bacteria ( reviewed by frazzon and dean , 2003 ) . many of the general steps of the pathway are common to all kingdoms of life , but it seems that the situation is more complicated in eukaryotes , in which fe - s proteins are functional and necessary in multiple subcellular compartments , including mitochondria , plastids , cytosol and nucleus . the initial stage of fe - s cluster biogenesis is accomplished by a multimeric protein complex in which a dimer of a cysteine desulfurase [ called iscs ( escherichia coli ) , nfs1 ( s. cerevisiae ) or nfs1 ( mammals ) ] forms a core to which two monomers of a dedicated scaffold protein ( iscu in bacteria , isu1 or isu2 in yeast and iscu in humans ) bind at either end ( shi et al . , 2010 ) pyridoxal phosphate , nfs1 supplies sulfur by removing it from cysteine residues , and iscu provides the backbone structure and the cysteine ligands upon which a new cluster consisting of covalently bound iron and inorganic sulfur is synthesized ( raulfs et al . , 2008 ; py and barras , 2010 ; bandyopadhyay et al . , 2008 ; lill , 2009 ) . the source of the iron in the nascent fe - s cluster has not been clearly identified , but suggested sources include iron that is bound to acidic patches on frataxin ( see below ) ( stemmler et al . , 2010 ) , or iron that is donated from a complex of glutathione and glutaredoxin that tethers an fe - s cluster ( qi and cowan , 2011 ) . in eukaryotes , the stability of the cysteine desulfurase depends on its binding to a small partner protein called isd11 ( wiedemann et al . , 2006 ) ( adam et al . , 2006 ) , which is found in the mitochondrial matrix of s. cerevisiae , but has been detected in both the mitochondrial matrix and the cytosolic and/or nuclear compartments of mammalian cells ( shi et al . , isd11 apparently became an indispensable binding partner for the eukaryotic cysteine desulfurase early in the evolution of eukaryotes ( richards and van der giezen , 2006 ) . the discovery that the isd11 protein is present in the mammalian nucleus supported previous reports that human nfs1 ( land and rouault , 1998 ) , iscu ( tong and rouault , 2000 ) and an alternative scaffold protein called nfu1 ( tong et al . , 2003 ) are present and active in the cytosolic and/or nuclear compartments of mammalian cells ( tong and rouault , 2006 ) . structural modeling suggests that the protein frataxin ( yfh1 in yeast ) then binds in a pocket - like region between nfs1 and iscu ( fig . 1 ) , where it might either repress , stabilize or enhance activity of the fe - s cluster biogenesis core complex ( prischi et al . , 2010 ; schmucker et al . , 2011 ; tsai and barondeau , 2010 ) . structural data demonstrating the inclusion of frataxin in the initial multimeric complex detracts somewhat from previous hypotheses that the role of frataxin was to deliver bound iron to the complex ( cook et al . , rather , it now seems that frataxin binding to the nfs1-iscu complex drives a useful conformational change that enhances initial cluster formation ( bridwell - rabb et al . , 2011 ) . assembly of the nascent clusters from the iron and sulfur building blocks available in cells relies on a source of electrons to achieve the electronic configurations that are observed in fe - s clusters . candidates for the provision of electrons to nascent fe - s clusters include glutaredoxin 5 ( glrx5 ) ( lillig et al . , 2008 ) and ferredoxin ( ewen et al . , 2011 ; shi et al . , 2011 ) ; ferredoxin has been shown to facilitate reductive coupling of two distinct [ 2fe-2s ] clusters into a single [ 4fe-4s ] cluster on bacterial iscu in vitro ( chandramouli et al . , 2007 ) . glutathione , which reduces glutaredoxins , might also serve as a previously unrecognized iron source ( hider and kong , 2011 ) . notably , a mutation in human mitochondrial glrx5 causes sideroblastic anemia and impairs fe - s cluster biogenesis , but the function of glrx5 is still not clear . glrx5 can dimerize and ligate a single bridging fe - s cluster in vitro , using a cysteine from each glrx5 monomer and two glutathiones as the ligands of a [ 2fe-2s ] cluster ( ye et al . , 2010 ) . a general scheme for biogenesis of fe - s clusters in mammalian cells . ( a ) nfs1 is a cysteine desulfurase that forms a dimer to which monomers of the primary scaffold protein iscu bind near the top and bottom of the complex . in eukaryotes nfs1 also binds the cofactor pyridoxal phosphate ( not shown ) . structural and biochemical studies suggest that frataxin forms part of the initial fe - s cluster biogenesis complex , potentially occupying a pocket between nfs1 and iscu . nfs1 donates inorganic sulfur , and cysteines from iscu provide the sulfur ligands that directly bind iron in the nascent fe - s cluster . ( b ) once the fe - s cluster is assembled , it must be transferred to recipient proteins . work in bacteria and yeast model systems suggests that a dedicated chaperone co - chaperone pair of proteins participates in cluster transfer from the primary scaffold , iscu , to recipient fe - s proteins . the co - chaperone is known to be hsc20 ( a dnaj protein ) , whereas the chaperone is an hsp70 homolog that has not yet been clearly identified in mammalian cells . hsc20 binds iscu , and the hsc20-iscu complex probably then binds to its hsc70 partner through two different binding sites : hsc20 contacts the n - terminus of hsc70 and its binding partner , iscu , binds to the c - terminal substrate - binding domain region of hsc70 . the j domain region of hsc20 contains three residues [ his ( h ) pro ( p ) and asp ( d ) ; hpd ] that activate the atpase activity of hsc70 . upon activation , a conformational change is proposed to occur in the substrate - binding domain of hsc70 that affects bound iscu , resulting in extrusion of a peptide containing the residues lppvk from the iscu globular protein . the lppvk peptide then binds to a groove in the substrate - binding domain of hsc70 , which consolidates or perhaps further enhances the conformational change in iscu , which might convert it to a conformation that facilitates donation of its cluster to recipient proteins . in this model , hsc20 helps protect the vulnerable fe - s cluster bound to iscu as it dissociates from the multimeric assembly complex , and hsc20 then escorts iscu to form a trimeric complex with hsc70 . the consumption of atp probably provides a powerful impetus to drive conformational changes of iscu and the substrate - binding domain of hsc70 ; these changes might facilitate release of the fe - s cluster from iscu . by capturing the energy released by atp hydrolysis and coupling it to conformational changes , the chaperone co - chaperone pair help the fe - s cluster to reach its target proteins . target proteins could include some direct targets , or proteins such as nfu1 , bola3 , nubpl or glrx5 that might function as intermediary scaffolds that then donate fe - s clusters to specific subsets of recipient proteins . ( c ) mutations in proteins acting at different points in the biogenesis pathway cause diseases with markedly different phenotypes ( described in table 1 ) . after an fe - s cluster is formed by the core complex ( composed of nfs1 , isd11 , frataxin and iscu in mammalian cells ) ( tsai and barondeau , 2010 ; schmucker et al . , 2011 ; bridwell - rabb et al . , 2011 although many details of this transfer process remain undefined , much evidence indicates that a highly conserved chaperone co - chaperone system participates in the next steps . a co - chaperone known as hsc20 in mammalian cells ( uhrigshardt et al . , 2010 ) , hscb in bacteria ( vickery and cupp - vickery , 2007 ) and jac1 in s. cerevisiae ( craig and marszalek , 2002 ) binds to iscu . in bacteria , hscb has been shown to form a complex with iscu and a co - chaperone partner , hsca ( ssq1 in s. cerevisiae ) , a member of the hsp70 heat shock protein family . the mammalian hsp70 homolog with this function has not yet been identified , so references to mammalian hsc70 below and in fig . homologs use the energy released by hydrolysis of atp to drive conformational changes and the refolding of target proteins . recent studies indicate that bacterial hscb binds to iscu using highly conserved residues in a hydrophobic patch in the c - terminal portion of hscb ( fuzery et al . , 2011 ) . together , hsca ( hsc70 ) and hscb ( hsc20 ) might facilitate fe - s cluster transfer from iscu to target apoproteins or to other secondary scaffolds by facilitating dissociation from the initial scaffold protein complex ( vickery and cupp - vickery , 2007 ; bonomi et al . , 2011 ) it remains unclear how fe - s cluster target proteins are identified , because the process seems to be selective and might depend on specific interactions between apoprotein targets and binding surfaces of the chaperone co - chaperone complex , particularly along the extensive potential interaction area provided by the unusually large and distinctive c - terminus of hsc20 ( kampinga and craig , 2010 ) . because fe - s clusters are notoriously vulnerable to oxidative stress , the biogenesis system might have evolved to protect and enshroud fe - s clusters during most of the biogenesis and transfer process . interestingly , mammalian hsc20 contains a cysteine - rich n - terminus that binds zinc in the crystal structure ( bitto et al . , 2008 ) , which could potentially bind an fe - s cluster ( uhrigshardt et al . it is likely that binding of human hsc20 protects the nascent cluster on iscu , and leads iscu into a new multisubunit complex that contains the hsp70 homolog along with hsc20 and iscu . using the energy derived from atpase activity of the hsp70 homolog , a conformational change in this complex might facilitate protected transfer of the fe - s cluster from iscu directly to recipient apoproteins or to secondary scaffold proteins that then facilitate delivery of the fe - s cluster to a defined group of recipient proteins . target apoproteins are likely to be tethered to the chaperone co - chaperone complex when the fe - s cluster of iscu is released so that cluster release can be coupled to fe - s acquisition by apoproteins . as discussed , the fe - s cluster biogenesis pathway is fundamental to a variety of cellular processes : fe - s proteins are required for the function of two proteins of the citric acid cycle , succinate dehydrogenase and aconitase ; for respiratory chain complexes i iii ; and for numerous other proteins in the mitochondria , cytosol and nucleus of mammalian cells . thus , it is not surprising that disruption of fe - s cluster biogenesis causes human disease . however , defects in different parts of the pathway cause very different disease phenotypes ( fig . 1 , table 1 ) . for example , markedly diminished expression of frataxin causes frda , which is characterized by cardiac failure and the death of specific neuronal cell types , including the dorsal root ganglia , which are responsible for sensory perception and maintenance of balance [ see table 1 and the review by martelli et al . in this issue of disease models & mechanisms ( martelli et al . , 2012 ) ] . by contrast , iscu myopathy , a disease resulting from a splicing mutation of iscu , causes pathology almost exclusively in skeletal muscles , and cardiac involvement is rare , developing only when there is a specific inactivating mutation on one allele ( kollberg et al . , 2009 ) . tissue - specific factors might exacerbate the loss of protein function in affected tissues ; indeed , it has been suggested that tissue - specific splicing factors drive abnormal splicing of iscu in the skeletal muscles of individuals with iscu myopathy ( nordin et al . , 2011 ) . it seems likely that the cells in affected tissues in frda express less frataxin than unaffected tissues , and that the fe - s cluster biogenesis pathway is therefore more compromised at those sites . however , this observation has not been closely examined , possibly because of challenges associated with obtaining tissues from patients or autopsies . because there has only been a single patient with sideroblastic anemia caused by glrx5 mutation , it is not possible to verify proposed causes of tissue specificity for this disease ( table 1 ) . studies of three newly described diseases caused by mutations in nubpl , nfu1 or bola3 suggest that transfer of fe - s clusters to specific target proteins downstream of the iscu chaperone co - chaperone complex depends on specific pathways that shepherd fe - s clusters to subsets of recipient proteins ( also discussed below ) . for instance , mutations in the complex - i - dedicated chaperone protein nubpl have been reported to cause mitochondrial encephalomyopathy , a severe multisystem infantile disease attributable to loss of complex i function . by contrast , mutations in nfu1 or bola3 interfere quite specifically with activity of an fe - s enzyme , lipoate synthase , which provides lipoic acid modifications to a small group of specific enzymes , including the e2 subunits of pyruvate dehydrogenase and -ketoglutarate dehydrogenase , and to enzymes involved in branched chain amino acid metabolism and glycine degradation ( cameron et al . , 2011 ; navarro - sastre et al . , 2011 ) . the small number of individuals with nfu1 or bola3 mutations died within the first year of life , and their main symptoms of lactic acidosis and failure to thrive have thus far not been reported to be highly tissue specific ( see table 1 ) . to understand the diseases that result from defects in the fe - s cluster biogenesis machinery , it is important to fully understand how this process occurs in mammalian cells . insights into mammalian fe - s cluster biogenesis have been confounded by the fact that a dominating paradigm has been developed in the model system s. cerevisiae , in which it was proposed that fe - s cluster biogenesis occurred or was initiated solely in the mitochondria , but not cytosolic or nuclear compartments ( lill , 2009 ) . specifically , it was proposed that the mitochondrial exporter atm1 exported either fully formed fe - s clusters or a special form of sulfur that was required for cytosolic fe - s cluster biogenesis ( lill , 2009 ) , and that loss of atm1 transporter activity led to the accumulation of iron within the mitochondrial matrix . in s. sulfur compound from mitochondria has been proposed to contribute to cytosolic fe - s cluster biogenesis by proteins known as tah18 and dre2 ( netz et al . , 2010 ) . it was proposed that a cluster is transferred from dre2 to other members of the cytosolic fe - s cluster assembly ( cia ) proteins ( sharma et al . , 2010 ) . there are mammalian homologs of yeast cia proteins , including ndor1 and ciapin1 ( homologs of tah18 and dre2 , respectively ) , nubp1 and nubp2 [ homologs of yeast nbp35 and cfd1 , respectively ( stehling et al . , 2008 ) ] , narfl ( homolog of yeast nar1 ) and ciao1 ( homolog of yeast cia1 ) ( srinivasan et al . , 2007 ) . however , the model purporting that cytosolic fe - s cluster biogenesis depends on export of a mitochondrial product was recently called into question because genetic manipulations that were initially used to inactivate atm1 also introduced wild - type leu2 into a leu2-null strain , which unknowingly resulted in repressed transcription of leu1 relative to the control strain . leu1 encodes a cytosolic fe - s protein that was being used as a readout to quantify cytosolic fe - s cluster biogenesis . because leu1 activity was assessed as being low in atm1-null strains , this was misinterpreted as evidence that the mitochondrial exporter , atm1 , contributed an essential component to cytosolic fe - s cluster biogenesis . recently , when the experiment was performed with appropriate controls , there was no evidence that loss of atm1 adversely affected leu1 activity ( bedekovics et al . , 2011 ) . thus , the model that fe - s clusters are assembled solely in mitochondria of yeast cells lacks crucial experimental support , and some models based on work in s. cerevisiae must be reconsidered . in any case , there is now substantial evidence that the paradigm that fe - s clusters are assembled or initiated solely in mitochondria is not true in mammalian cells , an issue that must be clarified to allow insights into disease pathophysiology . it is well accepted that the basic fe - s cluster biosynthetic proteins are highly expressed in mammalian mitochondria , where they supply fe - s clusters to proteins of the citric acid cycle , the respiratory chain complexes and other pathways ( described above ) . less appreciated and somewhat controversial , however , is the idea that some of these proteins are also present in the cytosolic and/or nuclear compartments of mammalian cells ( fig . it has been reported that small amounts of the cysteine desulfurase nfs1 and its binding partner isd11 are expressed in the cytosolic and/or nuclear compartments of mammalian cells ( land and rouault , 1998 ; shi et al . , 2009 ) , where small amounts of the scaffold proteins iscu and nfu1 are also present ( tong et al . , 2003 ; tong and rouault , 2006 ) . the observed extra - mitochondrial localization of these proteins is consistent with the fact that there are nuclear localization signals in mammalian nfs1 ( land and rouault , 1998 ) and isd11 ( shi et al . , 2009 ) , as well as in nfs1 of s. cerevisiae ( nakai et al . , 2001 ) ; however , s. cerevisiae nfs1 is not believed to participate in extra - mitochondrial fe - s cluster biogenesis ( biederbick et al . , 2006 ) . the extra - mitochondrial isoforms of mammalian iscu and nfu1 are encoded by alternatively spliced transcripts that lack a recognizable mitochondrial targeting signal ( tong et al . , 2003 ; tong and rouault , 2006 ) , and there is functional evidence that the cytosolic iscu functions as a scaffold and source for extra - mitochondrial fe - s clusters in mammalian cells ( tong and rouault , 2006 ) . moreover , the co - chaperone hsc20 has been detected in cytosolic fractions ( uhrigshardt et al . , however , because proteins are synthesized in the cytosol , it is possible that some of the fe - s cluster biogenesis proteins found in this compartment represent proteins that were inefficiently imported into and processed by mitochondria . notably , there is almost always much more of each protein in the mitochondrial matrix than in the extra - mitochondrial fractions . proposed differences in the localization of fe - s cluster biogenesis proteins in s. cerevisiae versus mammalian cells . ( a ) yeast mitochondria contain the basic fe - s cluster biogenesis proteins , whereas cytosolic fe - s cluster biogenesis has been proposed to depend on the export of a sulfur compound from mitochondria through the transporter atm1 ( abcb7 in human cells ) . further biogenesis in the cytoplasm depends on a distinct set of proteins , including tah18 , dre2 , nbp35 , cfd1 , nar1 and cia1 , which are collectively referred to as cia ( cytosolic iron - sulfur assembly ) proteins . ( b ) by contrast , in mammalian cells , it seems that most of the basic fe - s cluster biogenesis proteins are expressed in mitochondria as well as in the cytosolic and/or nuclear compartment ( nuclear localization of these proteins not represented in the figure ) . in addition , there are human counterparts to the proteins implicated in cytosolic fe - s cluster biogenesis in yeast , including ndor1 ( homolog of tah18 ) , ciapin1 , ( homolog of dre2 ) , nubp1 and nubp2 ( homologs of nbp35 and cfd1 , respectively ) , and narfl and ciao1 ( homologs of nar1 and cia1 , respectively ) . to explain mitochondrial iron overload ( which occurs as a consequence of disrupted fe - s cluster biogenesis ) , it is possible that mitochondria depend on fe - s cluster biogenesis to synthesize a molecule that functions as a regulatory signal . in the absence of this signal ( i.e. when fe - s cluster biogenesis is impaired ) , remodeling of nuclear transcription leads to mitochondrial iron overload , as the cell attempts to compensate for possible mitochondrial iron deficiency . because mitochondrial iron overload probably contributes to disease pathogenesis in frda , iscu myopathy and possibly several other diseases , elucidation of the details of this pathway might pave the way to new therapeutic interventions . understanding the pathway by which fe - s clusters could be assembled outside the mitochondrial compartment is becoming more important as the list of recognized cytosolic and nuclear fe - s proteins grows . indeed , there might be many as - yet - undiscovered fe - s proteins in the mitochondrial , cytosolic and nuclear compartments of mammalian cells . however , fe - s proteins lack a distinctive motif that permits easy recognition , and they tend to lose their sensitive prosthetic groups during purification . importantly , a significant number of dna repair enzymes are fe - s proteins , including the protein responsible for excision - repair of uv damage that causes the disease xeroderma pigmentosum ( xpd ) when defective , and a related helicase that causes fanconi anemia when defective ( rudolf et al . , 2006 ) . in addition , telomere maintenance depends on the nuclear fe - s protein rtel-1 ( uringa et al . , 2011 ) , and eukaryotic dna polymerases depend on fe - s clusters for function ( netz et al . , 2011 ) . some of the cytosolic fe - s assembly proteins previously identified in yeast might function in mammalian cells as secondary scaffold proteins that transfer clusters to specific classes of protein targets such as dna repair enzymes . it is well known that fully formed fe - s clusters can readily move from one protein to another ; this mobility of transfer between proteins is one of the most distinctive features of fe - s clusters ( beinert et al . , 1997 ) . thus , it is perhaps not surprising that mutations of nubp1 and nubp2 impair cell division ( christodoulou et al . , 2006 ) , because these proteins are implicated in cytosolic fe - s cluster biogenesis and some of their likely targets are involved in chromosomal duplication and in maintenance of nuclear integrity , processes that are necessary for proper cell division ( netz et al . , 2010 ) . in summary , although the scenario in yeast remains in dispute , the accumulating evidence that fe - s cluster biogenesis can occur de novo in the mitochondrial matrix as well as in extra - mitochondrial compartments of mammalian cells is strong and consistent . it is crucial to correctly analyze the basics of the mammalian fe - s cluster biogenesis pathway , because it sets the stage for understanding one of the most common complications of impaired fe - s cluster biogenesis : the propensity to develop marked mitochondrial iron overload and subsequent iron - related mitochondrial damage . evidence suggests that interruption of mitochondrial fe - s cluster biogenesis leads to mitochondrial iron overload and cytosolic iron depletion in mammalian cells , which is associated with disease pathology . for example , mitochondrial iron overload is a distinctive feature of the heart and some neurons of frda patients ( koeppen , 2011 ) , of the skeletal muscle cells of individuals with iscu myopathy ( mochel et al . , 2008 ) , and of the immature red blood cells of a patient with sideroblastic anemia attributable to deficiency of glrx5 ( ye et al . , 2010 ) . in yeast , mutations of frataxin ( babcock et al . , 1997 ) , of the chaperone ssq1 ( hsp70 homolog ) ( knight et al . , 1998 ) or the j - domain co - chaperone jac1 ( hsc20 homolog ) ( kim et al . , 2001 ) compromise the biogenesis of fe - s clusters , and also cause mitochondrial iron overload . in mammalian cells , mitochondrial iron overload has been observed to occur in concert with cytosolic iron depletion , as judged by activities of cytosolic iron regulatory proteins ( li et al . , 2008 ; shi et al . , 2009 ) . in both yeast and human cells , this mitochondrial iron overload can be readily reversed by restoration of the missing fe - s cluster biogenesis protein ( babcock et al . , 1997 ; ye et al . , 2010 one early hypothesis referred to above was that iron was able to leave mitochondria only when it had been incorporated into an fe - s cluster ( kispal et al . , 1999 ) . subsequently , this hypothesis was modified to suggest that abcb7 ( the mammalian homolog of yeast atm1 ) exports a special type of sulfur , but not iron , from mitochondria ( lill , 2009 ; netz et al . , 2010 ) . however , the revised theory does not offer an explanation for why iron accumulates in mitochondria : the idea that iron accumulates in mitochondria passively because it can not be incorporated into fe - s clusters does not take into account the possibility that mitochondrial iron metabolism is a highly regulated process that might be imbalanced in cells with defective fe - s cluster biogenesis . in support of this idea , transcriptional expression of the mitochondrial iron importer mitoferrin is increased in frataxin - deficient mouse hearts ( huang et al . , remodeling transcription of this nuclear gene might represent a response to signals of iron deficiency from mitochondria . how might mitochondria generate a signal that could instruct the nucleus to upregulate the expression of mitoferrin ? one possibility is that mitochondria export a product that is made only when the fe - s cluster biogenesis pathway is intact , and absence of this signal triggers a coordinated nuclear transcriptional response . mitochondrial iron overload also occurs in individuals with x - linked sideroblastic anemia and ataxia ( allikmets et al . , 1999 ) caused by mutations in the abcb7 mitochondrial iron transporter , as well as in individuals with sideroblastic anemia caused by mutations in the red - blood - cell - specific amino - levulinate synthase alas2 , a mitochondrial protein that is involved in heme biosynthesis ( table 2 ) . it is possible that both alas2 and abcb7 participate in a signaling pathway that regulates mitochondrial iron homeostasis , and , furthermore , that abcb7 transports a crucial regulatory molecule related to the product of alas2 . because the abcb7 substrate ( or that of its yeast homolog atm1 ) ( kuhnke et al . , 2006 ) has not yet been identified , it might be time to think more broadly about the regulation of mitochondrial iron homeostasis and to integrate the phenotypes observed upon mutation of alas2 and abcb7 into a single overarching model for mitochondrial iron regulation . for instance , it will be important to determine whether mitochondrial iron overload occurs in cells from individuals with multiple mitochondrial dysfunctions syndrome caused by mutations in nfu1 or bola3 ( cameron et al . , 2011 ) ( table 1 ) . nfu1 probably acts as a scaffold downstream of iscu during fe - s cluster biogenesis ( navarro - sastre et al . , 2011 ) , whereas bola3 is related to proteins that bind glutaredoxins , which play an unknown role in fe - s cluster biogenesis . mutations in either nfu1 or bola3 disrupt the function of lipoic acid synthetase ( lias ) , which depends on a [ 4fe-4s ] cluster for activity . lias adds a lipoate moiety to key subunits of pyruvate dehydrogenase and several other enzymes ( table 1 ) . the fact that a specific subset of fe - s proteins is affected in these newly described diseases suggests that target specificity is achieved through use of specific scaffold proteins that function as intermediates between iscu and target proteins . because loss of glrx5 causes mitochondrial iron overload , and glrx5 might function as a bola3 partner ( huynen et al . , 2005 ) , it seems likely that the mitochondrial iron overload phenotype will also be observed in this newly described disease . proposed roles of proteins encoded by genes associated with human sideroblastic anemias ( characterized by mitochondrial iron overload ) mitochondrial iron overload develops in several other diseases that do not involve defects in fe - s cluster biogenesis . these diseases include those caused by mutations in an enzyme known as pus1 , which is expressed in both mitochondria and the nucleus and is required for pseudouridylate processing on transfer rnas ( trnas ; see table 2 ) , and by mutations in yars2 , a gene that encodes a mitochondrial tyrosine trna synthetase . these results imply that a molecule produced by the mitochondrial protein synthesis apparatus also contributes to regulation of mitochondrial iron homeostasis . the list of diseases attributable to defects in fe - s cluster biogenesis is growing . in the human genome , many of the genes encoding fe - s cluster biogenesis proteins , including hsc20 and iscu , are represented only once . the lack of duplication of these essential genes might enhance the likelihood that deleterious phenotypes will arise from mutations that affect fe - s cluster biogenesis proteins . notably , mutations in nfu1 , bola3 and nubpl cause severe disease and mortality in early infancy , and there is no evidence of significant tissue specificity . identification of new disease genes in humans should accelerate progress in understanding fe - s cluster biogenesis , and should shed light on how specific target proteins are identified . better characterization of several newly recognized diseases offers the prospect of making progress on details of the second part of the fe - s biogenesis pathway , particularly downstream of the chaperone co - chaperone proteins , when fe - s clusters are ligated to specific recipient apoproteins . further work will help to clarify how mitochondrial iron homeostasis is regulated , and learning what drives mitochondrial iron overload should yield better therapeutic approaches for diseases in which this feature contributes to pathology .

iron - sulfur ( fe - s ) clusters are ubiquitous cofactors composed of iron and inorganic sulfur . they are required for the function of proteins involved in a wide range of activities , including electron transport in respiratory chain complexes , regulatory sensing , photosynthesis and dna repair . the proteins involved in the biogenesis of fe - s clusters are evolutionarily conserved from bacteria to humans , and many insights into the process of fe - s cluster biogenesis have come from studies of model organisms , including bacteria , fungi and plants . it is now clear that several rare and seemingly dissimilar human diseases are attributable to defects in the basic process of fe - s cluster biogenesis . although these diseases which include friedreich s ataxia ( frda ) , iscu myopathy , a rare form of sideroblastic anemia , an encephalomyopathy caused by dysfunction of respiratory chain complex i and multiple mitochondrial dysfunctions syndrome affect different tissues , a feature common to many of them is that mitochondrial iron overload develops as a secondary consequence of a defect in fe - s cluster biogenesis . this commentary outlines the basic steps of fe - s cluster biogenesis as they have been defined in model organisms . in addition , it draws attention to refinements of the process that might be specific to the subcellular compartmentalization of fe - s cluster biogenesis proteins in some eukaryotes , including mammals . finally , it outlines several important unresolved questions in the field that , once addressed , should offer important clues into how mitochondrial iron homeostasis is regulated , and how dysfunction in fe - s cluster biogenesis can contribute to disease .

Introduction Basic biochemistry and function of Fe-S clusters Fe-S cluster biogenesis: the basic pathway Defects in Fe-S cluster biogenesis as causes of human disease Subcellular distribution of Fe-S cluster biogenesis proteins in eukaryotic cells A unifying hypothesis to integrate defective Fe-S cluster biogenesis with mitochondrial iron overload Perspective

iron - sulfur ( fe - s ) clusters are important prosthetic groups with unusual chemical properties that enable the proteins that contain them ( fe - s proteins ) to function in pathways ranging from metabolism to dna repair . they are evolutionarily ancient and are present in essentially all organisms , including archaea , bacteria , plants and animals . however , as plants oxygenated the earth s atmosphere , fe - s clusters were no longer as readily assembled or as stable owing to the oxidation of iron and of fe - s clusters themselves ( imlay , 2006 ) . despite their inherent vulnerability to oxidation and degradation , fe - s clusters are crucial for facilitating enzyme activities in all kingdoms of life because they can bind electron - rich enzymatic substrates , accept or donate single electrons and stabilize specific protein conformations that are important to the activities of numerous proteins . their importance for normal mammalian physiology is illustrated by the fact that mutations in proteins involved in fe - s cluster biogenesis cause at least five distinctive human diseases ( table 1 ) , including friedreich s ataxia ( frda ) , iscu myopathy and a specific sideroblastic anemia , all of which seem to be caused by mutations in proteins in the main fe - s cluster biogenesis pathway ( reviewed by ye and rouault , 2010 ) . , 2010 ) , also known as ind1 , a protein involved in transferring fe - s clusters to complex i ( bych et al . , 2009 ) , were recently discovered to cause deficiency of respiratory chain complex i , resulting in mitochondrial encephalomyopathy . finally , two forms of a disease called multiple mitochondrial dysfunctions syndrome , caused by mutations in either nfu1 or bola3 , indicate that some fe - s proteins might acquire their clusters from a specific and essential subset of donor proteins ( cameron et al . careful phenotyping of humans with problems caused by defects in fe - s cluster biogenesis should help to clarify aspects of the pathway , particularly regarding how target fe - s proteins are identified , that have remained elusive in studies of bacteria and yeast model systems . diseases caused by defects in fe - s cluster biogenesis insights into the pathophysiology of these diseases depends on gaining a better understanding of fe - s cluster biogenesis and its regulation in eukaryotic cells , particularly in the mitochondria , which must synthesize fe - s clusters for incorporation into the citric acid cycle enzymes aconitase and succinate dehydrogenase , and into respiratory chain complexes i iii . many studies have been performed to investigate fe - s cluster biogenesis in the model organism saccharomyces cerevisiae ; however , there are apparently differences between aspects of mammalian and yeast fe - s cluster biogenesis , including multicellularity , the role of frataxin ( an important component of the initial fe - s biogenesis complex ; see below ) ( bridwell - rabb et al . , 2011 ) and differences in the cytosolic fe - s cluster biogenesis machinery . notably , in mammalian cells , accumulating evidence suggests that several key fe - s cluster biogenesis proteins are present not only in mitochondria but also in cytosolic and/or nuclear compartments , whereas their yeast counterparts are alleged to reside only in mitochondria [ with the exception of cysteine desulfurase ( biederbick et al . this commentary focuses on how advances in understanding the steps of basic fe - s cluster biogenesis can enhance our understanding of the pathogenesis of a relatively new class of human diseases . in addition , it highlights important features of the compartmentalization of the fe - s biogenesis machinery in mammalian cells , which seem to differ from those of the lower eukaryote s. cerevisiae . fe - s clusters are cofactors that are generally ligated to cysteine residues of proteins , where they facilitate numerous types of reactions . composed of iron and inorganic sulfur , they are most frequently found in a cubane form that contains four iron and four inorganic sulfur atoms ( meyer , 2008 ) . fe - s clusters are highly chemically versatile because both iron and sulfur can readily donate or accept multiple electrons ( beinert , 2000 ) , and these chemical features can synergize so that the affinity of an fe - s cluster for electrons can be fine - tuned across an extremely broad electrochemical range by its surrounding protein residues . fe - s clusters are versatile in other ways , because they can directly facilitate chemical reactions by binding to an fe - s protein s substrate , as in the enzyme aconitase , which binds and interconverts citrate and isocitrate in the citric acid cycle . in addition , fe - s proteins have been found to function as sensors , as in the bacterial fnr and iscr proteins ( kiley and beinert , 2003 ) , and in mammalian irp1 , which regulates cytosolic iron metabolism in mammalian cells ( rouault , 2006 ; muckenthaler et al . the highly conserved general fe - s cluster biogenesis pathway has been the subject of intense study in numerous species of bacteria , plants , yeast and mammals since it was first described in bacteria ( reviewed by frazzon and dean , 2003 ) . many of the general steps of the pathway are common to all kingdoms of life , but it seems that the situation is more complicated in eukaryotes , in which fe - s proteins are functional and necessary in multiple subcellular compartments , including mitochondria , plastids , cytosol and nucleus . the initial stage of fe - s cluster biogenesis is accomplished by a multimeric protein complex in which a dimer of a cysteine desulfurase [ called iscs ( escherichia coli ) , nfs1 ( s. cerevisiae ) or nfs1 ( mammals ) ] forms a core to which two monomers of a dedicated scaffold protein ( iscu in bacteria , isu1 or isu2 in yeast and iscu in humans ) bind at either end ( shi et al . the source of the iron in the nascent fe - s cluster has not been clearly identified , but suggested sources include iron that is bound to acidic patches on frataxin ( see below ) ( stemmler et al . 1 ) , where it might either repress , stabilize or enhance activity of the fe - s cluster biogenesis core complex ( prischi et al . assembly of the nascent clusters from the iron and sulfur building blocks available in cells relies on a source of electrons to achieve the electronic configurations that are observed in fe - s clusters . notably , a mutation in human mitochondrial glrx5 causes sideroblastic anemia and impairs fe - s cluster biogenesis , but the function of glrx5 is still not clear . a general scheme for biogenesis of fe - s clusters in mammalian cells . structural and biochemical studies suggest that frataxin forms part of the initial fe - s cluster biogenesis complex , potentially occupying a pocket between nfs1 and iscu . nfs1 donates inorganic sulfur , and cysteines from iscu provide the sulfur ligands that directly bind iron in the nascent fe - s cluster . , 2011 ) it remains unclear how fe - s cluster target proteins are identified , because the process seems to be selective and might depend on specific interactions between apoprotein targets and binding surfaces of the chaperone co - chaperone complex , particularly along the extensive potential interaction area provided by the unusually large and distinctive c - terminus of hsc20 ( kampinga and craig , 2010 ) . because fe - s clusters are notoriously vulnerable to oxidative stress , the biogenesis system might have evolved to protect and enshroud fe - s clusters during most of the biogenesis and transfer process . using the energy derived from atpase activity of the hsp70 homolog , a conformational change in this complex might facilitate protected transfer of the fe - s cluster from iscu directly to recipient apoproteins or to secondary scaffold proteins that then facilitate delivery of the fe - s cluster to a defined group of recipient proteins . as discussed , the fe - s cluster biogenesis pathway is fundamental to a variety of cellular processes : fe - s proteins are required for the function of two proteins of the citric acid cycle , succinate dehydrogenase and aconitase ; for respiratory chain complexes i iii ; and for numerous other proteins in the mitochondria , cytosol and nucleus of mammalian cells . thus , it is not surprising that disruption of fe - s cluster biogenesis causes human disease . by contrast , iscu myopathy , a disease resulting from a splicing mutation of iscu , causes pathology almost exclusively in skeletal muscles , and cardiac involvement is rare , developing only when there is a specific inactivating mutation on one allele ( kollberg et al . it seems likely that the cells in affected tissues in frda express less frataxin than unaffected tissues , and that the fe - s cluster biogenesis pathway is therefore more compromised at those sites . studies of three newly described diseases caused by mutations in nubpl , nfu1 or bola3 suggest that transfer of fe - s clusters to specific target proteins downstream of the iscu chaperone co - chaperone complex depends on specific pathways that shepherd fe - s clusters to subsets of recipient proteins ( also discussed below ) . for instance , mutations in the complex - i - dedicated chaperone protein nubpl have been reported to cause mitochondrial encephalomyopathy , a severe multisystem infantile disease attributable to loss of complex i function . by contrast , mutations in nfu1 or bola3 interfere quite specifically with activity of an fe - s enzyme , lipoate synthase , which provides lipoic acid modifications to a small group of specific enzymes , including the e2 subunits of pyruvate dehydrogenase and -ketoglutarate dehydrogenase , and to enzymes involved in branched chain amino acid metabolism and glycine degradation ( cameron et al . to understand the diseases that result from defects in the fe - s cluster biogenesis machinery , it is important to fully understand how this process occurs in mammalian cells . insights into mammalian fe - s cluster biogenesis have been confounded by the fact that a dominating paradigm has been developed in the model system s. cerevisiae , in which it was proposed that fe - s cluster biogenesis occurred or was initiated solely in the mitochondria , but not cytosolic or nuclear compartments ( lill , 2009 ) . specifically , it was proposed that the mitochondrial exporter atm1 exported either fully formed fe - s clusters or a special form of sulfur that was required for cytosolic fe - s cluster biogenesis ( lill , 2009 ) , and that loss of atm1 transporter activity led to the accumulation of iron within the mitochondrial matrix . in s. sulfur compound from mitochondria has been proposed to contribute to cytosolic fe - s cluster biogenesis by proteins known as tah18 and dre2 ( netz et al . however , the model purporting that cytosolic fe - s cluster biogenesis depends on export of a mitochondrial product was recently called into question because genetic manipulations that were initially used to inactivate atm1 also introduced wild - type leu2 into a leu2-null strain , which unknowingly resulted in repressed transcription of leu1 relative to the control strain . leu1 encodes a cytosolic fe - s protein that was being used as a readout to quantify cytosolic fe - s cluster biogenesis . thus , the model that fe - s clusters are assembled solely in mitochondria of yeast cells lacks crucial experimental support , and some models based on work in s. cerevisiae must be reconsidered . in any case , there is now substantial evidence that the paradigm that fe - s clusters are assembled or initiated solely in mitochondria is not true in mammalian cells , an issue that must be clarified to allow insights into disease pathophysiology . it is well accepted that the basic fe - s cluster biosynthetic proteins are highly expressed in mammalian mitochondria , where they supply fe - s clusters to proteins of the citric acid cycle , the respiratory chain complexes and other pathways ( described above ) . , 2003 ; tong and rouault , 2006 ) , and there is functional evidence that the cytosolic iscu functions as a scaffold and source for extra - mitochondrial fe - s clusters in mammalian cells ( tong and rouault , 2006 ) . , however , because proteins are synthesized in the cytosol , it is possible that some of the fe - s cluster biogenesis proteins found in this compartment represent proteins that were inefficiently imported into and processed by mitochondria . proposed differences in the localization of fe - s cluster biogenesis proteins in s. cerevisiae versus mammalian cells . ( a ) yeast mitochondria contain the basic fe - s cluster biogenesis proteins , whereas cytosolic fe - s cluster biogenesis has been proposed to depend on the export of a sulfur compound from mitochondria through the transporter atm1 ( abcb7 in human cells ) . further biogenesis in the cytoplasm depends on a distinct set of proteins , including tah18 , dre2 , nbp35 , cfd1 , nar1 and cia1 , which are collectively referred to as cia ( cytosolic iron - sulfur assembly ) proteins . ( b ) by contrast , in mammalian cells , it seems that most of the basic fe - s cluster biogenesis proteins are expressed in mitochondria as well as in the cytosolic and/or nuclear compartment ( nuclear localization of these proteins not represented in the figure ) . in addition , there are human counterparts to the proteins implicated in cytosolic fe - s cluster biogenesis in yeast , including ndor1 ( homolog of tah18 ) , ciapin1 , ( homolog of dre2 ) , nubp1 and nubp2 ( homologs of nbp35 and cfd1 , respectively ) , and narfl and ciao1 ( homologs of nar1 and cia1 , respectively ) . to explain mitochondrial iron overload ( which occurs as a consequence of disrupted fe - s cluster biogenesis ) , it is possible that mitochondria depend on fe - s cluster biogenesis to synthesize a molecule that functions as a regulatory signal . when fe - s cluster biogenesis is impaired ) , remodeling of nuclear transcription leads to mitochondrial iron overload , as the cell attempts to compensate for possible mitochondrial iron deficiency . because mitochondrial iron overload probably contributes to disease pathogenesis in frda , iscu myopathy and possibly several other diseases , elucidation of the details of this pathway might pave the way to new therapeutic interventions . indeed , there might be many as - yet - undiscovered fe - s proteins in the mitochondrial , cytosolic and nuclear compartments of mammalian cells . importantly , a significant number of dna repair enzymes are fe - s proteins , including the protein responsible for excision - repair of uv damage that causes the disease xeroderma pigmentosum ( xpd ) when defective , and a related helicase that causes fanconi anemia when defective ( rudolf et al . , 2011 ) , and eukaryotic dna polymerases depend on fe - s clusters for function ( netz et al . it is well known that fully formed fe - s clusters can readily move from one protein to another ; this mobility of transfer between proteins is one of the most distinctive features of fe - s clusters ( beinert et al . , 2006 ) , because these proteins are implicated in cytosolic fe - s cluster biogenesis and some of their likely targets are involved in chromosomal duplication and in maintenance of nuclear integrity , processes that are necessary for proper cell division ( netz et al . in summary , although the scenario in yeast remains in dispute , the accumulating evidence that fe - s cluster biogenesis can occur de novo in the mitochondrial matrix as well as in extra - mitochondrial compartments of mammalian cells is strong and consistent . it is crucial to correctly analyze the basics of the mammalian fe - s cluster biogenesis pathway , because it sets the stage for understanding one of the most common complications of impaired fe - s cluster biogenesis : the propensity to develop marked mitochondrial iron overload and subsequent iron - related mitochondrial damage . evidence suggests that interruption of mitochondrial fe - s cluster biogenesis leads to mitochondrial iron overload and cytosolic iron depletion in mammalian cells , which is associated with disease pathology . for example , mitochondrial iron overload is a distinctive feature of the heart and some neurons of frda patients ( koeppen , 2011 ) , of the skeletal muscle cells of individuals with iscu myopathy ( mochel et al . , 2008 ) , and of the immature red blood cells of a patient with sideroblastic anemia attributable to deficiency of glrx5 ( ye et al . , 2001 ) compromise the biogenesis of fe - s clusters , and also cause mitochondrial iron overload . in both yeast and human cells , this mitochondrial iron overload can be readily reversed by restoration of the missing fe - s cluster biogenesis protein ( babcock et al . however , the revised theory does not offer an explanation for why iron accumulates in mitochondria : the idea that iron accumulates in mitochondria passively because it can not be incorporated into fe - s clusters does not take into account the possibility that mitochondrial iron metabolism is a highly regulated process that might be imbalanced in cells with defective fe - s cluster biogenesis . one possibility is that mitochondria export a product that is made only when the fe - s cluster biogenesis pathway is intact , and absence of this signal triggers a coordinated nuclear transcriptional response . , 1999 ) caused by mutations in the abcb7 mitochondrial iron transporter , as well as in individuals with sideroblastic anemia caused by mutations in the red - blood - cell - specific amino - levulinate synthase alas2 , a mitochondrial protein that is involved in heme biosynthesis ( table 2 ) . it is possible that both alas2 and abcb7 participate in a signaling pathway that regulates mitochondrial iron homeostasis , and , furthermore , that abcb7 transports a crucial regulatory molecule related to the product of alas2 . for instance , it will be important to determine whether mitochondrial iron overload occurs in cells from individuals with multiple mitochondrial dysfunctions syndrome caused by mutations in nfu1 or bola3 ( cameron et al . nfu1 probably acts as a scaffold downstream of iscu during fe - s cluster biogenesis ( navarro - sastre et al . , 2011 ) , whereas bola3 is related to proteins that bind glutaredoxins , which play an unknown role in fe - s cluster biogenesis . proposed roles of proteins encoded by genes associated with human sideroblastic anemias ( characterized by mitochondrial iron overload ) mitochondrial iron overload develops in several other diseases that do not involve defects in fe - s cluster biogenesis . these diseases include those caused by mutations in an enzyme known as pus1 , which is expressed in both mitochondria and the nucleus and is required for pseudouridylate processing on transfer rnas ( trnas ; see table 2 ) , and by mutations in yars2 , a gene that encodes a mitochondrial tyrosine trna synthetase . the list of diseases attributable to defects in fe - s cluster biogenesis is growing . in the human genome , many of the genes encoding fe - s cluster biogenesis proteins , including hsc20 and iscu , are represented only once . the lack of duplication of these essential genes might enhance the likelihood that deleterious phenotypes will arise from mutations that affect fe - s cluster biogenesis proteins . identification of new disease genes in humans should accelerate progress in understanding fe - s cluster biogenesis , and should shed light on how specific target proteins are identified . further work will help to clarify how mitochondrial iron homeostasis is regulated , and learning what drives mitochondrial iron overload should yield better therapeutic approaches for diseases in which this feature contributes to pathology .

iron - sulfur ( fe - s ) clusters are important prosthetic groups with unusual chemical properties that enable the proteins that contain them ( fe - s proteins ) to function in pathways ranging from metabolism to dna repair . despite their inherent vulnerability to oxidation and degradation , fe - s clusters are crucial for facilitating enzyme activities in all kingdoms of life because they can bind electron - rich enzymatic substrates , accept or donate single electrons and stabilize specific protein conformations that are important to the activities of numerous proteins . their importance for normal mammalian physiology is illustrated by the fact that mutations in proteins involved in fe - s cluster biogenesis cause at least five distinctive human diseases ( table 1 ) , including friedreich s ataxia ( frda ) , iscu myopathy and a specific sideroblastic anemia , all of which seem to be caused by mutations in proteins in the main fe - s cluster biogenesis pathway ( reviewed by ye and rouault , 2010 ) . finally , two forms of a disease called multiple mitochondrial dysfunctions syndrome , caused by mutations in either nfu1 or bola3 , indicate that some fe - s proteins might acquire their clusters from a specific and essential subset of donor proteins ( cameron et al . careful phenotyping of humans with problems caused by defects in fe - s cluster biogenesis should help to clarify aspects of the pathway , particularly regarding how target fe - s proteins are identified , that have remained elusive in studies of bacteria and yeast model systems . diseases caused by defects in fe - s cluster biogenesis insights into the pathophysiology of these diseases depends on gaining a better understanding of fe - s cluster biogenesis and its regulation in eukaryotic cells , particularly in the mitochondria , which must synthesize fe - s clusters for incorporation into the citric acid cycle enzymes aconitase and succinate dehydrogenase , and into respiratory chain complexes i iii . many studies have been performed to investigate fe - s cluster biogenesis in the model organism saccharomyces cerevisiae ; however , there are apparently differences between aspects of mammalian and yeast fe - s cluster biogenesis , including multicellularity , the role of frataxin ( an important component of the initial fe - s biogenesis complex ; see below ) ( bridwell - rabb et al . notably , in mammalian cells , accumulating evidence suggests that several key fe - s cluster biogenesis proteins are present not only in mitochondria but also in cytosolic and/or nuclear compartments , whereas their yeast counterparts are alleged to reside only in mitochondria [ with the exception of cysteine desulfurase ( biederbick et al . thus , there is a need to clarify the situation in the mammalian system to obtain a better understanding of the role of the fe - s synthetic machinery in normal and diseased physiology . this commentary focuses on how advances in understanding the steps of basic fe - s cluster biogenesis can enhance our understanding of the pathogenesis of a relatively new class of human diseases . in addition , it highlights important features of the compartmentalization of the fe - s biogenesis machinery in mammalian cells , which seem to differ from those of the lower eukaryote s. cerevisiae . fe - s clusters are highly chemically versatile because both iron and sulfur can readily donate or accept multiple electrons ( beinert , 2000 ) , and these chemical features can synergize so that the affinity of an fe - s cluster for electrons can be fine - tuned across an extremely broad electrochemical range by its surrounding protein residues . fe - s clusters are versatile in other ways , because they can directly facilitate chemical reactions by binding to an fe - s protein s substrate , as in the enzyme aconitase , which binds and interconverts citrate and isocitrate in the citric acid cycle . in addition , fe - s proteins have been found to function as sensors , as in the bacterial fnr and iscr proteins ( kiley and beinert , 2003 ) , and in mammalian irp1 , which regulates cytosolic iron metabolism in mammalian cells ( rouault , 2006 ; muckenthaler et al . the highly conserved general fe - s cluster biogenesis pathway has been the subject of intense study in numerous species of bacteria , plants , yeast and mammals since it was first described in bacteria ( reviewed by frazzon and dean , 2003 ) . many of the general steps of the pathway are common to all kingdoms of life , but it seems that the situation is more complicated in eukaryotes , in which fe - s proteins are functional and necessary in multiple subcellular compartments , including mitochondria , plastids , cytosol and nucleus . the initial stage of fe - s cluster biogenesis is accomplished by a multimeric protein complex in which a dimer of a cysteine desulfurase [ called iscs ( escherichia coli ) , nfs1 ( s. cerevisiae ) or nfs1 ( mammals ) ] forms a core to which two monomers of a dedicated scaffold protein ( iscu in bacteria , isu1 or isu2 in yeast and iscu in humans ) bind at either end ( shi et al . , 2010 ) pyridoxal phosphate , nfs1 supplies sulfur by removing it from cysteine residues , and iscu provides the backbone structure and the cysteine ligands upon which a new cluster consisting of covalently bound iron and inorganic sulfur is synthesized ( raulfs et al . the source of the iron in the nascent fe - s cluster has not been clearly identified , but suggested sources include iron that is bound to acidic patches on frataxin ( see below ) ( stemmler et al . , 2006 ) , which is found in the mitochondrial matrix of s. cerevisiae , but has been detected in both the mitochondrial matrix and the cytosolic and/or nuclear compartments of mammalian cells ( shi et al . the discovery that the isd11 protein is present in the mammalian nucleus supported previous reports that human nfs1 ( land and rouault , 1998 ) , iscu ( tong and rouault , 2000 ) and an alternative scaffold protein called nfu1 ( tong et al . structural data demonstrating the inclusion of frataxin in the initial multimeric complex detracts somewhat from previous hypotheses that the role of frataxin was to deliver bound iron to the complex ( cook et al . hsc20 binds iscu , and the hsc20-iscu complex probably then binds to its hsc70 partner through two different binding sites : hsc20 contacts the n - terminus of hsc70 and its binding partner , iscu , binds to the c - terminal substrate - binding domain region of hsc70 . upon activation , a conformational change is proposed to occur in the substrate - binding domain of hsc70 that affects bound iscu , resulting in extrusion of a peptide containing the residues lppvk from the iscu globular protein . the lppvk peptide then binds to a groove in the substrate - binding domain of hsc70 , which consolidates or perhaps further enhances the conformational change in iscu , which might convert it to a conformation that facilitates donation of its cluster to recipient proteins . in this model , hsc20 helps protect the vulnerable fe - s cluster bound to iscu as it dissociates from the multimeric assembly complex , and hsc20 then escorts iscu to form a trimeric complex with hsc70 . the consumption of atp probably provides a powerful impetus to drive conformational changes of iscu and the substrate - binding domain of hsc70 ; these changes might facilitate release of the fe - s cluster from iscu . together , hsca ( hsc70 ) and hscb ( hsc20 ) might facilitate fe - s cluster transfer from iscu to target apoproteins or to other secondary scaffolds by facilitating dissociation from the initial scaffold protein complex ( vickery and cupp - vickery , 2007 ; bonomi et al . , 2011 ) it remains unclear how fe - s cluster target proteins are identified , because the process seems to be selective and might depend on specific interactions between apoprotein targets and binding surfaces of the chaperone co - chaperone complex , particularly along the extensive potential interaction area provided by the unusually large and distinctive c - terminus of hsc20 ( kampinga and craig , 2010 ) . using the energy derived from atpase activity of the hsp70 homolog , a conformational change in this complex might facilitate protected transfer of the fe - s cluster from iscu directly to recipient apoproteins or to secondary scaffold proteins that then facilitate delivery of the fe - s cluster to a defined group of recipient proteins . target apoproteins are likely to be tethered to the chaperone co - chaperone complex when the fe - s cluster of iscu is released so that cluster release can be coupled to fe - s acquisition by apoproteins . as discussed , the fe - s cluster biogenesis pathway is fundamental to a variety of cellular processes : fe - s proteins are required for the function of two proteins of the citric acid cycle , succinate dehydrogenase and aconitase ; for respiratory chain complexes i iii ; and for numerous other proteins in the mitochondria , cytosol and nucleus of mammalian cells . for example , markedly diminished expression of frataxin causes frda , which is characterized by cardiac failure and the death of specific neuronal cell types , including the dorsal root ganglia , which are responsible for sensory perception and maintenance of balance [ see table 1 and the review by martelli et al . by contrast , iscu myopathy , a disease resulting from a splicing mutation of iscu , causes pathology almost exclusively in skeletal muscles , and cardiac involvement is rare , developing only when there is a specific inactivating mutation on one allele ( kollberg et al . tissue - specific factors might exacerbate the loss of protein function in affected tissues ; indeed , it has been suggested that tissue - specific splicing factors drive abnormal splicing of iscu in the skeletal muscles of individuals with iscu myopathy ( nordin et al . it seems likely that the cells in affected tissues in frda express less frataxin than unaffected tissues , and that the fe - s cluster biogenesis pathway is therefore more compromised at those sites . studies of three newly described diseases caused by mutations in nubpl , nfu1 or bola3 suggest that transfer of fe - s clusters to specific target proteins downstream of the iscu chaperone co - chaperone complex depends on specific pathways that shepherd fe - s clusters to subsets of recipient proteins ( also discussed below ) . for instance , mutations in the complex - i - dedicated chaperone protein nubpl have been reported to cause mitochondrial encephalomyopathy , a severe multisystem infantile disease attributable to loss of complex i function . by contrast , mutations in nfu1 or bola3 interfere quite specifically with activity of an fe - s enzyme , lipoate synthase , which provides lipoic acid modifications to a small group of specific enzymes , including the e2 subunits of pyruvate dehydrogenase and -ketoglutarate dehydrogenase , and to enzymes involved in branched chain amino acid metabolism and glycine degradation ( cameron et al . the small number of individuals with nfu1 or bola3 mutations died within the first year of life , and their main symptoms of lactic acidosis and failure to thrive have thus far not been reported to be highly tissue specific ( see table 1 ) . to understand the diseases that result from defects in the fe - s cluster biogenesis machinery , it is important to fully understand how this process occurs in mammalian cells . insights into mammalian fe - s cluster biogenesis have been confounded by the fact that a dominating paradigm has been developed in the model system s. cerevisiae , in which it was proposed that fe - s cluster biogenesis occurred or was initiated solely in the mitochondria , but not cytosolic or nuclear compartments ( lill , 2009 ) . specifically , it was proposed that the mitochondrial exporter atm1 exported either fully formed fe - s clusters or a special form of sulfur that was required for cytosolic fe - s cluster biogenesis ( lill , 2009 ) , and that loss of atm1 transporter activity led to the accumulation of iron within the mitochondrial matrix . there are mammalian homologs of yeast cia proteins , including ndor1 and ciapin1 ( homologs of tah18 and dre2 , respectively ) , nubp1 and nubp2 [ homologs of yeast nbp35 and cfd1 , respectively ( stehling et al . however , the model purporting that cytosolic fe - s cluster biogenesis depends on export of a mitochondrial product was recently called into question because genetic manipulations that were initially used to inactivate atm1 also introduced wild - type leu2 into a leu2-null strain , which unknowingly resulted in repressed transcription of leu1 relative to the control strain . thus , the model that fe - s clusters are assembled solely in mitochondria of yeast cells lacks crucial experimental support , and some models based on work in s. cerevisiae must be reconsidered . in any case , there is now substantial evidence that the paradigm that fe - s clusters are assembled or initiated solely in mitochondria is not true in mammalian cells , an issue that must be clarified to allow insights into disease pathophysiology . it is well accepted that the basic fe - s cluster biosynthetic proteins are highly expressed in mammalian mitochondria , where they supply fe - s clusters to proteins of the citric acid cycle , the respiratory chain complexes and other pathways ( described above ) . the observed extra - mitochondrial localization of these proteins is consistent with the fact that there are nuclear localization signals in mammalian nfs1 ( land and rouault , 1998 ) and isd11 ( shi et al . , 2003 ; tong and rouault , 2006 ) , and there is functional evidence that the cytosolic iscu functions as a scaffold and source for extra - mitochondrial fe - s clusters in mammalian cells ( tong and rouault , 2006 ) . , however , because proteins are synthesized in the cytosol , it is possible that some of the fe - s cluster biogenesis proteins found in this compartment represent proteins that were inefficiently imported into and processed by mitochondria . ( a ) yeast mitochondria contain the basic fe - s cluster biogenesis proteins , whereas cytosolic fe - s cluster biogenesis has been proposed to depend on the export of a sulfur compound from mitochondria through the transporter atm1 ( abcb7 in human cells ) . further biogenesis in the cytoplasm depends on a distinct set of proteins , including tah18 , dre2 , nbp35 , cfd1 , nar1 and cia1 , which are collectively referred to as cia ( cytosolic iron - sulfur assembly ) proteins . ( b ) by contrast , in mammalian cells , it seems that most of the basic fe - s cluster biogenesis proteins are expressed in mitochondria as well as in the cytosolic and/or nuclear compartment ( nuclear localization of these proteins not represented in the figure ) . in addition , there are human counterparts to the proteins implicated in cytosolic fe - s cluster biogenesis in yeast , including ndor1 ( homolog of tah18 ) , ciapin1 , ( homolog of dre2 ) , nubp1 and nubp2 ( homologs of nbp35 and cfd1 , respectively ) , and narfl and ciao1 ( homologs of nar1 and cia1 , respectively ) . to explain mitochondrial iron overload ( which occurs as a consequence of disrupted fe - s cluster biogenesis ) , it is possible that mitochondria depend on fe - s cluster biogenesis to synthesize a molecule that functions as a regulatory signal . importantly , a significant number of dna repair enzymes are fe - s proteins , including the protein responsible for excision - repair of uv damage that causes the disease xeroderma pigmentosum ( xpd ) when defective , and a related helicase that causes fanconi anemia when defective ( rudolf et al . some of the cytosolic fe - s assembly proteins previously identified in yeast might function in mammalian cells as secondary scaffold proteins that transfer clusters to specific classes of protein targets such as dna repair enzymes . , 2006 ) , because these proteins are implicated in cytosolic fe - s cluster biogenesis and some of their likely targets are involved in chromosomal duplication and in maintenance of nuclear integrity , processes that are necessary for proper cell division ( netz et al . in summary , although the scenario in yeast remains in dispute , the accumulating evidence that fe - s cluster biogenesis can occur de novo in the mitochondrial matrix as well as in extra - mitochondrial compartments of mammalian cells is strong and consistent . it is crucial to correctly analyze the basics of the mammalian fe - s cluster biogenesis pathway , because it sets the stage for understanding one of the most common complications of impaired fe - s cluster biogenesis : the propensity to develop marked mitochondrial iron overload and subsequent iron - related mitochondrial damage . evidence suggests that interruption of mitochondrial fe - s cluster biogenesis leads to mitochondrial iron overload and cytosolic iron depletion in mammalian cells , which is associated with disease pathology . for example , mitochondrial iron overload is a distinctive feature of the heart and some neurons of frda patients ( koeppen , 2011 ) , of the skeletal muscle cells of individuals with iscu myopathy ( mochel et al . subsequently , this hypothesis was modified to suggest that abcb7 ( the mammalian homolog of yeast atm1 ) exports a special type of sulfur , but not iron , from mitochondria ( lill , 2009 ; netz et al . however , the revised theory does not offer an explanation for why iron accumulates in mitochondria : the idea that iron accumulates in mitochondria passively because it can not be incorporated into fe - s clusters does not take into account the possibility that mitochondrial iron metabolism is a highly regulated process that might be imbalanced in cells with defective fe - s cluster biogenesis . in support of this idea , transcriptional expression of the mitochondrial iron importer mitoferrin is increased in frataxin - deficient mouse hearts ( huang et al . , 1999 ) caused by mutations in the abcb7 mitochondrial iron transporter , as well as in individuals with sideroblastic anemia caused by mutations in the red - blood - cell - specific amino - levulinate synthase alas2 , a mitochondrial protein that is involved in heme biosynthesis ( table 2 ) . , 2006 ) has not yet been identified , it might be time to think more broadly about the regulation of mitochondrial iron homeostasis and to integrate the phenotypes observed upon mutation of alas2 and abcb7 into a single overarching model for mitochondrial iron regulation . the fact that a specific subset of fe - s proteins is affected in these newly described diseases suggests that target specificity is achieved through use of specific scaffold proteins that function as intermediates between iscu and target proteins . these diseases include those caused by mutations in an enzyme known as pus1 , which is expressed in both mitochondria and the nucleus and is required for pseudouridylate processing on transfer rnas ( trnas ; see table 2 ) , and by mutations in yars2 , a gene that encodes a mitochondrial tyrosine trna synthetase . better characterization of several newly recognized diseases offers the prospect of making progress on details of the second part of the fe - s biogenesis pathway , particularly downstream of the chaperone co - chaperone proteins , when fe - s clusters are ligated to specific recipient apoproteins .

the ability to evolve through the combined effects of inherited variation and natural selection is the unifying principle of biology . the most elementary cellular system capable of darwinian evolution requires two components : a self - replicating informational molecule , such as an rna polymerase ribozyme ( replicase ) , and a mechanism for spatial localization such as compartmentalization . without compartmentalization or some other mechanism for restricting the diffusion of the genetic material , a superior mutant replicase would act to propagate unrelated sequences more efficiently , while having no effect on its own fitness . the physical proximity of related sequences , which can be readily maintained by the random segregation of sequences within replicating compartments , is therefore necessary for the evolution of improved replicase activity through natural selection . membranes provide the major mechanism for compartmentalization in modern biology and also mediate the chemical fluxes between the cell and its environment . to construct the simplest possible protocell , the membrane boundary should be capable of growth and division without complex biochemical machinery and should allow the entry of ions and small substrates by passive diffusion . to have potential prebiotic relevance , the membrane should also be formed from chemicals that can be synthesized abiotically . fatty acids are simple amphiphiles that form bilayer membrane vesicles when the solution ph is near the apparent pka of the membrane - incorporated fatty acid . they can be synthesized under a variety of conditions , including simulated hydrothermal vent chemistry , and have been detected in carbonaceous chondrite meteorites . clay surfaces that catalyze rna polymerization also promote the self - assembly of fatty acid micelles into vesicles , suggesting a route for enhancing rna encapsulation by colocalizing vesicles and rna . in addition to their chemical simplicity , fatty acid membranes have dynamic properties that are not exhibited by phospholipid membranes . for example , fatty acid vesicles show autocatalytic growth , and they can undergo repeated cycles of growth and division in the laboratory . these vesicles can also exhibit a primitive form of competition for limited resources , as vesicles encapsulating a high concentration of rna grow larger at the expense of empty vesicles ; this is referred to below as the exchange pathway for growth . under certain conditions , growth can drive the formation of a transmembrane ph gradient , thereby storing energy for several hours . these protocellular behaviors are facilitated by the fast time scales of monomermicellevesicle transitions and the relatively small energetic barriers between these phases , as compared to phospholipid membranes , which do not share these dynamic properties . the major disadvantage of using fatty acid membranes to build protocells is their instability under the ionic conditions necessary for catalytic rna activity . for example , decanoic acid vesicles aggregate at moderate concentrations of monovalent ions ( 0.2 m nacl ) , and divalent cations cause aggregation and precipitation even at quite low concentrations ( < 1.5 mm mgcl2 , or 1:65 ratio of cation to lipid ) . this is an important problem because divalent cations are often required for the formation of tertiary contacts in rna folding and also commonly participate directly in the catalytic mechanism . consequently , the activity of many ribozymes , such as the hammerhead ribozyme , is highly dependent on divalent cation concentration . for a protocell to use rna - based biochemistry , vesicle membranes must be stable under conditions enabling ribozyme activity . we therefore sought to encapsulate catalytically active rna in simple vesicles that were stable and capable of growth . the addition of fatty acid glycerol esters stabilizes fatty acid vesicles in the presence of ionic solutes . although a variety of cations can be utilized by ribozymes , we focused on mg because of its common use by ribozymes and its abundance on the earth , particularly in seawater . we characterized the growth properties , mg tolerance , and mg permeability of such mixed composition vesicles . by varying the composition of mixed vesicles , we were able to increase the tolerance of vesicles to mg and to demonstrate that mg could rapidly enter vesicles by passive diffusion . remarkably , the presence of mg increased the permeability of the membrane to small , negatively charged solutes such as nucleotides . on the other hand , vesicle growth by the exchange pathway was slowed , and vesicle growth following micelle addition was only possible after further modification of the membrane composition . finally , we encapsulated a hammerhead ribozyme in mixed vesicles and showed that the ribozyme is active under conditions consistent with vesicle stability and growth . ( i.e. , monomyristolein , the glycerol ester of myristoleic acid ) were purchased from nuchek prep ( elysian , mn ) . pyrene was from fluka ( buchs sg , switzerland ) ; n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine ( nbd - pe ) , lissamine rhodamine b 1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine ( rh - dhpe ) , calcein , and mag - fura-2 ( tetrapotassium salt ) were from molecular probes ( eugene , or ) . ( austin , tx ) , and rnasin was from promega ( madison , wi ) . rna labeled with fluorescein ( 5-6-fam - aaaaaaaaaa-3 ) was synthesized at the w. m. keck facility at yale university . all other chemicals were purchased from sigma - aldrich ( st . louis , mo ) . micelles were prepared by dissolving neat myristoleic acid oil in 1 equiv of naoh solution ( 80 mm ) , as described . vesicles 100 nm in diameter were prepared by resuspending 1 equiv of the oil in 0.2 m bicine buffer , ph 8.5 , to which 0.5 equiv of naoh had been added ( resulting in a final ph of 8.5 ) . resuspension was followed by several freezethaw cycles and extrusion to 100 nm diameter using the mini - extruder system ( avanti polar lipids , inc . ) , as described . mixed composition micelles and vesicles were prepared by mixing the appropriate neat oils before resuspension . vesicles labeled by the fret dyes ( nbd - pe and rh - dhpe ) were prepared by mixing the dyes with fatty acid / ester in methanol , and then removing the solvent by rotary evaporation before resuspension . encapsulation of rna or water - soluble fluorescent dyes was achieved by mixing the solute with the resuspension buffer before adding the oil . vesicles were purified from unencapsulated solutes on a gravity - flow size exclusion column ( sepharose 4b ) . the fluorescent dyes ( 1 kd ) and rna ( 19 kd ) used in these experiments elute at the low molecular weight range of the resin ( 30 kd ) , while vesicles ( 100 nm in diameter ) elute with the void volume . purification and dilution buffers contained a concentration of amphiphiles approximately equal to the critical aggregate concentration , to avoid disintegration of the vesicles . vesicle growth . vesicles labeled with fret dyes were diluted to 3 mm amphiphile and added to a cuvette . growth from addition of micelles was initiated by adding micelles from an 80 mm stock solution , with mixing by pipetting for 5 s. fret was measured using the fluorescence ratio between donor ( em 530 nm ) and acceptor ( em 586 nm ) dyes , with excitation at 430 nm , as described . to convert fret signal into relative surface area , a standard curve was prepared for each vesicle composition ( figures s1 , s2 ) . growth by intermembrane transfer of amphiphiles from empty vesicles to vesicles encapsulating trna was performed as described previously , with the addition of 3.2 mm mgcl2 to the reaction buffer containing 2 mm amphiphile . the concentration of free mg in this solution was 2 mm , as measured by mag - fura-2 fluorescence . dye leakage assay . extruded vesicles encapsulating calcein or h - ump were added to a small volume of a stock solution of mgcl2 ( 100 mm ) to achieve the desired concentration of mg . to determine the extent of dye leakage , vesicles were separated from free dye by size - exclusion chromatography as described above . the integrated fluorescence intensity of each peak was determined by summing the fluorescence intensity of the fractions , as measured on a spectramax gemini em ( molecular devices ) . for vesicles of this size ( 100 nm ) , the concentration of vesicles was low enough ( < 10 mm lipid ) to avoid effects on the fluorescence from turbidity . radioactivity was measured on a beckman ls 6500 scintillation counter . for time courses , the percent of free dye in the initial time point was used as the background and subtracted from subsequent time points . calcein fluorescence was measured at ex 415 nm , em 600 nm ; when appropriate , values were corrected for self - quenching using a standard curve . initial rates of leakage were determined by fitting a straight line to the data for leakage over time . mg concentration was related to the ratio of the fluorescence intensity at ex 340 nm and ex 370 nm ( at constant em 500 nm ) through a standard curve prepared with varying concentrations of mgcl2 in 0.2 m bicine , ph 8.5 . to determine mg permeability , vesicles containing encapsulated mag - fura-2 were diluted to 4 mm amphiphile and mixed by stopped - flow with 1/25 volume of 50 mm mgcl2 ( final [ mg ] = 2 mm ) , as described . because the counter - transport of na , required for charge neutrality , is fast relative to the transport of mg , the mg permeability coefficient was calculated by p = k*v / a , where k is the first - order rate constant , and v and a are the volume and surface area of the vesicle , respectively . the pyrene excimer / monomer ratio was determined from the ratio of fluorescence intensity at em 374 nm ( monomer ) and em 470 nm ( excimer ) , as described . the transcription template for the hammerhead ribozyme n15min7 ( 5- gggacgcagtttcgcttagctcatcagagtaaattcctttcggaatttac tgactgcgt - ccctatagtgagtcgtattacagatc-3 ) was pcr amplified using the primers ( 5-gatctgtaatacgactcac-3 and 5-gggacgcagtttcgcttag-3 ) for 20 cycles . the pcr product was purified using agarose gel electrophoresis , ethanol precipitated , dissolved , and stored in 10 mm tris - cl , ph 8.5 . rna was transcribed from double - stranded n15min7 template by t7 rna polymerase in a solution containing 0.5 mm ntps , 20 ci -p - utp , 40 mm tris - hcl , ph 7.9 , 6 mm mgcl2 , 2 mm spermidine , 10 mm dtt , and 0.2 u/l rnasin . the reaction also included 25 m of two oligonucleotides complementary to the ribozyme sequence , to block ribozyme self - cleavage during transcription ( 5-tcgcttagctcatcag-3 and 5-ggaatttactgactgc-3 ) . transcribed rna was purified by denaturing page , ethanol precipitated , dissolved in water , and stored at 80 c . the reaction was stopped at each time point by removing an aliquot and adding 1.12.3 volumes of 10 mm tris - cl , ph 7.5 , containing 314 mm edta . for encapsulated reactions , the amphiphile concentration was 15 mm and the rna concentration was 40300 nm ( less than one molecule per vesicle on average ) . vesicles were disrupted by the addition of 0.3% triton x-100 , and the rna was precipitated in 0.3 m sodium acetate , ph 7 , 73% ethanol , with 10 g of glycoblue . the extent of cleavage observed before addition of mgcl2 ( t = 0 ) was subtracted from subsequent time points . vesicles were prepared by mixing ma : gmm : dodecane ( 2:1:0.3 ) oil with buffer consisting of 0.2 m bicine , ph 8.5 , 3 mm mgcl2 , and 40 nm n15min7 ( final amphiphile concentration = 20 mm ) . vesicle suspensions were allowed to equilibrate for at least 1 h before staining with 10 mm rhodamine 6 g ( max , ex = 528 nm ; max , em = 551 nm ) for microscopy . conventional epifluorescence images were collected using a zeiss axioplan 2 microscope ( 100 objective lens ; rhodamine filter ) . confocal images were collected using a leica tcs sp confocal laser scanning microscope equipped with ar , arkr , and hene lasers ( 100 objective lens ; 514 nm laser line ) . we assessed the stability of vesicles composed of myristoleic acid ( ma ) and glycerol monomyristoleate ( gmm ) in the presence of mg by monitoring the retention of encapsulated dye and macroscopic turbidity changes . pure ma vesicles showed low mg tolerance , but vesicles of mixed composition , ma : gmm ( 2:1 ) , showed increased stability in the presence of mg , as previously reported on the basis of light microscopy . an intermediate composition of ma : gmm ( 4:1 ) showed intermediate stability ( table 1 ) . higher proportions of gmm led to the appearance of oil droplets mixed with vesicles . table 1mgcl2 tolerance of simple vesiclesma : gmm ratio[mgcl2 ] tolerated , assayed by dye leakage ( mm)[mgcl2 ] at turbidity change ( mm ) 1:0 0.5 1 4:1 2 3 2:1 4 6 dye leakage was assessed < 1 h after addition of mgcl2 , and onset of leakage defined the maximum [ mgcl2 ] tolerated . turbidity change was visible as a sudden increase in the opacity of the solution . mgcl2 tolerance of simple vesicles dye leakage was assessed < 1 h after addition of mgcl2 , and onset of leakage defined the maximum [ mgcl2 ] tolerated . turbidity change was visible as a sudden increase in the opacity of the solution . to determine whether vesicles exposed to relatively high concentrations of mgcl2 are only transiently destabilized by the sudden addition of mgcl2 , we followed the leakage of encapsulated calcein over time . dye leakage continued to increase over the course of at least 1 day ( figure 1a , b ) , indicating that the vesicles remained permeabilized throughout the experiment . the initial rate of dye leakage from ma : gmm ( 2:1 ) vesicles increased by a factor of 27 in the presence of 4 mm mg . to determine whether this enhanced permeability was due to large - scale membrane destabilization by mg , we encapsulated a fluorescently labeled rna decamer in vesicles exposed to 4 mm mg . we expected that large - scale membrane defects would cause rna leakage at a rate similar to small molecule leakage , but no rna leakage was detected after 19 h ( figure 1c ) . h - ump showed a 4-fold permeability enhancement in the presence of 4 mm mg ( figure 1d ) . thus , exposure to mg increased the permeability of an rna mononucleotide but not of rna itself , perhaps by neutralizing the negative charge and/or stabilizing the interaction between the solute and small - scale transient defects in the membrane . even so , more than 95% of encapsulated calcein was retained in ma : gmm ( 2:1 ) vesicles for at least 3 h. this is much longer than the hammerhead ribozyme cleavage reactions described below , which were completed within a few minutes . figure 1leakage from ma : gmm vesicles ( 2:1 ) in 0.2 m bicine , ph 8.5 , 4 mm mgcl2 . ( a ) vesicles were initially purified away from unencapsulated calcein , and dye leakage was measured over time with ( blue ) or without ( black ) the addition of 4 mm mgcl2 . ( b ) calcein leakage assayed by size - exclusion chromatography after 22 h in the presence of 4 mm mgcl2 . ( c ) rna leakage assayed by size - exclusion chromatography after 19 h , with ( red ) or without ( black ) the addition of 4 mm mgcl2 . ( d ) vesicles encapsulating 0.1 mm 5-ump and a trace amount of h - ump were initially purified , and then leakage of h - ump was measured over time , with ( red ) or without ( black ) the addition of 4 mm mgcl2 . leakage from ma : gmm vesicles ( 2:1 ) in 0.2 m bicine , ph 8.5 , 4 mm mgcl2 . ( a ) vesicles were initially purified away from unencapsulated calcein , and dye leakage was measured over time with ( blue ) or without ( black ) the addition of 4 mm mgcl2 . ( b ) calcein leakage assayed by size - exclusion chromatography after 22 h in the presence of 4 mm mgcl2 . ( c ) rna leakage assayed by size - exclusion chromatography after 19 h , with ( red ) or without ( black ) the addition of 4 mm mgcl2 . ( d ) vesicles encapsulating 0.1 mm 5-ump and a trace amount of h - ump were initially purified , and then leakage of h - ump was measured over time , with ( red ) or without ( black ) the addition of 4 mm mgcl2 . because we wished to eventually measure the activity of encapsulated rna , we also determined the permeability of mg through the vesicle membrane . we encapsulated mag - fura-2 , a magnesium - sensitive fluorescent dye ( figure 2a ) , in ma : gmm vesicles ( 2:1 ) and added mgcl2 using a stopped - flow device . the internal concentration of mg equilibrated within several seconds ( permeability coefficient = 2 10 cm / s ) to 1.4 mm , a concentration lower than the added concentration ( 2 mm ; figure 2b ) . because ma is partially negatively charged at ph 8.5 , it may associate with mg , thereby reducing the amount of free mg detected by the mag - fura-2 dye . using size - exclusion chromatography , we verified that < 5% of encapsulated mag - fura-2 leaked from vesicles after 30 min of exposure to 2 mm mg ( figure 2c ) , consistent with the results obtained for calcein leakage . when we mixed phospholipid ( popc ) vesicles with mgcl2 , we saw no detectable change in the internal mg concentration over several hours ( figure 2d ) , demonstrating that equilibration of both mg and mag - fura-2 across popc membranes is quite slow . ( a ) standard curve of mag - fura-2 fluorescence ratio as a function of [ mg ] in 0.2 m bicine , ph 8.5 . mgcl2 was added to a final concentration of 2 mm , by stopped - flow mixing , to ma : gmm ( 2:1 ) vesicles ( b ) or popc vesicles ( d ) encapsulating mag - fura-2 . the internal concentration of mg at each time point was calculated from the standard curve . the solid line ( b ) indicates a curve fit to a single - exponential equation , where k = 0.11 s. ( c ) size - exclusion chromatography shows that no mag - fura-2 dye has leaked out of the ma : gmm vesicles after equilibration of mg . ( a ) standard curve of mag - fura-2 fluorescence ratio as a function of [ mg ] in 0.2 m bicine , ph 8.5 . mgcl2 was added to a final concentration of 2 mm , by stopped - flow mixing , to ma : gmm ( 2:1 ) vesicles ( b ) or popc vesicles ( d ) encapsulating mag - fura-2 . the internal concentration of mg at each time point was calculated from the standard curve . the solid line ( b ) indicates a curve fit to a single - exponential equation , where k = 0.11 s. ( c ) size - exclusion chromatography shows that no mag - fura-2 dye has leaked out of the ma : gmm vesicles after equilibration of mg . although the addition of gmm to ma vesicles increased their tolerance for mg , we suspected that the increase in the stability of the vesicles might come at the expense of dampened dynamic behaviors , such as growth , that rely on the rapid insertion and dissociation of molecules from the membrane . growth of the surface area of vesicles can be monitored using nonexchanging fret dyes , whose fluorescence depends on the surface density of the dyes . we have previously studied the growth of osmotically swollen vesicles due to intermembrane transfer of amphiphiles from empty vesicles . while transfer was slower in ma : gmm ( 2:1 ) membranes , the total amount of transfer was comparable in ma : gmm ( 2:1 ) and pure fatty acid membranes . to determine whether mg affected the intermembrane transfer of amphiphiles , we measured the transfer between ma : gmm ( 2:1 ) vesicles ( 2 mm amphiphile ) in the presence of 3.2 mm mgcl2 ( 2 mm free mg ) . the rate ( k = 0.05 min ) and amount of transfer ( 28% ) were not substantially affected by the presence of mg . another way to achieve vesicle growth is by the addition of micelles , prepared at high ph , to preformed vesicles buffered at ph 8.5 . when 0.5 equiv of pure fatty acid in micelle form is rapidly added to preformed vesicles , the surface area of preformed vesicles grows by 50% , indicating a very high yield of incorporation . in contrast , 4:1 ma : gmm vesicles showed only 35% growth upon addition of 0.5 equiv of micelles of the same composition . dynamic light scattering ( dls ) indicated that ma : gmm ( 4:1 ) micelle solutions contained particles 30 nm in diameter , much larger than pure fatty acid micelles ( diameter = 3 nm ) . aggregation can also be detected by the excimer / monomer ratio of the hydrophobic dye pyrene when present at low molar ratio ( 1:200 ) . when dissolved in small micelles , pyrene is distributed among the separate volumes and exists primarily as a monomer . however , lipid aggregation allows multiple pyrene molecules to share a contiguous volume , forming excimers with a red - shifted fluorescence emission spectrum . consistent with the dls data , pyrene fluorescence indicated the presence of large aggregates in ma : gmm ( 4:1 ) micelles ( figure 3a ) . ( a ) pyrene fluorescence shifts from monomer to excimer if aggregates are present . shown are the spectra of pyrene in pure ma micelles ( green ) , pure ma vesicles ( blue ) , and ma : gmm ( 4:1 ) micelles ( red ) . ( b ) growth of 2:1:0.3 ma : gmm : dodecane vesicles over time , after addition of 1 equiv of micelles , in 0.2 m bicine , ph 8.5 , 1 mm mgcl2 . the solid line indicates a curve fit to a single - exponential equation , with k = 1.5 min . a similar yield was obtained if 1 equiv of micelles was added at once or in up to five separate aliquots . ( a ) pyrene fluorescence shifts from monomer to excimer if aggregates are present . shown are the spectra of pyrene in pure ma micelles ( green ) , pure ma vesicles ( blue ) , and ma : gmm ( 4:1 ) micelles ( red ) . ( b ) growth of 2:1:0.3 ma : gmm : dodecane vesicles over time , after addition of 1 equiv of micelles , in 0.2 m bicine , ph 8.5 , 1 mm mgcl2 . the solid line indicates a curve fit to a single - exponential equation , with k = 1.5 min . a similar yield was obtained if 1 equiv of micelles was added at once or in up to five separate aliquots . ma : gmm ( 2:1 ) vesicles , which had the greatest magnesium tolerance , showed no growth when micelles of the same composition were added , either rapidly or slowly over 4 h. however , these vesicles were able to grow following the rapid addition of pure fatty acid micelles . thus , 2:1 ma : gmm micelles ( which are also large by the pyrene excimer assay and by dls ) appeared to be unusually stable , perhaps due to the large headgroup of gmm , which may stabilize areas of high curvature in micellar aggregates and which would also provide h - bond donors at high ph . to destabilize these micelles , we added dodecane ( 9 mol % ) to act as a hydrophobic spacer , reducing the preferred curvature and decreasing the surface charge density . the rapid addition of 0.5 equiv of 2:1:0.3 ma : gmm : dodecane micelles to vesicles of the same composition resulted in 20% growth , and the rapid addition of 1 equiv of micelles resulted in 40% growth ( figure 3b ) . the rate and total amount of growth were similar in the presence or absence of 1 mm free mg . we determined the activity of a self - cleaving hammerhead ribozyme encapsulated in ma : gmm : dodecane vesicles . the hammerhead ribozyme is widespread in nature and also arises during in vitro selection for self - cleaving rnas . this ribozyme can catalyze both cleavage and ligation of rnas , activities that may be important for simple rna - based replicating systems . furthermore , because of its small size , the hammerhead ribozyme may function robustly under error - prone replication conditions . n15min7 is an artificially evolved 62 nucleotide hammerhead ribozyme , which is cleaved into two fragments ( 11 and 51 nucleotides ) in the presence of mg . n15min7 was encapsulated in 2:1:0.3 ma : gmm : dodecane vesicles during amphiphile resuspension . multiple freezethaw cycles , used to equilibrate vesicle formation , did not affect self - cleavage activity . the self - cleavage reaction was initiated by adding mgcl2 ( 4 mm ) , and the extent of self - cleavage was monitored as a function of time ( figure 4a , b ) . we found that 60% of the rna was cleaved whether the ribozyme was encapsulated or free in solution . the observed first - order rate constant of the encapsulated reaction was reduced 4-fold as compared to the unencapsulated reaction at both 1 and 4 mm added mgcl2 ( table 2 ) . as expected , the rate of the encapsulated reaction in 4 mm added mgcl2 was 5-fold higher than that in 1 mm added mgcl2 . the top band corresponds to the uncleaved ribozyme ; the bottom band is the cleavage product . ( a ) unencapsulated ribozyme activity in 0.2 m bicine , ph 8.5 , 4 mm mgcl2 . time points , from left to right : 0 ( no mgcl2 ) , 0.2 , 0.4 , 0.6 , 0.8 , 1.1 min . the solid line indicates a curve fit to a single - exponential equation , y = a(1 e ) , where the extent of cleavage a = 0.66 , and the observed rate constant k = 7.9 min . ( b ) activity of ribozymes encapsulated in 2:1:0.3 ma : gmm : dodecane vesicles ( 15 mm amphiphile ) , in 0.2 m bicine , ph 8.5 , 4 mm mgcl2 . time points , from left to right : 0 ( no mgcl2 ) , 0.33 , 0.63 , 1 , 1.3 , 1.7 , 3 , 4.7 min . ( c ) size - exclusion chromatography of ma : gmm : dodecane vesicles shows that all radiolabeled n15min7 rna remained encapsulated 15 min after the addition of mgcl2.table 2hammerhead cleavage in vesicles[mgcl2 ] ( mm)kunencapsulated ( min)kencapsulated ( min ) 1 1.3 0.4 4 7.9 1.7 self - cleavage activity of the hammerhead ribozyme n15min7 . the top band corresponds to the uncleaved ribozyme ; the bottom band is the cleavage product . ( a ) unencapsulated ribozyme activity in 0.2 m bicine , ph 8.5 , 4 mm mgcl2 . time points , from left to right : 0 ( no mgcl2 ) , 0.2 , 0.4 , 0.6 , 0.8 , 1.1 min . the solid line indicates a curve fit to a single - exponential equation , y = a(1 e ) , where the extent of cleavage a = 0.66 , and the observed rate constant k = 7.9 min . ( b ) activity of ribozymes encapsulated in 2:1:0.3 ma : gmm : dodecane vesicles ( 15 mm amphiphile ) , in 0.2 m bicine , ph 8.5 , 4 mm mgcl2 . time points , from left to right : 0 ( no mgcl2 ) , 0.33 , 0.63 , 1 , 1.3 , 1.7 , 3 , 4.7 min . curve fit : a = 0.6 , and k = 1.7 min . ( c ) size - exclusion chromatography of ma : gmm : dodecane vesicles shows that all radiolabeled n15min7 rna remained encapsulated 15 min after the addition of mgcl2 . hammerhead cleavage in vesicles this decreased rate of self - cleavage of the encapsulated ribozyme may be partly due to the time scale of mg permeation into gmm - ma - dodecane vesicles ( k 1 min ) , particularly in 4 mm mgcl2 . however , for the encapsulated ribozyme in 1 mm mgcl2 , the rate of mg entry is faster than the observed self - cleavage , indicating that mg entry was not the rate - limiting step in this case . the decrease in free mg concentration ( kd 10 mm amphiphile , considering both the inner and the outer leaflets of the bilayer ) , as determined by mag - fura-2 fluorescence , accounts for about one - half of the observed decrease in rate . to confirm that the observed self - cleavage activity reflected encapsulated ribozyme , rather than ribozyme adsorbed to the membrane , n15min7 rna after purification by size - exclusion chromatography , these preparations contained < 3% of the radioactivity of the encapsulated reaction , indicating that only a small amount of rna was adsorbed to the membrane . we also used size - exclusion chromatography to confirm that the ribozyme still co - purified with vesicles after the self - cleavage reaction ( figure 4c ) . we confirmed that the 2:1:0.3 ma : gmm : dodecane mixture formed vesicles in the presence of mg and rna by fluorescence microscopy before extrusion ( figure 5 ) . dls of these vesicles after extrusion indicated an average particle diameter of 65 nm , similar to the size of extruded vesicles made from myristoleic acid alone . furthermore , self - cleavage of n15min7 was neither catalyzed nor inhibited by the presence of vesicles alone . in the absence of mg , no self - cleavage was detected after 30 min , and the self - cleavage rate of unencapsulated n15min7 upon addition of mg was not affected by the presence of vesicles . figure 5fluorescence microscopy of 2:1:0.3 ma : gmm : dodecane vesicles containing hammerhead ribozyme in the presence of 3 mm mgcl2 , prior to extrusion . membranes were stained using rhodamine 6 g for visualization by ( a ) epifluorescence or ( b ) laser scanning confocal microscopy . fluorescence microscopy of 2:1:0.3 ma : gmm : dodecane vesicles containing hammerhead ribozyme in the presence of 3 mm mgcl2 , prior to extrusion . membranes were stained using rhodamine 6 g for visualization by ( a ) epifluorescence or ( b ) laser scanning confocal microscopy . the [ mg]1/2 ( concentration of mg corresponding to half - maximal activity ) of the n15 construct used in our studies is 9 mm , and hammerhead ribozymes of biological origin have even lower mg optima of < 1 mm . other complex natural ribozymes that function under physiological conditions ( < 1 mm ) have evolved low mg requirements . for example , the [ mg]1/2 for folding and catalysis of a number of self - splicing group i introns is 4 mm . an rna polymerase ribozyme with a similarly low mg requirement could function inside ma : gmm vesicles . however , currently available ribozymes with polymerase or ligase activity have much higher mg requirements . for example , the class i ligase , discovered by in vitro evolution in the presence of high concentrations of mg , has a [ mg]1/2 of 70100 mm , as does a variant that can catalyze limited rna polymerization . the discrepancy between the mg requirement of this ribozyme and the mg tolerance of the simple vesicles studied here suggests that observing ribozyme polymerase activity inside vesicles that can grow and divide may not be trivial in practice . further optimization of existing ribozymes , or isolation of new ones , may yield rna polymerases active at lower mg concentrations that could be compatible with the vesicles described here . an independent reason for focusing on low to moderate mg concentrations is that high mg concentrations promote rna degradation . mg - catalyzed rna degradation is in fact thought to limit the extent of polymerization obtainable by the polymerase derived from the class i ligase . we have shown that vesicles composed of chemically simple amphiphiles can be stable , grow , and retain contents in the presence of sufficient mg to activate encapsulated ribozymes . because these membranes , unlike phospholipid membranes , are quite permeable to mg , encapsulated ribozymes can be activated by the external addition of mg . other rna - catalyzed reactions , including rna replication , may ultimately be possible within similar vesicles , enabling the experimental construction of evolvable protocells . the permeability of these simple membranes to mg means that the internal mg concentration of a population of growing and dividing vesicles could be maintained indefinitely , from generation to generation , solely by passive trans - membrane diffusion in the absence of permeases . our experiments also illustrate that there are tradeoffs in the search for conditions compatible with both rna and vesicle replication . while the addition of gmm confers stability on ma vesicles , it also causes a decrease in the yield of growth following micelle addition . this decrease could be partially offset by the addition of another simple component , dodecane . on the other hand , the yield of growth of osmotically swollen vesicles was not affected by the addition of gmm . indeed , this mode of growth may be more likely to occur in natural situations , because the source of lipid , empty vesicles , is the same physical phase as the growing vesicles , readily permitting their coexistence . an unexpected effect of mg was to increase the permeability of small , negatively charged solutes , including uridine monophosphate . this effect would be advantageous to an encapsulated replicating rna that was polymerizing nucleic acid monomers into longer strands of rna . the increase in monomer permeability might enhance polymerization , while the polymeric product would continue to be retained . many selective pressures are likely to have influenced the evolution of the phospholipid membranes of modern biology , which are much more stable to divalent cations and can maintain ph and ionic gradients for long periods . however , the evolution of such membranes would have required the co - evolution of numerous ion and substrate - specific permeases , as well as the biochemical machinery for membrane growth . our results show that membranes made from simple amphiphiles can be stable enough to retain rna , yet dynamic enough to grow and allow the spontaneous entry of mg and mononucleotides . we are continuing to search for membrane compositions with similar dynamic properties , but greater tolerance to mg and possibly ca ( another abundant divalent cation on earth ) , as well as ribozyme polymerases active at lower mg concentrations , with the goal of observing rna - catalyzed rna synthesis within model protocell vesicles . standard curve for fret signal prepared using 4:1 ma : gmm vesicles ( figure s1 ) and 2:1 ma : gmm vesicles ( figure s2 ) .

we are engaged in a long - term effort to synthesize chemical systems capable of darwinian evolution , based on the encapsulation of self - replicating nucleic acids in self - replicating membrane vesicles . here , we address the issue of the compatibility of these two replicating systems . fatty acids form vesicles that are able to grow and divide , but vesicles composed solely of fatty acids are incompatible with the folding and activity of most ribozymes , because low concentrations of divalent cations ( e.g. , mg2 + ) cause fatty acids to precipitate . furthermore , vesicles that grow and divide must be permeable to the cations and substrates required for internal metabolism . we used a mixture of myristoleic acid and its glycerol monoester to construct vesicles that were mg2 + -tolerant and found that mg2 + cations can permeate the membrane and equilibrate within a few minutes . in vesicles encapsulating a hammerhead ribozyme , the addition of external mg2 + led to the activation and self - cleavage of the ribozyme molecules . vesicles composed of these amphiphiles grew spontaneously through osmotically driven competition between vesicles , and further modification of the membrane composition allowed growth following mixed micelle addition . our results show that membranes made from simple amphiphiles can form vesicles that are stable enough to retain encapsulated rnas in the presence of divalent cations , yet dynamic enough to grow spontaneously and allow the passage of mg2 + and mononucleotides without specific macromolecular transporters . this combination of stability and dynamics is critical for building model protocells in the laboratory and may have been important for early cellular evolution .

Introduction Materials and Methods Results and Discussion Conclusions None Supplementary Material

the most elementary cellular system capable of darwinian evolution requires two components : a self - replicating informational molecule , such as an rna polymerase ribozyme ( replicase ) , and a mechanism for spatial localization such as compartmentalization . to construct the simplest possible protocell , the membrane boundary should be capable of growth and division without complex biochemical machinery and should allow the entry of ions and small substrates by passive diffusion . fatty acids are simple amphiphiles that form bilayer membrane vesicles when the solution ph is near the apparent pka of the membrane - incorporated fatty acid . clay surfaces that catalyze rna polymerization also promote the self - assembly of fatty acid micelles into vesicles , suggesting a route for enhancing rna encapsulation by colocalizing vesicles and rna . for example , fatty acid vesicles show autocatalytic growth , and they can undergo repeated cycles of growth and division in the laboratory . these vesicles can also exhibit a primitive form of competition for limited resources , as vesicles encapsulating a high concentration of rna grow larger at the expense of empty vesicles ; this is referred to below as the exchange pathway for growth . for example , decanoic acid vesicles aggregate at moderate concentrations of monovalent ions ( 0.2 m nacl ) , and divalent cations cause aggregation and precipitation even at quite low concentrations ( < 1.5 mm mgcl2 , or 1:65 ratio of cation to lipid ) . this is an important problem because divalent cations are often required for the formation of tertiary contacts in rna folding and also commonly participate directly in the catalytic mechanism . consequently , the activity of many ribozymes , such as the hammerhead ribozyme , is highly dependent on divalent cation concentration . we therefore sought to encapsulate catalytically active rna in simple vesicles that were stable and capable of growth . the addition of fatty acid glycerol esters stabilizes fatty acid vesicles in the presence of ionic solutes . although a variety of cations can be utilized by ribozymes , we focused on mg because of its common use by ribozymes and its abundance on the earth , particularly in seawater . by varying the composition of mixed vesicles , we were able to increase the tolerance of vesicles to mg and to demonstrate that mg could rapidly enter vesicles by passive diffusion . remarkably , the presence of mg increased the permeability of the membrane to small , negatively charged solutes such as nucleotides . on the other hand , vesicle growth by the exchange pathway was slowed , and vesicle growth following micelle addition was only possible after further modification of the membrane composition . finally , we encapsulated a hammerhead ribozyme in mixed vesicles and showed that the ribozyme is active under conditions consistent with vesicle stability and growth . , monomyristolein , the glycerol ester of myristoleic acid ) were purchased from nuchek prep ( elysian , mn ) . the fluorescent dyes ( 1 kd ) and rna ( 19 kd ) used in these experiments elute at the low molecular weight range of the resin ( 30 kd ) , while vesicles ( 100 nm in diameter ) elute with the void volume . purification and dilution buffers contained a concentration of amphiphiles approximately equal to the critical aggregate concentration , to avoid disintegration of the vesicles . growth by intermembrane transfer of amphiphiles from empty vesicles to vesicles encapsulating trna was performed as described previously , with the addition of 3.2 mm mgcl2 to the reaction buffer containing 2 mm amphiphile . for vesicles of this size ( 100 nm ) , the concentration of vesicles was low enough ( < 10 mm lipid ) to avoid effects on the fluorescence from turbidity . for time courses , the percent of free dye in the initial time point was used as the background and subtracted from subsequent time points . mg concentration was related to the ratio of the fluorescence intensity at ex 340 nm and ex 370 nm ( at constant em 500 nm ) through a standard curve prepared with varying concentrations of mgcl2 in 0.2 m bicine , ph 8.5 . because the counter - transport of na , required for charge neutrality , is fast relative to the transport of mg , the mg permeability coefficient was calculated by p = k*v / a , where k is the first - order rate constant , and v and a are the volume and surface area of the vesicle , respectively . rna was transcribed from double - stranded n15min7 template by t7 rna polymerase in a solution containing 0.5 mm ntps , 20 ci -p - utp , 40 mm tris - hcl , ph 7.9 , 6 mm mgcl2 , 2 mm spermidine , 10 mm dtt , and 0.2 u/l rnasin . the reaction also included 25 m of two oligonucleotides complementary to the ribozyme sequence , to block ribozyme self - cleavage during transcription ( 5-tcgcttagctcatcag-3 and 5-ggaatttactgactgc-3 ) . vesicles were disrupted by the addition of 0.3% triton x-100 , and the rna was precipitated in 0.3 m sodium acetate , ph 7 , 73% ethanol , with 10 g of glycoblue . we assessed the stability of vesicles composed of myristoleic acid ( ma ) and glycerol monomyristoleate ( gmm ) in the presence of mg by monitoring the retention of encapsulated dye and macroscopic turbidity changes . pure ma vesicles showed low mg tolerance , but vesicles of mixed composition , ma : gmm ( 2:1 ) , showed increased stability in the presence of mg , as previously reported on the basis of light microscopy . higher proportions of gmm led to the appearance of oil droplets mixed with vesicles . to determine whether vesicles exposed to relatively high concentrations of mgcl2 are only transiently destabilized by the sudden addition of mgcl2 , we followed the leakage of encapsulated calcein over time . the initial rate of dye leakage from ma : gmm ( 2:1 ) vesicles increased by a factor of 27 in the presence of 4 mm mg . h - ump showed a 4-fold permeability enhancement in the presence of 4 mm mg ( figure 1d ) . thus , exposure to mg increased the permeability of an rna mononucleotide but not of rna itself , perhaps by neutralizing the negative charge and/or stabilizing the interaction between the solute and small - scale transient defects in the membrane . even so , more than 95% of encapsulated calcein was retained in ma : gmm ( 2:1 ) vesicles for at least 3 h. this is much longer than the hammerhead ribozyme cleavage reactions described below , which were completed within a few minutes . ( a ) vesicles were initially purified away from unencapsulated calcein , and dye leakage was measured over time with ( blue ) or without ( black ) the addition of 4 mm mgcl2 . ( b ) calcein leakage assayed by size - exclusion chromatography after 22 h in the presence of 4 mm mgcl2 . ( c ) rna leakage assayed by size - exclusion chromatography after 19 h , with ( red ) or without ( black ) the addition of 4 mm mgcl2 . ( d ) vesicles encapsulating 0.1 mm 5-ump and a trace amount of h - ump were initially purified , and then leakage of h - ump was measured over time , with ( red ) or without ( black ) the addition of 4 mm mgcl2 . ( a ) vesicles were initially purified away from unencapsulated calcein , and dye leakage was measured over time with ( blue ) or without ( black ) the addition of 4 mm mgcl2 . ( b ) calcein leakage assayed by size - exclusion chromatography after 22 h in the presence of 4 mm mgcl2 . ( c ) rna leakage assayed by size - exclusion chromatography after 19 h , with ( red ) or without ( black ) the addition of 4 mm mgcl2 . ( d ) vesicles encapsulating 0.1 mm 5-ump and a trace amount of h - ump were initially purified , and then leakage of h - ump was measured over time , with ( red ) or without ( black ) the addition of 4 mm mgcl2 . because we wished to eventually measure the activity of encapsulated rna , we also determined the permeability of mg through the vesicle membrane . when we mixed phospholipid ( popc ) vesicles with mgcl2 , we saw no detectable change in the internal mg concentration over several hours ( figure 2d ) , demonstrating that equilibration of both mg and mag - fura-2 across popc membranes is quite slow . although the addition of gmm to ma vesicles increased their tolerance for mg , we suspected that the increase in the stability of the vesicles might come at the expense of dampened dynamic behaviors , such as growth , that rely on the rapid insertion and dissociation of molecules from the membrane . to determine whether mg affected the intermembrane transfer of amphiphiles , we measured the transfer between ma : gmm ( 2:1 ) vesicles ( 2 mm amphiphile ) in the presence of 3.2 mm mgcl2 ( 2 mm free mg ) . consistent with the dls data , pyrene fluorescence indicated the presence of large aggregates in ma : gmm ( 4:1 ) micelles ( figure 3a ) . ma : gmm ( 2:1 ) vesicles , which had the greatest magnesium tolerance , showed no growth when micelles of the same composition were added , either rapidly or slowly over 4 h. however , these vesicles were able to grow following the rapid addition of pure fatty acid micelles . the rapid addition of 0.5 equiv of 2:1:0.3 ma : gmm : dodecane micelles to vesicles of the same composition resulted in 20% growth , and the rapid addition of 1 equiv of micelles resulted in 40% growth ( figure 3b ) . we determined the activity of a self - cleaving hammerhead ribozyme encapsulated in ma : gmm : dodecane vesicles . the hammerhead ribozyme is widespread in nature and also arises during in vitro selection for self - cleaving rnas . this ribozyme can catalyze both cleavage and ligation of rnas , activities that may be important for simple rna - based replicating systems . furthermore , because of its small size , the hammerhead ribozyme may function robustly under error - prone replication conditions . n15min7 is an artificially evolved 62 nucleotide hammerhead ribozyme , which is cleaved into two fragments ( 11 and 51 nucleotides ) in the presence of mg . the self - cleavage reaction was initiated by adding mgcl2 ( 4 mm ) , and the extent of self - cleavage was monitored as a function of time ( figure 4a , b ) . we found that 60% of the rna was cleaved whether the ribozyme was encapsulated or free in solution . the observed first - order rate constant of the encapsulated reaction was reduced 4-fold as compared to the unencapsulated reaction at both 1 and 4 mm added mgcl2 ( table 2 ) . ( c ) size - exclusion chromatography of ma : gmm : dodecane vesicles shows that all radiolabeled n15min7 rna remained encapsulated 15 min after the addition of mgcl2.table 2hammerhead cleavage in vesicles[mgcl2 ] ( mm)kunencapsulated ( min)kencapsulated ( min ) 1 1.3 0.4 4 7.9 1.7 self - cleavage activity of the hammerhead ribozyme n15min7 . ( c ) size - exclusion chromatography of ma : gmm : dodecane vesicles shows that all radiolabeled n15min7 rna remained encapsulated 15 min after the addition of mgcl2 . hammerhead cleavage in vesicles this decreased rate of self - cleavage of the encapsulated ribozyme may be partly due to the time scale of mg permeation into gmm - ma - dodecane vesicles ( k 1 min ) , particularly in 4 mm mgcl2 . however , for the encapsulated ribozyme in 1 mm mgcl2 , the rate of mg entry is faster than the observed self - cleavage , indicating that mg entry was not the rate - limiting step in this case . to confirm that the observed self - cleavage activity reflected encapsulated ribozyme , rather than ribozyme adsorbed to the membrane , n15min7 rna after purification by size - exclusion chromatography , these preparations contained < 3% of the radioactivity of the encapsulated reaction , indicating that only a small amount of rna was adsorbed to the membrane . we also used size - exclusion chromatography to confirm that the ribozyme still co - purified with vesicles after the self - cleavage reaction ( figure 4c ) . we confirmed that the 2:1:0.3 ma : gmm : dodecane mixture formed vesicles in the presence of mg and rna by fluorescence microscopy before extrusion ( figure 5 ) . dls of these vesicles after extrusion indicated an average particle diameter of 65 nm , similar to the size of extruded vesicles made from myristoleic acid alone . furthermore , self - cleavage of n15min7 was neither catalyzed nor inhibited by the presence of vesicles alone . in the absence of mg , no self - cleavage was detected after 30 min , and the self - cleavage rate of unencapsulated n15min7 upon addition of mg was not affected by the presence of vesicles . figure 5fluorescence microscopy of 2:1:0.3 ma : gmm : dodecane vesicles containing hammerhead ribozyme in the presence of 3 mm mgcl2 , prior to extrusion . fluorescence microscopy of 2:1:0.3 ma : gmm : dodecane vesicles containing hammerhead ribozyme in the presence of 3 mm mgcl2 , prior to extrusion . for example , the [ mg]1/2 for folding and catalysis of a number of self - splicing group i introns is 4 mm . for example , the class i ligase , discovered by in vitro evolution in the presence of high concentrations of mg , has a [ mg]1/2 of 70100 mm , as does a variant that can catalyze limited rna polymerization . the discrepancy between the mg requirement of this ribozyme and the mg tolerance of the simple vesicles studied here suggests that observing ribozyme polymerase activity inside vesicles that can grow and divide may not be trivial in practice . further optimization of existing ribozymes , or isolation of new ones , may yield rna polymerases active at lower mg concentrations that could be compatible with the vesicles described here . we have shown that vesicles composed of chemically simple amphiphiles can be stable , grow , and retain contents in the presence of sufficient mg to activate encapsulated ribozymes . because these membranes , unlike phospholipid membranes , are quite permeable to mg , encapsulated ribozymes can be activated by the external addition of mg . the permeability of these simple membranes to mg means that the internal mg concentration of a population of growing and dividing vesicles could be maintained indefinitely , from generation to generation , solely by passive trans - membrane diffusion in the absence of permeases . while the addition of gmm confers stability on ma vesicles , it also causes a decrease in the yield of growth following micelle addition . this decrease could be partially offset by the addition of another simple component , dodecane . on the other hand , the yield of growth of osmotically swollen vesicles was not affected by the addition of gmm . indeed , this mode of growth may be more likely to occur in natural situations , because the source of lipid , empty vesicles , is the same physical phase as the growing vesicles , readily permitting their coexistence . many selective pressures are likely to have influenced the evolution of the phospholipid membranes of modern biology , which are much more stable to divalent cations and can maintain ph and ionic gradients for long periods . our results show that membranes made from simple amphiphiles can be stable enough to retain rna , yet dynamic enough to grow and allow the spontaneous entry of mg and mononucleotides . we are continuing to search for membrane compositions with similar dynamic properties , but greater tolerance to mg and possibly ca ( another abundant divalent cation on earth ) , as well as ribozyme polymerases active at lower mg concentrations , with the goal of observing rna - catalyzed rna synthesis within model protocell vesicles .

the most elementary cellular system capable of darwinian evolution requires two components : a self - replicating informational molecule , such as an rna polymerase ribozyme ( replicase ) , and a mechanism for spatial localization such as compartmentalization . without compartmentalization or some other mechanism for restricting the diffusion of the genetic material , a superior mutant replicase would act to propagate unrelated sequences more efficiently , while having no effect on its own fitness . the physical proximity of related sequences , which can be readily maintained by the random segregation of sequences within replicating compartments , is therefore necessary for the evolution of improved replicase activity through natural selection . membranes provide the major mechanism for compartmentalization in modern biology and also mediate the chemical fluxes between the cell and its environment . to construct the simplest possible protocell , the membrane boundary should be capable of growth and division without complex biochemical machinery and should allow the entry of ions and small substrates by passive diffusion . fatty acids are simple amphiphiles that form bilayer membrane vesicles when the solution ph is near the apparent pka of the membrane - incorporated fatty acid . clay surfaces that catalyze rna polymerization also promote the self - assembly of fatty acid micelles into vesicles , suggesting a route for enhancing rna encapsulation by colocalizing vesicles and rna . these vesicles can also exhibit a primitive form of competition for limited resources , as vesicles encapsulating a high concentration of rna grow larger at the expense of empty vesicles ; this is referred to below as the exchange pathway for growth . these protocellular behaviors are facilitated by the fast time scales of monomermicellevesicle transitions and the relatively small energetic barriers between these phases , as compared to phospholipid membranes , which do not share these dynamic properties . for example , decanoic acid vesicles aggregate at moderate concentrations of monovalent ions ( 0.2 m nacl ) , and divalent cations cause aggregation and precipitation even at quite low concentrations ( < 1.5 mm mgcl2 , or 1:65 ratio of cation to lipid ) . this is an important problem because divalent cations are often required for the formation of tertiary contacts in rna folding and also commonly participate directly in the catalytic mechanism . consequently , the activity of many ribozymes , such as the hammerhead ribozyme , is highly dependent on divalent cation concentration . we therefore sought to encapsulate catalytically active rna in simple vesicles that were stable and capable of growth . although a variety of cations can be utilized by ribozymes , we focused on mg because of its common use by ribozymes and its abundance on the earth , particularly in seawater . we characterized the growth properties , mg tolerance , and mg permeability of such mixed composition vesicles . by varying the composition of mixed vesicles , we were able to increase the tolerance of vesicles to mg and to demonstrate that mg could rapidly enter vesicles by passive diffusion . remarkably , the presence of mg increased the permeability of the membrane to small , negatively charged solutes such as nucleotides . on the other hand , vesicle growth by the exchange pathway was slowed , and vesicle growth following micelle addition was only possible after further modification of the membrane composition . finally , we encapsulated a hammerhead ribozyme in mixed vesicles and showed that the ribozyme is active under conditions consistent with vesicle stability and growth . pyrene was from fluka ( buchs sg , switzerland ) ; n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine ( nbd - pe ) , lissamine rhodamine b 1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine ( rh - dhpe ) , calcein , and mag - fura-2 ( tetrapotassium salt ) were from molecular probes ( eugene , or ) . vesicles 100 nm in diameter were prepared by resuspending 1 equiv of the oil in 0.2 m bicine buffer , ph 8.5 , to which 0.5 equiv of naoh had been added ( resulting in a final ph of 8.5 ) . vesicles labeled by the fret dyes ( nbd - pe and rh - dhpe ) were prepared by mixing the dyes with fatty acid / ester in methanol , and then removing the solvent by rotary evaporation before resuspension . the fluorescent dyes ( 1 kd ) and rna ( 19 kd ) used in these experiments elute at the low molecular weight range of the resin ( 30 kd ) , while vesicles ( 100 nm in diameter ) elute with the void volume . growth by intermembrane transfer of amphiphiles from empty vesicles to vesicles encapsulating trna was performed as described previously , with the addition of 3.2 mm mgcl2 to the reaction buffer containing 2 mm amphiphile . mg concentration was related to the ratio of the fluorescence intensity at ex 340 nm and ex 370 nm ( at constant em 500 nm ) through a standard curve prepared with varying concentrations of mgcl2 in 0.2 m bicine , ph 8.5 . because the counter - transport of na , required for charge neutrality , is fast relative to the transport of mg , the mg permeability coefficient was calculated by p = k*v / a , where k is the first - order rate constant , and v and a are the volume and surface area of the vesicle , respectively . rna was transcribed from double - stranded n15min7 template by t7 rna polymerase in a solution containing 0.5 mm ntps , 20 ci -p - utp , 40 mm tris - hcl , ph 7.9 , 6 mm mgcl2 , 2 mm spermidine , 10 mm dtt , and 0.2 u/l rnasin . we assessed the stability of vesicles composed of myristoleic acid ( ma ) and glycerol monomyristoleate ( gmm ) in the presence of mg by monitoring the retention of encapsulated dye and macroscopic turbidity changes . pure ma vesicles showed low mg tolerance , but vesicles of mixed composition , ma : gmm ( 2:1 ) , showed increased stability in the presence of mg , as previously reported on the basis of light microscopy . table 1mgcl2 tolerance of simple vesiclesma : gmm ratio[mgcl2 ] tolerated , assayed by dye leakage ( mm)[mgcl2 ] at turbidity change ( mm ) 1:0 0.5 1 4:1 2 3 2:1 4 6 dye leakage was assessed < 1 h after addition of mgcl2 , and onset of leakage defined the maximum [ mgcl2 ] tolerated . to determine whether vesicles exposed to relatively high concentrations of mgcl2 are only transiently destabilized by the sudden addition of mgcl2 , we followed the leakage of encapsulated calcein over time . to determine whether this enhanced permeability was due to large - scale membrane destabilization by mg , we encapsulated a fluorescently labeled rna decamer in vesicles exposed to 4 mm mg . thus , exposure to mg increased the permeability of an rna mononucleotide but not of rna itself , perhaps by neutralizing the negative charge and/or stabilizing the interaction between the solute and small - scale transient defects in the membrane . even so , more than 95% of encapsulated calcein was retained in ma : gmm ( 2:1 ) vesicles for at least 3 h. this is much longer than the hammerhead ribozyme cleavage reactions described below , which were completed within a few minutes . ( d ) vesicles encapsulating 0.1 mm 5-ump and a trace amount of h - ump were initially purified , and then leakage of h - ump was measured over time , with ( red ) or without ( black ) the addition of 4 mm mgcl2 . ( d ) vesicles encapsulating 0.1 mm 5-ump and a trace amount of h - ump were initially purified , and then leakage of h - ump was measured over time , with ( red ) or without ( black ) the addition of 4 mm mgcl2 . because we wished to eventually measure the activity of encapsulated rna , we also determined the permeability of mg through the vesicle membrane . using size - exclusion chromatography , we verified that < 5% of encapsulated mag - fura-2 leaked from vesicles after 30 min of exposure to 2 mm mg ( figure 2c ) , consistent with the results obtained for calcein leakage . when we mixed phospholipid ( popc ) vesicles with mgcl2 , we saw no detectable change in the internal mg concentration over several hours ( figure 2d ) , demonstrating that equilibration of both mg and mag - fura-2 across popc membranes is quite slow . although the addition of gmm to ma vesicles increased their tolerance for mg , we suspected that the increase in the stability of the vesicles might come at the expense of dampened dynamic behaviors , such as growth , that rely on the rapid insertion and dissociation of molecules from the membrane . growth of the surface area of vesicles can be monitored using nonexchanging fret dyes , whose fluorescence depends on the surface density of the dyes . to determine whether mg affected the intermembrane transfer of amphiphiles , we measured the transfer between ma : gmm ( 2:1 ) vesicles ( 2 mm amphiphile ) in the presence of 3.2 mm mgcl2 ( 2 mm free mg ) . when 0.5 equiv of pure fatty acid in micelle form is rapidly added to preformed vesicles , the surface area of preformed vesicles grows by 50% , indicating a very high yield of incorporation . ( b ) growth of 2:1:0.3 ma : gmm : dodecane vesicles over time , after addition of 1 equiv of micelles , in 0.2 m bicine , ph 8.5 , 1 mm mgcl2 . ma : gmm ( 2:1 ) vesicles , which had the greatest magnesium tolerance , showed no growth when micelles of the same composition were added , either rapidly or slowly over 4 h. however , these vesicles were able to grow following the rapid addition of pure fatty acid micelles . thus , 2:1 ma : gmm micelles ( which are also large by the pyrene excimer assay and by dls ) appeared to be unusually stable , perhaps due to the large headgroup of gmm , which may stabilize areas of high curvature in micellar aggregates and which would also provide h - bond donors at high ph . to destabilize these micelles , we added dodecane ( 9 mol % ) to act as a hydrophobic spacer , reducing the preferred curvature and decreasing the surface charge density . the rapid addition of 0.5 equiv of 2:1:0.3 ma : gmm : dodecane micelles to vesicles of the same composition resulted in 20% growth , and the rapid addition of 1 equiv of micelles resulted in 40% growth ( figure 3b ) . we determined the activity of a self - cleaving hammerhead ribozyme encapsulated in ma : gmm : dodecane vesicles . the self - cleavage reaction was initiated by adding mgcl2 ( 4 mm ) , and the extent of self - cleavage was monitored as a function of time ( figure 4a , b ) . the observed first - order rate constant of the encapsulated reaction was reduced 4-fold as compared to the unencapsulated reaction at both 1 and 4 mm added mgcl2 ( table 2 ) . as expected , the rate of the encapsulated reaction in 4 mm added mgcl2 was 5-fold higher than that in 1 mm added mgcl2 . ( b ) activity of ribozymes encapsulated in 2:1:0.3 ma : gmm : dodecane vesicles ( 15 mm amphiphile ) , in 0.2 m bicine , ph 8.5 , 4 mm mgcl2 . ( c ) size - exclusion chromatography of ma : gmm : dodecane vesicles shows that all radiolabeled n15min7 rna remained encapsulated 15 min after the addition of mgcl2.table 2hammerhead cleavage in vesicles[mgcl2 ] ( mm)kunencapsulated ( min)kencapsulated ( min ) 1 1.3 0.4 4 7.9 1.7 self - cleavage activity of the hammerhead ribozyme n15min7 . ( b ) activity of ribozymes encapsulated in 2:1:0.3 ma : gmm : dodecane vesicles ( 15 mm amphiphile ) , in 0.2 m bicine , ph 8.5 , 4 mm mgcl2 . hammerhead cleavage in vesicles this decreased rate of self - cleavage of the encapsulated ribozyme may be partly due to the time scale of mg permeation into gmm - ma - dodecane vesicles ( k 1 min ) , particularly in 4 mm mgcl2 . however , for the encapsulated ribozyme in 1 mm mgcl2 , the rate of mg entry is faster than the observed self - cleavage , indicating that mg entry was not the rate - limiting step in this case . the decrease in free mg concentration ( kd 10 mm amphiphile , considering both the inner and the outer leaflets of the bilayer ) , as determined by mag - fura-2 fluorescence , accounts for about one - half of the observed decrease in rate . to confirm that the observed self - cleavage activity reflected encapsulated ribozyme , rather than ribozyme adsorbed to the membrane , n15min7 rna after purification by size - exclusion chromatography , these preparations contained < 3% of the radioactivity of the encapsulated reaction , indicating that only a small amount of rna was adsorbed to the membrane . we also used size - exclusion chromatography to confirm that the ribozyme still co - purified with vesicles after the self - cleavage reaction ( figure 4c ) . we confirmed that the 2:1:0.3 ma : gmm : dodecane mixture formed vesicles in the presence of mg and rna by fluorescence microscopy before extrusion ( figure 5 ) . in the absence of mg , no self - cleavage was detected after 30 min , and the self - cleavage rate of unencapsulated n15min7 upon addition of mg was not affected by the presence of vesicles . the [ mg]1/2 ( concentration of mg corresponding to half - maximal activity ) of the n15 construct used in our studies is 9 mm , and hammerhead ribozymes of biological origin have even lower mg optima of < 1 mm . for example , the class i ligase , discovered by in vitro evolution in the presence of high concentrations of mg , has a [ mg]1/2 of 70100 mm , as does a variant that can catalyze limited rna polymerization . the discrepancy between the mg requirement of this ribozyme and the mg tolerance of the simple vesicles studied here suggests that observing ribozyme polymerase activity inside vesicles that can grow and divide may not be trivial in practice . we have shown that vesicles composed of chemically simple amphiphiles can be stable , grow , and retain contents in the presence of sufficient mg to activate encapsulated ribozymes . the permeability of these simple membranes to mg means that the internal mg concentration of a population of growing and dividing vesicles could be maintained indefinitely , from generation to generation , solely by passive trans - membrane diffusion in the absence of permeases . indeed , this mode of growth may be more likely to occur in natural situations , because the source of lipid , empty vesicles , is the same physical phase as the growing vesicles , readily permitting their coexistence . many selective pressures are likely to have influenced the evolution of the phospholipid membranes of modern biology , which are much more stable to divalent cations and can maintain ph and ionic gradients for long periods . however , the evolution of such membranes would have required the co - evolution of numerous ion and substrate - specific permeases , as well as the biochemical machinery for membrane growth . our results show that membranes made from simple amphiphiles can be stable enough to retain rna , yet dynamic enough to grow and allow the spontaneous entry of mg and mononucleotides . we are continuing to search for membrane compositions with similar dynamic properties , but greater tolerance to mg and possibly ca ( another abundant divalent cation on earth ) , as well as ribozyme polymerases active at lower mg concentrations , with the goal of observing rna - catalyzed rna synthesis within model protocell vesicles .

insulin resistance , the key component of type 2 diabetes mellitus ( t2 dm ) , is linked to a multitude of diseases like obesity , hypertension , coronary artery disease ( cad ) , and dyslipidemia . these diseases exert a major toll on resources across the world with wide ramifications on physical , psychological , and financial well - being . the prevalence of diabetes is projected to be 7.7% in 2030 , affecting 439 million adults . the enormity of the problem has necessitated a shift in focus from treatment of diabetes and related diseases to primary prevention . intrauterine environment provides an individual with a sneak preview of the conditions one may be exposed to during childhood growth and adult life . this information in turn leads to an adaptive response in metabolism , which provides a survival advantage . some brilliant epidemiologic studies from the latter part of the last century were instrumental in bringing out the association of body size at birth with various adult disorders including diabetes and cardiovascular disease . in this article , we have attempted to give a comprehensive review of the existing literature on developmental origins of adult disease ( doad ) . the pathophysiological effect of adverse intrauterine life on adult health was a relatively unresearched area , till epidemiological studies revealed an association between intrauterine fetal health and adult life . infants who were subjected to mid or late gestation calorie restriction had a lower birth weight and impaired glucose tolerance ( igt ) as adults . infants of mothers who endured the famine in early gestation had a normal birth weights , but had obesity and atherogenic lipid prole as adults . studies from hertfordshire , uk , and helsinki , finland , showed that poor fetal nutrition and low birth weight are related to adult diseases , namely , cad , obesity , and insulin resistance . in the leningrad siege study of the leningrad famine which extended for 800 days the leningrad cohort did not show increased incidence of diabetes , hypertension , and cad . based on the overwhelming epidemiological evidence , barker and associates proposed the barker hypothesis which suggested that an environment which produces poor fetal and infant growth is followed by an adult environment that determines high risk for ischemic heart disease . this hypothesis came to be known as fetal origins of adult disease or developmental origins of adult disease.[5710 ] low birth weight has been consistently associated with adult - onset diabetes in various published literature from the developed world and asia.[1114 ] however , a study done from mysore in south india suggested that shorter body length and higher ponderal index at birth were related to igt and type 2 diabetes at age 45 , while low birth weight was not . the birth weight in turn is dependent upon a variety of factors like maternal nutrition , parity , mother 's birth weight , mother 's adult size , and mother 's birth weight . the evolution of the human species through a process of survival of the fittest has necessitated a complex interaction between maternal and fetal genotypes . the thrifty genotype theory suggests that the relative scarcity of food during the early periods of human evolution leads to an adaptive response of enriched thrifty genes , which is conducive in a nutritionally poor environment . however , with relative easy availability of food and nutritional enrichment , this adaptation becomes counterproductive . the epidemiologic studies mentioned earlier revealed that this adaptive response is detrimental as low birth weight was associated with increased risk of obesity , dyslipidemia , hypertension , ischemic heart disease , type 2 diabetes , and polycystic ovarian syndrome ( pcos ) in adults . thrifty phenotype hypothesis suggested that in a nutritionally deprived environment , the fetus undergoes intrauterine programming and channels the nutrient resources toward the development of vital organs like brain at the expense of other organs like beta cell islets . the response to these adaptations in later life is based on an environmental mismatch with respect to the nutritional availability and requirement . the intrauterine programming in a nutritionally challenged environment provides a survival advantage in a nutrient - deficient extrauterine environment and becomes detrimental in a nutrient - rich environment . hence , there is a developmental plasticity by which a single genotype may give rise to different phenotypes by adjusting their phenotype to match the environment they have to survive in . the complex interactions of environment and nutrition with maternal and fetal genotypes influence maternal and fetal well - being . we also do know that nutrition , stress , and other environmental factors can modify the epigenome . there are excellent review articles published recently which highlight the relationship between epigenetic changes and metabolic diseases like insulin resistance , obesity , hypertension , and cardiovascular disease . twin studies have revealed that the heritability of birth weight may range from 30 to 70% . the avon longitudinal study of parents and children ( asplac ) , which was a longitudinal study of pregnancy and childhood , reported a mitochondrial variant associated with thinner babies at birth . the two key concepts to be considered while discussing the genetic factors influencing birth weight and postnatal metabolic diseases are epigenetics and genomic imprinting . epigenetics is defined as the study of heritable changes other than those in the dna sequence that encompass two major modifications of dna or chromatin namely dna methylation , the covalent modification of cytosine , and post - translational modification of histones including methylation , acetylation , phosphorylation , and sumoylation . dna methylations are instrumental in long - term silencing of gene expressions . on the other hand , gametogenesis studies reveal a differential methylation , which is before birth in the male germ line and after birth in the female germ line . in a recent article , gluckman has reviewed the genetic machinery of the epigenetic modification of gene expression . in an active gene , the cpg dinucleotides in the gene promoter area are unmethylated , which gives the transcription factors and rna polymerase a free run and exon transcription will occur [ figure 1 ] . in the presence of dna methyltransferases ( dnmts ) , methylation of cpgs occurs , which in turn leads to the binding of methyl cpg binding protein 2 . these events bring forward the histone modifying enzymes ( hmes ) , namely , the histone deacetylase and histone methyltransferase , which deacetylates the histones and methylates the lysine residues , respectively . the end result is a conformational change in the chromatin strand which prevents the action of transcription factors and rna polymerases . tf : transcription factors , p : promoter , hme : histone modifying enzymes , m : methyl group , ac : acetyl group , mcp : methyl cpg binding protein there are many animal models illustrating how specific environmental situations induce epigenetic modifications , like agouti viable yellow ( a ) mouse . hypermethylation of the gene promoter decreases agouti expression which by its effects on hypothalamus gives rise to mice with normal weight and brown coat color . agouti - related protein is an orexegenic peptide which plays an important role in hypothalamic control of feeding behavior . hypomethylation results in increased agouti gene expression and mouse develops obesity and yellow colored coat . hypomethylation occurs with maternal deficiency of methyl donors and cofactors like folate , while hypermethylation can be induced by maternal intake of soy isoflavone genistein . in another animal model low - protein diet induced promoter hypomethylation of peroxisome proliferator - activated receptor ( ppar) and was associated with decreased methylation of cpg dinucleotides . this modification resulted in overexpression of ppar receptors in the offspring . in the adipose tissue ppar and receptors are involved in fatty acid oxidation and regulation of insulin sensitivity . these epigenetic modifications can result in increased triglycerides and nonesterified fatty acids in offspring , and finally insulin resistance . histone modifications and promoter hypomethylation result in overexpression of the hepatic glucocorticoid receptors . in the liver , increased glucocorticoid activity can induce phosphoenol pyruvate kinase expression which induces gluconeogenesis . the pancreatic growth factor insulin - like growth factor 2 ( igf2 ) was shown to be reduced in neonatal rats when mothers were exposed to low - protein diet . in fact , taurine deficiency was also proposed to play a role .. low - protein diet also reduced the methylation of the promoter gene of type 1b adrenal angiotensin receptor and in turn overexpression of the receptor . constellations of these changes are known to produce hypertension and endothelial dysfunction in the offspring . in a recent article in normal pancreatic cells , the promoter for pdx1 is in an unmethylated open chromatin state , which in turn promotes usf-1 and is associated with acetylated h3 , h4 , and trimethylated h3k4 . in iugr islets , hdac1dnmt1 repressor complex is recruited with gradual loss of histone acetylation and trimethylated h3k4 . on the other hand , dimethylated h3k9 starts appearing from birth . when one looks at the iugr adult islets , there is an inactive chromatin with dimethylated h3k9 and high dna methylation which is not conducive for transcription and hence pdx gene is silenced . a key initiator in this methylation process could possibly be the oxidative stress found in iugr . iugr increases mef2d binding to glut-4 promoter which is an inhibitor and decreases mef2a and myod ( activator and co - activator , respectively ) binding to the glut-4 promoter in skeletal muscle tissue , which in turn promotes insulin resistance . placental insufficiency can decrease the production of pancreatic transcription factor product of pdx1 by cpg methylation in animals and they can develop diabetes at a later date . the role of genomic imprinting in birth weight can be understood by studying two fetal growth disorders associated with 11p15 h19/igf2 imprinted domain . methylation of this imprinting region can result in silencing of h19 and the expression of igf2 , and if methylation does not occur , silencing of igf2 and the expression of h19 is the result . normally methylation occurs in the male germ line and is prevented in the female germ line . absent dna methylation in the paternal allele leads to biallelic expression of h19 with silencing of igf2 , which in turn results in iugr and russell silver syndrome . on the other hand , a gain of dna methylation on the maternal allele results in silencing of h19 and biallelic expression of igf2 with fetal overgrowth and beckwith wiedemann syndrome . wiedemann is more commonly seen associated with assisted reproductive technology as a result of epigenetic changes . epidemiological studies have recorded a subnormal mitochondrial function in patients with hypertension and diabetes . since mitochondrial dna is inherited from the mother , it may be a player that determines the transfer of maternal nutritional stress to fetus . reactive oxygen species ( ros ) can induce several intramitochondrial changes like shutting down of mitochondrial energy production and various other cellular changes . the antioxidant machinery of cell is suboptimal , which makes it more prone for free radical induced injury . complexes i and iii of the electron transport chain are shown to function below par . iugr is associated with decreased mitochondrial gene content and progressive damage of the mitochondrial dna content with free radicals . these dysfunctions can manifest functionally as decreased glucose - mediated insulin secretion and cell death , with diabetes as the end result . human studies , though few , are also remarkable in showing the effect of nutritional modification of epigenome . in a study by yajnik et al . , increased insulin resistance was observed in children whose mothers were vitamin b12 deficient with folate sufficiency . in fact , folate levels during pregnancy were positively associated with child 's adiposity and insulin resistance , while vitamin b12 levels negatively correlated with the same . these findings demonstrate the power of methyl donors and their cofactors to change the epigenome and ultimately affect the adult phenotype . in subjects with hyperhomocysteinemia , s - adenosylhomocysteine , this leads to lower levels of dna methylation and a shift from monoallelic to biallelic expression for some imprinted genes ( including h19 ) . there is evidence that tells us that the effect of nutritional modifications can persist through a number of generations despite nutritional correctional measures . india is rapidly undergoing a demographic , economical , and health transition as it is advancing through the 21 century . there is a reduction in the crude death rate , birth rate , infant mortality rate and there is an increase in the population of age group between 15 and 59 years . there is also a steady increase in per capita income , with reduction of poverty . the rural urban migration has resulted in a steady growth of urban population and creation of megacities . the maternal and perinatal problems in india are still high and non - communicable diseases like diabetes and cardiovascular disease are rapidly increasing . over the years rural populations consume more cereals , fewer pulses , and less oil , fat , and sugars , compared with urban populations . with the advent of fast food operators with high - energy density food and sedentary lifestyle in urban population , osmani and sen have discussed the hidden penalties of gender inequality on fetal origins of adult disease in an excellent review . in the developing countries and especially in india , gender inequality influences birth rate , mortality , and male female ratio which is steeped against female gender . the world is moving through an epidemiological transition by which mortality and morbidity through infectious diseases are decreasing while the mortality and morbidity due to non - infectious diseases are slowly increasing . in india , there is a paradox and the population suffers from high incidence of infectious and non - communicable diseases . statistical evidence shows that though girls and boys have similar nutrition at birth , as they grow up , an inequality creeps in with girls falling behind boys . as these girls reach the reproductive age group , they suffer from undernourishment and anemia . multiparity and relatively young age at the time of marriage also contribute to the low birth weight of children born to these mothers . if undernutrition persists during childhood , it puts the patient at high risk for infectious diseases . however , the economic and social transition combined with relative nutritional affluence during childhood puts these patients at risk for obesity , cad , and insulin resistance , as adults . various studies across the globe have shown that excessive postnatal weight gain can have additional adverse influences on adult health . considerable body of evidence has come from studying and following up children who were born short for gestational age ( sga ) . most of these children exhibit good catch - up growth in the initial few years of life , while some of them have a smaller degree of catch - up growth and remain short . sga children have elevated levels of growth hormone , insulin like growth factor binding proteins 1 and 2 ( igfbp1 , and igfbp2 ) , with low levels of igf-1 and igfbp3 , which is a pattern consistent with growth hormone insensitivity and insulin resistance.[5658 ] in most of these children , growth hormone dynamics normalizes in the initial few months of life . sga children who have weight gain are at risk of developing insulin resistance , pcos , premature adrenarche during adolescent period , and hypertension , diabetes , and cardiovascular disease in adulthood . the abdominal adipose tissue of these infants shows resistance to the actions of insulin and catecholamine hyperresponsiveness . low birth weight may be associated with decreased number of glomeruli , which when exposed to hyperfiltration associated with weight gain can accelerate glomerulosclerosis and contribute to hypertension . pro12ala polymorphism of the ppar2 gene has been shown to increase the risk of type 2 diabetes in iugr patients . it is important to note that those infants who do not have significant catch - up growth are not at risk for most of these diseases . in the pune maternal nutrition study , indian babies were found to be lighter , shorter , and thinner , compared to british babies . however , there was a relative preservation of subscapular fat and a paucity of non - adipose tissue . in another study by the same group , the cord leptin concentrations in the british babies and indian babies were found to be similar despite the fact that indian babies were thinner and lighter . these findings suggest a higher adiposity in the indian babies , paving way for the concept of thin fat indian baby . yajnik and group also studied body size , glucose tolerance , insulin resistance , and cardiovascular risk factors in children . at 4 years of age , glucose , insulin , and igf-1 levels were inversely related to birth weight after considering the effect of current weight . however , they also found that growth velocity of children from 4 to 8 years was a stronger predictor of insulin resistance and cardiovascular risk than measurements at 8 years . these concepts become important in children undergoing rural to urban migration , adoption , and those stepping up on the economic and affluence ladder , which ultimately increases nutritional availability . although majority of the evidence points out insulin resistance as the primary pathology behind diabetes in children with iugr , some evidence also points toward decreased insulin secretion . studies have thrown mixed results with some showing an association between low birth weight and defective insulin secretion while others not showing a similar association . an earlier study reported that the pancreatic cell number was reduced in infants with iugr . in an interesting experiment , insulin secretion and insulin sensitivity of young men , who were small for gestational age , although insulin sensitivity was normal , insulin secretion was decreased by 30% when controlled for insulin sensitivity . hence , it is also possible that there is an insulin secretory defect compounded by insulin resistance , which comes into play when they accumulate body fat . the adaptational response in a fetus to a nutritionally challenging environment is preservation of its vital organ function , namely the brain , at the expense of other organs in the body . the mechanisms by which the fetus achieves this include blood and nutritional diversion to the brain , along with insulin and growth hormone resistance in the periphery . if energy is stored when bmr is low , there is a relative preference to fat storage . the insulin resistance associated with reduced lean muscle mass along with hyperinsulinemia directs the excess glucose toward fat storage in adipose tissue.[7779 ] though fat can act as an energy reservoir for vital organ functions , in a nutritionally rich environment with limited energy expenditure , fat deposition occurs . this fat deposition promotes insulin resistance , inflammation , and finally diabetes and coronary vascular disease . haguenau cohort of sga and appropriate for gestational age ( aga ) subjects looked at metabolic syndrome components at 22 years of age . metabolic syndrome was six times more common in sga individuals when compared to aga individuals . last few decades have shown a high activity in decoding molecular and cellular mechanisms underlying doad . the hormonal programming concept points out that an adverse intrauterine environment leads to metabolic and endocrine adaptations programmed toward energy conservation . these adaptations prove counterproductive in adulthood as the body finds it difficult to adjust to a nutritionally enriched environment and excess nutrients become deposited as adipose tissue . abnormalities in gh - igf axis have already been described in an earlier part of the article . experimental studies in animals have shown that those that are born small are prone to adult - onset hyperglycemia , blood pressure , anxiety , and increased hypothalamic pituitary adrenal ( hpa ) axis activity . however , the hpa axis activity may vary depending on the sex of the fetus , and time and nature of the stimulus . placental corticotropin - releasing hormone ( crh ) , sympathoadrenal system , and glucocorticoid and mineralocorticoid receptors in the hippocampus and other areas of brain are supposed to play a role in the programming of hpa axis . one of the key regulators of fetal cortisol levels is placental 11 beta hydroxy steroid dehydrogenase 2 ( 11hsd2 ) enzyme which converts cortisol to cortisone and keeps fetal cortisol levels at a lower level . high morning cortisol levels are seen in adults who are born as preterm or with low normal gestational age . it is interesting that different , but normal environmental changes can result in individual variations of hpa axis to stress . studies using stimulated cortisol have shown an association between low birth weight and hyperactive hpa axis , with adult cardiovascular disease , glucose intolerance , and higher systolic and diastolic pressures.[9395 ] recent researches have brought out an exciting interplay of adipocytokines and subsequent development of adult disease in iugr . subsequently , when they progress to childhood and adulthood , the leptin levels are higher compared to normal cohort . this leptin resistance may be an adaptive response which may serve as a stimulus for food intake and promote catch - up growth . studies examining adiponectin levels have failed to show any significant differences between iugr neonates and normal cohort , although conflicting reports exist.[98100 ] however , sga infants show a shift to a higher molecular form adiponectin , which is more related to insulin sensitivity . the idea behind this shift is an enhancement of insulin sensitivity and promotion of neonatal catch - up growth . ghrelin levels are elevated in iugr , which again may help in postnatal catch - up growth . other adipocytokines under active investigation for their role in iugr and subsequent adult - onset adiposity and insulin resistance include tumor necrosis factor - alpha ( tnf- ) , interleukin-6 ( il-6 ) , resistin , apelin , and visfatin . current evidence points toward the fact that maternal diabetes predisposes to adult - onset obesity and diabetes in the fetus even after other influencing factors are adjusted for . evidence for the influence of intrauterine metabolic derangements secondary to maternal diabetes comes from the study on pima indians . children born to mothers who were diabetic at the time of pregnancy were found to be more obese than their siblings who were born before the onset of diabetes . it was also seen that diabetes was more common in children who were born to mothers who had diabetes at the time of pregnancy when compared to infants who were born to mothers who did not have diabetes at the time of pregnancy , though they developed diabetes subsequently . infants with high birth weight are also prone to develop obesity and insulin resistance later in life . a study by yajnik et al . showed that higher birth weight in children was associated with adiposity in the parents and metabolic syndrome in mother 8 years after the child birth . even obese women with normal glucose tolerance were shown to beget babies who had increased percentage of body fat . the metabolic abnormalities that are present in the obese and diabetic mothers are transferred to the fetus during intrauterine period , conferring a risk of subsequent obesity . studies have shown that the levels of endothelial progenitor cells are reduced in neonates born to mothers with type 1 diabetes mellitus . endothelial progenitor cells play an important role in vascular repair and neovascular formation , and endothelial dysfunction plays a pivotal role in the development of cardiovascular diseases . existing literature gives us some insight on islet cell function in an environment of hypercholesterolemia . hypercholesterolemia has been shown to induce islet cell apoptosis and glucokinase downregulation , resulting in decreased insulin secretion . further studies are required to highlight the effects of hypercholesterolemia and a nutrient - rich environment on fetal insulin secretion . the alarming rise in diabetes and other communicable diseases has necessitated a focus shift from treating the non - communicable diseases to their prevention . the mysteries behind these intrauterine origins are solved by current understanding of epigenetic modifications and hormonal adaptations . the future research on fetal origins of adult disease needs to be focused on expanding our current knowledge on molecular mechanisms . ultimately , the horizon may open up preventive nutritional modifications with genomic and hormonal targets for disease prevention .

there is considerable evidence for the fact that early life environment in human beings are associated with future development of various metabolic diseases . fetal programming and perinatal events appear to exert effects on later life that are independent of environmental risk factors in adults . our understanding of the underlying mechanisms are limited and remains unclear . however several animal models and epidemiological studies have shown this association , and it is assumed secondary to the penalties of developmental plasticity . in this review , we amalgamate facts from several disciplines to support this hypothesis .

B E G S I R M C

insulin resistance , the key component of type 2 diabetes mellitus ( t2 dm ) , is linked to a multitude of diseases like obesity , hypertension , coronary artery disease ( cad ) , and dyslipidemia . these diseases exert a major toll on resources across the world with wide ramifications on physical , psychological , and financial well - being . the prevalence of diabetes is projected to be 7.7% in 2030 , affecting 439 million adults . the enormity of the problem has necessitated a shift in focus from treatment of diabetes and related diseases to primary prevention . intrauterine environment provides an individual with a sneak preview of the conditions one may be exposed to during childhood growth and adult life . some brilliant epidemiologic studies from the latter part of the last century were instrumental in bringing out the association of body size at birth with various adult disorders including diabetes and cardiovascular disease . in this article , we have attempted to give a comprehensive review of the existing literature on developmental origins of adult disease ( doad ) . the pathophysiological effect of adverse intrauterine life on adult health was a relatively unresearched area , till epidemiological studies revealed an association between intrauterine fetal health and adult life . infants who were subjected to mid or late gestation calorie restriction had a lower birth weight and impaired glucose tolerance ( igt ) as adults . studies from hertfordshire , uk , and helsinki , finland , showed that poor fetal nutrition and low birth weight are related to adult diseases , namely , cad , obesity , and insulin resistance . in the leningrad siege study of the leningrad famine which extended for 800 days the leningrad cohort did not show increased incidence of diabetes , hypertension , and cad . based on the overwhelming epidemiological evidence , barker and associates proposed the barker hypothesis which suggested that an environment which produces poor fetal and infant growth is followed by an adult environment that determines high risk for ischemic heart disease . this hypothesis came to be known as fetal origins of adult disease or developmental origins of adult disease. [5710 ] low birth weight has been consistently associated with adult - onset diabetes in various published literature from the developed world and asia. the birth weight in turn is dependent upon a variety of factors like maternal nutrition , parity , mother 's birth weight , mother 's adult size , and mother 's birth weight . the evolution of the human species through a process of survival of the fittest has necessitated a complex interaction between maternal and fetal genotypes . the thrifty genotype theory suggests that the relative scarcity of food during the early periods of human evolution leads to an adaptive response of enriched thrifty genes , which is conducive in a nutritionally poor environment . however , with relative easy availability of food and nutritional enrichment , this adaptation becomes counterproductive . the epidemiologic studies mentioned earlier revealed that this adaptive response is detrimental as low birth weight was associated with increased risk of obesity , dyslipidemia , hypertension , ischemic heart disease , type 2 diabetes , and polycystic ovarian syndrome ( pcos ) in adults . thrifty phenotype hypothesis suggested that in a nutritionally deprived environment , the fetus undergoes intrauterine programming and channels the nutrient resources toward the development of vital organs like brain at the expense of other organs like beta cell islets . the response to these adaptations in later life is based on an environmental mismatch with respect to the nutritional availability and requirement . the intrauterine programming in a nutritionally challenged environment provides a survival advantage in a nutrient - deficient extrauterine environment and becomes detrimental in a nutrient - rich environment . hence , there is a developmental plasticity by which a single genotype may give rise to different phenotypes by adjusting their phenotype to match the environment they have to survive in . the complex interactions of environment and nutrition with maternal and fetal genotypes influence maternal and fetal well - being . we also do know that nutrition , stress , and other environmental factors can modify the epigenome . there are excellent review articles published recently which highlight the relationship between epigenetic changes and metabolic diseases like insulin resistance , obesity , hypertension , and cardiovascular disease . twin studies have revealed that the heritability of birth weight may range from 30 to 70% . the avon longitudinal study of parents and children ( asplac ) , which was a longitudinal study of pregnancy and childhood , reported a mitochondrial variant associated with thinner babies at birth . the two key concepts to be considered while discussing the genetic factors influencing birth weight and postnatal metabolic diseases are epigenetics and genomic imprinting . epigenetics is defined as the study of heritable changes other than those in the dna sequence that encompass two major modifications of dna or chromatin namely dna methylation , the covalent modification of cytosine , and post - translational modification of histones including methylation , acetylation , phosphorylation , and sumoylation . dna methylations are instrumental in long - term silencing of gene expressions . in a recent article , gluckman has reviewed the genetic machinery of the epigenetic modification of gene expression . in an active gene , the cpg dinucleotides in the gene promoter area are unmethylated , which gives the transcription factors and rna polymerase a free run and exon transcription will occur [ figure 1 ] . in the presence of dna methyltransferases ( dnmts ) , methylation of cpgs occurs , which in turn leads to the binding of methyl cpg binding protein 2 . these events bring forward the histone modifying enzymes ( hmes ) , namely , the histone deacetylase and histone methyltransferase , which deacetylates the histones and methylates the lysine residues , respectively . tf : transcription factors , p : promoter , hme : histone modifying enzymes , m : methyl group , ac : acetyl group , mcp : methyl cpg binding protein there are many animal models illustrating how specific environmental situations induce epigenetic modifications , like agouti viable yellow ( a ) mouse . hypermethylation of the gene promoter decreases agouti expression which by its effects on hypothalamus gives rise to mice with normal weight and brown coat color . agouti - related protein is an orexegenic peptide which plays an important role in hypothalamic control of feeding behavior . in another animal model low - protein diet induced promoter hypomethylation of peroxisome proliferator - activated receptor ( ppar) and was associated with decreased methylation of cpg dinucleotides . these epigenetic modifications can result in increased triglycerides and nonesterified fatty acids in offspring , and finally insulin resistance . histone modifications and promoter hypomethylation result in overexpression of the hepatic glucocorticoid receptors . in the liver , increased glucocorticoid activity can induce phosphoenol pyruvate kinase expression which induces gluconeogenesis . the pancreatic growth factor insulin - like growth factor 2 ( igf2 ) was shown to be reduced in neonatal rats when mothers were exposed to low - protein diet . in fact , taurine deficiency was also proposed to play a role .. low - protein diet also reduced the methylation of the promoter gene of type 1b adrenal angiotensin receptor and in turn overexpression of the receptor . in a recent article in normal pancreatic cells , the promoter for pdx1 is in an unmethylated open chromatin state , which in turn promotes usf-1 and is associated with acetylated h3 , h4 , and trimethylated h3k4 . in iugr islets , hdac1dnmt1 repressor complex is recruited with gradual loss of histone acetylation and trimethylated h3k4 . on the other hand , dimethylated h3k9 starts appearing from birth . when one looks at the iugr adult islets , there is an inactive chromatin with dimethylated h3k9 and high dna methylation which is not conducive for transcription and hence pdx gene is silenced . a key initiator in this methylation process could possibly be the oxidative stress found in iugr . iugr increases mef2d binding to glut-4 promoter which is an inhibitor and decreases mef2a and myod ( activator and co - activator , respectively ) binding to the glut-4 promoter in skeletal muscle tissue , which in turn promotes insulin resistance . the role of genomic imprinting in birth weight can be understood by studying two fetal growth disorders associated with 11p15 h19/igf2 imprinted domain . methylation of this imprinting region can result in silencing of h19 and the expression of igf2 , and if methylation does not occur , silencing of igf2 and the expression of h19 is the result . normally methylation occurs in the male germ line and is prevented in the female germ line . on the other hand , a gain of dna methylation on the maternal allele results in silencing of h19 and biallelic expression of igf2 with fetal overgrowth and beckwith wiedemann syndrome . wiedemann is more commonly seen associated with assisted reproductive technology as a result of epigenetic changes . epidemiological studies have recorded a subnormal mitochondrial function in patients with hypertension and diabetes . reactive oxygen species ( ros ) can induce several intramitochondrial changes like shutting down of mitochondrial energy production and various other cellular changes . complexes i and iii of the electron transport chain are shown to function below par . iugr is associated with decreased mitochondrial gene content and progressive damage of the mitochondrial dna content with free radicals . these dysfunctions can manifest functionally as decreased glucose - mediated insulin secretion and cell death , with diabetes as the end result . , increased insulin resistance was observed in children whose mothers were vitamin b12 deficient with folate sufficiency . in fact , folate levels during pregnancy were positively associated with child 's adiposity and insulin resistance , while vitamin b12 levels negatively correlated with the same . in subjects with hyperhomocysteinemia , s - adenosylhomocysteine , this leads to lower levels of dna methylation and a shift from monoallelic to biallelic expression for some imprinted genes ( including h19 ) . there is evidence that tells us that the effect of nutritional modifications can persist through a number of generations despite nutritional correctional measures . india is rapidly undergoing a demographic , economical , and health transition as it is advancing through the 21 century . there is a reduction in the crude death rate , birth rate , infant mortality rate and there is an increase in the population of age group between 15 and 59 years . there is also a steady increase in per capita income , with reduction of poverty . the rural urban migration has resulted in a steady growth of urban population and creation of megacities . the maternal and perinatal problems in india are still high and non - communicable diseases like diabetes and cardiovascular disease are rapidly increasing . over the years rural populations consume more cereals , fewer pulses , and less oil , fat , and sugars , compared with urban populations . with the advent of fast food operators with high - energy density food and sedentary lifestyle in urban population , osmani and sen have discussed the hidden penalties of gender inequality on fetal origins of adult disease in an excellent review . in the developing countries and especially in india , gender inequality influences birth rate , mortality , and male female ratio which is steeped against female gender . in india , there is a paradox and the population suffers from high incidence of infectious and non - communicable diseases . statistical evidence shows that though girls and boys have similar nutrition at birth , as they grow up , an inequality creeps in with girls falling behind boys . multiparity and relatively young age at the time of marriage also contribute to the low birth weight of children born to these mothers . if undernutrition persists during childhood , it puts the patient at high risk for infectious diseases . however , the economic and social transition combined with relative nutritional affluence during childhood puts these patients at risk for obesity , cad , and insulin resistance , as adults . various studies across the globe have shown that excessive postnatal weight gain can have additional adverse influences on adult health . sga children have elevated levels of growth hormone , insulin like growth factor binding proteins 1 and 2 ( igfbp1 , and igfbp2 ) , with low levels of igf-1 and igfbp3 , which is a pattern consistent with growth hormone insensitivity and insulin resistance. [5658 ] in most of these children , growth hormone dynamics normalizes in the initial few months of life . sga children who have weight gain are at risk of developing insulin resistance , pcos , premature adrenarche during adolescent period , and hypertension , diabetes , and cardiovascular disease in adulthood . the abdominal adipose tissue of these infants shows resistance to the actions of insulin and catecholamine hyperresponsiveness . low birth weight may be associated with decreased number of glomeruli , which when exposed to hyperfiltration associated with weight gain can accelerate glomerulosclerosis and contribute to hypertension . pro12ala polymorphism of the ppar2 gene has been shown to increase the risk of type 2 diabetes in iugr patients . it is important to note that those infants who do not have significant catch - up growth are not at risk for most of these diseases . in the pune maternal nutrition study , indian babies were found to be lighter , shorter , and thinner , compared to british babies . however , there was a relative preservation of subscapular fat and a paucity of non - adipose tissue . in another study by the same group , the cord leptin concentrations in the british babies and indian babies were found to be similar despite the fact that indian babies were thinner and lighter . these findings suggest a higher adiposity in the indian babies , paving way for the concept of thin fat indian baby . yajnik and group also studied body size , glucose tolerance , insulin resistance , and cardiovascular risk factors in children . at 4 years of age , glucose , insulin , and igf-1 levels were inversely related to birth weight after considering the effect of current weight . however , they also found that growth velocity of children from 4 to 8 years was a stronger predictor of insulin resistance and cardiovascular risk than measurements at 8 years . these concepts become important in children undergoing rural to urban migration , adoption , and those stepping up on the economic and affluence ladder , which ultimately increases nutritional availability . although majority of the evidence points out insulin resistance as the primary pathology behind diabetes in children with iugr , some evidence also points toward decreased insulin secretion . studies have thrown mixed results with some showing an association between low birth weight and defective insulin secretion while others not showing a similar association . in an interesting experiment , insulin secretion and insulin sensitivity of young men , who were small for gestational age , although insulin sensitivity was normal , insulin secretion was decreased by 30% when controlled for insulin sensitivity . hence , it is also possible that there is an insulin secretory defect compounded by insulin resistance , which comes into play when they accumulate body fat . the adaptational response in a fetus to a nutritionally challenging environment is preservation of its vital organ function , namely the brain , at the expense of other organs in the body . the mechanisms by which the fetus achieves this include blood and nutritional diversion to the brain , along with insulin and growth hormone resistance in the periphery . if energy is stored when bmr is low , there is a relative preference to fat storage . the insulin resistance associated with reduced lean muscle mass along with hyperinsulinemia directs the excess glucose toward fat storage in adipose tissue. [7779 ] though fat can act as an energy reservoir for vital organ functions , in a nutritionally rich environment with limited energy expenditure , fat deposition occurs . this fat deposition promotes insulin resistance , inflammation , and finally diabetes and coronary vascular disease . haguenau cohort of sga and appropriate for gestational age ( aga ) subjects looked at metabolic syndrome components at 22 years of age . last few decades have shown a high activity in decoding molecular and cellular mechanisms underlying doad . abnormalities in gh - igf axis have already been described in an earlier part of the article . experimental studies in animals have shown that those that are born small are prone to adult - onset hyperglycemia , blood pressure , anxiety , and increased hypothalamic pituitary adrenal ( hpa ) axis activity . however , the hpa axis activity may vary depending on the sex of the fetus , and time and nature of the stimulus . placental corticotropin - releasing hormone ( crh ) , sympathoadrenal system , and glucocorticoid and mineralocorticoid receptors in the hippocampus and other areas of brain are supposed to play a role in the programming of hpa axis . one of the key regulators of fetal cortisol levels is placental 11 beta hydroxy steroid dehydrogenase 2 ( 11hsd2 ) enzyme which converts cortisol to cortisone and keeps fetal cortisol levels at a lower level . high morning cortisol levels are seen in adults who are born as preterm or with low normal gestational age . it is interesting that different , but normal environmental changes can result in individual variations of hpa axis to stress . studies using stimulated cortisol have shown an association between low birth weight and hyperactive hpa axis , with adult cardiovascular disease , glucose intolerance , and higher systolic and diastolic pressures. [9395 ] recent researches have brought out an exciting interplay of adipocytokines and subsequent development of adult disease in iugr . this leptin resistance may be an adaptive response which may serve as a stimulus for food intake and promote catch - up growth . studies examining adiponectin levels have failed to show any significant differences between iugr neonates and normal cohort , although conflicting reports exist. the idea behind this shift is an enhancement of insulin sensitivity and promotion of neonatal catch - up growth . other adipocytokines under active investigation for their role in iugr and subsequent adult - onset adiposity and insulin resistance include tumor necrosis factor - alpha ( tnf- ) , interleukin-6 ( il-6 ) , resistin , apelin , and visfatin . current evidence points toward the fact that maternal diabetes predisposes to adult - onset obesity and diabetes in the fetus even after other influencing factors are adjusted for . evidence for the influence of intrauterine metabolic derangements secondary to maternal diabetes comes from the study on pima indians . children born to mothers who were diabetic at the time of pregnancy were found to be more obese than their siblings who were born before the onset of diabetes . infants with high birth weight are also prone to develop obesity and insulin resistance later in life . showed that higher birth weight in children was associated with adiposity in the parents and metabolic syndrome in mother 8 years after the child birth . the metabolic abnormalities that are present in the obese and diabetic mothers are transferred to the fetus during intrauterine period , conferring a risk of subsequent obesity . studies have shown that the levels of endothelial progenitor cells are reduced in neonates born to mothers with type 1 diabetes mellitus . endothelial progenitor cells play an important role in vascular repair and neovascular formation , and endothelial dysfunction plays a pivotal role in the development of cardiovascular diseases . hypercholesterolemia has been shown to induce islet cell apoptosis and glucokinase downregulation , resulting in decreased insulin secretion . the mysteries behind these intrauterine origins are solved by current understanding of epigenetic modifications and hormonal adaptations . the future research on fetal origins of adult disease needs to be focused on expanding our current knowledge on molecular mechanisms . ultimately , the horizon may open up preventive nutritional modifications with genomic and hormonal targets for disease prevention .

insulin resistance , the key component of type 2 diabetes mellitus ( t2 dm ) , is linked to a multitude of diseases like obesity , hypertension , coronary artery disease ( cad ) , and dyslipidemia . these diseases exert a major toll on resources across the world with wide ramifications on physical , psychological , and financial well - being . the prevalence of diabetes is projected to be 7.7% in 2030 , affecting 439 million adults . the enormity of the problem has necessitated a shift in focus from treatment of diabetes and related diseases to primary prevention . intrauterine environment provides an individual with a sneak preview of the conditions one may be exposed to during childhood growth and adult life . some brilliant epidemiologic studies from the latter part of the last century were instrumental in bringing out the association of body size at birth with various adult disorders including diabetes and cardiovascular disease . in this article , we have attempted to give a comprehensive review of the existing literature on developmental origins of adult disease ( doad ) . the pathophysiological effect of adverse intrauterine life on adult health was a relatively unresearched area , till epidemiological studies revealed an association between intrauterine fetal health and adult life . infants who were subjected to mid or late gestation calorie restriction had a lower birth weight and impaired glucose tolerance ( igt ) as adults . infants of mothers who endured the famine in early gestation had a normal birth weights , but had obesity and atherogenic lipid prole as adults . studies from hertfordshire , uk , and helsinki , finland , showed that poor fetal nutrition and low birth weight are related to adult diseases , namely , cad , obesity , and insulin resistance . in the leningrad siege study of the leningrad famine which extended for 800 days the leningrad cohort did not show increased incidence of diabetes , hypertension , and cad . based on the overwhelming epidemiological evidence , barker and associates proposed the barker hypothesis which suggested that an environment which produces poor fetal and infant growth is followed by an adult environment that determines high risk for ischemic heart disease . this hypothesis came to be known as fetal origins of adult disease or developmental origins of adult disease. [5710 ] low birth weight has been consistently associated with adult - onset diabetes in various published literature from the developed world and asia. [1114 ] however , a study done from mysore in south india suggested that shorter body length and higher ponderal index at birth were related to igt and type 2 diabetes at age 45 , while low birth weight was not . the birth weight in turn is dependent upon a variety of factors like maternal nutrition , parity , mother 's birth weight , mother 's adult size , and mother 's birth weight . the evolution of the human species through a process of survival of the fittest has necessitated a complex interaction between maternal and fetal genotypes . the thrifty genotype theory suggests that the relative scarcity of food during the early periods of human evolution leads to an adaptive response of enriched thrifty genes , which is conducive in a nutritionally poor environment . however , with relative easy availability of food and nutritional enrichment , this adaptation becomes counterproductive . the epidemiologic studies mentioned earlier revealed that this adaptive response is detrimental as low birth weight was associated with increased risk of obesity , dyslipidemia , hypertension , ischemic heart disease , type 2 diabetes , and polycystic ovarian syndrome ( pcos ) in adults . thrifty phenotype hypothesis suggested that in a nutritionally deprived environment , the fetus undergoes intrauterine programming and channels the nutrient resources toward the development of vital organs like brain at the expense of other organs like beta cell islets . the response to these adaptations in later life is based on an environmental mismatch with respect to the nutritional availability and requirement . the intrauterine programming in a nutritionally challenged environment provides a survival advantage in a nutrient - deficient extrauterine environment and becomes detrimental in a nutrient - rich environment . hence , there is a developmental plasticity by which a single genotype may give rise to different phenotypes by adjusting their phenotype to match the environment they have to survive in . the complex interactions of environment and nutrition with maternal and fetal genotypes influence maternal and fetal well - being . we also do know that nutrition , stress , and other environmental factors can modify the epigenome . there are excellent review articles published recently which highlight the relationship between epigenetic changes and metabolic diseases like insulin resistance , obesity , hypertension , and cardiovascular disease . twin studies have revealed that the heritability of birth weight may range from 30 to 70% . the avon longitudinal study of parents and children ( asplac ) , which was a longitudinal study of pregnancy and childhood , reported a mitochondrial variant associated with thinner babies at birth . the two key concepts to be considered while discussing the genetic factors influencing birth weight and postnatal metabolic diseases are epigenetics and genomic imprinting . epigenetics is defined as the study of heritable changes other than those in the dna sequence that encompass two major modifications of dna or chromatin namely dna methylation , the covalent modification of cytosine , and post - translational modification of histones including methylation , acetylation , phosphorylation , and sumoylation . dna methylations are instrumental in long - term silencing of gene expressions . on the other hand , gametogenesis studies reveal a differential methylation , which is before birth in the male germ line and after birth in the female germ line . in a recent article , gluckman has reviewed the genetic machinery of the epigenetic modification of gene expression . in an active gene , the cpg dinucleotides in the gene promoter area are unmethylated , which gives the transcription factors and rna polymerase a free run and exon transcription will occur [ figure 1 ] . in the presence of dna methyltransferases ( dnmts ) , methylation of cpgs occurs , which in turn leads to the binding of methyl cpg binding protein 2 . these events bring forward the histone modifying enzymes ( hmes ) , namely , the histone deacetylase and histone methyltransferase , which deacetylates the histones and methylates the lysine residues , respectively . the end result is a conformational change in the chromatin strand which prevents the action of transcription factors and rna polymerases . tf : transcription factors , p : promoter , hme : histone modifying enzymes , m : methyl group , ac : acetyl group , mcp : methyl cpg binding protein there are many animal models illustrating how specific environmental situations induce epigenetic modifications , like agouti viable yellow ( a ) mouse . hypermethylation of the gene promoter decreases agouti expression which by its effects on hypothalamus gives rise to mice with normal weight and brown coat color . agouti - related protein is an orexegenic peptide which plays an important role in hypothalamic control of feeding behavior . hypomethylation occurs with maternal deficiency of methyl donors and cofactors like folate , while hypermethylation can be induced by maternal intake of soy isoflavone genistein . in another animal model low - protein diet induced promoter hypomethylation of peroxisome proliferator - activated receptor ( ppar) and was associated with decreased methylation of cpg dinucleotides . in the adipose tissue ppar and receptors are involved in fatty acid oxidation and regulation of insulin sensitivity . these epigenetic modifications can result in increased triglycerides and nonesterified fatty acids in offspring , and finally insulin resistance . in the liver , increased glucocorticoid activity can induce phosphoenol pyruvate kinase expression which induces gluconeogenesis . the pancreatic growth factor insulin - like growth factor 2 ( igf2 ) was shown to be reduced in neonatal rats when mothers were exposed to low - protein diet . in fact , taurine deficiency was also proposed to play a role .. low - protein diet also reduced the methylation of the promoter gene of type 1b adrenal angiotensin receptor and in turn overexpression of the receptor . in a recent article in normal pancreatic cells , the promoter for pdx1 is in an unmethylated open chromatin state , which in turn promotes usf-1 and is associated with acetylated h3 , h4 , and trimethylated h3k4 . in iugr islets , hdac1dnmt1 repressor complex is recruited with gradual loss of histone acetylation and trimethylated h3k4 . when one looks at the iugr adult islets , there is an inactive chromatin with dimethylated h3k9 and high dna methylation which is not conducive for transcription and hence pdx gene is silenced . a key initiator in this methylation process could possibly be the oxidative stress found in iugr . iugr increases mef2d binding to glut-4 promoter which is an inhibitor and decreases mef2a and myod ( activator and co - activator , respectively ) binding to the glut-4 promoter in skeletal muscle tissue , which in turn promotes insulin resistance . placental insufficiency can decrease the production of pancreatic transcription factor product of pdx1 by cpg methylation in animals and they can develop diabetes at a later date . the role of genomic imprinting in birth weight can be understood by studying two fetal growth disorders associated with 11p15 h19/igf2 imprinted domain . methylation of this imprinting region can result in silencing of h19 and the expression of igf2 , and if methylation does not occur , silencing of igf2 and the expression of h19 is the result . normally methylation occurs in the male germ line and is prevented in the female germ line . absent dna methylation in the paternal allele leads to biallelic expression of h19 with silencing of igf2 , which in turn results in iugr and russell silver syndrome . on the other hand , a gain of dna methylation on the maternal allele results in silencing of h19 and biallelic expression of igf2 with fetal overgrowth and beckwith wiedemann syndrome . wiedemann is more commonly seen associated with assisted reproductive technology as a result of epigenetic changes . since mitochondrial dna is inherited from the mother , it may be a player that determines the transfer of maternal nutritional stress to fetus . reactive oxygen species ( ros ) can induce several intramitochondrial changes like shutting down of mitochondrial energy production and various other cellular changes . the antioxidant machinery of cell is suboptimal , which makes it more prone for free radical induced injury . complexes i and iii of the electron transport chain are shown to function below par . iugr is associated with decreased mitochondrial gene content and progressive damage of the mitochondrial dna content with free radicals . these dysfunctions can manifest functionally as decreased glucose - mediated insulin secretion and cell death , with diabetes as the end result . human studies , though few , are also remarkable in showing the effect of nutritional modification of epigenome . , increased insulin resistance was observed in children whose mothers were vitamin b12 deficient with folate sufficiency . in fact , folate levels during pregnancy were positively associated with child 's adiposity and insulin resistance , while vitamin b12 levels negatively correlated with the same . these findings demonstrate the power of methyl donors and their cofactors to change the epigenome and ultimately affect the adult phenotype . in subjects with hyperhomocysteinemia , s - adenosylhomocysteine , this leads to lower levels of dna methylation and a shift from monoallelic to biallelic expression for some imprinted genes ( including h19 ) . there is evidence that tells us that the effect of nutritional modifications can persist through a number of generations despite nutritional correctional measures . there is a reduction in the crude death rate , birth rate , infant mortality rate and there is an increase in the population of age group between 15 and 59 years . the rural urban migration has resulted in a steady growth of urban population and creation of megacities . the maternal and perinatal problems in india are still high and non - communicable diseases like diabetes and cardiovascular disease are rapidly increasing . over the years rural populations consume more cereals , fewer pulses , and less oil , fat , and sugars , compared with urban populations . with the advent of fast food operators with high - energy density food and sedentary lifestyle in urban population , osmani and sen have discussed the hidden penalties of gender inequality on fetal origins of adult disease in an excellent review . in the developing countries and especially in india , gender inequality influences birth rate , mortality , and male female ratio which is steeped against female gender . in india , there is a paradox and the population suffers from high incidence of infectious and non - communicable diseases . multiparity and relatively young age at the time of marriage also contribute to the low birth weight of children born to these mothers . however , the economic and social transition combined with relative nutritional affluence during childhood puts these patients at risk for obesity , cad , and insulin resistance , as adults . various studies across the globe have shown that excessive postnatal weight gain can have additional adverse influences on adult health . considerable body of evidence has come from studying and following up children who were born short for gestational age ( sga ) . most of these children exhibit good catch - up growth in the initial few years of life , while some of them have a smaller degree of catch - up growth and remain short . sga children have elevated levels of growth hormone , insulin like growth factor binding proteins 1 and 2 ( igfbp1 , and igfbp2 ) , with low levels of igf-1 and igfbp3 , which is a pattern consistent with growth hormone insensitivity and insulin resistance. [5658 ] in most of these children , growth hormone dynamics normalizes in the initial few months of life . sga children who have weight gain are at risk of developing insulin resistance , pcos , premature adrenarche during adolescent period , and hypertension , diabetes , and cardiovascular disease in adulthood . the abdominal adipose tissue of these infants shows resistance to the actions of insulin and catecholamine hyperresponsiveness . low birth weight may be associated with decreased number of glomeruli , which when exposed to hyperfiltration associated with weight gain can accelerate glomerulosclerosis and contribute to hypertension . pro12ala polymorphism of the ppar2 gene has been shown to increase the risk of type 2 diabetes in iugr patients . it is important to note that those infants who do not have significant catch - up growth are not at risk for most of these diseases . in the pune maternal nutrition study , indian babies were found to be lighter , shorter , and thinner , compared to british babies . however , there was a relative preservation of subscapular fat and a paucity of non - adipose tissue . in another study by the same group , the cord leptin concentrations in the british babies and indian babies were found to be similar despite the fact that indian babies were thinner and lighter . these findings suggest a higher adiposity in the indian babies , paving way for the concept of thin fat indian baby . yajnik and group also studied body size , glucose tolerance , insulin resistance , and cardiovascular risk factors in children . at 4 years of age , glucose , insulin , and igf-1 levels were inversely related to birth weight after considering the effect of current weight . however , they also found that growth velocity of children from 4 to 8 years was a stronger predictor of insulin resistance and cardiovascular risk than measurements at 8 years . these concepts become important in children undergoing rural to urban migration , adoption , and those stepping up on the economic and affluence ladder , which ultimately increases nutritional availability . although majority of the evidence points out insulin resistance as the primary pathology behind diabetes in children with iugr , some evidence also points toward decreased insulin secretion . in an interesting experiment , insulin secretion and insulin sensitivity of young men , who were small for gestational age , although insulin sensitivity was normal , insulin secretion was decreased by 30% when controlled for insulin sensitivity . the adaptational response in a fetus to a nutritionally challenging environment is preservation of its vital organ function , namely the brain , at the expense of other organs in the body . the mechanisms by which the fetus achieves this include blood and nutritional diversion to the brain , along with insulin and growth hormone resistance in the periphery . the insulin resistance associated with reduced lean muscle mass along with hyperinsulinemia directs the excess glucose toward fat storage in adipose tissue. abnormalities in gh - igf axis have already been described in an earlier part of the article . experimental studies in animals have shown that those that are born small are prone to adult - onset hyperglycemia , blood pressure , anxiety , and increased hypothalamic pituitary adrenal ( hpa ) axis activity . however , the hpa axis activity may vary depending on the sex of the fetus , and time and nature of the stimulus . placental corticotropin - releasing hormone ( crh ) , sympathoadrenal system , and glucocorticoid and mineralocorticoid receptors in the hippocampus and other areas of brain are supposed to play a role in the programming of hpa axis . one of the key regulators of fetal cortisol levels is placental 11 beta hydroxy steroid dehydrogenase 2 ( 11hsd2 ) enzyme which converts cortisol to cortisone and keeps fetal cortisol levels at a lower level . studies using stimulated cortisol have shown an association between low birth weight and hyperactive hpa axis , with adult cardiovascular disease , glucose intolerance , and higher systolic and diastolic pressures. [9395 ] recent researches have brought out an exciting interplay of adipocytokines and subsequent development of adult disease in iugr . [98100 ] however , sga infants show a shift to a higher molecular form adiponectin , which is more related to insulin sensitivity . the idea behind this shift is an enhancement of insulin sensitivity and promotion of neonatal catch - up growth . other adipocytokines under active investigation for their role in iugr and subsequent adult - onset adiposity and insulin resistance include tumor necrosis factor - alpha ( tnf- ) , interleukin-6 ( il-6 ) , resistin , apelin , and visfatin . current evidence points toward the fact that maternal diabetes predisposes to adult - onset obesity and diabetes in the fetus even after other influencing factors are adjusted for . evidence for the influence of intrauterine metabolic derangements secondary to maternal diabetes comes from the study on pima indians . children born to mothers who were diabetic at the time of pregnancy were found to be more obese than their siblings who were born before the onset of diabetes . it was also seen that diabetes was more common in children who were born to mothers who had diabetes at the time of pregnancy when compared to infants who were born to mothers who did not have diabetes at the time of pregnancy , though they developed diabetes subsequently . showed that higher birth weight in children was associated with adiposity in the parents and metabolic syndrome in mother 8 years after the child birth . the metabolic abnormalities that are present in the obese and diabetic mothers are transferred to the fetus during intrauterine period , conferring a risk of subsequent obesity . studies have shown that the levels of endothelial progenitor cells are reduced in neonates born to mothers with type 1 diabetes mellitus . endothelial progenitor cells play an important role in vascular repair and neovascular formation , and endothelial dysfunction plays a pivotal role in the development of cardiovascular diseases . the alarming rise in diabetes and other communicable diseases has necessitated a focus shift from treating the non - communicable diseases to their prevention .

in computational geometry , many fundamental properties of finite point sets do not depend on the actual coordinates of each point in real space , but rather on the relative position of the points among each other . in their landmark paper , goodman and pollack capture this idea by defining the order type of a point set . in the plane , two point sets have the same order type if there is a bijection between the sets s.t . for every triple p , q , r of the first set , the corresponding points (p),(q ) , and (r ) have the same orientation ( i.e. , are both oriented clockwise or counterclockwise).3 this orientation can be tested by the inequalitydet(pxpy1qxqy1rxry1)>0 , which indicates whether r is to the left of the directed line through p and q , i.e. , whether the triple is oriented counterclockwise . the sign of the determinant therefore gives a predicate (p , q , r ) that is true iff the triple is oriented counterclockwise . this mapping of all triples of a set to their orientation is also called the chirotope of the set ( cf . remark 1.6 in and [ 3 , p. 95 many combinatorial properties of a set of points only depend on its order type , like its convex hull , the set of its crossing - free graphs , etc . we implicitly assume throughout this paper that all sets are in general position , i.e. , do not contain collinear triples . metric properties of a point set that are not determined by the order type . this includes the sets delaunay triangulation ; it is straightforward to construct two sets of the same order type whose delaunay triangulations are different . guibas and stolfi separate topological from geometric aspects , using a predicate incircle(p , q , r , s ) that is true iff the triple ( p , q , r ) is oriented counterclockwise and the point s lies inside the circle defined by the first three points . their delaunay triangulation algorithm depends almost entirely on this predicate , making it a robust approach , that is intended to be easy to implement and to prove . he defines five axioms over a ternary predicate p and calls sets of triples obeying them cc systems . axiom 4interiorityp(t , p , q)p(t , q , r)p(t , r , p)p(p , q , r ) . axiom 5transitivityp(p , q , r)p(p , q , s)p(p , q , t)p(p , r , s)p(p , s , t ) p(p , r , t ) . p(t , p , q)p(t , q , r)p(t , r , p)p(p , q , r ) . p(p , q , r)p(p , q , s)p(p , q , t)p(p , r , s)p(p , s , t ) p(p , r , t ) . these are fulfilled by all point sets in the euclidean plane , with p= defined as point triple orientation . for these sets , however , there exist cc systems that can not be constructed as point sets in r2 . cc systems are equivalent to abstract order types , which , for example , can be used in so - called abstract order type extension . every abstract order type can be mapped to an arrangement of pseudo - lines in the projective plane . a stretchable arrangement corresponds to a set of realizable order types [ 3 , pp . goodman and pollack showed that all cc systems of up to 8 elements are realizable as point sets . the concept of the convex hull of a point set generalizes to all cc systems . the axiomatic approach can also be extended to cover delaunay triangulations . in the axiomatic settings , knuth provides o(nlogn ) time algorithms for both problems , where the time bound for the latter holds in the expected case . he points out that the algorithm of guibas and stolfi uses the coordinate representation to find a line that partitions the point set into two equally sized subsets ( cf . 9798 ] therefore asks for an algorithm to find such a partition of a cc system in linear time . the problem is straightforward when given an extreme point of the set ( i.e. , an element of the convex hull boundary ) . proving the existence of a linear - time algorithm for finding a single extreme point is also explicitly part of open problem 1 . in this work , we answer both parts of the open problem in the affirmative . in section 2 , we give a simple o(n ) time algorithm that , given a point c of a set s of size n , finds a halving edge through c ; more specifically , it finds a second point cs s.t . not more than n22 points are on each side of the supporting line of c and c. we then describe in section 3 an algorithm that , given two points p and q , returns the edge of the convex hull that is crossed by the ray from p through q. we first show that the algorithm runs in o(n ) time for realizable sets . we then show that the time bound is also correct for non - realizable sets , that is , for all cc systems . our main motivation is to show that the asymptotic running time for solving these problems does not depend on the representation by coordinates . while an arbitrary halving edge can easily be found by picking a point with median , say , x - coordinate , the problem is more sophisticated when the halving line should pass through a predefined point . e.g. , the linear time ham - sandwich - cut algorithm of lo , matouek and steiger can be adapted to find a halving line through a point . the straightforward way of finding an extreme point of a set given by coordinates is selecting the one with , say , lowest x - coordinate . finding a convex hull edge that is traversed by a given line in linear time is a subroutine of the so - called ultimate convex hull algorithm of kirkpatrick and seidel . there , the median of the slopes of an arbitrary matching of the points is used for the prune and search approach . recent algorithms that operate only on triple orientations involve computing the convex hull of disks . also note that there are models that allow for more abstract order types to be realized than by point sets in the euclidean plane . a preliminary version of this work appeared as . in this section we describe an algorithm to compute a halving edge through a given point c of a finite point set s of size n in the plane . while we give the algorithm for realizable order types , note throughout the proof that all assumptions also hold in the abstract setting . the idea is to split the point set evenly by two double wedges centered at c. we identify the double wedge that must contain a halving edge , and continue with the points therein . imagine a directed line rotated by 180 clockwise around c , starting at p1 . let p2, ,pn1 be the remaining points in the order passes through them , either with its front or its tail . we denote this order by , which is actually the order described in [ 3 , p. 16 for each pair of s{c } , we can compute the relative order in which passes through them in constant time , using the predicate . thus the median m = pn2 with respect to can be computed in o(n ) time . ( note that the number of points to the right of may not change monotonically while rotating . ) the supporting lines of cp1 and cm define two double wedges , containing s1={ps{c}:pm } and s2={ps{c}:mp } , respectively , each having not more than n22 points . more than n22 points to the right of 1 and thus less than n22 points to the right of n . due to our general position assumption , while rotating only one point changes sides at the same time . if less than n22 points are right of n2 , then one of the points in s1 forms a halving edge with c ( recall that there are w.l.o.g . . otherwise one of the points in s2 does . this way we can decide which subset contains a halving edge , exclude the other subset and iterate . observe that all the points we exclude belong to two ( open ) wedges centered at c , one entirely to the left and one entirely to the right of the non - removed lines of 2, ,n1 . the closure of each wedge contains a ray of 1 . we store how many points each of these wedges contained . now , if we want to compute the number of points to the right of some line i in subsequent iterations , then we only need to consider the remaining points and add the number of points belonging to the excluded wedge to the right of i . the algorithm has linear running time , as we exclude approximately half of the points in each linear - time iteration . theorem 2given a point set sr2 of size n in general position and a point cs , a halving edge of s incident to c can be found in linear time using only the predicate . given a point set sr2 of size n in general position and a point cs , a halving edge of s incident to c can be found in linear time using only the predicate . now we argue that the above approach does not require that s is realizable , that is , it works for any cc system ( using the predicate p ) . the order around c we use is also defined for cc systems by signing the points in a way that the positive points are to the right of 1 and the negative ones are to the left of 1 , where each negative sign will change the orientation of a triple containing c [ 3 , p. 16 this allows us to give a purely combinatorial formulation of our algorithm : let s be a variable initialized to 0 and let ri be the number of negative points preceding point pi plus the number of positive points succeeding pi with respect to in the current iteration ( this corresponds to the points to the right of i ) . if the sum of rm and s is less than n22 , we remove the points that succeed m , and add the number of removed positive points to s. otherwise , we remove the points that precede m and add the number of removed negative points to s. note that for every point pi , the value of ri+s is an invariant over all iterations . eventually , we end up with an element pj such that rj+s=n22. in the combinatorial formulation of the algorithm , the crucial properties of the sequence r we use is that subsequent elements change by one and that the required value is between r1 and rn . therefore , the algorithm is also adaptable to more sophisticated problems than finding a halving edge . one example is the search for a balanced line , that is , a line that separates a set consisting of n white points and n black points in a way that the difference between the black and white points on each side of the line is 0 ( additional care has to be taken when defining the sequence r since a balanced line does in general not pass directly through a given point c , i.e. , there is no explicit representation of the line as a supporting line of two points ) . there , it is shown that certain crossing - free matchings on a point set are not unique by constructing a balanced line crossing a segment of a given matching . for realizable sets , for the oriented hyperplane h through the points ( p1, ,pd ) , (p1, corollary 3given a point set srd in general position , d constant , and distinct points c1, ,cd1s , one can find a halving hyperplane through c1, ,cd1 in linear time using only . given a point set srd in general position , d constant , and distinct points c1, ,cd1s , one can find a halving hyperplane through c1, in this section we show how to compute a convex hull edge of a set in linear time , only using the orientation of triples . we first present the algorithm for point sets and then extend it to general cc systems . as a first step we describe an algorithm called basicmin , which plays a crucial role as a subroutine . let s be a point set in the plane and suppose we are given two points p , rs . let r be the coordinate origin and let p be on the positive part of the x - axis . let m be an arbitrary perfect matching between the points above and below the x - axis , i.e. , for any edge s = abm we have (p , r , a)(p , r , b ) . let be the binary operator that accepts two edges s , sm as input and returns the edge on the convex hull boundary of ss that crosses the x - axis at the smallest x - coordinate , i.e. , the pair of endpoints whose upward - directed supporting line has all other points of s and s to the right . the relevant property of the operator is that the crossing of (s , s ) with the x - axis is not to the right of the crossings of s and s with the x - axis . basicmin takes a point set s and two points p and r as input , partitions s arbitrarily into the matching m={s1, ,s(n22 ) } , and computes a special edge m = m(n22 ) iteratively viam1=s1,m(i+1)=(mi , s(i+1 ) ) . obviously , the running time of basicmin is linear in n. note that m does not need to be on the convex hull of the whole set . also , m may depend on the ( undefined ) order in which the elements of m are processed by basicmin . lemma 4let be a line directed upwards , crossing the x - axis left of the crossing of m with the x - axis . then at least n22 points of s are to the right of . proofnote that the crossing of with the x - axis is further to the left than any other such crossing of m. assume , for the purpose of contradiction , that there is an edge in m for which both points lie to the left of . then also the crossing with the x - axis is to the left of the crossing of m , a contradiction . hence , for each of the n22 edges in m at least one endpoint lies to the right of . let be a line directed upwards , crossing the x - axis left of the crossing of m with the x - axis . then at least n22 points of s are to the right of . note that the crossing of with the x - axis is further to the left than any other such crossing of m. assume , for the purpose of contradiction , that there is an edge in m for which both points lie to the left of . then also the crossing with the x - axis is to the left of the crossing of m , a contradiction . hence , for each of the n22 edges in m at least one endpoint lies to the right of . note that , even though the argumentation of lemma 4 involves relative positions of crossings , the constant - time operation can be expressed by using only . now we are ready to give the main algorithm . theorem 5given two points p , q of a point set sr2 of size n in general position , one can find the edge e of the sets convex hull that passes through the ray pq in o(n ) time using the predicate . proofw.l.o.g . let pq be horizontal with q left of p. note first that the case where q is a vertex of the convex hull can be identified in linear time . we therefore concentrate on the setting where one endpoint of e is below pq and the other one is above . let u be the set strictly above pq , whereas l is the set strictly below pq . w.l.o.g . let |u|n22.consider the endpoint u1u of e. if we remove u1 , the ray pq intersects the boundary of the ( new ) convex hull at a new edge e with an endpoint u2u . note that the other endpoint of e might now be q. in any case , iteratively removing points from u of the intersected convex hull edge induces an order on u ( see fig . 4 ) . note that this corresponds to the order in which the points of u are traversed by a tangent t of ch(l{p , q } ) that is rotated clockwise around that hull , starting at e and ending at pq . the main observation is that in the search for u1 , given a point ui and the tangent t passing through ui , we can discard all points of u to the right of ui with respect to the point l where t touches ch(l{p , q } ) , since none of these points can be u1 . the support ll{q } of t can be found in linear time , since the radial order of l{p , q } around any ui is linear.note that these observations already imply the following randomized approach . the other support l of the tangent ( recall that this might as well be q ) can be found in linear time . we discard the points of u right of lui and iterate . however , consecutive bad choices of ui result in overall quadratic worst - case behavior . we therefore have to make a good choice of ui in order to discard a linear number of points per iteration.the points of u are ordered linearly around p. let ur be the median of this order , which we select in linear time . let m be an arbitrary perfect matching between the points of u to the left and to the right of pur ( maybe omitting one point ) , see fig . now we apply basicmin on m with r = ur , which results in an edge m = uaub . by construction , all edges of m as well as uaub cross the ray pur . now , find the tangents ta and tb of ch(l{p , q } ) through ua and ub , respectively ( we consider them being directed upwards ) . let =ta if ub is to the right of ta , otherwise let =tb . note that p is right of since q is included in the set that we place the tangent on . if crosses the ray pur at a point x , then the crossing of uaub is on the ray between p and x ( by definition of ) . due to lemma 4 , at least half of the points of m are to the right of . otherwise , if does not cross the ray pur , then all points of u to the right of the ray are also to the right of . in both cases , we can discard at least half of the points , which is at least a quarter of the overall set s ( recall that u was w.l.o.g . larger than l ; in each iteration , the process is applied to the larger of the two sets ) . we can therefore in linear time reduce this problem to constant size such that it then can be solved by a brute - force approach . given two points p , q of a point set sr2 of size n in general position , one can find the edge e of the sets convex hull that passes through the ray pq in o(n ) time using the predicate . w.l.o.g . let pq be horizontal with q left of p. note first that the case where q is a vertex of the convex hull can be identified in linear time . we therefore concentrate on the setting where one endpoint of e is below pq and the other one is above . let u be the set strictly above pq , whereas l is the set strictly below pq . consider the endpoint u1u of e. if we remove u1 , the ray pq intersects the boundary of the ( new ) convex hull at a new edge e with an endpoint u2u . note that the other endpoint of e might now be q. in any case , iteratively removing points from u of the intersected convex hull edge induces an order on u ( see fig . 4 ) . note that this corresponds to the order in which the points of u are traversed by a tangent t of ch(l{p , q } ) that is rotated clockwise around that hull , starting at e and ending at pq . the main observation is that in the search for u1 , given a point ui and the tangent t passing through ui , we can discard all points of u to the right of ui with respect to the point l where t touches ch(l{p , q } ) , since none of these points can be u1 . the support ll{q } of t can be found in linear time , since the radial order of l{p , q } around any ui is linear . the other support l of the tangent ( recall that this might as well be q ) can be found in linear time . we discard the points of u right of lui and iterate . however , consecutive bad choices of ui result in overall quadratic worst - case behavior . we therefore have to make a good choice of ui in order to discard a linear number of points per iteration . the points of u are ordered linearly around p. let ur be the median of this order , which we select in linear time . let m be an arbitrary perfect matching between the points of u to the left and to the right of pur ( maybe omitting one point ) , see fig . 5 . now we apply basicmin on m with r = ur , which results in an edge m = uaub . by construction , all edges of m as well as uaub cross the ray pur . now , find the tangents ta and tb of ch(l{p , q } ) through ua and ub , respectively ( we consider them being directed upwards ) . let =ta if ub is to the right of ta , otherwise let =tb . note that p is right of since q is included in the set that we place the tangent on . if crosses the ray pur at a point x , then the crossing of uaub is on the ray between p and x ( by definition of ) . due to lemma 4 , at least half of the points of m are to the right of . otherwise , if does not cross the ray pur , then all points of u to the right of the ray are also to the right of . in both cases , we can discard at least half of the points , which is at least a quarter of the overall set s ( recall that u was w.l.o.g . larger than l ; in each iteration , the process is applied to the larger of the two sets ) . we can therefore in linear time reduce this problem to constant size such that it then can be solved by a brute - force approach . note that the transitivity of the order on u directly follows from the definition of the convex hull and carries over to general cc systems . also , the transitivity of l{p , q } around any point of u holds for cc systems due to axiom 5 . however , the linear time bound depends on the number of points that are to the right of . in order to show that the bound also holds for abstract order types , we need to prove that basicmin also works as expected on non - realizable sets , and that then also has at least half of the points of u to the right . there exist several equivalent definitions for the convex hull of a finite point set s in the plane . for example , the convex hull can be defined as the convex polygon of smallest area that contains all points of the set . equivalently , the boundary of the convex hull of s is the sequence of pairs uv with { u , v}s such that all points s{u , v } are to the left of uv . this latter definition is more combinatorial and carries over to the abstract setting , where we are no longer concerned with geometric artefacts like area or perimeter.4 in this subsection , we will give a brief summary of the relevant properties of abstract order types , following goodman and pollack , which we suggest to the reader for further information on that topic , in addition to . throughout this section , let us again start in the euclidean plane by considering a point set s , which we still require , for ease of presentation , to be in general position ( see on how to handle other cases ) . let be a directed line not orthogonal to any supporting line of s. project the points orthogonally on , which gives us a sequence of the points along . if we rotate in counterclockwise direction , then eventually two points , let them be u and v , will happen to be projected to the same point on . when continuing the rotation process , u and v will have changed their position in the sequence along . after a rotation of 180 , the initial sequence of the projected points on will be reversed . continuing the rotation gives an infinite periodic sequence of transpositions like the one of u and v. it is called the circular sequence of permutations associated to s , or the circular sequence of s , for short . one can observe that after a reversal of u and v all other pairs are reversed until u and v are reversed again . the circular sequence allows us to observe several geometric properties of s. a point that precedes all others in some permutation ( on ) in the circular sequence is an extreme point . note that all the information we can get from the circular sequence is encoded in one half - period . we therefore can identify the vertices of the convex hull as those points that are topmost or bottommost on at some point , when we look at such that it is directed downwards . for every subset of s , the circular sequence therefore gives the point on its convex hull . consider again the moment when u and v change their position in the permutation , say from uv to vu . then the triple uvw is oriented counterclockwise if and only if w is projected below ( i.e. , after ) both u and v when they change their position ( assuming counterclockwise rotation of ) . note that this requires that at the beginning of the half - period u is above ( before ) v. it is common and convenient to identify the points of s with their order at the initial position of . for s={p1, ,pn } , we therefore start with pi above pj on iff i < j . while the starting position of in the half - period is arbitrary , this labeling is useful when comparing point triples . after a half - period , the points are given in the sequence pn, ,p1. the circular sequence contains strictly more information than the order type of a set . while the order type is implied by the circular sequence , it is rather straightforward to construct point sets with different circular sequences that have the same order type . consider an arrangement of pseudo - lines in the projective plane p2 ( a set of closed curves not separating p2 that pairwise intersect in exactly one point ) . an arrangement is simple if no three pseudo - lines intersect in one point . one important property of pseudo - line arrangements , shown by levi , is that they can be extended by additional pseudo - lines . lemma 6levi enlargement lemmagiven a pseudo - line arrangement a in p2 and two points that do not both lie on the same element of a , there exists a pseudo - line arrangement a{ } such that the pseudo - line passes through these two points . given a pseudo - line arrangement a in p2 and two points that do not both lie on the same element of a , there exists a pseudo - line arrangement a{ } such that the pseudo - line passes through these two points . using this lemma , choose a pseudo - line not part of the arrangement as the line at infinity . place a point on . if we choose as the point at vertical infinity , we can identify the arrangement of pseudo - lines with an arrangement of x - monotone pseudo - lines in the euclidean plane ( where the curves are no longer closed ) . this requires the application of the levi enlargement lemma to get x - monotonicity , details are given in . if we sweep this arrangement from left to right , we get again a half - period of a circular sequence , where a change of a pair happens as we sweep across the intersection of two pseudo - lines . goodman and pollack show that any circular sequence of a point set describes the topology of an arrangement of pseudo - lines in p2 . actually , for every point set in the euclidean plane , the standard duality transform for point sets gives an arrangement of straight lines whose crossings , when sweeping from left to right , give a half - period of the circular sequence of the set . there are , however , arrangements of pseudo - lines in the projective plane that are not stretchable . for them , no point set in the euclidean plane exists that implies any sequence derived from such an arrangements by any choice of . recall that we were able to derive the orientation of a point triple uvw by the position of the points in a half - period of the circular sequence . given a direction around a point at infinity allows us to give a triple of pseudo - lines an orientation , even if there does not exist a corresponding point set order type in the euclidean plane ( i.e. , the sequence is non - realizable ) ; we still get a predicate a that gives each triple an orientation , that is , an abstract order type . knuth shows that , for any simple arrangement , a fulfills the five axioms defining cc systems , and from any cc system a corresponding arrangement can be derived ; hence , the terms cc system and abstract order type are merely synonyms . we can use either the axioms or a pseudo - line arrangement ( together with a direction around a point at infinity ) for arguing about abstract order types . in the next section , we will use the model of x - monotone pseudo - lines in the euclidean plane . at first sight , the identification of abstract order types with pseudo - line arrangements in the projective plane might feel like a detour to x - monotone arrangements in the euclidean plane as representation of circular sequences . this might be the case for the application of the concept in this paper , however , it is profound in the whole theory about abstract order types . recall that different circular sequences might correspond to two point sets having the same order type . ( on the other hand , for arrangements of pseudo - lines in the projective plane , there are transformations that are order - type - preserving and some that are not . ) if two pairs uv and st of four different points change their position in the sequence and uv happens right before st in the half - period , then changing st right before uv results in the same order type . ( to reduce the cases to consider , we will nevertheless make use of that relative position of pseudo - line crossings in the next section . ) there is another aspect that is not directly captured by x - monotone arrangements in the euclidean plane . when mapping the arrangement from the projective to the euclidean plane , we chose the line at infinity through arbitrarily ; however , the topology of the arrangement in the projective plane does not depend on the choice of this line . analogously , the circular sequence of a point set does not depend on the actual starting position of . in terms of pseudo - lines in the euclidean plane , the following can be shown ( more details are given in ) . consider a crossing of two pseudo - lines that has no further crossings to the left from its initial position . then we can move this crossing to the right end of the arrangement , such that the resulting arrangement still represents the same abstract order type . in other words , we untangle the crossing at the left end and introduce a new one to the right ( note that this corresponds to removing the position change uv at the beginning by a change vu at the end ) . for every abstract order type and any crossing therein , we can therefore choose a x - monotone representation where that crossing is leftmost . to see why care has to be taken when we deal with non - realizable order types , note that in general the order type does not capture the relative position of the supporting lines of point pairs and the crossings of these lines . however , abstract order types capture some of the information that is related to crossings of supporting lines , which allows us to show that the properties needed for basicmin are also present in the abstract setting.5 we use the dual representation of abstract order types in the euclidean plane by x - monotone pseudo - lines ; this allows us to use the obvious meaning of terms like above and below when describing an arrangement a ( e.g. , a pseudo - line a is above a point b when the point on a that has the same x - coordinate as b has a larger y - coordinate than b ) . we are given a set s of n elements ( which we call points , even though s might not be a realizable point set ) , containing two special points p and q. the set s is separated by pq into a set u to the right of pq , and a set l to the left of it ( where left and right are indicated by a predicate a ) . we want to obtain the pair l1u1,l1l , u1u that is consecutive on the convex hull of s , where p is to the right of l1u1 . this is done by obtaining a pair =ljui such that at least half of the points of u ( minus a constant ) are to the right of and no point of l is to the left of . consider the dual x - monotone pseudo - line arrangement a representing the abstract order type of s. keep in mind that p is an extreme point of the set u{p } , and that r = ur is the median of the points in u ordered radially around p. we can represent the arrangement such that the crossing of the pseudo - lines p and r ( which corresponds to the supporting line of pr in the primal6 ) is the leftmost crossing in the x - monotone representation of a. the linear order of the points around p in the primal splits the set u{ur } into left and right points , separated by pr . in the dual , the right pseudo - lines pass above the crossing pr , and the left pseudo - lines pass below . the operator accepts two point pairs , each pair consisting of a left and a right point . the output of the operator is a pair z consisting of a left and a right point such that all other points are to the right of the oriented line through these points . this pair z is well - defined in the abstract setting as well ( and there is always a geometric representation due to theorem 1 ) . recall that we compute a special pair m = m(n22 ) iteratively viam1=s1,m(i+1)=(mi , s(i+1 ) ) . the crucial property of the pair m = uaub ( and later the line tangent to ch(l{p , q } ) through ua or ub ) is that at least one endpoint of each pair sim lies on the same side of m as the pivot p. ( we assume that m and the elements of m are directed from the left point to the right point and hence p is to the right of m. ) in the dual , each element of m is represented as the crossing of a right and a left pseudo - line in a ( this corresponds to the supporting line of the matched pair of points in the primal ) . the crucial property for m in the dual therefore is to have at least one pseudo - line of each pair sim passing below its crossing in a. for realizable point sets , we were able to argue for the correctness of basicmin using the intersection of a matched pair s with the supporting line of p and r. in the dual , this intersection corresponds again to a pseudo - line that can be added to a ; this pseudo - line passes through the crossing pr and s. also for non - realizable sets , such a pseudo - line exists due to the levi enlargement lemma . in fact , in non - realizable settings the intersections behave in the same transitive manner as in the realizable setting ; see fig . 7 for an illustration of the following statement . observation 7let z=(a , b ) and z=(c , d ) be two pairs such that the point p is to the right of both pairs ( i.e. , in the dual the pseudo - line p is below the crossings z and z ) . let ( ) be a dual pseudo - line through the crossings pr and z ( z ) . if none of a and b is to the left of the primal line z , then the dual pseudo - line is above in the part of the dual arrangement that is to the right of the dual crossing pr . let z=(a , b ) and z=(c , d ) be two pairs such that the point p is to the right of both pairs ( i.e. , in the dual the pseudo - line p is below the crossings z and z ) . let ( ) be a dual pseudo - line through the crossings pr and z ( z ) . if none of a and b is to the left of the primal line z , then the dual pseudo - line is above in the part of the dual arrangement that is to the right of the dual crossing pr . hence , after applying basicmin to the matching m , we obtain in the dual a pseudo - line crossing m on a pseudo - line m that passes through the crossing pr , such that no crossing of m is above m . suppose , for the sake of contradiction , that there is a pair ( a , b ) such that both dual pseudo - lines a and b pass above the crossing m , but the crossing ab is below m . if the crossing ab is to the left of m in the x - monotone arrangement , then , when traversing a from left to right , one would have to pass below m and then go above it again before m. otherwise , if the crossing ab is to the right of m , then b would have to intersect m to be above it at m and then has to be below m again to reach the crossing ab . both cases contradict the fact that a pair of pseudo - lines intersects exactly once in the x - monotone arrangement . hence , half of the pseudo - lines are below the pseudo - line crossing m in a. this corresponds to at least half of the points being on the same side of m as p. recall that in the proof of theorem 5 , we chose a directed line supported by a point ll{q } and either ua or ub such that no elements of l{p , q , ua , ub } is to the left of it . we now proceed to show that at least one point of each pair ( v , w ) in m is to the right of the directed line , as demanded in the proof of theorem 5 . the points involved are { p , q , r , l , ua , ub , v , w } , where l and q might be the same point . since these are at most eight points , we are allowed to use geometric arguments due to theorem 1 . however , we must not rely on the positions of the crossing points on the ray pr . suppose , for the sake of contradiction , that neither of v and w is right of . at least one of v or w has to be to the right of uaub . these observations imply that vw is an edge of the convex hull of { p , ua , ub , v , w}. however , this means that the crossing of the pseudo - lines v and w is above ua and ub in a , which contradicts the fact that uaub is m. thus , we conclude theorem 8theorem 5 also holds for non - realizable cc systems , i.e. , abstract order types . theorem 5 also holds for non - realizable cc systems , i.e. , abstract order types . as a first step we describe an algorithm called basicmin , which plays a crucial role as a subroutine . let s be a point set in the plane and suppose we are given two points p , rs . let r be the coordinate origin and let p be on the positive part of the x - axis . let m be an arbitrary perfect matching between the points above and below the x - axis , i.e. , for any edge s = abm we have (p , r , a)(p , r , b ) . let be the binary operator that accepts two edges s , sm as input and returns the edge on the convex hull boundary of ss that crosses the x - axis at the smallest x - coordinate , i.e. , the pair of endpoints whose upward - directed supporting line has all other points of s and s to the right . the relevant property of the operator is that the crossing of (s , s ) with the x - axis is not to the right of the crossings of s and s with the x - axis . basicmin takes a point set s and two points p and r as input , partitions s arbitrarily into the matching m={s1, ,s(n22 ) } , and computes a special edge m = m(n22 ) iteratively viam1=s1,m(i+1)=(mi , s(i+1 ) ) . obviously , the running time of basicmin is linear in n. note that m does not need to be on the convex hull of the whole set . also , m may depend on the ( undefined ) order in which the elements of m are processed by basicmin . lemma 4let be a line directed upwards , crossing the x - axis left of the crossing of m with the x - axis . then at least n22 points of s are to the right of . proofnote that the crossing of with the x - axis is further to the left than any other such crossing of m. assume , for the purpose of contradiction , that there is an edge in m for which both points lie to the left of . then also the crossing with the x - axis is to the left of the crossing of m , a contradiction . hence , for each of the n22 edges in m at least one endpoint lies to the right of . let be a line directed upwards , crossing the x - axis left of the crossing of m with the x - axis . then at least n22 points of s are to the right of . note that the crossing of with the x - axis is further to the left than any other such crossing of m. assume , for the purpose of contradiction , that there is an edge in m for which both points lie to the left of . then also the crossing with the x - axis is to the left of the crossing of m , a contradiction . hence , for each of the n22 edges in m at least one endpoint lies to the right of . note that , even though the argumentation of lemma 4 involves relative positions of crossings , the constant - time operation can be expressed by using only . now we are ready to give the main algorithm . theorem 5given two points p , q of a point set sr2 of size n in general position , one can find the edge e of the sets convex hull that passes through the ray pq in o(n ) time using the predicate . proofw.l.o.g . let pq be horizontal with q left of p. note first that the case where q is a vertex of the convex hull can be identified in linear time . we therefore concentrate on the setting where one endpoint of e is below pq and the other one is above . let u be the set strictly above pq , whereas l is the set strictly below pq . w.l.o.g . let |u|n22.consider the endpoint u1u of e. if we remove u1 , the ray pq intersects the boundary of the ( new ) convex hull at a new edge e with an endpoint u2u . note that the other endpoint of e might now be q. in any case , iteratively removing points from u of the intersected convex hull edge induces an order on u ( see fig . 4 ) . note that this corresponds to the order in which the points of u are traversed by a tangent t of ch(l{p , q } ) that is rotated clockwise around that hull , starting at e and ending at pq . the main observation is that in the search for u1 , given a point ui and the tangent t passing through ui , we can discard all points of u to the right of ui with respect to the point l where t touches ch(l{p , q } ) , since none of these points can be u1 . the support ll{q } of t can be found in linear time , since the radial order of l{p , q } around any ui is linear.note that these observations already imply the following randomized approach . the other support l of the tangent ( recall that this might as well be q ) can be found in linear time . we discard the points of u right of lui and iterate . however , consecutive bad choices of ui result in overall quadratic worst - case behavior . we therefore have to make a good choice of ui in order to discard a linear number of points per iteration.the points of u are ordered linearly around p. let ur be the median of this order , which we select in linear time . let m be an arbitrary perfect matching between the points of u to the left and to the right of pur ( maybe omitting one point ) , see fig . 5 . now we apply basicmin on m with r = ur , which results in an edge m = uaub . by construction , all edges of m as well as uaub cross the ray pur . now , find the tangents ta and tb of ch(l{p , q } ) through ua and ub , respectively ( we consider them being directed upwards ) . let =ta if ub is to the right of ta , otherwise let =tb . note that p is right of since q is included in the set that we place the tangent on . if crosses the ray pur at a point x , then the crossing of uaub is on the ray between p and x ( by definition of ) . due to lemma 4 , at least half of the points of m are to the right of . otherwise , if does not cross the ray pur , then all points of u to the right of the ray are also to the right of . in both cases , we can discard at least half of the points , which is at least a quarter of the overall set s ( recall that u was w.l.o.g . larger than l ; in each iteration , the process is applied to the larger of the two sets ) . we can therefore in linear time reduce this problem to constant size such that it then can be solved by a brute - force approach . given two points p , q of a point set sr2 of size n in general position , one can find the edge e of the sets convex hull that passes through the ray pq in o(n ) time using the predicate . w.l.o.g . let pq be horizontal with q left of p. note first that the case where q is a vertex of the convex hull can be identified in linear time . we therefore concentrate on the setting where one endpoint of e is below pq and the other one is above . let u be the set strictly above pq , whereas l is the set strictly below pq . consider the endpoint u1u of e. if we remove u1 , the ray pq intersects the boundary of the ( new ) convex hull at a new edge e with an endpoint u2u . note that the other endpoint of e might now be q. in any case , iteratively removing points from u of the intersected convex hull edge induces an order on u ( see fig . 4 ) . note that this corresponds to the order in which the points of u are traversed by a tangent t of ch(l{p , q } ) that is rotated clockwise around that hull , starting at e and ending at pq . the main observation is that in the search for u1 , given a point ui and the tangent t passing through ui , we can discard all points of u to the right of ui with respect to the point l where t touches ch(l{p , q } ) , since none of these points can be u1 . the support ll{q } of t can be found in linear time , since the radial order of l{p , q } around any ui is linear . the other support l of the tangent ( recall that this might as well be q ) can be found in linear time . however , consecutive bad choices of ui result in overall quadratic worst - case behavior . we therefore have to make a good choice of ui in order to discard a linear number of points per iteration . the points of u are ordered linearly around p. let ur be the median of this order , which we select in linear time . let m be an arbitrary perfect matching between the points of u to the left and to the right of pur ( maybe omitting one point ) , see fig . 5 . now we apply basicmin on m with r = ur , which results in an edge m = uaub . by construction , all edges of m as well as uaub cross the ray pur . now , find the tangents ta and tb of ch(l{p , q } ) through ua and ub , respectively ( we consider them being directed upwards ) . let =ta if ub is to the right of ta , otherwise let =tb . note that p is right of since q is included in the set that we place the tangent on . there are two cases to consider . if crosses the ray pur at a point x , then the crossing of uaub is on the ray between p and x ( by definition of ) . due to lemma 4 , at least half of the points of m are to the right of . otherwise , if does not cross the ray pur , then all points of u to the right of the ray are also to the right of . in both cases , we can discard at least half of the points , which is at least a quarter of the overall set s ( recall that u was w.l.o.g . larger than l ; in each iteration , the process is applied to the larger of the two sets ) . we can therefore in linear time reduce this problem to constant size such that it then can be solved by a brute - force approach . note that the transitivity of the order on u directly follows from the definition of the convex hull and carries over to general cc systems . also , the transitivity of l{p , q } around any point of u holds for cc systems due to axiom 5 . however , the linear time bound depends on the number of points that are to the right of . in order to show that the bound also holds for abstract order types , we need to prove that basicmin also works as expected on non - realizable sets , and that then also has at least half of the points of u to the right . there exist several equivalent definitions for the convex hull of a finite point set s in the plane . for example , the convex hull can be defined as the convex polygon of smallest area that contains all points of the set . equivalently , the boundary of the convex hull of s is the sequence of pairs uv with { u , v}s such that all points s{u , v } are to the left of uv . this latter definition is more combinatorial and carries over to the abstract setting , where we are no longer concerned with geometric artefacts like area or perimeter.4 in this subsection , we will give a brief summary of the relevant properties of abstract order types , following goodman and pollack , which we suggest to the reader for further information on that topic , in addition to . throughout this section , let us again start in the euclidean plane by considering a point set s , which we still require , for ease of presentation , to be in general position ( see on how to handle other cases ) . let be a directed line not orthogonal to any supporting line of s. project the points orthogonally on , which gives us a sequence of the points along . if we rotate in counterclockwise direction , then eventually two points , let them be u and v , will happen to be projected to the same point on . when continuing the rotation process , u and v will have changed their position in the sequence along . after a rotation of 180 , the initial sequence of the projected points on will be reversed . continuing the rotation gives an infinite periodic sequence of transpositions like the one of u and v. it is called the circular sequence of permutations associated to s , or the circular sequence of s , for short . one can observe that after a reversal of u and v all other pairs are reversed until u and v are reversed again . the circular sequence allows us to observe several geometric properties of s. a point that precedes all others in some permutation ( on ) in the circular sequence is an extreme point . note that all the information we can get from the circular sequence is encoded in one half - period . we therefore can identify the vertices of the convex hull as those points that are topmost or bottommost on at some point , when we look at such that it is directed downwards . for every subset of s , the circular sequence therefore gives the point on its convex hull . consider again the moment when u and v change their position in the permutation , say from uv to vu . then the triple uvw is oriented counterclockwise if and only if w is projected below ( i.e. , after ) both u and v when they change their position ( assuming counterclockwise rotation of ) . note that this requires that at the beginning of the half - period u is above ( before ) v. it is common and convenient to identify the points of s with their order at the initial position of . for s={p1, ,pn } , we therefore start with pi above pj on iff i < j . while the starting position of in the half - period is arbitrary , this labeling is useful when comparing point triples . after a half - period , the points are given in the sequence pn, ,p1. the circular sequence contains strictly more information than the order type of a set . while the order type is implied by the circular sequence , it is rather straightforward to construct point sets with different circular sequences that have the same order type . consider an arrangement of pseudo - lines in the projective plane p2 ( a set of closed curves not separating p2 that pairwise intersect in exactly one point ) . an arrangement is simple if no three pseudo - lines intersect in one point . one important property of pseudo - line arrangements , shown by levi , is that they can be extended by additional pseudo - lines . lemma 6levi enlargement lemmagiven a pseudo - line arrangement a in p2 and two points that do not both lie on the same element of a , there exists a pseudo - line arrangement a{ } such that the pseudo - line passes through these two points . given a pseudo - line arrangement a in p2 and two points that do not both lie on the same element of a , there exists a pseudo - line arrangement a{ } such that the pseudo - line passes through these two points . using this lemma , choose a pseudo - line not part of the arrangement as the line at infinity . place a point on . if we choose as the point at vertical infinity , we can identify the arrangement of pseudo - lines with an arrangement of x - monotone pseudo - lines in the euclidean plane ( where the curves are no longer closed ) . this requires the application of the levi enlargement lemma to get x - monotonicity , details are given in . if we sweep this arrangement from left to right , we get again a half - period of a circular sequence , where a change of a pair happens as we sweep across the intersection of two pseudo - lines . goodman and pollack show that any circular sequence of a point set describes the topology of an arrangement of pseudo - lines in p2 . actually , for every point set in the euclidean plane , the standard duality transform for point sets gives an arrangement of straight lines whose crossings , when sweeping from left to right , give a half - period of the circular sequence of the set . there are , however , arrangements of pseudo - lines in the projective plane that are not stretchable . for them , no point set in the euclidean plane exists that implies any sequence derived from such an arrangements by any choice of . recall that we were able to derive the orientation of a point triple uvw by the position of the points in a half - period of the circular sequence . given a direction around a point at infinity allows us to give a triple of pseudo - lines an orientation , even if there does not exist a corresponding point set order type in the euclidean plane ( i.e. , the sequence is non - realizable ) ; we still get a predicate a that gives each triple an orientation , that is , an abstract order type . knuth shows that , for any simple arrangement , a fulfills the five axioms defining cc systems , and from any cc system a corresponding arrangement can be derived ; hence , the terms cc system and abstract order type are merely synonyms . we can use either the axioms or a pseudo - line arrangement ( together with a direction around a point at infinity ) for arguing about abstract order types . in the next section , we will use the model of x - monotone pseudo - lines in the euclidean plane . at first sight , the identification of abstract order types with pseudo - line arrangements in the projective plane might feel like a detour to x - monotone arrangements in the euclidean plane as representation of circular sequences . this might be the case for the application of the concept in this paper , however , it is profound in the whole theory about abstract order types . recall that different circular sequences might correspond to two point sets having the same order type . ( on the other hand , for arrangements of pseudo - lines in the projective plane , there are transformations that are order - type - preserving and some that are not . ) if two pairs uv and st of four different points change their position in the sequence and uv happens right before st in the half - period , then changing st right before uv results in the same order type . ( to reduce the cases to consider , we will nevertheless make use of that relative position of pseudo - line crossings in the next section . ) there is another aspect that is not directly captured by x - monotone arrangements in the euclidean plane . when mapping the arrangement from the projective to the euclidean plane , we chose the line at infinity through arbitrarily ; however , the topology of the arrangement in the projective plane does not depend on the choice of this line . analogously , the circular sequence of a point set does not depend on the actual starting position of . in terms of pseudo - lines in the euclidean plane , the following can be shown ( more details are given in ) . consider a crossing of two pseudo - lines that has no further crossings to the left from its initial position . then we can move this crossing to the right end of the arrangement , such that the resulting arrangement still represents the same abstract order type . in other words , we untangle the crossing at the left end and introduce a new one to the right ( note that this corresponds to removing the position change uv at the beginning by a change vu at the end ) . for every abstract order type and any crossing therein , we can therefore choose a x - monotone representation where that crossing is leftmost . to see why care has to be taken when we deal with non - realizable order types , note that in general the order type does not capture the relative position of the supporting lines of point pairs and the crossings of these lines . however , abstract order types capture some of the information that is related to crossings of supporting lines , which allows us to show that the properties needed for basicmin are also present in the abstract setting.5 we use the dual representation of abstract order types in the euclidean plane by x - monotone pseudo - lines ; this allows us to use the obvious meaning of terms like above and below when describing an arrangement a ( e.g. , a pseudo - line a is above a point b when the point on a that has the same x - coordinate as b has a larger y - coordinate than b ) . we are given a set s of n elements ( which we call points , even though s might not be a realizable point set ) , containing two special points p and q. the set s is separated by pq into a set u to the right of pq , and a set l to the left of it ( where left and right are indicated by a predicate a ) . we want to obtain the pair l1u1,l1l , u1u that is consecutive on the convex hull of s , where p is to the right of l1u1 . this is done by obtaining a pair =ljui such that at least half of the points of u ( minus a constant ) are to the right of and no point of l is to the left of . consider the dual x - monotone pseudo - line arrangement a representing the abstract order type of s. keep in mind that p is an extreme point of the set u{p } , and that r = ur is the median of the points in u ordered radially around p. we can represent the arrangement such that the crossing of the pseudo - lines p and r ( which corresponds to the supporting line of pr in the primal6 ) is the leftmost crossing in the x - monotone representation of a. the linear order of the points around p in the primal splits the set u{ur } into left and right points , separated by pr . in the dual , the right pseudo - lines pass above the crossing pr , and the left pseudo - lines pass below . the operator accepts two point pairs , each pair consisting of a left and a right point . the output of the operator is a pair z consisting of a left and a right point such that all other points are to the right of the oriented line through these points . this pair z is well - defined in the abstract setting as well ( and there is always a geometric representation due to theorem 1 ) . recall that we compute a special pair m = m(n22 ) iteratively viam1=s1,m(i+1)=(mi , s(i+1 ) ) . the crucial property of the pair m = uaub ( and later the line tangent to ch(l{p , q } ) through ua or ub ) is that at least one endpoint of each pair sim lies on the same side of m as the pivot p. ( we assume that m and the elements of m are directed from the left point to the right point and hence p is to the right of m. ) in the dual , each element of m is represented as the crossing of a right and a left pseudo - line in a ( this corresponds to the supporting line of the matched pair of points in the primal ) . the crucial property for m in the dual therefore is to have at least one pseudo - line of each pair sim passing below its crossing in a. for realizable point sets , we were able to argue for the correctness of basicmin using the intersection of a matched pair s with the supporting line of p and r. in the dual , this intersection corresponds again to a pseudo - line that can be added to a ; this pseudo - line passes through the crossing pr and s. also for non - realizable sets , such a pseudo - line exists due to the levi enlargement lemma . in fact , in non - realizable settings the intersections behave in the same transitive manner as in the realizable setting ; see fig . 7 for an illustration of the following statement . observation 7let z=(a , b ) and z=(c , d ) be two pairs such that the point p is to the right of both pairs ( i.e. , in the dual the pseudo - line p is below the crossings z and z ) . let ( ) be a dual pseudo - line through the crossings pr and z ( z ) . if none of a and b is to the left of the primal line z , then the dual pseudo - line is above in the part of the dual arrangement that is to the right of the dual crossing pr . let z=(a , b ) and z=(c , d ) be two pairs such that the point p is to the right of both pairs ( i.e. , in the dual the pseudo - line p is below the crossings z and z ) . let ( ) be a dual pseudo - line through the crossings pr and z ( z ) . if none of a and b is to the left of the primal line z , then the dual pseudo - line is above in the part of the dual arrangement that is to the right of the dual crossing pr . hence , after applying basicmin to the matching m , we obtain in the dual a pseudo - line crossing m on a pseudo - line m that passes through the crossing pr , such that no crossing of m is above m . suppose , for the sake of contradiction , that there is a pair ( a , b ) such that both dual pseudo - lines a and b pass above the crossing m , but the crossing ab is below m . if the crossing ab is to the left of m in the x - monotone arrangement , then , when traversing a from left to right , one would have to pass below m and then go above it again before m. otherwise , if the crossing ab is to the right of m , then b would have to intersect m to be above it at m and then has to be below m again to reach the crossing ab . both cases contradict the fact that a pair of pseudo - lines intersects exactly once in the x - monotone arrangement . hence , half of the pseudo - lines are below the pseudo - line crossing m in a. this corresponds to at least half of the points being on the same side of m as p. recall that in the proof of theorem 5 , we chose a directed line supported by a point ll{q } and either ua or ub such that no elements of l{p , q , ua , ub } is to the left of it . we now proceed to show that at least one point of each pair ( v , w ) in m is to the right of the directed line , as demanded in the proof of theorem 5 . the points involved are { p , q , r , l , ua , ub , v , w } , where l and q might be the same point . since these are at most eight points , we are allowed to use geometric arguments due to theorem 1 . however , we must not rely on the positions of the crossing points on the ray pr . suppose , for the sake of contradiction , that neither of v and w is right of . at least one of v or w has to be to the right of uaub . these observations imply that vw is an edge of the convex hull of { p , ua , ub , v , w}. however , this means that the crossing of the pseudo - lines v and w is above ua and ub in a , which contradicts the fact that uaub is m. thus , we conclude theorem 8theorem 5 also holds for non - realizable cc systems , i.e. , abstract order types . theorem 5 also holds for non - realizable cc systems , i.e. , abstract order types . we presented two algorithms that only use the information whether a point triple is oriented clockwise or counterclockwise . both , a halving edge through a given point and a convex hull edge crossing a specified ray , can be found in linear time , without being given the coordinate representation . we showed that the algorithms also work for general cc systems ( i.e. , abstract order types ) , and thus answer a long - standing open problem of knuth in the affirmative . note that the parts of the so - called ultimate convex hull algorithm by kirkpatrick and seidel that depend on coordinates are essentially the one that find the convex hull edge on the ray that separates a subproblem into two parts . also , chans output - sensitive algorithm can be implemented in our setting using theorem 5 . both allow to improve the time bound given in for realizable point sets regarding output - sensitivity to o(nlogh ) for h extreme points .

many properties of finite point sets only depend on the relative position of the points , e.g. , on the order type of the set . however , many fundamental algorithms in computational geometry rely on coordinate representations . this includes the straightforward algorithms for finding a halving line for a given planar point set , as well as finding a point on the convex hull , both in linear time . in his monograph axioms and hulls , knuth asks whether these problems can be solved in linear time in a more abstract setting , given only the orientation of each point triple , i.e. , the sets chirotope , as a source of information . we answer this question in the affirmative . more precisely , we can find a halving line through any given point , as well as the vertices of the convex hull edges that are intersected by the supporting line of any two given points of the set in linear time . we first give a proof for sets realizable in the euclidean plane and then extend the result to non - realizable abstract order types .

Introduction Computing a halving edge in linear time Computing a convex hull edge in linear time Realizable point sets Non-realizable sets A general proof of the time bound Conclusion

in computational geometry , many fundamental properties of finite point sets do not depend on the actual coordinates of each point in real space , but rather on the relative position of the points among each other . in their landmark paper , goodman and pollack capture this idea by defining the order type of a point set . , are both oriented clockwise or counterclockwise).3 this orientation can be tested by the inequalitydet(pxpy1qxqy1rxry1)>0 , which indicates whether r is to the left of the directed line through p and q , i.e. remark 1.6 in and [ 3 , p. 95 many combinatorial properties of a set of points only depend on its order type , like its convex hull , the set of its crossing - free graphs , etc . metric properties of a point set that are not determined by the order type . these are fulfilled by all point sets in the euclidean plane , with p= defined as point triple orientation . in section 2 , we give a simple o(n ) time algorithm that , given a point c of a set s of size n , finds a halving edge through c ; more specifically , it finds a second point cs s.t . not more than n22 points are on each side of the supporting line of c and c. we then describe in section 3 an algorithm that , given two points p and q , returns the edge of the convex hull that is crossed by the ray from p through q. we first show that the algorithm runs in o(n ) time for realizable sets . , the linear time ham - sandwich - cut algorithm of lo , matouek and steiger can be adapted to find a halving line through a point . finding a convex hull edge that is traversed by a given line in linear time is a subroutine of the so - called ultimate convex hull algorithm of kirkpatrick and seidel . also note that there are models that allow for more abstract order types to be realized than by point sets in the euclidean plane . in this section we describe an algorithm to compute a halving edge through a given point c of a finite point set s of size n in the plane . theorem 2given a point set sr2 of size n in general position and a point cs , a halving edge of s incident to c can be found in linear time using only the predicate . given a point set sr2 of size n in general position and a point cs , a halving edge of s incident to c can be found in linear time using only the predicate . the order around c we use is also defined for cc systems by signing the points in a way that the positive points are to the right of 1 and the negative ones are to the left of 1 , where each negative sign will change the orientation of a triple containing c [ 3 , p. 16 this allows us to give a purely combinatorial formulation of our algorithm : let s be a variable initialized to 0 and let ri be the number of negative points preceding point pi plus the number of positive points succeeding pi with respect to in the current iteration ( this corresponds to the points to the right of i ) . one example is the search for a balanced line , that is , a line that separates a set consisting of n white points and n black points in a way that the difference between the black and white points on each side of the line is 0 ( additional care has to be taken when defining the sequence r since a balanced line does in general not pass directly through a given point c , i.e. for realizable sets , for the oriented hyperplane h through the points ( p1, ,pd ) , (p1, corollary 3given a point set srd in general position , d constant , and distinct points c1, ,cd1s , one can find a halving hyperplane through c1, ,cd1 in linear time using only . given a point set srd in general position , d constant , and distinct points c1, ,cd1s , one can find a halving hyperplane through c1, in this section we show how to compute a convex hull edge of a set in linear time , only using the orientation of triples . let be the binary operator that accepts two edges s , sm as input and returns the edge on the convex hull boundary of ss that crosses the x - axis at the smallest x - coordinate , i.e. obviously , the running time of basicmin is linear in n. note that m does not need to be on the convex hull of the whole set . theorem 5given two points p , q of a point set sr2 of size n in general position , one can find the edge e of the sets convex hull that passes through the ray pq in o(n ) time using the predicate . let pq be horizontal with q left of p. note first that the case where q is a vertex of the convex hull can be identified in linear time . the main observation is that in the search for u1 , given a point ui and the tangent t passing through ui , we can discard all points of u to the right of ui with respect to the point l where t touches ch(l{p , q } ) , since none of these points can be u1 . given two points p , q of a point set sr2 of size n in general position , one can find the edge e of the sets convex hull that passes through the ray pq in o(n ) time using the predicate . let pq be horizontal with q left of p. note first that the case where q is a vertex of the convex hull can be identified in linear time . the main observation is that in the search for u1 , given a point ui and the tangent t passing through ui , we can discard all points of u to the right of ui with respect to the point l where t touches ch(l{p , q } ) , since none of these points can be u1 . in order to show that the bound also holds for abstract order types , we need to prove that basicmin also works as expected on non - realizable sets , and that then also has at least half of the points of u to the right . for example , the convex hull can be defined as the convex polygon of smallest area that contains all points of the set . this latter definition is more combinatorial and carries over to the abstract setting , where we are no longer concerned with geometric artefacts like area or perimeter.4 in this subsection , we will give a brief summary of the relevant properties of abstract order types , following goodman and pollack , which we suggest to the reader for further information on that topic , in addition to . we therefore can identify the vertices of the convex hull as those points that are topmost or bottommost on at some point , when we look at such that it is directed downwards . if we choose as the point at vertical infinity , we can identify the arrangement of pseudo - lines with an arrangement of x - monotone pseudo - lines in the euclidean plane ( where the curves are no longer closed ) . actually , for every point set in the euclidean plane , the standard duality transform for point sets gives an arrangement of straight lines whose crossings , when sweeping from left to right , give a half - period of the circular sequence of the set . recall that we were able to derive the orientation of a point triple uvw by the position of the points in a half - period of the circular sequence . given a direction around a point at infinity allows us to give a triple of pseudo - lines an orientation , even if there does not exist a corresponding point set order type in the euclidean plane ( i.e. at first sight , the identification of abstract order types with pseudo - line arrangements in the projective plane might feel like a detour to x - monotone arrangements in the euclidean plane as representation of circular sequences . when mapping the arrangement from the projective to the euclidean plane , we chose the line at infinity through arbitrarily ; however , the topology of the arrangement in the projective plane does not depend on the choice of this line . analogously , the circular sequence of a point set does not depend on the actual starting position of . to see why care has to be taken when we deal with non - realizable order types , note that in general the order type does not capture the relative position of the supporting lines of point pairs and the crossings of these lines . however , abstract order types capture some of the information that is related to crossings of supporting lines , which allows us to show that the properties needed for basicmin are also present in the abstract setting.5 we use the dual representation of abstract order types in the euclidean plane by x - monotone pseudo - lines ; this allows us to use the obvious meaning of terms like above and below when describing an arrangement a ( e.g. consider the dual x - monotone pseudo - line arrangement a representing the abstract order type of s. keep in mind that p is an extreme point of the set u{p } , and that r = ur is the median of the points in u ordered radially around p. we can represent the arrangement such that the crossing of the pseudo - lines p and r ( which corresponds to the supporting line of pr in the primal6 ) is the leftmost crossing in the x - monotone representation of a. the linear order of the points around p in the primal splits the set u{ur } into left and right points , separated by pr . the crucial property of the pair m = uaub ( and later the line tangent to ch(l{p , q } ) through ua or ub ) is that at least one endpoint of each pair sim lies on the same side of m as the pivot p. ( we assume that m and the elements of m are directed from the left point to the right point and hence p is to the right of m. ) in the dual , each element of m is represented as the crossing of a right and a left pseudo - line in a ( this corresponds to the supporting line of the matched pair of points in the primal ) . the crucial property for m in the dual therefore is to have at least one pseudo - line of each pair sim passing below its crossing in a. for realizable point sets , we were able to argue for the correctness of basicmin using the intersection of a matched pair s with the supporting line of p and r. in the dual , this intersection corresponds again to a pseudo - line that can be added to a ; this pseudo - line passes through the crossing pr and s. also for non - realizable sets , such a pseudo - line exists due to the levi enlargement lemma . hence , half of the pseudo - lines are below the pseudo - line crossing m in a. this corresponds to at least half of the points being on the same side of m as p. recall that in the proof of theorem 5 , we chose a directed line supported by a point ll{q } and either ua or ub such that no elements of l{p , q , ua , ub } is to the left of it . however , this means that the crossing of the pseudo - lines v and w is above ua and ub in a , which contradicts the fact that uaub is m. thus , we conclude theorem 8theorem 5 also holds for non - realizable cc systems , i.e. obviously , the running time of basicmin is linear in n. note that m does not need to be on the convex hull of the whole set . theorem 5given two points p , q of a point set sr2 of size n in general position , one can find the edge e of the sets convex hull that passes through the ray pq in o(n ) time using the predicate . let pq be horizontal with q left of p. note first that the case where q is a vertex of the convex hull can be identified in linear time . the main observation is that in the search for u1 , given a point ui and the tangent t passing through ui , we can discard all points of u to the right of ui with respect to the point l where t touches ch(l{p , q } ) , since none of these points can be u1 . given two points p , q of a point set sr2 of size n in general position , one can find the edge e of the sets convex hull that passes through the ray pq in o(n ) time using the predicate . let pq be horizontal with q left of p. note first that the case where q is a vertex of the convex hull can be identified in linear time . the main observation is that in the search for u1 , given a point ui and the tangent t passing through ui , we can discard all points of u to the right of ui with respect to the point l where t touches ch(l{p , q } ) , since none of these points can be u1 . we can therefore in linear time reduce this problem to constant size such that it then can be solved by a brute - force approach . in order to show that the bound also holds for abstract order types , we need to prove that basicmin also works as expected on non - realizable sets , and that then also has at least half of the points of u to the right . for example , the convex hull can be defined as the convex polygon of smallest area that contains all points of the set . this latter definition is more combinatorial and carries over to the abstract setting , where we are no longer concerned with geometric artefacts like area or perimeter.4 in this subsection , we will give a brief summary of the relevant properties of abstract order types , following goodman and pollack , which we suggest to the reader for further information on that topic , in addition to . we therefore can identify the vertices of the convex hull as those points that are topmost or bottommost on at some point , when we look at such that it is directed downwards . if we choose as the point at vertical infinity , we can identify the arrangement of pseudo - lines with an arrangement of x - monotone pseudo - lines in the euclidean plane ( where the curves are no longer closed ) . actually , for every point set in the euclidean plane , the standard duality transform for point sets gives an arrangement of straight lines whose crossings , when sweeping from left to right , give a half - period of the circular sequence of the set . recall that we were able to derive the orientation of a point triple uvw by the position of the points in a half - period of the circular sequence . given a direction around a point at infinity allows us to give a triple of pseudo - lines an orientation , even if there does not exist a corresponding point set order type in the euclidean plane ( i.e. at first sight , the identification of abstract order types with pseudo - line arrangements in the projective plane might feel like a detour to x - monotone arrangements in the euclidean plane as representation of circular sequences . when mapping the arrangement from the projective to the euclidean plane , we chose the line at infinity through arbitrarily ; however , the topology of the arrangement in the projective plane does not depend on the choice of this line . analogously , the circular sequence of a point set does not depend on the actual starting position of . to see why care has to be taken when we deal with non - realizable order types , note that in general the order type does not capture the relative position of the supporting lines of point pairs and the crossings of these lines . however , abstract order types capture some of the information that is related to crossings of supporting lines , which allows us to show that the properties needed for basicmin are also present in the abstract setting.5 we use the dual representation of abstract order types in the euclidean plane by x - monotone pseudo - lines ; this allows us to use the obvious meaning of terms like above and below when describing an arrangement a ( e.g. consider the dual x - monotone pseudo - line arrangement a representing the abstract order type of s. keep in mind that p is an extreme point of the set u{p } , and that r = ur is the median of the points in u ordered radially around p. we can represent the arrangement such that the crossing of the pseudo - lines p and r ( which corresponds to the supporting line of pr in the primal6 ) is the leftmost crossing in the x - monotone representation of a. the linear order of the points around p in the primal splits the set u{ur } into left and right points , separated by pr . the crucial property of the pair m = uaub ( and later the line tangent to ch(l{p , q } ) through ua or ub ) is that at least one endpoint of each pair sim lies on the same side of m as the pivot p. ( we assume that m and the elements of m are directed from the left point to the right point and hence p is to the right of m. ) in the dual , each element of m is represented as the crossing of a right and a left pseudo - line in a ( this corresponds to the supporting line of the matched pair of points in the primal ) . the crucial property for m in the dual therefore is to have at least one pseudo - line of each pair sim passing below its crossing in a. for realizable point sets , we were able to argue for the correctness of basicmin using the intersection of a matched pair s with the supporting line of p and r. in the dual , this intersection corresponds again to a pseudo - line that can be added to a ; this pseudo - line passes through the crossing pr and s. also for non - realizable sets , such a pseudo - line exists due to the levi enlargement lemma . hence , half of the pseudo - lines are below the pseudo - line crossing m in a. this corresponds to at least half of the points being on the same side of m as p. recall that in the proof of theorem 5 , we chose a directed line supported by a point ll{q } and either ua or ub such that no elements of l{p , q , ua , ub } is to the left of it . however , we must not rely on the positions of the crossing points on the ray pr . however , this means that the crossing of the pseudo - lines v and w is above ua and ub in a , which contradicts the fact that uaub is m. thus , we conclude theorem 8theorem 5 also holds for non - realizable cc systems , i.e. both , a halving edge through a given point and a convex hull edge crossing a specified ray , can be found in linear time , without being given the coordinate representation .

remark 1.6 in and [ 3 , p. 95 many combinatorial properties of a set of points only depend on its order type , like its convex hull , the set of its crossing - free graphs , etc . not more than n22 points are on each side of the supporting line of c and c. we then describe in section 3 an algorithm that , given two points p and q , returns the edge of the convex hull that is crossed by the ray from p through q. we first show that the algorithm runs in o(n ) time for realizable sets . observe that all the points we exclude belong to two ( open ) wedges centered at c , one entirely to the left and one entirely to the right of the non - removed lines of 2, ,n1 . now , if we want to compute the number of points to the right of some line i in subsequent iterations , then we only need to consider the remaining points and add the number of points belonging to the excluded wedge to the right of i . the order around c we use is also defined for cc systems by signing the points in a way that the positive points are to the right of 1 and the negative ones are to the left of 1 , where each negative sign will change the orientation of a triple containing c [ 3 , p. 16 this allows us to give a purely combinatorial formulation of our algorithm : let s be a variable initialized to 0 and let ri be the number of negative points preceding point pi plus the number of positive points succeeding pi with respect to in the current iteration ( this corresponds to the points to the right of i ) . if the sum of rm and s is less than n22 , we remove the points that succeed m , and add the number of removed positive points to s. otherwise , we remove the points that precede m and add the number of removed negative points to s. note that for every point pi , the value of ri+s is an invariant over all iterations . in the combinatorial formulation of the algorithm , the crucial properties of the sequence r we use is that subsequent elements change by one and that the required value is between r1 and rn . one example is the search for a balanced line , that is , a line that separates a set consisting of n white points and n black points in a way that the difference between the black and white points on each side of the line is 0 ( additional care has to be taken when defining the sequence r since a balanced line does in general not pass directly through a given point c , i.e. for realizable sets , for the oriented hyperplane h through the points ( p1, ,pd ) , (p1, corollary 3given a point set srd in general position , d constant , and distinct points c1, ,cd1s , one can find a halving hyperplane through c1, ,cd1 in linear time using only . given a point set srd in general position , d constant , and distinct points c1, ,cd1s , one can find a halving hyperplane through c1, in this section we show how to compute a convex hull edge of a set in linear time , only using the orientation of triples . the relevant property of the operator is that the crossing of (s , s ) with the x - axis is not to the right of the crossings of s and s with the x - axis . proofnote that the crossing of with the x - axis is further to the left than any other such crossing of m. assume , for the purpose of contradiction , that there is an edge in m for which both points lie to the left of . note that the crossing of with the x - axis is further to the left than any other such crossing of m. assume , for the purpose of contradiction , that there is an edge in m for which both points lie to the left of . theorem 5given two points p , q of a point set sr2 of size n in general position , one can find the edge e of the sets convex hull that passes through the ray pq in o(n ) time using the predicate . the main observation is that in the search for u1 , given a point ui and the tangent t passing through ui , we can discard all points of u to the right of ui with respect to the point l where t touches ch(l{p , q } ) , since none of these points can be u1 . given two points p , q of a point set sr2 of size n in general position , one can find the edge e of the sets convex hull that passes through the ray pq in o(n ) time using the predicate . consider the endpoint u1u of e. if we remove u1 , the ray pq intersects the boundary of the ( new ) convex hull at a new edge e with an endpoint u2u . the main observation is that in the search for u1 , given a point ui and the tangent t passing through ui , we can discard all points of u to the right of ui with respect to the point l where t touches ch(l{p , q } ) , since none of these points can be u1 . in order to show that the bound also holds for abstract order types , we need to prove that basicmin also works as expected on non - realizable sets , and that then also has at least half of the points of u to the right . this latter definition is more combinatorial and carries over to the abstract setting , where we are no longer concerned with geometric artefacts like area or perimeter.4 in this subsection , we will give a brief summary of the relevant properties of abstract order types , following goodman and pollack , which we suggest to the reader for further information on that topic , in addition to . lemma 6levi enlargement lemmagiven a pseudo - line arrangement a in p2 and two points that do not both lie on the same element of a , there exists a pseudo - line arrangement a{ } such that the pseudo - line passes through these two points . given a pseudo - line arrangement a in p2 and two points that do not both lie on the same element of a , there exists a pseudo - line arrangement a{ } such that the pseudo - line passes through these two points . if we choose as the point at vertical infinity , we can identify the arrangement of pseudo - lines with an arrangement of x - monotone pseudo - lines in the euclidean plane ( where the curves are no longer closed ) . when mapping the arrangement from the projective to the euclidean plane , we chose the line at infinity through arbitrarily ; however , the topology of the arrangement in the projective plane does not depend on the choice of this line . however , abstract order types capture some of the information that is related to crossings of supporting lines , which allows us to show that the properties needed for basicmin are also present in the abstract setting.5 we use the dual representation of abstract order types in the euclidean plane by x - monotone pseudo - lines ; this allows us to use the obvious meaning of terms like above and below when describing an arrangement a ( e.g. we are given a set s of n elements ( which we call points , even though s might not be a realizable point set ) , containing two special points p and q. the set s is separated by pq into a set u to the right of pq , and a set l to the left of it ( where left and right are indicated by a predicate a ) . consider the dual x - monotone pseudo - line arrangement a representing the abstract order type of s. keep in mind that p is an extreme point of the set u{p } , and that r = ur is the median of the points in u ordered radially around p. we can represent the arrangement such that the crossing of the pseudo - lines p and r ( which corresponds to the supporting line of pr in the primal6 ) is the leftmost crossing in the x - monotone representation of a. the linear order of the points around p in the primal splits the set u{ur } into left and right points , separated by pr . the crucial property of the pair m = uaub ( and later the line tangent to ch(l{p , q } ) through ua or ub ) is that at least one endpoint of each pair sim lies on the same side of m as the pivot p. ( we assume that m and the elements of m are directed from the left point to the right point and hence p is to the right of m. ) in the dual , each element of m is represented as the crossing of a right and a left pseudo - line in a ( this corresponds to the supporting line of the matched pair of points in the primal ) . the crucial property for m in the dual therefore is to have at least one pseudo - line of each pair sim passing below its crossing in a. for realizable point sets , we were able to argue for the correctness of basicmin using the intersection of a matched pair s with the supporting line of p and r. in the dual , this intersection corresponds again to a pseudo - line that can be added to a ; this pseudo - line passes through the crossing pr and s. also for non - realizable sets , such a pseudo - line exists due to the levi enlargement lemma . if none of a and b is to the left of the primal line z , then the dual pseudo - line is above in the part of the dual arrangement that is to the right of the dual crossing pr . if none of a and b is to the left of the primal line z , then the dual pseudo - line is above in the part of the dual arrangement that is to the right of the dual crossing pr . hence , after applying basicmin to the matching m , we obtain in the dual a pseudo - line crossing m on a pseudo - line m that passes through the crossing pr , such that no crossing of m is above m . if the crossing ab is to the left of m in the x - monotone arrangement , then , when traversing a from left to right , one would have to pass below m and then go above it again before m. otherwise , if the crossing ab is to the right of m , then b would have to intersect m to be above it at m and then has to be below m again to reach the crossing ab . hence , half of the pseudo - lines are below the pseudo - line crossing m in a. this corresponds to at least half of the points being on the same side of m as p. recall that in the proof of theorem 5 , we chose a directed line supported by a point ll{q } and either ua or ub such that no elements of l{p , q , ua , ub } is to the left of it . however , this means that the crossing of the pseudo - lines v and w is above ua and ub in a , which contradicts the fact that uaub is m. thus , we conclude theorem 8theorem 5 also holds for non - realizable cc systems , i.e. the relevant property of the operator is that the crossing of (s , s ) with the x - axis is not to the right of the crossings of s and s with the x - axis . note that the crossing of with the x - axis is further to the left than any other such crossing of m. assume , for the purpose of contradiction , that there is an edge in m for which both points lie to the left of . theorem 5given two points p , q of a point set sr2 of size n in general position , one can find the edge e of the sets convex hull that passes through the ray pq in o(n ) time using the predicate . the main observation is that in the search for u1 , given a point ui and the tangent t passing through ui , we can discard all points of u to the right of ui with respect to the point l where t touches ch(l{p , q } ) , since none of these points can be u1 . given two points p , q of a point set sr2 of size n in general position , one can find the edge e of the sets convex hull that passes through the ray pq in o(n ) time using the predicate . consider the endpoint u1u of e. if we remove u1 , the ray pq intersects the boundary of the ( new ) convex hull at a new edge e with an endpoint u2u . the main observation is that in the search for u1 , given a point ui and the tangent t passing through ui , we can discard all points of u to the right of ui with respect to the point l where t touches ch(l{p , q } ) , since none of these points can be u1 . this latter definition is more combinatorial and carries over to the abstract setting , where we are no longer concerned with geometric artefacts like area or perimeter.4 in this subsection , we will give a brief summary of the relevant properties of abstract order types , following goodman and pollack , which we suggest to the reader for further information on that topic , in addition to . lemma 6levi enlargement lemmagiven a pseudo - line arrangement a in p2 and two points that do not both lie on the same element of a , there exists a pseudo - line arrangement a{ } such that the pseudo - line passes through these two points . given a pseudo - line arrangement a in p2 and two points that do not both lie on the same element of a , there exists a pseudo - line arrangement a{ } such that the pseudo - line passes through these two points . if we choose as the point at vertical infinity , we can identify the arrangement of pseudo - lines with an arrangement of x - monotone pseudo - lines in the euclidean plane ( where the curves are no longer closed ) . when mapping the arrangement from the projective to the euclidean plane , we chose the line at infinity through arbitrarily ; however , the topology of the arrangement in the projective plane does not depend on the choice of this line . however , abstract order types capture some of the information that is related to crossings of supporting lines , which allows us to show that the properties needed for basicmin are also present in the abstract setting.5 we use the dual representation of abstract order types in the euclidean plane by x - monotone pseudo - lines ; this allows us to use the obvious meaning of terms like above and below when describing an arrangement a ( e.g. we are given a set s of n elements ( which we call points , even though s might not be a realizable point set ) , containing two special points p and q. the set s is separated by pq into a set u to the right of pq , and a set l to the left of it ( where left and right are indicated by a predicate a ) . consider the dual x - monotone pseudo - line arrangement a representing the abstract order type of s. keep in mind that p is an extreme point of the set u{p } , and that r = ur is the median of the points in u ordered radially around p. we can represent the arrangement such that the crossing of the pseudo - lines p and r ( which corresponds to the supporting line of pr in the primal6 ) is the leftmost crossing in the x - monotone representation of a. the linear order of the points around p in the primal splits the set u{ur } into left and right points , separated by pr . the crucial property of the pair m = uaub ( and later the line tangent to ch(l{p , q } ) through ua or ub ) is that at least one endpoint of each pair sim lies on the same side of m as the pivot p. ( we assume that m and the elements of m are directed from the left point to the right point and hence p is to the right of m. ) in the dual , each element of m is represented as the crossing of a right and a left pseudo - line in a ( this corresponds to the supporting line of the matched pair of points in the primal ) . the crucial property for m in the dual therefore is to have at least one pseudo - line of each pair sim passing below its crossing in a. for realizable point sets , we were able to argue for the correctness of basicmin using the intersection of a matched pair s with the supporting line of p and r. in the dual , this intersection corresponds again to a pseudo - line that can be added to a ; this pseudo - line passes through the crossing pr and s. also for non - realizable sets , such a pseudo - line exists due to the levi enlargement lemma . if none of a and b is to the left of the primal line z , then the dual pseudo - line is above in the part of the dual arrangement that is to the right of the dual crossing pr . if none of a and b is to the left of the primal line z , then the dual pseudo - line is above in the part of the dual arrangement that is to the right of the dual crossing pr . hence , after applying basicmin to the matching m , we obtain in the dual a pseudo - line crossing m on a pseudo - line m that passes through the crossing pr , such that no crossing of m is above m . if the crossing ab is to the left of m in the x - monotone arrangement , then , when traversing a from left to right , one would have to pass below m and then go above it again before m. otherwise , if the crossing ab is to the right of m , then b would have to intersect m to be above it at m and then has to be below m again to reach the crossing ab . hence , half of the pseudo - lines are below the pseudo - line crossing m in a. this corresponds to at least half of the points being on the same side of m as p. recall that in the proof of theorem 5 , we chose a directed line supported by a point ll{q } and either ua or ub such that no elements of l{p , q , ua , ub } is to the left of it . however , this means that the crossing of the pseudo - lines v and w is above ua and ub in a , which contradicts the fact that uaub is m. thus , we conclude theorem 8theorem 5 also holds for non - realizable cc systems , i.e.

cancer represents a set of genetic diseases caused by several mutations in specific genes , leading to genomic instability and consequently to accelerated and uncontrolled cell proliferation.1 the changes occurring in cancerous cells culminate in the production of danger signals resulting in the expression of altered proteins that can be recognized as foreign to the immune system , generating an antitumour response.2 the immune system consists of two main lines of response : innate immunity and adaptive or acquired immunity . innate immunity is composed of cells such as macrophages , dendritic cells ( dcs ) , natural killer ( nk ) cells , neutrophils , basophils , eosinophils , and mast cells , which can be characterized by their relatively fast response , however , with absence of induction of immunologic memory.3,4 stimulation of the innate immune response leads to activation of acquired immunity , composed of cytotoxic t cells ( cd8 + ) , t helper cells ( cd4 + ) , and b cells . these cells are characterized by presenting a wide variety of antigen receptors and their ability to generate immunologic memory.3,5 dcs are considered to be the most important antigen - presenting cell . through a cross - presentation mechanism , they can activate cytotoxic t lymphocytes ( cd8 + ) as well as t helper cells ( cd4 + ) , with t helper cells being critical in the production of cytokines involved in acquired immunity.68 although the main focus of studies involving the antitumour immune response have been aimed mainly at cytotoxic t lymphocytes , there have been reports that t helper cells ( cd4 + ) can also exhibit a wide range of functions in this type of immune response . some epitopes derived from human tumour antigens recognized by human cd4 + t cells have already been described.9,10 tumour cells can also suppress dc function , thereby causing them to be incapable of stimulating and activating t cells efficiently.11,12 given the importance of these cells , clinical studies related to immunotherapy involving dcs have been performed for various tumour types , with noteworthy results1315 and it was shown that the vaccine was generally well tolerated without significant adverse effects . therefore , given the complexity of inducing an antitumour response , it is critical that we improve our understanding of immune cell behaviour , particularly t lymphocytes , so that possible immunological mechanisms responsible for tumour regression can be inferred . furthermore , such research can enable the development of new and effective immunotherapeutic protocols for the treatment of cancer . this study aimed to elucidate the immune response of t lymphocytes in cancer patients during immunotherapy with autologous dcs . for this purpose , we evaluated peripheral blood samples from the patients before and during immunotherapy for total t lymphocytes , t helper cells , cytotoxic t cells , and the cytokines produced by them [ interleukin ( il)-2 , il-12 , interferon ( ifn)- , tumour necrosis factor ( tnf)- , and il-10 . we also assessed levels of the transcription factor foxp3 and the cell surface marker cd25 , which is essential for identification of regulatory t cells . seven female patients diagnosed with cancer were selected at the research institute for oncology ( ipon)/discipline of gynecology and obstetrics at uftm . all individuals involved were fully informed about the intentions of the research study and signed a consent form to confirm their participation ( case number 683 - 2006 ) , in accordance with the research ethics committee requirements . some of the participating patients had previously been treated with conventional methods , such as chemotherapy and/or radiation therapy . as such , in order to participate in the current study and undergo immunotherapy with dcs , such patients were required to wait at least 2 months prior to starting the new treatment . table 1 shows a summary of the general characteristics for each patient participating in the study . mononuclear cells were obtained from peripheral blood samples from the patients recruited to participate in the study . the mononuclear cells were isolated from peripheral blood cells by density gradient and centrifugation in ficoll - hypaque ( bd biosciences ) solution . the cells were re - suspended in 15 ml of imdm , enriched with 5% fetal bovine serum , 1% penicillin / streptomycin , and 1% gentamicin . mononuclear cell cultures were maintained in vitro at 37 c and 5% co2 for 7 days in gm - csf and il-4 to support dc differentiation . the neoplastic cells were lysed by cycles of freezing and used at a concentration of 100 ug / ml . differentiated cells were placed in contact with tumour antigens , obtained from each patient , and subjected to electroporation . dcs activated with tumour antigens were infused by subcutaneous injection into the forearm ( 510 10 cells autologous dcs / patient ) and the procedure was repeated at a mean interval of 15 days . over the course of the study , peripheral blood samples were drawn from the patients and cells were evaluated by flow cytometry ( bd facs calibur cytometer and cell sorter ) . cytometry protocols were deployed in accordance with those suggested by the manufacturer ( bd biosciences ) . the peripheral blood cells were collected for the following markers in all patients : total t ( cd3 + ) , helper t ( cd4 + ) , cytotoxic t cells ( cd8 + ) , and regulatory t cells ( cd25+/foxp3 + ) . the procedure was performed prior to initiating therapy with dcs ( pre - treatment analysis ) and further analysis was performed every 15 days . leukocytes were isolated from peripheral blood samples via centrifugation at 4 c using a standard cell lysing protocol ( bd biosciences for t cell tagging , initially cells were re - suspended in pbs for extracellular tagging . for tagging , -cd3 pe antibodies were used for total t cells , -cd4 pe for t helper cells , -cd8 pe for cytotoxic t cells , and -cd25 pe for regulatory t cells . after extracellular tagging was completed , cells were incubated at 4 c for 30 min , and then rinsed twice by centrifugation with pbs . following the rinses , a fixation and permeabilization solution was added ( bd cytofix / cytoperm ) for duration of 20 min at 4 c . the cells were rinsed twice again with perm / wash ( bd biosciences ) buffer prior to the second tagging . for intracellular t helper cells were also tagged with -il-12 fitc , -il-10 fitc , -ifn- fitc and -tnf- fitc antibodies . following intracellular tagging , cells were incubated once more at 4 c for a 30 min duration . finally , cells were re - suspended in 500 l of pbs for cytometric analysis using bd facscalibur. for an accurate determination of the cells , corresponds to lymphocytes and not other cell types , we determine the region to be analysed by constructing a gate according to a chart control for relative size ( forward scatter fsc ) and granularity and complexity ( side scatter ssc ) in each experiment and for each patient . figure 1 is an illustrative example of how the determination was performed by flow cytometry data . the data are shown as median values , with error bars representing the range from the minimum value ( lower bar ) to the maximum value ( upper bar ) . statistical analysis and graphing were performed using graphpad prism version 5.0 ( graphpad software , inc . ) . seven female patients diagnosed with cancer were selected at the research institute for oncology ( ipon)/discipline of gynecology and obstetrics at uftm . all individuals involved were fully informed about the intentions of the research study and signed a consent form to confirm their participation ( case number 683 - 2006 ) , in accordance with the research ethics committee requirements . some of the participating patients had previously been treated with conventional methods , such as chemotherapy and/or radiation therapy . as such , in order to participate in the current study and undergo immunotherapy with dcs , such patients were required to wait at least 2 months prior to starting the new treatment . table 1 shows a summary of the general characteristics for each patient participating in the study . mononuclear cells were obtained from peripheral blood samples from the patients recruited to participate in the study . the mononuclear cells were isolated from peripheral blood cells by density gradient and centrifugation in ficoll - hypaque ( bd biosciences ) solution . the cells were re - suspended in 15 ml of imdm , enriched with 5% fetal bovine serum , 1% penicillin / streptomycin , and 1% gentamicin . mononuclear cell cultures were maintained in vitro at 37 c and 5% co2 for 7 days in gm - csf and il-4 to support dc differentiation . the neoplastic cells were lysed by cycles of freezing and used at a concentration of 100 ug / ml . differentiated cells were placed in contact with tumour antigens , obtained from each patient , and subjected to electroporation . dcs activated with tumour antigens were infused by subcutaneous injection into the forearm ( 510 10 cells autologous dcs / patient ) and the procedure was repeated at a mean interval of 15 days . over the course of the study , peripheral blood samples were drawn from the patients and cells were evaluated by flow cytometry ( bd facs calibur cytometer and cell sorter ) . cytometry protocols were deployed in accordance with those suggested by the manufacturer ( bd biosciences ) . the peripheral blood cells were collected for the following markers in all patients : total t ( cd3 + ) , helper t ( cd4 + ) , cytotoxic t cells ( cd8 + ) , and regulatory t cells ( cd25+/foxp3 + ) . the procedure was performed prior to initiating therapy with dcs ( pre - treatment analysis ) and further analysis was performed every 15 days . leukocytes were isolated from peripheral blood samples via centrifugation at 4 c using a standard cell lysing protocol ( bd biosciences for t cell tagging , initially cells were re - suspended in pbs for extracellular tagging . for tagging , -cd3 pe antibodies were used for total t cells , -cd4 pe for t helper cells , -cd8 pe for cytotoxic t cells , and -cd25 pe for regulatory t cells . after extracellular tagging was completed , cells were incubated at 4 c for 30 min , and then rinsed twice by centrifugation with pbs . following the rinses , a fixation and permeabilization solution was added ( bd cytofix / cytoperm ) for duration of 20 min at 4 c . the cells were rinsed twice again with perm / wash ( bd biosciences ) buffer prior to the second tagging . for intracellular t helper cells were also tagged with -il-12 fitc , -il-10 fitc , -ifn- fitc and -tnf- fitc antibodies . following intracellular tagging , cells were incubated once more at 4 c for a 30 min duration . finally , cells were re - suspended in 500 l of pbs for cytometric analysis using bd facscalibur. for an accurate determination of the cells , corresponds to lymphocytes and not other cell types , we determine the region to be analysed by constructing a gate according to a chart control for relative size ( forward scatter fsc ) and granularity and complexity ( side scatter ssc ) in each experiment and for each patient . figure 1 is an illustrative example of how the determination was performed by flow cytometry data . the data are shown as median values , with error bars representing the range from the minimum value ( lower bar ) to the maximum value ( upper bar ) . the results were analyzed using the kruskal - wallis nonparametric test ( anova ) . statistical analysis and graphing immunotherapy with dcs was shown to be safe without any significant side effects or toxicity . only patient 2 , who was in treatment for metastatic vaginal melanoma showed worsening of a pre - existing condition ( vitiligo ) after commencing treatment . however , it was not possible to determine whether this worsening occurred as a result of the action of the vaccine or as a result of systemic activation of an immune response . a high percentage of t lymphocytes ( > 60% ) were observed to be cd3 + ( fluorescently labelled ) in the pretreatment analysis and that percentage increased following initiation of treatment until the twelfth analysis , after which a rapid reduction of fluorescent labelling was observed ( p = 0.3986 ) . this finding is contrary to the results observed for the other cell types investigated as cd4 + , cd8 + and cd25+foxp3 + ( described below ) , which fell to nearly to zero ( fig . cd4 + and cd8 + t lymphocytes showed similar patterns of il-2 expression , with an initial increase in the percentage of fluorescently labelled cells after initiation of treatment with dcs . the early increase persisted until the seventh and tenth analysis , respectively , and then was followed by a reduction . these treatment effects were highly significant for both cell populations ( p = 0.0044 ; p = 0.0191 , respectively ) ( fig . 2b and 2c ) . cd4 + t lymphocytes showed a trend toward an increase in percentage of ifn--positive ( fluorescent labelled ) cells upon initiation of immunotherapy , until the seventh analyse and that upward trend was followed by a reduction ( p = 0.5688 ) ( fig . similar patterns of results were obtained for the expression of tnf- and il-12 by cd4 + t cells ( fig . 1 ) , with a rapid initial increase that was significant for tnf- ( p = 0.0419 ) ( fig . 3b ) , but only a weak trend for il-12 ( p = 0.6774 ) ( fig . these cells exhibited a significant increase in il-10 expression , as evidenced by the percentage fluorescent cells , after treatment initiation ( p = 0.0111 ) , followed by a tendency toward reduction after the eighth analysis ( fig . 3d ) . with regard to the percentage of regulatory t cells ( cd25 + , foxp3 + ) , no considerable stimulation was observed ( p = 0.0278 ) ( fig . immunotherapy with dcs was shown to be safe without any significant side effects or toxicity . only patient 2 , who was in treatment for metastatic vaginal melanoma showed worsening of a pre - existing condition ( vitiligo ) after commencing treatment . however , it was not possible to determine whether this worsening occurred as a result of the action of the vaccine or as a result of systemic activation of an immune response . a high percentage of t lymphocytes ( > 60% ) were observed to be cd3 + ( fluorescently labelled ) in the pretreatment analysis and that percentage increased following initiation of treatment until the twelfth analysis , after which a rapid reduction of fluorescent labelling was observed ( p = 0.3986 ) . this finding is contrary to the results observed for the other cell types investigated as cd4 + , cd8 + and cd25+foxp3 + ( described below ) , which fell to nearly to zero ( fig . cd4 + and cd8 + t lymphocytes showed similar patterns of il-2 expression , with an initial increase in the percentage of fluorescently labelled cells after initiation of treatment with dcs . the early increase persisted until the seventh and tenth analysis , respectively , and then was followed by a reduction . these treatment effects were highly significant for both cell populations ( p = 0.0044 ; p = 0.0191 , respectively ) ( fig . 2b and 2c ) . cd4 + t lymphocytes showed a trend toward an increase in percentage of ifn--positive ( fluorescent labelled ) cells upon initiation of immunotherapy , until the seventh analyse and that upward trend was followed by a reduction ( p = 0.5688 ) ( fig . similar patterns of results were obtained for the expression of tnf- and il-12 by cd4 + t cells ( fig . 1 ) , with a rapid initial increase that was significant for tnf- ( p = 0.0419 ) ( fig . 3b ) , but only a weak trend for il-12 ( p = 0.6774 ) ( fig . these cells exhibited a significant increase in il-10 expression , as evidenced by the percentage fluorescent cells , after treatment initiation ( p = 0.0111 ) , followed by a tendency toward reduction after the eighth analysis ( fig . 3d ) . with regard to the percentage of regulatory t cells ( cd25 + , foxp3 + ) , no considerable stimulation was observed ( p = 0.0278 ) ( fig . in recent years , dcs immunogenic potential for immunotherapy has been the focus of a multitude of studies . the effects of these cells on the host s immunity hold the key to further development of this work . the study of immune response in cancer patients is difficult to be carried by several factors . the immune response is already a complex factor being studied in humans because this system is highly dependent on the genetics of each individual . the selection of patients depends on that all therapies have been used without success , the consent of the patient and/or family and a good general physical condition . another point is the variation in the levels of cytokines in humans , which is very variable , however , likely to demonstrate statistical significance the examination in some tests . the immunotherapy using autologous dcs developed in this study was well tolerated , and no significant side effects were observed to the vaccines . only one patient experienced a mild worsening of a pre - existing case of vitiligo , which is known to be a common autoimmune response against melanocytes during treatment of melanoma.16,17 with regards to the population of t helper cells ( cd4 + ) expressing ifn- , it was observed an increased percentage of fluorescently labelled ifn--positive cells , after the start of immunotherapy with dcs . the present study also showed that cd4 + t cells do exhibit a significant increase in tnf- expression after stimulation with dc immunotherapy . fong et al18 assessed the effects of dc vaccines via different routes of administration ( intradermal , intravenous and intralymphatic routes ) and detected the production of ifn- by intradermal and intralymphatic routes . however , they found low tnf- expression levels in 1/6 patients treated with an intravenous dc vaccine.18 mumberg et al19 deduced that cytokine ifn- plays a central role in the elimination of tumour cells by cd4 + t cells , however , this effect seemed to be indirect , since the tumour cells remained negative for mhc class ii molecules after stimulation with ifn-. tnf- is a multifunctional cytokine and a major mediator of inflammation . it can be detected in stromal and malignant cells in several types of cancer.20 chronic production of this cytokine in the neoplastic micro - environment can induce tumour development and spread . in contrast , tnf- is also important in cell destruction mediated by nk cells and cd8 + t cells.21,22 hence , tnf- is functionally pleiotropic , as it is able to engage in responses that are tumour promoting or tumour suppressing depending on the stimuli received by the cells . an interesting finding of this work and unpublished in the literature is that we observed a slight stimulation of il-12 expression by cd4 + t lymphocytes after the onset of dc immunotherapy . both the il-12 and ifn- cytokines are important for stimulation of the th1 immune response.23 in addition , the il-12 induces cytotoxic effects in nk and cd8 + cells , aside from stimulating their production of ifn-.24,25 il-10 , in turn , showed a considerable increase after commencement of the treatment . il-10 can be secreted by a variety of different cells,26,27 and although it is most often described in terms of its immunosuppressive action , there are reports describing its effector function on cd8 + t cells.28 il-10 , generally considered to be a th2 cytokine , is capable of stimulating b cell function . on the other hand , there is evidence suggesting that it may be produced by th1 cells,29,30 and that it may also exhibit an antitumour function.31,32 saraiva et al33 demonstrated that in vitro differentiation of il-10-expressing th1 cells was dependent upon the activation of transcription factor stat-4-induced by cytokine il-12 , through a powerful activation via t - cell receptors ( tcrs ) and phosphorylation of extracellular - signal - regulated kinases ( erk ) 1 and 2 . these findings further demonstrate the multifunctional role of il-10 and its dependence on il-12 for its production by th1 cells . in the current study , total t cells demonstrated elevated expression of the cd3 + marker practically throughout the entire period of treatment with dcs . our assessment of il-2 expression by cd4 + and cd8 + t cells also yielded similar results for these two cell types : an early increase in expression after initiation of immunotherapy followed by a decline to nearly zero by the end of the analysis . these findings demonstrate that activation of dcs in vitro can enhance immune effector responses in t cells upon vaccination , while inducing a significant increase in il-2 expression . il-2 is considered to be an important factor in proliferation of t lymphocytes against antigenic stimulation and plays a central role in the regulation of t cells . furthermore , its expression is considered to be indicative of cellular activation.34 our findings corroborate the literature . in a study in which autologous dc vaccination was administered to patients with metastatic breast cancer and renal cancer , avigan et al35 observed an increased in the percentage of cd4 + and cd8 + t cells that was related to increased expression of the cytokine ifn- , which apparently led to induction of antitumour activity . in a study examining advanced stage breast and ovarian cancer patients who were vaccinated with autologous dcs , brossart et al36 described observing a specific antigenic response in 5/10 patients , as evidenced by ifn- production by cytotoxic t lymphocytes . the tendency for there to be a sharp reduction in il-2-tagged cd4 + and cd8 + t cells after a certain time in treatment , despite sustained high levels of total t cell marker ( cd3 + ) expression , suggests that there may be another cd3 + cell population involved . two cells types can likely be t/3739 and nk t - cells.40,41 both populations are identified as having regulatory and protective functions in cancer.4246 thus , investigating these cell types may help explain the reduced immune response that is observed after some time of immunotherapeutic stimulation , as well as the conservation of marker cd3 + . we did not observe evidence of stimulation of cd25+/foxp3 + regulatory cells in this study . there was only a higher maximum value in the pre - therapy analysis , relative to the maximum values observed during treatment . thus , we can conclude that although a decrease in immune response was observed vis - - vis the assessed markers , which suggests that immunosuppression did occur , these cell types were not responsible for the findings . on the contrary , others have demonstrated an induction of regulatory t cells after immunotherapy with dcs . according to berntsen et al,47 significantly increased levels of cd4+/cd25+/foxp3 + treg cells ( vs. pretreatment levels ) were found in peripheral blood samples of patients with metastatic renal carcinomas treated with a dc vaccine combined with cytokine il-2 . our results indicate that there is a stimulation of the response of cd4 + t cells expressing il-2 , ifn- , and tnf- , as well as of il-2-expressing cd8 + t cells , suggesting that immunotherapy with dcs has the ability to induce a th1 response . as such , the immune system is probably acting as an effector against tumour cells . in the present study , some of the patients involved were treated for a prolonged period of time and given the vaccine and immunological stimulation on several occasions . one explanation for the diminished response over the course of treatment could be due to selection for tumour cell variants with immunoresistant phenotypes.48 additionally , after a certain period of vaccination , there could have been a loss in expression of mhc class i molecules by tumour antigens , a major mechanism of immune system evasion.49 the maturation stage of dcs is also a determining factor for directing a th1 response , as immature dcs secrete il-10 leading to a th2 response.50,51 as such , it is important to continuously review the therapeutic protocol being used and to seek innovative strategies to prevent the development of new tumour escape mechanisms in order to maintain a good response during the treatment period . it should be noted that the present study was based on the analysis of the percentage of fluorescently labelled cells for each assessed marker , and that the analysis of fluorescence intensity was important . thus , although there may be a numerical decline in tagged cells after a certain period of treatment , it is possible that there could still have been an increase in the numbers of assessed markers . in other words , even though there may be a smaller number of cells , each cell might be expressing a larger number of surface markers or producing a larger quantity of cytokines . given the important implications of this possibility , follow up studies are already underway . in conclusion , upon assessing immunologic response before and during treatment , we observed that immunotherapy with dcs was capable of stimulating the immune system in cancer patients . this finding was evidenced by observations of increased percentages fluorescently labelled cells for markers of the major cell populations studied . upon immunotherapeutic intervention with dcs , activated in vitro and pulsed with tumour specific antigens for each patient , an increase in the percent fluorescently labelled cells was observed for the majority of cell populations assessed . the increases were particularly significant for t helper cells expressing tnf- , il-2 , il-10 , and for cytotoxic t cells expressing il-2 . the findings of this study indicate that after a certain period of immunologic stimulation against tumour antigens , there is a reduction in the immunologic response .

introduction : cancer stems from mutations in specific genes that induce uncontrolled cell proliferation . dendritic cells ( dcs ) are important immunologic cells and play a crucial role in the induction of an antitumour response.patients and methods : we examined the immune response mediated by t lymphocytes , helper t cells , cytotoxic t cells , and regulatory t cells , as well as the cytokines [ interleukin ( il)-2 , il-12 , interferon ( ifn)- , tumour necrosis factor ( tnf)- and il-10 ] , produced by these cell populations , in cancer patients ( n = 7 ) undergoing immunotheraphy with autologous dcs.results:we observed an initial increase in t helper cells ( cd4 + ) expressing il-2 , ifn- , il-12 , tnf- , and il-10 after initiation of treatment , with statistically significant for the cytokines il-2 , tnf- and il-10 . a similar significant effect was observed for il-2-expressing cytotoxic t cells ( cd8 + ) . the percentage of total t cells ( cd3 + ) remained elevated throughout immunotherapy . regulatory t cells ( cd25+/foxp3 + ) only showed high percentage of their maximum value when analyzed the pretreatment levels , with statistically significant.conclusion:immunotherapy with dcs stimulated the immune response , as evidenced by an increase in percent fluorescence of most cell populations investigated during the specified treatment period .

Introduction Methods Patients Acquisition of mononuclear cells DC vaccines Flow cytometry Statistical analysis Results Safety and toxicity of the vaccine Cell population analysis using flow cytometry Discussion and Conclusion

cancer represents a set of genetic diseases caused by several mutations in specific genes , leading to genomic instability and consequently to accelerated and uncontrolled cell proliferation.1 the changes occurring in cancerous cells culminate in the production of danger signals resulting in the expression of altered proteins that can be recognized as foreign to the immune system , generating an antitumour response.2 the immune system consists of two main lines of response : innate immunity and adaptive or acquired immunity . innate immunity is composed of cells such as macrophages , dendritic cells ( dcs ) , natural killer ( nk ) cells , neutrophils , basophils , eosinophils , and mast cells , which can be characterized by their relatively fast response , however , with absence of induction of immunologic memory.3,4 stimulation of the innate immune response leads to activation of acquired immunity , composed of cytotoxic t cells ( cd8 + ) , t helper cells ( cd4 + ) , and b cells . through a cross - presentation mechanism , they can activate cytotoxic t lymphocytes ( cd8 + ) as well as t helper cells ( cd4 + ) , with t helper cells being critical in the production of cytokines involved in acquired immunity.68 although the main focus of studies involving the antitumour immune response have been aimed mainly at cytotoxic t lymphocytes , there have been reports that t helper cells ( cd4 + ) can also exhibit a wide range of functions in this type of immune response . some epitopes derived from human tumour antigens recognized by human cd4 + t cells have already been described.9,10 tumour cells can also suppress dc function , thereby causing them to be incapable of stimulating and activating t cells efficiently.11,12 given the importance of these cells , clinical studies related to immunotherapy involving dcs have been performed for various tumour types , with noteworthy results1315 and it was shown that the vaccine was generally well tolerated without significant adverse effects . therefore , given the complexity of inducing an antitumour response , it is critical that we improve our understanding of immune cell behaviour , particularly t lymphocytes , so that possible immunological mechanisms responsible for tumour regression can be inferred . furthermore , such research can enable the development of new and effective immunotherapeutic protocols for the treatment of cancer . this study aimed to elucidate the immune response of t lymphocytes in cancer patients during immunotherapy with autologous dcs . for this purpose , we evaluated peripheral blood samples from the patients before and during immunotherapy for total t lymphocytes , t helper cells , cytotoxic t cells , and the cytokines produced by them [ interleukin ( il)-2 , il-12 , interferon ( ifn)- , tumour necrosis factor ( tnf)- , and il-10 . we also assessed levels of the transcription factor foxp3 and the cell surface marker cd25 , which is essential for identification of regulatory t cells . all individuals involved were fully informed about the intentions of the research study and signed a consent form to confirm their participation ( case number 683 - 2006 ) , in accordance with the research ethics committee requirements . as such , in order to participate in the current study and undergo immunotherapy with dcs , such patients were required to wait at least 2 months prior to starting the new treatment . the cells were re - suspended in 15 ml of imdm , enriched with 5% fetal bovine serum , 1% penicillin / streptomycin , and 1% gentamicin . dcs activated with tumour antigens were infused by subcutaneous injection into the forearm ( 510 10 cells autologous dcs / patient ) and the procedure was repeated at a mean interval of 15 days . over the course of the study , peripheral blood samples were drawn from the patients and cells were evaluated by flow cytometry ( bd facs calibur cytometer and cell sorter ) . the peripheral blood cells were collected for the following markers in all patients : total t ( cd3 + ) , helper t ( cd4 + ) , cytotoxic t cells ( cd8 + ) , and regulatory t cells ( cd25+/foxp3 + ) . for tagging , -cd3 pe antibodies were used for total t cells , -cd4 pe for t helper cells , -cd8 pe for cytotoxic t cells , and -cd25 pe for regulatory t cells . after extracellular tagging was completed , cells were incubated at 4 c for 30 min , and then rinsed twice by centrifugation with pbs . for intracellular t helper cells were also tagged with -il-12 fitc , -il-10 fitc , -ifn- fitc and -tnf- fitc antibodies . for an accurate determination of the cells , corresponds to lymphocytes and not other cell types , we determine the region to be analysed by constructing a gate according to a chart control for relative size ( forward scatter fsc ) and granularity and complexity ( side scatter ssc ) in each experiment and for each patient . the data are shown as median values , with error bars representing the range from the minimum value ( lower bar ) to the maximum value ( upper bar ) . all individuals involved were fully informed about the intentions of the research study and signed a consent form to confirm their participation ( case number 683 - 2006 ) , in accordance with the research ethics committee requirements . as such , in order to participate in the current study and undergo immunotherapy with dcs , such patients were required to wait at least 2 months prior to starting the new treatment . differentiated cells were placed in contact with tumour antigens , obtained from each patient , and subjected to electroporation . dcs activated with tumour antigens were infused by subcutaneous injection into the forearm ( 510 10 cells autologous dcs / patient ) and the procedure was repeated at a mean interval of 15 days . the peripheral blood cells were collected for the following markers in all patients : total t ( cd3 + ) , helper t ( cd4 + ) , cytotoxic t cells ( cd8 + ) , and regulatory t cells ( cd25+/foxp3 + ) . the procedure was performed prior to initiating therapy with dcs ( pre - treatment analysis ) and further analysis was performed every 15 days . for tagging , -cd3 pe antibodies were used for total t cells , -cd4 pe for t helper cells , -cd8 pe for cytotoxic t cells , and -cd25 pe for regulatory t cells . after extracellular tagging was completed , cells were incubated at 4 c for 30 min , and then rinsed twice by centrifugation with pbs . for intracellular t helper cells were also tagged with -il-12 fitc , -il-10 fitc , -ifn- fitc and -tnf- fitc antibodies . the data are shown as median values , with error bars representing the range from the minimum value ( lower bar ) to the maximum value ( upper bar ) . statistical analysis and graphing immunotherapy with dcs was shown to be safe without any significant side effects or toxicity . however , it was not possible to determine whether this worsening occurred as a result of the action of the vaccine or as a result of systemic activation of an immune response . a high percentage of t lymphocytes ( > 60% ) were observed to be cd3 + ( fluorescently labelled ) in the pretreatment analysis and that percentage increased following initiation of treatment until the twelfth analysis , after which a rapid reduction of fluorescent labelling was observed ( p = 0.3986 ) . this finding is contrary to the results observed for the other cell types investigated as cd4 + , cd8 + and cd25+foxp3 + ( described below ) , which fell to nearly to zero ( fig . cd4 + and cd8 + t lymphocytes showed similar patterns of il-2 expression , with an initial increase in the percentage of fluorescently labelled cells after initiation of treatment with dcs . the early increase persisted until the seventh and tenth analysis , respectively , and then was followed by a reduction . these treatment effects were highly significant for both cell populations ( p = 0.0044 ; p = 0.0191 , respectively ) ( fig . cd4 + t lymphocytes showed a trend toward an increase in percentage of ifn--positive ( fluorescent labelled ) cells upon initiation of immunotherapy , until the seventh analyse and that upward trend was followed by a reduction ( p = 0.5688 ) ( fig . similar patterns of results were obtained for the expression of tnf- and il-12 by cd4 + t cells ( fig . 1 ) , with a rapid initial increase that was significant for tnf- ( p = 0.0419 ) ( fig . these cells exhibited a significant increase in il-10 expression , as evidenced by the percentage fluorescent cells , after treatment initiation ( p = 0.0111 ) , followed by a tendency toward reduction after the eighth analysis ( fig . with regard to the percentage of regulatory t cells ( cd25 + , foxp3 + ) , no considerable stimulation was observed ( p = 0.0278 ) ( fig . however , it was not possible to determine whether this worsening occurred as a result of the action of the vaccine or as a result of systemic activation of an immune response . a high percentage of t lymphocytes ( > 60% ) were observed to be cd3 + ( fluorescently labelled ) in the pretreatment analysis and that percentage increased following initiation of treatment until the twelfth analysis , after which a rapid reduction of fluorescent labelling was observed ( p = 0.3986 ) . this finding is contrary to the results observed for the other cell types investigated as cd4 + , cd8 + and cd25+foxp3 + ( described below ) , which fell to nearly to zero ( fig . cd4 + and cd8 + t lymphocytes showed similar patterns of il-2 expression , with an initial increase in the percentage of fluorescently labelled cells after initiation of treatment with dcs . the early increase persisted until the seventh and tenth analysis , respectively , and then was followed by a reduction . these treatment effects were highly significant for both cell populations ( p = 0.0044 ; p = 0.0191 , respectively ) ( fig . cd4 + t lymphocytes showed a trend toward an increase in percentage of ifn--positive ( fluorescent labelled ) cells upon initiation of immunotherapy , until the seventh analyse and that upward trend was followed by a reduction ( p = 0.5688 ) ( fig . similar patterns of results were obtained for the expression of tnf- and il-12 by cd4 + t cells ( fig . 1 ) , with a rapid initial increase that was significant for tnf- ( p = 0.0419 ) ( fig . these cells exhibited a significant increase in il-10 expression , as evidenced by the percentage fluorescent cells , after treatment initiation ( p = 0.0111 ) , followed by a tendency toward reduction after the eighth analysis ( fig . with regard to the percentage of regulatory t cells ( cd25 + , foxp3 + ) , no considerable stimulation was observed ( p = 0.0278 ) ( fig . the study of immune response in cancer patients is difficult to be carried by several factors . the immune response is already a complex factor being studied in humans because this system is highly dependent on the genetics of each individual . another point is the variation in the levels of cytokines in humans , which is very variable , however , likely to demonstrate statistical significance the examination in some tests . the immunotherapy using autologous dcs developed in this study was well tolerated , and no significant side effects were observed to the vaccines . only one patient experienced a mild worsening of a pre - existing case of vitiligo , which is known to be a common autoimmune response against melanocytes during treatment of melanoma.16,17 with regards to the population of t helper cells ( cd4 + ) expressing ifn- , it was observed an increased percentage of fluorescently labelled ifn--positive cells , after the start of immunotherapy with dcs . the present study also showed that cd4 + t cells do exhibit a significant increase in tnf- expression after stimulation with dc immunotherapy . however , they found low tnf- expression levels in 1/6 patients treated with an intravenous dc vaccine.18 mumberg et al19 deduced that cytokine ifn- plays a central role in the elimination of tumour cells by cd4 + t cells , however , this effect seemed to be indirect , since the tumour cells remained negative for mhc class ii molecules after stimulation with ifn-. it can be detected in stromal and malignant cells in several types of cancer.20 chronic production of this cytokine in the neoplastic micro - environment can induce tumour development and spread . in contrast , tnf- is also important in cell destruction mediated by nk cells and cd8 + t cells.21,22 hence , tnf- is functionally pleiotropic , as it is able to engage in responses that are tumour promoting or tumour suppressing depending on the stimuli received by the cells . an interesting finding of this work and unpublished in the literature is that we observed a slight stimulation of il-12 expression by cd4 + t lymphocytes after the onset of dc immunotherapy . both the il-12 and ifn- cytokines are important for stimulation of the th1 immune response.23 in addition , the il-12 induces cytotoxic effects in nk and cd8 + cells , aside from stimulating their production of ifn-.24,25 il-10 , in turn , showed a considerable increase after commencement of the treatment . il-10 can be secreted by a variety of different cells,26,27 and although it is most often described in terms of its immunosuppressive action , there are reports describing its effector function on cd8 + t cells.28 il-10 , generally considered to be a th2 cytokine , is capable of stimulating b cell function . on the other hand , there is evidence suggesting that it may be produced by th1 cells,29,30 and that it may also exhibit an antitumour function.31,32 saraiva et al33 demonstrated that in vitro differentiation of il-10-expressing th1 cells was dependent upon the activation of transcription factor stat-4-induced by cytokine il-12 , through a powerful activation via t - cell receptors ( tcrs ) and phosphorylation of extracellular - signal - regulated kinases ( erk ) 1 and 2 . in the current study , total t cells demonstrated elevated expression of the cd3 + marker practically throughout the entire period of treatment with dcs . our assessment of il-2 expression by cd4 + and cd8 + t cells also yielded similar results for these two cell types : an early increase in expression after initiation of immunotherapy followed by a decline to nearly zero by the end of the analysis . these findings demonstrate that activation of dcs in vitro can enhance immune effector responses in t cells upon vaccination , while inducing a significant increase in il-2 expression . il-2 is considered to be an important factor in proliferation of t lymphocytes against antigenic stimulation and plays a central role in the regulation of t cells . in a study in which autologous dc vaccination was administered to patients with metastatic breast cancer and renal cancer , avigan et al35 observed an increased in the percentage of cd4 + and cd8 + t cells that was related to increased expression of the cytokine ifn- , which apparently led to induction of antitumour activity . in a study examining advanced stage breast and ovarian cancer patients who were vaccinated with autologous dcs , brossart et al36 described observing a specific antigenic response in 5/10 patients , as evidenced by ifn- production by cytotoxic t lymphocytes . the tendency for there to be a sharp reduction in il-2-tagged cd4 + and cd8 + t cells after a certain time in treatment , despite sustained high levels of total t cell marker ( cd3 + ) expression , suggests that there may be another cd3 + cell population involved . two cells types can likely be t/3739 and nk t - cells.40,41 both populations are identified as having regulatory and protective functions in cancer.4246 thus , investigating these cell types may help explain the reduced immune response that is observed after some time of immunotherapeutic stimulation , as well as the conservation of marker cd3 + . we did not observe evidence of stimulation of cd25+/foxp3 + regulatory cells in this study . there was only a higher maximum value in the pre - therapy analysis , relative to the maximum values observed during treatment . thus , we can conclude that although a decrease in immune response was observed vis - - vis the assessed markers , which suggests that immunosuppression did occur , these cell types were not responsible for the findings . on the contrary , others have demonstrated an induction of regulatory t cells after immunotherapy with dcs . according to berntsen et al,47 significantly increased levels of cd4+/cd25+/foxp3 + treg cells ( vs. pretreatment levels ) were found in peripheral blood samples of patients with metastatic renal carcinomas treated with a dc vaccine combined with cytokine il-2 . our results indicate that there is a stimulation of the response of cd4 + t cells expressing il-2 , ifn- , and tnf- , as well as of il-2-expressing cd8 + t cells , suggesting that immunotherapy with dcs has the ability to induce a th1 response . in the present study , some of the patients involved were treated for a prolonged period of time and given the vaccine and immunological stimulation on several occasions . one explanation for the diminished response over the course of treatment could be due to selection for tumour cell variants with immunoresistant phenotypes.48 additionally , after a certain period of vaccination , there could have been a loss in expression of mhc class i molecules by tumour antigens , a major mechanism of immune system evasion.49 the maturation stage of dcs is also a determining factor for directing a th1 response , as immature dcs secrete il-10 leading to a th2 response.50,51 as such , it is important to continuously review the therapeutic protocol being used and to seek innovative strategies to prevent the development of new tumour escape mechanisms in order to maintain a good response during the treatment period . it should be noted that the present study was based on the analysis of the percentage of fluorescently labelled cells for each assessed marker , and that the analysis of fluorescence intensity was important . thus , although there may be a numerical decline in tagged cells after a certain period of treatment , it is possible that there could still have been an increase in the numbers of assessed markers . in other words , even though there may be a smaller number of cells , each cell might be expressing a larger number of surface markers or producing a larger quantity of cytokines . in conclusion , upon assessing immunologic response before and during treatment , we observed that immunotherapy with dcs was capable of stimulating the immune system in cancer patients . this finding was evidenced by observations of increased percentages fluorescently labelled cells for markers of the major cell populations studied . upon immunotherapeutic intervention with dcs , activated in vitro and pulsed with tumour specific antigens for each patient , an increase in the percent fluorescently labelled cells was observed for the majority of cell populations assessed . the increases were particularly significant for t helper cells expressing tnf- , il-2 , il-10 , and for cytotoxic t cells expressing il-2 . the findings of this study indicate that after a certain period of immunologic stimulation against tumour antigens , there is a reduction in the immunologic response .

cancer represents a set of genetic diseases caused by several mutations in specific genes , leading to genomic instability and consequently to accelerated and uncontrolled cell proliferation.1 the changes occurring in cancerous cells culminate in the production of danger signals resulting in the expression of altered proteins that can be recognized as foreign to the immune system , generating an antitumour response.2 the immune system consists of two main lines of response : innate immunity and adaptive or acquired immunity . innate immunity is composed of cells such as macrophages , dendritic cells ( dcs ) , natural killer ( nk ) cells , neutrophils , basophils , eosinophils , and mast cells , which can be characterized by their relatively fast response , however , with absence of induction of immunologic memory.3,4 stimulation of the innate immune response leads to activation of acquired immunity , composed of cytotoxic t cells ( cd8 + ) , t helper cells ( cd4 + ) , and b cells . these cells are characterized by presenting a wide variety of antigen receptors and their ability to generate immunologic memory.3,5 dcs are considered to be the most important antigen - presenting cell . through a cross - presentation mechanism , they can activate cytotoxic t lymphocytes ( cd8 + ) as well as t helper cells ( cd4 + ) , with t helper cells being critical in the production of cytokines involved in acquired immunity.68 although the main focus of studies involving the antitumour immune response have been aimed mainly at cytotoxic t lymphocytes , there have been reports that t helper cells ( cd4 + ) can also exhibit a wide range of functions in this type of immune response . some epitopes derived from human tumour antigens recognized by human cd4 + t cells have already been described.9,10 tumour cells can also suppress dc function , thereby causing them to be incapable of stimulating and activating t cells efficiently.11,12 given the importance of these cells , clinical studies related to immunotherapy involving dcs have been performed for various tumour types , with noteworthy results1315 and it was shown that the vaccine was generally well tolerated without significant adverse effects . therefore , given the complexity of inducing an antitumour response , it is critical that we improve our understanding of immune cell behaviour , particularly t lymphocytes , so that possible immunological mechanisms responsible for tumour regression can be inferred . furthermore , such research can enable the development of new and effective immunotherapeutic protocols for the treatment of cancer . this study aimed to elucidate the immune response of t lymphocytes in cancer patients during immunotherapy with autologous dcs . for this purpose , we evaluated peripheral blood samples from the patients before and during immunotherapy for total t lymphocytes , t helper cells , cytotoxic t cells , and the cytokines produced by them [ interleukin ( il)-2 , il-12 , interferon ( ifn)- , tumour necrosis factor ( tnf)- , and il-10 . we also assessed levels of the transcription factor foxp3 and the cell surface marker cd25 , which is essential for identification of regulatory t cells . seven female patients diagnosed with cancer were selected at the research institute for oncology ( ipon)/discipline of gynecology and obstetrics at uftm . all individuals involved were fully informed about the intentions of the research study and signed a consent form to confirm their participation ( case number 683 - 2006 ) , in accordance with the research ethics committee requirements . some of the participating patients had previously been treated with conventional methods , such as chemotherapy and/or radiation therapy . as such , in order to participate in the current study and undergo immunotherapy with dcs , such patients were required to wait at least 2 months prior to starting the new treatment . table 1 shows a summary of the general characteristics for each patient participating in the study . the cells were re - suspended in 15 ml of imdm , enriched with 5% fetal bovine serum , 1% penicillin / streptomycin , and 1% gentamicin . mononuclear cell cultures were maintained in vitro at 37 c and 5% co2 for 7 days in gm - csf and il-4 to support dc differentiation . dcs activated with tumour antigens were infused by subcutaneous injection into the forearm ( 510 10 cells autologous dcs / patient ) and the procedure was repeated at a mean interval of 15 days . over the course of the study , peripheral blood samples were drawn from the patients and cells were evaluated by flow cytometry ( bd facs calibur cytometer and cell sorter ) . the peripheral blood cells were collected for the following markers in all patients : total t ( cd3 + ) , helper t ( cd4 + ) , cytotoxic t cells ( cd8 + ) , and regulatory t cells ( cd25+/foxp3 + ) . the procedure was performed prior to initiating therapy with dcs ( pre - treatment analysis ) and further analysis was performed every 15 days . leukocytes were isolated from peripheral blood samples via centrifugation at 4 c using a standard cell lysing protocol ( bd biosciences for t cell tagging , initially cells were re - suspended in pbs for extracellular tagging . for tagging , -cd3 pe antibodies were used for total t cells , -cd4 pe for t helper cells , -cd8 pe for cytotoxic t cells , and -cd25 pe for regulatory t cells . for an accurate determination of the cells , corresponds to lymphocytes and not other cell types , we determine the region to be analysed by constructing a gate according to a chart control for relative size ( forward scatter fsc ) and granularity and complexity ( side scatter ssc ) in each experiment and for each patient . seven female patients diagnosed with cancer were selected at the research institute for oncology ( ipon)/discipline of gynecology and obstetrics at uftm . all individuals involved were fully informed about the intentions of the research study and signed a consent form to confirm their participation ( case number 683 - 2006 ) , in accordance with the research ethics committee requirements . some of the participating patients had previously been treated with conventional methods , such as chemotherapy and/or radiation therapy . as such , in order to participate in the current study and undergo immunotherapy with dcs , such patients were required to wait at least 2 months prior to starting the new treatment . table 1 shows a summary of the general characteristics for each patient participating in the study . mononuclear cells were obtained from peripheral blood samples from the patients recruited to participate in the study . the cells were re - suspended in 15 ml of imdm , enriched with 5% fetal bovine serum , 1% penicillin / streptomycin , and 1% gentamicin . mononuclear cell cultures were maintained in vitro at 37 c and 5% co2 for 7 days in gm - csf and il-4 to support dc differentiation . dcs activated with tumour antigens were infused by subcutaneous injection into the forearm ( 510 10 cells autologous dcs / patient ) and the procedure was repeated at a mean interval of 15 days . over the course of the study , peripheral blood samples were drawn from the patients and cells were evaluated by flow cytometry ( bd facs calibur cytometer and cell sorter ) . the peripheral blood cells were collected for the following markers in all patients : total t ( cd3 + ) , helper t ( cd4 + ) , cytotoxic t cells ( cd8 + ) , and regulatory t cells ( cd25+/foxp3 + ) . the procedure was performed prior to initiating therapy with dcs ( pre - treatment analysis ) and further analysis was performed every 15 days . for tagging , -cd3 pe antibodies were used for total t cells , -cd4 pe for t helper cells , -cd8 pe for cytotoxic t cells , and -cd25 pe for regulatory t cells . for an accurate determination of the cells , corresponds to lymphocytes and not other cell types , we determine the region to be analysed by constructing a gate according to a chart control for relative size ( forward scatter fsc ) and granularity and complexity ( side scatter ssc ) in each experiment and for each patient . only patient 2 , who was in treatment for metastatic vaginal melanoma showed worsening of a pre - existing condition ( vitiligo ) after commencing treatment . however , it was not possible to determine whether this worsening occurred as a result of the action of the vaccine or as a result of systemic activation of an immune response . a high percentage of t lymphocytes ( > 60% ) were observed to be cd3 + ( fluorescently labelled ) in the pretreatment analysis and that percentage increased following initiation of treatment until the twelfth analysis , after which a rapid reduction of fluorescent labelling was observed ( p = 0.3986 ) . this finding is contrary to the results observed for the other cell types investigated as cd4 + , cd8 + and cd25+foxp3 + ( described below ) , which fell to nearly to zero ( fig . cd4 + and cd8 + t lymphocytes showed similar patterns of il-2 expression , with an initial increase in the percentage of fluorescently labelled cells after initiation of treatment with dcs . these treatment effects were highly significant for both cell populations ( p = 0.0044 ; p = 0.0191 , respectively ) ( fig . cd4 + t lymphocytes showed a trend toward an increase in percentage of ifn--positive ( fluorescent labelled ) cells upon initiation of immunotherapy , until the seventh analyse and that upward trend was followed by a reduction ( p = 0.5688 ) ( fig . similar patterns of results were obtained for the expression of tnf- and il-12 by cd4 + t cells ( fig . 1 ) , with a rapid initial increase that was significant for tnf- ( p = 0.0419 ) ( fig . these cells exhibited a significant increase in il-10 expression , as evidenced by the percentage fluorescent cells , after treatment initiation ( p = 0.0111 ) , followed by a tendency toward reduction after the eighth analysis ( fig . with regard to the percentage of regulatory t cells ( cd25 + , foxp3 + ) , no considerable stimulation was observed ( p = 0.0278 ) ( fig . only patient 2 , who was in treatment for metastatic vaginal melanoma showed worsening of a pre - existing condition ( vitiligo ) after commencing treatment . however , it was not possible to determine whether this worsening occurred as a result of the action of the vaccine or as a result of systemic activation of an immune response . a high percentage of t lymphocytes ( > 60% ) were observed to be cd3 + ( fluorescently labelled ) in the pretreatment analysis and that percentage increased following initiation of treatment until the twelfth analysis , after which a rapid reduction of fluorescent labelling was observed ( p = 0.3986 ) . this finding is contrary to the results observed for the other cell types investigated as cd4 + , cd8 + and cd25+foxp3 + ( described below ) , which fell to nearly to zero ( fig . cd4 + and cd8 + t lymphocytes showed similar patterns of il-2 expression , with an initial increase in the percentage of fluorescently labelled cells after initiation of treatment with dcs . these treatment effects were highly significant for both cell populations ( p = 0.0044 ; p = 0.0191 , respectively ) ( fig . cd4 + t lymphocytes showed a trend toward an increase in percentage of ifn--positive ( fluorescent labelled ) cells upon initiation of immunotherapy , until the seventh analyse and that upward trend was followed by a reduction ( p = 0.5688 ) ( fig . similar patterns of results were obtained for the expression of tnf- and il-12 by cd4 + t cells ( fig . these cells exhibited a significant increase in il-10 expression , as evidenced by the percentage fluorescent cells , after treatment initiation ( p = 0.0111 ) , followed by a tendency toward reduction after the eighth analysis ( fig . with regard to the percentage of regulatory t cells ( cd25 + , foxp3 + ) , no considerable stimulation was observed ( p = 0.0278 ) ( fig . the effects of these cells on the host s immunity hold the key to further development of this work . the study of immune response in cancer patients is difficult to be carried by several factors . the selection of patients depends on that all therapies have been used without success , the consent of the patient and/or family and a good general physical condition . another point is the variation in the levels of cytokines in humans , which is very variable , however , likely to demonstrate statistical significance the examination in some tests . only one patient experienced a mild worsening of a pre - existing case of vitiligo , which is known to be a common autoimmune response against melanocytes during treatment of melanoma.16,17 with regards to the population of t helper cells ( cd4 + ) expressing ifn- , it was observed an increased percentage of fluorescently labelled ifn--positive cells , after the start of immunotherapy with dcs . the present study also showed that cd4 + t cells do exhibit a significant increase in tnf- expression after stimulation with dc immunotherapy . fong et al18 assessed the effects of dc vaccines via different routes of administration ( intradermal , intravenous and intralymphatic routes ) and detected the production of ifn- by intradermal and intralymphatic routes . however , they found low tnf- expression levels in 1/6 patients treated with an intravenous dc vaccine.18 mumberg et al19 deduced that cytokine ifn- plays a central role in the elimination of tumour cells by cd4 + t cells , however , this effect seemed to be indirect , since the tumour cells remained negative for mhc class ii molecules after stimulation with ifn-. it can be detected in stromal and malignant cells in several types of cancer.20 chronic production of this cytokine in the neoplastic micro - environment can induce tumour development and spread . in contrast , tnf- is also important in cell destruction mediated by nk cells and cd8 + t cells.21,22 hence , tnf- is functionally pleiotropic , as it is able to engage in responses that are tumour promoting or tumour suppressing depending on the stimuli received by the cells . an interesting finding of this work and unpublished in the literature is that we observed a slight stimulation of il-12 expression by cd4 + t lymphocytes after the onset of dc immunotherapy . both the il-12 and ifn- cytokines are important for stimulation of the th1 immune response.23 in addition , the il-12 induces cytotoxic effects in nk and cd8 + cells , aside from stimulating their production of ifn-.24,25 il-10 , in turn , showed a considerable increase after commencement of the treatment . il-10 can be secreted by a variety of different cells,26,27 and although it is most often described in terms of its immunosuppressive action , there are reports describing its effector function on cd8 + t cells.28 il-10 , generally considered to be a th2 cytokine , is capable of stimulating b cell function . on the other hand , there is evidence suggesting that it may be produced by th1 cells,29,30 and that it may also exhibit an antitumour function.31,32 saraiva et al33 demonstrated that in vitro differentiation of il-10-expressing th1 cells was dependent upon the activation of transcription factor stat-4-induced by cytokine il-12 , through a powerful activation via t - cell receptors ( tcrs ) and phosphorylation of extracellular - signal - regulated kinases ( erk ) 1 and 2 . these findings further demonstrate the multifunctional role of il-10 and its dependence on il-12 for its production by th1 cells . in the current study , total t cells demonstrated elevated expression of the cd3 + marker practically throughout the entire period of treatment with dcs . our assessment of il-2 expression by cd4 + and cd8 + t cells also yielded similar results for these two cell types : an early increase in expression after initiation of immunotherapy followed by a decline to nearly zero by the end of the analysis . these findings demonstrate that activation of dcs in vitro can enhance immune effector responses in t cells upon vaccination , while inducing a significant increase in il-2 expression . in a study in which autologous dc vaccination was administered to patients with metastatic breast cancer and renal cancer , avigan et al35 observed an increased in the percentage of cd4 + and cd8 + t cells that was related to increased expression of the cytokine ifn- , which apparently led to induction of antitumour activity . in a study examining advanced stage breast and ovarian cancer patients who were vaccinated with autologous dcs , brossart et al36 described observing a specific antigenic response in 5/10 patients , as evidenced by ifn- production by cytotoxic t lymphocytes . the tendency for there to be a sharp reduction in il-2-tagged cd4 + and cd8 + t cells after a certain time in treatment , despite sustained high levels of total t cell marker ( cd3 + ) expression , suggests that there may be another cd3 + cell population involved . two cells types can likely be t/3739 and nk t - cells.40,41 both populations are identified as having regulatory and protective functions in cancer.4246 thus , investigating these cell types may help explain the reduced immune response that is observed after some time of immunotherapeutic stimulation , as well as the conservation of marker cd3 + . thus , we can conclude that although a decrease in immune response was observed vis - - vis the assessed markers , which suggests that immunosuppression did occur , these cell types were not responsible for the findings . according to berntsen et al,47 significantly increased levels of cd4+/cd25+/foxp3 + treg cells ( vs. pretreatment levels ) were found in peripheral blood samples of patients with metastatic renal carcinomas treated with a dc vaccine combined with cytokine il-2 . our results indicate that there is a stimulation of the response of cd4 + t cells expressing il-2 , ifn- , and tnf- , as well as of il-2-expressing cd8 + t cells , suggesting that immunotherapy with dcs has the ability to induce a th1 response . in the present study , some of the patients involved were treated for a prolonged period of time and given the vaccine and immunological stimulation on several occasions . one explanation for the diminished response over the course of treatment could be due to selection for tumour cell variants with immunoresistant phenotypes.48 additionally , after a certain period of vaccination , there could have been a loss in expression of mhc class i molecules by tumour antigens , a major mechanism of immune system evasion.49 the maturation stage of dcs is also a determining factor for directing a th1 response , as immature dcs secrete il-10 leading to a th2 response.50,51 as such , it is important to continuously review the therapeutic protocol being used and to seek innovative strategies to prevent the development of new tumour escape mechanisms in order to maintain a good response during the treatment period . it should be noted that the present study was based on the analysis of the percentage of fluorescently labelled cells for each assessed marker , and that the analysis of fluorescence intensity was important . in conclusion , upon assessing immunologic response before and during treatment , we observed that immunotherapy with dcs was capable of stimulating the immune system in cancer patients .

years of intensive research on organic electronic devices have taught us that supramolecular interactions and organization have a major influence on their final performance . connected to the preparation of devices , e.g. , by vacuum deposition techniques or with solution methods , the sword of damocles of polymorphism is always present , definitely a widespread phenomenon when dealing with molecular crystals . polymorphism in bulk phases is a general issue in several fields , including pharmaceuticals and materials science . in thin films the suitable form for a realistic exploitation of electronic materials occurrence of polymorphism is further enhanced by the presence of the substrate surface and/or strong kinetic effects during growth . when these polymorphs are distinct from those observed in bulk crystals , one may speak of surface - induced phases ( sips ) . since the first discovery of polymorphism in pentancene thin films . the list of organic films displaying polymorphism and sips has grown considerably in length . a brief , nonexhaustive , summary of sips limited to the most promising compounds containing only c and h atoms already contains several paradigmatic examples . p - hexaphenyl ( 4,4,4,4-hexaphenyl ) , although scarcely prone to giving polymorphic bulk structures , shows thin film phases , some of them with hexaphenyl molecules arranged perpendicularly to the herringone layers . on the other hand , rubrene ( 5,6,11,12-tetraphenyltetracene ) bulk crystal polymorphs have been obtained by changing growth conditions or ambient pressure , but crystalline thin films show invariably the orthorhombic structure . pentacene shows a rich bulk polymorphism based on different triclinic crystal structures in space group p1 and characterized by herringbone packings of molecules . the first triclinic phase ( polymorph i ) has been described in 1961 and appeared again only recently . for many years thin films also show a variety of polymorphs , some of them displaying small differences in their diffraction pattern , quite often coexisting as interpenetrating microdomains or at different depth in the films . growth parameters or substrate nature such as sio2 , silicon , h - terminated or passivated silicon , metals , alkali metal halides , and polymer surfaces provide a thin film phase with upright molecules possibly with polymorph ii co - present . moreover , an orthorhombic phase has been observed in low thickness films grown on sio2 or polymers . up to now , there is no general or clear - cut agreement about the causes triggering the formation of sips . they may arise from kinetic phenomena under the influence of the substrate or of the strain induced by lattice mismatch at the film / substrate interface . sometimes , a thickness dependent behavior has been observed due to the formation of metastable phases . for pentacene , a clear thickness dependence of polymorphs is found with films grown on oxide substrates : the single crystal phase appears beyond a critical film thickness , and the critical thickness decreases on increasing the substrate temperature . large - amplitude molecular motions about the average positions may become possible on going from the constrained environment of the bulk to the surface , possibly triggering surface relaxation / reconstruction , even at low temperatures . despite these characteristics , judging from the scientific literature , while for inorganic surfaces a rich literature and a surface structure database are available , reports on surface reconstructions of molecular solids are sparse . we are aware of only a few examples concerning pyrene ( 001 ) , benzyl ( 001 ) , and benzophenone surfaces , while recent molecular mechanics minimizations ( i.e. , excluding thermal disorder ) on organic semiconductors crystals exhibiting the typical herringbone packing showed that surface relaxation of the low energy crystal faces is in general negligible . the recent study of morisaki et al . , conversely , has shown that surface relaxation in tetracene single crystals may have a deep impact on electron transport properties . in this respect , a deep study of the structure of molecular crystal surfaces is a fundamental step , especially when studying technologically relevant crystalline films . structural properties of thin films and film / substrate interfaces can be studied experimentally by ex situ and in situ scattering techniques ( grazing incidence x - ray diffraction , x - ray reflectivity , and electron diffraction ) . in parallel , computer modeling of crystals and crystal surfaces has substantially grown in importance over the past decade , thanks to more reliable force fields and increased computer power . molecular dynamics ( md ) simulations have provided a deeper understanding of the interactions and dynamical processes in organic semiconductor thin films , sometimes with a level of detail hardly accessible by experimental investigations . recent works exploring this approach include studies of growth of thin films and epitaxial structures , bulk crystals , and polymorphism . particularly pertinent to this work , sips have also become the subject of md simulations centered on the role of substrate surface on polymorph selection during thin film deposition . owing to the fact that polymorphism can be triggered by several mechanisms and parameters , in our opinion intrinsic properties of surfaces such as relaxation / reconstruction can not be excluded a priori as a factor responsible for the appearance of polymorphs in thin films . the present study is devoted to 2,2:6,2-ternaphthalene ( nnn ) , a molecule suitable for preparation of organic light - emitting diodes . the crystal structure of nnn has been solved recently for a thin film phase grown on thermally oxidized silicon . the same crystal structure has been observed on single crystals obtained by vacuum sublimation . unfortunately , the low quality of the single crystals hampered an accurate comparison of the two data sets , looking for minor differences in their crystal packing . this work aims to set up a background knowledge about surface features of nnn ( 001 ) , the low energy face displayed by thin films , by means of classical md . the role of md in this work is to investigate the stability of the ideal ( 001 ) surface and of surfaces containing point defects ( molecule vacancies ) , looking for possible surface reconstruction phenomena linked to molecular thermal motion . we deem this preliminary step necessary for a full understanding of possible surface induced polymorphism or phase transformations associated with thin film phases of nnn grown on different substrates , by organic molecular beam deposition or hot wall epitaxy . indeed , our md simulations of the nnn ( 001 ) surface give evidence of a rich structural behavior , with intramolecular effects coupled to intermolecular interactions and temperature as a determining variable for triggering surface reconstruction . to derive models of the nnn ( 001 ) surface , we started with the unit cell parameters and crystal structure published in ref ( 82 ) and illustrated in figure 1 . this belongs to the monoclinic p21/a space group , with two molecules per unit cell . the molecules are arranged in layers , parallel to the ab plane . within a stack , they adopt a classic herringbone arrangement , their long molecular axes being tilted by 23 with respect to the layer normal . left : unit cell content of nnn thin film and bulk phase ( room temperature ) . the modulus of is the tilt angle with respect to the surface normal ( c * ) . the sign of is the same as the inner product between the molecular axis and the a lattice vector . 1 and 2 are dihedral angles defined by the atoms in violet ; 1 is always on the outermost side of the molecule . the last three columns collect unit cell volumes , densities , and tilt angles ( ) . bulk values collected after the transition at 600 k , extending the first simulation by 1 ns . bulk values collected after the transition at 600 k , extending the first simulation by 5 ns , and replacing the berendsen with the parrinello rahman barostat . a force field for the nnn molecule the parameters for bonding and nonbonding interactions were equal to the standard ones for aromatic carbons and hydrogens . c bonds was increased to 1.48 in order to match the average value from ab initio geometry optimizations ( details are given later ) , and the stretching and bending force constants involving them were reduced by 30% with respect to the standard opls - aa ones , following the prescriptions of dubay et al . for conjugated oligomers and polymers . finally , the inter - ring intrinsic torsion potential was obtained from the energies of the conformers of 2,2-dinaphthalene ( nn ) . in practice , we fitted the molecular mechanics energies to those calculated ab initio with the gamess - us code . for the latter , we performed constrained geometry optimizations using the b3lyp density functional with the 6 - 311 g * * basis set and an empirical dispersion correction ( b3lyp - d3/6 - 311 g * * ) , followed by an analogous single point calculation with a larger basis including diffuse functions ( b3lyp - d3/6 - 311++g * * ) . it is qualitatively similar to that of biphenyl , but it is less symmetric due to the lower symmetry of the naphthyl units . plot of the potential energy curve obtained by fitting b3lyp - d3/6 - 311++g * * data ( squares ) , for the torsion about the central bond of 2,2-dinaphthalene . the structures superposed on the plot depict the molecule in the transoid ( at 38.7 ) and the cisoid ( at 138.0 ) conformation . a large supercell consisting of 10 10 8 unit cells along the a , b , and c axes ( 1600 molecules overall ) was constructed and employed to describe the bulk crystal by md , with three - dimensional periodic boundary conditions . to model the ( 001 ) surface , the supercell periodicity was kept within the ab plane , while , along c , it was elongated from 15.5 to 30 nm , thus creating a slab with a wide vacuum space between its two sides . in one set of simulations , the molecules occupying the outermost layer on the lower side of the slab were left untouched , leading to an ideal and defect - free surface ( excluding possible surface reconstructions and thermal disorder ) , while one molecule from the layer on the upper side of the slab was deleted to create a point defect ( vacancy ) . s0 and s1 surfaces ( corresponding to 100% and 99.5% of fractional coverage , respectively ) . in another set of simulations , we created two vacancies on both sides , by deleting two neighboring ( top layer ) or two far away molecules ( bottom layer ) . henceforth , we shall refer to them as s2-prox and s2-dist surfaces ( both corresponding to 99% of fractional coverage ) . an example of such surfaces , obtained from simulations performed at 500 k , is shown in figure 3 . in all cases , these surfaces are separated by a sufficiently thick layer of bulk - like crystal ( on one side ) and of vacuum space ( on the other side ) , so that they are effectively noninteracting and independent . snapshots of nnn slabs , from the md simulations at 500 k. left : full s0/s1 slab . here the lower surface ( s0 ) is defect - free , while the upper one ( s1 ) has one vacancy . right : the s2 slab showing two neighboring vacancies on the upper surface ( s2-prox ) and two distant vacancies on the lower one ( s2-dist ) . to better visualize the defects , the inner layers have been made transparent , and the molecules surrounding the defects have been highlighted . the simulations of the bulk crystals were carried out under constant temperature and pressure conditions ( npt ) , with a standard duration of 1 ns . the leapfrog algorithm with a time step of 1 fs was used to integrate the equations of motion . the pressure was maintained constant ( 1 atm ) and controlled via anisotropic coupling to the berendsen barostat , with a coupling constant of 1.0 ps . rahman barostat ( with a coupling costant of 10 ps ) was adopted in one npt simulation to check the stability of the new crystal structure ( see later ) . the temperature was controlled by coupling with a velocity rescaling thermostat , with a time constant of 0.1 ps . after equilibration of the bulk crystal at each temperature , the c axis of the supercell was increased to 30 nm to create the slabs and one or two molecules at the outer surfaces were removed to create the vacancies , as explained previously . the md simulations of the slabs were then carried out with fixed lattice parameters , under constant temperature and volume conditions ( nvt ) . electrostatic interactions were treated via the particle - mesh - ewald method with a fourier grid spacing of 0.12 nm . as detailed in later text , for the bulk structure at 600 k , a phase transition was observed after about 500 ps . in order to equilibrate the final structure , the former was generated starting from a trajectory taken at the early stage of the simulation ( 200 ps ) , whereas , the latter was obtained after equilibration . atomic coordinates from the md simulations were saved once every picosecond , to be visualized with vmd and analyzed with our own programs to extract information about the structural organization at the nnn ( 001 ) surfaces . relevant observables included the distributions of average distances and angles between the chosen molecules and the mean ( 001 ) plane , and intramolecular dihedral angles . voronoi diagrams were also calculated from the center - of - mass of the molecules in the top and the bottom layers , so as to highlight local changes in the structure , especially around the defects . in order to investigate nnn bulk dynamics and obtain suitable starting structures for the surface simulations , we performed preliminary npt simulations of the bulk crystal phase at 300 , 400 , 500 , 550 , and 600 k. each of these had a standard duration of 1 ns , enough to observe the fast and relatively minor changes in the crystal structure due to thermal expansion . for the npt simulation at 600 k , the overall simulation time table 1 compares the experimental ( at room temperature ) and the calculated average lattice parameters . in order to compare these parameters with those of the crystallographic unit cell , the average axes lengths of the md supercell were divided by 10 , 10 and 8 , respectively . at 300 k , the simulated lattice parameters are in good agreement with the experimental ones , demonstrating the reliability of the force field used in the simulations . the calculated cell parameters change gradually and regularly with temperature up to 550 k. the thermal expansion coefficient , v = ( v/t)p / v 180 10 k is comparable with that of other organic semiconducting materials , such as naphthalene ( 251(9 ) 10 k ) and anthracene ( 181(3 ) 10 k ) . further increasing the temperature to 600 k promotes a structural rearrangement associated with a solid - to - solid phase transition . the formation of the new crystal phase was observed starting at 0.5 ns and took the remaining simulation time to complete . in order to calculate the structural properties of the new polymorph compared to that at 550 k , the new cell shows longer a and c axes and a shorter b axis . the value of increased from 90.40 to 97.43 , whereas no change was observed for and . the cell volume increase across the phase transition is about three times that expected from thermal expansion only . the density of the new structure ( 1.188 g / cm ) is about 10% smaller than the experimental one at 300 k. accordingly , the distance between adjacent ( 001 ) planes increases from 1.939 to 2.077(7 ) . evolution of the angle of all molecules belonging to the bulk phase at 600 k over the first nanosecond . the starting ( a ) and final ( b ) structures are also shown on the right . we characterized the temperature dependence of the nnn crystal structure through the values of the angle defining the molecular orientation with respect to the ( 001 ) plane ( ) and the intramolecular dihedral angles determining their conformational state ( 1 and 2 ) . this planarity is a result of intermolecular interactions , as the most favorable conformation for an isolated molecule would be distorted from planarity by about 40 ( see again figure 2 for the conformational energy ) . it is worth mentioning that , in addition to the transoid conformation , in the gas phase there is also a cisoid conformation ( at about 140 ) with nearly identical energy . at ambient temperature and pressure , nnn molecules pack more efficiently when they are planar due to the intermolecular interactions , which provide about 18 kj / mol ( = 2 9 kj / mol , since in each nnn molecule there are two such torsions such as those in figure 1 ) . nonetheless , the md simulations clearly indicate that even at room temperature there can be appreciable instantaneous deviations from planarity . these deviations reach 30 at 300 k and , as expected , increase with temperature ( 50 at 550 k ) . also the orientation of the molecular axis with respect to the ab plane , expressed by the angle , changes with temperature , from about 19 at 300 k to about 17 at 550 k ( see table 1 ) . the phase transition observed at 600 k occurs with a significant molecular rearrangement and involves a substantial change in the average value of , with only a moderate broadening in the torsional angles . the entire process is induced by thermal motion and occurs gradually across adjacent molecular layers . figure 4 shows the evolution of over time . during the simulation , the value of , starting at 23 , gradually increases on the average up to about 15. at this point , corresponding to 550 ps , the molecules in a single molecular layer change simultaneously their orientation with respect to the ab plane , from negative values to positive values , centered at about 8. shortly after , after 1 ns , all molecules have changed their orientation ( see structure b in figure 4 ) . it is worth noting that , during the transition , the molecules quickly swap from negative to positive angles , thereby suggesting an energy maximum actually , a transition state at = 0 . torsional angles ( not shown for brevity ) are only marginally involved in the transition because the molecules in the new structure remain essentially planar . nonetheless , during the transition , the maximum instantaneous deviations from planarity increase to 60. starting at 0.7 ns , a small fraction of molecules exhibit a change in conformation , from the trans to a cisoid conformation , with values around 160 ( in the bulk , there is no distinction between 1 and 2 ) . the extension of this simulation by 1 ns revealed no further change in the structural parameters . these are reported in table 1 . to further confirm the stability of the new structure , we also performed a long npt run ( 5 ns ) using the parrinello in contrast to berendsen s , this barostat correctly samples the canonical ensemble but requires a well equilibrated system to avoid numerical instabilities . also in this case , the parameters collected in table 1 show no evidence of further structural changes . to further characterize the structural properties of the new phase , we calculated the radial distribution functions ( rdfs ) between the molecular centers of mass at different temperatures ( 300 , 550 , and 600 k ) . the presence of sharp peaks at both short and long range at all temperatures confirms the absence of liquid crystalline phases . increasing the temperature from 300 to 550 k broadens the peaks without significantly affecting their positions . the rdf profile for the new phase at 600 k clearly evidences a different distribution in peaks location and intensity . figure 5 also compares the torsional angle distributions ( 1 and 2 ) at 550 and 600 k , over the first nanosecond of the simulation . rahman barostat is equivalent to that observed right after the phase transition ( see above ) . also in this case , the torsional angles never exceeded 60 for the majority of molecules . the number of molecules with significant changes in conformation was small ( one to five molecules ) compared to the whole system ( 1600 molecules ) , and within the range of the statistical variability to be expected at this temperature . we , therefore , exclude the possibility of a plastic crystal phase at 600 k. plot of the pairwise radial distribution functions between the molecular centers of mass at 300 , 550 , and 600 k ( left panel ) for the bulk phase . on the right : evolution of the dihedral angles ( 1 and 2 ) at 550 ( top ) and 600 k ( bottom ) . we now turn to the discussion of the nnn slabs exposing the ( 001 ) surfaces . we are especially interested in the temperature dependence of their structure , as we are looking for evidence of single molecule rearrangements or collective surface reconstructions triggered either by thermal motion as observed earlier for the bulk structure and / or defects . at 300 , 400 , and 500 k figure 3 shows that , even at 500 k , the structure of the outer molecular layers is closely analogous to that of the bulk crystal . on the contrary , figure 6 demonstrates that at 550 k there is a clear reorientation of all of the molecules belonging to the outer layers , retaining at the same time a high degree of order . a similar outcome has recently been reported for tetracene , where surface relaxation was observed in the first monolayer of single crystals . snapshots from the final trajectories of the md simulations of the nnn slabs at 550 and 600 k. the snapshots at 600 k refer to slabs generated from the corresponding bulk structure after 200 ps ( e.g. , prior the phase transition ) . the bottom panel of figure 6 shows the snapshots at 600 k for the slabs obtained from the bulk prior the phase transition ( i.e. , at 200 ps ) . these trajectories are closely similar to those obtained for the bulk at the same temperature ( i.e. , structure b in figure 4 ) . a first indication of this analogy is given by comparing the distances between adjacent ( 001 ) planes , equal to 2.077(7 ) for the bulk and 2.082(16 ) for the s0/s1 slab . below , we shall show that the primitive cells extracted from the bulk and the slab share the same space group . at 600 k , the molecular rearrangement starts from the outer layers and propagates to the whole interior of the slab . these rearrangements occur on all of the surfaces , demonstrating that they are independent of the presence and type of vacancies . the main difference between the bulk and the slabs is the time required for the transition to occur . the process took about 500 ps to begin in the bulk , but only 50 ps in the slabs . also in this case , to characterize the structural changes at the surfaces , we calculated the inter- and intramolecular angles , 1 , and 2 . at relatively low temperatures ( see figure 7 for 500 k ) , these parameters for the molecules within the s0 and s1 surfaces fluctuate in a relatively narrow and sharply defined range , similar to those for the bulk at the same temperature . on the contrary , the molecules belonging to the two s2 surfaces have more orientational and conformational freedom , as evidenced by the broader distributions of their and angles . however , even in these cases the majority of molecules remains relatively unaffected . thus , one or two vacancies produce only a local perturbation in the film structure , without triggering any collective rearrangement . evolution of the orientations ( , left - hand side ) and of the torsion angles ( 1 and 2 , right - hand side ) of the molecules belonging to the surface layers , from the md simulations at t = 500 k. at 550 k , all of the slab systems undergo a very fast structural change . figure 8 shows that the time scale for these rearrangements is always of the order of 100 ps , independently of defectiveness of the surface . the structural change is clearly highlighted by the angle , which switches from 15 to + 10. the molecules in the outer layers switch from a tilted to a more vertical arrangement in the opposite direction , as already highlighted in figure 6 . this rearrangement does not involve a major change in conformation , as most molecules remain essentially planar . however , after the transition has occurred , we do see a broadening of the torsional distributions on the s0 surface , as the more vertical orientation of the molecules allows greater librational freedom . evolution of the orientations ( , left - hand side ) and of the torsion angles ( 1 and 2 , right - hand side ) of the molecules belonging to the surface layers , from the md simulations at t = 550 k. on the three defective surfaces , a small fraction of molecules also shows a change in conformation , from transoid to cisoid ( 1 , 2 160 ) . we have observed that this occurs especially when the molecules ends temporarily emerge from the surface and , as a consequence , the outer naphthyl group is freed from the packing restrictions imposed by the neighboring molecules . at 600 k , the dynamics of surface reconstruction occurs on a shorter time scale ( about 50 ps ) than at 550 k. the further propagation of the transformation wavefront to the slab interior ( figure 6 ) takes about 170 ps to complete in the s0/s1 case and only 110 ps in the s2 one . in order to test the reversibility of this process , we performed an additional nvt simulation by cooling the s0/s1 slab from 600 to 300 k. using a linear temperature gradient , and an overall annealing time of 10 ns , the original tilted herringbone structure was not restored , demonstrating a certain irreversibility for this transformation . after averaging over space and time the md configurations for the bulk at 600 k and ( 001 ) surface s0 at 550 k , it was possible to obtain the unit cell content after the phase transformation . both new phases , shown in figure 9 , share the monoclinic space group p21/a with a herringbone disposition of molecules , differing from the starting one for the length of the unit cell axes and , more relevant , the molecule tilt with respect to the ( 001 ) plane , as evidenced by the variation of the monoclinic angle in the three cases ( see table 2 ) . unit cell content for the bulk at 600 k ( a ) and ( 001 ) s0 surface at 550 k ( b ) . to describe the relative positions of the molecules and the structure of the defects , we performed a voronoi analysis . given a set of sites on a plane ( in this case , the centers of mass of the molecules at a surface ) , a cell is assigned to each site , such that the points within a cell are closer to that site than to any other . figures 10 and 11 show some representative voronoi diagrams for the surfaces containing two defects , at 550 k. far away from the defects , the molecules always have a well defined hexagonal close packing associated with the herringbone motif . in the s2-dist case ( figure 10 ) , the defects have a well defined structure , defined by six neighboring molecules . we do observe some variation in the defect structure shortly after the reconstruction , as exemplified by the diagram at 150 ps , but after a while this disorder disappears and the original defect pattern is restored . note that , by the end of the simulation , the defects have moved one step closer to each other . this demonstrates that at this temperature there is some defect mobility , which may eventually allow their coalescence . two neighboring defects ( figure 11 ) seem to have a more dynamic , amoeba - like structure . a full clarification of these points and a quantitative description of defect mobilities would require further , much longer simulations and a dedicated analysis , which are currently under way . representative voronoi diagrams for the s2-dist surface at 550 k. dots represent the molecules centers of mass . representative voronoi diagrams for the s2-prox surface at 550 k. dots represent the molecules centers of mass . we have applied classical md to characterize the structural properties and thermal behavior of bulk and ( 001 ) surfaces of nnn . our simulations on the bulk structure have revealed the occurrence of a crystal - to - crystal transition at high temperatures , which was previously undiscovered , possibly due to the difficulty in growing high quality crystalline samples of this material . the entire process is triggered by molecular thermal motion , and it occurs on a nanosecond time scale at 600 k. it involves the reorientation of the molecular long axes with respect to the ( 001 ) plane , by about 30. although showing significant instantaneous deviations from planarity , dihedral angles are only marginally involved in phase transition , as the majority of molecules remains essentially planar . simulations performed on slab models of the ( 001 ) surface with zero , one , or two vacancies have evidenced surface reconstruction phenomena starting at 550 k. at this temperature , the molecular rearrangement is characterized by the fast ( 100 ps ) reorientation of all molecules belonging to the outer layers , again with a change in the molecules tilt angle by about 30. further increasing the temperature to 600 k , we observe the propagation of this rearrangement to the slab interior , leading to a structure similar to that of the bulk crystals at the same temperature . increasing surface defectiveness accelerates the process , but it has a negligible effect on the final structures . the fluctuation of the torsional angles with temperature are similar to those observed for the bulk , with the more defective surfaces showing increased orientational and conformational freedom . the analogy between bulk and slab structures after phase transition is further enforced by the fact the unit cells extracted from our simulations share the monoclinic space group p21/a and have similar lattice parameters , with a herringbone disposition of molecules . similar to the results of a recent experimental and computational study of tetracene , surface reconstruction appears to be an intrinsic property of nnn crystals , not induced by a specific foreign substrate . these findings prompt us to undertake in the future further computational work devoted to the md simulation of true thin films on different substrates such as graphite ( 0001 ) and amorphous sio2 , along with a characterization of the change in electronic properties ( e.g. , charge carrier mobilities ) associated with these reconstructions . these studies , together with the present one , will allow us to decouple effects due to the substrate and kinetics effects during thin film growth from features which are inherent in the thermodynamic behavior of the crystalline phase of nnn , correctly assigning the role of the underlying substrate .

2,2:6,2-ternaphthalene ( nnn ) is a novel , blue - emitting material , suitable for preparation of organic light - emitting diodes . its crystal structure has been solved recently , but its thermal behavior and surface properties have not yet been explored , partly due to the difficulty in obtaining high quality crystals . in the present study we use classical molecular dynamics to investigate the thermal behavior of bulk and ( 001 ) surfaces of nnn . our bulk simulations indicate the occurrence of a phase transition at about 600 k. the transition is facilitated by the presence of a free ( 001 ) surface , since a reconstruction leading to a very similar structure occurs around 550 k in our surface models . this holds for both ideal and defective surface models , containing a small number of vacancies ( one or two missing molecules in the outermost layer ) . in all cases , the process is triggered by thermal motion and involves the reorientation of the molecules with respect to the ( 001 ) plane . both the bulk and surface phases share the monoclinic space group p21/a with a herringbone disposition of molecules . these findings and their implications for the use of nnn in organic electronics are discussed .

Introduction Model and Computational Methods Results and Discussion Summary and Conclusions

in thin films the suitable form for a realistic exploitation of electronic materials occurrence of polymorphism is further enhanced by the presence of the substrate surface and/or strong kinetic effects during growth . sometimes , a thickness dependent behavior has been observed due to the formation of metastable phases . large - amplitude molecular motions about the average positions may become possible on going from the constrained environment of the bulk to the surface , possibly triggering surface relaxation / reconstruction , even at low temperatures . molecular dynamics ( md ) simulations have provided a deeper understanding of the interactions and dynamical processes in organic semiconductor thin films , sometimes with a level of detail hardly accessible by experimental investigations . owing to the fact that polymorphism can be triggered by several mechanisms and parameters , in our opinion intrinsic properties of surfaces such as relaxation / reconstruction can not be excluded a priori as a factor responsible for the appearance of polymorphs in thin films . the present study is devoted to 2,2:6,2-ternaphthalene ( nnn ) , a molecule suitable for preparation of organic light - emitting diodes . the crystal structure of nnn has been solved recently for a thin film phase grown on thermally oxidized silicon . the same crystal structure has been observed on single crystals obtained by vacuum sublimation . this work aims to set up a background knowledge about surface features of nnn ( 001 ) , the low energy face displayed by thin films , by means of classical md . the role of md in this work is to investigate the stability of the ideal ( 001 ) surface and of surfaces containing point defects ( molecule vacancies ) , looking for possible surface reconstruction phenomena linked to molecular thermal motion . indeed , our md simulations of the nnn ( 001 ) surface give evidence of a rich structural behavior , with intramolecular effects coupled to intermolecular interactions and temperature as a determining variable for triggering surface reconstruction . to derive models of the nnn ( 001 ) surface , we started with the unit cell parameters and crystal structure published in ref ( 82 ) and illustrated in figure 1 . this belongs to the monoclinic p21/a space group , with two molecules per unit cell . the modulus of is the tilt angle with respect to the surface normal ( c * ) . 1 and 2 are dihedral angles defined by the atoms in violet ; 1 is always on the outermost side of the molecule . bulk values collected after the transition at 600 k , extending the first simulation by 1 ns . bulk values collected after the transition at 600 k , extending the first simulation by 5 ns , and replacing the berendsen with the parrinello rahman barostat . c bonds was increased to 1.48 in order to match the average value from ab initio geometry optimizations ( details are given later ) , and the stretching and bending force constants involving them were reduced by 30% with respect to the standard opls - aa ones , following the prescriptions of dubay et al . it is qualitatively similar to that of biphenyl , but it is less symmetric due to the lower symmetry of the naphthyl units . to model the ( 001 ) surface , the supercell periodicity was kept within the ab plane , while , along c , it was elongated from 15.5 to 30 nm , thus creating a slab with a wide vacuum space between its two sides . in one set of simulations , the molecules occupying the outermost layer on the lower side of the slab were left untouched , leading to an ideal and defect - free surface ( excluding possible surface reconstructions and thermal disorder ) , while one molecule from the layer on the upper side of the slab was deleted to create a point defect ( vacancy ) . in all cases , these surfaces are separated by a sufficiently thick layer of bulk - like crystal ( on one side ) and of vacuum space ( on the other side ) , so that they are effectively noninteracting and independent . the simulations of the bulk crystals were carried out under constant temperature and pressure conditions ( npt ) , with a standard duration of 1 ns . rahman barostat ( with a coupling costant of 10 ps ) was adopted in one npt simulation to check the stability of the new crystal structure ( see later ) . after equilibration of the bulk crystal at each temperature , the c axis of the supercell was increased to 30 nm to create the slabs and one or two molecules at the outer surfaces were removed to create the vacancies , as explained previously . as detailed in later text , for the bulk structure at 600 k , a phase transition was observed after about 500 ps . relevant observables included the distributions of average distances and angles between the chosen molecules and the mean ( 001 ) plane , and intramolecular dihedral angles . voronoi diagrams were also calculated from the center - of - mass of the molecules in the top and the bottom layers , so as to highlight local changes in the structure , especially around the defects . in order to investigate nnn bulk dynamics and obtain suitable starting structures for the surface simulations , we performed preliminary npt simulations of the bulk crystal phase at 300 , 400 , 500 , 550 , and 600 k. each of these had a standard duration of 1 ns , enough to observe the fast and relatively minor changes in the crystal structure due to thermal expansion . for the npt simulation at 600 k , the overall simulation time table 1 compares the experimental ( at room temperature ) and the calculated average lattice parameters . at 300 k , the simulated lattice parameters are in good agreement with the experimental ones , demonstrating the reliability of the force field used in the simulations . the calculated cell parameters change gradually and regularly with temperature up to 550 k. the thermal expansion coefficient , v = ( v/t)p / v 180 10 k is comparable with that of other organic semiconducting materials , such as naphthalene ( 251(9 ) 10 k ) and anthracene ( 181(3 ) 10 k ) . further increasing the temperature to 600 k promotes a structural rearrangement associated with a solid - to - solid phase transition . the density of the new structure ( 1.188 g / cm ) is about 10% smaller than the experimental one at 300 k. accordingly , the distance between adjacent ( 001 ) planes increases from 1.939 to 2.077(7 ) . evolution of the angle of all molecules belonging to the bulk phase at 600 k over the first nanosecond . we characterized the temperature dependence of the nnn crystal structure through the values of the angle defining the molecular orientation with respect to the ( 001 ) plane ( ) and the intramolecular dihedral angles determining their conformational state ( 1 and 2 ) . it is worth mentioning that , in addition to the transoid conformation , in the gas phase there is also a cisoid conformation ( at about 140 ) with nearly identical energy . at ambient temperature and pressure , nnn molecules pack more efficiently when they are planar due to the intermolecular interactions , which provide about 18 kj / mol ( = 2 9 kj / mol , since in each nnn molecule there are two such torsions such as those in figure 1 ) . also the orientation of the molecular axis with respect to the ab plane , expressed by the angle , changes with temperature , from about 19 at 300 k to about 17 at 550 k ( see table 1 ) . the phase transition observed at 600 k occurs with a significant molecular rearrangement and involves a substantial change in the average value of , with only a moderate broadening in the torsional angles . the entire process is induced by thermal motion and occurs gradually across adjacent molecular layers . during the simulation , the value of , starting at 23 , gradually increases on the average up to about 15. at this point , corresponding to 550 ps , the molecules in a single molecular layer change simultaneously their orientation with respect to the ab plane , from negative values to positive values , centered at about 8. shortly after , after 1 ns , all molecules have changed their orientation ( see structure b in figure 4 ) . it is worth noting that , during the transition , the molecules quickly swap from negative to positive angles , thereby suggesting an energy maximum actually , a transition state at = 0 . torsional angles ( not shown for brevity ) are only marginally involved in the transition because the molecules in the new structure remain essentially planar . nonetheless , during the transition , the maximum instantaneous deviations from planarity increase to 60. starting at 0.7 ns , a small fraction of molecules exhibit a change in conformation , from the trans to a cisoid conformation , with values around 160 ( in the bulk , there is no distinction between 1 and 2 ) . also in this case , the torsional angles never exceeded 60 for the majority of molecules . the number of molecules with significant changes in conformation was small ( one to five molecules ) compared to the whole system ( 1600 molecules ) , and within the range of the statistical variability to be expected at this temperature . we , therefore , exclude the possibility of a plastic crystal phase at 600 k. plot of the pairwise radial distribution functions between the molecular centers of mass at 300 , 550 , and 600 k ( left panel ) for the bulk phase . we now turn to the discussion of the nnn slabs exposing the ( 001 ) surfaces . we are especially interested in the temperature dependence of their structure , as we are looking for evidence of single molecule rearrangements or collective surface reconstructions triggered either by thermal motion as observed earlier for the bulk structure and / or defects . at 300 , 400 , and 500 k figure 3 shows that , even at 500 k , the structure of the outer molecular layers is closely analogous to that of the bulk crystal . on the contrary , figure 6 demonstrates that at 550 k there is a clear reorientation of all of the molecules belonging to the outer layers , retaining at the same time a high degree of order . snapshots from the final trajectories of the md simulations of the nnn slabs at 550 and 600 k. the snapshots at 600 k refer to slabs generated from the corresponding bulk structure after 200 ps ( e.g. the bottom panel of figure 6 shows the snapshots at 600 k for the slabs obtained from the bulk prior the phase transition ( i.e. a first indication of this analogy is given by comparing the distances between adjacent ( 001 ) planes , equal to 2.077(7 ) for the bulk and 2.082(16 ) for the s0/s1 slab . below , we shall show that the primitive cells extracted from the bulk and the slab share the same space group . at 600 k , the molecular rearrangement starts from the outer layers and propagates to the whole interior of the slab . these rearrangements occur on all of the surfaces , demonstrating that they are independent of the presence and type of vacancies . the main difference between the bulk and the slabs is the time required for the transition to occur . the process took about 500 ps to begin in the bulk , but only 50 ps in the slabs . at relatively low temperatures ( see figure 7 for 500 k ) , these parameters for the molecules within the s0 and s1 surfaces fluctuate in a relatively narrow and sharply defined range , similar to those for the bulk at the same temperature . on the contrary , the molecules belonging to the two s2 surfaces have more orientational and conformational freedom , as evidenced by the broader distributions of their and angles . thus , one or two vacancies produce only a local perturbation in the film structure , without triggering any collective rearrangement . evolution of the orientations ( , left - hand side ) and of the torsion angles ( 1 and 2 , right - hand side ) of the molecules belonging to the surface layers , from the md simulations at t = 500 k. at 550 k , all of the slab systems undergo a very fast structural change . the structural change is clearly highlighted by the angle , which switches from 15 to + 10. the molecules in the outer layers switch from a tilted to a more vertical arrangement in the opposite direction , as already highlighted in figure 6 . however , after the transition has occurred , we do see a broadening of the torsional distributions on the s0 surface , as the more vertical orientation of the molecules allows greater librational freedom . evolution of the orientations ( , left - hand side ) and of the torsion angles ( 1 and 2 , right - hand side ) of the molecules belonging to the surface layers , from the md simulations at t = 550 k. on the three defective surfaces , a small fraction of molecules also shows a change in conformation , from transoid to cisoid ( 1 , 2 160 ) . we have observed that this occurs especially when the molecules ends temporarily emerge from the surface and , as a consequence , the outer naphthyl group is freed from the packing restrictions imposed by the neighboring molecules . at 600 k , the dynamics of surface reconstruction occurs on a shorter time scale ( about 50 ps ) than at 550 k. the further propagation of the transformation wavefront to the slab interior ( figure 6 ) takes about 170 ps to complete in the s0/s1 case and only 110 ps in the s2 one . after averaging over space and time the md configurations for the bulk at 600 k and ( 001 ) surface s0 at 550 k , it was possible to obtain the unit cell content after the phase transformation . both new phases , shown in figure 9 , share the monoclinic space group p21/a with a herringbone disposition of molecules , differing from the starting one for the length of the unit cell axes and , more relevant , the molecule tilt with respect to the ( 001 ) plane , as evidenced by the variation of the monoclinic angle in the three cases ( see table 2 ) . unit cell content for the bulk at 600 k ( a ) and ( 001 ) s0 surface at 550 k ( b ) . given a set of sites on a plane ( in this case , the centers of mass of the molecules at a surface ) , a cell is assigned to each site , such that the points within a cell are closer to that site than to any other . figures 10 and 11 show some representative voronoi diagrams for the surfaces containing two defects , at 550 k. far away from the defects , the molecules always have a well defined hexagonal close packing associated with the herringbone motif . we do observe some variation in the defect structure shortly after the reconstruction , as exemplified by the diagram at 150 ps , but after a while this disorder disappears and the original defect pattern is restored . note that , by the end of the simulation , the defects have moved one step closer to each other . representative voronoi diagrams for the s2-dist surface at 550 k. dots represent the molecules centers of mass . representative voronoi diagrams for the s2-prox surface at 550 k. dots represent the molecules centers of mass . we have applied classical md to characterize the structural properties and thermal behavior of bulk and ( 001 ) surfaces of nnn . our simulations on the bulk structure have revealed the occurrence of a crystal - to - crystal transition at high temperatures , which was previously undiscovered , possibly due to the difficulty in growing high quality crystalline samples of this material . the entire process is triggered by molecular thermal motion , and it occurs on a nanosecond time scale at 600 k. it involves the reorientation of the molecular long axes with respect to the ( 001 ) plane , by about 30. although showing significant instantaneous deviations from planarity , dihedral angles are only marginally involved in phase transition , as the majority of molecules remains essentially planar . simulations performed on slab models of the ( 001 ) surface with zero , one , or two vacancies have evidenced surface reconstruction phenomena starting at 550 k. at this temperature , the molecular rearrangement is characterized by the fast ( 100 ps ) reorientation of all molecules belonging to the outer layers , again with a change in the molecules tilt angle by about 30. further increasing the temperature to 600 k , we observe the propagation of this rearrangement to the slab interior , leading to a structure similar to that of the bulk crystals at the same temperature . the fluctuation of the torsional angles with temperature are similar to those observed for the bulk , with the more defective surfaces showing increased orientational and conformational freedom . the analogy between bulk and slab structures after phase transition is further enforced by the fact the unit cells extracted from our simulations share the monoclinic space group p21/a and have similar lattice parameters , with a herringbone disposition of molecules . similar to the results of a recent experimental and computational study of tetracene , surface reconstruction appears to be an intrinsic property of nnn crystals , not induced by a specific foreign substrate . these findings prompt us to undertake in the future further computational work devoted to the md simulation of true thin films on different substrates such as graphite ( 0001 ) and amorphous sio2 , along with a characterization of the change in electronic properties ( e.g. these studies , together with the present one , will allow us to decouple effects due to the substrate and kinetics effects during thin film growth from features which are inherent in the thermodynamic behavior of the crystalline phase of nnn , correctly assigning the role of the underlying substrate .

in thin films the suitable form for a realistic exploitation of electronic materials occurrence of polymorphism is further enhanced by the presence of the substrate surface and/or strong kinetic effects during growth . a brief , nonexhaustive , summary of sips limited to the most promising compounds containing only c and h atoms already contains several paradigmatic examples . for many years thin films also show a variety of polymorphs , some of them displaying small differences in their diffraction pattern , quite often coexisting as interpenetrating microdomains or at different depth in the films . for pentacene , a clear thickness dependence of polymorphs is found with films grown on oxide substrates : the single crystal phase appears beyond a critical film thickness , and the critical thickness decreases on increasing the substrate temperature . large - amplitude molecular motions about the average positions may become possible on going from the constrained environment of the bulk to the surface , possibly triggering surface relaxation / reconstruction , even at low temperatures . despite these characteristics , judging from the scientific literature , while for inorganic surfaces a rich literature and a surface structure database are available , reports on surface reconstructions of molecular solids are sparse . in this respect , a deep study of the structure of molecular crystal surfaces is a fundamental step , especially when studying technologically relevant crystalline films . structural properties of thin films and film / substrate interfaces can be studied experimentally by ex situ and in situ scattering techniques ( grazing incidence x - ray diffraction , x - ray reflectivity , and electron diffraction ) . in parallel , computer modeling of crystals and crystal surfaces has substantially grown in importance over the past decade , thanks to more reliable force fields and increased computer power . molecular dynamics ( md ) simulations have provided a deeper understanding of the interactions and dynamical processes in organic semiconductor thin films , sometimes with a level of detail hardly accessible by experimental investigations . particularly pertinent to this work , sips have also become the subject of md simulations centered on the role of substrate surface on polymorph selection during thin film deposition . owing to the fact that polymorphism can be triggered by several mechanisms and parameters , in our opinion intrinsic properties of surfaces such as relaxation / reconstruction can not be excluded a priori as a factor responsible for the appearance of polymorphs in thin films . the present study is devoted to 2,2:6,2-ternaphthalene ( nnn ) , a molecule suitable for preparation of organic light - emitting diodes . unfortunately , the low quality of the single crystals hampered an accurate comparison of the two data sets , looking for minor differences in their crystal packing . this work aims to set up a background knowledge about surface features of nnn ( 001 ) , the low energy face displayed by thin films , by means of classical md . the role of md in this work is to investigate the stability of the ideal ( 001 ) surface and of surfaces containing point defects ( molecule vacancies ) , looking for possible surface reconstruction phenomena linked to molecular thermal motion . we deem this preliminary step necessary for a full understanding of possible surface induced polymorphism or phase transformations associated with thin film phases of nnn grown on different substrates , by organic molecular beam deposition or hot wall epitaxy . indeed , our md simulations of the nnn ( 001 ) surface give evidence of a rich structural behavior , with intramolecular effects coupled to intermolecular interactions and temperature as a determining variable for triggering surface reconstruction . to derive models of the nnn ( 001 ) surface , we started with the unit cell parameters and crystal structure published in ref ( 82 ) and illustrated in figure 1 . a force field for the nnn molecule the parameters for bonding and nonbonding interactions were equal to the standard ones for aromatic carbons and hydrogens . c bonds was increased to 1.48 in order to match the average value from ab initio geometry optimizations ( details are given later ) , and the stretching and bending force constants involving them were reduced by 30% with respect to the standard opls - aa ones , following the prescriptions of dubay et al . finally , the inter - ring intrinsic torsion potential was obtained from the energies of the conformers of 2,2-dinaphthalene ( nn ) . for the latter , we performed constrained geometry optimizations using the b3lyp density functional with the 6 - 311 g * * basis set and an empirical dispersion correction ( b3lyp - d3/6 - 311 g * * ) , followed by an analogous single point calculation with a larger basis including diffuse functions ( b3lyp - d3/6 - 311++g * * ) . plot of the potential energy curve obtained by fitting b3lyp - d3/6 - 311++g * * data ( squares ) , for the torsion about the central bond of 2,2-dinaphthalene . the structures superposed on the plot depict the molecule in the transoid ( at 38.7 ) and the cisoid ( at 138.0 ) conformation . a large supercell consisting of 10 10 8 unit cells along the a , b , and c axes ( 1600 molecules overall ) was constructed and employed to describe the bulk crystal by md , with three - dimensional periodic boundary conditions . to model the ( 001 ) surface , the supercell periodicity was kept within the ab plane , while , along c , it was elongated from 15.5 to 30 nm , thus creating a slab with a wide vacuum space between its two sides . in one set of simulations , the molecules occupying the outermost layer on the lower side of the slab were left untouched , leading to an ideal and defect - free surface ( excluding possible surface reconstructions and thermal disorder ) , while one molecule from the layer on the upper side of the slab was deleted to create a point defect ( vacancy ) . after equilibration of the bulk crystal at each temperature , the c axis of the supercell was increased to 30 nm to create the slabs and one or two molecules at the outer surfaces were removed to create the vacancies , as explained previously . in order to equilibrate the final structure , the former was generated starting from a trajectory taken at the early stage of the simulation ( 200 ps ) , whereas , the latter was obtained after equilibration . voronoi diagrams were also calculated from the center - of - mass of the molecules in the top and the bottom layers , so as to highlight local changes in the structure , especially around the defects . in order to investigate nnn bulk dynamics and obtain suitable starting structures for the surface simulations , we performed preliminary npt simulations of the bulk crystal phase at 300 , 400 , 500 , 550 , and 600 k. each of these had a standard duration of 1 ns , enough to observe the fast and relatively minor changes in the crystal structure due to thermal expansion . for the npt simulation at 600 k , the overall simulation time table 1 compares the experimental ( at room temperature ) and the calculated average lattice parameters . in order to compare these parameters with those of the crystallographic unit cell , the average axes lengths of the md supercell were divided by 10 , 10 and 8 , respectively . the calculated cell parameters change gradually and regularly with temperature up to 550 k. the thermal expansion coefficient , v = ( v/t)p / v 180 10 k is comparable with that of other organic semiconducting materials , such as naphthalene ( 251(9 ) 10 k ) and anthracene ( 181(3 ) 10 k ) . in order to calculate the structural properties of the new polymorph compared to that at 550 k , the new cell shows longer a and c axes and a shorter b axis . the density of the new structure ( 1.188 g / cm ) is about 10% smaller than the experimental one at 300 k. accordingly , the distance between adjacent ( 001 ) planes increases from 1.939 to 2.077(7 ) . we characterized the temperature dependence of the nnn crystal structure through the values of the angle defining the molecular orientation with respect to the ( 001 ) plane ( ) and the intramolecular dihedral angles determining their conformational state ( 1 and 2 ) . this planarity is a result of intermolecular interactions , as the most favorable conformation for an isolated molecule would be distorted from planarity by about 40 ( see again figure 2 for the conformational energy ) . at ambient temperature and pressure , nnn molecules pack more efficiently when they are planar due to the intermolecular interactions , which provide about 18 kj / mol ( = 2 9 kj / mol , since in each nnn molecule there are two such torsions such as those in figure 1 ) . also the orientation of the molecular axis with respect to the ab plane , expressed by the angle , changes with temperature , from about 19 at 300 k to about 17 at 550 k ( see table 1 ) . the phase transition observed at 600 k occurs with a significant molecular rearrangement and involves a substantial change in the average value of , with only a moderate broadening in the torsional angles . during the simulation , the value of , starting at 23 , gradually increases on the average up to about 15. at this point , corresponding to 550 ps , the molecules in a single molecular layer change simultaneously their orientation with respect to the ab plane , from negative values to positive values , centered at about 8. shortly after , after 1 ns , all molecules have changed their orientation ( see structure b in figure 4 ) . nonetheless , during the transition , the maximum instantaneous deviations from planarity increase to 60. starting at 0.7 ns , a small fraction of molecules exhibit a change in conformation , from the trans to a cisoid conformation , with values around 160 ( in the bulk , there is no distinction between 1 and 2 ) . to further confirm the stability of the new structure , we also performed a long npt run ( 5 ns ) using the parrinello in contrast to berendsen s , this barostat correctly samples the canonical ensemble but requires a well equilibrated system to avoid numerical instabilities . to further characterize the structural properties of the new phase , we calculated the radial distribution functions ( rdfs ) between the molecular centers of mass at different temperatures ( 300 , 550 , and 600 k ) . the number of molecules with significant changes in conformation was small ( one to five molecules ) compared to the whole system ( 1600 molecules ) , and within the range of the statistical variability to be expected at this temperature . we , therefore , exclude the possibility of a plastic crystal phase at 600 k. plot of the pairwise radial distribution functions between the molecular centers of mass at 300 , 550 , and 600 k ( left panel ) for the bulk phase . we are especially interested in the temperature dependence of their structure , as we are looking for evidence of single molecule rearrangements or collective surface reconstructions triggered either by thermal motion as observed earlier for the bulk structure and / or defects . on the contrary , figure 6 demonstrates that at 550 k there is a clear reorientation of all of the molecules belonging to the outer layers , retaining at the same time a high degree of order . snapshots from the final trajectories of the md simulations of the nnn slabs at 550 and 600 k. the snapshots at 600 k refer to slabs generated from the corresponding bulk structure after 200 ps ( e.g. a first indication of this analogy is given by comparing the distances between adjacent ( 001 ) planes , equal to 2.077(7 ) for the bulk and 2.082(16 ) for the s0/s1 slab . below , we shall show that the primitive cells extracted from the bulk and the slab share the same space group . also in this case , to characterize the structural changes at the surfaces , we calculated the inter- and intramolecular angles , 1 , and 2 . at relatively low temperatures ( see figure 7 for 500 k ) , these parameters for the molecules within the s0 and s1 surfaces fluctuate in a relatively narrow and sharply defined range , similar to those for the bulk at the same temperature . on the contrary , the molecules belonging to the two s2 surfaces have more orientational and conformational freedom , as evidenced by the broader distributions of their and angles . evolution of the orientations ( , left - hand side ) and of the torsion angles ( 1 and 2 , right - hand side ) of the molecules belonging to the surface layers , from the md simulations at t = 500 k. at 550 k , all of the slab systems undergo a very fast structural change . the structural change is clearly highlighted by the angle , which switches from 15 to + 10. the molecules in the outer layers switch from a tilted to a more vertical arrangement in the opposite direction , as already highlighted in figure 6 . however , after the transition has occurred , we do see a broadening of the torsional distributions on the s0 surface , as the more vertical orientation of the molecules allows greater librational freedom . evolution of the orientations ( , left - hand side ) and of the torsion angles ( 1 and 2 , right - hand side ) of the molecules belonging to the surface layers , from the md simulations at t = 550 k. on the three defective surfaces , a small fraction of molecules also shows a change in conformation , from transoid to cisoid ( 1 , 2 160 ) . we have observed that this occurs especially when the molecules ends temporarily emerge from the surface and , as a consequence , the outer naphthyl group is freed from the packing restrictions imposed by the neighboring molecules . at 600 k , the dynamics of surface reconstruction occurs on a shorter time scale ( about 50 ps ) than at 550 k. the further propagation of the transformation wavefront to the slab interior ( figure 6 ) takes about 170 ps to complete in the s0/s1 case and only 110 ps in the s2 one . in order to test the reversibility of this process , we performed an additional nvt simulation by cooling the s0/s1 slab from 600 to 300 k. using a linear temperature gradient , and an overall annealing time of 10 ns , the original tilted herringbone structure was not restored , demonstrating a certain irreversibility for this transformation . both new phases , shown in figure 9 , share the monoclinic space group p21/a with a herringbone disposition of molecules , differing from the starting one for the length of the unit cell axes and , more relevant , the molecule tilt with respect to the ( 001 ) plane , as evidenced by the variation of the monoclinic angle in the three cases ( see table 2 ) . to describe the relative positions of the molecules and the structure of the defects , we performed a voronoi analysis . given a set of sites on a plane ( in this case , the centers of mass of the molecules at a surface ) , a cell is assigned to each site , such that the points within a cell are closer to that site than to any other . figures 10 and 11 show some representative voronoi diagrams for the surfaces containing two defects , at 550 k. far away from the defects , the molecules always have a well defined hexagonal close packing associated with the herringbone motif . we do observe some variation in the defect structure shortly after the reconstruction , as exemplified by the diagram at 150 ps , but after a while this disorder disappears and the original defect pattern is restored . our simulations on the bulk structure have revealed the occurrence of a crystal - to - crystal transition at high temperatures , which was previously undiscovered , possibly due to the difficulty in growing high quality crystalline samples of this material . the entire process is triggered by molecular thermal motion , and it occurs on a nanosecond time scale at 600 k. it involves the reorientation of the molecular long axes with respect to the ( 001 ) plane , by about 30. although showing significant instantaneous deviations from planarity , dihedral angles are only marginally involved in phase transition , as the majority of molecules remains essentially planar . simulations performed on slab models of the ( 001 ) surface with zero , one , or two vacancies have evidenced surface reconstruction phenomena starting at 550 k. at this temperature , the molecular rearrangement is characterized by the fast ( 100 ps ) reorientation of all molecules belonging to the outer layers , again with a change in the molecules tilt angle by about 30. further increasing the temperature to 600 k , we observe the propagation of this rearrangement to the slab interior , leading to a structure similar to that of the bulk crystals at the same temperature . the analogy between bulk and slab structures after phase transition is further enforced by the fact the unit cells extracted from our simulations share the monoclinic space group p21/a and have similar lattice parameters , with a herringbone disposition of molecules . similar to the results of a recent experimental and computational study of tetracene , surface reconstruction appears to be an intrinsic property of nnn crystals , not induced by a specific foreign substrate . these findings prompt us to undertake in the future further computational work devoted to the md simulation of true thin films on different substrates such as graphite ( 0001 ) and amorphous sio2 , along with a characterization of the change in electronic properties ( e.g. these studies , together with the present one , will allow us to decouple effects due to the substrate and kinetics effects during thin film growth from features which are inherent in the thermodynamic behavior of the crystalline phase of nnn , correctly assigning the role of the underlying substrate .

in 2013 , there existed multiple pharmacologic interventions for the prevention and treatment of type 2 diabetes ( 1 ) . however , all evaluations of known efficacious interventions reveal that some patients respond to treatment while others do not . as recognized by the american diabetes association in its 2012 statement on the management of hyperglycemia , care in type 2 diabetes must become more patient - centered ( 2 ) , and the individualization of diabetes prevention and treatment based on genetic variation has great potential . narrative reviews have commented on the promise of pharmacogenomics of type 2 diabetes ( 36 ) , and prominent individual studies have found statistically significant pharmacogenetic interactions associated with diabetes risk and glycemic outcomes ( 711 ) . however , prior reviews have not systematically evaluated this literature to inform future research questions , and these reviews do not address the quality issues that affect the existing literature on diabetes pharmacogenetics . the clinical utility of genetic variation for tailoring diabetes medications rests on the identification of substantial and statistically significant pharmacogenetic interactions from internally valid studies and confirmation of their findings in varied populations based on race / ethnicity . we conducted a systematic review of observational and experimental studies to determine if the effect of diabetes medications on diabetes incidence , hba1c , fasting glucose ( fg ) , and postprandial glucose ( ppg ) varies by independent genetic variation in patients with impaired fg , impaired glucose tolerance , or type 2 diabetes . we hypothesized that 1 ) genetic variation associated with drug transporters , metabolizers , targets , and mechanisms of action would modify the effect of specific drugs and 2 ) the existing evidence would be insufficient to recommend clinical use of pharmacogenetic interactions because of a lack of well - conducted studies across diverse populations . senior members of the study were diabetes and obesity researchers with training in clinical epidemiology , clinical trials , and systematic review methodology ( e.b . , w.l.b . , the team also included an experienced project manager with expertise in the conduct of systematic reviews ( l.m.w . ) . we searched the pubmed , embase , cochrane electronic databases and also manually searched key review articles , key journals tables of contents , and the references of included articles . key journals were selected based on content area and ones that commonly published the included articles . the pubmed search and list of key journals the electronic search included dates of database inception through 13 march 2013 , and the manual search of tables of contents included january to march 2013 . we included original articles on the effect of food and drug administration ( fda)-approved diabetes medications ( supplementary table 3 ) on diabetes incidence , hba1c , fg , and ppg in adults with either type 2 diabetes or increased diabetes risk because of impaired fg ( fg 5.556.94 mmol / l ) or impaired glucose tolerance ( 2-h postload [ 75 g ] glucose 7.7711.04 mmol / l ) by common genetic variation . we considered any independent genetic variation ( e.g. , single nucleotide polymorphisms [ snps ] , copy number variants ) eligible and excluded variation such as haplotypes . eligible study designs were 1 ) controlled studies evaluating the effect of a drug for one allele / genotype versus another over time and 2 ) uncontrolled studies evaluating the effect ( change in outcome or incidence of outcome ) of a drug comparing one genotype / allele to another . we excluded studies of less than 24-h duration and did not include results for hba1c in studies shorter than 3 months . we excluded case reports , case series , and cross - sectional studies ; studies not written in english ( due to lack of availability of resources to interpret these articles ) ; and studies that included participants on more than one diabetes medication . abstracts and full - text articles were reviewed using a standardized and piloted eligibility criteria form , and disagreements were resolved through consensus . data abstraction forms were piloted extensively and included information on study design , study population characteristics , genetic variation under study , and study results on outcomes of interest . we developed quality abstraction forms based on the strengthening the reporting of genetic association studies guidelines for reporting of genetic association studies ( 12 ) . in the absence of guidelines for pharmacogenetic studies , we also incorporated recommendations from the hugenet ( human genome epidemiology network ) huge review handbook ( 13 ) and prior methodological papers ( 14 ) . we considered a study to be randomized if it randomized participants for the pharmacogenetic study and was not simply based on a prior randomized study . forms captured elements of quality control of genotyping , including method of genotyping and genotyping call rate , and we considered a call rate 95% to be acceptable . we considered selective reporting of interactions based on positive results and selection bias related to availability of genotyping ( supplementary data ) . two investigators evaluated the quality of each study independently , and disagreements were resolved through consensus . we were unable to perform quantitative syntheses with meta - analyses because too few studies contained the same snp drug interactions with common outcomes . funding sources had no role in the design , conduct , analysis , or interpretation of the study . of 7,279 citations , we included 34 articles from 21 studies ( 7,8,10,1545 ) comprised of 10,407 subjects ( fig . the included articles used one of three study designs : 1 ) subanalysis of prior randomized controlled trials ( rcts ; n = 13 ) ; 2 ) analysis of observational data ( n = 8) ; and 3 ) nonrandomized , experimental , pre post design without a control group ( n = 13 ) ( table 1 ) . none of the studies were de novo rcts specifically designed to evaluate pharmacogenetic interactions . with the exception of the stop non - insulin dependent diabetes mellitus ( stop - niddm ) trial ( 2023 ) and the diabetes prevention program ( dpp ) ( 7,8,1719,36,37,45 ) , all studies evaluated pharmacogenetic interactions in patients with diabetes . in the dpp , a randomized trial of metformin , a lifestyle intervention , and placebo for diabetes prevention , a broad candidate gene approach ( more than 1,590 candidate gene loci ) was taken to evaluate associations of snps with diabetes and interactions between genetic variants and the trial s interventions ( 1 to 3.2 years of follow - up ) ( 7,8,1719,36,37,45 ) . genetics of diabetes audit and research tayside ( godarts ) investigators performed retrospective analyses of observational data from patients with diabetes who had 12 to 18 months of follow - up using both a genome - wide ( 10 ) and candidate gene approach ( 15,16 ) . selection of included studies . description of included studies genetic interactions with metformin were reported in 14 articles ( table 2 ; supplementary table 4 ) ( 7,8,10,1519,3438,45 ) . interaction between metformin and selected snps for glycemic outcomes genes encoding the metformin transporters , slc22a1 , slc22a2 , and slc47a1 , were each studied in four articles that evaluated different outcomes . for the slc22a1 locus , rs683369 was associated with response to metformin with respect to diabetes risk in the dpp over 3.2 years ( 7 ) , and three snps were not associated with hba1c in people with diabetes in two other studies ( 16,38 ) . for slc22a2 , rs662301 was associated with risk of diabetes at 3.2 years in the metformin arm versus placebo in the dpp ( 7 ) , and two snps were not associated with response to metformin with fg and hba1c as outcomes in three other articles ( 34,36,38 ) . for the slc47a1 locus , rs8065082 was associated with response to metformin for diabetes risk ( 7 ) , and rs2289669 was not associated with metformin response in patients with diabetes ( hba1c as the outcome ) ( 34,38 ) . rs11212617 at the atm locus predicted response to metformin for diabetes risk at 1 year in the dpp ( 37 ) and attainment of hba1c < 7% ( 53 mmol / mol ) at 18 months in godarts ( 10 ) . however , results for hba1c and fg at 1 year were not significant in the dpp ( 37 ) . neither godarts or the dpp found a significant interaction for tcf7l2 snps for attainment of hba1c < 7% ( 53 mmol / mol ) ( 15 ) or diabetes incidence ( 17 ) . statistically significant interactions between metformin and genetic variants were also reported for genes encoding additional proteins associated with amp - activated protein kinase dependent inhibition of gluconeogenesis [ prkab2 , prkaa2 , prkaa1 , stk11 ( 46,47 ) , pck1 , ppara ( 48 ) , and ppargc1a ( 7,49 ) ] , insulin secretion [ kcnj11 ( 7,18 ) , abcc8 , cdkn2a / b ( 45 ) , hnf4a , and hnf1b ( 7 ) ] ; and insulin sensitivity [ adipor2 , enpp1 ( 8) , capn10 , and gck ( 7 ) ] ( table 2 ; supplementary table 4 ) . four studies evaluated the interaction between sulfonylureas and snps ( table 3 ; supplementary table 5 ) ( 15,2426 ) . of two studies evaluating snps in cyp2c9 ( 25,26 ) , the gene encoding the primary hepatic cytochrome p450 enzyme , which metabolizes sulfonylureas , one small study found a greater mean change from baseline in hba1c at 6 months by diplotype of rs1057910 ( 25 ) . notably , the sample size for the variant diplotype was very small ( n = 2 ) ( 25 ) . interaction between sulfonylureas , repaglinide , thiazolidinediones , and acarbose and selected snps for glycemic outcomes godarts evaluated the interaction between two tcf7l2 snps and sulfonylureas and reported a significant association with response to medication ( 15 ) . another study evaluating the interaction between two kcnj11 snps and sulfonylureas did not find any differences in the change in fg across genotypes at 12 months ( 24 ) . eight articles reported on genetic interactions with repaglinide ( table 3 ; supplementary table 6 ) ( 2730,3941,44 ) . of two snps evaluated in the slc30a8 gene ( 28,40 ) , rs13266634 was associated with response to repaglinide using hba1c , fg , and ppg at 8 weeks as outcomes ( 28 ) . similar , but nonsignificant , results were observed with the other snp evaluated ( d = 0.928 for rs168889462 and rs13266634 ) ( 28 ) . notably , rs13266634 has been one of the most replicated genetic risk variants in type 2 diabetes ( 50 ) . a single study reported a significantly different change in hba1c and ppg at 6 months ( and similar , nonsignificant results for fg ) by e23k genotype of kcnj11 , which encodes the potassium channel inhibited by binding of repaglinide to its receptor on the -cell ( 27 ) . snps in neurod1/beta2 , pax4 ( 39 ) , and upc2 ( 44 ) predicted response to repaglinide for some glycemic outcomes ( table 3 ) . three studies reported on interactions between pioglitazone and genetic variation ( 32,33,42 ) ( table 3 ; supplementary table 7 ) . the pro12ala variant was associated with pioglitazone response in one ( 42 ) of two studies evaluating this snp ( 32,42 ) . a single study reported a significant effect of ptprd rs17584499 genotype on ppg at 12 weeks but not on hba1c or fg ( 42 ) . four studies reported on response to rosiglitazone by genetic variation ( table 3 ; supplementary table 8) ( 30,31,40,43 ) . individual studies reported significant interactions between the kcnq1 ( 30 ) and rbp4 ( 43 ) loci and rosiglitazone for some , but not all , glycemic measures . interactions between acarbose and the ppara , hnf4a , lipc , pparg2 , and ppargc1a loci were evaluated in the stop - niddm trial with 3.3 years of follow - up for diabetes risk ( table 3 ; supplementary table 9 ) ( 2023 ) . two of 11 snps from the ppara locus were associated with response to acarbose ( 20 ) . of six snps from the hnf4a locus , two were associated with response to acarbose ( 21 ) . single snps at the lipc and the ppargc1a loci were also associated with response to acarbose ( 22,23 ) . none of the included studies was a prospective rct designed to evaluate a pharmacogenetic interaction , and only 13 of 34 ( 38% ) had a control group . twenty - six of 34 ( 76% ) studies did not report on losses to follow - up . pharmacogenetic analyses were prespecified in 24 of 34 ( 71% ) studies and were either not reported or not prespecified in the remainder of the studies . sixteen of 34 ( 47% ) studies addressed the issue of multiple comparisons ( or only looked at a single snp ) . thirty - one of 34 studies ( 91% ) addressed population stratification by adjusting for admixture or self - reported race / ethnicity or only included one race / ethnicity . only 14 of 34 ( 41% ) reported on genotyping or snp - specific call rate . most studies ( 24 of 34 [ 71% ] ) did not report on genotyping concordance . twenty - seven of 34 ( 79% ) reported on testing for hardy weinberg proportions . studies did not report on masking of genotyping personnel , and 41% declared some form of industry support . genetic interactions with metformin were reported in 14 articles ( table 2 ; supplementary table 4 ) ( 7,8,10,1519,3438,45 ) . interaction between metformin and selected snps for glycemic outcomes genes encoding the metformin transporters , slc22a1 , slc22a2 , and slc47a1 , were each studied in four articles that evaluated different outcomes . for the slc22a1 locus , rs683369 was associated with response to metformin with respect to diabetes risk in the dpp over 3.2 years ( 7 ) , and three snps were not associated with hba1c in people with diabetes in two other studies ( 16,38 ) . for slc22a2 , rs662301 was associated with risk of diabetes at 3.2 years in the metformin arm versus placebo in the dpp ( 7 ) , and two snps were not associated with response to metformin with fg and hba1c as outcomes in three other articles ( 34,36,38 ) . for the slc47a1 locus , rs8065082 was associated with response to metformin for diabetes risk ( 7 ) , and rs2289669 was not associated with metformin response in patients with diabetes ( hba1c as the outcome ) ( 34,38 ) . rs11212617 at the atm locus predicted response to metformin for diabetes risk at 1 year in the dpp ( 37 ) and attainment of hba1c < 7% ( 53 mmol / mol ) at 18 months in godarts ( 10 ) . however , results for hba1c and fg at 1 year were not significant in the dpp ( 37 ) . neither godarts or the dpp found a significant interaction for tcf7l2 snps for attainment of hba1c < 7% ( 53 mmol / mol ) ( 15 ) or diabetes incidence ( 17 ) . statistically significant interactions between metformin and genetic variants were also reported for genes encoding additional proteins associated with amp - activated protein kinase dependent inhibition of gluconeogenesis [ prkab2 , prkaa2 , prkaa1 , stk11 ( 46,47 ) , pck1 , ppara ( 48 ) , and ppargc1a ( 7,49 ) ] , insulin secretion [ kcnj11 ( 7,18 ) , abcc8 , cdkn2a / b ( 45 ) , hnf4a , and hnf1b ( 7 ) ] ; and insulin sensitivity [ adipor2 , enpp1 ( 8) , capn10 , and gck ( 7 ) ] ( table 2 ; supplementary table 4 ) . four studies evaluated the interaction between sulfonylureas and snps ( table 3 ; supplementary table 5 ) ( 15,2426 ) . of two studies evaluating snps in cyp2c9 ( 25,26 ) , the gene encoding the primary hepatic cytochrome p450 enzyme , which metabolizes sulfonylureas , one small study found a greater mean change from baseline in hba1c at 6 months by diplotype of rs1057910 ( 25 ) . notably , the sample size for the variant diplotype was very small ( n = 2 ) ( 25 ) . interaction between sulfonylureas , repaglinide , thiazolidinediones , and acarbose and selected snps for glycemic outcomes godarts evaluated the interaction between two tcf7l2 snps and sulfonylureas and reported a significant association with response to medication ( 15 ) . another study evaluating the interaction between two kcnj11 snps and sulfonylureas did not find any differences in the change in fg across genotypes at 12 months ( 24 ) . eight articles reported on genetic interactions with repaglinide ( table 3 ; supplementary table 6 ) ( 2730,3941,44 ) . of two snps evaluated in the slc30a8 gene ( 28,40 ) , rs13266634 was associated with response to repaglinide using hba1c , fg , and ppg at 8 weeks as outcomes ( 28 ) . similar , but nonsignificant , results were observed with the other snp evaluated ( d = 0.928 for rs168889462 and rs13266634 ) ( 28 ) . notably , rs13266634 has been one of the most replicated genetic risk variants in type 2 diabetes ( 50 ) . a single study reported a significantly different change in hba1c and ppg at 6 months ( and similar , nonsignificant results for fg ) by e23k genotype of kcnj11 , which encodes the potassium channel inhibited by binding of repaglinide to its receptor on the -cell ( 27 ) . snps in neurod1/beta2 , pax4 ( 39 ) , and upc2 ( 44 ) predicted response to repaglinide for some glycemic outcomes ( table 3 ) . three studies reported on interactions between pioglitazone and genetic variation ( 32,33,42 ) ( table 3 ; supplementary table 7 ) . the pro12ala variant was associated with pioglitazone response in one ( 42 ) of two studies evaluating this snp ( 32,42 ) . a single study reported a significant effect of ptprd rs17584499 genotype on ppg at 12 weeks but not on hba1c or fg ( 42 ) . four studies reported on response to rosiglitazone by genetic variation ( table 3 ; supplementary table 8) ( 30,31,40,43 ) . individual studies reported significant interactions between the kcnq1 ( 30 ) and rbp4 ( 43 ) loci and rosiglitazone for some , but not all , glycemic measures . interactions between acarbose and the ppara , hnf4a , lipc , pparg2 , and ppargc1a loci were evaluated in the stop - niddm trial with 3.3 years of follow - up for diabetes risk ( table 3 ; supplementary table 9 ) ( 2023 ) . two of 11 snps from the ppara locus were associated with response to acarbose ( 20 ) . of six snps from the hnf4a locus , two were associated with response to acarbose ( 21 ) . single snps at the lipc and the ppargc1a loci were also associated with response to acarbose ( 22,23 ) . we provide detailed results on the quality of included studies in supplementary table 10 . none of the included studies was a prospective rct designed to evaluate a pharmacogenetic interaction , and only 13 of 34 ( 38% ) had a control group . twenty - six of 34 ( 76% ) studies did not report on losses to follow - up . pharmacogenetic analyses were prespecified in 24 of 34 ( 71% ) studies and were either not reported or not prespecified in the remainder of the studies . sixteen of 34 ( 47% ) studies addressed the issue of multiple comparisons ( or only looked at a single snp ) . thirty - one of 34 studies ( 91% ) addressed population stratification by adjusting for admixture or self - reported race / ethnicity or only included one race / ethnicity . only 14 of 34 ( 41% ) reported on genotyping or snp - specific call rate . most studies ( 24 of 34 [ 71% ] ) did not report on genotyping concordance . twenty - seven of 34 ( 79% ) reported on testing for hardy weinberg proportions . studies did not report on masking of genotyping personnel , and 41% declared some form of industry support . in this systematic review , we identified 34 articles on the pharmacogenetics of diabetes medications , with several reporting statistically significant interactions between genetic variants and medications for glycemic outcomes . most pharmacogenetic interactions were only evaluated in a single study , did not use a control group , and/or did not report enough information to judge internal validity . however , our results do suggest specific , biologically plausible , gene medication interactions , and we recommend confirmation of the biologically plausible interactions as a priority , including those for drug transporters , metabolizers , and targets of action . in particular , we recommend follow - up of the 1 ) slc22a1 , slc22a2 , slc47a1 , prkab2 , prkaa2 , prkaa1 , and stk11 loci for metformin ; 2 ) cyp2c9 and tcf7l2 loci for sulfonylureas ; 3 ) kcnj11 , slc30a8 , neurod1/beta2 , ucp2 , and pax4 loci for repaglinide ; 4 ) pparg2 and ptprd for pioglitazone ; 5 ) kcnq1 and rbp4 loci for rosiglitazone ; and 6 ) ppara , hnf4a , lipc , and ppargc1a loci for acarbose . given the number of comparisons reported in the included studies and the lack of accounting for multiple comparisons in approximately 53% of studies , many of the reported findings may be false positives . however , we expect interactions between response to medications and genes encoding their transporters , metabolizers , targets , and components of their pathways for action as observed in the included studies . the dpp reported significant interactions between metformin and loci for its transporters ( slc22a1 , slc22a2 , and slc47a1 ) ( 7 ) . it deserves mention that positive findings were not replicated in other studies evaluating these loci ( 16,34,38 ) , but outcomes ( mean change in quantitative traits , achievement of hba1c < 7% ( 53 mmol / mol ) versus diabetes risk in the dpp ) and follow - up time ( 6 to 18 months vs. 3 years in the dpp ) differed in the other studies as well as did study design . the dpp also reported on significant interactions for loci associated with metformin pharmacodynamics ( prkab2 , prkaa2 , prkaa1 , and stk11 ) ( 7,46 ) . the primary action of metformin is the inhibition of hepatic glucose production through inhibition of gluconeogenesis , and interactions with loci associated within this pathway ( pck1 , ppara , and ppargc1a ) were reported ( 7,48,49 ) . sulfonylureas and repaglinide bind to the sulfonylurea receptor ( encoded by abcc8 ) , which then inhibits the function of the potassium channel encoded by kcnj11 and causes -cell depolarization and eventual insulin secretion . while we did identify interactions between repaglinide and kcnj11 ( 27 ) , this locus was not associated with sulfonylurea action in a single study that evaluated fg ( 24 ) . variation in cyp2c9 , which encodes an enzyme that metabolizes sulfonylureas , was associated with response to sulfonylureas in one ( 25 ) of two studies ( 25,26 ) . finally , the thiazolidinediones activate peroxisome proliferator - activated receptor receptors , which regulate expression of genes important for sensitivity to insulin . thus , variation in pparg would likely affect response to this class of medications , and this was suggested in one ( 42 ) of the two studies ( 32,42 ) evaluating this for pioglitazone and was not evaluated for rosiglitazone . variation in the hepatic cytochrome p450 enzymes , which metabolize diabetes medications , would be expected to impact their effects , including variation in cyp3a4 and cyp2c8 for repaglinide ( 51 ) , cyp2c8 and cyp2c9 for rosiglitazone ( 52 ) , and cyp2c8 and cyp3a4 for pioglitazone ( 53 ) . the transporter encoded by slcob1 transports repaglinide into hepatocytes for metabolism , and variation in this gene could affect the response to this medication ( 51 ) . acarbose primarily decreases intestinal glucose absorption by inhibiting brush border enzymes that hydrolyze carbohydrates and is mainly excreted fecally and does not seem to have obvious pharmacokinetic or pharmacodynamic targets . generally , we would also expect genetic variants that impact -cell function to affect the response to insulin secretagogues and genetic variants that impact insulin sensitivity to affect response to insulin sensitizing medications . also , because of its primary effect on ppg , genetic variation impacting glucose - stimulated insulin secretion would likely impact the response to acarbose . this rationale may explain other observed significant pharmacogenetic interactions ( e.g. , rosiglitazone kcnq1 , repaglinide prior work in this area has consisted of mainly narrative reviews , many of which have included the studies that we identified ( 36 ) . we add to this literature by using a thorough and systematic approach with double review at all levels to identify as many studies as possible that have reported some interaction between individual diabetes medications of interest and diabetes risk and glycemic outcomes . thus we present the state of the literature on the pharmacogenetics of type 2 diabetes , which lays the groundwork for directing future research efforts . another novel contribution of our systematic review is the collection of detailed quality information from included studies , which aids in the interpretation of prior studies and illuminates areas for improvement and standardization . the major limitation of the literature on the pharmacogenomics of type 2 diabetes is the lack of high - quality studies to identify and confirm findings for specific drug outcome combinations : 1 ) the rationale for selection of loci and interactions studied was often not clear , which raises the concern of selective reporting of results and publication bias ; in particular , we would be less suspicious of false - positive results in the setting of prespecified analyses based on prior evidence and/or biologic plausibility with adjustment for multiple comparisons . therefore , it is likely that positive results were reported and that null results were not . 2 ) the small size of many of the studies does not exclude the possibility that interactions exist but could not be identified because of lack of power ; we reported study results as significant based on a p value less than 0.05 but have noted results when p values were < 0.20 when possible . meta - analysis could help to address this issue , but the heterogeneity of studies with specific snp drug interactions , outcomes , and follow - up times differing across studies precluded quantitative synthesis with meta - analyses . while our qualitative synthesis summarizes the literature and therefore , our inferences often relied on the results of a single medication intervention on change in or incidence of outcomes by genotype ; these types of studies do not exclude the possibility that we are simply observing the effect of genotype and not specifically modification of the response to the medication . 4 ) studies did not generally provide information to determine the potential for selection bias based on availability of genotyping information , on losses to follow up , or on the amount and handling of missing data . regarding selection bias due to availability of genotyping , participant behaviors ( e.g. , adherence to intervention , follow - up ) and outcomes ( diabetes , death ) may have differed between those with and without genotyping information ; these kinds of differentiating characteristics in participants included in genetic analyses could impact the observed gene 5 ) while studies did provide information on methods for genotyping , information on snp - specific call rate was often not reported , and studies did not report on masking of personnel performing genotyping . 6 ) while none of the included studies were actually de novo rcts , which would limit selection bias and confounding most completely , several articles were based on data from prior , well - conducted randomized trials ( 7,8,1723,36,37,45 ) . in the case of these trials , we would not expect that participant characteristics correlated with genotype would be related to assigned intervention and thus can feel more confident about the robustness of these studies . to address these issues with quality , we did tailor our inclusion criteria and abstraction tools to limit the inclusion of poor - quality studies and to understand the important potential sources of bias . one limitation of our systematic review methodology is the exclusion of studies of patients on more than one diabetes medication . we sought to identify pharmacogenetic interactions that were based on a single drug and single genetic variant and wanted to avoid drug drug snp interactions . because of this exclusion , we did not include additional articles on the pharmacogenetics of diabetes in this review ( 11,5456 ) . drug interactions based on pharmacodynamics [ e.g. , pparg rosiglitazone ( 54 ) , slc22a1metformin ( 11 ) , irs1sulfonylurea ( 55 ) , and atm metformin ( 56 ) ] . these studies confirm our hypotheses regarding the possibility of gene drug interactions based on pharmacodynamics but are still individual studies different enough from the existing literature to preclude meta - analysis . we also excluded studies of non - fda - approved medications and therefore did not include the article by feng et al . evaluating interactions with gliclazide ; this study did demonstrate a significant interaction between gliclazide and abcc8 and kcnj11 snps consistent with the pharmacodynamics of sulfonylureas ( 57 ) . we limited our analyses to diabetes and glycemic outcomes ( hba1c , fg , ppg ) because these are more commonly and consistently measured and are also strongly associated with improvements in long - term complications and mortality ( 58 ) . future studies should evaluate other important efficacy and safety outcomes . finally , because of study resource limitations , we excluded non - english language studies from our initial search and can not estimate the number of otherwise - eligible studies that we excluded based on this . we recommend that guidelines for the design , analysis , and reporting of pharmacogenetic studies of diabetes medications be developed to improve study quality and enhance comparability among studies ; we have provided a prioritized list of quality and reporting items in supplementary table 11 . the incorporation of response to medications based on genetic variation into clinical practice can not occur without well - designed studies confirming significant pharmacogenetic interactions . based on the limitations of the current literature , we recommend the following for future studies : 1 ) a priori specification of the snps and medications to be studied , 2 ) the use of experimental designs , 3 ) inclusion of a concurrent comparison group when possible , 4 ) agreement in the diabetes pharmacogenetics community regarding standardized outcomes and follow - up ( e.g. , hba1c at 3 months ) , 5 ) sufficient power for the primary outcome , 6 ) adjustment for multiple comparisons if multiple snps are examined , and 7 ) controlling for population stratification and relatedness . in addition , independent replication is important . we recommend that diabetes pharmacogenetics studies use current guidelines for reporting of genetic association studies ( 12 ) and that these guidelines be extended to emphasize information relevant to pharmacogenetic studies , including prespecified reporting of analyses with rationale , estimates of type 2 error , standardized reporting of medication interventions , and reporting of differences between genotyped and nongenotyped subjects when possible . in conclusion , for all known efficacious diabetes preventive and therapeutic pharmacologic agents , some patients benefit or experience harm while others do not . in this systematic review , we find evidence of biologically plausible pharmacogenetic interactions for metformin , sulfonylureas , repaglinide , pioglitazone , rosiglitazone , and acarbose , but these results require confirmation in future studies to determine if an individual s genetic information can be used to individualize the choice of prediabetes and diabetes pharmacologic management . importantly , our results should guide the development of guidelines for the design , conduct , and reporting of studies of the pharmacogenetics of type 2 diabetes and other chronic conditions . these promising results show the potential of using genetic variation to tailor therapy for type 2 diabetes prevention and management . we recommend that guidelines for the design , analysis , and reporting of pharmacogenetic studies of diabetes medications be developed to improve study quality and enhance comparability among studies ; we have provided a prioritized list of quality and reporting items in supplementary table 11 . the incorporation of response to medications based on genetic variation into clinical practice can not occur without well - designed studies confirming significant pharmacogenetic interactions . based on the limitations of the current literature , we recommend the following for future studies : 1 ) a priori specification of the snps and medications to be studied , 2 ) the use of experimental designs , 3 ) inclusion of a concurrent comparison group when possible , 4 ) agreement in the diabetes pharmacogenetics community regarding standardized outcomes and follow - up ( e.g. , hba1c at 3 months ) , 5 ) sufficient power for the primary outcome , 6 ) adjustment for multiple comparisons if multiple snps are examined , and 7 ) controlling for population stratification and relatedness . in addition we recommend that diabetes pharmacogenetics studies use current guidelines for reporting of genetic association studies ( 12 ) and that these guidelines be extended to emphasize information relevant to pharmacogenetic studies , including prespecified reporting of analyses with rationale , estimates of type 2 error , standardized reporting of medication interventions , and reporting of differences between genotyped and nongenotyped subjects when possible . in conclusion , for all known efficacious diabetes preventive and therapeutic pharmacologic agents , some patients benefit or experience harm while others do not . in this systematic review , we find evidence of biologically plausible pharmacogenetic interactions for metformin , sulfonylureas , repaglinide , pioglitazone , rosiglitazone , and acarbose , but these results require confirmation in future studies to determine if an individual s genetic information can be used to individualize the choice of prediabetes and diabetes pharmacologic management . importantly , our results should guide the development of guidelines for the design , conduct , and reporting of studies of the pharmacogenetics of type 2 diabetes and other chronic conditions . these promising results show the potential of using genetic variation to tailor therapy for type 2 diabetes prevention and management .

objectivewe performed a systematic review to identify which genetic variants predict response to diabetes medications.research design and methodswe performed a search of electronic databases ( pubmed , embase , and cochrane database ) and a manual search to identify original , longitudinal studies of the effect of diabetes medications on incident diabetes , hba1c , fasting glucose , and postprandial glucose in prediabetes or type 2 diabetes by genetic variation . two investigators reviewed titles , abstracts , and articles independently . two investigators abstracted data sequentially and evaluated study quality independently . quality evaluations were based on the strengthening the reporting of genetic association studies guidelines and human genome epidemiology network guidance.resultsof 7,279 citations , we included 34 articles ( n = 10,407 ) evaluating metformin ( n = 14 ) , sulfonylureas ( n = 4 ) , repaglinide ( n = 8) , pioglitazone ( n = 3 ) , rosiglitazone ( n = 4 ) , and acarbose ( n = 4 ) . studies were not standalone randomized controlled trials , and most evaluated patients with diabetes . significant medication gene interactions for glycemic outcomes included 1 ) metformin and the slc22a1 , slc22a2 , slc47a1 , prkab2 , prkaa2 , prkaa1 , and stk11 loci ; 2 ) sulfonylureas and the cyp2c9 and tcf7l2 loci ; 3 ) repaglinide and the kcnj11 , slc30a8 , neurod1/beta2 , ucp2 , and pax4 loci ; 4 ) pioglitazone and the pparg2 and ptprd loci ; 5 ) rosiglitazone and the kcnq1 and rbp4 loci ; and 5 ) acarbose and the ppara , hnf4a , lipc , and ppargc1a loci . data were insufficient for meta-analysis.conclusionswe found evidence of pharmacogenetic interactions for metformin , sulfonylureas , repaglinide , thiazolidinediones , and acarbose consistent with their pharmacokinetics and pharmacodynamics . while high - quality controlled studies with prespecified analyses are still lacking , our results bring the promise of personalized medicine in diabetes one step closer to fruition .

Introduction Research Design and Methods Results Metformin Sulfonylureas Repaglinide Pioglitazone Rosiglitazone Acarbose Quality of Included Studies Conclusions Guidance for the Development of Future Evidence in Diabetes Pharmacogenetics Supplementary Material

as recognized by the american diabetes association in its 2012 statement on the management of hyperglycemia , care in type 2 diabetes must become more patient - centered ( 2 ) , and the individualization of diabetes prevention and treatment based on genetic variation has great potential . narrative reviews have commented on the promise of pharmacogenomics of type 2 diabetes ( 36 ) , and prominent individual studies have found statistically significant pharmacogenetic interactions associated with diabetes risk and glycemic outcomes ( 711 ) . the clinical utility of genetic variation for tailoring diabetes medications rests on the identification of substantial and statistically significant pharmacogenetic interactions from internally valid studies and confirmation of their findings in varied populations based on race / ethnicity . we conducted a systematic review of observational and experimental studies to determine if the effect of diabetes medications on diabetes incidence , hba1c , fasting glucose ( fg ) , and postprandial glucose ( ppg ) varies by independent genetic variation in patients with impaired fg , impaired glucose tolerance , or type 2 diabetes . we hypothesized that 1 ) genetic variation associated with drug transporters , metabolizers , targets , and mechanisms of action would modify the effect of specific drugs and 2 ) the existing evidence would be insufficient to recommend clinical use of pharmacogenetic interactions because of a lack of well - conducted studies across diverse populations . senior members of the study were diabetes and obesity researchers with training in clinical epidemiology , clinical trials , and systematic review methodology ( e.b . we searched the pubmed , embase , cochrane electronic databases and also manually searched key review articles , key journals tables of contents , and the references of included articles . the pubmed search and list of key journals the electronic search included dates of database inception through 13 march 2013 , and the manual search of tables of contents included january to march 2013 . we included original articles on the effect of food and drug administration ( fda)-approved diabetes medications ( supplementary table 3 ) on diabetes incidence , hba1c , fg , and ppg in adults with either type 2 diabetes or increased diabetes risk because of impaired fg ( fg 5.556.94 mmol / l ) or impaired glucose tolerance ( 2-h postload [ 75 g ] glucose 7.7711.04 mmol / l ) by common genetic variation . eligible study designs were 1 ) controlled studies evaluating the effect of a drug for one allele / genotype versus another over time and 2 ) uncontrolled studies evaluating the effect ( change in outcome or incidence of outcome ) of a drug comparing one genotype / allele to another . we developed quality abstraction forms based on the strengthening the reporting of genetic association studies guidelines for reporting of genetic association studies ( 12 ) . in the absence of guidelines for pharmacogenetic studies , we also incorporated recommendations from the hugenet ( human genome epidemiology network ) huge review handbook ( 13 ) and prior methodological papers ( 14 ) . of 7,279 citations , we included 34 articles from 21 studies ( 7,8,10,1545 ) comprised of 10,407 subjects ( fig . the included articles used one of three study designs : 1 ) subanalysis of prior randomized controlled trials ( rcts ; n = 13 ) ; 2 ) analysis of observational data ( n = 8) ; and 3 ) nonrandomized , experimental , pre post design without a control group ( n = 13 ) ( table 1 ) . none of the studies were de novo rcts specifically designed to evaluate pharmacogenetic interactions . with the exception of the stop non - insulin dependent diabetes mellitus ( stop - niddm ) trial ( 2023 ) and the diabetes prevention program ( dpp ) ( 7,8,1719,36,37,45 ) , all studies evaluated pharmacogenetic interactions in patients with diabetes . in the dpp , a randomized trial of metformin , a lifestyle intervention , and placebo for diabetes prevention , a broad candidate gene approach ( more than 1,590 candidate gene loci ) was taken to evaluate associations of snps with diabetes and interactions between genetic variants and the trial s interventions ( 1 to 3.2 years of follow - up ) ( 7,8,1719,36,37,45 ) . genetics of diabetes audit and research tayside ( godarts ) investigators performed retrospective analyses of observational data from patients with diabetes who had 12 to 18 months of follow - up using both a genome - wide ( 10 ) and candidate gene approach ( 15,16 ) . interaction between metformin and selected snps for glycemic outcomes genes encoding the metformin transporters , slc22a1 , slc22a2 , and slc47a1 , were each studied in four articles that evaluated different outcomes . for the slc22a1 locus , rs683369 was associated with response to metformin with respect to diabetes risk in the dpp over 3.2 years ( 7 ) , and three snps were not associated with hba1c in people with diabetes in two other studies ( 16,38 ) . for slc22a2 , rs662301 was associated with risk of diabetes at 3.2 years in the metformin arm versus placebo in the dpp ( 7 ) , and two snps were not associated with response to metformin with fg and hba1c as outcomes in three other articles ( 34,36,38 ) . for the slc47a1 locus , rs8065082 was associated with response to metformin for diabetes risk ( 7 ) , and rs2289669 was not associated with metformin response in patients with diabetes ( hba1c as the outcome ) ( 34,38 ) . statistically significant interactions between metformin and genetic variants were also reported for genes encoding additional proteins associated with amp - activated protein kinase dependent inhibition of gluconeogenesis [ prkab2 , prkaa2 , prkaa1 , stk11 ( 46,47 ) , pck1 , ppara ( 48 ) , and ppargc1a ( 7,49 ) ] , insulin secretion [ kcnj11 ( 7,18 ) , abcc8 , cdkn2a / b ( 45 ) , hnf4a , and hnf1b ( 7 ) ] ; and insulin sensitivity [ adipor2 , enpp1 ( 8) , capn10 , and gck ( 7 ) ] ( table 2 ; supplementary table 4 ) . interaction between sulfonylureas , repaglinide , thiazolidinediones , and acarbose and selected snps for glycemic outcomes godarts evaluated the interaction between two tcf7l2 snps and sulfonylureas and reported a significant association with response to medication ( 15 ) . of two snps evaluated in the slc30a8 gene ( 28,40 ) , rs13266634 was associated with response to repaglinide using hba1c , fg , and ppg at 8 weeks as outcomes ( 28 ) . snps in neurod1/beta2 , pax4 ( 39 ) , and upc2 ( 44 ) predicted response to repaglinide for some glycemic outcomes ( table 3 ) . four studies reported on response to rosiglitazone by genetic variation ( table 3 ; supplementary table 8) ( 30,31,40,43 ) . interactions between acarbose and the ppara , hnf4a , lipc , pparg2 , and ppargc1a loci were evaluated in the stop - niddm trial with 3.3 years of follow - up for diabetes risk ( table 3 ; supplementary table 9 ) ( 2023 ) . two of 11 snps from the ppara locus were associated with response to acarbose ( 20 ) . single snps at the lipc and the ppargc1a loci were also associated with response to acarbose ( 22,23 ) . interaction between metformin and selected snps for glycemic outcomes genes encoding the metformin transporters , slc22a1 , slc22a2 , and slc47a1 , were each studied in four articles that evaluated different outcomes . for the slc22a1 locus , rs683369 was associated with response to metformin with respect to diabetes risk in the dpp over 3.2 years ( 7 ) , and three snps were not associated with hba1c in people with diabetes in two other studies ( 16,38 ) . for slc22a2 , rs662301 was associated with risk of diabetes at 3.2 years in the metformin arm versus placebo in the dpp ( 7 ) , and two snps were not associated with response to metformin with fg and hba1c as outcomes in three other articles ( 34,36,38 ) . for the slc47a1 locus , rs8065082 was associated with response to metformin for diabetes risk ( 7 ) , and rs2289669 was not associated with metformin response in patients with diabetes ( hba1c as the outcome ) ( 34,38 ) . statistically significant interactions between metformin and genetic variants were also reported for genes encoding additional proteins associated with amp - activated protein kinase dependent inhibition of gluconeogenesis [ prkab2 , prkaa2 , prkaa1 , stk11 ( 46,47 ) , pck1 , ppara ( 48 ) , and ppargc1a ( 7,49 ) ] , insulin secretion [ kcnj11 ( 7,18 ) , abcc8 , cdkn2a / b ( 45 ) , hnf4a , and hnf1b ( 7 ) ] ; and insulin sensitivity [ adipor2 , enpp1 ( 8) , capn10 , and gck ( 7 ) ] ( table 2 ; supplementary table 4 ) . interaction between sulfonylureas , repaglinide , thiazolidinediones , and acarbose and selected snps for glycemic outcomes godarts evaluated the interaction between two tcf7l2 snps and sulfonylureas and reported a significant association with response to medication ( 15 ) . of two snps evaluated in the slc30a8 gene ( 28,40 ) , rs13266634 was associated with response to repaglinide using hba1c , fg , and ppg at 8 weeks as outcomes ( 28 ) . snps in neurod1/beta2 , pax4 ( 39 ) , and upc2 ( 44 ) predicted response to repaglinide for some glycemic outcomes ( table 3 ) . interactions between acarbose and the ppara , hnf4a , lipc , pparg2 , and ppargc1a loci were evaluated in the stop - niddm trial with 3.3 years of follow - up for diabetes risk ( table 3 ; supplementary table 9 ) ( 2023 ) . single snps at the lipc and the ppargc1a loci were also associated with response to acarbose ( 22,23 ) . in this systematic review , we identified 34 articles on the pharmacogenetics of diabetes medications , with several reporting statistically significant interactions between genetic variants and medications for glycemic outcomes . however , our results do suggest specific , biologically plausible , gene medication interactions , and we recommend confirmation of the biologically plausible interactions as a priority , including those for drug transporters , metabolizers , and targets of action . in particular , we recommend follow - up of the 1 ) slc22a1 , slc22a2 , slc47a1 , prkab2 , prkaa2 , prkaa1 , and stk11 loci for metformin ; 2 ) cyp2c9 and tcf7l2 loci for sulfonylureas ; 3 ) kcnj11 , slc30a8 , neurod1/beta2 , ucp2 , and pax4 loci for repaglinide ; 4 ) pparg2 and ptprd for pioglitazone ; 5 ) kcnq1 and rbp4 loci for rosiglitazone ; and 6 ) ppara , hnf4a , lipc , and ppargc1a loci for acarbose . the dpp reported significant interactions between metformin and loci for its transporters ( slc22a1 , slc22a2 , and slc47a1 ) ( 7 ) . the dpp also reported on significant interactions for loci associated with metformin pharmacodynamics ( prkab2 , prkaa2 , prkaa1 , and stk11 ) ( 7,46 ) . thus , variation in pparg would likely affect response to this class of medications , and this was suggested in one ( 42 ) of the two studies ( 32,42 ) evaluating this for pioglitazone and was not evaluated for rosiglitazone . variation in the hepatic cytochrome p450 enzymes , which metabolize diabetes medications , would be expected to impact their effects , including variation in cyp3a4 and cyp2c8 for repaglinide ( 51 ) , cyp2c8 and cyp2c9 for rosiglitazone ( 52 ) , and cyp2c8 and cyp3a4 for pioglitazone ( 53 ) . thus we present the state of the literature on the pharmacogenetics of type 2 diabetes , which lays the groundwork for directing future research efforts . the major limitation of the literature on the pharmacogenomics of type 2 diabetes is the lack of high - quality studies to identify and confirm findings for specific drug outcome combinations : 1 ) the rationale for selection of loci and interactions studied was often not clear , which raises the concern of selective reporting of results and publication bias ; in particular , we would be less suspicious of false - positive results in the setting of prespecified analyses based on prior evidence and/or biologic plausibility with adjustment for multiple comparisons . 2 ) the small size of many of the studies does not exclude the possibility that interactions exist but could not be identified because of lack of power ; we reported study results as significant based on a p value less than 0.05 but have noted results when p values were < 0.20 when possible . while our qualitative synthesis summarizes the literature and therefore , our inferences often relied on the results of a single medication intervention on change in or incidence of outcomes by genotype ; these types of studies do not exclude the possibility that we are simply observing the effect of genotype and not specifically modification of the response to the medication . 4 ) studies did not generally provide information to determine the potential for selection bias based on availability of genotyping information , on losses to follow up , or on the amount and handling of missing data . , adherence to intervention , follow - up ) and outcomes ( diabetes , death ) may have differed between those with and without genotyping information ; these kinds of differentiating characteristics in participants included in genetic analyses could impact the observed gene 5 ) while studies did provide information on methods for genotyping , information on snp - specific call rate was often not reported , and studies did not report on masking of personnel performing genotyping . 6 ) while none of the included studies were actually de novo rcts , which would limit selection bias and confounding most completely , several articles were based on data from prior , well - conducted randomized trials ( 7,8,1723,36,37,45 ) . we sought to identify pharmacogenetic interactions that were based on a single drug and single genetic variant and wanted to avoid drug drug snp interactions . , pparg rosiglitazone ( 54 ) , slc22a1metformin ( 11 ) , irs1sulfonylurea ( 55 ) , and atm metformin ( 56 ) ] . we recommend that guidelines for the design , analysis , and reporting of pharmacogenetic studies of diabetes medications be developed to improve study quality and enhance comparability among studies ; we have provided a prioritized list of quality and reporting items in supplementary table 11 . the incorporation of response to medications based on genetic variation into clinical practice can not occur without well - designed studies confirming significant pharmacogenetic interactions . based on the limitations of the current literature , we recommend the following for future studies : 1 ) a priori specification of the snps and medications to be studied , 2 ) the use of experimental designs , 3 ) inclusion of a concurrent comparison group when possible , 4 ) agreement in the diabetes pharmacogenetics community regarding standardized outcomes and follow - up ( e.g. , hba1c at 3 months ) , 5 ) sufficient power for the primary outcome , 6 ) adjustment for multiple comparisons if multiple snps are examined , and 7 ) controlling for population stratification and relatedness . we recommend that diabetes pharmacogenetics studies use current guidelines for reporting of genetic association studies ( 12 ) and that these guidelines be extended to emphasize information relevant to pharmacogenetic studies , including prespecified reporting of analyses with rationale , estimates of type 2 error , standardized reporting of medication interventions , and reporting of differences between genotyped and nongenotyped subjects when possible . in this systematic review , we find evidence of biologically plausible pharmacogenetic interactions for metformin , sulfonylureas , repaglinide , pioglitazone , rosiglitazone , and acarbose , but these results require confirmation in future studies to determine if an individual s genetic information can be used to individualize the choice of prediabetes and diabetes pharmacologic management . importantly , our results should guide the development of guidelines for the design , conduct , and reporting of studies of the pharmacogenetics of type 2 diabetes and other chronic conditions . we recommend that guidelines for the design , analysis , and reporting of pharmacogenetic studies of diabetes medications be developed to improve study quality and enhance comparability among studies ; we have provided a prioritized list of quality and reporting items in supplementary table 11 . the incorporation of response to medications based on genetic variation into clinical practice can not occur without well - designed studies confirming significant pharmacogenetic interactions . based on the limitations of the current literature , we recommend the following for future studies : 1 ) a priori specification of the snps and medications to be studied , 2 ) the use of experimental designs , 3 ) inclusion of a concurrent comparison group when possible , 4 ) agreement in the diabetes pharmacogenetics community regarding standardized outcomes and follow - up ( e.g. , hba1c at 3 months ) , 5 ) sufficient power for the primary outcome , 6 ) adjustment for multiple comparisons if multiple snps are examined , and 7 ) controlling for population stratification and relatedness . in addition we recommend that diabetes pharmacogenetics studies use current guidelines for reporting of genetic association studies ( 12 ) and that these guidelines be extended to emphasize information relevant to pharmacogenetic studies , including prespecified reporting of analyses with rationale , estimates of type 2 error , standardized reporting of medication interventions , and reporting of differences between genotyped and nongenotyped subjects when possible . in this systematic review , we find evidence of biologically plausible pharmacogenetic interactions for metformin , sulfonylureas , repaglinide , pioglitazone , rosiglitazone , and acarbose , but these results require confirmation in future studies to determine if an individual s genetic information can be used to individualize the choice of prediabetes and diabetes pharmacologic management . importantly , our results should guide the development of guidelines for the design , conduct , and reporting of studies of the pharmacogenetics of type 2 diabetes and other chronic conditions .

as recognized by the american diabetes association in its 2012 statement on the management of hyperglycemia , care in type 2 diabetes must become more patient - centered ( 2 ) , and the individualization of diabetes prevention and treatment based on genetic variation has great potential . narrative reviews have commented on the promise of pharmacogenomics of type 2 diabetes ( 36 ) , and prominent individual studies have found statistically significant pharmacogenetic interactions associated with diabetes risk and glycemic outcomes ( 711 ) . however , prior reviews have not systematically evaluated this literature to inform future research questions , and these reviews do not address the quality issues that affect the existing literature on diabetes pharmacogenetics . the clinical utility of genetic variation for tailoring diabetes medications rests on the identification of substantial and statistically significant pharmacogenetic interactions from internally valid studies and confirmation of their findings in varied populations based on race / ethnicity . we conducted a systematic review of observational and experimental studies to determine if the effect of diabetes medications on diabetes incidence , hba1c , fasting glucose ( fg ) , and postprandial glucose ( ppg ) varies by independent genetic variation in patients with impaired fg , impaired glucose tolerance , or type 2 diabetes . we hypothesized that 1 ) genetic variation associated with drug transporters , metabolizers , targets , and mechanisms of action would modify the effect of specific drugs and 2 ) the existing evidence would be insufficient to recommend clinical use of pharmacogenetic interactions because of a lack of well - conducted studies across diverse populations . senior members of the study were diabetes and obesity researchers with training in clinical epidemiology , clinical trials , and systematic review methodology ( e.b . we searched the pubmed , embase , cochrane electronic databases and also manually searched key review articles , key journals tables of contents , and the references of included articles . the pubmed search and list of key journals the electronic search included dates of database inception through 13 march 2013 , and the manual search of tables of contents included january to march 2013 . we included original articles on the effect of food and drug administration ( fda)-approved diabetes medications ( supplementary table 3 ) on diabetes incidence , hba1c , fg , and ppg in adults with either type 2 diabetes or increased diabetes risk because of impaired fg ( fg 5.556.94 mmol / l ) or impaired glucose tolerance ( 2-h postload [ 75 g ] glucose 7.7711.04 mmol / l ) by common genetic variation . eligible study designs were 1 ) controlled studies evaluating the effect of a drug for one allele / genotype versus another over time and 2 ) uncontrolled studies evaluating the effect ( change in outcome or incidence of outcome ) of a drug comparing one genotype / allele to another . in the absence of guidelines for pharmacogenetic studies , we also incorporated recommendations from the hugenet ( human genome epidemiology network ) huge review handbook ( 13 ) and prior methodological papers ( 14 ) . the included articles used one of three study designs : 1 ) subanalysis of prior randomized controlled trials ( rcts ; n = 13 ) ; 2 ) analysis of observational data ( n = 8) ; and 3 ) nonrandomized , experimental , pre post design without a control group ( n = 13 ) ( table 1 ) . with the exception of the stop non - insulin dependent diabetes mellitus ( stop - niddm ) trial ( 2023 ) and the diabetes prevention program ( dpp ) ( 7,8,1719,36,37,45 ) , all studies evaluated pharmacogenetic interactions in patients with diabetes . in the dpp , a randomized trial of metformin , a lifestyle intervention , and placebo for diabetes prevention , a broad candidate gene approach ( more than 1,590 candidate gene loci ) was taken to evaluate associations of snps with diabetes and interactions between genetic variants and the trial s interventions ( 1 to 3.2 years of follow - up ) ( 7,8,1719,36,37,45 ) . genetics of diabetes audit and research tayside ( godarts ) investigators performed retrospective analyses of observational data from patients with diabetes who had 12 to 18 months of follow - up using both a genome - wide ( 10 ) and candidate gene approach ( 15,16 ) . interaction between metformin and selected snps for glycemic outcomes genes encoding the metformin transporters , slc22a1 , slc22a2 , and slc47a1 , were each studied in four articles that evaluated different outcomes . for the slc22a1 locus , rs683369 was associated with response to metformin with respect to diabetes risk in the dpp over 3.2 years ( 7 ) , and three snps were not associated with hba1c in people with diabetes in two other studies ( 16,38 ) . for slc22a2 , rs662301 was associated with risk of diabetes at 3.2 years in the metformin arm versus placebo in the dpp ( 7 ) , and two snps were not associated with response to metformin with fg and hba1c as outcomes in three other articles ( 34,36,38 ) . for the slc47a1 locus , rs8065082 was associated with response to metformin for diabetes risk ( 7 ) , and rs2289669 was not associated with metformin response in patients with diabetes ( hba1c as the outcome ) ( 34,38 ) . rs11212617 at the atm locus predicted response to metformin for diabetes risk at 1 year in the dpp ( 37 ) and attainment of hba1c < 7% ( 53 mmol / mol ) at 18 months in godarts ( 10 ) . neither godarts or the dpp found a significant interaction for tcf7l2 snps for attainment of hba1c < 7% ( 53 mmol / mol ) ( 15 ) or diabetes incidence ( 17 ) . statistically significant interactions between metformin and genetic variants were also reported for genes encoding additional proteins associated with amp - activated protein kinase dependent inhibition of gluconeogenesis [ prkab2 , prkaa2 , prkaa1 , stk11 ( 46,47 ) , pck1 , ppara ( 48 ) , and ppargc1a ( 7,49 ) ] , insulin secretion [ kcnj11 ( 7,18 ) , abcc8 , cdkn2a / b ( 45 ) , hnf4a , and hnf1b ( 7 ) ] ; and insulin sensitivity [ adipor2 , enpp1 ( 8) , capn10 , and gck ( 7 ) ] ( table 2 ; supplementary table 4 ) . of two studies evaluating snps in cyp2c9 ( 25,26 ) , the gene encoding the primary hepatic cytochrome p450 enzyme , which metabolizes sulfonylureas , one small study found a greater mean change from baseline in hba1c at 6 months by diplotype of rs1057910 ( 25 ) . interaction between sulfonylureas , repaglinide , thiazolidinediones , and acarbose and selected snps for glycemic outcomes godarts evaluated the interaction between two tcf7l2 snps and sulfonylureas and reported a significant association with response to medication ( 15 ) . of two snps evaluated in the slc30a8 gene ( 28,40 ) , rs13266634 was associated with response to repaglinide using hba1c , fg , and ppg at 8 weeks as outcomes ( 28 ) . a single study reported a significantly different change in hba1c and ppg at 6 months ( and similar , nonsignificant results for fg ) by e23k genotype of kcnj11 , which encodes the potassium channel inhibited by binding of repaglinide to its receptor on the -cell ( 27 ) . interactions between acarbose and the ppara , hnf4a , lipc , pparg2 , and ppargc1a loci were evaluated in the stop - niddm trial with 3.3 years of follow - up for diabetes risk ( table 3 ; supplementary table 9 ) ( 2023 ) . interaction between metformin and selected snps for glycemic outcomes genes encoding the metformin transporters , slc22a1 , slc22a2 , and slc47a1 , were each studied in four articles that evaluated different outcomes . for the slc22a1 locus , rs683369 was associated with response to metformin with respect to diabetes risk in the dpp over 3.2 years ( 7 ) , and three snps were not associated with hba1c in people with diabetes in two other studies ( 16,38 ) . for slc22a2 , rs662301 was associated with risk of diabetes at 3.2 years in the metformin arm versus placebo in the dpp ( 7 ) , and two snps were not associated with response to metformin with fg and hba1c as outcomes in three other articles ( 34,36,38 ) . for the slc47a1 locus , rs8065082 was associated with response to metformin for diabetes risk ( 7 ) , and rs2289669 was not associated with metformin response in patients with diabetes ( hba1c as the outcome ) ( 34,38 ) . rs11212617 at the atm locus predicted response to metformin for diabetes risk at 1 year in the dpp ( 37 ) and attainment of hba1c < 7% ( 53 mmol / mol ) at 18 months in godarts ( 10 ) . statistically significant interactions between metformin and genetic variants were also reported for genes encoding additional proteins associated with amp - activated protein kinase dependent inhibition of gluconeogenesis [ prkab2 , prkaa2 , prkaa1 , stk11 ( 46,47 ) , pck1 , ppara ( 48 ) , and ppargc1a ( 7,49 ) ] , insulin secretion [ kcnj11 ( 7,18 ) , abcc8 , cdkn2a / b ( 45 ) , hnf4a , and hnf1b ( 7 ) ] ; and insulin sensitivity [ adipor2 , enpp1 ( 8) , capn10 , and gck ( 7 ) ] ( table 2 ; supplementary table 4 ) . of two studies evaluating snps in cyp2c9 ( 25,26 ) , the gene encoding the primary hepatic cytochrome p450 enzyme , which metabolizes sulfonylureas , one small study found a greater mean change from baseline in hba1c at 6 months by diplotype of rs1057910 ( 25 ) . interaction between sulfonylureas , repaglinide , thiazolidinediones , and acarbose and selected snps for glycemic outcomes godarts evaluated the interaction between two tcf7l2 snps and sulfonylureas and reported a significant association with response to medication ( 15 ) . of two snps evaluated in the slc30a8 gene ( 28,40 ) , rs13266634 was associated with response to repaglinide using hba1c , fg , and ppg at 8 weeks as outcomes ( 28 ) . a single study reported a significantly different change in hba1c and ppg at 6 months ( and similar , nonsignificant results for fg ) by e23k genotype of kcnj11 , which encodes the potassium channel inhibited by binding of repaglinide to its receptor on the -cell ( 27 ) . interactions between acarbose and the ppara , hnf4a , lipc , pparg2 , and ppargc1a loci were evaluated in the stop - niddm trial with 3.3 years of follow - up for diabetes risk ( table 3 ; supplementary table 9 ) ( 2023 ) . in this systematic review , we identified 34 articles on the pharmacogenetics of diabetes medications , with several reporting statistically significant interactions between genetic variants and medications for glycemic outcomes . however , our results do suggest specific , biologically plausible , gene medication interactions , and we recommend confirmation of the biologically plausible interactions as a priority , including those for drug transporters , metabolizers , and targets of action . in particular , we recommend follow - up of the 1 ) slc22a1 , slc22a2 , slc47a1 , prkab2 , prkaa2 , prkaa1 , and stk11 loci for metformin ; 2 ) cyp2c9 and tcf7l2 loci for sulfonylureas ; 3 ) kcnj11 , slc30a8 , neurod1/beta2 , ucp2 , and pax4 loci for repaglinide ; 4 ) pparg2 and ptprd for pioglitazone ; 5 ) kcnq1 and rbp4 loci for rosiglitazone ; and 6 ) ppara , hnf4a , lipc , and ppargc1a loci for acarbose . it deserves mention that positive findings were not replicated in other studies evaluating these loci ( 16,34,38 ) , but outcomes ( mean change in quantitative traits , achievement of hba1c < 7% ( 53 mmol / mol ) versus diabetes risk in the dpp ) and follow - up time ( 6 to 18 months vs. 3 years in the dpp ) differed in the other studies as well as did study design . the primary action of metformin is the inhibition of hepatic glucose production through inhibition of gluconeogenesis , and interactions with loci associated within this pathway ( pck1 , ppara , and ppargc1a ) were reported ( 7,48,49 ) . thus , variation in pparg would likely affect response to this class of medications , and this was suggested in one ( 42 ) of the two studies ( 32,42 ) evaluating this for pioglitazone and was not evaluated for rosiglitazone . variation in the hepatic cytochrome p450 enzymes , which metabolize diabetes medications , would be expected to impact their effects , including variation in cyp3a4 and cyp2c8 for repaglinide ( 51 ) , cyp2c8 and cyp2c9 for rosiglitazone ( 52 ) , and cyp2c8 and cyp3a4 for pioglitazone ( 53 ) . thus we present the state of the literature on the pharmacogenetics of type 2 diabetes , which lays the groundwork for directing future research efforts . the major limitation of the literature on the pharmacogenomics of type 2 diabetes is the lack of high - quality studies to identify and confirm findings for specific drug outcome combinations : 1 ) the rationale for selection of loci and interactions studied was often not clear , which raises the concern of selective reporting of results and publication bias ; in particular , we would be less suspicious of false - positive results in the setting of prespecified analyses based on prior evidence and/or biologic plausibility with adjustment for multiple comparisons . 2 ) the small size of many of the studies does not exclude the possibility that interactions exist but could not be identified because of lack of power ; we reported study results as significant based on a p value less than 0.05 but have noted results when p values were < 0.20 when possible . meta - analysis could help to address this issue , but the heterogeneity of studies with specific snp drug interactions , outcomes , and follow - up times differing across studies precluded quantitative synthesis with meta - analyses . while our qualitative synthesis summarizes the literature and therefore , our inferences often relied on the results of a single medication intervention on change in or incidence of outcomes by genotype ; these types of studies do not exclude the possibility that we are simply observing the effect of genotype and not specifically modification of the response to the medication . , adherence to intervention , follow - up ) and outcomes ( diabetes , death ) may have differed between those with and without genotyping information ; these kinds of differentiating characteristics in participants included in genetic analyses could impact the observed gene 5 ) while studies did provide information on methods for genotyping , information on snp - specific call rate was often not reported , and studies did not report on masking of personnel performing genotyping . we limited our analyses to diabetes and glycemic outcomes ( hba1c , fg , ppg ) because these are more commonly and consistently measured and are also strongly associated with improvements in long - term complications and mortality ( 58 ) . we recommend that guidelines for the design , analysis , and reporting of pharmacogenetic studies of diabetes medications be developed to improve study quality and enhance comparability among studies ; we have provided a prioritized list of quality and reporting items in supplementary table 11 . based on the limitations of the current literature , we recommend the following for future studies : 1 ) a priori specification of the snps and medications to be studied , 2 ) the use of experimental designs , 3 ) inclusion of a concurrent comparison group when possible , 4 ) agreement in the diabetes pharmacogenetics community regarding standardized outcomes and follow - up ( e.g. we recommend that diabetes pharmacogenetics studies use current guidelines for reporting of genetic association studies ( 12 ) and that these guidelines be extended to emphasize information relevant to pharmacogenetic studies , including prespecified reporting of analyses with rationale , estimates of type 2 error , standardized reporting of medication interventions , and reporting of differences between genotyped and nongenotyped subjects when possible . in this systematic review , we find evidence of biologically plausible pharmacogenetic interactions for metformin , sulfonylureas , repaglinide , pioglitazone , rosiglitazone , and acarbose , but these results require confirmation in future studies to determine if an individual s genetic information can be used to individualize the choice of prediabetes and diabetes pharmacologic management . importantly , our results should guide the development of guidelines for the design , conduct , and reporting of studies of the pharmacogenetics of type 2 diabetes and other chronic conditions . we recommend that guidelines for the design , analysis , and reporting of pharmacogenetic studies of diabetes medications be developed to improve study quality and enhance comparability among studies ; we have provided a prioritized list of quality and reporting items in supplementary table 11 . based on the limitations of the current literature , we recommend the following for future studies : 1 ) a priori specification of the snps and medications to be studied , 2 ) the use of experimental designs , 3 ) inclusion of a concurrent comparison group when possible , 4 ) agreement in the diabetes pharmacogenetics community regarding standardized outcomes and follow - up ( e.g. in addition we recommend that diabetes pharmacogenetics studies use current guidelines for reporting of genetic association studies ( 12 ) and that these guidelines be extended to emphasize information relevant to pharmacogenetic studies , including prespecified reporting of analyses with rationale , estimates of type 2 error , standardized reporting of medication interventions , and reporting of differences between genotyped and nongenotyped subjects when possible . in this systematic review , we find evidence of biologically plausible pharmacogenetic interactions for metformin , sulfonylureas , repaglinide , pioglitazone , rosiglitazone , and acarbose , but these results require confirmation in future studies to determine if an individual s genetic information can be used to individualize the choice of prediabetes and diabetes pharmacologic management .

bifidobacterium breve ucc2003 was grown at 37c in mrs broth ( difco , bd diagnostic systems , sparks , md , usa ) supplemented with 0.05% lcysteine ( w / v ) ( sigma , st louis , mo , usa ) ( mrsc ) in an anaerobic chamber ( mac 500 , don whitley scientific , west yorkshire , uk ) with an atmosphere of 5% co2 5% h2 90% n2 . analysis of bifidobacterium tolerance to toxic compounds was performed by supplementing mrsc before inoculation with different concentrations of oxgall ( oxoid limited , hampshire , uk ) , or a particular bile salt ( cholate , glycocholate , taurocholate , deoxycholate ) supplied by sigma . the dead / live labelling kit was supplied by invitrogen ( invitrogen , barcelona , spain ) . growth of bifidobacterium strains was monitored using a powerwave microplate spectrophotometer ( biotek , uk ) . bertani ( lb ) broth at 37c with agitation , and l. lactis was cultivated in m17 broth ( oxoid ) supplemented with 0.5% glucose ( gm17 ) at 30c . where appropriate , the growth medium contained chloramphenicol ( cm ; 5 g ml for l. lactis and 2 g ml for b. breve ) , erythromycin ( em ; 100 g ml for e. coli ) , kanamycin ( 50 g ml for e. coli ) , ampicillin ( 100 g ml for e. coli ) or tetracycline ( 10 g ml for e. coli or b. breve ) when required for plasmid maintenance . recombinant e. coli cells containing pori19838 were selected on lb agar containing em and supplemented with 40 g ml of xgal and 1 mm of iptg . restoration of bile survival phenotype in the complemented mutant strain was analysed by exposing early exponential growth phase cells to different concentrations of cholate in fresh mrsc , incubating samples at 37c in anaerobic conditions and determining colony counts after several hours of bile exposure . sequence data were obtained from genome annotations of b. breve ucc2003 ( o'connellmotherway et al . , 2011 ) . database searches were performed using blast at the national center for biotechnology information internet site ( http://www.ncbi.nlm.nih.gov ) and predictions of protein topology and domains were performed at expasy server ( http://expasy.org ) ( gasteiger et al . , 2003 ) . chromosomal dna from b. breve ucc2003 was obtained according to previously described procedures ( margolles and de los reyesgaviln , 2003 ) and sequences for cloning were amplified from b. breve chromosomal dna using highfidelity platinum pfx polymerase ( invitrogen ) . plasmid minipreparations from e. coli and l. lactis were performed using spinincolumn system ( sigma ) , according to the manufacturer 's instructions , with the incorporation of an initial lysis step for l. lactis , involving suspension of the cells in lysis buffer ( 20% sucrose , 10 mm trishcl ph 8.1 , 10 mm edta and 50 mm nacl ) supplemented with lysozyme ( 10 mg ml ) , followed by incubation at 37c for 30 min . plasmid preparations from b. breve were performed according to previously described procedures ( o'connellmotherway et al . , 2009 ) . electroporation of plasmid dna into e. coli was carried out as described by sambrook and colleagues ( 1989 ) , into l. lactis as described by wells and colleagues ( 1993 ) and into b. breve ucc2003 was performed as reported by maz and colleagues ( 2007 ) . the absence of pcrintroduced mutations was verified in all plasmid constructs created in this study by sequencing both dna strands on an abi prism sequencer ( applied biosystems , foster city , ca , usa ) . southern blot transfer of ecoridigested dna from b. breve ucc2003 and insertion mutants was performed according to standard procedures ( sambrook et al . , 1989 ) . hybridization and detection were carried out using the digoxigenin dnalabelling and detection kit ( roche molecular biochemicals , lewes , uk ) according to manufacturer 's instructions . internal 800 and 1000 bp fragments of bbr_0838 ( which corresponds to amino acid residues 100 to 357 , and 100 to 438 respectively ) were pcramplified using b. breve ucc2003 chromosomal dna as a template and primer combinations 838f and 838800r , or 838f and 8381000r . amplified products were digested with hindiii and xbai , and cloned into similarly digested pori19 , to generate pori19838800 and pori198381000 respectively . the tetracycline ( tet ) resistance antibiotic cassette , tet(w ) , from pam4 ( lvarezmartn et al . , 2008 ) was then cloned as a saci fragment into the unique saci site on each of these pori19 derivatives , yielding pori19838800tetw and pori198381000tetw respectively . these latter two plasmids were then introduced into e. coli ec101 harbouring pnzm.bbriim.bbriii in order to obtain methylated plasmid dna as previously described ( o'connellmotherway et al . , 2009 ) and selection for transformants was made on reinforced clostridial agar ( rca , oxoid ) plates containing tetracycline . transformants were checked by pcr and southern hybridization to confirm whether they contained either pori19838800tetw or pori198381000tetw inserted into their chromosome , and one verified insertion mutant for each of these events was retained for further use and designated ucc2003::838800 and ucc2003::8381000 respectively . the stability of these insertions was analysed following 50 generations of growth in mrsc in the absence of selective agents , by spreading cells with and without antibiotic . for mutant complementation , primers pbc838f and pbc838r were used to pcramplify the region comprising the coding sequence of bbr_0838 under the control of its own promoter from b. breve ucc2003 chromosomal dna . purified pcr product was hindiii and bamhi digested , and ligated into similarly digested pbc1.2 , yielding pbc838 , which was transformed into b. breve ucc2003::838800 by electroporation . differences in transcriptional response to bile stress between b. breve ucc2003 and its derivative b. breve ucc2003::838800 were determined by microarray experiments . verification of microarray results was carried out by quantitative reverse transcription pcr ( qrtpcr ) ( table 2 ) . volumes of 50 ml of b. breve ucc2003 and b. breve ucc2003::838800 cultures , grown until the od600 reached 0.5 , were exposed to subinhibitory concentrations of sodium cholate for 1 h , and samples for rna isolation were collected . methods for cell disruption , rna isolation , cdna synthesis and indirect labelling for microarray hybridizations were performed according to previously described procedures ( zomer et al . , 2009 ) . dna microarray data were processed as previously described ( zomer et al . , 2009 ) . a gene was considered differentially expressed between a test condition and a control when an expression ratio of > 3 or < 0.33 relative to the result for the control was obtained with a corresponding pvalue that was < 0.001 . methods for gene expression analysis through qrtpcr were performed according to gueimonde and colleagues ( 2009 ) . for northern hybridization analysis of bbr_0838 expression in b. breve ucc2003 , total rna ( 20 g ) obtained from sodium cholateexposed and control cultures , was separated on a 1% ( w / v ) agarose/0.66 m formaldehyde gel . blotting , hybridization and washing conditions were performed using a northernmax kit ( ambion applied biosystems ) , according to manufacturer 's instructions . a biotinylated antisenserna probe encompassing 951 bp of the bbr_0838 coding sequence was generated through in vitro transcription . to achieve this , an internal fragment of bbr_0838 was amplified using primers n838f and n838r ( table 2 ) . the amplified region was hindiii and xbai digested , and ligated into pbluescriptsk ( invitrogen ) . the resulting construct was isolated from e. coli , hindiii linearized , and used as a template for in vitro transcription with t7 rna polymerase incorporating biotinlabelled dutps ( roche ) . post hybridization signal detection was accomplished using streptavidin irdye 680 conjugate and visualized using the odyssey imaging system ( licor biosciences , lincoln , ne , usa ) . the key plasmids constructed in this work for bbr_0838 promoter characterization are listed in table 2 . dna fragments of different lengths encompassing various segments of the bbr_0838 promoter region were used to construct transcriptional fusions to the reporter gene gusa in the promoterless probe vector pnz272 ( platteeuw et al . , 1994 ) . the dna region comprising the complete bbr_0838 promoter was amplified from b. breve ucc2003 chromosomal dna using primers prom838rvpsti and prom838f2bglii . these primers are complementary to the dna sequence located in the 5 end of bbr_0838 coding sequence and 579 bp further upstream from this point respectively . amplified product was digested using unique bglii and psti restriction sites ( primerintroduced ) and ligated into the similarly digested promoter cloning vector pnz272 , resulting in the plasmid pnz272long . primers annealing 424 bp ( prom838f2bgliishort ) and 162 bp ( prom838fbglii ) upstream from the 5 end of bbr_0838 respectively , were used in combination with prom838rvpsti for pcr amplification , using b. breve ucc2003 chromosomal dna as a template . amplified fragments were bglii and psti digested and ligated into similarly digested pnz272 , yielding pnz272ss and pnz272short respectively . employing a similar approach , the promoter region was minimized from 3 end . primers annealing 71 bp ( prom838revbamhi + ir ) , 144 bp ( prom838revbamhiir ) , 190 bp ( prom838rv + irii50psti ) , 228 bp ( prom838rv + iriipsti ) , and 296 bp ( prom838rviriipsti ) upstream of bbr_0838 start codon respectively , were used in combination with prom838f2bglii for pcr amplification , using b. breve ucc2003 chromosomal dna as a template . amplified fragments were bglii and psti or bamhi digested , and ligated into similarly digested pnz272 , yielding to pnz27271 , pnz272144 , pnz272190 , pnz272228 and pnz272296 respectively . all these constructs were initially electroporated into l. lactis and dna integrity of inserts was verified through sequencing of plasmid minipreparations from positive clones . sequencedverified constructs isolated from the corresponding l. lactis clones were then introduced into electrocompetent cells of b. breve ucc2003 . dglucuronidase ( gusa ) activity was routinely determined in cultures grown until midexponential growth phase , in the presence of subinhibitory concentrations of sodium cholate ( 0.025% w / v ) . briefly , cells from 10 ml cultures were collected , resuspended in 0.5 ml of pbs and disrupted with glassbeads ( sigma ) using a fastprep , by five 15 s treatments , with cooling intervals of 45 s when samples were put on ice . after centrifugation , gusa activity was immediately determined : appropriate dilutions of the extracts ( 4 l ) were mixed with 88 l of sodium acetate 0.1 m and 8 l of a 20 mm solution of paranitrodglucuronid acid ( sigma ) , and the mixtures were incubated at 37c . after 10 min , 100 l of 1 m icecold sodium carbonate was added to stop the reaction . optical density at 420 nm was measured and gusa activity was determined measuring the paranitrophenol ( pnp ) released , quantified by a pnp standard curve . protein concentration in extracts was measured by using the bca protein assay kit ( pierce , rockford , il , usa ) and used to determine gusa specific activity . one unit of enzymatic activity was defined as the amount of protein that releases 1 nmol of pnp per minute . specific activities were expressed as units per mg of protein and were measured in triplicate for each extract . primer extension was performed by annealing 1 pmol of ird800 synthetic oligonucleotides pe3 or pe4 to 20 g of rna as described by ventura and colleagues ( 2005 ) . sequence ladders of the presumed bbr_0838 promoter regions , which were run alongside the primer extension products , were produced using the same primer as the primer extension reaction and a pcr product template generated using 838pseqfor and 838pseqrev . thermo sequenase primer cycle sequencing kit ( healthcare biosciences , buckinghamshire , uk ) was employed . signal detection and image capture was performed by means of a licor sequencing instrument ( licor biosciences ) . bifidobacterium breve ucc2003 was grown at 37c in mrs broth ( difco , bd diagnostic systems , sparks , md , usa ) supplemented with 0.05% lcysteine ( w / v ) ( sigma , st louis , mo , usa ) ( mrsc ) in an anaerobic chamber ( mac 500 , don whitley scientific , west yorkshire , uk ) with an atmosphere of 5% co2 5% h2 90% n2 . analysis of bifidobacterium tolerance to toxic compounds was performed by supplementing mrsc before inoculation with different concentrations of oxgall ( oxoid limited , hampshire , uk ) , or a particular bile salt ( cholate , glycocholate , taurocholate , deoxycholate ) supplied by sigma . the dead / live labelling kit was supplied by invitrogen ( invitrogen , barcelona , spain ) . growth of bifidobacterium strains was monitored using a powerwave microplate spectrophotometer ( biotek , uk ) . bertani ( lb ) broth at 37c with agitation , and l. lactis was cultivated in m17 broth ( oxoid ) supplemented with 0.5% glucose ( gm17 ) at 30c . where appropriate , the growth medium contained chloramphenicol ( cm ; 5 g ml for l. lactis and 2 g ml for b. breve ) , erythromycin ( em ; 100 g ml for e. coli ) , kanamycin ( 50 g ml for e. coli ) , ampicillin ( 100 g ml for e. coli ) or tetracycline ( 10 g ml for e. coli or b. breve ) when required for plasmid maintenance . recombinant e. coli cells containing pori19838 were selected on lb agar containing em and supplemented with 40 g ml of xgal and 1 mm of iptg . restoration of bile survival phenotype in the complemented mutant strain was analysed by exposing early exponential growth phase cells to different concentrations of cholate in fresh mrsc , incubating samples at 37c in anaerobic conditions and determining colony counts after several hours of bile exposure . sequence data were obtained from genome annotations of b. breve ucc2003 ( o'connellmotherway et al . , 2011 ) . database searches were performed using blast at the national center for biotechnology information internet site ( http://www.ncbi.nlm.nih.gov ) and predictions of protein topology and domains were performed at expasy server ( http://expasy.org ) ( gasteiger et al . , 2003 ) . chromosomal dna from b. breve ucc2003 was obtained according to previously described procedures ( margolles and de los reyesgaviln , 2003 ) and sequences for cloning were amplified from b. breve chromosomal dna using highfidelity platinum pfx polymerase ( invitrogen ) . plasmid minipreparations from e. coli and l. lactis were performed using spinincolumn system ( sigma ) , according to the manufacturer 's instructions , with the incorporation of an initial lysis step for l. lactis , involving suspension of the cells in lysis buffer ( 20% sucrose , 10 mm trishcl ph 8.1 , 10 mm edta and 50 mm nacl ) supplemented with lysozyme ( 10 mg ml ) , followed by incubation at 37c for 30 min . plasmid preparations from b. breve were performed according to previously described procedures ( o'connellmotherway et al . , 2009 ) . electroporation of plasmid dna into e. coli was carried out as described by sambrook and colleagues ( 1989 ) , into l. lactis as described by wells and colleagues ( 1993 ) and into b. breve ucc2003 was performed as reported by maz and colleagues ( 2007 ) . the absence of pcrintroduced mutations was verified in all plasmid constructs created in this study by sequencing both dna strands on an abi prism sequencer ( applied biosystems , foster city , ca , usa ) . southern blot transfer of ecoridigested dna from b. breve ucc2003 and insertion mutants was performed according to standard procedures ( sambrook et al . , 1989 ) . hybridization and detection were carried out using the digoxigenin dnalabelling and detection kit ( roche molecular biochemicals , lewes , uk ) according to manufacturer 's instructions . internal 800 and 1000 bp fragments of bbr_0838 ( which corresponds to amino acid residues 100 to 357 , and 100 to 438 respectively ) were pcramplified using b. breve ucc2003 chromosomal dna as a template and primer combinations 838f and 838800r , or 838f and 8381000r . amplified products were digested with hindiii and xbai , and cloned into similarly digested pori19 , to generate pori19838800 and pori198381000 respectively . the tetracycline ( tet ) resistance antibiotic cassette , tet(w ) , from pam4 ( lvarezmartn et al . , 2008 ) was then cloned as a saci fragment into the unique saci site on each of these pori19 derivatives , yielding pori19838800tetw and pori198381000tetw respectively . these latter two plasmids were then introduced into e. coli ec101 harbouring pnzm.bbriim.bbriii in order to obtain methylated plasmid dna as previously described ( o'connellmotherway et al . , 2009 ) and selection for transformants was made on reinforced clostridial agar ( rca , oxoid ) plates containing tetracycline . transformants were checked by pcr and southern hybridization to confirm whether they contained either pori19838800tetw or pori198381000tetw inserted into their chromosome , and one verified insertion mutant for each of these events was retained for further use and designated ucc2003::838800 and ucc2003::8381000 respectively . the stability of these insertions was analysed following 50 generations of growth in mrsc in the absence of selective agents , by spreading cells with and without antibiotic . stability was expressed as the proportion of viable cells retaining the insertion . for mutant complementation , primers pbc838f and pbc838r were used to pcramplify the region comprising the coding sequence of bbr_0838 under the control of its own promoter from b. breve ucc2003 chromosomal dna . purified pcr product was hindiii and bamhi digested , and ligated into similarly digested pbc1.2 , yielding pbc838 , which was transformed into b. breve ucc2003::838800 by electroporation . differences in transcriptional response to bile stress between b. breve ucc2003 and its derivative b. breve ucc2003::838800 were determined by microarray experiments . verification of microarray results was carried out by quantitative reverse transcription pcr ( qrtpcr ) ( table 2 ) . volumes of 50 ml of b. breve ucc2003 and b. breve ucc2003::838800 cultures , grown until the od600 reached 0.5 , were exposed to subinhibitory concentrations of sodium cholate for 1 h , and samples for rna isolation were collected . methods for cell disruption , rna isolation , cdna synthesis and indirect labelling for microarray hybridizations were performed according to previously described procedures ( zomer et al . , 2009 ) . dna microarray data were processed as previously described ( zomer et al . , 2009 ) . a gene was considered differentially expressed between a test condition and a control when an expression ratio of > 3 or < 0.33 relative to the result for the control was obtained with a corresponding pvalue that was < 0.001 . methods for gene expression analysis through qrtpcr were performed according to gueimonde and colleagues ( 2009 ) . for northern hybridization analysis of bbr_0838 expression in b. breve ucc2003 , total rna ( 20 g ) obtained from sodium cholateexposed and control cultures , was separated on a 1% ( w / v ) agarose/0.66 m formaldehyde gel . blotting , hybridization and washing conditions were performed using a northernmax kit ( ambion applied biosystems ) , according to manufacturer 's instructions . a biotinylated antisenserna probe encompassing 951 bp of the bbr_0838 coding sequence was generated through in vitro transcription . to achieve this , an internal fragment of bbr_0838 was amplified using primers n838f and n838r ( table 2 ) . the amplified region was hindiii and xbai digested , and ligated into pbluescriptsk ( invitrogen ) . the resulting construct was isolated from e. coli , hindiii linearized , and used as a template for in vitro transcription with t7 rna polymerase incorporating biotinlabelled dutps ( roche ) . post hybridization signal detection was accomplished using streptavidin irdye 680 conjugate and visualized using the odyssey imaging system ( licor biosciences , lincoln , ne , usa ) . the key plasmids constructed in this work for bbr_0838 promoter characterization are listed in table 2 . dna fragments of different lengths encompassing various segments of the bbr_0838 promoter region were used to construct transcriptional fusions to the reporter gene gusa in the promoterless probe vector pnz272 ( platteeuw et al . , 1994 ) . the dna region comprising the complete bbr_0838 promoter was amplified from b. breve ucc2003 chromosomal dna using primers prom838rvpsti and prom838f2bglii . these primers are complementary to the dna sequence located in the 5 end of bbr_0838 coding sequence and 579 bp further upstream from this point respectively . amplified product was digested using unique bglii and psti restriction sites ( primerintroduced ) and ligated into the similarly digested promoter cloning vector pnz272 , resulting in the plasmid pnz272long . primers annealing 424 bp ( prom838f2bgliishort ) and 162 bp ( prom838fbglii ) upstream from the 5 end of bbr_0838 respectively , were used in combination with prom838rvpsti for pcr amplification , using b. breve ucc2003 chromosomal dna as a template . amplified fragments were bglii and psti digested and ligated into similarly digested pnz272 , yielding pnz272ss and pnz272short respectively . employing a similar approach , the promoter region was minimized from 3 end . primers annealing 71 bp ( prom838revbamhi + ir ) , 144 bp ( prom838revbamhiir ) , 190 bp ( prom838rv + irii50psti ) , 228 bp ( prom838rv + iriipsti ) , and 296 bp ( prom838rviriipsti ) upstream of bbr_0838 start codon respectively , were used in combination with prom838f2bglii for pcr amplification , using b. breve ucc2003 chromosomal dna as a template . amplified fragments were bglii and psti or bamhi digested , and ligated into similarly digested pnz272 , yielding to pnz27271 , pnz272144 , pnz272190 , pnz272228 and pnz272296 respectively . all these constructs were initially electroporated into l. lactis and dna integrity of inserts was verified through sequencing of plasmid minipreparations from positive clones . sequencedverified constructs isolated from the corresponding l. lactis clones were then introduced into electrocompetent cells of b. breve ucc2003 . dglucuronidase ( gusa ) activity was routinely determined in cultures grown until midexponential growth phase , in the presence of subinhibitory concentrations of sodium cholate ( 0.025% w / v ) . briefly , cells from 10 ml cultures were collected , resuspended in 0.5 ml of pbs and disrupted with glassbeads ( sigma ) using a fastprep , by five 15 s treatments , with cooling intervals of 45 s when samples were put on ice . after centrifugation , gusa activity was immediately determined : appropriate dilutions of the extracts ( 4 l ) were mixed with 88 l of sodium acetate 0.1 m and 8 l of a 20 mm solution of paranitrodglucuronid acid ( sigma ) , and the mixtures were incubated at 37c . after 10 min , 100 l of 1 m icecold sodium carbonate was added to stop the reaction . optical density at 420 nm was measured and gusa activity was determined measuring the paranitrophenol ( pnp ) released , quantified by a pnp standard curve . protein concentration in extracts was measured by using the bca protein assay kit ( pierce , rockford , il , usa ) and used to determine gusa specific activity . one unit of enzymatic activity was defined as the amount of protein that releases 1 nmol of pnp per minute . specific activities were expressed as units per mg of protein and were measured in triplicate for each extract . primer extension was performed by annealing 1 pmol of ird800 synthetic oligonucleotides pe3 or pe4 to 20 g of rna as described by ventura and colleagues ( 2005 ) . sequence ladders of the presumed bbr_0838 promoter regions , which were run alongside the primer extension products , were produced using the same primer as the primer extension reaction and a pcr product template generated using 838pseqfor and 838pseqrev . thermo sequenase primer cycle sequencing kit ( healthcare biosciences , buckinghamshire , uk ) was employed . signal detection and image capture was performed by means of a licor sequencing instrument ( licor biosciences ) . additional supporting information may be found in the online version of this article : whole utr secondary structure . please note : wiley - blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors . any queries ( other than missing material ) should be directed to the corresponding author for the article .

summarybbr_0838 from bifidobacterium breve ucc2003 is predicted to encode a 683 residue membrane protein , containing both a permease domain that displays similarity to transporters belonging to the major facilitator superfamily , as well as a cbs ( cystathionine beta synthase ) domain . the high level of similarity to bile efflux pumps from other bifidobacteria suggests a significant and general role for bbr_0838 in bile tolerance . bbr_0838 transcription was shown to be monocistronic and strongly induced upon exposure to bile . further analysis delineated the transcriptional start site and the minimal region required for promoter activity and bile regulation . insertional inactivation of bbr_0838 in b. breve ucc2003 resulted in a strain , ucc2003:838800 , which exhibited reduced survival upon cholate exposure as compared with the parent strain , a phenotype that was reversed when a functional , plasmidencoded bbr_0838 gene was introduced into ucc2003:838800 . transcriptome analysis of ucc2003:838800 grown in the presence or absence of bile demonstrated that transcription of bbr_0832 , which is predicted to encode a macrolide efflux transporter gene , was significantly increased in the presence of bile , representing a likely compensatory mechanism for bile removal in the absence of bbr_0838 . this study represents the first indepth analysis of a bileinducible locus in bifidobacteria , identifying a key gene relevant for bifidobacterial bile tolerance .

Experimental procedures Bacterial strains, plasmids and growth conditions Nucleotide and protein sequence analysis DNA manipulations Construction of Transcriptional analysis Plasmid constructions for promoter characterization Measurement of Dglucuronidase activity Primer extension analysis Supporting Information

bifidobacterium breve ucc2003 was grown at 37c in mrs broth ( difco , bd diagnostic systems , sparks , md , usa ) supplemented with 0.05% lcysteine ( w / v ) ( sigma , st louis , mo , usa ) ( mrsc ) in an anaerobic chamber ( mac 500 , don whitley scientific , west yorkshire , uk ) with an atmosphere of 5% co2 5% h2 90% n2 . analysis of bifidobacterium tolerance to toxic compounds was performed by supplementing mrsc before inoculation with different concentrations of oxgall ( oxoid limited , hampshire , uk ) , or a particular bile salt ( cholate , glycocholate , taurocholate , deoxycholate ) supplied by sigma . where appropriate , the growth medium contained chloramphenicol ( cm ; 5 g ml for l. lactis and 2 g ml for b. breve ) , erythromycin ( em ; 100 g ml for e. coli ) , kanamycin ( 50 g ml for e. coli ) , ampicillin ( 100 g ml for e. coli ) or tetracycline ( 10 g ml for e. coli or b. breve ) when required for plasmid maintenance . restoration of bile survival phenotype in the complemented mutant strain was analysed by exposing early exponential growth phase cells to different concentrations of cholate in fresh mrsc , incubating samples at 37c in anaerobic conditions and determining colony counts after several hours of bile exposure . sequence data were obtained from genome annotations of b. breve ucc2003 ( o'connellmotherway et al . chromosomal dna from b. breve ucc2003 was obtained according to previously described procedures ( margolles and de los reyesgaviln , 2003 ) and sequences for cloning were amplified from b. breve chromosomal dna using highfidelity platinum pfx polymerase ( invitrogen ) . plasmid minipreparations from e. coli and l. lactis were performed using spinincolumn system ( sigma ) , according to the manufacturer 's instructions , with the incorporation of an initial lysis step for l. lactis , involving suspension of the cells in lysis buffer ( 20% sucrose , 10 mm trishcl ph 8.1 , 10 mm edta and 50 mm nacl ) supplemented with lysozyme ( 10 mg ml ) , followed by incubation at 37c for 30 min . plasmid preparations from b. breve were performed according to previously described procedures ( o'connellmotherway et al . electroporation of plasmid dna into e. coli was carried out as described by sambrook and colleagues ( 1989 ) , into l. lactis as described by wells and colleagues ( 1993 ) and into b. breve ucc2003 was performed as reported by maz and colleagues ( 2007 ) . the absence of pcrintroduced mutations was verified in all plasmid constructs created in this study by sequencing both dna strands on an abi prism sequencer ( applied biosystems , foster city , ca , usa ) . southern blot transfer of ecoridigested dna from b. breve ucc2003 and insertion mutants was performed according to standard procedures ( sambrook et al . internal 800 and 1000 bp fragments of bbr_0838 ( which corresponds to amino acid residues 100 to 357 , and 100 to 438 respectively ) were pcramplified using b. breve ucc2003 chromosomal dna as a template and primer combinations 838f and 838800r , or 838f and 8381000r . , 2008 ) was then cloned as a saci fragment into the unique saci site on each of these pori19 derivatives , yielding pori19838800tetw and pori198381000tetw respectively . these latter two plasmids were then introduced into e. coli ec101 harbouring pnzm.bbriim.bbriii in order to obtain methylated plasmid dna as previously described ( o'connellmotherway et al . the stability of these insertions was analysed following 50 generations of growth in mrsc in the absence of selective agents , by spreading cells with and without antibiotic . for mutant complementation , primers pbc838f and pbc838r were used to pcramplify the region comprising the coding sequence of bbr_0838 under the control of its own promoter from b. breve ucc2003 chromosomal dna . purified pcr product was hindiii and bamhi digested , and ligated into similarly digested pbc1.2 , yielding pbc838 , which was transformed into b. breve ucc2003::838800 by electroporation . differences in transcriptional response to bile stress between b. breve ucc2003 and its derivative b. breve ucc2003::838800 were determined by microarray experiments . volumes of 50 ml of b. breve ucc2003 and b. breve ucc2003::838800 cultures , grown until the od600 reached 0.5 , were exposed to subinhibitory concentrations of sodium cholate for 1 h , and samples for rna isolation were collected . a gene was considered differentially expressed between a test condition and a control when an expression ratio of > 3 or < 0.33 relative to the result for the control was obtained with a corresponding pvalue that was < 0.001 . for northern hybridization analysis of bbr_0838 expression in b. breve ucc2003 , total rna ( 20 g ) obtained from sodium cholateexposed and control cultures , was separated on a 1% ( w / v ) agarose/0.66 m formaldehyde gel . to achieve this , an internal fragment of bbr_0838 was amplified using primers n838f and n838r ( table 2 ) . the resulting construct was isolated from e. coli , hindiii linearized , and used as a template for in vitro transcription with t7 rna polymerase incorporating biotinlabelled dutps ( roche ) . the key plasmids constructed in this work for bbr_0838 promoter characterization are listed in table 2 . dna fragments of different lengths encompassing various segments of the bbr_0838 promoter region were used to construct transcriptional fusions to the reporter gene gusa in the promoterless probe vector pnz272 ( platteeuw et al . the dna region comprising the complete bbr_0838 promoter was amplified from b. breve ucc2003 chromosomal dna using primers prom838rvpsti and prom838f2bglii . these primers are complementary to the dna sequence located in the 5 end of bbr_0838 coding sequence and 579 bp further upstream from this point respectively . amplified product was digested using unique bglii and psti restriction sites ( primerintroduced ) and ligated into the similarly digested promoter cloning vector pnz272 , resulting in the plasmid pnz272long . primers annealing 424 bp ( prom838f2bgliishort ) and 162 bp ( prom838fbglii ) upstream from the 5 end of bbr_0838 respectively , were used in combination with prom838rvpsti for pcr amplification , using b. breve ucc2003 chromosomal dna as a template . primers annealing 71 bp ( prom838revbamhi + ir ) , 144 bp ( prom838revbamhiir ) , 190 bp ( prom838rv + irii50psti ) , 228 bp ( prom838rv + iriipsti ) , and 296 bp ( prom838rviriipsti ) upstream of bbr_0838 start codon respectively , were used in combination with prom838f2bglii for pcr amplification , using b. breve ucc2003 chromosomal dna as a template . sequencedverified constructs isolated from the corresponding l. lactis clones were then introduced into electrocompetent cells of b. breve ucc2003 . dglucuronidase ( gusa ) activity was routinely determined in cultures grown until midexponential growth phase , in the presence of subinhibitory concentrations of sodium cholate ( 0.025% w / v ) . after centrifugation , gusa activity was immediately determined : appropriate dilutions of the extracts ( 4 l ) were mixed with 88 l of sodium acetate 0.1 m and 8 l of a 20 mm solution of paranitrodglucuronid acid ( sigma ) , and the mixtures were incubated at 37c . sequence ladders of the presumed bbr_0838 promoter regions , which were run alongside the primer extension products , were produced using the same primer as the primer extension reaction and a pcr product template generated using 838pseqfor and 838pseqrev . signal detection and image capture was performed by means of a licor sequencing instrument ( licor biosciences ) . bifidobacterium breve ucc2003 was grown at 37c in mrs broth ( difco , bd diagnostic systems , sparks , md , usa ) supplemented with 0.05% lcysteine ( w / v ) ( sigma , st louis , mo , usa ) ( mrsc ) in an anaerobic chamber ( mac 500 , don whitley scientific , west yorkshire , uk ) with an atmosphere of 5% co2 5% h2 90% n2 . analysis of bifidobacterium tolerance to toxic compounds was performed by supplementing mrsc before inoculation with different concentrations of oxgall ( oxoid limited , hampshire , uk ) , or a particular bile salt ( cholate , glycocholate , taurocholate , deoxycholate ) supplied by sigma . where appropriate , the growth medium contained chloramphenicol ( cm ; 5 g ml for l. lactis and 2 g ml for b. breve ) , erythromycin ( em ; 100 g ml for e. coli ) , kanamycin ( 50 g ml for e. coli ) , ampicillin ( 100 g ml for e. coli ) or tetracycline ( 10 g ml for e. coli or b. breve ) when required for plasmid maintenance . restoration of bile survival phenotype in the complemented mutant strain was analysed by exposing early exponential growth phase cells to different concentrations of cholate in fresh mrsc , incubating samples at 37c in anaerobic conditions and determining colony counts after several hours of bile exposure . sequence data were obtained from genome annotations of b. breve ucc2003 ( o'connellmotherway et al . chromosomal dna from b. breve ucc2003 was obtained according to previously described procedures ( margolles and de los reyesgaviln , 2003 ) and sequences for cloning were amplified from b. breve chromosomal dna using highfidelity platinum pfx polymerase ( invitrogen ) . plasmid minipreparations from e. coli and l. lactis were performed using spinincolumn system ( sigma ) , according to the manufacturer 's instructions , with the incorporation of an initial lysis step for l. lactis , involving suspension of the cells in lysis buffer ( 20% sucrose , 10 mm trishcl ph 8.1 , 10 mm edta and 50 mm nacl ) supplemented with lysozyme ( 10 mg ml ) , followed by incubation at 37c for 30 min . plasmid preparations from b. breve were performed according to previously described procedures ( o'connellmotherway et al . electroporation of plasmid dna into e. coli was carried out as described by sambrook and colleagues ( 1989 ) , into l. lactis as described by wells and colleagues ( 1993 ) and into b. breve ucc2003 was performed as reported by maz and colleagues ( 2007 ) . the absence of pcrintroduced mutations was verified in all plasmid constructs created in this study by sequencing both dna strands on an abi prism sequencer ( applied biosystems , foster city , ca , usa ) . southern blot transfer of ecoridigested dna from b. breve ucc2003 and insertion mutants was performed according to standard procedures ( sambrook et al . internal 800 and 1000 bp fragments of bbr_0838 ( which corresponds to amino acid residues 100 to 357 , and 100 to 438 respectively ) were pcramplified using b. breve ucc2003 chromosomal dna as a template and primer combinations 838f and 838800r , or 838f and 8381000r . , 2008 ) was then cloned as a saci fragment into the unique saci site on each of these pori19 derivatives , yielding pori19838800tetw and pori198381000tetw respectively . these latter two plasmids were then introduced into e. coli ec101 harbouring pnzm.bbriim.bbriii in order to obtain methylated plasmid dna as previously described ( o'connellmotherway et al . the stability of these insertions was analysed following 50 generations of growth in mrsc in the absence of selective agents , by spreading cells with and without antibiotic . for mutant complementation , primers pbc838f and pbc838r were used to pcramplify the region comprising the coding sequence of bbr_0838 under the control of its own promoter from b. breve ucc2003 chromosomal dna . purified pcr product was hindiii and bamhi digested , and ligated into similarly digested pbc1.2 , yielding pbc838 , which was transformed into b. breve ucc2003::838800 by electroporation . differences in transcriptional response to bile stress between b. breve ucc2003 and its derivative b. breve ucc2003::838800 were determined by microarray experiments . volumes of 50 ml of b. breve ucc2003 and b. breve ucc2003::838800 cultures , grown until the od600 reached 0.5 , were exposed to subinhibitory concentrations of sodium cholate for 1 h , and samples for rna isolation were collected . a gene was considered differentially expressed between a test condition and a control when an expression ratio of > 3 or < 0.33 relative to the result for the control was obtained with a corresponding pvalue that was < 0.001 . for northern hybridization analysis of bbr_0838 expression in b. breve ucc2003 , total rna ( 20 g ) obtained from sodium cholateexposed and control cultures , was separated on a 1% ( w / v ) agarose/0.66 m formaldehyde gel . to achieve this , an internal fragment of bbr_0838 was amplified using primers n838f and n838r ( table 2 ) . the resulting construct was isolated from e. coli , hindiii linearized , and used as a template for in vitro transcription with t7 rna polymerase incorporating biotinlabelled dutps ( roche ) . the key plasmids constructed in this work for bbr_0838 promoter characterization are listed in table 2 . dna fragments of different lengths encompassing various segments of the bbr_0838 promoter region were used to construct transcriptional fusions to the reporter gene gusa in the promoterless probe vector pnz272 ( platteeuw et al . the dna region comprising the complete bbr_0838 promoter was amplified from b. breve ucc2003 chromosomal dna using primers prom838rvpsti and prom838f2bglii . these primers are complementary to the dna sequence located in the 5 end of bbr_0838 coding sequence and 579 bp further upstream from this point respectively . amplified product was digested using unique bglii and psti restriction sites ( primerintroduced ) and ligated into the similarly digested promoter cloning vector pnz272 , resulting in the plasmid pnz272long . primers annealing 424 bp ( prom838f2bgliishort ) and 162 bp ( prom838fbglii ) upstream from the 5 end of bbr_0838 respectively , were used in combination with prom838rvpsti for pcr amplification , using b. breve ucc2003 chromosomal dna as a template . primers annealing 71 bp ( prom838revbamhi + ir ) , 144 bp ( prom838revbamhiir ) , 190 bp ( prom838rv + irii50psti ) , 228 bp ( prom838rv + iriipsti ) , and 296 bp ( prom838rviriipsti ) upstream of bbr_0838 start codon respectively , were used in combination with prom838f2bglii for pcr amplification , using b. breve ucc2003 chromosomal dna as a template . sequencedverified constructs isolated from the corresponding l. lactis clones were then introduced into electrocompetent cells of b. breve ucc2003 . dglucuronidase ( gusa ) activity was routinely determined in cultures grown until midexponential growth phase , in the presence of subinhibitory concentrations of sodium cholate ( 0.025% w / v ) . after centrifugation , gusa activity was immediately determined : appropriate dilutions of the extracts ( 4 l ) were mixed with 88 l of sodium acetate 0.1 m and 8 l of a 20 mm solution of paranitrodglucuronid acid ( sigma ) , and the mixtures were incubated at 37c . sequence ladders of the presumed bbr_0838 promoter regions , which were run alongside the primer extension products , were produced using the same primer as the primer extension reaction and a pcr product template generated using 838pseqfor and 838pseqrev . signal detection and image capture was performed by means of a licor sequencing instrument ( licor biosciences ) . additional supporting information may be found in the online version of this article : whole utr secondary structure . any queries ( other than missing material ) should be directed to the corresponding author for the article .

bifidobacterium breve ucc2003 was grown at 37c in mrs broth ( difco , bd diagnostic systems , sparks , md , usa ) supplemented with 0.05% lcysteine ( w / v ) ( sigma , st louis , mo , usa ) ( mrsc ) in an anaerobic chamber ( mac 500 , don whitley scientific , west yorkshire , uk ) with an atmosphere of 5% co2 5% h2 90% n2 . analysis of bifidobacterium tolerance to toxic compounds was performed by supplementing mrsc before inoculation with different concentrations of oxgall ( oxoid limited , hampshire , uk ) , or a particular bile salt ( cholate , glycocholate , taurocholate , deoxycholate ) supplied by sigma . bertani ( lb ) broth at 37c with agitation , and l. lactis was cultivated in m17 broth ( oxoid ) supplemented with 0.5% glucose ( gm17 ) at 30c . where appropriate , the growth medium contained chloramphenicol ( cm ; 5 g ml for l. lactis and 2 g ml for b. breve ) , erythromycin ( em ; 100 g ml for e. coli ) , kanamycin ( 50 g ml for e. coli ) , ampicillin ( 100 g ml for e. coli ) or tetracycline ( 10 g ml for e. coli or b. breve ) when required for plasmid maintenance . recombinant e. coli cells containing pori19838 were selected on lb agar containing em and supplemented with 40 g ml of xgal and 1 mm of iptg . restoration of bile survival phenotype in the complemented mutant strain was analysed by exposing early exponential growth phase cells to different concentrations of cholate in fresh mrsc , incubating samples at 37c in anaerobic conditions and determining colony counts after several hours of bile exposure . chromosomal dna from b. breve ucc2003 was obtained according to previously described procedures ( margolles and de los reyesgaviln , 2003 ) and sequences for cloning were amplified from b. breve chromosomal dna using highfidelity platinum pfx polymerase ( invitrogen ) . plasmid minipreparations from e. coli and l. lactis were performed using spinincolumn system ( sigma ) , according to the manufacturer 's instructions , with the incorporation of an initial lysis step for l. lactis , involving suspension of the cells in lysis buffer ( 20% sucrose , 10 mm trishcl ph 8.1 , 10 mm edta and 50 mm nacl ) supplemented with lysozyme ( 10 mg ml ) , followed by incubation at 37c for 30 min . electroporation of plasmid dna into e. coli was carried out as described by sambrook and colleagues ( 1989 ) , into l. lactis as described by wells and colleagues ( 1993 ) and into b. breve ucc2003 was performed as reported by maz and colleagues ( 2007 ) . the absence of pcrintroduced mutations was verified in all plasmid constructs created in this study by sequencing both dna strands on an abi prism sequencer ( applied biosystems , foster city , ca , usa ) . hybridization and detection were carried out using the digoxigenin dnalabelling and detection kit ( roche molecular biochemicals , lewes , uk ) according to manufacturer 's instructions . internal 800 and 1000 bp fragments of bbr_0838 ( which corresponds to amino acid residues 100 to 357 , and 100 to 438 respectively ) were pcramplified using b. breve ucc2003 chromosomal dna as a template and primer combinations 838f and 838800r , or 838f and 8381000r . amplified products were digested with hindiii and xbai , and cloned into similarly digested pori19 , to generate pori19838800 and pori198381000 respectively . the tetracycline ( tet ) resistance antibiotic cassette , tet(w ) , from pam4 ( lvarezmartn et al . , 2008 ) was then cloned as a saci fragment into the unique saci site on each of these pori19 derivatives , yielding pori19838800tetw and pori198381000tetw respectively . these latter two plasmids were then introduced into e. coli ec101 harbouring pnzm.bbriim.bbriii in order to obtain methylated plasmid dna as previously described ( o'connellmotherway et al . , 2009 ) and selection for transformants was made on reinforced clostridial agar ( rca , oxoid ) plates containing tetracycline . transformants were checked by pcr and southern hybridization to confirm whether they contained either pori19838800tetw or pori198381000tetw inserted into their chromosome , and one verified insertion mutant for each of these events was retained for further use and designated ucc2003::838800 and ucc2003::8381000 respectively . the stability of these insertions was analysed following 50 generations of growth in mrsc in the absence of selective agents , by spreading cells with and without antibiotic . for mutant complementation , primers pbc838f and pbc838r were used to pcramplify the region comprising the coding sequence of bbr_0838 under the control of its own promoter from b. breve ucc2003 chromosomal dna . purified pcr product was hindiii and bamhi digested , and ligated into similarly digested pbc1.2 , yielding pbc838 , which was transformed into b. breve ucc2003::838800 by electroporation . differences in transcriptional response to bile stress between b. breve ucc2003 and its derivative b. breve ucc2003::838800 were determined by microarray experiments . verification of microarray results was carried out by quantitative reverse transcription pcr ( qrtpcr ) ( table 2 ) . volumes of 50 ml of b. breve ucc2003 and b. breve ucc2003::838800 cultures , grown until the od600 reached 0.5 , were exposed to subinhibitory concentrations of sodium cholate for 1 h , and samples for rna isolation were collected . methods for cell disruption , rna isolation , cdna synthesis and indirect labelling for microarray hybridizations were performed according to previously described procedures ( zomer et al . a gene was considered differentially expressed between a test condition and a control when an expression ratio of > 3 or < 0.33 relative to the result for the control was obtained with a corresponding pvalue that was < 0.001 . methods for gene expression analysis through qrtpcr were performed according to gueimonde and colleagues ( 2009 ) . for northern hybridization analysis of bbr_0838 expression in b. breve ucc2003 , total rna ( 20 g ) obtained from sodium cholateexposed and control cultures , was separated on a 1% ( w / v ) agarose/0.66 m formaldehyde gel . a biotinylated antisenserna probe encompassing 951 bp of the bbr_0838 coding sequence was generated through in vitro transcription . the resulting construct was isolated from e. coli , hindiii linearized , and used as a template for in vitro transcription with t7 rna polymerase incorporating biotinlabelled dutps ( roche ) . the key plasmids constructed in this work for bbr_0838 promoter characterization are listed in table 2 . dna fragments of different lengths encompassing various segments of the bbr_0838 promoter region were used to construct transcriptional fusions to the reporter gene gusa in the promoterless probe vector pnz272 ( platteeuw et al . the dna region comprising the complete bbr_0838 promoter was amplified from b. breve ucc2003 chromosomal dna using primers prom838rvpsti and prom838f2bglii . these primers are complementary to the dna sequence located in the 5 end of bbr_0838 coding sequence and 579 bp further upstream from this point respectively . amplified product was digested using unique bglii and psti restriction sites ( primerintroduced ) and ligated into the similarly digested promoter cloning vector pnz272 , resulting in the plasmid pnz272long . primers annealing 424 bp ( prom838f2bgliishort ) and 162 bp ( prom838fbglii ) upstream from the 5 end of bbr_0838 respectively , were used in combination with prom838rvpsti for pcr amplification , using b. breve ucc2003 chromosomal dna as a template . primers annealing 71 bp ( prom838revbamhi + ir ) , 144 bp ( prom838revbamhiir ) , 190 bp ( prom838rv + irii50psti ) , 228 bp ( prom838rv + iriipsti ) , and 296 bp ( prom838rviriipsti ) upstream of bbr_0838 start codon respectively , were used in combination with prom838f2bglii for pcr amplification , using b. breve ucc2003 chromosomal dna as a template . amplified fragments were bglii and psti or bamhi digested , and ligated into similarly digested pnz272 , yielding to pnz27271 , pnz272144 , pnz272190 , pnz272228 and pnz272296 respectively . dglucuronidase ( gusa ) activity was routinely determined in cultures grown until midexponential growth phase , in the presence of subinhibitory concentrations of sodium cholate ( 0.025% w / v ) . briefly , cells from 10 ml cultures were collected , resuspended in 0.5 ml of pbs and disrupted with glassbeads ( sigma ) using a fastprep , by five 15 s treatments , with cooling intervals of 45 s when samples were put on ice . after centrifugation , gusa activity was immediately determined : appropriate dilutions of the extracts ( 4 l ) were mixed with 88 l of sodium acetate 0.1 m and 8 l of a 20 mm solution of paranitrodglucuronid acid ( sigma ) , and the mixtures were incubated at 37c . one unit of enzymatic activity was defined as the amount of protein that releases 1 nmol of pnp per minute . sequence ladders of the presumed bbr_0838 promoter regions , which were run alongside the primer extension products , were produced using the same primer as the primer extension reaction and a pcr product template generated using 838pseqfor and 838pseqrev . bifidobacterium breve ucc2003 was grown at 37c in mrs broth ( difco , bd diagnostic systems , sparks , md , usa ) supplemented with 0.05% lcysteine ( w / v ) ( sigma , st louis , mo , usa ) ( mrsc ) in an anaerobic chamber ( mac 500 , don whitley scientific , west yorkshire , uk ) with an atmosphere of 5% co2 5% h2 90% n2 . analysis of bifidobacterium tolerance to toxic compounds was performed by supplementing mrsc before inoculation with different concentrations of oxgall ( oxoid limited , hampshire , uk ) , or a particular bile salt ( cholate , glycocholate , taurocholate , deoxycholate ) supplied by sigma . bertani ( lb ) broth at 37c with agitation , and l. lactis was cultivated in m17 broth ( oxoid ) supplemented with 0.5% glucose ( gm17 ) at 30c . where appropriate , the growth medium contained chloramphenicol ( cm ; 5 g ml for l. lactis and 2 g ml for b. breve ) , erythromycin ( em ; 100 g ml for e. coli ) , kanamycin ( 50 g ml for e. coli ) , ampicillin ( 100 g ml for e. coli ) or tetracycline ( 10 g ml for e. coli or b. breve ) when required for plasmid maintenance . restoration of bile survival phenotype in the complemented mutant strain was analysed by exposing early exponential growth phase cells to different concentrations of cholate in fresh mrsc , incubating samples at 37c in anaerobic conditions and determining colony counts after several hours of bile exposure . chromosomal dna from b. breve ucc2003 was obtained according to previously described procedures ( margolles and de los reyesgaviln , 2003 ) and sequences for cloning were amplified from b. breve chromosomal dna using highfidelity platinum pfx polymerase ( invitrogen ) . plasmid minipreparations from e. coli and l. lactis were performed using spinincolumn system ( sigma ) , according to the manufacturer 's instructions , with the incorporation of an initial lysis step for l. lactis , involving suspension of the cells in lysis buffer ( 20% sucrose , 10 mm trishcl ph 8.1 , 10 mm edta and 50 mm nacl ) supplemented with lysozyme ( 10 mg ml ) , followed by incubation at 37c for 30 min . electroporation of plasmid dna into e. coli was carried out as described by sambrook and colleagues ( 1989 ) , into l. lactis as described by wells and colleagues ( 1993 ) and into b. breve ucc2003 was performed as reported by maz and colleagues ( 2007 ) . internal 800 and 1000 bp fragments of bbr_0838 ( which corresponds to amino acid residues 100 to 357 , and 100 to 438 respectively ) were pcramplified using b. breve ucc2003 chromosomal dna as a template and primer combinations 838f and 838800r , or 838f and 8381000r . , 2008 ) was then cloned as a saci fragment into the unique saci site on each of these pori19 derivatives , yielding pori19838800tetw and pori198381000tetw respectively . transformants were checked by pcr and southern hybridization to confirm whether they contained either pori19838800tetw or pori198381000tetw inserted into their chromosome , and one verified insertion mutant for each of these events was retained for further use and designated ucc2003::838800 and ucc2003::8381000 respectively . the stability of these insertions was analysed following 50 generations of growth in mrsc in the absence of selective agents , by spreading cells with and without antibiotic . for mutant complementation , primers pbc838f and pbc838r were used to pcramplify the region comprising the coding sequence of bbr_0838 under the control of its own promoter from b. breve ucc2003 chromosomal dna . purified pcr product was hindiii and bamhi digested , and ligated into similarly digested pbc1.2 , yielding pbc838 , which was transformed into b. breve ucc2003::838800 by electroporation . differences in transcriptional response to bile stress between b. breve ucc2003 and its derivative b. breve ucc2003::838800 were determined by microarray experiments . volumes of 50 ml of b. breve ucc2003 and b. breve ucc2003::838800 cultures , grown until the od600 reached 0.5 , were exposed to subinhibitory concentrations of sodium cholate for 1 h , and samples for rna isolation were collected . methods for cell disruption , rna isolation , cdna synthesis and indirect labelling for microarray hybridizations were performed according to previously described procedures ( zomer et al . a gene was considered differentially expressed between a test condition and a control when an expression ratio of > 3 or < 0.33 relative to the result for the control was obtained with a corresponding pvalue that was < 0.001 . for northern hybridization analysis of bbr_0838 expression in b. breve ucc2003 , total rna ( 20 g ) obtained from sodium cholateexposed and control cultures , was separated on a 1% ( w / v ) agarose/0.66 m formaldehyde gel . the resulting construct was isolated from e. coli , hindiii linearized , and used as a template for in vitro transcription with t7 rna polymerase incorporating biotinlabelled dutps ( roche ) . dna fragments of different lengths encompassing various segments of the bbr_0838 promoter region were used to construct transcriptional fusions to the reporter gene gusa in the promoterless probe vector pnz272 ( platteeuw et al . primers annealing 424 bp ( prom838f2bgliishort ) and 162 bp ( prom838fbglii ) upstream from the 5 end of bbr_0838 respectively , were used in combination with prom838rvpsti for pcr amplification , using b. breve ucc2003 chromosomal dna as a template . primers annealing 71 bp ( prom838revbamhi + ir ) , 144 bp ( prom838revbamhiir ) , 190 bp ( prom838rv + irii50psti ) , 228 bp ( prom838rv + iriipsti ) , and 296 bp ( prom838rviriipsti ) upstream of bbr_0838 start codon respectively , were used in combination with prom838f2bglii for pcr amplification , using b. breve ucc2003 chromosomal dna as a template . dglucuronidase ( gusa ) activity was routinely determined in cultures grown until midexponential growth phase , in the presence of subinhibitory concentrations of sodium cholate ( 0.025% w / v ) . after centrifugation , gusa activity was immediately determined : appropriate dilutions of the extracts ( 4 l ) were mixed with 88 l of sodium acetate 0.1 m and 8 l of a 20 mm solution of paranitrodglucuronid acid ( sigma ) , and the mixtures were incubated at 37c .

interferons ( ifns ) represent a family of cytokines which were originally identified by their ability to mediate antiviral effects . since their discovery more than 54 years ago ( lindenmann et al . , 1957 ) , this class of proteins now embraces around 30 members . based on common structural , biochemical , and signaling properties as well as the source of cells producing these factors , ifns can be classed into three distinct subfamilies namely type i , type ii , and class iii ifns . while ifn- is the sole type ii ifn and the three different ifn-s constitute the type iii ifns , type i ifns are a highly divergent group of cytokines encompassing 13 different ifn- subtypes , ifn- , ifn- , ifn- , ifn- , ifn- , ifn- and three different ifn-s ( il-28a / b and il-29 ; noppert et al . , 2007 ) . consistent with the functional role of type i ifns in pathogen defense , induction of these cytokines is predominantly triggered by distinct pathogen - associated molecular patterns ( pamps ) which are recognized by specific pathogen recognition receptors ( prrs ) . as depicted in figure 1 , the surface toll - like receptor ( tlr ) 4 recognizing lipopolysaccharide from gram - negative bacteria as well as tlrs 3 , 7 , 8 , and 9 , which recognize pathogen - derived nucleic acids , induce type i ifns ( blasius and beutler , 2010 ) . tlr3 recognizes viral double - stranded rna ( dsrna ) while viral single - stranded rna ( ssrna ) is detected by tlr7 and tlr8 . viral or bacterial unmethylated dna , commonly referred to as cpg dna , is sensed by tlr 9 ( akira et al . overview of typical signaling cascades inducing type i interferon expression . upon ligand engagement , several toll - like receptors ( tlrs ) and rig - i like helicases ( rlhs ) induce transcription of type i interferons ( ifn ) . tlr4 located at the cell surface is typically induced extracellular while tlr3 , tlr 7/8 , and tlr9 sense pathogen - derived single - stranded rna ( ssrna ) , double - stranded rna ( dsrna ) , and unmethylated dna ( cpg dna ) within the cell sequestered from the cytoplasmic compartment . intracellular tlrs are localized , traffic , and initiate signaling cascades in membrane surrounded compartments like the endoplasmic reticulum , endosomes , lysosomes , and phagocytic vesicles . upon ligand downstream signaling inducing type i ifn is mediated by initial binding to either myd88 ( tlr7/8/9 ) or trif ( tlr3/4 ) , followed by recruitment of multicomponent protein complexes . typically a complex with tlr3 or tlr4 together with trif and traf3 activates the kinase tbk1 mediating phosphorylation of irf3 , which subsequently forms homodimers , translocates to the nucleus , and initiates type i ifn gene expression . myd88 recruited to tlr7/8/9 complexes with irak1 , traf6 , traf3 , and the kinases tak1 and ikk , which phosphorylate and thus activate irf7 to drive type i ifn expression . the cytoplasmic rlhs mda5 and rig - i recognize longer rnas like poly i : c or 5-3-p - rna respectively and engage ips at the mitochondrial membrane . recruitment of a complex containing tbk1 induces phosphorylation and thus dimerization of irf3 followed by type i ifn gene expression . independent from tlr and rlh intracellular , non - cpg dna , and cyclic - di - gmp are sensed in a sting dependent manner . sting interacts with rig - i and activates type i ifn transcription via the irf3 axis but is also capable to recruit stat6 to the er followed by tbk1 mediated stat6 phosphorylation . the localization of nucleic acid sensing tlrs at the endoplasmic reticulum and endosomal membranes limits the detection of viruses by tlrs to this specific compartment . in general , signal transduction for type i ifn induction via the tlrs mentioned above starts with the recruitment of either toll - il-1 receptor ( tir ) domain - containing factor ( trif ; for tlr4 , tlr3 ) and/or myeloid differentiation primary response gene 88 ( myd88 ; for tlr7 , tlr9 ) to the activated receptor . subsequent signaling events involving the molecules interleukin-1 receptor - associated kinase ( irak ) 1 , irak 4 , tumor necrosis associated factor ( traf ) 6 and traf3 activate the kinases tgf- activated kinase 1 ( tak1 ) and tank - binding kinase 1 ( tbk1 ) . while tak1 acts as a common activator of nuclear factor of kappa light polypeptide gene enhancer in b cells ( nf-b ) signaling via the i kappa b kinase ( ikk ) complex which mediates phosphorylation and degradation of the inhibitory component ib , tbk1 phosphorylates interferon regulatory factor ( irf ) 3 leading to its dimerization ( mori et al . , 2004 ; tamura et al . , 2008 ) . finally , dimeric irf3 and nf-b translocate into the nucleus to induce type i ifn expression . in addition , engagement of distinct tlrs also causes irf7 phosphorylation , which preferentially activates ifn- but also leads to ifn- induction ( honda and taniguchi , 2006 ) . alternative to the tlrs , the cell employs the retinoic acid - inducible gene - i ( rig - i ) like helicases ( rlhs ) rig - i and the melanoma differentiation - associated gene 5 ( mda5 ) to detect viral infection ( pichlmair and reis e sousa , 2007 ) . rig - i detects 5-triphosphate - containing rna which for example is typical for vesicular stomatitis virus ( vsv ; hornung et al . , 2006 ; schlee et al . , characteristic for mda5 is the recognition of longer rna molecules as exemplified by the synthetic dsrna poly i : c or picornavirus genomes ( kato et al . , 2006 ) . rlhs are characterized by a helicase domain for rna binding and a caspase recruitment domain ( card ) mediating protein interaction for downstream signaling . binding to viral rna induces homodimerization of these sensors resulting in the engagement of mavs ( ips-1 , visa , cardif ; kawai et al . , 2005 ; meylan et al . , 2005 ; seth et al . , 2005 ; xu et al . , 2005 ) located in the mitochondrial membrane followed by the recruitment of traf3 , traf6 , tbk1 , and ikk/. as a result of these signaling events , irf3 , irf7 , and nf-b are activated and together with cjun / atf2 and the coactivators cbp / p300 initiate the expression of type i ifns ( kawai and akira , 2011 ) . the observation that tlr9 deficient cells are still capable to efficiently mount type i ifn production when transfected with double - stranded dna ( dsdna ) led to the search of alternative dna sensing molecules especially in non - plasmacytoid dendritic cells ( pdcs ; kawai and akira , 2009 ) . these investigations led to the identification of the dna sensor endoplasmic reticulum ifn stimulator ( eris ) now better known as stimulator of interferon genes ( sting ) which is located in the er membrane and plays an important role in the induction of ifn by non - cpg intracellular dna species ( ishikawa and barber , 2008 ; zhong et al . , 2008 ) . analysis of sting/ mice revealed that the protein , which has no homology to other dna sensors , is essential for host defense against dna pathogens such as herpes simplex virus 1 ( hsv-1 ; ishikawa et al . , 2009 ) . recent work also provided strong evidence that sting also acts as a direct sensor for the bacterial second messenger cyclic - di - gmp , which was shown previously to elicit a type i ifn response in host cells ( mcwhirter et al . , 2009 ; woodward et al . it was recently shown that sting recruits stat6 to the endoplasmatic reticulum ( er ) followed by tbk - mediated phosphorylation . remarkably , this stat6 activation at the er by sting differs from cytokine induced stat6 phosphorylation at the plasma membrane as janus kinases ( jaks ) are not involved and phosphorylation is not restricted to distinct cell types . the virus - induced stat6 activation also occurs in ifnar2/ or irf3/ cells as well as in cells lacking individual jaks providing strong evidence that sting - triggered stat6 activation represents a novel signaling module . while the canonical stat6 activation is mavs independent this non - canonical stat6 activation required the presence of mavs and stat6 phosphorylation is mediated by tbk1 . consistent with a fundamental role of stat6 in antiviral activity , stat6 deficient mice exhibited exacerbated pathologies when challenged with either vsv , sendai virus , or herpes simplex virus ( chen et al . , 2011 ) . as a common feature all interferons activate the jak signal transducers of activation and transcription ( stat ) signaling pathway . a hallmark of type i ifns is that they all bind and signal via the interferon alpha receptor ( ifnar ) , which is a heterodimer composed of interferon receptor 1 ( ifnar1 ) and interferon receptor 2 ( ifnar2 ) . it is a unique feature compared to other cytokine receptors that ifnar can bind and mediate signaling of multiple ligands . type i ifns bind to the ifnar and as a consequence they activate the kinases jaks tyk 2 and jak 1 which are associated with ifnar1 and ifnar2 respectively and phosphorylate receptor tyrosine residues on the receptor ( de weerd et al . , 2007 ) . via sh2 domains stat phosphorylated stat1 forms heterodimers with stat2 and together with irf9 constitutes a transcription factor ( isgf3 ) which binds to interferon - stimulated response elements ( isres ) of interferon - stimulated target genes ( isgs ) inducing their expression . further complexity is added to the system by the formation of stat1 and stat3 homo- and heterodimers which activate gas elements controlling the expression of other isgs which are also inducible by ifn-. in addition , also stat independent pathways are activated ( boxel - dezaire et al . , 2006 ) . ultimately , hundreds of isgs , which might vary depending on the cell type , are induced and translated into the effector proteins responsible to mediate biological effects like antiviral activity , control of cellular proliferation , apoptosis , and immune regulation . interferon beta has proinflammatory properties and contributes to the pathology of autoimmune diseases like systemic lupus erythematosus ( sle ) , rheumatoid arthritis , and psoriasis ( preble et al . , 1982 ; baechler et al . , 2006 ) proinflammatory detrimental effects of ifn- were described for aicardi goutires syndrome ( ags ) and neuromyelitis optica ( nmo ) . the disease is characterized by increased lymphocyte numbers in the cerebrospinal fluid , demyelination of the white matter and calcification of basal ganglia thus mimicking pathological consequences of congenital infection which often leads to misdiagnoses ( rice et al . , 2007 ) . one hallmark of ags are increased levels of ifn- in serum and cerebrospinal fluid ( dussaix et al . , 1985 ) . increased levels of ifn- are also a key feature of sle and indeed , molecular mechanisms underlying ags and sle exhibit striking parallels allocating ags into the group of autoimmune diseases . furthermore , some children with ags also exhibit an early onset form of sle ( de laet et al . , 2005 ) . ags is inherited in an autosomal recessive trait and mutations in different genetic loci were identified . it was shown before that recessive mutations in the genes encoding either human 3repair exonuclease 1 ( trex1 ) , different components of the rnaseh2 complex or the sam - domain hd - domain - containing protein 1 ( samhd1 ) can cause this severe inflammatory disease ( crow et al . , trex1 was shown to be a 35 dna exonuclease preferentially binding and cleaving single - stranded dna ( ssdna ; mazur and perrino , 1999 , 2001 ) . initially appearing paradox for a dnase , trex1 is predominantly localized in the cytoplasm where the protein is associated with the endoplasmic reticulum . trex1 is part of the set complex and in response to oxidative stress the protein can translocate to the nucleus and is involved in granzyme a - mediated apoptosis ( martinvalet et al . , 2005 ; absence of trex in mice and humans results in the accumulation of cytoplasmic ssdna ( yang et al . it was reported that excessive and mislocalized ssdna originates from excision mediated dna repair ( yang et al . , 2007 ) . work by medzhitov s group identified trex1 as an important negative regulator of the interferon stimulatory dna ( isd ) response . they provided compelling evidence that the ssdna accumulating in the absence of trex arises from endogenous retroelements which are no longer degraded properly by the trex nuclease . as a consequence , the elevated levels of ssdna which might mimic viral infection are sensed by an unidentified dna sensor , initiate the isd pathway and activate irf3 resulting in the production of high levels of type i ifn and lymphocyte mobilization ( stetson et al . , 2008 ) . in line with an aberrant activation of the innate immune system the observed cardiomyopathy in trex1 deficient mice might be analogous to the encephalopathy in ags patients associated with an autoinflammatory response which arises in the absence of an infection . the observation that the phenotype of trex1/ mice can be rescued in the absence of either irf3 , ifnar , or the recombination activating gene ( rag ) further underlines that the autoimmune mechanism is triggered by activation of irf3 mediated type i ifn induction ( stetson et al . , 2008 ) but also shows that secondary mechanisms like leukocyte recruitment are essential for disease manifestation . in line with the central role of trex in the maintenance of immune homeostasis is the observation that mutations in this gene are also associated with sle ( lee - kirsch et al . , 2007 ) . the central role of trex1 as a negative regulator of type i ifn induction is exploited by the human immunodeficiency virus ( hiv ) type 1 . recent work showed that trex1 not only degrades dna from endogenous retroelements to prevent autoimmune activation . the nuclease is also active against hiv dna which arises during hiv infection after reverse transcription ( rt ; geijtenbeek , 2010 ; yan et al . , 2010 ) . apparently , the virus hijacks trex1 s specificity for retrovirus - derived dna to avoid activation of cytoplasmic nucleic acid sensors , which usually trigger the antiviral effector system . degradation of hiv dna thus represents one mechanism how hiv escapes immune recognition and helps to explain why infection of t cells and macrophages do not elicit an effective antiviral interferon response ( geijtenbeek , 2010 ) . interestingly , also another ags susceptibility gene , samhd1 , which is highly expressed in macrophages and dendritic cells and upregulated upon viral infection was proposed to act as a negative regulator of the innate immune response ( lafuse et al . , 1995 ; li et al . , 2000 ; rice et al . , 2009 ) . however , the molecular function until recently remained elusive . samhd1 function was now shown to be tightly linked to the control of retroviral infection ( hrecka et al . , 2011 ; hiv is incapable to transduce dendritic cells and impaired in the transduction of macrophages because efficient viral cdna synthesis is inhibited in these cells . in contrast , other retroviruses encoding vpx accessory proteins circumvent this inhibition ( ayinde et al . , 2010 ) . two recent publications identified samhd1 as the factor restricting hiv infection in myeloid and dendritic cells and showed that vpx proteins counteract this restriction by mediating proteasomal degradation of samhd1 ( hrecka et al . , 2011 ; laguette et al . , it was shown that the protein is an effective dgtp - stimulated deoxyguanosine triphosphate triphosphohydrolase , which catalyzes the conversion of dgtp to guanosine and inorganic triphosphate ( goldstone et al . , 2011 ) . samhd1 rapidly hydrolyzes dgtp but not datp , dctp , and dttp . however , samhd1 is allosterically activated by dgtp which binds to samhd1 leading to a more promiscuous substrate specificity which is extended to datp , dctp , and dttp . dgtp is thus both a substrate and an activator of the enzyme against the other dntps . the catalytic activity should strongly limit the dntp pool within the cytoplasm and thus exert a role in nucleic acid metabolism . it is reasonable to hypothesize that samhd1 limits the dntp pool and thus interferes with hiv replication by inhibiting endogenous rt . with respect to the etiology of ags , loss of samhd1 activity would no longer counteract rt of endogenous retroviral elements as the dntp pool is no longer restricted . as a consequence , the loss of rt restriction might promote the accumulation of cytoplasmic dna similar to what is observed in trex1 mutants . although further experimental evidence needs to be added , this model would be a straight forward explanation for the similar phenotypic manifestations arising from human mutations in samhd1 and trex genes . in summary , trex1 and samhd1 provide excellent examples for the intricate relationship between the control of retroviral infection and the control of cell intrinsic nucleic acids harboring the risk to trigger autoimmune inflammation induced by type i ifns . in addition , also mutations in subunits of the rnase h2 complex were shown to cause ags ( crow et al . , 2006b ) . the rnase h2 is the dominant cytoplasmic rnase in human cells and composed of a heterotrimeric complex in which all three components are needed to constitute the active enzyme . rnase h2 enzymes recognize rna : dna hybrids , degrade the polyribonucleotide strand and thus also play a central role in nucleic acid metabolism ( eder and walder , 1991 ; rydberg and game , 2002 ) . although direct experimental evidence is still missing , it is likely that rnase h2 mutations which impair degradation of rna : dna hybrids alter cytoplasmic nucleic acid homeostasis resulting in the activation of the isd response . as depicted in figure 2 , analysis of the molecular mechanisms underlying ags revealed a role of all the susceptibility genes mentioned above in the control of cytoplasmic nucleic acid homeostasis which needs to be tightly controlled in order to prevent autoimmunity induced by type i ifns . model for the role of cytoplasmic nucleic acid homeostasis in the etiology of aicardi goutieres syndrome ( ags ) . trex1 is an 53 exonuclease which degrades cytoplasmic single - stranded dna ( ssdna ) originating from reverse transcription of endogenous retrotransposons . lack of trex1 activity results in the congestion of ssdna thereby augmenting the pool of nucleic acids in the cytoplasm . samhd1 cleaves inorganic triphosphate from deoxynucleotides and thus restricts the cytoplasmic dntp concentrations hence constraining reverse transcription by limiting the amount of building blocks for dna synthesis . samhd1 mutations would eliminate this dntp control mechanism , enhance the dntp pool , and could thus fuel reverse transcription from endogenous retroelements resulting in increased levels of cytoplasmic ssdna . rnaseh2 is an endonuclease composed of three subunits and required to cleave ribonucleotides from rna : dna duplexes which arise from intracellular processes . lack of function mutations in rnaseh2 would thus also impair the control of nucleic acid homeostasis . thus mutations in any kind of the genes mentioned above which were found in ags patients would lead to the accumulation of nucleic acids in the cytoplasm . these aberrant levels are sensed by cytoplasmic dna sensors originally designated to detect viral infections . consequently the initiated signaling cascade activates interferon regulatory transcription factors ( irfs ) for type i ifn induction and drive autoimmune disease onset . neuromyelitis optica represents a neuroinflammatory disease previously considered to be a variant of relapsing remitting multiple sclerosis ( rrms ) . however , in contrast to rrms , nmo is characterized by demyelination in the spinal cord and optic nerve ( lucchinetti et al . , 2002 ) . autoantibodies directed against the aquaporin 4 water channel are found in these patients and occurrence of anti - aquaporin antibodies also is the major parameter in the diagnosis of nmo ( paul et al . , 2007 ) . driven by the supposed analogy to rrms , ifn- was tried as a therapeutic but it was quickly clear that ifn- treatment even worsens the disease and induces severe relapses ( warabi et al . , 2007 ; palace et al . , 2010 ; shimizu et al . , 2010 ; uzawa et al . , 2010 ) notably , the dominant infiltrating cells are granulocytes which is in sharp contrast to classical multiple sclerosis ( ms ) . interestingly , levels of il-17 were found to be elevated in the cerebrospinal fluid of people suffering from nmo and it has been proposed that il-17 mediated induction of il-8 , granulocyte stimulating factor ( g - csf ) and gro - alpha causes granulocyte recruitment ( axtell et al . , 2011 ) . as exemplified for sle and ags , type i ifns were frequently causally associated with the development of autoimmune pathologies . in contrast , type i ifns were also shown to exert anti - inflammatory effects and ifn- is used as a routine therapeutic for the treatment of rrms in patients ( jacobs et al . , 2000 ; ms is an autoimmune inflammatory disease of the cns of unclear etiology characterized by demyelination of axons in brain and spinal cord . characteristic for the disease is the infiltration of the cns with inflammatory cells like monocytes , th1 and th17 cells . in rrms , phases with no or only minor disease progression are followed by unpredictable acute relapses causing deficits which might at least partially resolve in times of remission . according to results from clinical trials , ifn- treatment reduces relapse rates by about 30% , decreases the formation of inflammatory lesions in the cns and extends remission periods ( schwid and panitch , 2007 ) . however , a major problem is that a high proportion of about 20% of the patients do not or only poorly respond to ifn- treatment . despite excessive attempts to define biomarkers for responders and non - responders it is still impossible to predict whether an individual patient will respond to ifn- therapy . mechanism underlying this variety include development of antibodies against ifn- , polymorphisms in components of the ifn signaling pathway and ifn effector genes as well as variable ms pathomechanisms ( killestein and polman , 2011 ) . by further dissecting type i ifn signatures in ms patients , ifn- non - responders were found to exhibit increased monocyte - specific type i ifn secretion upon innate immune stimuli via tlr 4 , by increased endogenous production of type i interferon , and by an elevated activation status of myeloid dendritic cells ( comabella et al . , 2009 ) . these findings indicate that perturbations of the type i ifn signaling pathway in monocytes are related to a lack of response to ifn- and type i ifn - regulated genes may be used as response markers in ifn- treatment in ms . consistent with the concept that genetic variants define the individual response is that induction of interferon response genes among different patients varies significantly but is remarkably stable within a long time period . a frequently used system to gain insight into the molecular and cellular events underlying ms is the model of experimental autoimmune encephalomyelitis ( eae ) . in this well - established mouse model active immunization with myelin components together with a strong adjuvant induces t - cell mediated neuroinflammation and demyelination resembling multiple aspects of ms ( owens et al . , 2001 ; consistent with the beneficial effect of ifn- in human ms , eae in rodents can be suppressed by injection of recombinant ifn-. in addition , mice lacking ifn- exhibit strongly exacerbated disease parameters in the eae model ( teige et al . , 2003 ) . furthermore , ifn is stronger induced in the cns than in the periphery . to trace down the cell type mediating the protective immune regulation of type i ifns via the type i receptor ( ifnar ) , mice lacking this receptor only on distinct cell types were used for eae experiments and ifn- treatment ( prinz et al . , 2008 ) . it could be shown that negative regulation of autoimmunity in the eae model relies on the presence of ifnar on cells of the myeloid lineage while absence of ifnar on t cells , b cells , and cells of neuroectodermal origin does not enhance disease severity . these experiments suggest a scenario where locally produced ifn- within the cns acts on invading myeloid cells to attenuate autoimmune damage especially in the effector phase of eae . another study which also employed eae in ifnar deficient mice provided evidence for a functional role of type i interferon in negatively regulating the development of th17 cells . numbers of encephalitogenic th17 cells were found by this group to be elevated after eae induction in ifnar1 and trif deficient mice ( guo et al . th17 cells develop from naive t cells which induced by tgf and il-6 secrete il-21 . this cytokine subsequently induces the transcription factor rort in an autocrine manner which triggers the th17 differentiation program ( veldhoen et al . , 2006 ; ifn- induces expression of il-27 which acts on naive cd4 + t cells as a negative regulator of th17 development ( prinz and kalinke , 2010 ) . il-27 also promotes il-10 secretion by t cells which was shown to suppress autoimmune inflammation of the cns ( fitzgerald et al . , 2007 ) . ifn- was furthermore shown to inhibit il-23 induced proliferation of th17 cells ( harrington et al . , 2005 ) . increased levels of th17 cells in ifnar/ mice would thus be explained by the lack of these inhibitory effects of type i ifns . disease symptoms and generation of antigen specific th17 cells were also enhanced in trif deficient mice suggesting that type i ifn production counteracting autoimmunity in eae is triggered by a trif dependent mechanism ( guo et al . , 2008 ) . interestingly , type i ifn - related genes were found to be strongly induced in toxic mouse models of demyelination whereas the presence of ifnar could neither modulate demyelination or myelin repair ( schmidt et al . , 2009 ) . just recently , also cytosolic rlhs were shown to act as negative regulators of sterile inflammation within the cns ( dann et al . , 2012 ) . mice lacking the rig - i like helicase adaptor ips-1 developed exacerbated disease symptoms . in a reciprocal experiment the same study showed that on the other hand activation of rlhs via ifn - inducing 5-triphosphate rna oligonucleotides ameliorated disease outcome providing compelling evidence that rlhs mediate signals counteracting encephalitogenic immune responses . while rlh stimulation did not affect t - cell differentiation , the maintenance and expansion of th1 and th17 cells was strongly suppressed upon rlh engagement ( figure 3 ) . repression of proinflammatory th1 and th17 cells could only be observed when the type i interferon receptor was present on dendritic cells by using cd11ccre mice crossed with conditional ifnar animals . remarkably , absence of ifnar1 on macrophages or microglia did not interfere with rlh mediated th1 or th17 repression ( dann et al . , 2012 ) . these results imply that engagement of rlhs might be a passable way to stimulate endogenous type i ifns for therapeutic intervention . replacing systemic administration with the endogenous induction of type i ifns should prevent the development of neutralizing antibodies . it might also be more effective than systemic administration as type i ifns are biologically designed to exert their effects in a spatially restricted area affecting cells in their direct neighborhood . model for negative regulation of sterile cns inflammation by rig - i like helicases ( rlhs ) . in dendritic cells activation of rlhs rig - i or mda5 by 5triphosphate rna or polyi : c complexed to liposomes ( cpolyi : c ) respectively , recruits ips-1 to the rlh . this initiates a signaling cascade which involves tbk1 mediated phosphorylation of irf3 and/or irf7 which upon dimerization enter the nucleus and lead to the expression of type i ifn . secreted type ifns ( ifn ) can either bind to type i interferon receptor ( ifnar ) in an autocrine or paracrine manner and stimulate the expression of interferon - stimulated target genes ( isgs ) . while lack of ips-1 leads to exacerbated disease pathology in the eae model of sterile inflammation , rlh stimulation improved clinical signs of disease and inhibits th1 and th17 expansion and survival . however , mice specifically lacking ifnar on monocytes , macrophages , microglia , or granulocytes like wildtype mice showed reduced symptoms upon rlh induction . ifnar signaling on these cells is thus dispensable for the suppressive effect of rlh engagement . in contrast , when mice specifically lacking ifnar on dendritic cells were used , rlh treatment did not ameliorate autoimmunity clearly showing that interferon stimulation in this particular celltype is essential for ifn mediated th1 and th17 inhibition and disease improvement . recognition of triphosphate rna by rig - i like helicases followed by ifn induction is largely independent from the sequence used for the dsrna ( hornung et al . , 2006 ) . thus , a specific short interfering rna ( rnai ) suitable to suppress expression of a specific target protein can be used . this , at least in principle , offers therapeutic strategies where activation of the type i ifn response can be combined with the suppression of harmful gene products . as a proof of principle for the feasibility of such an approach it was shown in a melanoma model that bcl2 specific sirna carrying 5 triphosphate ends efficiently activated type i ifn via rig - i and in parallel silenced bcl2 expression ( poeck et al . , 2008 ) . as a consequence , the synergistic activation of the innate immune response combined with suppression of a pro survival signal caused massive apoptosis of tumor cells , prolonged survival of the animals , and reduced tumor size . it might thus also be suitable to combine type i ifn induction with rnai mediated inhibition of proinflammatory factors or proteins triggering the differentiation of deleterious cells . such a strategy where for example type i ifn induction could be combined with rnai mediated inhibition of rort or t - bet to simultaneously repress th17 and th1 differentiation might potentially open new ways to modulate the immune response and counteract autoimmunity within the cns . despite extensively analyzed , the functions of type i ifns within the cns are still far from being well understood . one principal problem to generalize type i ifn effects is surely based on the fact that hundreds of genes , which even vary among different cell types , are controlled by this particular group of cytokines . in addition , the levels and kind of ifn target genes induced are differentially regulated depending on the situation , interaction and environment of the cell . further complexity is added to the system as multiple negative regulators of the ifn signaling system modulate the outcome and refractory mechanisms blunt restimulation . a major task for the future will be to identify the particular interferon effector genes responsible for the anti - inflammatory and therapeutic properties which even might considerably differ between various cell types . with respect to the etiology of type i ifn associated autoimmune disease like ags it will be interesting to see which particular molecules are involved in the pathways sensing enhanced levels of endogenous nucleic acids in the cytoplasm . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

type i interferons ( ifns ) were originally identified as antiviral effector molecules that exert pleiotropic physiological processes ranging from immune modulation , control of proliferation , apoptosis to antitumor activity . however , type i ifns were recently also shown to apply both beneficial and detrimental effects to the central nervous system ( cns ) and a tightly balanced equilibrium between cellular activation and inhibition seems to be essential to maintain homeostasis within the cns . in inflammatory pathologies affecting the cns , type i ifns are in the center of attention not only because interferon beta ( ifn- ) is used as a standard therapeutic in the treatment of relapsing remitting multiple sclerosis ( ms ) , but also as type i ifn expression is associated with distinct pathologies . despite the great efficiency of ifn- in reducing ms relapses and attenuation of novel inflammatory lesions is well documented , underlying molecular mechanisms and cellular target specificities are just beginning to emerge . in contrast to the curative effects , aberrant activation of the type i ifn response were also recently shown to be associated with detrimental effects exemplified by the aicardi goutires syndrome ( ags ) , a severe disabling autoimmune inflammatory encephalopathy . this review will highlight the dual role of type i interferons during chronic cns inflammation . recently uncovered molecular and cellular mechanisms in the etiology of ags and experimental autoimmune encephalomyelitis ( eae ) , the murine model of ms will be highlighted .

Type I Interferons and Their Induction IFN Signaling Inflammatory Brain Pathologies Associated with Aberrant Type I IFN Induction Neuromyelitis Optica (Devics Disease) and Type I IFNs Anti-Inflammatory Effects of Type I IFNs in RelapsingRemitting Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis Conclusion and Future Perspectives Conflict of Interest Statement

interferons ( ifns ) represent a family of cytokines which were originally identified by their ability to mediate antiviral effects . based on common structural , biochemical , and signaling properties as well as the source of cells producing these factors , ifns can be classed into three distinct subfamilies namely type i , type ii , and class iii ifns . while ifn- is the sole type ii ifn and the three different ifn-s constitute the type iii ifns , type i ifns are a highly divergent group of cytokines encompassing 13 different ifn- subtypes , ifn- , ifn- , ifn- , ifn- , ifn- , ifn- and three different ifn-s ( il-28a / b and il-29 ; noppert et al . consistent with the functional role of type i ifns in pathogen defense , induction of these cytokines is predominantly triggered by distinct pathogen - associated molecular patterns ( pamps ) which are recognized by specific pathogen recognition receptors ( prrs ) . as depicted in figure 1 , the surface toll - like receptor ( tlr ) 4 recognizing lipopolysaccharide from gram - negative bacteria as well as tlrs 3 , 7 , 8 , and 9 , which recognize pathogen - derived nucleic acids , induce type i ifns ( blasius and beutler , 2010 ) . upon ligand engagement , several toll - like receptors ( tlrs ) and rig - i like helicases ( rlhs ) induce transcription of type i interferons ( ifn ) . upon ligand downstream signaling inducing type i ifn is mediated by initial binding to either myd88 ( tlr7/8/9 ) or trif ( tlr3/4 ) , followed by recruitment of multicomponent protein complexes . typically a complex with tlr3 or tlr4 together with trif and traf3 activates the kinase tbk1 mediating phosphorylation of irf3 , which subsequently forms homodimers , translocates to the nucleus , and initiates type i ifn gene expression . myd88 recruited to tlr7/8/9 complexes with irak1 , traf6 , traf3 , and the kinases tak1 and ikk , which phosphorylate and thus activate irf7 to drive type i ifn expression . recruitment of a complex containing tbk1 induces phosphorylation and thus dimerization of irf3 followed by type i ifn gene expression . sting interacts with rig - i and activates type i ifn transcription via the irf3 axis but is also capable to recruit stat6 to the er followed by tbk1 mediated stat6 phosphorylation . in general , signal transduction for type i ifn induction via the tlrs mentioned above starts with the recruitment of either toll - il-1 receptor ( tir ) domain - containing factor ( trif ; for tlr4 , tlr3 ) and/or myeloid differentiation primary response gene 88 ( myd88 ; for tlr7 , tlr9 ) to the activated receptor . while tak1 acts as a common activator of nuclear factor of kappa light polypeptide gene enhancer in b cells ( nf-b ) signaling via the i kappa b kinase ( ikk ) complex which mediates phosphorylation and degradation of the inhibitory component ib , tbk1 phosphorylates interferon regulatory factor ( irf ) 3 leading to its dimerization ( mori et al . finally , dimeric irf3 and nf-b translocate into the nucleus to induce type i ifn expression . as a result of these signaling events , irf3 , irf7 , and nf-b are activated and together with cjun / atf2 and the coactivators cbp / p300 initiate the expression of type i ifns ( kawai and akira , 2011 ) . the observation that tlr9 deficient cells are still capable to efficiently mount type i ifn production when transfected with double - stranded dna ( dsdna ) led to the search of alternative dna sensing molecules especially in non - plasmacytoid dendritic cells ( pdcs ; kawai and akira , 2009 ) . these investigations led to the identification of the dna sensor endoplasmic reticulum ifn stimulator ( eris ) now better known as stimulator of interferon genes ( sting ) which is located in the er membrane and plays an important role in the induction of ifn by non - cpg intracellular dna species ( ishikawa and barber , 2008 ; zhong et al . recent work also provided strong evidence that sting also acts as a direct sensor for the bacterial second messenger cyclic - di - gmp , which was shown previously to elicit a type i ifn response in host cells ( mcwhirter et al . it was recently shown that sting recruits stat6 to the endoplasmatic reticulum ( er ) followed by tbk - mediated phosphorylation . as a common feature all interferons activate the jak signal transducers of activation and transcription ( stat ) signaling pathway . a hallmark of type i ifns is that they all bind and signal via the interferon alpha receptor ( ifnar ) , which is a heterodimer composed of interferon receptor 1 ( ifnar1 ) and interferon receptor 2 ( ifnar2 ) . type i ifns bind to the ifnar and as a consequence they activate the kinases jaks tyk 2 and jak 1 which are associated with ifnar1 and ifnar2 respectively and phosphorylate receptor tyrosine residues on the receptor ( de weerd et al . further complexity is added to the system by the formation of stat1 and stat3 homo- and heterodimers which activate gas elements controlling the expression of other isgs which are also inducible by ifn-. ultimately , hundreds of isgs , which might vary depending on the cell type , are induced and translated into the effector proteins responsible to mediate biological effects like antiviral activity , control of cellular proliferation , apoptosis , and immune regulation . interferon beta has proinflammatory properties and contributes to the pathology of autoimmune diseases like systemic lupus erythematosus ( sle ) , rheumatoid arthritis , and psoriasis ( preble et al . , 2006 ) proinflammatory detrimental effects of ifn- were described for aicardi goutires syndrome ( ags ) and neuromyelitis optica ( nmo ) . one hallmark of ags are increased levels of ifn- in serum and cerebrospinal fluid ( dussaix et al . increased levels of ifn- are also a key feature of sle and indeed , molecular mechanisms underlying ags and sle exhibit striking parallels allocating ags into the group of autoimmune diseases . it was shown before that recessive mutations in the genes encoding either human 3repair exonuclease 1 ( trex1 ) , different components of the rnaseh2 complex or the sam - domain hd - domain - containing protein 1 ( samhd1 ) can cause this severe inflammatory disease ( crow et al . , trex1 was shown to be a 35 dna exonuclease preferentially binding and cleaving single - stranded dna ( ssdna ; mazur and perrino , 1999 , 2001 ) . initially appearing paradox for a dnase , trex1 is predominantly localized in the cytoplasm where the protein is associated with the endoplasmic reticulum . trex1 is part of the set complex and in response to oxidative stress the protein can translocate to the nucleus and is involved in granzyme a - mediated apoptosis ( martinvalet et al . they provided compelling evidence that the ssdna accumulating in the absence of trex arises from endogenous retroelements which are no longer degraded properly by the trex nuclease . as a consequence , the elevated levels of ssdna which might mimic viral infection are sensed by an unidentified dna sensor , initiate the isd pathway and activate irf3 resulting in the production of high levels of type i ifn and lymphocyte mobilization ( stetson et al . in line with an aberrant activation of the innate immune system the observed cardiomyopathy in trex1 deficient mice might be analogous to the encephalopathy in ags patients associated with an autoinflammatory response which arises in the absence of an infection . the observation that the phenotype of trex1/ mice can be rescued in the absence of either irf3 , ifnar , or the recombination activating gene ( rag ) further underlines that the autoimmune mechanism is triggered by activation of irf3 mediated type i ifn induction ( stetson et al . in line with the central role of trex in the maintenance of immune homeostasis is the observation that mutations in this gene are also associated with sle ( lee - kirsch et al . the central role of trex1 as a negative regulator of type i ifn induction is exploited by the human immunodeficiency virus ( hiv ) type 1 . apparently , the virus hijacks trex1 s specificity for retrovirus - derived dna to avoid activation of cytoplasmic nucleic acid sensors , which usually trigger the antiviral effector system . interestingly , also another ags susceptibility gene , samhd1 , which is highly expressed in macrophages and dendritic cells and upregulated upon viral infection was proposed to act as a negative regulator of the innate immune response ( lafuse et al . however , the molecular function until recently remained elusive . samhd1 function was now shown to be tightly linked to the control of retroviral infection ( hrecka et al . with respect to the etiology of ags , loss of samhd1 activity would no longer counteract rt of endogenous retroviral elements as the dntp pool is no longer restricted . as a consequence , the loss of rt restriction might promote the accumulation of cytoplasmic dna similar to what is observed in trex1 mutants . in summary , trex1 and samhd1 provide excellent examples for the intricate relationship between the control of retroviral infection and the control of cell intrinsic nucleic acids harboring the risk to trigger autoimmune inflammation induced by type i ifns . in addition , also mutations in subunits of the rnase h2 complex were shown to cause ags ( crow et al . although direct experimental evidence is still missing , it is likely that rnase h2 mutations which impair degradation of rna : dna hybrids alter cytoplasmic nucleic acid homeostasis resulting in the activation of the isd response . as depicted in figure 2 , analysis of the molecular mechanisms underlying ags revealed a role of all the susceptibility genes mentioned above in the control of cytoplasmic nucleic acid homeostasis which needs to be tightly controlled in order to prevent autoimmunity induced by type i ifns . model for the role of cytoplasmic nucleic acid homeostasis in the etiology of aicardi goutieres syndrome ( ags ) . thus mutations in any kind of the genes mentioned above which were found in ags patients would lead to the accumulation of nucleic acids in the cytoplasm . consequently the initiated signaling cascade activates interferon regulatory transcription factors ( irfs ) for type i ifn induction and drive autoimmune disease onset . neuromyelitis optica represents a neuroinflammatory disease previously considered to be a variant of relapsing remitting multiple sclerosis ( rrms ) . however , in contrast to rrms , nmo is characterized by demyelination in the spinal cord and optic nerve ( lucchinetti et al . driven by the supposed analogy to rrms , ifn- was tried as a therapeutic but it was quickly clear that ifn- treatment even worsens the disease and induces severe relapses ( warabi et al . , 2010 ) notably , the dominant infiltrating cells are granulocytes which is in sharp contrast to classical multiple sclerosis ( ms ) . interestingly , levels of il-17 were found to be elevated in the cerebrospinal fluid of people suffering from nmo and it has been proposed that il-17 mediated induction of il-8 , granulocyte stimulating factor ( g - csf ) and gro - alpha causes granulocyte recruitment ( axtell et al . as exemplified for sle and ags , type i ifns were frequently causally associated with the development of autoimmune pathologies . in contrast , type i ifns were also shown to exert anti - inflammatory effects and ifn- is used as a routine therapeutic for the treatment of rrms in patients ( jacobs et al . , 2000 ; ms is an autoimmune inflammatory disease of the cns of unclear etiology characterized by demyelination of axons in brain and spinal cord . characteristic for the disease is the infiltration of the cns with inflammatory cells like monocytes , th1 and th17 cells . according to results from clinical trials , ifn- treatment reduces relapse rates by about 30% , decreases the formation of inflammatory lesions in the cns and extends remission periods ( schwid and panitch , 2007 ) . however , a major problem is that a high proportion of about 20% of the patients do not or only poorly respond to ifn- treatment . by further dissecting type i ifn signatures in ms patients , ifn- non - responders were found to exhibit increased monocyte - specific type i ifn secretion upon innate immune stimuli via tlr 4 , by increased endogenous production of type i interferon , and by an elevated activation status of myeloid dendritic cells ( comabella et al . these findings indicate that perturbations of the type i ifn signaling pathway in monocytes are related to a lack of response to ifn- and type i ifn - regulated genes may be used as response markers in ifn- treatment in ms . a frequently used system to gain insight into the molecular and cellular events underlying ms is the model of experimental autoimmune encephalomyelitis ( eae ) . , 2001 ; consistent with the beneficial effect of ifn- in human ms , eae in rodents can be suppressed by injection of recombinant ifn-. furthermore , ifn is stronger induced in the cns than in the periphery . to trace down the cell type mediating the protective immune regulation of type i ifns via the type i receptor ( ifnar ) , mice lacking this receptor only on distinct cell types were used for eae experiments and ifn- treatment ( prinz et al . it could be shown that negative regulation of autoimmunity in the eae model relies on the presence of ifnar on cells of the myeloid lineage while absence of ifnar on t cells , b cells , and cells of neuroectodermal origin does not enhance disease severity . these experiments suggest a scenario where locally produced ifn- within the cns acts on invading myeloid cells to attenuate autoimmune damage especially in the effector phase of eae . another study which also employed eae in ifnar deficient mice provided evidence for a functional role of type i interferon in negatively regulating the development of th17 cells . il-27 also promotes il-10 secretion by t cells which was shown to suppress autoimmune inflammation of the cns ( fitzgerald et al . increased levels of th17 cells in ifnar/ mice would thus be explained by the lack of these inhibitory effects of type i ifns . disease symptoms and generation of antigen specific th17 cells were also enhanced in trif deficient mice suggesting that type i ifn production counteracting autoimmunity in eae is triggered by a trif dependent mechanism ( guo et al . interestingly , type i ifn - related genes were found to be strongly induced in toxic mouse models of demyelination whereas the presence of ifnar could neither modulate demyelination or myelin repair ( schmidt et al . just recently , also cytosolic rlhs were shown to act as negative regulators of sterile inflammation within the cns ( dann et al . repression of proinflammatory th1 and th17 cells could only be observed when the type i interferon receptor was present on dendritic cells by using cd11ccre mice crossed with conditional ifnar animals . these results imply that engagement of rlhs might be a passable way to stimulate endogenous type i ifns for therapeutic intervention . replacing systemic administration with the endogenous induction of type i ifns should prevent the development of neutralizing antibodies . it might also be more effective than systemic administration as type i ifns are biologically designed to exert their effects in a spatially restricted area affecting cells in their direct neighborhood . in dendritic cells activation of rlhs rig - i or mda5 by 5triphosphate rna or polyi : c complexed to liposomes ( cpolyi : c ) respectively , recruits ips-1 to the rlh . this initiates a signaling cascade which involves tbk1 mediated phosphorylation of irf3 and/or irf7 which upon dimerization enter the nucleus and lead to the expression of type i ifn . while lack of ips-1 leads to exacerbated disease pathology in the eae model of sterile inflammation , rlh stimulation improved clinical signs of disease and inhibits th1 and th17 expansion and survival . this , at least in principle , offers therapeutic strategies where activation of the type i ifn response can be combined with the suppression of harmful gene products . as a proof of principle for the feasibility of such an approach it was shown in a melanoma model that bcl2 specific sirna carrying 5 triphosphate ends efficiently activated type i ifn via rig - i and in parallel silenced bcl2 expression ( poeck et al . as a consequence , the synergistic activation of the innate immune response combined with suppression of a pro survival signal caused massive apoptosis of tumor cells , prolonged survival of the animals , and reduced tumor size . it might thus also be suitable to combine type i ifn induction with rnai mediated inhibition of proinflammatory factors or proteins triggering the differentiation of deleterious cells . such a strategy where for example type i ifn induction could be combined with rnai mediated inhibition of rort or t - bet to simultaneously repress th17 and th1 differentiation might potentially open new ways to modulate the immune response and counteract autoimmunity within the cns . despite extensively analyzed , the functions of type i ifns within the cns are still far from being well understood . one principal problem to generalize type i ifn effects is surely based on the fact that hundreds of genes , which even vary among different cell types , are controlled by this particular group of cytokines . in addition , the levels and kind of ifn target genes induced are differentially regulated depending on the situation , interaction and environment of the cell . further complexity is added to the system as multiple negative regulators of the ifn signaling system modulate the outcome and refractory mechanisms blunt restimulation . with respect to the etiology of type i ifn associated autoimmune disease like ags it will be interesting to see which particular molecules are involved in the pathways sensing enhanced levels of endogenous nucleic acids in the cytoplasm . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

based on common structural , biochemical , and signaling properties as well as the source of cells producing these factors , ifns can be classed into three distinct subfamilies namely type i , type ii , and class iii ifns . while ifn- is the sole type ii ifn and the three different ifn-s constitute the type iii ifns , type i ifns are a highly divergent group of cytokines encompassing 13 different ifn- subtypes , ifn- , ifn- , ifn- , ifn- , ifn- , ifn- and three different ifn-s ( il-28a / b and il-29 ; noppert et al . consistent with the functional role of type i ifns in pathogen defense , induction of these cytokines is predominantly triggered by distinct pathogen - associated molecular patterns ( pamps ) which are recognized by specific pathogen recognition receptors ( prrs ) . as depicted in figure 1 , the surface toll - like receptor ( tlr ) 4 recognizing lipopolysaccharide from gram - negative bacteria as well as tlrs 3 , 7 , 8 , and 9 , which recognize pathogen - derived nucleic acids , induce type i ifns ( blasius and beutler , 2010 ) . upon ligand engagement , several toll - like receptors ( tlrs ) and rig - i like helicases ( rlhs ) induce transcription of type i interferons ( ifn ) . tlr4 located at the cell surface is typically induced extracellular while tlr3 , tlr 7/8 , and tlr9 sense pathogen - derived single - stranded rna ( ssrna ) , double - stranded rna ( dsrna ) , and unmethylated dna ( cpg dna ) within the cell sequestered from the cytoplasmic compartment . intracellular tlrs are localized , traffic , and initiate signaling cascades in membrane surrounded compartments like the endoplasmic reticulum , endosomes , lysosomes , and phagocytic vesicles . typically a complex with tlr3 or tlr4 together with trif and traf3 activates the kinase tbk1 mediating phosphorylation of irf3 , which subsequently forms homodimers , translocates to the nucleus , and initiates type i ifn gene expression . myd88 recruited to tlr7/8/9 complexes with irak1 , traf6 , traf3 , and the kinases tak1 and ikk , which phosphorylate and thus activate irf7 to drive type i ifn expression . the cytoplasmic rlhs mda5 and rig - i recognize longer rnas like poly i : c or 5-3-p - rna respectively and engage ips at the mitochondrial membrane . independent from tlr and rlh intracellular , non - cpg dna , and cyclic - di - gmp are sensed in a sting dependent manner . the localization of nucleic acid sensing tlrs at the endoplasmic reticulum and endosomal membranes limits the detection of viruses by tlrs to this specific compartment . in general , signal transduction for type i ifn induction via the tlrs mentioned above starts with the recruitment of either toll - il-1 receptor ( tir ) domain - containing factor ( trif ; for tlr4 , tlr3 ) and/or myeloid differentiation primary response gene 88 ( myd88 ; for tlr7 , tlr9 ) to the activated receptor . subsequent signaling events involving the molecules interleukin-1 receptor - associated kinase ( irak ) 1 , irak 4 , tumor necrosis associated factor ( traf ) 6 and traf3 activate the kinases tgf- activated kinase 1 ( tak1 ) and tank - binding kinase 1 ( tbk1 ) . while tak1 acts as a common activator of nuclear factor of kappa light polypeptide gene enhancer in b cells ( nf-b ) signaling via the i kappa b kinase ( ikk ) complex which mediates phosphorylation and degradation of the inhibitory component ib , tbk1 phosphorylates interferon regulatory factor ( irf ) 3 leading to its dimerization ( mori et al . alternative to the tlrs , the cell employs the retinoic acid - inducible gene - i ( rig - i ) like helicases ( rlhs ) rig - i and the melanoma differentiation - associated gene 5 ( mda5 ) to detect viral infection ( pichlmair and reis e sousa , 2007 ) . , characteristic for mda5 is the recognition of longer rna molecules as exemplified by the synthetic dsrna poly i : c or picornavirus genomes ( kato et al . , 2005 ) located in the mitochondrial membrane followed by the recruitment of traf3 , traf6 , tbk1 , and ikk/. as a result of these signaling events , irf3 , irf7 , and nf-b are activated and together with cjun / atf2 and the coactivators cbp / p300 initiate the expression of type i ifns ( kawai and akira , 2011 ) . the observation that tlr9 deficient cells are still capable to efficiently mount type i ifn production when transfected with double - stranded dna ( dsdna ) led to the search of alternative dna sensing molecules especially in non - plasmacytoid dendritic cells ( pdcs ; kawai and akira , 2009 ) . these investigations led to the identification of the dna sensor endoplasmic reticulum ifn stimulator ( eris ) now better known as stimulator of interferon genes ( sting ) which is located in the er membrane and plays an important role in the induction of ifn by non - cpg intracellular dna species ( ishikawa and barber , 2008 ; zhong et al . analysis of sting/ mice revealed that the protein , which has no homology to other dna sensors , is essential for host defense against dna pathogens such as herpes simplex virus 1 ( hsv-1 ; ishikawa et al . recent work also provided strong evidence that sting also acts as a direct sensor for the bacterial second messenger cyclic - di - gmp , which was shown previously to elicit a type i ifn response in host cells ( mcwhirter et al . remarkably , this stat6 activation at the er by sting differs from cytokine induced stat6 phosphorylation at the plasma membrane as janus kinases ( jaks ) are not involved and phosphorylation is not restricted to distinct cell types . the virus - induced stat6 activation also occurs in ifnar2/ or irf3/ cells as well as in cells lacking individual jaks providing strong evidence that sting - triggered stat6 activation represents a novel signaling module . while the canonical stat6 activation is mavs independent this non - canonical stat6 activation required the presence of mavs and stat6 phosphorylation is mediated by tbk1 . consistent with a fundamental role of stat6 in antiviral activity , stat6 deficient mice exhibited exacerbated pathologies when challenged with either vsv , sendai virus , or herpes simplex virus ( chen et al . a hallmark of type i ifns is that they all bind and signal via the interferon alpha receptor ( ifnar ) , which is a heterodimer composed of interferon receptor 1 ( ifnar1 ) and interferon receptor 2 ( ifnar2 ) . type i ifns bind to the ifnar and as a consequence they activate the kinases jaks tyk 2 and jak 1 which are associated with ifnar1 and ifnar2 respectively and phosphorylate receptor tyrosine residues on the receptor ( de weerd et al . via sh2 domains stat phosphorylated stat1 forms heterodimers with stat2 and together with irf9 constitutes a transcription factor ( isgf3 ) which binds to interferon - stimulated response elements ( isres ) of interferon - stimulated target genes ( isgs ) inducing their expression . further complexity is added to the system by the formation of stat1 and stat3 homo- and heterodimers which activate gas elements controlling the expression of other isgs which are also inducible by ifn-. ultimately , hundreds of isgs , which might vary depending on the cell type , are induced and translated into the effector proteins responsible to mediate biological effects like antiviral activity , control of cellular proliferation , apoptosis , and immune regulation . interferon beta has proinflammatory properties and contributes to the pathology of autoimmune diseases like systemic lupus erythematosus ( sle ) , rheumatoid arthritis , and psoriasis ( preble et al . the disease is characterized by increased lymphocyte numbers in the cerebrospinal fluid , demyelination of the white matter and calcification of basal ganglia thus mimicking pathological consequences of congenital infection which often leads to misdiagnoses ( rice et al . increased levels of ifn- are also a key feature of sle and indeed , molecular mechanisms underlying ags and sle exhibit striking parallels allocating ags into the group of autoimmune diseases . it was shown before that recessive mutations in the genes encoding either human 3repair exonuclease 1 ( trex1 ) , different components of the rnaseh2 complex or the sam - domain hd - domain - containing protein 1 ( samhd1 ) can cause this severe inflammatory disease ( crow et al . initially appearing paradox for a dnase , trex1 is predominantly localized in the cytoplasm where the protein is associated with the endoplasmic reticulum . trex1 is part of the set complex and in response to oxidative stress the protein can translocate to the nucleus and is involved in granzyme a - mediated apoptosis ( martinvalet et al . they provided compelling evidence that the ssdna accumulating in the absence of trex arises from endogenous retroelements which are no longer degraded properly by the trex nuclease . as a consequence , the elevated levels of ssdna which might mimic viral infection are sensed by an unidentified dna sensor , initiate the isd pathway and activate irf3 resulting in the production of high levels of type i ifn and lymphocyte mobilization ( stetson et al . in line with an aberrant activation of the innate immune system the observed cardiomyopathy in trex1 deficient mice might be analogous to the encephalopathy in ags patients associated with an autoinflammatory response which arises in the absence of an infection . the observation that the phenotype of trex1/ mice can be rescued in the absence of either irf3 , ifnar , or the recombination activating gene ( rag ) further underlines that the autoimmune mechanism is triggered by activation of irf3 mediated type i ifn induction ( stetson et al . in line with the central role of trex in the maintenance of immune homeostasis is the observation that mutations in this gene are also associated with sle ( lee - kirsch et al . the central role of trex1 as a negative regulator of type i ifn induction is exploited by the human immunodeficiency virus ( hiv ) type 1 . apparently , the virus hijacks trex1 s specificity for retrovirus - derived dna to avoid activation of cytoplasmic nucleic acid sensors , which usually trigger the antiviral effector system . degradation of hiv dna thus represents one mechanism how hiv escapes immune recognition and helps to explain why infection of t cells and macrophages do not elicit an effective antiviral interferon response ( geijtenbeek , 2010 ) . interestingly , also another ags susceptibility gene , samhd1 , which is highly expressed in macrophages and dendritic cells and upregulated upon viral infection was proposed to act as a negative regulator of the innate immune response ( lafuse et al . two recent publications identified samhd1 as the factor restricting hiv infection in myeloid and dendritic cells and showed that vpx proteins counteract this restriction by mediating proteasomal degradation of samhd1 ( hrecka et al . with respect to the etiology of ags , loss of samhd1 activity would no longer counteract rt of endogenous retroviral elements as the dntp pool is no longer restricted . as a consequence , the loss of rt restriction might promote the accumulation of cytoplasmic dna similar to what is observed in trex1 mutants . in summary , trex1 and samhd1 provide excellent examples for the intricate relationship between the control of retroviral infection and the control of cell intrinsic nucleic acids harboring the risk to trigger autoimmune inflammation induced by type i ifns . the rnase h2 is the dominant cytoplasmic rnase in human cells and composed of a heterotrimeric complex in which all three components are needed to constitute the active enzyme . as depicted in figure 2 , analysis of the molecular mechanisms underlying ags revealed a role of all the susceptibility genes mentioned above in the control of cytoplasmic nucleic acid homeostasis which needs to be tightly controlled in order to prevent autoimmunity induced by type i ifns . model for the role of cytoplasmic nucleic acid homeostasis in the etiology of aicardi goutieres syndrome ( ags ) . lack of trex1 activity results in the congestion of ssdna thereby augmenting the pool of nucleic acids in the cytoplasm . thus mutations in any kind of the genes mentioned above which were found in ags patients would lead to the accumulation of nucleic acids in the cytoplasm . however , in contrast to rrms , nmo is characterized by demyelination in the spinal cord and optic nerve ( lucchinetti et al . autoantibodies directed against the aquaporin 4 water channel are found in these patients and occurrence of anti - aquaporin antibodies also is the major parameter in the diagnosis of nmo ( paul et al . interestingly , levels of il-17 were found to be elevated in the cerebrospinal fluid of people suffering from nmo and it has been proposed that il-17 mediated induction of il-8 , granulocyte stimulating factor ( g - csf ) and gro - alpha causes granulocyte recruitment ( axtell et al . as exemplified for sle and ags , type i ifns were frequently causally associated with the development of autoimmune pathologies . in contrast , type i ifns were also shown to exert anti - inflammatory effects and ifn- is used as a routine therapeutic for the treatment of rrms in patients ( jacobs et al . characteristic for the disease is the infiltration of the cns with inflammatory cells like monocytes , th1 and th17 cells . according to results from clinical trials , ifn- treatment reduces relapse rates by about 30% , decreases the formation of inflammatory lesions in the cns and extends remission periods ( schwid and panitch , 2007 ) . mechanism underlying this variety include development of antibodies against ifn- , polymorphisms in components of the ifn signaling pathway and ifn effector genes as well as variable ms pathomechanisms ( killestein and polman , 2011 ) . by further dissecting type i ifn signatures in ms patients , ifn- non - responders were found to exhibit increased monocyte - specific type i ifn secretion upon innate immune stimuli via tlr 4 , by increased endogenous production of type i interferon , and by an elevated activation status of myeloid dendritic cells ( comabella et al . these findings indicate that perturbations of the type i ifn signaling pathway in monocytes are related to a lack of response to ifn- and type i ifn - regulated genes may be used as response markers in ifn- treatment in ms . consistent with the concept that genetic variants define the individual response is that induction of interferon response genes among different patients varies significantly but is remarkably stable within a long time period . a frequently used system to gain insight into the molecular and cellular events underlying ms is the model of experimental autoimmune encephalomyelitis ( eae ) . in this well - established mouse model active immunization with myelin components together with a strong adjuvant induces t - cell mediated neuroinflammation and demyelination resembling multiple aspects of ms ( owens et al . to trace down the cell type mediating the protective immune regulation of type i ifns via the type i receptor ( ifnar ) , mice lacking this receptor only on distinct cell types were used for eae experiments and ifn- treatment ( prinz et al . it could be shown that negative regulation of autoimmunity in the eae model relies on the presence of ifnar on cells of the myeloid lineage while absence of ifnar on t cells , b cells , and cells of neuroectodermal origin does not enhance disease severity . these experiments suggest a scenario where locally produced ifn- within the cns acts on invading myeloid cells to attenuate autoimmune damage especially in the effector phase of eae . disease symptoms and generation of antigen specific th17 cells were also enhanced in trif deficient mice suggesting that type i ifn production counteracting autoimmunity in eae is triggered by a trif dependent mechanism ( guo et al . interestingly , type i ifn - related genes were found to be strongly induced in toxic mouse models of demyelination whereas the presence of ifnar could neither modulate demyelination or myelin repair ( schmidt et al . in a reciprocal experiment the same study showed that on the other hand activation of rlhs via ifn - inducing 5-triphosphate rna oligonucleotides ameliorated disease outcome providing compelling evidence that rlhs mediate signals counteracting encephalitogenic immune responses . while rlh stimulation did not affect t - cell differentiation , the maintenance and expansion of th1 and th17 cells was strongly suppressed upon rlh engagement ( figure 3 ) . in dendritic cells activation of rlhs rig - i or mda5 by 5triphosphate rna or polyi : c complexed to liposomes ( cpolyi : c ) respectively , recruits ips-1 to the rlh . as a proof of principle for the feasibility of such an approach it was shown in a melanoma model that bcl2 specific sirna carrying 5 triphosphate ends efficiently activated type i ifn via rig - i and in parallel silenced bcl2 expression ( poeck et al . as a consequence , the synergistic activation of the innate immune response combined with suppression of a pro survival signal caused massive apoptosis of tumor cells , prolonged survival of the animals , and reduced tumor size . such a strategy where for example type i ifn induction could be combined with rnai mediated inhibition of rort or t - bet to simultaneously repress th17 and th1 differentiation might potentially open new ways to modulate the immune response and counteract autoimmunity within the cns . a major task for the future will be to identify the particular interferon effector genes responsible for the anti - inflammatory and therapeutic properties which even might considerably differ between various cell types . with respect to the etiology of type i ifn associated autoimmune disease like ags it will be interesting to see which particular molecules are involved in the pathways sensing enhanced levels of endogenous nucleic acids in the cytoplasm .

it has been thirty years since the war on cancer was declared , yet in 2008 , the most recent year for which incidence and mortality rates are available , almost 12.7 million people were diagnosed with cancer and more than 7.5 million died of the disease . enormous progress has been made in the understanding of the molecular basis of carcinogenesis and the complete sequencing of the human genome represents a milestone in this quest . the situation though is far more complex than a simple catalogue of genes and despite this progress the discovery of anticancer drugs remains a highly challenging endeavor and cancer a hard - to - cure disease . traditionally , the development of cancer is thought to be largely due to the accumulation of genetic defects such as mutations , amplifications , deletions , and translocations affecting the cancer cell machinery and providing the cancer cell with the advantage to survive and metastasize . in addition , interactions between cancer cells and their microenvironment further support these processes . of equal importance is a second system that cells use to determine when and where a particular gene will be expressed during development . this system is overlaid on dna in the form of epigenetic marks that are heritable during cell division but do not alter the dna sequence . the pattern of these chemical tags is called the epigenome of the cell , whereas epigenetics is the study of these marks that lead to changes in gene expression in the absence of corresponding structural changes in the genome . it is now well recognized that tumorigenesis is a multistep process involving multiple genetic and epigenetic alterations , with the latter often termed epimutations that contribute to the progressive transformation of normal cells towards a malignant phenotype , so that cancer is nowadays consider to be both a genetic and an epigenetic disease [ 5 , 6 ] . epigenetic abnormalities are reversible and as a result novel therapies that work by reversing epigenetic effects are being increasingly explored . more recently , increasing evidence suggests that genetic and epigenetic mechanisms intertwine and take advantage of each other during malignant transformation . there are many chemical modifications that affect not only dna , but also rna and proteins , and create different epigenetic layers . the most well studied epigenetic modification in humans is dna methylation ; however , it becomes increasingly acknowledged that dna methylation does not work alone , but rather is linked to other modifications , such as histone modifications . this paper will discuss the most well studied epigenetic modifications and how these are linked to cancer , give a brief overview of the clinical use of epigenetics as biomarkers , and focus in more detail on epigenetic drugs and their use in solid and blood cancers . dna methylation consists of the addition of a methyl group to carbon 5 of the cytosine within the dinucleotide cpg . regions of dna in the human genome , ranging from 0.5 to 5 kb , that are cg rich are called cpg islands and are usually found in the promoters of genes . approximately half of all gene promoters have cpg islands that when methylated lead to transcriptional silencing . de nov dna methylation is brought about by dna methyltransferases ( dnmt ) 3a and 3b that convert cytosine residues of cpg dinucleotides into 5-methylcytosine , whereas dna methylation is maintained by dnmt1 . 5-methylcytosine can be further converted into 5-hydroxymethyl-2-deoxycytidine by the ten - eleven - translocation ( tet ) family enzymes . the function and significance of 5-hydroxymethylation are still unclear and under investigation . although methylation of dna in 5 promoters has been well studied and has been shown to suppress gene expression , recently dna methylation was described downstream of the promoters in intra- and inter - genic regions as well in cpg shores , that is regions with lower cpg density neighboring cpg islands . a nucleosome is the basic unit of dna packaging within the nucleus and consists of 147 base pairs of genomic dna wrapped twice around a highly conserved histone octamer , consisting of 2 copies each of the core histones h2a , h2b , h3 , and h4 . histones , however , are not only packaging elements , but also critical regulators of gene expression . histone tails may undergo many posttranslational chemical modifications , such as acetylation , methylation , phosphorylation , ubiquitylation , and sumoylation that constitute a code , named the histone code . these modifications can alter the chromatin structure , from an open to a closed , condensed form and vice versa . histone modifications act , except for chromatin packaging , on various other biological processes including transcriptional repression , gene activation , and dna repair . three classes of histone interacting proteins have been described thus far , based on their function : the writers that place histone modifications , the erasers that can remove these modifications , and finally the readers that recognize the histone modifications and can deliver nucleosome , histone , or dna - modifying enzymes . histone acetylation occurs at either arginine-(r ) or lysine-(k ) residues and is a dynamic and reversible process that is regulated by two enzyme families , histone acetyltransferases ( hat ) and histone deacetylases ( hdac ) . hats catalyse the transfer of an acetyl group to the -amino group of the lysine residue on the histone protein and use acetyl - coa as a cofactor . as a result chromatin adopts a more relaxed form ( euchromatin ) allowing the recruitment of transcription factors . hdacs reverse the acetylation of lysine residues and the local chromatin architecture becomes condensed ( heterochromatin ) . acetylation of lysine 16 of histone 4 ( h4k16 ) appears to be crucial in chromatin folding and in the switch from the euchromatin state to heterochromatin . histone acetylation can also promote transcription by providing binding sites to proteins that are involved in gene activation , such as the bromodomain - containing family of proteins . lysine residues can be monomethylated , dimethylated , or trimethylated whereas arginine residues can be mono- or dimethylated . methyl marks are written by s - adenosylmethionine ( sam)- dependent methyltransferases and erased by either the jumonji family of demethylases or the lysine - specific histone demethylases 1 ( lsd1 ) and 2 ( lsd2 ) . histone methylation at lysine and arginine residues does not alter the chromatic structure , but rather acts as binding sites for other proteins that may condense chromatin or have other effects . the different levels of lysine methylation are recognized by different methyl - lysine - binding domains and may be associated with either transcription activation or repression . arginine methylation of histone proteins has recently been shown to antagonize other histone marks , further increasing the histone code complexity . in cancer , a global process of genomic hypomethylation occurs mostly at dna - repetitive regions which results in activation of genes with growth and tumour promoting functions and loss of genome stability and imprinting . in contrast , there are site - specific increases in cpg methylation in areas of the genome with a high density of cpg , termed cpg islands causing transcriptional silencing of tumour suppressor genes ( tsg ) , such as brca1 , hmlh1 , vhl , bik , and mgmt [ 22 , 23 ] . cancer contains not only dna methylation aberrations , but also major disruption of the histone modification landscape . histone modifiers have been shown to be targets of aberrations and/or mutations in cancer such as mutated deacetylases , and amplified histone methyltransferases and demethylases . deregulation of the epigenetic machinery can also occur due to activation or inactivation of the epigenetic regulatory proteins . in other words , the enzymes that maintain and modify the epigenome are themselves frequent targets for mutation and/or epimutation in neoplasia ; for example , dna methyltransferases themselves have been found to be genetically altered in malignancies , such as dnmt3a and dnmt3b in pancreatic and breast cancer cells . somatic dnmt3a mutations have been described in approximately 20% of acute myeloid leukemia ( aml ) patients , especially in those with an intermediate risk cytogenetic profile and although they did not affect the 5-methylcytosine content they were associated with poor clinical outcome [ 30 , 31 ] . how the lack of effect of dnmt3a mutations on 5-methylcytosine content is linked to an otherwise poor clinical outcome it has been suggested that the r882 dnmt3a mutations alter functions of dnmt3a such as its ability to bind other proteins involved in transcriptional regulation and localization to chromatin regions containing methylated dna . loss - of - function tet2 mutations were also identified in myeloid neoplasms in 2030% [ 32 , 33 ] and have been associated with both good and bad prognoses . genome sequencing has also revealed the presence of metabolic mutations in patients with myelodysplastic syndromes ( mds ) and aml related to the isocitrate dehydrogenase ( idh ) 1 and idh2 genes . these mutations have been reported in approximately 30% of patients with normal karyotype aml [ 37 , 38 ] and have been linked to the disruption of various processes such as bone marrow microenvironment changes and impaired differentiation suggesting a proleukemogenic effect . in an aml cohort , idh1 and idh2 mutations were mutually exclusive with tet2 mutations while they shared the similar epigenetic defects with the tet2 mutants . epigenetic profiling revealed that aml patients with idh1/2 mutations displayed global hypermethylation and a specific hypermethylation signature . mll is another epigenetic modifier that is commonly mutated in acute leukemias and mainly due to translocations . in normal karyotype aml cases the incidence of mll partial tandem duplications ( mll - ptd ) is up to 8% whereas in cases of trisomy 11 the incidence reaches 25% . favorable amls such as those with t(8 ; 21 ) are mll - ptd negative . as mll is a h3 k4 methyltransferase , translocations that replace the methyltransferase domain affect its function and have been linked with leukaemic transformation . mutations affecting the polycomb repressive complex ( prc ) components , such as ezh2 , can also affect histone modifications and have recently been reported . ezh2 is the enzymatic component of the prc2 complex and is a h3 k27 methyltransferase . overexpression of ezh2 has been reported in various epithelial neoplasms and several types of leukemia [ 4345 ] and has been shown to be due to , at least in part , the loss of transcriptional repression of specific micrornas . activating mutations of ezh2 have been reported in b - cell lymphomas whereas missense , nonsense , and frameshift mutations have been reported in various myeloid malignancies [ 47 , 48 ] . in aml , at present , there are two major areas of interest in the clinical use of epigenetics , namely , biomarkers and therapeutics . dna has a number of properties , which make it an attractive molecule for biomarker utility . first , it is stable in biofluids such as blood , urine , and saliva . second , in the majority of cases methylation in cpg is acquired during malignant transformation and is therefore specific to neoplasia . third , the techniques used for detection of methylated dna are readily amenable to automation . several studies have explored the methylation status of gene promoters and its association with clinical parameters in primary patient samples from patients with haematological malignancies and solid tumours . various methodologies have been used such as methylation - specific pcr ( msp ) , methylation - specific restriction enzyme digestion , hpaii tiny fragment enrichment by ligation - mediated pcr ( help ) , bisulphite sequencing , and pyrosequencing . either single genes or panels of genes in microarrays were studied . in mds and aml methylation of several genes has been reported such as meg3 , snrpn , plk2 , cyclin - dependent kinase inhibitors , e - cadherin , and various others reviewed in . in multiple myeloma , methylation of the vhl promoter has been shown to correlate with bone disease and methylation of the bcl-2 interacting killer ( bik ) promoter has been shown to predict relapsed / refractory disease , while methylated fhit has been shown to be an independent adverse prognostic factor . in a study by shen et al . quantitative pyrosequencing in a large cohort showed that patients with higher levels of methylation for these genes had shorter median overall and progressive - free survival ( pfs ) independent of age , sex , and the international prognostic scoring system ( ipss ) . a substantial body of experimental evidence exists mechanistically associating acquired chemotherapy resistance with changes in the cancer cell epigenome and a number of genes have been identified , in which increased cpg island methylation and transcriptional downregulation are associated with resistance to specific agents such hmlh1 and plk2 in ovarian cancer . of note , methylation - dependent silencing of the methyl transferase mgmt in glioblastoma multiforme confers sensitivity to the alkylating agent temozolomide but as with many such candidate biomarkers , clinical application to inform patient management is not yet routine . the list of genes reported to be methylated in haematological neoplasms is extensive , and although several have been linked to clinical parameters and have been associated with survival or response to treatment , none of these markers has been used so far in the clinic to guide diagnosis or treatment , as opposed to gene mutations such as npm1 and flt3 that are now widely used to risk classify aml patients . one of the major goals of investigators in oncology is that of individualized cancer therapy . the challenge now is to translate these findings into clinically usable tests to inform optimal deployment of anticancer drugs . it remains unlikely that a single gene methylation test will be sufficiently informative to guide individual patient management and it is more likely that panels of genes will be required . both epigenetic proteins and protein markers are good targets for the development of new anticancer treatments . the proof - of - concept for epigenetic therapies is the fda and emea approval of demethylating agents and histone acetylase ( hdac ) inhibitors for the treatment of mds , aml and certain types of lymphomas , respectively . however , we should not forget that these agents are nonselective and their side effects are not clearly known . the two most well studied and in clinical use dna methyltransferase inhibitors ( dnmti ) are the azanucleosides azacytidine ( 5-azacytidine ) and decitabine ( 5-aza-2-deoxycytidine ) . both are approved for use in the myelodysplastic syndromes and low - blast count aml and have improved the survival of patients with these diseases . unfortunately , clinical trials with dnmti in solid tumours did not have the same results . a phase 1 study of decitabine with interleukin-2 in melanoma and renal cell carcinoma showed that decitabine caused grade 4 neutropenia in most patients . however , in a phase ii trial low - dose decitabine was found to restore sensitivity to carboplatin in patients with heavily pretreated ovarian cancer resulting in a high response rate ( rr ) and prolonged pfs . in both studies , there was evidence that decitabine induced dose - dependent demethylation in marker genes such as mlh1 , rassf1a , hoxa10 , and hoxa11 . it is possible that such an approach could efficiently be coupled with the use of epigenetic biomarkers predictive of chemosensitivity . a major likely reason for the disappointing activity of demethylating agents in solid tumours is limited incorporation into cells , which are proliferating relatively slowly . these limitations may be less relevant for newer dnmtis which are independent of replication for incorporation into dna . a second explanation for these results is that agents such as azacytidine , which cause global hypomethylation , likely reactivate expression of multiple silenced genes including oncogenes and tumour suppressors in different cell types and in different cancers . for example , the oncogene nt5e is overexpressed in aggressive metastatic melanomas , yet transcriptionally silenced by methylation in breast cancer with more favorable prognosis . a third and key possible explanation why dnmti have advanced less rapidly in the clinic in solid tumours than in haematological malignancies is that of toxicity . both decitabine and azacytidine are active in haematological malignancy at lower ( less toxic ) doses than are required for demethylation in epithelial malignancies . it is clearly of interest , therefore , that transient exposure of cells to low ( relatively non - toxic ) doses of these agents could induce a response with sustained reduction in cpg island methylation and reactivation of expression of previously silenced genes . these observations imply that low - dose decitabine and azacytidine may have wider uses in management of neoplastic disease than previously believed . in a recently reported phase ii trial matei et al . showed that pretreatment with low - dose azacytidine restored sensitivity to carboplatin in patients with drug resistant epithelial ovarian cancer and resulted in a high response rate and significantly improved clinical outcomes . this study clearly attests to the utility of low - dose azacytidine in solid tumours and sets the scene for further studies . newer azanucleosides are zebularine , s-110 , and sgi-1027 that have shown antiproliferative activity in cell lines [ 63 , 64 ] , but have not entered the clinical trial setting yet . the hdacs catalyse removal of acetyl groups from lysine residues in the histones and functionally are transcriptional repressors . hdacs are divided into five classes : class i comprises hdac1 , hdac2 , hdac3 , and hdac8 ; class iia comprises hdac4 , hdac5 , hdac7 , and hdac9 ; class iib contains hdac6 and hdac10 ; class iii comprises the sirtuins sirt1-sirt7 while class iv contains only hdac11 . sodium butyrate was the first hdaci described to induce acetylation , and later on trichostatin ( tsa ) , a fungal antibiotic , currently used in in vitro experiments , and valproic acid , a widely used antiepileptic , were identified . valproic acid , in particular , has been used in combination with dnmti and/or chemotherapy in patients with haematological malignancies [ 67 , 68 ] . currently hdaci that have been developed focus on class i and class ii hdacs and can be further distinguished into chemically distinct subgroups based on their structure : aliphatic acids ( phenylbutyrate , valproic acid ) , benzamides ( entinostat ) , cyclic peptides ( romidepsin ) , and hydroxamates ( tsa , vorinostat / saha ) . several hdaci are currently being tested in phase ii - iii trials , while two of them , vorinostat and romidepsin are the first fda and emea approved agents for the treatment of progressive or recurrent cutaneous t cell lymphoma ( ctcl ) as second lines of treatment in 2006 and 2009 , respectively , but convincing clinical evidence of activity of these agents in other cancer types is still lacking . in non - small - cell lung cancer a number of hdaci such as entinostat , vorinostat , pivanex , and ci-994 are in early phases of clinical development and first results have been reported [ 70 , 71 ] . however , it appears that hdaci may need rational combinations to counterbalance the inherent potential of these compounds to reactivate tumor - progression genes . givinostat has been shown to selectively target cells harboring the jak2 v617f mutation and has been tested in combination with hydroxyurea in patients with polycythemia vera in a phase ii study ( nct00928707 ) . panobinostat ( lbh589 ) has shown activity as monotherapy in patients with hodgkin 's lymphoma , who relapsed or were refractory to autologous transplantation but limited activity in mds . however , in solid tumors the results of panobinostat monotherapy or in combination with other agents were rather disappointing [ 76 , 77 ] . second generation hdaci , such as acy-1215 , are more selective and have recently entered the clinical trial setting . it would be really interesting to see the efficacy and safety profile of such compounds . it becomes increasingly recognized that hdaci deacetylase other nonhistone proteins that are transcription factors , signal transducers , or even the products of oncogenes or tsg that are involved in oncogenesis . this could partly explain the unacceptable toxicity as well as the lack of efficacy of some compounds . the recognition that a subset of tsgs are silenced by a combination of cpg hypermethylation and histone hypoacetylation has prompted testing of combinations of the two classes of agents and trials of these are in progress . there is initial evidence to suggest that such combinations may greatly increase clinical efficacy without unacceptable toxicity . for example , in multiply pretreated metastatic non - small - cell lung cancer patients , the combination of azacytidine and the histone deacetylase inhibitor entinostat produced objective clinical responses and , importantly , four of 19 treated patients had therapeutic responses to further agents given immediately after epigenetic therapy . evidence that demethylation is key to the responses was shown by analysis from peripheral blood samples of a set of four marker genes . the combination of decitabine and pegylated interferon alfa-2b was tested in patients with unresectable or metastatic solid tumours ( nct00701298 ) . in ongoing trials , the combination of azacytidine and entinostat is undergoing testing in resected stage i non - small - cell lung cancer ( nct01207726 ) and oral azacytidine in combination with carboplatin or abraxane ( nanoparticle paclitaxel ) is being evaluated in patients with refractory solid tumours ( nct01478685 ) . in elderly previously untreated aml patients and high - risk mds patients the combination of azacytidine and lenalidomide , an immunomodulator drug , is currently under investigation ( nct01442714 ) . both drugs as monotherapies have already shown efficacy in this group of patients so their combination seems very promising . sequential treatment of azacytidine and lenalidomide in elderly patients with aml also showed encouraging clinical and biologic activity . in a recent phase i study decitabine was combined with bortezomib for the treatment of elderly poor risk aml patients and the combination showed good preliminary activity since response rates were very encouraging . the use of epidrugs on the intent to restore sensitivity to cytotoxic or hormonal drugs is a major goal in the setting of solid tumors [ 8587 ] . restoring hormonal sensitivity in breast cancer is of uppermost clinical importance and has been intensively studied over the last decades . in total 25% of breast cancers have the estrogen receptor - alpha ( er alpha ) repressed mainly due to hypermethylation of the er promoter and do not respond to endocrine therapy , and almost all hormone - sensitive tumors turn to be refractory at some point . it appears now that epigenetic therapy seems to offer a promising tool to restore / reverse hormonal sensitivity . recent studies found that decitabine and histone hdaci such as trichostatin a , entinostat , and scriptaid can restore expression of er mrna and functional protein and aromatase , along with the enzymatic activity of aromatase , indicating a potential to restore long term responsiveness of a subset of er - negative tumors to endocrine therapy [ 8789 ] . given the complexity and heterogeneity of the cancer cell epigenome , it is highly likely that some form of epigenomic profiling of individual cancers will be required to inform optimal use of the available agents , which induce modification of the cancer cell epigenome . for example , it would clearly be important to determine the epigenome of chemotherapy resistant cancer cells , to identify potentially deleterious silenced genes , before deploying epigenetic therapeutic strategies in an attempt to pharmacologically reverse resistance . malignant melanoma is an interesting example of such an approach . in this tumor type , loss of 5-hydroxymethylcytosine ( 5-hmc ) has diagnostic and prognostic implications , which relates to downregulation of idh2 and tet family enzymes . reintroducing active tet2 or idh2 was found to suppress melanoma growth and increase tumor - free survival in animal models . identifying the epigenetically modified genes , which are principally involved in tumor resistance , can be achieved by comparative analysis of diagnostic ( pretreatment ) biopsy with a second biopsy at disease relapse . such rebiopsying is rapidly becoming the standard of care in oncology , for example , in breast cancer . the ability of the physician to exploit therapeutic opportunities created by epigenetic changes in the cancer cell epigenome may also offer new approaches to cancer management . for example , ass1 , which encodes arginine succinate synthetase , the rate - limiting enzyme in arginine biosynthesis , is silenced by methylation in some cancer types including renal cell carcinoma , hepatocellular carcinoma , malignant melanoma , glioblastoma multiforme ( gbm ) , and platinum - resistant epithelial ovarian cancer . asl encoding arginine succinate lyase ( a second key enzyme in arginine biosynthesis ) is also silenced by cpg island methylation in gbm . these observations imply a further form of epigenetic therapy in which biochemical abnormalities resulting from epigenetic changes can be targeted for clinical benefit . as we previously discussed , several epigenetic modifiers such as ezh2 , idh1/2 , and dnmt3a are genetically altered in cancer . what seems to be needed though is a better selection of patients who will benefit from such treatments as well as identification of new druggable targets and compounds such as histone kinases or inhibitors of histone methyltransferases and sirtuins . the biggest clinical impact of epigenetic modifying agents in neoplastic disorders thus far has been in haematological malignancies and the efficacy of dnmtis and hdaci in blood cancers clearly attests to the principle that therapeutic modification of the cancer cell epigenome can produce clinical benefit . although the efficacy of epigenetic therapy in solid tumours remains as yet unproven , there is every reason to believe that more rational use of existing agents , perhaps informed by individual patient epigenetic profiling , will improve the therapeutic index of this approach . furthermore , an increasing number of viable new therapeutic targets are emerging from increased understanding of the epigenetic regulatory circuitry and its derangement in neoplasia .

cancer is nowadays considered to be both a genetic and an epigenetic disease . the most well studied epigenetic modification in humans is dna methylation ; however it becomes increasingly acknowledged that dna methylation does not work alone , but rather is linked to other modifications , such as histone modifications . epigenetic abnormalities are reversible and as a result novel therapies that work by reversing epigenetic effects are being increasingly explored . the biggest clinical impact of epigenetic modifying agents in neoplastic disorders thus far has been in haematological malignancies , and the efficacy of dnmt inhibitors and hdac inhibitors in blood cancers clearly attests to the principle that therapeutic modification of the cancer cell epigenome can produce clinical benefit . this paper will discuss the most well studied epigenetic modifications and how these are linked to cancer , will give a brief overview of the clinical use of epigenetics as biomarkers , and will focus in more detail on epigenetic drugs and their use in solid and blood cancers .

1. Introduction 2. DNA Methylation 3. Histone Modifications 4. Cancer and Epigenetic Modifications 5. Clinical Use of Epigenetics 6. Future Promise: Therapeutics 7. Conclusions

it has been thirty years since the war on cancer was declared , yet in 2008 , the most recent year for which incidence and mortality rates are available , almost 12.7 million people were diagnosed with cancer and more than 7.5 million died of the disease . enormous progress has been made in the understanding of the molecular basis of carcinogenesis and the complete sequencing of the human genome represents a milestone in this quest . traditionally , the development of cancer is thought to be largely due to the accumulation of genetic defects such as mutations , amplifications , deletions , and translocations affecting the cancer cell machinery and providing the cancer cell with the advantage to survive and metastasize . in addition , interactions between cancer cells and their microenvironment further support these processes . this system is overlaid on dna in the form of epigenetic marks that are heritable during cell division but do not alter the dna sequence . the pattern of these chemical tags is called the epigenome of the cell , whereas epigenetics is the study of these marks that lead to changes in gene expression in the absence of corresponding structural changes in the genome . it is now well recognized that tumorigenesis is a multistep process involving multiple genetic and epigenetic alterations , with the latter often termed epimutations that contribute to the progressive transformation of normal cells towards a malignant phenotype , so that cancer is nowadays consider to be both a genetic and an epigenetic disease [ 5 , 6 ] . epigenetic abnormalities are reversible and as a result novel therapies that work by reversing epigenetic effects are being increasingly explored . more recently , increasing evidence suggests that genetic and epigenetic mechanisms intertwine and take advantage of each other during malignant transformation . there are many chemical modifications that affect not only dna , but also rna and proteins , and create different epigenetic layers . the most well studied epigenetic modification in humans is dna methylation ; however , it becomes increasingly acknowledged that dna methylation does not work alone , but rather is linked to other modifications , such as histone modifications . this paper will discuss the most well studied epigenetic modifications and how these are linked to cancer , give a brief overview of the clinical use of epigenetics as biomarkers , and focus in more detail on epigenetic drugs and their use in solid and blood cancers . dna methylation consists of the addition of a methyl group to carbon 5 of the cytosine within the dinucleotide cpg . de nov dna methylation is brought about by dna methyltransferases ( dnmt ) 3a and 3b that convert cytosine residues of cpg dinucleotides into 5-methylcytosine , whereas dna methylation is maintained by dnmt1 . although methylation of dna in 5 promoters has been well studied and has been shown to suppress gene expression , recently dna methylation was described downstream of the promoters in intra- and inter - genic regions as well in cpg shores , that is regions with lower cpg density neighboring cpg islands . a nucleosome is the basic unit of dna packaging within the nucleus and consists of 147 base pairs of genomic dna wrapped twice around a highly conserved histone octamer , consisting of 2 copies each of the core histones h2a , h2b , h3 , and h4 . histones , however , are not only packaging elements , but also critical regulators of gene expression . histone tails may undergo many posttranslational chemical modifications , such as acetylation , methylation , phosphorylation , ubiquitylation , and sumoylation that constitute a code , named the histone code . histone modifications act , except for chromatin packaging , on various other biological processes including transcriptional repression , gene activation , and dna repair . three classes of histone interacting proteins have been described thus far , based on their function : the writers that place histone modifications , the erasers that can remove these modifications , and finally the readers that recognize the histone modifications and can deliver nucleosome , histone , or dna - modifying enzymes . hats catalyse the transfer of an acetyl group to the -amino group of the lysine residue on the histone protein and use acetyl - coa as a cofactor . as a result chromatin adopts a more relaxed form ( euchromatin ) allowing the recruitment of transcription factors . hdacs reverse the acetylation of lysine residues and the local chromatin architecture becomes condensed ( heterochromatin ) . acetylation of lysine 16 of histone 4 ( h4k16 ) appears to be crucial in chromatin folding and in the switch from the euchromatin state to heterochromatin . histone acetylation can also promote transcription by providing binding sites to proteins that are involved in gene activation , such as the bromodomain - containing family of proteins . lysine residues can be monomethylated , dimethylated , or trimethylated whereas arginine residues can be mono- or dimethylated . histone methylation at lysine and arginine residues does not alter the chromatic structure , but rather acts as binding sites for other proteins that may condense chromatin or have other effects . in cancer , a global process of genomic hypomethylation occurs mostly at dna - repetitive regions which results in activation of genes with growth and tumour promoting functions and loss of genome stability and imprinting . in contrast , there are site - specific increases in cpg methylation in areas of the genome with a high density of cpg , termed cpg islands causing transcriptional silencing of tumour suppressor genes ( tsg ) , such as brca1 , hmlh1 , vhl , bik , and mgmt [ 22 , 23 ] . cancer contains not only dna methylation aberrations , but also major disruption of the histone modification landscape . histone modifiers have been shown to be targets of aberrations and/or mutations in cancer such as mutated deacetylases , and amplified histone methyltransferases and demethylases . deregulation of the epigenetic machinery can also occur due to activation or inactivation of the epigenetic regulatory proteins . in other words , the enzymes that maintain and modify the epigenome are themselves frequent targets for mutation and/or epimutation in neoplasia ; for example , dna methyltransferases themselves have been found to be genetically altered in malignancies , such as dnmt3a and dnmt3b in pancreatic and breast cancer cells . how the lack of effect of dnmt3a mutations on 5-methylcytosine content is linked to an otherwise poor clinical outcome it has been suggested that the r882 dnmt3a mutations alter functions of dnmt3a such as its ability to bind other proteins involved in transcriptional regulation and localization to chromatin regions containing methylated dna . genome sequencing has also revealed the presence of metabolic mutations in patients with myelodysplastic syndromes ( mds ) and aml related to the isocitrate dehydrogenase ( idh ) 1 and idh2 genes . these mutations have been reported in approximately 30% of patients with normal karyotype aml [ 37 , 38 ] and have been linked to the disruption of various processes such as bone marrow microenvironment changes and impaired differentiation suggesting a proleukemogenic effect . favorable amls such as those with t(8 ; 21 ) are mll - ptd negative . mutations affecting the polycomb repressive complex ( prc ) components , such as ezh2 , can also affect histone modifications and have recently been reported . ezh2 is the enzymatic component of the prc2 complex and is a h3 k27 methyltransferase . overexpression of ezh2 has been reported in various epithelial neoplasms and several types of leukemia [ 4345 ] and has been shown to be due to , at least in part , the loss of transcriptional repression of specific micrornas . activating mutations of ezh2 have been reported in b - cell lymphomas whereas missense , nonsense , and frameshift mutations have been reported in various myeloid malignancies [ 47 , 48 ] . in aml , at present , there are two major areas of interest in the clinical use of epigenetics , namely , biomarkers and therapeutics . first , it is stable in biofluids such as blood , urine , and saliva . several studies have explored the methylation status of gene promoters and its association with clinical parameters in primary patient samples from patients with haematological malignancies and solid tumours . various methodologies have been used such as methylation - specific pcr ( msp ) , methylation - specific restriction enzyme digestion , hpaii tiny fragment enrichment by ligation - mediated pcr ( help ) , bisulphite sequencing , and pyrosequencing . in mds and aml methylation of several genes has been reported such as meg3 , snrpn , plk2 , cyclin - dependent kinase inhibitors , e - cadherin , and various others reviewed in . in multiple myeloma , methylation of the vhl promoter has been shown to correlate with bone disease and methylation of the bcl-2 interacting killer ( bik ) promoter has been shown to predict relapsed / refractory disease , while methylated fhit has been shown to be an independent adverse prognostic factor . quantitative pyrosequencing in a large cohort showed that patients with higher levels of methylation for these genes had shorter median overall and progressive - free survival ( pfs ) independent of age , sex , and the international prognostic scoring system ( ipss ) . a substantial body of experimental evidence exists mechanistically associating acquired chemotherapy resistance with changes in the cancer cell epigenome and a number of genes have been identified , in which increased cpg island methylation and transcriptional downregulation are associated with resistance to specific agents such hmlh1 and plk2 in ovarian cancer . of note , methylation - dependent silencing of the methyl transferase mgmt in glioblastoma multiforme confers sensitivity to the alkylating agent temozolomide but as with many such candidate biomarkers , clinical application to inform patient management is not yet routine . the list of genes reported to be methylated in haematological neoplasms is extensive , and although several have been linked to clinical parameters and have been associated with survival or response to treatment , none of these markers has been used so far in the clinic to guide diagnosis or treatment , as opposed to gene mutations such as npm1 and flt3 that are now widely used to risk classify aml patients . one of the major goals of investigators in oncology is that of individualized cancer therapy . however , we should not forget that these agents are nonselective and their side effects are not clearly known . the two most well studied and in clinical use dna methyltransferase inhibitors ( dnmti ) are the azanucleosides azacytidine ( 5-azacytidine ) and decitabine ( 5-aza-2-deoxycytidine ) . both are approved for use in the myelodysplastic syndromes and low - blast count aml and have improved the survival of patients with these diseases . unfortunately , clinical trials with dnmti in solid tumours did not have the same results . in both studies , there was evidence that decitabine induced dose - dependent demethylation in marker genes such as mlh1 , rassf1a , hoxa10 , and hoxa11 . it is possible that such an approach could efficiently be coupled with the use of epigenetic biomarkers predictive of chemosensitivity . a major likely reason for the disappointing activity of demethylating agents in solid tumours is limited incorporation into cells , which are proliferating relatively slowly . a second explanation for these results is that agents such as azacytidine , which cause global hypomethylation , likely reactivate expression of multiple silenced genes including oncogenes and tumour suppressors in different cell types and in different cancers . a third and key possible explanation why dnmti have advanced less rapidly in the clinic in solid tumours than in haematological malignancies is that of toxicity . both decitabine and azacytidine are active in haematological malignancy at lower ( less toxic ) doses than are required for demethylation in epithelial malignancies . this study clearly attests to the utility of low - dose azacytidine in solid tumours and sets the scene for further studies . newer azanucleosides are zebularine , s-110 , and sgi-1027 that have shown antiproliferative activity in cell lines [ 63 , 64 ] , but have not entered the clinical trial setting yet . hdacs are divided into five classes : class i comprises hdac1 , hdac2 , hdac3 , and hdac8 ; class iia comprises hdac4 , hdac5 , hdac7 , and hdac9 ; class iib contains hdac6 and hdac10 ; class iii comprises the sirtuins sirt1-sirt7 while class iv contains only hdac11 . sodium butyrate was the first hdaci described to induce acetylation , and later on trichostatin ( tsa ) , a fungal antibiotic , currently used in in vitro experiments , and valproic acid , a widely used antiepileptic , were identified . valproic acid , in particular , has been used in combination with dnmti and/or chemotherapy in patients with haematological malignancies [ 67 , 68 ] . currently hdaci that have been developed focus on class i and class ii hdacs and can be further distinguished into chemically distinct subgroups based on their structure : aliphatic acids ( phenylbutyrate , valproic acid ) , benzamides ( entinostat ) , cyclic peptides ( romidepsin ) , and hydroxamates ( tsa , vorinostat / saha ) . several hdaci are currently being tested in phase ii - iii trials , while two of them , vorinostat and romidepsin are the first fda and emea approved agents for the treatment of progressive or recurrent cutaneous t cell lymphoma ( ctcl ) as second lines of treatment in 2006 and 2009 , respectively , but convincing clinical evidence of activity of these agents in other cancer types is still lacking . in non - small - cell lung cancer a number of hdaci such as entinostat , vorinostat , pivanex , and ci-994 are in early phases of clinical development and first results have been reported [ 70 , 71 ] . givinostat has been shown to selectively target cells harboring the jak2 v617f mutation and has been tested in combination with hydroxyurea in patients with polycythemia vera in a phase ii study ( nct00928707 ) . however , in solid tumors the results of panobinostat monotherapy or in combination with other agents were rather disappointing [ 76 , 77 ] . second generation hdaci , such as acy-1215 , are more selective and have recently entered the clinical trial setting . it would be really interesting to see the efficacy and safety profile of such compounds . it becomes increasingly recognized that hdaci deacetylase other nonhistone proteins that are transcription factors , signal transducers , or even the products of oncogenes or tsg that are involved in oncogenesis . this could partly explain the unacceptable toxicity as well as the lack of efficacy of some compounds . the recognition that a subset of tsgs are silenced by a combination of cpg hypermethylation and histone hypoacetylation has prompted testing of combinations of the two classes of agents and trials of these are in progress . for example , in multiply pretreated metastatic non - small - cell lung cancer patients , the combination of azacytidine and the histone deacetylase inhibitor entinostat produced objective clinical responses and , importantly , four of 19 treated patients had therapeutic responses to further agents given immediately after epigenetic therapy . evidence that demethylation is key to the responses was shown by analysis from peripheral blood samples of a set of four marker genes . in a recent phase i study decitabine was combined with bortezomib for the treatment of elderly poor risk aml patients and the combination showed good preliminary activity since response rates were very encouraging . the use of epidrugs on the intent to restore sensitivity to cytotoxic or hormonal drugs is a major goal in the setting of solid tumors [ 8587 ] . restoring hormonal sensitivity in breast cancer is of uppermost clinical importance and has been intensively studied over the last decades . in total 25% of breast cancers have the estrogen receptor - alpha ( er alpha ) repressed mainly due to hypermethylation of the er promoter and do not respond to endocrine therapy , and almost all hormone - sensitive tumors turn to be refractory at some point . recent studies found that decitabine and histone hdaci such as trichostatin a , entinostat , and scriptaid can restore expression of er mrna and functional protein and aromatase , along with the enzymatic activity of aromatase , indicating a potential to restore long term responsiveness of a subset of er - negative tumors to endocrine therapy [ 8789 ] . given the complexity and heterogeneity of the cancer cell epigenome , it is highly likely that some form of epigenomic profiling of individual cancers will be required to inform optimal use of the available agents , which induce modification of the cancer cell epigenome . the ability of the physician to exploit therapeutic opportunities created by epigenetic changes in the cancer cell epigenome may also offer new approaches to cancer management . for example , ass1 , which encodes arginine succinate synthetase , the rate - limiting enzyme in arginine biosynthesis , is silenced by methylation in some cancer types including renal cell carcinoma , hepatocellular carcinoma , malignant melanoma , glioblastoma multiforme ( gbm ) , and platinum - resistant epithelial ovarian cancer . these observations imply a further form of epigenetic therapy in which biochemical abnormalities resulting from epigenetic changes can be targeted for clinical benefit . as we previously discussed , several epigenetic modifiers such as ezh2 , idh1/2 , and dnmt3a are genetically altered in cancer . what seems to be needed though is a better selection of patients who will benefit from such treatments as well as identification of new druggable targets and compounds such as histone kinases or inhibitors of histone methyltransferases and sirtuins . the biggest clinical impact of epigenetic modifying agents in neoplastic disorders thus far has been in haematological malignancies and the efficacy of dnmtis and hdaci in blood cancers clearly attests to the principle that therapeutic modification of the cancer cell epigenome can produce clinical benefit . although the efficacy of epigenetic therapy in solid tumours remains as yet unproven , there is every reason to believe that more rational use of existing agents , perhaps informed by individual patient epigenetic profiling , will improve the therapeutic index of this approach . furthermore , an increasing number of viable new therapeutic targets are emerging from increased understanding of the epigenetic regulatory circuitry and its derangement in neoplasia .

it has been thirty years since the war on cancer was declared , yet in 2008 , the most recent year for which incidence and mortality rates are available , almost 12.7 million people were diagnosed with cancer and more than 7.5 million died of the disease . enormous progress has been made in the understanding of the molecular basis of carcinogenesis and the complete sequencing of the human genome represents a milestone in this quest . the situation though is far more complex than a simple catalogue of genes and despite this progress the discovery of anticancer drugs remains a highly challenging endeavor and cancer a hard - to - cure disease . traditionally , the development of cancer is thought to be largely due to the accumulation of genetic defects such as mutations , amplifications , deletions , and translocations affecting the cancer cell machinery and providing the cancer cell with the advantage to survive and metastasize . this system is overlaid on dna in the form of epigenetic marks that are heritable during cell division but do not alter the dna sequence . the pattern of these chemical tags is called the epigenome of the cell , whereas epigenetics is the study of these marks that lead to changes in gene expression in the absence of corresponding structural changes in the genome . it is now well recognized that tumorigenesis is a multistep process involving multiple genetic and epigenetic alterations , with the latter often termed epimutations that contribute to the progressive transformation of normal cells towards a malignant phenotype , so that cancer is nowadays consider to be both a genetic and an epigenetic disease [ 5 , 6 ] . epigenetic abnormalities are reversible and as a result novel therapies that work by reversing epigenetic effects are being increasingly explored . more recently , increasing evidence suggests that genetic and epigenetic mechanisms intertwine and take advantage of each other during malignant transformation . the most well studied epigenetic modification in humans is dna methylation ; however , it becomes increasingly acknowledged that dna methylation does not work alone , but rather is linked to other modifications , such as histone modifications . this paper will discuss the most well studied epigenetic modifications and how these are linked to cancer , give a brief overview of the clinical use of epigenetics as biomarkers , and focus in more detail on epigenetic drugs and their use in solid and blood cancers . dna methylation consists of the addition of a methyl group to carbon 5 of the cytosine within the dinucleotide cpg . regions of dna in the human genome , ranging from 0.5 to 5 kb , that are cg rich are called cpg islands and are usually found in the promoters of genes . although methylation of dna in 5 promoters has been well studied and has been shown to suppress gene expression , recently dna methylation was described downstream of the promoters in intra- and inter - genic regions as well in cpg shores , that is regions with lower cpg density neighboring cpg islands . a nucleosome is the basic unit of dna packaging within the nucleus and consists of 147 base pairs of genomic dna wrapped twice around a highly conserved histone octamer , consisting of 2 copies each of the core histones h2a , h2b , h3 , and h4 . three classes of histone interacting proteins have been described thus far , based on their function : the writers that place histone modifications , the erasers that can remove these modifications , and finally the readers that recognize the histone modifications and can deliver nucleosome , histone , or dna - modifying enzymes . hats catalyse the transfer of an acetyl group to the -amino group of the lysine residue on the histone protein and use acetyl - coa as a cofactor . hdacs reverse the acetylation of lysine residues and the local chromatin architecture becomes condensed ( heterochromatin ) . acetylation of lysine 16 of histone 4 ( h4k16 ) appears to be crucial in chromatin folding and in the switch from the euchromatin state to heterochromatin . histone acetylation can also promote transcription by providing binding sites to proteins that are involved in gene activation , such as the bromodomain - containing family of proteins . the different levels of lysine methylation are recognized by different methyl - lysine - binding domains and may be associated with either transcription activation or repression . in cancer , a global process of genomic hypomethylation occurs mostly at dna - repetitive regions which results in activation of genes with growth and tumour promoting functions and loss of genome stability and imprinting . in contrast , there are site - specific increases in cpg methylation in areas of the genome with a high density of cpg , termed cpg islands causing transcriptional silencing of tumour suppressor genes ( tsg ) , such as brca1 , hmlh1 , vhl , bik , and mgmt [ 22 , 23 ] . histone modifiers have been shown to be targets of aberrations and/or mutations in cancer such as mutated deacetylases , and amplified histone methyltransferases and demethylases . deregulation of the epigenetic machinery can also occur due to activation or inactivation of the epigenetic regulatory proteins . in other words , the enzymes that maintain and modify the epigenome are themselves frequent targets for mutation and/or epimutation in neoplasia ; for example , dna methyltransferases themselves have been found to be genetically altered in malignancies , such as dnmt3a and dnmt3b in pancreatic and breast cancer cells . somatic dnmt3a mutations have been described in approximately 20% of acute myeloid leukemia ( aml ) patients , especially in those with an intermediate risk cytogenetic profile and although they did not affect the 5-methylcytosine content they were associated with poor clinical outcome [ 30 , 31 ] . how the lack of effect of dnmt3a mutations on 5-methylcytosine content is linked to an otherwise poor clinical outcome it has been suggested that the r882 dnmt3a mutations alter functions of dnmt3a such as its ability to bind other proteins involved in transcriptional regulation and localization to chromatin regions containing methylated dna . loss - of - function tet2 mutations were also identified in myeloid neoplasms in 2030% [ 32 , 33 ] and have been associated with both good and bad prognoses . genome sequencing has also revealed the presence of metabolic mutations in patients with myelodysplastic syndromes ( mds ) and aml related to the isocitrate dehydrogenase ( idh ) 1 and idh2 genes . these mutations have been reported in approximately 30% of patients with normal karyotype aml [ 37 , 38 ] and have been linked to the disruption of various processes such as bone marrow microenvironment changes and impaired differentiation suggesting a proleukemogenic effect . in an aml cohort , idh1 and idh2 mutations were mutually exclusive with tet2 mutations while they shared the similar epigenetic defects with the tet2 mutants . in normal karyotype aml cases the incidence of mll partial tandem duplications ( mll - ptd ) is up to 8% whereas in cases of trisomy 11 the incidence reaches 25% . overexpression of ezh2 has been reported in various epithelial neoplasms and several types of leukemia [ 4345 ] and has been shown to be due to , at least in part , the loss of transcriptional repression of specific micrornas . activating mutations of ezh2 have been reported in b - cell lymphomas whereas missense , nonsense , and frameshift mutations have been reported in various myeloid malignancies [ 47 , 48 ] . in aml , at present , there are two major areas of interest in the clinical use of epigenetics , namely , biomarkers and therapeutics . second , in the majority of cases methylation in cpg is acquired during malignant transformation and is therefore specific to neoplasia . several studies have explored the methylation status of gene promoters and its association with clinical parameters in primary patient samples from patients with haematological malignancies and solid tumours . various methodologies have been used such as methylation - specific pcr ( msp ) , methylation - specific restriction enzyme digestion , hpaii tiny fragment enrichment by ligation - mediated pcr ( help ) , bisulphite sequencing , and pyrosequencing . in mds and aml methylation of several genes has been reported such as meg3 , snrpn , plk2 , cyclin - dependent kinase inhibitors , e - cadherin , and various others reviewed in . in multiple myeloma , methylation of the vhl promoter has been shown to correlate with bone disease and methylation of the bcl-2 interacting killer ( bik ) promoter has been shown to predict relapsed / refractory disease , while methylated fhit has been shown to be an independent adverse prognostic factor . quantitative pyrosequencing in a large cohort showed that patients with higher levels of methylation for these genes had shorter median overall and progressive - free survival ( pfs ) independent of age , sex , and the international prognostic scoring system ( ipss ) . a substantial body of experimental evidence exists mechanistically associating acquired chemotherapy resistance with changes in the cancer cell epigenome and a number of genes have been identified , in which increased cpg island methylation and transcriptional downregulation are associated with resistance to specific agents such hmlh1 and plk2 in ovarian cancer . of note , methylation - dependent silencing of the methyl transferase mgmt in glioblastoma multiforme confers sensitivity to the alkylating agent temozolomide but as with many such candidate biomarkers , clinical application to inform patient management is not yet routine . the list of genes reported to be methylated in haematological neoplasms is extensive , and although several have been linked to clinical parameters and have been associated with survival or response to treatment , none of these markers has been used so far in the clinic to guide diagnosis or treatment , as opposed to gene mutations such as npm1 and flt3 that are now widely used to risk classify aml patients . one of the major goals of investigators in oncology is that of individualized cancer therapy . the proof - of - concept for epigenetic therapies is the fda and emea approval of demethylating agents and histone acetylase ( hdac ) inhibitors for the treatment of mds , aml and certain types of lymphomas , respectively . the two most well studied and in clinical use dna methyltransferase inhibitors ( dnmti ) are the azanucleosides azacytidine ( 5-azacytidine ) and decitabine ( 5-aza-2-deoxycytidine ) . both are approved for use in the myelodysplastic syndromes and low - blast count aml and have improved the survival of patients with these diseases . a phase 1 study of decitabine with interleukin-2 in melanoma and renal cell carcinoma showed that decitabine caused grade 4 neutropenia in most patients . however , in a phase ii trial low - dose decitabine was found to restore sensitivity to carboplatin in patients with heavily pretreated ovarian cancer resulting in a high response rate ( rr ) and prolonged pfs . in both studies , there was evidence that decitabine induced dose - dependent demethylation in marker genes such as mlh1 , rassf1a , hoxa10 , and hoxa11 . a second explanation for these results is that agents such as azacytidine , which cause global hypomethylation , likely reactivate expression of multiple silenced genes including oncogenes and tumour suppressors in different cell types and in different cancers . for example , the oncogene nt5e is overexpressed in aggressive metastatic melanomas , yet transcriptionally silenced by methylation in breast cancer with more favorable prognosis . a third and key possible explanation why dnmti have advanced less rapidly in the clinic in solid tumours than in haematological malignancies is that of toxicity . it is clearly of interest , therefore , that transient exposure of cells to low ( relatively non - toxic ) doses of these agents could induce a response with sustained reduction in cpg island methylation and reactivation of expression of previously silenced genes . showed that pretreatment with low - dose azacytidine restored sensitivity to carboplatin in patients with drug resistant epithelial ovarian cancer and resulted in a high response rate and significantly improved clinical outcomes . this study clearly attests to the utility of low - dose azacytidine in solid tumours and sets the scene for further studies . hdacs are divided into five classes : class i comprises hdac1 , hdac2 , hdac3 , and hdac8 ; class iia comprises hdac4 , hdac5 , hdac7 , and hdac9 ; class iib contains hdac6 and hdac10 ; class iii comprises the sirtuins sirt1-sirt7 while class iv contains only hdac11 . sodium butyrate was the first hdaci described to induce acetylation , and later on trichostatin ( tsa ) , a fungal antibiotic , currently used in in vitro experiments , and valproic acid , a widely used antiepileptic , were identified . valproic acid , in particular , has been used in combination with dnmti and/or chemotherapy in patients with haematological malignancies [ 67 , 68 ] . currently hdaci that have been developed focus on class i and class ii hdacs and can be further distinguished into chemically distinct subgroups based on their structure : aliphatic acids ( phenylbutyrate , valproic acid ) , benzamides ( entinostat ) , cyclic peptides ( romidepsin ) , and hydroxamates ( tsa , vorinostat / saha ) . several hdaci are currently being tested in phase ii - iii trials , while two of them , vorinostat and romidepsin are the first fda and emea approved agents for the treatment of progressive or recurrent cutaneous t cell lymphoma ( ctcl ) as second lines of treatment in 2006 and 2009 , respectively , but convincing clinical evidence of activity of these agents in other cancer types is still lacking . in non - small - cell lung cancer a number of hdaci such as entinostat , vorinostat , pivanex , and ci-994 are in early phases of clinical development and first results have been reported [ 70 , 71 ] . however , it appears that hdaci may need rational combinations to counterbalance the inherent potential of these compounds to reactivate tumor - progression genes . givinostat has been shown to selectively target cells harboring the jak2 v617f mutation and has been tested in combination with hydroxyurea in patients with polycythemia vera in a phase ii study ( nct00928707 ) . however , in solid tumors the results of panobinostat monotherapy or in combination with other agents were rather disappointing [ 76 , 77 ] . the recognition that a subset of tsgs are silenced by a combination of cpg hypermethylation and histone hypoacetylation has prompted testing of combinations of the two classes of agents and trials of these are in progress . for example , in multiply pretreated metastatic non - small - cell lung cancer patients , the combination of azacytidine and the histone deacetylase inhibitor entinostat produced objective clinical responses and , importantly , four of 19 treated patients had therapeutic responses to further agents given immediately after epigenetic therapy . in ongoing trials , the combination of azacytidine and entinostat is undergoing testing in resected stage i non - small - cell lung cancer ( nct01207726 ) and oral azacytidine in combination with carboplatin or abraxane ( nanoparticle paclitaxel ) is being evaluated in patients with refractory solid tumours ( nct01478685 ) . in elderly previously untreated aml patients and high - risk mds patients the combination of azacytidine and lenalidomide , an immunomodulator drug , is currently under investigation ( nct01442714 ) . in a recent phase i study decitabine was combined with bortezomib for the treatment of elderly poor risk aml patients and the combination showed good preliminary activity since response rates were very encouraging . the use of epidrugs on the intent to restore sensitivity to cytotoxic or hormonal drugs is a major goal in the setting of solid tumors [ 8587 ] . in total 25% of breast cancers have the estrogen receptor - alpha ( er alpha ) repressed mainly due to hypermethylation of the er promoter and do not respond to endocrine therapy , and almost all hormone - sensitive tumors turn to be refractory at some point . recent studies found that decitabine and histone hdaci such as trichostatin a , entinostat , and scriptaid can restore expression of er mrna and functional protein and aromatase , along with the enzymatic activity of aromatase , indicating a potential to restore long term responsiveness of a subset of er - negative tumors to endocrine therapy [ 8789 ] . given the complexity and heterogeneity of the cancer cell epigenome , it is highly likely that some form of epigenomic profiling of individual cancers will be required to inform optimal use of the available agents , which induce modification of the cancer cell epigenome . for example , it would clearly be important to determine the epigenome of chemotherapy resistant cancer cells , to identify potentially deleterious silenced genes , before deploying epigenetic therapeutic strategies in an attempt to pharmacologically reverse resistance . identifying the epigenetically modified genes , which are principally involved in tumor resistance , can be achieved by comparative analysis of diagnostic ( pretreatment ) biopsy with a second biopsy at disease relapse . the ability of the physician to exploit therapeutic opportunities created by epigenetic changes in the cancer cell epigenome may also offer new approaches to cancer management . for example , ass1 , which encodes arginine succinate synthetase , the rate - limiting enzyme in arginine biosynthesis , is silenced by methylation in some cancer types including renal cell carcinoma , hepatocellular carcinoma , malignant melanoma , glioblastoma multiforme ( gbm ) , and platinum - resistant epithelial ovarian cancer . what seems to be needed though is a better selection of patients who will benefit from such treatments as well as identification of new druggable targets and compounds such as histone kinases or inhibitors of histone methyltransferases and sirtuins . the biggest clinical impact of epigenetic modifying agents in neoplastic disorders thus far has been in haematological malignancies and the efficacy of dnmtis and hdaci in blood cancers clearly attests to the principle that therapeutic modification of the cancer cell epigenome can produce clinical benefit . although the efficacy of epigenetic therapy in solid tumours remains as yet unproven , there is every reason to believe that more rational use of existing agents , perhaps informed by individual patient epigenetic profiling , will improve the therapeutic index of this approach .

ra is also characterized by autoantibody production ( antibody of anti - citrullinated protein and rheumatoid factor ) , hyperplasia and synovial inflammation , and systemic features ( e.g. , pulmonary , cardiovascular , psychological , and skeletal disorders ) . even though some patients have mild self - limited disease , many of them have experienced joint destruction , severe physical disability , and multiple co - morbidities [ 57 ] . unfortunately , patients with ra may develop various systemic symptoms such as fatigue , fever , anemia , weight loss , anorexia , muscle weakness , and osteoporosis . death rates are more than twice as high in ra patients as in the general population , and this gap seems to be widening . the specific cause of ra is unknown , but it has been suspected that a complex interplay among environmental factors , genotype , and chance contributes to the occurrence of ra . unrestrained cytokine production has been correlated with various diseases , including ra . in ra , chronic inflammation in ra is induced by the imbalance between cytokines of pro- and anti - inflammation and the induction of autoimmunity . potential triggers of ra include autoantibodies such as anti - citrullinated peptides antibodies and rheumatoid triggers , and pro - inflammatory cytokines like tumor necrosis factor- ( tnf- ) and interleukin 6 ( il-6 ) . cytokines modulate a large range of inflammatory processes correlated with the pathogenesis of ra , and appear abundantly in the serum and the arthritic synovial fluid of patients with ra . after synovitis or synovial inflammation develops from autoimmunity in ra , additional triggers , including the production of pro - inflammatory cytokines ( e.g. , tnf- and il-6 ) , further develop bone erosion and osteoclastogenesis , which relates to the regression of bone - resorbing osteoclasts . tnf- , secreted by activated macrophages and t - cells , exerts pro - inflammatory effects through binding to one of its receptors , p55 ( tnf - ri ) or p75 ( tnf - rii ) , and plays a vital role in production of other cytokines and the induction of chronic inflammation . il-6 , as another pro - inflammatory cytokine , drives the activation of local synovial leukocyte and antibody production . evidence from previous studies suggest that tnf- and il-6 play a significant role in the occurrence and development of ra due to their pro - inflammatory effects . however , studies have also indicated that il-6 and tnf- can have anti - inflammatory functions under some circumstances . consequently , the purpose of our meta - analysis was to investigate the association between ra and serum levels of il-6 and tnf- via a meta - analysis . electronic databases ebsco , pubmed , ovid , springerlink , web of science , wiley , wanfang data , and china national knowledge infrastructure ( cnki)were searched to collect all relevant published studies ( last updated search in october , 2014 ) . the following key words and subject terms were used in the searches : ra , il-6 , and tnf-. we also selected ( tumor necrosis factor - alpha or tnf - alpha or tumor necrosis factor or tnf ) , ( interleukin-6 or il-6 or differentiation factor-2 , b - cell or b cell stimulatory factor-2 ) , ( arthritis , rheumatoid or rheumatoid arthritis or ra ) in the search strategy . studies were qualified for inclusion if they met the following criteria : ( 1 ) the study type was case - control ; ( 2 ) the research topic mentioned the association between ra and serum levels of il-6 and tnf- ; ( 3 ) all subjects were patients clinically diagnosed with ra ; ( 4 ) the detection method was enzyme - linked immunosorbent assay ( elisa ) ; and ( 5 ) complete data was supplied in included studies . when the extracted studies were published by the same authors , only the complete or latest study was included . studies were excluded if : ( 1 ) data were incomplete ; ( 2 ) the differences in baseline characteristics between the case group and the control group were too great ; ( 3 ) the article was published repeatedly ; or ( 4 ) the diagnostic criteria for study subjects were ambiguous . relevant data eligible for the final analyses in retrieved papers were extracted by 2 investigators separately on the basis of the inclusion criteria , including surname of first author , country of origin , publication year , ethnicity , language , disease , detection method , age , sex , study design , and sample size . review reports from the 2 investigators were compared to identify inconsistency , and disputes were settled through further discussion and reexaminations among all investigators . the methodological quality of the included cases was evaluated by more than 2 investigators under the methodological index for non - randomized studies ( minors ) criteria . minors is an effective scoring tool , with a 12-item assessment , and each item can be scored from 0 to 2 , with an ideal score of 16 for non - comparative studies and a score of 24 for comparative studies . the details of 12 criteria were displayed : the aim was clearly stated ( minors 01 ) , consecutive inclusion of patients or not ( minors 02 ) , collection of prospective data or not ( minors 03 ) , the endpoints were appropriate for the aim of the study ( minors 04 ) , unbiased assessment of endpoint or not ( minors 05 ) , appropriate follow - up period or not ( minors 06 ) , less than 5% loss of follow - up or not ( minors 07 ) , prospectively calculated study size or not ( minors 08 ) , adequate control group or not ( minors 09 ) , contemporary groups or not ( minors 10 ) , equivalent baseline of groups or not ( minors 11 ) , and adequate statistical analyses or not ( minors 12 ) . stata 12.0 software ( stata corp , college station , tx , usa ) was used for statistical analyses . the differences of serum levels of inflammatory cytokines ( il-6 and tnf- ) between the case and control groups were estimated by the standardized mean difference ( smds ) with 95% confidence interval ( ci ) . cochran s q statistic ( p<0.05 was considered significant ) and the i test ( 0% , no heterogeneity ; 100% , maximal heterogeneity ) were also used to reflect the heterogeneity among studies . there was great heterogeneity among studies at p<0.05 or i > 50% ; therefore , a random - effects model was used ; otherwise , a fixed - effects model was used . univariate and multiple meta - regression analyses were conducted to evaluate the potential source of heterogeneity , and monte carlo simulation ( mcs ) was used to correct and verify the result . sensitivity analysis was achieved by deleting each included study one at a time to assess the effect of each study on the overall outcome . a funnel plot was constructed to evaluate publication bias that might influence the reliability of the results . electronic databases ebsco , pubmed , ovid , springerlink , web of science , wiley , wanfang data , and china national knowledge infrastructure ( cnki)were searched to collect all relevant published studies ( last updated search in october , 2014 ) . the following key words and subject terms were used in the searches : ra , il-6 , and tnf-. we also selected ( tumor necrosis factor - alpha or tnf - alpha or tumor necrosis factor or tnf ) , ( interleukin-6 or il-6 or differentiation factor-2 , b - cell or b cell stimulatory factor-2 ) , ( arthritis , rheumatoid or rheumatoid arthritis or ra ) in the search strategy . studies were qualified for inclusion if they met the following criteria : ( 1 ) the study type was case - control ; ( 2 ) the research topic mentioned the association between ra and serum levels of il-6 and tnf- ; ( 3 ) all subjects were patients clinically diagnosed with ra ; ( 4 ) the detection method was enzyme - linked immunosorbent assay ( elisa ) ; and ( 5 ) complete data was supplied in included studies . when the extracted studies were published by the same authors , only the complete or latest study was included . studies were excluded if : ( 1 ) data were incomplete ; ( 2 ) the differences in baseline characteristics between the case group and the control group were too great ; ( 3 ) the article was published repeatedly ; or ( 4 ) the diagnostic criteria for study subjects were ambiguous . relevant data eligible for the final analyses in retrieved papers were extracted by 2 investigators separately on the basis of the inclusion criteria , including surname of first author , country of origin , publication year , ethnicity , language , disease , detection method , age , sex , study design , and sample size . review reports from the 2 investigators were compared to identify inconsistency , and disputes were settled through further discussion and reexaminations among all investigators . the methodological quality of the included cases was evaluated by more than 2 investigators under the methodological index for non - randomized studies ( minors ) criteria . minors is an effective scoring tool , with a 12-item assessment , and each item can be scored from 0 to 2 , with an ideal score of 16 for non - comparative studies and a score of 24 for comparative studies . the details of 12 criteria were displayed : the aim was clearly stated ( minors 01 ) , consecutive inclusion of patients or not ( minors 02 ) , collection of prospective data or not ( minors 03 ) , the endpoints were appropriate for the aim of the study ( minors 04 ) , unbiased assessment of endpoint or not ( minors 05 ) , appropriate follow - up period or not ( minors 06 ) , less than 5% loss of follow - up or not ( minors 07 ) , prospectively calculated study size or not ( minors 08 ) , adequate control group or not ( minors 09 ) , contemporary groups or not ( minors 10 ) , equivalent baseline of groups or not ( minors 11 ) , and adequate statistical analyses or not ( minors 12 ) . stata 12.0 software ( stata corp , college station , tx , usa ) was used for statistical analyses . the differences of serum levels of inflammatory cytokines ( il-6 and tnf- ) between the case and control groups were estimated by the standardized mean difference ( smds ) with 95% confidence interval ( ci ) . cochran s q statistic ( p<0.05 was considered significant ) and the i test ( 0% , no heterogeneity ; 100% , maximal heterogeneity ) were also used to reflect the heterogeneity among studies . there was great heterogeneity among studies at p<0.05 or i > 50% ; therefore , a random - effects model was used ; otherwise , a fixed - effects model was used . univariate and multiple meta - regression analyses were conducted to evaluate the potential source of heterogeneity , and monte carlo simulation ( mcs ) was used to correct and verify the result . sensitivity analysis was achieved by deleting each included study one at a time to assess the effect of each study on the overall outcome . a funnel plot was constructed to evaluate publication bias that might influence the reliability of the results . a total of 849 studies were chosen from the 8 databases via screening both title and key words . followed by removing reviews , letters , meta - analyses ( n=5 ) , duplicates ( n=21 ) , non - human studies ( n=14 ) , and studies not relevant to the research topics ( n=760 ) , the remaining studies ( n=49 ) were checked and an additional 31 studies were excluded because they were not case - control or cohort studies ( n=7 ) , not related to tnf- ( n=12 ) , or not relevant to il-6 ( n=12 ) . after the remaining 18 studies were further reviewed , 14 studies [ 23,3244 ] were enrolled in the analysis . during the final selection process , there were 890 patients with ra in the case group and 441 controls in this meta - analysis . heterogeneity was found among included studies according to the heterogeneity test ( i=97.6% , p<0.001 ) ; thus , a random - effects model was used . the results of this meta - analysis indicated that the serum il-6 level of ra patients was evidently higher than in the controls , suggesting a statistically significant difference between the case and control groups ( smd=2.40 , 95% ci=1.57~3.24 , p<0.001 ) ( figure 1a ) . in ethnic subgroups , the serum il-6 level of patients with ra was also significantly higher compared with the controls in asians and caucasians ( asians : smd=3.64 , 95% ci=2.16~5.12 , p<0.001 ; caucasians : smd=0.75 , 95% ci=0.47~1.02 , p<0.001 ) ( figure 2a ) . the result of univariate meta - regression analysis suggests that ethnicity might be the source of heterogeneity ( p=0.036 ) , but the year of publication , language , and sample size were not associated with the heterogeneity ( year of publication : p=0.504 ; language : p=0.318 ; sample size : p=0.545 ) as presented in figure 3a3d . nevertheless , in multiple meta - regression analysis , ethnicity , year of publication , sample size , and language were not potential sources of heterogeneity ( table 1 ) . a random - effects model was used due to heterogeneity among included studies ( i=96.8% , p<0.001 ) according to the heterogeneity test . as presented in figure 1b , the results indicated that the serum tnf- level of patients with ra was clearly higher than in the controls , and there was a statistically significant difference between the case and control groups ( smd=1.93 , 95% ci=1.23~2.64 , p<0.001 ) . on the basis of analyses of the ethnic subgroups , the serum tnf- level of ra patients was also significantly higher compared with the controls in asians and caucasians ( asians : smd=2.74 , 95% ci=1.58~3.91 , p<0.001 ; caucasians : smd=0.81 , 95% ci=0.50~1.11 , p<0.001 ) ( figure 2b ) . the univariate meta - regression analysis suggested that the main source of heterogeneity might be ethnicity ( p=0.016 ) , but not the year of publication , language , or sample size ( year of publication : p=0.660 ; language : p=0.079 ; sample size : p=0.899 ) as shown in figure 3e3h . additionally , in multiple meta - regression analysis , ethnicity , year of publication , language , and sample size were not sources of heterogeneity ( table 2 ) . the sensitivity analysis suggests that all included studies had no evident influence on the pooled smds of serum il-6 and tnf- level in ra patients and the controls , and the shapes of funnel plots of the differences in serum level of il-6 and tnf- showed asymmetry ( figure 4 ) . thus , egger s test was used to further provide statistical evidence of funnel plot asymmetry , and also showed publication bias among included studies ( il-6 : p<0.001 ; tnf- : p=0.007 ) . a total of 849 studies were chosen from the 8 databases via screening both title and key words . followed by removing reviews , letters , meta - analyses ( n=5 ) , duplicates ( n=21 ) , non - human studies ( n=14 ) , and studies not relevant to the research topics ( n=760 ) , the remaining studies ( n=49 ) were checked and an additional 31 studies were excluded because they were not case - control or cohort studies ( n=7 ) , not related to tnf- ( n=12 ) , or not relevant to il-6 ( n=12 ) . after the remaining 18 studies were further reviewed , 14 studies [ 23,3244 ] were enrolled in the analysis . during the final selection process , there were 890 patients with ra in the case group and 441 controls in this meta - analysis . heterogeneity was found among included studies according to the heterogeneity test ( i=97.6% , p<0.001 ) ; thus , a random - effects model was used . the results of this meta - analysis indicated that the serum il-6 level of ra patients was evidently higher than in the controls , suggesting a statistically significant difference between the case and control groups ( smd=2.40 , 95% ci=1.57~3.24 , p<0.001 ) ( figure 1a ) . in ethnic subgroups , the serum il-6 level of patients with ra was also significantly higher compared with the controls in asians and caucasians ( asians : smd=3.64 , 95% ci=2.16~5.12 , p<0.001 ; caucasians : smd=0.75 , 95% ci=0.47~1.02 , p<0.001 ) ( figure 2a ) . the result of univariate meta - regression analysis suggests that ethnicity might be the source of heterogeneity ( p=0.036 ) , but the year of publication , language , and sample size were not associated with the heterogeneity ( year of publication : p=0.504 ; language : p=0.318 ; sample size : p=0.545 ) as presented in figure 3a3d . nevertheless , in multiple meta - regression analysis , ethnicity , year of publication , sample size , and language were not potential sources of heterogeneity ( table 1 ) . a random - effects model was used due to heterogeneity among included studies ( i=96.8% , p<0.001 ) according to the heterogeneity test . as presented in figure 1b , the results indicated that the serum tnf- level of patients with ra was clearly higher than in the controls , and there was a statistically significant difference between the case and control groups ( smd=1.93 , 95% ci=1.23~2.64 , p<0.001 ) . on the basis of analyses of the ethnic subgroups , the serum tnf- level of ra patients was also significantly higher compared with the controls in asians and caucasians ( asians : smd=2.74 , 95% ci=1.58~3.91 , p<0.001 ; caucasians : smd=0.81 , 95% ci=0.50~1.11 , p<0.001 ) ( figure 2b ) . the univariate meta - regression analysis suggested that the main source of heterogeneity might be ethnicity ( p=0.016 ) , but not the year of publication , language , or sample size ( year of publication : p=0.660 ; language : p=0.079 ; sample size : p=0.899 ) as shown in figure 3e3h . additionally , in multiple meta - regression analysis , ethnicity , year of publication , language , and sample size were not sources of heterogeneity ( table 2 ) . the sensitivity analysis suggests that all included studies had no evident influence on the pooled smds of serum il-6 and tnf- level in ra patients and the controls , and the shapes of funnel plots of the differences in serum level of il-6 and tnf- showed asymmetry ( figure 4 ) . thus , egger s test was used to further provide statistical evidence of funnel plot asymmetry , and also showed publication bias among included studies ( il-6 : p<0.001 ; tnf- : p=0.007 ) . one of the characteristics of ra was consistent chronic inflammation resulting in damage to synovial tissue , which led to irreversible tissue or joint damage . it has been reported that the high level of inflammation in vivo is correlated with serious joint damage and the poor prognosis of ra . il-6 and tnf- are 2 well - known inflammatory cytokines with critical roles in modulating tissue inflammation ; moreover , the plasma concentrations of il-6 and tnf- can reflect the severity of inflammation in vivo . we investigated the association between ra and the serum levels of il-6 and tnf- on the basis of previous studies . we found that compared with the controls , the serum levels of il-6 and tnf- were significantly higher in patients with ra , suggesting that il-6 and tnf- might play important roles in the pathogenesis of ra . in ra , tnf- is secreted by various cell types , predominantly by macrophages and dendritic cells in reaction to the interactions between pathogen - associated molecular patterns ( pamps ) or damage - associated molecular patterns ( damps ) and pattern - recognition receptors ( prrs ) or to the cytokine environment . il-6 is a multifunctional cytokine with biological activities that include the modulation of inflammation , immune response , and hematopoiesis . varieties of innate effector cells , such as mast cells , macrophages , and natural killer cells , are discovered in the synovial membrane , but neutrophils reside mostly in synovial fluid . granulocyte colony - stimulating factor , macrophage colony - stimulating factor , and granulocyte macrophage colony - stimulating factor ( gm - csf ) promote the maturation of the above innate effector cells , trafficking to the synovium , and their efflux from the bone marrow . importantly , macrophages act as central effectors of synovitis and are effective biologic agents that could reduce macrophage infiltration consistently in the synovium . macrophages act via release of cytokines , for instance , tnf- and il-1 , -6 , -12 , -15 , -18 , and -23 , with tnf- and il-6 being the most predominant mediators , eventually resulting in the breakdown of extracellular matrix of bone and cartilage . as the pathology of ra was unclear , most studies speculated that synovial hyperplasia and progressive joint destruction was involved in the possible mechanism of the immune system attacking the joints . the inflammatory reaction involved in the synovial hyperplasia and joint destruction could be enhanced by il-6 , which amplifies the inflammatory cell infiltration . moreover , the synovial fibroblastic cells secreted il-6 once it was stimulated by inflammatory cytokines such as il-1 and tnf- and , in turn , il-6 enhanced the proliferation of synovial fibroblastic cells in the presence of soluble il-6 receptor . the explanation could be that il-6 and tnf- affect the progression in synovial hyperplasia , resulting in development and progression of ra . therefore , it was reasonable to think that the serum levels of il-6 and tnf- in ra patients were evidently higher compared with the controls , which is consistent with some previous studies that revealed that il-6 and tnf- may contribute to the development of ra due to their pro - inflammatory effects . although ra was characterized by persistent synovitis and continuous joint destruction , anemia was the most common symptom during the early stage of the disease . research evidence shows that il-6 injection contribute to induce of anemia , and vice versa , suggesting that il-6 , but not tnf- , plays a crucial role in anemia in monkey collagen - induced arthritis , whose pathogenesis was similar with ra , by inhibiting the il-6-induced hepcidin production . a study by godfrin - valnet et al . identified a positive trend of il-6 and the serum c - terminal cross - linking telopeptide of type i collagen ( ctx ) levels in patients with osteoporosis , revealing that increased level of il-6 was correlated with accelerated bone resorption from increased osteoclastogenesis and reduced bone formation . moreover , elevated vascular endothelial growth factor ( vegf ) level was found in ra patients , suggesting that vegf is involved in ra pathogenesis . release of tnf- and il-6 from synovial tissue modifies the function of distant tissue , including vascular endothelial tissue , which could lead to insulin resistance , abnormal blood fats , and endothelial dysfunction . to avoid the influence of other factors on the overall results , we selected ethnic subgroups analyses to further validate our results . we found that the serum il-6 and tnf- levels of ra patients were also significantly higher than in the controls in asians and caucasians , indicating that ethnicity did not affect our final results . additionally , ethnicity , year of publication , language , and sample size also did not affect the results of this meta - analysis . for instance , because the inclusion process was performed manually , there might be some deviation that could impact the results ; data on gender and age were lost in several included studies , which might be correlated with the serum levels of il-6 and tnf- and thereby might influence the result of this meta - analysis . in addition , according to the baseline characteristics of included studies , the sample size was too small to make firm conclusions about the correlation between ra and serum levels of il-6 and tnf-. il-6 and tnf- , as 2 inflammatory cytokines , might play a vital role in the pathogenesis of ra through their pro - inflammatory effects .

backgroundwe aimed to investigate the association of rheumatoid arthritis ( ra ) with interleukin 6 ( il-6 ) and tumor necrosis factor- ( tnf- ) through a meta-analysis.material/methodsthe case - control studies that investigated the association between ra and serum levels of il-6 and tnf- were retrieved strictly according to the inclusion and exclusion criteria . the statistical analysis was performed using stata statistical software ( version 12.0 , stata corporation , college station , tx , usa).resultsfourteen studies were enrolled in our meta - analysis , with a total of 890 patients with ra and 441 healthy people as the controls . the results of this meta - analysis revealed that the serum il-6 and tnf- levels of ra patients were significantly higher than in the controls , and this difference was statistically significant ( il-6 : smd=2.40 , 95% ci=1.57~3.24 , p<0.001 ; tnf- : smd=1.93 , 95% ci=1.23~2.64 , p<0.001 ) . according to ethnic subgroup analysis , the serum il-6 and tnf- levels of ra patients were also significantly higher compared with the controls in asians and caucasians ( il-6 : asians : smd=3.64 , 95% ci=2.16~5.12 , p<0.001 ; caucasians : smd=0.75 , 95% ci=0.47~1.02 , p<0.001 ; tnf- : asians : smd=2.74 , 95%ci=1.58~3.91 , p<0.001 ; caucasians : smd=0.81 , 95% ci=0.50~1.11 , p<0.001).conclusionsil-6 and tnf- may play crucial roles in the activity and severity of ra .

Background Material and Methods Search strategy Inclusion and exclusion criteria Data extraction and quality evaluation Statistical analysis Results The baseline characteristics of included studies Differences in serum levels of IL-6 Differences in serum levels of TNF- Sensitiveness analysis and publication bias Discussion Conclusions

unfortunately , patients with ra may develop various systemic symptoms such as fatigue , fever , anemia , weight loss , anorexia , muscle weakness , and osteoporosis . death rates are more than twice as high in ra patients as in the general population , and this gap seems to be widening . the specific cause of ra is unknown , but it has been suspected that a complex interplay among environmental factors , genotype , and chance contributes to the occurrence of ra . potential triggers of ra include autoantibodies such as anti - citrullinated peptides antibodies and rheumatoid triggers , and pro - inflammatory cytokines like tumor necrosis factor- ( tnf- ) and interleukin 6 ( il-6 ) . cytokines modulate a large range of inflammatory processes correlated with the pathogenesis of ra , and appear abundantly in the serum and the arthritic synovial fluid of patients with ra . , tnf- and il-6 ) , further develop bone erosion and osteoclastogenesis , which relates to the regression of bone - resorbing osteoclasts . evidence from previous studies suggest that tnf- and il-6 play a significant role in the occurrence and development of ra due to their pro - inflammatory effects . however , studies have also indicated that il-6 and tnf- can have anti - inflammatory functions under some circumstances . consequently , the purpose of our meta - analysis was to investigate the association between ra and serum levels of il-6 and tnf- via a meta - analysis . the following key words and subject terms were used in the searches : ra , il-6 , and tnf-. we also selected ( tumor necrosis factor - alpha or tnf - alpha or tumor necrosis factor or tnf ) , ( interleukin-6 or il-6 or differentiation factor-2 , b - cell or b cell stimulatory factor-2 ) , ( arthritis , rheumatoid or rheumatoid arthritis or ra ) in the search strategy . studies were qualified for inclusion if they met the following criteria : ( 1 ) the study type was case - control ; ( 2 ) the research topic mentioned the association between ra and serum levels of il-6 and tnf- ; ( 3 ) all subjects were patients clinically diagnosed with ra ; ( 4 ) the detection method was enzyme - linked immunosorbent assay ( elisa ) ; and ( 5 ) complete data was supplied in included studies . relevant data eligible for the final analyses in retrieved papers were extracted by 2 investigators separately on the basis of the inclusion criteria , including surname of first author , country of origin , publication year , ethnicity , language , disease , detection method , age , sex , study design , and sample size . minors is an effective scoring tool , with a 12-item assessment , and each item can be scored from 0 to 2 , with an ideal score of 16 for non - comparative studies and a score of 24 for comparative studies . the details of 12 criteria were displayed : the aim was clearly stated ( minors 01 ) , consecutive inclusion of patients or not ( minors 02 ) , collection of prospective data or not ( minors 03 ) , the endpoints were appropriate for the aim of the study ( minors 04 ) , unbiased assessment of endpoint or not ( minors 05 ) , appropriate follow - up period or not ( minors 06 ) , less than 5% loss of follow - up or not ( minors 07 ) , prospectively calculated study size or not ( minors 08 ) , adequate control group or not ( minors 09 ) , contemporary groups or not ( minors 10 ) , equivalent baseline of groups or not ( minors 11 ) , and adequate statistical analyses or not ( minors 12 ) . stata 12.0 software ( stata corp , college station , tx , usa ) was used for statistical analyses . the differences of serum levels of inflammatory cytokines ( il-6 and tnf- ) between the case and control groups were estimated by the standardized mean difference ( smds ) with 95% confidence interval ( ci ) . cochran s q statistic ( p<0.05 was considered significant ) and the i test ( 0% , no heterogeneity ; 100% , maximal heterogeneity ) were also used to reflect the heterogeneity among studies . univariate and multiple meta - regression analyses were conducted to evaluate the potential source of heterogeneity , and monte carlo simulation ( mcs ) was used to correct and verify the result . the following key words and subject terms were used in the searches : ra , il-6 , and tnf-. we also selected ( tumor necrosis factor - alpha or tnf - alpha or tumor necrosis factor or tnf ) , ( interleukin-6 or il-6 or differentiation factor-2 , b - cell or b cell stimulatory factor-2 ) , ( arthritis , rheumatoid or rheumatoid arthritis or ra ) in the search strategy . studies were qualified for inclusion if they met the following criteria : ( 1 ) the study type was case - control ; ( 2 ) the research topic mentioned the association between ra and serum levels of il-6 and tnf- ; ( 3 ) all subjects were patients clinically diagnosed with ra ; ( 4 ) the detection method was enzyme - linked immunosorbent assay ( elisa ) ; and ( 5 ) complete data was supplied in included studies . minors is an effective scoring tool , with a 12-item assessment , and each item can be scored from 0 to 2 , with an ideal score of 16 for non - comparative studies and a score of 24 for comparative studies . the details of 12 criteria were displayed : the aim was clearly stated ( minors 01 ) , consecutive inclusion of patients or not ( minors 02 ) , collection of prospective data or not ( minors 03 ) , the endpoints were appropriate for the aim of the study ( minors 04 ) , unbiased assessment of endpoint or not ( minors 05 ) , appropriate follow - up period or not ( minors 06 ) , less than 5% loss of follow - up or not ( minors 07 ) , prospectively calculated study size or not ( minors 08 ) , adequate control group or not ( minors 09 ) , contemporary groups or not ( minors 10 ) , equivalent baseline of groups or not ( minors 11 ) , and adequate statistical analyses or not ( minors 12 ) . stata 12.0 software ( stata corp , college station , tx , usa ) was used for statistical analyses . the differences of serum levels of inflammatory cytokines ( il-6 and tnf- ) between the case and control groups were estimated by the standardized mean difference ( smds ) with 95% confidence interval ( ci ) . cochran s q statistic ( p<0.05 was considered significant ) and the i test ( 0% , no heterogeneity ; 100% , maximal heterogeneity ) were also used to reflect the heterogeneity among studies . univariate and multiple meta - regression analyses were conducted to evaluate the potential source of heterogeneity , and monte carlo simulation ( mcs ) was used to correct and verify the result . a total of 849 studies were chosen from the 8 databases via screening both title and key words . followed by removing reviews , letters , meta - analyses ( n=5 ) , duplicates ( n=21 ) , non - human studies ( n=14 ) , and studies not relevant to the research topics ( n=760 ) , the remaining studies ( n=49 ) were checked and an additional 31 studies were excluded because they were not case - control or cohort studies ( n=7 ) , not related to tnf- ( n=12 ) , or not relevant to il-6 ( n=12 ) . after the remaining 18 studies were further reviewed , 14 studies [ 23,3244 ] were enrolled in the analysis . during the final selection process , there were 890 patients with ra in the case group and 441 controls in this meta - analysis . heterogeneity was found among included studies according to the heterogeneity test ( i=97.6% , p<0.001 ) ; thus , a random - effects model was used . the results of this meta - analysis indicated that the serum il-6 level of ra patients was evidently higher than in the controls , suggesting a statistically significant difference between the case and control groups ( smd=2.40 , 95% ci=1.57~3.24 , p<0.001 ) ( figure 1a ) . in ethnic subgroups , the serum il-6 level of patients with ra was also significantly higher compared with the controls in asians and caucasians ( asians : smd=3.64 , 95% ci=2.16~5.12 , p<0.001 ; caucasians : smd=0.75 , 95% ci=0.47~1.02 , p<0.001 ) ( figure 2a ) . the result of univariate meta - regression analysis suggests that ethnicity might be the source of heterogeneity ( p=0.036 ) , but the year of publication , language , and sample size were not associated with the heterogeneity ( year of publication : p=0.504 ; language : p=0.318 ; sample size : p=0.545 ) as presented in figure 3a3d . nevertheless , in multiple meta - regression analysis , ethnicity , year of publication , sample size , and language were not potential sources of heterogeneity ( table 1 ) . a random - effects model was used due to heterogeneity among included studies ( i=96.8% , p<0.001 ) according to the heterogeneity test . as presented in figure 1b , the results indicated that the serum tnf- level of patients with ra was clearly higher than in the controls , and there was a statistically significant difference between the case and control groups ( smd=1.93 , 95% ci=1.23~2.64 , p<0.001 ) . on the basis of analyses of the ethnic subgroups , the serum tnf- level of ra patients was also significantly higher compared with the controls in asians and caucasians ( asians : smd=2.74 , 95% ci=1.58~3.91 , p<0.001 ; caucasians : smd=0.81 , 95% ci=0.50~1.11 , p<0.001 ) ( figure 2b ) . the univariate meta - regression analysis suggested that the main source of heterogeneity might be ethnicity ( p=0.016 ) , but not the year of publication , language , or sample size ( year of publication : p=0.660 ; language : p=0.079 ; sample size : p=0.899 ) as shown in figure 3e3h . additionally , in multiple meta - regression analysis , ethnicity , year of publication , language , and sample size were not sources of heterogeneity ( table 2 ) . the sensitivity analysis suggests that all included studies had no evident influence on the pooled smds of serum il-6 and tnf- level in ra patients and the controls , and the shapes of funnel plots of the differences in serum level of il-6 and tnf- showed asymmetry ( figure 4 ) . thus , egger s test was used to further provide statistical evidence of funnel plot asymmetry , and also showed publication bias among included studies ( il-6 : p<0.001 ; tnf- : p=0.007 ) . a total of 849 studies were chosen from the 8 databases via screening both title and key words . followed by removing reviews , letters , meta - analyses ( n=5 ) , duplicates ( n=21 ) , non - human studies ( n=14 ) , and studies not relevant to the research topics ( n=760 ) , the remaining studies ( n=49 ) were checked and an additional 31 studies were excluded because they were not case - control or cohort studies ( n=7 ) , not related to tnf- ( n=12 ) , or not relevant to il-6 ( n=12 ) . after the remaining 18 studies were further reviewed , 14 studies [ 23,3244 ] were enrolled in the analysis . during the final selection process , there were 890 patients with ra in the case group and 441 controls in this meta - analysis . heterogeneity was found among included studies according to the heterogeneity test ( i=97.6% , p<0.001 ) ; thus , a random - effects model was used . the results of this meta - analysis indicated that the serum il-6 level of ra patients was evidently higher than in the controls , suggesting a statistically significant difference between the case and control groups ( smd=2.40 , 95% ci=1.57~3.24 , p<0.001 ) ( figure 1a ) . in ethnic subgroups , the serum il-6 level of patients with ra was also significantly higher compared with the controls in asians and caucasians ( asians : smd=3.64 , 95% ci=2.16~5.12 , p<0.001 ; caucasians : smd=0.75 , 95% ci=0.47~1.02 , p<0.001 ) ( figure 2a ) . the result of univariate meta - regression analysis suggests that ethnicity might be the source of heterogeneity ( p=0.036 ) , but the year of publication , language , and sample size were not associated with the heterogeneity ( year of publication : p=0.504 ; language : p=0.318 ; sample size : p=0.545 ) as presented in figure 3a3d . nevertheless , in multiple meta - regression analysis , ethnicity , year of publication , sample size , and language were not potential sources of heterogeneity ( table 1 ) . a random - effects model was used due to heterogeneity among included studies ( i=96.8% , p<0.001 ) according to the heterogeneity test . as presented in figure 1b , the results indicated that the serum tnf- level of patients with ra was clearly higher than in the controls , and there was a statistically significant difference between the case and control groups ( smd=1.93 , 95% ci=1.23~2.64 , p<0.001 ) . on the basis of analyses of the ethnic subgroups , the serum tnf- level of ra patients was also significantly higher compared with the controls in asians and caucasians ( asians : smd=2.74 , 95% ci=1.58~3.91 , p<0.001 ; caucasians : smd=0.81 , 95% ci=0.50~1.11 , p<0.001 ) ( figure 2b ) . additionally , in multiple meta - regression analysis , ethnicity , year of publication , language , and sample size were not sources of heterogeneity ( table 2 ) . the sensitivity analysis suggests that all included studies had no evident influence on the pooled smds of serum il-6 and tnf- level in ra patients and the controls , and the shapes of funnel plots of the differences in serum level of il-6 and tnf- showed asymmetry ( figure 4 ) . thus , egger s test was used to further provide statistical evidence of funnel plot asymmetry , and also showed publication bias among included studies ( il-6 : p<0.001 ; tnf- : p=0.007 ) . it has been reported that the high level of inflammation in vivo is correlated with serious joint damage and the poor prognosis of ra . il-6 and tnf- are 2 well - known inflammatory cytokines with critical roles in modulating tissue inflammation ; moreover , the plasma concentrations of il-6 and tnf- can reflect the severity of inflammation in vivo . we investigated the association between ra and the serum levels of il-6 and tnf- on the basis of previous studies . we found that compared with the controls , the serum levels of il-6 and tnf- were significantly higher in patients with ra , suggesting that il-6 and tnf- might play important roles in the pathogenesis of ra . in ra , tnf- is secreted by various cell types , predominantly by macrophages and dendritic cells in reaction to the interactions between pathogen - associated molecular patterns ( pamps ) or damage - associated molecular patterns ( damps ) and pattern - recognition receptors ( prrs ) or to the cytokine environment . varieties of innate effector cells , such as mast cells , macrophages , and natural killer cells , are discovered in the synovial membrane , but neutrophils reside mostly in synovial fluid . granulocyte colony - stimulating factor , macrophage colony - stimulating factor , and granulocyte macrophage colony - stimulating factor ( gm - csf ) promote the maturation of the above innate effector cells , trafficking to the synovium , and their efflux from the bone marrow . macrophages act via release of cytokines , for instance , tnf- and il-1 , -6 , -12 , -15 , -18 , and -23 , with tnf- and il-6 being the most predominant mediators , eventually resulting in the breakdown of extracellular matrix of bone and cartilage . as the pathology of ra was unclear , most studies speculated that synovial hyperplasia and progressive joint destruction was involved in the possible mechanism of the immune system attacking the joints . moreover , the synovial fibroblastic cells secreted il-6 once it was stimulated by inflammatory cytokines such as il-1 and tnf- and , in turn , il-6 enhanced the proliferation of synovial fibroblastic cells in the presence of soluble il-6 receptor . the explanation could be that il-6 and tnf- affect the progression in synovial hyperplasia , resulting in development and progression of ra . therefore , it was reasonable to think that the serum levels of il-6 and tnf- in ra patients were evidently higher compared with the controls , which is consistent with some previous studies that revealed that il-6 and tnf- may contribute to the development of ra due to their pro - inflammatory effects . identified a positive trend of il-6 and the serum c - terminal cross - linking telopeptide of type i collagen ( ctx ) levels in patients with osteoporosis , revealing that increased level of il-6 was correlated with accelerated bone resorption from increased osteoclastogenesis and reduced bone formation . we found that the serum il-6 and tnf- levels of ra patients were also significantly higher than in the controls in asians and caucasians , indicating that ethnicity did not affect our final results . additionally , ethnicity , year of publication , language , and sample size also did not affect the results of this meta - analysis . for instance , because the inclusion process was performed manually , there might be some deviation that could impact the results ; data on gender and age were lost in several included studies , which might be correlated with the serum levels of il-6 and tnf- and thereby might influence the result of this meta - analysis . in addition , according to the baseline characteristics of included studies , the sample size was too small to make firm conclusions about the correlation between ra and serum levels of il-6 and tnf-. il-6 and tnf- , as 2 inflammatory cytokines , might play a vital role in the pathogenesis of ra through their pro - inflammatory effects .

death rates are more than twice as high in ra patients as in the general population , and this gap seems to be widening . the specific cause of ra is unknown , but it has been suspected that a complex interplay among environmental factors , genotype , and chance contributes to the occurrence of ra . in ra , chronic inflammation in ra is induced by the imbalance between cytokines of pro- and anti - inflammation and the induction of autoimmunity . potential triggers of ra include autoantibodies such as anti - citrullinated peptides antibodies and rheumatoid triggers , and pro - inflammatory cytokines like tumor necrosis factor- ( tnf- ) and interleukin 6 ( il-6 ) . cytokines modulate a large range of inflammatory processes correlated with the pathogenesis of ra , and appear abundantly in the serum and the arthritic synovial fluid of patients with ra . after synovitis or synovial inflammation develops from autoimmunity in ra , additional triggers , including the production of pro - inflammatory cytokines ( e.g. , tnf- and il-6 ) , further develop bone erosion and osteoclastogenesis , which relates to the regression of bone - resorbing osteoclasts . tnf- , secreted by activated macrophages and t - cells , exerts pro - inflammatory effects through binding to one of its receptors , p55 ( tnf - ri ) or p75 ( tnf - rii ) , and plays a vital role in production of other cytokines and the induction of chronic inflammation . evidence from previous studies suggest that tnf- and il-6 play a significant role in the occurrence and development of ra due to their pro - inflammatory effects . consequently , the purpose of our meta - analysis was to investigate the association between ra and serum levels of il-6 and tnf- via a meta - analysis . electronic databases ebsco , pubmed , ovid , springerlink , web of science , wiley , wanfang data , and china national knowledge infrastructure ( cnki)were searched to collect all relevant published studies ( last updated search in october , 2014 ) . we also selected ( tumor necrosis factor - alpha or tnf - alpha or tumor necrosis factor or tnf ) , ( interleukin-6 or il-6 or differentiation factor-2 , b - cell or b cell stimulatory factor-2 ) , ( arthritis , rheumatoid or rheumatoid arthritis or ra ) in the search strategy . studies were qualified for inclusion if they met the following criteria : ( 1 ) the study type was case - control ; ( 2 ) the research topic mentioned the association between ra and serum levels of il-6 and tnf- ; ( 3 ) all subjects were patients clinically diagnosed with ra ; ( 4 ) the detection method was enzyme - linked immunosorbent assay ( elisa ) ; and ( 5 ) complete data was supplied in included studies . studies were excluded if : ( 1 ) data were incomplete ; ( 2 ) the differences in baseline characteristics between the case group and the control group were too great ; ( 3 ) the article was published repeatedly ; or ( 4 ) the diagnostic criteria for study subjects were ambiguous . relevant data eligible for the final analyses in retrieved papers were extracted by 2 investigators separately on the basis of the inclusion criteria , including surname of first author , country of origin , publication year , ethnicity , language , disease , detection method , age , sex , study design , and sample size . minors is an effective scoring tool , with a 12-item assessment , and each item can be scored from 0 to 2 , with an ideal score of 16 for non - comparative studies and a score of 24 for comparative studies . the details of 12 criteria were displayed : the aim was clearly stated ( minors 01 ) , consecutive inclusion of patients or not ( minors 02 ) , collection of prospective data or not ( minors 03 ) , the endpoints were appropriate for the aim of the study ( minors 04 ) , unbiased assessment of endpoint or not ( minors 05 ) , appropriate follow - up period or not ( minors 06 ) , less than 5% loss of follow - up or not ( minors 07 ) , prospectively calculated study size or not ( minors 08 ) , adequate control group or not ( minors 09 ) , contemporary groups or not ( minors 10 ) , equivalent baseline of groups or not ( minors 11 ) , and adequate statistical analyses or not ( minors 12 ) . the differences of serum levels of inflammatory cytokines ( il-6 and tnf- ) between the case and control groups were estimated by the standardized mean difference ( smds ) with 95% confidence interval ( ci ) . univariate and multiple meta - regression analyses were conducted to evaluate the potential source of heterogeneity , and monte carlo simulation ( mcs ) was used to correct and verify the result . electronic databases ebsco , pubmed , ovid , springerlink , web of science , wiley , wanfang data , and china national knowledge infrastructure ( cnki)were searched to collect all relevant published studies ( last updated search in october , 2014 ) . we also selected ( tumor necrosis factor - alpha or tnf - alpha or tumor necrosis factor or tnf ) , ( interleukin-6 or il-6 or differentiation factor-2 , b - cell or b cell stimulatory factor-2 ) , ( arthritis , rheumatoid or rheumatoid arthritis or ra ) in the search strategy . studies were qualified for inclusion if they met the following criteria : ( 1 ) the study type was case - control ; ( 2 ) the research topic mentioned the association between ra and serum levels of il-6 and tnf- ; ( 3 ) all subjects were patients clinically diagnosed with ra ; ( 4 ) the detection method was enzyme - linked immunosorbent assay ( elisa ) ; and ( 5 ) complete data was supplied in included studies . studies were excluded if : ( 1 ) data were incomplete ; ( 2 ) the differences in baseline characteristics between the case group and the control group were too great ; ( 3 ) the article was published repeatedly ; or ( 4 ) the diagnostic criteria for study subjects were ambiguous . relevant data eligible for the final analyses in retrieved papers were extracted by 2 investigators separately on the basis of the inclusion criteria , including surname of first author , country of origin , publication year , ethnicity , language , disease , detection method , age , sex , study design , and sample size . the details of 12 criteria were displayed : the aim was clearly stated ( minors 01 ) , consecutive inclusion of patients or not ( minors 02 ) , collection of prospective data or not ( minors 03 ) , the endpoints were appropriate for the aim of the study ( minors 04 ) , unbiased assessment of endpoint or not ( minors 05 ) , appropriate follow - up period or not ( minors 06 ) , less than 5% loss of follow - up or not ( minors 07 ) , prospectively calculated study size or not ( minors 08 ) , adequate control group or not ( minors 09 ) , contemporary groups or not ( minors 10 ) , equivalent baseline of groups or not ( minors 11 ) , and adequate statistical analyses or not ( minors 12 ) . the differences of serum levels of inflammatory cytokines ( il-6 and tnf- ) between the case and control groups were estimated by the standardized mean difference ( smds ) with 95% confidence interval ( ci ) . univariate and multiple meta - regression analyses were conducted to evaluate the potential source of heterogeneity , and monte carlo simulation ( mcs ) was used to correct and verify the result . followed by removing reviews , letters , meta - analyses ( n=5 ) , duplicates ( n=21 ) , non - human studies ( n=14 ) , and studies not relevant to the research topics ( n=760 ) , the remaining studies ( n=49 ) were checked and an additional 31 studies were excluded because they were not case - control or cohort studies ( n=7 ) , not related to tnf- ( n=12 ) , or not relevant to il-6 ( n=12 ) . during the final selection process , there were 890 patients with ra in the case group and 441 controls in this meta - analysis . the results of this meta - analysis indicated that the serum il-6 level of ra patients was evidently higher than in the controls , suggesting a statistically significant difference between the case and control groups ( smd=2.40 , 95% ci=1.57~3.24 , p<0.001 ) ( figure 1a ) . in ethnic subgroups , the serum il-6 level of patients with ra was also significantly higher compared with the controls in asians and caucasians ( asians : smd=3.64 , 95% ci=2.16~5.12 , p<0.001 ; caucasians : smd=0.75 , 95% ci=0.47~1.02 , p<0.001 ) ( figure 2a ) . the result of univariate meta - regression analysis suggests that ethnicity might be the source of heterogeneity ( p=0.036 ) , but the year of publication , language , and sample size were not associated with the heterogeneity ( year of publication : p=0.504 ; language : p=0.318 ; sample size : p=0.545 ) as presented in figure 3a3d . nevertheless , in multiple meta - regression analysis , ethnicity , year of publication , sample size , and language were not potential sources of heterogeneity ( table 1 ) . as presented in figure 1b , the results indicated that the serum tnf- level of patients with ra was clearly higher than in the controls , and there was a statistically significant difference between the case and control groups ( smd=1.93 , 95% ci=1.23~2.64 , p<0.001 ) . on the basis of analyses of the ethnic subgroups , the serum tnf- level of ra patients was also significantly higher compared with the controls in asians and caucasians ( asians : smd=2.74 , 95% ci=1.58~3.91 , p<0.001 ; caucasians : smd=0.81 , 95% ci=0.50~1.11 , p<0.001 ) ( figure 2b ) . the univariate meta - regression analysis suggested that the main source of heterogeneity might be ethnicity ( p=0.016 ) , but not the year of publication , language , or sample size ( year of publication : p=0.660 ; language : p=0.079 ; sample size : p=0.899 ) as shown in figure 3e3h . additionally , in multiple meta - regression analysis , ethnicity , year of publication , language , and sample size were not sources of heterogeneity ( table 2 ) . the sensitivity analysis suggests that all included studies had no evident influence on the pooled smds of serum il-6 and tnf- level in ra patients and the controls , and the shapes of funnel plots of the differences in serum level of il-6 and tnf- showed asymmetry ( figure 4 ) . thus , egger s test was used to further provide statistical evidence of funnel plot asymmetry , and also showed publication bias among included studies ( il-6 : p<0.001 ; tnf- : p=0.007 ) . followed by removing reviews , letters , meta - analyses ( n=5 ) , duplicates ( n=21 ) , non - human studies ( n=14 ) , and studies not relevant to the research topics ( n=760 ) , the remaining studies ( n=49 ) were checked and an additional 31 studies were excluded because they were not case - control or cohort studies ( n=7 ) , not related to tnf- ( n=12 ) , or not relevant to il-6 ( n=12 ) . during the final selection process , there were 890 patients with ra in the case group and 441 controls in this meta - analysis . the results of this meta - analysis indicated that the serum il-6 level of ra patients was evidently higher than in the controls , suggesting a statistically significant difference between the case and control groups ( smd=2.40 , 95% ci=1.57~3.24 , p<0.001 ) ( figure 1a ) . in ethnic subgroups , the serum il-6 level of patients with ra was also significantly higher compared with the controls in asians and caucasians ( asians : smd=3.64 , 95% ci=2.16~5.12 , p<0.001 ; caucasians : smd=0.75 , 95% ci=0.47~1.02 , p<0.001 ) ( figure 2a ) . the result of univariate meta - regression analysis suggests that ethnicity might be the source of heterogeneity ( p=0.036 ) , but the year of publication , language , and sample size were not associated with the heterogeneity ( year of publication : p=0.504 ; language : p=0.318 ; sample size : p=0.545 ) as presented in figure 3a3d . nevertheless , in multiple meta - regression analysis , ethnicity , year of publication , sample size , and language were not potential sources of heterogeneity ( table 1 ) . as presented in figure 1b , the results indicated that the serum tnf- level of patients with ra was clearly higher than in the controls , and there was a statistically significant difference between the case and control groups ( smd=1.93 , 95% ci=1.23~2.64 , p<0.001 ) . on the basis of analyses of the ethnic subgroups , the serum tnf- level of ra patients was also significantly higher compared with the controls in asians and caucasians ( asians : smd=2.74 , 95% ci=1.58~3.91 , p<0.001 ; caucasians : smd=0.81 , 95% ci=0.50~1.11 , p<0.001 ) ( figure 2b ) . the univariate meta - regression analysis suggested that the main source of heterogeneity might be ethnicity ( p=0.016 ) , but not the year of publication , language , or sample size ( year of publication : p=0.660 ; language : p=0.079 ; sample size : p=0.899 ) as shown in figure 3e3h . additionally , in multiple meta - regression analysis , ethnicity , year of publication , language , and sample size were not sources of heterogeneity ( table 2 ) . the sensitivity analysis suggests that all included studies had no evident influence on the pooled smds of serum il-6 and tnf- level in ra patients and the controls , and the shapes of funnel plots of the differences in serum level of il-6 and tnf- showed asymmetry ( figure 4 ) . il-6 and tnf- are 2 well - known inflammatory cytokines with critical roles in modulating tissue inflammation ; moreover , the plasma concentrations of il-6 and tnf- can reflect the severity of inflammation in vivo . we investigated the association between ra and the serum levels of il-6 and tnf- on the basis of previous studies . we found that compared with the controls , the serum levels of il-6 and tnf- were significantly higher in patients with ra , suggesting that il-6 and tnf- might play important roles in the pathogenesis of ra . in ra , tnf- is secreted by various cell types , predominantly by macrophages and dendritic cells in reaction to the interactions between pathogen - associated molecular patterns ( pamps ) or damage - associated molecular patterns ( damps ) and pattern - recognition receptors ( prrs ) or to the cytokine environment . varieties of innate effector cells , such as mast cells , macrophages , and natural killer cells , are discovered in the synovial membrane , but neutrophils reside mostly in synovial fluid . granulocyte colony - stimulating factor , macrophage colony - stimulating factor , and granulocyte macrophage colony - stimulating factor ( gm - csf ) promote the maturation of the above innate effector cells , trafficking to the synovium , and their efflux from the bone marrow . macrophages act via release of cytokines , for instance , tnf- and il-1 , -6 , -12 , -15 , -18 , and -23 , with tnf- and il-6 being the most predominant mediators , eventually resulting in the breakdown of extracellular matrix of bone and cartilage . as the pathology of ra was unclear , most studies speculated that synovial hyperplasia and progressive joint destruction was involved in the possible mechanism of the immune system attacking the joints . moreover , the synovial fibroblastic cells secreted il-6 once it was stimulated by inflammatory cytokines such as il-1 and tnf- and , in turn , il-6 enhanced the proliferation of synovial fibroblastic cells in the presence of soluble il-6 receptor . therefore , it was reasonable to think that the serum levels of il-6 and tnf- in ra patients were evidently higher compared with the controls , which is consistent with some previous studies that revealed that il-6 and tnf- may contribute to the development of ra due to their pro - inflammatory effects . research evidence shows that il-6 injection contribute to induce of anemia , and vice versa , suggesting that il-6 , but not tnf- , plays a crucial role in anemia in monkey collagen - induced arthritis , whose pathogenesis was similar with ra , by inhibiting the il-6-induced hepcidin production . identified a positive trend of il-6 and the serum c - terminal cross - linking telopeptide of type i collagen ( ctx ) levels in patients with osteoporosis , revealing that increased level of il-6 was correlated with accelerated bone resorption from increased osteoclastogenesis and reduced bone formation . moreover , elevated vascular endothelial growth factor ( vegf ) level was found in ra patients , suggesting that vegf is involved in ra pathogenesis . release of tnf- and il-6 from synovial tissue modifies the function of distant tissue , including vascular endothelial tissue , which could lead to insulin resistance , abnormal blood fats , and endothelial dysfunction . we found that the serum il-6 and tnf- levels of ra patients were also significantly higher than in the controls in asians and caucasians , indicating that ethnicity did not affect our final results . for instance , because the inclusion process was performed manually , there might be some deviation that could impact the results ; data on gender and age were lost in several included studies , which might be correlated with the serum levels of il-6 and tnf- and thereby might influence the result of this meta - analysis . in addition , according to the baseline characteristics of included studies , the sample size was too small to make firm conclusions about the correlation between ra and serum levels of il-6 and tnf-.

these cells normally line body cavities , specifically pleura , peritoneum , pericardium , and testis . they can be localized or diffuse . initially it occurred in miners and millworkers who were directly exposed to asbestos in their work environment . subsequently , mesothelioma was diagnosed in plumbers , carpenters , defence personnel , and insulation installers . furthermore people living in industrial areas were inadvertently exposed to asbestos fibres released in the atmosphere . elsewhere , for example , in countries where asbestos is still unregulated , the prospects are even worse . there is widespread interest in this disease by doctors and the general public , because millions of people have been exposed to asbestos fibres . in addition to its substantial personal and health care costs , malignant mesothelioma is associated with considerable compensation costs . the predicted total economic burden of malignant mesothelioma related to compensation for asbestos exposure in the next 40 years is up to $ 200 billion for the united states and $ 80 billion for europe . malignant pleural mesothelioma ( mpm ) has caused and is still causing violent debate between protagonists and antagonists of the three options in relation to treatment . surgery , radiotherapy , and chemotherapy are endlessly discussed , often with limited scientific evidence to support strongly held viewpoints . diagnosis and palliation will be part of the surgeon 's role but everyone 's big hope and question is is there a chance of surgical cure ? until recently it was thought that local radiotherapy directed to surgical sites prevents spreading of tumour and radiotherapy can provide relief of chest - wall pain . however , an up - to - date systematic review paper by price concluded that there is currently no evidence to support the routine role of radiotherapy in patients with mesothelioma . surgery became part of management options in the 1970s and large series have been published in relevant journals [ 14 , 15 ] . the two surgical procedures commonly used are pleurectomy with decortication ( p / d ) and extrapleural pneumonectomy ( epp ) . p / d is a more limited procedure and requires less cardiorespiratory reserve ; it has a morbidity of 25% and a mortality of 2% . data from the uk thoracic surgical register of the society for cardiothoracic surgery in great britain and ireland showed that a limited number of patients underwent surgery for mesothelioma in the past decade . single centre retrospective studies reported favourable results with epp . in some hospitals , this procedure , within a multimodal treatment strategy , became the standard of care . the median survival after epp within mars is consistent with 10 , 12 , 13 , and 14 months in larger observational studies . a best evidence topic paper addressing the question of whether p / d is superior to palliative care in the treatment of patients with malignant pleural mesothelioma came to the following conclusion : p / d is a morbid operation that is associated with significant peri - operative mortality and complication rates . the antagonists of surgery argue that , when a patient commences trimodality ( chemotherapy , radiotherapy , and surgery ) treatment , it will take six to nine months to complete it . this is a time during which the patient is in and out of hospital and sometimes travelling long distances from home . the primary objective of the current review is to determine if surgery improves all - cause mortality and/or quality of life in patients with mpm . we will discuss the different surgical options ( epp versus p / d ) and their relevant advantages and disadvantages including mortality and morbidity . this is a combination of chemotherapy either before ( neoadjuvant ) or after ( adjuvant ) surgery , an operation that aims to remove the tumour and radiotherapy . the evidence around the use of different chemotherapeutic regimes will be discussed as well as the evidence on radiotherapy approaches . the overall success rates of the trimodality therapy will be discussed in relation to survival , freedom from recurrence , and quality of life ( symptom relief from breathlessness and pain ) . electronic search was performed of pubmed , ovid , cochrane central register of controlled trials , cochrane database of systematic reviews , and web of science from january 1990 to january 2013 . in order to identify all potentially relevant studies we combined mesothelioma and pneumonectomy or mesothelioma and pleurectomy / decortication as keywords . we reviewed abstracts from the recent european association of cardiothoracic surgery meetings and i searched the british thoracic society website for relevant material . the reference lists of all retrieved studies were reviewed for further identification of relevant papers . the articles that were found were screened using the inclusion and exclusion criteria ( previously described in detail in the protocol of this review ) . eligible studies for the present review included those in which patients with histologically proven mpm were treated with surgery ( epp or p / d ) either as the only treatment or as part of a protocol involving adjuvant or neoadjuvant chemotherapy and/or radiotherapy . we did not exclude any forms of chemotherapy or radiotherapy and there was considerable variability between studies in this respect . patient selection may have varied between institutions and within an institution at different time periods . we attempted to identify all relevant randomised controlled trials ( rcts ) ; however , it became clear from the initial search that we would have to include retrospective series and nonrandomised phase i and ii trials as the number of rcts was small . we included studies with more than 20 participants , age greater than 18 ( adults ) , life expectancy at least 12 weeks , and at least moderate performance status either by who or karnofsky criteria . review articles were also excluded to avoid duplication of results ; however , their references were reviewed . we reviewed their references and included some of the studies that were relevant to my systematic review . we reviewed their references and we also used similar headlines ( to theirs ) for the summary tables that we constructed . we did not find any systematic review looking at the role of both epp and p / d in the management of mpp . recurrence - free survival , symptomatic status , and complications were not always reported or measured and we will discuss this below . the time period that the studies cover extends from 1980 to 2012 . over those two decades the diagnostic modalities have evolved ( earlier studies did not have modern scanners available , e.g. ) , the management has changed from single treatment approach to triple therapy ( surgery , chemotherapy , and radiotherapy ) , the chemotherapeutic agents and radiotherapy strategies have improved or changed , even surgical technique has become more consistent , and surgeons have acquired better skill with experience . furthermore the postoperative hospital care and support has improved . this evolution has made it possible , in our days , to have diagnosis of mesothelioma at earlier stage and protocol and multidisciplinary team guided management , with better drugs and more skilful surgeons on the background of modern health care institutions with specialist interest . all these factors influence operative mortality and survival , which are the hard endpoints of this review . the staging system for mesothelioma has changed throughout the years and the definitions of p / d and epp have only recently started to become more consistent . these factors make meaningful comparison of reported results an extremely difficult and sometimes impossible task . as the main focus of this review , from an intervention point of view , is surgery for mesothelioma we have classified relevant studies into p / d and epp . we will critically summarise first the studies that include p / d and epp results , then studies involving only p / d , and lastly studies which focus only on epp . the characteristics of each publication included as well as their main findings are depicted in the relevant tables . ( tables 1 and 2 for p / d publications and tables 3 and 4 for epp publications ) . the study by schipper et al . was a retrospective review of a single institution over an eighteen - year period . they classified p / d into total and subtotal depending on how extensive the surgical resection was . epp was associated with acceptable mortality and had a median survival significantly better than that for subtotal p / d . they documented their concern regarding the fact that improved survival for epp came at a cost of high morbidity . in the paper by luckraz et al . the surgeon who first described surgical management of pleural mesothelioma in the 1970s ( butchart ) is one of the authors . they did statistical analysis of their results and found that the use of adjuvant therapy ( radiotherapy , chemotherapy , or both ) was associated with an increased postoperative survival . on univariate analysis , p / d combined with chemotherapy and radiotherapy was the strongest predictor of prolonged survival . an important limitation is that , during the study period , the chemotherapy and radiotherapy regimens changed significantly so it was impossible to make meaningful comparisons . they conclude that despite the serious limitations in their study their overall results represent real life their recommendation was to undertake a randomised controlled trial to determine the relative role of each procedure . . attempted to perform a straightforward comparison of epp and p / d in their institution , by retrospectively reviewing their results . the selection of which patient will undergo epp and which will undergo p / d depended on stage of the mpm , the overall medical fitness of the patient , and requirements of several prospective clinical trials performed during that time period . in patients not participating in protocols that predefined either epp or p / d , the decision to perform an epp or p / d was based on surgeons ' intraoperative judgement on which procedure was appropriate in order to completely remove the tumour . also tumour stage and patients ' medical condition influenced the decision for epp or p / d . the patients had chemotherapy or radiotherapy according to which clinical trial they were participating in . univariate analysis showed that more aggressive tumour histology and higher stage were associated with significantly worse survival . a limitation of this study is that the investigators did not have comorbidity data . whether a patient received epp or p / d was very much based on how fit the patient was , so this introduced selection bias in the study . the authors were not able to support the use of one or the other surgical approach . their choice of whether to perform an epp or a p / d was based on the degree of the tumour invasion into the lung as well as fitness of the patient for each surgical procedure . they concluded that older age , nonepithelial histology , and pathologic stages iii - iv disease had a significantly negative impact on survival . their findings show that younger patients with a stages i - ii epithelial tumour were good candidates for radical surgery , either p / d or epp . aziz et al . reported a retrospective review of their 10-year experience in their centre . p / d was considered for locally extensive disease but only for relief of pain or shortness of breath . the decision to perform an epp as opposed to a p / d was based on how much the tumour had spread within the lung and whether patients were well enough to go through major surgery . during the more recent years they also decided not to operate on patients older than 60 . this was because they believed that younger patients would be able to tolerate the operation better and therefore have better operative mortality results . they did not find any significant difference in survival between patients undergoing p / d or having no surgery at all . systemic chemotherapy was not offered to patients who underwent p / d because this was essentially an attempt to reduce the tumour load in the lung , not to completely remove it , so chemotherapy seemed futile as an option . lang - lazdunski et al . in a ground - breaking and certainly controversial paper compared p / d with hyperthermic pleural lavage against epp . this is the first prospective but not randomised study which is hinting on the possibility of treating patients with mesothelioma with p / d as a surgical curative attempt ( complete resection ) . the authors did multivariate analysis and concluded that epithelioid histology , p / d , and completeness of resection were independent prognostic factors of completion of trimodality therapy . this means that patients who had p / d were well enough after the surgery to undergo chemotherapy and radiotherapy . rena and casadio published their single centre 11-year experience with epp and p / d . importantly they included a quality - of - life ( qol ) assessment in their comparison . it is the only study of its kind and their findings agree with lang - lazdunski et al . in supporting superiority of p / d over epp . qol parameters were similar at baseline for patients undergoing either procedure but p / d patients had a better qol at 6 and 12 months . all parameters were improved among p / d patients with the exception of postoperative pain , which was similar in the two groups . the authors thought that this was because both epp and p / d use a posterolateral thoracotomy incision to get to the lung , which is associated with a lot of pain after the surgery . patients with lower stage mpm ( i and ii ) who had p / d had a significantly better quality of life when compared with that of those submitted to epp . patients with histological diagnosis of epithelial mesothelioma had similar survival regardless of which surgical procedure they had . they also found that patients who underwent epp had worse long - term survival than those who underwent p / d . this finding needs further exploration as in theory epp achieves complete removal of mpm whereas p / d does not . therefore we would expect epp patients to live longer but the opposite was found in this study . maintained a prospectively updated database which they used to analyse patients that underwent p / d only . exclusion criteria that made them unfit for epp were clinical stage t4 or m1 ( cancer had extensive spread within the lung or had given metastases elsewhere in the body ) , mediastinoscopy proven stage n2 ( cancer had spread to lymph nodes of the opposite side ) , age > 70 years , poor lung function due to smoking or other lung problems ( predicted postoperative fev1 < 40% ) , and right or left ventricular systolic dysfunction ( poor myocardial contractility ) . p / d was classified into radical ( r ) and nonradical ( nr ) depending on how extensive the resection of the pleura and tumour were . they found that patients undergoing radical p / d had a survival advantage over those who underwent nonradical p / d . there was a difference though in the distribution of the histological subtypes of the disease : there were proportionately more patients with epithelioid cell type ( a less aggressive type ) in group r ( 40 epithelioid , 4 sarcomatoid , and 7 biphasic ) than in group nr ( 28 epithelioid , 11 sarcomatoid , and 12 biphasic ) . in contrast to the majority of the other studies included in this review , nakas et al . believe that with radical p / d they can achieve complete macroscopic clearance of tumour . most surgeons think that p / d results in high rates of local recurrence of mpm . an important limitation in their paper is that they did not have an established protocol regarding adjuvant treatment following p / d since these decisions were at the discretion of referring oncologists . martin - ucar et al . performed p / d for symptomatic control of patients diagnosed with mpm . indications included shortness of breath , chest pain , and empyema ( collection of pus in the chest cavity ) . radiotherapy and chemotherapy were not given to patients undergoing p / d unless there was clinical evidence of disease progression . they found that at 3 weeks and 6 months after p / d the patients felt less breathlessness and less chest pain . however , despite good symptom control in those who survived , mortality outweighed the benefits after 3 months . in multivariate analysis , an aggressive cell type and weight loss were predictors of poor symptom control . patients with epithelial cell type and no weight loss were significantly more likely to retain symptomatic control than those with more aggressive cell type and loss of weight . a recent paper by lang - lazdunski et al . looked at patients unsuitable to undergo epp or those that refused epp . they developed an alternative multimodality therapy plan for these patients based on p / d and hyperthermic pleural lavage ( hot water at 40c mixed with betadine was used to wash the pleural cavity ) followed by prophylactic radiotherapy and adjuvant chemotherapy . a large proportion of patients were referred to their hospitals for enrolment into the mesothelioma and radical surgery ( mars ) trial involving epp during the 20052008 period . those patients not wishing to undergo epp or to enrol on the mars trial were offered the alternative treatment described above . selection bias is therefore an issue in this study as well as small sample size and nonrandomisation . those patients that had complete macroscopical removal of the mpm tumour had better survival than those who underwent an incomplete resection . the authors concluded that treatment with p / d and hyperthermic pleural lavage was associated with low morbidity and mortality and therefore it could represent an alternative to the classical trimodality regimen . have published one of the largest series of patients treated with trimodality therapy for mesothelioma . this is one of the earliest publications on this topic and one of the most quoted papers that we encountered in our literature search . they found that multimodality therapy including epp is feasible in selected patients , microscopic resection margins affect long - term survival and patients with epithelial subtype ( less aggressive ) , margin - negative ( complete removal of mpm with surgery ) , extrapleural node - negative ( tumour not spread to lymph nodes elsewhere in the body ) resection had extended survival . they also proposed a revision to the staging system that was used at that time and it has since been revised . krug et al . conducted a multicentre phase ii study in the us to assess performance of a treatment regime which consisted of neo - adjuvant chemotherapy , followed by epp , followed by radiotherapy in patients with stages i to iii disease . a rigorous assessment of each patient 's fitness was done before enrolment to determine respiratory and cardiac function . among those who underwent epp , median survival was 21.9 months . among patients who completed radiotherapy , median survival was 29.1 months , 1-year survival was 90.0% , and 2-year survival was 61.2% . remarkably the parameters histological type of the tumour , gender , clinical stage of mpm , and lymph node spread did not influence survival . relapse - free rates among epp patients were 63.8% at 1 year and 38.9% at 2 years . one limitation of this study is that a mediastinoscopy to assess whether tumour had spread to lymph nodes outside the lung was not required for staging and data regarding how many patients underwent mediastinoscopy was not recorded . furthermore these patients were highly selected on the basis of stage of disease ( early stages ) , good performance status , and satisfactory heart and lung function . the authors concluded that the treatment algorithm with induction chemotherapy , epp , and then hemithoracic radiation is feasible and effective , but only a subgroup of patients experience long survival . did a retrospective review of 46 patients treated with trimodality therapy over a ten - year period in a single centre . median survival for stages 0 , ii , iii , and iv patients was 17 , 33 , 21 , and 24 months , respectively . if the mpm involved lymph nodes , that was a poor prognostic finding , with a median survival of 11 months , compared with 34 months if node - negative . the authors estimate that , for every patient deemed acceptable of trimodality therapy , about another 4 mesothelioma patients were turned down because of advanced disease or comorbidities . a limitation is that the choice of chemotherapy agent , dose , and schedule were at the discretion of the oncologist . during the last 4 years , also patients were referred to epp after they had already started on or completed chemotherapy at other institutions . de perrot et al . conducted a retrospective analysis of 60 patients that were selected to undergo trimodality therapy for mesothelioma in canada . disease - free survival was influenced by whether mpm had invaded lymph nodes or not ( pathologic nodal status ) and to a lesser extent by the histologic type of mpm . the median disease - free survival was 12 months for patients with n2 disease ( mpm spread to lymph nodes outside the affected lung ) , 44 months for patients with n1 disease ( mpm spread to lymph nodes of the affected lung ) , and not reached for patients with n0 disease ( no lymph node involvement ) . after multivariate analysis , the presence of n2 disease remained a significant prognostic marker of worse outcome despite completion of the trimodality therapy . histological type , extent of lung invasion ( t status ) , gender , and age were not significant . weder et al . did a multicentre trial of patients undergoing trimodality therapy for mesothelioma and they assessed quality - of - life parameters as well as survival . the levels of distress did not change over time ( from 0 to 6 months ) . physical symptoms and activity showed worsening after surgery , but there was a recovery back to normal after 36 months . the authors concluded that qol impairment was not a major issue ; however , they stressed the importance of a specialised experienced team . only 22 patients managed to complete the full intervention including chemotherapy , epp , and radiotherapy . they reduced the total radiation dose administered to patients and included additional cardiopulmonary and respiratory function tests which led to a more careful selection of patients . however this amendment made meaningful interpretation of results impossible as the baseline characteristics of the groups before and after the change in protocol were very different . van schil et al . published a study , which was original in that their primary endpoint was success of treatment . this was defined as a patient who received the full protocol treatment within the defined time frames and was still alive 90 days after the end of protocol treatment without progression or evidence of high toxicity related to the chemotherapy or the radiotherapy . only 24 ( 42.1% ) patients met the primary endpoint definition of success . median progression - free survival for all the 57 patients who were eligible and started treatment was 13.9 months and 1-year survival rate was 54.4% . trimodality treatment was completed in 37 ( 64.9% ) patients and median treatment duration was 184 days . embarked in a very ambitious project in 2005 to conduct a randomised controlled trial ( mars ) across 12 centres in the uk , which would definitively determine the role of epp in the management of mesothelioma in the context of trimodality treatment . 12-month recurrence - free survival in the epp group was 34.8% and median recurrence - free survival was 7.6 months . median quality - of - life scores seemed to be lower for the epp group than the no epp group , with the lowest median score shortly after surgery ; however , there were no statistically significant differences between treatment groups . at the time mars was being planned , pemetrexed was not yet the standard of care in the uk . unfortunately during recruitment , the chemotherapy standard of care for mesothelioma changed , and patients recruited later were more likely to receive cisplatin and pemetrexed than those recruited earlier in the study . at the start of the trial the authors calculated that 670 patients would be needed to identify any statistically significant difference between epp and no epp with overall survival as the primary outcome . because of the anticipated difficulty in recruitment of such a high number of patients to a trial comparing epp with a nonsurgical approach , the mars researchers designed a feasibility trial with the objective of randomly assigning 50 patients within 1 year to assess the possibility of completing a larger trial to clarify the role of epp . the study was therefore not designed to test the benefit of epp for patient outcome and any conclusions were speculative . moreover it could be argued that this feasibility study was partly unsuccessful , because it took 3 years to compile 50 patients . . reviewed retrospectively 56 patients across three centres in italy who had undergone epp followed by different modalities of radiotherapy . recurrent tumour ( considered as the first site of relapse ) was local in two cases ( within the lung cavity ) , nodal in three cases ( into regional or distant lymph nodes ) , and distant ( metastasis in other organs ) in 13 cases . baseline characteristics , patient selection , and follow - up periods varied between institutions . tables 2 and 4 summarise survival and peri - operative outcomes for p / d and epp , respectively . for p / d median survival ranged from 8.1 to 32 months . mortality ranged from 0% to 9.8% and morbidity ranged from 5.9% to 55% . for epp median survival ranged from 6.9 to 46.9 months . this large variability makes it difficult to reach safe conclusions in relation to the efficacy and safety of each procedure and echoes the issues that i have discussed at the beginning of section 3 . the principles of surgical management for cancer are similar for all types of solid tumours . surgery for mpm aims at removing all the tumour that can be seen during the operation ( macroscopic disease ) whereas chemotherapy and radiotherapy aim to kill any remaining cancer cells locally ( in the lung cavity ) or in other organs ( microscopic disease ) . two surgical techniques exist.epp involves resection of the ipsilateral ( same side as the tumour ) lung , visceral and parietal pleura ( mesothelial linings of lung and chest wall ) , ipsilateral hemidiaphragm , and pericardium with reconstruction of the latter two structures to prevent cardiac and visceral herniation ( movement of the heart or abdominal organs into the chest cavity after the lung has been removed ) . the description of the epp surgical technique is common knowledge in the thoracic surgical community without much variation . because of this uniformity in the definition of epp , results from different hospitals can be compared , differences in outcomes can be studied , and meaningful conclusions can be reached.p / d is an operation that has not been standardised yet . the reasons behind this are probably multifactorial . from the current review it has become clear that p / d was considered a sort of compromise procedure reserved for patients who were not fit enough from cardiovascular or respiratory point of view to undergo the definitive epp procedure . however in recent years some surgeons tend to favour it over epp mainly due to the fact that most studies have shown no superiority of epp in terms of survival and also much more complications related to epp . epp involves resection of the ipsilateral ( same side as the tumour ) lung , visceral and parietal pleura ( mesothelial linings of lung and chest wall ) , ipsilateral hemidiaphragm , and pericardium with reconstruction of the latter two structures to prevent cardiac and visceral herniation ( movement of the heart or abdominal organs into the chest cavity after the lung has been removed ) . the description of the epp surgical technique is common knowledge in the thoracic surgical community without much variation . because of this uniformity in the definition of epp , results from different hospitals can be compared , differences in outcomes can be studied , and meaningful conclusions can be reached . it has become clear that p / d was considered a sort of compromise procedure reserved for patients who were not fit enough from cardiovascular or respiratory point of view to undergo the definitive epp procedure . however in recent years some surgeons tend to favour it over epp mainly due to the fact that most studies have shown no superiority of epp in terms of survival and also much more complications related to epp . the international association for the study of lung cancer staging committee ( iaslc ) recommended that the following terminology should be used in the mesothelioma staging project.extrapleural pneumonectomy ( epp ) : en bloc resection of the parietal and visceral pleura with the ipsilateral lung , pericardium , and diaphragm.extended pleurectomy / decortication ( epd ) : parietal and visceral pleurectomy to remove all gross tumour with resection of the diaphragm and/or pericardium.pleurectomy/decortication ( p / d ) : parietal and visceral pleurectomy to remove all gross tumour without diaphragm or pericardial resection.partial pleurectomy : partial removal of parietal and/or visceral pleura for diagnostic or palliative purposes but leaving gross tumour behind . extrapleural pneumonectomy ( epp ) : en bloc resection of the parietal and visceral pleura with the ipsilateral lung , pericardium , and diaphragm . extended pleurectomy / decortication ( epd ) : parietal and visceral pleurectomy to remove all gross tumour with resection of the diaphragm and/or pericardium . pleurectomy / decortication ( p / d ) : parietal and visceral pleurectomy to remove all gross tumour without diaphragm or pericardial resection . partial pleurectomy : partial removal of parietal and/or visceral pleura for diagnostic or palliative purposes but leaving gross tumour behind . in addition , this will also provide uniform descriptors to be used in future research and improve pathologic staging and efforts to provide accurate prognosis to patients . current evidence and the iaslc report led several international mesothelioma interest group members ( latest meeting in boston 2012 ) to conclude that both surgical options ( p / d or epp ) are valid as long as they aim at complete removal of the cancer . furthermore they should be performed as part of a multimodality treatment for mpm . the choice of which one to use for each patient depends on the spread of mpm within the lung cavity , the preference and experience of the surgeon , and also which one is favoured at different hospitals . furthermore , it was collectively agreed that multimodality treatment should be performed in centers of high expertise and by surgeons who have achieved morbidity and mortality rates similar to what is reported in the current literature . for the time being , there is no evidence - based answer as to which procedure p / d or epp is the more appropriate technique to achieve long - term survival in patients with mpm . epp has been reported to have better survival but comes at a higher cost of peri - operative mortality and morbidity . p / d is associated with not only less peri - operative risk , but also less long - term success in controlling the spread or recurrence of mpm . the largest report comparing both procedures in a retrospective multicentre study on 663 patients , combining the experience of three large centers in the united states , concluded that the study emphasises the similarities in outcome after epp or p / d . the studies that were reviewed in this paper are highlighting the fact that nowadays the challenge is how to carefully select the patients that would benefit from each procedure ( epp or p / d ) . the timing of the operation and whether chemotherapy should come before or after surgery also vary between centres . the controversy around this issue remains and the debate in the scientific community is ongoing . there is one statement however that scientists seem to agree on : patients with histologically proven mpm and resectable tumour who could tolerate the three treatment modalities should be considered for a multimodal approach and be included in a trial if possible . despite more than two decades of intensive research into possible treatments for mpm , a landmark randomised controlled trial with the acronym emphacis demonstrated the superiority of cisplatin combined with pemetrexed in the management of patients with mpm . survival increased from 9.3 to 12.1 months and this was also associated with quality - of - life benefit . since publication of this study the combination of cisplatin and pemetrexed has replaced all previous chemotherapeutic agents as the main therapy for patients who are having chemotherapy as part of multimodality therapy and for those who have too high risk to undergo a surgical procedure . previously , worldwide research into drug development used to rely on empirical testing of new agents in clinical trials . in vitro or in vivo molecular targets were used extensively . in the current century genomic medicine was based on our efforts to identify targets which may have some clinical significance . some of the cell changes are called passenger mutations because although they are present they do not give a growth advantage to the cancer cell . unfortunately , no mutations of clinical value ( that can be targeted by drugs or other interventions ) have yet been identified in mesothelioma , and certainly none have been transferred into practical clinical application . traditionally , adjuvant rt has been given through fields from in front of as well as behind the patient in order to include the hemithorax where the mpm is located in its entirety . the heart , liver , kidneys , and stomach are protected by using radiotherapy shields over them . however in this way the dose that the chest receives at the edges of the shields is uncertain . therefore sometimes the radiologists end up giving too much or too little radiation to the chest . intensity - modulated radiation therapy ( imrt ) is a complex technique that was designed to overcome the problems associated with the traditional technique . with imrt normal organs are protected against receiving any radiation , and higher doses are delivered to the chest . therefore this is safer for the patient and more precise way of delivering rt to the mpm . current research focuses on arc therapy and helical tomotherapy which are rotational radiotherapy techniques that deliver radiation from more angles than imrt . they have been shown to achieve good results in preventing spread of the mpm ; however this comes with a cost : pneumonitis ( reactive inflammation of the lung which has been radiated ) is a common complication . research is ongoing in current trials to determine if theoretical advantages of new radiotherapy techniques can be translated into clinical benefit for mesothelioma patients . this systematic review has shown that epp and p / d for patients with mpm can be performed with an acceptable perioperative mortality rate in specialised centres . however , the evidence for long - term survival in patients operated - on in the context of tmt in the current literature is inconsistent . these encouraging results demonstrate the potential benefit that surgery can offer for patients treated by a multidisciplinary approach in specialist high volume centres .

introduction . malignant pleural mesothelioma ( mpm ) is an aggressive cancer arising from pleural mesothelium . surgery aims to either cure the disease or control the symptoms . two surgical procedures exist : extrapleural pneumonectomy ( epp ) and pleurectomy / decortication ( p / d ) . in this systematic review we assess current evidence on safety and efficacy of surgery . methods . five electronic databases were reviewed from january 1990 to january 2013 . studies were selected according to a predefined protocol . primary endpoint was overall survival . secondary endpoints included quality of life , disease - free survival , disease recurrence , morbidity , and length of hospital stay . results . sixteen studies were included . median survival ranged from 8.1 to 32 months for p / d and from 6.9 to 46.9 months for epp . perioperative mortality was 0%9.8% and 3.2%12.5% , respectively . perioperative morbidity was 5.9%55% for p / d and 10%82.6% for epp . average length of stay was 7 days for p / d and 9 days for epp . conclusion . current evidence can not definitively answer which procedure ( epp or p / d ) is more beneficial in terms of survival and operative risks . this systematic review suggests that surgery in the context of trimodality therapy offers acceptable perioperative outcomes and long - term survival . centres specialising in mpm management have better results .

1. Introduction 2. Methods 3. Results 4. Discussion: Current and Future Research 5. Conclusion

these cells normally line body cavities , specifically pleura , peritoneum , pericardium , and testis . there is widespread interest in this disease by doctors and the general public , because millions of people have been exposed to asbestos fibres . malignant pleural mesothelioma ( mpm ) has caused and is still causing violent debate between protagonists and antagonists of the three options in relation to treatment . surgery , radiotherapy , and chemotherapy are endlessly discussed , often with limited scientific evidence to support strongly held viewpoints . the two surgical procedures commonly used are pleurectomy with decortication ( p / d ) and extrapleural pneumonectomy ( epp ) . p / d is a more limited procedure and requires less cardiorespiratory reserve ; it has a morbidity of 25% and a mortality of 2% . data from the uk thoracic surgical register of the society for cardiothoracic surgery in great britain and ireland showed that a limited number of patients underwent surgery for mesothelioma in the past decade . the median survival after epp within mars is consistent with 10 , 12 , 13 , and 14 months in larger observational studies . a best evidence topic paper addressing the question of whether p / d is superior to palliative care in the treatment of patients with malignant pleural mesothelioma came to the following conclusion : p / d is a morbid operation that is associated with significant peri - operative mortality and complication rates . the antagonists of surgery argue that , when a patient commences trimodality ( chemotherapy , radiotherapy , and surgery ) treatment , it will take six to nine months to complete it . the primary objective of the current review is to determine if surgery improves all - cause mortality and/or quality of life in patients with mpm . we will discuss the different surgical options ( epp versus p / d ) and their relevant advantages and disadvantages including mortality and morbidity . the overall success rates of the trimodality therapy will be discussed in relation to survival , freedom from recurrence , and quality of life ( symptom relief from breathlessness and pain ) . electronic search was performed of pubmed , ovid , cochrane central register of controlled trials , cochrane database of systematic reviews , and web of science from january 1990 to january 2013 . in order to identify all potentially relevant studies we combined mesothelioma and pneumonectomy or mesothelioma and pleurectomy / decortication as keywords . the reference lists of all retrieved studies were reviewed for further identification of relevant papers . eligible studies for the present review included those in which patients with histologically proven mpm were treated with surgery ( epp or p / d ) either as the only treatment or as part of a protocol involving adjuvant or neoadjuvant chemotherapy and/or radiotherapy . we did not exclude any forms of chemotherapy or radiotherapy and there was considerable variability between studies in this respect . we did not find any systematic review looking at the role of both epp and p / d in the management of mpp . recurrence - free survival , symptomatic status , and complications were not always reported or measured and we will discuss this below . the staging system for mesothelioma has changed throughout the years and the definitions of p / d and epp have only recently started to become more consistent . as the main focus of this review , from an intervention point of view , is surgery for mesothelioma we have classified relevant studies into p / d and epp . we will critically summarise first the studies that include p / d and epp results , then studies involving only p / d , and lastly studies which focus only on epp . the characteristics of each publication included as well as their main findings are depicted in the relevant tables . ( tables 1 and 2 for p / d publications and tables 3 and 4 for epp publications ) . they classified p / d into total and subtotal depending on how extensive the surgical resection was . epp was associated with acceptable mortality and had a median survival significantly better than that for subtotal p / d . in the paper by luckraz et al . the surgeon who first described surgical management of pleural mesothelioma in the 1970s ( butchart ) is one of the authors . on univariate analysis , p / d combined with chemotherapy and radiotherapy was the strongest predictor of prolonged survival . attempted to perform a straightforward comparison of epp and p / d in their institution , by retrospectively reviewing their results . the selection of which patient will undergo epp and which will undergo p / d depended on stage of the mpm , the overall medical fitness of the patient , and requirements of several prospective clinical trials performed during that time period . in patients not participating in protocols that predefined either epp or p / d , the decision to perform an epp or p / d was based on surgeons ' intraoperative judgement on which procedure was appropriate in order to completely remove the tumour . also tumour stage and patients ' medical condition influenced the decision for epp or p / d . whether a patient received epp or p / d was very much based on how fit the patient was , so this introduced selection bias in the study . their choice of whether to perform an epp or a p / d was based on the degree of the tumour invasion into the lung as well as fitness of the patient for each surgical procedure . their findings show that younger patients with a stages i - ii epithelial tumour were good candidates for radical surgery , either p / d or epp . p / d was considered for locally extensive disease but only for relief of pain or shortness of breath . the decision to perform an epp as opposed to a p / d was based on how much the tumour had spread within the lung and whether patients were well enough to go through major surgery . they did not find any significant difference in survival between patients undergoing p / d or having no surgery at all . systemic chemotherapy was not offered to patients who underwent p / d because this was essentially an attempt to reduce the tumour load in the lung , not to completely remove it , so chemotherapy seemed futile as an option . in a ground - breaking and certainly controversial paper compared p / d with hyperthermic pleural lavage against epp . this is the first prospective but not randomised study which is hinting on the possibility of treating patients with mesothelioma with p / d as a surgical curative attempt ( complete resection ) . the authors did multivariate analysis and concluded that epithelioid histology , p / d , and completeness of resection were independent prognostic factors of completion of trimodality therapy . this means that patients who had p / d were well enough after the surgery to undergo chemotherapy and radiotherapy . rena and casadio published their single centre 11-year experience with epp and p / d . in supporting superiority of p / d over epp . qol parameters were similar at baseline for patients undergoing either procedure but p / d patients had a better qol at 6 and 12 months . all parameters were improved among p / d patients with the exception of postoperative pain , which was similar in the two groups . the authors thought that this was because both epp and p / d use a posterolateral thoracotomy incision to get to the lung , which is associated with a lot of pain after the surgery . patients with lower stage mpm ( i and ii ) who had p / d had a significantly better quality of life when compared with that of those submitted to epp . they also found that patients who underwent epp had worse long - term survival than those who underwent p / d . this finding needs further exploration as in theory epp achieves complete removal of mpm whereas p / d does not . maintained a prospectively updated database which they used to analyse patients that underwent p / d only . exclusion criteria that made them unfit for epp were clinical stage t4 or m1 ( cancer had extensive spread within the lung or had given metastases elsewhere in the body ) , mediastinoscopy proven stage n2 ( cancer had spread to lymph nodes of the opposite side ) , age > 70 years , poor lung function due to smoking or other lung problems ( predicted postoperative fev1 < 40% ) , and right or left ventricular systolic dysfunction ( poor myocardial contractility ) . p / d was classified into radical ( r ) and nonradical ( nr ) depending on how extensive the resection of the pleura and tumour were . they found that patients undergoing radical p / d had a survival advantage over those who underwent nonradical p / d . there was a difference though in the distribution of the histological subtypes of the disease : there were proportionately more patients with epithelioid cell type ( a less aggressive type ) in group r ( 40 epithelioid , 4 sarcomatoid , and 7 biphasic ) than in group nr ( 28 epithelioid , 11 sarcomatoid , and 12 biphasic ) . believe that with radical p / d they can achieve complete macroscopic clearance of tumour . most surgeons think that p / d results in high rates of local recurrence of mpm . an important limitation in their paper is that they did not have an established protocol regarding adjuvant treatment following p / d since these decisions were at the discretion of referring oncologists . performed p / d for symptomatic control of patients diagnosed with mpm . indications included shortness of breath , chest pain , and empyema ( collection of pus in the chest cavity ) . radiotherapy and chemotherapy were not given to patients undergoing p / d unless there was clinical evidence of disease progression . they found that at 3 weeks and 6 months after p / d the patients felt less breathlessness and less chest pain . in multivariate analysis , an aggressive cell type and weight loss were predictors of poor symptom control . they developed an alternative multimodality therapy plan for these patients based on p / d and hyperthermic pleural lavage ( hot water at 40c mixed with betadine was used to wash the pleural cavity ) followed by prophylactic radiotherapy and adjuvant chemotherapy . those patients not wishing to undergo epp or to enrol on the mars trial were offered the alternative treatment described above . the authors concluded that treatment with p / d and hyperthermic pleural lavage was associated with low morbidity and mortality and therefore it could represent an alternative to the classical trimodality regimen . have published one of the largest series of patients treated with trimodality therapy for mesothelioma . they found that multimodality therapy including epp is feasible in selected patients , microscopic resection margins affect long - term survival and patients with epithelial subtype ( less aggressive ) , margin - negative ( complete removal of mpm with surgery ) , extrapleural node - negative ( tumour not spread to lymph nodes elsewhere in the body ) resection had extended survival . conducted a multicentre phase ii study in the us to assess performance of a treatment regime which consisted of neo - adjuvant chemotherapy , followed by epp , followed by radiotherapy in patients with stages i to iii disease . among those who underwent epp , median survival was 21.9 months . among patients who completed radiotherapy , median survival was 29.1 months , 1-year survival was 90.0% , and 2-year survival was 61.2% . median survival for stages 0 , ii , iii , and iv patients was 17 , 33 , 21 , and 24 months , respectively . the authors estimate that , for every patient deemed acceptable of trimodality therapy , about another 4 mesothelioma patients were turned down because of advanced disease or comorbidities . conducted a retrospective analysis of 60 patients that were selected to undergo trimodality therapy for mesothelioma in canada . disease - free survival was influenced by whether mpm had invaded lymph nodes or not ( pathologic nodal status ) and to a lesser extent by the histologic type of mpm . the median disease - free survival was 12 months for patients with n2 disease ( mpm spread to lymph nodes outside the affected lung ) , 44 months for patients with n1 disease ( mpm spread to lymph nodes of the affected lung ) , and not reached for patients with n0 disease ( no lymph node involvement ) . after multivariate analysis , the presence of n2 disease remained a significant prognostic marker of worse outcome despite completion of the trimodality therapy . histological type , extent of lung invasion ( t status ) , gender , and age were not significant . they reduced the total radiation dose administered to patients and included additional cardiopulmonary and respiratory function tests which led to a more careful selection of patients . published a study , which was original in that their primary endpoint was success of treatment . median progression - free survival for all the 57 patients who were eligible and started treatment was 13.9 months and 1-year survival rate was 54.4% . embarked in a very ambitious project in 2005 to conduct a randomised controlled trial ( mars ) across 12 centres in the uk , which would definitively determine the role of epp in the management of mesothelioma in the context of trimodality treatment . 12-month recurrence - free survival in the epp group was 34.8% and median recurrence - free survival was 7.6 months . unfortunately during recruitment , the chemotherapy standard of care for mesothelioma changed , and patients recruited later were more likely to receive cisplatin and pemetrexed than those recruited earlier in the study . recurrent tumour ( considered as the first site of relapse ) was local in two cases ( within the lung cavity ) , nodal in three cases ( into regional or distant lymph nodes ) , and distant ( metastasis in other organs ) in 13 cases . baseline characteristics , patient selection , and follow - up periods varied between institutions . tables 2 and 4 summarise survival and peri - operative outcomes for p / d and epp , respectively . for p / d median survival ranged from 8.1 to 32 months . mortality ranged from 0% to 9.8% and morbidity ranged from 5.9% to 55% . for epp median survival ranged from 6.9 to 46.9 months . surgery for mpm aims at removing all the tumour that can be seen during the operation ( macroscopic disease ) whereas chemotherapy and radiotherapy aim to kill any remaining cancer cells locally ( in the lung cavity ) or in other organs ( microscopic disease ) . two surgical techniques exist.epp involves resection of the ipsilateral ( same side as the tumour ) lung , visceral and parietal pleura ( mesothelial linings of lung and chest wall ) , ipsilateral hemidiaphragm , and pericardium with reconstruction of the latter two structures to prevent cardiac and visceral herniation ( movement of the heart or abdominal organs into the chest cavity after the lung has been removed ) . because of this uniformity in the definition of epp , results from different hospitals can be compared , differences in outcomes can be studied , and meaningful conclusions can be reached.p / d is an operation that has not been standardised yet . from the current review it has become clear that p / d was considered a sort of compromise procedure reserved for patients who were not fit enough from cardiovascular or respiratory point of view to undergo the definitive epp procedure . however in recent years some surgeons tend to favour it over epp mainly due to the fact that most studies have shown no superiority of epp in terms of survival and also much more complications related to epp . the description of the epp surgical technique is common knowledge in the thoracic surgical community without much variation . because of this uniformity in the definition of epp , results from different hospitals can be compared , differences in outcomes can be studied , and meaningful conclusions can be reached . it has become clear that p / d was considered a sort of compromise procedure reserved for patients who were not fit enough from cardiovascular or respiratory point of view to undergo the definitive epp procedure . however in recent years some surgeons tend to favour it over epp mainly due to the fact that most studies have shown no superiority of epp in terms of survival and also much more complications related to epp . the international association for the study of lung cancer staging committee ( iaslc ) recommended that the following terminology should be used in the mesothelioma staging project.extrapleural pneumonectomy ( epp ) : en bloc resection of the parietal and visceral pleura with the ipsilateral lung , pericardium , and diaphragm.extended pleurectomy / decortication ( epd ) : parietal and visceral pleurectomy to remove all gross tumour with resection of the diaphragm and/or pericardium.pleurectomy/decortication ( p / d ) : parietal and visceral pleurectomy to remove all gross tumour without diaphragm or pericardial resection.partial pleurectomy : partial removal of parietal and/or visceral pleura for diagnostic or palliative purposes but leaving gross tumour behind . extrapleural pneumonectomy ( epp ) : en bloc resection of the parietal and visceral pleura with the ipsilateral lung , pericardium , and diaphragm . extended pleurectomy / decortication ( epd ) : parietal and visceral pleurectomy to remove all gross tumour with resection of the diaphragm and/or pericardium . pleurectomy / decortication ( p / d ) : parietal and visceral pleurectomy to remove all gross tumour without diaphragm or pericardial resection . current evidence and the iaslc report led several international mesothelioma interest group members ( latest meeting in boston 2012 ) to conclude that both surgical options ( p / d or epp ) are valid as long as they aim at complete removal of the cancer . the choice of which one to use for each patient depends on the spread of mpm within the lung cavity , the preference and experience of the surgeon , and also which one is favoured at different hospitals . for the time being , there is no evidence - based answer as to which procedure p / d or epp is the more appropriate technique to achieve long - term survival in patients with mpm . p / d is associated with not only less peri - operative risk , but also less long - term success in controlling the spread or recurrence of mpm . the largest report comparing both procedures in a retrospective multicentre study on 663 patients , combining the experience of three large centers in the united states , concluded that the study emphasises the similarities in outcome after epp or p / d . the studies that were reviewed in this paper are highlighting the fact that nowadays the challenge is how to carefully select the patients that would benefit from each procedure ( epp or p / d ) . the controversy around this issue remains and the debate in the scientific community is ongoing . they have been shown to achieve good results in preventing spread of the mpm ; however this comes with a cost : pneumonitis ( reactive inflammation of the lung which has been radiated ) is a common complication . this systematic review has shown that epp and p / d for patients with mpm can be performed with an acceptable perioperative mortality rate in specialised centres . however , the evidence for long - term survival in patients operated - on in the context of tmt in the current literature is inconsistent .

there is widespread interest in this disease by doctors and the general public , because millions of people have been exposed to asbestos fibres . in addition to its substantial personal and health care costs , malignant mesothelioma is associated with considerable compensation costs . the predicted total economic burden of malignant mesothelioma related to compensation for asbestos exposure in the next 40 years is up to $ 200 billion for the united states and $ 80 billion for europe . malignant pleural mesothelioma ( mpm ) has caused and is still causing violent debate between protagonists and antagonists of the three options in relation to treatment . diagnosis and palliation will be part of the surgeon 's role but everyone 's big hope and question is is there a chance of surgical cure ? until recently it was thought that local radiotherapy directed to surgical sites prevents spreading of tumour and radiotherapy can provide relief of chest - wall pain . however , an up - to - date systematic review paper by price concluded that there is currently no evidence to support the routine role of radiotherapy in patients with mesothelioma . surgery became part of management options in the 1970s and large series have been published in relevant journals [ 14 , 15 ] . data from the uk thoracic surgical register of the society for cardiothoracic surgery in great britain and ireland showed that a limited number of patients underwent surgery for mesothelioma in the past decade . the median survival after epp within mars is consistent with 10 , 12 , 13 , and 14 months in larger observational studies . a best evidence topic paper addressing the question of whether p / d is superior to palliative care in the treatment of patients with malignant pleural mesothelioma came to the following conclusion : p / d is a morbid operation that is associated with significant peri - operative mortality and complication rates . the antagonists of surgery argue that , when a patient commences trimodality ( chemotherapy , radiotherapy , and surgery ) treatment , it will take six to nine months to complete it . the primary objective of the current review is to determine if surgery improves all - cause mortality and/or quality of life in patients with mpm . we will discuss the different surgical options ( epp versus p / d ) and their relevant advantages and disadvantages including mortality and morbidity . this is a combination of chemotherapy either before ( neoadjuvant ) or after ( adjuvant ) surgery , an operation that aims to remove the tumour and radiotherapy . the evidence around the use of different chemotherapeutic regimes will be discussed as well as the evidence on radiotherapy approaches . the overall success rates of the trimodality therapy will be discussed in relation to survival , freedom from recurrence , and quality of life ( symptom relief from breathlessness and pain ) . electronic search was performed of pubmed , ovid , cochrane central register of controlled trials , cochrane database of systematic reviews , and web of science from january 1990 to january 2013 . in order to identify all potentially relevant studies we combined mesothelioma and pneumonectomy or mesothelioma and pleurectomy / decortication as keywords . we reviewed abstracts from the recent european association of cardiothoracic surgery meetings and i searched the british thoracic society website for relevant material . eligible studies for the present review included those in which patients with histologically proven mpm were treated with surgery ( epp or p / d ) either as the only treatment or as part of a protocol involving adjuvant or neoadjuvant chemotherapy and/or radiotherapy . we attempted to identify all relevant randomised controlled trials ( rcts ) ; however , it became clear from the initial search that we would have to include retrospective series and nonrandomised phase i and ii trials as the number of rcts was small . we included studies with more than 20 participants , age greater than 18 ( adults ) , life expectancy at least 12 weeks , and at least moderate performance status either by who or karnofsky criteria . we reviewed their references and we also used similar headlines ( to theirs ) for the summary tables that we constructed . we did not find any systematic review looking at the role of both epp and p / d in the management of mpp . , the management has changed from single treatment approach to triple therapy ( surgery , chemotherapy , and radiotherapy ) , the chemotherapeutic agents and radiotherapy strategies have improved or changed , even surgical technique has become more consistent , and surgeons have acquired better skill with experience . this evolution has made it possible , in our days , to have diagnosis of mesothelioma at earlier stage and protocol and multidisciplinary team guided management , with better drugs and more skilful surgeons on the background of modern health care institutions with specialist interest . the staging system for mesothelioma has changed throughout the years and the definitions of p / d and epp have only recently started to become more consistent . as the main focus of this review , from an intervention point of view , is surgery for mesothelioma we have classified relevant studies into p / d and epp . we will critically summarise first the studies that include p / d and epp results , then studies involving only p / d , and lastly studies which focus only on epp . epp was associated with acceptable mortality and had a median survival significantly better than that for subtotal p / d . the surgeon who first described surgical management of pleural mesothelioma in the 1970s ( butchart ) is one of the authors . they did statistical analysis of their results and found that the use of adjuvant therapy ( radiotherapy , chemotherapy , or both ) was associated with an increased postoperative survival . on univariate analysis , p / d combined with chemotherapy and radiotherapy was the strongest predictor of prolonged survival . they conclude that despite the serious limitations in their study their overall results represent real life their recommendation was to undertake a randomised controlled trial to determine the relative role of each procedure . the selection of which patient will undergo epp and which will undergo p / d depended on stage of the mpm , the overall medical fitness of the patient , and requirements of several prospective clinical trials performed during that time period . in patients not participating in protocols that predefined either epp or p / d , the decision to perform an epp or p / d was based on surgeons ' intraoperative judgement on which procedure was appropriate in order to completely remove the tumour . also tumour stage and patients ' medical condition influenced the decision for epp or p / d . univariate analysis showed that more aggressive tumour histology and higher stage were associated with significantly worse survival . their choice of whether to perform an epp or a p / d was based on the degree of the tumour invasion into the lung as well as fitness of the patient for each surgical procedure . they concluded that older age , nonepithelial histology , and pathologic stages iii - iv disease had a significantly negative impact on survival . their findings show that younger patients with a stages i - ii epithelial tumour were good candidates for radical surgery , either p / d or epp . the decision to perform an epp as opposed to a p / d was based on how much the tumour had spread within the lung and whether patients were well enough to go through major surgery . systemic chemotherapy was not offered to patients who underwent p / d because this was essentially an attempt to reduce the tumour load in the lung , not to completely remove it , so chemotherapy seemed futile as an option . in a ground - breaking and certainly controversial paper compared p / d with hyperthermic pleural lavage against epp . this is the first prospective but not randomised study which is hinting on the possibility of treating patients with mesothelioma with p / d as a surgical curative attempt ( complete resection ) . the authors did multivariate analysis and concluded that epithelioid histology , p / d , and completeness of resection were independent prognostic factors of completion of trimodality therapy . all parameters were improved among p / d patients with the exception of postoperative pain , which was similar in the two groups . the authors thought that this was because both epp and p / d use a posterolateral thoracotomy incision to get to the lung , which is associated with a lot of pain after the surgery . patients with lower stage mpm ( i and ii ) who had p / d had a significantly better quality of life when compared with that of those submitted to epp . exclusion criteria that made them unfit for epp were clinical stage t4 or m1 ( cancer had extensive spread within the lung or had given metastases elsewhere in the body ) , mediastinoscopy proven stage n2 ( cancer had spread to lymph nodes of the opposite side ) , age > 70 years , poor lung function due to smoking or other lung problems ( predicted postoperative fev1 < 40% ) , and right or left ventricular systolic dysfunction ( poor myocardial contractility ) . p / d was classified into radical ( r ) and nonradical ( nr ) depending on how extensive the resection of the pleura and tumour were . there was a difference though in the distribution of the histological subtypes of the disease : there were proportionately more patients with epithelioid cell type ( a less aggressive type ) in group r ( 40 epithelioid , 4 sarcomatoid , and 7 biphasic ) than in group nr ( 28 epithelioid , 11 sarcomatoid , and 12 biphasic ) . patients with epithelial cell type and no weight loss were significantly more likely to retain symptomatic control than those with more aggressive cell type and loss of weight . they developed an alternative multimodality therapy plan for these patients based on p / d and hyperthermic pleural lavage ( hot water at 40c mixed with betadine was used to wash the pleural cavity ) followed by prophylactic radiotherapy and adjuvant chemotherapy . those patients that had complete macroscopical removal of the mpm tumour had better survival than those who underwent an incomplete resection . the authors concluded that treatment with p / d and hyperthermic pleural lavage was associated with low morbidity and mortality and therefore it could represent an alternative to the classical trimodality regimen . this is one of the earliest publications on this topic and one of the most quoted papers that we encountered in our literature search . they found that multimodality therapy including epp is feasible in selected patients , microscopic resection margins affect long - term survival and patients with epithelial subtype ( less aggressive ) , margin - negative ( complete removal of mpm with surgery ) , extrapleural node - negative ( tumour not spread to lymph nodes elsewhere in the body ) resection had extended survival . conducted a multicentre phase ii study in the us to assess performance of a treatment regime which consisted of neo - adjuvant chemotherapy , followed by epp , followed by radiotherapy in patients with stages i to iii disease . among patients who completed radiotherapy , median survival was 29.1 months , 1-year survival was 90.0% , and 2-year survival was 61.2% . remarkably the parameters histological type of the tumour , gender , clinical stage of mpm , and lymph node spread did not influence survival . relapse - free rates among epp patients were 63.8% at 1 year and 38.9% at 2 years . one limitation of this study is that a mediastinoscopy to assess whether tumour had spread to lymph nodes outside the lung was not required for staging and data regarding how many patients underwent mediastinoscopy was not recorded . furthermore these patients were highly selected on the basis of stage of disease ( early stages ) , good performance status , and satisfactory heart and lung function . median survival for stages 0 , ii , iii , and iv patients was 17 , 33 , 21 , and 24 months , respectively . if the mpm involved lymph nodes , that was a poor prognostic finding , with a median survival of 11 months , compared with 34 months if node - negative . disease - free survival was influenced by whether mpm had invaded lymph nodes or not ( pathologic nodal status ) and to a lesser extent by the histologic type of mpm . the median disease - free survival was 12 months for patients with n2 disease ( mpm spread to lymph nodes outside the affected lung ) , 44 months for patients with n1 disease ( mpm spread to lymph nodes of the affected lung ) , and not reached for patients with n0 disease ( no lymph node involvement ) . after multivariate analysis , the presence of n2 disease remained a significant prognostic marker of worse outcome despite completion of the trimodality therapy . however this amendment made meaningful interpretation of results impossible as the baseline characteristics of the groups before and after the change in protocol were very different . this was defined as a patient who received the full protocol treatment within the defined time frames and was still alive 90 days after the end of protocol treatment without progression or evidence of high toxicity related to the chemotherapy or the radiotherapy . median progression - free survival for all the 57 patients who were eligible and started treatment was 13.9 months and 1-year survival rate was 54.4% . embarked in a very ambitious project in 2005 to conduct a randomised controlled trial ( mars ) across 12 centres in the uk , which would definitively determine the role of epp in the management of mesothelioma in the context of trimodality treatment . 12-month recurrence - free survival in the epp group was 34.8% and median recurrence - free survival was 7.6 months . median quality - of - life scores seemed to be lower for the epp group than the no epp group , with the lowest median score shortly after surgery ; however , there were no statistically significant differences between treatment groups . unfortunately during recruitment , the chemotherapy standard of care for mesothelioma changed , and patients recruited later were more likely to receive cisplatin and pemetrexed than those recruited earlier in the study . at the start of the trial the authors calculated that 670 patients would be needed to identify any statistically significant difference between epp and no epp with overall survival as the primary outcome . because of the anticipated difficulty in recruitment of such a high number of patients to a trial comparing epp with a nonsurgical approach , the mars researchers designed a feasibility trial with the objective of randomly assigning 50 patients within 1 year to assess the possibility of completing a larger trial to clarify the role of epp . recurrent tumour ( considered as the first site of relapse ) was local in two cases ( within the lung cavity ) , nodal in three cases ( into regional or distant lymph nodes ) , and distant ( metastasis in other organs ) in 13 cases . this large variability makes it difficult to reach safe conclusions in relation to the efficacy and safety of each procedure and echoes the issues that i have discussed at the beginning of section 3 . surgery for mpm aims at removing all the tumour that can be seen during the operation ( macroscopic disease ) whereas chemotherapy and radiotherapy aim to kill any remaining cancer cells locally ( in the lung cavity ) or in other organs ( microscopic disease ) . two surgical techniques exist.epp involves resection of the ipsilateral ( same side as the tumour ) lung , visceral and parietal pleura ( mesothelial linings of lung and chest wall ) , ipsilateral hemidiaphragm , and pericardium with reconstruction of the latter two structures to prevent cardiac and visceral herniation ( movement of the heart or abdominal organs into the chest cavity after the lung has been removed ) . the description of the epp surgical technique is common knowledge in the thoracic surgical community without much variation . because of this uniformity in the definition of epp , results from different hospitals can be compared , differences in outcomes can be studied , and meaningful conclusions can be reached.p / d is an operation that has not been standardised yet . epp involves resection of the ipsilateral ( same side as the tumour ) lung , visceral and parietal pleura ( mesothelial linings of lung and chest wall ) , ipsilateral hemidiaphragm , and pericardium with reconstruction of the latter two structures to prevent cardiac and visceral herniation ( movement of the heart or abdominal organs into the chest cavity after the lung has been removed ) . because of this uniformity in the definition of epp , results from different hospitals can be compared , differences in outcomes can be studied , and meaningful conclusions can be reached . the international association for the study of lung cancer staging committee ( iaslc ) recommended that the following terminology should be used in the mesothelioma staging project.extrapleural pneumonectomy ( epp ) : en bloc resection of the parietal and visceral pleura with the ipsilateral lung , pericardium , and diaphragm.extended pleurectomy / decortication ( epd ) : parietal and visceral pleurectomy to remove all gross tumour with resection of the diaphragm and/or pericardium.pleurectomy/decortication ( p / d ) : parietal and visceral pleurectomy to remove all gross tumour without diaphragm or pericardial resection.partial pleurectomy : partial removal of parietal and/or visceral pleura for diagnostic or palliative purposes but leaving gross tumour behind . current evidence and the iaslc report led several international mesothelioma interest group members ( latest meeting in boston 2012 ) to conclude that both surgical options ( p / d or epp ) are valid as long as they aim at complete removal of the cancer . the choice of which one to use for each patient depends on the spread of mpm within the lung cavity , the preference and experience of the surgeon , and also which one is favoured at different hospitals . for the time being , there is no evidence - based answer as to which procedure p / d or epp is the more appropriate technique to achieve long - term survival in patients with mpm . p / d is associated with not only less peri - operative risk , but also less long - term success in controlling the spread or recurrence of mpm . the largest report comparing both procedures in a retrospective multicentre study on 663 patients , combining the experience of three large centers in the united states , concluded that the study emphasises the similarities in outcome after epp or p / d . the studies that were reviewed in this paper are highlighting the fact that nowadays the challenge is how to carefully select the patients that would benefit from each procedure ( epp or p / d ) . there is one statement however that scientists seem to agree on : patients with histologically proven mpm and resectable tumour who could tolerate the three treatment modalities should be considered for a multimodal approach and be included in a trial if possible . despite more than two decades of intensive research into possible treatments for mpm , a landmark randomised controlled trial with the acronym emphacis demonstrated the superiority of cisplatin combined with pemetrexed in the management of patients with mpm . since publication of this study the combination of cisplatin and pemetrexed has replaced all previous chemotherapeutic agents as the main therapy for patients who are having chemotherapy as part of multimodality therapy and for those who have too high risk to undergo a surgical procedure . however , the evidence for long - term survival in patients operated - on in the context of tmt in the current literature is inconsistent .

inflammatory bowel disease ( ibd ) is considered an autoimmune disease that causes chronic inflammation in the gastro - intestinal tract . ulcerative colitis ( uc ) is one of the ibds ' reflecting chronic relapsing inflammatory disorders of the intestine . among the cell types , epithelial cells , myeloid innate cells , effector t cells , regulatory cells , and b cells excessive effector t - cell cytokine secretion or defective regulatory t cells lead to the disease propagation of ibd . the intestinal barrier performs two important tasks to keep the balance between health and disease . first , it must mediate effective absorption of fluids , nutrients , and minerals from the lumen across the epithelium and into the microcirculation and microvilli . second , the barrier must be impermeable to prevent the transfer of potentially pathogenic microbes and infectious agents . dysfunction in this intestinal barrier leads to the dysfunction in the intestinal immune system , which has been implicated as the major mechanism by which chronic inflammation occurs in colitis . hence a common feature of ibd pathogenesis is the dysregulated effector t cell response to commensal microbiota . because the intestinal mucosa is constantly exposed to several antigens , the mucosal immune system have evolved several strategies to avoid an unnecessary and uncontrolled inflammatory reaction . once the antigen from the commensal microbiota has been eradicated , t lymphocytes of the intestinal mucosa require a method to attenuate the local immune response . one of the regulatory methods , downregulation of activated t lymphocytes via apoptosis , is a very potent and effective strategy , now considered as a key controlling mechanism of ibd . failure to regulate t - cell responses in the intestinal or colonic mucosa leads to an inappropriate and sustained injurious immunologic reaction [ 6 , 7 ] . because of their heightened activation and activity in ibd pathogenesis , effector t cells are considered an excellent target for therapeutics . cells undergoing apoptosis are characterized by the shrinkage and condensation of their cytoplasm , increased mitochondrial permeability , chromatin condensation into caps at the edge of the nucleus , dna fragmentation , and the appearance of plasma membrane blebs , often referred to as there are two signaling pathways for the cell death by apoptosis : the intrinsic and extrinsic pathways [ 9 , 10 ] . activation of the extrinsic pathway occurs by ligand - induced cell surface receptor ( e.g. , tumor necrosis factor receptor 1 tnfr1 , fas , and death receptor 5 ) activation . the intrinsic pathway is activated with growth factor deprivation , oncogene activation , or when dna damage is detected by cellular sensors such as ataxia telangiectasia mutated ( atm ) , ataxia telangiectasia and rad3-related ( atr ) , and tumor protein 53 ( p53 ) . inflammation - induced reactive oxygen species and nitric oxide lead to p53 stabilization and accumulation [ 12 , 13 ] . this activates p53 to eliminate the damaged cells by apoptosis [ 12 , 13 ] . this is an intrinsic approach for the apoptosis of damaged cells , where activated p53 plays an important role in mediating apoptosis in epithelial and inflammatory cells during the process of colitis [ 4 , 12 , 1417 ] . the post - translational modification ( often phosphorylation at serine 15 ) tends to elevate the level of wild - type ( wt ) p53 during inflammation . p53 plays at least 2 separate roles in the responses to therapeutic agents it is an important component of cellular checkpoints ( cell cycle arrest ) , and it can mediate apoptosis . for example , loss of p53 function can cause resistance to 5-fluorouracil ( 5-fu ) , but increased sensitivity to dna damaging agent such as adriamycin . this provides a clear indication that some drugs may exert an apoptosis - inducing effect through a p53-dependent while other drugs effect through a p53-independent pathway . cancer is one of the scenarios where too little apoptosis occurs , resulting in malignant cells that will not die and continue to proliferate . four cellular functions are inappropriately regulated in the cancer cells : cellular proliferation , differentiation , chromosomal and genetic organization , and apoptosis ( reviewed in ) . abnormal cell proliferation leading to accumulation of clonal cells is seen in cancer . as mentioned this could be due to the defects with the cell cycle control . upon sensing dna damage , p53 is activated , resulting in either a g1 cell cycle arrest [ 20 , 21 ] or apoptosis [ 22 , 23 ] . cells undergo a g1 cell cycle arrest to allow the dna repair before replication and if the dna damage is beyond repair , the cells tend to undergo apoptosis . cells can also undergo a g1 arrest due to targeting cyclins and cyclin - dependent kinases . p53 mutation is observed in most cancers , which often tend to relax this g1-s cell cycle transition because p53 could not be activated . american ginseng ( ag , panax quinquefolius ) is grown in the eastern temperate forest areas of north america , from british columbia , southern quebec , ontario , minnesota , and wisconsin in the north , to oklahoma , the ozark plateau , and georgia in the south . ag is an obligate shade perennial native of north america and its root is the commonly used component . ag has been reported to have a wide range of pharmacological effects , including effects on the central nervous system , blood - sugar levels , cardiovascular system , endocrine system , immune system , and cancer [ 4 , 26 , 27 ] . recently we have shown ag extract suppresses colitis by the accumulation and activation of p53 to induce apoptosis of inflammatory cells . to further delineate the active anti - inflammatory and proapoptotic components present in the ag a hexane fraction of ag ( hag ) showed increased anti - inflammatory and pro - apoptotic properties in the chemically induced mouse model of colitis . the elevation of wt p53 levels during inflammation resulting in apoptosis of inflammatory and damaged cells [ 4 , 29 , 30 ] led us to the notion that the active anti - inflammatory components present in hag might suppress colitis through the p53 pathway . here , we tested this hypothesis . the p. quinquefolius extract has been described previously in detail by our laboratory . as well , we have recently described the generation of the hag used in the present study . dextran sulfate sodium ( dss ) was purchased from mp biomedicals ( solon , oh : molecular weight , 36,00050,000 ) . tk6 ( p53 ) and nh32 ( p53 ) cell lines were a kind gift from curtis harris ( national cancer institute ) , originally derived from dr . tk6 cells are a lymphoblastoid cell line derived from the spleen more than 30 years ago . nh32 cells are an isogenic derivative of tk6 cells in which both alleles of the p53 gene were knocked out . jurkat t cells are an immortalized line of t lymphocyte cells derived in the late 1970s from the peripheral blood of a 14-year - old male with t - cell leukemia . jurkat t cells have a defective p53 pathway due to a mutation in the cooh - terminal domain responsible for transactivation [ 34 , 35 ] . tk6 , nh32 , and jurkat t cells were maintained in exponentially growing suspension culture at 37c in a humidified 5% co2 atmosphere in rpmi 1640 supplemented with 10% heat - inactivated calf serum , 100 units / ml penicillin , 100 g / ml streptomycin , and 2 mmol / l l - glutamine . cd4/cd25 cells from c57bl/6 mice were purified from the spleens using nylon wool columns ( polysciences , warrington , pa , usa ) followed by depletion of b cells and macrophages . cd4/cd25 t cells were then isolated using an macs miniseparator and cd4 and cd25 microbeads according to the manufacturer 's instructions ( miltenyi biotec , auburn , ca , usa ) by depletion of cd4cd25 t cells ( negative selection ) . the purity of t cells was 88.6% ( supplementary figure 1 available online at doi:10.1155/2012/785739 ) as determined by flow cytometry ( cytomics fc 500 , beckman coulter , brea , ca , usa ) . briefly , isolated cd4/cd25 t cells were washed with pbs and 2 10 cells were re - suspended with 100 l of pbs and incubated with anti - mouse cd4 antibody conjugated to allophycocyanin ( apc ) ( clone : gk1.5 ; isotype : rat igg2b , kappa , ebioscience , san diego , ca , usa ) ( 0.125 g/100 l of cell suspension ) . the anti - mouse cd25 antibody conjugated to r - phycoerythrin ( pe ) ( clone : 7d4 ; isotype : rat igm , miltenyi biotec , auburn , ca , usa ) was incubated during the isolation of cd4/cd25 effector t cells according to the manufacturer 's instructions ( miltenyi biotec , auburn , ca , usa ) . the purity of cd4 t cells was determined by obtaining a dot plot of fl-4 ( apc ) versus fl-2 ( pe ) and individual histogram plots of fl-4 and fl-2 versus the number of events ( supplementary figure 1 ) . isolated cd4/cd25 effector t cells ( 1 10 ) were cultured in six - well plates overnight followed by experimentation as indicated . all cells were maintained in exponentially growing suspension culture at 37c in a humidified , 5% co2 atmosphere in rpmi 1640 supplemented with 10% heat - inactivated calf serum , 100 units / ml penicillin , 100 g / ml streptomycin , and 2 mm l - glutamine . hct-116 , human epithelial colon cancer cells line , proficient in p53 , and isogenic hct-116 p53 cells were maintained in exponentially growing adherent culture at 37c in a humidified 5% co2 atmosphere in rpmi 1640 supplemented with 10% heat - inactivated calf serum , 100 units / ml penicillin , 100 g / ml streptomycin , and 2 mmol / l l - glutamine . we followed our previous protocol for our dss ( mp biomedicals , solon , oh : 36,00050,000 mw ) mouse model of colitis . briefly ( figure 1 ) , 810-week - old c57bl/6 mice received either water ad libitum or 1% dss . 11.9 mg / kg of hag was dissolved in 100 l 1 pbs per mouse and administered daily by oral gavage ( per os , po ) . 11.9 mg / kg daily is the human equivalent dose of 58 mg daily . of note , currently the use of ginseng in human clinical trials can range anywhere from 200 mg to 9 g daily [ 38 , 39 ] . the control group of mice was given 100 l of 1 pbs by oral gavage . all procedures performed were in accordance with the guide for the care and use of laboratory animals ( national research council , washington , dc , usa ) and approved by the animal resource facility , university of south carolina , institutional animal care and use committee . to determine whether the hag can prevent the onset of colitis , mice were fed 1% dss for 2.5 cycles ( 7 days dss , 7 days water making 1 cycle ) . the vehicle or the hag was administered daily by oral gavage 7 days prior to the first dss exposure and continued throughout the course of the experiment . mice were euthanized at 2.5 cycles ( figure 1 ) . for pathology , colon tissue samples were washed with phosphate - buffered saline ( pbs ; mediatech , herndon , va , usa ) , cut longitudinally , swissrolled , then formalin - fixed overnight , and paraffin - embedded . paraffin - embedded tissues were serially sectioned , and one section from each mouse was stained with h&e . sections were microscopically examined by two blinded investigators ( d.p and x.c ) for histopathologic changes using a scoring system previously used and validated by ourselves and others [ 4 , 28 , 40 , 41 ] . histology score was determined by multiplying the percent involvement for each of the three following histologic features by the percent area of involvement : inflammation severity ( 0 , none ; 1 , minimal ; 2 , moderate ; 3 , severe ) , inflammation extent ( 0 , none ; 1 , mucosa ; 2 , mucosa and submucosa ; 3 , transmural ) , and extent of crypt damage ( 0 , none ; 1 , one third of crypt damaged ; 2 , two thirds of crypt damaged ; 3 , crypts lost , surface epithelium intact ; 4 , crypts lost , surface epithelium lost ) . percent area involvement was defined as : 0 , 0% ; 1 , 125% ; 2 , 2650% ; 3 , 5175% ; 4 , 76100% . therefore , the minimal score is 0 and the maximal score is 40 . since , dss - induced colitis in mice leads to the damage in colonic epithelial barrier and is characterized by extent and depth of inflammation , a grading or scoring system of inflammation with all these parameters provides an excellent measure of histologic assessment of dss - induced colitis . antibodies used include : p53 ( mouse monoclonal , do-1 , diluted 1 in 500 , cat # op43 t ; calbiochem , gibbstown , nj , usa ) and gapdh ( rabbit monoclonal , diluted 1 in 1000 , cat # 5174 p ; cell signaling technology , danvers , ma , usa ) . horseradish peroxidase - conjugated anti - mouse and anti - rabbit secondary antibodies were purchased from amersham biosciences ( piscataway , nj , usa ) . all antibodies were diluted in 5% milk / pbst ( 0.1% tween 20 in 1 pbs ) . western blot signal was detected by pierce ecl western blotting substrate ( thermo scientific , rockford , il , usa ) and developed onto hyperfilm ( ge healthcare life sciences , pittsburgh , pa , usa ) . briefly , after treating blot with the chemiluminescent substrate ( pierce ecl ) for a minute , the blot was exposed to the hyperfilm in the dark ( exposure time was optimized based on the band signal obtained ) and the film was developed in an automatic x - ray film processor ( futura classic e automatic x - ray film processor , fisher industry , geneva , il , usa ) . cd4/cd25 effector t cells were seeded at 1 10 per well into six - well dishes for 24 h in triplicate ( n = 3 ) . fresh medium or fresh medium containing freshly dissolved indicated concentrations of hag ( 0300 g / ml ) was added for 24 h , as indicated . cells were then harvested for annexin v according to instructions provided by the kit manufacturer ( bd biosciences , san diego , ca , usa ) . annexin v / propidium iodide ( pi ) staining was examined using a beckman coulter cytomics fc500 flow cytometer . 1 10 cells / wells of hct-116 wt and hct-116 p53 cells were incubated in 1.0% nbcs supplemented rpmi-1640 media for 24 hrs in 6-well culture plate . the media was changed and the cells were treated with hexane fraction of ag ( 0500 g / ml ) . the cells were harvested after 24 hrs of treatment and cell cycle assay was performed by labeling the dsdna of the cells with dapi ( 4,6-diamidino-2-phenylindole ) ( sigma - aldrich , mo , usa ) . briefly , the harvested cells were fixed by gently vortexing and adding 70% ethanol dropwise . the cells were washed with pbs/1% bsa and stained with 1 g / ml of dapi ( in pbs/0.1% triton x-100 ) for 10 min . at room temperature . 20,000 cells / events were directly analyzed by bd - lsr - ii flow - cytometer ( bd biosciences , san jose , ca , usa ) . based on the dna content , the different phases of the cell cycle was determined by using bd facsdiva software ( bd biosciences , san jose , ca , usa ) . tk6 ( p53 wt ) , nh32 ( isogenic p53 ) , and jurkat t ( dysfunctional p53 ) cells were incubated in 0.1% nbcs supplemented rpmi-1640 media for 24 hrs . the media was changed and the cells were treated with hexane fraction of ag ( 0500 g / ml ) as indicated in figure 2 . cells were harvested after 24 hours of treatment and tunel assay was performed as described by vendor ( roche diagnostics , in ) in triplicate ( n = 3 ) . briefly , 1 10 cells were fixed using a 100 l of fixation solution ( 2% paraformaldehyde ) and permeabilized using a permeabilization solution ( 0.1% triton x-100 in 0.1% sodium citrate ) . cells were washed and incubated with tunel reaction mixture ( label solution and enzyme solution ) ( roche diagnostics , in , usa ) . apoptosis in the samples were analyzed by flow cytometry ( beckman coulter , ca , usa ) . the fluorescence was evaluated using the excitation wavelength of 488 nm and detected in the range of 515565 nm ( green , fl-1 channel ) . the dot plot of fs versus fl-1 and histogram plot of ( number of event ) versus ( fl-1 channel ) were plotted to obtain a percentage increase in the apoptosis of the hexane fraction of ag treated cells . positive control for apoptosis is the fixed and permeabilized cells treated with dnase i recombinant ( 3 u / ml in 50 mm tris - hcl , ph7.5 , and 1 mg / ml bsa ) ( invitrogen , ca , usa ) to induce dna strand breaks prior to labeling ( following the vendors protocol ) . isogenic enzyme control is the fixed and permeabilized cells with the labeling solution but without the terminal transferase enzyme . statistical analysis was done using one - way anova with scheffe 's post hoc test for comparison of endpoint data between mouse groups , as well as in vitro endpoints . the results were analyzed using the stat - view ii statistical program ( abacus concepts , inc . , piscataway , nj , usa ) and microsoft excel ( microsoft , bellevue , wa , usa ) for macintosh computers . in uc , intestinal immune responses are often characterized by activation of lamina propria t lymphocytes ( lpl ) with potent effector functions . among its regulatory mechanisms , down - regulation of activated t lymphocytes via apoptosis is a very potent and effective strategy , now considered as a key controlling mechanism of uc . to this end , lymphoblastoid cell lines tk6 ( wt p53 ) , nh32 ( isogenic to tk6 , but p53 ) , and jurkat t cells ( which have a dysfunctional p53 pathway ) were tested for the apoptosis inducing property of hag . previously , we showed the whole ag extract induces apoptosis of tk6 , but not nh32 cells . interestingly , here , we show hag was able to induce apoptosis ( tunel ) not only in tk6 cells but ( albeit to a lesser extent ) also in nh32 cells ( figures 2(a ) and 2(b ) ) . hag induced a 3.9-fold increase in apoptosis of tk6 cells at 500 g / ml of hag when compared to the untreated cells . g / ml of hag when compared to the non - treated cells ( figures 2(a ) and 2(b ) ) . also , at 100 g / ml , the hag induced 2.6-fold increase in apoptosis in the tk6 cells , but a 2-fold increase in apoptosis in the nh32 cells . interestingly , the jurkat t - cell line ( which has a defective p53 pathway ) was somewhat resistant to hag - induced apoptosis ( figure 2(c ) ) , which is consistent with what we have previously observed with the whole ag extract . figure 2(d ) provides confirmation of the p53 status of each of the cell lines used in these experiments . overly aggressive cd4/cd25 t cells are thought to contribute to colitis , and defects in mucosal t - cell apoptosis are likely to be critical in the pathogenesis of colitis [ 4 , 6 , 7 ] . recently we have shown hag induces apoptosis of cd4/cd25 t cells derived from c57/bl6 wt mice during the suppression of colitis . in the present paper , consistent with results from tk6 ( p53 ) and nh32 ( p53 ) cells , hag induced apoptosis of the cd4/cd25 t cells isolated from both wt and p53 c57/bl6 mice ( table 1 ) . however , the induction of apoptosis was suppressed in the absence of p53 . there was 2.6-fold increase from ( 5.1 0.9% ) to ( 13.2 2.8% ) in apoptosis ( mean s.e . ) of effector t cells isolated from p53 c57/bl6 mice and a 4.6-fold increase from ( 4.5 0.5% ) to ( 20.7 8.9% ) in the apoptosis was observed in cd4/cd25 t cells isolated from wt c57/bl6 mice upon treatment with increasing concentration of hag ( 0300 g / ml ) . we have recently shown that hag suppresses chemically ( dss mouse model ) induced colitis in the wt p53 c57/bl6 mice . previously we have also shown that ag whole extract suppresses chemically induced colitis only in the wt p53 c57/bl6 mice and not in the p53 c57/bl6 mice . this led us to the conclusion that the suppression of colitis by ag extract is p53 dependent . as shown in the figure 3 , the hag was able to suppress the dss - induced colitis in both the p53 and wt p53 mice . however , consistent with our results on apoptosis of inflammatory cells in vitro , the suppression was more prominent in wt mice than in p53 mice . the h&e stained swissrolled colon inflammation score of wt c57/bl6 mice was reduced from 15 0.86 to 4.4 0.32 ( average s.e . ) ( a reduction of 71% ) in the hag - treated mice ( figure 3 ) . similar results were obtained with the p53 c57/bl6 mice , where the score dropped from 14.5 2.12 to 7.8 1.78 ( a reduction of 46% ) in the hag - treated mice . this result is consistent with the notion that the hag seems to act not only through a p53 pathway but also through a p53-independent pathway while suppressing colitis . the mitogenic stimuli triggered signal transduction pathways eventually converge on the cell cycle checkpoint that controls the g1 to s phase transition and activate appropriate cyclin - dependent kinases . this anomaly increases proliferation of the mutated cells to increase the cancer growth and progression . thus , one way to cause cancer to subside is to prevent this abnormal cancer - cell growth by inducing cell cycle arrest . we tested this hypothesis in vitro by using two different cell lines , hct-116 p53 wt and hct-116 p53 , and treated these cells with increasing concentrations of hag for 24 hr . in three repeated experiments , hag was able to increase a g1 cell cycle arrest in both the wt and p53 hct-116 cells . ( 34% to 56.7% ) at 100 g / ml , hag treatment and the percentage of cells in g1 remained above 50% with the other concentrations ( up to 500 g / ml ) ( figure 4(a ) ) . similarly there was a 1.5-fold increase ( 34.1% to 51.2% ) of hct-116 p53 cells in g1 phase after treatment with 100 g / ml of hag ( figure 4(a ) ) , and this checkpoint remaining with the remaining concentrations . figure 4(b ) provides confirmation of the p53 status of hct-116 cells used in this experiment . our hag is extracted from ag whole extract by non - polar solvent n - hexane . mostly polyacetylenes ( panaxynol , panaxydol , and panaxydiol ) and fatty acids ( fa ) present in the ag whole extract were extracted in this fraction . since hag is more effective at suppressing colitis than the whole ag extract and based on our previous study , where ag whole extract suppressed colitis through p53-mediated apoptosis of inflammatory cells , we hypothesized that hag also induces apoptosis of inflammatory cells through a p53-mediated mechanism . interestingly , we found that , unlike the whole ag extract , hag was able to induce modest apoptosis of p53 cd4/cd25 effector t cells and p53 lymphoblastoid cells in addition to their p53 wt counterpart cells ( figure 2 , table 1 ) . this indicates that hag mechanistically acts differently than the ag whole extract , where it shows a modest involvement of p53-dependent apoptosis . this still raises the question , what is the most active component present in the hag ? recently we have identified the different components present in the hag , with fas comprising 43% w / w , polyacetylenes 26.52% w / w ( 7.39% panaxydiol , 8.92% panaxydol , and 10.21% panaxynol ) , and less than 0.1% w / w ginsenosides . this indicates that specific fa ingredients or specific polyacetylenes , or both are responsible for the apoptotic property of hag through both p53-dependent and p53-independent mechanisms . have shown that asian ginseng extracted with ethanol induces a g2-m arrest and apoptosis via modulation of mapk and p53 pathway in llc-1 cells ( mouse lewis lung carcinoma cells ) . the ag whole extract is also extracted with ethanol and the ginsenoside composition is for the most part , similar in asian and american ginseng . [ 45 , 46 ] have shown that the ginsenosides - rs3 and -rs4 selectively elevate protein levels of p53 and p21 , and downregulate the activities of the cyclin - dependent kinases , resulting in cell cycle arrest at the g1/s boundary and apoptosis of sk - hep-1 cells ( immortalized human hepatoma cells ) [ 45 , 46 ] . all these observations suggest that ginsenosides may be a key player in modulating apoptosis and cell cycle through the p53 pathway and are consistent with the findings here and our previous findings that our whole ag extract has 10.1% ginsenoside ( w / w ) compared to the hag which has 0.074% ginsenoside ( w / w ) content . interestingly , ginsenoside rd , which is present in ag whole extract ( extracted with aqueous ethanol ) and absent in the hag , has been shown to attenuate the inflammatory response to tnbs ( 2,4,6-trinotrobenzenesulfonic acid- ) induced colitis by down - regulating multiple pro - inflammatory cytokines through the modulation of jnk ( c - jun n - terminal kinase ) and p38 activation . similarly another ginsenoside not present in hag , rb1 , and its metabolite compound k after oral administration blocked tnbs - induced expression of inos ( inducible - nitric oxide synthase ) , cox-2 ( cyclooxygenase-2 ) , and nf- ( nuclear transcription factor ) activation in mice . ginsenoside rb1 and its metabolite compound inhibited the activation of key inflammatory mediators irak-1 ( interleukin-1 receptor associated kinase-1 ) , ikk- ( inhibitor of nf- kinase ) , nf- , and mapk ( mitogen - activated protein kinases ) while suppressing colitis . these studies suggest that different ginsenosides may help attenuate inflammation , and our finding that n - hexane does not extract these ginsenosides from ginseng should not rule out that ginsenosides are not the active components of ginseng . taken together , these observations suggest that multiple components of ag , including ginsenosides , fas , and polyacetylenes , are responsible for the activity of ag in the suppression of colitis in animals . have shown that during the g1/s cell cycle arrest , protein levels of p21 , p16 , p53 , prb ( retinoblastoma protein ) , and e2f-1 ( transcription factor e2f1 ) were not changed after exposure to the polyacetylene , panaxydol ( isolated from p. ginseng ) , in the human malignant melanoma cell line , sk - mel-1 . human promyelocytic leukemia hl60 cells do not express the p53 protein due to a large deletion in the gene . the polyacetylenes , panaxynol , and panaxydol ( isolated from the lipophilic fractions of panax notoginseng ) have been shown to inhibit the proliferation of hl60 cells in a time and dose - dependent manner via an apoptotic pathway . these studies indicate that polyacetylenes may act independently of p53 while inducing apoptosis of certain cell lines . hag - induced apoptosis indicated by cleaved parp in tk6 cells , appears to change the protein levels of wt p53 and activated form of p53 ( phospho - ser15 ) very little . after 24 hr of hag treatment ( 300 g / ml ) , we observed a 2.6-fold increase in apoptosis of p53 cd4/cd25 t cells and a 4.6-fold increase in apoptosis of p53 cd4/cd25 t cells when compared to untreated cells ( table 1 ) . figures 2(a ) , 2(b ) and 2(c ) , and our recent study are consistent and indicate that p53 has a limited role in inducing hag - mediated apoptosis of inflammatory cells both in vitro and ex vivo . a clearly increased p53 , phospho - p53 ser-15 , and cleaved parp protein levels along with the increased apoptosis were reported with the ag whole extract , suggesting p53 plays a key role in apoptosis of inflammatory cells induced by the whole ag extract . however , an increased cleaved parp protein expression and only a slight change in the p53 , phospho - p53 ( ser-15 ) , and p53-upregulated mediator of apoptosis ( puma ) protein expression of tk6 cells after treatment with hag are consistent with our current results that hag induces apoptosis but this induction of apoptosis is likely mediated through both p53-dependent and p53-independent mechanisms . the other high volume components present in hag are fas , where 19% w / w is linoleic acid , a polyunsaturated fatty acid . have reported that linoleic acid treatment resulted in a concentration - dependent growth inhibition of ags cells ( human gastric adenocarcinoma cells ) by inducing apoptosis in a p53-independent manner with an elevated fas and fas ligand expression . this , then , is consistent with our results regarding a limited role for p53 in the cells undergoing apoptosis after treatment with hag . we further tested our compound in the in vivo animal study . as indicated ( figure 1 ) , both the p53 and p53 wt c57/bl6 mice were subjected to 2.5 cycles of 1% dss to induce colitis . in this preventive dss model , 75 ppm ( 11.9 mg / kg ) of hag or vehicle ( 1 pbs ) were administered to the mice daily ( po ) and continued throughout the course of the experiment . in our previous published studies , ag whole extract was effective in suppressing colitis only in the p53 wt c57/bl6 mice where whole ag extract prevented colon epithelial cells from the dna damage due to dss and induced apoptosis of lymphocytes in vivo . in the present study , with the same protocol , the hag was able to suppress dss - induced colonic inflammation to approximately one - third ( from 15 0.86 to 4.4 0.32 ) . interestingly , the hag also showed a modest suppression of inflammation to about one - half ( from 14.5 2.12 to 7.8 1.78 ) in the p53 c57/bl6 mice ( figure 3 ) . this provides evidence that the hag is anti - inflammatory and p53 plays a limited role in the hag - mediated suppression of colitis . two major problems are associated with apoptosis ; too much apoptosis is associated with various degenerative diseases and too little to no apoptosis is associated with carcinogenesis . hence one way to tackle the cancerous cells is to induce apoptosis of such cells . colon cancer is associated with long standing uc and hence inflammation seems to drive the progression of cancer . interestingly , both hct-116 p53 and p53 colon cancer cells were relatively resistant to apoptosis induced by hag ( data not shown ) . however , there was a g1 cell cycle checkpoint with hag treatment ( figure 4(a ) ) . it is more likely that one or more ingredients in hag target modulators of the cell cycle ( e.g. , cyclins or cyclin - dependent kinases ) . have shown that lipid soluble ginseng extract ( red ginseng extracted with n - hexane ) induces a cell cycle arrest at the g1 phase in nci - h460 cells ( human lung cancer cells ) . these separate observations suggest that the hag may halt the progression of cell cycle at g1 phase in multiple cancer cell lines . we have shown here that the hag can induce apoptosis in lymphoblastoid cells and cd4/cd25 effector t cells and cause a g1 checkpoint in colon cancer cell lines . it also suppressed chemically induced colitis in c57/bl6 mice . in all the above observations , we simultaneously tested the pro - apoptotic and anti - inflammatory properties of hag with the wt p53 and its p53 counterpart cells and mice . it is evident that hag can perform its pro - apoptotic , anti - inflammatory , and cell cycle arrest activities even in the absence of p53 . however , the hag has a more robust effect in the presence of p53 . these observations suggest that there is a role of p53 in the hag - mediated apoptosis of inflammatory cells and suppression of colitis ; but this role is limited . this entails that the hag affects other pathways independent of p53 in the suppression of colitis . for example , is there one component in the whole ag extract that is particularly powerful in inducing p53-mediated apoptosis that is missing from the hag ? does this component inhibit the ability of components only seen in the hag fraction to drive p53-independent apoptosis and suppress p53-independent colitis ? because mutation in p53 is observed in most cancers , including colitis - driven colon cancer , the hag may be particularly effective in targeting both p53 and p53 inflammatory cells and cancer cells and hence particularly effective in inhibiting the colitis - to - cancer sequence .

ulcerative colitis ( uc ) is debilitating and carries a high colon cancer risk . apoptosis of inflammatory cells is a key mechanism regulating uc . we have recently shown that american ginseng ( ag ) , and to a greater extent , a hexane fraction of ag ( hag ) can cause apoptosis and suppress mouse colitis through a p53-mediated mechanism . here , we tested the hypothesis that hag suppresses colitis through a p53 mechanism . we found only a limited impact of p53 in the ability of hag to induce inflammatory cell apoptosis and suppress mouse colitis in vitro and in vivo . finally , we asked whether hag could cause cell cycle arrest of hct116 colon cancer cells in vitro . interestingly , hag caused a g1 arrest of such cells independent of p53 status . findings are significant because hag suppresses colitis and associated colon cancer , and mutation in p53 is observed in most colitis - driven colon cancers . therefore , hag might be very effective in targeting the inflammatory cells and cancer cells since it induces apoptosis of inflammatory cells and cell cycle arrest in both p53/ and wt p53 colon cancer cells .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusion

inflammatory bowel disease ( ibd ) is considered an autoimmune disease that causes chronic inflammation in the gastro - intestinal tract . ulcerative colitis ( uc ) is one of the ibds ' reflecting chronic relapsing inflammatory disorders of the intestine . one of the regulatory methods , downregulation of activated t lymphocytes via apoptosis , is a very potent and effective strategy , now considered as a key controlling mechanism of ibd . the intrinsic pathway is activated with growth factor deprivation , oncogene activation , or when dna damage is detected by cellular sensors such as ataxia telangiectasia mutated ( atm ) , ataxia telangiectasia and rad3-related ( atr ) , and tumor protein 53 ( p53 ) . this is an intrinsic approach for the apoptosis of damaged cells , where activated p53 plays an important role in mediating apoptosis in epithelial and inflammatory cells during the process of colitis [ 4 , 12 , 1417 ] . p53 plays at least 2 separate roles in the responses to therapeutic agents it is an important component of cellular checkpoints ( cell cycle arrest ) , and it can mediate apoptosis . for example , loss of p53 function can cause resistance to 5-fluorouracil ( 5-fu ) , but increased sensitivity to dna damaging agent such as adriamycin . four cellular functions are inappropriately regulated in the cancer cells : cellular proliferation , differentiation , chromosomal and genetic organization , and apoptosis ( reviewed in ) . abnormal cell proliferation leading to accumulation of clonal cells is seen in cancer . upon sensing dna damage , p53 is activated , resulting in either a g1 cell cycle arrest [ 20 , 21 ] or apoptosis [ 22 , 23 ] . cells undergo a g1 cell cycle arrest to allow the dna repair before replication and if the dna damage is beyond repair , the cells tend to undergo apoptosis . cells can also undergo a g1 arrest due to targeting cyclins and cyclin - dependent kinases . p53 mutation is observed in most cancers , which often tend to relax this g1-s cell cycle transition because p53 could not be activated . american ginseng ( ag , panax quinquefolius ) is grown in the eastern temperate forest areas of north america , from british columbia , southern quebec , ontario , minnesota , and wisconsin in the north , to oklahoma , the ozark plateau , and georgia in the south . ag has been reported to have a wide range of pharmacological effects , including effects on the central nervous system , blood - sugar levels , cardiovascular system , endocrine system , immune system , and cancer [ 4 , 26 , 27 ] . recently we have shown ag extract suppresses colitis by the accumulation and activation of p53 to induce apoptosis of inflammatory cells . to further delineate the active anti - inflammatory and proapoptotic components present in the ag a hexane fraction of ag ( hag ) showed increased anti - inflammatory and pro - apoptotic properties in the chemically induced mouse model of colitis . the elevation of wt p53 levels during inflammation resulting in apoptosis of inflammatory and damaged cells [ 4 , 29 , 30 ] led us to the notion that the active anti - inflammatory components present in hag might suppress colitis through the p53 pathway . here , we tested this hypothesis . as well , we have recently described the generation of the hag used in the present study . jurkat t cells have a defective p53 pathway due to a mutation in the cooh - terminal domain responsible for transactivation [ 34 , 35 ] . hct-116 , human epithelial colon cancer cells line , proficient in p53 , and isogenic hct-116 p53 cells were maintained in exponentially growing adherent culture at 37c in a humidified 5% co2 atmosphere in rpmi 1640 supplemented with 10% heat - inactivated calf serum , 100 units / ml penicillin , 100 g / ml streptomycin , and 2 mmol / l l - glutamine . for pathology , colon tissue samples were washed with phosphate - buffered saline ( pbs ; mediatech , herndon , va , usa ) , cut longitudinally , swissrolled , then formalin - fixed overnight , and paraffin - embedded . histology score was determined by multiplying the percent involvement for each of the three following histologic features by the percent area of involvement : inflammation severity ( 0 , none ; 1 , minimal ; 2 , moderate ; 3 , severe ) , inflammation extent ( 0 , none ; 1 , mucosa ; 2 , mucosa and submucosa ; 3 , transmural ) , and extent of crypt damage ( 0 , none ; 1 , one third of crypt damaged ; 2 , two thirds of crypt damaged ; 3 , crypts lost , surface epithelium intact ; 4 , crypts lost , surface epithelium lost ) . since , dss - induced colitis in mice leads to the damage in colonic epithelial barrier and is characterized by extent and depth of inflammation , a grading or scoring system of inflammation with all these parameters provides an excellent measure of histologic assessment of dss - induced colitis . the media was changed and the cells were treated with hexane fraction of ag ( 0500 g / ml ) . the cells were harvested after 24 hrs of treatment and cell cycle assay was performed by labeling the dsdna of the cells with dapi ( 4,6-diamidino-2-phenylindole ) ( sigma - aldrich , mo , usa ) . tk6 ( p53 wt ) , nh32 ( isogenic p53 ) , and jurkat t ( dysfunctional p53 ) cells were incubated in 0.1% nbcs supplemented rpmi-1640 media for 24 hrs . the media was changed and the cells were treated with hexane fraction of ag ( 0500 g / ml ) as indicated in figure 2 . the dot plot of fs versus fl-1 and histogram plot of ( number of event ) versus ( fl-1 channel ) were plotted to obtain a percentage increase in the apoptosis of the hexane fraction of ag treated cells . positive control for apoptosis is the fixed and permeabilized cells treated with dnase i recombinant ( 3 u / ml in 50 mm tris - hcl , ph7.5 , and 1 mg / ml bsa ) ( invitrogen , ca , usa ) to induce dna strand breaks prior to labeling ( following the vendors protocol ) . among its regulatory mechanisms , down - regulation of activated t lymphocytes via apoptosis is a very potent and effective strategy , now considered as a key controlling mechanism of uc . to this end , lymphoblastoid cell lines tk6 ( wt p53 ) , nh32 ( isogenic to tk6 , but p53 ) , and jurkat t cells ( which have a dysfunctional p53 pathway ) were tested for the apoptosis inducing property of hag . previously , we showed the whole ag extract induces apoptosis of tk6 , but not nh32 cells . interestingly , here , we show hag was able to induce apoptosis ( tunel ) not only in tk6 cells but ( albeit to a lesser extent ) also in nh32 cells ( figures 2(a ) and 2(b ) ) . hag induced a 3.9-fold increase in apoptosis of tk6 cells at 500 g / ml of hag when compared to the untreated cells . interestingly , the jurkat t - cell line ( which has a defective p53 pathway ) was somewhat resistant to hag - induced apoptosis ( figure 2(c ) ) , which is consistent with what we have previously observed with the whole ag extract . overly aggressive cd4/cd25 t cells are thought to contribute to colitis , and defects in mucosal t - cell apoptosis are likely to be critical in the pathogenesis of colitis [ 4 , 6 , 7 ] . recently we have shown hag induces apoptosis of cd4/cd25 t cells derived from c57/bl6 wt mice during the suppression of colitis . in the present paper , consistent with results from tk6 ( p53 ) and nh32 ( p53 ) cells , hag induced apoptosis of the cd4/cd25 t cells isolated from both wt and p53 c57/bl6 mice ( table 1 ) . however , the induction of apoptosis was suppressed in the absence of p53 . of effector t cells isolated from p53 c57/bl6 mice and a 4.6-fold increase from ( 4.5 0.5% ) to ( 20.7 8.9% ) in the apoptosis was observed in cd4/cd25 t cells isolated from wt c57/bl6 mice upon treatment with increasing concentration of hag ( 0300 g / ml ) . we have recently shown that hag suppresses chemically ( dss mouse model ) induced colitis in the wt p53 c57/bl6 mice . previously we have also shown that ag whole extract suppresses chemically induced colitis only in the wt p53 c57/bl6 mice and not in the p53 c57/bl6 mice . as shown in the figure 3 , the hag was able to suppress the dss - induced colitis in both the p53 and wt p53 mice . however , consistent with our results on apoptosis of inflammatory cells in vitro , the suppression was more prominent in wt mice than in p53 mice . this result is consistent with the notion that the hag seems to act not only through a p53 pathway but also through a p53-independent pathway while suppressing colitis . thus , one way to cause cancer to subside is to prevent this abnormal cancer - cell growth by inducing cell cycle arrest . we tested this hypothesis in vitro by using two different cell lines , hct-116 p53 wt and hct-116 p53 , and treated these cells with increasing concentrations of hag for 24 hr . in three repeated experiments , hag was able to increase a g1 cell cycle arrest in both the wt and p53 hct-116 cells . ( 34% to 56.7% ) at 100 g / ml , hag treatment and the percentage of cells in g1 remained above 50% with the other concentrations ( up to 500 g / ml ) ( figure 4(a ) ) . similarly there was a 1.5-fold increase ( 34.1% to 51.2% ) of hct-116 p53 cells in g1 phase after treatment with 100 g / ml of hag ( figure 4(a ) ) , and this checkpoint remaining with the remaining concentrations . mostly polyacetylenes ( panaxynol , panaxydol , and panaxydiol ) and fatty acids ( fa ) present in the ag whole extract were extracted in this fraction . since hag is more effective at suppressing colitis than the whole ag extract and based on our previous study , where ag whole extract suppressed colitis through p53-mediated apoptosis of inflammatory cells , we hypothesized that hag also induces apoptosis of inflammatory cells through a p53-mediated mechanism . interestingly , we found that , unlike the whole ag extract , hag was able to induce modest apoptosis of p53 cd4/cd25 effector t cells and p53 lymphoblastoid cells in addition to their p53 wt counterpart cells ( figure 2 , table 1 ) . recently we have identified the different components present in the hag , with fas comprising 43% w / w , polyacetylenes 26.52% w / w ( 7.39% panaxydiol , 8.92% panaxydol , and 10.21% panaxynol ) , and less than 0.1% w / w ginsenosides . [ 45 , 46 ] have shown that the ginsenosides - rs3 and -rs4 selectively elevate protein levels of p53 and p21 , and downregulate the activities of the cyclin - dependent kinases , resulting in cell cycle arrest at the g1/s boundary and apoptosis of sk - hep-1 cells ( immortalized human hepatoma cells ) [ 45 , 46 ] . all these observations suggest that ginsenosides may be a key player in modulating apoptosis and cell cycle through the p53 pathway and are consistent with the findings here and our previous findings that our whole ag extract has 10.1% ginsenoside ( w / w ) compared to the hag which has 0.074% ginsenoside ( w / w ) content . interestingly , ginsenoside rd , which is present in ag whole extract ( extracted with aqueous ethanol ) and absent in the hag , has been shown to attenuate the inflammatory response to tnbs ( 2,4,6-trinotrobenzenesulfonic acid- ) induced colitis by down - regulating multiple pro - inflammatory cytokines through the modulation of jnk ( c - jun n - terminal kinase ) and p38 activation . similarly another ginsenoside not present in hag , rb1 , and its metabolite compound k after oral administration blocked tnbs - induced expression of inos ( inducible - nitric oxide synthase ) , cox-2 ( cyclooxygenase-2 ) , and nf- ( nuclear transcription factor ) activation in mice . ginsenoside rb1 and its metabolite compound inhibited the activation of key inflammatory mediators irak-1 ( interleukin-1 receptor associated kinase-1 ) , ikk- ( inhibitor of nf- kinase ) , nf- , and mapk ( mitogen - activated protein kinases ) while suppressing colitis . taken together , these observations suggest that multiple components of ag , including ginsenosides , fas , and polyacetylenes , are responsible for the activity of ag in the suppression of colitis in animals . have shown that during the g1/s cell cycle arrest , protein levels of p21 , p16 , p53 , prb ( retinoblastoma protein ) , and e2f-1 ( transcription factor e2f1 ) were not changed after exposure to the polyacetylene , panaxydol ( isolated from p. ginseng ) , in the human malignant melanoma cell line , sk - mel-1 . human promyelocytic leukemia hl60 cells do not express the p53 protein due to a large deletion in the gene . the polyacetylenes , panaxynol , and panaxydol ( isolated from the lipophilic fractions of panax notoginseng ) have been shown to inhibit the proliferation of hl60 cells in a time and dose - dependent manner via an apoptotic pathway . these studies indicate that polyacetylenes may act independently of p53 while inducing apoptosis of certain cell lines . hag - induced apoptosis indicated by cleaved parp in tk6 cells , appears to change the protein levels of wt p53 and activated form of p53 ( phospho - ser15 ) very little . after 24 hr of hag treatment ( 300 g / ml ) , we observed a 2.6-fold increase in apoptosis of p53 cd4/cd25 t cells and a 4.6-fold increase in apoptosis of p53 cd4/cd25 t cells when compared to untreated cells ( table 1 ) . figures 2(a ) , 2(b ) and 2(c ) , and our recent study are consistent and indicate that p53 has a limited role in inducing hag - mediated apoptosis of inflammatory cells both in vitro and ex vivo . a clearly increased p53 , phospho - p53 ser-15 , and cleaved parp protein levels along with the increased apoptosis were reported with the ag whole extract , suggesting p53 plays a key role in apoptosis of inflammatory cells induced by the whole ag extract . however , an increased cleaved parp protein expression and only a slight change in the p53 , phospho - p53 ( ser-15 ) , and p53-upregulated mediator of apoptosis ( puma ) protein expression of tk6 cells after treatment with hag are consistent with our current results that hag induces apoptosis but this induction of apoptosis is likely mediated through both p53-dependent and p53-independent mechanisms . this , then , is consistent with our results regarding a limited role for p53 in the cells undergoing apoptosis after treatment with hag . we further tested our compound in the in vivo animal study . as indicated ( figure 1 ) , both the p53 and p53 wt c57/bl6 mice were subjected to 2.5 cycles of 1% dss to induce colitis . in our previous published studies , ag whole extract was effective in suppressing colitis only in the p53 wt c57/bl6 mice where whole ag extract prevented colon epithelial cells from the dna damage due to dss and induced apoptosis of lymphocytes in vivo . interestingly , the hag also showed a modest suppression of inflammation to about one - half ( from 14.5 2.12 to 7.8 1.78 ) in the p53 c57/bl6 mice ( figure 3 ) . this provides evidence that the hag is anti - inflammatory and p53 plays a limited role in the hag - mediated suppression of colitis . hence one way to tackle the cancerous cells is to induce apoptosis of such cells . interestingly , both hct-116 p53 and p53 colon cancer cells were relatively resistant to apoptosis induced by hag ( data not shown ) . however , there was a g1 cell cycle checkpoint with hag treatment ( figure 4(a ) ) . it is more likely that one or more ingredients in hag target modulators of the cell cycle ( e.g. have shown that lipid soluble ginseng extract ( red ginseng extracted with n - hexane ) induces a cell cycle arrest at the g1 phase in nci - h460 cells ( human lung cancer cells ) . we have shown here that the hag can induce apoptosis in lymphoblastoid cells and cd4/cd25 effector t cells and cause a g1 checkpoint in colon cancer cell lines . in all the above observations , we simultaneously tested the pro - apoptotic and anti - inflammatory properties of hag with the wt p53 and its p53 counterpart cells and mice . it is evident that hag can perform its pro - apoptotic , anti - inflammatory , and cell cycle arrest activities even in the absence of p53 . however , the hag has a more robust effect in the presence of p53 . these observations suggest that there is a role of p53 in the hag - mediated apoptosis of inflammatory cells and suppression of colitis ; but this role is limited . this entails that the hag affects other pathways independent of p53 in the suppression of colitis . does this component inhibit the ability of components only seen in the hag fraction to drive p53-independent apoptosis and suppress p53-independent colitis ? because mutation in p53 is observed in most cancers , including colitis - driven colon cancer , the hag may be particularly effective in targeting both p53 and p53 inflammatory cells and cancer cells and hence particularly effective in inhibiting the colitis - to - cancer sequence .

among the cell types , epithelial cells , myeloid innate cells , effector t cells , regulatory cells , and b cells excessive effector t - cell cytokine secretion or defective regulatory t cells lead to the disease propagation of ibd . first , it must mediate effective absorption of fluids , nutrients , and minerals from the lumen across the epithelium and into the microcirculation and microvilli . dysfunction in this intestinal barrier leads to the dysfunction in the intestinal immune system , which has been implicated as the major mechanism by which chronic inflammation occurs in colitis . hence a common feature of ibd pathogenesis is the dysregulated effector t cell response to commensal microbiota . because the intestinal mucosa is constantly exposed to several antigens , the mucosal immune system have evolved several strategies to avoid an unnecessary and uncontrolled inflammatory reaction . once the antigen from the commensal microbiota has been eradicated , t lymphocytes of the intestinal mucosa require a method to attenuate the local immune response . one of the regulatory methods , downregulation of activated t lymphocytes via apoptosis , is a very potent and effective strategy , now considered as a key controlling mechanism of ibd . failure to regulate t - cell responses in the intestinal or colonic mucosa leads to an inappropriate and sustained injurious immunologic reaction [ 6 , 7 ] . because of their heightened activation and activity in ibd pathogenesis , effector t cells are considered an excellent target for therapeutics . cells undergoing apoptosis are characterized by the shrinkage and condensation of their cytoplasm , increased mitochondrial permeability , chromatin condensation into caps at the edge of the nucleus , dna fragmentation , and the appearance of plasma membrane blebs , often referred to as there are two signaling pathways for the cell death by apoptosis : the intrinsic and extrinsic pathways [ 9 , 10 ] . the intrinsic pathway is activated with growth factor deprivation , oncogene activation , or when dna damage is detected by cellular sensors such as ataxia telangiectasia mutated ( atm ) , ataxia telangiectasia and rad3-related ( atr ) , and tumor protein 53 ( p53 ) . this is an intrinsic approach for the apoptosis of damaged cells , where activated p53 plays an important role in mediating apoptosis in epithelial and inflammatory cells during the process of colitis [ 4 , 12 , 1417 ] . the post - translational modification ( often phosphorylation at serine 15 ) tends to elevate the level of wild - type ( wt ) p53 during inflammation . p53 plays at least 2 separate roles in the responses to therapeutic agents it is an important component of cellular checkpoints ( cell cycle arrest ) , and it can mediate apoptosis . four cellular functions are inappropriately regulated in the cancer cells : cellular proliferation , differentiation , chromosomal and genetic organization , and apoptosis ( reviewed in ) . cells undergo a g1 cell cycle arrest to allow the dna repair before replication and if the dna damage is beyond repair , the cells tend to undergo apoptosis . american ginseng ( ag , panax quinquefolius ) is grown in the eastern temperate forest areas of north america , from british columbia , southern quebec , ontario , minnesota , and wisconsin in the north , to oklahoma , the ozark plateau , and georgia in the south . ag has been reported to have a wide range of pharmacological effects , including effects on the central nervous system , blood - sugar levels , cardiovascular system , endocrine system , immune system , and cancer [ 4 , 26 , 27 ] . recently we have shown ag extract suppresses colitis by the accumulation and activation of p53 to induce apoptosis of inflammatory cells . to further delineate the active anti - inflammatory and proapoptotic components present in the ag a hexane fraction of ag ( hag ) showed increased anti - inflammatory and pro - apoptotic properties in the chemically induced mouse model of colitis . the elevation of wt p53 levels during inflammation resulting in apoptosis of inflammatory and damaged cells [ 4 , 29 , 30 ] led us to the notion that the active anti - inflammatory components present in hag might suppress colitis through the p53 pathway . as well , we have recently described the generation of the hag used in the present study . jurkat t cells are an immortalized line of t lymphocyte cells derived in the late 1970s from the peripheral blood of a 14-year - old male with t - cell leukemia . tk6 , nh32 , and jurkat t cells were maintained in exponentially growing suspension culture at 37c in a humidified 5% co2 atmosphere in rpmi 1640 supplemented with 10% heat - inactivated calf serum , 100 units / ml penicillin , 100 g / ml streptomycin , and 2 mmol / l l - glutamine . the purity of cd4 t cells was determined by obtaining a dot plot of fl-4 ( apc ) versus fl-2 ( pe ) and individual histogram plots of fl-4 and fl-2 versus the number of events ( supplementary figure 1 ) . hct-116 , human epithelial colon cancer cells line , proficient in p53 , and isogenic hct-116 p53 cells were maintained in exponentially growing adherent culture at 37c in a humidified 5% co2 atmosphere in rpmi 1640 supplemented with 10% heat - inactivated calf serum , 100 units / ml penicillin , 100 g / ml streptomycin , and 2 mmol / l l - glutamine . of note , currently the use of ginseng in human clinical trials can range anywhere from 200 mg to 9 g daily [ 38 , 39 ] . to determine whether the hag can prevent the onset of colitis , mice were fed 1% dss for 2.5 cycles ( 7 days dss , 7 days water making 1 cycle ) . histology score was determined by multiplying the percent involvement for each of the three following histologic features by the percent area of involvement : inflammation severity ( 0 , none ; 1 , minimal ; 2 , moderate ; 3 , severe ) , inflammation extent ( 0 , none ; 1 , mucosa ; 2 , mucosa and submucosa ; 3 , transmural ) , and extent of crypt damage ( 0 , none ; 1 , one third of crypt damaged ; 2 , two thirds of crypt damaged ; 3 , crypts lost , surface epithelium intact ; 4 , crypts lost , surface epithelium lost ) . since , dss - induced colitis in mice leads to the damage in colonic epithelial barrier and is characterized by extent and depth of inflammation , a grading or scoring system of inflammation with all these parameters provides an excellent measure of histologic assessment of dss - induced colitis . briefly , after treating blot with the chemiluminescent substrate ( pierce ecl ) for a minute , the blot was exposed to the hyperfilm in the dark ( exposure time was optimized based on the band signal obtained ) and the film was developed in an automatic x - ray film processor ( futura classic e automatic x - ray film processor , fisher industry , geneva , il , usa ) . the cells were harvested after 24 hrs of treatment and cell cycle assay was performed by labeling the dsdna of the cells with dapi ( 4,6-diamidino-2-phenylindole ) ( sigma - aldrich , mo , usa ) . based on the dna content , the different phases of the cell cycle was determined by using bd facsdiva software ( bd biosciences , san jose , ca , usa ) . the dot plot of fs versus fl-1 and histogram plot of ( number of event ) versus ( fl-1 channel ) were plotted to obtain a percentage increase in the apoptosis of the hexane fraction of ag treated cells . positive control for apoptosis is the fixed and permeabilized cells treated with dnase i recombinant ( 3 u / ml in 50 mm tris - hcl , ph7.5 , and 1 mg / ml bsa ) ( invitrogen , ca , usa ) to induce dna strand breaks prior to labeling ( following the vendors protocol ) . among its regulatory mechanisms , down - regulation of activated t lymphocytes via apoptosis is a very potent and effective strategy , now considered as a key controlling mechanism of uc . to this end , lymphoblastoid cell lines tk6 ( wt p53 ) , nh32 ( isogenic to tk6 , but p53 ) , and jurkat t cells ( which have a dysfunctional p53 pathway ) were tested for the apoptosis inducing property of hag . interestingly , here , we show hag was able to induce apoptosis ( tunel ) not only in tk6 cells but ( albeit to a lesser extent ) also in nh32 cells ( figures 2(a ) and 2(b ) ) . hag induced a 3.9-fold increase in apoptosis of tk6 cells at 500 g / ml of hag when compared to the untreated cells . also , at 100 g / ml , the hag induced 2.6-fold increase in apoptosis in the tk6 cells , but a 2-fold increase in apoptosis in the nh32 cells . interestingly , the jurkat t - cell line ( which has a defective p53 pathway ) was somewhat resistant to hag - induced apoptosis ( figure 2(c ) ) , which is consistent with what we have previously observed with the whole ag extract . overly aggressive cd4/cd25 t cells are thought to contribute to colitis , and defects in mucosal t - cell apoptosis are likely to be critical in the pathogenesis of colitis [ 4 , 6 , 7 ] . in the present paper , consistent with results from tk6 ( p53 ) and nh32 ( p53 ) cells , hag induced apoptosis of the cd4/cd25 t cells isolated from both wt and p53 c57/bl6 mice ( table 1 ) . of effector t cells isolated from p53 c57/bl6 mice and a 4.6-fold increase from ( 4.5 0.5% ) to ( 20.7 8.9% ) in the apoptosis was observed in cd4/cd25 t cells isolated from wt c57/bl6 mice upon treatment with increasing concentration of hag ( 0300 g / ml ) . previously we have also shown that ag whole extract suppresses chemically induced colitis only in the wt p53 c57/bl6 mice and not in the p53 c57/bl6 mice . as shown in the figure 3 , the hag was able to suppress the dss - induced colitis in both the p53 and wt p53 mice . however , consistent with our results on apoptosis of inflammatory cells in vitro , the suppression was more prominent in wt mice than in p53 mice . the mitogenic stimuli triggered signal transduction pathways eventually converge on the cell cycle checkpoint that controls the g1 to s phase transition and activate appropriate cyclin - dependent kinases . we tested this hypothesis in vitro by using two different cell lines , hct-116 p53 wt and hct-116 p53 , and treated these cells with increasing concentrations of hag for 24 hr . ( 34% to 56.7% ) at 100 g / ml , hag treatment and the percentage of cells in g1 remained above 50% with the other concentrations ( up to 500 g / ml ) ( figure 4(a ) ) . similarly there was a 1.5-fold increase ( 34.1% to 51.2% ) of hct-116 p53 cells in g1 phase after treatment with 100 g / ml of hag ( figure 4(a ) ) , and this checkpoint remaining with the remaining concentrations . mostly polyacetylenes ( panaxynol , panaxydol , and panaxydiol ) and fatty acids ( fa ) present in the ag whole extract were extracted in this fraction . since hag is more effective at suppressing colitis than the whole ag extract and based on our previous study , where ag whole extract suppressed colitis through p53-mediated apoptosis of inflammatory cells , we hypothesized that hag also induces apoptosis of inflammatory cells through a p53-mediated mechanism . interestingly , we found that , unlike the whole ag extract , hag was able to induce modest apoptosis of p53 cd4/cd25 effector t cells and p53 lymphoblastoid cells in addition to their p53 wt counterpart cells ( figure 2 , table 1 ) . recently we have identified the different components present in the hag , with fas comprising 43% w / w , polyacetylenes 26.52% w / w ( 7.39% panaxydiol , 8.92% panaxydol , and 10.21% panaxynol ) , and less than 0.1% w / w ginsenosides . [ 45 , 46 ] have shown that the ginsenosides - rs3 and -rs4 selectively elevate protein levels of p53 and p21 , and downregulate the activities of the cyclin - dependent kinases , resulting in cell cycle arrest at the g1/s boundary and apoptosis of sk - hep-1 cells ( immortalized human hepatoma cells ) [ 45 , 46 ] . all these observations suggest that ginsenosides may be a key player in modulating apoptosis and cell cycle through the p53 pathway and are consistent with the findings here and our previous findings that our whole ag extract has 10.1% ginsenoside ( w / w ) compared to the hag which has 0.074% ginsenoside ( w / w ) content . interestingly , ginsenoside rd , which is present in ag whole extract ( extracted with aqueous ethanol ) and absent in the hag , has been shown to attenuate the inflammatory response to tnbs ( 2,4,6-trinotrobenzenesulfonic acid- ) induced colitis by down - regulating multiple pro - inflammatory cytokines through the modulation of jnk ( c - jun n - terminal kinase ) and p38 activation . similarly another ginsenoside not present in hag , rb1 , and its metabolite compound k after oral administration blocked tnbs - induced expression of inos ( inducible - nitric oxide synthase ) , cox-2 ( cyclooxygenase-2 ) , and nf- ( nuclear transcription factor ) activation in mice . ginsenoside rb1 and its metabolite compound inhibited the activation of key inflammatory mediators irak-1 ( interleukin-1 receptor associated kinase-1 ) , ikk- ( inhibitor of nf- kinase ) , nf- , and mapk ( mitogen - activated protein kinases ) while suppressing colitis . these studies suggest that different ginsenosides may help attenuate inflammation , and our finding that n - hexane does not extract these ginsenosides from ginseng should not rule out that ginsenosides are not the active components of ginseng . taken together , these observations suggest that multiple components of ag , including ginsenosides , fas , and polyacetylenes , are responsible for the activity of ag in the suppression of colitis in animals . have shown that during the g1/s cell cycle arrest , protein levels of p21 , p16 , p53 , prb ( retinoblastoma protein ) , and e2f-1 ( transcription factor e2f1 ) were not changed after exposure to the polyacetylene , panaxydol ( isolated from p. ginseng ) , in the human malignant melanoma cell line , sk - mel-1 . the polyacetylenes , panaxynol , and panaxydol ( isolated from the lipophilic fractions of panax notoginseng ) have been shown to inhibit the proliferation of hl60 cells in a time and dose - dependent manner via an apoptotic pathway . hag - induced apoptosis indicated by cleaved parp in tk6 cells , appears to change the protein levels of wt p53 and activated form of p53 ( phospho - ser15 ) very little . after 24 hr of hag treatment ( 300 g / ml ) , we observed a 2.6-fold increase in apoptosis of p53 cd4/cd25 t cells and a 4.6-fold increase in apoptosis of p53 cd4/cd25 t cells when compared to untreated cells ( table 1 ) . figures 2(a ) , 2(b ) and 2(c ) , and our recent study are consistent and indicate that p53 has a limited role in inducing hag - mediated apoptosis of inflammatory cells both in vitro and ex vivo . a clearly increased p53 , phospho - p53 ser-15 , and cleaved parp protein levels along with the increased apoptosis were reported with the ag whole extract , suggesting p53 plays a key role in apoptosis of inflammatory cells induced by the whole ag extract . however , an increased cleaved parp protein expression and only a slight change in the p53 , phospho - p53 ( ser-15 ) , and p53-upregulated mediator of apoptosis ( puma ) protein expression of tk6 cells after treatment with hag are consistent with our current results that hag induces apoptosis but this induction of apoptosis is likely mediated through both p53-dependent and p53-independent mechanisms . have reported that linoleic acid treatment resulted in a concentration - dependent growth inhibition of ags cells ( human gastric adenocarcinoma cells ) by inducing apoptosis in a p53-independent manner with an elevated fas and fas ligand expression . in our previous published studies , ag whole extract was effective in suppressing colitis only in the p53 wt c57/bl6 mice where whole ag extract prevented colon epithelial cells from the dna damage due to dss and induced apoptosis of lymphocytes in vivo . in the present study , with the same protocol , the hag was able to suppress dss - induced colonic inflammation to approximately one - third ( from 15 0.86 to 4.4 0.32 ) . interestingly , the hag also showed a modest suppression of inflammation to about one - half ( from 14.5 2.12 to 7.8 1.78 ) in the p53 c57/bl6 mice ( figure 3 ) . have shown that lipid soluble ginseng extract ( red ginseng extracted with n - hexane ) induces a cell cycle arrest at the g1 phase in nci - h460 cells ( human lung cancer cells ) . we have shown here that the hag can induce apoptosis in lymphoblastoid cells and cd4/cd25 effector t cells and cause a g1 checkpoint in colon cancer cell lines . in all the above observations , we simultaneously tested the pro - apoptotic and anti - inflammatory properties of hag with the wt p53 and its p53 counterpart cells and mice . these observations suggest that there is a role of p53 in the hag - mediated apoptosis of inflammatory cells and suppression of colitis ; but this role is limited . because mutation in p53 is observed in most cancers , including colitis - driven colon cancer , the hag may be particularly effective in targeting both p53 and p53 inflammatory cells and cancer cells and hence particularly effective in inhibiting the colitis - to - cancer sequence .

they have antipruritic , anti - inflammatory , anti - proliferative and pigment - lightening activity on the skin . a recent prescription audit from a dermatology opd revealed that close to 30% of all prescriptions contained a tc . an important reason for such widespread use is their ability to produce rapid alleviation of unpleasant signs and symptoms on the skin . symptoms such as itch , stinging and tenderness are rapidly relieved regardless of the cause , as are redness , scaling and hyperpigmentation . unfortunately , this improvement may be short - lived and can be followed by worsening of the original condition if tcs are not used correctly or used in conditions where they are not indicated at all . the natural instinct of every physician is to give rapid relief to a patient who is distressed and tcs are ideally suited for such a role . this is because tcs are highly susceptible to misuse by patients , pharmacists and physicians themselves as several studies have shown . if not properly counseled , patients may not return for a follow - up and may continue to use the tc for prolonged periods . such improper use can have devastating consequences , both local and systemic , in the long run . it is therefore essential that we develop a clear understanding of the ethics of tc prescribing so that they are used optimally for the benefit of our patients . in our country , easy availability of mild to superpotent tcs even without a prescription coupled with rampant sharing of tcs by patients has created a situation where they seem to be more misused then used correctly . on the other hand , steroid phobia is becoming increasingly common in the internet - savvy population in the cities creating new problems in prescription of tcs . in this backdrop , ethical and careful prescription of these powerful agents is the need of the hour . awareness of these issues is essential so that pitfalls can be avoided and a good doctor - patient relationship be established . autonomy : this refers to respect for the individuals right to make informed decisions about their personal matters . in other words , the patient has the right to refuse or choose their treatment ( voluntas aegroti suprema lex . ) this is in contrast to the older paternalistic style of medicine where patients were simply advised on what to take without any discussion of alternatives , risk vs. benefit etc . , with regards to tcs , this law applies when we are faced with a steroid - phobic patient . in this situation , is often a good approach whereby such a patient should be counseled about the potential benefits of tc use , the harm that can be caused by non - use and how common adverse effects can be avoided . however , they should also be informed about alternative therapies e.g. topical calcineurin inhibitors or calcipotriol and their efficacy vis - - vis tcs . after this , whatever their ultimate decision be , it should be respected.non-maleficence : this is the principle of primum non nocere ( first , do no harm ) a common scenario where non - maleficence needs to be remembered is a patient who is obsessed with a fair complexion and asks for a tc prescription which he / she may have heard about from a friend . it is absolutely essential to avoid the temptation to give in to the patient 's request in this situation . non - maleficence also becomes relevant when we are dealing with an undiagnosed rash and a demanding , distressed patient . several studies have been reported on the misuse of tcs , especially on the face where the initial prescription was given for an undiagnosed facial rash which then led to prolonged misuse of tcs leading to considerable morbidity . in such a scenario , pending a diagnosis , tcs should be avoided and relief given with safer options like topical antipruritics and oral antihistamines . in the absence of a clear diagnosis and treatment plan , a physician should not hesitate to refer a patient to a senior colleague or an expert instead of using tcs to suppress unpleasant symptoms indefinitely.beneficence vs. autonomy : this kind of conflict occurs when patients disagree with recommendations that doctors believe are in the patients best interest . an appropriate example would be a patient with widespread eczema who is applying potent tcs over a large area leading to adverse effects . the appropriate approach in such a situation would be to start a systemic steroid - sparing drug like azathioprine and gradually taper the tc . appropriate counseling about the dangers of potent tc use over large areas along with laboratory demonstration of adrenal suppression etc . autonomy : this refers to respect for the individuals right to make informed decisions about their personal matters . in other words , the patient has the right to refuse or choose their treatment ( voluntas aegroti suprema lex . ) this is in contrast to the older paternalistic style of medicine where patients were simply advised on what to take without any discussion of alternatives , risk vs. benefit etc . , with regards to tcs , this law applies when we are faced with a steroid - phobic patient . in this situation , is often a good approach whereby such a patient should be counseled about the potential benefits of tc use , the harm that can be caused by non - use and how common adverse effects can be avoided . however , they should also be informed about alternative therapies e.g. topical calcineurin inhibitors or calcipotriol and their efficacy vis - - vis tcs . non - maleficence : this is the principle of primum non nocere ( first , do no harm ) . a common scenario where non - maleficence needs to be remembered is a patient who is obsessed with a fair complexion and asks for a tc prescription which he / she may have heard about from a friend . it is absolutely essential to avoid the temptation to give in to the patient 's request in this situation . non - maleficence also becomes relevant when we are dealing with an undiagnosed rash and a demanding , distressed patient . several studies have been reported on the misuse of tcs , especially on the face where the initial prescription was given for an undiagnosed facial rash which then led to prolonged misuse of tcs leading to considerable morbidity . in such a scenario , pending a diagnosis , tcs should be avoided and relief given with safer options like topical antipruritics and oral antihistamines . in the absence of a clear diagnosis and treatment plan , a physician should not hesitate to refer a patient to a senior colleague or an expert instead of using tcs to suppress unpleasant symptoms indefinitely . beneficence vs. autonomy : this kind of conflict occurs when patients disagree with recommendations that doctors believe are in the patients best interest . an appropriate example would be a patient with widespread eczema who is applying potent tcs over a large area leading to adverse effects . the appropriate approach in such a situation would be to start a systemic steroid - sparing drug like azathioprine and gradually taper the tc . appropriate counseling about the dangers of potent tc use over large areas along with laboratory demonstration of adrenal suppression etc . in fact , effective communication is the key to resolving almost all ethical dilemmas faced by a physician prescribing tcs . not only doctor - patient , but communication between doctors of different specialties and between doctors and society in general is essential in resolving these ethical issues . correct use of tcs is predicated on the fulfilling of the following conditions : the right diagnosis : there are relatively few conditions where there is good evidence of efficacy of tcs . various eczemas , psoriasis , lichen planus , immunobullous diseases in their localized form , skin manifestations of collagen vascular diseases like lupus erythematosus or dermatomyositis are the best established indications . other conditions where careful , short - term use may be warranted are superficial fungal or bacterial infections associated with significant inflammation , localized itch of any origin and certain idiopathic diseases characterized by dermal inflammation e.g. superficial variants of pyoderma gangrenosum . in the absence of a diagnosis , it is very important to avoid tc use so that conditions like tinea incognito and majocchi 's granuloma are not produced . if a patient is already applying tc thereby obscuring the clinical features of a disease , an appropriate treatment - free interval should be given after explaining to the patient that his / her symptoms may flare up temporarily , to facilitate a diagnosis.the right molecule and delivery system : since they were first introduced in the early 1950s , tcs have grown tremendously as a class and today , more than 20 different molecules of varying potency are available worldwide , ranging from mild to superpotent . in addition , there are varying concentrations and dosage forms available , namely creams , gels , lotions , ointments , foams , muco - adherent gels , aerosols and tapes . it is important for the prescriber to be familiar with at least a few of the molecules , preferably of differing potencies . the characteristics of the various dosage forms should also be thoroughly understood so that the correct drug in the correct vehicle can be given in a particular situation . although it is generally good to err on the side of caution when prescribing tcs , in many situations , it is better to prescribe a more potent molecule to control the disease quickly and stop treatment fast instead of prolonged treatment with a mild formulation . this is especially true of self - limited conditions like acute irritant dermatitis , paederous dermatitis etc.the right patient : the age , sex and occupation of the patient play an important role in determining tc use . very young and very old patients have impaired epidermal barrier function and relatively thinner skin . in addition , infants have a very high surface area to weight ratio that can lead to disproportionately high systemic absorption from topical application . women who do housework often have damaged skin due to frequent wet - dry cycling . this creates the need for more frequent application , as do certain occupations involving manual labor . other factors like the anatomical location and extent of the disease also dictate which formulation is optimal . thin skin areas like eyelids , face and scrotum need milder products than back , palms and soles . widespread dermatoses demand easily spreadable dosage forms and milder potencies to guard against undesirable systemic absorption . cosmetic considerations are paramount when tcs are applied on the face or other visible parts and creams , gels and lotions are preferred here due to their elegance . intertriginous areas are particularly susceptible to stronger than expected effect due to the occlusion and maceration of the skin and therefore we should err on the side of caution when deciding the potency on these sites.the right amount , frequency and duration : perhaps the commonest omission in a tc prescription is advice regarding the right amount to be applied in a particular situation . in this regard , the fingertip unit ( ftu ) devised by long and finlay is the simplest way to explain to the patient how much tc is to be applied . an ftu is the amount of ointment that occupies the space from the fingertip to the first skin crease when squeezed out of a 5 mm nozzle . this amount is enough to cover a palm - sized area on the skin in the case of creams and about 20% more with ointments due to their increased spreadability . standard recommendations have been developed to guide how many ftus are required to cover particular anatomical areas . this should be clearly conveyed to the patient to prevent over- or underuse.the frequency of application is dependent on patient factors such as site and occupation . hands and feet often need twice or thrice a day application due to the propensity of being rubbed off . otherwise , most experts agree that once daily application is optimal for all other sites since more frequent application has shown no added benefit and adherence is also expected to be higher with this regimen.the duration of treatment needs to be clearly told to the patient orally as well as in writing and he / she should also be apprised of the dangers of overuse at this point . in general , a follow - up visit should be planned after 2 to 4 weeks to review the progress and institute the exit strategy.the right exit strategy : it is extremely important to have a clear plan in mind about tapering and stopping tc use after adequate control / remission has been achieved . except for self - limiting conditions , tc use is often needed for extended periods , much longer than recommended safe durations . this can be done safely by tapering down to progressively less potent preparations or instituting alternate - day or weekends - only treatment . emollients and/or steroid - sparing drugs like calcineurin inhibitors are prescribed on tc - free days . finding the right exit strategy can involve a lot of empiricism but is a worthwhile skill to learn for every physician.focusing on prevention and modifiable factors : in many conditions , lasting remission or cure is only achievable if preventive actions are taken or behavioral modifications are done . recurrent irritant hand dermatitis , pseudofolliculitis barbae and intertrigo are good examples of these conditions . physicians should ensure that the quick relief provided by tcs does not make them complacent about tackling deeper issues . they should explain to the patients that relief will be transient if preventive actions like skin protection and moisturizing ( hand eczema ) , changing shaving habits ( pseudofolliculitis barbae ) and keeping the skin dry , losing weight and wearing loose natural fabrics ( intertrigo ) are not done.being aware of corticosteroid allergy : many times we are faced with a patient who does not show the desired improvement even if appropriate tc is being used . at other times , control is achieved , only to lead to episodes of increased disease activity which are controlled with increasingly higher potency corticosteroids . in these situations this can happen both due to the preservatives / other excipients or the active molecule per se . in these cases , appropriate patch testing followed by prescription of an allergen - free product should be done.effective communication : involving and informing the patient at every step of diagnosis and treatment is the key to using tcs safely , ethically and effectively . a few scenarios are presented below to exemplify this point.patients often self - treat themselves with over - the - counter tcs before coming to the physician , altering the appearance of the skin and making diagnosis difficult . in such a situation , it behooves us to explain the situation to the patient and withdraw all tcs while relevant investigations are done . even if no investigations are done , a steroid - free interval often renders the disease recognizable , allowing proper treatment to be started.patients often neglect to come for follow - up when asked to , either due to financial constraints such as loss of wages / inability to pay re - visit fees or simply due to negligence . to minimize the chances of adverse effects of tc , partial control of the treatment should be handed over to the patient , thus making them partners in their well - being , not just the recipients of instructions . to this end , they can be told at which point they should start alternate day treatment , introduce a calcineurin inhibitor or switch over to a milder tc . with this , they should also be told the warning signs of common adverse effects like folliculitis which would necessitate a re - visit immediately.many patients are unsure how long it would take for the effect of tcs to start and therefore either become anxious too quickly or continue to use an ineffective tc preparation till the next scheduled visit . to avoid this , patients should be clearly told approximately how long it would take for the beneficial effects of the tc to kick in . they should be counseled to walk in if they are not feeling substantially better at this point.corticosteroid phobia is an increasingly common problem in urban and internet - savvy patients . a lot of counseling and reassurance is needed if these patients or their dependents are not to be denied the benefits of tcs . they also need to be told about the non - tc options available and their relative efficacy . if they still choose not to use tcs , their choice should be respected and an appropriate regimen devised . the right diagnosis : there are relatively few conditions where there is good evidence of efficacy of tcs . various eczemas , psoriasis , lichen planus , immunobullous diseases in their localized form , skin manifestations of collagen vascular diseases like lupus erythematosus or dermatomyositis are the best established indications . other conditions where careful , short - term use may be warranted are superficial fungal or bacterial infections associated with significant inflammation , localized itch of any origin and certain idiopathic diseases characterized by dermal inflammation e.g. superficial variants of pyoderma gangrenosum . in the absence of a diagnosis , it is very important to avoid tc use so that conditions like tinea incognito and majocchi 's granuloma are not produced . if a patient is already applying tc thereby obscuring the clinical features of a disease , an appropriate treatment - free interval should be given after explaining to the patient that his / her symptoms may flare up temporarily , to facilitate a diagnosis . the right molecule and delivery system : since they were first introduced in the early 1950s , tcs have grown tremendously as a class and today , more than 20 different molecules of varying potency are available worldwide , ranging from mild to superpotent . in addition , there are varying concentrations and dosage forms available , namely creams , gels , lotions , ointments , foams , muco - adherent gels , aerosols and tapes . it is important for the prescriber to be familiar with at least a few of the molecules , preferably of differing potencies . the characteristics of the various dosage forms should also be thoroughly understood so that the correct drug in the correct vehicle can be given in a particular situation . although it is generally good to err on the side of caution when prescribing tcs , in many situations , it is better to prescribe a more potent molecule to control the disease quickly and stop treatment fast instead of prolonged treatment with a mild formulation . this is especially true of self - limited conditions like acute irritant dermatitis , paederous dermatitis etc . the right patient : the age , sex and occupation of the patient play an important role in determining tc use . very young and very old patients have impaired epidermal barrier function and relatively thinner skin . in addition , infants have a very high surface area to weight ratio that can lead to disproportionately high systemic absorption from topical application . women who do housework often have damaged skin due to frequent wet - dry cycling . this creates the need for more frequent application , as do certain occupations involving manual labor . other factors like the anatomical location and extent of the disease also dictate which formulation is optimal . thin skin areas like eyelids , face and scrotum need milder products than back , palms and soles . widespread dermatoses demand easily spreadable dosage forms and milder potencies to guard against undesirable systemic absorption . cosmetic considerations are paramount when tcs are applied on the face or other visible parts and creams , gels and lotions are preferred here due to their elegance . intertriginous areas are particularly susceptible to stronger than expected effect due to the occlusion and maceration of the skin and therefore we should err on the side of caution when deciding the potency on these sites . the right amount , frequency and duration : perhaps the commonest omission in a tc prescription is advice regarding the right amount to be applied in a particular situation . in this regard , the fingertip unit ( ftu ) devised by long and finlay is the simplest way to explain to the patient how much tc is to be applied . an ftu is the amount of ointment that occupies the space from the fingertip to the first skin crease when squeezed out of a 5 mm nozzle . this amount is enough to cover a palm - sized area on the skin in the case of creams and about 20% more with ointments due to their increased spreadability . standard recommendations have been developed to guide how many ftus are required to cover particular anatomical areas . hands and feet often need twice or thrice a day application due to the propensity of being rubbed off . otherwise , most experts agree that once daily application is optimal for all other sites since more frequent application has shown no added benefit and adherence is also expected to be higher with this regimen . the duration of treatment needs to be clearly told to the patient orally as well as in writing and he / she should also be apprised of the dangers of overuse at this point . in general , a follow - up visit should be planned after 2 to 4 weeks to review the progress and institute the exit strategy . the right exit strategy : it is extremely important to have a clear plan in mind about tapering and stopping tc use after adequate control / remission has been achieved . except for self - limiting conditions , tc use is often needed for extended periods , much longer than recommended safe durations . this can be done safely by tapering down to progressively less potent preparations or instituting alternate - day or weekends - only treatment . emollients and/or steroid - sparing drugs like calcineurin inhibitors are prescribed on tc - free days . finding the right exit strategy can involve a lot of empiricism but is a worthwhile skill to learn for every physician . focusing on prevention and modifiable factors : in many conditions , lasting remission or cure is only achievable if preventive actions are taken or behavioral modifications are done . recurrent irritant hand dermatitis , pseudofolliculitis barbae and intertrigo are good examples of these conditions . physicians should ensure that the quick relief provided by tcs does not make them complacent about tackling deeper issues . they should explain to the patients that relief will be transient if preventive actions like skin protection and moisturizing ( hand eczema ) , changing shaving habits ( pseudofolliculitis barbae ) and keeping the skin dry , losing weight and wearing loose natural fabrics ( intertrigo ) are not done . being aware of corticosteroid allergy : many times we are faced with a patient who does not show the desired improvement even if appropriate tc is being used . at other times , control is achieved , only to lead to episodes of increased disease activity which are controlled with increasingly higher potency corticosteroids . in these situations this can happen both due to the preservatives / other excipients or the active molecule per se . in these cases , appropriate patch testing followed by prescription of an allergen - free product should be done . effective communication : involving and informing the patient at every step of diagnosis and treatment is the key to using tcs safely , ethically and effectively . patients often self - treat themselves with over - the - counter tcs before coming to the physician , altering the appearance of the skin and making diagnosis difficult . in such a situation , it behooves us to explain the situation to the patient and withdraw all tcs while relevant investigations are done . even if no investigations are done , a steroid - free interval often renders the disease recognizable , allowing proper treatment to be started . patients often neglect to come for follow - up when asked to , either due to financial constraints such as loss of wages / inability to pay re - visit fees or simply due to negligence . to minimize the chances of adverse effects of tc , partial control of the treatment should be handed over to the patient , thus making them partners in their well - being , not just the recipients of instructions . to this end , they can be told at which point they should start alternate day treatment , introduce a calcineurin inhibitor or switch over to a milder tc . with this , they should also be told the warning signs of common adverse effects like folliculitis which would necessitate a re - visit immediately . many patients are unsure how long it would take for the effect of tcs to start and therefore either become anxious too quickly or continue to use an ineffective tc preparation till the next scheduled visit . to avoid this , patients should be clearly told approximately how long it would take for the beneficial effects of the tc to kick in . they should be counseled to walk in if they are not feeling substantially better at this point . a lot of counseling and reassurance is needed if these patients or their dependents are not to be denied the benefits of tcs . they also need to be told about the non - tc options available and their relative efficacy . if they still choose not to use tcs , their choice should be respected and an appropriate regimen devised . today , both patients and physicians contribute to the issues associated with rational and ethical use of topical corticosteroids . patients are prone to misuse them due to their unrestricted availability and poor awareness about their adverse effects . a smaller but growing section of patients is gravitating toward the other extreme , i.e. an unwarranted fear of topical corticosteroids largely driven by an alarmist media . dermatologists who number about 7000 in our country are grossly insufficient to care for our vast population and are consequently overworked and often lack the time and skills to counsel the patients regarding safe use . other specialists and general practitioners who probably see more dermatology patients than dermatologists suffer from grossly inadequate dermatology training . therefore , it is imperative that increased awareness about ethical use of these agents is created amongst all caregivers so that they are used to their fullest extent and with maximum safety for the benefit of humanity . ethical conflicts are common when prescribing topical corticosteroids and we need to be aware of them to avoid pitfalls.examples of various clinical scenarios are used to exemplify common issues in topical corticosteroid prescriptions.practical pointers are given to help physicians use these agents in the best possible manner i.e. maximizing efficacy and minimizing adverse effects . ethical conflicts are common when prescribing topical corticosteroids and we need to be aware of them to avoid pitfalls . examples of various clinical scenarios are used to exemplify common issues in topical corticosteroid prescriptions . practical pointers are given to help physicians use these agents in the best possible manner i.e. maximizing efficacy and minimizing adverse effects .

dermatologists rely very heavily on corticosteroids for treating many common dermatoses . concerns about their incorrect use are widely expressed both in lay public and specialist discourse . from the point of view of medical ethics , issues of autonomy , beneficence and non - maleficence are all raised frequently when we prescribe topical corticosteroids to our patients . we need to be aware of situations when conflicts between these issues arise and have a clear thought process about resolving them . this can only be achieved if we have a thorough understanding of the skin disease being treated coupled with expertise in the use of the varied potencies and available dosage forms of topical corticosteroids . a good understanding of human psychology and effective communication is also needed to use these agents optimally .

Introduction What are The Ethical Issues That are Relevant to Topical Corticosteroid Use? What are The Parameters of Proper Use of Topical Corticosteroids? Conclusion

they have antipruritic , anti - inflammatory , anti - proliferative and pigment - lightening activity on the skin . a recent prescription audit from a dermatology opd revealed that close to 30% of all prescriptions contained a tc . an important reason for such widespread use is their ability to produce rapid alleviation of unpleasant signs and symptoms on the skin . symptoms such as itch , stinging and tenderness are rapidly relieved regardless of the cause , as are redness , scaling and hyperpigmentation . unfortunately , this improvement may be short - lived and can be followed by worsening of the original condition if tcs are not used correctly or used in conditions where they are not indicated at all . the natural instinct of every physician is to give rapid relief to a patient who is distressed and tcs are ideally suited for such a role . if not properly counseled , patients may not return for a follow - up and may continue to use the tc for prolonged periods . such improper use can have devastating consequences , both local and systemic , in the long run . it is therefore essential that we develop a clear understanding of the ethics of tc prescribing so that they are used optimally for the benefit of our patients . in our country , easy availability of mild to superpotent tcs even without a prescription coupled with rampant sharing of tcs by patients has created a situation where they seem to be more misused then used correctly . on the other hand , steroid phobia is becoming increasingly common in the internet - savvy population in the cities creating new problems in prescription of tcs . in this backdrop , ethical and careful prescription of these powerful agents is the need of the hour . awareness of these issues is essential so that pitfalls can be avoided and a good doctor - patient relationship be established . autonomy : this refers to respect for the individuals right to make informed decisions about their personal matters . this is in contrast to the older paternalistic style of medicine where patients were simply advised on what to take without any discussion of alternatives , risk vs. benefit etc . , with regards to tcs , this law applies when we are faced with a steroid - phobic patient . in this situation , is often a good approach whereby such a patient should be counseled about the potential benefits of tc use , the harm that can be caused by non - use and how common adverse effects can be avoided . topical calcineurin inhibitors or calcipotriol and their efficacy vis - - vis tcs . after this , whatever their ultimate decision be , it should be respected.non-maleficence : this is the principle of primum non nocere ( first , do no harm ) a common scenario where non - maleficence needs to be remembered is a patient who is obsessed with a fair complexion and asks for a tc prescription which he / she may have heard about from a friend . it is absolutely essential to avoid the temptation to give in to the patient 's request in this situation . non - maleficence also becomes relevant when we are dealing with an undiagnosed rash and a demanding , distressed patient . in the absence of a clear diagnosis and treatment plan , a physician should not hesitate to refer a patient to a senior colleague or an expert instead of using tcs to suppress unpleasant symptoms indefinitely.beneficence vs. autonomy : this kind of conflict occurs when patients disagree with recommendations that doctors believe are in the patients best interest . an appropriate example would be a patient with widespread eczema who is applying potent tcs over a large area leading to adverse effects . the appropriate approach in such a situation would be to start a systemic steroid - sparing drug like azathioprine and gradually taper the tc . autonomy : this refers to respect for the individuals right to make informed decisions about their personal matters . in other words , the patient has the right to refuse or choose their treatment ( voluntas aegroti suprema lex . ) , with regards to tcs , this law applies when we are faced with a steroid - phobic patient . in this situation , is often a good approach whereby such a patient should be counseled about the potential benefits of tc use , the harm that can be caused by non - use and how common adverse effects can be avoided . topical calcineurin inhibitors or calcipotriol and their efficacy vis - - vis tcs . non - maleficence : this is the principle of primum non nocere ( first , do no harm ) . a common scenario where non - maleficence needs to be remembered is a patient who is obsessed with a fair complexion and asks for a tc prescription which he / she may have heard about from a friend . non - maleficence also becomes relevant when we are dealing with an undiagnosed rash and a demanding , distressed patient . several studies have been reported on the misuse of tcs , especially on the face where the initial prescription was given for an undiagnosed facial rash which then led to prolonged misuse of tcs leading to considerable morbidity . in such a scenario , pending a diagnosis , tcs should be avoided and relief given with safer options like topical antipruritics and oral antihistamines . in the absence of a clear diagnosis and treatment plan , a physician should not hesitate to refer a patient to a senior colleague or an expert instead of using tcs to suppress unpleasant symptoms indefinitely . beneficence vs. autonomy : this kind of conflict occurs when patients disagree with recommendations that doctors believe are in the patients best interest . appropriate counseling about the dangers of potent tc use over large areas along with laboratory demonstration of adrenal suppression etc . in fact , effective communication is the key to resolving almost all ethical dilemmas faced by a physician prescribing tcs . correct use of tcs is predicated on the fulfilling of the following conditions : the right diagnosis : there are relatively few conditions where there is good evidence of efficacy of tcs . in the absence of a diagnosis , it is very important to avoid tc use so that conditions like tinea incognito and majocchi 's granuloma are not produced . if a patient is already applying tc thereby obscuring the clinical features of a disease , an appropriate treatment - free interval should be given after explaining to the patient that his / her symptoms may flare up temporarily , to facilitate a diagnosis.the right molecule and delivery system : since they were first introduced in the early 1950s , tcs have grown tremendously as a class and today , more than 20 different molecules of varying potency are available worldwide , ranging from mild to superpotent . in addition , there are varying concentrations and dosage forms available , namely creams , gels , lotions , ointments , foams , muco - adherent gels , aerosols and tapes . it is important for the prescriber to be familiar with at least a few of the molecules , preferably of differing potencies . the characteristics of the various dosage forms should also be thoroughly understood so that the correct drug in the correct vehicle can be given in a particular situation . this is especially true of self - limited conditions like acute irritant dermatitis , paederous dermatitis etc.the right patient : the age , sex and occupation of the patient play an important role in determining tc use . in addition , infants have a very high surface area to weight ratio that can lead to disproportionately high systemic absorption from topical application . women who do housework often have damaged skin due to frequent wet - dry cycling . this creates the need for more frequent application , as do certain occupations involving manual labor . other factors like the anatomical location and extent of the disease also dictate which formulation is optimal . widespread dermatoses demand easily spreadable dosage forms and milder potencies to guard against undesirable systemic absorption . intertriginous areas are particularly susceptible to stronger than expected effect due to the occlusion and maceration of the skin and therefore we should err on the side of caution when deciding the potency on these sites.the right amount , frequency and duration : perhaps the commonest omission in a tc prescription is advice regarding the right amount to be applied in a particular situation . in this regard , the fingertip unit ( ftu ) devised by long and finlay is the simplest way to explain to the patient how much tc is to be applied . an ftu is the amount of ointment that occupies the space from the fingertip to the first skin crease when squeezed out of a 5 mm nozzle . this amount is enough to cover a palm - sized area on the skin in the case of creams and about 20% more with ointments due to their increased spreadability . standard recommendations have been developed to guide how many ftus are required to cover particular anatomical areas . otherwise , most experts agree that once daily application is optimal for all other sites since more frequent application has shown no added benefit and adherence is also expected to be higher with this regimen.the duration of treatment needs to be clearly told to the patient orally as well as in writing and he / she should also be apprised of the dangers of overuse at this point . in general , a follow - up visit should be planned after 2 to 4 weeks to review the progress and institute the exit strategy.the right exit strategy : it is extremely important to have a clear plan in mind about tapering and stopping tc use after adequate control / remission has been achieved . this can be done safely by tapering down to progressively less potent preparations or instituting alternate - day or weekends - only treatment . recurrent irritant hand dermatitis , pseudofolliculitis barbae and intertrigo are good examples of these conditions . they should explain to the patients that relief will be transient if preventive actions like skin protection and moisturizing ( hand eczema ) , changing shaving habits ( pseudofolliculitis barbae ) and keeping the skin dry , losing weight and wearing loose natural fabrics ( intertrigo ) are not done.being aware of corticosteroid allergy : many times we are faced with a patient who does not show the desired improvement even if appropriate tc is being used . in these situations this can happen both due to the preservatives / other excipients or the active molecule per se . in these cases , appropriate patch testing followed by prescription of an allergen - free product should be done.effective communication : involving and informing the patient at every step of diagnosis and treatment is the key to using tcs safely , ethically and effectively . a few scenarios are presented below to exemplify this point.patients often self - treat themselves with over - the - counter tcs before coming to the physician , altering the appearance of the skin and making diagnosis difficult . in such a situation , it behooves us to explain the situation to the patient and withdraw all tcs while relevant investigations are done . even if no investigations are done , a steroid - free interval often renders the disease recognizable , allowing proper treatment to be started.patients often neglect to come for follow - up when asked to , either due to financial constraints such as loss of wages / inability to pay re - visit fees or simply due to negligence . to minimize the chances of adverse effects of tc , partial control of the treatment should be handed over to the patient , thus making them partners in their well - being , not just the recipients of instructions . to this end , they can be told at which point they should start alternate day treatment , introduce a calcineurin inhibitor or switch over to a milder tc . with this , they should also be told the warning signs of common adverse effects like folliculitis which would necessitate a re - visit immediately.many patients are unsure how long it would take for the effect of tcs to start and therefore either become anxious too quickly or continue to use an ineffective tc preparation till the next scheduled visit . to avoid this , patients should be clearly told approximately how long it would take for the beneficial effects of the tc to kick in . a lot of counseling and reassurance is needed if these patients or their dependents are not to be denied the benefits of tcs . they also need to be told about the non - tc options available and their relative efficacy . if they still choose not to use tcs , their choice should be respected and an appropriate regimen devised . other conditions where careful , short - term use may be warranted are superficial fungal or bacterial infections associated with significant inflammation , localized itch of any origin and certain idiopathic diseases characterized by dermal inflammation e.g. in the absence of a diagnosis , it is very important to avoid tc use so that conditions like tinea incognito and majocchi 's granuloma are not produced . if a patient is already applying tc thereby obscuring the clinical features of a disease , an appropriate treatment - free interval should be given after explaining to the patient that his / her symptoms may flare up temporarily , to facilitate a diagnosis . the right molecule and delivery system : since they were first introduced in the early 1950s , tcs have grown tremendously as a class and today , more than 20 different molecules of varying potency are available worldwide , ranging from mild to superpotent . in addition , there are varying concentrations and dosage forms available , namely creams , gels , lotions , ointments , foams , muco - adherent gels , aerosols and tapes . it is important for the prescriber to be familiar with at least a few of the molecules , preferably of differing potencies . the characteristics of the various dosage forms should also be thoroughly understood so that the correct drug in the correct vehicle can be given in a particular situation . although it is generally good to err on the side of caution when prescribing tcs , in many situations , it is better to prescribe a more potent molecule to control the disease quickly and stop treatment fast instead of prolonged treatment with a mild formulation . the right patient : the age , sex and occupation of the patient play an important role in determining tc use . in addition , infants have a very high surface area to weight ratio that can lead to disproportionately high systemic absorption from topical application . women who do housework often have damaged skin due to frequent wet - dry cycling . this creates the need for more frequent application , as do certain occupations involving manual labor . other factors like the anatomical location and extent of the disease also dictate which formulation is optimal . widespread dermatoses demand easily spreadable dosage forms and milder potencies to guard against undesirable systemic absorption . cosmetic considerations are paramount when tcs are applied on the face or other visible parts and creams , gels and lotions are preferred here due to their elegance . intertriginous areas are particularly susceptible to stronger than expected effect due to the occlusion and maceration of the skin and therefore we should err on the side of caution when deciding the potency on these sites . the right amount , frequency and duration : perhaps the commonest omission in a tc prescription is advice regarding the right amount to be applied in a particular situation . in this regard , the fingertip unit ( ftu ) devised by long and finlay is the simplest way to explain to the patient how much tc is to be applied . an ftu is the amount of ointment that occupies the space from the fingertip to the first skin crease when squeezed out of a 5 mm nozzle . this amount is enough to cover a palm - sized area on the skin in the case of creams and about 20% more with ointments due to their increased spreadability . standard recommendations have been developed to guide how many ftus are required to cover particular anatomical areas . hands and feet often need twice or thrice a day application due to the propensity of being rubbed off . otherwise , most experts agree that once daily application is optimal for all other sites since more frequent application has shown no added benefit and adherence is also expected to be higher with this regimen . the duration of treatment needs to be clearly told to the patient orally as well as in writing and he / she should also be apprised of the dangers of overuse at this point . the right exit strategy : it is extremely important to have a clear plan in mind about tapering and stopping tc use after adequate control / remission has been achieved . except for self - limiting conditions , tc use is often needed for extended periods , much longer than recommended safe durations . this can be done safely by tapering down to progressively less potent preparations or instituting alternate - day or weekends - only treatment . focusing on prevention and modifiable factors : in many conditions , lasting remission or cure is only achievable if preventive actions are taken or behavioral modifications are done . they should explain to the patients that relief will be transient if preventive actions like skin protection and moisturizing ( hand eczema ) , changing shaving habits ( pseudofolliculitis barbae ) and keeping the skin dry , losing weight and wearing loose natural fabrics ( intertrigo ) are not done . being aware of corticosteroid allergy : many times we are faced with a patient who does not show the desired improvement even if appropriate tc is being used . in these situations this can happen both due to the preservatives / other excipients or the active molecule per se . in these cases , appropriate patch testing followed by prescription of an allergen - free product should be done . effective communication : involving and informing the patient at every step of diagnosis and treatment is the key to using tcs safely , ethically and effectively . patients often self - treat themselves with over - the - counter tcs before coming to the physician , altering the appearance of the skin and making diagnosis difficult . in such a situation , it behooves us to explain the situation to the patient and withdraw all tcs while relevant investigations are done . even if no investigations are done , a steroid - free interval often renders the disease recognizable , allowing proper treatment to be started . to minimize the chances of adverse effects of tc , partial control of the treatment should be handed over to the patient , thus making them partners in their well - being , not just the recipients of instructions . with this , they should also be told the warning signs of common adverse effects like folliculitis which would necessitate a re - visit immediately . many patients are unsure how long it would take for the effect of tcs to start and therefore either become anxious too quickly or continue to use an ineffective tc preparation till the next scheduled visit . to avoid this , patients should be clearly told approximately how long it would take for the beneficial effects of the tc to kick in . a lot of counseling and reassurance is needed if these patients or their dependents are not to be denied the benefits of tcs . they also need to be told about the non - tc options available and their relative efficacy . if they still choose not to use tcs , their choice should be respected and an appropriate regimen devised . today , both patients and physicians contribute to the issues associated with rational and ethical use of topical corticosteroids . patients are prone to misuse them due to their unrestricted availability and poor awareness about their adverse effects . an unwarranted fear of topical corticosteroids largely driven by an alarmist media . other specialists and general practitioners who probably see more dermatology patients than dermatologists suffer from grossly inadequate dermatology training . therefore , it is imperative that increased awareness about ethical use of these agents is created amongst all caregivers so that they are used to their fullest extent and with maximum safety for the benefit of humanity . ethical conflicts are common when prescribing topical corticosteroids and we need to be aware of them to avoid pitfalls.examples of various clinical scenarios are used to exemplify common issues in topical corticosteroid prescriptions.practical pointers are given to help physicians use these agents in the best possible manner i.e. ethical conflicts are common when prescribing topical corticosteroids and we need to be aware of them to avoid pitfalls . examples of various clinical scenarios are used to exemplify common issues in topical corticosteroid prescriptions . practical pointers are given to help physicians use these agents in the best possible manner i.e.

they have antipruritic , anti - inflammatory , anti - proliferative and pigment - lightening activity on the skin . a recent prescription audit from a dermatology opd revealed that close to 30% of all prescriptions contained a tc . symptoms such as itch , stinging and tenderness are rapidly relieved regardless of the cause , as are redness , scaling and hyperpigmentation . unfortunately , this improvement may be short - lived and can be followed by worsening of the original condition if tcs are not used correctly or used in conditions where they are not indicated at all . the natural instinct of every physician is to give rapid relief to a patient who is distressed and tcs are ideally suited for such a role . if not properly counseled , patients may not return for a follow - up and may continue to use the tc for prolonged periods . it is therefore essential that we develop a clear understanding of the ethics of tc prescribing so that they are used optimally for the benefit of our patients . in our country , easy availability of mild to superpotent tcs even without a prescription coupled with rampant sharing of tcs by patients has created a situation where they seem to be more misused then used correctly . on the other hand , steroid phobia is becoming increasingly common in the internet - savvy population in the cities creating new problems in prescription of tcs . in this backdrop , ethical and careful prescription of these powerful agents is the need of the hour . awareness of these issues is essential so that pitfalls can be avoided and a good doctor - patient relationship be established . in other words , the patient has the right to refuse or choose their treatment ( voluntas aegroti suprema lex . ) this is in contrast to the older paternalistic style of medicine where patients were simply advised on what to take without any discussion of alternatives , risk vs. benefit etc . , with regards to tcs , this law applies when we are faced with a steroid - phobic patient . in this situation , is often a good approach whereby such a patient should be counseled about the potential benefits of tc use , the harm that can be caused by non - use and how common adverse effects can be avoided . topical calcineurin inhibitors or calcipotriol and their efficacy vis - - vis tcs . after this , whatever their ultimate decision be , it should be respected.non-maleficence : this is the principle of primum non nocere ( first , do no harm ) a common scenario where non - maleficence needs to be remembered is a patient who is obsessed with a fair complexion and asks for a tc prescription which he / she may have heard about from a friend . it is absolutely essential to avoid the temptation to give in to the patient 's request in this situation . non - maleficence also becomes relevant when we are dealing with an undiagnosed rash and a demanding , distressed patient . several studies have been reported on the misuse of tcs , especially on the face where the initial prescription was given for an undiagnosed facial rash which then led to prolonged misuse of tcs leading to considerable morbidity . in such a scenario , pending a diagnosis , tcs should be avoided and relief given with safer options like topical antipruritics and oral antihistamines . in the absence of a clear diagnosis and treatment plan , a physician should not hesitate to refer a patient to a senior colleague or an expert instead of using tcs to suppress unpleasant symptoms indefinitely.beneficence vs. autonomy : this kind of conflict occurs when patients disagree with recommendations that doctors believe are in the patients best interest . the appropriate approach in such a situation would be to start a systemic steroid - sparing drug like azathioprine and gradually taper the tc . appropriate counseling about the dangers of potent tc use over large areas along with laboratory demonstration of adrenal suppression etc . in other words , the patient has the right to refuse or choose their treatment ( voluntas aegroti suprema lex . ) this is in contrast to the older paternalistic style of medicine where patients were simply advised on what to take without any discussion of alternatives , risk vs. benefit etc . , with regards to tcs , this law applies when we are faced with a steroid - phobic patient . in this situation , is often a good approach whereby such a patient should be counseled about the potential benefits of tc use , the harm that can be caused by non - use and how common adverse effects can be avoided . topical calcineurin inhibitors or calcipotriol and their efficacy vis - - vis tcs . non - maleficence : this is the principle of primum non nocere ( first , do no harm ) . a common scenario where non - maleficence needs to be remembered is a patient who is obsessed with a fair complexion and asks for a tc prescription which he / she may have heard about from a friend . it is absolutely essential to avoid the temptation to give in to the patient 's request in this situation . non - maleficence also becomes relevant when we are dealing with an undiagnosed rash and a demanding , distressed patient . several studies have been reported on the misuse of tcs , especially on the face where the initial prescription was given for an undiagnosed facial rash which then led to prolonged misuse of tcs leading to considerable morbidity . in such a scenario , pending a diagnosis , tcs should be avoided and relief given with safer options like topical antipruritics and oral antihistamines . in the absence of a clear diagnosis and treatment plan , a physician should not hesitate to refer a patient to a senior colleague or an expert instead of using tcs to suppress unpleasant symptoms indefinitely . beneficence vs. autonomy : this kind of conflict occurs when patients disagree with recommendations that doctors believe are in the patients best interest . the appropriate approach in such a situation would be to start a systemic steroid - sparing drug like azathioprine and gradually taper the tc . appropriate counseling about the dangers of potent tc use over large areas along with laboratory demonstration of adrenal suppression etc . in fact , effective communication is the key to resolving almost all ethical dilemmas faced by a physician prescribing tcs . not only doctor - patient , but communication between doctors of different specialties and between doctors and society in general is essential in resolving these ethical issues . correct use of tcs is predicated on the fulfilling of the following conditions : the right diagnosis : there are relatively few conditions where there is good evidence of efficacy of tcs . various eczemas , psoriasis , lichen planus , immunobullous diseases in their localized form , skin manifestations of collagen vascular diseases like lupus erythematosus or dermatomyositis are the best established indications . other conditions where careful , short - term use may be warranted are superficial fungal or bacterial infections associated with significant inflammation , localized itch of any origin and certain idiopathic diseases characterized by dermal inflammation e.g. in the absence of a diagnosis , it is very important to avoid tc use so that conditions like tinea incognito and majocchi 's granuloma are not produced . if a patient is already applying tc thereby obscuring the clinical features of a disease , an appropriate treatment - free interval should be given after explaining to the patient that his / her symptoms may flare up temporarily , to facilitate a diagnosis.the right molecule and delivery system : since they were first introduced in the early 1950s , tcs have grown tremendously as a class and today , more than 20 different molecules of varying potency are available worldwide , ranging from mild to superpotent . in addition , there are varying concentrations and dosage forms available , namely creams , gels , lotions , ointments , foams , muco - adherent gels , aerosols and tapes . it is important for the prescriber to be familiar with at least a few of the molecules , preferably of differing potencies . the characteristics of the various dosage forms should also be thoroughly understood so that the correct drug in the correct vehicle can be given in a particular situation . although it is generally good to err on the side of caution when prescribing tcs , in many situations , it is better to prescribe a more potent molecule to control the disease quickly and stop treatment fast instead of prolonged treatment with a mild formulation . this is especially true of self - limited conditions like acute irritant dermatitis , paederous dermatitis etc.the right patient : the age , sex and occupation of the patient play an important role in determining tc use . in addition , infants have a very high surface area to weight ratio that can lead to disproportionately high systemic absorption from topical application . other factors like the anatomical location and extent of the disease also dictate which formulation is optimal . cosmetic considerations are paramount when tcs are applied on the face or other visible parts and creams , gels and lotions are preferred here due to their elegance . intertriginous areas are particularly susceptible to stronger than expected effect due to the occlusion and maceration of the skin and therefore we should err on the side of caution when deciding the potency on these sites.the right amount , frequency and duration : perhaps the commonest omission in a tc prescription is advice regarding the right amount to be applied in a particular situation . in this regard , the fingertip unit ( ftu ) devised by long and finlay is the simplest way to explain to the patient how much tc is to be applied . an ftu is the amount of ointment that occupies the space from the fingertip to the first skin crease when squeezed out of a 5 mm nozzle . this amount is enough to cover a palm - sized area on the skin in the case of creams and about 20% more with ointments due to their increased spreadability . this should be clearly conveyed to the patient to prevent over- or underuse.the frequency of application is dependent on patient factors such as site and occupation . otherwise , most experts agree that once daily application is optimal for all other sites since more frequent application has shown no added benefit and adherence is also expected to be higher with this regimen.the duration of treatment needs to be clearly told to the patient orally as well as in writing and he / she should also be apprised of the dangers of overuse at this point . in general , a follow - up visit should be planned after 2 to 4 weeks to review the progress and institute the exit strategy.the right exit strategy : it is extremely important to have a clear plan in mind about tapering and stopping tc use after adequate control / remission has been achieved . except for self - limiting conditions , tc use is often needed for extended periods , much longer than recommended safe durations . emollients and/or steroid - sparing drugs like calcineurin inhibitors are prescribed on tc - free days . finding the right exit strategy can involve a lot of empiricism but is a worthwhile skill to learn for every physician.focusing on prevention and modifiable factors : in many conditions , lasting remission or cure is only achievable if preventive actions are taken or behavioral modifications are done . recurrent irritant hand dermatitis , pseudofolliculitis barbae and intertrigo are good examples of these conditions . they should explain to the patients that relief will be transient if preventive actions like skin protection and moisturizing ( hand eczema ) , changing shaving habits ( pseudofolliculitis barbae ) and keeping the skin dry , losing weight and wearing loose natural fabrics ( intertrigo ) are not done.being aware of corticosteroid allergy : many times we are faced with a patient who does not show the desired improvement even if appropriate tc is being used . at other times , control is achieved , only to lead to episodes of increased disease activity which are controlled with increasingly higher potency corticosteroids . in these situations this can happen both due to the preservatives / other excipients or the active molecule per se . in these cases , appropriate patch testing followed by prescription of an allergen - free product should be done.effective communication : involving and informing the patient at every step of diagnosis and treatment is the key to using tcs safely , ethically and effectively . a few scenarios are presented below to exemplify this point.patients often self - treat themselves with over - the - counter tcs before coming to the physician , altering the appearance of the skin and making diagnosis difficult . even if no investigations are done , a steroid - free interval often renders the disease recognizable , allowing proper treatment to be started.patients often neglect to come for follow - up when asked to , either due to financial constraints such as loss of wages / inability to pay re - visit fees or simply due to negligence . to minimize the chances of adverse effects of tc , partial control of the treatment should be handed over to the patient , thus making them partners in their well - being , not just the recipients of instructions . with this , they should also be told the warning signs of common adverse effects like folliculitis which would necessitate a re - visit immediately.many patients are unsure how long it would take for the effect of tcs to start and therefore either become anxious too quickly or continue to use an ineffective tc preparation till the next scheduled visit . to avoid this , patients should be clearly told approximately how long it would take for the beneficial effects of the tc to kick in . they should be counseled to walk in if they are not feeling substantially better at this point.corticosteroid phobia is an increasingly common problem in urban and internet - savvy patients . a lot of counseling and reassurance is needed if these patients or their dependents are not to be denied the benefits of tcs . the right diagnosis : there are relatively few conditions where there is good evidence of efficacy of tcs . various eczemas , psoriasis , lichen planus , immunobullous diseases in their localized form , skin manifestations of collagen vascular diseases like lupus erythematosus or dermatomyositis are the best established indications . other conditions where careful , short - term use may be warranted are superficial fungal or bacterial infections associated with significant inflammation , localized itch of any origin and certain idiopathic diseases characterized by dermal inflammation e.g. in the absence of a diagnosis , it is very important to avoid tc use so that conditions like tinea incognito and majocchi 's granuloma are not produced . if a patient is already applying tc thereby obscuring the clinical features of a disease , an appropriate treatment - free interval should be given after explaining to the patient that his / her symptoms may flare up temporarily , to facilitate a diagnosis . the right molecule and delivery system : since they were first introduced in the early 1950s , tcs have grown tremendously as a class and today , more than 20 different molecules of varying potency are available worldwide , ranging from mild to superpotent . in addition , there are varying concentrations and dosage forms available , namely creams , gels , lotions , ointments , foams , muco - adherent gels , aerosols and tapes . it is important for the prescriber to be familiar with at least a few of the molecules , preferably of differing potencies . the characteristics of the various dosage forms should also be thoroughly understood so that the correct drug in the correct vehicle can be given in a particular situation . although it is generally good to err on the side of caution when prescribing tcs , in many situations , it is better to prescribe a more potent molecule to control the disease quickly and stop treatment fast instead of prolonged treatment with a mild formulation . the right patient : the age , sex and occupation of the patient play an important role in determining tc use . in addition , infants have a very high surface area to weight ratio that can lead to disproportionately high systemic absorption from topical application . other factors like the anatomical location and extent of the disease also dictate which formulation is optimal . cosmetic considerations are paramount when tcs are applied on the face or other visible parts and creams , gels and lotions are preferred here due to their elegance . intertriginous areas are particularly susceptible to stronger than expected effect due to the occlusion and maceration of the skin and therefore we should err on the side of caution when deciding the potency on these sites . the right amount , frequency and duration : perhaps the commonest omission in a tc prescription is advice regarding the right amount to be applied in a particular situation . in this regard , the fingertip unit ( ftu ) devised by long and finlay is the simplest way to explain to the patient how much tc is to be applied . an ftu is the amount of ointment that occupies the space from the fingertip to the first skin crease when squeezed out of a 5 mm nozzle . this amount is enough to cover a palm - sized area on the skin in the case of creams and about 20% more with ointments due to their increased spreadability . the duration of treatment needs to be clearly told to the patient orally as well as in writing and he / she should also be apprised of the dangers of overuse at this point . in general , a follow - up visit should be planned after 2 to 4 weeks to review the progress and institute the exit strategy . the right exit strategy : it is extremely important to have a clear plan in mind about tapering and stopping tc use after adequate control / remission has been achieved . except for self - limiting conditions , tc use is often needed for extended periods , much longer than recommended safe durations . emollients and/or steroid - sparing drugs like calcineurin inhibitors are prescribed on tc - free days . they should explain to the patients that relief will be transient if preventive actions like skin protection and moisturizing ( hand eczema ) , changing shaving habits ( pseudofolliculitis barbae ) and keeping the skin dry , losing weight and wearing loose natural fabrics ( intertrigo ) are not done . being aware of corticosteroid allergy : many times we are faced with a patient who does not show the desired improvement even if appropriate tc is being used . at other times , control is achieved , only to lead to episodes of increased disease activity which are controlled with increasingly higher potency corticosteroids . effective communication : involving and informing the patient at every step of diagnosis and treatment is the key to using tcs safely , ethically and effectively . patients often self - treat themselves with over - the - counter tcs before coming to the physician , altering the appearance of the skin and making diagnosis difficult . patients often neglect to come for follow - up when asked to , either due to financial constraints such as loss of wages / inability to pay re - visit fees or simply due to negligence . to minimize the chances of adverse effects of tc , partial control of the treatment should be handed over to the patient , thus making them partners in their well - being , not just the recipients of instructions . many patients are unsure how long it would take for the effect of tcs to start and therefore either become anxious too quickly or continue to use an ineffective tc preparation till the next scheduled visit . to avoid this , patients should be clearly told approximately how long it would take for the beneficial effects of the tc to kick in . today , both patients and physicians contribute to the issues associated with rational and ethical use of topical corticosteroids . therefore , it is imperative that increased awareness about ethical use of these agents is created amongst all caregivers so that they are used to their fullest extent and with maximum safety for the benefit of humanity . ethical conflicts are common when prescribing topical corticosteroids and we need to be aware of them to avoid pitfalls.examples of various clinical scenarios are used to exemplify common issues in topical corticosteroid prescriptions.practical pointers are given to help physicians use these agents in the best possible manner i.e.

signal transducer and activator of transcription 5 ( stat5 ) is widely expressed throughout the hematopoietic system , both in stem and progenitor cells as well as in committed erythroid , myeloid and lymphoid cells . indeed , it is not surprising that stat5 can be activated by a wide variety of cytokines and growth factors . these include cytokines and growth factors that can signal through the interleukin 3 ( il3)-receptor family [ il3 , il5 , granulocyte - macrophage colony stimulating factor ( gm - csf ) ] , through the common -chain receptor family ( il2 , il7 , il9 , il12 , il15 ) , through single chain receptors [ erythropoietin ( epo ) , thrombopoietin ( tpo ) , growth hormone ( gh ) , prolactin , granulocyte - colony stimulating factor ( g - csf ) ] , through class ii receptors [ interferon ( ifn- ) , ifn- , il22 ] or through tyrosine kinase receptors [ stem cell factor ( scf ) , platelet derived growth factor ( pdgf ) , epidermal growth factor ( egf ) ] ( fig . 1 ) . in most cases , janus kinase ( jak ) tyrosine kinase activity mediates stat5 tyrosine phosphorylation , and stat5 can be activated by jak1 , 2 or 3 , depending on the cytokine - activated receptor complex . alternatively , the tyrosine kinase receptor family can also induce stat5 phosphorylation in a jak - independent manner . while stat5 is expressed in the majority of hematopoietic cell types , thus , specific cytokines are able to induce stat5 activity in subsets of cell types only . for example , cytokines that activate stat5 in the most immature human hematopoietic stem compartment include scf and tpo . these cytokines have been shown to promote long - term hematopoiesis in vitro , and hypersensitivity to tpo in lnk mice resulted in elevated stem cell self - renewal , which coincided with increased levels of stat5 activity . within the erythroid compartment , stat5 is activated by epo , where stat5 fulfills an important anti - apoptotic role by upregulating bcl - xl , although a more direct role in initiating erythroid commitment might exist as well . in myeloid cells , stat5 can be activated by a variety of cytokines , including il3 , il5 , gm - csf and csf1 ( reviewed in ref . although initially in stat5ab mice myelopoiesis appeared to be relatively unaffected , it is likely that in myeloid cells many of the signals initiated by e.g. , il-3 and gm - csf are , at least in part , mediated by stat5 , thereby regulating myeloproliferation or anti - apoptosis . during myelosuppression , mice completely deficient of stat5ab failed to produce enhanced levels of neutrophils and were unable to respond to gm - csf . stat5 activation is required for il2-induced t cell proliferation and the production of nk cells , or for il7-mediated b cell expansion . as summarized in table 1 and figure 1b , a wide variety of genetic defects in myeloid leukemias and myeloproliferative diseases ( mpds ) result in activation of the stat5 pathway , including mutations in flt3 and ckit receptors , jak2 mutations , translocations such as tel - pdgfra , and bcr - abl , but also as a result of increased cytokine signaling . numerous functional studies have indicated that aberrant activation of stat5 can contribute to the process of leukemic transformation . downstream of flt3-itd ( internal tandem duplication ) mutations , stat5 is strongly activated via two tyrosine residues within the flt3 receptor , y589 and y592 that act as docking sites for the sh2 domain of stat5 molecules . mutation of these residues into phenylalanines completely abrogated activation of stat5 , and importantly completely impaired induction of a myeloproliferative disease in vivo in a murine transplantation model . thus , it is likely that stat5 signaling is essential for the transforming potential of flt3-itd . although in human cells introduction of flt3-itd did not result in a myeloproliferative disease in transplanted nod - scid mice , the activated stem cell phenotype imposed on cb cd34 cells , as revealed by the formation of early cobblestone area forming cells ( cafcs ) , was impaired by coexpression of a dominant negative stat5a ( y694f ) mutant , suggesting that also in human cells stat5 is an important mediator of flt3-itd - induced signaling . in studies in which stat5 expression was targeted in primary acute myeloid leukemia ( aml ) cd34 cells using a lentiviral approach , it was observed that long - term expansion and the formation of leukemic cafcs was strongly impaired by downmodulation of stat5 . although the presence of flt3-itds was not the exclusive genetic mutation that induced constitutive stat5 signaling in the samples that were studied , these data clearly underscore the important role that stat5 fulfills in long - term expansion and self - renewal of primary aml stem / progenitor cells as well . in chronic myeloid leukemia ( cml ) induced by bcr - abl a number of studies have shown that stat5 is efficiently activated downstream of bcr - abl , and interference with stat5 activation negatively impacts the survival and proliferation of bcr - abl - expressing cells . bcr - abl p210-transduced stat5 murine bone marrow ( bm ) cells developed cml with low frequencies , with a delayed onset of disease . the induction of acute lymphoid leukemia ( all ) was not impaired in these animals . complete abrogation of stat5 expression in stat5ab mice also impaired lymphoid transformation induced by bcr - abl - expressing murine bm . in primary human cml cells , it was demonstrated that downmodulation of stat5 expression by rnai impaired bcr - abl - dependent proliferation and also reduced colony formation in methylcellulose . inhibition of stat5 by pimozide reduced colony formation of cml cd34 cells , also in tyrosine kinase - resistant patient samples . in myeloproliferative diseases it has been demonstrated in mouse models that bone marrow ( bm)-transduced with tel - jak2 no longer induced disease in recipient mice when the oncogene was introduced in a stat5 background . finally , enhanced stat5 activity has been observed in polycythemia vera ( pv ) , caused by the activating jak2 v617f mutation . inhibition of jak2 kinase activity abrogated the activation of stat5 , which coincided with a suppression of erythropoiesis in vitro and in vivo . the most direct evidence for stat5 acting as an oncogene arises from murine bm transplantation studies in which constitutively activated stat5 ( s711f ) mutants were overexpressed . lethally irradiated recipients receiving activated stat5-transduced bm died within 6 weeks after transplantation of a multilineage leukemia . it was demonstrated that a tryptophan residue in the n - terminal region of stat5 is required for tetramerization of stat5 dimers , and tetramer - deficient stat5 mutants were unable to induce leukemia in mice . another activating mutant of stat5 , stat5a(1 * 6 ) that contains two point mutations ( h299r and s711f ) was earlier shown to induce myeloid hyperproliferation , but not leukemia , in a murine retroviral overexpression model . this was later confirmed by others , and a fatal mpd was observed by overexpression of these stat5 mutants , but only when the most primitive cd34lin ckitsca1 ( lsk ) population was transduced and used for transplantation to irradiated recipients , suggesting that the stem cell , but not a committed progenitor is the target cell for transformation induced by activated stat5 . intriguingly , while these examples clearly demonstrate that stat5 can transform murine hematopoietic stem cells ( hscs ) , no in vivo stat5-induced transformation has been reported in human cell populations . while enhanced self - renewal and long - term stem cell maintenance can be achieved by introduction of activated stat5 in human cd34 cells , a myeloproliferative disease or leukemia does not occur in non - obese diabetic / severe combined immunodeficiency ( nod - scid ) transplantations models . it is plausible that the nod - scid xenograft model is not suitable to completely recapitulate human disease , or alternatively it is possible that species - specific differences in stat5 signaling exist . in line with these observations , introduction of bcr - abl in murine bm resulted in a rapid and lethal mpd whereby recipients die within 3 weeks after transplantation . introduction of bcr - abl in human cd34 cells does not result in a rapid leukemia or mpd in engrafted nod - scid mice , and only after 5 mo progression to an early stage disease was observed in some animals . collectively , these data indicate that stat5 is frequently activated in various hematological malignancies , whereby it strongly affects processes such as self - renewal and lineage fate determination . whether stat5 target genes in normal and leukemic stem cells are identical , or whether leukemic stem cell - specific stat5 target genes exist remains to be determined . also , it will be informative to study how stat5 might cooperate with additional leukemic oncogenes in a multi - hit approach to model the development of human leukemias . loss - of - function and gain - of - function experiments have revealed critical roles for stat5 in the hematopoietic stem / progenitor compartment . stat5ab mice have been used to assess stem cell function in the absence of wt stat5 signaling . these mice were characterized by normal hsc numbers and stem cells isolated from the bone marrow or fetal liver were capable of engrafting irradiated recipients . yet , competitive repopulating capacity of stat5ab hscs was severely impaired . the underlying mechanisms are not fully elucidated yet , but it has been observed that the responsiveness of stat5ab hscs to early - acting cytokines such as il3 and scf was reduced , while the sensitivity to 5-fluoroacil was enhanced . loss of protection against apoptosis most likely does not explain the stat5ab hscs phenotypes , as overexpression of bcl2 was not sufficient to rescue repopulating defects . although homing of stat5ab bm cells into lethally irradiated recipients was not impaired , retention in the bone marrow was reduced under non - myeloablative conditions , leaving open the possibility that competition for the niche might play a role . more recently , using an mx1-cre inducible mouse model , it was shown that conditional deletion of stat5 results in a loss of stem cell quiescence , associated with reduced survival and gradual loss of the hsc pool . in order to study stat5 signaling in human hematopoietic stem / progenitor cells , we have used a lentiviral shrna approach in cord blood ( cb ) cd34 cells . downmodulation of stat5 to about 30% of the endogenous levels reduced progenitor frequencies as determined by colony forming cell ( cfc ) assays in methylcellulose as well as stem cell frequencies as determined by long - term culture - initiating cell ( ltc - ic ) assays in limiting dilution . this resulted in reduced long - term expansion on ms5 bone marrow stroma upon downmodulation of stat5 expression , whereby the myeloid and erythroid differentiation were unaffected . single - cell assays using transduced cd34/cd38 cells revealed that cell cycle progression induced by early - acting cytokines scf and tpo was impaired by stat5 downmodulation . reversely , activating mutants of stat5 have been introduced in murine cd34lsk cells and the effects on stem and progenitor cells were assessed in vitro and in vivo . introduction of stat5a(1 * 6 ) or stat5a(1 * 7 ) mutants resulted in a strong ex vivo expansion of immature cfu - nmem progenitors , without affecting the symmetry of stem cell divisions as determined in paired - daughter cell assays . importantly , long - term repopulating hscs could be maintained under ex vivo culture conditions as cd34lsk cells expressing activated stat5 had a strong competitive repopulating advantage over wild type cells after 7 d and 10 d ex vivo culturing in the presence of scf or scf and tpo . overexpression of stat5a(1 * 6 ) in human cd34 cord blood cells resulted in enhanced stem cell self - renewal . this enhanced self - renewal was only observed in bone marrow stromal cocultures , but not in cytokine - driven liquid culture conditions . these data argued that stat5-induced hsc cell self - renewal depends on the presence of a bone marrow microenvironment , and it was indeed observed that stat5a(1 * 6)-expressing cd34 cells have a strongly enhanced interaction with bone marrow stromal cells , resulting in the appearance of early cafcs underneath the stroma within 1 week after plating . these cafcs contained self - renewal potential as demonstrated by their capacity to give rise to second cafcs upon harvest and replating onto new stroma , as well as by their capacity to engraft in sublethally irradiated nod - scid mice . upon serial replating , long - term cultures could be established by overexpression of activated stat5 for over 20 weeks , giving rise to new cafcs upon each replating as well as to progeny in suspension . hematopoietic progenitors could be maintained long - term in these culture conditions and the suspension cells retained an immature blast - like morphology . when stat5a(1 * 6 ) mutants were expressed in murine embryonic stem ( es ) cells , the generation of hematopoietic stem cells was greatly facilitated as studied on op9 bone marrow stromal cells . importantly , these cafcs could be serially passaged onto new op9 stroma , giving rise to second and third cafcs that were able to sustain long - term hematopoiesis and generate high numbers hematopoietic progenitors , indicative of hsc self - renewal in vitro . also , the cafcs generated by activation of stat5 could engraft sublethally irradiated nod - scid mice , indicating that stat5 facilitates the generation of es - derived hscs that can contribute to hematopoiesis in vivo as well . although various stat5 target genes have been identified , the mechanisms by which stat5 acts on hscs remain to be elucidated . using cell lines or heterogeneous stem / progenitor cell populations , enhanced cell growth is one of the most dominant phenotypes that is frequently observed in various studies , and several genes that are regulated by stat5 associate with cell proliferation and cell cycle progression , including cyclin d1 , pim1 and c - myc . pim serine / threonine kinases act as mediators of cytokine - induced cell growth by promoting acceleration of cell - cycle progression both at the g1/s and g2/m transitions by phosphorylating and activating the phosphatases cdc25a and cdc25c , respectively . c - myc controls the balance between self - renewal and differentiation of hscs . however , when more purified stem cell populations were studied using a conditional deletion mx1-cre model it was observed that stat5 was required to maintain hsc quiescence . upon stat5 deletion a decrease in the percentage of cells in g0 within the long - term and short - term hsc compartments was observed , coinciding with a decrease in expression of quiescence - associated genes such as p57 and tie2 . in erythroid cells , it has been convincingly demonstrated that stat5 contributes to cell survival by upregulating the anti - apoptosis gene bcl - xl . survival of flt3-itd aml cells has been shown to depend on stat5-mediated expression of mcl1 . also , bcl2 can be upregulated by stat5 and is required to prevent apoptosis during terminal differentiation of myeloid cells . whether prevention of apoptosis contributes to stat5-induced hsc self - renewal is currently unclear , but bcl2 overexpression was not sufficient to rescue the repopulation defects of stat5ab hscs , suggesting that protection against apoptosis is not the main role of stat5 signaling in hscs . in our co - cultures , despite strong reductions in ltc - ic and cfc frequencies , we also did not detect an increased rate of apoptosis in stat5 rnai - transduced cd34 cb cells , and no decreased expression of the bcl - xl gene was observed . the basic helix - loop - helix transcriptional inhibitor id1 is also upregulated by stat5 , and id1-deficient hscs fail to self - renew , leading to low steady - state hsc numbers and premature hsc exhaustion . little evidence exists that stat5 affects the expression of other known hsc self - renewal regulators such as bmi1 or hoxb4 . recently , we observed that stat5 binds to and activates the promoter of hypoxia induced factor 2 ( hif2 ) in human cd34/cd38 hscs . functional studies indicated that stat5-induced long - term expansion and elevated ltc - ic and cfc frequencies were reduced upon downmodulation of hif2. glucose uptake was enhanced in cells expression activated stat5 , coinciding with a hif2-dependent upregulation glucose metabolism genes , suggesting that pathways normally active under hypoxia might be utilized by stat5 under normoxic conditions as well to maintain stem cell properties . the phenotype imposed on cells by stat5 might well depend on the actual level of stat5 activity that is induced . using a 4-hydroxytamoxifen inducible system that allowed titration of stat5 activity in human stem and progenitor cells we have demonstrated that the stem cell maintenance properties require intermediate stat5 activation . on the other hand , high stat5 activation levels resulted in erythroid differentiation at the expense of hsc self - renewal . c / ebp levels were reduced upon stat5 activation , which reached maximum reduction levels at intermediate stat5 activation . in line with these observations , in mice c / ebp deficiency resulted in hyperproliferation of hematopoietic progenitor cells and enhancement of hematopoietic stem cell repopulating capacity and self - renewal . reintroduction of c / ebp in stat5a(1 * 6)-transduced human cd34 cells was sufficient to impair hsc self - renewal capacity . the mechanisms by which stat5 affects c / ebp expression levels are still under investigation , but these observations leave open the possibility that enhanced hsc self - renewal might in part be explained by reduction in c / ebp expression levels . it is remarkable that the effects of stat5 on hsc self - renewal are confined to intermediate stat5 activation levels . this dosage effect of stat5 on self - renewal is consistent with the observed constitutive activation of stat5 in aml samples , which is typically lower then cytokine - induced stat5 activation . such a dosage effect of transcription factors is at present not well understood , but besides stat5 this has also been observed for the myeloid transcription factor pu.1 , which at 20% expression gives rise to self - renewing murine myeloid leukemias , whereas 50% or 100% reduction in expression do not have such a dramatic effect . recently , also for wnt signaling it was demonstrated that intermediate activation levels enhance self - renewal of hscs . collectively , these examples clearly underscore the role of transcription factor dosage in regulating hsc self - renewal . the observation that stat5 drives cell cycle progression in various cell types and anti - apoptosis in others , while stat5 is also required to maintain quiescence of hematopoietic stem cells , suggests that the cell - biological consequences of stat5 signaling might be highly cell type - specific . we have addressed this issue by introducing a 4-hydroxytamoxifen ( 4oht)-inducible stat5-er fusion in human stem and progenitor cells . activation of stat5 specifically in hsc , common myeloid ( cmp ) , granulocyte - macrophage ( gmp ) or megakaryocyte - erythroid progenitor ( mep ) populations resulted in rather distinct phenotypes . long - term self - renewal and enhanced cobblestone formation could only be imposed on hscs , but not on committed progenitor subpopulations . erythroid differentiation could be induced in hsc , cmp and mep populations , but not in gmps . gene expression profiling revealed that rather distinct gene expression profiles were induced in hsc as compared with more committed progenitor subpopulations . for instance , tubb1 , hif2 , sod2 , il8 and also the cell cycle inhibitor cdkn1a / p21 were particularly upregulated in hscs but not in committed progenitors ( fig . 2 ) . in contrast , osm , pim1 and the negative feedback regulators cish and socs2 were upregulated both in hscs and mpps . the underlying mechanisms are currently unclear , but a number of possibilities might be hypothesized . first , it has been shown that several cofactors such as p300/cbp , but also interactions with other transcription factors such as foxo3a , can modulate and fine - tune the stat5 response . cell type - specific interaction with such cofactors would then dictate a cell type - specific stat5 activation pattern of target genes . seen from this perspective , the modulation of stat5 signaling by p300/cbp could add to a stem vs. progenitor - specific component of stat5 . it has been observed that , unlike p300 , cbp is essential for hsc self - renewal maintenance , while p300 is suggested to play a role in differentiation . interaction of various transcription factors with p300/cbp is facilitated by the p300/cbp interacting protein cited2 which has been shown to be a target gene of stat5 and has differential expression and functions in hematopoietic stem vs. progenitor cells ( and our own observations ) . in part , such interactions can be mediated by posttranslational modifications such as serine phosphorylation or glycosylation of stat5 . thus , besides the induction of stat5 tyrosine phosphorylation required for dimerization , nuclear translocation and dna binding , the simultaneous activation of pathways that mediate stat5 serine phosphorylation or glycosylation would be required . ( 2 ) epigenetic factors that influence stat5 dna binding . ( 3 ) expression of receptors and ligands . ( 4 ) niche interactions . hypermethylation of specific promoters or polycomb - mediated condensation of chromatin might prevent stat5 association with regulatory promoter elements and thus transactivation of certain genes . clearly , such differences in epigenetic status and cofactor expression might also be dictated by different responses to extracellular stimuli . thus , the repertoire of specific cytokine and growth factor receptors that is expressed on a cell , as well as direct interactions between hematopoietic stem cells and their bone marrow niche , might ultimately determine the specific stat5 response ( fig . 2 ) . single cell tyrosine phospho - stat5 analysis revealed that within the normal hematopoietic stem cell and progenitor compartment highly distinct cytokine - induced stat5 activation patterns are observed . also in primary aml patient samples , rather heterogeneous responses toward a series of cytokines were observed , not directly linked to whether or not the cognate receptor was expressesed . there was clear heterogeneity between different patient samples , but also different responses could be observed within distinct cellular compartments within a single patient . for instance , in some patient samples strong il3 and gm - csf responses were observed , but only in the cd34 subpopulation , while in other cases strong tpo responses were observed within cd34/cd38 and cd34/cd38 compartments . these observations clearly indicate that strong differences exist in how cytokine and growth factor signals are mediated within a certain cell type , both normal as well as leukemic . although elucidation of molecular mechanisms by which cell type specific stat5 signaling is orchestrated needs further studies , cell type - specific stat5 target genes clearly do exist . the observation that p21 is upregulated by stat5 , particularly in hscs , is remarkable ( our unpublished observations and ref . it will be interesting to analyze whether the enhanced long - term self - renewal that is observed upon activation of stat5 in hematopoietic stem cells involves improved stem cell maintenance by keeping the hscs pool in a relatively quiescent state via upregulation of p21 . knockout studies in mice have indicated that p21 is required during stress hematopoiesis , and although p21 was also initially downregulated in stat5 depleted lsk cells , this downmodulation was not maintained . on the other hand , in murine embryonic fibroblasts it has also been shown that stat5 can negatively regulate cell cycle progression through activation of p21 . inhibition of jak2/stat5 signaling by the specific jak2 inhibitor az960 stimulated cell cycling in cd34/cd38 cells in conjunction with downregulation of p21 . further , activation of p21 has been shown to be critical in preventing excess dna - damage accumulation and functional exhaustion of leukemic stem cells , and it will be interesting to further reveal its role downstream of stat5 in hscs . furthermore , hif2 was upregulated in hscs and cmps by stat5 , but not in meps and gmps . under normoxic conditions , proline residues of hypoxia - induced factor 2 are hydroxylated resulting in a reduction in protein levels via vhl - mediated proteasomal degradation . under hypoxic conditions , such as in the presumed endosteal quiescent stem cell niche , it is currently unknown whether and which hif - induced target genes are essential to maintain stemness of normal hscs , but it was recently shown that in hif1 mice hscs numbers decrease during stress which was associated with a loss of hsc quiescence . another report indicated that hscs in the quiescence niche utilize glycolysis for their energy demands , which depended on a meis1-induced hif1 signaling network . whether hif1 and hif2 display similar or distinct functions in hscs remains to be established . our understanding of the mechanisms that determine whether , where and when a stem cell will self - renew or differentiate is still limited , but recent advances have indicated that the stem cell microenvironment provides essential cues that direct these cell fate decisions . it is remarkable that stat5-induced long - term self - renewal is typically observed when cells are cultured in direct contact with stromal cells , in contrast to , e.g. , bmi1-induced self - renewal , which occurred in a more microenvironment - independent manner . thus , altered interactions with the stem cells niche might also underlie the enhanced self - renewal properties imposed on hscs by activated stat5 . although the mechanisms by which the interaction with the microenvironment of stat5a(1 * 6)-expressing cd34 cells are still unclear , our ongoing studies in which gene - expression profiling was performed in hscs and progenitor subsets revealed that the list of stat5-targets is significantly enriched for membrane ( associated ) proteins . one of the stat5 targets that has been identified is muc1 which is a ( proto)oncogene involved in adhesion and transendothelial migration , and has been associated with initiation of various intracellular signal transduction pathways including -catenin , p53 and nfb pathways . also , muc1 has been shown to mediate an oscillatory calcium signal upon binding to icam1 . within the endosteal region of the bone marrow where stem cells are thought to reside , ca levels are high , and hsc retention within the niche depends on the calcium - sensing receptor ( car ) . thus , stat5 might exert its phenotype , at least in part , by influencing interactions between hscs and their niche . in both murine and human model systems it has been convincingly shown that stat5 fulfills an important role in hematopoietic stem cell self - renewal . although the precise mechanisms by which hsc self - renewal is orchestrated by stat5 remain elusive to date , an increasing number of stat5 target genes have been identified that are currently under investigation . in myeloproliferative diseases and leukemias , a number of oncogenes have been identified that are capable of inducing stat5 activity , and accumulating evidence has indicated that stat5 participates in self - renewal of leukemic stem cells as well . thus , it appears likely that stat5 will become an important diagnostic marker in the near future , and specific targeting of stat5 should be focus of therapeutical intervention strategies to improve treatment of hematological malignancies .

the level of transcription factor activity critically regulates cell fate decisions such as hematopoietic stem cell self - renewal and differentiation . the balance between hematopoietic stem cell self - renewal and differentiation needs to be tightly controlled , as a shift toward differentiation might exhaust the stem cell pool , while a shift toward self - renewal might mark the onset of leukemic transformation . a number of transcription factors have been proposed to be critically involved in governing stem cell fate and lineage commitment , such as hox transcription factors , c - myc , notch1 , -catenin , c / ebp , pu.1 and stat5 . it is therefore no surprise that dysregulation of these transcription factors can also contribute to the development of leukemias . this review will discuss the role of stat5 in both normal and leukemic hematopoietic stem cells as well as mechanisms by which stat5 might contribute to the development of human leukemias .

Introduction STAT5 as a Stem Cell Self-Renewal Factor Mechanisms Involved in STAT5-Induced HSC Self-Renewal Cell Type-Specific STAT5 Signaling: Differential Role of STAT5 in Hematopoietic Stem and Progenitor Cells? Conclusions and Future Perspectives

signal transducer and activator of transcription 5 ( stat5 ) is widely expressed throughout the hematopoietic system , both in stem and progenitor cells as well as in committed erythroid , myeloid and lymphoid cells . these include cytokines and growth factors that can signal through the interleukin 3 ( il3)-receptor family [ il3 , il5 , granulocyte - macrophage colony stimulating factor ( gm - csf ) ] , through the common -chain receptor family ( il2 , il7 , il9 , il12 , il15 ) , through single chain receptors [ erythropoietin ( epo ) , thrombopoietin ( tpo ) , growth hormone ( gh ) , prolactin , granulocyte - colony stimulating factor ( g - csf ) ] , through class ii receptors [ interferon ( ifn- ) , ifn- , il22 ] or through tyrosine kinase receptors [ stem cell factor ( scf ) , platelet derived growth factor ( pdgf ) , epidermal growth factor ( egf ) ] ( fig . in most cases , janus kinase ( jak ) tyrosine kinase activity mediates stat5 tyrosine phosphorylation , and stat5 can be activated by jak1 , 2 or 3 , depending on the cytokine - activated receptor complex . alternatively , the tyrosine kinase receptor family can also induce stat5 phosphorylation in a jak - independent manner . for example , cytokines that activate stat5 in the most immature human hematopoietic stem compartment include scf and tpo . these cytokines have been shown to promote long - term hematopoiesis in vitro , and hypersensitivity to tpo in lnk mice resulted in elevated stem cell self - renewal , which coincided with increased levels of stat5 activity . although initially in stat5ab mice myelopoiesis appeared to be relatively unaffected , it is likely that in myeloid cells many of the signals initiated by e.g. as summarized in table 1 and figure 1b , a wide variety of genetic defects in myeloid leukemias and myeloproliferative diseases ( mpds ) result in activation of the stat5 pathway , including mutations in flt3 and ckit receptors , jak2 mutations , translocations such as tel - pdgfra , and bcr - abl , but also as a result of increased cytokine signaling . numerous functional studies have indicated that aberrant activation of stat5 can contribute to the process of leukemic transformation . downstream of flt3-itd ( internal tandem duplication ) mutations , stat5 is strongly activated via two tyrosine residues within the flt3 receptor , y589 and y592 that act as docking sites for the sh2 domain of stat5 molecules . mutation of these residues into phenylalanines completely abrogated activation of stat5 , and importantly completely impaired induction of a myeloproliferative disease in vivo in a murine transplantation model . although in human cells introduction of flt3-itd did not result in a myeloproliferative disease in transplanted nod - scid mice , the activated stem cell phenotype imposed on cb cd34 cells , as revealed by the formation of early cobblestone area forming cells ( cafcs ) , was impaired by coexpression of a dominant negative stat5a ( y694f ) mutant , suggesting that also in human cells stat5 is an important mediator of flt3-itd - induced signaling . in studies in which stat5 expression was targeted in primary acute myeloid leukemia ( aml ) cd34 cells using a lentiviral approach , it was observed that long - term expansion and the formation of leukemic cafcs was strongly impaired by downmodulation of stat5 . although the presence of flt3-itds was not the exclusive genetic mutation that induced constitutive stat5 signaling in the samples that were studied , these data clearly underscore the important role that stat5 fulfills in long - term expansion and self - renewal of primary aml stem / progenitor cells as well . in chronic myeloid leukemia ( cml ) induced by bcr - abl a number of studies have shown that stat5 is efficiently activated downstream of bcr - abl , and interference with stat5 activation negatively impacts the survival and proliferation of bcr - abl - expressing cells . in primary human cml cells , it was demonstrated that downmodulation of stat5 expression by rnai impaired bcr - abl - dependent proliferation and also reduced colony formation in methylcellulose . inhibition of stat5 by pimozide reduced colony formation of cml cd34 cells , also in tyrosine kinase - resistant patient samples . it was demonstrated that a tryptophan residue in the n - terminal region of stat5 is required for tetramerization of stat5 dimers , and tetramer - deficient stat5 mutants were unable to induce leukemia in mice . this was later confirmed by others , and a fatal mpd was observed by overexpression of these stat5 mutants , but only when the most primitive cd34lin ckitsca1 ( lsk ) population was transduced and used for transplantation to irradiated recipients , suggesting that the stem cell , but not a committed progenitor is the target cell for transformation induced by activated stat5 . intriguingly , while these examples clearly demonstrate that stat5 can transform murine hematopoietic stem cells ( hscs ) , no in vivo stat5-induced transformation has been reported in human cell populations . while enhanced self - renewal and long - term stem cell maintenance can be achieved by introduction of activated stat5 in human cd34 cells , a myeloproliferative disease or leukemia does not occur in non - obese diabetic / severe combined immunodeficiency ( nod - scid ) transplantations models . collectively , these data indicate that stat5 is frequently activated in various hematological malignancies , whereby it strongly affects processes such as self - renewal and lineage fate determination . whether stat5 target genes in normal and leukemic stem cells are identical , or whether leukemic stem cell - specific stat5 target genes exist remains to be determined . also , it will be informative to study how stat5 might cooperate with additional leukemic oncogenes in a multi - hit approach to model the development of human leukemias . loss - of - function and gain - of - function experiments have revealed critical roles for stat5 in the hematopoietic stem / progenitor compartment . stat5ab mice have been used to assess stem cell function in the absence of wt stat5 signaling . these mice were characterized by normal hsc numbers and stem cells isolated from the bone marrow or fetal liver were capable of engrafting irradiated recipients . the underlying mechanisms are not fully elucidated yet , but it has been observed that the responsiveness of stat5ab hscs to early - acting cytokines such as il3 and scf was reduced , while the sensitivity to 5-fluoroacil was enhanced . more recently , using an mx1-cre inducible mouse model , it was shown that conditional deletion of stat5 results in a loss of stem cell quiescence , associated with reduced survival and gradual loss of the hsc pool . downmodulation of stat5 to about 30% of the endogenous levels reduced progenitor frequencies as determined by colony forming cell ( cfc ) assays in methylcellulose as well as stem cell frequencies as determined by long - term culture - initiating cell ( ltc - ic ) assays in limiting dilution . this resulted in reduced long - term expansion on ms5 bone marrow stroma upon downmodulation of stat5 expression , whereby the myeloid and erythroid differentiation were unaffected . reversely , activating mutants of stat5 have been introduced in murine cd34lsk cells and the effects on stem and progenitor cells were assessed in vitro and in vivo . introduction of stat5a(1 * 6 ) or stat5a(1 * 7 ) mutants resulted in a strong ex vivo expansion of immature cfu - nmem progenitors , without affecting the symmetry of stem cell divisions as determined in paired - daughter cell assays . overexpression of stat5a(1 * 6 ) in human cd34 cord blood cells resulted in enhanced stem cell self - renewal . this enhanced self - renewal was only observed in bone marrow stromal cocultures , but not in cytokine - driven liquid culture conditions . these data argued that stat5-induced hsc cell self - renewal depends on the presence of a bone marrow microenvironment , and it was indeed observed that stat5a(1 * 6)-expressing cd34 cells have a strongly enhanced interaction with bone marrow stromal cells , resulting in the appearance of early cafcs underneath the stroma within 1 week after plating . these cafcs contained self - renewal potential as demonstrated by their capacity to give rise to second cafcs upon harvest and replating onto new stroma , as well as by their capacity to engraft in sublethally irradiated nod - scid mice . upon serial replating , long - term cultures could be established by overexpression of activated stat5 for over 20 weeks , giving rise to new cafcs upon each replating as well as to progeny in suspension . when stat5a(1 * 6 ) mutants were expressed in murine embryonic stem ( es ) cells , the generation of hematopoietic stem cells was greatly facilitated as studied on op9 bone marrow stromal cells . importantly , these cafcs could be serially passaged onto new op9 stroma , giving rise to second and third cafcs that were able to sustain long - term hematopoiesis and generate high numbers hematopoietic progenitors , indicative of hsc self - renewal in vitro . also , the cafcs generated by activation of stat5 could engraft sublethally irradiated nod - scid mice , indicating that stat5 facilitates the generation of es - derived hscs that can contribute to hematopoiesis in vivo as well . although various stat5 target genes have been identified , the mechanisms by which stat5 acts on hscs remain to be elucidated . using cell lines or heterogeneous stem / progenitor cell populations , enhanced cell growth is one of the most dominant phenotypes that is frequently observed in various studies , and several genes that are regulated by stat5 associate with cell proliferation and cell cycle progression , including cyclin d1 , pim1 and c - myc . c - myc controls the balance between self - renewal and differentiation of hscs . however , when more purified stem cell populations were studied using a conditional deletion mx1-cre model it was observed that stat5 was required to maintain hsc quiescence . whether prevention of apoptosis contributes to stat5-induced hsc self - renewal is currently unclear , but bcl2 overexpression was not sufficient to rescue the repopulation defects of stat5ab hscs , suggesting that protection against apoptosis is not the main role of stat5 signaling in hscs . the basic helix - loop - helix transcriptional inhibitor id1 is also upregulated by stat5 , and id1-deficient hscs fail to self - renew , leading to low steady - state hsc numbers and premature hsc exhaustion . little evidence exists that stat5 affects the expression of other known hsc self - renewal regulators such as bmi1 or hoxb4 . glucose uptake was enhanced in cells expression activated stat5 , coinciding with a hif2-dependent upregulation glucose metabolism genes , suggesting that pathways normally active under hypoxia might be utilized by stat5 under normoxic conditions as well to maintain stem cell properties . the phenotype imposed on cells by stat5 might well depend on the actual level of stat5 activity that is induced . using a 4-hydroxytamoxifen inducible system that allowed titration of stat5 activity in human stem and progenitor cells we have demonstrated that the stem cell maintenance properties require intermediate stat5 activation . on the other hand , high stat5 activation levels resulted in erythroid differentiation at the expense of hsc self - renewal . c / ebp levels were reduced upon stat5 activation , which reached maximum reduction levels at intermediate stat5 activation . in line with these observations , in mice c / ebp deficiency resulted in hyperproliferation of hematopoietic progenitor cells and enhancement of hematopoietic stem cell repopulating capacity and self - renewal . reintroduction of c / ebp in stat5a(1 * 6)-transduced human cd34 cells was sufficient to impair hsc self - renewal capacity . the mechanisms by which stat5 affects c / ebp expression levels are still under investigation , but these observations leave open the possibility that enhanced hsc self - renewal might in part be explained by reduction in c / ebp expression levels . it is remarkable that the effects of stat5 on hsc self - renewal are confined to intermediate stat5 activation levels . this dosage effect of stat5 on self - renewal is consistent with the observed constitutive activation of stat5 in aml samples , which is typically lower then cytokine - induced stat5 activation . such a dosage effect of transcription factors is at present not well understood , but besides stat5 this has also been observed for the myeloid transcription factor pu.1 , which at 20% expression gives rise to self - renewing murine myeloid leukemias , whereas 50% or 100% reduction in expression do not have such a dramatic effect . recently , also for wnt signaling it was demonstrated that intermediate activation levels enhance self - renewal of hscs . collectively , these examples clearly underscore the role of transcription factor dosage in regulating hsc self - renewal . the observation that stat5 drives cell cycle progression in various cell types and anti - apoptosis in others , while stat5 is also required to maintain quiescence of hematopoietic stem cells , suggests that the cell - biological consequences of stat5 signaling might be highly cell type - specific . activation of stat5 specifically in hsc , common myeloid ( cmp ) , granulocyte - macrophage ( gmp ) or megakaryocyte - erythroid progenitor ( mep ) populations resulted in rather distinct phenotypes . long - term self - renewal and enhanced cobblestone formation could only be imposed on hscs , but not on committed progenitor subpopulations . the underlying mechanisms are currently unclear , but a number of possibilities might be hypothesized . first , it has been shown that several cofactors such as p300/cbp , but also interactions with other transcription factors such as foxo3a , can modulate and fine - tune the stat5 response . it has been observed that , unlike p300 , cbp is essential for hsc self - renewal maintenance , while p300 is suggested to play a role in differentiation . interaction of various transcription factors with p300/cbp is facilitated by the p300/cbp interacting protein cited2 which has been shown to be a target gene of stat5 and has differential expression and functions in hematopoietic stem vs. progenitor cells ( and our own observations ) . in part , such interactions can be mediated by posttranslational modifications such as serine phosphorylation or glycosylation of stat5 . thus , the repertoire of specific cytokine and growth factor receptors that is expressed on a cell , as well as direct interactions between hematopoietic stem cells and their bone marrow niche , might ultimately determine the specific stat5 response ( fig . single cell tyrosine phospho - stat5 analysis revealed that within the normal hematopoietic stem cell and progenitor compartment highly distinct cytokine - induced stat5 activation patterns are observed . these observations clearly indicate that strong differences exist in how cytokine and growth factor signals are mediated within a certain cell type , both normal as well as leukemic . although elucidation of molecular mechanisms by which cell type specific stat5 signaling is orchestrated needs further studies , cell type - specific stat5 target genes clearly do exist . it will be interesting to analyze whether the enhanced long - term self - renewal that is observed upon activation of stat5 in hematopoietic stem cells involves improved stem cell maintenance by keeping the hscs pool in a relatively quiescent state via upregulation of p21 . further , activation of p21 has been shown to be critical in preventing excess dna - damage accumulation and functional exhaustion of leukemic stem cells , and it will be interesting to further reveal its role downstream of stat5 in hscs . under hypoxic conditions , such as in the presumed endosteal quiescent stem cell niche , it is currently unknown whether and which hif - induced target genes are essential to maintain stemness of normal hscs , but it was recently shown that in hif1 mice hscs numbers decrease during stress which was associated with a loss of hsc quiescence . whether hif1 and hif2 display similar or distinct functions in hscs remains to be established . our understanding of the mechanisms that determine whether , where and when a stem cell will self - renew or differentiate is still limited , but recent advances have indicated that the stem cell microenvironment provides essential cues that direct these cell fate decisions . it is remarkable that stat5-induced long - term self - renewal is typically observed when cells are cultured in direct contact with stromal cells , in contrast to , e.g. , bmi1-induced self - renewal , which occurred in a more microenvironment - independent manner . thus , altered interactions with the stem cells niche might also underlie the enhanced self - renewal properties imposed on hscs by activated stat5 . although the mechanisms by which the interaction with the microenvironment of stat5a(1 * 6)-expressing cd34 cells are still unclear , our ongoing studies in which gene - expression profiling was performed in hscs and progenitor subsets revealed that the list of stat5-targets is significantly enriched for membrane ( associated ) proteins . one of the stat5 targets that has been identified is muc1 which is a ( proto)oncogene involved in adhesion and transendothelial migration , and has been associated with initiation of various intracellular signal transduction pathways including -catenin , p53 and nfb pathways . within the endosteal region of the bone marrow where stem cells are thought to reside , ca levels are high , and hsc retention within the niche depends on the calcium - sensing receptor ( car ) . in both murine and human model systems it has been convincingly shown that stat5 fulfills an important role in hematopoietic stem cell self - renewal . although the precise mechanisms by which hsc self - renewal is orchestrated by stat5 remain elusive to date , an increasing number of stat5 target genes have been identified that are currently under investigation . in myeloproliferative diseases and leukemias , a number of oncogenes have been identified that are capable of inducing stat5 activity , and accumulating evidence has indicated that stat5 participates in self - renewal of leukemic stem cells as well . thus , it appears likely that stat5 will become an important diagnostic marker in the near future , and specific targeting of stat5 should be focus of therapeutical intervention strategies to improve treatment of hematological malignancies .

signal transducer and activator of transcription 5 ( stat5 ) is widely expressed throughout the hematopoietic system , both in stem and progenitor cells as well as in committed erythroid , myeloid and lymphoid cells . these include cytokines and growth factors that can signal through the interleukin 3 ( il3)-receptor family [ il3 , il5 , granulocyte - macrophage colony stimulating factor ( gm - csf ) ] , through the common -chain receptor family ( il2 , il7 , il9 , il12 , il15 ) , through single chain receptors [ erythropoietin ( epo ) , thrombopoietin ( tpo ) , growth hormone ( gh ) , prolactin , granulocyte - colony stimulating factor ( g - csf ) ] , through class ii receptors [ interferon ( ifn- ) , ifn- , il22 ] or through tyrosine kinase receptors [ stem cell factor ( scf ) , platelet derived growth factor ( pdgf ) , epidermal growth factor ( egf ) ] ( fig . while stat5 is expressed in the majority of hematopoietic cell types , thus , specific cytokines are able to induce stat5 activity in subsets of cell types only . these cytokines have been shown to promote long - term hematopoiesis in vitro , and hypersensitivity to tpo in lnk mice resulted in elevated stem cell self - renewal , which coincided with increased levels of stat5 activity . in myeloid cells , stat5 can be activated by a variety of cytokines , including il3 , il5 , gm - csf and csf1 ( reviewed in ref . stat5 activation is required for il2-induced t cell proliferation and the production of nk cells , or for il7-mediated b cell expansion . as summarized in table 1 and figure 1b , a wide variety of genetic defects in myeloid leukemias and myeloproliferative diseases ( mpds ) result in activation of the stat5 pathway , including mutations in flt3 and ckit receptors , jak2 mutations , translocations such as tel - pdgfra , and bcr - abl , but also as a result of increased cytokine signaling . downstream of flt3-itd ( internal tandem duplication ) mutations , stat5 is strongly activated via two tyrosine residues within the flt3 receptor , y589 and y592 that act as docking sites for the sh2 domain of stat5 molecules . mutation of these residues into phenylalanines completely abrogated activation of stat5 , and importantly completely impaired induction of a myeloproliferative disease in vivo in a murine transplantation model . although in human cells introduction of flt3-itd did not result in a myeloproliferative disease in transplanted nod - scid mice , the activated stem cell phenotype imposed on cb cd34 cells , as revealed by the formation of early cobblestone area forming cells ( cafcs ) , was impaired by coexpression of a dominant negative stat5a ( y694f ) mutant , suggesting that also in human cells stat5 is an important mediator of flt3-itd - induced signaling . in studies in which stat5 expression was targeted in primary acute myeloid leukemia ( aml ) cd34 cells using a lentiviral approach , it was observed that long - term expansion and the formation of leukemic cafcs was strongly impaired by downmodulation of stat5 . although the presence of flt3-itds was not the exclusive genetic mutation that induced constitutive stat5 signaling in the samples that were studied , these data clearly underscore the important role that stat5 fulfills in long - term expansion and self - renewal of primary aml stem / progenitor cells as well . in chronic myeloid leukemia ( cml ) induced by bcr - abl a number of studies have shown that stat5 is efficiently activated downstream of bcr - abl , and interference with stat5 activation negatively impacts the survival and proliferation of bcr - abl - expressing cells . bcr - abl p210-transduced stat5 murine bone marrow ( bm ) cells developed cml with low frequencies , with a delayed onset of disease . in primary human cml cells , it was demonstrated that downmodulation of stat5 expression by rnai impaired bcr - abl - dependent proliferation and also reduced colony formation in methylcellulose . inhibition of jak2 kinase activity abrogated the activation of stat5 , which coincided with a suppression of erythropoiesis in vitro and in vivo . it was demonstrated that a tryptophan residue in the n - terminal region of stat5 is required for tetramerization of stat5 dimers , and tetramer - deficient stat5 mutants were unable to induce leukemia in mice . this was later confirmed by others , and a fatal mpd was observed by overexpression of these stat5 mutants , but only when the most primitive cd34lin ckitsca1 ( lsk ) population was transduced and used for transplantation to irradiated recipients , suggesting that the stem cell , but not a committed progenitor is the target cell for transformation induced by activated stat5 . intriguingly , while these examples clearly demonstrate that stat5 can transform murine hematopoietic stem cells ( hscs ) , no in vivo stat5-induced transformation has been reported in human cell populations . while enhanced self - renewal and long - term stem cell maintenance can be achieved by introduction of activated stat5 in human cd34 cells , a myeloproliferative disease or leukemia does not occur in non - obese diabetic / severe combined immunodeficiency ( nod - scid ) transplantations models . it is plausible that the nod - scid xenograft model is not suitable to completely recapitulate human disease , or alternatively it is possible that species - specific differences in stat5 signaling exist . in line with these observations , introduction of bcr - abl in murine bm resulted in a rapid and lethal mpd whereby recipients die within 3 weeks after transplantation . introduction of bcr - abl in human cd34 cells does not result in a rapid leukemia or mpd in engrafted nod - scid mice , and only after 5 mo progression to an early stage disease was observed in some animals . collectively , these data indicate that stat5 is frequently activated in various hematological malignancies , whereby it strongly affects processes such as self - renewal and lineage fate determination . whether stat5 target genes in normal and leukemic stem cells are identical , or whether leukemic stem cell - specific stat5 target genes exist remains to be determined . loss - of - function and gain - of - function experiments have revealed critical roles for stat5 in the hematopoietic stem / progenitor compartment . the underlying mechanisms are not fully elucidated yet , but it has been observed that the responsiveness of stat5ab hscs to early - acting cytokines such as il3 and scf was reduced , while the sensitivity to 5-fluoroacil was enhanced . loss of protection against apoptosis most likely does not explain the stat5ab hscs phenotypes , as overexpression of bcl2 was not sufficient to rescue repopulating defects . although homing of stat5ab bm cells into lethally irradiated recipients was not impaired , retention in the bone marrow was reduced under non - myeloablative conditions , leaving open the possibility that competition for the niche might play a role . more recently , using an mx1-cre inducible mouse model , it was shown that conditional deletion of stat5 results in a loss of stem cell quiescence , associated with reduced survival and gradual loss of the hsc pool . in order to study stat5 signaling in human hematopoietic stem / progenitor cells , we have used a lentiviral shrna approach in cord blood ( cb ) cd34 cells . downmodulation of stat5 to about 30% of the endogenous levels reduced progenitor frequencies as determined by colony forming cell ( cfc ) assays in methylcellulose as well as stem cell frequencies as determined by long - term culture - initiating cell ( ltc - ic ) assays in limiting dilution . this resulted in reduced long - term expansion on ms5 bone marrow stroma upon downmodulation of stat5 expression , whereby the myeloid and erythroid differentiation were unaffected . reversely , activating mutants of stat5 have been introduced in murine cd34lsk cells and the effects on stem and progenitor cells were assessed in vitro and in vivo . introduction of stat5a(1 * 6 ) or stat5a(1 * 7 ) mutants resulted in a strong ex vivo expansion of immature cfu - nmem progenitors , without affecting the symmetry of stem cell divisions as determined in paired - daughter cell assays . importantly , long - term repopulating hscs could be maintained under ex vivo culture conditions as cd34lsk cells expressing activated stat5 had a strong competitive repopulating advantage over wild type cells after 7 d and 10 d ex vivo culturing in the presence of scf or scf and tpo . this enhanced self - renewal was only observed in bone marrow stromal cocultures , but not in cytokine - driven liquid culture conditions . these data argued that stat5-induced hsc cell self - renewal depends on the presence of a bone marrow microenvironment , and it was indeed observed that stat5a(1 * 6)-expressing cd34 cells have a strongly enhanced interaction with bone marrow stromal cells , resulting in the appearance of early cafcs underneath the stroma within 1 week after plating . these cafcs contained self - renewal potential as demonstrated by their capacity to give rise to second cafcs upon harvest and replating onto new stroma , as well as by their capacity to engraft in sublethally irradiated nod - scid mice . when stat5a(1 * 6 ) mutants were expressed in murine embryonic stem ( es ) cells , the generation of hematopoietic stem cells was greatly facilitated as studied on op9 bone marrow stromal cells . importantly , these cafcs could be serially passaged onto new op9 stroma , giving rise to second and third cafcs that were able to sustain long - term hematopoiesis and generate high numbers hematopoietic progenitors , indicative of hsc self - renewal in vitro . also , the cafcs generated by activation of stat5 could engraft sublethally irradiated nod - scid mice , indicating that stat5 facilitates the generation of es - derived hscs that can contribute to hematopoiesis in vivo as well . using cell lines or heterogeneous stem / progenitor cell populations , enhanced cell growth is one of the most dominant phenotypes that is frequently observed in various studies , and several genes that are regulated by stat5 associate with cell proliferation and cell cycle progression , including cyclin d1 , pim1 and c - myc . pim serine / threonine kinases act as mediators of cytokine - induced cell growth by promoting acceleration of cell - cycle progression both at the g1/s and g2/m transitions by phosphorylating and activating the phosphatases cdc25a and cdc25c , respectively . c - myc controls the balance between self - renewal and differentiation of hscs . upon stat5 deletion a decrease in the percentage of cells in g0 within the long - term and short - term hsc compartments was observed , coinciding with a decrease in expression of quiescence - associated genes such as p57 and tie2 . in erythroid cells , it has been convincingly demonstrated that stat5 contributes to cell survival by upregulating the anti - apoptosis gene bcl - xl . whether prevention of apoptosis contributes to stat5-induced hsc self - renewal is currently unclear , but bcl2 overexpression was not sufficient to rescue the repopulation defects of stat5ab hscs , suggesting that protection against apoptosis is not the main role of stat5 signaling in hscs . in our co - cultures , despite strong reductions in ltc - ic and cfc frequencies , we also did not detect an increased rate of apoptosis in stat5 rnai - transduced cd34 cb cells , and no decreased expression of the bcl - xl gene was observed . the basic helix - loop - helix transcriptional inhibitor id1 is also upregulated by stat5 , and id1-deficient hscs fail to self - renew , leading to low steady - state hsc numbers and premature hsc exhaustion . little evidence exists that stat5 affects the expression of other known hsc self - renewal regulators such as bmi1 or hoxb4 . recently , we observed that stat5 binds to and activates the promoter of hypoxia induced factor 2 ( hif2 ) in human cd34/cd38 hscs . glucose uptake was enhanced in cells expression activated stat5 , coinciding with a hif2-dependent upregulation glucose metabolism genes , suggesting that pathways normally active under hypoxia might be utilized by stat5 under normoxic conditions as well to maintain stem cell properties . using a 4-hydroxytamoxifen inducible system that allowed titration of stat5 activity in human stem and progenitor cells we have demonstrated that the stem cell maintenance properties require intermediate stat5 activation . in line with these observations , in mice c / ebp deficiency resulted in hyperproliferation of hematopoietic progenitor cells and enhancement of hematopoietic stem cell repopulating capacity and self - renewal . the mechanisms by which stat5 affects c / ebp expression levels are still under investigation , but these observations leave open the possibility that enhanced hsc self - renewal might in part be explained by reduction in c / ebp expression levels . this dosage effect of stat5 on self - renewal is consistent with the observed constitutive activation of stat5 in aml samples , which is typically lower then cytokine - induced stat5 activation . such a dosage effect of transcription factors is at present not well understood , but besides stat5 this has also been observed for the myeloid transcription factor pu.1 , which at 20% expression gives rise to self - renewing murine myeloid leukemias , whereas 50% or 100% reduction in expression do not have such a dramatic effect . collectively , these examples clearly underscore the role of transcription factor dosage in regulating hsc self - renewal . the observation that stat5 drives cell cycle progression in various cell types and anti - apoptosis in others , while stat5 is also required to maintain quiescence of hematopoietic stem cells , suggests that the cell - biological consequences of stat5 signaling might be highly cell type - specific . activation of stat5 specifically in hsc , common myeloid ( cmp ) , granulocyte - macrophage ( gmp ) or megakaryocyte - erythroid progenitor ( mep ) populations resulted in rather distinct phenotypes . long - term self - renewal and enhanced cobblestone formation could only be imposed on hscs , but not on committed progenitor subpopulations . seen from this perspective , the modulation of stat5 signaling by p300/cbp could add to a stem vs. progenitor - specific component of stat5 . it has been observed that , unlike p300 , cbp is essential for hsc self - renewal maintenance , while p300 is suggested to play a role in differentiation . interaction of various transcription factors with p300/cbp is facilitated by the p300/cbp interacting protein cited2 which has been shown to be a target gene of stat5 and has differential expression and functions in hematopoietic stem vs. progenitor cells ( and our own observations ) . thus , besides the induction of stat5 tyrosine phosphorylation required for dimerization , nuclear translocation and dna binding , the simultaneous activation of pathways that mediate stat5 serine phosphorylation or glycosylation would be required . thus , the repertoire of specific cytokine and growth factor receptors that is expressed on a cell , as well as direct interactions between hematopoietic stem cells and their bone marrow niche , might ultimately determine the specific stat5 response ( fig . single cell tyrosine phospho - stat5 analysis revealed that within the normal hematopoietic stem cell and progenitor compartment highly distinct cytokine - induced stat5 activation patterns are observed . for instance , in some patient samples strong il3 and gm - csf responses were observed , but only in the cd34 subpopulation , while in other cases strong tpo responses were observed within cd34/cd38 and cd34/cd38 compartments . it will be interesting to analyze whether the enhanced long - term self - renewal that is observed upon activation of stat5 in hematopoietic stem cells involves improved stem cell maintenance by keeping the hscs pool in a relatively quiescent state via upregulation of p21 . further , activation of p21 has been shown to be critical in preventing excess dna - damage accumulation and functional exhaustion of leukemic stem cells , and it will be interesting to further reveal its role downstream of stat5 in hscs . under hypoxic conditions , such as in the presumed endosteal quiescent stem cell niche , it is currently unknown whether and which hif - induced target genes are essential to maintain stemness of normal hscs , but it was recently shown that in hif1 mice hscs numbers decrease during stress which was associated with a loss of hsc quiescence . our understanding of the mechanisms that determine whether , where and when a stem cell will self - renew or differentiate is still limited , but recent advances have indicated that the stem cell microenvironment provides essential cues that direct these cell fate decisions . thus , altered interactions with the stem cells niche might also underlie the enhanced self - renewal properties imposed on hscs by activated stat5 . although the mechanisms by which the interaction with the microenvironment of stat5a(1 * 6)-expressing cd34 cells are still unclear , our ongoing studies in which gene - expression profiling was performed in hscs and progenitor subsets revealed that the list of stat5-targets is significantly enriched for membrane ( associated ) proteins . one of the stat5 targets that has been identified is muc1 which is a ( proto)oncogene involved in adhesion and transendothelial migration , and has been associated with initiation of various intracellular signal transduction pathways including -catenin , p53 and nfb pathways . within the endosteal region of the bone marrow where stem cells are thought to reside , ca levels are high , and hsc retention within the niche depends on the calcium - sensing receptor ( car ) . in myeloproliferative diseases and leukemias , a number of oncogenes have been identified that are capable of inducing stat5 activity , and accumulating evidence has indicated that stat5 participates in self - renewal of leukemic stem cells as well . thus , it appears likely that stat5 will become an important diagnostic marker in the near future , and specific targeting of stat5 should be focus of therapeutical intervention strategies to improve treatment of hematological malignancies .

an effectively designed and properly implemented clinical trial protocol is the most definitive method of determining whether an intervention has a hypothesized effect . recruitment and retention of an appropriate and representative study sample enhance the validity of the results and heighten the potential public health impact . despite the benefits of clinical research , many people approached to participate decline to do so because of deeply rooted myths about research . physician - scientists and basic / translational scientists need to work together to dispel these misconceptions so that enhanced participant enrollment can be attained , a step that is vital to conducting a successful clinical trial . the philosophical underpinnings of conducting clinical trial research are centered around three basic ethical principles : respect for persons , beneficence , and justice . those unable to act autonomously should be considered vulnerable populations , including children , prisoners , persons with physical handicaps / mental disabilities , and pregnant women . the informed consent process is vital to protecting human subjects by ensuring that subjects enter into research voluntarily and with complete information . the beneficence principle assesses the risks and benefits of research protection with the two principles : do no harm and maximize benefits and minimize harms . the justice principle is related to the selection of research subjects , addressing the question of who should bear the burden of research and who should benefit . an injustice occurs when a benefit is denied without good reason or when there is an unfair burden . the justice principle prohibits exploiting disadvantaged populations for the benefit of other more advantaged populations . additionally , the principle prohibits researchers from selecting samples that are easily accessible ( college students ) , in compromised positions ( prisoners , those confined to institutions ) or those easily manipulated rather than choosing a population directly related to the study . this principle also requires that everyone benefit from research , not just those who are able to afford it . all three principles must be adhered to during every phase of clinical trial design and conduct in order to safeguard participants and preserve research integrity . enrolling patients into a clinical trial is clearly an important step in the success of a research protocol . many individuals mention some mistrust about their willingness to be involved in a clinical trial . to counter this issue , financial and other incentives have been more frequently used to recruit subjects into clinical trials . when patients come from disadvantaged or low - income populations , this practice clearly raises questions about coercion , manipulation and concerns about true informed consent . institutional review boards are tasked to review these concerns and have rigorous criteria in which to determine acceptance of the use of financial and other incentives in the conduct of research trials , especially related to minority and underrepresented populations . beyond the challenges of patient recruitment exists the issue of participant retention and ensuring that those individuals who enroll successfully complete the study . low retention rates can impair the accuracy of study results and undermine fiscal and human resources ; therefore , emphasis must be placed on maintaining high levels of participation . it is widely known that special barriers and challenges affect the successful enrollment and retention of study participants in clinical trials targeting minority populations . one way of dealing with these issues is to investigate related challenges , consider action plans or strategies that represent evidence - based research and continue seeking methods for improvement . in this paper , we report such experiences in a clinical trial that we are currently conducting in an underserved minority population in the southern united states . a unique approach is that we are utilizing the principles of the socio - ecological model ( sem ) to effectively achieve and maintain high participant recruitment and retention rates . although previous literature[10 - 12 ] has described the use of the sem to promote study recruitment , limited information exists which explores the utility of this model in participant retention , especially , in a minority and socio - economically disadvantaged setting . this research study is a randomized double - blind controlled clinical trial that tests the effi cacy of high - strength ( 4 mg ) as compared to lower - strength / standard of care ( 0.8 mg ) folic acid to prevent fetal body and brain size reduction in pregnant women who smoke . experimental ( 4 mg ) and control ( 0.8 mg ) groups are assigned ( 165 pregnant women to each arm ) . the specific aims of this study are to : assess folic acid reserves in women who smoke during pregnancy ; anddetermine whether higher - strength folic acid in combination with smoking cessation programs will prevent reduction in fetal body and brain size among smoking mothers . assess folic acid reserves in women who smoke during pregnancy ; and determine whether higher - strength folic acid in combination with smoking cessation programs will prevent reduction in fetal body and brain size among smoking mothers . we hypothesize that higher - strength folic acid in combination with a smoking cessation program will prevent reduction in fetal body and brain size . this 5-year clinical trial is being implemented within an underserved minority community in florida , usa and is now in its fifth year . to our knowledge , this is the first human study to determine the utility of higher - strength folic acid in preventing smoking - induced fetal body and brain size reduction ( clinicaltrials.gov registration # nct01248260 ) . women who receive care at this community health center and deliver at the affiliated hospital are typically of low socioeconomic status.very few patients have private health insurance and most are either uninsured or covered by medicaid , the public health insurance program in us that covers the poor and underprivileged members of the society . the racial / ethnic distribution includes 42% blacks , 30% hispanics , and 28% whites . about 15% of the women at the study site smoke actively during pregnancy without quitting , thus this study is approved by the university of south florida institutional review board and each participant signs an approved informed consent form . eligible consented women are enrolled 20 weeks gestation and screened for smoking habits . maternal blood is drawn at baseline for assessment of biomarkers including folate , vitamin b12 , homocysteine levels , and maternal methylenetetrahydrofolatereductase ( mthfr ) enzyme polymorphism . survey instruments are administered to participants during three subsequent antenatal study visits to assess changes in smoking habits , dietary factors , quality of sleep , stress levels and other antenatal psycho - social markers . all study participants are followed until delivery at which time fetal body and brain growth anthropological measures are collected . umbilical cord blood is also collected for genetic and epigenetic tests on markers of fetal brain growth and development . non - smokers are also randomly selected for blood draw to compare baseline folate levels . previous literature has proposed the use of the sem in examining barriers related specifically to enrollment in clinical trials.[10 - 12 ] the sem provides a very useful theoretical framework for addressing the numerous and varied obstacles in participant recruitment and retention . the sem suggests that an individual s behavior is integrated in a dynamic network of intrapersonal characteristics , interpersonal processes , institutional factors , community features and public policy . the model assumes that interactions between individuals and their environment are reciprocal , implying that an individual is influenced by his / her environment and the environment is infl uenced by the individual . rct consort flow diagram the intrapersonal level encompasses the research participant s knowledge , awareness , attitudes , beliefs and perceptions . the participant s family , friends and health care providers comprise important components of the interpersonal level . the healthcare institution s rules , regulations and general attitude toward research shape the institutional level within the model . features of the community that may influence participant retention include convenience and acceptance of prenatal care , local cultural attitudes about smoking , the availability of public transportation and safety of the neighborhood . the public policy level is shaped by local , state and federal laws regarding socio - behavioral and biomedical research . the sem takes into account socio - cultural factors , as well as environmental factors , and their linkages to biologic factors . in order to effectively examine factors that influence participant recruitment and retention in our socio - behavioral , genetic - epigenetic clinical trial among pregnant smokers , we will identify the barriers we have encountered in our trial and use their placement within the sem to identify the reasons for these obstacles ( perceived and/or real ) and viable solutions . taking into account all these level - specific influential factors will help ensure our success in assessing the effi cacy of higher - strength folic acid in preventing the fetal body and brain growth inhibitory effect of prenatal smoking . swift identification and remediation of issues is critical to the success of any clinical research study . based on our knowledge of the sem first , we were aware that our study population , because of its nature , may be experiencing higher levels of stress , depression and other markers of psychosocial dysfunction . with this in mind , the trial was designed to collect information on and provide referrals for individuals who suffer from abnormal levels of stress , depression and sleep disturbances ( factors found on the intrapersonal level of the sem ) . additionally , we question women about their nutritional intake or access to nutritious food ( community level factor ) . as the trial progresses , each issue we encounter is identified and addressed using the sem in the following sections and in figure 2 . application of the socio - ecological model ( sem ) to enhance participation in a clinical trial the intrapersonal level of the sem is made up of individualknowledge , attitudes , beliefs and perceptions that infl uence behavior . fear , distrust , and/or suspicions of research have proven to be substantial barriers to participation in research . there is the belief among many prospective participants that entering a randomized trial will mean a loss of control because they will not be able to choose their treatment or that clinical research will cause them harm others fear being treated like guinea pigs or distrust of the medical community due to past experiences , particularly in racial and ethnic minority populations . many believe signing the informed consent provides legal protection to physicians or researchers rather than study participants . the national cancer institute reports that african americans are less likely to participate because of knowledge of historical mistreatment of research participants such as the tuskegee syphilis study . one study reported that one - third of african - american women avoided participating in clinical trials because they believed that scientists could not be trusted . educating the public and health professionals on human subjects protection and legislation is essential to eliminating this mistrust and demonstrating how the tuskegee incident would be prevented today . health care providers and research staff should communicate safety measures to patients to help build their trust and overcome suspicions . the target population for the project is comprised of minority socio - economically disadvantaged individuals and a mistrust of research could be a reason for declining to participate . we are aware of these challenges and are developing strategies to limit this as a recruitment barrier . study staff has held meetings with clinic staff to discuss ways to eliminate mistrust of researchers , particularly among minority patients . currently , some clinic providers explain the trial to their patients prior to referring them to our study . in order to ease potential suspicions , research staff gives a potential participant time to read through the informed consent document and to discuss with her partner or family . additionally , trained research staff reviews the consent document section by section with each prospective participant . the research staff is careful to explain fully the following issues : the voluntary nature of the research , its distinction from clinical care , the right to withdraw without penalty at any time , and the steps to be taken to protect confidentiality of information . research staff also confirms the participant s correct understanding of each of these issues through appropriate questioning . to reduce social and psychological consequences about answering questions on high risk behaviors ( smoking ) during pregnancy , participants are assured that responses are kept confidential and are for research purposes only . language or literacy barriers may also create obstacles to participation which also fall under the intrapersonal level of the sem . in order to address this issue in our study , only trained research staff is permitted to recruit participants . the participant s thorough understanding of the informed consent form is confi rmed through appropriate questioning . additionally , two of the study staff members speak spanish and english , which is extremely useful because of the high population of spanish - speaking patients at the study site . although the majority of patients have reported taking their folic acid on a regular basis , a few have reported unwelcome side effects such as nausea and/or constipation . literature has suggested that counseling women to understand that side effects will subside is one way to prevent side effect - related non - compliance . in our trial , we suggest that participants take their multivitamins with food to prevent gastrointestinal discomfort and to increase their water and fiber intake . one particular belief that is most common in latino cultures is that an individual must accept god s will and that their health is in god s hands . individuals who hold this belief may be less likely to take actions to promote their health , such as taking folic acid / multivitamins in a research study . although we have not investigated individual health belief models and how they impact compliance to medication during pregnancy , we believe understanding the patient s intrapersonal perspective within the sem framework is a potential solution to medication adherence . a strong point of the study design is that both self - report of dietary folic acid intake as well as biologic blood levels are measured and assessed in the analysis . the interpersonal level of the sem comprises the participant s social network including family , friends , peers , and health care providers . several sociodemographic factors such as maternal age and household income influence multivitamin use . provider influence can have a signifi ca nt impact on patient enrollment . the extent to which the patient trusts her doctor can infl uence whether she will participate in the study . physicians must recognize these concerns , teach the patient that the decision to participate is entirely that of the patient , not the doctor , and confidently recommend the most suitable studies . some physicians may feel they are losing control of their patient s care by referring a patient to a clinical trial . provider attitudes and perceptions , such as mistrust of researchers and provider lack of awareness of the study are the leading barriers that prevent providers from referring or enrolling patients in clinical trials that target minority populations . we have encountered many providers at the clinic who seem to have a mistrust of research and do not want to refer their patients to our study . this obstacle was addressed by setting up a formal meeting with the medical director and clinic manager to discuss our study objectives and open the lines of communication . during the meeting , additional topics of discussion included patient fl ow , the average wait time for new obstetrical appointments , and the most effective use of the patient s wait time . in addition , due to limited space in the clinic , discussions covered the most ideal days for recruitment particularly , days with the highest numbers of new appointments . educating staff and demonstrating that they will not face additional burden is important , however , this is easier to accomplish in theory than in practice . prior to initiating recruitment , research staff presented the study objectives to nursing personnel at a clinic staff meeting . gaining the support of the medical director and the clinic manager was very instrumental in bridging the gap between research and clinic staff who were initially less cooperative . study staff provides monthly emails to the director and manager about recruitment numbers and/or any important updates . establishing and maintaining these open lines of communication has provided a strong foundation for recruiting in the clinic . the institutional level of the sem involves the health care system , policies , and structures working together to assist or hinder the clinical trial . one barrier we have had to overcome at this level includes policies and structures in place within the health care system that influence or limit participation . during the initial phase of participant recruitment one challenge was to fi nd the best time and place to consent and interview participants that afforded adequate privacy without interrupting clinic flow . for instance , although women spend more time in the waiting room than in the examination room , there is little privacy to complete the interview process in this setting . research staff quickly discovered that the most ideal opportunity to consent and interview women is following triage when the women are sitting in the examination room waiting for the provider . the examination room allows for privacy and the window of time is lengthy enough to answer the potential participant s questions regarding the study and to complete the consenting process . depending on clinic fl ow , the recruiter is usually able to complete the interview process during this time as well . biological specimen collection for research use can be a complicated process and requires detailed protocols . collection of blood , urine , saliva , and extracted dna and rna should be carefully planned prior to research initiation , closely monitored , and handled consistently to guarantee samples will provide maximum value to the study . most of the challenges related to biological specimen collection fall under the institutional level of the sem . when establishing collaborations with the laboratories , we discovered that the rna had to be extracted within four hours of draw if collected and transported to the lab in regular edta tubes . this proved to be a major institutional barrier when working with a lab that is only open during normal business hours , as women can deliver at all hours of the night and on weekends . this problem was remedied by purchasing paxgene blood rna tubes , which contain an rna preservative . these tubes are about eight times the cost of regular tubes , but allow the blood to be kept at room temperature for up to three days . therefore , these tubes are ideal for our study and allow for rna extraction at a later date it was challenging to introduce new research staff members into a unit that has limited space . we hired a staff member who spends time daily on the labor and delivery unit . after establishing our presence , the cooperation of the hospital staff has been more forthcoming and is improving with time . finally , the transition of the hospital medical record system from paper to an electronic system as well as a change in hospital leadership also falls under the institutional level . prior to this change in health information systems , it was diffi cult to collect the appropriate samples and identify when patients would arrive at the hospital for their delivery . after the transition , the new electronic medical record system has become a continuous link of patient information from the antenatal period through delivery to the postnatal period . this new system has facilitated the conduct of the study by enabling identifi cation of patients at delivery and timely biological specimen collection . without this cohesion of institutional systems working together , we would not have been able to successfully retain as many patients or collect as many biological samples . although we anticipated barriers in recruiting minority participants into the trial , we quickly realized that the true challenge would be retaining the women s interest in participating over multiple study visits . we understood that in addition to providing a small monetary incentive to compensate them for their time , we also had to ensure that their participation did not create an additional burden . with this in mind , study visits are combined with routine prenatal appointments to reduce the scheduling hassles for participants . this effort helps keep our retention rate high and our loss to follow - up rate low . currently , our loss to follow - up rate is less than 6% and this can be attributed to our understanding of the factors that influence participation in research studies based on the sem community level . because our study population is comprised of socio - economically disadvantaged individuals , access to nutritious foods is often limited . folic acid is important for fetal development and the prevention of neural tube defects . since many women do not obtain the recommended daily intake ( rdi ) of folic acid , the food and drug administration began folic acid fortification of cereal and other grain products . in spite of this , many women still do not meet the rdi for folic acid . non - hispanic black women and hispanic women are less likely to meet the rdi , regardless of folate source , than non - hispanic white women . both arms of the study are likely to be exposed to these sources of dietary folic acid since the study design employs randomization . this paper describes a clinical research study utilizing a population made up primarily of minority , socio - economically disadvantaged individuals . this population is often poorly represented in clinical research studies . due to the heterogeneous nature of the global population , it is vital to have an equally signifi ca nt number of participants enrolled in clinical research from different ethnic and racial groups . additionally , inclusion of underrepresented groups in clinical trials is important because sex , race , and co - morbid conditions could play vital roles in intervention studies , especially those related to response to medications / drugs . as is the case for this study , using different dosage levels of folic acid to prevent fetal body and brain size reduction in pregnant women who smoke is important for a variety of reasons . certainly , babies born with reduced body and brain development have many challenges early in their lives and can continue into adolescence and adulthood . secondly , determining more effi cacious drug dosages has signifi ca nt preventive outcomes and fi nancial implications for this and other populations . finally , folic acid is an inexpensive intervention that has minimal risk and is easily available . it is important to determine patient compliance issues to maximize the likelihood of this population taking folic acid during the entire pregnancy but especially as early as possible after the pregnancy is identifi ed and diagnosed . the population in this study is often seen with poor nutritional behaviors . providing a folic acid supplement is again an important way to enhance the health of the neonate . with the challenges evident in this population to consider folic acid supplementation , a clear analysis of the challenges and barriers with such an intervention is essential . using the sem as a theoretical framework , we have been able to address many barriers and challenges that could have reduced the effectiveness and utility of our intervention . we have faced barriers but have succeeded in overcoming them through a thorough analysis of the intrapersonal , interpersonal , institutional , and community levels . from this analysis , the implementation strategies have evolved with greater anticipated outcomes in patient adherence to folic acid and to retention in the study . this is important because we continue to have insuffi cient data about what works and does not especially related to drug effi cacy in individuals of various racial and ethnic backgrounds . if suffi cient populations of minority subjects are underrepresented in the trial , that subset s information on drug use and effi cacy are not valid and therefore , the study may not be generalizable to the entire population . this success is due mostly to an understanding of the usefulness of a theoretical model for structure and problem solving , strong leadership , cohesion across study staff , and excellent insight into the relationship dynamics that exist in the clinical research setting including clinic staff , research personnel and study participants . our experiences have widespread implications as the sem approach is adaptable to developed and developing regions . the principles of the model can be used to address challenges related to religious , cultural or political considerations . effective implementation of this approach has the potential to increase recruitment and retention of hard - to - reach populations who are typically under - represented in clinical trials . with the use of the sem , we believe we have laid the groundwork for continued success for effectively securing valid and reliable outcomes for the clinical trial research study we currently have underway . despite the benefi ts of clinical research , many people approached to participate decline to do so because of deeply rooted myths about research.it is vital to have an equally significant number of participants enrolled in clinical research from different ethnic and racial groups.using the sem as a theoretical framework , we have been able to address many barriers and challenges that could have reduced the effectiveness and utility of our intervention . we have faced barriers but have succeeded in overcoming them through a thorough analysisof the intrapersonal , interpersonal , institutional , and community levels . despite the benefi ts of clinical research , many people approached to participate decline to do so because of deeply rooted myths about research . it is vital to have an equally significant number of participants enrolled in clinical research from different ethnic and racial groups . using the sem as a theoretical framework , we have been able to address many barriers and challenges that could have reduced the effectiveness and utility of our intervention . we have faced barriers but have succeeded in overcoming them through a thorough analysis of the intrapersonal , interpersonal , institutional , and community levels .

background : numerous barriers and challenges can hinder the successful enrollment and retention of study participants in clinical trials targeting minority populations . to conduct quality research , it is important to investigate these challenges , determine appropriate strategies that are evidence - based and continue seeking methods of improvement.methods:in this paper , we report such experiences in a registered clinical trial in an underserved minority population in the southern part of united states . this research study is a randomized double - blind controlled clinical trial that tests the efficacy of higher - strength as compared to low - strength / standard of care folic acid to prevent fetal body and brain size reduction in pregnant women who smoke . a unique approach in this socio - behavioral , genetic - epigenetic clinical trial is that we have adopted the socio - ecological model as a functional platform to effectively achieve and maintain high participant recruitment and retention rates.results:we highlight the barriers we have encountered in our trial and describe how we have successfully applied the socio - ecological model to overcome these obstacles.conclusions and global health implications : our positive experience will be of utility to other researchers globally . our fi ndings have far - reaching implications as the socio - ecological model approach is adaptable to developed and developing regions and has the potential to increase recruitment and retention of hard - to - reach populations who are typically under - represented in clinical trials .

Background Description of the Clinical Trial The Socio-ecological Model as Conceptual Framework Application of the SEM to Clinical Trials Intrapersonal Factors Interpersonal Factors Institutional Factors Community Factors Conclusion and Global Health Implications

an effectively designed and properly implemented clinical trial protocol is the most definitive method of determining whether an intervention has a hypothesized effect . recruitment and retention of an appropriate and representative study sample enhance the validity of the results and heighten the potential public health impact . despite the benefits of clinical research , many people approached to participate decline to do so because of deeply rooted myths about research . physician - scientists and basic / translational scientists need to work together to dispel these misconceptions so that enhanced participant enrollment can be attained , a step that is vital to conducting a successful clinical trial . the philosophical underpinnings of conducting clinical trial research are centered around three basic ethical principles : respect for persons , beneficence , and justice . those unable to act autonomously should be considered vulnerable populations , including children , prisoners , persons with physical handicaps / mental disabilities , and pregnant women . the informed consent process is vital to protecting human subjects by ensuring that subjects enter into research voluntarily and with complete information . the beneficence principle assesses the risks and benefits of research protection with the two principles : do no harm and maximize benefits and minimize harms . the justice principle is related to the selection of research subjects , addressing the question of who should bear the burden of research and who should benefit . an injustice occurs when a benefit is denied without good reason or when there is an unfair burden . the justice principle prohibits exploiting disadvantaged populations for the benefit of other more advantaged populations . additionally , the principle prohibits researchers from selecting samples that are easily accessible ( college students ) , in compromised positions ( prisoners , those confined to institutions ) or those easily manipulated rather than choosing a population directly related to the study . this principle also requires that everyone benefit from research , not just those who are able to afford it . all three principles must be adhered to during every phase of clinical trial design and conduct in order to safeguard participants and preserve research integrity . enrolling patients into a clinical trial is clearly an important step in the success of a research protocol . many individuals mention some mistrust about their willingness to be involved in a clinical trial . to counter this issue , financial and other incentives have been more frequently used to recruit subjects into clinical trials . when patients come from disadvantaged or low - income populations , this practice clearly raises questions about coercion , manipulation and concerns about true informed consent . institutional review boards are tasked to review these concerns and have rigorous criteria in which to determine acceptance of the use of financial and other incentives in the conduct of research trials , especially related to minority and underrepresented populations . beyond the challenges of patient recruitment exists the issue of participant retention and ensuring that those individuals who enroll successfully complete the study . low retention rates can impair the accuracy of study results and undermine fiscal and human resources ; therefore , emphasis must be placed on maintaining high levels of participation . it is widely known that special barriers and challenges affect the successful enrollment and retention of study participants in clinical trials targeting minority populations . one way of dealing with these issues is to investigate related challenges , consider action plans or strategies that represent evidence - based research and continue seeking methods for improvement . in this paper , we report such experiences in a clinical trial that we are currently conducting in an underserved minority population in the southern united states . a unique approach is that we are utilizing the principles of the socio - ecological model ( sem ) to effectively achieve and maintain high participant recruitment and retention rates . although previous literature[10 - 12 ] has described the use of the sem to promote study recruitment , limited information exists which explores the utility of this model in participant retention , especially , in a minority and socio - economically disadvantaged setting . this research study is a randomized double - blind controlled clinical trial that tests the effi cacy of high - strength ( 4 mg ) as compared to lower - strength / standard of care ( 0.8 mg ) folic acid to prevent fetal body and brain size reduction in pregnant women who smoke . experimental ( 4 mg ) and control ( 0.8 mg ) groups are assigned ( 165 pregnant women to each arm ) . the specific aims of this study are to : assess folic acid reserves in women who smoke during pregnancy ; anddetermine whether higher - strength folic acid in combination with smoking cessation programs will prevent reduction in fetal body and brain size among smoking mothers . assess folic acid reserves in women who smoke during pregnancy ; and determine whether higher - strength folic acid in combination with smoking cessation programs will prevent reduction in fetal body and brain size among smoking mothers . we hypothesize that higher - strength folic acid in combination with a smoking cessation program will prevent reduction in fetal body and brain size . this 5-year clinical trial is being implemented within an underserved minority community in florida , usa and is now in its fifth year . to our knowledge , this is the first human study to determine the utility of higher - strength folic acid in preventing smoking - induced fetal body and brain size reduction ( clinicaltrials.gov registration # nct01248260 ) . women who receive care at this community health center and deliver at the affiliated hospital are typically of low socioeconomic status.very few patients have private health insurance and most are either uninsured or covered by medicaid , the public health insurance program in us that covers the poor and underprivileged members of the society . about 15% of the women at the study site smoke actively during pregnancy without quitting , thus this study is approved by the university of south florida institutional review board and each participant signs an approved informed consent form . eligible consented women are enrolled 20 weeks gestation and screened for smoking habits . survey instruments are administered to participants during three subsequent antenatal study visits to assess changes in smoking habits , dietary factors , quality of sleep , stress levels and other antenatal psycho - social markers . all study participants are followed until delivery at which time fetal body and brain growth anthropological measures are collected . umbilical cord blood is also collected for genetic and epigenetic tests on markers of fetal brain growth and development . previous literature has proposed the use of the sem in examining barriers related specifically to enrollment in clinical trials. [10 - 12 ] the sem provides a very useful theoretical framework for addressing the numerous and varied obstacles in participant recruitment and retention . the sem suggests that an individual s behavior is integrated in a dynamic network of intrapersonal characteristics , interpersonal processes , institutional factors , community features and public policy . the participant s family , friends and health care providers comprise important components of the interpersonal level . the healthcare institution s rules , regulations and general attitude toward research shape the institutional level within the model . features of the community that may influence participant retention include convenience and acceptance of prenatal care , local cultural attitudes about smoking , the availability of public transportation and safety of the neighborhood . the public policy level is shaped by local , state and federal laws regarding socio - behavioral and biomedical research . the sem takes into account socio - cultural factors , as well as environmental factors , and their linkages to biologic factors . in order to effectively examine factors that influence participant recruitment and retention in our socio - behavioral , genetic - epigenetic clinical trial among pregnant smokers , we will identify the barriers we have encountered in our trial and use their placement within the sem to identify the reasons for these obstacles ( perceived and/or real ) and viable solutions . taking into account all these level - specific influential factors will help ensure our success in assessing the effi cacy of higher - strength folic acid in preventing the fetal body and brain growth inhibitory effect of prenatal smoking . swift identification and remediation of issues is critical to the success of any clinical research study . based on our knowledge of the sem first , we were aware that our study population , because of its nature , may be experiencing higher levels of stress , depression and other markers of psychosocial dysfunction . with this in mind , the trial was designed to collect information on and provide referrals for individuals who suffer from abnormal levels of stress , depression and sleep disturbances ( factors found on the intrapersonal level of the sem ) . additionally , we question women about their nutritional intake or access to nutritious food ( community level factor ) . as the trial progresses , each issue we encounter is identified and addressed using the sem in the following sections and in figure 2 . application of the socio - ecological model ( sem ) to enhance participation in a clinical trial the intrapersonal level of the sem is made up of individualknowledge , attitudes , beliefs and perceptions that infl uence behavior . there is the belief among many prospective participants that entering a randomized trial will mean a loss of control because they will not be able to choose their treatment or that clinical research will cause them harm others fear being treated like guinea pigs or distrust of the medical community due to past experiences , particularly in racial and ethnic minority populations . many believe signing the informed consent provides legal protection to physicians or researchers rather than study participants . the national cancer institute reports that african americans are less likely to participate because of knowledge of historical mistreatment of research participants such as the tuskegee syphilis study . one study reported that one - third of african - american women avoided participating in clinical trials because they believed that scientists could not be trusted . health care providers and research staff should communicate safety measures to patients to help build their trust and overcome suspicions . the target population for the project is comprised of minority socio - economically disadvantaged individuals and a mistrust of research could be a reason for declining to participate . we are aware of these challenges and are developing strategies to limit this as a recruitment barrier . study staff has held meetings with clinic staff to discuss ways to eliminate mistrust of researchers , particularly among minority patients . in order to ease potential suspicions , research staff gives a potential participant time to read through the informed consent document and to discuss with her partner or family . the research staff is careful to explain fully the following issues : the voluntary nature of the research , its distinction from clinical care , the right to withdraw without penalty at any time , and the steps to be taken to protect confidentiality of information . research staff also confirms the participant s correct understanding of each of these issues through appropriate questioning . in order to address this issue in our study , only trained research staff is permitted to recruit participants . although the majority of patients have reported taking their folic acid on a regular basis , a few have reported unwelcome side effects such as nausea and/or constipation . literature has suggested that counseling women to understand that side effects will subside is one way to prevent side effect - related non - compliance . in our trial , we suggest that participants take their multivitamins with food to prevent gastrointestinal discomfort and to increase their water and fiber intake . one particular belief that is most common in latino cultures is that an individual must accept god s will and that their health is in god s hands . individuals who hold this belief may be less likely to take actions to promote their health , such as taking folic acid / multivitamins in a research study . although we have not investigated individual health belief models and how they impact compliance to medication during pregnancy , we believe understanding the patient s intrapersonal perspective within the sem framework is a potential solution to medication adherence . a strong point of the study design is that both self - report of dietary folic acid intake as well as biologic blood levels are measured and assessed in the analysis . provider influence can have a signifi ca nt impact on patient enrollment . the extent to which the patient trusts her doctor can infl uence whether she will participate in the study . some physicians may feel they are losing control of their patient s care by referring a patient to a clinical trial . provider attitudes and perceptions , such as mistrust of researchers and provider lack of awareness of the study are the leading barriers that prevent providers from referring or enrolling patients in clinical trials that target minority populations . we have encountered many providers at the clinic who seem to have a mistrust of research and do not want to refer their patients to our study . this obstacle was addressed by setting up a formal meeting with the medical director and clinic manager to discuss our study objectives and open the lines of communication . in addition , due to limited space in the clinic , discussions covered the most ideal days for recruitment particularly , days with the highest numbers of new appointments . educating staff and demonstrating that they will not face additional burden is important , however , this is easier to accomplish in theory than in practice . establishing and maintaining these open lines of communication has provided a strong foundation for recruiting in the clinic . the institutional level of the sem involves the health care system , policies , and structures working together to assist or hinder the clinical trial . one barrier we have had to overcome at this level includes policies and structures in place within the health care system that influence or limit participation . during the initial phase of participant recruitment one challenge was to fi nd the best time and place to consent and interview participants that afforded adequate privacy without interrupting clinic flow . for instance , although women spend more time in the waiting room than in the examination room , there is little privacy to complete the interview process in this setting . research staff quickly discovered that the most ideal opportunity to consent and interview women is following triage when the women are sitting in the examination room waiting for the provider . the examination room allows for privacy and the window of time is lengthy enough to answer the potential participant s questions regarding the study and to complete the consenting process . depending on clinic fl ow , the recruiter is usually able to complete the interview process during this time as well . biological specimen collection for research use can be a complicated process and requires detailed protocols . collection of blood , urine , saliva , and extracted dna and rna should be carefully planned prior to research initiation , closely monitored , and handled consistently to guarantee samples will provide maximum value to the study . when establishing collaborations with the laboratories , we discovered that the rna had to be extracted within four hours of draw if collected and transported to the lab in regular edta tubes . this problem was remedied by purchasing paxgene blood rna tubes , which contain an rna preservative . we hired a staff member who spends time daily on the labor and delivery unit . after establishing our presence , the cooperation of the hospital staff has been more forthcoming and is improving with time . finally , the transition of the hospital medical record system from paper to an electronic system as well as a change in hospital leadership also falls under the institutional level . prior to this change in health information systems , it was diffi cult to collect the appropriate samples and identify when patients would arrive at the hospital for their delivery . without this cohesion of institutional systems working together , we would not have been able to successfully retain as many patients or collect as many biological samples . although we anticipated barriers in recruiting minority participants into the trial , we quickly realized that the true challenge would be retaining the women s interest in participating over multiple study visits . we understood that in addition to providing a small monetary incentive to compensate them for their time , we also had to ensure that their participation did not create an additional burden . this effort helps keep our retention rate high and our loss to follow - up rate low . currently , our loss to follow - up rate is less than 6% and this can be attributed to our understanding of the factors that influence participation in research studies based on the sem community level . because our study population is comprised of socio - economically disadvantaged individuals , access to nutritious foods is often limited . folic acid is important for fetal development and the prevention of neural tube defects . since many women do not obtain the recommended daily intake ( rdi ) of folic acid , the food and drug administration began folic acid fortification of cereal and other grain products . in spite of this , many women still do not meet the rdi for folic acid . non - hispanic black women and hispanic women are less likely to meet the rdi , regardless of folate source , than non - hispanic white women . both arms of the study are likely to be exposed to these sources of dietary folic acid since the study design employs randomization . this paper describes a clinical research study utilizing a population made up primarily of minority , socio - economically disadvantaged individuals . this population is often poorly represented in clinical research studies . due to the heterogeneous nature of the global population , it is vital to have an equally signifi ca nt number of participants enrolled in clinical research from different ethnic and racial groups . additionally , inclusion of underrepresented groups in clinical trials is important because sex , race , and co - morbid conditions could play vital roles in intervention studies , especially those related to response to medications / drugs . as is the case for this study , using different dosage levels of folic acid to prevent fetal body and brain size reduction in pregnant women who smoke is important for a variety of reasons . certainly , babies born with reduced body and brain development have many challenges early in their lives and can continue into adolescence and adulthood . finally , folic acid is an inexpensive intervention that has minimal risk and is easily available . it is important to determine patient compliance issues to maximize the likelihood of this population taking folic acid during the entire pregnancy but especially as early as possible after the pregnancy is identifi ed and diagnosed . the population in this study is often seen with poor nutritional behaviors . providing a folic acid supplement is again an important way to enhance the health of the neonate . with the challenges evident in this population to consider folic acid supplementation , a clear analysis of the challenges and barriers with such an intervention is essential . using the sem as a theoretical framework , we have been able to address many barriers and challenges that could have reduced the effectiveness and utility of our intervention . we have faced barriers but have succeeded in overcoming them through a thorough analysis of the intrapersonal , interpersonal , institutional , and community levels . from this analysis , the implementation strategies have evolved with greater anticipated outcomes in patient adherence to folic acid and to retention in the study . this is important because we continue to have insuffi cient data about what works and does not especially related to drug effi cacy in individuals of various racial and ethnic backgrounds . if suffi cient populations of minority subjects are underrepresented in the trial , that subset s information on drug use and effi cacy are not valid and therefore , the study may not be generalizable to the entire population . this success is due mostly to an understanding of the usefulness of a theoretical model for structure and problem solving , strong leadership , cohesion across study staff , and excellent insight into the relationship dynamics that exist in the clinical research setting including clinic staff , research personnel and study participants . our experiences have widespread implications as the sem approach is adaptable to developed and developing regions . the principles of the model can be used to address challenges related to religious , cultural or political considerations . effective implementation of this approach has the potential to increase recruitment and retention of hard - to - reach populations who are typically under - represented in clinical trials . with the use of the sem , we believe we have laid the groundwork for continued success for effectively securing valid and reliable outcomes for the clinical trial research study we currently have underway . despite the benefi ts of clinical research , many people approached to participate decline to do so because of deeply rooted myths about research.it is vital to have an equally significant number of participants enrolled in clinical research from different ethnic and racial groups.using the sem as a theoretical framework , we have been able to address many barriers and challenges that could have reduced the effectiveness and utility of our intervention . we have faced barriers but have succeeded in overcoming them through a thorough analysisof the intrapersonal , interpersonal , institutional , and community levels . despite the benefi ts of clinical research , many people approached to participate decline to do so because of deeply rooted myths about research . it is vital to have an equally significant number of participants enrolled in clinical research from different ethnic and racial groups . using the sem as a theoretical framework , we have been able to address many barriers and challenges that could have reduced the effectiveness and utility of our intervention . we have faced barriers but have succeeded in overcoming them through a thorough analysis of the intrapersonal , interpersonal , institutional , and community levels .

an effectively designed and properly implemented clinical trial protocol is the most definitive method of determining whether an intervention has a hypothesized effect . recruitment and retention of an appropriate and representative study sample enhance the validity of the results and heighten the potential public health impact . despite the benefits of clinical research , many people approached to participate decline to do so because of deeply rooted myths about research . physician - scientists and basic / translational scientists need to work together to dispel these misconceptions so that enhanced participant enrollment can be attained , a step that is vital to conducting a successful clinical trial . the philosophical underpinnings of conducting clinical trial research are centered around three basic ethical principles : respect for persons , beneficence , and justice . those unable to act autonomously should be considered vulnerable populations , including children , prisoners , persons with physical handicaps / mental disabilities , and pregnant women . the beneficence principle assesses the risks and benefits of research protection with the two principles : do no harm and maximize benefits and minimize harms . the justice principle is related to the selection of research subjects , addressing the question of who should bear the burden of research and who should benefit . additionally , the principle prohibits researchers from selecting samples that are easily accessible ( college students ) , in compromised positions ( prisoners , those confined to institutions ) or those easily manipulated rather than choosing a population directly related to the study . all three principles must be adhered to during every phase of clinical trial design and conduct in order to safeguard participants and preserve research integrity . enrolling patients into a clinical trial is clearly an important step in the success of a research protocol . many individuals mention some mistrust about their willingness to be involved in a clinical trial . to counter this issue , financial and other incentives have been more frequently used to recruit subjects into clinical trials . when patients come from disadvantaged or low - income populations , this practice clearly raises questions about coercion , manipulation and concerns about true informed consent . institutional review boards are tasked to review these concerns and have rigorous criteria in which to determine acceptance of the use of financial and other incentives in the conduct of research trials , especially related to minority and underrepresented populations . beyond the challenges of patient recruitment exists the issue of participant retention and ensuring that those individuals who enroll successfully complete the study . low retention rates can impair the accuracy of study results and undermine fiscal and human resources ; therefore , emphasis must be placed on maintaining high levels of participation . it is widely known that special barriers and challenges affect the successful enrollment and retention of study participants in clinical trials targeting minority populations . one way of dealing with these issues is to investigate related challenges , consider action plans or strategies that represent evidence - based research and continue seeking methods for improvement . in this paper , we report such experiences in a clinical trial that we are currently conducting in an underserved minority population in the southern united states . a unique approach is that we are utilizing the principles of the socio - ecological model ( sem ) to effectively achieve and maintain high participant recruitment and retention rates . although previous literature[10 - 12 ] has described the use of the sem to promote study recruitment , limited information exists which explores the utility of this model in participant retention , especially , in a minority and socio - economically disadvantaged setting . this research study is a randomized double - blind controlled clinical trial that tests the effi cacy of high - strength ( 4 mg ) as compared to lower - strength / standard of care ( 0.8 mg ) folic acid to prevent fetal body and brain size reduction in pregnant women who smoke . experimental ( 4 mg ) and control ( 0.8 mg ) groups are assigned ( 165 pregnant women to each arm ) . the specific aims of this study are to : assess folic acid reserves in women who smoke during pregnancy ; anddetermine whether higher - strength folic acid in combination with smoking cessation programs will prevent reduction in fetal body and brain size among smoking mothers . assess folic acid reserves in women who smoke during pregnancy ; and determine whether higher - strength folic acid in combination with smoking cessation programs will prevent reduction in fetal body and brain size among smoking mothers . we hypothesize that higher - strength folic acid in combination with a smoking cessation program will prevent reduction in fetal body and brain size . to our knowledge , this is the first human study to determine the utility of higher - strength folic acid in preventing smoking - induced fetal body and brain size reduction ( clinicaltrials.gov registration # nct01248260 ) . women who receive care at this community health center and deliver at the affiliated hospital are typically of low socioeconomic status.very few patients have private health insurance and most are either uninsured or covered by medicaid , the public health insurance program in us that covers the poor and underprivileged members of the society . about 15% of the women at the study site smoke actively during pregnancy without quitting , thus this study is approved by the university of south florida institutional review board and each participant signs an approved informed consent form . maternal blood is drawn at baseline for assessment of biomarkers including folate , vitamin b12 , homocysteine levels , and maternal methylenetetrahydrofolatereductase ( mthfr ) enzyme polymorphism . survey instruments are administered to participants during three subsequent antenatal study visits to assess changes in smoking habits , dietary factors , quality of sleep , stress levels and other antenatal psycho - social markers . umbilical cord blood is also collected for genetic and epigenetic tests on markers of fetal brain growth and development . non - smokers are also randomly selected for blood draw to compare baseline folate levels . previous literature has proposed the use of the sem in examining barriers related specifically to enrollment in clinical trials. [10 - 12 ] the sem provides a very useful theoretical framework for addressing the numerous and varied obstacles in participant recruitment and retention . the sem suggests that an individual s behavior is integrated in a dynamic network of intrapersonal characteristics , interpersonal processes , institutional factors , community features and public policy . the model assumes that interactions between individuals and their environment are reciprocal , implying that an individual is influenced by his / her environment and the environment is infl uenced by the individual . rct consort flow diagram the intrapersonal level encompasses the research participant s knowledge , awareness , attitudes , beliefs and perceptions . the participant s family , friends and health care providers comprise important components of the interpersonal level . the healthcare institution s rules , regulations and general attitude toward research shape the institutional level within the model . features of the community that may influence participant retention include convenience and acceptance of prenatal care , local cultural attitudes about smoking , the availability of public transportation and safety of the neighborhood . the public policy level is shaped by local , state and federal laws regarding socio - behavioral and biomedical research . the sem takes into account socio - cultural factors , as well as environmental factors , and their linkages to biologic factors . in order to effectively examine factors that influence participant recruitment and retention in our socio - behavioral , genetic - epigenetic clinical trial among pregnant smokers , we will identify the barriers we have encountered in our trial and use their placement within the sem to identify the reasons for these obstacles ( perceived and/or real ) and viable solutions . taking into account all these level - specific influential factors will help ensure our success in assessing the effi cacy of higher - strength folic acid in preventing the fetal body and brain growth inhibitory effect of prenatal smoking . swift identification and remediation of issues is critical to the success of any clinical research study . based on our knowledge of the sem first , we were aware that our study population , because of its nature , may be experiencing higher levels of stress , depression and other markers of psychosocial dysfunction . with this in mind , the trial was designed to collect information on and provide referrals for individuals who suffer from abnormal levels of stress , depression and sleep disturbances ( factors found on the intrapersonal level of the sem ) . additionally , we question women about their nutritional intake or access to nutritious food ( community level factor ) . as the trial progresses , each issue we encounter is identified and addressed using the sem in the following sections and in figure 2 . application of the socio - ecological model ( sem ) to enhance participation in a clinical trial the intrapersonal level of the sem is made up of individualknowledge , attitudes , beliefs and perceptions that infl uence behavior . fear , distrust , and/or suspicions of research have proven to be substantial barriers to participation in research . there is the belief among many prospective participants that entering a randomized trial will mean a loss of control because they will not be able to choose their treatment or that clinical research will cause them harm others fear being treated like guinea pigs or distrust of the medical community due to past experiences , particularly in racial and ethnic minority populations . the national cancer institute reports that african americans are less likely to participate because of knowledge of historical mistreatment of research participants such as the tuskegee syphilis study . one study reported that one - third of african - american women avoided participating in clinical trials because they believed that scientists could not be trusted . educating the public and health professionals on human subjects protection and legislation is essential to eliminating this mistrust and demonstrating how the tuskegee incident would be prevented today . health care providers and research staff should communicate safety measures to patients to help build their trust and overcome suspicions . the target population for the project is comprised of minority socio - economically disadvantaged individuals and a mistrust of research could be a reason for declining to participate . we are aware of these challenges and are developing strategies to limit this as a recruitment barrier . study staff has held meetings with clinic staff to discuss ways to eliminate mistrust of researchers , particularly among minority patients . in order to ease potential suspicions , research staff gives a potential participant time to read through the informed consent document and to discuss with her partner or family . the research staff is careful to explain fully the following issues : the voluntary nature of the research , its distinction from clinical care , the right to withdraw without penalty at any time , and the steps to be taken to protect confidentiality of information . to reduce social and psychological consequences about answering questions on high risk behaviors ( smoking ) during pregnancy , participants are assured that responses are kept confidential and are for research purposes only . language or literacy barriers may also create obstacles to participation which also fall under the intrapersonal level of the sem . additionally , two of the study staff members speak spanish and english , which is extremely useful because of the high population of spanish - speaking patients at the study site . although the majority of patients have reported taking their folic acid on a regular basis , a few have reported unwelcome side effects such as nausea and/or constipation . literature has suggested that counseling women to understand that side effects will subside is one way to prevent side effect - related non - compliance . in our trial , we suggest that participants take their multivitamins with food to prevent gastrointestinal discomfort and to increase their water and fiber intake . individuals who hold this belief may be less likely to take actions to promote their health , such as taking folic acid / multivitamins in a research study . although we have not investigated individual health belief models and how they impact compliance to medication during pregnancy , we believe understanding the patient s intrapersonal perspective within the sem framework is a potential solution to medication adherence . a strong point of the study design is that both self - report of dietary folic acid intake as well as biologic blood levels are measured and assessed in the analysis . the interpersonal level of the sem comprises the participant s social network including family , friends , peers , and health care providers . several sociodemographic factors such as maternal age and household income influence multivitamin use . the extent to which the patient trusts her doctor can infl uence whether she will participate in the study . physicians must recognize these concerns , teach the patient that the decision to participate is entirely that of the patient , not the doctor , and confidently recommend the most suitable studies . provider attitudes and perceptions , such as mistrust of researchers and provider lack of awareness of the study are the leading barriers that prevent providers from referring or enrolling patients in clinical trials that target minority populations . we have encountered many providers at the clinic who seem to have a mistrust of research and do not want to refer their patients to our study . this obstacle was addressed by setting up a formal meeting with the medical director and clinic manager to discuss our study objectives and open the lines of communication . during the meeting , additional topics of discussion included patient fl ow , the average wait time for new obstetrical appointments , and the most effective use of the patient s wait time . in addition , due to limited space in the clinic , discussions covered the most ideal days for recruitment particularly , days with the highest numbers of new appointments . educating staff and demonstrating that they will not face additional burden is important , however , this is easier to accomplish in theory than in practice . gaining the support of the medical director and the clinic manager was very instrumental in bridging the gap between research and clinic staff who were initially less cooperative . the institutional level of the sem involves the health care system , policies , and structures working together to assist or hinder the clinical trial . one barrier we have had to overcome at this level includes policies and structures in place within the health care system that influence or limit participation . during the initial phase of participant recruitment one challenge was to fi nd the best time and place to consent and interview participants that afforded adequate privacy without interrupting clinic flow . for instance , although women spend more time in the waiting room than in the examination room , there is little privacy to complete the interview process in this setting . research staff quickly discovered that the most ideal opportunity to consent and interview women is following triage when the women are sitting in the examination room waiting for the provider . the examination room allows for privacy and the window of time is lengthy enough to answer the potential participant s questions regarding the study and to complete the consenting process . collection of blood , urine , saliva , and extracted dna and rna should be carefully planned prior to research initiation , closely monitored , and handled consistently to guarantee samples will provide maximum value to the study . most of the challenges related to biological specimen collection fall under the institutional level of the sem . when establishing collaborations with the laboratories , we discovered that the rna had to be extracted within four hours of draw if collected and transported to the lab in regular edta tubes . this proved to be a major institutional barrier when working with a lab that is only open during normal business hours , as women can deliver at all hours of the night and on weekends . these tubes are about eight times the cost of regular tubes , but allow the blood to be kept at room temperature for up to three days . therefore , these tubes are ideal for our study and allow for rna extraction at a later date it was challenging to introduce new research staff members into a unit that has limited space . after establishing our presence , the cooperation of the hospital staff has been more forthcoming and is improving with time . finally , the transition of the hospital medical record system from paper to an electronic system as well as a change in hospital leadership also falls under the institutional level . prior to this change in health information systems , it was diffi cult to collect the appropriate samples and identify when patients would arrive at the hospital for their delivery . after the transition , the new electronic medical record system has become a continuous link of patient information from the antenatal period through delivery to the postnatal period . this new system has facilitated the conduct of the study by enabling identifi cation of patients at delivery and timely biological specimen collection . without this cohesion of institutional systems working together , we would not have been able to successfully retain as many patients or collect as many biological samples . although we anticipated barriers in recruiting minority participants into the trial , we quickly realized that the true challenge would be retaining the women s interest in participating over multiple study visits . with this in mind , study visits are combined with routine prenatal appointments to reduce the scheduling hassles for participants . currently , our loss to follow - up rate is less than 6% and this can be attributed to our understanding of the factors that influence participation in research studies based on the sem community level . since many women do not obtain the recommended daily intake ( rdi ) of folic acid , the food and drug administration began folic acid fortification of cereal and other grain products . in spite of this , many women still do not meet the rdi for folic acid . non - hispanic black women and hispanic women are less likely to meet the rdi , regardless of folate source , than non - hispanic white women . both arms of the study are likely to be exposed to these sources of dietary folic acid since the study design employs randomization . this paper describes a clinical research study utilizing a population made up primarily of minority , socio - economically disadvantaged individuals . due to the heterogeneous nature of the global population , it is vital to have an equally signifi ca nt number of participants enrolled in clinical research from different ethnic and racial groups . additionally , inclusion of underrepresented groups in clinical trials is important because sex , race , and co - morbid conditions could play vital roles in intervention studies , especially those related to response to medications / drugs . as is the case for this study , using different dosage levels of folic acid to prevent fetal body and brain size reduction in pregnant women who smoke is important for a variety of reasons . certainly , babies born with reduced body and brain development have many challenges early in their lives and can continue into adolescence and adulthood . secondly , determining more effi cacious drug dosages has signifi ca nt preventive outcomes and fi nancial implications for this and other populations . it is important to determine patient compliance issues to maximize the likelihood of this population taking folic acid during the entire pregnancy but especially as early as possible after the pregnancy is identifi ed and diagnosed . with the challenges evident in this population to consider folic acid supplementation , a clear analysis of the challenges and barriers with such an intervention is essential . using the sem as a theoretical framework , we have been able to address many barriers and challenges that could have reduced the effectiveness and utility of our intervention . we have faced barriers but have succeeded in overcoming them through a thorough analysis of the intrapersonal , interpersonal , institutional , and community levels . from this analysis , the implementation strategies have evolved with greater anticipated outcomes in patient adherence to folic acid and to retention in the study . if suffi cient populations of minority subjects are underrepresented in the trial , that subset s information on drug use and effi cacy are not valid and therefore , the study may not be generalizable to the entire population . this success is due mostly to an understanding of the usefulness of a theoretical model for structure and problem solving , strong leadership , cohesion across study staff , and excellent insight into the relationship dynamics that exist in the clinical research setting including clinic staff , research personnel and study participants . our experiences have widespread implications as the sem approach is adaptable to developed and developing regions . the principles of the model can be used to address challenges related to religious , cultural or political considerations . effective implementation of this approach has the potential to increase recruitment and retention of hard - to - reach populations who are typically under - represented in clinical trials . with the use of the sem , we believe we have laid the groundwork for continued success for effectively securing valid and reliable outcomes for the clinical trial research study we currently have underway . despite the benefi ts of clinical research , many people approached to participate decline to do so because of deeply rooted myths about research.it is vital to have an equally significant number of participants enrolled in clinical research from different ethnic and racial groups.using the sem as a theoretical framework , we have been able to address many barriers and challenges that could have reduced the effectiveness and utility of our intervention . using the sem as a theoretical framework , we have been able to address many barriers and challenges that could have reduced the effectiveness and utility of our intervention .

over the past two decades , the prevalence of metabolic abnormalities such as type 2 diabetes and metabolic syndrome ( mets ) has been increasing worldwide together with the escalating obesity pandemic [ 13 ] . abdominal obesity , in particular , substantially increases the risk of developing type 2 diabetes , mets , and fatty liver . according to the american association for the study of liver diseases ( aasld ) , fatty liver in the absence of a chronic increase in alcohol intake ( i.e. , alcohol intake is < 20 g ethanol / day ) is referred to as nonalcoholic fatty liver disease ( nafld ) . according to the aasld 's practice guidelines for nafld , nafld is histologically subdivided into nonalcoholic fatty liver ( nafl ) and a more severe condition , nonalcoholic steatohepatitis ( nash ) , which sometimes advances over several decades to life - threatening hepatic cirrhosis and hepatocellular carcinoma . the prevalence of nafld , as detected by ultrasound , is up to 3046% in developed countries and nearly 10% in developing nations , making nafld the most common liver disorder worldwide [ 5 , 6 ] . lifestyle interventions such as diet and moderate exercise , which lead to weight loss , are fundamental for the treatment of nafld . paradoxically , nafld has also been reported in nonobese people [ 79 ] . in india , individuals with a normal bmi ( 18.524.9 kg / m ) have a 2-fold higher risk of developing nafld compared with those with a bmi of < 18.5 therefore , nafld is expected to become a major burden in asian countries where the prevalence of obesity is less than that in western countries [ 10 , 11 ] . notably , nafld appears to be an early predictor of metabolic disorders , particularly among normal - weight individuals . this is because nafld may be more tightly associated with insulin resistance and with markers of oxidative stress and endothelial dysfunction than with the adult treatment panel iii criteria for mets in nonobese , nondiabetic subjects . therefore , although obese people are predisposed to develop nafld , normal weight and overweight people may , through the development of insulin resistance , also show the pathogenic characteristics of nafld . the clinical relevance of nafld is still poorly understood because some investigators [ 1215 ] , but not all [ 16 , 17 ] , have shown that nafld is associated with higher overall mortality and cardiovascular disease . since nafld is closely associated with obesity , diabetes , and mets , it is unknown whether the relationship between nafld and all - cause mortality and cardiovascular death , if any , is independent of cardiometabolic risk factors ( figure 1 ) such as mets and type 2 diabetes . taken together , nafld and nash are multidisciplinary liver diseases that require interventions targeting the cardiometabolic and liver disorders for the effective treatment of patients with these diseases . therefore , it is likely that mild nafld will require predominantly cardiometabolic pharmacotherapies , whereas moderate to severe nafld and nash will require pharmacotherapies targeting the hepatic disorders . however , since many of the candidate drugs are likely to have broad therapeutic effects targeting multiple aspects of these diseases , distinct classifications are unavailable . ectopic fat deposition in organs other than fat tissue , such as the liver and skeletal muscle , reflects severe energy overaccumulation or disturbed fat distribution . however , hepatocytes can , under physiological conditions , store small amounts of triglyceride in a transient manner . low physical activity as a result of a sedentary state , other unfavorable lifestyle behaviors ( e.g. , diet and habitual smoking ) , and sympathetic overdrive as a result of physical / mental stress may lead to insulin resistance independently of obesity . in turn , insulin resistance suppresses the influx of glucose and free fatty acids ( ffas ) into adipose tissue , increasing ffa influx into the liver . the pathogenic characteristics described above are often observed in metabolically obese young women with a normal body weight [ 19 , 20 ] . proposed that reduced ectopic fat in the liver may be more important than reduced visceral fat for the discrimination of benign obesity , in other words metabolically normal obesity with good insulin sensitivity . ectopic fat deposition in the liver may be an initial feature of peripheral insulin resistance , particularly in adipose tissue , which predominantly accumulates surplus energy as fat . for many years , it was thought that simple steatosis ( i.e. , fatty liver ) remains benign throughout life . however , studies published in the last two decades have shown that such conditions , without appropriate treatment or intervention , provoke other events , including oxidative stress , inflammation , and fibrosis . these events lead to the degeneration and impaired functioning of liver tissue and ultimately result in nash . unfortunately , similar to other cardiometabolic diseases , most people with asymptomatic nafld remain untreated until the results of blood test or imaging studies indicate the presence of nafld . serum hepatic enzymes , particularly alanine aminotransferase ( alt ) , are often elevated beyond the normal range [ 2224 ] . however , they may remain within the normal ranges [ 23 , 25 ] and may be overlooked until abdominal ultrasound , computed tomography , or magnetic resonance imaging are performed . in clinical practice , people with hypertrophic abdominal fat cells often develop nafld at a later date . this phenomenon may be caused by direct influx , via the portal vein , of long - chain ffas , glycerol , and proinflammatory cytokines ( e.g. , tumor necrosis factor [ tnf ] , interleukin [ il]-6 , and il-1 ) from the visceral and upper body fat depots , which exhibit insulin resistance and activation of hormone - sensitive lipase and adipose tissue triacylglycerol lipase [ 2628 ] . ffas derived from the lipolysis of visceral and subcutaneous fat account for approximately 60% of the total hepatic ffa content . besides increased ffa influx , hyperinsulinemia increases hepatic lipogenesis by activating sterol regulatory element binding protein 1c ( srebp-1c ) , a key regulator of lipogenic gene expression . similarly , hyperglycemia caused by inadequate insulin action results in the activation of carbohydrate response element binding protein , which activates l - type pyruvate kinase and lipogenic genes in hepatocytes . nevertheless , it seems unexpected that hepatic lipogenesis is not attenuated by decreased insulin sensitivity or insulin resistance . recent studies have suggested that srebp-1c and lipogenesis are activated secondary to enhanced endoplasmic reticulum ( er ) stress . er stress activates the cleavage of srebp-1c independently of insulin , which may explain the paradoxical stimulation of lipogenesis in insulin - resistant liver . consistently , reducing er stress in obese animals decreases srebp-1c activation and lipogenesis , and improves hepatic steatosis and insulin sensitivity . moreover , er stress in response to increased hepatic lipid content attenuates triglyceride secretion by limiting apolipoprotein b secretion , thus increasing hepatic triglyceride accumulation . alternatively , reduced mitochondrial -oxidation , following inhibition of carnitine palmitoyl transferase-1 ( cpt-1 ) , may contribute to enhanced fatty acid synthesis . several animal studies have also demonstrated a relationship between decreased microsomal triglyceride transfer protein ( mtp ) expression and steatosis . for example , patients with nash were reported to show reduced very low - density lipoprotein ( vldl ) synthesis and impaired hepatic mtp mrna expression , which was likely due to single nucleotide polymorphisms of the mtp gene . in terms of preventing liver damage , the priority for pharmacotherapy of nafld is to prevent transformation of nafld into nash and to improve the pathophysiology . therefore , in theory , antioxidative , antiinflammatory , and antifibrotic agents may constitute first - line treatments for nafld . at the same time the order of the drugs is mostly based on the recommendation for use [ 46 ] . metformin ( biguanide ) is an insulin - sensitizer that is considered to be the first - line treatment of type 2 diabetes because it has relatively few side effects and is inexpensive . besides reducing hepatic glucose output , metformin also activates amp kinase , which inhibits the production of glucose , cholesterol , and triglycerides , and stimulates fatty acid oxidation . nevertheless , the outcomes of clinical trials in nafld are conflicting [ 38 , 39 ] . metformin was also better than a prescriptive diet or vitamin e intake for the treatment of nafld in patients receiving nutritional counseling . in a clinical study , serum alt levels were consistently lower in patients treated with metformin compared with those treated with placebo . the effects of metformin in that study were considered to be mediated by weight loss . moreover , 6 months of treatment with metformin was no better than placebo in terms of improving liver histology in patients with nafld . further well - designed studies are needed to elucidate the significance of metformin treatment for nafld . a number of clinical trials , including randomized controlled trials , have shown that thiazolidinediones ( peroxisome proliferator activated receptor [ ppar]- agonists ) improve liver enzyme levels and liver histology in patients with nafld and nash [ 39 , 45 ] . ppar , which belongs to the nuclear hormone receptor family , is predominantly expressed in adipose tissues and plays a key role in adipogenesis and glucose homeostasis . since ppar is also expressed in cardiovascular tissues , such as vascular endothelial cells , smooth muscle cells , macrophages , and cardiomyocytes , altered ppar activity may be involved in the etiology of cardiovascular diseases , particularly atherosclerosis . it has been shown that treatment with pioglitazone for 1 - 2 years improves the overall pathogenic characteristics of nafld and nash [ 4850 ] , suggesting that ppar agonists may improve the pathophysiology of the liver as well as clinical factors in patients with nafld and nash . ppar regulates several key activities , including adipocyte differentiation and fibroblast differentiation into mature adipocyte types [ 51 , 52 ] . therefore , the improvements in nafld may be due to improvements in the visceral tissue and upper subcutaneous fat , as thiazolidinediones redirect fat accumulation from the liver or muscle into adipose tissues and confine it in adipose tissue at the expense of adipose cell expansion , which unfortunately leads to weight gain . thiazolidinediones also activate amp - activated protein kinase and inhibit lipolysis , at least in part by inhibiting the translocation of hormone - sensitive lipase to lipid droplets . mayerson et al . showed that treatment with rosiglitazone significantly reduced hepatic triglyceride content and suppresses adipocyte lipolysis . in this way , rosiglitazone increased intramyocellular fat storage as triglycerides , which was accompanied by improved muscle insulin sensitivity . however , this apparent improvement in insulin resistance often results in adverse outcomes , such as an increase in the number of adipose cells through enhanced differentiation , eventually leading to weight gain and systematic edema [ 39 , 50 ] . consequently , long - term treatment with thiazolidinediones may be poorly tolerated in some people with diabetes because of their adverse effects , although pioglitazone has fewer side effects than rosiglitazone [ 56 , 57 ] ( troglitazone was excluded from the market because of hepatic toxicity ) . some adverse effects of thiazolidinediones , particularly weight gain and edema , could be prevented when administered in low dosages . indeed , even low - dose pioglitazone ( 15 mg / day ) improved liver enzymes without overt adverse effects in a study of 12 patients with biopsy - confirmed nash . some intervention studies in patients with nafld have shown that vitamin e , a lipophilic antioxidant , may improve some of the pathogenic characteristics of nafld . vitamin e exerts its antioxidative effects by reducing lipid peroxidation , preventing free radical reactions , and stabilizing cellular phospholipid membranes . vitamin e also inhibits hepatic transforming growth factor-1 expression , attenuates cytokine stimulation of stellate cells , and protects against hepatic fibrosis , suggesting that vitamin e is more beneficial for nash rather than nafld . some studies , most of them involving small numbers of patients , showed positive outcomes in terms of the treatment of nafld [ 41 , 48 , 61 , 62 ] . however , other studies showed negative results or no significant improvement with vitamin e compared with placebo or lifestyle interventions , suggesting that vitamin e alone is insufficient to treat nafld [ 38 , 63 ] . lavine et al . reported that vitamin e and metformin were not superior to placebo in terms of sustained reductions in alt levels in children with nafld . it was also reported that simple lifestyle interventions ( i.e. , diet and physical exercise ) in children with nafld can significantly improve liver function , glucose metabolism , and lipid levels beyond those achieved with antioxidant therapy ( 600 iu / day vitamin e plus 500 mg / day vitamin c ) . several studies have shown that high - dose vitamin e supplementation may be harmful in some patients because of unexpected adverse outcomes , such as increased cardiovascular disease mortality [ 64 , 65 ] , although other studies found no such risk [ 66 , 67 ] . among nondiabetic patients with biopsy - confirmed nash , current recommendations advocate the administration of 800 iu / day vitamin e , as this dose may reduce serum alt levels and improve steatosis , inflammation , and hepatocyte ballooning . udca is a cytoprotective antiinflammatory agent that is widely used to treat liver diseases , as well as gallstones . long - term clinical studies have revealed that udca safely and effectively improves hepatic enzyme levels , serum fibrosis markers , and selected metabolic parameters . oral administration of taurine - conjugated udca decreased hepatic steatosis in ob / ob mice by cooperatively regulating multiple metabolic pathways , including reduced expression of genes that regulate de novo lipogenesis . overall , however , there were no significant differences between udca and placebo in several clinical trials . n-3 pufas are mainly consumed in the form of marine oils from fatty fish or other seafood . fish oil contains both docosahexaenoic acid ( dha , c22:6 n-3 ) and eicosapentaenoic acid ( epa , c20:5 n-3 ) . n-3 pufa consumption lowers plasma triglycerides and blood pressure and may reduce inflammation and improve vascular function . these effects occur because n-3 pufas are natural ligands for several nuclear receptors and transcription factors that regulate gene expression in various tissues . considering these properties of n-3 pufas , several clinical trials have investigated the potential effects of fish oil or n-3 pufa consumption on the outcomes of cardiovascular disease . meta - analyses of these studies have shown that fish oil and n-3 pufa consumption , but not -linolenic acid , reduce the risk of cardiovascular events and the risk of coronary heart disease - related mortality [ 71 , 73 ] . regarding nafld , dietary supplementation with long - chain n-3 pufas appears to safely reduce nutritional hepatic steatosis in adults . reported that the levels of n-3 pufas were decreased while the n-6/n-3 fatty acid ratio was increased in nafld patients compared with controls , probably because of defective desaturation of pufa by inadequate intake of their precursors and increased peroxidation of pufa . reported that increased erythrocyte n-3 and n-6 pufa levels are significantly associated with a lower prevalence of steatosis in patients with type 2 diabetes . in addition , the dietary records of middle - aged healthy japanese men revealed that dietary epa and epa + dha may help to prevent nafld . furthermore , sato et al . reported that the antiobesity effects of epa in high - fat / high - sucrose - induced obese mice were associated with the suppression of hepatic lipogenesis and steatosis . enjoji and nakamuta proposed that excess cholesterol intake appears to be one of the main factors associated with nafld , particularly in nonobese subjects , because excess cholesterol consumption stimulates the liver x receptor-srebp-1c pathway and enhances fatty acid synthesis . indeed , it was reported that low - dose ( 2.0% of total energy ) fish oil diets improve hepatic lipid accumulation in mice fed a high - cholesterol diet . however , studies examining the effects of epa / fish oil on dietary cholesterol - induced nafld in humans are still lacking . statins ( 3-hydroxy-3-methyglutaryl - coenzyme a reductase inhibitors ) are used worldwide to treat lipid disorders , particularly elevated low - density lipoprotein - cholesterol ( ldl - c ) and substantially reduce cardiovascular events and mortality . statins also have pleiotropic effects , including antiinflammatory actions , as they greatly reduce the levels of proinflammatory cytokines , such as c - reactive protein ( crp ) and tnf . although statins are putatively associated with some adverse events , including elevated hepatic enzymes and liver dysfunction , an elevated serum alt level at baseline attributable to nafld is unlikely to increase the risk of statin - associated elevations in alt . similarly , an elevated baseline serum alt was not associated with an increased risk of hepatotoxicity in patients treated with lovastatin . in clinical studies , simvastatin and atorvastatin [ 8486 ] were associated with a reduction in hepatic steatosis and may inhibit the progression to nash . four years of treatment with atorvastatin ( 20 mg ) combined with vitamins c and e reduced hepatic steatosis by 71% in people with nafld at baseline . taken together with a previous report showing that excess cholesterol consumption may accelerate nafld , statins may be promising candidates for the treatment of nafld . additionally , some statins improved surrogate markers of hepatic steatosis , such as serum glyceraldehyde - derived advanced glycation end - products . on the other hand , considering that statins are associated with worsening of glucose metabolism [ 89 , 90 ] and that multifactorial medications , such as antihypertensive and antidiabetic drugs , were used in most of the previous studies of statin therapy , well - designed , randomized , placebo - controlled studies are needed to determination the suitability of statins as monotherapy or combination therapy for nafld . fibrates , ppar agonists , increase ffa oxidation in the liver , alter tg synthesis , and reduce hepatic synthesis of vldl [ 93 , 94 ] , and theoretically improve the pathogenic characteristics of mets and nafld . unlike the striking outcomes of numerous clinical trials using statins , the results of several large studies of fibrates were inconsistent and had varying outcomes , including the incidence of cardiovascular mortality and events [ 9599 ] . in all of these studies , treatment with a fibrate was associated with a large , although nonsignificant , reduction in cardiovascular events in people with type 2 diabetes or components of the metabolic syndrome . to date , however , few clinical studies , except for a small pilot study [ 100 , 101 ] , have examined the effects of fibrates on the pathophysiology of nafld . fibrates are expected to ameliorate the pathogenic characteristics of nafld because they reduce the levels of inflammatory biomarkers , such as crp and il-6 , and may improve insulin resistance [ 102104 ] , via mechanisms that differ from those of statins . fenofibrate is commonly used in clinical practice because it is generally well tolerated when used as monotherapy and as combination therapy in a wide range of individuals [ 103 , 105 ] . no clinical studies have examined the effects or safety of a statin in combination with a fibrate compared with monotherapy for treating nafld . however , some benefits of combination therapy on cardiovascular and microvascular outcomes were observed in specific subgroups of patients , such as patients with low hdl levels or hypertriglyceridemia . ezetimibe , an npc1l1 inhibitor , was reported to reduce circulating ldl - c levels and improve clinical outcomes in patients at increased risk for cardiovascular events when administered alone or in combination with a statin . unlike in rodents in which npc1l1 is mainly expressed in the intestine , npc1l1 is highly expressed in the liver in humans [ 108 , 109 ] . this suggests that hepatic npc1l1 may facilitate hepatic cholesterol accumulation and that ezetimibe may be a potential candidate for nafld , especially nafld induced by a high - cholesterol diet . in clinical trials , albeit on a small scale , ezetimibe improved biochemical parameters and hepatic enzyme levels , as well as the histological abnormalities of nafld [ 110112 ] . the ras plays key roles in the regulation of blood pressure and fluid balance , as well as in the pathogenesis of insulin resistance and nafld [ 113 , 114 ] . in addition , inhibition of the ras may improve the intracellular insulin signaling pathway , offering better control of adipose tissue proliferation and adipokine production . the two main classes of ras blockers , angiotensin ii receptor blockers ( arbs ) and angiotensin - converting enzyme inhibitors ( aceis ) , are efficient drugs that significantly reduce cardiovascular events and mortality [ 116118 ] . as expected , arbs and aceis improve insulin resistance and possibly lipid profiles , suggesting that these agents may be suitable treatments for nafld and nash . of interest , telmisartan was reported to be a partial agonist of ppar [ 119 , 120 ] , a property that does not appear to be shared by other arbs . nevertheless , despite many animal studies showing the beneficial effects of arbs and aceis , clinical studies for nafld are still lacking . beneficial effects of spironolactone and eplerenone , aldosterone antagonists , were recently shown in a mouse model of nafld [ 121123 ] . additionally , spironolactone in combination with vitamin e was reported to improve insulin resistance in patients with nafld . however , these effects of aldosterone antagonists have only been shown in mouse and small - scale clinical studies . aliskiren , a direct renin inhibitor used to treat hypertension , provides an organ - protective effect by attenuating oxidative stress and improving insulin resistance in mice models [ 125127 ] . therefore , although aliskiren is a promising drug in terms of improving insulin resistance and oxidative stress , again , human studies are lacking . treatment with incretin modulators , glucagon - like peptide-1 ( glp-1 ) analogs , and dipeptidyl peptidase-4 inhibitors reduces weight gain , minimizes hypoglycemia , decreases inflammation , and is cardioprotective in preclinical studies [ 128 , 129 ] . the most common adverse events are mild gastrointestinal tract symptoms such as nausea , vomiting , and diarrhea , the incidence and severity of which generally decreased during continued therapy . regarding the beneficial effects of incretin modulators , weight loss ( or weight neutrality ) and reduced inflammation appear to be particularly promising for the treatment of nafld because these effects facilitate improvements in insulin resistance and metabolic abnormalities . . reported that administration of exendin-4 , a glp-1 receptor agonist , induces the regression of hepatic steatosis in ob / ob mice by improving insulin sensitivity . glp-1 appears to protect hepatocytes from fatty acid - related death by suppressing dysfunctional er stress responses . in addition , glp-1 suppresses hepatic lipogenesis by activating the amp kinase pathway and reduces hepatic fat accumulation and nutrient - induced hepatic proinflammatory responses . reported that 44 weeks of exenatide therapy decreased liver fat measured by liver spectroscopy from 15.8% to 4.3% and improved hepatic enzymes in a patient with type 2 diabetes . taken together , there are promising results in animal models and limited human reports , but clinical studies examining the effects of incretin - based agents on hepatic steatosis have not been performed . theoretically , improving nafld via weight loss is an ideal approach in obese or overweight people because other complications are simultaneously ameliorated . of several commonly used antiobesity medications , orlistat and sibutramine are available for long - term prescription [ 135 , 136 ] . orlistat inhibits dietary triglyceride hydrolysis in the gut , occasionally with mild gastrointestinal symptoms , resulting in a substantial decrease in fat absorption . . showed that orlistat improves serum alt levels and steatosis determined by ultrasound in patients with nafld , beyond its weight - lowering effects . harrison et al . reported that subjects who lost 5% of their body weight over 9 months experienced improvements in insulin resistance and steatosis , while subjects who lost 9% of their body weight also experienced improvements in hepatic histology . sibutramine , a combined norepinephrine and serotonin reuptake inhibitor , reduces food intake and body weight . both orlistat and sibutramine had beneficial effects on body weight , lipid profiles , glucose metabolism , and inflammatory markers in many trials [ 135 , 137 ] . however , there is still insufficient safety data regarding the long - term outcomes of antiobesity therapy . indeed , sibutramine was reported to increase blood pressure and heart rate , which may limit its use in clinical practice . mazindol , a tetracyclic chemical , has been approved in several countries , including japan . however , long - term treatment with mazindol is not currently permitted , although treatment with mazindol for a few weeks to several months is allowed in severely obese individuals ( e.g. , bmi 35 kg / m in japan ) . mazindol exerts antiobesity effects by inhibiting the appetite and activating thermogenesis , as well as having antidiabetic effects . however , clinical studies of mazindol have not been conducted in patients with nafld , partly because of the restriction for short - term administration . rimonabant , a selective cannabinoid-1 ( cb1 ) receptor blocker , was shown to reduce body weight and improve cardiovascular risk factors in obese patients by regulating the energy balance and body composition . furthermore , administration of 20 mg rimonabant daily in combination with a hypocaloric diet for 1 year , significantly decreased body weight and waist circumference . based on several trials showing its weight loss benefits , rimonabant entered the european market for the treatment of obesity . however , the drug was withdrawn worldwide in 2008 - 2009 because of the emergence of significant side effects , particularly psychiatric disorders ( e.g. , depression and anxiety ) [ 143 , 144 ] . the cb1 receptor is expressed not only in the central nervous system but also in the gastrointestinal tract , adipose tissue , and cardiovascular system . consistently , animal studies have shown that cb1 receptor antagonists improve glucose homeostasis , insulin levels , fatty liver , and plasma lipid profiles by blocking the cb1 receptor in peripheral tissues , including the liver and visceral fat [ 146 , 147 ] . despite these promising effects , the clinical application of cb1 receptor antagonists will be restricted until their critical adverse effects on the central nervous system can be overcome . polyphenols , ubiquitous dietary components that mainly include flavonoids and tannins , are considered dietary supplements rather than drugs . several polyphenols obtained from plants may be promising candidate treatments for nafld and nash because they are effective scavengers of reactive oxygen and reactive nitrogen species . of several polyphenols described to date , resveratrol , a component of several grape species , appears to be particularly relevant in the context of liver disease . for example , resveratrol has antiinflammatory effects mediated through a decrease in proinflammatory cytokines , including tnf . in a study using an animal model of steatosis , resveratrol appeared to reduce liver oxidative stress by increasing the expression of cpt-1a and acyl - coenzyme a oxidase . another study revealed that resveratrol can also protect the liver from nafld by reducing ffa availability . resveratrol also decreased the severity of nafld in rats , at least in part , through its antioxidant effects and by inhibiting tnf . resveratrol was also reported to protect the liver from nafld by enhancing amp - activated protein kinase phosphorylation . habitual alcohol intake , even light consumption , interferes with the development and progression of critical diseases . intriguingly , light to moderate alcohol consumption is often associated with a low prevalence of fatty liver . clinical studies have suggested that light or moderate ( < 1020 g ethanol / day ) alcohol intake protects against nafld . light alcohol consumption ( 20 g ethanol on 13 days / week or 40140 g ethanol / week ) and moderate alcohol consumption ( 140280 g ethanol / week ) were independently associated with a low prevalence of fatty liver [ 155 , 156 ] , whereas moderate to heavy drinking ( > 60 g ethanol / week ) was associated with the progression of hepatic steatosis and fibrosis [ 157 , 158 ] . in addition , habitual alcohol consumption generally impairs fatty acid oxidation and stimulates lipogenesis [ 160 , 161 ] . however , the specific mechanisms by which alcohol ( alone or in combination with obesity / metabolic abnormalities ) causes liver injury are poorly understood . in clinical studies , the causality between light to moderate alcohol consumption and reduced prevalence of fatty liver or nafld remains unknown . thus , patients with nafld should not consume heavy amounts of alcohol [ 5 , 162 ] . since hepatic dysfunction is usually closely associated with systemic disorders , there are no specific medications that ameliorate only the pathogenic characteristics of fatty liver without affecting other organs and tissues . in other words , drugs used , or expected to be used , in the treatment of type 2 diabetes , mets ( i.e. , hypertension and dyslipidemia ) , hypercholesterolemia , and obesity may be candidate drugs for the treatment of nafld and nash . these agents can be more effective than monotherapy , although further evidence from animal and cellular studies , as well as large clinical trials , is needed to examine this possibility .

nonalcoholic fatty liver disease ( nafld ) and non - alcoholic steatohepatitis ( nash ) are multidisciplinary liver diseases that often accompany type 2 diabetes or metabolic syndrome , which are characterized by insulin resistance . therefore , effective treatment of type 2 diabetes and metabolic syndrome should target not only the cardiometabolic abnormalities , but also the associated liver disorders . in the last decade , it has been shown that metformin , thiazolidinediones , vitamin e , ezetimibe , n-3 polyunsaturated fatty acids , renin - angiotensin system ( ras ) blockers , and antiobesity drugs may improve hepatic pathophysiological disorders as well as clinical parameters . accordingly , insulin sensitizers , antioxidative agents , niemann - pick c1-like 1 ( npc1l1 ) inhibitors , ras blockers , and drugs that target the central nervous system may represent candidate pharmacotherapies for nafld and possibly nash . however , the efficacy , safety , and tolerability of long - term treatment ( potentially for many years ) with these drugs have not been fully established . furthermore , clinical trials have not comprehensively examined the efficacy of lipid - lowering drugs ( i.e. , statins , fibrates , and npc1l1 inhibitors ) for the treatment of nafld . although clinical evidence for ras blockers and incretin - based agents ( glp-1 analogs and dipeptidyl peptidase-4 inhibitors ) is also lacking , these agents are promising in terms of their insulin - sensitizing and anti - inflammatory effects without causing weight gain .

1. Introduction 2. Liver-Specific Pathogenic Characteristics of NAFLD and NASH 3. Pharmacotherapeutic Candidates for Multidisciplinary Treatment of NAFLD and NASH 4. Conclusion

over the past two decades , the prevalence of metabolic abnormalities such as type 2 diabetes and metabolic syndrome ( mets ) has been increasing worldwide together with the escalating obesity pandemic [ 13 ] . abdominal obesity , in particular , substantially increases the risk of developing type 2 diabetes , mets , and fatty liver . according to the american association for the study of liver diseases ( aasld ) , fatty liver in the absence of a chronic increase in alcohol intake ( i.e. , alcohol intake is < 20 g ethanol / day ) is referred to as nonalcoholic fatty liver disease ( nafld ) . according to the aasld 's practice guidelines for nafld , nafld is histologically subdivided into nonalcoholic fatty liver ( nafl ) and a more severe condition , nonalcoholic steatohepatitis ( nash ) , which sometimes advances over several decades to life - threatening hepatic cirrhosis and hepatocellular carcinoma . the prevalence of nafld , as detected by ultrasound , is up to 3046% in developed countries and nearly 10% in developing nations , making nafld the most common liver disorder worldwide [ 5 , 6 ] . lifestyle interventions such as diet and moderate exercise , which lead to weight loss , are fundamental for the treatment of nafld . in india , individuals with a normal bmi ( 18.524.9 kg / m ) have a 2-fold higher risk of developing nafld compared with those with a bmi of < 18.5 therefore , nafld is expected to become a major burden in asian countries where the prevalence of obesity is less than that in western countries [ 10 , 11 ] . therefore , although obese people are predisposed to develop nafld , normal weight and overweight people may , through the development of insulin resistance , also show the pathogenic characteristics of nafld . the clinical relevance of nafld is still poorly understood because some investigators [ 1215 ] , but not all [ 16 , 17 ] , have shown that nafld is associated with higher overall mortality and cardiovascular disease . since nafld is closely associated with obesity , diabetes , and mets , it is unknown whether the relationship between nafld and all - cause mortality and cardiovascular death , if any , is independent of cardiometabolic risk factors ( figure 1 ) such as mets and type 2 diabetes . taken together , nafld and nash are multidisciplinary liver diseases that require interventions targeting the cardiometabolic and liver disorders for the effective treatment of patients with these diseases . therefore , it is likely that mild nafld will require predominantly cardiometabolic pharmacotherapies , whereas moderate to severe nafld and nash will require pharmacotherapies targeting the hepatic disorders . however , since many of the candidate drugs are likely to have broad therapeutic effects targeting multiple aspects of these diseases , distinct classifications are unavailable . however , hepatocytes can , under physiological conditions , store small amounts of triglyceride in a transient manner . , diet and habitual smoking ) , and sympathetic overdrive as a result of physical / mental stress may lead to insulin resistance independently of obesity . in turn , insulin resistance suppresses the influx of glucose and free fatty acids ( ffas ) into adipose tissue , increasing ffa influx into the liver . proposed that reduced ectopic fat in the liver may be more important than reduced visceral fat for the discrimination of benign obesity , in other words metabolically normal obesity with good insulin sensitivity . ectopic fat deposition in the liver may be an initial feature of peripheral insulin resistance , particularly in adipose tissue , which predominantly accumulates surplus energy as fat . for many years , it was thought that simple steatosis ( i.e. however , studies published in the last two decades have shown that such conditions , without appropriate treatment or intervention , provoke other events , including oxidative stress , inflammation , and fibrosis . however , they may remain within the normal ranges [ 23 , 25 ] and may be overlooked until abdominal ultrasound , computed tomography , or magnetic resonance imaging are performed . this phenomenon may be caused by direct influx , via the portal vein , of long - chain ffas , glycerol , and proinflammatory cytokines ( e.g. , tumor necrosis factor [ tnf ] , interleukin [ il]-6 , and il-1 ) from the visceral and upper body fat depots , which exhibit insulin resistance and activation of hormone - sensitive lipase and adipose tissue triacylglycerol lipase [ 2628 ] . similarly , hyperglycemia caused by inadequate insulin action results in the activation of carbohydrate response element binding protein , which activates l - type pyruvate kinase and lipogenic genes in hepatocytes . nevertheless , it seems unexpected that hepatic lipogenesis is not attenuated by decreased insulin sensitivity or insulin resistance . er stress activates the cleavage of srebp-1c independently of insulin , which may explain the paradoxical stimulation of lipogenesis in insulin - resistant liver . in terms of preventing liver damage , the priority for pharmacotherapy of nafld is to prevent transformation of nafld into nash and to improve the pathophysiology . therefore , in theory , antioxidative , antiinflammatory , and antifibrotic agents may constitute first - line treatments for nafld . metformin ( biguanide ) is an insulin - sensitizer that is considered to be the first - line treatment of type 2 diabetes because it has relatively few side effects and is inexpensive . besides reducing hepatic glucose output , metformin also activates amp kinase , which inhibits the production of glucose , cholesterol , and triglycerides , and stimulates fatty acid oxidation . nevertheless , the outcomes of clinical trials in nafld are conflicting [ 38 , 39 ] . metformin was also better than a prescriptive diet or vitamin e intake for the treatment of nafld in patients receiving nutritional counseling . moreover , 6 months of treatment with metformin was no better than placebo in terms of improving liver histology in patients with nafld . a number of clinical trials , including randomized controlled trials , have shown that thiazolidinediones ( peroxisome proliferator activated receptor [ ppar]- agonists ) improve liver enzyme levels and liver histology in patients with nafld and nash [ 39 , 45 ] . since ppar is also expressed in cardiovascular tissues , such as vascular endothelial cells , smooth muscle cells , macrophages , and cardiomyocytes , altered ppar activity may be involved in the etiology of cardiovascular diseases , particularly atherosclerosis . it has been shown that treatment with pioglitazone for 1 - 2 years improves the overall pathogenic characteristics of nafld and nash [ 4850 ] , suggesting that ppar agonists may improve the pathophysiology of the liver as well as clinical factors in patients with nafld and nash . therefore , the improvements in nafld may be due to improvements in the visceral tissue and upper subcutaneous fat , as thiazolidinediones redirect fat accumulation from the liver or muscle into adipose tissues and confine it in adipose tissue at the expense of adipose cell expansion , which unfortunately leads to weight gain . however , this apparent improvement in insulin resistance often results in adverse outcomes , such as an increase in the number of adipose cells through enhanced differentiation , eventually leading to weight gain and systematic edema [ 39 , 50 ] . consequently , long - term treatment with thiazolidinediones may be poorly tolerated in some people with diabetes because of their adverse effects , although pioglitazone has fewer side effects than rosiglitazone [ 56 , 57 ] ( troglitazone was excluded from the market because of hepatic toxicity ) . some adverse effects of thiazolidinediones , particularly weight gain and edema , could be prevented when administered in low dosages . some intervention studies in patients with nafld have shown that vitamin e , a lipophilic antioxidant , may improve some of the pathogenic characteristics of nafld . vitamin e exerts its antioxidative effects by reducing lipid peroxidation , preventing free radical reactions , and stabilizing cellular phospholipid membranes . vitamin e also inhibits hepatic transforming growth factor-1 expression , attenuates cytokine stimulation of stellate cells , and protects against hepatic fibrosis , suggesting that vitamin e is more beneficial for nash rather than nafld . some studies , most of them involving small numbers of patients , showed positive outcomes in terms of the treatment of nafld [ 41 , 48 , 61 , 62 ] . however , other studies showed negative results or no significant improvement with vitamin e compared with placebo or lifestyle interventions , suggesting that vitamin e alone is insufficient to treat nafld [ 38 , 63 ] . reported that vitamin e and metformin were not superior to placebo in terms of sustained reductions in alt levels in children with nafld . it was also reported that simple lifestyle interventions ( i.e. , diet and physical exercise ) in children with nafld can significantly improve liver function , glucose metabolism , and lipid levels beyond those achieved with antioxidant therapy ( 600 iu / day vitamin e plus 500 mg / day vitamin c ) . several studies have shown that high - dose vitamin e supplementation may be harmful in some patients because of unexpected adverse outcomes , such as increased cardiovascular disease mortality [ 64 , 65 ] , although other studies found no such risk [ 66 , 67 ] . among nondiabetic patients with biopsy - confirmed nash , current recommendations advocate the administration of 800 iu / day vitamin e , as this dose may reduce serum alt levels and improve steatosis , inflammation , and hepatocyte ballooning . udca is a cytoprotective antiinflammatory agent that is widely used to treat liver diseases , as well as gallstones . long - term clinical studies have revealed that udca safely and effectively improves hepatic enzyme levels , serum fibrosis markers , and selected metabolic parameters . overall , however , there were no significant differences between udca and placebo in several clinical trials . fish oil contains both docosahexaenoic acid ( dha , c22:6 n-3 ) and eicosapentaenoic acid ( epa , c20:5 n-3 ) . considering these properties of n-3 pufas , several clinical trials have investigated the potential effects of fish oil or n-3 pufa consumption on the outcomes of cardiovascular disease . meta - analyses of these studies have shown that fish oil and n-3 pufa consumption , but not -linolenic acid , reduce the risk of cardiovascular events and the risk of coronary heart disease - related mortality [ 71 , 73 ] . regarding nafld , dietary supplementation with long - chain n-3 pufas appears to safely reduce nutritional hepatic steatosis in adults . reported that increased erythrocyte n-3 and n-6 pufa levels are significantly associated with a lower prevalence of steatosis in patients with type 2 diabetes . indeed , it was reported that low - dose ( 2.0% of total energy ) fish oil diets improve hepatic lipid accumulation in mice fed a high - cholesterol diet . however , studies examining the effects of epa / fish oil on dietary cholesterol - induced nafld in humans are still lacking . statins ( 3-hydroxy-3-methyglutaryl - coenzyme a reductase inhibitors ) are used worldwide to treat lipid disorders , particularly elevated low - density lipoprotein - cholesterol ( ldl - c ) and substantially reduce cardiovascular events and mortality . taken together with a previous report showing that excess cholesterol consumption may accelerate nafld , statins may be promising candidates for the treatment of nafld . fibrates , ppar agonists , increase ffa oxidation in the liver , alter tg synthesis , and reduce hepatic synthesis of vldl [ 93 , 94 ] , and theoretically improve the pathogenic characteristics of mets and nafld . unlike the striking outcomes of numerous clinical trials using statins , the results of several large studies of fibrates were inconsistent and had varying outcomes , including the incidence of cardiovascular mortality and events [ 9599 ] . in all of these studies , treatment with a fibrate was associated with a large , although nonsignificant , reduction in cardiovascular events in people with type 2 diabetes or components of the metabolic syndrome . to date , however , few clinical studies , except for a small pilot study [ 100 , 101 ] , have examined the effects of fibrates on the pathophysiology of nafld . fibrates are expected to ameliorate the pathogenic characteristics of nafld because they reduce the levels of inflammatory biomarkers , such as crp and il-6 , and may improve insulin resistance [ 102104 ] , via mechanisms that differ from those of statins . this suggests that hepatic npc1l1 may facilitate hepatic cholesterol accumulation and that ezetimibe may be a potential candidate for nafld , especially nafld induced by a high - cholesterol diet . in clinical trials , albeit on a small scale , ezetimibe improved biochemical parameters and hepatic enzyme levels , as well as the histological abnormalities of nafld [ 110112 ] . the ras plays key roles in the regulation of blood pressure and fluid balance , as well as in the pathogenesis of insulin resistance and nafld [ 113 , 114 ] . in addition , inhibition of the ras may improve the intracellular insulin signaling pathway , offering better control of adipose tissue proliferation and adipokine production . the two main classes of ras blockers , angiotensin ii receptor blockers ( arbs ) and angiotensin - converting enzyme inhibitors ( aceis ) , are efficient drugs that significantly reduce cardiovascular events and mortality [ 116118 ] . as expected , arbs and aceis improve insulin resistance and possibly lipid profiles , suggesting that these agents may be suitable treatments for nafld and nash . nevertheless , despite many animal studies showing the beneficial effects of arbs and aceis , clinical studies for nafld are still lacking . beneficial effects of spironolactone and eplerenone , aldosterone antagonists , were recently shown in a mouse model of nafld [ 121123 ] . additionally , spironolactone in combination with vitamin e was reported to improve insulin resistance in patients with nafld . however , these effects of aldosterone antagonists have only been shown in mouse and small - scale clinical studies . therefore , although aliskiren is a promising drug in terms of improving insulin resistance and oxidative stress , again , human studies are lacking . treatment with incretin modulators , glucagon - like peptide-1 ( glp-1 ) analogs , and dipeptidyl peptidase-4 inhibitors reduces weight gain , minimizes hypoglycemia , decreases inflammation , and is cardioprotective in preclinical studies [ 128 , 129 ] . the most common adverse events are mild gastrointestinal tract symptoms such as nausea , vomiting , and diarrhea , the incidence and severity of which generally decreased during continued therapy . regarding the beneficial effects of incretin modulators , weight loss ( or weight neutrality ) and reduced inflammation appear to be particularly promising for the treatment of nafld because these effects facilitate improvements in insulin resistance and metabolic abnormalities . reported that 44 weeks of exenatide therapy decreased liver fat measured by liver spectroscopy from 15.8% to 4.3% and improved hepatic enzymes in a patient with type 2 diabetes . taken together , there are promising results in animal models and limited human reports , but clinical studies examining the effects of incretin - based agents on hepatic steatosis have not been performed . of several commonly used antiobesity medications , orlistat and sibutramine are available for long - term prescription [ 135 , 136 ] . showed that orlistat improves serum alt levels and steatosis determined by ultrasound in patients with nafld , beyond its weight - lowering effects . reported that subjects who lost 5% of their body weight over 9 months experienced improvements in insulin resistance and steatosis , while subjects who lost 9% of their body weight also experienced improvements in hepatic histology . however , there is still insufficient safety data regarding the long - term outcomes of antiobesity therapy . however , long - term treatment with mazindol is not currently permitted , although treatment with mazindol for a few weeks to several months is allowed in severely obese individuals ( e.g. mazindol exerts antiobesity effects by inhibiting the appetite and activating thermogenesis , as well as having antidiabetic effects . however , clinical studies of mazindol have not been conducted in patients with nafld , partly because of the restriction for short - term administration . furthermore , administration of 20 mg rimonabant daily in combination with a hypocaloric diet for 1 year , significantly decreased body weight and waist circumference . based on several trials showing its weight loss benefits , rimonabant entered the european market for the treatment of obesity . however , the drug was withdrawn worldwide in 2008 - 2009 because of the emergence of significant side effects , particularly psychiatric disorders ( e.g. the cb1 receptor is expressed not only in the central nervous system but also in the gastrointestinal tract , adipose tissue , and cardiovascular system . consistently , animal studies have shown that cb1 receptor antagonists improve glucose homeostasis , insulin levels , fatty liver , and plasma lipid profiles by blocking the cb1 receptor in peripheral tissues , including the liver and visceral fat [ 146 , 147 ] . despite these promising effects , the clinical application of cb1 receptor antagonists will be restricted until their critical adverse effects on the central nervous system can be overcome . several polyphenols obtained from plants may be promising candidate treatments for nafld and nash because they are effective scavengers of reactive oxygen and reactive nitrogen species . of several polyphenols described to date , resveratrol , a component of several grape species , appears to be particularly relevant in the context of liver disease . resveratrol also decreased the severity of nafld in rats , at least in part , through its antioxidant effects and by inhibiting tnf . light alcohol consumption ( 20 g ethanol on 13 days / week or 40140 g ethanol / week ) and moderate alcohol consumption ( 140280 g ethanol / week ) were independently associated with a low prevalence of fatty liver [ 155 , 156 ] , whereas moderate to heavy drinking ( > 60 g ethanol / week ) was associated with the progression of hepatic steatosis and fibrosis [ 157 , 158 ] . however , the specific mechanisms by which alcohol ( alone or in combination with obesity / metabolic abnormalities ) causes liver injury are poorly understood . in clinical studies , the causality between light to moderate alcohol consumption and reduced prevalence of fatty liver or nafld remains unknown . since hepatic dysfunction is usually closely associated with systemic disorders , there are no specific medications that ameliorate only the pathogenic characteristics of fatty liver without affecting other organs and tissues . in other words , drugs used , or expected to be used , in the treatment of type 2 diabetes , mets ( i.e. , hypertension and dyslipidemia ) , hypercholesterolemia , and obesity may be candidate drugs for the treatment of nafld and nash . these agents can be more effective than monotherapy , although further evidence from animal and cellular studies , as well as large clinical trials , is needed to examine this possibility .

over the past two decades , the prevalence of metabolic abnormalities such as type 2 diabetes and metabolic syndrome ( mets ) has been increasing worldwide together with the escalating obesity pandemic [ 13 ] . abdominal obesity , in particular , substantially increases the risk of developing type 2 diabetes , mets , and fatty liver . according to the american association for the study of liver diseases ( aasld ) , fatty liver in the absence of a chronic increase in alcohol intake ( i.e. according to the aasld 's practice guidelines for nafld , nafld is histologically subdivided into nonalcoholic fatty liver ( nafl ) and a more severe condition , nonalcoholic steatohepatitis ( nash ) , which sometimes advances over several decades to life - threatening hepatic cirrhosis and hepatocellular carcinoma . the prevalence of nafld , as detected by ultrasound , is up to 3046% in developed countries and nearly 10% in developing nations , making nafld the most common liver disorder worldwide [ 5 , 6 ] . lifestyle interventions such as diet and moderate exercise , which lead to weight loss , are fundamental for the treatment of nafld . in india , individuals with a normal bmi ( 18.524.9 kg / m ) have a 2-fold higher risk of developing nafld compared with those with a bmi of < 18.5 therefore , nafld is expected to become a major burden in asian countries where the prevalence of obesity is less than that in western countries [ 10 , 11 ] . notably , nafld appears to be an early predictor of metabolic disorders , particularly among normal - weight individuals . this is because nafld may be more tightly associated with insulin resistance and with markers of oxidative stress and endothelial dysfunction than with the adult treatment panel iii criteria for mets in nonobese , nondiabetic subjects . therefore , although obese people are predisposed to develop nafld , normal weight and overweight people may , through the development of insulin resistance , also show the pathogenic characteristics of nafld . the clinical relevance of nafld is still poorly understood because some investigators [ 1215 ] , but not all [ 16 , 17 ] , have shown that nafld is associated with higher overall mortality and cardiovascular disease . since nafld is closely associated with obesity , diabetes , and mets , it is unknown whether the relationship between nafld and all - cause mortality and cardiovascular death , if any , is independent of cardiometabolic risk factors ( figure 1 ) such as mets and type 2 diabetes . taken together , nafld and nash are multidisciplinary liver diseases that require interventions targeting the cardiometabolic and liver disorders for the effective treatment of patients with these diseases . therefore , it is likely that mild nafld will require predominantly cardiometabolic pharmacotherapies , whereas moderate to severe nafld and nash will require pharmacotherapies targeting the hepatic disorders . however , since many of the candidate drugs are likely to have broad therapeutic effects targeting multiple aspects of these diseases , distinct classifications are unavailable . ectopic fat deposition in organs other than fat tissue , such as the liver and skeletal muscle , reflects severe energy overaccumulation or disturbed fat distribution . , diet and habitual smoking ) , and sympathetic overdrive as a result of physical / mental stress may lead to insulin resistance independently of obesity . in turn , insulin resistance suppresses the influx of glucose and free fatty acids ( ffas ) into adipose tissue , increasing ffa influx into the liver . proposed that reduced ectopic fat in the liver may be more important than reduced visceral fat for the discrimination of benign obesity , in other words metabolically normal obesity with good insulin sensitivity . ectopic fat deposition in the liver may be an initial feature of peripheral insulin resistance , particularly in adipose tissue , which predominantly accumulates surplus energy as fat . however , studies published in the last two decades have shown that such conditions , without appropriate treatment or intervention , provoke other events , including oxidative stress , inflammation , and fibrosis . unfortunately , similar to other cardiometabolic diseases , most people with asymptomatic nafld remain untreated until the results of blood test or imaging studies indicate the presence of nafld . this phenomenon may be caused by direct influx , via the portal vein , of long - chain ffas , glycerol , and proinflammatory cytokines ( e.g. , tumor necrosis factor [ tnf ] , interleukin [ il]-6 , and il-1 ) from the visceral and upper body fat depots , which exhibit insulin resistance and activation of hormone - sensitive lipase and adipose tissue triacylglycerol lipase [ 2628 ] . ffas derived from the lipolysis of visceral and subcutaneous fat account for approximately 60% of the total hepatic ffa content . besides increased ffa influx , hyperinsulinemia increases hepatic lipogenesis by activating sterol regulatory element binding protein 1c ( srebp-1c ) , a key regulator of lipogenic gene expression . similarly , hyperglycemia caused by inadequate insulin action results in the activation of carbohydrate response element binding protein , which activates l - type pyruvate kinase and lipogenic genes in hepatocytes . er stress activates the cleavage of srebp-1c independently of insulin , which may explain the paradoxical stimulation of lipogenesis in insulin - resistant liver . moreover , er stress in response to increased hepatic lipid content attenuates triglyceride secretion by limiting apolipoprotein b secretion , thus increasing hepatic triglyceride accumulation . alternatively , reduced mitochondrial -oxidation , following inhibition of carnitine palmitoyl transferase-1 ( cpt-1 ) , may contribute to enhanced fatty acid synthesis . for example , patients with nash were reported to show reduced very low - density lipoprotein ( vldl ) synthesis and impaired hepatic mtp mrna expression , which was likely due to single nucleotide polymorphisms of the mtp gene . in terms of preventing liver damage , the priority for pharmacotherapy of nafld is to prevent transformation of nafld into nash and to improve the pathophysiology . therefore , in theory , antioxidative , antiinflammatory , and antifibrotic agents may constitute first - line treatments for nafld . metformin ( biguanide ) is an insulin - sensitizer that is considered to be the first - line treatment of type 2 diabetes because it has relatively few side effects and is inexpensive . metformin was also better than a prescriptive diet or vitamin e intake for the treatment of nafld in patients receiving nutritional counseling . in a clinical study , serum alt levels were consistently lower in patients treated with metformin compared with those treated with placebo . moreover , 6 months of treatment with metformin was no better than placebo in terms of improving liver histology in patients with nafld . further well - designed studies are needed to elucidate the significance of metformin treatment for nafld . a number of clinical trials , including randomized controlled trials , have shown that thiazolidinediones ( peroxisome proliferator activated receptor [ ppar]- agonists ) improve liver enzyme levels and liver histology in patients with nafld and nash [ 39 , 45 ] . ppar , which belongs to the nuclear hormone receptor family , is predominantly expressed in adipose tissues and plays a key role in adipogenesis and glucose homeostasis . since ppar is also expressed in cardiovascular tissues , such as vascular endothelial cells , smooth muscle cells , macrophages , and cardiomyocytes , altered ppar activity may be involved in the etiology of cardiovascular diseases , particularly atherosclerosis . it has been shown that treatment with pioglitazone for 1 - 2 years improves the overall pathogenic characteristics of nafld and nash [ 4850 ] , suggesting that ppar agonists may improve the pathophysiology of the liver as well as clinical factors in patients with nafld and nash . therefore , the improvements in nafld may be due to improvements in the visceral tissue and upper subcutaneous fat , as thiazolidinediones redirect fat accumulation from the liver or muscle into adipose tissues and confine it in adipose tissue at the expense of adipose cell expansion , which unfortunately leads to weight gain . thiazolidinediones also activate amp - activated protein kinase and inhibit lipolysis , at least in part by inhibiting the translocation of hormone - sensitive lipase to lipid droplets . however , this apparent improvement in insulin resistance often results in adverse outcomes , such as an increase in the number of adipose cells through enhanced differentiation , eventually leading to weight gain and systematic edema [ 39 , 50 ] . consequently , long - term treatment with thiazolidinediones may be poorly tolerated in some people with diabetes because of their adverse effects , although pioglitazone has fewer side effects than rosiglitazone [ 56 , 57 ] ( troglitazone was excluded from the market because of hepatic toxicity ) . indeed , even low - dose pioglitazone ( 15 mg / day ) improved liver enzymes without overt adverse effects in a study of 12 patients with biopsy - confirmed nash . some intervention studies in patients with nafld have shown that vitamin e , a lipophilic antioxidant , may improve some of the pathogenic characteristics of nafld . vitamin e also inhibits hepatic transforming growth factor-1 expression , attenuates cytokine stimulation of stellate cells , and protects against hepatic fibrosis , suggesting that vitamin e is more beneficial for nash rather than nafld . some studies , most of them involving small numbers of patients , showed positive outcomes in terms of the treatment of nafld [ 41 , 48 , 61 , 62 ] . however , other studies showed negative results or no significant improvement with vitamin e compared with placebo or lifestyle interventions , suggesting that vitamin e alone is insufficient to treat nafld [ 38 , 63 ] . reported that vitamin e and metformin were not superior to placebo in terms of sustained reductions in alt levels in children with nafld . , diet and physical exercise ) in children with nafld can significantly improve liver function , glucose metabolism , and lipid levels beyond those achieved with antioxidant therapy ( 600 iu / day vitamin e plus 500 mg / day vitamin c ) . several studies have shown that high - dose vitamin e supplementation may be harmful in some patients because of unexpected adverse outcomes , such as increased cardiovascular disease mortality [ 64 , 65 ] , although other studies found no such risk [ 66 , 67 ] . among nondiabetic patients with biopsy - confirmed nash , current recommendations advocate the administration of 800 iu / day vitamin e , as this dose may reduce serum alt levels and improve steatosis , inflammation , and hepatocyte ballooning . long - term clinical studies have revealed that udca safely and effectively improves hepatic enzyme levels , serum fibrosis markers , and selected metabolic parameters . oral administration of taurine - conjugated udca decreased hepatic steatosis in ob / ob mice by cooperatively regulating multiple metabolic pathways , including reduced expression of genes that regulate de novo lipogenesis . considering these properties of n-3 pufas , several clinical trials have investigated the potential effects of fish oil or n-3 pufa consumption on the outcomes of cardiovascular disease . meta - analyses of these studies have shown that fish oil and n-3 pufa consumption , but not -linolenic acid , reduce the risk of cardiovascular events and the risk of coronary heart disease - related mortality [ 71 , 73 ] . regarding nafld , dietary supplementation with long - chain n-3 pufas appears to safely reduce nutritional hepatic steatosis in adults . reported that the levels of n-3 pufas were decreased while the n-6/n-3 fatty acid ratio was increased in nafld patients compared with controls , probably because of defective desaturation of pufa by inadequate intake of their precursors and increased peroxidation of pufa . reported that increased erythrocyte n-3 and n-6 pufa levels are significantly associated with a lower prevalence of steatosis in patients with type 2 diabetes . in addition , the dietary records of middle - aged healthy japanese men revealed that dietary epa and epa + dha may help to prevent nafld . reported that the antiobesity effects of epa in high - fat / high - sucrose - induced obese mice were associated with the suppression of hepatic lipogenesis and steatosis . enjoji and nakamuta proposed that excess cholesterol intake appears to be one of the main factors associated with nafld , particularly in nonobese subjects , because excess cholesterol consumption stimulates the liver x receptor-srebp-1c pathway and enhances fatty acid synthesis . indeed , it was reported that low - dose ( 2.0% of total energy ) fish oil diets improve hepatic lipid accumulation in mice fed a high - cholesterol diet . however , studies examining the effects of epa / fish oil on dietary cholesterol - induced nafld in humans are still lacking . statins ( 3-hydroxy-3-methyglutaryl - coenzyme a reductase inhibitors ) are used worldwide to treat lipid disorders , particularly elevated low - density lipoprotein - cholesterol ( ldl - c ) and substantially reduce cardiovascular events and mortality . although statins are putatively associated with some adverse events , including elevated hepatic enzymes and liver dysfunction , an elevated serum alt level at baseline attributable to nafld is unlikely to increase the risk of statin - associated elevations in alt . similarly , an elevated baseline serum alt was not associated with an increased risk of hepatotoxicity in patients treated with lovastatin . in clinical studies , simvastatin and atorvastatin [ 8486 ] were associated with a reduction in hepatic steatosis and may inhibit the progression to nash . four years of treatment with atorvastatin ( 20 mg ) combined with vitamins c and e reduced hepatic steatosis by 71% in people with nafld at baseline . taken together with a previous report showing that excess cholesterol consumption may accelerate nafld , statins may be promising candidates for the treatment of nafld . on the other hand , considering that statins are associated with worsening of glucose metabolism [ 89 , 90 ] and that multifactorial medications , such as antihypertensive and antidiabetic drugs , were used in most of the previous studies of statin therapy , well - designed , randomized , placebo - controlled studies are needed to determination the suitability of statins as monotherapy or combination therapy for nafld . fibrates , ppar agonists , increase ffa oxidation in the liver , alter tg synthesis , and reduce hepatic synthesis of vldl [ 93 , 94 ] , and theoretically improve the pathogenic characteristics of mets and nafld . unlike the striking outcomes of numerous clinical trials using statins , the results of several large studies of fibrates were inconsistent and had varying outcomes , including the incidence of cardiovascular mortality and events [ 9599 ] . in all of these studies , treatment with a fibrate was associated with a large , although nonsignificant , reduction in cardiovascular events in people with type 2 diabetes or components of the metabolic syndrome . to date , however , few clinical studies , except for a small pilot study [ 100 , 101 ] , have examined the effects of fibrates on the pathophysiology of nafld . fibrates are expected to ameliorate the pathogenic characteristics of nafld because they reduce the levels of inflammatory biomarkers , such as crp and il-6 , and may improve insulin resistance [ 102104 ] , via mechanisms that differ from those of statins . no clinical studies have examined the effects or safety of a statin in combination with a fibrate compared with monotherapy for treating nafld . however , some benefits of combination therapy on cardiovascular and microvascular outcomes were observed in specific subgroups of patients , such as patients with low hdl levels or hypertriglyceridemia . ezetimibe , an npc1l1 inhibitor , was reported to reduce circulating ldl - c levels and improve clinical outcomes in patients at increased risk for cardiovascular events when administered alone or in combination with a statin . unlike in rodents in which npc1l1 is mainly expressed in the intestine , npc1l1 is highly expressed in the liver in humans [ 108 , 109 ] . this suggests that hepatic npc1l1 may facilitate hepatic cholesterol accumulation and that ezetimibe may be a potential candidate for nafld , especially nafld induced by a high - cholesterol diet . the ras plays key roles in the regulation of blood pressure and fluid balance , as well as in the pathogenesis of insulin resistance and nafld [ 113 , 114 ] . in addition , inhibition of the ras may improve the intracellular insulin signaling pathway , offering better control of adipose tissue proliferation and adipokine production . treatment with incretin modulators , glucagon - like peptide-1 ( glp-1 ) analogs , and dipeptidyl peptidase-4 inhibitors reduces weight gain , minimizes hypoglycemia , decreases inflammation , and is cardioprotective in preclinical studies [ 128 , 129 ] . regarding the beneficial effects of incretin modulators , weight loss ( or weight neutrality ) and reduced inflammation appear to be particularly promising for the treatment of nafld because these effects facilitate improvements in insulin resistance and metabolic abnormalities . reported that 44 weeks of exenatide therapy decreased liver fat measured by liver spectroscopy from 15.8% to 4.3% and improved hepatic enzymes in a patient with type 2 diabetes . taken together , there are promising results in animal models and limited human reports , but clinical studies examining the effects of incretin - based agents on hepatic steatosis have not been performed . however , long - term treatment with mazindol is not currently permitted , although treatment with mazindol for a few weeks to several months is allowed in severely obese individuals ( e.g. however , clinical studies of mazindol have not been conducted in patients with nafld , partly because of the restriction for short - term administration . rimonabant , a selective cannabinoid-1 ( cb1 ) receptor blocker , was shown to reduce body weight and improve cardiovascular risk factors in obese patients by regulating the energy balance and body composition . consistently , animal studies have shown that cb1 receptor antagonists improve glucose homeostasis , insulin levels , fatty liver , and plasma lipid profiles by blocking the cb1 receptor in peripheral tissues , including the liver and visceral fat [ 146 , 147 ] . despite these promising effects , the clinical application of cb1 receptor antagonists will be restricted until their critical adverse effects on the central nervous system can be overcome . light alcohol consumption ( 20 g ethanol on 13 days / week or 40140 g ethanol / week ) and moderate alcohol consumption ( 140280 g ethanol / week ) were independently associated with a low prevalence of fatty liver [ 155 , 156 ] , whereas moderate to heavy drinking ( > 60 g ethanol / week ) was associated with the progression of hepatic steatosis and fibrosis [ 157 , 158 ] . however , the specific mechanisms by which alcohol ( alone or in combination with obesity / metabolic abnormalities ) causes liver injury are poorly understood .

our hospital 's institutional review board approved the research protocols , and all patients gave informed written consent . thirty - eight patients who had undergone two - phase contrast - enhanced ecg - gated mdct for ischemic heart disease between october 2004 and june 2005 were retrospectively identified . among these 38 patients , there were 17 patients with acute myocardial infarction ( ami : infarction occurring less than one week before the ct study ) and six patients with chronic myocardial infarction ( cmi : infarction occurring more than one month before the ct study ) . the patient population was characterized as follows : 20 males and three females ( mean age 6311 years , range 34 - 82 years ) . a complete patient history , electrocardiography , echocardiography , and recent laboratory tests were available for all the patients . among these 23 patients , six patients ( 5 ami and 1 cmi ) underwent tl single photon emission computed tomography ( spect ) . catheter angiography was performed on 19 patients ( 17 ami and 2 cmi ) for diagnosis and percutaneous coronary intervention . acute myocardial infarction was diagnosed if the patient had 1 ) clinical symptoms , 2 ) electrocardiographic changes indicative of mi , and 3 ) characteristic elevation of the serum enzyme levels . cmi was diagnosed if the patient had 1 ) a history of previous mi , 2 ) diagnostic q waves on the electrocardiogram or ecg registration of the acute event , and 3 ) normal enzyme levels during the observation period . the patients with st segment elevation mi ( n = 10 ) were treated with thrombolytics before their ct scans . the average time between two - phase contrast - enhanced mdct and catheter angiography was 43 days . each patient 's heart rate ( hr ) was measured prior to the examination . patients with a pre - scan hr65 beats per minute ( bpm ) were given 25 - 50 mg of atenolol ( tenormin , hyundai ) per os 1 h before the scan . two - phase contrast - enhanced ecg - gated mdct was performed using a sensation 16 scanner ( siemens , forchheim , germany ) . the acquisition protocol included both early - phase acquisition for analysis of the coronary arteries and myocardial perfusion of the left ventricle , and late - phase acquisition that was performed 7 minutes after this first - pass acquisition for analysis of the delayed enhancement patterns . the following parameters were employed : 160.75 mm collimation , 0.37s rotation time , table feed 6.8 mm / rotation , 120 kv , and 620 mas , resulting in a total scan time of about 20s to cover the entire heart , and the scan was acquired a single breath hold with using retrospective ecg gating . a contrast injection protocol with injection of 100 ml of non - ionic contrast material ( ultravist 370 , schering , berlin , germany ) at a flow rate of 3.5 ml / s followed by a saline chaser bolus of 50 ml at the same flow rate was used . ct scanning started with real - time bolus tracking ( care bolus , siemens , forchheim , germany ) using a region of interest in the ascending aorta for monitoring a threshold of + 100 hu above the baseline attenuation . a late - phase acquisition was obtained using the same protocol , except for reducing the mas to 310 . mdct evaluation was done on a workstation ( syngo , wizard ; simens medical solutions ) . mdct coronary angiography was used for detecting coronary stenosis in four cmi patients who did not have catheter angiography performed . a series of maximum intensity projection images with a 4-mm thick slab were generated in the short - axis , horizontal long - axis and vertical long - axis plane of the heart from early - phase axial images . the presence of significant coronary stenosis was visually defined as more than 50% luminal narrowing . the contrast enhancement patterns were assessed with the multiplanar vertical long - axis and shot - axis views by using 5-mm - thick slab sections of the heart from the early and late - phase axial images taken at mid - diastole . the two - phase multiplanar reformation ( mpr ) images of each patient were analyzed by two experienced radiologists in consensus and unaware of any clinical information and the results of both the catheter and mdct coronary angiography . assessment of myocardial enhancement patterns was based on those used in a previous study with using two - phase contrast - enhanced mdct ( 14 ) and as follows : the presence of a hypoenhanced area on the early - phase images as an early perfusion defect , the presence of persistent hypoenhanced area in the subendocardium surrounded by partially enhanced myocardium on the latephase images as a residual perfusion defect , and the presence of a hyperenhanced area on the late - phase images as late enhancement . regions of interest of 2 - 5 mm in size were placed over the center of the area of each finding and the surrounding normal myocardium . for all the perfusion defects and delayed enhancement detected on the two - phase mpr images , the location of each detected mi within the left ventricular myocardium was defined with using a 17-segment model in the cardiac short axis , as recently suggested by the american heart association ( aha ) ( 16 ) . the depth of myocardial involvement was classified into three regions as subendocardial : involvement of less than 1/2 of the left inner ventricle wall thickness , subepicardial : involvement of less than 1/2 of the outer wall thickness , and transmural : involvement of more than 50% of the wall thickness . the typical window width and level settings ranged between 150 - 350 and 60 - 200 hu , respectively . the presence , location , and patterns of myocardial enhancement on two - phase mdct images were compared with infarcted myocardial territories determined by using electrocardiogram , echocardiography , tl spect , catheter and mdct coronary angiography . comparison of the ct attenuation numbers of the normal and abnormally enhanced myocardial segments on the two - phase mdct images were done with using paired student 's t - tests . a p value of 0.05 or less was considered to indicate a statistically significant difference . for statistical analysis , a commercially available windows - based software product was used ( spss 12.0.1 , 2003 ) . our hospital 's institutional review board approved the research protocols , and all patients gave informed written consent . thirty - eight patients who had undergone two - phase contrast - enhanced ecg - gated mdct for ischemic heart disease between october 2004 and june 2005 were retrospectively identified . among these 38 patients , there were 17 patients with acute myocardial infarction ( ami : infarction occurring less than one week before the ct study ) and six patients with chronic myocardial infarction ( cmi : infarction occurring more than one month before the ct study ) . the patient population was characterized as follows : 20 males and three females ( mean age 6311 years , range 34 - 82 years ) . a complete patient history , electrocardiography , echocardiography , and recent laboratory tests were available for all the patients . among these 23 patients , six patients ( 5 ami and 1 cmi ) underwent tl single photon emission computed tomography ( spect ) . catheter angiography was performed on 19 patients ( 17 ami and 2 cmi ) for diagnosis and percutaneous coronary intervention . acute myocardial infarction was diagnosed if the patient had 1 ) clinical symptoms , 2 ) electrocardiographic changes indicative of mi , and 3 ) characteristic elevation of the serum enzyme levels . cmi was diagnosed if the patient had 1 ) a history of previous mi , 2 ) diagnostic q waves on the electrocardiogram or ecg registration of the acute event , and 3 ) normal enzyme levels during the observation period . the patients with st segment elevation mi ( n = 10 ) were treated with thrombolytics before their ct scans . the average time between two - phase contrast - enhanced mdct and catheter angiography was 43 days . patients with a pre - scan hr65 beats per minute ( bpm ) were given 25 - 50 mg of atenolol ( tenormin , hyundai ) per os 1 h before the scan . two - phase contrast - enhanced ecg - gated mdct was performed using a sensation 16 scanner ( siemens , forchheim , germany ) . the acquisition protocol included both early - phase acquisition for analysis of the coronary arteries and myocardial perfusion of the left ventricle , and late - phase acquisition that was performed 7 minutes after this first - pass acquisition for analysis of the delayed enhancement patterns . the following parameters were employed : 160.75 mm collimation , 0.37s rotation time , table feed 6.8 mm / rotation , 120 kv , and 620 mas , resulting in a total scan time of about 20s to cover the entire heart , and the scan was acquired a single breath hold with using retrospective ecg gating . a contrast injection protocol with injection of 100 ml of non - ionic contrast material ( ultravist 370 , schering , berlin , germany ) at a flow rate of 3.5 ml / s followed by a saline chaser bolus of 50 ml at the same flow rate was used . ct scanning started with real - time bolus tracking ( care bolus , siemens , forchheim , germany ) using a region of interest in the ascending aorta for monitoring a threshold of + 100 hu above the baseline attenuation . a late - phase acquisition was obtained using the same protocol , except for reducing the mas to 310 . mdct evaluation was done on a workstation ( syngo , wizard ; simens medical solutions ) . mdct coronary angiography was used for detecting coronary stenosis in four cmi patients who did not have catheter angiography performed . a series of maximum intensity projection images with a 4-mm thick slab were generated in the short - axis , horizontal long - axis and vertical long - axis plane of the heart from early - phase axial images . the presence of significant coronary stenosis was visually defined as more than 50% luminal narrowing . the contrast enhancement patterns were assessed with the multiplanar vertical long - axis and shot - axis views by using 5-mm - thick slab sections of the heart from the early and late - phase axial images taken at mid - diastole . the two - phase multiplanar reformation ( mpr ) images of each patient were analyzed by two experienced radiologists in consensus and unaware of any clinical information and the results of both the catheter and mdct coronary angiography . assessment of myocardial enhancement patterns was based on those used in a previous study with using two - phase contrast - enhanced mdct ( 14 ) and as follows : the presence of a hypoenhanced area on the early - phase images as an early perfusion defect , the presence of persistent hypoenhanced area in the subendocardium surrounded by partially enhanced myocardium on the latephase images as a residual perfusion defect , and the presence of a hyperenhanced area on the late - phase images as late enhancement . regions of interest of 2 - 5 mm in size were placed over the center of the area of each finding and the surrounding normal myocardium . for all the perfusion defects and delayed enhancement detected on the two - phase mpr images , the location of each detected mi within the left ventricular myocardium was defined with using a 17-segment model in the cardiac short axis , as recently suggested by the american heart association ( aha ) ( 16 ) . the depth of myocardial involvement was classified into three regions as subendocardial : involvement of less than 1/2 of the left inner ventricle wall thickness , subepicardial : involvement of less than 1/2 of the outer wall thickness , and transmural : involvement of more than 50% of the wall thickness . the typical window width and level settings ranged between 150 - 350 and 60 - 200 hu , respectively . the presence , location , and patterns of myocardial enhancement on two - phase mdct images were compared with infarcted myocardial territories determined by using electrocardiogram , echocardiography , tl spect , catheter and mdct coronary angiography . comparison of the ct attenuation numbers of the normal and abnormally enhanced myocardial segments on the two - phase mdct images were done with using paired student 's t - tests . a p value of 0.05 or less was considered to indicate a statistically significant difference . for statistical analysis , a commercially available windows - based software product was used ( spss 12.0.1 , 2003 ) . fifteen of 23 ( 65% ) patients received a beta - blocker regimen before their scan ( 5 patients had an hr < 65 bpm and 3 patients had a blood pressure < 100/65 mmhg ) . beta - blocker ( 25 - 50 mg of atenolol ) decreased the hr 12 bpm in our study . of the 23 patients who were analyzed , 21 had significant coronary stenoses on catheter or mdct coronary angiography . by clinical assessment , a total of 27 infarct territories ( 19 ami and 8 cmi ) were identified in 23 patients . in the 19 patients who underwent catheter angiography , 21 infarcted myocardial territories were clinically identified and 17 infarct - related arteries were detected . ten of these infarct - related arteries were in the left anterior descending coronary artery ( lad ) , two in the left circumflex coronary artery ( lcx ) and five in the right coronary artery ( rca ) . two patients with ami had double anterior and inferior mis in which the infarct - related arteries were assigned to the lad because each rca was totally occluded along with the presence of collaterals from the lad . two infarcted territories ( 1 lad and 1 lcx ) that were not associated with significant coronary stenoses were noted in two patients with reperfused ami . all 23 patients showed 30 hypoenhanced territories of the left ventricle myocardium on the early - phase mdct scan . twenty - seven of 30 territories corresponded to clinically assessed mi territories ( table 1 ) . in three ( 1 lad and 2 lcx ) of 30 territories , the areas of decreased myocardial attenuation were not corresponded to infarcted myocardial territories and this discrepancy was caused by artifacts that were seen as transverse dark bands crossing the left ventricle on the short - axis images . early perfusion defects were noted in the 19 territories of 17 patients with ami ( figs . 2a , 3a , 4a ) and 8 territories of six patients with cmi ( fig . the attenuation of the early perfusion defects ( 40.3 hu 13.2 ) was significantly lower than that of the noninfarcted areas ( 120.3 hu12.8 , p < 0.001 ) . among the 27 territories of the early perfusion defects , a total of 25 of the 27 ( 93% ) territories with early perfusion defects were consistent with the territories of significant coronary stenoses , as observed on the catheter or mdct coronary angiography . delayed enhancement was observed in 16 of 17 ( 94 % ) ami patients and in all six ( 100 % ) cmi patients . a total of 25 territories ( 17 ami and 8 cmi ) with delayed enhancement were identified on the mdct scans , which corresponded to clinically assessed infarct territories . late enhancement ( n = 19 , 98.8 hu10.1 ) had higher attenuation compared with the residual perfusion defects ( n = 12 , 40.5 hu11.7 , p < 0.001 ) and the remote noninfarcted areas ( 80.5 hu5.7 ; p < 0.001 ) that were observed on the late - phase images . two territories ( lad and rca ) with subendocardial perfusion defects in one ami patient did not show delayed enhancement , but they showed fixed perfusion defects on tl spect . the delayed enhancement patterns were variable ( table 3 ) : 1 ) five territories of subendocardial late enhancement ( fig . 1b ) , 2 ) eight territories of transmural late enhancement ( figs . 2b , 4b ) , 3 ) six territories of subendocardial residual perfusion defect , 4 ) six territories of subendocardial residual perfusion defect with subepicardial late enhancement ( fig . 3b ) . for the association of the delayed enhancement patterns with the depth of myocardial involvement of the early perfusion defect , the subendocardial perfusion defects ( n = 15)showed various delayed enhancement patterns , but the transmural perfusion defects ( n = 12 ) mainly showed transmural late enhancement ( n = 5 ) and subendocardial residual defect with subepicardial late enhancement ( n = 5 ) ( table 4 ) . single - slice nonspiral ct has not been widely used for evaluating mi because of the low temporal and spatial resolution even though this modality was able to detect and characterize mi in both animal models and human subjects ( 4 - 6 ) . electron beam ct could assess regional myocardial perfusion , non - invasively detect coronary artery disease , and assess the risk of future coronary events by quantifying calcification of the coronary arteries . but this modality failed to gain popularity because of its high cost , limited availability , high radiation exposure and the limited extra information compared to the other established imaging modalities ( 17 - 19 ) . in recent years , mdct has been proposed as a useful non - invasive imaging method for evaluating both coronary artery stenosis and the cardiac morphology at the same time . in addition , mdct can provide information about myocardial perfusion because mdct coronary angiographic scanning is performed during maximal enhancement of the coronary artery . ( 14 , 15 ) reported that the early perfusion defects seen on two - phase contrast - enhanced mdct might reflect a decrease in the myocardial blood flow , and so this might correspond to myocardial necrosis with extensive capillary disorder , or to myocardial necrosis in those patients with reperfused ami . on the cardiac mr image , early perfusion defect indicates myocardial microcirculatory occlusion and edema in an area of mi ( 20 - 24 ) . in our study , all 23 patients showed 30 hypoenhanced territories of the left ventricle myocardium on the early - phase mdct scan . in 3 of the 30 territories , these artifacts were seen as transverse dark bands crossing the left ventricle on short - axis images and they might be associated with arrythmia and higher hr . two territories with perfusion defects were noted in two patients with reperfused ami who did not have a significant coronary stenosis ; this possibly corresponded to resolution of thrombus in the coronary artery . in one study using an animal model to evaluate the usefulness of mdct for reperfused mi , the areas showing hyperenhancement on the delayed ct scans were significantly correlated with the histopathologic results ( 11 ) . on mr studies , delayed hyperenhancement obtained within 5 to 10 minutes after injection of contrast medium mainly reflects the volume of the interstitial space and this correlates with the localization and extent of the myocardial infarction . persistent hypoenhancement is caused by microvascular obstruction and this is associated with non - viability ( 2 , 23 , 25 - 28 ) . ( 14 , 15 ) classified patients with reperfused ami into 3 groups according to the myocardial enhancement patterns with using two - phase contrast - enhanced mdct . in group 3 that had early perfusion defect , residual defect and late enhancement , the wall thickness of the chronic infarcted stage was significantly thinner than that of the acute infarcted stage , and the deterioration of left ventricular function was more severe than that observed in group 2 , which did not have residual defect . that study 's results demonstrated that the variability in the enhancement patterns indicated variability in the extent of microvascular damage , and so assessment of myocardial enhancement patterns could serve as a predictor of the changes in wall motion and thickness , the left ventricular function , and the myocardial viability in patients with reperfused ami . in our study , transmural early perfusion defects were closely associated with transmural late enhancement and subendocardial residual defect with subepicardial late enhancement . delayed enhancement was not present in 2 of the 19 territories of early perfusion defects in the ami patients . the false negative results on the late - phase mdct images were probably due to weak contrast between the infarcted myocardium and the noninfarcted myocardium . residual defects are usually observed in large areas of infarcted myocardium as smaller dark subendocardial regions surrounded by a partially hyperenhanced zone and might correspond to myocardial necrosis with extensive capillary damage . among the 12 territories of the subendocardial residual defects , 6 territories were accompanied with subepicardial late enhancement but the other 6 territories were not . the difference between these enhancement patterns was possibly due to the poor tissue contrast resolution that was insufficient to differentiate the subtle differences in hounsfield units and an increase in the noise resulted from decreasing the mas to 310 . the time after injecting the contrast medium may be important for the detecting delayed enhancement . we adopted a standard delay of 7 minutes after injection , based on previous studies ( 14 , 15 ) . in an animal model , even though the contrast between the infarcted myocardium and the normal myocardium was equally prominent at both 5 min and 10 min on the delayed - phase mdct images ( 11 ) , a different time interval between the injection of contrast medium and mdct acquisition may cause different outcomes , and further studies are needed to determine the optimal delay time . paul et al . ( 29 ) lowered the tube voltage to 80 kv for delayed acquisition and they obtained better contrast enhancement on the delayed contrast - enhanced mdct and decreased radiation exposure to the patients . also , the noise was reduced by using 8-mm - thick mprs , and making consensual assessment of myocardial enhancement was possible in all cases . the delayed enhancement technique of mdct technique requires a second scan that increases the radiation dose to the patient and prolongs the examination times . this is why two - phase mdct has been not commonly used for assessing mi . in our study , the effective radiation dose for both studies was around 20 msv even though we lowered the mas to 310 for the delayed acquisition . when one considers the important information obtained from the delayed enhancement technique , particularly if the functional imaging techniques are not available , the radiation dose could be acceptable for ami patients . the standard reference modalities for the assessment of myocardial perfusion and delayed enhancement were in short . comparing the two - phase contrast - enhanced mdct with cardiac mr imaging or nuclear imaging techniques would be of great interest . we had lowered the mas to 310 for the late - phase acquisition to reduce the radiation dose , which caused an increase in noise . in our study population , with 15 of 23 ( 65% ) patients receiving a beta - blocker regimen before the scan , the mean hr was 67.76.6 bpm and it ranged from 57 to 78 bpm . a higher hr ( > 70 bpm ) might cause cardiac motion artifacts that hamper the evaluation of abnormal myocardial enhancement . interobserver variability was not assessed because the weak contrast resolution , the increase in noise , and the artifacts may have caused difficulty in assessing the myocardial enhancement patterns . finally , the number of patients investigated in this study was rather small . in conclusion , myocardial infarction showed early perfusion defect , late enhancement , residual perfusion defect , or subendocardial residual defect with subepicardial late enhancement on two - phase contrast - enhanced mdct . the delayed enhancement imaging of mdct could provide additional information on the location and residual perfusion defect of the infarcted myocardium , and it could be useful when planning appropriate therapeutic strategies for patients with ami .

objectivethe purpose of this study was to describe the myocardial enhancement patterns in patients with myocardial infarction using two - phase contrast - enhanced multidetector - row computed tomography ( mdct).materials and methodstwenty - three patients with clinically proven myocardial infarction ( 17 acute myocardial infarction [ ami ] and 6 chronic myocardial infarction [ cmi ] ) were examined with two - phase contrast - enhanced ecg - gated mdct . the presence , location , and patterns of myocardial enhancement on two - phase mdct images were compared with infarcted myocardial territories determined by using electrocardiogram , echocardiography , thallium-201 single photon emission computed tomography , catheter and mdct coronary angiography.resultsafter clinical assessment , the presence of myocardial infarctions were found in 27 territories ( 19 ami and 8 cmi ) of 23 patients . early perfusion defects were observed in 30 territories of all 23 patients . three territories not corresponding to a myocardial infarction were detected in three patients with ami and were associated with artifacts . fourteen of perfusion defects were in the left anterior descending artery territory , four in the left circumflex artery territory , and nine in the right coronary artery territory . delayed enhancement was observed in 25 territories ( 17 ami and 8 cmi ) of 21 patients . delayed enhancement patterns were variable . transmural early perfusion defects ( n = 12 ) were closely associated with transmural late enhancement ( n = 5 ) and subendocardial residual defect with subepicardial late enhancement ( n = 5).conclusionmyocardial infarction showed early perfusion defects and variable delayed enhancement patterns on two - phase contrast - enhanced mdct . delayed enhancement technique of mdct could provide additional information of the location and extent of infarcted myocardium , and could be useful to plan appropriate therapeutic strategies in patients with ami .

MATERIALS AND METHODS Patient Population and Clinical Assessment Two-Phase Contrast-Enhanced MDCT Technique and Evaluation Data and Statistical Analysis RESULTS DISCUSSION

thirty - eight patients who had undergone two - phase contrast - enhanced ecg - gated mdct for ischemic heart disease between october 2004 and june 2005 were retrospectively identified . among these 38 patients , there were 17 patients with acute myocardial infarction ( ami : infarction occurring less than one week before the ct study ) and six patients with chronic myocardial infarction ( cmi : infarction occurring more than one month before the ct study ) . a complete patient history , electrocardiography , echocardiography , and recent laboratory tests were available for all the patients . among these 23 patients , six patients ( 5 ami and 1 cmi ) underwent tl single photon emission computed tomography ( spect ) . catheter angiography was performed on 19 patients ( 17 ami and 2 cmi ) for diagnosis and percutaneous coronary intervention . acute myocardial infarction was diagnosed if the patient had 1 ) clinical symptoms , 2 ) electrocardiographic changes indicative of mi , and 3 ) characteristic elevation of the serum enzyme levels . cmi was diagnosed if the patient had 1 ) a history of previous mi , 2 ) diagnostic q waves on the electrocardiogram or ecg registration of the acute event , and 3 ) normal enzyme levels during the observation period . the patients with st segment elevation mi ( n = 10 ) were treated with thrombolytics before their ct scans . the average time between two - phase contrast - enhanced mdct and catheter angiography was 43 days . patients with a pre - scan hr65 beats per minute ( bpm ) were given 25 - 50 mg of atenolol ( tenormin , hyundai ) per os 1 h before the scan . two - phase contrast - enhanced ecg - gated mdct was performed using a sensation 16 scanner ( siemens , forchheim , germany ) . the acquisition protocol included both early - phase acquisition for analysis of the coronary arteries and myocardial perfusion of the left ventricle , and late - phase acquisition that was performed 7 minutes after this first - pass acquisition for analysis of the delayed enhancement patterns . mdct coronary angiography was used for detecting coronary stenosis in four cmi patients who did not have catheter angiography performed . a series of maximum intensity projection images with a 4-mm thick slab were generated in the short - axis , horizontal long - axis and vertical long - axis plane of the heart from early - phase axial images . the presence of significant coronary stenosis was visually defined as more than 50% luminal narrowing . the contrast enhancement patterns were assessed with the multiplanar vertical long - axis and shot - axis views by using 5-mm - thick slab sections of the heart from the early and late - phase axial images taken at mid - diastole . the two - phase multiplanar reformation ( mpr ) images of each patient were analyzed by two experienced radiologists in consensus and unaware of any clinical information and the results of both the catheter and mdct coronary angiography . assessment of myocardial enhancement patterns was based on those used in a previous study with using two - phase contrast - enhanced mdct ( 14 ) and as follows : the presence of a hypoenhanced area on the early - phase images as an early perfusion defect , the presence of persistent hypoenhanced area in the subendocardium surrounded by partially enhanced myocardium on the latephase images as a residual perfusion defect , and the presence of a hyperenhanced area on the late - phase images as late enhancement . for all the perfusion defects and delayed enhancement detected on the two - phase mpr images , the location of each detected mi within the left ventricular myocardium was defined with using a 17-segment model in the cardiac short axis , as recently suggested by the american heart association ( aha ) ( 16 ) . the depth of myocardial involvement was classified into three regions as subendocardial : involvement of less than 1/2 of the left inner ventricle wall thickness , subepicardial : involvement of less than 1/2 of the outer wall thickness , and transmural : involvement of more than 50% of the wall thickness . the presence , location , and patterns of myocardial enhancement on two - phase mdct images were compared with infarcted myocardial territories determined by using electrocardiogram , echocardiography , tl spect , catheter and mdct coronary angiography . comparison of the ct attenuation numbers of the normal and abnormally enhanced myocardial segments on the two - phase mdct images were done with using paired student 's t - tests . thirty - eight patients who had undergone two - phase contrast - enhanced ecg - gated mdct for ischemic heart disease between october 2004 and june 2005 were retrospectively identified . among these 38 patients , there were 17 patients with acute myocardial infarction ( ami : infarction occurring less than one week before the ct study ) and six patients with chronic myocardial infarction ( cmi : infarction occurring more than one month before the ct study ) . a complete patient history , electrocardiography , echocardiography , and recent laboratory tests were available for all the patients . among these 23 patients , six patients ( 5 ami and 1 cmi ) underwent tl single photon emission computed tomography ( spect ) . catheter angiography was performed on 19 patients ( 17 ami and 2 cmi ) for diagnosis and percutaneous coronary intervention . acute myocardial infarction was diagnosed if the patient had 1 ) clinical symptoms , 2 ) electrocardiographic changes indicative of mi , and 3 ) characteristic elevation of the serum enzyme levels . cmi was diagnosed if the patient had 1 ) a history of previous mi , 2 ) diagnostic q waves on the electrocardiogram or ecg registration of the acute event , and 3 ) normal enzyme levels during the observation period . the patients with st segment elevation mi ( n = 10 ) were treated with thrombolytics before their ct scans . the average time between two - phase contrast - enhanced mdct and catheter angiography was 43 days . patients with a pre - scan hr65 beats per minute ( bpm ) were given 25 - 50 mg of atenolol ( tenormin , hyundai ) per os 1 h before the scan . two - phase contrast - enhanced ecg - gated mdct was performed using a sensation 16 scanner ( siemens , forchheim , germany ) . the acquisition protocol included both early - phase acquisition for analysis of the coronary arteries and myocardial perfusion of the left ventricle , and late - phase acquisition that was performed 7 minutes after this first - pass acquisition for analysis of the delayed enhancement patterns . mdct coronary angiography was used for detecting coronary stenosis in four cmi patients who did not have catheter angiography performed . a series of maximum intensity projection images with a 4-mm thick slab were generated in the short - axis , horizontal long - axis and vertical long - axis plane of the heart from early - phase axial images . the presence of significant coronary stenosis was visually defined as more than 50% luminal narrowing . the contrast enhancement patterns were assessed with the multiplanar vertical long - axis and shot - axis views by using 5-mm - thick slab sections of the heart from the early and late - phase axial images taken at mid - diastole . the two - phase multiplanar reformation ( mpr ) images of each patient were analyzed by two experienced radiologists in consensus and unaware of any clinical information and the results of both the catheter and mdct coronary angiography . assessment of myocardial enhancement patterns was based on those used in a previous study with using two - phase contrast - enhanced mdct ( 14 ) and as follows : the presence of a hypoenhanced area on the early - phase images as an early perfusion defect , the presence of persistent hypoenhanced area in the subendocardium surrounded by partially enhanced myocardium on the latephase images as a residual perfusion defect , and the presence of a hyperenhanced area on the late - phase images as late enhancement . for all the perfusion defects and delayed enhancement detected on the two - phase mpr images , the location of each detected mi within the left ventricular myocardium was defined with using a 17-segment model in the cardiac short axis , as recently suggested by the american heart association ( aha ) ( 16 ) . the depth of myocardial involvement was classified into three regions as subendocardial : involvement of less than 1/2 of the left inner ventricle wall thickness , subepicardial : involvement of less than 1/2 of the outer wall thickness , and transmural : involvement of more than 50% of the wall thickness . the presence , location , and patterns of myocardial enhancement on two - phase mdct images were compared with infarcted myocardial territories determined by using electrocardiogram , echocardiography , tl spect , catheter and mdct coronary angiography . comparison of the ct attenuation numbers of the normal and abnormally enhanced myocardial segments on the two - phase mdct images were done with using paired student 's t - tests . of the 23 patients who were analyzed , 21 had significant coronary stenoses on catheter or mdct coronary angiography . by clinical assessment , a total of 27 infarct territories ( 19 ami and 8 cmi ) were identified in 23 patients . in the 19 patients who underwent catheter angiography , 21 infarcted myocardial territories were clinically identified and 17 infarct - related arteries were detected . ten of these infarct - related arteries were in the left anterior descending coronary artery ( lad ) , two in the left circumflex coronary artery ( lcx ) and five in the right coronary artery ( rca ) . two patients with ami had double anterior and inferior mis in which the infarct - related arteries were assigned to the lad because each rca was totally occluded along with the presence of collaterals from the lad . two infarcted territories ( 1 lad and 1 lcx ) that were not associated with significant coronary stenoses were noted in two patients with reperfused ami . all 23 patients showed 30 hypoenhanced territories of the left ventricle myocardium on the early - phase mdct scan . twenty - seven of 30 territories corresponded to clinically assessed mi territories ( table 1 ) . in three ( 1 lad and 2 lcx ) of 30 territories , the areas of decreased myocardial attenuation were not corresponded to infarcted myocardial territories and this discrepancy was caused by artifacts that were seen as transverse dark bands crossing the left ventricle on the short - axis images . early perfusion defects were noted in the 19 territories of 17 patients with ami ( figs . 2a , 3a , 4a ) and 8 territories of six patients with cmi ( fig . the attenuation of the early perfusion defects ( 40.3 hu 13.2 ) was significantly lower than that of the noninfarcted areas ( 120.3 hu12.8 , p < 0.001 ) . among the 27 territories of the early perfusion defects , a total of 25 of the 27 ( 93% ) territories with early perfusion defects were consistent with the territories of significant coronary stenoses , as observed on the catheter or mdct coronary angiography . delayed enhancement was observed in 16 of 17 ( 94 % ) ami patients and in all six ( 100 % ) cmi patients . a total of 25 territories ( 17 ami and 8 cmi ) with delayed enhancement were identified on the mdct scans , which corresponded to clinically assessed infarct territories . late enhancement ( n = 19 , 98.8 hu10.1 ) had higher attenuation compared with the residual perfusion defects ( n = 12 , 40.5 hu11.7 , p < 0.001 ) and the remote noninfarcted areas ( 80.5 hu5.7 ; p < 0.001 ) that were observed on the late - phase images . two territories ( lad and rca ) with subendocardial perfusion defects in one ami patient did not show delayed enhancement , but they showed fixed perfusion defects on tl spect . the delayed enhancement patterns were variable ( table 3 ) : 1 ) five territories of subendocardial late enhancement ( fig . 1b ) , 2 ) eight territories of transmural late enhancement ( figs . 2b , 4b ) , 3 ) six territories of subendocardial residual perfusion defect , 4 ) six territories of subendocardial residual perfusion defect with subepicardial late enhancement ( fig . for the association of the delayed enhancement patterns with the depth of myocardial involvement of the early perfusion defect , the subendocardial perfusion defects ( n = 15)showed various delayed enhancement patterns , but the transmural perfusion defects ( n = 12 ) mainly showed transmural late enhancement ( n = 5 ) and subendocardial residual defect with subepicardial late enhancement ( n = 5 ) ( table 4 ) . electron beam ct could assess regional myocardial perfusion , non - invasively detect coronary artery disease , and assess the risk of future coronary events by quantifying calcification of the coronary arteries . but this modality failed to gain popularity because of its high cost , limited availability , high radiation exposure and the limited extra information compared to the other established imaging modalities ( 17 - 19 ) . in addition , mdct can provide information about myocardial perfusion because mdct coronary angiographic scanning is performed during maximal enhancement of the coronary artery . ( 14 , 15 ) reported that the early perfusion defects seen on two - phase contrast - enhanced mdct might reflect a decrease in the myocardial blood flow , and so this might correspond to myocardial necrosis with extensive capillary disorder , or to myocardial necrosis in those patients with reperfused ami . in our study , all 23 patients showed 30 hypoenhanced territories of the left ventricle myocardium on the early - phase mdct scan . in 3 of the 30 territories , these artifacts were seen as transverse dark bands crossing the left ventricle on short - axis images and they might be associated with arrythmia and higher hr . two territories with perfusion defects were noted in two patients with reperfused ami who did not have a significant coronary stenosis ; this possibly corresponded to resolution of thrombus in the coronary artery . in one study using an animal model to evaluate the usefulness of mdct for reperfused mi , the areas showing hyperenhancement on the delayed ct scans were significantly correlated with the histopathologic results ( 11 ) . on mr studies , delayed hyperenhancement obtained within 5 to 10 minutes after injection of contrast medium mainly reflects the volume of the interstitial space and this correlates with the localization and extent of the myocardial infarction . ( 14 , 15 ) classified patients with reperfused ami into 3 groups according to the myocardial enhancement patterns with using two - phase contrast - enhanced mdct . in group 3 that had early perfusion defect , residual defect and late enhancement , the wall thickness of the chronic infarcted stage was significantly thinner than that of the acute infarcted stage , and the deterioration of left ventricular function was more severe than that observed in group 2 , which did not have residual defect . that study 's results demonstrated that the variability in the enhancement patterns indicated variability in the extent of microvascular damage , and so assessment of myocardial enhancement patterns could serve as a predictor of the changes in wall motion and thickness , the left ventricular function , and the myocardial viability in patients with reperfused ami . in our study , transmural early perfusion defects were closely associated with transmural late enhancement and subendocardial residual defect with subepicardial late enhancement . delayed enhancement was not present in 2 of the 19 territories of early perfusion defects in the ami patients . the false negative results on the late - phase mdct images were probably due to weak contrast between the infarcted myocardium and the noninfarcted myocardium . residual defects are usually observed in large areas of infarcted myocardium as smaller dark subendocardial regions surrounded by a partially hyperenhanced zone and might correspond to myocardial necrosis with extensive capillary damage . among the 12 territories of the subendocardial residual defects , 6 territories were accompanied with subepicardial late enhancement but the other 6 territories were not . the difference between these enhancement patterns was possibly due to the poor tissue contrast resolution that was insufficient to differentiate the subtle differences in hounsfield units and an increase in the noise resulted from decreasing the mas to 310 . the time after injecting the contrast medium may be important for the detecting delayed enhancement . in an animal model , even though the contrast between the infarcted myocardium and the normal myocardium was equally prominent at both 5 min and 10 min on the delayed - phase mdct images ( 11 ) , a different time interval between the injection of contrast medium and mdct acquisition may cause different outcomes , and further studies are needed to determine the optimal delay time . ( 29 ) lowered the tube voltage to 80 kv for delayed acquisition and they obtained better contrast enhancement on the delayed contrast - enhanced mdct and decreased radiation exposure to the patients . also , the noise was reduced by using 8-mm - thick mprs , and making consensual assessment of myocardial enhancement was possible in all cases . the delayed enhancement technique of mdct technique requires a second scan that increases the radiation dose to the patient and prolongs the examination times . this is why two - phase mdct has been not commonly used for assessing mi . in our study , the effective radiation dose for both studies was around 20 msv even though we lowered the mas to 310 for the delayed acquisition . when one considers the important information obtained from the delayed enhancement technique , particularly if the functional imaging techniques are not available , the radiation dose could be acceptable for ami patients . the standard reference modalities for the assessment of myocardial perfusion and delayed enhancement were in short . comparing the two - phase contrast - enhanced mdct with cardiac mr imaging or nuclear imaging techniques would be of great interest . in our study population , with 15 of 23 ( 65% ) patients receiving a beta - blocker regimen before the scan , the mean hr was 67.76.6 bpm and it ranged from 57 to 78 bpm . interobserver variability was not assessed because the weak contrast resolution , the increase in noise , and the artifacts may have caused difficulty in assessing the myocardial enhancement patterns . finally , the number of patients investigated in this study was rather small . in conclusion , myocardial infarction showed early perfusion defect , late enhancement , residual perfusion defect , or subendocardial residual defect with subepicardial late enhancement on two - phase contrast - enhanced mdct . the delayed enhancement imaging of mdct could provide additional information on the location and residual perfusion defect of the infarcted myocardium , and it could be useful when planning appropriate therapeutic strategies for patients with ami .

thirty - eight patients who had undergone two - phase contrast - enhanced ecg - gated mdct for ischemic heart disease between october 2004 and june 2005 were retrospectively identified . among these 38 patients , there were 17 patients with acute myocardial infarction ( ami : infarction occurring less than one week before the ct study ) and six patients with chronic myocardial infarction ( cmi : infarction occurring more than one month before the ct study ) . the patient population was characterized as follows : 20 males and three females ( mean age 6311 years , range 34 - 82 years ) . among these 23 patients , six patients ( 5 ami and 1 cmi ) underwent tl single photon emission computed tomography ( spect ) . catheter angiography was performed on 19 patients ( 17 ami and 2 cmi ) for diagnosis and percutaneous coronary intervention . acute myocardial infarction was diagnosed if the patient had 1 ) clinical symptoms , 2 ) electrocardiographic changes indicative of mi , and 3 ) characteristic elevation of the serum enzyme levels . cmi was diagnosed if the patient had 1 ) a history of previous mi , 2 ) diagnostic q waves on the electrocardiogram or ecg registration of the acute event , and 3 ) normal enzyme levels during the observation period . the patients with st segment elevation mi ( n = 10 ) were treated with thrombolytics before their ct scans . patients with a pre - scan hr65 beats per minute ( bpm ) were given 25 - 50 mg of atenolol ( tenormin , hyundai ) per os 1 h before the scan . the acquisition protocol included both early - phase acquisition for analysis of the coronary arteries and myocardial perfusion of the left ventricle , and late - phase acquisition that was performed 7 minutes after this first - pass acquisition for analysis of the delayed enhancement patterns . the following parameters were employed : 160.75 mm collimation , 0.37s rotation time , table feed 6.8 mm / rotation , 120 kv , and 620 mas , resulting in a total scan time of about 20s to cover the entire heart , and the scan was acquired a single breath hold with using retrospective ecg gating . a contrast injection protocol with injection of 100 ml of non - ionic contrast material ( ultravist 370 , schering , berlin , germany ) at a flow rate of 3.5 ml / s followed by a saline chaser bolus of 50 ml at the same flow rate was used . ct scanning started with real - time bolus tracking ( care bolus , siemens , forchheim , germany ) using a region of interest in the ascending aorta for monitoring a threshold of + 100 hu above the baseline attenuation . mdct coronary angiography was used for detecting coronary stenosis in four cmi patients who did not have catheter angiography performed . a series of maximum intensity projection images with a 4-mm thick slab were generated in the short - axis , horizontal long - axis and vertical long - axis plane of the heart from early - phase axial images . the contrast enhancement patterns were assessed with the multiplanar vertical long - axis and shot - axis views by using 5-mm - thick slab sections of the heart from the early and late - phase axial images taken at mid - diastole . the two - phase multiplanar reformation ( mpr ) images of each patient were analyzed by two experienced radiologists in consensus and unaware of any clinical information and the results of both the catheter and mdct coronary angiography . assessment of myocardial enhancement patterns was based on those used in a previous study with using two - phase contrast - enhanced mdct ( 14 ) and as follows : the presence of a hypoenhanced area on the early - phase images as an early perfusion defect , the presence of persistent hypoenhanced area in the subendocardium surrounded by partially enhanced myocardium on the latephase images as a residual perfusion defect , and the presence of a hyperenhanced area on the late - phase images as late enhancement . regions of interest of 2 - 5 mm in size were placed over the center of the area of each finding and the surrounding normal myocardium . for all the perfusion defects and delayed enhancement detected on the two - phase mpr images , the location of each detected mi within the left ventricular myocardium was defined with using a 17-segment model in the cardiac short axis , as recently suggested by the american heart association ( aha ) ( 16 ) . the depth of myocardial involvement was classified into three regions as subendocardial : involvement of less than 1/2 of the left inner ventricle wall thickness , subepicardial : involvement of less than 1/2 of the outer wall thickness , and transmural : involvement of more than 50% of the wall thickness . the presence , location , and patterns of myocardial enhancement on two - phase mdct images were compared with infarcted myocardial territories determined by using electrocardiogram , echocardiography , tl spect , catheter and mdct coronary angiography . comparison of the ct attenuation numbers of the normal and abnormally enhanced myocardial segments on the two - phase mdct images were done with using paired student 's t - tests . thirty - eight patients who had undergone two - phase contrast - enhanced ecg - gated mdct for ischemic heart disease between october 2004 and june 2005 were retrospectively identified . among these 38 patients , there were 17 patients with acute myocardial infarction ( ami : infarction occurring less than one week before the ct study ) and six patients with chronic myocardial infarction ( cmi : infarction occurring more than one month before the ct study ) . the patient population was characterized as follows : 20 males and three females ( mean age 6311 years , range 34 - 82 years ) . among these 23 patients , six patients ( 5 ami and 1 cmi ) underwent tl single photon emission computed tomography ( spect ) . catheter angiography was performed on 19 patients ( 17 ami and 2 cmi ) for diagnosis and percutaneous coronary intervention . acute myocardial infarction was diagnosed if the patient had 1 ) clinical symptoms , 2 ) electrocardiographic changes indicative of mi , and 3 ) characteristic elevation of the serum enzyme levels . cmi was diagnosed if the patient had 1 ) a history of previous mi , 2 ) diagnostic q waves on the electrocardiogram or ecg registration of the acute event , and 3 ) normal enzyme levels during the observation period . patients with a pre - scan hr65 beats per minute ( bpm ) were given 25 - 50 mg of atenolol ( tenormin , hyundai ) per os 1 h before the scan . the acquisition protocol included both early - phase acquisition for analysis of the coronary arteries and myocardial perfusion of the left ventricle , and late - phase acquisition that was performed 7 minutes after this first - pass acquisition for analysis of the delayed enhancement patterns . the following parameters were employed : 160.75 mm collimation , 0.37s rotation time , table feed 6.8 mm / rotation , 120 kv , and 620 mas , resulting in a total scan time of about 20s to cover the entire heart , and the scan was acquired a single breath hold with using retrospective ecg gating . a contrast injection protocol with injection of 100 ml of non - ionic contrast material ( ultravist 370 , schering , berlin , germany ) at a flow rate of 3.5 ml / s followed by a saline chaser bolus of 50 ml at the same flow rate was used . ct scanning started with real - time bolus tracking ( care bolus , siemens , forchheim , germany ) using a region of interest in the ascending aorta for monitoring a threshold of + 100 hu above the baseline attenuation . mdct coronary angiography was used for detecting coronary stenosis in four cmi patients who did not have catheter angiography performed . a series of maximum intensity projection images with a 4-mm thick slab were generated in the short - axis , horizontal long - axis and vertical long - axis plane of the heart from early - phase axial images . the contrast enhancement patterns were assessed with the multiplanar vertical long - axis and shot - axis views by using 5-mm - thick slab sections of the heart from the early and late - phase axial images taken at mid - diastole . the two - phase multiplanar reformation ( mpr ) images of each patient were analyzed by two experienced radiologists in consensus and unaware of any clinical information and the results of both the catheter and mdct coronary angiography . assessment of myocardial enhancement patterns was based on those used in a previous study with using two - phase contrast - enhanced mdct ( 14 ) and as follows : the presence of a hypoenhanced area on the early - phase images as an early perfusion defect , the presence of persistent hypoenhanced area in the subendocardium surrounded by partially enhanced myocardium on the latephase images as a residual perfusion defect , and the presence of a hyperenhanced area on the late - phase images as late enhancement . regions of interest of 2 - 5 mm in size were placed over the center of the area of each finding and the surrounding normal myocardium . for all the perfusion defects and delayed enhancement detected on the two - phase mpr images , the location of each detected mi within the left ventricular myocardium was defined with using a 17-segment model in the cardiac short axis , as recently suggested by the american heart association ( aha ) ( 16 ) . the depth of myocardial involvement was classified into three regions as subendocardial : involvement of less than 1/2 of the left inner ventricle wall thickness , subepicardial : involvement of less than 1/2 of the outer wall thickness , and transmural : involvement of more than 50% of the wall thickness . the presence , location , and patterns of myocardial enhancement on two - phase mdct images were compared with infarcted myocardial territories determined by using electrocardiogram , echocardiography , tl spect , catheter and mdct coronary angiography . comparison of the ct attenuation numbers of the normal and abnormally enhanced myocardial segments on the two - phase mdct images were done with using paired student 's t - tests . fifteen of 23 ( 65% ) patients received a beta - blocker regimen before their scan ( 5 patients had an hr < 65 bpm and 3 patients had a blood pressure < 100/65 mmhg ) . beta - blocker ( 25 - 50 mg of atenolol ) decreased the hr 12 bpm in our study . by clinical assessment , a total of 27 infarct territories ( 19 ami and 8 cmi ) were identified in 23 patients . in the 19 patients who underwent catheter angiography , 21 infarcted myocardial territories were clinically identified and 17 infarct - related arteries were detected . ten of these infarct - related arteries were in the left anterior descending coronary artery ( lad ) , two in the left circumflex coronary artery ( lcx ) and five in the right coronary artery ( rca ) . two patients with ami had double anterior and inferior mis in which the infarct - related arteries were assigned to the lad because each rca was totally occluded along with the presence of collaterals from the lad . two infarcted territories ( 1 lad and 1 lcx ) that were not associated with significant coronary stenoses were noted in two patients with reperfused ami . in three ( 1 lad and 2 lcx ) of 30 territories , the areas of decreased myocardial attenuation were not corresponded to infarcted myocardial territories and this discrepancy was caused by artifacts that were seen as transverse dark bands crossing the left ventricle on the short - axis images . early perfusion defects were noted in the 19 territories of 17 patients with ami ( figs . the attenuation of the early perfusion defects ( 40.3 hu 13.2 ) was significantly lower than that of the noninfarcted areas ( 120.3 hu12.8 , p < 0.001 ) . among the 27 territories of the early perfusion defects , a total of 25 of the 27 ( 93% ) territories with early perfusion defects were consistent with the territories of significant coronary stenoses , as observed on the catheter or mdct coronary angiography . delayed enhancement was observed in 16 of 17 ( 94 % ) ami patients and in all six ( 100 % ) cmi patients . a total of 25 territories ( 17 ami and 8 cmi ) with delayed enhancement were identified on the mdct scans , which corresponded to clinically assessed infarct territories . late enhancement ( n = 19 , 98.8 hu10.1 ) had higher attenuation compared with the residual perfusion defects ( n = 12 , 40.5 hu11.7 , p < 0.001 ) and the remote noninfarcted areas ( 80.5 hu5.7 ; p < 0.001 ) that were observed on the late - phase images . two territories ( lad and rca ) with subendocardial perfusion defects in one ami patient did not show delayed enhancement , but they showed fixed perfusion defects on tl spect . the delayed enhancement patterns were variable ( table 3 ) : 1 ) five territories of subendocardial late enhancement ( fig . 2b , 4b ) , 3 ) six territories of subendocardial residual perfusion defect , 4 ) six territories of subendocardial residual perfusion defect with subepicardial late enhancement ( fig . for the association of the delayed enhancement patterns with the depth of myocardial involvement of the early perfusion defect , the subendocardial perfusion defects ( n = 15)showed various delayed enhancement patterns , but the transmural perfusion defects ( n = 12 ) mainly showed transmural late enhancement ( n = 5 ) and subendocardial residual defect with subepicardial late enhancement ( n = 5 ) ( table 4 ) . single - slice nonspiral ct has not been widely used for evaluating mi because of the low temporal and spatial resolution even though this modality was able to detect and characterize mi in both animal models and human subjects ( 4 - 6 ) . electron beam ct could assess regional myocardial perfusion , non - invasively detect coronary artery disease , and assess the risk of future coronary events by quantifying calcification of the coronary arteries . but this modality failed to gain popularity because of its high cost , limited availability , high radiation exposure and the limited extra information compared to the other established imaging modalities ( 17 - 19 ) . in recent years , mdct has been proposed as a useful non - invasive imaging method for evaluating both coronary artery stenosis and the cardiac morphology at the same time . ( 14 , 15 ) reported that the early perfusion defects seen on two - phase contrast - enhanced mdct might reflect a decrease in the myocardial blood flow , and so this might correspond to myocardial necrosis with extensive capillary disorder , or to myocardial necrosis in those patients with reperfused ami . on the cardiac mr image , early perfusion defect indicates myocardial microcirculatory occlusion and edema in an area of mi ( 20 - 24 ) . in our study , all 23 patients showed 30 hypoenhanced territories of the left ventricle myocardium on the early - phase mdct scan . in 3 of the 30 territories , these artifacts were seen as transverse dark bands crossing the left ventricle on short - axis images and they might be associated with arrythmia and higher hr . two territories with perfusion defects were noted in two patients with reperfused ami who did not have a significant coronary stenosis ; this possibly corresponded to resolution of thrombus in the coronary artery . in one study using an animal model to evaluate the usefulness of mdct for reperfused mi , the areas showing hyperenhancement on the delayed ct scans were significantly correlated with the histopathologic results ( 11 ) . on mr studies , delayed hyperenhancement obtained within 5 to 10 minutes after injection of contrast medium mainly reflects the volume of the interstitial space and this correlates with the localization and extent of the myocardial infarction . in group 3 that had early perfusion defect , residual defect and late enhancement , the wall thickness of the chronic infarcted stage was significantly thinner than that of the acute infarcted stage , and the deterioration of left ventricular function was more severe than that observed in group 2 , which did not have residual defect . that study 's results demonstrated that the variability in the enhancement patterns indicated variability in the extent of microvascular damage , and so assessment of myocardial enhancement patterns could serve as a predictor of the changes in wall motion and thickness , the left ventricular function , and the myocardial viability in patients with reperfused ami . in our study , transmural early perfusion defects were closely associated with transmural late enhancement and subendocardial residual defect with subepicardial late enhancement . delayed enhancement was not present in 2 of the 19 territories of early perfusion defects in the ami patients . among the 12 territories of the subendocardial residual defects , 6 territories were accompanied with subepicardial late enhancement but the other 6 territories were not . the difference between these enhancement patterns was possibly due to the poor tissue contrast resolution that was insufficient to differentiate the subtle differences in hounsfield units and an increase in the noise resulted from decreasing the mas to 310 . in an animal model , even though the contrast between the infarcted myocardium and the normal myocardium was equally prominent at both 5 min and 10 min on the delayed - phase mdct images ( 11 ) , a different time interval between the injection of contrast medium and mdct acquisition may cause different outcomes , and further studies are needed to determine the optimal delay time . ( 29 ) lowered the tube voltage to 80 kv for delayed acquisition and they obtained better contrast enhancement on the delayed contrast - enhanced mdct and decreased radiation exposure to the patients . in our study , the effective radiation dose for both studies was around 20 msv even though we lowered the mas to 310 for the delayed acquisition . when one considers the important information obtained from the delayed enhancement technique , particularly if the functional imaging techniques are not available , the radiation dose could be acceptable for ami patients . in our study population , with 15 of 23 ( 65% ) patients receiving a beta - blocker regimen before the scan , the mean hr was 67.76.6 bpm and it ranged from 57 to 78 bpm . in conclusion , myocardial infarction showed early perfusion defect , late enhancement , residual perfusion defect , or subendocardial residual defect with subepicardial late enhancement on two - phase contrast - enhanced mdct . the delayed enhancement imaging of mdct could provide additional information on the location and residual perfusion defect of the infarcted myocardium , and it could be useful when planning appropriate therapeutic strategies for patients with ami .

the innate immune response is the first line of defense against microbial pathogens such as viruses , bacteria , fungi , and protozoa . a critical component of the innate immune response is the nfb family of transcription factors . the toll - like receptors ( tlrs ) are critical components of the innate immune system that regulate nfb activation . in general , the tlrs recognize macromolecules that are associated with pathogens and with cell stress . these pathogen - associated molecular patterns ( pamps ) and their corresponding tlrs include : lipopeptides ( tlr2 ) , double - stranded rna ( tlr3 ) , lipopolysaccharide ( lps , tlr4 ) , bacterial flagellin ( tlr5 ) , guanine and uridine - rich single - stranded rna ( tlr7 , 8) , and hypo - methylated cpg rich dna ( tlr9 ) . innate immune cells use pattern recognition receptors ( prrs ) such as tlrs to promote a rapid response to perceived threats before pathogen - specific adaptive immunity can be established . indeed , this rapid response , involving multiple components of the innate immune system , has been recognized to guide the type of adaptive immune response that is most effective for the specific pathogenic threat . the discovery that certain small molecules could serve as specific innate immune receptor ligands has expanded the possibility of designing drugs that may modulate the immune response at its earliest stages . notable examples of such small molecules are the tlr7 agonists imiquimod , isotorabine , and the more recent 8-oxo-9-benzyladenines , as well as the tlr7/8 agonist resiquimod . a useful application of this concept is the incorporation of adjuvants into vaccines . adjuvants are added to antigens in a vaccine setting to provide enhanced response to poorly immunogenic antigens , to increase seroconversion rates in populations with reduced responsiveness due to age ( infants and the elderly ) or disease ( diabetes , renal failure ) , to facilitate the use of smaller doses of antigen , and to permit immunization with fewer doses of vaccine . macromolecular activators of the innate immune system , such as monophosphoryl lipid a ( mpla ) , a tlr4 ligand , are utilized as adjuvants , but development of small molecular weight nonlipid ligands might have several advantages to address different immunological requirements for vaccines or immune therapeutics . as part of our studies on small molecules that can activate tlrs , we conducted a high through - put screening ( hts ) campaign in which a commercially available library of compounds was screened in a human cell - based nfb activation assay . many innate signaling pathways converge on the transcription factor nfb . hence it was used in the primary screen as a broad indicator of small molecule agonism of innate immunity . the specific innate receptor for the lead compounds was determined using genetically modified cells and primary mouse and human blood or bone marrow mononuclear cells . here we report our discovery and initial structure activity relationship ( sar ) studies of the pyrimido[5,4-b]indole class of ligands and their characterization as tlr4/md-2 agonists . a library of compounds was acquired from the university of california , san francisco , small molecule discovery center consisting of about 170000 compounds from eight suppliers ( https://smdc.ucsf.edu ) . the library was screened in three phases at a commercial hts screening facility using the thp-1 human monocytic leukemia cell line , which contained a -lactamase reporter gene under the control of the nfb response element that had been stably integrated into the cells . all screens were performed in activator mode using lps as a positive control , achieving typical z values above 0.75 . the three - phase screening process consisted of ( 1 ) a pilot screen of about 10000 compounds selected as representative of the entire primary library , ( 2 ) the primary screen of the entire library , and ( 3 ) a confirmation screen of about 2000 hits found in the primary screen . an analysis of the cluster enrichment methods for hit selection has been recently reported . following the cluster enrichment analysis , 225 compounds were selected for further in vitro biological evaluation involving cytokine induction assays in primary cells , including human peripheral blood mononuclear cells ( hpbmc ) , mouse splenocytes , mouse bone marrow derived dendritic cells ( mbmdc ) , and mouse bone marrow derived macrophages ( mbmdm ) . these cells were incubated in triplicate , with each of the 225 compounds at a single concentration ( 1 m for splenocytes and 5 m for all other mouse cells and human cells ) , and the supernatants were tested for the presence of nfb dependent cytokines , il-8 or il-6 , released from the human or mouse cells , respectively . thirty - nine of the 225 compounds stimulated the human and mouse cells to secrete il-8 or il-6 above the detectable limit . to further confirm activity , these compounds were repurchased and retested by stimulating hpbmc and mbmdc with titrated doses and assaying for il-8 and il-6 . a few structurally diverse library scaffolds were identified in these cytokine assays as having reproducible responses in mouse and human stimulation assays . among these scaffolds , the pyrimido[5,4-b]indoles emerged as the most potent and diverse class of compounds in the mouse cell assays with clear evidence of sar . within this scaffold cluster , the leading hit from the initial primary and secondary screens was a substituted acetamide attached to the pyrimidoindole ring system through a thioether linkage ( figure 1 ) . we used hek293 cells stably transfected with individual human ( h ) tlrs : tlr2 , tlr3 , tlr4/md-2/cd14 , tlr5 , tlr7 , tlr8 , or tlr9 expressing hek293 cells , along with an nfb activation reporter producing secreted embryonic alkaline phosphatase ( seap ) . among the tested tlr transfected cells , only those expressing tlr4/md-2/cd14 responded to pyrimidoindoles , as shown in figure 2a for compound 1 . because tlr4/md-2/cd14 was the receptor complex for the active compounds in this series , it was important to rule out the possibility that activity might have been caused by lps contamination . therefore , compound 1 ( and all active derivatives ) was assayed for lps ( endotoxin ) levels using a commercially available detection system and found to contain less than 10 endotoxin units ( eu)/mol compound . to further exclude contamination , compound 1 both samples of compound 1 displayed indistinguishable physicochemical and biological properties , indicating that the positive biological activity was not due to lps or another contaminant . full titration curves of the resynthesized compound were performed using the seap assay with mouse and human tlr4 transfected hek293 cell lines , confirming dose - dependent activation ( figure 2b , c ) . target identification of compound 1 using human tlr hek293 reporter cell lines and genetically deficient cells . ( a ) human tlr2 , tlr3 , tlr4/md-2/cd14 , tlr5 , tlr7 , tlr8 , and tlr9 hek 293 blue cells or nf-b / seaporter cells were incubated with compound 1 ( 10 m ) for 2024 h , and activation was evaluated by seap secretion in the culture supernatants by colorimetric assay at od405 . data shown are mean sem of triplicates and representative of two to three independent experiments showing similar results . p < 0.05 was considered significant compared to the vehicle control using student s t test . ( b , c ) mouse ( b ) or human ( c ) tlr4 hek transfectomas were incubated with graded concentrations of compound 1 . tlr4 mediated nfb activation was measured by seap secretion in the culture supernatant . ( d ) wt and tlr4 mbmdc were incubated with compound 1 ( 10 m ) for18 h. il-6 in the culture supernatants was measured by elisa . ( e ) mouse tlr4 transfectoma cells were incubated with 2.5 m compound 1 in the presence or absence of tlr4 antagonist lps - rs ( 12 , 111 , 1000 ng / ml ) . activation of the tlr4/nfb pathway was evaluated by seap secretion in the culture supernatants . * denotes p < 0.05 considered as significant compared to vehicle using one way anova with dunnett s post hoc testing . ( f , g ) wt and cd14 mbmdc were incubated with compound 1 ( 3.7 m ) overnight . il-6 in the culture supernatants was measured by elisa ( f ) and type i ifn ( ifn- ) measured by luciferase release in from an isre reporter cell line ( g ) . p < 0.05 was considered as significant compared to vehicle using student s t test . data shown are mean sem of triplicates and representative of two independent experiments showing similar results . reagents and conditions : ( a ) brch2cooet , nahco3 , etoh , reflux ; ( b ) tert - buok , thf , < 30 c ; ( c ) r - ncs , etoh , reflux ; ( d ) ppa , 110 c ; ( e ) clch2cooh , koh / etoh , reflux ; ( f ) r - nh2 ( for r substitutions refer to table 1 ) , hatu , dmf , room temp . to confirm that tlr4 was indeed the receptor , compound 1 was assayed for il-6 production in mbmdcs from wild - type and tlr4 deficient mice ( figure 2d ) . the binding of compound 1 to the tlr4/md-2/cd14 complex was further confirmed using a competitive antagonist for the tlr4 binding complex , lps - rs ( lps from rhodococcus sphaeroides ) . lps - rs inhibited the activation by compound 1 in a dose - dependent manner , indicating that compound 1 bound to the tlr4 complex ( figure 2e ) . tlr4 signals through two distinct pathways , leading respectively to nfb dependent cytokines and type i interferon ( ifn ) production . several naturally occurring tlr4 ligands and mpla have been reported to require cd14 to activate the type i ifn regulatory pathway . the htlr4 transfected hek293 cell line ( figure 2a ) also overexpresses md-2 and cd14 , which are tlr4 accessory proteins . compound 1 , however , was not dependent on cd14 for either il-6 or type i ifn production , as demonstrated using cd14 deficient cells ( figure 2f , g ) . the supernatants from mbmdcs stimulated with graded doses of compound 1 were also tested for ip-10 as a surrogate marker of type i ifn release . results showed a dose - dependent response for type i ifn ( figure 3a ) production , which paralleled that of ip-10 ( figure 3b ) . the levels of type i ifn were determined using an l929-isre luciferase reporter cell line and a mifn standard ( a ) . data shown are mean sem of triplicates and representative of two independent experiments showing similar results . * denotes p < 0.05 compared to vehicle using one way anova with dunnett s post hoc testing . the above assays were utilized to compare the derivatives of the lead pyrimidoindole for their ability to activate mouse and human tlr4 . mouse tlr4 activation was assessed using primary mbmdc and il-6 release ( figure 4 ) and confirmed using mtlr4 transfected hek293 cells ( figure 5a , c ) . the values shown in the tables 13 for il-6 release , and mtlr4 activation are area under the curve ( auc ) values for titrated doses of compounds from 312 nm to 10 m . each cytokine induction curve was first converted to a percent activity curve , and then the auc of the percent activity curve was calculated . the process of converting to a percent activity curve allowed subtracting background and adjusting for plate - to - plate variation . finally , the auc values were normalized to the activity of compound 1 within each experiment , set at 100 . human tlr4 activation is shown for stimulation of pbmc ( il-8 ) and htlr4 hek293 transfectomas ( figure 5b , d ) at 10 m , as these assays were not sufficiently sensitive at lower concentrations to make auc comparisons . the levels of htlr4 activation by sar derivatives are expressed relative to compound 1 , set at 100 . the cells were incubated with graded concentrations of the indicated compounds 1 , 36 , and 39 ( a ) or 11 and 12 ( b ) for 18 h. dmso 0.5% served as the vehicle control . mouse tlr4 ( a , c ) , and htlr4 ( b , d ) hek transfectomas were incubated with graded concentrations of the indicated compounds 1 , 36 , and 39 ( a ) or 11 and 12 ( b ) for 18 h. dmso 0.5% served as the vehicle control . the specific activation of the reporter cell lines was measured by seap activity in the supernatant by absorption at 630 nm . the primary hts library contained a family of 452 compounds in the pyrimido[5,4-b]indole class and therefore represented a valuable initial indication of structural features important for activation of nfb . upon inspection of the nfb activation values in the initial hts relative to the various structural features , several trends were apparent . there was a requirement for a hydrophobic moiety in the region of the cyclohexyl group of compound 1 . in this same region , furthermore , a hydrophobic group at n-3 , preferably a phenyl group , was also required for activity . substitutions on the n-3 phenyl , other than fluorine atoms , resulted in loss of activity . removal of the benzo ring of the indole portion of the scaffold also resulted in loss of activity . when the c-4 oxo was replaced by nh to form a triazine ring , loss of activity was observed . finally , exchange of the entire acetamide moiety on the c-2 thiol with the n-3 phenyl group , such that the acetamide was attached at n-3 and the phenyl was attached at the c-2 thiol , resulted in loss of activity . with these sar features as an initial guide , we elected to investigate modifications of hit compound 1 at three regions while maintaining the core pyrimido[5,4-b]indole ring system : the n - substitution of the s - acetamide , the n-3 substituent , and the n-5 substituent ( figure 6 ) . n - substitutions at the carboxamide moiety were first undertaken to probe the limitations of the hydrophobic group requirement at this position with respect to optimization of cytokine induction ( table 1 ) . while keeping all other structural features of hit compound 1 constant , we prepared a series of carboxamides substituted with various alkyl , cycloalkyl , aromatic , and heteroaromatic groups . the synthesis began with construction of the appropriately substituted pyrimido[5,4-b]indole ring system as shown in scheme 1 . 2-aminobenzonitrile ( 2 ) was reacted with ethyl bromoacetate to yield ethyl 2-((2-cyanophenyl)amino)acetate ( 3 ) followed by base catalyzed ring closure to the aminoindole ( 4 ) . at this point in the synthesis , a variety of substituents , represented by r in the scheme , may be introduced that will determine the respective n-3 substituent following annulation of the pyrimidine ring . thus , reaction of compound 4 with an isothiocyanate , such as phenylisothiocyanate , provided the substituted thioureidoindole ( 5 ) , where rwas phenyl in this example . ring closure of 5 using polyphosphoric acid yielded the pyrimido[5,4-b]indole ( 6 ) bearing the n-3 substituent r. alkylation of the 2-thioxo function with chloroacetic acid provided the versatile intermediate 7 , which was then used to prepare the final test compounds ( 8) bearing a variety of n - substitutions at the acetamide moiety , designated as r in scheme 1 . compounds 1 , 13 , 14 , 15 , 16 , 17 , as well as the n-3 derivatives 37 and 38 , discussed later , were registered with chemical abstracts service , but no literature references were found for any of these compounds . the hit compound 1 was resynthesized according to scheme 1 , as discussed above . thus , by this synthetic method , compounds shown in table 1 , with r held constant as a phenyl group , were prepared and evaluated in a cytokine induction assay for il-6 in mbmdc and compared to compound 1 . examples of the data obtained from typical cytokine induction assays and nfb activation assays are shown in figures 4 and 5 , wherein compounds 1 , 11 , 12 , 36 , and 39 are compared for il-6 production by mbmdc and hpbmc . stimulation with lps ( 10 ng / ml ) , and 5 m of 1 released an average of 20.6 4.8 and 10.5 1.3 ng / ml of il-6 , respectively . all compounds tested at 5 m , values normalized to 1 . stimulation with lps 10 ng / ml and 5 m of 1 released averages of 234.8 8.6 and 590 11 pg / ml of ip-10 , respectively . tlr4 cell activation stimulated with 10 ng / ml lps and 5 m compound 1 resulted in od630 of 1.69 0.08 and 1.52 0.03 , respectively . stimulation with lps 10 ng / ml and 10 m of 1 released averages of 14.5 0.6 and 7.8 0.8 ng / ml of il-8 , respectively . all compounds tested at 10 m , values normalized to 1 . tlr4 cell activation stimulated with 10 ng / ml lps and 10 m compound 1 resulted in od630 of 1.90 0.04 and 0.88 0.02 , respectively . inspection of the il-6 auc values relative to the hydrophobic group r revealed that in general , compounds bearing the larger cycloalkyl groups , such as cyclooctyl ( 9 ) , cycloheptyl ( 10 ) , and cyclohexyl ( 1 ) , were the most active , followed by branched alkyls and then straight chain alkyls and aromatic and heteroaromatic groups . a notable exception would be the p - fluorophenyl ( 14 ) and o - fluorophenyl ( 15 ) compounds . for the r group hence , the 3,3-dimethylbutyl compound ( 28 ) was among the most active of the alkyls , with isopentyl ( 27 ) , butyl ( 23 ) , and isobutyl ( 26 ) being somewhat less active . interestingly , when the alkyl chain length of r was extended to more than five carbons or reduced to three or fewer carbons , whether branched or not , significant loss of activity was observed ( table 1 ) . encouraged by the sar trends at the carboxamide moiety , we then addressed the n-3 position ( table 2 ) . as mentioned above , data from the nfb primary screen indicated that a hydrophobic group at n-3 , preferably an unsubstituted phenyl , was required for activity . however , to draw that conclusion with confidence , more examples of compounds bearing similar hydrophobic groups than were represented in the primary compound collection were needed . we therefore elected to prepare and evaluate a few additional derivatives of hit compound 1 with variation only at n-3 . thus , the r group in compound 5 was varied by reaction of 4 with the appropriate isothiocyanate , and the remaining three steps for each derivative were completed as outlined in scheme 1 while maintaining the r group as cyclohexyl in each case . as the il-6 data indicate , replacing the n-3 phenyl in compound 1 with a cyclohexyl group ( 36 ) caused some loss of activity , while substitution on the n-3 phenyl ( 37 and 38 ) resulted in greater loss of activity . replacing n-3 phenyl with a larger aromatic , such as naphthyl ( 39 ) , or extending the phenyl with an alkyl chain , as in phenethyl ( 40 ) , yielded total loss of activity . the combined substitution of r with cyclohexyl and r with cyclopentyl ( 41 ) resulted in partial loss of activity . stimulation with lps ( 10 ng / ml ) and 5 m of 1 released an average of 20.6 4.8 and 10.5 1.3 ng / ml of il-6 , respectively . all compounds tested at 5 m , values normalized to 1 . stimulation with lps 10 ng / ml and 5 m of 1 released averages of 234.8 8.6 and 590 11 pg / ml of ip-10 , respectively . tlr4 cell activation stimulated with 10 ng / ml lps and 5 m compound 1 resulted in od630 of 1.69 0.08 and 1.52 0.03 , respectively . stimulation with lps 10 ng / ml and 10 m of 1 released averages of 14.5 0.6 and 7.8 0.8 ng / ml of il-8 , respectively . tlr4 cell activation stimulated with 10 ng / ml lps and 10 m compound 1 resulted in od630 of 1.90 0.04 and 0.88 0.02 , respectively . none of the compounds in the original hts library had modifications at this position to guide the sar . the n-5 indole - like nitrogen can be alkylated if the proton is first removed by a strong base , such as sodium hydride . starting from compound 1 , scheme 2 shows the preparation of the n-5 methyl derivative ( 42 ) that also produced a dimethyl side product ( 43 ) wherein the second methylation occurred at the carboxamide function , yielding the n , n - disubstituted derivative . the n-5 methyl butyrate ester ( 44 ) of compound 1 was also prepared by this method . table 3 compares the il-6 inducing activity of the n-5 derivatives relative to compound 1 . interestingly , simple methylation at n-5 did not abrogate the immune stimulatory activity of compound 1 . moreover , methylation at n-5 decreased the toxicity of compound 1 ( figure 7 ) as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) viability assay . dimethylation , on the other hand , resulted in total loss of il-6 activity ( compound 43 ) . to further probe the n-5 position , we elected to study a few n-5 alkylated derivatives of compound 1 while avoiding dialkylation in the process . accordingly , we prepared the t - butyl ester of 7a ( compound 45 ) and then alkylated the n-5 position with several primary alkyl halides , as depicted in scheme 3 . the resulting t - butyl esters ( 46a d ) were easily hydrolyzed to the corresponding free carboxylic acids ( 47a d ) and then converted to the n - cyclohexylcarboxamides ( 4851 ) by the same method as described for compound 1 . thus , using this strategy , the n-5 n - propyl , n - pentyl , n - dodecyl , and cyanomethyl derivatives were prepared . finally , the cyanomethyl derivative ( 51 ) was converted to the n-5 acetamide derivative ( 52 ) . all n-5 alkyl derivatives were found to be less active than the n-5 methyl ( 42 ) , with the n - propyl ( 48 ) being the next most active of the series . the trend in the toxicity profile for these n-5 alkyl derivatives was confirmed , at least for the shorter chain alkyls , in that the n-5 methyl ( 42 ) , cyanomethyl ( 51 ) , and n - propyl ( 48 ) derivatives did not reduce cell viability at concentrations up to 10 m ( figure 7 , shown for 42 , data not shown for 48 and 51 ) . stimulation with lps ( 10 ng / ml ) and 5 m of 1 released an average of 20.6 4.8 and 10.5 1.3 ng / ml of il-6 , respectively . all compounds tested at 5 m , values normalized to 1 . stimulation with lps 10 ng / ml and 5 m of 1 released averages of 234.8 8.6 and 590 11 pg / ml of ip-10 , respectively . tlr4 cell activation stimulated with 10 ng / ml lps and 5 m compound 1 resulted in od630 of 1.69 0.08 and 1.52 0.03 , respectively . stimulation with lps 10 ng / ml and 10 m of 1 released averages of 14.5 0.6 and 7.8 0.8 ng / ml of il-8 , respectively . tlr4 cell activation stimulated with 10 ng / ml lps and 10 m compound 1 resulted in od630 of 1.90 0.04 and 0.88 0.02 , respectively . cytotoxicity of the compounds was evaluated by mtt assay as a measure of viability . mbmdc ( 10/well ) were incubated with graded concentrations of the compounds for 18 h and compared with vehicle ( 0.5% dmso ) and mpla ( 1 g / ml ) . ( ( b ) the cells were lysed after the overnight incubation with mtt reagents , and absorbance at 570 nm was measured , subtracting the reference absorbance at 650 nm . data shown are mean sem of triplicates and are representative of two independent experiments showing similar results . ( a ) nah , dmf , room temp , then ch3i ; ( b ) nah , dmf , then br(ch2)3cooch3 ( a ) clch2coo t - bu , koh , dma , h2o ; ( b ) nah , dmf , then rx ( iodopropane or iodopentane or bromododecane or bromoacetonitrile ) ; ( c ) tfa , dcm or ch3cn ; ( d ) cyclohexyl - nh2 , hatu , dmf , room temp ; ( e ) compound 51 , h2so4 , h2o . the tlr4 transfectoma lines transmit their reporter signal after activation of the proinflammatory transcription factor , nfb . as one might expect , the il-6 secretion induced by the compounds in primary mbmdcs correlated well with their respective activities in mouse tlr4 transfectoma cell lines . with few exceptions , the most active compounds in the il-6 assays were also those that showed the highest activity in the mtlr4 transfectoma line ( tables 13 ) . in addition to stimulating the production of inflammatory cytokines ( e.g. , il-6 ) by nfb activation , tlr4 signaling can induce the production of type i ifn via the trif pathway . this pathway results in the activation of interferon regulatory factors ( irf ) , which are nuclear transcription factors that promote the transcription of ifns . type i ifn is important for the activation of antigen presenting dendritic cells , leading to good adjuvant activity , and also promotes cellular defenses against a variety of pathogens , particularly rna viruses . this host defense is aided by circulating interferon inducible factors such as ip-10 . the tlr4 transfectoma reporter lines in this study did not utilize an interferon reporter system ; hence , the ip-10 data does not correlate as closely as the il-6 data . interestingly , several of the compounds , namely 12 , 13 , 15 , 16 , 17 , 29 , 30 , and 33 , induced diminished il-6 release compared to compound 1 but had nearly equivalent induction of ip-10 ( tables 1 and 2 ) . these results suggest that there may be a possibility of separating the two activities on the basis of structural features . the correlation of il-6 and ip-10 production of the selected compounds is shown graphically in figure 8 . two distinct groups of compounds are observed , shown as circled regions of the plot . thus , the group at the upper right area ( dotted circle ) comprises compounds that induce high il-6 and high ip-10 production , including compounds 1 , 9 , 10 , 28 , and 42 . the other group is composed of compounds that induce low il-6 but relatively higher ip-10 production . these two clusters were selected based on mean sd of the normalized il-6 and ip-10 values of all sar compounds . the cluster in the dotted circle includes those compounds whose values were at least one sd above the mean for both cytokines . the other cluster includes those compounds whose values were below the mean for il-6 and were above the mean for ip-10 . it was notable that when the carboxamide substituent was phenyl or substituted phenyl ( 13 , 15 , 16 , 17 ) , with the exception of p - fluorophenyl ( 14 ) , il-6 release was reduced while ip-10 production was maintained at a relatively higher level . a few compounds bearing branched aliphatic carboxamide substituents showed a similar trend , as noted above for 29 , 30 , and 33 . two distinct groups , compounds in the dotted circled ( upper right ) and in solid ( left ) circled areas , were selected based on the induction of il-6 and ip-10 . these clusters were selected based on mean sd of the normalized il-6 and ip-10 values of all sar compounds . for il-6 . the group at the upper right area comprises compounds that induce high il-6 and high ip-10 production , including compounds 1 , 9 , 10 , 28 , and 42 , whose values were at least one sd above the mean for both cytokines . the other cluster is composed of compounds ( 13 , 16 , 17 , 29 , 30 , and 33 ) that induce low il-6 but relatively higher ip-10 production , whose values were below the mean for il-6 and were above the mean for ip-10 . in general , the active compounds exhibited greater activity in the murine cell systems than in human cells . although the innate immune system is highly conserved among species , there are differences between human and mouse tlr4 . similar to the active compounds reported here nonsynthetic ligands have been noted to have species specific behavior . notably , tetraacylated lipid iva , a synthetic lipid a precursor , has been reported to act as a weak agonist to mouse tlr4/md-2 but as an antagonist to human tlr4/md-2 . recently mouse and human tlr4/md2 crystal structures with this ligand have suggested that the different charge distributions of mouse and human tlr4/md-2 affect the positions of the phosphate groups of lipid iva , orienting them in a manner that would limit receptor dimerization by human tlr4 . tlr4 , in association with md-2 , is responsible for the physiological recognition of lps . the structural basis of receptor specificity and of the mechanism of activation by lps have recently been elucidated by determining the crystal structure of the tlr4/md-2-lps complex at 3.1 a resolution . binding of agonistic ligands such as lps causes dimerization of the extracellular domains to form a tlr4/md-2-lps macromolecular complex . like the extracellular domains of other tlrs , tlr4 contains leucine - rich repeats and adopts a characteristic horseshoe - like shape . md-2 is noncovalently bound to the side of the horseshoe ring and also directly interfaces with the ligand . md-2 has a -cup fold structure composed of two antiparallel -sheets forming a large hydrophobic pocket for ligand binding . lps binds to this pocket and directly mediates dimerization of the two tlr4/md-2 complexes . tlr4 can be activated by structurally diverse lps molecules , which have been predicted to occupy this pocket in md-2 . small molecule hit compounds discovered in the present study were also thought to bind to the tlr4/md-2 complex in such a way as to facilitate dimerization . accordingly , we examined the predicted binding mode(s ) of one of the most active compounds to the murine tlr4/md-2 complex by conducting molecular docking of compound 28 to the crystal structure of the mouse complex ( pdb 2z64 ) using the programs hex and ampac . we selected the best configurations of 28 bound to this complex based on molecular surface shape complementarity and the most favorable intermolecular energy of interactions . it is noteworthy that the best docking position for 28 was within the lps pocket . figure 9 shows the predicted binding mode of compound 28 in the tlr4/md-2 model , while the set of binding interactions that may keep the compound in the md-2 pocket bound to both tlr4 and md-2 is depicted in figure 10 . there is a set of favorable electrostatic interactions resulting in possible hydrogen bonds formed by the residues glu439 of tlr4 and arg90 of md-2 with compound 28 , although the former would approach a hydrogen bond interaction due to the flexibility of the glu439 side chain . there are also multiple potential hydrophobic interactions with md-2 and tlr4 . such interactions of the compound with two proteins can improve the free energy of complex formation by approximately 810 kcal / mol . predicted binding mode of compound 28 to mouse tlr4/md-2 complex . predicted binding interactions of compound 28 with mouse tlr4/md-2 complex ( pdb 2z64 ) . interestingly , the predicted binding model for the pyrimidoindoles is similar to that proposed for tricyclic antidepressant , amitriptyline . this molecule also has some tlr4/md-2 binding and has a similar three - ring scaffold . other small molecules such as paclitaxel , opioids , and a peptide have also been reported to bind to the md-2 binding pocket . in the course of an hts designed to identify activators of innate immunity , a series of substituted pyrimido[5,4-b]indoles were discovered as selective tlr4 ligands . small molecules of this class are unique among tlr4 activators in that they are non - lipid - like . structure activity evaluation in both mouse and human cells revealed that , to maintain activity , the carboxamide region of this scaffold must contain a hydrophobic moiety of significant volume . interestingly , a subset of the compounds bearing phenyl and substituted phenyl carboxamides induced lower nfb - dependent inflammatory cytokine release while maintaining interferon - dependent ip-10 production . varying the substituents at n-3 indicated an even greater restriction for a hydrophobic moiety at this position , with a phenyl group being preferred for activity . finally , n-5 substitution revealed that short alkyl substituents at this position attenuate cell toxicity relative to the corresponding nonsubstituted derivative while maintaining tlr4 activity . both the inflammatory cytokine and type i ifn inducing activities of these compounds were cd14-independent . computational studies with one of the active compounds predicted binding primarily to md-2 in the murine tlr4/md-2 complex . lead optimization studies to improve the activity of the compounds using computational methods are currently underway in our laboratories . to date , no single adjuvant can elicit the optimal immune response to all pathogens in vaccines . here we have described a panel of small molecules that stimulate immune cells to produce distinct profiles of nfb and interferon associated cytokines . this panel of molecules may allow for differential analysis of the relative pathway induction needed for adequate immunoprotection and immunotherapy for diverse pathogens . reagents were purchased as at least reagent grade from sigma - aldrich ( st . solvents were purchased from fischer scientific ( pittsburgh , pa ) and were either used as purchased or redistilled with an appropriate drying agent . hts compound library was obtained from ucsf small molecule discovery center ( san francisco , ca ) . compounds 13 , 14 , 15 , 16 , 17 , 37 , and 38 were purchased from life chemicals ( burlington , on , canada ) . all synthesized compounds , intermediates , and purchased compounds from life chemicals were determined to be > 95% pure by hplc utilizing an agilent 1100 lc / msd . endotoxin levels of active compounds were measured with endosafe - pts ( charles river , wilmington , ma ) and found to have less than 10 eu/mol . analytical tlc was performed using precoated tlc silica gel 60 f254 aluminum sheets purchased from emd ( gibbstown , nj ) and visualized using uv light . flash chromatography was carried out on emd silica gel 60 ( 4063 m ) system or with a biotage isolera one ( charlotte , nc ) using the specified solvent . reactions were monitored using an agilent 1100 lc / msd ( santa clara , ca ) with either a supelco discovery hs c18 column ( sigma - aldrich ) or an onyx monolithic c18 ( phenomenex , torrance , ca ) with purity above 98% by percent area . all synthesized compounds and intermediates were analyzed by high - resolution ms using an agilent 6230 esi - tofms ( santa clara , ca ) . selected compounds were analyzed by ir ( kbr window method ) to confirm presence of functional groups having useful diagnostic frequencies using a perkin - elmer 1600 series . h nmr spectra were obtained on a varian mercury 300 or 400 ( varian , inc . , the chemical shifts are expressed in parts per million ( ppm ) using suitable deuterated nmr solvents in reference to tms at 0 ppm . the 3d structures were prepared and optimized using the ampac semiempirical quantum chemistry program ( accelrys , san diego , ca ) . to a solution of compound 4 ( 1 equiv ) in warm etoh was added the appropriate isothiocyanate ( 1.1 equiv ) dropwise with stirring . solids were filtered , washed with etoh , and dried overnight in vacuo to give compound 5 . compound 5 was dissolved in polyphosphoric acid and stirred at 110 c for 3.5 h. solution was added to ice - cold water and extracted with etoac and dried over mgso4 . the solid was then dried in vacuo overnight to give compound 6 . in a flame - dried flask , compound 6a ( 1 equiv ) and koh ( 2 equiv ) were dissolved in anhydrous etoh with heat . in a separate flame - dried flask , chloroacetic acid ( 1 equiv ) chloroacetic acid solution was then added to the reaction mixture and refluxed for 6 h. the reaction was concentrated by half and acidified with 3 m hcl to ph 4 . the solids were collected , washed with water , and dried in vacuo to give compound 7 . compound 7 ( 1 equiv ) , triethylamine ( 2 equiv ) , and the appropriate amine ( 1.1 equiv ) were dissolved in anhydrous dmf . to this solution , hatu ( 1.1 equiv ) dissolved in dmf was added and stirred until complete and concentrated in vacuo . the crude material was then recrystallized in meoh to give compound 8 . nah ( 1.1 equiv ) was added to a solution of compound 45 ( 1 equiv ) in dmf . the reaction mixture was stirred for 5 min , and then the appropriate alkyl halide ( 1.1 equiv ) was added and stirred until complete . compound 46 was dissolved in 1:1 acetonitrile / triflouroacetic acid and stirred at room temperature overnight . depending on the n - alkyl substitution , crude material either precipitated as pure product or was purified by chromatography to give compound 47 . compound 7a ( 50 mg , 0.14 mmol ) , triethylamine ( 40 l , 0.28 mmol ) , and cyclohexylamine ( 18 l , 0.16 mmol ) were dissolved in anhydrous dmf ( 1 ml ) . hatu ( 59.5 mg , 0.16 mmol ) dissolved in 0.2 ml of dmf was added to the reaction mixture and stirred for 20 min and then concentrated in vacuo . the crude material was recrystallized with meoh to give 61 mg in near quantitative yield . h nmr ( 300 mhz , dmso - d6 ) ppm 0.971.33 ( m , 4 h ) , 1.51 ( d , j = 8.53 hz , 1 h ) , 1.561.80 ( m , 4 h ) , 3.48 ( m , 1 h ) , 3.87 ( s , 2 h ) , 7.24 ( t , j = 7.29 hz , 1 h ) , 7.387.67 ( m , 6 h ) , 8.05 ( d , j = 7.98 hz , 1 h ) , 8.15 ( d , j = 7.70 hz , 1 h ) , 12.09 ( s , 1 h ) . hrms calcd for c24h24n4o2sna ( m + na ) , 455.1512 ; found , 455.1511 . anthranilonitrile ( 30.29 g , 256 mmol ) , ethyl bromoacetate ( 29.46 ml , 267 mmol ) , and sodium bicarbonate ( 25.6 g , 300 mmol ) were combined in anhydrous etoh ( 90 ml ) and refluxed for 42 h. after cooling slightly , solution was decanted from precipitate into a prewarmed flask . further cooling of the decantate yielded crystals , which were then filtered and washed with cold water to give 23.6 g of white crystalline product . h nmr ( 500 mhz , dmso - d6 ) ppm 1.131.27 ( m , 3 h ) , 4.04 ( d , j = 6.41 hz , 2 h ) , 4.12 ( q , j = 7.22 hz , 2 h ) , 6.36 ( t , j = 6.25 hz , 1 h ) , 6.64 ( d , j = 8.54 hz , 1 h ) , 6.69 ( t , j = 7.47 hz , 1 h ) , 7.41 ( t , j = 7.78 hz , 1 h ) , 7.49 ( dd , j = 7.93 , 1.53 hz , 1 h ) . hrms calcd for c11h12n2o2na ( m + na ) , 227.0791 ; found , 227.0794 . in a flame - dried flask , a suspension of potassium t - butoxide ( 7.124 g , 63.5 mmol ) in anhydrous thf was stirred and maintained below 30 c under argon . to this solution was added a solution of compound 3 ( 16 g , 63.5 mmol ) in anhydrous thf over 45 min and stirred for an additional 2 h. the reaction was then poured into ice water , extracted with etoac , and dried over mgso4 . the solid was then dissolved in minimal etoh , and water was added dropwise until just cloudy and allowed to precipitate at room temperature . precipitate was filtered and washed with cold etoh to give 6.6 g of compound 4 in 51% yield . h nmr ( 500 mhz , dmso - d6 ) ppm 1.33 ( t , j = 7.02 hz , 3 h ) , 4.29 ( q , j = 7.12 hz , 2 h ) , 5.68 ( s , 2 h ) , 6.88 ( ddd , j = 8.08 , 5.80 , 1.98 hz , 1 h ) , 7.157.25 ( m , 2 h ) , 7.74 ( d , j = 7.93 hz , 1 h ) , 10.34 ( s , 1 h ) . hrms calcd for c11h12n2o2 ( m + h ) , 205.0972 ; found , 205.0973 . compound 4 ( 5.21 g , 25.5 mmol ) was reacted with phenyl isothiocyanate ( 3.36 ml , 28.1 mmol ) in warm etoh ( 75 ml ) according to general procedure a to give 6.732 g compound 5a in 75% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 1.32 ( t , j = 7.07 hz , 3 h ) , 4.32 ( q , j = 7.31 hz , 2 h ) , 7.09 ( dt , j = 14.75 , 7.50 hz , 2 h ) , 7.227.38 ( m , 3 h ) , 7.43 ( d , j = 8.29 hz , 1 h ) , 7.51 ( d , j = 7.80 hz , 2 h ) , 7.56 ( d , j = 8.29 hz , 1 h ) , 9.40 ( s , 1 h ) , 9.69 ( br s , 1 h ) , 11.79 ( s , 1 h ) . hrms calcd for c18h17n3o2sna ( m + na ) , 362.0934 ; found , 362.0935 . compound 4 ( 100 mg , 0.49 mmol ) was reacted with cyclohexyl isothiocyanate ( 169.15 mg , 0.54 mmol ) in warm etoh ( 750 l ) according to general procedure a to give 104 mg of compound 5b in 61.8% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 1.011.42 ( m , 9 h ) , 1.491.62 ( m , 1 h ) , 1.621.74 ( m , 2 h ) , 1.771.96 ( m , 2 h ) , 4.13 ( br s , 1 h ) , 4.29 ( q , j = 7.09 hz , 2 h ) , 7.05 ( t , j = 7.33 hz , 1 h ) , 7.25 ( t , j = 8.07 hz , 1 h ) , 7.41 ( d , j = 8.07 hz , 1 h ) , 7.49 ( d , j = 8.07 hz , 1 h ) , 8.91 ( s , 1 h ) , 11.71 ( br s , 1 h ) . hrms calcd for c18h23n3o2s ( m + h ) , 345.1511 ; found , 345.15142 . compound 4 ( 50 mg , 0.28 mmol ) was reacted with 1-naphthyl isothiocyanate ( 49.9 mg , 0.27 mmol ) in warm etoh ( 750 l ) according to general procedure a to give 58.6 mg of compound 5c in 61.5% yield . hrms calcd for c22h19n3o2sna ( m + na ) , 412.1090 ; found , 412.1091 . compound 4 ( 100 mg , 0.49 mmol ) was reacted with phenethyl isothiocyanate ( 80.4 l , 0.54 mmol ) in warm etoh ( 1.5 ml ) according to general procedure a to give 147 mg of compound 5d in 81.8% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 1.31 ( t , j = 6.97 hz , 3 h ) , 4.27 ( q , j = 7.21 hz , 2 h ) , 7.06 ( t , j = 7.70 hz , 1 h ) , 7.097.33 ( m , 7 h ) , 7.43 ( dd , j = 8.07 , 4.03 hz , 2 h ) , 7.56 ( br s , 1 h ) , 9.12 ( br s , 1 h ) , 11.79 ( br s , 1 h ) . hrms calcd for c20h22n3o2s ( m + h ) , 368.1427 ; found , 368.1431 to a flame - dried flask with cold anhydrous etoh ( 75 ml ) was added cold acetyl chloride ( 7 ml , 98.5 mmol ) under argon with stirring . in a separate flame - dried flask charged with argon , compound 5a ( 6.5 g , 19 mmol ) was dissolved in anhydrous etoh ( 25 ml ) and added to the acetyl chloride solution . precipitate was filtered and recrystallized with etoh to give 3.77 g of compound 6a in 75% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 7.097.31 ( m , 3 h ) , 7.317.61 ( m , 6 h ) , 8.20 ( d , j = 8.07 hz , 1 h ) , 12.17 ( br s , 1 h ) . hrms calcd for c16h12n3os ( m + h ) , 294.0696 ; found , 294.0698 . compound 5b ( 70 mg , 0.2 mmol ) and 1 ml of polyphosphoric acid was reacted according to general procedure b to give 60.6 mg of compound 6b in quantitative yield . h nmr ( 400 mhz , dmso - d6 ) ppm 1.071.49 ( m , 5 h ) , 1.531.70 ( m , 1 h ) , 1.701.84 ( m , 2 h ) , 2.01 ( s , 2 h ) , 3.864.09 ( m , 1 h ) , 7.11 ( t , j = 8.07 hz , 1 h ) , 7.297.54 ( m , 2 h ) , 7.87 ( d , j = 8.07 hz , 1 h ) , 8.06 ( d , j = 6.97 hz , 1 h ) , 11.60 ( s , 1 h ) . hrms calcd for c16h18n3os ( m + h ) , 300.1165 ; found , 300.1169 . compound 5c ( 150 mg , 0.38 mmol ) and 1 ml of polyphosphoric acid was reacted according to general procedure b to give 132 mg of compound 6b in quantitative yield . h nmr ( 400 mhz , dmso - d6 ) ppm 7.027.18 ( m , 1 h ) , 7.337.48 ( m , 2 h ) , 7.487.67 ( m , 3 h ) , 7.74 ( d , j = 8.07 hz , 1 h ) , 7.87 ( d , j = 8.07 hz , 1 h ) , 7.84 ( d , j = 7.70 hz , 1 h ) , 7.948.03 ( m , 1 h ) , 8.038.18 ( m , 1 h ) , 11.83 ( s , 1 h ) . hrms calcd for c20h14n3os ( m + h ) , 344.0852 ; found , 344.0855 . compound 5d ( 50.7 mg , 0.138 mmol ) and 1 ml of polyphosphoric acid was reacted according to general procedure b. crude product was purified by automated flash chromatography ( biotage ) ( dcm / meoh = 99:1 ) to give 27.1 mg of compound 6d in 61.1% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 2.95 ( t , j = 7.33 hz , 2 h ) , 3.67 ( t , j = 6.20 hz , 2 h ) , 6.967.63 ( m , 8 h ) , 7.91 ( d , j = 7.70 hz , 1 h ) , 8.31 ( d , j = 4.77 hz , 1 h ) , 11.5011.89 ( m , 1 h ) . hrms calcd for c18h15n3os ( m + h ) , 321.0936 ; found , 321.0939 . compound 6a ( 3.77 g , 12.9 mmol ) , koh ( 1.44 g , 25.8 mmol ) , chloroacetic acid ( 1.25 g , 12.9 mmol , 6.5 ml of anhydrous etoh ) , and 115 ml of anhydrous etoh were reacted according to general procedure c to give 3.66 g of compound 7a in 81% yield . ir : 3251 ( oh ) , 1702 ( co carboxyl ) , 1660 ( co amide ) cm . h nmr ( 400 mhz , dmso - d6 ) ppm 3.94 ( s , 2 h ) , 7.147.36 ( m , 1 h ) , 7.387.84 ( m , 7 h ) , 7.93 ( d , j = 7.70 hz , 1 h ) , 12.09 ( br s , 1 h ) . hrms calcd for c18h13n3o3sna ( m + na ) , 374.0570 ; found , 374.0572 . compound 6b ( 165.8 mg , 0.55 mmol ) , koh ( 62 mg , 1.1 mmol ) , chloroacetic acid ( 52.3 mg , 0.55 mmol , 3 ml of anhydrous etoh ) , and 62 ml of anhydrous etoh were reacted according to general procedure c to give 35.6 mg of compound 7b in 17.9% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 1.051.52 ( m , 4 h ) , 1.592.05 ( m , 6 h ) , 3.94 ( s , 2 h ) , 4.204.46 ( m , 1 h ) , 7.15 ( t , j = 7.33 hz , 1 h ) , 7.307.59 ( m , 2 h ) , 7.88 ( d , j = 8.07 hz , 1 h ) , 11.79 ( br s , 1 h ) . hrms calcd for c18h19n3o3sna ( m + na ) , 380.1039 ; found , 380.1042 . compound 6c ( 120 mg , 0.35 mmol ) , koh ( 39.1 mg , 70 mmol ) , chloroacetic acid ( 33 mg , 0.35 mmol , 3 ml of anhydrous etoh ) , and 62 ml of anhydrous etoh were reacted according to general procedure c to give 67.1 mg of compound 7c in 47.8% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 3.90 ( s , 2 h ) , 7.26 ( t , j = 7.40 hz , 1 h ) , 7.337.66 ( m , 5 h ) , 7.677.80 ( m , 2 h ) , 7.98 ( d , j = 7.70 hz , 1 h ) , 8.09 ( d , j = 8.43 hz , 1 h ) , 8.17 ( t , j = 4.77 hz , 1 h ) , 12.14 ( br s , 1 h ) . hrms calcd for c22h15n3o3sna ( m + na ) , 424.0726 ; found , 424.0730 . compound 6d ( 271 mg , 0.84 mmol ) , koh ( 94.5 mg , 1.69 mmol ) , chloroacetic acid ( 79.8 mg , 0.84 mmol , 3 ml of anhydrous etoh ) , and 62 ml of anhydrous etoh were reacted according to general procedure c to give 160 mg of compound 7d in 50% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 3.02 ( t , j = 7.80 hz , 2 h ) , 4.02 ( s , 2 h ) , 4.35 ( t , j = 7.31 hz , 2 h ) , 6.877.83 ( m , 8 h ) , 7.95 ( d , j = 7.31 hz , 1 h ) , 11.92 ( br s , 1 h ) . hrms calcd for c20h17n3o3sna ( m + na ) , 402.0883 ; found , 402.0885 . compound 7a ( 50 mg , 0.14 mmol ) , cycloheptylamine ( 39.86 l , 0.16 mmol ) , triethylamine ( 39.63 l , 0.28 mmol ) , and hatu ( 59.52 mg , 0.16 mmol ) were reacted according to general procedure d to give 12.46 mg in 18.5% yield . h nmr ( 300 mhz , dmso - d6 ) ppm 1.181.65 ( m , 10 h ) , 1.661.88 ( m , 2 h ) , 3.593.77 ( m , 1 h ) , 3.87 ( s , 2 h ) , 7.25 ( t , j = 7.01 hz , 1 h ) , 7.407.55 ( m , 4 h ) , 7.567.68 ( m , 3 h ) , 8.06 ( d , j = 7.98 hz , 1 h ) , 8.20 ( d , j = 7.70 hz , 1 h ) , 12.10 ( s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 24.12 , 28.28 , 34.69 , 37.17 , 50.51 , 113.31 , 119.7 , 120.48 , 120.78 , 120.81 , 127.77 , 130.00 , 130.01 , 130.32 , 136.51 , 137.68 , 139.35 , 153.01 , 155.39 , 165.92 . hrms calcd for c25h26n4o2sna ( m + na ) , 469.1669 ; found , 469.1670 . compound 7a ( 25 mg , 0.07 mmol ) , cyclooctylamine ( 24.15 l , 0.08 mmol ) , triethylamine ( 19.82 l , 0.14 mmol ) , and hatu ( 29.76 mg , 0.08 mmol ) were reacted according to general procedure d to give 30.77 mg in quantitative yield . h nmr ( 400 mhz , dmso - d6 ) ppm 1.231.89 ( m , 14 h ) , 3.683.82 ( m , 1 h ) , 3.87 ( s , 2 h ) , 7.24 ( t , j = 6.97 hz , 1 h ) , 7.377.75 ( m , 7 h ) , 8.06 ( d , j = 8.07 hz , 1 h ) , 8.18 ( d , j = 7.70 hz , 1 h ) , 12.10 ( br s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 23.75 , 25.47 , 27.25 , 31.87 , 37.16 , 49.49 , 113.32 , 119.7 , 120.48 , 120.78 , 120.81 , 127.78 , 130.00 , 130.01 , 130.32 , 136.51 , 137.68 , 139.36 , 153.02 , 155.39 , 165.90 . hrms calcd for c26h29n4o2s ( m + h ) , 461.2006 ; found , 461.2010 . compound 7a ( 50 mg , 0.14 mmol ) , cyclopentylamine ( 31 l , 0.16 mmol ) , triethylamine ( 39.63 l , 0.28 mmol ) , and hatu ( 59.52 mg , 0.16 mmol ) were reacted according to general procedure d to give 48.9 mg in 76% yield . h nmr ( 300 mhz , dmso - d6 ) ppm 1.321.55 ( m , 4 h ) , 1.551.70 ( m , 2 h ) , 1.711.86 ( m , 2 h ) , 3.87 ( s , 2 h ) , 3.97 ( dd , j = 13.07 , 6.46 hz , 1 h ) , 7.25 ( t , j = 7.29 hz , 1 h ) , 7.357.72 ( m , 7 h ) , 8.05 ( d , j = 7.98 hz , 1 h ) , 8.26 ( d , j = 6.88 hz , 1 h ) , 12.10 ( s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 23.95 , 32.74 , 37.13 , 51.12 , 113.32 , 119.69 , 120.5 , 120.76 , 120.79 , 127.77 , 129.99 , 130.01 , 130.32 , 136.50 , 137.67 , 139.35 , 153.02 , 155.39 , 166.64 . hrms calcd for c25h26n4o2sna ( m + na ) , 441.1356 ; found , 441.1367 . compound 7a ( 50 mg , 0.14 mmol ) , cyclobutylamine ( 26.73 l , 0.16 mmol ) , triethylamine ( 39.63 l , 0.28 mmol ) , and hatu ( 59.52 mg , 0.16 mmol ) were reacted according to general procedure d to give 41.6 mg in 73.5% yield . h nmr ( 300 mhz , dmso - d6 ) ppm 1.481.72 ( m , 2 h ) , 1.93 ( q , j = 9.70 hz , 2 h ) , 2.052.26 ( m , 2 h ) , 3.85 ( s , 2 h ) , 4.084.26 ( m , 1 h ) , 7.26 ( t , j = 7.29 hz , 1 h ) , 7.377.70 ( m , 6 h ) , 8.07 ( d , j = 7.98 hz , 1 h ) , 8.56 ( d , j = 7.70 hz , 1 h ) , 12.10 ( s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 15.11 , 30.72 , 37.03 , 44.72 , 113.32 , 119.70 , 120.52 , 120.81 , 127.78 , 130.00 , 130.01 , 130.33 , 136.49 , 137.69 , 139.35 , 152.95 , 155.4 , 166.21 . hrms calcd for c22h20n4o2sna ( m + na ) , 427.1199 ; found , 427.1200 . compound 7a ( 50 mg , 0.14 mmol ) , 2-aminonaphthalene ( 44.82 mg , 0.16 mmol ) , triethylamine ( 39.63 l , 0.28 mmol ) , and hatu ( 59.52 mg , 0.16 mmol ) were reacted according to general procedure d to give 40.8 mg in 61% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 4.16 ( br s , 2 h ) , 7.11 ( t , j = 7.70 hz , 1 h ) , 7.268.07 ( m , 14 h ) , 8.31 ( br s , 1 h ) , 10.65 ( br s , 1 h ) , 12.10 ( br s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 37.99 , 113.27 , 115.48 , 119.71 , 120.21 , 120.49 , 120.68 , 120.72 , 125.07 , 126.92 , 127.71 , 127.74 , 127.90 , 128.89 , 130.01 , 130.06 , 130.18 , 130.41 , 133.84 , 136.49 , 137.16 , 137.63 , 139.30 , 152.89 , 155.37 , 166.96 . hrms calcd for c28h20n4o2sna ( m + na ) , 499.1199 ; found , 499.1201 . compound 7a ( 50 mg , 0.14 mmol ) , furfurylamine ( 27.66 l , 0.16 mmol ) , triethylamine ( 39.63 l , 0.28 mmol ) , and hatu ( 59.52 mg , 0.16 mmol ) were reacted according to general procedure d to give 50.37 mg in 83.6% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 3.95 ( s , 2 h ) , 4.29 ( d , j = 5.50 hz , 2 h ) , 6.25 ( d , j = 3.30 hz , 1 h ) , 6.32 ( t , j = 2.57 hz , 1 h ) , 7.24 ( t , j = 6.97 hz , 1 h ) , 7.357.72 ( m , 7 h ) , 7.97 ( d , j = 7.70 hz , 1 h ) , 8.73 ( t , j = 5.32 hz , 1 h ) , 12.10 ( br s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 36.46 , 36.76 , 107.46 , 107.48 , 107.49 , 110.86 , 110.87 , 110.89 , 113.23 , 119.71 , 120.60 , 120.80 , 120.88 , 127.76 , 130.01 , 130.02 , 130.34 , 136.50 , 137.69 , 139.34 , 142.64 , 152.32 , 152.81 , 155.41 , 167.44 . hrms calcd for c23h18n4o3sna ( m + na ) , 453.0992 ; found , 453.0995 . compound 7a ( 50 mg , 0.14 mmol ) , 2-aminoindazole ( 41.68 mg , 0.16 mmol ) , triethylamine ( 39.63 l , 0.28 mmol ) , and hatu ( 59.52 mg , 0.16 mmol ) were reacted according to general procedure d to give 32.7 mg in 50% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 4.11 ( s , 2 h ) , 7.09 ( t , j = 7.33 hz , 1 h ) , 7.17 ( d , j = 8.43 hz , 1 h ) , 7.327.77 ( m , 9 h ) , 7.878.01 ( m , 2 h ) , 8.11 ( s , 1 h ) , 10.55 ( br s , 1 h ) , 12.07 ( br s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 38.00 , 99.22 , 113.29 , 114.43 , 119.60 , 119.65 , 120.55 , 120.69 , 121.03 , 121.24 , 127.81 , 129.95 , 130.10 , 130.45 , 133.80 , 136.42 , 137.66 , 137.73 , 139.31 , 140.74 , 148.09 , 152.88 , 155.37 , 162.89 , 167.00 . hrms calcd for c25h18n6o2sna ( m + na ) , 489.1104 ; found , 489.1106 . compound 7a ( 50 mg , 0.14 mmol ) , 2-aminothiazole ( 31.35 mg , 0.16 mmol ) , triethylamine ( 39.63 l , 0.28 mmol ) , and hatu ( 59.52 mg , 0.16 mmol ) were reacted according to general procedure d to give 12.82 mg in 21.1% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 4.17 ( s , 2 h ) , 6.907.27 ( m , 2 h ) , 7.307.78 ( m , 8 h ) , 7.83 ( d , j = 7.70 hz , 1 h ) , 12.0312.16 ( m , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 20.55 , 28.58 , 36.97 , 46.95 , 113.30 , 119.71 , 120.54 , 120.77 , 120.80 , 127.76 , 130.01 , 130.32 , 136.49 , 137.68 , 139.35 , 152.91 , 155.40 , 167.27 . hrms calcd for c21h15n5o2s2na ( m + na ) , 456.0559 ; found , 456.0562 . compound 7a ( 50 mg , 0.14 mmol ) , ethylamine ( 17.73 l , 0.16 mmol ) , triethylamine ( 39.63 l , 0.28 mmol ) , and hatu ( 59.52 mg , 0.16 mmol ) were reacted according to general procedure d to give 22.9 mg in 43.3% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 1.02 ( t , j = 7.15 hz , 3 h ) , 3.043.16 ( m , 2 h ) , 3.88 ( s , 2 h ) , 7.26 ( t , j = 7.33 hz , 1 h ) , 7.397.54 ( m , 3 h ) , 7.567.69 ( m , 3 h ) , 8.05 ( d , j = 8.07 hz , 1 h ) , 8.26 ( m , 1 h ) , 12.11 ( s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 15.12 , 34.34 , 37.01 , 113.30 , 119.70 , 120.59 , 120.74 , 120.81 , 127.76 , 130.01 , 130.32 , 136.49 , 137.68 , 139.35 , 152.88 , 155.41 , 167.02 . hrms calcd for c20h18n4o2sna ( m + na ) , 401.1043 ; found , 401.1046 . compound 7a ( 50 mg , 0.14 mmol ) , butylamine ( 30.94 l , 0.16 mmol ) , triethylamine ( 39.63 l , 0.28 mmol ) , and hatu ( 59.52 mg , 0.16 mmol ) were reacted according to general procedure d to give 18.6 mg in 44.7% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.78 ( t , j = 7.31 hz , 3 h ) , 1.171.30 ( m , 2 h ) , 1.321.46 ( m , 2 h ) , 2.983.13 ( m , 2 h ) , 3.89 ( s , 2 h ) , 7.25 ( t , j = 6.83 hz , 1 h ) , 7.397.55 ( m , 4 h ) , 7.60 ( m , j = 5.40 , 5.40 hz , 3 h ) , 8.04 ( d , j = 7.80 hz , 1 h ) , 8.178.25 ( m , 1 h ) , 12.10 ( s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 13.61 , 19.53 , 31.2 , 36.52 , 38.7 , 112.84 , 119.26 , 120.08 , 120.32 , 120.38 , 127.3 , 129.54 , 129.55 , 129.86 , 136.05 , 137.25 , 138.93 , 152.43 , 154.96 , 166.7 . hrms calcd for c22h22n4o2sna ( m + na ) , 429.1356 ; found , 429.1359 . compound 7a ( 25 mg , 0.07 mmol ) , n - amylamine ( 20 l , 0.16 mmol ) , triethylamine ( 19.82 l , 0.14 mmol ) , and hatu ( 29.76 mg , 0.08 mmol ) were reacted according to general procedure d to give 19.5 mg in 66% yield . h nmr ( 300 mhz , dmso - d6 ) ppm 0.74 ( t , j = 7.15 hz , 3 h ) , 1.071.25 ( m , 4 h ) , 1.311.45 ( m , 2 h ) , 2.983.15 ( m , 2 h ) , 3.88 ( s , 2 h ) , 7.24 ( t , j = 6.88 hz , 1 h ) , 7.407.54 ( m , 3 h ) , 7.567.68 ( m , 3 h ) , 8.04 ( d , j = 7.98 hz , 1 h ) , 8.20 ( t , j = 5.36 hz , 1 h ) , 12.10 ( s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 14.26 , 22.29 , 29.03 , 29.25 , 36.92 , 113.27 , 119.69 , 120.52 , 120.78 , 120.81 , 127.76 , 130.00 , 130.32 , 136.48 , 137.69 , 139.35 , 152.85 , 155.40 , 167.16 . hrms calcd for c23h25n4o2s ( m + h ) , 421.1693 ; found , 421.1689 . compound 7a ( 25 mg , 0.07 mmol ) , hexylamine ( 21 l , 0.16 mmol ) , triethylamine ( 19.82 l , 0.14 mmol ) , and hatu ( 29.76 mg , 0.08 mmol ) were reacted according to general procedure d to give 20 mg in 65% yield . h nmr ( 300 mhz , dmso - d6 ) ppm 0.76 ( t , j = 6.46 hz , 3 h ) , 1.011.49 ( m , 8 h ) , 3.06 ( q , j = 6.60 hz , 2 h ) , 3.87 ( s , 2 h ) , 7.24 ( t , j = 6.80 hz , 1 h ) , 7.417.55 ( m , 3 h ) , 7.557.66 ( m , 3 h ) , 8.05 ( d , j = 7.70 hz , 1 h ) , 8.19 ( t , j = 5.23 hz , 1 h ) , 12.10 ( s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 14.29 , 22.40 , 26.49 , 29.51 , 31.40 , 36.82 , 113.28 , 119.63 , 120.55 , 120.79 , 120.81 , 127.82 , 129.94 , 130.04 , 130.36 , 136.41 , 137.72 , 139.36 , 152.80 , 155.41 , 167.33 . hrms calcd for c24h27n4o2s ( m + h ) , 435.1849 ; found , 435.1846 . compound 7a ( 50 mg , 0.14 mmol ) , isobutylamine ( 31.11 l , 0.16 mmol ) , triethylamine ( 39.63 l , 0.28 mmol ) , and hatu ( 59.52 mg , 0.16 mmol ) were reacted according to general procedure d to give 17.71 mg in 31% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.720.92 ( m , 6 h ) , 1.68 ( dt , j = 13.47 , 6.64 hz , 1 h ) , 2.91 ( t , j = 6.60 hz , 2 h ) , 3.93 ( s , 2 h ) , 7.25 ( t , j = 6.97 hz , 1 h ) , 7.407.56 ( m , 3 h ) , 7.61 ( d , j = 6.23 hz , 3 h ) , 8.04 ( d , j = 7.70 hz , 1 h ) , 8.23 ( t , j = 6.23 hz , 1 h ) , 12.11 ( br s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 20.55 , 28.58 , 36.97 , 46.95 , 113.30 , 119.71 , 120.54 , 120.77 , 120.80 , 127.76 , 130.01 , 130.32 , 136.49 , 137.68 , 139.35 , 152.91 , 155.40 , 167.27 . hrms calcd for c22h22n4o2sna ( m + na ) , 429.1356 ; found , 429.1359 . compound 7a ( 25 mg , 0.07 mmol ) , isopentylamine ( 20 l , 0.16 mmol ) , triethylamine ( 19.82 l , 0.14 mmol ) , and hatu ( 29.76 mg , 0.08 mmol ) were reacted according to general procedure d to give 20 mg in 67% yield . h nmr ( 300 mhz , dmso - d6 ) ppm 0.78 ( d , j = 6.60 hz , 6 h ) , 1.29 ( q , j = 6.88 hz , 2 h ) , 1.54 ( tt , j = 13.38 , 6.84 hz , 1 h ) , 3.08 ( s , 2 h ) , 3.88 ( s , 2 h ) , 7.24 ( t , j = 7.01 hz , 1 h ) , 7.367.68 ( m , 6 h ) , 8.04 ( d , j = 7.98 hz , 1 h ) , 8.17 ( t , j = 5.50 hz , 1 h ) , 12.10 ( s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 22.71 , 25.45 , 36.86 , 37.68 , 38.43 , 113.29 , 119.64 , 120.55 , 120.77 , 120.80 , 127.83 , 129.95 , 130.03 , 130.35 , 136.42 , 137.71 , 139.34 , 152.82 , 155.40 , 167.23 . hrms calcd for c23h25n4o2s ( m + h ) , 421.1693 ; found , 421.1689 . compound 7a ( 25 mg , 0.07 mmol ) ) , 3,3-dimethylbutylamine ( 20 l , 0.16 mmol ) , triethylamine ( 19.82 l , 0.14 mmol ) , and hatu ( 29.76 mg , 0.08 mmol ) were reacted according to general procedure d and purified by reverse - phase chromatography ( c18 , 10:90 to 90:10 water : methanol gradient ) to give 18 mg in 60% yield . h nmr ( 300 mhz , dmso - d6 ) ppm 0.83 ( s , 9 h ) , 1.261.47 ( m , 2 h ) , 3.003.16 ( m , 2 h ) , 3.86 ( s , 2 h ) , 7.24 ( t , j = 7.29 hz , 1 h ) , 7.397.55 ( m , 3 h ) , 7.567.70 ( m , 3 h ) , 8.04 ( d , j = 7.98 hz , 1 h ) , 8.17 ( t , j = 5.50 hz , 1 h ) , 12.10 ( s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 24.12 , 28.28 , 34.68 , 37.17 , 50.51 , 113.31 , 119.70 , 120.48 , 120.78 , 120.81 , 127.77 , 130.00 , 130.01 , 130.32 , 136.51 , 137.68 , 139.35 , 153.01 , 155.39 , 165.92 . hrms calcd for c24h27n4o2s ( m + h ) , 435.1849 ; found , 435.1846 . compound 7a ( 25 mg , 0.07 mmol ) , 3-methylpentylamine ( 20 l , 0.16 mmol ) , triethylamine ( 19.82 l , 0.14 mmol ) and hatu ( 29.76 mg , 0.08 mmol ) according togeneral procedure d and purified by reverse - phase chromatography ( c18 , 10:90 to 90:10 water : methanol gradient ) to give 17 mg in 55% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.670.85 ( m , 6 h ) , 0.961.11 ( m , 1 h ) , 1.131.26 ( m , 2 h ) , 1.31 ( dd , j = 12.65 , 6.42 hz , 1 h ) , 1.371.51 ( m , 1 h ) , 2.993.17 ( m , 2 h ) , 3.87 ( s , 2 h ) , 7.24 ( t , j = 7.52 hz , 1 h ) , 7.417.55 ( m , 4 h ) , 7.567.69 ( m , 3 h ) , 8.04 ( d , j = 8.07 hz , 1 h ) , 8.17 ( t , j = 5.50 hz , 1 h ) , 12.10 ( s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 11.42 , 19.22 , 29.22 , 31.70 , 36.16 , 37.49 , 113.27 , 119.69 , 120.50 , 120.81 , 127.77 , 130.00 , 130.01 , 130.33 , 136.48 , 137.69 , 139.36 , 152.84 , 155.40 , 167.11 . hrms calcd for c24h26n4o2sna ( m + na ) , 457.1669 ; found , 457.1670 . compound 7a ( 25 mg , 0.07 mmol ) , 2-aminopentane ( 18.54 l , 0.16 mmol ) , triethylamine ( 19.82 l , 0.14 mmol ) , and hatu ( 29.76 mg , 0.08 mmol ) according to general procedure d to give 19.1 mg in 65% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.74 ( t , j = 6.97 hz , 3 h ) , 1.01 ( d , j = 6.60 hz , 3 h ) , 1.131.62 ( m , 4 h ) , 3.643.81 ( m , 1 h ) , 3.89 ( s , 2 h ) , 7.24 ( t , j = 7.33 hz , 1 h ) , 7.417.72 ( m , 6 h ) , 8.05 ( t , j = 6.60 hz , 2 h ) , 12.09 ( br s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 14.25 , 19.32 , 21.23 , 37.11 , 38.84 , 44.94 , 113.28 , 119.68 , 120.47 , 120.77 , 120.84 , 127.75 , 130.01 , 130.32 , 136.51 , 137.68 , 139.35 , 152.97 , 155.39 , 166.36 . hrms calcd for c23h24n4o2sna ( m + na ) , 443.1512 ; found , 443.1514 . compound 7a ( 25 mg , 0.07 mmol ) , 3-aminopentane ( 18.24 l , 0.16 mmol ) , triethylamine ( 19.82 l , 0.14 mmol ) , and hatu ( 29.76 mg , 0.08 mmol ) were reacted according to general procedure d to give 19.44 mg in 65% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.77 ( d , j = 6.83 hz , 6 h ) , 1.211.62 ( m , 4 h ) , 3.473.58 ( m , 1 h ) , 3.93 ( s , 1 h ) , 7.24 ( t , j = 6.83 hz , 1 h ) , 7.377.76 ( m , 6 h ) , 7.93 ( d , j = 7.31 hz , 1 h ) , 8.05 ( d , j = 7.80 hz , 1 h ) , 12.10 ( br s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 10.87 , 27.40 , 37.00 , 52.36 , 113.29 , 119.64 , 120.50 , 120.73 , 120.86 , 127.82 , 129.95 , 130.05 , 130.36 , 136.43 , 137.71 , 139.34 , 152.98 , 155.40 , 167.06 . hrms calcd for c23h25n4o2s ( m + h ) , 421.1693 ; found , 421.1695 . compound 7a ( 50 mg , 0.14 mmol ) , t - butylamine ( 32.9 l , 0.16 mmol ) , triethylamine ( 39.63 l , 0.28 mmol ) , and hatu ( 59.52 mg , 0.16 mmol ) were reacted according to general procedure d to give 22.6 mg in 39.7% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 1.151.37 ( m , 9 h ) , 3.85 ( s , 2 h ) , 7.26 ( t , j = 6.83 hz , 1 h ) , 7.417.56 ( m , 4 h ) , 7.577.71 ( m , 3 h ) , 7.93 ( s , 1 h ) , 8.07 ( d , j = 7.80 hz , 1 h ) , 12.11 ( s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 28.86 , 37.86 , 50.89 , 113.34 , 119.68 , 120.54 , 120.74 , 120.78 , 127.76 , 129.99 , 130.00 , 130.31 , 136.54 , 137.68 , 139.37 , 153.13 , 155.40 , 166.60 . hrms calcd for c22h22n4o2sna ( m + na ) , 429.1356 ; found , 429.1359 . compound 7a ( 50 mg , 0.14 mmol ) , tetrahydrofurfurylamine ( 32.31 l , 0.16 mmol ) , triethylamine ( 39.63 l , 0.28 mmol ) and hatu ( 59.52 mg , 0.16 mmol ) were reacted according to general procedure d to give 35 mg in 57.5% yield . ir : 3372 , 3193 ( nh ) , 1651 ( co ) cm . h nmr ( 400 mhz , dmso - d6 ) ppm 1.321.61 ( m , 1 h ) , 1.632.06 ( m , 3 h ) , 2.963.26 ( m , 2 h ) , 3.523.64 ( m , 1 h ) , 3.643.75 ( m , 1 h ) , 3.763.86 ( m , 1 h ) , 3.92 ( s , 2 h ) , 7.25 ( t , j = 6.97 hz , 1 h ) , 7.357.71 ( m , 6 h ) , 8.07 ( d , j = 8.06 hz , 1 h ) , 8.30 ( t , j = 5.50 hz , 1 h ) , 12.10 ( br s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 25.12 , 28.4 , 36.38 , 43.03 , 67.15 , 77.11 , 112.81 , 119.25 , 120.11 , 120.39 , 127.32 , 129.55 , 129.57 , 129.88 , 136.05 , 137.25 , 138.92 , 152.4 , 154.95 , 167.14 . hrms calcd for c23h22n4o3sna ( m + na ) , 457.1305 ; found , 457.1308 . compound 7a ( 25 mg , 0.07 mmol ) , 4-aminopiperidine ( 16.6 ul , 0.16 mmol ) , triethylamine ( 19.82 l , 0.14 mmol ) , and hatu ( 29.76 mg , 0.08 mmol ) were reacted according to general procedure d to give 17 mg in 65% yield . ir : 3372 , 3193 ( nh ) , 1651 ( co ) cm . h nmr ( 400 mhz , dmso - d6 ) ppm 1.27 ( d , j = 11.36 hz , 1 h ) , 1.46 ( d , j = 9.90 hz , 1 h ) , 1.772.05 ( m , 2 h ) , 2.642.85 ( m , 1 h ) , 4.004.43 ( m , 3 h ) , 7.147.38 ( m , 2 h ) , 7.417.80 ( m , 6 h ) , 8.04 ( d , j = 7.70 hz , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 33.41 , 35.16 , 36.00 , 36.46 , 40.91 , 44.69 , 45.37 , 48.25 , 113.30 , 119.70 , 120.67 , 120.75 , 120.79 , 127.76 , 130.00 , 130.03 , 130.33 , 136.51 , 137.63 , 137.68 , 139.36 , 152.93 , 152.97 , 155.40 , 165.44 , 166.23 . hrms calcd for c23h24n5o2s ( m + h ) , 434.1645 ; found , 434.1648 . compound 7a ( 50 mg , 0.14 mmol ) , dope ( 32.9 l , 0.16 mmol ) , triethylamine ( 39.63 l , 0.28 mmol ) , and hatu ( 59.52 mg , 0.16 mmol ) were reacted according to general procedure d to give 16.4 mg in 10.9% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.84 ( t , j = 6.83 hz , 6 h ) , 1.051.75 ( m , 50 h ) , 1.96 ( br s , 8 h ) , 2.23 ( t , j = 7.31 hz , 4 h ) , 3.02 ( s , 3 h ) , 3.143.26 ( m , 2 h ) , 3.593.83 ( m , 3 h ) , 3.90 ( s , 2 h ) , 4.024.17 ( m , 1 h ) , 4.26 ( d , j = 9.26 hz , 1 h ) , 5.06 ( br s , 1 h ) , 5.30 ( br s , 3 h ) , 7.24 ( t , j = 7.31 hz , 1 h ) , 7.387.75 ( m , 6 h ) , 8.09 ( d , j = 7.80 hz , 1 h ) , 8.87 ( br s , 1 h ) , 10.12 ( br s , 1 h ) , 12.08 ( br s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 8.84 , 14.38 , 14.39 , 22.58 , 24.85 , 24.91 , 27.02 , 27.05 , 28.88 , 28.91 , 28.98 , 29.00 , 29.07 , 29.12 , 29.19 , 29.33 , 29.43 , 29.45 , 29.57 , 31.77 , 33.82 , 34.00 , 37.11 , 45.62 , 62.78 , 63.05 , 70.84 , 70.89 , 113.19 , 119.66 , 120.61 , 120.86 , 120.89 , 127.62 , 129.96 , 130.00 , 130.27 , 136.50 , 137.72 , 139.36 , 152.72 , 155.40 , 167.22 , 172.67 , 172.91 . hrms calcd for c59h89n4o10psna ( m + na ) , 1099.5929 ; found , 1099.5930 . compound 7b ( 17.7 mg , 0.05 mmol ) , triethylamine ( 13.8 l , 0.1 mmol ) , and cyclohexylamine ( 6.2 l , 0.06 mmol ) were dissolved in anhydrous dmf ( 0.5 ml ) . hatu ( 20.74 mg , 0.06 mmol ) dissolved in 0.2 ml of dmf was added to the reaction mixture and stirred for 20 min and concentrated in vacuo . the crude material was recrystallized in meoh to give 9.1 mg in 41% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.992.17 ( m , 16 h ) , 2.602.88 ( m , 4 h ) , 3.99 ( br s , 2 h ) , 4.174.45 ( m , 1 h ) , 7.20 ( t , j = 7.33 hz , 1 h ) , 7.347.65 ( m , 2 h ) , 8.01 ( d , j = 8.07 hz , 1 h ) , 8.22 ( d , j = 6.97 hz , 1 h ) , 11.86 ( br s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 19.02 , 24.92 , 25.28 , 25.63 , 26.36 , 28.79 , 32.87 , 37.21 , 48.49 , 56.47 , 61.95 , 113.22 , 120.27 , 120.56 , 120.72 , 120.82 , 127.59 , 136.47 , 139.26 , 151.90 , 155.96 , 166.22 . hrms calcd for c24h30n4o2sna ( m + na ) , 461.1982 ; found , 461.1983 . compound 7c ( 26.1 mg , 0.065 mmol ) , triethylamine ( 18.1 l , 0.13 mmol ) , and cyclohexylamine ( 8.2 l , 0.071 mmol ) were dissolved in anhydrous dmf ( 0.1 ml ) . hatu ( 27.2 mg , 0.071 mmol ) dissolved in 0.2 ml of dmf was added to the reaction mixture and stirred for 20 min and concentrated in vacuo . the crude material was recrystallized in meoh to give 27 mg in 86% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.761.38 ( m , 5 h ) , 1.421.95 ( m , 5 h ) , 3.433.55 ( m , 1 h ) , 3.744.00 ( m , 2 h ) , 7.29 ( t , j = 6.97 hz , 1 h ) , 7.41 ( d , j = 8.43 hz , 1 h ) , 7.477.66 ( m , 4 h ) , 7.687.81 ( m , 2 h ) , 8.018.36 ( m , 4 h ) , 12.17 ( s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 24.82 , 25.57 , 31.23 , 32.72 , 32.75 , 36.27 , 36.97 , 48.36 , 113.39 , 119.55 , 120.69 , 120.81 , 120.84 , 122.13 , 126.31 , 127.24 , 127.99 , 128.25 , 128.88 , 129.03 , 130.07 , 130.92 , 132.83 , 134.46 , 138.05 , 139.48 , 153.40 , 155.47 , 162.87 , 166.19 . hrms calcd for c28h26n4o2sna ( m + na ) , 505.1669 ; found , 505.1671 . compound 7d ( 25 mg , 0.066 mmol ) , triethylamine ( 19.5 l , 0.13 mmol ) , and cyclohexylamine ( 8.2 l , 0.073 mmol ) were dissolved in anhydrous dmf ( 0.1 ml ) . hatu ( 27.2 mg , 0.073 mmol ) dissolved in 0.2 ml of dmf was added to the reaction mixture and stirred for 20 min and concentrated in vacuo . the crude material was recrystallized in meoh to give 18.28 mg in 60% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.831.42 ( m , 5 h ) , 1.442.08 ( m , 5 h ) , 2.873.17 ( m , 2 h ) , 3.954.24 ( m , 2 h ) , 4.254.62 ( m , 2 h ) , 7.33 ( br s , 6 h ) , 7.878.48 ( m , 3 h ) , 11.99 ( s , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 24.91 , 25.63 , 32.83 , 34.09 , 36.63 , 45.66 , 48.51 , 113.26 , 119.40 , 120.37 , 120.67 , 120.84 , 127.16 , 127.76 , 129.10 , 137.30 , 138.26 , 139.36 , 151.68 , 154.92 , 166.19 . hrms calcd for c26h28n4o2sna ( m + na ) , 483.1825 ; found , 483.1827 . compound 7b ( 25 mg , 0.07 mmol ) , triethylamine ( 19.5 l , 0.14 mmol ) , and cyclopentylamine ( 8.82 l , 0.08 mmol ) were dissolved in anhydrous dmf ( 0.5 ml ) . hatu ( 29.3 mg , 0.08 mmol ) dissolved in 0.2 ml of dmf was added to the reaction mixture and stirred for 20 min and concentrated in vacuo . the crude material was recrystallized in meoh to give 9.3 mg in 30.3% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.972.09 ( m , 18 h ) , 2.612.83 ( m , 4 h ) , 4.02 ( br s , 1 h ) , 4.174.37 ( m , 1 h ) , 7.20 ( t , j = 7.30 hz , 1 h ) , 7.367.56 ( m , 2 h ) , 8.00 ( d , j = 8.06 hz , 1 h ) , 8.32 ( d , j = 7.33 hz , 1 h ) , 11.86 ( br s , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 23.97 , 25.25 , 26.34 , 28.78 , 32.73 , 37.02 , 51.21 , 61.99 , 113.23 , 120.31 , 120.54 , 120.67 , 120.79 , 127.66 , 136.48 , 139.24 , 151.93 , 155.94 , 166.85 . hrms calcd for c23h28n4o2sna ( m + na ) , 447.1825 ; found , 447.1827 . to a solution of compound 1 ( 50 mg , 0.12 mmol ) in dmf ( 1 ml ) in a flame - dried flask was added nah 60% dispersion in mineral oil ( 9.24 mg , 0.24 mmol ) and stirred at room temperature for 10 min . methyl iodide ( 14.3 l , 0.24 mmol ) was then added to the solution and stirred overnight . reaction mixture was concentrated and purified by preparative thin - layer chromatography ( 40:60 ethyl acetate : hexane ) to give 18.18 mg in 33.9% yield of compound 42 and 5.3 mg in 9.6% yield of compound 43 . 42 : h nmr ( 400 mhz , dmso - d6 ) ppm 0.931.37 ( m , 5 h ) , 1.401.90 ( m , 5 h ) , 3.443.58 ( m , 1 h ) , 3.88 ( s , 2 h ) , 4.15 ( s , 3 h ) , 7.31 ( t , j = 7.33 hz , 1 h ) , 7.397.53 ( m , 2 h ) , 7.537.78 ( m , 5 h ) , 8.058.27 ( m , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 24.90 , 25.62 , 31.47 , 32.82 , 37.14 , 48.41 , 111.31 , 119.12 , 120.09 , 120.74 , 120.92 , 128.03 , 129.99 , 130.34 , 136.35 , 137.47 , 140.40 , 153.27 , 155.75 , 166.14 . hrms calcd for c25h27n4o2s ( m + h ) , 447.1849 ; found , 447.1852 . 43 : h nmr ( 400 mhz , dmso - d6 ) ppm 0.761.98 ( m , 10 h ) , 2.10 ( s , 3 h ) , 3.97 ( s , 2 h ) , 4.23 ( s , 3 h ) , 7.16 ( t , j = 7.52 hz , 1 h ) , 7.267.85 ( m , 8 h ) , 8.07 ( d , j = 7.70 hz , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 24.85 , 25.60 , 31.43 , 32.28 , 38.02 , 50.24 , 62.03 , 111.44 , 119.63 , 120.34 , 120.85 , 121.08 , 128.20 , 129.74 , 130.25 , 135.43 , 136.73 , 139.34 , 155.99 , 156.50 , 169.99 . hrms calcd for c26h29n4o2s ( m + h ) , 461.2006 ; found , 461.2007 . to a flame - dried flask , bromobutyric acid ( 50 l , 0.43 mmol ) was dissolved in anhydrous meoh ( 3 ml ) followed by the addition of tmscl ( 272.45 l , 2.15 mmol ) . the solution was stirred overnight at room temperature and concentrated in vacuo to remove all trace of meoh and hcl . in a separate flame - dried flask , compound 1 ( 25 mg , 0.06 mmol ) was dissolved in dmf ( 1 ml ) . nah 60% dispersion ( 2.31 mg , 0.06 mmol ) was added to the solution . after stirring at room temperature for 10 min , methyl iodide ( 3.6 l , 0.06 mmol ) was added and stirred for an additional 12 h. the reaction mixture was concentrated in vacuo and purified by preparative thin - layer chromatography to give 1.3 mg in 4% yield . ir : 3293 ( nh ) , 1732 ( co ester ) , 1683 ( co amide ) cm . h nmr ( 400 mhz , dmso - d6 ) ppm 0.991.32 ( m , 5 h ) , 1.52 ( d , j = 12.19 hz , 1 h ) , 1.571.80 ( m , 4 h ) , 1.912.10 ( m , 2 h ) , 2.29 ( t , j = 7.31 hz , 2 h ) , 3.47 ( s , 3 h ) , 3.47 ( br s , 1 h ) , 3.89 ( s , 2 h ) , 4.62 ( t , j = 6.83 hz , 2 h ) , 7.30 ( t , j = 7.56 hz , 1 h ) , 7.397.51 ( m , 2 h ) , 7.527.68 ( m , 4 h ) , 7.74 ( s , 1 h ) , 8.11 ( d , j = 7.80 hz , 1 h ) , 8.17 ( d , j = 7.80 hz , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 24.88 , 25.59 , 26.00 , 30.71 , 32.79 , 37.02 , 51.71 , 111.34 , 118.74 , 120.19 , 120.80 , 121.09 , 128.18 , 129.93 , 130.02 , 130.37 , 136.29 , 137.81 , 139.77 , 153.45 , 155.47 , 166.28 , 173.19 . hrms calcd for c29h32n4o4sna ( m + na ) , 555.2036 ; found , 555.2036 . compound 6a ( 100 mg , 0.34 mmol ) and koh ( 38.2 g , 0.68 mmol ) were suspended in 2 ml of dimethylacetamide in a flame - dried flask with stirring . t - butyl chloroacetate ( 49 l , 0.34 mmol ) was immediately added to the reaction mixture and stirred at room temperature monitoring with thin - layer chromatography ( 1:99 meoh : dcm ) . upon completion , reaction mixture was extracted with ethyl acetate ( 20 ml ) and water ( 40 ml ) , dried over mgso4 , and concentrated in vacuo . etoh ( 5 ml ) was added to the resulting viscous liquid , and pure product was filtered to give 55 mg in 40% yield . ir : 3192 ( nh ) , 1732 ( co ester ) , 1674 ( co amide ) cm . h nmr ( 400 mhz , dmso - d6 ) ppm 1.321.48 ( m , 9 h ) , 3.91 ( s , 2 h ) , 7.26 ( t , j = 7.31 hz , 1 h ) , 7.427.55 ( m , 4 h ) , 7.567.69 ( m , 3 h ) , 7.96 ( d , j = 8.29 hz , 1 h ) , 12.13 ( s , 1 h ) . hrms calcd for c22h21n3o3sna ( m + na ) , 430.1196 ; found , 430.1197 . compound 45 ( 50 mg , 0.12 mmol ) , nah 60% dispersion in mineral oil ( 3.3 mg , 0.14 mmol ) , iodopropane ( 13.1 l , 0.14 mmol ) , and dmf ( 1 ml ) were reacted according to general procedure e and purified by silica gel chromatography ( 10:90 ethyl acetate : hexane ) to give 32.8 mg in 61% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.82 ( t , j = 7.31 hz , 3 h ) , 1.40 ( s , 9 h ) , 1.661.95 ( m , 2 h ) , 3.91 ( s , 2 h ) , 4.56 ( t , j = 6.58 hz , 2 h ) , 7.30 ( br s , 1 h ) , 7.397.68 ( m , 6 h ) , 7.75 ( d , j = 8.29 hz , 1 h ) , 7.99 ( d , j = 7.80 hz , 1 h ) . hrms calcd for c25h27n3o3sna ( m + na ) , 472.1665 ; found , 472.1668 . compound 45 ( 50 mg , 0.12 mmol ) , nah 60% dispersion in mineral oil ( 3.3 mg , 0.14 mmol ) , iodopentane ( 17.64 l , 0.14 mmol ) , and dmf ( 1 ml ) were reacted according to general procedure e and purified by silica gel chromatography ( 10:90 ethyl acetate : hexane ) to give 33.8 mg in 59% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.80 ( t , j = 6.83 hz , 3 h ) , 1.25 ( d , j = 6.83 hz , 4 h ) , 1.321.55 ( m , 9 h ) , 1.641.83 ( m , 2 h ) , 3.91 ( s , 2 h ) , 4.59 ( t , j = 7.31 hz , 2 h ) , 7.30 ( t , j = 7.56 hz , 1 h ) , 7.46 ( dd , j = 7.56 , 1.71 hz , 2 h ) , 7.54 ( t , j = 7.31 hz , 1 h ) , 7.577.67 ( m , 3 h ) , 7.72 ( d , j = 8.78 hz , 1 h ) , 7.99 ( d , j = 7.80 hz , 1 h ) . hrms calcd for c27h31n3o3sna ( m + na ) , 500.1978 ; found , 500.1975 . compound 45 ( 50 mg , 0.12 mmol ) , nah 60% dispersion in mineral oil ( 3.3 mg , 0.14 mmol ) , bromododecane ( 32.4 l , 0.14 mmol ) , and dmf ( 1 ml ) were reacted according to general procedure e and purified by silica gel chromatography ( 10:90 ethyl acetate : hexane ) to give 37.4 mg in 54% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.84 ( t , j = 6.83 hz , 3 h ) , 1.21 ( br s , 18 h ) , 1.40 ( s , 9 h ) , 1.641.86 ( m , 3 h ) , 3.91 ( s , 2 h ) , 4.59 ( t , j = 7.07 hz , 2 h ) , 7.29 ( t , j = 7.80 hz , 1 h ) , 7.45 ( dd , j = 7.31 , 1.95 hz , 2 h ) , 7.54 ( t , j = 7.80 hz , 1 h ) , 7.587.67 ( m , 3 h ) , 7.72 ( d , j = 8.29 hz , 1 h ) , 7.98 ( d , j = 7.80 hz , 1 h ) . hrms calcd for c34h45n3o3sna ( m + na ) , 598.3074 ; found , 598.3070 . compound 45 ( 318 mg , 0.78 mmol ) , nah 60% dispersion ( 38.16 mg , 0.86 mmol ) , bromoacetonitrile ( 65.5 l , 0.86 mmol ) , and dmf ( 20 ml ) were reacted according to general procedure e to give 247.8 mg in 71% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 1.41 ( s , 9 h ) , 3.94 ( s , 2 h ) , 5.87 ( s , 2 h ) , 7.43 ( t , j = 7.31 hz , 1 h ) , 7.477.55 ( m , 2 h ) , 7.587.72 ( m , 4 h ) , 7.88 ( d , j = 8.78 hz , 1 h ) , 8.03 ( d , j = 8.29 hz , 1 h ) . hrms calcd for c24h22n4o3sna ( m + na ) , 469.1305 ; found , 469.1303 . compound 46a ( 29.74 mg , 0.07 mmol ) , acetonitrile ( 1 ml ) , and trifluoroacetic acid ( 1 ml ) were reacted according to general procedure f to give 26 mg in quantitative yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.81 ( t , j = 6.58 hz , 3 h ) , 1.75 ( m , 2 h ) , 3.96 ( s , 2 h ) , 4.56 ( t , j = 7.07 hz , 2 h ) , 7.29 ( t , j = 7.31 hz , 1 h ) , 7.387.88 ( m , 7 h ) , 7.99 ( d , j = 8.29 hz , 1 h ) . hrms calcd for c21h19n3o3sna ( m + na ) , 416.1039 ; found , 416.1036 . compound 46b ( 30.98 mg , 0.065 mmol ) , acetonitrile ( 1 ml ) , and trifluoroacetic acid ( 1 ml ) were reacted according to general procedure f to give 27 mg in quantitative yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.81 ( t , j = 6.58 hz , 3 h ) , 0.991.43 ( m , 4 h ) , 1.621.84 ( m , 2 h ) , 3.96 ( s , 2 h ) , 4.59 ( t , j = 7.07 hz , 2 h ) , 7.29 ( t , j = 7.31 hz , 1 h ) , 7.45 ( br s , 2 h ) , 7.517.68 ( m , 4 h ) , 7.72 ( d , j = 8.29 hz , 1 h ) , 7.98 ( d , j = 8.29 hz , 1 h ) . hrms calcd for c23h23n3o3sna ( m + na ) , 444.1352 ; found , 444.1351 . compound 46c ( 34.81 mg , 0.06 mmol ) , acetonitrile ( 1 ml ) , and trifluoroacetic acid ( 1 ml ) were reacted according to general procedure f to give 28.6 mg in 92% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.82 ( t , j = 6.83 hz , 3 h ) , 1.19 ( d , j = 7.80 hz , 18 h ) , 1.71 ( d , j = 6.34 hz , 2 h ) , 3.94 ( s , 2 h ) , 4.56 ( t , j = 7.07 hz , 2 h ) , 7.27 ( t , j = 7.80 hz , 1 h ) , 7.367.47 ( m , 2 h ) , 7.59 ( s , 4 h ) , 7.70 ( d , j = 8.29 hz , 1 h ) , 7.96 ( d , j = 7.80 hz , 1 h ) , 12.74 ( br s , 1 h ) . hrms calcd for c30h37n3o3sna ( m + na ) , 542.2448 ; found , 542.2444 . compound 46a ( 29.74 mg , 0.07 mmol ) , acetonitrile ( 1 ml ) , and trifluoroacetic acid ( 1 ml ) were reacted according to general procedure f to give 26 mg in quantitative yield . h nmr ( 400 mhz , dmso - d6 ) ppm 3.844.15 ( m , 2 h ) , 5.87 ( s , 2 h ) , 7.42 ( t , j = 7.56 hz , 1 h ) , 7.477.56 ( m , 2 h ) , 7.577.74 ( m , 4 h ) , 7.88 ( d , j = 8.29 hz , 1 h ) , 8.03 ( d , j = 7.80 hz , 1 h ) , 12.89 ( br s , 1 h ) . hrms calcd for c20h14n4o3sna ( m + na ) , 413.0679 ; found , 413.0676 . compound 47a ( 29.4 mg , 0.075 mmol ) , hatu ( 31.3 mg , 0.082 mmol ) , triethylamine ( 20.8 l , 0.15 mmol ) , cyclohexylamine ( 9.43 l , 0.082 mmol ) , and dmf ( 1 ml ) were reacted according to general procedure d to give 4 mg in 11% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.82 ( t , j = 7.33 hz , 3 h ) , 1.001.37 ( m , 5 h ) , 1.421.99 ( m , 7 h ) , 3.88 ( s , 2 h ) , 4.56 ( t , j = 6.97 hz , 2 h ) , 7.29 ( t , j = 7.70 hz , 1 h ) , 7.417.50 ( m , 1 h ) , 7.507.68 ( m , 4 h ) , 7.74 ( d , j = 8.43 hz , 1 h ) , 8.10 ( d , j = 8.07 hz , 1 h ) , 8.17 ( d , j = 8.07 hz , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 11.35 , 24.15 , 24.89 , 25.61 , 32.83 , 37.12 , 45.80 , 48.40 , 111.49 , 118.75 , 120.09 , 120.61 , 120.99 , 127.98 , 129.96 , 129.98 , 130.29 , 136.38 , 137.68 , 139.83 , 153.35 , 155.43 , 166.14 . hrms calcd for c27h31n4o2s ( m + h ) , 475.2162 ; found , 475.2164 . compound 47b ( 34 mg , 0.08 mmol ) , hatu ( 34 mg , 0.088 mmol ) , triethylamine ( 22.5 l , 0.16 mmol ) , cyclohexylamine ( 10.2 l , 0.088 mmol ) , and dmf ( 1 ml ) were reacted according to general procedure d to give 21 mg in 52% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.80 ( t , j = 6.97 hz , 3 h ) , 1.001.40 ( m , 9 h ) , 1.431.92 ( m , 7 h ) , 3.443.58 ( m , 1 h ) , 3.88 ( s , 2 h ) , 4.58 ( t , j = 7.15 hz , 2 h ) , 7.29 ( t , j = 7.33 hz , 1 h ) , 7.407.68 ( m , 6 h ) , 7.72 ( d , j = 8.43 hz , 1 h ) , 8.038.32 ( m , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 14.35 , 22.29 , 24.91 , 25.62 , 28.70 , 30.56 , 32.84 , 37.11 , 44.38 , 48.42 , 111.45 , 118.70 , 120.13 , 120.64 , 121.02 , 128.02 , 129.99 , 130.31 , 136.39 , 137.70 , 139.74 , 153.36 , 155.44 , 166.16 . hrms calcd for c29h34n4o2s ( m + h ) , 502.24025 ; found , 502.24018 . compound 47c ( 28.6 mg , 0.055 mmol ) , hatu ( 23 mg , 0.061 mmol ) , triethylamine ( 15.33 l , 0.110 mmol ) , cyclohexylamine ( 7 l , 0.061 mmol ) , and dmf ( 1 ml ) were reacted according to general procedure d and purified by silica gel chromatography ( 20:80 ethyl acetate : hexane ) to give 22 mg in 67% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.730.92 ( m , 4 h ) , 1.20 ( d , j = 6.97 hz , 22 h ) , 1.411.89 ( m , 7 h ) , 3.453.61 ( m , 1 h ) , 3.88 ( s , 2 h ) , 4.58 ( t , j = 6.60 hz , 2 h ) , 7.28 ( t , j = 7.52 hz , 1 h ) , 7.367.80 ( m , 7 h ) , 8.058.26 ( m , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 11.85 , 14.50 , 22.63 , 24.97 , 25.70 , 26.59 , 29.19 , 29.44 , 30.88 , 31.82 , 32.90 , 37.20 , 44.48 , 48.42 , 111.54 , 118.70 , 120.22 , 120.69 , 121.09 , 128.06 , 130.05 , 130.39 , 136.46 , 137.79 , 139.83 , 153.41 , 155.50 , 166.17 . hrms calcd for c36h49n4o2s ( m + h ) , 601.3571 ; found , 601.3570 . compound 47d ( 150 mg , 0.39 mmol ) , hatu ( 161 mg , 0.42 mmol ) , triethylamine ( 59 l , 0.77 mmol ) , cyclohexylamine ( 49 l , 0.42 mmol ) , and dmf ( 5 ml ) were reacted according to general procedure d to give 157.3 mg in 86.7% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.971.33 ( m , 5 h ) , 1.451.87 ( m , 5 h ) , 3.453.58 ( m , 1 h ) , 3.90 ( s , 2 h ) , 5.87 ( s , 2 h ) , 7.42 ( t , j = 7.80 hz , 1 h ) , 7.477.56 ( m , 2 h ) , 7.577.74 ( m , 4 h ) , 7.88 ( d , j = 8.29 hz , 1 h ) , 8.17 ( dd , j = 13.16 , 7.80 hz , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 24.92 , 25.62 , 32.84 , 33.04 , 37.21 , 48.45 , 111.45 , 116.75 , 118.20 , 121.01 , 121.35 , 122.12 , 129.02 , 129.90 , 130.08 , 130.56 , 135.95 , 138.98 , 139.65 , 155.44 , 155.50 , 166.02 . hrms calcd for c26h26n5o2s ( m + h ) , 472.1802 ; found , 472.1803 . a koh ( 6 mg , 0.11 mmol ) solution in water ( 50 l ) was added to a solution of compound 51 in dimethylacetamide ( 200 l ) and stirred at rt overnight . reaction mixture was acidified with 3 m hcl , extracted with ethyl acetate and water , dried over mgso4 , and concentrated to dryness in vacuo . h nmr ( 400 mhz , dmso - d6 ) ppm 0.941.36 ( m , 5 h ) , 1.431.89 ( m , 5 h ) , 3.433.62 ( m , 1 h ) , 3.89 ( s , 2 h ) , 5.24 ( s , 2 h ) , 7.127.22 ( m , 1 h ) , 7.30 ( t , j = 7.33 hz , 1 h ) , 7.42 ( m , j = 7.30 , 2.20 hz , 2 h ) , 7.487.67 ( m , 5 h ) , 8.10 ( d , j = 7.70 hz , 1 h ) , 8.18 ( d , j = 7.70 hz , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) ppm 24.90 , 25.59 , 32.82 , 37.05 , 46.85 , 48.48 , 111.36 , 119.30 , 120.36 , 120.91 , 120.95 , 128.13 , 129.87 , 130.05 , 130.41 , 136.14 , 137.82 , 140.64 , 153.47 , 155.65 , 166.26 , 169.77 . hrms calcd for c26h27n5o3sna ( m + na ) , 512.1727 ; found , 512.1726 . nine - week - old c57bl/6 ( wild - type , wt ) and cd14 ( c57bl/6 background ) were purchased from the jackson laboratories ( bar harbor , ma ) . shizuo akira ( osaka university , japan ) and backcrossed for 10 generations onto the c57bl/6 background at university of california , san diego ( ucsd ) . all animal experiments were approved by the ucsd institutional animal care and use committee . bmdc ( 10cells per well ) were plated in 96-well plates in 200 l of complete rpmi1640 supplemented with 10% fetal calf serum ( fcs ; sigma aldrich ) , 100 u / ml penicillin , and 100 g / ml streptomycin ( invitrogen ) . the cells were incubated with graded concentrations of the compounds for 18 h at 37 c , 5% co2 . after 18 h incubation , lps ( purified lps , invivogen , san diego , ca ) or mpla ( 1 g / ml synthetic mpla , invivogen , san diego , ca ) were used as positive controls . the levels of il-6 in the culture supernatants were determined by elisa ( bd biosciences , la jolla , ca ) . each cytokine induction curve was first converted to a percent activity curve , and then the auc of the percent activity curve was calculated . the process of converting to a percent activity curve allowed subtracting background and adjusting for plate - to - plate variation . finally , the auc values were normalized to the activity of compound 1 within each experiment , set at 100 . the cultures stimulated with lps 10 ng / ml , and 5 m compound 1 released an average of 20.6 ng / ml 4.7 sd and 10.5 ng / ml 1.3 sd of il-6 , respectively . murine or humantlr4 hek blue cells ( invivogen , 2.5 10 cells per well of a 96-well plate ) , or nfb / seaporter hek 293 cells ( imgenex , san diego , ca ) for human tlr2 , tlr3 , tlr5 , tlr7 , tlr8 , or tlr9 ( 5 10 cells per well of 96 well plate ) were incubated with graded doses of compound 1 . seap activity in the supernatants was determined by a colorimetric assay , using either the seaporter assay kit ( imgenex ) , with absorbance read at 405 nm , or quantiblue ( invivogen ) , with absorbance read at 630 nm . stimulation of the human tlr4 cells with 10 ng / ml lps resulted in od630 of 1.90 relative to 0.88 of cells stimulated with 10 m compound 1 . the relative reporter activation for the murine tlr4 cells for cells stimulated with 10 ng / ml lps and 5 m compound 1 was 1.70 0.08 and 1.52 0.03 , respectively . human pbmc were isolated from buffy coats obtained from the san diego blood bank ( san diego , ca ) as described previously . pbmc ( 1 10/ml ) were incubated with various compounds in complete rpmi for 18 h at 37 c , 5% co2 , and culture supernatants were collected . the levels of il-8 in the supernatants were determined by elisa ( bd biosciences , la jolla , ca ) . human cell cultures were treated with 10 ng / ml lps as the positive control . cultures treated with lps 10 ng / ml and compound 1 10 m released averages of 14.5 0.6 ng / ml sd and 7.8 0.8 ng / ml sd of il-8 , respectively . l929 cells stably expressing an interferon sensitive response element ( isre ) luciferase reporter construct were kindly provided by dr . the bioactivity of type i ifn in mbmdc supernatants was measured by luciferase assay using l929-isre cells as described previously . l929-isre cells were plated at 5 10 cells per well in dulbecco s modified eagle medium ( invitrogen ) supplemented with 10% fcs , 100 u / ml penicillin , and 100 g / ml streptomycin ( dmem-10 ) in a 96-well white - walled clear - bottom plate . thus , 50 l of mbmdc supernatant was incubated with l929-isre cells in 50 l of dmem for 6 h. mu - ifn beta standard ( pbl interferon source , piscataway , nj ) was used as a standard . the luciferase activities were measured by steady - glo luciferase assay buffer ( promega , madison , wi ) . in addition , the levels of ip-10 , a surrogate marker of type i ifn , in the supernatants were determined by elisa ( r&d systems , minneapolis , mn ) . the cultures stimulated with lps 10 ng / ml , and 5 m compound 1 released an average of 234.8 8.6 and 590.0 10.5 the data are represented as mean standard error of the mean ( sem ) . areas - under - curve ( aucs ) were calculated using the trapezoid method . prism 5 ( graphpad software ) statistical software was used to obtain p - values for comparison between groups ( p < 0.05 was considered significant ) . for the in vitro studies , two - tailed student s t test was used to compare two groups , and one - way anova dunnett s test was used to compare multiple groups .

a cell - based high - throughput screen to identify small molecular weight stimulators of the innate immune system revealed substituted pyrimido[5,4-b]indoles as potent nfb activators . the most potent hit compound selectively stimulated toll - like receptor 4 ( tlr4 ) in human and mouse cells . synthetic modifications of the pyrimido[5,4-b]indole scaffold at the carboxamide , n-3 , and n-5 positions revealed differential tlr4 dependent production of nfb and type i interferon associated cytokines , il-6 and interferon -induced protein 10 ( ip-10 ) respectively . specifically , a subset of compounds bearing phenyl and substituted phenyl carboxamides induced lower il-6 release while maintaining higher ip-10 production , skewing toward the type i interferon pathway . substitution at n-5 with short alkyl substituents reduced the cytotoxicity of the leading hit compound . computational studies supported that active compounds appeared to bind primarily to md-2 in the tlr4/md-2 complex . these small molecules , which stimulate innate immune cells with minimal toxicity , could potentially be used as adjuvants or immune modulators .

Introduction Results and Discussion Conclusion Experimental Section

the innate immune response is the first line of defense against microbial pathogens such as viruses , bacteria , fungi , and protozoa . a critical component of the innate immune response is the nfb family of transcription factors . the toll - like receptors ( tlrs ) are critical components of the innate immune system that regulate nfb activation . these pathogen - associated molecular patterns ( pamps ) and their corresponding tlrs include : lipopeptides ( tlr2 ) , double - stranded rna ( tlr3 ) , lipopolysaccharide ( lps , tlr4 ) , bacterial flagellin ( tlr5 ) , guanine and uridine - rich single - stranded rna ( tlr7 , 8) , and hypo - methylated cpg rich dna ( tlr9 ) . innate immune cells use pattern recognition receptors ( prrs ) such as tlrs to promote a rapid response to perceived threats before pathogen - specific adaptive immunity can be established . indeed , this rapid response , involving multiple components of the innate immune system , has been recognized to guide the type of adaptive immune response that is most effective for the specific pathogenic threat . the discovery that certain small molecules could serve as specific innate immune receptor ligands has expanded the possibility of designing drugs that may modulate the immune response at its earliest stages . macromolecular activators of the innate immune system , such as monophosphoryl lipid a ( mpla ) , a tlr4 ligand , are utilized as adjuvants , but development of small molecular weight nonlipid ligands might have several advantages to address different immunological requirements for vaccines or immune therapeutics . as part of our studies on small molecules that can activate tlrs , we conducted a high through - put screening ( hts ) campaign in which a commercially available library of compounds was screened in a human cell - based nfb activation assay . here we report our discovery and initial structure activity relationship ( sar ) studies of the pyrimido[5,4-b]indole class of ligands and their characterization as tlr4/md-2 agonists . the library was screened in three phases at a commercial hts screening facility using the thp-1 human monocytic leukemia cell line , which contained a -lactamase reporter gene under the control of the nfb response element that had been stably integrated into the cells . the three - phase screening process consisted of ( 1 ) a pilot screen of about 10000 compounds selected as representative of the entire primary library , ( 2 ) the primary screen of the entire library , and ( 3 ) a confirmation screen of about 2000 hits found in the primary screen . following the cluster enrichment analysis , 225 compounds were selected for further in vitro biological evaluation involving cytokine induction assays in primary cells , including human peripheral blood mononuclear cells ( hpbmc ) , mouse splenocytes , mouse bone marrow derived dendritic cells ( mbmdc ) , and mouse bone marrow derived macrophages ( mbmdm ) . these cells were incubated in triplicate , with each of the 225 compounds at a single concentration ( 1 m for splenocytes and 5 m for all other mouse cells and human cells ) , and the supernatants were tested for the presence of nfb dependent cytokines , il-8 or il-6 , released from the human or mouse cells , respectively . thirty - nine of the 225 compounds stimulated the human and mouse cells to secrete il-8 or il-6 above the detectable limit . among these scaffolds , the pyrimido[5,4-b]indoles emerged as the most potent and diverse class of compounds in the mouse cell assays with clear evidence of sar . within this scaffold cluster , the leading hit from the initial primary and secondary screens was a substituted acetamide attached to the pyrimidoindole ring system through a thioether linkage ( figure 1 ) . ( a ) human tlr2 , tlr3 , tlr4/md-2/cd14 , tlr5 , tlr7 , tlr8 , and tlr9 hek 293 blue cells or nf-b / seaporter cells were incubated with compound 1 ( 10 m ) for 2024 h , and activation was evaluated by seap secretion in the culture supernatants by colorimetric assay at od405 . il-6 in the culture supernatants was measured by elisa ( f ) and type i ifn ( ifn- ) measured by luciferase release in from an isre reporter cell line ( g ) . tlr4 signals through two distinct pathways , leading respectively to nfb dependent cytokines and type i interferon ( ifn ) production . several naturally occurring tlr4 ligands and mpla have been reported to require cd14 to activate the type i ifn regulatory pathway . compound 1 , however , was not dependent on cd14 for either il-6 or type i ifn production , as demonstrated using cd14 deficient cells ( figure 2f , g ) . results showed a dose - dependent response for type i ifn ( figure 3a ) production , which paralleled that of ip-10 ( figure 3b ) . the values shown in the tables 13 for il-6 release , and mtlr4 activation are area under the curve ( auc ) values for titrated doses of compounds from 312 nm to 10 m . the cells were incubated with graded concentrations of the indicated compounds 1 , 36 , and 39 ( a ) or 11 and 12 ( b ) for 18 h. dmso 0.5% served as the vehicle control . mouse tlr4 ( a , c ) , and htlr4 ( b , d ) hek transfectomas were incubated with graded concentrations of the indicated compounds 1 , 36 , and 39 ( a ) or 11 and 12 ( b ) for 18 h. dmso 0.5% served as the vehicle control . the specific activation of the reporter cell lines was measured by seap activity in the supernatant by absorption at 630 nm . the primary hts library contained a family of 452 compounds in the pyrimido[5,4-b]indole class and therefore represented a valuable initial indication of structural features important for activation of nfb . upon inspection of the nfb activation values in the initial hts relative to the various structural features , several trends were apparent . there was a requirement for a hydrophobic moiety in the region of the cyclohexyl group of compound 1 . in this same region , furthermore , a hydrophobic group at n-3 , preferably a phenyl group , was also required for activity . with these sar features as an initial guide , we elected to investigate modifications of hit compound 1 at three regions while maintaining the core pyrimido[5,4-b]indole ring system : the n - substitution of the s - acetamide , the n-3 substituent , and the n-5 substituent ( figure 6 ) . n - substitutions at the carboxamide moiety were first undertaken to probe the limitations of the hydrophobic group requirement at this position with respect to optimization of cytokine induction ( table 1 ) . while keeping all other structural features of hit compound 1 constant , we prepared a series of carboxamides substituted with various alkyl , cycloalkyl , aromatic , and heteroaromatic groups . at this point in the synthesis , a variety of substituents , represented by r in the scheme , may be introduced that will determine the respective n-3 substituent following annulation of the pyrimidine ring . ring closure of 5 using polyphosphoric acid yielded the pyrimido[5,4-b]indole ( 6 ) bearing the n-3 substituent r. alkylation of the 2-thioxo function with chloroacetic acid provided the versatile intermediate 7 , which was then used to prepare the final test compounds ( 8) bearing a variety of n - substitutions at the acetamide moiety , designated as r in scheme 1 . inspection of the il-6 auc values relative to the hydrophobic group r revealed that in general , compounds bearing the larger cycloalkyl groups , such as cyclooctyl ( 9 ) , cycloheptyl ( 10 ) , and cyclohexyl ( 1 ) , were the most active , followed by branched alkyls and then straight chain alkyls and aromatic and heteroaromatic groups . for the r group hence , the 3,3-dimethylbutyl compound ( 28 ) was among the most active of the alkyls , with isopentyl ( 27 ) , butyl ( 23 ) , and isobutyl ( 26 ) being somewhat less active . encouraged by the sar trends at the carboxamide moiety , we then addressed the n-3 position ( table 2 ) . however , to draw that conclusion with confidence , more examples of compounds bearing similar hydrophobic groups than were represented in the primary compound collection were needed . thus , the r group in compound 5 was varied by reaction of 4 with the appropriate isothiocyanate , and the remaining three steps for each derivative were completed as outlined in scheme 1 while maintaining the r group as cyclohexyl in each case . none of the compounds in the original hts library had modifications at this position to guide the sar . starting from compound 1 , scheme 2 shows the preparation of the n-5 methyl derivative ( 42 ) that also produced a dimethyl side product ( 43 ) wherein the second methylation occurred at the carboxamide function , yielding the n , n - disubstituted derivative . the trend in the toxicity profile for these n-5 alkyl derivatives was confirmed , at least for the shorter chain alkyls , in that the n-5 methyl ( 42 ) , cyanomethyl ( 51 ) , and n - propyl ( 48 ) derivatives did not reduce cell viability at concentrations up to 10 m ( figure 7 , shown for 42 , data not shown for 48 and 51 ) . cytotoxicity of the compounds was evaluated by mtt assay as a measure of viability . with few exceptions , the most active compounds in the il-6 assays were also those that showed the highest activity in the mtlr4 transfectoma line ( tables 13 ) . , il-6 ) by nfb activation , tlr4 signaling can induce the production of type i ifn via the trif pathway . this pathway results in the activation of interferon regulatory factors ( irf ) , which are nuclear transcription factors that promote the transcription of ifns . type i ifn is important for the activation of antigen presenting dendritic cells , leading to good adjuvant activity , and also promotes cellular defenses against a variety of pathogens , particularly rna viruses . interestingly , several of the compounds , namely 12 , 13 , 15 , 16 , 17 , 29 , 30 , and 33 , induced diminished il-6 release compared to compound 1 but had nearly equivalent induction of ip-10 ( tables 1 and 2 ) . the correlation of il-6 and ip-10 production of the selected compounds is shown graphically in figure 8 . two distinct groups of compounds are observed , shown as circled regions of the plot . thus , the group at the upper right area ( dotted circle ) comprises compounds that induce high il-6 and high ip-10 production , including compounds 1 , 9 , 10 , 28 , and 42 . the other group is composed of compounds that induce low il-6 but relatively higher ip-10 production . these two clusters were selected based on mean sd of the normalized il-6 and ip-10 values of all sar compounds . it was notable that when the carboxamide substituent was phenyl or substituted phenyl ( 13 , 15 , 16 , 17 ) , with the exception of p - fluorophenyl ( 14 ) , il-6 release was reduced while ip-10 production was maintained at a relatively higher level . a few compounds bearing branched aliphatic carboxamide substituents showed a similar trend , as noted above for 29 , 30 , and 33 . two distinct groups , compounds in the dotted circled ( upper right ) and in solid ( left ) circled areas , were selected based on the induction of il-6 and ip-10 . the group at the upper right area comprises compounds that induce high il-6 and high ip-10 production , including compounds 1 , 9 , 10 , 28 , and 42 , whose values were at least one sd above the mean for both cytokines . the other cluster is composed of compounds ( 13 , 16 , 17 , 29 , 30 , and 33 ) that induce low il-6 but relatively higher ip-10 production , whose values were below the mean for il-6 and were above the mean for ip-10 . in general , the active compounds exhibited greater activity in the murine cell systems than in human cells . although the innate immune system is highly conserved among species , there are differences between human and mouse tlr4 . small molecule hit compounds discovered in the present study were also thought to bind to the tlr4/md-2 complex in such a way as to facilitate dimerization . accordingly , we examined the predicted binding mode(s ) of one of the most active compounds to the murine tlr4/md-2 complex by conducting molecular docking of compound 28 to the crystal structure of the mouse complex ( pdb 2z64 ) using the programs hex and ampac . figure 9 shows the predicted binding mode of compound 28 in the tlr4/md-2 model , while the set of binding interactions that may keep the compound in the md-2 pocket bound to both tlr4 and md-2 is depicted in figure 10 . other small molecules such as paclitaxel , opioids , and a peptide have also been reported to bind to the md-2 binding pocket . in the course of an hts designed to identify activators of innate immunity , a series of substituted pyrimido[5,4-b]indoles were discovered as selective tlr4 ligands . interestingly , a subset of the compounds bearing phenyl and substituted phenyl carboxamides induced lower nfb - dependent inflammatory cytokine release while maintaining interferon - dependent ip-10 production . finally , n-5 substitution revealed that short alkyl substituents at this position attenuate cell toxicity relative to the corresponding nonsubstituted derivative while maintaining tlr4 activity . both the inflammatory cytokine and type i ifn inducing activities of these compounds were cd14-independent . computational studies with one of the active compounds predicted binding primarily to md-2 in the murine tlr4/md-2 complex . here we have described a panel of small molecules that stimulate immune cells to produce distinct profiles of nfb and interferon associated cytokines . to a solution of compound 4 ( 1 equiv ) in warm etoh was added the appropriate isothiocyanate ( 1.1 equiv ) dropwise with stirring . further cooling of the decantate yielded crystals , which were then filtered and washed with cold water to give 23.6 g of white crystalline product . in a flame - dried flask , a suspension of potassium t - butoxide ( 7.124 g , 63.5 mmol ) in anhydrous thf was stirred and maintained below 30 c under argon . compound 4 ( 5.21 g , 25.5 mmol ) was reacted with phenyl isothiocyanate ( 3.36 ml , 28.1 mmol ) in warm etoh ( 75 ml ) according to general procedure a to give 6.732 g compound 5a in 75% yield . compound 4 ( 100 mg , 0.49 mmol ) was reacted with cyclohexyl isothiocyanate ( 169.15 mg , 0.54 mmol ) in warm etoh ( 750 l ) according to general procedure a to give 104 mg of compound 5b in 61.8% yield . compound 4 ( 50 mg , 0.28 mmol ) was reacted with 1-naphthyl isothiocyanate ( 49.9 mg , 0.27 mmol ) in warm etoh ( 750 l ) according to general procedure a to give 58.6 mg of compound 5c in 61.5% yield . compound 4 ( 100 mg , 0.49 mmol ) was reacted with phenethyl isothiocyanate ( 80.4 l , 0.54 mmol ) in warm etoh ( 1.5 ml ) according to general procedure a to give 147 mg of compound 5d in 81.8% yield . stimulation of the human tlr4 cells with 10 ng / ml lps resulted in od630 of 1.90 relative to 0.88 of cells stimulated with 10 m compound 1 . l929-isre cells were plated at 5 10 cells per well in dulbecco s modified eagle medium ( invitrogen ) supplemented with 10% fcs , 100 u / ml penicillin , and 100 g / ml streptomycin ( dmem-10 ) in a 96-well white - walled clear - bottom plate . in addition , the levels of ip-10 , a surrogate marker of type i ifn , in the supernatants were determined by elisa ( r&d systems , minneapolis , mn ) . the cultures stimulated with lps 10 ng / ml , and 5 m compound 1 released an average of 234.8 8.6 and 590.0 10.5 the data are represented as mean standard error of the mean ( sem ) .

these pathogen - associated molecular patterns ( pamps ) and their corresponding tlrs include : lipopeptides ( tlr2 ) , double - stranded rna ( tlr3 ) , lipopolysaccharide ( lps , tlr4 ) , bacterial flagellin ( tlr5 ) , guanine and uridine - rich single - stranded rna ( tlr7 , 8) , and hypo - methylated cpg rich dna ( tlr9 ) . indeed , this rapid response , involving multiple components of the innate immune system , has been recognized to guide the type of adaptive immune response that is most effective for the specific pathogenic threat . adjuvants are added to antigens in a vaccine setting to provide enhanced response to poorly immunogenic antigens , to increase seroconversion rates in populations with reduced responsiveness due to age ( infants and the elderly ) or disease ( diabetes , renal failure ) , to facilitate the use of smaller doses of antigen , and to permit immunization with fewer doses of vaccine . macromolecular activators of the innate immune system , such as monophosphoryl lipid a ( mpla ) , a tlr4 ligand , are utilized as adjuvants , but development of small molecular weight nonlipid ligands might have several advantages to address different immunological requirements for vaccines or immune therapeutics . as part of our studies on small molecules that can activate tlrs , we conducted a high through - put screening ( hts ) campaign in which a commercially available library of compounds was screened in a human cell - based nfb activation assay . here we report our discovery and initial structure activity relationship ( sar ) studies of the pyrimido[5,4-b]indole class of ligands and their characterization as tlr4/md-2 agonists . the library was screened in three phases at a commercial hts screening facility using the thp-1 human monocytic leukemia cell line , which contained a -lactamase reporter gene under the control of the nfb response element that had been stably integrated into the cells . the three - phase screening process consisted of ( 1 ) a pilot screen of about 10000 compounds selected as representative of the entire primary library , ( 2 ) the primary screen of the entire library , and ( 3 ) a confirmation screen of about 2000 hits found in the primary screen . following the cluster enrichment analysis , 225 compounds were selected for further in vitro biological evaluation involving cytokine induction assays in primary cells , including human peripheral blood mononuclear cells ( hpbmc ) , mouse splenocytes , mouse bone marrow derived dendritic cells ( mbmdc ) , and mouse bone marrow derived macrophages ( mbmdm ) . these cells were incubated in triplicate , with each of the 225 compounds at a single concentration ( 1 m for splenocytes and 5 m for all other mouse cells and human cells ) , and the supernatants were tested for the presence of nfb dependent cytokines , il-8 or il-6 , released from the human or mouse cells , respectively . we used hek293 cells stably transfected with individual human ( h ) tlrs : tlr2 , tlr3 , tlr4/md-2/cd14 , tlr5 , tlr7 , tlr8 , or tlr9 expressing hek293 cells , along with an nfb activation reporter producing secreted embryonic alkaline phosphatase ( seap ) . ( a ) human tlr2 , tlr3 , tlr4/md-2/cd14 , tlr5 , tlr7 , tlr8 , and tlr9 hek 293 blue cells or nf-b / seaporter cells were incubated with compound 1 ( 10 m ) for 2024 h , and activation was evaluated by seap secretion in the culture supernatants by colorimetric assay at od405 . reagents and conditions : ( a ) brch2cooet , nahco3 , etoh , reflux ; ( b ) tert - buok , thf , < 30 c ; ( c ) r - ncs , etoh , reflux ; ( d ) ppa , 110 c ; ( e ) clch2cooh , koh / etoh , reflux ; ( f ) r - nh2 ( for r substitutions refer to table 1 ) , hatu , dmf , room temp . the binding of compound 1 to the tlr4/md-2/cd14 complex was further confirmed using a competitive antagonist for the tlr4 binding complex , lps - rs ( lps from rhodococcus sphaeroides ) . lps - rs inhibited the activation by compound 1 in a dose - dependent manner , indicating that compound 1 bound to the tlr4 complex ( figure 2e ) . human tlr4 activation is shown for stimulation of pbmc ( il-8 ) and htlr4 hek293 transfectomas ( figure 5b , d ) at 10 m , as these assays were not sufficiently sensitive at lower concentrations to make auc comparisons . mouse tlr4 ( a , c ) , and htlr4 ( b , d ) hek transfectomas were incubated with graded concentrations of the indicated compounds 1 , 36 , and 39 ( a ) or 11 and 12 ( b ) for 18 h. dmso 0.5% served as the vehicle control . the primary hts library contained a family of 452 compounds in the pyrimido[5,4-b]indole class and therefore represented a valuable initial indication of structural features important for activation of nfb . finally , exchange of the entire acetamide moiety on the c-2 thiol with the n-3 phenyl group , such that the acetamide was attached at n-3 and the phenyl was attached at the c-2 thiol , resulted in loss of activity . with these sar features as an initial guide , we elected to investigate modifications of hit compound 1 at three regions while maintaining the core pyrimido[5,4-b]indole ring system : the n - substitution of the s - acetamide , the n-3 substituent , and the n-5 substituent ( figure 6 ) . at this point in the synthesis , a variety of substituents , represented by r in the scheme , may be introduced that will determine the respective n-3 substituent following annulation of the pyrimidine ring . ring closure of 5 using polyphosphoric acid yielded the pyrimido[5,4-b]indole ( 6 ) bearing the n-3 substituent r. alkylation of the 2-thioxo function with chloroacetic acid provided the versatile intermediate 7 , which was then used to prepare the final test compounds ( 8) bearing a variety of n - substitutions at the acetamide moiety , designated as r in scheme 1 . thus , by this synthetic method , compounds shown in table 1 , with r held constant as a phenyl group , were prepared and evaluated in a cytokine induction assay for il-6 in mbmdc and compared to compound 1 . examples of the data obtained from typical cytokine induction assays and nfb activation assays are shown in figures 4 and 5 , wherein compounds 1 , 11 , 12 , 36 , and 39 are compared for il-6 production by mbmdc and hpbmc . inspection of the il-6 auc values relative to the hydrophobic group r revealed that in general , compounds bearing the larger cycloalkyl groups , such as cyclooctyl ( 9 ) , cycloheptyl ( 10 ) , and cyclohexyl ( 1 ) , were the most active , followed by branched alkyls and then straight chain alkyls and aromatic and heteroaromatic groups . for the r group hence , the 3,3-dimethylbutyl compound ( 28 ) was among the most active of the alkyls , with isopentyl ( 27 ) , butyl ( 23 ) , and isobutyl ( 26 ) being somewhat less active . thus , the r group in compound 5 was varied by reaction of 4 with the appropriate isothiocyanate , and the remaining three steps for each derivative were completed as outlined in scheme 1 while maintaining the r group as cyclohexyl in each case . as the il-6 data indicate , replacing the n-3 phenyl in compound 1 with a cyclohexyl group ( 36 ) caused some loss of activity , while substitution on the n-3 phenyl ( 37 and 38 ) resulted in greater loss of activity . starting from compound 1 , scheme 2 shows the preparation of the n-5 methyl derivative ( 42 ) that also produced a dimethyl side product ( 43 ) wherein the second methylation occurred at the carboxamide function , yielding the n , n - disubstituted derivative . moreover , methylation at n-5 decreased the toxicity of compound 1 ( figure 7 ) as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) viability assay . the resulting t - butyl esters ( 46a d ) were easily hydrolyzed to the corresponding free carboxylic acids ( 47a d ) and then converted to the n - cyclohexylcarboxamides ( 4851 ) by the same method as described for compound 1 . the trend in the toxicity profile for these n-5 alkyl derivatives was confirmed , at least for the shorter chain alkyls , in that the n-5 methyl ( 42 ) , cyanomethyl ( 51 ) , and n - propyl ( 48 ) derivatives did not reduce cell viability at concentrations up to 10 m ( figure 7 , shown for 42 , data not shown for 48 and 51 ) . ( a ) nah , dmf , room temp , then ch3i ; ( b ) nah , dmf , then br(ch2)3cooch3 ( a ) clch2coo t - bu , koh , dma , h2o ; ( b ) nah , dmf , then rx ( iodopropane or iodopentane or bromododecane or bromoacetonitrile ) ; ( c ) tfa , dcm or ch3cn ; ( d ) cyclohexyl - nh2 , hatu , dmf , room temp ; ( e ) compound 51 , h2so4 , h2o . interestingly , several of the compounds , namely 12 , 13 , 15 , 16 , 17 , 29 , 30 , and 33 , induced diminished il-6 release compared to compound 1 but had nearly equivalent induction of ip-10 ( tables 1 and 2 ) . it was notable that when the carboxamide substituent was phenyl or substituted phenyl ( 13 , 15 , 16 , 17 ) , with the exception of p - fluorophenyl ( 14 ) , il-6 release was reduced while ip-10 production was maintained at a relatively higher level . the group at the upper right area comprises compounds that induce high il-6 and high ip-10 production , including compounds 1 , 9 , 10 , 28 , and 42 , whose values were at least one sd above the mean for both cytokines . the other cluster is composed of compounds ( 13 , 16 , 17 , 29 , 30 , and 33 ) that induce low il-6 but relatively higher ip-10 production , whose values were below the mean for il-6 and were above the mean for ip-10 . recently mouse and human tlr4/md2 crystal structures with this ligand have suggested that the different charge distributions of mouse and human tlr4/md-2 affect the positions of the phosphate groups of lipid iva , orienting them in a manner that would limit receptor dimerization by human tlr4 . the structural basis of receptor specificity and of the mechanism of activation by lps have recently been elucidated by determining the crystal structure of the tlr4/md-2-lps complex at 3.1 a resolution . accordingly , we examined the predicted binding mode(s ) of one of the most active compounds to the murine tlr4/md-2 complex by conducting molecular docking of compound 28 to the crystal structure of the mouse complex ( pdb 2z64 ) using the programs hex and ampac . figure 9 shows the predicted binding mode of compound 28 in the tlr4/md-2 model , while the set of binding interactions that may keep the compound in the md-2 pocket bound to both tlr4 and md-2 is depicted in figure 10 . there is a set of favorable electrostatic interactions resulting in possible hydrogen bonds formed by the residues glu439 of tlr4 and arg90 of md-2 with compound 28 , although the former would approach a hydrogen bond interaction due to the flexibility of the glu439 side chain . h nmr ( 400 mhz , dmso - d6 ) ppm 0.761.38 ( m , 5 h ) , 1.421.95 ( m , 5 h ) , 3.433.55 ( m , 1 h ) , 3.744.00 ( m , 2 h ) , 7.29 ( t , j = 6.97 hz , 1 h ) , 7.41 ( d , j = 8.43 hz , 1 h ) , 7.477.66 ( m , 4 h ) , 7.687.81 ( m , 2 h ) , 8.018.36 ( m , 4 h ) , 12.17 ( s , 1 h ) . h nmr ( 400 mhz , dmso - d6 ) ppm 0.831.42 ( m , 5 h ) , 1.442.08 ( m , 5 h ) , 2.873.17 ( m , 2 h ) , 3.954.24 ( m , 2 h ) , 4.254.62 ( m , 2 h ) , 7.33 ( br s , 6 h ) , 7.878.48 ( m , 3 h ) , 11.99 ( s , 2 h ) . compound 45 ( 50 mg , 0.12 mmol ) , nah 60% dispersion in mineral oil ( 3.3 mg , 0.14 mmol ) , iodopropane ( 13.1 l , 0.14 mmol ) , and dmf ( 1 ml ) were reacted according to general procedure e and purified by silica gel chromatography ( 10:90 ethyl acetate : hexane ) to give 32.8 mg in 61% yield . compound 45 ( 50 mg , 0.12 mmol ) , nah 60% dispersion in mineral oil ( 3.3 mg , 0.14 mmol ) , bromododecane ( 32.4 l , 0.14 mmol ) , and dmf ( 1 ml ) were reacted according to general procedure e and purified by silica gel chromatography ( 10:90 ethyl acetate : hexane ) to give 37.4 mg in 54% yield . h nmr ( 400 mhz , dmso - d6 ) ppm 0.80 ( t , j = 6.97 hz , 3 h ) , 1.001.40 ( m , 9 h ) , 1.431.92 ( m , 7 h ) , 3.443.58 ( m , 1 h ) , 3.88 ( s , 2 h ) , 4.58 ( t , j = 7.15 hz , 2 h ) , 7.29 ( t , j = 7.33 hz , 1 h ) , 7.407.68 ( m , 6 h ) , 7.72 ( d , j = 8.43 hz , 1 h ) , 8.038.32 ( m , 2 h ) . h nmr ( 400 mhz , dmso - d6 ) ppm 0.730.92 ( m , 4 h ) , 1.20 ( d , j = 6.97 hz , 22 h ) , 1.411.89 ( m , 7 h ) , 3.453.61 ( m , 1 h ) , 3.88 ( s , 2 h ) , 4.58 ( t , j = 6.60 hz , 2 h ) , 7.28 ( t , j = 7.52 hz , 1 h ) , 7.367.80 ( m , 7 h ) , 8.058.26 ( m , 2 h ) . h nmr ( 400 mhz , dmso - d6 ) ppm 0.971.33 ( m , 5 h ) , 1.451.87 ( m , 5 h ) , 3.453.58 ( m , 1 h ) , 3.90 ( s , 2 h ) , 5.87 ( s , 2 h ) , 7.42 ( t , j = 7.80 hz , 1 h ) , 7.477.56 ( m , 2 h ) , 7.577.74 ( m , 4 h ) , 7.88 ( d , j = 8.29 hz , 1 h ) , 8.17 ( dd , j = 13.16 , 7.80 hz , 2 h ) . h nmr ( 400 mhz , dmso - d6 ) ppm 0.941.36 ( m , 5 h ) , 1.431.89 ( m , 5 h ) , 3.433.62 ( m , 1 h ) , 3.89 ( s , 2 h ) , 5.24 ( s , 2 h ) , 7.127.22 ( m , 1 h ) , 7.30 ( t , j = 7.33 hz , 1 h ) , 7.42 ( m , j = 7.30 , 2.20 hz , 2 h ) , 7.487.67 ( m , 5 h ) , 8.10 ( d , j = 7.70 hz , 1 h ) , 8.18 ( d , j = 7.70 hz , 1 h ) .

in some important ways , mh both replaced and replicated the social and economic networks that families , at their most basic level , constitute . nineteenth - century missionaries were slow to make converts of zulu - speakers and some of the earliest to come to the mission stations and undergo baptism were those who had fled from or been rejected by their families , homesteads or communities . nor were african patriarchs and matriarchs immediately keen for their daughters to become nurses , for their labour was needed for homestead production , and guarantees of their virtue uncertain outside the direct control of the family , including peer groups who provided companionship , and who monitored sexuality and reproduction . the first converts amakholwa often stressed in similar ways to their western counterparts the gendered propriety of conduct of sons and daughters and it is not surprising therefore that it was the first and second generations of kholwa families which would encourage their daughters to take up schooling and then , as positions gradually opened , the professions of teaching and nursing . thus , from the first intake of trainee nurses in 1909 to the 1960s , the majority of mh nurses were recruited from inanda seminary , the american board s ground - breaking school for girls . of the first african women to be trained ( from 1911 ) by margaret mccord and martha s. macneill three , nomhlatuzi bhengu , julia magwaza and edna mzoneli , had been pupils at inanda seminary . the first nurse recruit , elizabeth njapa however , had been brought to the notice of the mccords earlier , since she had been forced to leave school when it was discovered that she had at the age of fourteen had an illegitimate half - caste child , and who was then rejected as rejection by natal family or adopted community and subsequent acceptance at a new mission - based site , though with the requirement of submitting to the authority of a new familial order echoed that of many of the first converts , other nurse trainees increasingly came from lineages of prominent kholwa families , whose fathers were preachers within the congregationalist churches ( after 1897 the zulu congregational church ) , or lawyers , teachers , traders and artisans , and later , doctors and political leaders . amongst the most prominent of these families by the 1940s were those of goba , ngcobo , gcabashe , msimang , and luthuli . beatrice gcabashe ( ne msimang ) was the first african registered nurse in natal to obtain joint general nursing and midwifery registration . the daughter of a prominent politically active amakholwa family , she was at mh from 1927 . her son , dr v m gcabashe , was later an intern at mh in the 1950s . to further illustrate the interconnections between such families and mh , selby ncgobo ( a political activist and academic who received a master s degree from yale university in 1940 and a phd in economics from the university of london in 1964 ) and chief albert luthuli ( who is most well - known for his role as president - general of the african national congress from 1952 to 1967 ) were both members of the mh board by 1946 and taylor and luthuli shared a close relationship . when the latter went to oslo in december 1961 to receive the nobel peace prize , it was taylor who lent him a warm coat . during luthuli s detention in pretoria the previous year , when this was granted , and after taylor s suggestion that luthuli be transferred to mh for treatment was declined , taylor confined the content of his letter to assurances of support for luthuli from his friends and passing on news about luthuli s daughters , hilda and eleanor , who trained as nurses at mh . in his letters , taylor manages to convey his own in loco parentis role and the wider support for luthuli in natal s liberal circles : they have taken your trouble very well , he added , and like the rest of us look forward to your return in time. in a letter franked by the prison authorities on 6 june , after sending personal greetings to dr and mrs taylor and asking that these be extended to the mh staff and to the mission council of churches and congregations , luthuli wrote : i do nt know if i am professionally correct to ask you , a senior , but may i ask you to send my greetings and best wishes to my two daughters hilda and eleanor . assure them i am not dying. soon after this correspondence , hilda was to marry thulani gcabashe at groutville . white guests were barred from attending the wedding , however . it could be argued that the notion of the mccord family was hierarchically constructed and in practice recognised as equals only fellow missionaries , and other formally appointed senior staff matrons , later additional doctors and health professionals ( none of whom until the mid - twentieth century were black ) while the largely female , black nursing , cleaning and other staff were merely subordinates , or children. lived experience , however , was far more complex and involved feelings , frictions and reciprocities that were played out through the notion of being part of a family. for instance , especially in the late nineteenth and early decades of the twentieth century , the missionary doctors and their families were also cut off from their own natal and extended families . nor , for many years after their arrival , were james and margaret mccord welcomed into durban s elitist and racist professional or social circles . while an instant community could be found with their fellow missionaries and congregationalists , these circles were not without their difficulties and rivalries . family was significant for the mccords themselves as well as for the young women who came to train as nurses , the first group of whom were more - or - less the same age as the mccords elder daughters , who had not lived with their parents for some years . as was the practice at the time , the mccords children ( except their youngest , also margaret , or peggy ) were sent home to the usa for their education . in 1911 for instance , the mccords return to durban after their first furlough leave was saddened by leaving their three eldest children , jessie , mary and robert , behind to receive their schooling . their next home leave , in 191819 , was the final occasion on which all the mccord family would ever be together , for both jessie and laura died in 1919 . although according to katie makanya s reminiscences neither james nor margaret ever mentioned their dead daughters again , it is impossible to read james s account of the death of edna mzoneli of influenza , during the mccords absence from durban , without imagining that this poignant description was in part drawn from the death of his own daughters . one further account of the relationship between james and margaret mccord and edna mzoneli is suggestive of how the close association between these self - identified parental figures and young person could be influential in the moulding of a sense of self . as a school girl and young woman , edna who had a cast in one eye , was regarded by both missionaries and her own father as weak and stupid , and she was subject to bouts of hysteria . demented , margaret physically shook her and then instructed her to go to her room and not to return until she could behave as a nurse. thereafter , edna devoted herself to her studies and was later appointed assistant matron at mh . there , she saved her wages and in a remarkable statement of affirmation told margaret : i am a person you have always been a person . now i am a nurse. even so , when during a severe outbreak of measles she addressed the people of her natal family home on basic preventive and hygiene measures she felt it necessary to subdue this sense of personhood : in the closing chapters of his memoir , my patients were zulus , james mccord devotes some pages to considering the significance of the mccord family. central to this , he believed was the mccord nurse , a composite picture of whom , he said , was a woman who is well - balanced conscious of her duty to her patients ( and ) of her own personal dignity. indeed , she was neither self - conscious nor servile ( and ) the only walls or bars to restrain her were her own dignity and self - respect , her knowledge that as a trusted member of the hospital family , she must be worthy of trust. sixty years later , these sentiments were strongly echoed during interviews with the researcher penny watts . for instance , in 2006 sister mary jane molefe spoke for many mh nursing staff when she stated that in her view what made mccord different was ( 1 ) prayer ; ( 2 ) staff selection ; ( 3 ) discipline . in their view , mh s staff selection of nurses largely from inanda seminary , and later of medical and surgical staff who may have been of faiths other than christian , but who nonetheless had a strong sense of spiritual calling and service made for a dedicated and hard - working staff complement in which nurses were valued and who valued themselves . sister molefe went on to add that ( at inanda in the 1950s and 1960s ) they did not just give us academics but they were able to give us ammunition to be assertive , to work hard , the ethos of standing up you know inanda seminary gave us that. thus , discipline applied both to an internal disposition and to outward manifestations of control , and both mccord s memoir and the oral testimony of long - service mh senior nurses , record a strong sense of pride in being a mh nurse , and an active embrace of its moral and behavioural codes . as at other hospitals , discipline was predictably strict , went hand - in - hand with spiritual instruction and was intimately connected to the vision of mh as a family. for instance in 1955 long - time mh gardener and sometime patient aide , baba dube recalled that : the strictness enforced on the nurses and myself was rigid and unbending . on sundays it was part of my duty to escort the nurses to and from the church , with dr on his motorcycle likely to be around the next corner . he told me openly that the parents of these children had entrusted them to his care . and it was his duty that his girls must be properly looked after as they would be in their own homes . he did his duty with the able assistance of mrs mccord . particularly under the stewardship of taylor , discipline could be harsh . in a letter written by taylor to friends dated 16 december 1945 , quoted in shula marks s book divided sisterhood : mine is more than the medical superintendent to nurse relationship . it is that and it is the father - daughter relationship as well it has numerous ramifications which i would gladly pass on to the matron if we had one who knew the language and customs as well as i do . with 125 daughters all potentially amorous and capable of making a bad match , to watch one comes perforce either a cynic or a philosopher or a better christian . martha the midwife was expelled from her course only three weeks before its end because she was staying over with a boyfriend and was already pregnant taylor explained that he had not done this lightly and , he said , would have preferred to work something out , but that he and the matron felt they had little alternative since martha expressed no contrition or signs of change of heart. tellingly , however , martha s condition had only come to their notice after an anonymous letter had reported the misbehaviour of one of our nurses who was shaming herself , the hospital and her fellow nurses. if , as seems likely , this letter had been written by one of martha s peers , it points to the conservative expectations of many women themselves at the time . indeed , when , a year earlier , another young woman had been sent home from mh by taylor when he discovered that she had married a man that has a wife , her mother ( or sister ; it is unclear which ) wrote thanking him for all the fatherly trouble ( he ) had taken in this matter of disgrace to our family. dismissal and discipline were not however necessarily mh s immediate or only means of interacting with wayward or fractious staff ; and nor were more senior white women always favoured over their black subordinates . we can see this in another letter from taylor , in june 1955 , which is full of praise for several nurses , and then goes on to describe how he had intervened in a disagreement between two white sisters and a number of african nurses . after this , sister evard had agreed to apologise to nurse sarah keswa and they had then all prayed together. the same afternoon , he noted , he was called to another indaba , this time between nurses mary mselku , mildred mnene , miriam ngidi and atchison kuzwayo . lectured them he said , but was pleased that these interviews ended on a higher note than treats [ sic possibly he meant threats ] and punishments. even before the hospital itself had been built , prayer the third element identified by sister molefe as being central to mh had been a part of daily practice . in the early years of the twentieth century , zulu - speaking evangelists worked with the mccords at their cottage hospital and dispensary in the centre of durban to persuade sometimes dubious patients of the value of biomedicine . the practices of preaching and praying continued even after the hospital had been established and expanded , with evangelists and nurses also leading prayers in the out patients department and wards . in turn , this christian ethos did much to help build the unifying going against the growing trend towards secularisation underway in many other mission hospitals in south africa during the 1950s and 1960s , where the mission s goal of healing the soul had become secondary to the growing pressures of providing professional medical services to heal the body , mh continued to place as much , if not more emphasis , on the importance of the christian faith in healing . although professing to be a non - denominational hospital that did not restrict staff appointments to professing christians , in practice during these middle decades of the twentieth century , the hospital continued to choose christian candidates as their preferred trainees and employees . from the election of hospital board members for their christian backgrounds , who started and ended every meeting with prayers ; to the appointment of christian medical superintendents , as well as medical and nursing staff ; to a hospital constitution , which emphasised the hospital s duty to carry on a ministry of health and healing to make known to hospital workers and patients by word and by life , the gospel of christ , christianity remained a driving force shaping the identity and work of the hospital . during the 1940s through to the 1970s , medical superintendents taylor and orchard were particularly responsible for the strong christian influences they brought to the hospital . of taylor , mccord wrote that his religion : was nt something ( he ) accepted on sunday and shed on monday . and when he took charge of the hospital , religion and healing went hand in hand . we had always had religious services for the staff and he continued this , but he also allowed religion to play an even larger role in the hospital without question his influence ma(de ) the nurses and the staff look upon their work as both spiritual and medical . professor sam fehrsen , a doctor who had worked at mh during the early 1960s , concurred . for him , the strong christian family ethos was created by the senior staff , who he said actually lived what they believed in , who infused christianity into the hospital s daily routines , and importantly , their long periods of service provided the continuity needed to nurture and sustain this christian culture . in addition to the efforts of these medical superintendents , as watts argues , mh s nurses who formed the majority of the staff , who had the most interaction with patients on a daily basis , and many of whom worked for decades at mh formed the backbone of the expanding christian mission at mh . indeed , the medical superintendents , matrons and nurse tutors devoted much effort to nurturing and sustaining within their nurse trainees a deep sense of christian devotion and every morning before classes commenced , prayers were held. christian messages , themes and stories , including those about the importance of family , were published in isibuko newsletters ; formed the core of plays and other entertainment events ; appeared on the back covers of nurses song books , and formed a cornerstone of the candle - lighting service at the graduation ceremony held annually at the nurses home . christian beliefs were also extended to patients and other workers and medical staff . in 1966 for instance , dr lasbrey described how the heart of the hospital is its religious life . zulu prayers for workers , ward services for patients , and a service in the out - patient department for nurses and staff , are held every morning . then there are the sunday night services in the nurses home , the staff fellowship , the nurses christian fellowship and quiet time for prayer . quiet time ( or hour ) was introduced by taylor in the 1930s ; it was held at 5:30 a.m. on a weekday and , as mccord recounted : dr . taylor would pass around slips with perhaps five scriptural questions from one of the books of the bible . for fifteen minutes , while the room glowed brighter with the rising sun , the staff and nurses would meditate on the questions ; then for another quarter hour discuss problems they had raised . prayers closed the meeting at six , and the nurses would then slip from the room , their faced composed and at peace . attendance at quiet hour was optional , but any of the staff , medical aides or nurses not on duty were expected to be present at the ( daily ) eight oclock morning services . the staff and nurses left ( these morning services ) with an inner calm and a spirit of friendship for each other and for their patients . what this passage does not relate is how such sessions were also spaces in which staff and nurses could explore their own spiritual and individual identities , even to raise questions about their faith or public or private conduct . one nurse asked the following existential , political and spiritual questions out loud during quiet time in may 1960 : why not an insect a flower a tree : if i am to be a person then why not a white , or a brown , or yellow person ? or if i am to be an african , why not an african in angola or in the congo ? if i am to be an african in south africa , then why not a man or a rich person or a school teacher , etc . ? why s i can only believe god knows , and that he has a plan for my happiness , and through me , for the happiness of others. taylor himself was a staunch , but soul - searching , christian and , perhaps more so in his later years , neither immune to self - doubt nor the need to identify his own short - comings . in one letter , he wrote of the centrality of prayer and quiet time in his own life as well as revealing some of the strains experienced within the mccord family : more than anything else were the times i had to go to god to get the answers to the problems presenting here in the hospital . yesterday i had occasion to count up and realized that last year among eleven ( interns ) there were seven who were real problems outside of their work to be more explicit two got to taking dope , three got into triangles ( tangles ? ) and two others got involved in a love affair that shook the hospital . it was a case of praying for them and for myself at nearly every quiet time . it is these decisions that have to be taken as an executive affecting others that i find hardest . formerly , i could harden my heart and cut people off from the hospital without too much trouble . ( now ) there is always a sense of conviction of failure if that has to be done . to stretch the analogy , such reflections are testament to the inevitable ruptures and conflicts that are the almost daily experience of families . further confidential correspondence reveals too how mh nurses and doctors were just as prey to the full range of social and individual troubles as anyone else . in letters between taylor and dr paul keen of the non - european hospital in johannesburg written in 1961 , harrowing details of drug addictions , overdoses , alcoholism and suicides amongst black doctors , nurses and midwives several of them mh graduates are itemised . for a small number of staff and patients who came to train or work , or convalesce at mh , the pervasive conservative christian orientation was experienced as restrictive , even suffocating . not all patients in particular succumbed to the proselytising ; and some nurse trainees objected to the prison - like discipline and regimented structures within the hospital , as well as regular monthly weigh - ins and strict monitoring of their movements and their visitors , which invaded their personal privacy and treated them like children . although weigh - ins were ostensibly to track bodily changes that could suggest serious illnesses , such as tuberculosis , in a strict christian mission hospital , such measuring techniques could also alert nurse supervisors to illegitimate pregnancies , which remained strictly forbidden amongst mh staff . on the other hand , there is much evidence in correspondence , board minutes , isibuko newsletters and interviews that many of mh trainees and staff actively embraced this ethos . this is apparent in the numerous references to the taylors and orchards as the parents or fathers and mothers of the big home. familial terms of endearment were also extended to another long - serving senior doctor on the staff , dr aldyth lasbrey , who was affectionately known as auntie. the deep effect this family atmosphere had on some individuals who trained at mh , even extended to attempts to propagate it elsewhere , such as mrs athee pillay when she worked at the friends of the sick association ( fosa ) tuberculosis settlement after her nurse training ; in the efforts of the young dr cecil orchard , who tried to build a mini mccords in the rural district of hammanskraal in the then transvaal after completing his internship in the 1950s ; and dr mohammed mayat , whose private hospital , shifa , was created as a if james mccord and alan b. taylor were patriarchs tasked with the spiritual , moral , educational and physical well - being of their staff , a complementary role as mother was often fulfilled by their marital counterparts , margaret mccord and mary taylor , and arguably in a less direct manner in later decades by ruth christofersen and then mavis orchard . while margaret mccord herself assumed the role of matron ( as well as nurse and fund - raiser , and occasionally dispenser and anaesthetist ) , in part reflecting shifting twentieth - century ideologies of the appropriate role of middle class wives , mary , ruth and mavis worked in other , though equally important ways , to build the mccord family. these included organising hospital social functions , such as annual christmas lunches where the medical superintendent , the matron , doctors and nurse supervisors acted as waiters to the great delight of the nurses as well as annual easter parties and usa - inspired thanksgiving dinners , regular hamburger suppers and braais ( barbeques ) , sunday teas , annual prize - giving and candle - lighting services , and many fund - raising events . often , these events were hosted in the homes or gardens of the incumbent superintendent and his wife . indeed , as recently , as 2009 , mavis orchard recalled how she had frequently worked herself to a frazzle in entertaining the numerous visitors who came to mh , as well as the hospital s staff who were often invited to their home so as to create a family atmosphere. at the 2009 mh centenary celebrations , dr zweli mkhize , spoke of mavis orchard as being like a mother to us. the for , not only did discipline and hard work characterise mh , but so , too , did the regular expression of gratitude , grace , joy and celebration . while these were often articulated via religious commemoration , the hospital has long had a vibrant culture of levity . this dates back at least to both the mccords and taylors own sometimes jocular and sometimes wry sense of fun , which on occasion , such as the new year s eve festivities in 1953 , saw alan taylor wearing his wife s dress and a wig in a sketch he had designed with other doctors to make his contribution in the way of fun to the party , and also from the interactions between mh s first superintendent of nurses martha s. macneill and the first nurse trainees . not much older than her charges when she took her position at mh in 1911 , james b. mccord described mac as sometimes being more likely than the probationers to giggle at amusing mishaps ; she relieved discipline by mischievous pranks ; and she was more effective than an older , more dignified superintendent would have been with young girls. to lighten routine , mac staged plays that frequently satirised the more serious parts of hospital life . and she planned and rehearsed plays and other entertainments with fully as much zest as the student nurses . while the humour displayed in these mh skits on hospital life may be part of what mikhail bakhtin has dubbed the carnivalesque release of tensions , in a context where few other avenues existed to release personal and larger structurally - induced tensions between different people , they also highlight a close sense of familiarity and certain level of comfort between staff members , which enabled them to tease and laugh at each other . these became traditions within the hospital and while decorous by most standards , they permitted both light - hearted and sometimes more trenchant commentary on the characteristics even the prudishness of the senior staff , most especially the superintendents . a bolder form of challenge to authority was exercised when regular holidays were shared by mh doctors and nurses at umnini holiday camp , located about 40 km south of durban . opened in september 1952 on mnini tribal trust land and with sponsorship from charitable organisations such as the rotary club , this seaside holiday camp , though not exclusively used by mh staff , provided a safe space where , in defiance of racially separatist apartheid laws , mh s doctors and nurses could relax and spend time away together . staff members would spend weekends there to mark special occasions , such as the end of year nurse trainee results , or to encourage bonding amongst new nurse recruits . though their leisure time was structured and supervised , such as being given a lecture on fractures by dr taylor , nurses enjoyed their experiences there immensely , as nurse martha diaho remembered : we certainly enjoyed our swim times ! it was fun to see baba taylor trussed up in his life saving gear , waiting for one of us to drown ! in the evening we played games by the seaside until 8:30 pm , after which we had our evening prayers and sang choruses . it really was a very pleasant weekend and we would love to repeat the experience while the number of staff remained relatively small , the medical superintendents and nurse supervisors kept a personal interest in all of their trainees , who were known by name and whose families , careers , marriages ( hospital romances were no less frequent then than now , perhaps more so given the limited opportunities for young people from respectable conservative families to meet eligible partners ) , children , illnesses and fortunes were tracked . the mccords and taylors , as well as other mh members of staff , were often invited to funerals and weddings of existing or former employees , to share in their sense of loss but also moments of happy celebration . bongi dlomo , who worked at mh between 1953 and 1973 asserted that when she got married dr taylor was one of the guest speakers on my behalf and most of the staff members came so it was really a family it was special. in addition , the hospital s historical papers contain many photographs of weddings as well as individual portrait photographs of the nurse probationers . this was a deliberate way in which taylor nurtured a sense of family - belonging and involved taking and keeping photographs of each of his staff , as a proud father would do of each of his children , as dr aldyth lasbrey wrote of the nurses in one of her letters to her friends in america : in ( our hospital ) she matters as an individual . she knows that her picture is mounted in his gallery of mccord nurses , and he has given her a print to keep or send home to her parents. these carefully planned and implemented activities and routines did much to strengthen mh s particular family - inspired organisational culture , which was so vital to the functioning of this mission hospital over the long term , with its high patient volumes , long hours of work and low pay . however , these photograph album - building exercises and the multi - racial family social get - togethers at mh , or umnini , also importantly helped to build a strong sense of camaraderie , harmony and team spirit , which , many felt , served to chip away , or at least deflect , some of the worst excesses of racial inequalities and tensions that plagued other hospitals at the time . the middle decades of the twentieth century were turbulent ones for mh as it had to weather the storm of political change that accompanied the election of a more conservative government , which after 1948 , promulgated legislative measures which promoted the greater segregation of the country s race groups , worsened race relations , and worked to oppress and further control the country s black majority . individuals and organisations which did not toe the apartheid - party line would feel the full wrath of the law . in the health care sector , these policies did not amount simply to idle threats . during this period , only about one - third of mh s total annual expenses were covered by government grants and subsidies ; a scenario very different from most other mission hospitals whose heavy or complete reliance on state subsidies led to their being nationalised and thus losing their operational independence . in addition to placing enormous strain on the hospital s managers to continue finding the money from other sources to cover expenses , they were also forced to walk a fine line between promoting a more liberal agenda , and facing state subsidy withdrawals if they went too far . on several occasions during the 1960s and early 1970s , mh faced very real threats of closure under the notorious group areas act for being wrongly sited in a white residential area , and for violating numerous apartheid policies . nor was mh immune from the negative effects of race - based policies . sometimes , white staff and their families were made targets of racial abuse and even assault because they or someone they knew worked at this kaffir hospital. black staff and their families not living on the premises had no option but to live in segregated townships , far from their work place and in inferior , under - serviced accommodation , and were often harassed , humiliated and even arrested by the police if they did not carry their passes. in addition , within the hospital s corridors , racial inequalities and discriminations were replicated . this is evident in numerous ways . as a black hospital , though one established many years prior to apartheid and with different humanitarian aims , its race - based admissions policies dove - tailed with the state s separate development aims . its practice , until the 1970s , of having white upper - level managers also kept decision - making power in white hands . paternalistic references made to adult african men and women employees as boys and girls respectively in mh documentary evidence also infantilised the staff . furthermore , for many years , differential salary scales based on racial criteria , as well as the perpetuation of hurtful racial segregation practices , including the existence of separate residential , dining and toilet facilities for black and white staff members , operated at mh . however , mh s greater operational independence as a state - aided not state - controlled institution a status it was able to maintain throughout the apartheid era enabled it to remain a school of liberalism in trying times . with regard to race - based admissions , mh was also able to flout rigid apartheid policies . although it had started out with the aim to serve zulu patients , mh quickly accepted and treated african patients from a variety of ethnic backgrounds , as well as patients who were legally classified as being indian and coloured , much to the chagrin of the state . in 1950 11% of its in - patients were made up of indians and coloureds : and this had increased dramatically to about 40% by 1975 . occasionally , white patients were also treated , such as members of missionary families . and , unlike other provincial hospitals that were run on a racially - segregated basis , mh s wards remained racially - mixed , enabling its patients to fraternise across racial lines . although discrimination in pay and in some other policies remained in place during the segregation and early apartheid years , by the 1960s , mh managers worked hard to achieve parity in the doctors and nurses salary scales across racial lines , and tried to eliminate petty apartheid practices in the hospital , such as segregated eating and ablution facilities . in fact , in 1959 , taylor had persuaded the mh board to vote in favour of paying all of its first year medical interns on the natal provincial administration ( npa)-determined white salary scale. taylor s equal pay for equal work policy for interns was taken up and extended by dr cecil orchard when he took over as medical superintendent in 1965 . balancing limited hospital finances and a contentious 1966 npa decision to increase the salaries of white interns and doctors by 20% ( a move which threatened to provoke staff unrest at mh similar to that which was disrupting health services at other provincial hospitals at the time ) , orchard , however , knew that compromises had to be made . in june 1967 , he persuaded the hospital board to accept the middle figure salary scale that had been stipulated by the npa for indian medical employees , for all interns and doctors working at mh . for its nurses , who were mostly made up of african women , the hospital decision - makers decided to follow the scale set out by the npa for african nurses . thus , although mh s managers were not able to remove the race issue from affecting salaries at their hospital completely , their carefully thought - out salary compromises , which resulted in the same salaries paid for the same levels of qualification and work done , helped prevent the divisive race issue from having an all - determining effect , allowing for the development of closer interracial staff working relationships . what is more , captured in numerous official hospital documents , but also in the words and opinions of countless staff members employed at various levels of the hospital s hierarchy , was a sense of belonging and value they felt as employees in a hospital that provided an inclusive and supportive space where opportunities abounded for both professional promotions and friendships . following 1930s and 1940s firsts , such as the appointment of one of the first african sisters in the country to assume responsibility for a major hospital ward , and using an african sister tutor to conduct nurse training , in 1953 mh appointed durban s first african theatre sister . two years later , mh had african sisters in charge of the whole hospital at night , of the children s ward , of one of the two maternity wards , of the operating theatres , of the large out - patient department and all district midwifery services. by 1958 , dr taylor could boast : we have more senior non - europeans in proportion to the european staff than the provincial hospitals , and by 1962 , of the ten sisters posts available at mh , seven were held by african sisters , two by whites and one by an indian. mh was also one of the first hospitals in the country to open up internship positions and employment opportunities for black interns and doctors from the late 1940s onwards . for many medical interns and doctors , the work experiences they had at mh were life - changing . in 1962 , taylor wrote : again and again , interns tell us they leave ( or later ) , how much their year at mccords has meant to them not only professionally , but in achieving racial understanding and goodwill. having come from different cultural backgrounds , often from diverse locations across the country , and having been brought up in racially - segregated environments , many doctors and nurses remember establishing some of their first close friendships across racial lines at mh . as one african doctor who did his internship in 1981 aptly captured it : at mccords everybody was community , nobody was seeing each other as different and it was ideal . i ca nt think of a better situation for one to have worked in than mccords because there was no racial issue at all . even the number of interns they took was a balance of all the races . orchard was intentional about making sure that he created an environment that ( was ) as close to normal as possible and he did well . sometimes these relationships led to open displays of anti - apartheid solidarity , such as mh nurses marching together in public protest against the nursing act in 1957 , which instituted further racial inequalities in the nursing profession , and which would bring additional heat down on mh managers . by the 1960s , increasingly the hospital s white managers knew that they were being watched by security police , and , threatened on occasion with closure . at times , the development of interracial friendships led to deep personal levels of support for individuals going through difficult times in their private lives . during the 1960s , sister bongi dlomo s husband , a laboratory technician , was arrested for his involvement in anti - apartheid terrorist activities and sentenced to three years imprisonment on robben island . according to sister dlomo : that is when i appreciated mccord s ( be)cause they ( mh supervisors ) were very , very supportive at the time he was detained , mccord ( hospital ) would give transport to take food down ( to the jail ) or take him , you know , a change of clean clothing . and during the trial , they would make sure i attended without saying you know you must pay back the time and it proved that as a family they were very , very supportive in problems like that they supported those people that were in trouble , that had problems like me . provided a possible answer to south africa s ( race ) problem that staff strikes , industrial disputes or serious disciplinary measures were not a serious feature of the mh landscape until the 1970s , says much about the strength of bonds forged by this sense of family also extended beyond mh staff to incorporate many people who appeared to have tenuous links to the hospital , because of actual distance or because they no longer worked or trained at this institution . by the 1940s , however , by which time several hundred nurse and midwife trainees , as well as a number of medical interns had been trained at mh and had moved on to live and work in other places , the close in - person family connections initially built within the walls of the hospital could no longer be maintained so intimately ; something which was lamented by the mccords and taylor in their correspondence and reminiscences . nonetheless , an extensive network of letter writing , both to and from the hospital , ensured the continued circulation of news , and the preservation for those far away of an interest and concern for their beloved mccords. taylor was very much a family man and worked hard to maintain close links with all his biological children by writing weekly letters , because as adults , they lived far from their parents either in the usa or rhodesia ( now zimbabwe ) , where his son boardman went to practise as a doctor . his value of these family - building letter - writing exercises can be seen in his weekly letters to the kids , which amount to hundreds of pages which he carefully preserved almost in lieu of a diary . often composed late at night or in the early hours of the morning , either in his home or in his office , and sometimes waiting to be called in to perform a surgery , these letters were written whenever he could carve a moment to write in his busy schedule . in 1969 , reflecting back on his working with dr taylor , dr mohammed mayat highlighted how many a time as i crept exhausted to bed after a hard day s work with dr taylor , doing many operations , i would notice the light in his office still burning and see him setting up the typewriter trying to catch up with correspondence he had had no time to attend to in the day these letters kept his family informed of the people and goings - on at mh , including people they had met as children growing up in durban . however , they also served to show how despite missing them enormously , both he and their mother had managed to become part of a surrogate mccord family at the hospital , which helped them to feel a sense of belonging too . the familial investments they placed on getting to know their staff , and their conceptualisation of staff doctors and nurses as their sons and daughters , helped soften , to some extent , their feelings of loss of their own sons and daughters . what is more , during the 1950s and 1960s , dr taylor wrote many letters addressed to the mccord family in the hospital s isibuko newsletter , which accompanied other newsworthy articles , stories and interest pieces to keep graduates , ex - employees and other supporters of the hospital around the country and overseas informed about matters affecting the hospital . for example , in 1962 , taylor wrote in an editorial entitled greetings to the mccord family everywhere about the strong bonds members of the mccord family shared , despite the distance : it is a grand feeling to know that though we may , some of us , have left the hospital , we have not left the family and so wherever you are this christmas - time i would like to wish you a very happy christmas. some of his letters also made a concerted effort to keep the mccord family informed of developments in his own family , including career achievements , marriages and the arrival of new grandchildren . the taylors shared the news of their family s lives with many people whose lives they regarded as intertwined with their own . many nurses living hundreds if not thousands of kilometres away from mh greatly appreciated this written umbilicus , capturing their internalisation of the mccord family ethos in their numerous supportive letters to the editor section in successive editions of the isibuko . for example , in 1959 , mavis zondi ( nee ndaba ) wrote how she still regarded mh as her home , despite starting a new life as a sister in a small hospital in pietermaritzburg , some ninety kilometres ( fifty - five miles ) away . further afield , in london , muthulumi pillay , who was undertaking a post - graduate course in paediatric nursing , wrote in 1964 of how much she had enjoyed seeing the taylors who were visiting london at the time , especially pop , who in her opinion , hasnt changed a bit. for many of these nurses , while making them feel home sick remembering all the happy days spent at mccords , reception of regular copies of this mh newsletter provided them with a vital connection to this hospital . furthermore , on various trips , including after their retirement in 1964 , the familial affection that the taylors had helped mould at mh saw members of their extended family ( by then working across the wider southern african region ) greet them with gusto . josephine matondo , writing from luanshya hospital , zambia , in a 1964 edition of the isibuko exclaimed : we were so delighted to see dr and mrs taylor before they left and it was very kind of them to visit the family so widely spread here in rhodesia and zambia. finally , although this wide network helped to preserve for many an interest in mh , its purpose was not only the communication of news or the ideological extension of the mccord family ethos . over the years , the mccords and taylors actively crafted an extensive network of personal and professional friends for mh , some of whom were wealthy and influential , such as government officials , legal or financial experts and powerful business - people . the taylors particularly built and maintained many friendships with various people both in south africa and overseas by writing numerous letters to friends , and , for those based in the durban region , or visiting the durban area , inviting them to formal or informal social gatherings hosted at the hospital or at the medical superintendent s home . here taylor , whose affable personality was often noted , served to charm innumerable guests to the work he and his staff were doing at mh . carefully chosen because of their interest in non - european affairs , in christian missionary work , or because they could assist the hospital in their professional capacities ; some of these mccord friends also provided considerable material support for the hospital . receipts of private donations from sponsors , as well as the provision of political and legal support services when needed , were just some of the actual benefits accrued . having an extended family of loyal supporters played an enormous role in enabling the hospital to survive and expand in difficult times . for more than a century , mccord hospital has self - consciously moulded its own identity and that of those who have studied , worked and been tended to there in christianity and in public service . it has also actively constructed and nurtured a skein of relationships that were based on the ethos of the middle - class family that had first emerged in the west in the nineteenth century . within this familial order , gender , generation and rank were to be observed and there was great investment in creating the belief that the family and the home were private spheres , distinct from the public world of politics and power . similarly , hospitals and their hierarchies were strongly influenced by the ideology of the domestic , particularly with regard to the position and value of nurses , most of whom in south africa ( but unlike much of other colonised regions of the continent ) were women . over the last several decades however , historians have shown just how intimately connected were the public and the private ; they have demonstrated too that hospitals in africa as elsewhere reflected and reinforced the wider social orders of which they were a part . and , as has been observed by marks , amongst others , in south africa s missionary - initiated hospitals and nurse training programmes , this world could be deeply conservative and reinforcing of patriarchal and patronising behaviours and attitudes we have however argued that in considering the different ways in which the notion of the mccord family has been experienced it seems that the world within mh was more complex . generally more respectful of the value of professional service and individual human dignity than was usually the case at state hospitals where white bodies and where working conditions were highly discriminatory and exploitative , mccord hospital also offered to many a space and an identity where they could feel part of a larger while mh has certainly not been without its internal inequalities , frictions , frauds , strikes , protests , rivalries , tragedies , and critics some of whom regard it as having a conservative ethos ( or being prepared to work closely with those who do ; for instance , pepfar under george w. bush ) this does not mean that the majority of doctors or nurses were or are depoliticised and see the individual rather than the social origins of ill health. rather , mh management and medical staff have long been at the forefront of progressive health care in the region ; and with its extended family this impact has been wider still . even as the discourse of the family with its implications of a naturalised sense of hierarchy has increasingly , in recent decades , given way to that of the team with its own resonances of professionalised equals competing with their peers , importantly , it is clear that even many patients whose testimonies are so seldom directly detectable in the records have continued to identify with the central tenets of mh , of christianity and family . as one wrote recently : thank you kindly for the extremely positive experience that we had with mccord hospital on monday , 8 february 2010 . this was visible from the security guards at the parking entrance and right through to our site visit in the hospital . we both experienced a feeling of being home. mccord definitely displays a calmness , being contended , working together for a larger cause , helping one another and care as a heart matter . being both reborn christians , our visit to mccord brought us back to basics . with the impending state takeover of mh significantly , however , at a time when both south african government officials at the highest level are calling for a return to traditional values in nursing and medicine , and nursing and management ( including human relations ) professionals and scholars are investigating how to build supportive environments for health care workers and patients , in a context of the hyper - commodification of hospital - based care , rather than being irrelevant , or a reflection of an out - dated ideology or organisational culture , it may be that a critical history of the ways in which individuals , hospitals , faith and families intersect may be of value for the future of hospitals in africa as well as of interest in their past .

for more than a century , mccord hospital , a partly private and partly state - subsidised mission hospital has provided affordable health - care services , as well as work and professional training opportunities for thousands of people in durban , a city on the east coast of south africa . this article focuses on one important aspect of the hospital s longevity and particular character , or organisational culture : the ethos of a mccord family , integral to which were faith and a commitment to service . while recognising that families including hospital families like that at mccord are contentious social constructs , with deeply embedded hierarchies and inequalities based on race , class and gender , we also consider however how the notion of a mccord family was experienced and shared in complex ways . indeed , during the twentieth century , this ethos was avidly promoted by the hospital s founders and managers and by a wide variety of employees and trainees . it also extended to people at a far geographical remove from durban . moreover , this ethos became so powerful that many patients felt that it shaped their convalescence experience positively . this article considers how this family ethos was constructed and what made it so attractive to this hospital s staff , trainees and patients . furthermore , we consider what work it did for this mission hospital , especially in promoting bonds of multi - racial unity in the contexts of segregation and apartheid society . more broadly , it suggests that critical histories of the ways in which individuals , hospitals , faith and families intersect may be of value for the future of hospitals as well as of interest in their past .

McCord Hospital and Family (Dis)Connections What made McCord different? (1) Prayer; (2) Staff selection; (3) Discipline. A Sense of Belonging: Fun, Friends and Family The McCord Family and Apartheid Extended family Networks Conclusion

the first nurse recruit , elizabeth njapa however , had been brought to the notice of the mccords earlier , since she had been forced to leave school when it was discovered that she had at the age of fourteen had an illegitimate half - caste child , and who was then rejected as rejection by natal family or adopted community and subsequent acceptance at a new mission - based site , though with the requirement of submitting to the authority of a new familial order echoed that of many of the first converts , other nurse trainees increasingly came from lineages of prominent kholwa families , whose fathers were preachers within the congregationalist churches ( after 1897 the zulu congregational church ) , or lawyers , teachers , traders and artisans , and later , doctors and political leaders . it could be argued that the notion of the mccord family was hierarchically constructed and in practice recognised as equals only fellow missionaries , and other formally appointed senior staff matrons , later additional doctors and health professionals ( none of whom until the mid - twentieth century were black ) while the largely female , black nursing , cleaning and other staff were merely subordinates , or children. for instance , especially in the late nineteenth and early decades of the twentieth century , the missionary doctors and their families were also cut off from their own natal and extended families . family was significant for the mccords themselves as well as for the young women who came to train as nurses , the first group of whom were more - or - less the same age as the mccords elder daughters , who had not lived with their parents for some years . although according to katie makanya s reminiscences neither james nor margaret ever mentioned their dead daughters again , it is impossible to read james s account of the death of edna mzoneli of influenza , during the mccords absence from durban , without imagining that this poignant description was in part drawn from the death of his own daughters . one further account of the relationship between james and margaret mccord and edna mzoneli is suggestive of how the close association between these self - identified parental figures and young person could be influential in the moulding of a sense of self . indeed , she was neither self - conscious nor servile ( and ) the only walls or bars to restrain her were her own dignity and self - respect , her knowledge that as a trusted member of the hospital family , she must be worthy of trust. sister molefe went on to add that ( at inanda in the 1950s and 1960s ) they did not just give us academics but they were able to give us ammunition to be assertive , to work hard , the ethos of standing up you know inanda seminary gave us that. in the early years of the twentieth century , zulu - speaking evangelists worked with the mccords at their cottage hospital and dispensary in the centre of durban to persuade sometimes dubious patients of the value of biomedicine . in turn , this christian ethos did much to help build the unifying going against the growing trend towards secularisation underway in many other mission hospitals in south africa during the 1950s and 1960s , where the mission s goal of healing the soul had become secondary to the growing pressures of providing professional medical services to heal the body , mh continued to place as much , if not more emphasis , on the importance of the christian faith in healing . although professing to be a non - denominational hospital that did not restrict staff appointments to professing christians , in practice during these middle decades of the twentieth century , the hospital continued to choose christian candidates as their preferred trainees and employees . from the election of hospital board members for their christian backgrounds , who started and ended every meeting with prayers ; to the appointment of christian medical superintendents , as well as medical and nursing staff ; to a hospital constitution , which emphasised the hospital s duty to carry on a ministry of health and healing to make known to hospital workers and patients by word and by life , the gospel of christ , christianity remained a driving force shaping the identity and work of the hospital . during the 1940s through to the 1970s , medical superintendents taylor and orchard were particularly responsible for the strong christian influences they brought to the hospital . and when he took charge of the hospital , religion and healing went hand in hand . for him , the strong christian family ethos was created by the senior staff , who he said actually lived what they believed in , who infused christianity into the hospital s daily routines , and importantly , their long periods of service provided the continuity needed to nurture and sustain this christian culture . christian messages , themes and stories , including those about the importance of family , were published in isibuko newsletters ; formed the core of plays and other entertainment events ; appeared on the back covers of nurses song books , and formed a cornerstone of the candle - lighting service at the graduation ceremony held annually at the nurses home . in one letter , he wrote of the centrality of prayer and quiet time in his own life as well as revealing some of the strains experienced within the mccord family : more than anything else were the times i had to go to god to get the answers to the problems presenting here in the hospital . for a small number of staff and patients who came to train or work , or convalesce at mh , the pervasive conservative christian orientation was experienced as restrictive , even suffocating . not all patients in particular succumbed to the proselytising ; and some nurse trainees objected to the prison - like discipline and regimented structures within the hospital , as well as regular monthly weigh - ins and strict monitoring of their movements and their visitors , which invaded their personal privacy and treated them like children . on the other hand , there is much evidence in correspondence , board minutes , isibuko newsletters and interviews that many of mh trainees and staff actively embraced this ethos . familial terms of endearment were also extended to another long - serving senior doctor on the staff , dr aldyth lasbrey , who was affectionately known as auntie. the deep effect this family atmosphere had on some individuals who trained at mh , even extended to attempts to propagate it elsewhere , such as mrs athee pillay when she worked at the friends of the sick association ( fosa ) tuberculosis settlement after her nurse training ; in the efforts of the young dr cecil orchard , who tried to build a mini mccords in the rural district of hammanskraal in the then transvaal after completing his internship in the 1950s ; and dr mohammed mayat , whose private hospital , shifa , was created as a if james mccord and alan b. taylor were patriarchs tasked with the spiritual , moral , educational and physical well - being of their staff , a complementary role as mother was often fulfilled by their marital counterparts , margaret mccord and mary taylor , and arguably in a less direct manner in later decades by ruth christofersen and then mavis orchard . while margaret mccord herself assumed the role of matron ( as well as nurse and fund - raiser , and occasionally dispenser and anaesthetist ) , in part reflecting shifting twentieth - century ideologies of the appropriate role of middle class wives , mary , ruth and mavis worked in other , though equally important ways , to build the mccord family. these included organising hospital social functions , such as annual christmas lunches where the medical superintendent , the matron , doctors and nurse supervisors acted as waiters to the great delight of the nurses as well as annual easter parties and usa - inspired thanksgiving dinners , regular hamburger suppers and braais ( barbeques ) , sunday teas , annual prize - giving and candle - lighting services , and many fund - raising events . indeed , as recently , as 2009 , mavis orchard recalled how she had frequently worked herself to a frazzle in entertaining the numerous visitors who came to mh , as well as the hospital s staff who were often invited to their home so as to create a family atmosphere. these became traditions within the hospital and while decorous by most standards , they permitted both light - hearted and sometimes more trenchant commentary on the characteristics even the prudishness of the senior staff , most especially the superintendents . the mccords and taylors , as well as other mh members of staff , were often invited to funerals and weddings of existing or former employees , to share in their sense of loss but also moments of happy celebration . in addition , the hospital s historical papers contain many photographs of weddings as well as individual portrait photographs of the nurse probationers . this was a deliberate way in which taylor nurtured a sense of family - belonging and involved taking and keeping photographs of each of his staff , as a proud father would do of each of his children , as dr aldyth lasbrey wrote of the nurses in one of her letters to her friends in america : in ( our hospital ) she matters as an individual . these carefully planned and implemented activities and routines did much to strengthen mh s particular family - inspired organisational culture , which was so vital to the functioning of this mission hospital over the long term , with its high patient volumes , long hours of work and low pay . however , these photograph album - building exercises and the multi - racial family social get - togethers at mh , or umnini , also importantly helped to build a strong sense of camaraderie , harmony and team spirit , which , many felt , served to chip away , or at least deflect , some of the worst excesses of racial inequalities and tensions that plagued other hospitals at the time . the middle decades of the twentieth century were turbulent ones for mh as it had to weather the storm of political change that accompanied the election of a more conservative government , which after 1948 , promulgated legislative measures which promoted the greater segregation of the country s race groups , worsened race relations , and worked to oppress and further control the country s black majority . furthermore , for many years , differential salary scales based on racial criteria , as well as the perpetuation of hurtful racial segregation practices , including the existence of separate residential , dining and toilet facilities for black and white staff members , operated at mh . although it had started out with the aim to serve zulu patients , mh quickly accepted and treated african patients from a variety of ethnic backgrounds , as well as patients who were legally classified as being indian and coloured , much to the chagrin of the state . although discrimination in pay and in some other policies remained in place during the segregation and early apartheid years , by the 1960s , mh managers worked hard to achieve parity in the doctors and nurses salary scales across racial lines , and tried to eliminate petty apartheid practices in the hospital , such as segregated eating and ablution facilities . what is more , captured in numerous official hospital documents , but also in the words and opinions of countless staff members employed at various levels of the hospital s hierarchy , was a sense of belonging and value they felt as employees in a hospital that provided an inclusive and supportive space where opportunities abounded for both professional promotions and friendships . provided a possible answer to south africa s ( race ) problem that staff strikes , industrial disputes or serious disciplinary measures were not a serious feature of the mh landscape until the 1970s , says much about the strength of bonds forged by this sense of family also extended beyond mh staff to incorporate many people who appeared to have tenuous links to the hospital , because of actual distance or because they no longer worked or trained at this institution . by the 1940s , however , by which time several hundred nurse and midwife trainees , as well as a number of medical interns had been trained at mh and had moved on to live and work in other places , the close in - person family connections initially built within the walls of the hospital could no longer be maintained so intimately ; something which was lamented by the mccords and taylor in their correspondence and reminiscences . in 1969 , reflecting back on his working with dr taylor , dr mohammed mayat highlighted how many a time as i crept exhausted to bed after a hard day s work with dr taylor , doing many operations , i would notice the light in his office still burning and see him setting up the typewriter trying to catch up with correspondence he had had no time to attend to in the day these letters kept his family informed of the people and goings - on at mh , including people they had met as children growing up in durban . however , they also served to show how despite missing them enormously , both he and their mother had managed to become part of a surrogate mccord family at the hospital , which helped them to feel a sense of belonging too . what is more , during the 1950s and 1960s , dr taylor wrote many letters addressed to the mccord family in the hospital s isibuko newsletter , which accompanied other newsworthy articles , stories and interest pieces to keep graduates , ex - employees and other supporters of the hospital around the country and overseas informed about matters affecting the hospital . for example , in 1962 , taylor wrote in an editorial entitled greetings to the mccord family everywhere about the strong bonds members of the mccord family shared , despite the distance : it is a grand feeling to know that though we may , some of us , have left the hospital , we have not left the family and so wherever you are this christmas - time i would like to wish you a very happy christmas. many nurses living hundreds if not thousands of kilometres away from mh greatly appreciated this written umbilicus , capturing their internalisation of the mccord family ethos in their numerous supportive letters to the editor section in successive editions of the isibuko . finally , although this wide network helped to preserve for many an interest in mh , its purpose was not only the communication of news or the ideological extension of the mccord family ethos . the taylors particularly built and maintained many friendships with various people both in south africa and overseas by writing numerous letters to friends , and , for those based in the durban region , or visiting the durban area , inviting them to formal or informal social gatherings hosted at the hospital or at the medical superintendent s home . carefully chosen because of their interest in non - european affairs , in christian missionary work , or because they could assist the hospital in their professional capacities ; some of these mccord friends also provided considerable material support for the hospital . receipts of private donations from sponsors , as well as the provision of political and legal support services when needed , were just some of the actual benefits accrued . for more than a century , mccord hospital has self - consciously moulded its own identity and that of those who have studied , worked and been tended to there in christianity and in public service . it has also actively constructed and nurtured a skein of relationships that were based on the ethos of the middle - class family that had first emerged in the west in the nineteenth century . similarly , hospitals and their hierarchies were strongly influenced by the ideology of the domestic , particularly with regard to the position and value of nurses , most of whom in south africa ( but unlike much of other colonised regions of the continent ) were women . and , as has been observed by marks , amongst others , in south africa s missionary - initiated hospitals and nurse training programmes , this world could be deeply conservative and reinforcing of patriarchal and patronising behaviours and attitudes we have however argued that in considering the different ways in which the notion of the mccord family has been experienced it seems that the world within mh was more complex . generally more respectful of the value of professional service and individual human dignity than was usually the case at state hospitals where white bodies and where working conditions were highly discriminatory and exploitative , mccord hospital also offered to many a space and an identity where they could feel part of a larger while mh has certainly not been without its internal inequalities , frictions , frauds , strikes , protests , rivalries , tragedies , and critics some of whom regard it as having a conservative ethos ( or being prepared to work closely with those who do ; for instance , pepfar under george w. bush ) this does not mean that the majority of doctors or nurses were or are depoliticised and see the individual rather than the social origins of ill health. even as the discourse of the family with its implications of a naturalised sense of hierarchy has increasingly , in recent decades , given way to that of the team with its own resonances of professionalised equals competing with their peers , importantly , it is clear that even many patients whose testimonies are so seldom directly detectable in the records have continued to identify with the central tenets of mh , of christianity and family . with the impending state takeover of mh significantly , however , at a time when both south african government officials at the highest level are calling for a return to traditional values in nursing and medicine , and nursing and management ( including human relations ) professionals and scholars are investigating how to build supportive environments for health care workers and patients , in a context of the hyper - commodification of hospital - based care , rather than being irrelevant , or a reflection of an out - dated ideology or organisational culture , it may be that a critical history of the ways in which individuals , hospitals , faith and families intersect may be of value for the future of hospitals in africa as well as of interest in their past .

nor were african patriarchs and matriarchs immediately keen for their daughters to become nurses , for their labour was needed for homestead production , and guarantees of their virtue uncertain outside the direct control of the family , including peer groups who provided companionship , and who monitored sexuality and reproduction . the first converts amakholwa often stressed in similar ways to their western counterparts the gendered propriety of conduct of sons and daughters and it is not surprising therefore that it was the first and second generations of kholwa families which would encourage their daughters to take up schooling and then , as positions gradually opened , the professions of teaching and nursing . of the first african women to be trained ( from 1911 ) by margaret mccord and martha s. macneill three , nomhlatuzi bhengu , julia magwaza and edna mzoneli , had been pupils at inanda seminary . the first nurse recruit , elizabeth njapa however , had been brought to the notice of the mccords earlier , since she had been forced to leave school when it was discovered that she had at the age of fourteen had an illegitimate half - caste child , and who was then rejected as rejection by natal family or adopted community and subsequent acceptance at a new mission - based site , though with the requirement of submitting to the authority of a new familial order echoed that of many of the first converts , other nurse trainees increasingly came from lineages of prominent kholwa families , whose fathers were preachers within the congregationalist churches ( after 1897 the zulu congregational church ) , or lawyers , teachers , traders and artisans , and later , doctors and political leaders . to further illustrate the interconnections between such families and mh , selby ncgobo ( a political activist and academic who received a master s degree from yale university in 1940 and a phd in economics from the university of london in 1964 ) and chief albert luthuli ( who is most well - known for his role as president - general of the african national congress from 1952 to 1967 ) were both members of the mh board by 1946 and taylor and luthuli shared a close relationship . during luthuli s detention in pretoria the previous year , when this was granted , and after taylor s suggestion that luthuli be transferred to mh for treatment was declined , taylor confined the content of his letter to assurances of support for luthuli from his friends and passing on news about luthuli s daughters , hilda and eleanor , who trained as nurses at mh . in a letter franked by the prison authorities on 6 june , after sending personal greetings to dr and mrs taylor and asking that these be extended to the mh staff and to the mission council of churches and congregations , luthuli wrote : i do nt know if i am professionally correct to ask you , a senior , but may i ask you to send my greetings and best wishes to my two daughters hilda and eleanor . it could be argued that the notion of the mccord family was hierarchically constructed and in practice recognised as equals only fellow missionaries , and other formally appointed senior staff matrons , later additional doctors and health professionals ( none of whom until the mid - twentieth century were black ) while the largely female , black nursing , cleaning and other staff were merely subordinates , or children. family was significant for the mccords themselves as well as for the young women who came to train as nurses , the first group of whom were more - or - less the same age as the mccords elder daughters , who had not lived with their parents for some years . even so , when during a severe outbreak of measles she addressed the people of her natal family home on basic preventive and hygiene measures she felt it necessary to subdue this sense of personhood : in the closing chapters of his memoir , my patients were zulus , james mccord devotes some pages to considering the significance of the mccord family. thus , discipline applied both to an internal disposition and to outward manifestations of control , and both mccord s memoir and the oral testimony of long - service mh senior nurses , record a strong sense of pride in being a mh nurse , and an active embrace of its moral and behavioural codes . in turn , this christian ethos did much to help build the unifying going against the growing trend towards secularisation underway in many other mission hospitals in south africa during the 1950s and 1960s , where the mission s goal of healing the soul had become secondary to the growing pressures of providing professional medical services to heal the body , mh continued to place as much , if not more emphasis , on the importance of the christian faith in healing . from the election of hospital board members for their christian backgrounds , who started and ended every meeting with prayers ; to the appointment of christian medical superintendents , as well as medical and nursing staff ; to a hospital constitution , which emphasised the hospital s duty to carry on a ministry of health and healing to make known to hospital workers and patients by word and by life , the gospel of christ , christianity remained a driving force shaping the identity and work of the hospital . christian messages , themes and stories , including those about the importance of family , were published in isibuko newsletters ; formed the core of plays and other entertainment events ; appeared on the back covers of nurses song books , and formed a cornerstone of the candle - lighting service at the graduation ceremony held annually at the nurses home . in letters between taylor and dr paul keen of the non - european hospital in johannesburg written in 1961 , harrowing details of drug addictions , overdoses , alcoholism and suicides amongst black doctors , nurses and midwives several of them mh graduates are itemised . not all patients in particular succumbed to the proselytising ; and some nurse trainees objected to the prison - like discipline and regimented structures within the hospital , as well as regular monthly weigh - ins and strict monitoring of their movements and their visitors , which invaded their personal privacy and treated them like children . the deep effect this family atmosphere had on some individuals who trained at mh , even extended to attempts to propagate it elsewhere , such as mrs athee pillay when she worked at the friends of the sick association ( fosa ) tuberculosis settlement after her nurse training ; in the efforts of the young dr cecil orchard , who tried to build a mini mccords in the rural district of hammanskraal in the then transvaal after completing his internship in the 1950s ; and dr mohammed mayat , whose private hospital , shifa , was created as a if james mccord and alan b. taylor were patriarchs tasked with the spiritual , moral , educational and physical well - being of their staff , a complementary role as mother was often fulfilled by their marital counterparts , margaret mccord and mary taylor , and arguably in a less direct manner in later decades by ruth christofersen and then mavis orchard . while margaret mccord herself assumed the role of matron ( as well as nurse and fund - raiser , and occasionally dispenser and anaesthetist ) , in part reflecting shifting twentieth - century ideologies of the appropriate role of middle class wives , mary , ruth and mavis worked in other , though equally important ways , to build the mccord family. these included organising hospital social functions , such as annual christmas lunches where the medical superintendent , the matron , doctors and nurse supervisors acted as waiters to the great delight of the nurses as well as annual easter parties and usa - inspired thanksgiving dinners , regular hamburger suppers and braais ( barbeques ) , sunday teas , annual prize - giving and candle - lighting services , and many fund - raising events . this dates back at least to both the mccords and taylors own sometimes jocular and sometimes wry sense of fun , which on occasion , such as the new year s eve festivities in 1953 , saw alan taylor wearing his wife s dress and a wig in a sketch he had designed with other doctors to make his contribution in the way of fun to the party , and also from the interactions between mh s first superintendent of nurses martha s. macneill and the first nurse trainees . while the humour displayed in these mh skits on hospital life may be part of what mikhail bakhtin has dubbed the carnivalesque release of tensions , in a context where few other avenues existed to release personal and larger structurally - induced tensions between different people , they also highlight a close sense of familiarity and certain level of comfort between staff members , which enabled them to tease and laugh at each other . it really was a very pleasant weekend and we would love to repeat the experience while the number of staff remained relatively small , the medical superintendents and nurse supervisors kept a personal interest in all of their trainees , who were known by name and whose families , careers , marriages ( hospital romances were no less frequent then than now , perhaps more so given the limited opportunities for young people from respectable conservative families to meet eligible partners ) , children , illnesses and fortunes were tracked . this was a deliberate way in which taylor nurtured a sense of family - belonging and involved taking and keeping photographs of each of his staff , as a proud father would do of each of his children , as dr aldyth lasbrey wrote of the nurses in one of her letters to her friends in america : in ( our hospital ) she matters as an individual . however , these photograph album - building exercises and the multi - racial family social get - togethers at mh , or umnini , also importantly helped to build a strong sense of camaraderie , harmony and team spirit , which , many felt , served to chip away , or at least deflect , some of the worst excesses of racial inequalities and tensions that plagued other hospitals at the time . the middle decades of the twentieth century were turbulent ones for mh as it had to weather the storm of political change that accompanied the election of a more conservative government , which after 1948 , promulgated legislative measures which promoted the greater segregation of the country s race groups , worsened race relations , and worked to oppress and further control the country s black majority . black staff and their families not living on the premises had no option but to live in segregated townships , far from their work place and in inferior , under - serviced accommodation , and were often harassed , humiliated and even arrested by the police if they did not carry their passes. although discrimination in pay and in some other policies remained in place during the segregation and early apartheid years , by the 1960s , mh managers worked hard to achieve parity in the doctors and nurses salary scales across racial lines , and tried to eliminate petty apartheid practices in the hospital , such as segregated eating and ablution facilities . balancing limited hospital finances and a contentious 1966 npa decision to increase the salaries of white interns and doctors by 20% ( a move which threatened to provoke staff unrest at mh similar to that which was disrupting health services at other provincial hospitals at the time ) , orchard , however , knew that compromises had to be made . thus , although mh s managers were not able to remove the race issue from affecting salaries at their hospital completely , their carefully thought - out salary compromises , which resulted in the same salaries paid for the same levels of qualification and work done , helped prevent the divisive race issue from having an all - determining effect , allowing for the development of closer interracial staff working relationships . what is more , captured in numerous official hospital documents , but also in the words and opinions of countless staff members employed at various levels of the hospital s hierarchy , was a sense of belonging and value they felt as employees in a hospital that provided an inclusive and supportive space where opportunities abounded for both professional promotions and friendships . following 1930s and 1940s firsts , such as the appointment of one of the first african sisters in the country to assume responsibility for a major hospital ward , and using an african sister tutor to conduct nurse training , in 1953 mh appointed durban s first african theatre sister . two years later , mh had african sisters in charge of the whole hospital at night , of the children s ward , of one of the two maternity wards , of the operating theatres , of the large out - patient department and all district midwifery services. by 1958 , dr taylor could boast : we have more senior non - europeans in proportion to the european staff than the provincial hospitals , and by 1962 , of the ten sisters posts available at mh , seven were held by african sisters , two by whites and one by an indian. provided a possible answer to south africa s ( race ) problem that staff strikes , industrial disputes or serious disciplinary measures were not a serious feature of the mh landscape until the 1970s , says much about the strength of bonds forged by this sense of family also extended beyond mh staff to incorporate many people who appeared to have tenuous links to the hospital , because of actual distance or because they no longer worked or trained at this institution . by the 1940s , however , by which time several hundred nurse and midwife trainees , as well as a number of medical interns had been trained at mh and had moved on to live and work in other places , the close in - person family connections initially built within the walls of the hospital could no longer be maintained so intimately ; something which was lamented by the mccords and taylor in their correspondence and reminiscences . in 1969 , reflecting back on his working with dr taylor , dr mohammed mayat highlighted how many a time as i crept exhausted to bed after a hard day s work with dr taylor , doing many operations , i would notice the light in his office still burning and see him setting up the typewriter trying to catch up with correspondence he had had no time to attend to in the day these letters kept his family informed of the people and goings - on at mh , including people they had met as children growing up in durban . what is more , during the 1950s and 1960s , dr taylor wrote many letters addressed to the mccord family in the hospital s isibuko newsletter , which accompanied other newsworthy articles , stories and interest pieces to keep graduates , ex - employees and other supporters of the hospital around the country and overseas informed about matters affecting the hospital . for example , in 1962 , taylor wrote in an editorial entitled greetings to the mccord family everywhere about the strong bonds members of the mccord family shared , despite the distance : it is a grand feeling to know that though we may , some of us , have left the hospital , we have not left the family and so wherever you are this christmas - time i would like to wish you a very happy christmas. similarly , hospitals and their hierarchies were strongly influenced by the ideology of the domestic , particularly with regard to the position and value of nurses , most of whom in south africa ( but unlike much of other colonised regions of the continent ) were women . and , as has been observed by marks , amongst others , in south africa s missionary - initiated hospitals and nurse training programmes , this world could be deeply conservative and reinforcing of patriarchal and patronising behaviours and attitudes we have however argued that in considering the different ways in which the notion of the mccord family has been experienced it seems that the world within mh was more complex . generally more respectful of the value of professional service and individual human dignity than was usually the case at state hospitals where white bodies and where working conditions were highly discriminatory and exploitative , mccord hospital also offered to many a space and an identity where they could feel part of a larger while mh has certainly not been without its internal inequalities , frictions , frauds , strikes , protests , rivalries , tragedies , and critics some of whom regard it as having a conservative ethos ( or being prepared to work closely with those who do ; for instance , pepfar under george w. bush ) this does not mean that the majority of doctors or nurses were or are depoliticised and see the individual rather than the social origins of ill health. even as the discourse of the family with its implications of a naturalised sense of hierarchy has increasingly , in recent decades , given way to that of the team with its own resonances of professionalised equals competing with their peers , importantly , it is clear that even many patients whose testimonies are so seldom directly detectable in the records have continued to identify with the central tenets of mh , of christianity and family . with the impending state takeover of mh significantly , however , at a time when both south african government officials at the highest level are calling for a return to traditional values in nursing and medicine , and nursing and management ( including human relations ) professionals and scholars are investigating how to build supportive environments for health care workers and patients , in a context of the hyper - commodification of hospital - based care , rather than being irrelevant , or a reflection of an out - dated ideology or organisational culture , it may be that a critical history of the ways in which individuals , hospitals , faith and families intersect may be of value for the future of hospitals in africa as well as of interest in their past .

nearly all of the photoactive iridium(iii ) complexes that are used as emitters in electroluminescent devices , as dyes in solar cells , in nonlinear optics ( nlo ) , as photoredox catalysts , as solar fuels , and in bioimaging contain conjugated five - membered chelated ligands , such as the commonly used 2-phenylpyridine ( ppyh ) , 2,2-bipyridine ( bpy ) , acetylacetonate ( acac ) , and picolinate ( pic ) . photoactive iridium complexes containing a six - membered cyclometalating chelate are very rare , and the few reported examples can be categorized into two families of complexes : those containing conjugated or nonconjugated bidentate cyclometalating ligands . for instance , in 2008 , song et al . obtained a phosphorescent ir(iii ) complex [ ir(dfb - pz)2(fptz ) ] ( where ( dfb - pz)h = 2,4-difluorobenzyl - n - pyrazole and fptz = 3-trifluoromethyl-5-(2-pyridyl)triazole ) containing a nonconjugated n - benzylpyrazole ligand to form a six - membered chelated framework . this complex is a blue emitter in dichloromethane with em = 437 and 460 nm ( pl = 10% and e = 0.10 s ) . the methylene spacer of the cyclometalated ligand effectively interrupts the -conjugation to produce a significant blue shift , compared to [ ir(dfppy)2(fptz ) ] ( where dfppyh = 2-(2,4-difluorophenyl)pyridine , which uses a five - membered ring chelate c^n ligand and the same ancillary ligand ( em = 460 and 489 nm in dichloromethane ) . complex [ ir(bis[2-(n - carbazolyl)pyridinato - n , c)picolinate ] ( [ ir(cpy)2(pic ) ] ) containing a six - membered chelating framework where the ligand is fully conjugated , leading to yellow luminescence in the recrystallized solid state with em = 538 nm ( pl = 5% ) . in an ongoing effort in our group to develop charged blue - emitting phosphors for solution - processed light - emitting electrochemical cells ( leecs ) and organic light emitting diodes ( oleds ) , we investigated the coordination of 2-benzhydrylpyridine ( bnpyh2 ) derivatives with ir(iii ) in order to access six - membered chelate complexes t1t3 ( figure 1a ) . surprisingly , given the prior art , upon sequential treatment of ircl36h2o with bnpyh2 and then 4,4-di - tert - butyl-2,2-bipyridine ( dtbubpy ) in a one - pot reaction , t1 was not obtained . instead the formation of 1 arises from a highly unusual double c h bond activation of the bnpyh2 ligand , which binds to the iridium in a tripodal fashion . following a similar synthetic protocol , analogues 2 and 3 , functionalized with either electron - donating tert - butyl or electron - withdrawing trifluoromethyl groups meta to the ir this is the first report of an iridium complex of the form [ ir(c^n^c)(n^n)cl ] and one in which the tridentate bis - cyclometalated ligand is a tripod featuring two six - membered metallacycles . indeed , the complex with the closest binding motif is [ ir(bppy)(bpy)cl ] , where bppy is 2-([1,1-biphenyl]-3-yl)pyridine . this previous complex possesses a related [ ir(c^c^n)(n^n)cl ] structure , but the bppy ligand coordinates to the iridium in the more commonly observed planar five - membered chelate fashion . it is a very poorly luminescent near - ir emitter in ch2cl2 ( em = 725 nm , pl = 8.4 10% ) . ir(iii ) complexes bearing monocyclometalating planar tridentate bis(five - membered ) chelate ligands ( e.g. , n^n^c or n^c^n ) and a cyclometalating bidentate ligand , c^n , have on the other hand been more widely explored , while kozhevnikov has reported dinuclear ir(iii ) complexes with a bridging ligand featuring two n^c^n motifs . ( a ) scheme for the proposed synthesis of the initial targets ( t1t3 ) and the synthesis of complexes 13 . ( a , i ) ircl36h2o , 2-ethoxyethanol / h2o ( 3:1 ) , reflux , 19 h ; ( ii ) dtbubpy , reflux , 6 h. ( b ) aq . nh4pf6 . compounds l1 , l2 , and l3 ( scheme 1 ) were synthesized in two steps via a grignard reaction followed by a reduction step and obtained as solids in good yields . a mixture of the corresponding proligand and ircl36h2o in 2-ethoxyethanol / h2o ( 3:1 ) was refluxed . after 19 h , dtbubpy 4,4-di - tert - butyl-2,2-bipyridyl was added at once , and heating was continued for 6 h to give the neutral complexes 13 as solids in a one - pot synthesis in 45% , 51% , and 55% yield , respectively ( figure 1a ) . complexes 13 were characterized by h , c , and , for 3 , f nmr spectroscopy ; esi - hr mass spectra ; elemental analysis ; and melting point determination ( see figures s21s30 in the supporting information ( si ) for nmr and esi - hr mass spectra ) . ( a , 1 ) mg , 1,2-dibromoethane , thf , n2 , reflux , 4 h. ( 2 ) methyl picolinate , thf , 0 c to r.t . , 90 min ; ( b , 1 ) hoac , 57% hi . ( 2 ) naohaq . , 0 c to r.t . ; ( c , 1 ) pbr3 , reflux , 2 h. ( 2 ) zn , hoac , ( 3 ) naohaq . , 0 c to r.t . the structures of 13 were determined by single - crystal x - ray diffraction ( figure 1b , table s1 ) . all three complexes possess a distorted octahedral geometry with the tridentate tripod ligand coordinated to the iridium to form two six - membered chelated rings . both cyclometalating carbon atoms are trans to the pyridine rings of the dtbubpy , and the pyridyl unit of the bnpy - type ligands is trans to the chloride . cl bond [ 2.375(3 ) for 1 , 2.3612(8 ) for 2 , and 2.369(2 ) for 3 ] is in the same range as that found for [ ir(tpy)(dmbpy)cl ] ( 2.357 , where tpy = 2,2:6,2-terpyridine and dmbpy = 4,4-dimethyl-2,2-bipyridine ) but is significantly shorter ( by ca . 0.1 ) than the ir cl bond in other cyclometalated tridentate ir(iii ) complexes . cc^n^c bonds [ 2.048(13 ) and 2.064(6 ) for 1 , 2.028(4 ) and 2.031(3 ) for 2 , and 2.017(7 ) and 2.027 for 3 ] , this leads also to a correspondingly shorter ir nc^n^c bond [ 2.055(11 ) for 1 , 2.044(3 ) for 2 , and 2.032(7 ) for 3 ] compared to the ir ndtbubpy bonds [ 2.158(10 ) and 2.159(11 ) for 1 , 2.127(3 ) and 2.140(3 ) for 2 , and 2.122(6 ) and 2.133(5 ) for 3 ] . the bite angle of the n^n ligand is unremarkable at 75.60(4) for 1 , 75.85(12) for 2 , and 76.1(2) for 3 and in line with cationic ir(iii ) complexes of the form [ ir(c^n)2(n^n ) ] . owing to the presence of the six - membered chelates , the c ir c bond angle is significantly larger ( 85.60(5) for 1 , 85.65(15) for 2 , and 84.(3 ) for 3 ) than the ndtbubpy ir the electrochemical behavior for 13 was evaluated by cyclic voltammetry ( cv ) and differential pulse voltammetry ( dpv ) in deaerated ch2cl2 solution at 298 k at a scan rate of 50 mv s using fc / fc as the internal reference and referenced with respect to sce . the electrochemistry data can be found in table 1 , and the voltammograms are shown in figure 2 . all complexes exhibit a quasi - reversible single electron oxidation peak , which is attributed to the ir(iii)/ir(iv ) redox couple with contributions from the bnpy - type ligand . complex 2 displays a lower oxidation potential ( 0.80 v ) than 1 ( 0.87 v ) , both of which are notably lower than [ ir(mesppy)2(dtbubpy)]pf6 ( e1/2;ox . = 1.17 v in deaerated ch2cl2 , where mesppy is 2-phenyl-4-mesityl - pyridinato ) . conversely , 3 shows a significantly anodically shifted oxidation potential at 1.14 v. the cvs of 13 show irreversible reduction waves that are monoelectronic as inferred from the respective dpvs . dft calculations ( figure 3a ) indicate that both the homo and homo1 , which are close in energy ( see figure s34 in the si ) , involve the iridium and chloride atoms and the two phenyl rings of the bnpy ligand . the lumo is almost exclusively localized on the dtbubpy ligand , while the lumo+1 is primarily on the pyridyl ring of the bnpy ligand . the reduction potentials of 1 and 2 are each found at 1.82 v , while the reduction wave of 3 at 1.62 v is anodically shifted by 200 mv compared to 1 and 2 . all three complexes are significantly more difficult to reduce than [ ir(mesppy)2(dtbubpy)]pf6 ( e1/2;red . cyclic voltammograms ( in solid lines ) and differential pulse voltammetry ( in dotted lines ) carried out in degassed ch2cl2 at a scan rate of 50 mv s , with fc / fc as the internal reference , referenced to sce ( 0.46 v vs sce ) . ( a ) representation of the four frontier mos of 1 . ( b ) side and top views of the spin density distribution for the lowest triplet state ( t1 ) of 1 . [ ru(bpy)3]pf6 in mecn as reference ( pl = 1.8% in aerated mecn at 298 k ) . measurements were carried out in degassed ch2cl2 at a scan rate of 50 mv s with fc / fc used as the internal reference , and referenced with respect to sce ( fc / fc = 0.46 v in ch2cl2 ) . the normalized uv vis absorption spectra of 13 recorded in ch2cl2 at 298 k are depicted in figure 4 and the data summarized in table s2 in the si . the invariance of the intense high - energy ( on the order of ( 11.5 ) 10 m cm ) absorption bands below 300 nm are ascribed to * ligand - centered ( lc ) transitions localized on the dtbubpy ligand . two moderately intense bands ( on the order of ( 35 ) 10 m cm ) in the region of 340360 nm and 390405 nm are assigned to mixed charge - transfer transitions with the former consisting of more metal - to - ligand / ligand - to - ligand charge - transfer ( mlct / llct ) character while the latter , according to td - dft calculations , implicate an intraligand ct ( ilct ) from the phenyl rings to the pyridyl heterocycle of the bnpy - type ligand ( see figure s34 and table s3 in the si ) . weak bands ( on the order of 10 m cm ) with onsets between 470 and 510 nm and tailing to 580 nm are attributed to a mixture of mlct / llct and spin - forbidden mlct / llct transitions involving the dtbubpy ligand . introduction of the tert - butyl groups in 2 results in a small red - shift of the ct bands below 340 nm , whereas the trifluoromethyl groups in 3 produce a significant blue - shift of these bands , trends that are corroborated by td - dft calculations ( figure s35 in the si ) . normalized uv vis absorption and photoluminescence spectra of 13 in ch2cl2 at 298 k. the normalized photoluminescence ( pl ) spectra of 13 in degassed ch2cl2 are shown in figure 4 , and the data are summarized in table 1 . upon photoexcitation at 420 nm , all complexes show a broad and unstructured profile , indicative of an emission with mixed ct character . in line with the trends observed in the absorption spectra and the oxidation potentials in the cvs , the emission maxima are 581 , 619 , and 630 nm for 3 , 1 , and 2 , respectively . these emission maxima match very closely to the vertical phosphorescence energies calculated by spin - unrestricted dft , which predicts emissions at 573 , 613 , and 622 nm , respectively . the calculations reveal that the emissive triplet state is localized on the iridium , chlorine , and dtbubpy but does not include significant contributions from the bnpy ligand ( figures 3b and s36 in the si ) . the photoluminescence quantum yield ( pl ) of 1 is 8% , which is lower than that of the yellow - emitting [ ir(ppy)2(dtbubpy)]pf6 ( pl = 64% in ch2cl2 , em = 570 nm , where ppyh is 2-phenylpyridine ) . the radiative rate constants , kr , for 1 and 2 are similar ( 2.52 vs 2.51 10 s , respectively ) . however , 2 exhibits a significantly larger nonradiative rate constant , knr ( 39.33 10 s ) , compared to 1 ( 28.93 10 s ) . complex 3 possesses both the largest kr ( 3.62 10 s ) and the smallest knr values ( 10.31 10 s ) in accordance with the energy gap law . in conclusion , a new family of luminescent iridium(iii ) complexes bearing an unprecedented tripodal bis(six - membered ) chelate tridentate ligand has been prepared through a highly unusual double cyclometalation reaction . current efforts are focused on further modulating the electronics through a combination of modifications of the ancillary di - imine and the monodentate chloride ligands . this unprecedented tripodal ligand opens new perspectives for the design of tridentate ir luminophores . an oven - dried flask was charged under a nitrogen atmosphere with magnesium turnings ( 0.911 g , 37.50 mmol , 5 equiv ) and thf ( 80 ml ) followed by 2 ml of 1,2-dibromoethane . after the observation of gas evolution , the corresponding bromo derivative ( 22.50 mmol , 3 equiv ) in thf ( 40 ml ) was added dropwise . the reaction mixture was heated under stirring and kept at reflux for 4 h , resulting in a color change of the solution to gray . the reaction mixture was then cooled in an ice bath , and a solution of methyl picolinate ( 7.50 mmol , 1.00 equiv ) in thf ( 40 ml ) was added carefully . the solution was allowed to warm to room temperature and was stirred for 90 min . the combined organic layers were dried over mgso4 , and the solvent was evaporated , leaving a residue , which was purified over silica ( 10% etoac in petroleum ether as the solvent ) . the desired fractions were combined , and the solvent was evaporated , leaving the title compound . compound a1 was prepared according to the general procedure and was obtained as a colorless solid ( 1.686 g , 6.45 mmol ) . h nmr ( 400 mhz , cdcl3 ) : 8.60 ( d , j = 4.7 hz , 1h ) , 7.64 ( td , j = 7.8 , 1.8 hz , 1h ) , 7.407.21 ( m , 11h ) , 7.12 ( d , j = 7.9 hz , 1h ) , 6.28 ( s , 1h ) . c nmr ( 101 mhz , cdcl3 ) : 163.2 , 147.7 , 146.1 , 136.4 , 128.1 , 127.9 , 127.3 , 122.9 , 122.3 , 80.8 . hr - ms ( ftms ) [ m + h ] calculated ( c18h15noh ) : 262.1226 . found : 262.1226 . chn calcd for c18h15no : c , 82.73 ; h , 5.79 ; n , 5.36 . found : c , 82.68 ; h , 5.73 ; n , 5.41 . compound a2 was prepared according to the general procedure and was obtained as a colorless solid ( 1.994 g , 5.34 mmol ) . h nmr ( 400 mhz , cdcl3 ) : 8.58 ( d , j = 3.2 hz , 1h ) , 7.677.60 ( m , 1h ) , 7.337.28 ( m , 4h ) , 7.247.12 ( m , 6h ) , 6.19 ( s , 1h ) , 1.30 ( s , 18h ) . c nmr ( 101 mhz , cdcl3 ) : 163.8 , 150.1 , 147.7 , 143.3 , 136.4 , 127.9 , 124.9 , 123.1 , 122.3 , 80.6 , 34.6 , 31.5 . hr - ms ( ftms ) [ m + h ] calculated ( c26h31noh ) : 374.2478 . found : 374.2476 . chn calcd for c26h31no : c , 83.60 ; h , 8.37 ; n , 3.75 . found : c , 83.45 ; h , 8.51 ; n , 3.88 . compound a3 was prepared according to the general procedure and was obtained as a beige solid ( 1.893 g , 4.76 mmol ) . h nmr ( 400 mhz , cdcl3 ) : 8.64 ( d , j = 7.4 hz , 1h ) , 7.757.69 ( m , 1h ) , 7.60 ( s , 4h ) , 7.44 ( s , 4h ) , 7.31 ( d , j = 8.5 hz , 1h ) , 7.13 ( d , j = 9.7 hz , 1h ) , 6.50 ( s , 1h ) . c nmr ( 101 mhz , cdcl3 ) : 161.5 , 149.4 , 148.2 , 136.9 , 130.4 , 130.1 , 129.7 , 129.4 , 128.5 , 125.2 , 125.1 , 125.1 , 125.1 , 123.1 , 122.7 , 80.4 , 77.4 , 77.1 , 76.7 . hr - ms ( ftms ) [ m + h ] calculated ( c20h13f6noh ) : 398.0974 . found : 398.0965 . chn calcd for c20h13f6no : c , 60.46 ; h , 3.30 ; n , 3.53 . found : c , 60.51 ; h , 3.36 ; a mixture of a1 ( 0.837 g , 3.21 mmol ) , aqueous 57% hi ( 2.5 ml ) , and hoac ( 13 ml ) was heated to 100 c for 4 h. the resulting mixture was then cooled to 0 c and basified to ph 9 with an aqueous naoh solution ( 2 m ) . ethyl acetate ( 100 ml ) was added , and the mixture was washed successively with an aqueous nahso3 solution and brine . the combined organic layers were dried over mgso4 , and the solvent was evaporated . the residue was purified over silica ( 10% etoac in petroleum ether as the solvent ) . the desired fractions were combined and the solvent evaporated leaving a beige solid ( 0.788 g , 3.21 mmol ) . h nmr ( 400 mhz , cdcl3 ) : 8.64 ( d , j = 4.1 hz , 1h ) , 7.667.59 ( m , 1h ) , 7.33 ( t , j = 7.3 hz , 4h ) , 7.24 ( dd , j = 21.8 , 7.2 hz , 6h ) , 7.187.10 ( m , 2h ) , 5.76 ( s , 1h ) . c nmr ( 101 mhz , cdcl3 ) : 163.2 , 149.5 , 142.7 , 136.4 , 129.4 , 128.4 , 126.5 , 123.8 , 121.4 , 59.4 . hr - ms ( ftms ) [ m + h ] calculated ( c18h15nh ) : 246.1277 . found : 246.1277 . a mixture of the a2 ( 0.900 g , 2.41 mmol ) , aqueous 57% hi ( 2.70 ml ) , and hoac ( 13.20 ml ) was heated to 100 c for 4 h. the resulting mixture was then cooled to 0 c and basified to ph 9 with an aqueous naoh solution ( 2 m ) . ethyl acetate ( 100 ml ) was added , and the mixture was washed successively with an aqueous nahso3 solution and brine . the combined organic layers were dried over mgso4 , and the solvent was evaporated . the residue was purified over silica ( 10% etoac in petroleum ether as the solvent ) . the desired fractions were combined and the solvent evaporated , yielding the title compound as oil ( 0.655 g , 1.83 mmol ) . h nmr ( 400 mhz , cdcl3 ) : 8.628.56 ( m , 1h ) , 7.627.56 ( m , 1h ) , 7.31 ( s , 4h ) , 7.10 ( s , 6h ) , 5.62 ( s , 1h ) , 1.29 ( s , 18h ) . c nmr ( 126 mhz , cdcl3 ) : 163.8 , 149.6 , 149.2 , 139.9 , 136.5 , 129.0 , 125.4 , 123.9 , 121.4 , 58.7 , 34.5 , 31.5 . hr - ms ( asap ) [ m + h ] calculated ( c26h31nh ) : 358.2535 . found : 358.2534 . a mixture of a3 ( 0.500 g , 1.26 mmol , 1 equiv ) and pbr3 ( 25 ml ) was vigorously stirred and heated and kept at 110 c for 2 h. the mixture was then cooled to r.t . and was carefully poured onto ice , and aqueous naoh ( 2 m ) was added until the ph was neutral . the organic layer was dried over mgso4 , and the solvent was evaporated , leaving a residue which was dissolved in acetic acid ( 50 ml ) . then , zinc dust ( 0.799 g , 12.60 mmol , 10 equiv ) was added . after 1 h , 20 ml of water was carefully added , and aqueous naoh ( 2 m ) was added until the ph was neutral . the organic layer was dried over mgso4 , and the solvent was evaporated , leaving a residue which was purified over silica ( 10% etoac in petroleum ether as the solvent ) . the desired fractions were combined , and the solvent was evaporated , leaving colorless oil ( 0.117 mg , 0.31 mmol ) . yield : 24% . h nmr ( 400 mhz , cdcl3 ) : 8.63 ( ddd , j = 4.9 , 1.9 , 0.9 hz , 1h ) , 7.66 ( td , j = 7.7 , 1.9 hz , 1h ) , 7.57 ( d , j = 8.2 hz , 4h ) , 7.30 ( d , j = 8.1 hz , 4h ) , 7.20 ( ddd , j = 7.6 , 4.8 , 1.1 hz , 1h ) , 7.10 ( dt , j = 7.9 , 1.1 hz , 1h ) , 5.75 ( s , 1h ) . c nmr ( 126 mhz , cdcl3 ) : 161.4 , 150.1 , 146.0 , 137.0 , 129.8 , 129.3 , 125.7 , 125.3 , 124.0 , 122.2 , 58.9 hr - ms ( ftms ) [ m + h ] calculated ( c20h13f6nh ) : 382.1030 . found : 382.1023 . a mixture of the corresponding ligand ( 1.2 equiv ) and ircl36h2o ( 1 equiv ) in 2-ethoxyethanol ( 15 ml ) and h2o ( 5 ml ) was heated under stirring to 125 c . after 19 h , 4,4-di - tert - butyl-2,2-bipyridine ( 1.5 equiv ) was added , and heating was continued . after 6 h , the solvent was evaporated , leaving a solid , which was filtered over silica ( 1% meoh in ch2cl2 ) . the desired fractions were combined , and the solvent was evaporated , leaving a solid which was washed with diethyl ether . the general one - pot protocol using 0.114 g ( 0.38 mmol ) of ircl36h2o was followed , and 1 was obtained as a red solid ( 0.127 g , 0.0.13 mmol ) . h nmr ( 400 mhz , cd2cl2 ) : 8.75 ( d , j = 5.8 hz , 2h ) , 8.42 ( d , j = 1.7 hz , 2h ) , 7.70 ( dd , j = 7.3 , 1.4 hz , 2h ) , 7.53 ( d , j = 7.5 hz , 1h ) , 7.49 ( dd , j = 5.9 , 1.9 hz , 3h ) , 7.24 ( dd , j = 7.1 , 1.6 hz , 2h ) , 7.19 ( d , j = 5.9 hz , 1h ) , 6.90 ( dtd , j = 26.4 , 7.3 , 1.5 hz , 4h ) , 6.456.39 ( m , 1h ) , 5.28 ( s , 1h ) , 1.50 ( s , 18h ) . c nmr ( 126 mhz , cd2cl2 ) : 165.4 , 162.6 , 157.4 , 151.8 , 151.3 , 143.9 , 141.4 , 138.7 , 137.0 , 125.8 , 124.0 , 123.8 , 123.4 , 122.7 , 122.1 , 120.8 , 69.7 , 35.9 , 30.8 . hr - ms ( asap ) [ m cl ] calculated ( c36h37irn3 ) : 704.2618 . found : 704.2618 . chn calcd for c36h37clirn33/2 h2o : c , 56.42 ; h , 5.26 ; n , 5.48 . found : c , 56.45 ; h , 5.24 ; n , 5.28 . the general one - pot protocol using 0.088 g ( 0.30 mmol ) of ircl36h2o was followed , and 2 was obtained as a red solid ( 0.129 g , 0.15 mmol ) . h nmr ( 400 mhz , cd2cl2 ) : 8.79 ( d , j = 5.9 hz , 2h ) , 8.42 ( d , j = 2.0 hz , 2h ) , 7.80 ( d , j = 2.1 hz , 2h ) , 7.497.42 ( m , 4h ) , 7.13 ( d , j = 7.7 hz , 3h ) , 6.89 ( dd , j = 7.7 , 2.1 hz , 2h ) , 6.38 ( ddd , j = 7.0 , 5.9 , 2.0 hz , 1h ) , 5.22 ( s , 1h ) , 1.51 ( s , 18h ) , 1.32 ( s , 18h ) . c nmr ( 126 mhz , cd2cl2 ) : 166.0 , 162.6 , 157.5 , 151.7 , 151.3 , 147.8 , 141.1 , 140.4 , 136.8 , 135.9 , 123.7 , 123.6 , 122.7 , 122.4 , 120.8 , 118.8 , 68.6 , 35.9 , 34.9 , 32.1 , 30.9 . [ m cl ] calculated ( c44h53irn3 ) 816.3869 . found : 816.3867 . chn calcd for c44h53clirn3 : c , 62.06 ; h , 6.27 ; n , 4.93 . found : c , 61.96 ; h , 6.31 ; n , 5.02 . the general one - pot protocol using 0.060 g ( 0.20 mmol ) of ircl36h2o was followed , and 3 was obtained as a yellow solid ( 0.096 g , 0.11 mmol ) . h nmr ( 400 mhz , cd2cl2 ) : 8.59 ( d , j = 5.8 hz , 2h ) , 8.44 ( d , j = 2.0 hz , 2h ) , 7.98 ( d , j = 2.0 hz , 2h ) , 7.627.48 ( m , 4h ) , 7.37 ( d , j = 7.7 hz , 2h ) , 7.207.15 ( m , 3h ) , 6.50 ( ddd , j = 7.5 , 5.9 , 1.7 hz , 1h ) , 5.46 ( s , 1h ) , 1.51 ( s , 18h ) . c nmr ( 126 mhz , cd2cl2 ) : 163.5 , 163.3 , 157.3 , 151.9 , 151.1 , 147.4 , 142.2 , 137.6 , 134.7 , 127.6 , 126.7 , 124.5 , 124.3 , 123.5 , 123.4 , 121.2 , 119.4 , 69.2 , 36.0 , 30.8 . f nmr ( 376 mhz , cd2cl2 ) : 61.58 . [ m cl ] calculated ( c38h35f6irn3 ) : 840.2364 . found : 840.2379 . chn calcd for c38h35f6irn3 : c , 52.14 ; h , 4.03 ; n , 4.80 . found : c , 52.10 ; h , 4.16 ; n , 4.74 .

a new family consisting of three luminescent neutral ir(iii ) complexes with the unprecedented [ ir(c^n^c)(n^n)cl ] architecture , where c^n^c is a bis(six - membered ) chelating tridentate tripod ligand derived from 2-benzhydrylpyridine ( bnpy ) and n^n is 4,4-di - tert - butyl-2,2-bipyridine ( dtbubpy ) , is reported . x - ray crystallography reveals an unexpected and unusual double c h bond activation of the two distal nonconjugated phenyl rings of the bnpy coupled with a very short ir cl bond trans to the pyridine of the bnpy ligand . depending on the substitution on the bnpy ligand , phosphorescence , ranging from yellow to red , is observed in dichloromethane solution . a combined study using density functional theory ( dft ) and time - dependent dft ( td - dft ) corroborates the mixed charge - transfer nature of the related excited states .

Introduction Results and Discussion Conclusions Experimental Section

nearly all of the photoactive iridium(iii ) complexes that are used as emitters in electroluminescent devices , as dyes in solar cells , in nonlinear optics ( nlo ) , as photoredox catalysts , as solar fuels , and in bioimaging contain conjugated five - membered chelated ligands , such as the commonly used 2-phenylpyridine ( ppyh ) , 2,2-bipyridine ( bpy ) , acetylacetonate ( acac ) , and picolinate ( pic ) . obtained a phosphorescent ir(iii ) complex [ ir(dfb - pz)2(fptz ) ] ( where ( dfb - pz)h = 2,4-difluorobenzyl - n - pyrazole and fptz = 3-trifluoromethyl-5-(2-pyridyl)triazole ) containing a nonconjugated n - benzylpyrazole ligand to form a six - membered chelated framework . this complex is a blue emitter in dichloromethane with em = 437 and 460 nm ( pl = 10% and e = 0.10 s ) . the methylene spacer of the cyclometalated ligand effectively interrupts the -conjugation to produce a significant blue shift , compared to [ ir(dfppy)2(fptz ) ] ( where dfppyh = 2-(2,4-difluorophenyl)pyridine , which uses a five - membered ring chelate c^n ligand and the same ancillary ligand ( em = 460 and 489 nm in dichloromethane ) . in an ongoing effort in our group to develop charged blue - emitting phosphors for solution - processed light - emitting electrochemical cells ( leecs ) and organic light emitting diodes ( oleds ) , we investigated the coordination of 2-benzhydrylpyridine ( bnpyh2 ) derivatives with ir(iii ) in order to access six - membered chelate complexes t1t3 ( figure 1a ) . surprisingly , given the prior art , upon sequential treatment of ircl36h2o with bnpyh2 and then 4,4-di - tert - butyl-2,2-bipyridine ( dtbubpy ) in a one - pot reaction , t1 was not obtained . instead the formation of 1 arises from a highly unusual double c h bond activation of the bnpyh2 ligand , which binds to the iridium in a tripodal fashion . following a similar synthetic protocol , analogues 2 and 3 , functionalized with either electron - donating tert - butyl or electron - withdrawing trifluoromethyl groups meta to the ir this is the first report of an iridium complex of the form [ ir(c^n^c)(n^n)cl ] and one in which the tridentate bis - cyclometalated ligand is a tripod featuring two six - membered metallacycles . indeed , the complex with the closest binding motif is [ ir(bppy)(bpy)cl ] , where bppy is 2-([1,1-biphenyl]-3-yl)pyridine . this previous complex possesses a related [ ir(c^c^n)(n^n)cl ] structure , but the bppy ligand coordinates to the iridium in the more commonly observed planar five - membered chelate fashion . it is a very poorly luminescent near - ir emitter in ch2cl2 ( em = 725 nm , pl = 8.4 10% ) . ir(iii ) complexes bearing monocyclometalating planar tridentate bis(five - membered ) chelate ligands ( e.g. , n^n^c or n^c^n ) and a cyclometalating bidentate ligand , c^n , have on the other hand been more widely explored , while kozhevnikov has reported dinuclear ir(iii ) complexes with a bridging ligand featuring two n^c^n motifs . ( a ) scheme for the proposed synthesis of the initial targets ( t1t3 ) and the synthesis of complexes 13 . ( a , i ) ircl36h2o , 2-ethoxyethanol / h2o ( 3:1 ) , reflux , 19 h ; ( ii ) dtbubpy , reflux , 6 h. ( b ) aq . a mixture of the corresponding proligand and ircl36h2o in 2-ethoxyethanol / h2o ( 3:1 ) was refluxed . after 19 h , dtbubpy 4,4-di - tert - butyl-2,2-bipyridyl was added at once , and heating was continued for 6 h to give the neutral complexes 13 as solids in a one - pot synthesis in 45% , 51% , and 55% yield , respectively ( figure 1a ) . the structures of 13 were determined by single - crystal x - ray diffraction ( figure 1b , table s1 ) . all three complexes possess a distorted octahedral geometry with the tridentate tripod ligand coordinated to the iridium to form two six - membered chelated rings . both cyclometalating carbon atoms are trans to the pyridine rings of the dtbubpy , and the pyridyl unit of the bnpy - type ligands is trans to the chloride . cl bond [ 2.375(3 ) for 1 , 2.3612(8 ) for 2 , and 2.369(2 ) for 3 ] is in the same range as that found for [ ir(tpy)(dmbpy)cl ] ( 2.357 , where tpy = 2,2:6,2-terpyridine and dmbpy = 4,4-dimethyl-2,2-bipyridine ) but is significantly shorter ( by ca . 0.1 ) than the ir cl bond in other cyclometalated tridentate ir(iii ) complexes . cc^n^c bonds [ 2.048(13 ) and 2.064(6 ) for 1 , 2.028(4 ) and 2.031(3 ) for 2 , and 2.017(7 ) and 2.027 for 3 ] , this leads also to a correspondingly shorter ir nc^n^c bond [ 2.055(11 ) for 1 , 2.044(3 ) for 2 , and 2.032(7 ) for 3 ] compared to the ir ndtbubpy bonds [ 2.158(10 ) and 2.159(11 ) for 1 , 2.127(3 ) and 2.140(3 ) for 2 , and 2.122(6 ) and 2.133(5 ) for 3 ] . the bite angle of the n^n ligand is unremarkable at 75.60(4) for 1 , 75.85(12) for 2 , and 76.1(2) for 3 and in line with cationic ir(iii ) complexes of the form [ ir(c^n)2(n^n ) ] . owing to the presence of the six - membered chelates , the c ir c bond angle is significantly larger ( 85.60(5) for 1 , 85.65(15) for 2 , and 84. (3 ) for 3 ) than the ndtbubpy ir the electrochemical behavior for 13 was evaluated by cyclic voltammetry ( cv ) and differential pulse voltammetry ( dpv ) in deaerated ch2cl2 solution at 298 k at a scan rate of 50 mv s using fc / fc as the internal reference and referenced with respect to sce . all complexes exhibit a quasi - reversible single electron oxidation peak , which is attributed to the ir(iii)/ir(iv ) redox couple with contributions from the bnpy - type ligand . complex 2 displays a lower oxidation potential ( 0.80 v ) than 1 ( 0.87 v ) , both of which are notably lower than [ ir(mesppy)2(dtbubpy)]pf6 ( e1/2;ox . = 1.17 v in deaerated ch2cl2 , where mesppy is 2-phenyl-4-mesityl - pyridinato ) . dft calculations ( figure 3a ) indicate that both the homo and homo1 , which are close in energy ( see figure s34 in the si ) , involve the iridium and chloride atoms and the two phenyl rings of the bnpy ligand . the lumo is almost exclusively localized on the dtbubpy ligand , while the lumo+1 is primarily on the pyridyl ring of the bnpy ligand . cyclic voltammograms ( in solid lines ) and differential pulse voltammetry ( in dotted lines ) carried out in degassed ch2cl2 at a scan rate of 50 mv s , with fc / fc as the internal reference , referenced to sce ( 0.46 v vs sce ) . ( a ) representation of the four frontier mos of 1 . the invariance of the intense high - energy ( on the order of ( 11.5 ) 10 m cm ) absorption bands below 300 nm are ascribed to * ligand - centered ( lc ) transitions localized on the dtbubpy ligand . two moderately intense bands ( on the order of ( 35 ) 10 m cm ) in the region of 340360 nm and 390405 nm are assigned to mixed charge - transfer transitions with the former consisting of more metal - to - ligand / ligand - to - ligand charge - transfer ( mlct / llct ) character while the latter , according to td - dft calculations , implicate an intraligand ct ( ilct ) from the phenyl rings to the pyridyl heterocycle of the bnpy - type ligand ( see figure s34 and table s3 in the si ) . weak bands ( on the order of 10 m cm ) with onsets between 470 and 510 nm and tailing to 580 nm are attributed to a mixture of mlct / llct and spin - forbidden mlct / llct transitions involving the dtbubpy ligand . introduction of the tert - butyl groups in 2 results in a small red - shift of the ct bands below 340 nm , whereas the trifluoromethyl groups in 3 produce a significant blue - shift of these bands , trends that are corroborated by td - dft calculations ( figure s35 in the si ) . in line with the trends observed in the absorption spectra and the oxidation potentials in the cvs , the emission maxima are 581 , 619 , and 630 nm for 3 , 1 , and 2 , respectively . these emission maxima match very closely to the vertical phosphorescence energies calculated by spin - unrestricted dft , which predicts emissions at 573 , 613 , and 622 nm , respectively . the calculations reveal that the emissive triplet state is localized on the iridium , chlorine , and dtbubpy but does not include significant contributions from the bnpy ligand ( figures 3b and s36 in the si ) . the photoluminescence quantum yield ( pl ) of 1 is 8% , which is lower than that of the yellow - emitting [ ir(ppy)2(dtbubpy)]pf6 ( pl = 64% in ch2cl2 , em = 570 nm , where ppyh is 2-phenylpyridine ) . complex 3 possesses both the largest kr ( 3.62 10 s ) and the smallest knr values ( 10.31 10 s ) in accordance with the energy gap law . in conclusion , a new family of luminescent iridium(iii ) complexes bearing an unprecedented tripodal bis(six - membered ) chelate tridentate ligand has been prepared through a highly unusual double cyclometalation reaction . current efforts are focused on further modulating the electronics through a combination of modifications of the ancillary di - imine and the monodentate chloride ligands . an oven - dried flask was charged under a nitrogen atmosphere with magnesium turnings ( 0.911 g , 37.50 mmol , 5 equiv ) and thf ( 80 ml ) followed by 2 ml of 1,2-dibromoethane . the reaction mixture was heated under stirring and kept at reflux for 4 h , resulting in a color change of the solution to gray . h nmr ( 400 mhz , cdcl3 ) : 8.60 ( d , j = 4.7 hz , 1h ) , 7.64 ( td , j = 7.8 , 1.8 hz , 1h ) , 7.407.21 ( m , 11h ) , 7.12 ( d , j = 7.9 hz , 1h ) , 6.28 ( s , 1h ) . compound a2 was prepared according to the general procedure and was obtained as a colorless solid ( 1.994 g , 5.34 mmol ) . compound a3 was prepared according to the general procedure and was obtained as a beige solid ( 1.893 g , 4.76 mmol ) . h nmr ( 400 mhz , cdcl3 ) : 8.64 ( d , j = 7.4 hz , 1h ) , 7.757.69 ( m , 1h ) , 7.60 ( s , 4h ) , 7.44 ( s , 4h ) , 7.31 ( d , j = 8.5 hz , 1h ) , 7.13 ( d , j = 9.7 hz , 1h ) , 6.50 ( s , 1h ) . found : c , 60.51 ; h , 3.36 ; a mixture of a1 ( 0.837 g , 3.21 mmol ) , aqueous 57% hi ( 2.5 ml ) , and hoac ( 13 ml ) was heated to 100 c for 4 h. the resulting mixture was then cooled to 0 c and basified to ph 9 with an aqueous naoh solution ( 2 m ) . a mixture of the a2 ( 0.900 g , 2.41 mmol ) , aqueous 57% hi ( 2.70 ml ) , and hoac ( 13.20 ml ) was heated to 100 c for 4 h. the resulting mixture was then cooled to 0 c and basified to ph 9 with an aqueous naoh solution ( 2 m ) . a mixture of a3 ( 0.500 g , 1.26 mmol , 1 equiv ) and pbr3 ( 25 ml ) was vigorously stirred and heated and kept at 110 c for 2 h. the mixture was then cooled to r.t . h nmr ( 400 mhz , cdcl3 ) : 8.63 ( ddd , j = 4.9 , 1.9 , 0.9 hz , 1h ) , 7.66 ( td , j = 7.7 , 1.9 hz , 1h ) , 7.57 ( d , j = 8.2 hz , 4h ) , 7.30 ( d , j = 8.1 hz , 4h ) , 7.20 ( ddd , j = 7.6 , 4.8 , 1.1 hz , 1h ) , 7.10 ( dt , j = 7.9 , 1.1 hz , 1h ) , 5.75 ( s , 1h ) . a mixture of the corresponding ligand ( 1.2 equiv ) and ircl36h2o ( 1 equiv ) in 2-ethoxyethanol ( 15 ml ) and h2o ( 5 ml ) was heated under stirring to 125 c . after 19 h , 4,4-di - tert - butyl-2,2-bipyridine ( 1.5 equiv ) was added , and heating was continued . h nmr ( 400 mhz , cd2cl2 ) : 8.75 ( d , j = 5.8 hz , 2h ) , 8.42 ( d , j = 1.7 hz , 2h ) , 7.70 ( dd , j = 7.3 , 1.4 hz , 2h ) , 7.53 ( d , j = 7.5 hz , 1h ) , 7.49 ( dd , j = 5.9 , 1.9 hz , 3h ) , 7.24 ( dd , j = 7.1 , 1.6 hz , 2h ) , 7.19 ( d , j = 5.9 hz , 1h ) , 6.90 ( dtd , j = 26.4 , 7.3 , 1.5 hz , 4h ) , 6.456.39 ( m , 1h ) , 5.28 ( s , 1h ) , 1.50 ( s , 18h ) . h nmr ( 400 mhz , cd2cl2 ) : 8.79 ( d , j = 5.9 hz , 2h ) , 8.42 ( d , j = 2.0 hz , 2h ) , 7.80 ( d , j = 2.1 hz , 2h ) , 7.497.42 ( m , 4h ) , 7.13 ( d , j = 7.7 hz , 3h ) , 6.89 ( dd , j = 7.7 , 2.1 hz , 2h ) , 6.38 ( ddd , j = 7.0 , 5.9 , 2.0 hz , 1h ) , 5.22 ( s , 1h ) , 1.51 ( s , 18h ) , 1.32 ( s , 18h ) .

nearly all of the photoactive iridium(iii ) complexes that are used as emitters in electroluminescent devices , as dyes in solar cells , in nonlinear optics ( nlo ) , as photoredox catalysts , as solar fuels , and in bioimaging contain conjugated five - membered chelated ligands , such as the commonly used 2-phenylpyridine ( ppyh ) , 2,2-bipyridine ( bpy ) , acetylacetonate ( acac ) , and picolinate ( pic ) . photoactive iridium complexes containing a six - membered cyclometalating chelate are very rare , and the few reported examples can be categorized into two families of complexes : those containing conjugated or nonconjugated bidentate cyclometalating ligands . obtained a phosphorescent ir(iii ) complex [ ir(dfb - pz)2(fptz ) ] ( where ( dfb - pz)h = 2,4-difluorobenzyl - n - pyrazole and fptz = 3-trifluoromethyl-5-(2-pyridyl)triazole ) containing a nonconjugated n - benzylpyrazole ligand to form a six - membered chelated framework . the methylene spacer of the cyclometalated ligand effectively interrupts the -conjugation to produce a significant blue shift , compared to [ ir(dfppy)2(fptz ) ] ( where dfppyh = 2-(2,4-difluorophenyl)pyridine , which uses a five - membered ring chelate c^n ligand and the same ancillary ligand ( em = 460 and 489 nm in dichloromethane ) . complex [ ir(bis[2-(n - carbazolyl)pyridinato - n , c)picolinate ] ( [ ir(cpy)2(pic ) ] ) containing a six - membered chelating framework where the ligand is fully conjugated , leading to yellow luminescence in the recrystallized solid state with em = 538 nm ( pl = 5% ) . in an ongoing effort in our group to develop charged blue - emitting phosphors for solution - processed light - emitting electrochemical cells ( leecs ) and organic light emitting diodes ( oleds ) , we investigated the coordination of 2-benzhydrylpyridine ( bnpyh2 ) derivatives with ir(iii ) in order to access six - membered chelate complexes t1t3 ( figure 1a ) . surprisingly , given the prior art , upon sequential treatment of ircl36h2o with bnpyh2 and then 4,4-di - tert - butyl-2,2-bipyridine ( dtbubpy ) in a one - pot reaction , t1 was not obtained . instead the formation of 1 arises from a highly unusual double c h bond activation of the bnpyh2 ligand , which binds to the iridium in a tripodal fashion . following a similar synthetic protocol , analogues 2 and 3 , functionalized with either electron - donating tert - butyl or electron - withdrawing trifluoromethyl groups meta to the ir this is the first report of an iridium complex of the form [ ir(c^n^c)(n^n)cl ] and one in which the tridentate bis - cyclometalated ligand is a tripod featuring two six - membered metallacycles . indeed , the complex with the closest binding motif is [ ir(bppy)(bpy)cl ] , where bppy is 2-([1,1-biphenyl]-3-yl)pyridine . this previous complex possesses a related [ ir(c^c^n)(n^n)cl ] structure , but the bppy ligand coordinates to the iridium in the more commonly observed planar five - membered chelate fashion . , n^n^c or n^c^n ) and a cyclometalating bidentate ligand , c^n , have on the other hand been more widely explored , while kozhevnikov has reported dinuclear ir(iii ) complexes with a bridging ligand featuring two n^c^n motifs . ( a ) scheme for the proposed synthesis of the initial targets ( t1t3 ) and the synthesis of complexes 13 . ( a , i ) ircl36h2o , 2-ethoxyethanol / h2o ( 3:1 ) , reflux , 19 h ; ( ii ) dtbubpy , reflux , 6 h. ( b ) aq . compounds l1 , l2 , and l3 ( scheme 1 ) were synthesized in two steps via a grignard reaction followed by a reduction step and obtained as solids in good yields . after 19 h , dtbubpy 4,4-di - tert - butyl-2,2-bipyridyl was added at once , and heating was continued for 6 h to give the neutral complexes 13 as solids in a one - pot synthesis in 45% , 51% , and 55% yield , respectively ( figure 1a ) . complexes 13 were characterized by h , c , and , for 3 , f nmr spectroscopy ; esi - hr mass spectra ; elemental analysis ; and melting point determination ( see figures s21s30 in the supporting information ( si ) for nmr and esi - hr mass spectra ) . ( a , 1 ) mg , 1,2-dibromoethane , thf , n2 , reflux , 4 h. ( 2 ) methyl picolinate , thf , 0 c to r.t . ; ( c , 1 ) pbr3 , reflux , 2 h. ( 2 ) zn , hoac , ( 3 ) naohaq . the structures of 13 were determined by single - crystal x - ray diffraction ( figure 1b , table s1 ) . all three complexes possess a distorted octahedral geometry with the tridentate tripod ligand coordinated to the iridium to form two six - membered chelated rings . both cyclometalating carbon atoms are trans to the pyridine rings of the dtbubpy , and the pyridyl unit of the bnpy - type ligands is trans to the chloride . cl bond [ 2.375(3 ) for 1 , 2.3612(8 ) for 2 , and 2.369(2 ) for 3 ] is in the same range as that found for [ ir(tpy)(dmbpy)cl ] ( 2.357 , where tpy = 2,2:6,2-terpyridine and dmbpy = 4,4-dimethyl-2,2-bipyridine ) but is significantly shorter ( by ca . 0.1 ) than the ir cl bond in other cyclometalated tridentate ir(iii ) complexes . cc^n^c bonds [ 2.048(13 ) and 2.064(6 ) for 1 , 2.028(4 ) and 2.031(3 ) for 2 , and 2.017(7 ) and 2.027 for 3 ] , this leads also to a correspondingly shorter ir nc^n^c bond [ 2.055(11 ) for 1 , 2.044(3 ) for 2 , and 2.032(7 ) for 3 ] compared to the ir ndtbubpy bonds [ 2.158(10 ) and 2.159(11 ) for 1 , 2.127(3 ) and 2.140(3 ) for 2 , and 2.122(6 ) and 2.133(5 ) for 3 ] . the bite angle of the n^n ligand is unremarkable at 75.60(4) for 1 , 75.85(12) for 2 , and 76.1(2) for 3 and in line with cationic ir(iii ) complexes of the form [ ir(c^n)2(n^n ) ] . owing to the presence of the six - membered chelates , the c ir c bond angle is significantly larger ( 85.60(5) for 1 , 85.65(15) for 2 , and 84. (3 ) for 3 ) than the ndtbubpy ir the electrochemical behavior for 13 was evaluated by cyclic voltammetry ( cv ) and differential pulse voltammetry ( dpv ) in deaerated ch2cl2 solution at 298 k at a scan rate of 50 mv s using fc / fc as the internal reference and referenced with respect to sce . the electrochemistry data can be found in table 1 , and the voltammograms are shown in figure 2 . all complexes exhibit a quasi - reversible single electron oxidation peak , which is attributed to the ir(iii)/ir(iv ) redox couple with contributions from the bnpy - type ligand . conversely , 3 shows a significantly anodically shifted oxidation potential at 1.14 v. the cvs of 13 show irreversible reduction waves that are monoelectronic as inferred from the respective dpvs . dft calculations ( figure 3a ) indicate that both the homo and homo1 , which are close in energy ( see figure s34 in the si ) , involve the iridium and chloride atoms and the two phenyl rings of the bnpy ligand . the lumo is almost exclusively localized on the dtbubpy ligand , while the lumo+1 is primarily on the pyridyl ring of the bnpy ligand . cyclic voltammograms ( in solid lines ) and differential pulse voltammetry ( in dotted lines ) carried out in degassed ch2cl2 at a scan rate of 50 mv s , with fc / fc as the internal reference , referenced to sce ( 0.46 v vs sce ) . ( b ) side and top views of the spin density distribution for the lowest triplet state ( t1 ) of 1 . measurements were carried out in degassed ch2cl2 at a scan rate of 50 mv s with fc / fc used as the internal reference , and referenced with respect to sce ( fc / fc = 0.46 v in ch2cl2 ) . the normalized uv vis absorption spectra of 13 recorded in ch2cl2 at 298 k are depicted in figure 4 and the data summarized in table s2 in the si . the invariance of the intense high - energy ( on the order of ( 11.5 ) 10 m cm ) absorption bands below 300 nm are ascribed to * ligand - centered ( lc ) transitions localized on the dtbubpy ligand . two moderately intense bands ( on the order of ( 35 ) 10 m cm ) in the region of 340360 nm and 390405 nm are assigned to mixed charge - transfer transitions with the former consisting of more metal - to - ligand / ligand - to - ligand charge - transfer ( mlct / llct ) character while the latter , according to td - dft calculations , implicate an intraligand ct ( ilct ) from the phenyl rings to the pyridyl heterocycle of the bnpy - type ligand ( see figure s34 and table s3 in the si ) . weak bands ( on the order of 10 m cm ) with onsets between 470 and 510 nm and tailing to 580 nm are attributed to a mixture of mlct / llct and spin - forbidden mlct / llct transitions involving the dtbubpy ligand . introduction of the tert - butyl groups in 2 results in a small red - shift of the ct bands below 340 nm , whereas the trifluoromethyl groups in 3 produce a significant blue - shift of these bands , trends that are corroborated by td - dft calculations ( figure s35 in the si ) . normalized uv vis absorption and photoluminescence spectra of 13 in ch2cl2 at 298 k. the normalized photoluminescence ( pl ) spectra of 13 in degassed ch2cl2 are shown in figure 4 , and the data are summarized in table 1 . in line with the trends observed in the absorption spectra and the oxidation potentials in the cvs , the emission maxima are 581 , 619 , and 630 nm for 3 , 1 , and 2 , respectively . the calculations reveal that the emissive triplet state is localized on the iridium , chlorine , and dtbubpy but does not include significant contributions from the bnpy ligand ( figures 3b and s36 in the si ) . the photoluminescence quantum yield ( pl ) of 1 is 8% , which is lower than that of the yellow - emitting [ ir(ppy)2(dtbubpy)]pf6 ( pl = 64% in ch2cl2 , em = 570 nm , where ppyh is 2-phenylpyridine ) . in conclusion , a new family of luminescent iridium(iii ) complexes bearing an unprecedented tripodal bis(six - membered ) chelate tridentate ligand has been prepared through a highly unusual double cyclometalation reaction . current efforts are focused on further modulating the electronics through a combination of modifications of the ancillary di - imine and the monodentate chloride ligands . after the observation of gas evolution , the corresponding bromo derivative ( 22.50 mmol , 3 equiv ) in thf ( 40 ml ) was added dropwise . h nmr ( 400 mhz , cdcl3 ) : 8.58 ( d , j = 3.2 hz , 1h ) , 7.677.60 ( m , 1h ) , 7.337.28 ( m , 4h ) , 7.247.12 ( m , 6h ) , 6.19 ( s , 1h ) , 1.30 ( s , 18h ) . h nmr ( 400 mhz , cdcl3 ) : 8.64 ( d , j = 7.4 hz , 1h ) , 7.757.69 ( m , 1h ) , 7.60 ( s , 4h ) , 7.44 ( s , 4h ) , 7.31 ( d , j = 8.5 hz , 1h ) , 7.13 ( d , j = 9.7 hz , 1h ) , 6.50 ( s , 1h ) . found : c , 60.51 ; h , 3.36 ; a mixture of a1 ( 0.837 g , 3.21 mmol ) , aqueous 57% hi ( 2.5 ml ) , and hoac ( 13 ml ) was heated to 100 c for 4 h. the resulting mixture was then cooled to 0 c and basified to ph 9 with an aqueous naoh solution ( 2 m ) . a mixture of the a2 ( 0.900 g , 2.41 mmol ) , aqueous 57% hi ( 2.70 ml ) , and hoac ( 13.20 ml ) was heated to 100 c for 4 h. the resulting mixture was then cooled to 0 c and basified to ph 9 with an aqueous naoh solution ( 2 m ) . a mixture of a3 ( 0.500 g , 1.26 mmol , 1 equiv ) and pbr3 ( 25 ml ) was vigorously stirred and heated and kept at 110 c for 2 h. the mixture was then cooled to r.t . h nmr ( 400 mhz , cdcl3 ) : 8.63 ( ddd , j = 4.9 , 1.9 , 0.9 hz , 1h ) , 7.66 ( td , j = 7.7 , 1.9 hz , 1h ) , 7.57 ( d , j = 8.2 hz , 4h ) , 7.30 ( d , j = 8.1 hz , 4h ) , 7.20 ( ddd , j = 7.6 , 4.8 , 1.1 hz , 1h ) , 7.10 ( dt , j = 7.9 , 1.1 hz , 1h ) , 5.75 ( s , 1h ) . a mixture of the corresponding ligand ( 1.2 equiv ) and ircl36h2o ( 1 equiv ) in 2-ethoxyethanol ( 15 ml ) and h2o ( 5 ml ) was heated under stirring to 125 c . h nmr ( 400 mhz , cd2cl2 ) : 8.79 ( d , j = 5.9 hz , 2h ) , 8.42 ( d , j = 2.0 hz , 2h ) , 7.80 ( d , j = 2.1 hz , 2h ) , 7.497.42 ( m , 4h ) , 7.13 ( d , j = 7.7 hz , 3h ) , 6.89 ( dd , j = 7.7 , 2.1 hz , 2h ) , 6.38 ( ddd , j = 7.0 , 5.9 , 2.0 hz , 1h ) , 5.22 ( s , 1h ) , 1.51 ( s , 18h ) , 1.32 ( s , 18h ) . h nmr ( 400 mhz , cd2cl2 ) : 8.59 ( d , j = 5.8 hz , 2h ) , 8.44 ( d , j = 2.0 hz , 2h ) , 7.98 ( d , j = 2.0 hz , 2h ) , 7.627.48 ( m , 4h ) , 7.37 ( d , j = 7.7 hz , 2h ) , 7.207.15 ( m , 3h ) , 6.50 ( ddd , j = 7.5 , 5.9 , 1.7 hz , 1h ) , 5.46 ( s , 1h ) , 1.51 ( s , 18h ) .

among the various types of fuel cells , polymer electrolyte fuel cells ( pefcs ) have attracted the most attention for transportation and portable applications . advantages include the following : rapid start - up and shut down , low temperature operation ( 80 c ) , high power density , and fully solid state components . current state - of - the - art technology uses platinum ( pt ) or platinum alloys supported on carbon for both anode and cathode electrodes . it is estimated that the catalyst layers contribute to as much as 39% of the total fuel cell stack cost , making it one of the major challenges for pefc commercialization . in order to meet the u.s . department of energy ( doe ) targets for 2015 , it is necessary to reduce the amount of pt catalyst used by three - quarters , while ensuring the same performance . another major challenge is insufficient durability ; current state - of - the art performance under realistic operating conditions is just half of the target set by the doe . it is generally accepted that catalyst activity and durability depends on pt particle size and dispersion ( which can be affected by the support material ) , as well as the interaction between the catalyst particle and the support . the most widely used catalyst support is carbon black vulcan xc-72r , which has high surface area and good electrical conductivity . however , pt particles can get buried inside the pores and hence reduce the active triple - phase boundary accessible for electrochemical reaction . furthermore , vulcan xc-72r is electrochemically unstable at high potential , leading to corrosion after extended operation in acidic media and varying potentials . as the carbon corrodes , pt nanoparticles agglomerate into larger particles and/or detach from the support material , consequently reducing the electrochemical surface area ( ecsa ) and catalytic activity . to solve this issue , much effort has been directed toward the development of alternative , chemically stable catalyst supports . in recent years , numerous conductive ceramics or oxide containing composites have been studied ; these include ito , wo3 , tio2 , ceo2 and ceo2zro2 , wc , ti4o7 , nbo2 or nb2o5 , tin , tib2 , and sic . various graphitized carbon support materials with special pore structures have also been explored , such as carbon nanotubes ( cnts ) , nanofibers ( cnfs ) , nanohorns ( cnhs ) , and nanocoils ( cncs ) . despite having promising performance and durability , there are concerns over their costly and complex synthesis methods . the graphitic structure has been shown to be effective at increasing durability ; however , its inert surface has a limited amount of active sites where the pt nanoparticle can stably anchor . these are often associated with structural electronic defects that yield higher interaction energy with the metal nanoparticle . a reasonable balance between catalyst dispersion and catalyst support interaction is necessary to meet the fuel cell performance requirement . an emerging trend is to dope the carbon support materials with oxygen or nitrogen , which act as tethers or anchors by increasing the interaction of the catalyst with the supporting materials , which leads to increased pt nanoparticle dispersions . unfortunately , surface oxygen functional groups on carbon are known to reduce the durability of fuel cell catalysts by promoting carbon corrosion . on the other hand , a growing body of literature suggests that the presence of nitrogen in carbon supports improves durability , as well as enhancing the intrinsic catalytic activity for both oxygen reduction reaction ( orr ) and methanol oxidation reaction ( mor ) . the incorporation of nitrogen into carbon leads to a reduction in detrimental surface oxygen groups and thus provides enhanced tolerance toward oxidation . the role of the n - surface species in catalytic enhancement is less studied and has only been hypothesized based on experimental observation and physicochemical reasoning . n defects near the catalyst particle interface adsorb oxygen containing intermediates that would otherwise block catalyst active sites , and thereby facilitate their removal and increase the rate of electrochemical reaction . it has been shown that the n - dopant alters the catalyst electronic structure , resulting in a higher binding energy , which may decrease the specific interaction between the pt nanoparticles and poisoning intermediates . this is further supported by an x - ray photoelectron spectroscopy ( xps ) study and theoretical calculations , where it was observed that pt nanoparticles nucleate strongly and experience a strong tethering n - doped carbon materials also have higher specific capacitance , suggesting the presence of additional charged species within the helmholtz layer , which might provide sites for surface groups to participate in the removal of strongly adsorbed poisoning intermediates . given the potential of n - doped carbon materials , graphitic carbon nitride material was chosen as a suitable candidate for a catalyst support in this study . polymeric solids with high n : c ratios formed by reactions of nitrogen - rich molecules were first reported by berzelius and later studied by liebig , franklin , pauling , and others . a fully condensed end member has the composition c3n4 with a planar structure related to graphite or graphene . however , most graphitic carbon nitrides are composed of c and n , along with residual amounts of h , and they are attracting new interest due to their unique structural and optoelectronic properties . the structures are based on triazine ( c3n3 ) or heptazine ( c6n7 ) ring units linked by n = or nh bridges to form sheets or zigzag chains of monomer units linked by hydrogen bonds to give a 2d array . typical examples include liebig s melon , melem , or highly condensed cxnyhz graphitic structures formed by continued elimination of nh3 component ( figure 1a c ) . recent discussions of graphitic carbon nitride structures and their properties have been based mainly on polymerized heptazine models that are shown to more thermodynamically stable , but structures based on condensation of s - triazine rings can also be produced under different synthesis conditions , and both structure types may be present . structural motifs found in graphitic carbon nitrides : ( a ) liebig s melon ( [ c6n7(nh2)(nh)]n ) contains zigzag chains of heptazine ( tri - s - triazine ) units linked by bridging nh groups and decorated on their edges by n h groups , ( b ) fully condensed c3n4 layer based on heptazine units , and ( c ) graphitic carbon nitride based on triazine ring units . graphitic carbon nitrides are semiconductors with an intrinsic band gap near 2.7 ev and optical absorption extending into the visible range . these materials exhibit catalytic and photocatalytic activity and are also of interest for their intercalation , ion exchange , and redox properties . because of their high nitrogen content , tunability reminiscent of polymer chemistry , and facile synthesis procedure , they may provide a good balance between activity , durability , and cost in pefc operation . carbon composites could provide comparable orr catalytic activity to that achieved by commercial pt / c in alkaline electrolyte with enhanced durability and high carbon monoxide ( co ) tolerance . although nitrogen in n - doped graphite / graphene introduces a charge carrier ( n - type polaron ) , this charge is not necessarily localized on the nitrogen . in the case of graphitic carbon nitride materials , they are stoichiometric materials and contain abundant lewis acid and base sites ( terminal and bridging nh groups and lone pairs of n in triazine / heptazine rings , respectively ) that are potential anchoring sites for pt as well as adsorption sites for co. to our knowledge , the first application of graphitic carbon nitride as a catalyst support can be attributed to yu et al . in 2007 for the direct methanol fuel cell ( dmfc ) operation . it was shown that ptru supported on graphitic carbon nitride in dmfc exhibits 7883% higher power density than on vulcan xc-72 . previously , it was shown that pt deposited on graphitic carbon nitride is more stable than commercial pt / vulcan under acidic accelerated carbon corrosion protocol ( after 1000 scans ) and has higher methanol oxidation activity per electrochemical surface area . in this study , three different graphitic carbon nitride materials ( polymeric carbon nitride , crystalline poly(triazine ) amide , and boron - doped graphitic carbon nitride ) were prepared , and their electrochemical durability was investigated in comparison to conventional carbon black , vulcan xc-72 . pt nanoparticles were deposited on each material , and their electrochemical properties and potential applications in pefc were investigated . polymeric carbon nitride ( gcnm ) was prepared by thermolysis and condensation reactions of a 1:1 molar ratio mixture of dicyandiamide ( dcda , c2n4h4 ) and melamine ( c3n6h9 ) at 550 c . a finely ground sample was loaded in an alumina boat into a quartz tube in a tubular furnace under nitrogen flow . the temperature was raised to 550 c at 5 c / min for 15 h. crystalline poly(triazine)imide ( pti / licl ) was synthesized from dcda in molten eutectic licl / kcl ( 45:55 wt % ) mixtures heated at 400 c under n2 ( g ) for 6 h and then sealed under vacuum and heated to 600 c for 12 h. b - doped graphitic carbon nitride ( b - gcnm ) was prepared by using ionic liquids . in a typical synthesis , 1-butyl-3-methylimidazolium tetrafluoroborate ( bmimbf4 ) was dissolved in water and stirred for 5 min . then dcda was added , and the mixture was heated at 100 c in an oil bath until the water completely evaporated . the resulting solid was then heated in an alumina crucible for 2 h at 350 c and kept at this temperature for 4 h. then , the temperature was raised to 550 c for another 4 h and finally cooled to room temperature . the pt catalyst was deposited onto gcnm using the ethylene glycol reduction method . ground gcnm ( 0.09 g ) was dispersed in 200 ml of ethylene glycol ( fisher scientific ) , and chloroplatinic acid ( 39.82% pt basis , 0.1507 g , sigma aldrich ) was added to the suspension . the mixture was stirred for 4 h under inert atmosphere and then heated to 140 c for 3 h , resulting in a dark brown mixture . the solid product was collected via vacuum filtration and dried at 60 c in a vacuum oven . the pt loading was 40 wt % for pt / gcnm and 20 wt % for pt / pti - licl and pt / b - gcnm . c , n , h analyses for gcnms were performed using a carlo - erba ea1108 system . sem was performed with a jeol jsm-6301f field emission imaging system at 5 kv . tem images were taken using a jeol tem1010 instrument operating at 80 kv and hrtem images were taken using a jeol tem2010 instrument operating at 200 kv . the x - ray diffraction data were obtained using a bruker - axs d4 system for the support materials and stoe powder diffractometer for the supported catalyst materials . bet measurements were carried out using a micrometrics asap 2420 surface area / porosity analyzer . the electrochemical measurements were carried out in a conventional three - electrode cell connected to an autolab pgstat32 potentiostat . a glassy carbon ( gc ) electrode with a surface area of 0.196 cm was employed as a working electrode , a pt mesh was used as a counter electrode , and a hydrogen reference electrode ( gaskatel ) , joined to the main chamber via a luggin capillary , was used as a reference electrode . a catalyst ink of 1.4 mg ml was obtained by dispersing the catalyst in a mixture of acetone , isopropyl alocohol , and nafion ultrasonically for 1 h. the ink was deposited on the gc and dried at room temperature , resulting in a loading of 35 g cm . the working electrode was electrochemically cleaned prior to each measurement via potential cycling between 0 and 1.2 v for 10 cycles , or until a steady state was reached , at a scan rate of 20 mv s. all electrochemical measurements were carried out at 25 c in 0.1 m hclo4 , except for mor which was carried out in 1 m ch3oh + 0.1 m hclo4 . all chemicals used were analytical grade , and solutions were prepared with deionized water ( millipore , 18.2 mcm ) an accelerated start stop cycling protocol was used for the carbon corrosion test . one cycle involves applying a voltage hold at 1.0 v for 30 s followed by two sequences of voltage cycling between 1.0 and 1.5 at 0.5 v s. the change in cyclic voltammogram ( capacitance and electrochemical surface area ) was monitored after 1 , 10 , 20 , 50 , 100 , 200 , 500 , 1000 , and 2000 cycles . in this study , different graphitic carbon nitride phases were prepared with the aim to address the effect of catalyst support properties such as crystallinity , porosity , and composition on the electrochemical performance in pefc . polymeric carbon nitride was prepared using a typical method of thermolysis and condensation . electronically modified graphitic carbon nitride with high porosity was prepared using ionic liquid under the same conditions , whereas highly crystalline graphitic carbon nitride was prepared via ionothermal method . layered carbon nitride was prepared by thermolysis and condensation reactions of 1:1 molar ratio mixtures of dicyandiamide ( c2n4h4 ) and melamine ( c3n6h9 ) at 550 c for 15 h. finely ground samples were heated in a tubular furnace under n2 flow at 5 c / min and allowed to cool to room temperature before being removed . upon heating , the condensation process takes place by removal of nh3 species leading to materials with different c : n : h stoichiometry depending on the synthesis temperature . x - ray diffraction pattern ( a ) and sem image ( b ) of a typical layered carbon nitride prepared by thermal condensation of dcda / melamine 1:1 molar ratio . a typical x - ray diffraction pattern ( xrd ) is shown in figure 2a . the strong peak at 27.5 2 corresponds to a repeat distance of 0.32 nm that correlates with the 002 reflection usually observed for graphitic materials . the feature around 12.5 2 corresponds to an in - plane repeat distance of 0.70 nm that matches with the dimensions of polyheptazine or polytriazine structures within the layers . scanning electron microscope ( sem ) examination ( figure 2b ) indicated that gcnm exhibits a latticework of interlocking planar microstructures with individual layer thicknesses on the order of 23 nm that give rise to porous aggregates with pore sizes on the order of a few nanometers . this material is not highly condensed , and its structure is believed to be close to that of liebig s melon . the aggregates are fused together to give rise to much larger pores ( 12 m ) in the resulting solid . the bet measurements showed a surface area of 28 m g. poly(triazine ) imide carbon nitride ( pti - licl ) was prepared using an ionothermal route . this method allowed us to obtain a highly crystalline carbon nitride using relatively mild conditions . in a typical synthesis , dcda was mixed with a eutectic mixture of licl / kcl ( 45:55 wt % ) and heated at 400 c for 6 h under n2 ( g ) followed by a thermal treatment under high vacuum at 600 c for 12 h. the pti - licl compound exhibits a sharp series of peaks in the x - ray diffraction pattern consistent with a p63 cm unit cell ( figure 3a ) . the hexagonal symmetry of the pti - licl can be clearly seen in the sem images of the hexagonal - shaped crystallites ( figure 3b ) . this material is a triazine - based ( c3n3)2(nh)3.licl structure with li and cl intercalated both within and between the graphitic layers . x - ray diffraction pattern ( a ) and sem image ( b ) of a poly(triazine)imide carbon nitride prepared by ionothermal route . 1-butyl-3-methylimidazolium tetrafluoroborate ( bmimbf4 ) ionic liquid was used to modify the electronic structure of gcnm by substituting c ( [ he]2s2p ) by b ( [ he]2s2p ) . the x - ray diffraction pattern ( figure 4a ) is dominated by the ( 002 ) interlayer - stacking reflection usually observed in gcnms . the higher porosity of b - gcnm compared to gcnm and pti - licl is clearly seen in the sem images ( figure 4b ) . a homogeneous porosity with pores of about 5075 nm were detected . x - ray diffraction pattern ( a ) and sem image ( b ) of b - gcnm doped with 10 wt % b. platinum was deposited onto the supports via the ethylene glycol method . the xrd patterns ( figure 5a d ) confirm the presence of platinum in each sample as characterized by the peaks at 39.8 2 and 46.5 2 corresponding to pt 111 and 200 reflections , respectively . the tem images shown in figure 6a d indicate varying extents of pt nanoparticle dispersion in each sample , with large agglomeration seen in all samples except for the commercial catalyst . x - ray diffraction pattern of supported pt electrocatalysts : ( a ) pt / vulcan , ( b ) pt / gcnm , ( c ) pt / pti - licl , and ( d ) pt / b - gcnm . the average pt crystallite sizes calculated using the scherrer equation and the particle size estimated from tem images are summarized in table 1 . all pt on graphitic carbon nitride materials have larger particle sizes ( 4.28.0 nm ) compared to commercial pt / vulcan ( 3.5 nm ) . this is due to the agglomeration in all the samples , indicating that the method used for catalyst deposition is not optimized . tem images of ( a ) pt / vulcan , ( b ) pt / gcnm , ( c ) pt / pti - licl , and ( d ) pt / b - gcnm . estimated from tem image based on the average of 100 particles . calculated from 111 signal widths of xrd using the scherrer equation . the stability of the carbon nitrides was determined by observing the change in double - capacitance in the cyclic voltammetry measurements while performing the carbon corrosion test . capacitance increases with the number of scans due to an increase in surface area and concentration of hydrophilic carbon corrosion products with oxygen functionalities at the surface of the carbon support . the presence of oxygen functionalities reduces the durability of pefc catalysts by promoting carbon corrosion . in addition , a more hydrophilic surface may affect water management in fuel cells and potentially contribute to performance instability and variability . figure 7 displays the degree of corrosion behavior / surface modification of graphitic carbon nitride materials , in comparison to a commercial vulcan carbon support . after 2000 cycles , all materials exhibit a higher degree of tolerance to cycling , compared to commercial carbon black ( vulcan ) , with b - gcnm showing the best tolerance at only a 6% increase in capacitance at the end of the cycle . change in double - layer capacitance ( calculated at 0.40 v ) of the support materials as a result of accelerated carbon corrosion cycling . all carbon nitride supported pt catalysts have lower initial electrochemical surface area ( ecsa ) compared to commercial pt catalyst . this is due to higher degree of agglomeration and larger particle size , as shown in figure 6 . this also indicates that each graphitic carbon nitride material has a different ability to accommodate catalyst particles . in addition , carbon nitride materials have 1 order of magnitude less bet surface area compared to vulcan carbon , and hence the same mass percentage loading of pt nanoparticles on vulcan carbon would result in higher pt particle density on graphitic carbon nitride . change in ecsa ( calculated from hydrogen adsorption / desorption ) of the supported pt electrocatalsysts as a result of accelerated carbon corrosion cycling . the durability of the materials in the presence of pt nanoparticles was evaluated using the same accelerated protocol , with cv and ecsa recorded at regular intervals as part of the diagnostic ( figure 8) . the decrease in ecsa is believed to be due to platinum agglomeration and dissolution as a result of substrate corrosion . at the end of the 2000 cycles , commercial pt / vulcan exhibits a 36.3% decrease in ecsa ( table 2 ) . pt / gcnm and pt / b - gcnm show higher ecsa loss at 81.0% and 100% loss , respectively , despite each support having higher degree of corrosion tolerance , as displayed in figure 7 . in addition , it is also observed that graphitic carbon nitride supported pt catalysts with higher initial ecsa exhibit higher electrochemical durability , indicating there is a link between good metal support material that provides strong adsorption and anchoring sites for the pt nanoparticles will increase particle dispersion and limit leaching and agglomeration processes during the accelerated test . the catalytic activities of graphitic carbon nitride supported pt catalysts were investigated in 1 m methanol + 0.1 m hclo4 solution at 25 c . it is generally accepted that low overpotential and high peak current density are an indication of good methanol oxidation reaction ( mor ) activity . the current density is normalized to the ecsa of each respective material due to the inherent differences in ecsa . all graphitic carbon nitride supported catalysts exhibit lower overpotential and higher peak current density compared to pt / vulcan . in addition , pt / pti - licl exhibits the lowest overpotential whereas pt / b - gcnm has the highest peak current density . mor overpotential may be influenced by particle size effects : smaller pt nanoparticles enhance the oxidation of poisoning intermediates and , hence , decrease the overpotential . given the difference in pt particle size in all materials with pt / vulcan having the smallest particle size , particle size effect can be eliminated from this observation . this suggests that the presence of nitrogen on / within the support material could lead to intrinsic mor catalytic enhancement . furthermore , each of the graphitic carbon nitride materials has different structural properties and therefore would require a different synthesis approach . methanol oxidation reaction of supported pt electrocatalysts in 1 m ch3oh + 0.1 m hclo4 at 25 c with a scan rate of 2 mv s. for the first time , three different graphitic carbon nitride materials ( polymeric gcnm , pti - licl , and b - doped gcnm ) were prepared and tested as catalyst support materials for pefcs . the results show that all graphitic carbon nitride materials prepared in this study exhibit significantly improved durability compared to commercial carbon black ( vulcan xc-72r ) and , therefore , are promising catalyst support materials for pefc applications . interestingly , b - gcnm and pti / licl exhibit the highest stability . as shown on the x - ray diffraction patterns , b - gcnm and pti / licl are more crystalline than vulcan and gcnm , suggesting that crystallinity may play an important role in the stability of the material against carbon corrosion . this also suggests that the presence of dopants such as boron in b - gcnm , and li and cl in pti / licl , may enhance the stability of the support materials . the durability of graphitic carbon nitride supported pt electrocatalysts is highly dependent on the initial ecsa . the pt / b - gcnm , with the lowest ecsa of all carbon nitride supported catalyst , exhibits the lowest durability with a loss of 100% after 2000 cycles . the pt - pti / licl has the best durability of all pt - supported catalyst with an ecsa loss of only 19% after 2000 scans . this value is even lower than pt / vulcan with an ecsa loss of 36% . in addition , all graphitic carbon nitride supported pt electrocatalysts have higher methanol oxidation activity per ecsa , compared to pt / vulcan . this work shows that graphitic carbon nitrides are promising catalyst supports that can potentially replace conventional carbon support . the performance of pti / licl is highly encouraging , and further research is already being developed to optimize catalyst particle dispersion and utilization .

graphitic carbon nitrides are investigated for developing highly durable pt electrocatalyst supports for polymer electrolyte fuel cells ( pefcs ) . three different graphitic carbon nitride materials were synthesized with the aim to address the effect of crystallinity , porosity , and composition on the catalyst support properties : polymeric carbon nitride ( gcnm ) , poly(triazine ) imide carbon nitride ( pti / li+cl ) , and boron - doped graphitic carbon nitride ( b - gcnm ) . following accelerated corrosion testing , all graphitic carbon nitride materials are found to be more electrochemically stable compared to conventional carbon black ( vulcan xc-72r ) with b - gcnm support showing the best stability . for the supported catalysts , pt / pti - li+cl catalyst exhibits better durability with only 19% electrochemical surface area ( ecsa ) loss versus 36% for pt / vulcan after 2000 scans . superior methanol oxidation activity is observed for all graphitic carbon nitride supported pt catalysts on the basis of the catalyst ecsa .

Introduction Experimental Section Results Conclusion

among the various types of fuel cells , polymer electrolyte fuel cells ( pefcs ) have attracted the most attention for transportation and portable applications . advantages include the following : rapid start - up and shut down , low temperature operation ( 80 c ) , high power density , and fully solid state components . it is estimated that the catalyst layers contribute to as much as 39% of the total fuel cell stack cost , making it one of the major challenges for pefc commercialization . another major challenge is insufficient durability ; current state - of - the art performance under realistic operating conditions is just half of the target set by the doe . it is generally accepted that catalyst activity and durability depends on pt particle size and dispersion ( which can be affected by the support material ) , as well as the interaction between the catalyst particle and the support . the most widely used catalyst support is carbon black vulcan xc-72r , which has high surface area and good electrical conductivity . however , pt particles can get buried inside the pores and hence reduce the active triple - phase boundary accessible for electrochemical reaction . as the carbon corrodes , pt nanoparticles agglomerate into larger particles and/or detach from the support material , consequently reducing the electrochemical surface area ( ecsa ) and catalytic activity . in recent years , numerous conductive ceramics or oxide containing composites have been studied ; these include ito , wo3 , tio2 , ceo2 and ceo2zro2 , wc , ti4o7 , nbo2 or nb2o5 , tin , tib2 , and sic . various graphitized carbon support materials with special pore structures have also been explored , such as carbon nanotubes ( cnts ) , nanofibers ( cnfs ) , nanohorns ( cnhs ) , and nanocoils ( cncs ) . the graphitic structure has been shown to be effective at increasing durability ; however , its inert surface has a limited amount of active sites where the pt nanoparticle can stably anchor . these are often associated with structural electronic defects that yield higher interaction energy with the metal nanoparticle . a reasonable balance between catalyst dispersion and catalyst support interaction is necessary to meet the fuel cell performance requirement . an emerging trend is to dope the carbon support materials with oxygen or nitrogen , which act as tethers or anchors by increasing the interaction of the catalyst with the supporting materials , which leads to increased pt nanoparticle dispersions . on the other hand , a growing body of literature suggests that the presence of nitrogen in carbon supports improves durability , as well as enhancing the intrinsic catalytic activity for both oxygen reduction reaction ( orr ) and methanol oxidation reaction ( mor ) . the role of the n - surface species in catalytic enhancement is less studied and has only been hypothesized based on experimental observation and physicochemical reasoning . n defects near the catalyst particle interface adsorb oxygen containing intermediates that would otherwise block catalyst active sites , and thereby facilitate their removal and increase the rate of electrochemical reaction . given the potential of n - doped carbon materials , graphitic carbon nitride material was chosen as a suitable candidate for a catalyst support in this study . polymeric solids with high n : c ratios formed by reactions of nitrogen - rich molecules were first reported by berzelius and later studied by liebig , franklin , pauling , and others . however , most graphitic carbon nitrides are composed of c and n , along with residual amounts of h , and they are attracting new interest due to their unique structural and optoelectronic properties . recent discussions of graphitic carbon nitride structures and their properties have been based mainly on polymerized heptazine models that are shown to more thermodynamically stable , but structures based on condensation of s - triazine rings can also be produced under different synthesis conditions , and both structure types may be present . structural motifs found in graphitic carbon nitrides : ( a ) liebig s melon ( [ c6n7(nh2)(nh)]n ) contains zigzag chains of heptazine ( tri - s - triazine ) units linked by bridging nh groups and decorated on their edges by n h groups , ( b ) fully condensed c3n4 layer based on heptazine units , and ( c ) graphitic carbon nitride based on triazine ring units . graphitic carbon nitrides are semiconductors with an intrinsic band gap near 2.7 ev and optical absorption extending into the visible range . these materials exhibit catalytic and photocatalytic activity and are also of interest for their intercalation , ion exchange , and redox properties . although nitrogen in n - doped graphite / graphene introduces a charge carrier ( n - type polaron ) , this charge is not necessarily localized on the nitrogen . in the case of graphitic carbon nitride materials , they are stoichiometric materials and contain abundant lewis acid and base sites ( terminal and bridging nh groups and lone pairs of n in triazine / heptazine rings , respectively ) that are potential anchoring sites for pt as well as adsorption sites for co. to our knowledge , the first application of graphitic carbon nitride as a catalyst support can be attributed to yu et al . it was shown that ptru supported on graphitic carbon nitride in dmfc exhibits 7883% higher power density than on vulcan xc-72 . previously , it was shown that pt deposited on graphitic carbon nitride is more stable than commercial pt / vulcan under acidic accelerated carbon corrosion protocol ( after 1000 scans ) and has higher methanol oxidation activity per electrochemical surface area . in this study , three different graphitic carbon nitride materials ( polymeric carbon nitride , crystalline poly(triazine ) amide , and boron - doped graphitic carbon nitride ) were prepared , and their electrochemical durability was investigated in comparison to conventional carbon black , vulcan xc-72 . pt nanoparticles were deposited on each material , and their electrochemical properties and potential applications in pefc were investigated . polymeric carbon nitride ( gcnm ) was prepared by thermolysis and condensation reactions of a 1:1 molar ratio mixture of dicyandiamide ( dcda , c2n4h4 ) and melamine ( c3n6h9 ) at 550 c . the temperature was raised to 550 c at 5 c / min for 15 h. crystalline poly(triazine)imide ( pti / licl ) was synthesized from dcda in molten eutectic licl / kcl ( 45:55 wt % ) mixtures heated at 400 c under n2 ( g ) for 6 h and then sealed under vacuum and heated to 600 c for 12 h. b - doped graphitic carbon nitride ( b - gcnm ) was prepared by using ionic liquids . ground gcnm ( 0.09 g ) was dispersed in 200 ml of ethylene glycol ( fisher scientific ) , and chloroplatinic acid ( 39.82% pt basis , 0.1507 g , sigma aldrich ) was added to the suspension . the pt loading was 40 wt % for pt / gcnm and 20 wt % for pt / pti - licl and pt / b - gcnm . the x - ray diffraction data were obtained using a bruker - axs d4 system for the support materials and stoe powder diffractometer for the supported catalyst materials . bet measurements were carried out using a micrometrics asap 2420 surface area / porosity analyzer . a glassy carbon ( gc ) electrode with a surface area of 0.196 cm was employed as a working electrode , a pt mesh was used as a counter electrode , and a hydrogen reference electrode ( gaskatel ) , joined to the main chamber via a luggin capillary , was used as a reference electrode . a catalyst ink of 1.4 mg ml was obtained by dispersing the catalyst in a mixture of acetone , isopropyl alocohol , and nafion ultrasonically for 1 h. the ink was deposited on the gc and dried at room temperature , resulting in a loading of 35 g cm . all chemicals used were analytical grade , and solutions were prepared with deionized water ( millipore , 18.2 mcm ) an accelerated start stop cycling protocol was used for the carbon corrosion test . one cycle involves applying a voltage hold at 1.0 v for 30 s followed by two sequences of voltage cycling between 1.0 and 1.5 at 0.5 v s. the change in cyclic voltammogram ( capacitance and electrochemical surface area ) was monitored after 1 , 10 , 20 , 50 , 100 , 200 , 500 , 1000 , and 2000 cycles . in this study , different graphitic carbon nitride phases were prepared with the aim to address the effect of catalyst support properties such as crystallinity , porosity , and composition on the electrochemical performance in pefc . polymeric carbon nitride was prepared using a typical method of thermolysis and condensation . electronically modified graphitic carbon nitride with high porosity was prepared using ionic liquid under the same conditions , whereas highly crystalline graphitic carbon nitride was prepared via ionothermal method . layered carbon nitride was prepared by thermolysis and condensation reactions of 1:1 molar ratio mixtures of dicyandiamide ( c2n4h4 ) and melamine ( c3n6h9 ) at 550 c for 15 h. finely ground samples were heated in a tubular furnace under n2 flow at 5 c / min and allowed to cool to room temperature before being removed . upon heating , the condensation process takes place by removal of nh3 species leading to materials with different c : n : h stoichiometry depending on the synthesis temperature . x - ray diffraction pattern ( a ) and sem image ( b ) of a typical layered carbon nitride prepared by thermal condensation of dcda / melamine 1:1 molar ratio . the strong peak at 27.5 2 corresponds to a repeat distance of 0.32 nm that correlates with the 002 reflection usually observed for graphitic materials . this material is not highly condensed , and its structure is believed to be close to that of liebig s melon . the bet measurements showed a surface area of 28 m g. poly(triazine ) imide carbon nitride ( pti - licl ) was prepared using an ionothermal route . this method allowed us to obtain a highly crystalline carbon nitride using relatively mild conditions . in a typical synthesis , dcda was mixed with a eutectic mixture of licl / kcl ( 45:55 wt % ) and heated at 400 c for 6 h under n2 ( g ) followed by a thermal treatment under high vacuum at 600 c for 12 h. the pti - licl compound exhibits a sharp series of peaks in the x - ray diffraction pattern consistent with a p63 cm unit cell ( figure 3a ) . the hexagonal symmetry of the pti - licl can be clearly seen in the sem images of the hexagonal - shaped crystallites ( figure 3b ) . x - ray diffraction pattern ( a ) and sem image ( b ) of a poly(triazine)imide carbon nitride prepared by ionothermal route . the higher porosity of b - gcnm compared to gcnm and pti - licl is clearly seen in the sem images ( figure 4b ) . x - ray diffraction pattern ( a ) and sem image ( b ) of b - gcnm doped with 10 wt % b. platinum was deposited onto the supports via the ethylene glycol method . x - ray diffraction pattern of supported pt electrocatalysts : ( a ) pt / vulcan , ( b ) pt / gcnm , ( c ) pt / pti - licl , and ( d ) pt / b - gcnm . all pt on graphitic carbon nitride materials have larger particle sizes ( 4.28.0 nm ) compared to commercial pt / vulcan ( 3.5 nm ) . tem images of ( a ) pt / vulcan , ( b ) pt / gcnm , ( c ) pt / pti - licl , and ( d ) pt / b - gcnm . the stability of the carbon nitrides was determined by observing the change in double - capacitance in the cyclic voltammetry measurements while performing the carbon corrosion test . capacitance increases with the number of scans due to an increase in surface area and concentration of hydrophilic carbon corrosion products with oxygen functionalities at the surface of the carbon support . in addition , a more hydrophilic surface may affect water management in fuel cells and potentially contribute to performance instability and variability . figure 7 displays the degree of corrosion behavior / surface modification of graphitic carbon nitride materials , in comparison to a commercial vulcan carbon support . after 2000 cycles , all materials exhibit a higher degree of tolerance to cycling , compared to commercial carbon black ( vulcan ) , with b - gcnm showing the best tolerance at only a 6% increase in capacitance at the end of the cycle . all carbon nitride supported pt catalysts have lower initial electrochemical surface area ( ecsa ) compared to commercial pt catalyst . this also indicates that each graphitic carbon nitride material has a different ability to accommodate catalyst particles . in addition , carbon nitride materials have 1 order of magnitude less bet surface area compared to vulcan carbon , and hence the same mass percentage loading of pt nanoparticles on vulcan carbon would result in higher pt particle density on graphitic carbon nitride . change in ecsa ( calculated from hydrogen adsorption / desorption ) of the supported pt electrocatalsysts as a result of accelerated carbon corrosion cycling . at the end of the 2000 cycles , commercial pt / vulcan exhibits a 36.3% decrease in ecsa ( table 2 ) . pt / gcnm and pt / b - gcnm show higher ecsa loss at 81.0% and 100% loss , respectively , despite each support having higher degree of corrosion tolerance , as displayed in figure 7 . in addition , it is also observed that graphitic carbon nitride supported pt catalysts with higher initial ecsa exhibit higher electrochemical durability , indicating there is a link between good metal support material that provides strong adsorption and anchoring sites for the pt nanoparticles will increase particle dispersion and limit leaching and agglomeration processes during the accelerated test . the catalytic activities of graphitic carbon nitride supported pt catalysts were investigated in 1 m methanol + 0.1 m hclo4 solution at 25 c . all graphitic carbon nitride supported catalysts exhibit lower overpotential and higher peak current density compared to pt / vulcan . in addition , pt / pti - licl exhibits the lowest overpotential whereas pt / b - gcnm has the highest peak current density . given the difference in pt particle size in all materials with pt / vulcan having the smallest particle size , particle size effect can be eliminated from this observation . furthermore , each of the graphitic carbon nitride materials has different structural properties and therefore would require a different synthesis approach . methanol oxidation reaction of supported pt electrocatalysts in 1 m ch3oh + 0.1 m hclo4 at 25 c with a scan rate of 2 mv s. for the first time , three different graphitic carbon nitride materials ( polymeric gcnm , pti - licl , and b - doped gcnm ) were prepared and tested as catalyst support materials for pefcs . the results show that all graphitic carbon nitride materials prepared in this study exhibit significantly improved durability compared to commercial carbon black ( vulcan xc-72r ) and , therefore , are promising catalyst support materials for pefc applications . interestingly , b - gcnm and pti / licl exhibit the highest stability . as shown on the x - ray diffraction patterns , b - gcnm and pti / licl are more crystalline than vulcan and gcnm , suggesting that crystallinity may play an important role in the stability of the material against carbon corrosion . this also suggests that the presence of dopants such as boron in b - gcnm , and li and cl in pti / licl , may enhance the stability of the support materials . the durability of graphitic carbon nitride supported pt electrocatalysts is highly dependent on the initial ecsa . the pt / b - gcnm , with the lowest ecsa of all carbon nitride supported catalyst , exhibits the lowest durability with a loss of 100% after 2000 cycles . the pt - pti / licl has the best durability of all pt - supported catalyst with an ecsa loss of only 19% after 2000 scans . this value is even lower than pt / vulcan with an ecsa loss of 36% . in addition , all graphitic carbon nitride supported pt electrocatalysts have higher methanol oxidation activity per ecsa , compared to pt / vulcan . this work shows that graphitic carbon nitrides are promising catalyst supports that can potentially replace conventional carbon support . the performance of pti / licl is highly encouraging , and further research is already being developed to optimize catalyst particle dispersion and utilization .

among the various types of fuel cells , polymer electrolyte fuel cells ( pefcs ) have attracted the most attention for transportation and portable applications . current state - of - the - art technology uses platinum ( pt ) or platinum alloys supported on carbon for both anode and cathode electrodes . it is estimated that the catalyst layers contribute to as much as 39% of the total fuel cell stack cost , making it one of the major challenges for pefc commercialization . department of energy ( doe ) targets for 2015 , it is necessary to reduce the amount of pt catalyst used by three - quarters , while ensuring the same performance . another major challenge is insufficient durability ; current state - of - the art performance under realistic operating conditions is just half of the target set by the doe . it is generally accepted that catalyst activity and durability depends on pt particle size and dispersion ( which can be affected by the support material ) , as well as the interaction between the catalyst particle and the support . however , pt particles can get buried inside the pores and hence reduce the active triple - phase boundary accessible for electrochemical reaction . as the carbon corrodes , pt nanoparticles agglomerate into larger particles and/or detach from the support material , consequently reducing the electrochemical surface area ( ecsa ) and catalytic activity . to solve this issue , much effort has been directed toward the development of alternative , chemically stable catalyst supports . in recent years , numerous conductive ceramics or oxide containing composites have been studied ; these include ito , wo3 , tio2 , ceo2 and ceo2zro2 , wc , ti4o7 , nbo2 or nb2o5 , tin , tib2 , and sic . various graphitized carbon support materials with special pore structures have also been explored , such as carbon nanotubes ( cnts ) , nanofibers ( cnfs ) , nanohorns ( cnhs ) , and nanocoils ( cncs ) . despite having promising performance and durability , there are concerns over their costly and complex synthesis methods . the graphitic structure has been shown to be effective at increasing durability ; however , its inert surface has a limited amount of active sites where the pt nanoparticle can stably anchor . these are often associated with structural electronic defects that yield higher interaction energy with the metal nanoparticle . a reasonable balance between catalyst dispersion and catalyst support interaction is necessary to meet the fuel cell performance requirement . an emerging trend is to dope the carbon support materials with oxygen or nitrogen , which act as tethers or anchors by increasing the interaction of the catalyst with the supporting materials , which leads to increased pt nanoparticle dispersions . unfortunately , surface oxygen functional groups on carbon are known to reduce the durability of fuel cell catalysts by promoting carbon corrosion . on the other hand , a growing body of literature suggests that the presence of nitrogen in carbon supports improves durability , as well as enhancing the intrinsic catalytic activity for both oxygen reduction reaction ( orr ) and methanol oxidation reaction ( mor ) . the incorporation of nitrogen into carbon leads to a reduction in detrimental surface oxygen groups and thus provides enhanced tolerance toward oxidation . the role of the n - surface species in catalytic enhancement is less studied and has only been hypothesized based on experimental observation and physicochemical reasoning . n defects near the catalyst particle interface adsorb oxygen containing intermediates that would otherwise block catalyst active sites , and thereby facilitate their removal and increase the rate of electrochemical reaction . it has been shown that the n - dopant alters the catalyst electronic structure , resulting in a higher binding energy , which may decrease the specific interaction between the pt nanoparticles and poisoning intermediates . this is further supported by an x - ray photoelectron spectroscopy ( xps ) study and theoretical calculations , where it was observed that pt nanoparticles nucleate strongly and experience a strong tethering n - doped carbon materials also have higher specific capacitance , suggesting the presence of additional charged species within the helmholtz layer , which might provide sites for surface groups to participate in the removal of strongly adsorbed poisoning intermediates . given the potential of n - doped carbon materials , graphitic carbon nitride material was chosen as a suitable candidate for a catalyst support in this study . polymeric solids with high n : c ratios formed by reactions of nitrogen - rich molecules were first reported by berzelius and later studied by liebig , franklin , pauling , and others . however , most graphitic carbon nitrides are composed of c and n , along with residual amounts of h , and they are attracting new interest due to their unique structural and optoelectronic properties . recent discussions of graphitic carbon nitride structures and their properties have been based mainly on polymerized heptazine models that are shown to more thermodynamically stable , but structures based on condensation of s - triazine rings can also be produced under different synthesis conditions , and both structure types may be present . structural motifs found in graphitic carbon nitrides : ( a ) liebig s melon ( [ c6n7(nh2)(nh)]n ) contains zigzag chains of heptazine ( tri - s - triazine ) units linked by bridging nh groups and decorated on their edges by n h groups , ( b ) fully condensed c3n4 layer based on heptazine units , and ( c ) graphitic carbon nitride based on triazine ring units . these materials exhibit catalytic and photocatalytic activity and are also of interest for their intercalation , ion exchange , and redox properties . because of their high nitrogen content , tunability reminiscent of polymer chemistry , and facile synthesis procedure , they may provide a good balance between activity , durability , and cost in pefc operation . carbon composites could provide comparable orr catalytic activity to that achieved by commercial pt / c in alkaline electrolyte with enhanced durability and high carbon monoxide ( co ) tolerance . although nitrogen in n - doped graphite / graphene introduces a charge carrier ( n - type polaron ) , this charge is not necessarily localized on the nitrogen . in the case of graphitic carbon nitride materials , they are stoichiometric materials and contain abundant lewis acid and base sites ( terminal and bridging nh groups and lone pairs of n in triazine / heptazine rings , respectively ) that are potential anchoring sites for pt as well as adsorption sites for co. to our knowledge , the first application of graphitic carbon nitride as a catalyst support can be attributed to yu et al . previously , it was shown that pt deposited on graphitic carbon nitride is more stable than commercial pt / vulcan under acidic accelerated carbon corrosion protocol ( after 1000 scans ) and has higher methanol oxidation activity per electrochemical surface area . in this study , three different graphitic carbon nitride materials ( polymeric carbon nitride , crystalline poly(triazine ) amide , and boron - doped graphitic carbon nitride ) were prepared , and their electrochemical durability was investigated in comparison to conventional carbon black , vulcan xc-72 . polymeric carbon nitride ( gcnm ) was prepared by thermolysis and condensation reactions of a 1:1 molar ratio mixture of dicyandiamide ( dcda , c2n4h4 ) and melamine ( c3n6h9 ) at 550 c . the temperature was raised to 550 c at 5 c / min for 15 h. crystalline poly(triazine)imide ( pti / licl ) was synthesized from dcda in molten eutectic licl / kcl ( 45:55 wt % ) mixtures heated at 400 c under n2 ( g ) for 6 h and then sealed under vacuum and heated to 600 c for 12 h. b - doped graphitic carbon nitride ( b - gcnm ) was prepared by using ionic liquids . the pt loading was 40 wt % for pt / gcnm and 20 wt % for pt / pti - licl and pt / b - gcnm . the x - ray diffraction data were obtained using a bruker - axs d4 system for the support materials and stoe powder diffractometer for the supported catalyst materials . a glassy carbon ( gc ) electrode with a surface area of 0.196 cm was employed as a working electrode , a pt mesh was used as a counter electrode , and a hydrogen reference electrode ( gaskatel ) , joined to the main chamber via a luggin capillary , was used as a reference electrode . a catalyst ink of 1.4 mg ml was obtained by dispersing the catalyst in a mixture of acetone , isopropyl alocohol , and nafion ultrasonically for 1 h. the ink was deposited on the gc and dried at room temperature , resulting in a loading of 35 g cm . the working electrode was electrochemically cleaned prior to each measurement via potential cycling between 0 and 1.2 v for 10 cycles , or until a steady state was reached , at a scan rate of 20 mv s. all electrochemical measurements were carried out at 25 c in 0.1 m hclo4 , except for mor which was carried out in 1 m ch3oh + 0.1 m hclo4 . one cycle involves applying a voltage hold at 1.0 v for 30 s followed by two sequences of voltage cycling between 1.0 and 1.5 at 0.5 v s. the change in cyclic voltammogram ( capacitance and electrochemical surface area ) was monitored after 1 , 10 , 20 , 50 , 100 , 200 , 500 , 1000 , and 2000 cycles . in this study , different graphitic carbon nitride phases were prepared with the aim to address the effect of catalyst support properties such as crystallinity , porosity , and composition on the electrochemical performance in pefc . layered carbon nitride was prepared by thermolysis and condensation reactions of 1:1 molar ratio mixtures of dicyandiamide ( c2n4h4 ) and melamine ( c3n6h9 ) at 550 c for 15 h. finely ground samples were heated in a tubular furnace under n2 flow at 5 c / min and allowed to cool to room temperature before being removed . upon heating , the condensation process takes place by removal of nh3 species leading to materials with different c : n : h stoichiometry depending on the synthesis temperature . x - ray diffraction pattern ( a ) and sem image ( b ) of a typical layered carbon nitride prepared by thermal condensation of dcda / melamine 1:1 molar ratio . scanning electron microscope ( sem ) examination ( figure 2b ) indicated that gcnm exhibits a latticework of interlocking planar microstructures with individual layer thicknesses on the order of 23 nm that give rise to porous aggregates with pore sizes on the order of a few nanometers . the bet measurements showed a surface area of 28 m g. poly(triazine ) imide carbon nitride ( pti - licl ) was prepared using an ionothermal route . in a typical synthesis , dcda was mixed with a eutectic mixture of licl / kcl ( 45:55 wt % ) and heated at 400 c for 6 h under n2 ( g ) followed by a thermal treatment under high vacuum at 600 c for 12 h. the pti - licl compound exhibits a sharp series of peaks in the x - ray diffraction pattern consistent with a p63 cm unit cell ( figure 3a ) . the hexagonal symmetry of the pti - licl can be clearly seen in the sem images of the hexagonal - shaped crystallites ( figure 3b ) . x - ray diffraction pattern ( a ) and sem image ( b ) of a poly(triazine)imide carbon nitride prepared by ionothermal route . 1-butyl-3-methylimidazolium tetrafluoroborate ( bmimbf4 ) ionic liquid was used to modify the electronic structure of gcnm by substituting c ( [ he]2s2p ) by b ( [ he]2s2p ) . the x - ray diffraction pattern ( figure 4a ) is dominated by the ( 002 ) interlayer - stacking reflection usually observed in gcnms . the higher porosity of b - gcnm compared to gcnm and pti - licl is clearly seen in the sem images ( figure 4b ) . x - ray diffraction pattern ( a ) and sem image ( b ) of b - gcnm doped with 10 wt % b. platinum was deposited onto the supports via the ethylene glycol method . the xrd patterns ( figure 5a d ) confirm the presence of platinum in each sample as characterized by the peaks at 39.8 2 and 46.5 2 corresponding to pt 111 and 200 reflections , respectively . the tem images shown in figure 6a d indicate varying extents of pt nanoparticle dispersion in each sample , with large agglomeration seen in all samples except for the commercial catalyst . x - ray diffraction pattern of supported pt electrocatalysts : ( a ) pt / vulcan , ( b ) pt / gcnm , ( c ) pt / pti - licl , and ( d ) pt / b - gcnm . this is due to the agglomeration in all the samples , indicating that the method used for catalyst deposition is not optimized . tem images of ( a ) pt / vulcan , ( b ) pt / gcnm , ( c ) pt / pti - licl , and ( d ) pt / b - gcnm . the stability of the carbon nitrides was determined by observing the change in double - capacitance in the cyclic voltammetry measurements while performing the carbon corrosion test . capacitance increases with the number of scans due to an increase in surface area and concentration of hydrophilic carbon corrosion products with oxygen functionalities at the surface of the carbon support . in addition , a more hydrophilic surface may affect water management in fuel cells and potentially contribute to performance instability and variability . figure 7 displays the degree of corrosion behavior / surface modification of graphitic carbon nitride materials , in comparison to a commercial vulcan carbon support . after 2000 cycles , all materials exhibit a higher degree of tolerance to cycling , compared to commercial carbon black ( vulcan ) , with b - gcnm showing the best tolerance at only a 6% increase in capacitance at the end of the cycle . in addition , carbon nitride materials have 1 order of magnitude less bet surface area compared to vulcan carbon , and hence the same mass percentage loading of pt nanoparticles on vulcan carbon would result in higher pt particle density on graphitic carbon nitride . change in ecsa ( calculated from hydrogen adsorption / desorption ) of the supported pt electrocatalsysts as a result of accelerated carbon corrosion cycling . the durability of the materials in the presence of pt nanoparticles was evaluated using the same accelerated protocol , with cv and ecsa recorded at regular intervals as part of the diagnostic ( figure 8) . pt / gcnm and pt / b - gcnm show higher ecsa loss at 81.0% and 100% loss , respectively , despite each support having higher degree of corrosion tolerance , as displayed in figure 7 . in addition , it is also observed that graphitic carbon nitride supported pt catalysts with higher initial ecsa exhibit higher electrochemical durability , indicating there is a link between good metal support material that provides strong adsorption and anchoring sites for the pt nanoparticles will increase particle dispersion and limit leaching and agglomeration processes during the accelerated test . in addition , pt / pti - licl exhibits the lowest overpotential whereas pt / b - gcnm has the highest peak current density . given the difference in pt particle size in all materials with pt / vulcan having the smallest particle size , particle size effect can be eliminated from this observation . methanol oxidation reaction of supported pt electrocatalysts in 1 m ch3oh + 0.1 m hclo4 at 25 c with a scan rate of 2 mv s. for the first time , three different graphitic carbon nitride materials ( polymeric gcnm , pti - licl , and b - doped gcnm ) were prepared and tested as catalyst support materials for pefcs . the results show that all graphitic carbon nitride materials prepared in this study exhibit significantly improved durability compared to commercial carbon black ( vulcan xc-72r ) and , therefore , are promising catalyst support materials for pefc applications . as shown on the x - ray diffraction patterns , b - gcnm and pti / licl are more crystalline than vulcan and gcnm , suggesting that crystallinity may play an important role in the stability of the material against carbon corrosion . this also suggests that the presence of dopants such as boron in b - gcnm , and li and cl in pti / licl , may enhance the stability of the support materials . the pt / b - gcnm , with the lowest ecsa of all carbon nitride supported catalyst , exhibits the lowest durability with a loss of 100% after 2000 cycles . the pt - pti / licl has the best durability of all pt - supported catalyst with an ecsa loss of only 19% after 2000 scans .

primary sjgren 's syndrome ( pss ) is a systemic autoimmune disorder that affects secretory organs and is characterized by ocular and mouth dryness , fatigue , and pain , as well as extra - glandular manifestations that reveal the severity of this disorder [ 1 , 2 ] . patients with pss also present broad spectrum analytical features ( cytopenias , hypergammaglobulinemia , and cryoglobulins ) . biological signatures of the disease are b - lymphocyte activation , which could be triggered by the dysregulation of b - cell activating factor ( baff ) . one of the objective classification criteria for pss is serum ssa / ssb antibodies ( abs ) . recent research studies suggest that these antibodies may also be the biomarkers of disease activity . some studies indicate that anti - ssa / ssb seropositive patients have the increased amount of b - cell activation markers , such as baff , free immunoglobulin light chain , beta-2 microglobulin , and igg [ 37 ] . thus , the spectrum of the disease ranges widely from minimal local symptoms of the eyes and oral mucosa to systemic involvement and development of malignant lymphoma ; the latter are being the most worrisome complication of pss . recently , much attention has been focused on the relationship between innate responses and subsequent activation of specific adaptive - immunity in an attempt to understand subsequent immune dysregulation [ 810 ] . specific cytotoxic lymphocyte populations can lead to the formation of autoimmune diseases , whereas suppressive / regulatory cell populations may lead to suppression of autoimmunity and disease remission [ 11 , 12 ] . the aim of the study was to perform a detailed quantitative analysis of peripheral blood cd4 and cd8 t lymphocyte subpopulations in patients with sjgren 's syndrome with special emphasis on treg , th17 , nkt lymphocytes , nk cells , and b cells and expression of cd57 and cd27 markers on cd8 lymphocytes . in total , 52 patients with pss and 28 healthy controls were recruited at the state research institute center for innovative medicine for this study . patients with pss were grouped in two groups : pss abs group , 29 without anti - ssa and/or anti - ssb abs , and pss abs group , 23 patients with anti - ssa and/or anti - ssb abs . the average age of the patients groups and healthy controls was accordingly : 57 13 years , 56 13 years , and 53 11 years . the majority of enrolled patients in our study were lithuanian women . only 1 lithuanian man ( 1 in pss abs group ) nobody of the control group had connective tissue diseases , anti - ssa or anti - ssb abs . primary ss was diagnosed according to american - european consensus group classification criteria for sjgren 's syndrome . all patients underwent serologic evaluations , which included test for the presence of antibodies against ssa and ssb , schirmer 's i test , unstimulated whole salivary flow test , and histology of minor salivary glands . disease activity was assessed using eular sjgren 's syndrome disease activity index ( essdai ) and eular sjgren 's syndrome patient reported index ( esspri ) . the characteristics of the pss patients included in the study are summarized in table 1 . informed and written consent was obtained from all participants of this study . the study has been approved by the lithuanian bioethics committee ( no . 158200 - 03 - 299 - 73 ) . absolute counts of white blood cells ( wbc ) were determined with a haemocytometer and used for calculation of absolute numbers of lymphocyte populations ( numbers of cells/l peripheral blood ) . for cell surface staining , the following mabs were used : anti - cd3 fitc ( exbio , czech ) ; anti - cd4 percp ( bd , usa ) ; anti - cd8-percp ( bd ) ; anti - cd-16 + 56-pe ( exbio , czech ) ; anti - cd-19 percp ( exbio , czech ) ; anti - cd57 fitc ( bd , usa ) ; and anti - cd27-apc ( exbio , czech ) . for isotype controls staining , mouse anti - igg1-fitc ( bd , usa ) , igg1-percp ( bd , usa ) , and igg2a - pe ( bd , usa ) were used . cell staining was followed by red blood cell lysis using pharm lyse ( bd , usa ) lysing solution for 15 min at room temperature in the dark . leukocytes were then centrifuged ( 500 g for 10 min ) and washed two times with cellwash ( bd , usa ) and resuspended in fbs ( bd , usa ) . samples were examined immediately after staining without fixation . for the evaluation of intracellular cytokine il-17a of cd4 t cells , 1 ml of whole heparinized blood was diluted 1 : 2 in rpmi-1640 supplemented with 80 mg / l gentamycin and 2 nm glutamine . cells were stimulated using 50 ng / ml phorbol - myristate - acetate ( sigma aldrich , st . louis , mo , usa ) and 1 ng / ml ionomycin ( sigma aldrich ) in the presence of 0.7 l / ml monensin ( golgistop ( bd , usa ) ) for 4.5 h at 37c in an atmosphere containing 5% co2 . following stimulation , cells were stained for cd4 for 30 min at room temperature . cell staining was followed by red blood cell lysis using pharm lyse ( bd , usa ) lysing solution for 15 min at room temperature in the dark . leucocytes were then centrifuged ( at 500 g for 10 min ) and washed two times with cellwash ( bd ) . ( bd , usa ) solution for 20 min , washed two times with perm / wash ( bd , usa ) solution , and incubated further for 30 min in the dark with the specific mabs anti - il17a apc ( bd , usa ) and isotypic control anti - igg1 apc ( bd , usa ) . following the incubation with mabs , the cells were washed two times with perm / wash solution and resuspended in fbs ( bd , usa ) . for the evaluation of intracellular foxp3 marker of cd4 and cd8 t cells , cells were stained with anti - cd4 percp ( bd , usa ) , anti - cd25 fitc ( exbio , czech ) , and anti - cd8-percp ( bd , usa ) for 30 min at room temperature . cell staining was followed by red blood cell lysis using pharm lyse ( bd , usa ) solution for 15 min at room temperature in the dark . leucocytes were then centrifuged ( at 500 g for 10 min ) and washed two times with cellwash ( bd , usa ) . then , cells were fixed and permeabilized with cytofix / cytoperm ( bd , usa ) solution for 20 min , washed two times with perm / wash ( bd , usa ) solution , and incubated further for 30 min in the dark with the specific mabs anti - foxp3-pe ( bd , usa ) and isotypic controls anti - igg1-fitc ( bd , usa ) , igg1-percp ( bd , usa ) , and igg2a - pe ( bd , usa ) . following the incubation with mabs , ( bd , usa ) solution and resuspended in fbs ( bd , usa ) . flow cytometry was performed on facscalibur flow cytometer ( bd biosciences , san jose , ca , usa ) calibrated with calibrite beads ( bd biosciences , san jose , ca , usa ) using cell - quest software ( bd biosciences , san jose , ca , usa ) . serum samples were analyzed using commercial baff , soluble ( human ) elisa kit ( hypersensitive ) ( adipogen , switzerland ) . from collected blood samples , serum dilutions and enzyme - linked immunoassay was carried out in the strict accordance with the manufacturer 's instructions and sets recommendations . a regression analysis was performed to derive an equation that was then used to predict the concentration of the unknown samples with gen5 microplate data collection & analysis software ( biotek instruments , usa ) . correlations were assessed by the spearman 's rank test using standard program graphpad prism 5.0 software ( graphpad software , san diego , ca , usa ) . we found that absolute count of cd3 t cell population was significantly decreased in pss patients in comparison to healthy controls . no differences were observed between pss groups . significant decrease of cd3 cells was found in pss abs ( p = 0.027 ) and pss abs ( p = 0.0002 ) groups when compared to controls ; however , no differences in the cd3 cells proportion of wbc were found between pss groups . analysis of cd4 t cell population showed significant proportion differences in pss abs patient 's blood when compared to pss abs ( p = 0.036 ) and control group ( p = 0.0036 ) . but absolute counts of cd4 cells were significantly lower in both pss groups than in control group , pss abs ( p = 0.015 ) and pss abs ( p < 0.0001 ) , and also absolute counts of cd4 cells were significantly lower in pss abs than in pss abs ( p = 0.01 ) patients ' blood . no significant differences in the proportion of cd8 t cell population were found between pss patients and controls . but analysis of absolute counts showed significantly lower counts of cd8 t cells in pss abs ( p = 0.014 ) and pss abs ( p = 0.006 ) in comparison to controls ; no differences were found between pss groups ( table 2 , figure 1 ) . analysis of nkt ( cd3cd16/56 ) and nk ( cd3cd16/56 ) cells showed only decreased absolute counts in pss abs group ( resp . , p = 0.009 and p = 0.036 ) in comparison to controls . increased proportion of cd3cd19 ( b cells ) in pss abs ( p = 0.045 ) was found when compared to controls , but no differences were found analyzing absolute counts among pss groups and controls ( table 2 ) . analysis of cd4 lymphocyte subpopulations according to their expression of cd25 and foxp3 markers showed significant reduced absolute counts of cd4cd25 ( p = 0.036 ) and cd4 cd25foxp3 ( p = 0.017 ) cells when comparing pss abs with controls . no significant differences were observed analyzing other cd4 subpopulations according to their expression of cd25 and foxp3 markers ( table 3 ) . analysis of cd4il-17a ( th17 ) cells showed significant lower proportion and absolute counts of these cells in pss patients in comparison to control group 's results ( figure 2 ) . proportion was significantly altered in pss abs ( p = 0.0003 ) and pss abs ( p = 0.004 ) , and also absolute counts of th17 cell were significantly lower in pss abs ( p < 0.0001 ) and pss abs ( p no significant differences were found analyzing proportion and absolute counts of this subpopulation between pss groups ( table 3 , figure 1 ) . cd8 lymphocyte population was differentiated to subpopulations according to the markers cd57 and cd27 that defines replicative senescence ( figure 1 ) . analysis of cd8cd57cd27 subpopulation showed no differences in proportion and absolute counts among pss and control groups . in pss patients with abs , we observed significantly increased proportion ( p = 0.032 ) and absolute counts ( p = 0.011 ) of cd8cd57cd27 subpopulation in comparison to pss without abs . no significant differences were found when comparing results of pss abs and pss abs to controls . cd8cd57cd27 population 's proportion was significantly reduced only in pss without abs patients ' blood ( p = 0.026 ) when compared to controls and no significant differences between pss groups were observed . absolute counts of this subpopulation were significantly reduced both pss abs ( p = 0.0003 ) and pss abs ( p = 0.005 ) in comparison to controls . proportion of cd8 subpopulation lacking cd57 and cd27 markers ( cd8cd57cd27 ) was significantly increased only in pss abs patients ' blood when compared to controls . differences in proportion or absolute counts of subpopulation expressing foxp3 marker were not observed ( table 4 ) . focus score correlated with absolute counts of cd3 ( p = 0.047 , r = 0.474 ) , cd4 ( p = 0.033 , r = 0.503 ) , b cell ( p = 0.023 , r = 0.532 ) , and cd8cd57cd27 ( p = 0.016 , r = 0.560 ) cell populations . essdai correlated with nk ( p = 0.043 , r = 0.482 ) and cd4cd25foxp3 ( p = 0.047 , r = 0.474 ) cell absolute counts . in pss patients without abs , we found that schirmer 's i test correlated with proportion ( p = 0.033 , r = 0.504 ) and absolute counts serum baff concentration correlated with proportion of cd8 ( p = 0.032 , r = 0.415 ) , nk ( p = 0.009 , r = 0.491 ) , and cd8cd57cd27 ( p = 0.014 , r = 0.469 ) cell populations and negatively correlated with b cells ( p = 0.026 , r = 0.429 ) , cd8cd57cd27 ( p = 0.036 , r = 0.404 ) , and cd8cd57cd27 ( p = 0.009 , r = 0.493 ) . analyzing absolute counts of cells populations observed negative correlation between baff and cd4 ( p = 0.025 , r = 0.430 ) , b cell ( p = 0.007 , r = 0.509 ) , cd8cd57cd27 ( p = 0.028 , r = 0.424 ) , and cd8cd57cd27 ( p = 0.017 , r = 0.454 ) cells . schirmer 's i test correlated with proportion of b cells ( p = 0.038 , r = 0.467 ) and cd8cd57cd27 ( p = 0.006 , r = 0.589 ) and negatively correlated with cd3 ( p = 0.006 , r = 0.590 ) and cd8cd57cd27 ( p = 0.033 , r = 0.479 ) cells . also correlation between schirmer 's i test and absolute count of cd8cd57cd27 ( p = 0.021 , r = 0.512 ) and negative correlation with cd8cd57cd27 ( p = 0.017 , r = 0.525 ) cell counts were observed . unstimulated salivary flow rate correlated with proportion of cd8 ( p = 0.017 , r = 0.525 ) and th17 ( p = 0.015 , r = 0.535 ) and also negatively correlated with absolute counts of cd3 ( p = 0.011 , r = 0.555 ) and cd4 ( p = 0.017 , r = 0.526 ) cells . focus score negatively correlated with proportion and absolute counts of cd8cd57cd27 cells , respectively , ( p = 0.020 , r = 0.517 ) and ( p = 0.040 , r = 0.462 ) . esspri correlated with proportion of cd3 ( p = 0.015 , r = 0.535 ) , cd4foxp3 ( p = 0.044 , r = 0.454 ) , and cd4cd25foxp3 ( p = 0.041 , r = 0.461 ) cells and negatively correlated with proportion of cd4cd25 ( p = 0.026 , r = 0.496 ) and cd4cd25 ( p = 0.043 , r = 0.456 ) cells . essdai negatively correlated with proportion of th17 ( p = 0.001 , r = 0.675 ) cells and also with absolute counts of cd8 ( p = 0.042 , r = 0.459 ) , th17 ( p = 0.001 , r = 0.673 ) , and cd8cd57cd27 ( p = 0.028 , r = 0.492 ) cells . no correlation was observed between serum baff concentration and cell populations changes in pss patients with abs . despite systemic b - cell hyperactivity , t and b cells constitute the vast majority of infiltrating mononuclear cells at the minor salivary glands inflammatory lesions of pss , with their prevalence varying according to the severity of the infiltrates . cd8 t cells with cytotoxic activity , as manifested by their expression of granzymes , constitute around 15% of infiltrating cells . t cells predominate in mild lesions , whereas in severe lesions b cells constitute the main population . the prevalence of cd4 t cells decreases with lesion severity , whereas the prevalence of cd8 t cells remains unchanged . the prevalence of regulatory t cells associates with lesion severity , with the higher values to be observed at intermediate lesions . nk cells comprise a small but considerable portion of the infiltrating mononuclear cells , and their percentage correlates with the grade of the lesions [ 15 , 16 ] . in our study , t lymphocyte identification by cd4 and cd8 markers showed a statistically significant decrease in the absolute counts of cd4 and cd8 t lymphocytes in the peripheral blood of pss patients in comparison to the control group . this shows that the decline of cd3 t lymphocyte population in the peripheral blood of pss patients is influenced by a decrease of both cd4 and cd8 t lymphocyte absolute counts . the decrease of the total amount of cd4 t lymphocytes in the peripheral blood of pss patients is also confirmed by other authors . in some pss patients , low counts of cd4 lymphocytes or their dysfunction in peripheral blood maybe due to anti - cd4 antibodies . the fact is that the proportion of cd4 was lower only in our pss patients with abs , and no differences in proportion of cd3 and cd8 in all pss patients were observed ; let us think that this lymphopenia can also be genetically determined . apoptosis may also play a role in the pathogenesis of some extraglandular manifestations of pss and peripheral cd4 lymphocytopenia [ 19 , 20 ] . studies in patients with pss and animal models of pss have identified the presence of il-17 in the lymphocytic infiltrates of the exocrine glands , as well as higher levels of circulating il-17 in both serum and saliva [ 8 , 21 , 22 ] . on the one hand , our finding that th17 lymphocyte counts decreased in the peripheral blood of patients with sjgren 's syndrome is quite unexpected . on the other hand , this result may be explained by the redistribution of th17 lymphocytes , that is , increasing their concentration in tissues ( salivary glands ) and decreasing concentration in peripheral blood [ 8 , 10 ] . treg lymphocytes are characterized by autoimmune reaction - inhibiting properties [ 15 , 23 ] . however , in this study , we have not find more distinct treg cells changes in the peripheral blood of pss patients . no statistical significant differences were found analyzing cd4cd25foxp3 cells , cd4cd25 , or cd4cd25foxp3 cells . sometimes it is hard to define what is what , when conflicting results have been reported . one of the problems is that different authors as treg population define different pools of cd4 cells . some researchers uses two markers cd4 and cd25 to identify treg cells , while others also uses foxp3 marker . this is why we have checked more pools of cd4 that some authors define as treg cells . the role of cytotoxic t cells in pss pathogenesis has not been studied in detail . cd8 t - cell deficiency is a feature of many chronic autoimmune diseases , including multiple sclerosis , rheumatoid arthritis , systemic lupus erythematosus , sjgren 's syndrome , systemic sclerosis , ulcerative colitis , crohn 's disease , psoriasis , etc . it also occurs in blood of healthy relatives of patients with autoimmune diseases , suggesting that it is genetically determined . these cells play critical roles in purging acute infections , limiting persistent infections , and conferring life - long protective immunity . cd8 t cell deficiency can prompt the development of chronic autoimmune diseases by impairing cd8 t cell control of virus infection . it is known that viral infections are the best candidates for the role of environmental triggers to autoimmune reactions . it is proposed that , after activation in peripheral lymphoid organs by cross - reacting foreign antigens , autoreactive t cells enter the target organ where they are reactivated by b cells which provide costimulatory survival signals , thereby inhibiting the activation - induced t - cell apoptosis which normally occurs when autoreactive t cells enter the target organ . understanding cd8 t memory effector cells differentiation is essential for studying how virus - specific cd8 t cells control viral infection . distinct stages of virus - specific cd8 t memory effector cells differentiation have been extensively characterized by phenotypic and functional analyses . primed virus - specific cd8 t cells typically differentiate from the least mature memory stage ( cd27 ) to the most mature effector stage when they start to lose cd27 and obtain cd57 marker and eventually become terminally differentiated effector cells which can be further defined by cd57 expression . according to some studies , last stage of cd8 cells differentiation seems to be cd8cd27cd57 t cells subset with high perforin and killing activity . is this the true end - stage or terminally - differentiated state of cytotoxic t cells ? we observed increased proportion of this population in pss patients , but significant differences were observed only in pss without abs group . in pss with abs group , negative correlation between cd8cd27cd57 t cells subset absolute count and schirmer 's i test results was observed . this connection proposes that this subset can be involved in a pathogenic process which appears in the glandular tissue . impaired proportion and absolute counts of cd8cd27cd57 t cells in blood of pss patients can be the reason of lower counts of cd8 in blood of pss patients . it is not known whether the proportion and absolute counts of this population are downregulated in blood by migration to inflammatory sites , or this can be due to increased apoptosis of these cells . there is hypothesis that more mature effector stage ( cd8cd27cd57 and cd8cd27cd57 ) cells with lower capacity of proliferation are more resistant to apoptosis than least mature memory stage cd8cd27cd57 t cells [ 2931 ] . all these observed changes in cd8 t cell subpopulations rearrangement prove that these subpopulations actively participate in pathological processes of pss . nkt lymphocytes and nk cells might function as regulatory t cells and are one of the autoimmune process preventing chains [ 32 , 33 ] . according to literature , in patients with autoimmune rheumatic diseases , the decreased nkt and nk cell counts and functional characteristics published results where they identified higher proportion of these cells in blood of pss patients than in control group . our investigation of these populations showed a significant decrease of nkt and nk - cell absolute counts in the peripheral blood of pss patients with abs ; however , the fact that a proportion of these cells were similar with the controls can indicate that lower absolute counts can be due to genetically determined lymphopenia . there is also possibility that low counts of these cell populations in periphery can be by reason of overall lymphocyte population migration to inflammatory sites or / and apoptosis . in conjunction with the classical cd4 tregs , we were also investigating cd8 suppressor cells that express foxp3 marker , as foxp3 confers suppressive properties and is confined to regulatory t cells . cd8foxp3 cells represent a new regulatory population and ability of these cd8foxp3 treg to suppress cd8 responses far more effectively than cd4foxp3 treg . our study results did not show any significant differences in proportion or absolute count changes on these cells in pss patients ' peripheral blood in comparison to healthy controls . we found negative correlation between baff and t and b cells in pss patients without abs . increase of baff in serum can be due to negative regulation of baff secretion by monocytes . this fact can indicate tight control of baff secretion . whereas we do not found correlation between baff and lymphocyte populations changes in pss patients with abs group , what can be the indication of uncontrolled baff secretion and its homeostasis disturbance ? despite recent knowledge , in many respects , the role of t cells and their subsets in pss remains unexplained . are cells in the infiltrate specific , or maybe many of them are just bystanders ( with nonactivated phenotype ) recruited from the periphery to the inflammatory sites ? t cells undergo expansion within the gland , or does this occur elsewhere with subsequent migration ? is there migration in and out of the gland , or do t cells remain in the infiltrates once they arrive ? pointers to these questions could help us understand which processes are going on periphery . one of future projects should be the immunohistochemistry for assessing cell populations ' changes in salivary glands in parallel with blood analysis and apoptosis markers . such analysis could help better to define changes of cell populations in periphery , is this due migration to the inflammatory sites or increased apoptosis , or maybe both .

purpose of this study was to evaluate the lymphocyte populations ' distribution changes in peripheral blood of patients with primary sjgren 's syndrome ( pss ) . lymphocyte populations ' distribution changes in peripheral blood of pss patients were investigated in 52 patients with pss and in 28 healthy controls by flow cytometry . we found decreased absolute count of cd3 + t cell population in pss patients . analysis of cd4 + t cell population showed significant proportion and absolute count differences in pss patient 's blood with ssa / ssb antibodies ( abs ) in comparison to controls . no significant differences were observed analyzing cd4 + and cd8 + treg subpopulation . proportion and absolute counts of th17 cells were significantly lower in pss patient 's blood . absolute counts of cd8 + t cells were significantly lower in pss patients in comparison to controls and also impaired proportion and absolute counts of cd8 + subpopulations according to cd27 + and cd57 + were observed . absolute counts of nkt and nk cells were decreased in pss with abs . b cells proportion was increased only in blood of pss with abs . lymphocyte distribution impairment can be due to genetically determined lymphopenia or lymphocyte migration from periphery to inflammatory sites or / and increased susceptibility to apoptosis .

1. Introduction 2. Patients and Methods 3. Results 4. Discussion

primary sjgren 's syndrome ( pss ) is a systemic autoimmune disorder that affects secretory organs and is characterized by ocular and mouth dryness , fatigue , and pain , as well as extra - glandular manifestations that reveal the severity of this disorder [ 1 , 2 ] . patients with pss also present broad spectrum analytical features ( cytopenias , hypergammaglobulinemia , and cryoglobulins ) . one of the objective classification criteria for pss is serum ssa / ssb antibodies ( abs ) . some studies indicate that anti - ssa / ssb seropositive patients have the increased amount of b - cell activation markers , such as baff , free immunoglobulin light chain , beta-2 microglobulin , and igg [ 37 ] . thus , the spectrum of the disease ranges widely from minimal local symptoms of the eyes and oral mucosa to systemic involvement and development of malignant lymphoma ; the latter are being the most worrisome complication of pss . the aim of the study was to perform a detailed quantitative analysis of peripheral blood cd4 and cd8 t lymphocyte subpopulations in patients with sjgren 's syndrome with special emphasis on treg , th17 , nkt lymphocytes , nk cells , and b cells and expression of cd57 and cd27 markers on cd8 lymphocytes . in total , 52 patients with pss and 28 healthy controls were recruited at the state research institute center for innovative medicine for this study . patients with pss were grouped in two groups : pss abs group , 29 without anti - ssa and/or anti - ssb abs , and pss abs group , 23 patients with anti - ssa and/or anti - ssb abs . the majority of enrolled patients in our study were lithuanian women . primary ss was diagnosed according to american - european consensus group classification criteria for sjgren 's syndrome . disease activity was assessed using eular sjgren 's syndrome disease activity index ( essdai ) and eular sjgren 's syndrome patient reported index ( esspri ) . informed and written consent was obtained from all participants of this study . absolute counts of white blood cells ( wbc ) were determined with a haemocytometer and used for calculation of absolute numbers of lymphocyte populations ( numbers of cells/l peripheral blood ) . for the evaluation of intracellular cytokine il-17a of cd4 t cells , 1 ml of whole heparinized blood was diluted 1 : 2 in rpmi-1640 supplemented with 80 mg / l gentamycin and 2 nm glutamine . following stimulation , cells were stained for cd4 for 30 min at room temperature . following the incubation with mabs , the cells were washed two times with perm / wash solution and resuspended in fbs ( bd , usa ) . for the evaluation of intracellular foxp3 marker of cd4 and cd8 t cells , cells were stained with anti - cd4 percp ( bd , usa ) , anti - cd25 fitc ( exbio , czech ) , and anti - cd8-percp ( bd , usa ) for 30 min at room temperature . then , cells were fixed and permeabilized with cytofix / cytoperm ( bd , usa ) solution for 20 min , washed two times with perm / wash ( bd , usa ) solution , and incubated further for 30 min in the dark with the specific mabs anti - foxp3-pe ( bd , usa ) and isotypic controls anti - igg1-fitc ( bd , usa ) , igg1-percp ( bd , usa ) , and igg2a - pe ( bd , usa ) . flow cytometry was performed on facscalibur flow cytometer ( bd biosciences , san jose , ca , usa ) calibrated with calibrite beads ( bd biosciences , san jose , ca , usa ) using cell - quest software ( bd biosciences , san jose , ca , usa ) . we found that absolute count of cd3 t cell population was significantly decreased in pss patients in comparison to healthy controls . no differences were observed between pss groups . significant decrease of cd3 cells was found in pss abs ( p = 0.027 ) and pss abs ( p = 0.0002 ) groups when compared to controls ; however , no differences in the cd3 cells proportion of wbc were found between pss groups . analysis of cd4 t cell population showed significant proportion differences in pss abs patient 's blood when compared to pss abs ( p = 0.036 ) and control group ( p = 0.0036 ) . but absolute counts of cd4 cells were significantly lower in both pss groups than in control group , pss abs ( p = 0.015 ) and pss abs ( p < 0.0001 ) , and also absolute counts of cd4 cells were significantly lower in pss abs than in pss abs ( p = 0.01 ) patients ' blood . no significant differences in the proportion of cd8 t cell population were found between pss patients and controls . but analysis of absolute counts showed significantly lower counts of cd8 t cells in pss abs ( p = 0.014 ) and pss abs ( p = 0.006 ) in comparison to controls ; no differences were found between pss groups ( table 2 , figure 1 ) . analysis of nkt ( cd3cd16/56 ) and nk ( cd3cd16/56 ) cells showed only decreased absolute counts in pss abs group ( resp . , p = 0.009 and p = 0.036 ) in comparison to controls . increased proportion of cd3cd19 ( b cells ) in pss abs ( p = 0.045 ) was found when compared to controls , but no differences were found analyzing absolute counts among pss groups and controls ( table 2 ) . analysis of cd4 lymphocyte subpopulations according to their expression of cd25 and foxp3 markers showed significant reduced absolute counts of cd4cd25 ( p = 0.036 ) and cd4 cd25foxp3 ( p = 0.017 ) cells when comparing pss abs with controls . no significant differences were observed analyzing other cd4 subpopulations according to their expression of cd25 and foxp3 markers ( table 3 ) . analysis of cd4il-17a ( th17 ) cells showed significant lower proportion and absolute counts of these cells in pss patients in comparison to control group 's results ( figure 2 ) . proportion was significantly altered in pss abs ( p = 0.0003 ) and pss abs ( p = 0.004 ) , and also absolute counts of th17 cell were significantly lower in pss abs ( p < 0.0001 ) and pss abs ( p no significant differences were found analyzing proportion and absolute counts of this subpopulation between pss groups ( table 3 , figure 1 ) . cd8 lymphocyte population was differentiated to subpopulations according to the markers cd57 and cd27 that defines replicative senescence ( figure 1 ) . analysis of cd8cd57cd27 subpopulation showed no differences in proportion and absolute counts among pss and control groups . in pss patients with abs , we observed significantly increased proportion ( p = 0.032 ) and absolute counts ( p = 0.011 ) of cd8cd57cd27 subpopulation in comparison to pss without abs . no significant differences were found when comparing results of pss abs and pss abs to controls . cd8cd57cd27 population 's proportion was significantly reduced only in pss without abs patients ' blood ( p = 0.026 ) when compared to controls and no significant differences between pss groups were observed . absolute counts of this subpopulation were significantly reduced both pss abs ( p = 0.0003 ) and pss abs ( p = 0.005 ) in comparison to controls . proportion of cd8 subpopulation lacking cd57 and cd27 markers ( cd8cd57cd27 ) was significantly increased only in pss abs patients ' blood when compared to controls . differences in proportion or absolute counts of subpopulation expressing foxp3 marker were not observed ( table 4 ) . focus score correlated with absolute counts of cd3 ( p = 0.047 , r = 0.474 ) , cd4 ( p = 0.033 , r = 0.503 ) , b cell ( p = 0.023 , r = 0.532 ) , and cd8cd57cd27 ( p = 0.016 , r = 0.560 ) cell populations . essdai correlated with nk ( p = 0.043 , r = 0.482 ) and cd4cd25foxp3 ( p = 0.047 , r = 0.474 ) cell absolute counts . in pss patients without abs , we found that schirmer 's i test correlated with proportion ( p = 0.033 , r = 0.504 ) and absolute counts serum baff concentration correlated with proportion of cd8 ( p = 0.032 , r = 0.415 ) , nk ( p = 0.009 , r = 0.491 ) , and cd8cd57cd27 ( p = 0.014 , r = 0.469 ) cell populations and negatively correlated with b cells ( p = 0.026 , r = 0.429 ) , cd8cd57cd27 ( p = 0.036 , r = 0.404 ) , and cd8cd57cd27 ( p = 0.009 , r = 0.493 ) . analyzing absolute counts of cells populations observed negative correlation between baff and cd4 ( p = 0.025 , r = 0.430 ) , b cell ( p = 0.007 , r = 0.509 ) , cd8cd57cd27 ( p = 0.028 , r = 0.424 ) , and cd8cd57cd27 ( p = 0.017 , r = 0.454 ) cells . also correlation between schirmer 's i test and absolute count of cd8cd57cd27 ( p = 0.021 , r = 0.512 ) and negative correlation with cd8cd57cd27 ( p = 0.017 , r = 0.525 ) cell counts were observed . unstimulated salivary flow rate correlated with proportion of cd8 ( p = 0.017 , r = 0.525 ) and th17 ( p = 0.015 , r = 0.535 ) and also negatively correlated with absolute counts of cd3 ( p = 0.011 , r = 0.555 ) and cd4 ( p = 0.017 , r = 0.526 ) cells . focus score negatively correlated with proportion and absolute counts of cd8cd57cd27 cells , respectively , ( p = 0.020 , r = 0.517 ) and ( p = 0.040 , r = 0.462 ) . esspri correlated with proportion of cd3 ( p = 0.015 , r = 0.535 ) , cd4foxp3 ( p = 0.044 , r = 0.454 ) , and cd4cd25foxp3 ( p = 0.041 , r = 0.461 ) cells and negatively correlated with proportion of cd4cd25 ( p = 0.026 , r = 0.496 ) and cd4cd25 ( p = 0.043 , r = 0.456 ) cells . essdai negatively correlated with proportion of th17 ( p = 0.001 , r = 0.675 ) cells and also with absolute counts of cd8 ( p = 0.042 , r = 0.459 ) , th17 ( p = 0.001 , r = 0.673 ) , and cd8cd57cd27 ( p = 0.028 , r = 0.492 ) cells . no correlation was observed between serum baff concentration and cell populations changes in pss patients with abs . despite systemic b - cell hyperactivity , t and b cells constitute the vast majority of infiltrating mononuclear cells at the minor salivary glands inflammatory lesions of pss , with their prevalence varying according to the severity of the infiltrates . cd8 t cells with cytotoxic activity , as manifested by their expression of granzymes , constitute around 15% of infiltrating cells . t cells predominate in mild lesions , whereas in severe lesions b cells constitute the main population . the prevalence of cd4 t cells decreases with lesion severity , whereas the prevalence of cd8 t cells remains unchanged . the prevalence of regulatory t cells associates with lesion severity , with the higher values to be observed at intermediate lesions . nk cells comprise a small but considerable portion of the infiltrating mononuclear cells , and their percentage correlates with the grade of the lesions [ 15 , 16 ] . in our study , t lymphocyte identification by cd4 and cd8 markers showed a statistically significant decrease in the absolute counts of cd4 and cd8 t lymphocytes in the peripheral blood of pss patients in comparison to the control group . this shows that the decline of cd3 t lymphocyte population in the peripheral blood of pss patients is influenced by a decrease of both cd4 and cd8 t lymphocyte absolute counts . the decrease of the total amount of cd4 t lymphocytes in the peripheral blood of pss patients is also confirmed by other authors . in some pss patients , low counts of cd4 lymphocytes or their dysfunction in peripheral blood maybe due to anti - cd4 antibodies . the fact is that the proportion of cd4 was lower only in our pss patients with abs , and no differences in proportion of cd3 and cd8 in all pss patients were observed ; let us think that this lymphopenia can also be genetically determined . apoptosis may also play a role in the pathogenesis of some extraglandular manifestations of pss and peripheral cd4 lymphocytopenia [ 19 , 20 ] . studies in patients with pss and animal models of pss have identified the presence of il-17 in the lymphocytic infiltrates of the exocrine glands , as well as higher levels of circulating il-17 in both serum and saliva [ 8 , 21 , 22 ] . on the one hand , our finding that th17 lymphocyte counts decreased in the peripheral blood of patients with sjgren 's syndrome is quite unexpected . on the other hand , this result may be explained by the redistribution of th17 lymphocytes , that is , increasing their concentration in tissues ( salivary glands ) and decreasing concentration in peripheral blood [ 8 , 10 ] . however , in this study , we have not find more distinct treg cells changes in the peripheral blood of pss patients . no statistical significant differences were found analyzing cd4cd25foxp3 cells , cd4cd25 , or cd4cd25foxp3 cells . one of the problems is that different authors as treg population define different pools of cd4 cells . this is why we have checked more pools of cd4 that some authors define as treg cells . the role of cytotoxic t cells in pss pathogenesis has not been studied in detail . cd8 t - cell deficiency is a feature of many chronic autoimmune diseases , including multiple sclerosis , rheumatoid arthritis , systemic lupus erythematosus , sjgren 's syndrome , systemic sclerosis , ulcerative colitis , crohn 's disease , psoriasis , etc . it also occurs in blood of healthy relatives of patients with autoimmune diseases , suggesting that it is genetically determined . cd8 t cell deficiency can prompt the development of chronic autoimmune diseases by impairing cd8 t cell control of virus infection . it is proposed that , after activation in peripheral lymphoid organs by cross - reacting foreign antigens , autoreactive t cells enter the target organ where they are reactivated by b cells which provide costimulatory survival signals , thereby inhibiting the activation - induced t - cell apoptosis which normally occurs when autoreactive t cells enter the target organ . primed virus - specific cd8 t cells typically differentiate from the least mature memory stage ( cd27 ) to the most mature effector stage when they start to lose cd27 and obtain cd57 marker and eventually become terminally differentiated effector cells which can be further defined by cd57 expression . according to some studies , last stage of cd8 cells differentiation seems to be cd8cd27cd57 t cells subset with high perforin and killing activity . is this the true end - stage or terminally - differentiated state of cytotoxic t cells ? we observed increased proportion of this population in pss patients , but significant differences were observed only in pss without abs group . in pss with abs group , negative correlation between cd8cd27cd57 t cells subset absolute count and schirmer 's i test results was observed . impaired proportion and absolute counts of cd8cd27cd57 t cells in blood of pss patients can be the reason of lower counts of cd8 in blood of pss patients . it is not known whether the proportion and absolute counts of this population are downregulated in blood by migration to inflammatory sites , or this can be due to increased apoptosis of these cells . there is hypothesis that more mature effector stage ( cd8cd27cd57 and cd8cd27cd57 ) cells with lower capacity of proliferation are more resistant to apoptosis than least mature memory stage cd8cd27cd57 t cells [ 2931 ] . all these observed changes in cd8 t cell subpopulations rearrangement prove that these subpopulations actively participate in pathological processes of pss . nkt lymphocytes and nk cells might function as regulatory t cells and are one of the autoimmune process preventing chains [ 32 , 33 ] . according to literature , in patients with autoimmune rheumatic diseases , the decreased nkt and nk cell counts and functional characteristics published results where they identified higher proportion of these cells in blood of pss patients than in control group . our investigation of these populations showed a significant decrease of nkt and nk - cell absolute counts in the peripheral blood of pss patients with abs ; however , the fact that a proportion of these cells were similar with the controls can indicate that lower absolute counts can be due to genetically determined lymphopenia . there is also possibility that low counts of these cell populations in periphery can be by reason of overall lymphocyte population migration to inflammatory sites or / and apoptosis . in conjunction with the classical cd4 tregs , we were also investigating cd8 suppressor cells that express foxp3 marker , as foxp3 confers suppressive properties and is confined to regulatory t cells . our study results did not show any significant differences in proportion or absolute count changes on these cells in pss patients ' peripheral blood in comparison to healthy controls . we found negative correlation between baff and t and b cells in pss patients without abs . increase of baff in serum can be due to negative regulation of baff secretion by monocytes . whereas we do not found correlation between baff and lymphocyte populations changes in pss patients with abs group , what can be the indication of uncontrolled baff secretion and its homeostasis disturbance ? despite recent knowledge , in many respects , the role of t cells and their subsets in pss remains unexplained . are cells in the infiltrate specific , or maybe many of them are just bystanders ( with nonactivated phenotype ) recruited from the periphery to the inflammatory sites ? t cells undergo expansion within the gland , or does this occur elsewhere with subsequent migration ? is there migration in and out of the gland , or do t cells remain in the infiltrates once they arrive ? one of future projects should be the immunohistochemistry for assessing cell populations ' changes in salivary glands in parallel with blood analysis and apoptosis markers . such analysis could help better to define changes of cell populations in periphery , is this due migration to the inflammatory sites or increased apoptosis , or maybe both .

primary sjgren 's syndrome ( pss ) is a systemic autoimmune disorder that affects secretory organs and is characterized by ocular and mouth dryness , fatigue , and pain , as well as extra - glandular manifestations that reveal the severity of this disorder [ 1 , 2 ] . biological signatures of the disease are b - lymphocyte activation , which could be triggered by the dysregulation of b - cell activating factor ( baff ) . some studies indicate that anti - ssa / ssb seropositive patients have the increased amount of b - cell activation markers , such as baff , free immunoglobulin light chain , beta-2 microglobulin , and igg [ 37 ] . thus , the spectrum of the disease ranges widely from minimal local symptoms of the eyes and oral mucosa to systemic involvement and development of malignant lymphoma ; the latter are being the most worrisome complication of pss . recently , much attention has been focused on the relationship between innate responses and subsequent activation of specific adaptive - immunity in an attempt to understand subsequent immune dysregulation [ 810 ] . specific cytotoxic lymphocyte populations can lead to the formation of autoimmune diseases , whereas suppressive / regulatory cell populations may lead to suppression of autoimmunity and disease remission [ 11 , 12 ] . the aim of the study was to perform a detailed quantitative analysis of peripheral blood cd4 and cd8 t lymphocyte subpopulations in patients with sjgren 's syndrome with special emphasis on treg , th17 , nkt lymphocytes , nk cells , and b cells and expression of cd57 and cd27 markers on cd8 lymphocytes . in total , 52 patients with pss and 28 healthy controls were recruited at the state research institute center for innovative medicine for this study . patients with pss were grouped in two groups : pss abs group , 29 without anti - ssa and/or anti - ssb abs , and pss abs group , 23 patients with anti - ssa and/or anti - ssb abs . the average age of the patients groups and healthy controls was accordingly : 57 13 years , 56 13 years , and 53 11 years . only 1 lithuanian man ( 1 in pss abs group ) nobody of the control group had connective tissue diseases , anti - ssa or anti - ssb abs . all patients underwent serologic evaluations , which included test for the presence of antibodies against ssa and ssb , schirmer 's i test , unstimulated whole salivary flow test , and histology of minor salivary glands . disease activity was assessed using eular sjgren 's syndrome disease activity index ( essdai ) and eular sjgren 's syndrome patient reported index ( esspri ) . the characteristics of the pss patients included in the study are summarized in table 1 . absolute counts of white blood cells ( wbc ) were determined with a haemocytometer and used for calculation of absolute numbers of lymphocyte populations ( numbers of cells/l peripheral blood ) . for cell surface staining , the following mabs were used : anti - cd3 fitc ( exbio , czech ) ; anti - cd4 percp ( bd , usa ) ; anti - cd8-percp ( bd ) ; anti - cd-16 + 56-pe ( exbio , czech ) ; anti - cd-19 percp ( exbio , czech ) ; anti - cd57 fitc ( bd , usa ) ; and anti - cd27-apc ( exbio , czech ) . for isotype controls staining , mouse anti - igg1-fitc ( bd , usa ) , igg1-percp ( bd , usa ) , and igg2a - pe ( bd , usa ) were used . for the evaluation of intracellular cytokine il-17a of cd4 t cells , 1 ml of whole heparinized blood was diluted 1 : 2 in rpmi-1640 supplemented with 80 mg / l gentamycin and 2 nm glutamine . louis , mo , usa ) and 1 ng / ml ionomycin ( sigma aldrich ) in the presence of 0.7 l / ml monensin ( golgistop ( bd , usa ) ) for 4.5 h at 37c in an atmosphere containing 5% co2 . ( bd , usa ) solution for 20 min , washed two times with perm / wash ( bd , usa ) solution , and incubated further for 30 min in the dark with the specific mabs anti - il17a apc ( bd , usa ) and isotypic control anti - igg1 apc ( bd , usa ) . for the evaluation of intracellular foxp3 marker of cd4 and cd8 t cells , cells were stained with anti - cd4 percp ( bd , usa ) , anti - cd25 fitc ( exbio , czech ) , and anti - cd8-percp ( bd , usa ) for 30 min at room temperature . then , cells were fixed and permeabilized with cytofix / cytoperm ( bd , usa ) solution for 20 min , washed two times with perm / wash ( bd , usa ) solution , and incubated further for 30 min in the dark with the specific mabs anti - foxp3-pe ( bd , usa ) and isotypic controls anti - igg1-fitc ( bd , usa ) , igg1-percp ( bd , usa ) , and igg2a - pe ( bd , usa ) . from collected blood samples , serum dilutions and enzyme - linked immunoassay was carried out in the strict accordance with the manufacturer 's instructions and sets recommendations . we found that absolute count of cd3 t cell population was significantly decreased in pss patients in comparison to healthy controls . significant decrease of cd3 cells was found in pss abs ( p = 0.027 ) and pss abs ( p = 0.0002 ) groups when compared to controls ; however , no differences in the cd3 cells proportion of wbc were found between pss groups . analysis of cd4 t cell population showed significant proportion differences in pss abs patient 's blood when compared to pss abs ( p = 0.036 ) and control group ( p = 0.0036 ) . but absolute counts of cd4 cells were significantly lower in both pss groups than in control group , pss abs ( p = 0.015 ) and pss abs ( p < 0.0001 ) , and also absolute counts of cd4 cells were significantly lower in pss abs than in pss abs ( p = 0.01 ) patients ' blood . no significant differences in the proportion of cd8 t cell population were found between pss patients and controls . but analysis of absolute counts showed significantly lower counts of cd8 t cells in pss abs ( p = 0.014 ) and pss abs ( p = 0.006 ) in comparison to controls ; no differences were found between pss groups ( table 2 , figure 1 ) . analysis of nkt ( cd3cd16/56 ) and nk ( cd3cd16/56 ) cells showed only decreased absolute counts in pss abs group ( resp . increased proportion of cd3cd19 ( b cells ) in pss abs ( p = 0.045 ) was found when compared to controls , but no differences were found analyzing absolute counts among pss groups and controls ( table 2 ) . analysis of cd4 lymphocyte subpopulations according to their expression of cd25 and foxp3 markers showed significant reduced absolute counts of cd4cd25 ( p = 0.036 ) and cd4 cd25foxp3 ( p = 0.017 ) cells when comparing pss abs with controls . no significant differences were observed analyzing other cd4 subpopulations according to their expression of cd25 and foxp3 markers ( table 3 ) . analysis of cd4il-17a ( th17 ) cells showed significant lower proportion and absolute counts of these cells in pss patients in comparison to control group 's results ( figure 2 ) . proportion was significantly altered in pss abs ( p = 0.0003 ) and pss abs ( p = 0.004 ) , and also absolute counts of th17 cell were significantly lower in pss abs ( p < 0.0001 ) and pss abs ( p no significant differences were found analyzing proportion and absolute counts of this subpopulation between pss groups ( table 3 , figure 1 ) . in pss patients with abs , we observed significantly increased proportion ( p = 0.032 ) and absolute counts ( p = 0.011 ) of cd8cd57cd27 subpopulation in comparison to pss without abs . cd8cd57cd27 population 's proportion was significantly reduced only in pss without abs patients ' blood ( p = 0.026 ) when compared to controls and no significant differences between pss groups were observed . absolute counts of this subpopulation were significantly reduced both pss abs ( p = 0.0003 ) and pss abs ( p = 0.005 ) in comparison to controls . proportion of cd8 subpopulation lacking cd57 and cd27 markers ( cd8cd57cd27 ) was significantly increased only in pss abs patients ' blood when compared to controls . focus score correlated with absolute counts of cd3 ( p = 0.047 , r = 0.474 ) , cd4 ( p = 0.033 , r = 0.503 ) , b cell ( p = 0.023 , r = 0.532 ) , and cd8cd57cd27 ( p = 0.016 , r = 0.560 ) cell populations . essdai correlated with nk ( p = 0.043 , r = 0.482 ) and cd4cd25foxp3 ( p = 0.047 , r = 0.474 ) cell absolute counts . in pss patients without abs , we found that schirmer 's i test correlated with proportion ( p = 0.033 , r = 0.504 ) and absolute counts serum baff concentration correlated with proportion of cd8 ( p = 0.032 , r = 0.415 ) , nk ( p = 0.009 , r = 0.491 ) , and cd8cd57cd27 ( p = 0.014 , r = 0.469 ) cell populations and negatively correlated with b cells ( p = 0.026 , r = 0.429 ) , cd8cd57cd27 ( p = 0.036 , r = 0.404 ) , and cd8cd57cd27 ( p = 0.009 , r = 0.493 ) . analyzing absolute counts of cells populations observed negative correlation between baff and cd4 ( p = 0.025 , r = 0.430 ) , b cell ( p = 0.007 , r = 0.509 ) , cd8cd57cd27 ( p = 0.028 , r = 0.424 ) , and cd8cd57cd27 ( p = 0.017 , r = 0.454 ) cells . schirmer 's i test correlated with proportion of b cells ( p = 0.038 , r = 0.467 ) and cd8cd57cd27 ( p = 0.006 , r = 0.589 ) and negatively correlated with cd3 ( p = 0.006 , r = 0.590 ) and cd8cd57cd27 ( p = 0.033 , r = 0.479 ) cells . also correlation between schirmer 's i test and absolute count of cd8cd57cd27 ( p = 0.021 , r = 0.512 ) and negative correlation with cd8cd57cd27 ( p = 0.017 , r = 0.525 ) cell counts were observed . unstimulated salivary flow rate correlated with proportion of cd8 ( p = 0.017 , r = 0.525 ) and th17 ( p = 0.015 , r = 0.535 ) and also negatively correlated with absolute counts of cd3 ( p = 0.011 , r = 0.555 ) and cd4 ( p = 0.017 , r = 0.526 ) cells . focus score negatively correlated with proportion and absolute counts of cd8cd57cd27 cells , respectively , ( p = 0.020 , r = 0.517 ) and ( p = 0.040 , r = 0.462 ) . esspri correlated with proportion of cd3 ( p = 0.015 , r = 0.535 ) , cd4foxp3 ( p = 0.044 , r = 0.454 ) , and cd4cd25foxp3 ( p = 0.041 , r = 0.461 ) cells and negatively correlated with proportion of cd4cd25 ( p = 0.026 , r = 0.496 ) and cd4cd25 ( p = 0.043 , r = 0.456 ) cells . essdai negatively correlated with proportion of th17 ( p = 0.001 , r = 0.675 ) cells and also with absolute counts of cd8 ( p = 0.042 , r = 0.459 ) , th17 ( p = 0.001 , r = 0.673 ) , and cd8cd57cd27 ( p = 0.028 , r = 0.492 ) cells . despite systemic b - cell hyperactivity , t and b cells constitute the vast majority of infiltrating mononuclear cells at the minor salivary glands inflammatory lesions of pss , with their prevalence varying according to the severity of the infiltrates . the prevalence of cd4 t cells decreases with lesion severity , whereas the prevalence of cd8 t cells remains unchanged . the prevalence of regulatory t cells associates with lesion severity , with the higher values to be observed at intermediate lesions . nk cells comprise a small but considerable portion of the infiltrating mononuclear cells , and their percentage correlates with the grade of the lesions [ 15 , 16 ] . in our study , t lymphocyte identification by cd4 and cd8 markers showed a statistically significant decrease in the absolute counts of cd4 and cd8 t lymphocytes in the peripheral blood of pss patients in comparison to the control group . this shows that the decline of cd3 t lymphocyte population in the peripheral blood of pss patients is influenced by a decrease of both cd4 and cd8 t lymphocyte absolute counts . the decrease of the total amount of cd4 t lymphocytes in the peripheral blood of pss patients is also confirmed by other authors . the fact is that the proportion of cd4 was lower only in our pss patients with abs , and no differences in proportion of cd3 and cd8 in all pss patients were observed ; let us think that this lymphopenia can also be genetically determined . apoptosis may also play a role in the pathogenesis of some extraglandular manifestations of pss and peripheral cd4 lymphocytopenia [ 19 , 20 ] . studies in patients with pss and animal models of pss have identified the presence of il-17 in the lymphocytic infiltrates of the exocrine glands , as well as higher levels of circulating il-17 in both serum and saliva [ 8 , 21 , 22 ] . on the one hand , our finding that th17 lymphocyte counts decreased in the peripheral blood of patients with sjgren 's syndrome is quite unexpected . on the other hand , this result may be explained by the redistribution of th17 lymphocytes , that is , increasing their concentration in tissues ( salivary glands ) and decreasing concentration in peripheral blood [ 8 , 10 ] . however , in this study , we have not find more distinct treg cells changes in the peripheral blood of pss patients . the role of cytotoxic t cells in pss pathogenesis has not been studied in detail . cd8 t - cell deficiency is a feature of many chronic autoimmune diseases , including multiple sclerosis , rheumatoid arthritis , systemic lupus erythematosus , sjgren 's syndrome , systemic sclerosis , ulcerative colitis , crohn 's disease , psoriasis , etc . it also occurs in blood of healthy relatives of patients with autoimmune diseases , suggesting that it is genetically determined . cd8 t cell deficiency can prompt the development of chronic autoimmune diseases by impairing cd8 t cell control of virus infection . it is proposed that , after activation in peripheral lymphoid organs by cross - reacting foreign antigens , autoreactive t cells enter the target organ where they are reactivated by b cells which provide costimulatory survival signals , thereby inhibiting the activation - induced t - cell apoptosis which normally occurs when autoreactive t cells enter the target organ . understanding cd8 t memory effector cells differentiation is essential for studying how virus - specific cd8 t cells control viral infection . distinct stages of virus - specific cd8 t memory effector cells differentiation have been extensively characterized by phenotypic and functional analyses . primed virus - specific cd8 t cells typically differentiate from the least mature memory stage ( cd27 ) to the most mature effector stage when they start to lose cd27 and obtain cd57 marker and eventually become terminally differentiated effector cells which can be further defined by cd57 expression . according to some studies , last stage of cd8 cells differentiation seems to be cd8cd27cd57 t cells subset with high perforin and killing activity . we observed increased proportion of this population in pss patients , but significant differences were observed only in pss without abs group . impaired proportion and absolute counts of cd8cd27cd57 t cells in blood of pss patients can be the reason of lower counts of cd8 in blood of pss patients . it is not known whether the proportion and absolute counts of this population are downregulated in blood by migration to inflammatory sites , or this can be due to increased apoptosis of these cells . all these observed changes in cd8 t cell subpopulations rearrangement prove that these subpopulations actively participate in pathological processes of pss . according to literature , in patients with autoimmune rheumatic diseases , the decreased nkt and nk cell counts and functional characteristics published results where they identified higher proportion of these cells in blood of pss patients than in control group . our investigation of these populations showed a significant decrease of nkt and nk - cell absolute counts in the peripheral blood of pss patients with abs ; however , the fact that a proportion of these cells were similar with the controls can indicate that lower absolute counts can be due to genetically determined lymphopenia . there is also possibility that low counts of these cell populations in periphery can be by reason of overall lymphocyte population migration to inflammatory sites or / and apoptosis . in conjunction with the classical cd4 tregs , we were also investigating cd8 suppressor cells that express foxp3 marker , as foxp3 confers suppressive properties and is confined to regulatory t cells . our study results did not show any significant differences in proportion or absolute count changes on these cells in pss patients ' peripheral blood in comparison to healthy controls . whereas we do not found correlation between baff and lymphocyte populations changes in pss patients with abs group , what can be the indication of uncontrolled baff secretion and its homeostasis disturbance ? despite recent knowledge , in many respects , the role of t cells and their subsets in pss remains unexplained . one of future projects should be the immunohistochemistry for assessing cell populations ' changes in salivary glands in parallel with blood analysis and apoptosis markers .

it is now clear that individual neurons are highly compartmentalized with specific functions and/or signaling that occur in restricted subcellular domains . extrinsic signals are often spatially localized such that they are seen by restricted parts of a neuron , such as synaptic input to a specific dendritic spine or a guidance cue encountered by a growth cone . twenty - five years ago , when the first issue of neuron was published , it was well appreciated that the neurons were capable of local information processing , but the potential cellular mechanisms that established and regulated local compartments were not well understood . dendritic spines had been proposed as biochemical and/or electrical compartments ( harris and kater , 1994 ; koch and zador , 1993 ) , and polyribosomes had been identified at the base of spines ( steward and levy , 1982 ) . however , the view that dominated until nearly the end of the twentieth century was that the central dogma ( dna - rna - protein ) was carried out centrally in the nuclei and somata of neurons . in that context , the localization of mrna observed in some cells was thought to represent a specialized mechanism that operated in unique biological systems , such as egg cells , where storage of mrnas is needed for subsequent patterning of the early embryo ( see martin and ephrussi , 2009 for review ) . evidence from a number of studies in the last decade , particularly in neurons , has led to a revolution in our thinking . although the field is still young , it is becoming clear that rna - based mechanisms provide a highly adaptable link between extrinsic signals in the environment and the functional responses of a neuron or parts of a neuron . this is accomplished by the localization of both protein - coding and noncoding rna in neuronal processes and the subsequent regulated local translation of mrna into protein . here we discuss some of the key findings that lead us to the view that mrna localization and rna - regulated and localized translation underlie many fundamental cellular processes that are regulated by extrinsic signals in neurons , such as memory , dendrite and arbor branching , synapse formation , axon steering , survival , and likely proteostasis . the dynamic regulation of protein synthesis is essential for all cells , including neurons . over 50 years ago , in vivo experiments ( in a variety of species ) established a clear functional link between protein synthesis and long - term memory ( see davis and squire , 1984 for review ) , indicating that proteome remodeling underlies behavioral plasticity . these observations were paralleled by in vitro studies of synaptic plasticity demonstrating a clear requirement for newly synthesized proteins in the long - term modification of synaptic function ( see sutton and schuman , 2006 for review ; also , tanaka et al . , 2008 ) . this link between protein synthesis and long - term plasticity is most recently reinforced by studies showing that targeted genetic disruption of signaling molecules that regulate protein translation interfere with long - term synaptic or behavioral memories ( costa - mattioli et al . , 2009 ) . the above studies , while indicating a requirement for protein synthesis , do not address the location . we now know dendrites and axons of neurons represent specialized cellular outposts that can function with a high degree of autonomy at long distances from the soma , as illustrated by the remarkable ability of growing axons to navigate correctly after soma removal ( harris et al . , 1987 ) or isolated synapses to undergo plasticity ( kang and schuman , 1996 ; vickers et al . , 2005 ) . the identification of polyribosomes at the base or in spines ( steward and levy , 1982 ) together with metabolic labeling experiments that provided the first evidence of de novo synthesis of specific proteins in axons and dendrites ( feig and lipton , 1993 ; giuditta et al . , 1968 ; koenig , 1967 ; torre and steward , 1992 ) indicated the competence of these compartments for translation . subsequent studies demonstrated that specific subsets of mrnas localize to synaptic sites ( steward et al . , 1998 ) and directly linked synaptic plasticity with local translation in dendrites ( aakalu et al . , 2001 ; huber et al . , 2000 ; kang and schuman , 1996 ; martin et al . , 1997 ; vickers et al . , 2005 ) , providing definitive proof that dendrites are a source of protein during plasticity . in axons , the idea of local protein synthesis has been slower to find acceptance , no doubt hindered by the classical view of axons as information transmitters rather than receivers ; so , why would local protein synthesis be required ? although ribosomes were identified in growth cones in early ultrastructural studies ( bunge , 1973 ; tennyson , 1970 ) , they were rarely observed in adult axons . it is now thought that at least part of the explanation for their apparent paucity lies in their localization close to the plasma membrane in axons ( sotelo - silveira et al . , 2008 ) where ribosomal subunits can associate directly with surface receptors ( tcherkezian et al . , 2010 ) in addition , evidence indicates that myelinated axons can tap into an external supply of ribosomes by the translocation of ribosomal proteins from schwann cells ( court et al . , 2011 ) . growing and navigating axons are clearly information receivers , like dendrites , since their growth cones steer using extrinsic signals . indeed , the first functional evidence for local protein synthesis in axons came from studies that showed that cue - induced directional steering is abolished by inhibitors of protein synthesis , including rapamycin , in surgically isolated axons ( campbell and holt , 2001 ) . , 2005 ; yao et al . , 2006 ) and revealed that local protein synthesis underlies growth - cone adaptation , gradient sensing , and directional turning in growing axons ( leung et al . , 2006 ; ming et al . , 2002 ; piper et al . , 2005 ; in addition , axonal protein synthesis is elicited in response to injury and plays key roles in axon regeneration and maintenance ( jung et al . , 2012 ; perry et al . , 2012 ; verma et al . , 2005 ; increasing evidence indicates that the complex morphology of neurons has created biological compartments that subdivide the neuron into spatially distinct signaling domains important for neuronal function ( hanus and schuman , 2013 ) . dendritic spines represent a specialized ( classical ) cellular compartment in which subsets of specific proteins ( e.g. receptors , channels , signaling molecules , and scaffolds ) are collected together with a common function for receiving and processing electrical and chemical input . spines have a distinct structural morphology and , as such , are easy to classify as a compartment . although spines are small ( 1 m ) , they can still be subdivided into further functional compartments ( see chen and sabatini , 2012 for review ) with multiple microdomains , raising the question of how a compartment is defined . for example , a recent superresolution imaging study demonstrated that , within synapses , ampa receptors are clustered into small nanodomains ( 70 nm in diameter ) that contain on average 20 receptors ( nair et al . , 2013 ) . these nanodomains are dynamic in both their shape and position and may have a limited lifetime . anatomically and functionally distinct compartments also exist in axons , such as the growth cone , the axon initial segment , and terminal arbor . equally , there are examples of compartments that exhibit no obvious anatomical specializations . in axons , for example , some membrane proteins are localized to restricted segments of the axon ( fasciclins , tag1/l1 , robo ) ( bastiani et al . , 1987 ; in addition , second - messenger signaling molecules such as calcium and cyclic nucleotides , once thought to signal extensively throughout a cell , are now known to be highly regulated such that increases in concentration can be confined to a small space , creating a signaling compartment . selective activation of a single spine on a dendrite , for example , can provide the receiving neuron with information about a specific stimulus ( varga et al . , 2011 ) . compartments may be overlapping or distinct and range in size depending on the biological function . as such , future experiments aimed at understanding how different compartments emerge and what mechanisms generate such spatially precise intracellular patterning will be very informative . compartmentalized signaling presents several challenges to the cell , a prime one being the localization of its component parts . one of the remarkable features of rna is its ability to be spatially localized and , therefore , potentially contribute to neuronal compartmentalization . historically , localized mrnas have been studied during development ( see martin and ephrussi , 2009 ) . that localized rna is more often the rule than the exception is spectacularly illustrated by the finding that 71% of the drosophila embryo transcriptome is localized to specific subcellular compartments ( lcuyer et al . , 2007 ) . the proteins encoded by localized mrnas are also concentrated at the site suggesting that mrna localization and the ensuing local translation plays an important role in positioning proteins for cellular functions . mrnas tend to be abundantly localized to the peripheral domains and motile parts of neurons where they are optimally positioned for the arrival of external signals , e.g. , in dendrites ( synaptic activation ) and growth cones . subcellular asymmetry can lead to highly polarized dynamics and cell morphology that can operate on a remarkably fine scale . to navigate in retinal growth cones , for example , which are only 5 m in diameter , a polarized external gradient of netrin-1 triggers increases in both the transport and translation of -actin mrna on the gradient near side ( leung et al . , 2006 ; yao et al . , 2006 ) . this polarized translation leads to a rapid ( 5 min ) polarized increase in -actin protein that helps to drive axon turning towards the gradient source . interestingly , different cues show specificity in their effects on mrna transport and translation . different growth factors , for example , trigger the transport of a specific repertoire of mrnas in axons ( willis et al . , 2005 , 2007 ; zhang et al . , 1999 ) , and different guidance cues elicit the translation of specific subsets of mrnas ( leung et al . , 2006 ; piper et al . , 2006 ; shigeoka et al . , 2013 ; -actin mrna translation is triggered by netrin-1 but not sema3a , whereas rhoa and cofilin mrna translation is induced by sema3a but not netrin-1 . this has given rise to the differential translation model suggesting that translation - dependent repulsive and attractive turning in growth cones depends on the differential translation of mrnas involved in assembly or disassembly of the actin cytoskeleton ( lin and holt , 2007 ) . several aspects of this translation - driven cue - induced turning remain to be understood , such as how receptor activation signals mrna recruitment and , critically , how specific subsets of mrna are translated . navigating growth cones encounter a series of patterned molecular cues along the pathway from which they must read out their spatial position . although there are several examples of stimulus - induced local translation in axons in vitro ( shigeoka et al . , 2013 ) , it has only recently become possible to investigate translation in neuronal compartments in vivo . early studies by flanagan and colleagues showing compartmentalized expression of epha2 , recapitulated by a translation reporter , in the post - midline crossing segment of commissural spinal cord axons introduced the idea that the growing tip of the axon is stimulated by a regionally expressed cue ( e.g. , at the midline ) that triggers the region - specific translation of proteins needed for pathfinding ( brittis et al . , 2002 ) . a recent study provides direct evidence for this type of mechanism in the control of robo expression and midline guidance ( colak et al . , 2013 ) . two robo3 receptor isoforms have opposing roles in guiding axons to and away from the midline , and their expression is compartmentalized in pre - crossing ( robo3.1 ) and postcrossing ( robo3.2 ) axonal segments ( chen et al . , 2008 ) . the switch to robo3.2 expression at the midline ( the transcript of which contains a premature termination codon ) is controlled by midline - induced axonal protein synthesis coupled with nonsense - mediated mrna decay . this provides an elegant mechanism for turning on synthesis time linked to the crossing event ( colak et al . , 2013 ) . it was not previously technically possible to inhibit translation of a specific transcript in a compartment - specific manner . recently , however , new tools have been developed that allow separate manipulation of specific neuronal compartments in vivo such as targeted delivery of sirnas or antisense morpholinos and conditional targeting of 3utrs ( perry et al . these subcellular - directed approaches are beginning to yield information suggesting that local translation is involved in regulating multiple aspects of axonal and dendritic biology . guidance cues induce immediate steering responses in growth cones via classical signaling pathways that involve receptor activation and phosphorylation of downstream signaling molecules ( bashaw and klein , 2010 ) . some of these immediate steering responses also involve local translation , as discussed above . thus , local translation can provide new proteins on demand at subcellular sites for immediate use . interestingly , local translation in response to extrinsic cues has recently been shown to provide proteins for delayed use in axon growth and regeneration . examples of this are the de novo synthesis of proteins that shuttle back to the nucleus where they influence transcriptional output ( cox et al . , 2008 ; 2012 ) , and another is the de novo synthesis of surface receptor proteins that are employed later in a growth cone s journey ( leung et al . , 2013 ) . recent advances in experimental procedures , allowing the stimulation of individual synapses , have shown that synapses can be independently regulated by synaptic activity ( matsuzaki et al . , 2004 ) . on the other hand , other studies emphasize the consideration of the dendritic branch as a computational unit ( govindarajan et al . , 2011 ) . taken together , it seems reasonable to consider a range of spatial domains over which signaling can occur , which would span the scale from subdomains in spines to dendritic branches to the entire neuron . these data can be compared to what we know about the quantitative localization of the protein - synthesis machinery . indeed , it is clear that many synapses possess a polyribosome nearby ( ostroff et al . , 2002 ) . moreover , recent high - resolution in situ hybridization data suggest that mrna molecules are distributed in local domains ( cajigas et al . , 2012 ) , but not necessarily specific to individual synapses . preliminary estimates of mrna numbers indicate that there may not be sufficient copies of individual mrna species for each synapse to have an exclusive and dedicated molecular toolbox . these data imply that there is local sharing of cell biological machineries , including the machinery for protein synthesis and degradation . it remains unclear , however , over what spatial scale local translation can be regulated and stimulated in dendrites . for example , is stimulation of a single spine sufficient to regulate local translation , and , if so , over what spatial domain do the newly synthesized proteins function ? the past view that rna acts primarily as an inert intermediate between genes and proteins has undergone a revolution in recent years with discoveries of both new classes of rnas ( e.g. , noncoding rnas , ( see ulitsky and bartel , 2013 for review ) and new rna - based mechanisms of gene regulation ( e.g. , microrna and rnai silencing ) ( see mcneill and van vactor , 2012 for review ) . indeed , given the relatively constrained diversity of proteomes across cells and organisms , rna - based mechanisms ( diverse rna species and rna functions ) represent a unique platform to diversify and specialize cells , especially neurons . numerous new roles for rna have been found in recent years , expanding the role of rna to controlling many and diverse cellular processes , including stimulus - induced local translation that underlie adaptive responses in neurons ( e.g. , memory , axon guidance , and maintenance ) . in addition , rna s role may not be limited to the cells where it is synthesized , as new studies indicate it can be transferred between cells ( via exosomes ) ( sharma et al . , 2013 ) and even between organisms ( sarkies and miska , 2013 ) , bringing a whole new era of rna function in cellular communication into focus . the demonstrations that local protein translation functions during synaptic development and plasticity led to the hunt for specific mrnas that could be translated in these local compartments . for many years , in situ hybridization was the method of choice , and several individual mrnas were visualized in dendrites , including the mrna for the ca - calmodulin - dependent protein kinase alpha subunit , camkii ( burgin et al . 1996 ) , map2 ( garner et al . , 1988 ) , shank ( bckers et al . , 2004 ) , and -actin ( tiruchinapalli et al . , 2003 ) . in growth cones and axons , in situ hybridization provided evidence for several different mrnas , including -actin ( bassell et al . , 1998 recent microarray approaches and deep rna sequencing have dramatically expanded the local transcriptome in both dendrites and axons ( poon et al . , 2006 ; zhong et al . , 2006 ) . one of the most surprising findings to come out of these studies is the vast number of mrnas that are present in these neuronal compartments . , 2010 ) , while dendrites have > 2500 mrnas ( cajigas et al . , 2012 ) . the mrnas resident in these compartments span many different functional classes of molecules : metabolism , translation , degradation , receptors / channels , cytoskeleton , etc . many functional categories are shared between the two compartments , although there are numerous distinct compartment - specific subsets of mrnas , e.g. , gap43 mrna in axons and neurotransmitter receptor subunits in dendrites . the localization of mrna to cellular compartments involves recognition of information that is contained in the 3 and/or 5 untranslated ( utr ) sequences . the use of mrna localization to achieve protein localization may arise from the fact that , at least theoretically , unlimited address information can be built into the 3 and/or 5 utrs of mrna without altering its gene - coding function , whereas there is a tight limit to how much additional coding sequence can be added to a protein without ramifications for function . the family of proteins that bind , transport , localize , and regulate the translation of mrnas are known as rna - binding proteins ( rbps ) ( see darnell , 2013 for review ) . rna - binding proteins complexed with mrna , other rna species , and accessory proteins are thought to be assembled in the cell body and form rna granules ( kiebler and bassell , 2006 ) . during transport on microtubules and microfilaments to its destination ( e.g. , hirokawa , 2006 and czaplinski and singer , 2006 ) , the mrna cargo is thought to be silenced by translational repressors ( krichevsky and kosik , 2001 ) . once transported , it is unclear how or whether mrnas are anchored near translational sites or if they show continued dynamics . both stationary and anchored particles have been observed in dynamic mrna imaging experiments ( lionnet et al . , 2011 ) . rna - binding proteins are an important class of regulatory molecule that recognizes specific nucleotide sequences in rna ( ray et al . , 2013 ) . ip - seq analysis has revealed , unexpectedly , that some rbps can bind hundreds of different mrnas ( see darnell , 2013 for review ) . some rbps , however , appear to be cell - type specific , such as hermes ( rpbms2 ) that is expressed exclusively in retinal ganglion cells in the cns and its knockdown causes severe defects in axon terminal branching ( hrnberg et al . , 2013 ) . the number of mrna - binding proteins identified by known rna - binding domains is relatively small ( around 270 ) given the increasingly large number of transcripts found in axons and dendrites . recent work using interactome capture in embryonic stem cells has significantly expanded the number of rbps , adding a further 280 proteins to the repertoire , including , remarkably , many enzymes such as e3 ubiquitin ligases with previously unknown rna - binding function ( kwon et al . , 2013 ) . several rbps have been implicated in neurological disorders , such as fmrp in fragile x syndrome and survival of motor neuron protein ( smn ) in spinal muscular atrophy ( bear et al . , 2008 ; liu - yesucevitz et al . , 2011 ) , and translation dysregulation has recently been implicated as a major factor in autism ( gkogkas et al . , 2013 ; santini et al . , 2013 ) . in recent years the discovery of noncoding rnas , including mirnas ( which use sequence complementarity to recognize target mrna ) , has revealed unanticipated and enormous potential for the regulation of mrna stability and translation , as well as other functions . given the huge and unanticipated number of mrnas detected in axons and dendrites , it is perhaps not surprising that these noncoding rnas also exist and are even enriched in neuronal compartments . one might even argue the complex morphology and functional specialization of neurons provides a hotbed for mrna regulation that can potentially be mediated by noncoding rnas . indeed , an analysis of 100 different mirnas discovered the differential distribution of some mirnas in dendrites versus somata and copy numbers in individual neurons as high as 10,000equivalent to the number of synapses a typical pyramidal neuron possesses ( kye et al . , 2007 ) . recently , the differential distribution of mirnas has been also reported in axons versus soma ( natera - naranjo et al . , 2010 ; 2013 ) and recently emerged as regulators of axon growth and branching ( kaplan et al . , 2013 ) . moreover , the enrichment of mirnas in synaptosomes isolated from specific brain regions has also been reported ( pichardo - casas et al . , 2012 ) . mirnas have now been shown to regulate many synaptic functions ( see schratt , 2009 for review ) . in addition , mirnas themselves are regulated by behavioral experience ( krol et al . , 2010 ) as well as more recently , the appreciation of other types of noncoding rnas have come into focus , though very little is known about their function in neurons . this includes small - nucleolar rna - derived and transfer rna - derived small rnas , firstly identified as degradation products , and long noncoding rna known as regulators of gene transcription , that may regulate gene expression posttranscriptionally . a recent study demonstrated , for example , that a long noncoding rna that is anti - sense to a k channel subunit ( kcna2 ) is upregulated following peripheral nerve injury , leading to a downregulation of the k channel and a resulting increase in the excitability of drg neurons , increasing neuropathic pain ( zhao et al . , 2013 ) . in the early years , the study of local translation was hampered by the technical difficulty of obtaining pure and sufficient quantities of dendrites and axons for analysis . pioneering studies used metabolic labeling to demonstrate the synthesis of specific proteins such as tubulin in axons ( giuditta et al . , 1968 ; koenig , 2009 ) , but the possibility that the signal arose from cell - body contamination could not be eliminated due to these technical limitations . localized translation was convincingly demonstrated by surgically severing the soma from its processes ( aakalu et al . , 2001 ; campbell and holt , 2001 ; kang and schuman , 1996 ) and , more recently , by the use of chambers in which the processes ( dendrites or axons ) are fluidically isolated from cell bodies ( eng et al . , 1999 ; taylor et al . , other methods for isolating neuronal processes include substrates with limited pore size that allow axons to penetrate but not cell bodies ( torre and steward , 1992 ; zheng et al . , 2001 ) and laser capture microdissection ( zivraj et al . , 2010 ) . these methods combined with the rapid increase in the sensitivity of profiling techniques have enabled genome - wide transcriptome analyses to be performed on axons and dendrites in a variety of neurons ( see below ) . the visualization and identification of newly synthesized proteins has also been a hurdle due to issues of sensitivity ( detecting low levels of newly synthesized proteins ) as well as difficulties in distinguishing between the movement of existing proteins and the synthesis of new proteins . puromycin , a trna analog , can be used together with fluorescent tags ( smith et al . , 2005 ) or antibodies ( schmidt et al . , 2009 ) to label sites of protein synthesis . fluorescent reporters , such as photo - switchable kaede , fused to the 3utr regulatory region of mrnas of interest have enabled de novo protein synthesis to be monitored live in neuronal processes ( aakalu et al . , 2001 ; brittis et al . , 2002 ; leung et al . , in addition , new methods have been developed to selectively label the pool of newly synthesized proteins , to ascertain a given cell type or cellular compartment as the site of synthesis , and to visualize the newly synthesized proteins . these methods make use of noncanonical amino acids that cross cell membranes and get charged onto trnas by the cell s own trna synthetases and then incorporated into new protein during protein synthesis . these techniques , bio - orthogonal noncanonical amino acid tagging ( boncat ) and fluorescent noncanonical amino acid tagging ( funcat ) can be used to selectively identify ( dieterich et al . , 2006 ) or visualize ( dieterich et al . , 2010 ) newly synthesized proteins . a modification of the ncat method , which in principle enables one to label newly synthesized proteins in specific cell types , has also recently been developed ( ngo et al . , 2012 ) , and ncat can be used in combination with 2d difference gel electrophoresis ( dige - ncat ) to compare the proteomes of specific subcellular ( e.g. axonal ) compartments ( yoon et al . , 2012 ) . there are many questions for the future , as noted below . we know that some compartments ( like spines ) have plasma membrane as a boundary that can serve to compartmentalize chemical and electrical signals . other compartments could be determined by the spatial arrangement of molecules , cytoskeleton , or limited diffusion . are compartments static when bounded by anatomy ( e.g. , a spine ) but dynamic when determined by signaling molecule volumes ? what defines a subcellular compartment such that mrnas contain specific addresses to target them there ? some mrnas are targeted specifically to axons and dendrites and even to the growth cone how is this targeting achieved ? while we have in hand several zip codes , there are certainly many messages for which a clear consensus sequence in the utr has not emerged . in addition , in some cases the signal for recognition by an rna - binding protein may reside in the secondary structure of the mrna , rather than the nucleotide sequence . the fact that current secondary structure prediction techniques are limited to small stretches of nucleotides ( 100 ) complicates our ability to identify binding motifs in 3utrs . adding to the complexity is the recent observation that low - complexity regions of rna - binding proteins are sufficient to create reversible rna granule - like structures ( kato et al . , 2012 ) . the expanded identification of rbps as well as the ability to define the binding sites with methods like hits - clip ( licatalosi et al . , 2008 ) should dramatically enhance our knowledge of the binding sites . future studies should focus on the dynamics of the rna - protein interactions in cellular contexts . in addition , the possibility that rna might be delivered from extracellular sources ( e.g. , via exosomes from neighboring neurons or glia ) is a recently suggested exciting idea . unbiased genome - wide analyses have shown that the mrna repertoire is dynamically regulated with the mrna repertoire changing over time ( gumy et al . , 2011 ; zivraj et al . , in addition , it is clear that synaptic activity can lead to the regulated trafficking of mrna to the distal processes ( e.g. , steward et al . , 1998 ) . is this regulated at the level of transcription , or is there some gating mechanism that regulates the trafficking of specific transcripts into dendrites / axons ? evidence with ephrinb in rgcs indicates that although the transcripts are present in somas early in development , they do not move into axons until later , suggesting that some kind of specific gating mechanism may exist . currently , little is known about the quantitative aspects of mrna localization and translation in neurons . for example , how many rna molecules are needed to provide a functionally significant amount of protein ? one might speculate that some classes of proteins , such as cytoskeletal , would be translated much more than others such as receptors or channels and transcript abundance could reflect this difference . in theory , just a few new channel or receptor proteins could be sufficient to alter signaling characteristics within a neuronal microdomain . in addition , a low abundant transcript could be stable and translated with high efficiency . thus , low - abundance transcripts could exert a significant physiological effect and should not be overlooked in profiling analyses . this also raises the intriguing question of whether translation from monosomes , rather than polysomes , may be more common in distal neuronal compartments where there could be demand for a few highly localized proteins . new high - resolution single molecule detection methods ( cajigas et al . , 2012 ; park et al . , 2012 ) and live - imaging methods for translation ( chao et al . , 2012 ) will be valuable when answering these sorts of questions . with the advent of trap ( translating affinity purification ) technology ( heiman et al . , 2008 ) it will be possible in the future to answer this question in specific neuronal compartments of specific subsets of neurons . for example , cell - type specific cre - driver lines can be crossed with the ribotag mouse ( sanz et al . , 2009 ) , which expresses ha - tagged endogenous ribosomal protein ( rrl22 ) , thereby generating mice with specific neurons expressing ha - tagged ribosomes . these can be isolated from mouse brains by immunoprecipitation at different ages and under different conditions ( and diseased ) , and rna - seq analysis can identify the ribosome - protected , and therefore , actively translating transcripts . this will be of huge importance in characterizing and understanding the translatome of neuronal compartments . thus , current technology now offers the exciting possibility of being able to discover differences in the dendritic or axonal translatome of diseased ( e.g. , autosomal models ) individuals . how does the spatial morphology of the dendrite , axon , or spine contribute to or constrain protein synthesis ? it was recently shown that spines enhance the cooperative interaction among multiple inputs ( harnett et al . , 2012 ) . these observations suggest that the amplifying and coordinating properties of dendritic spines have an effect on neuronal input processing and may influence information storage by promoting the induction of clustered forms of synaptic and dendritic plasticity among coactive spines . this could allow spines to enhance the ability of neurons to detect , uniquely respond to , and store distinct synaptic input patterns ( harnett et al . , 2012 ) . different patterns of synapse activation can lead to protein synthesis - dependent or -independent plasticity ( govindarajan et al . , however , the importance and mechanism of specific protein translation remains to be examined in this cooperativity . since there are mrnas that are differentially distributed in the length of the dendrites , it is tempting to speculate that there is a role for protein synthesis in regulating the functional compartment in dendrites and spines . thus , while it is clear that protein synthesis occurs in the dendrite and that it is regulated by neuronal activity , the extent to which the activity of single synapses or synaptic regions stimulates protein synthesis , or alters protein localization , remains unknown . moreover , the importance and impact of synapse location along the dendrite or axon for protein synthesis is unknown . in the small cytoplasmic volume of a dendritic spine or growth cone , there is a limit to the amount of protein that can fit into the space before molecular crowding becomes a problem . while it is clear that changes in synaptic transmission involve extensive regulation of the synaptic proteome via the regulated synthesis and degradation of proteins ( fonseca et al . , 2006 ; wang et al . , 2009 ) , it is not well understood how these two processes are coordinately regulated to achieve the desired level of individual proteins at synapses . indeed , this is another level of homeostatic control that must exist in order for synapses to maintain the desired level of receptors , scaffolds , and signaling molecules . changes in the steady - state level of a protein have to be particularly fast and fine - tuned in neurons , due to the fast nature of synaptic transmission and the rapid induction of plasticity . how are specific mrnas translated and studies using either global activity manipulations ( ttx / apv ) ( sutton et al . , 2004 ) or application of an d1/d5 agonist ( hodas et al . , 2012 ) have suggested large - scale ( at least 100 distinct proteins synthesized ) changes in the dendritic proteome . similarly , global cue stimulation of axons elicits the de novo translation of hundreds of new proteins ( yoon et al . , 2012 ) . in these studies , however , the stimulation was applied to the entire network ( dish of cultured neurons or brain slice ) . under physiological conditions the spatial and temporal profile of synaptic and cue stimulation is on a much finer scale and the translational readout is likely limited . indeed , we know that different cues can trigger translation of specific subsets of mrnas in the growth cone ( lin and holt , 2007 ) . the mechanisms by which specific patterns of synaptic signals ( e.g. , different frequencies of stimulation , different concentrations or gradients of agonists ) and receptor activation lead to activation of the translation machinery are not well understood . mechanistically , it is clear that elements contained in the 5 and 3utr of mrnas can regulate their translation initiation . in addition , it is probably the case that the spatial proximity of an mrna to an active translation site plays a role . the use of high - resolution imaging techniques and focal stimulation should provide answers to these questions . in neurons , the mirna function has been explored both individually and on a population level , but a broad conceptual understanding is still lacking . moreover , if mirnas regulate mrna translation and expression in different neuronal compartments , what regulates the expression of mirna themselves ? the accessibility of deep sequencing has enabled the detection of other noncoding rna species in neurons . these additional rna classes can directly regulate translation , regulate mirna function , or serve as scaffolds for other molecules , making the levels of regulation and interactions potentially extremely complicated . in addition , the recent appreciation of the abundance and regulatory potential of other noncoding rnas , mostly in nonneuronal cell types , adds another level of complexity , including the recent demonstration of regulation by circular rnas that may serve as either shuttles , assembly factories , or sponges for mirnas and/or rbps ( hentze and preiss , 2013 ) . based on this , it is likely that a real understanding of the complexity of rna function in neurons will require not only investigation of individual molecules but also a systems biology perspective where the entire network of rna molecules and their targets can be considered together ( see pelez and carthew , 2012 ) . while ribosomes are readily visible in dendrites spines ( ostroff et al . , 2002 ) and growth cones ( bassell et al . , 1998 ; bunge , 1973 ) how they are transported and whether they are sequestered or anchored is not well understood . a mechanism that could provide specificity or docking would be the specialization of ribosomes by accessory proteins or subunits . one of the most intriguing questions raised by recent work is whether ribosomes are tuned to translating specific mrnas . this possibility is suggested by recent studies showing that haplo - insufficiency of several different ribosomal proteins give rise to specific phenotypes rather than affecting all cells ubiquitously ( kondrashov et al . , 2011 ; uechi et al . ribocode that suggests heterogeneity in the composition of ribosomes , enabling ribosomes to be tuned to translate specific mrnas via specific ribosomal proteins ( xue and barna , 2012 ) . in addition , a striking and curious feature of many recent sequencing studies is the detection of many ribosomal subunits in dendritic or axonal fractions . indeed , the single most abundant class of mrnas encode ribosomal proteins in axons ( andreassi et al . , 2010 ; gumy et al . , 2011 ; taylor et al . , 2009 ; thus , an additional intriguing possibility suggested by the abundance of ribosomal protein mrna in axons and dendrites is that ribosomal proteins may be synthesized de novo . this could provide proteins for in situ repair of ribosomes , or even more interestingly could provide onsite tuning of translation ( lee et al . , 2013 ) . one of the most exciting clinically relevant findings to emerge from recent work is the link between dysregulated synaptic protein synthesis and neurological disorders ( bear et al . , 2008 ; darnell and klann , 2013 ; liu - yesucevitz et al . , 2011 ) . mouse models of neurodevelopmental disorders such as autism spectrum disorder ( asd ) show significant improvement on treatment with reagents that target the protein - synthesis pathway ( bear et al . , 2008 ; much of the focus has been on the postsynaptic side of the synapse , the predominant site of plasticity and learning . recent evidence indicates that regulated protein synthesis in the presynaptic compartment is also important for synapse formation ( taylor et al . , 2013 ) and axon arborization ( hrnberg and holt , 2013 ; hrnberg et al . , 2013 ; 2013 ) , raising the question of whether defects in axonal protein synthesis contribute to the miswiring aspects of neurodevelopmental disorders . dysregulated protein synthesis may also underlie a broad range of neurodegenerative disorders ( fallini et al . 2011 ) consistent with axonal protein synthesis being required for axon maintenance ( hillefors et al . 2012 ) . indeed , the first effective oral drug treatment that prevents neurodegeneration in a prion disease / alzheimer s mouse model targets a kinase ( perk ) that shuts down protein synthesis as part of the unfolded protein response ( moreno et al . , recent years have witnessed a transformation in our appreciation of rna function in dendrites / axons on the one hand and of neuronal compartments as spatially distinct signaling / processing units on the other . here we have highlighted the convergence of these two areas and have sought to define some of the many interesting questions and challenges that lie ahead . as technical approaches become increasingly sensitive for unbiased profiling there is the promise of improved understanding of the qualitative concepts that govern the various active rna species and formation and function of compartments as well as quantitative details on the stoichiometries of all of the players positioned within the morphological framework of the neuron and its remarkable dendritic and axonal arbor .

the elaborate morphology of neurons together with the information processing that occurs in remote dendritic and axonal compartments makes the use of decentralized cell biological machines necessary . recent years have witnessed a revolution in our understanding of signaling in neuronal compartments and the manifold functions of a variety of rna molecules that regulate protein translation and other cellular functions . here we discuss the view that mrna localization and rna - regulated and localized translation underlie many fundamental neuronal processes and highlight key issues for future experiments .

Main Text

extrinsic signals are often spatially localized such that they are seen by restricted parts of a neuron , such as synaptic input to a specific dendritic spine or a guidance cue encountered by a growth cone . twenty - five years ago , when the first issue of neuron was published , it was well appreciated that the neurons were capable of local information processing , but the potential cellular mechanisms that established and regulated local compartments were not well understood . however , the view that dominated until nearly the end of the twentieth century was that the central dogma ( dna - rna - protein ) was carried out centrally in the nuclei and somata of neurons . in that context , the localization of mrna observed in some cells was thought to represent a specialized mechanism that operated in unique biological systems , such as egg cells , where storage of mrnas is needed for subsequent patterning of the early embryo ( see martin and ephrussi , 2009 for review ) . evidence from a number of studies in the last decade , particularly in neurons , has led to a revolution in our thinking . although the field is still young , it is becoming clear that rna - based mechanisms provide a highly adaptable link between extrinsic signals in the environment and the functional responses of a neuron or parts of a neuron . this is accomplished by the localization of both protein - coding and noncoding rna in neuronal processes and the subsequent regulated local translation of mrna into protein . here we discuss some of the key findings that lead us to the view that mrna localization and rna - regulated and localized translation underlie many fundamental cellular processes that are regulated by extrinsic signals in neurons , such as memory , dendrite and arbor branching , synapse formation , axon steering , survival , and likely proteostasis . over 50 years ago , in vivo experiments ( in a variety of species ) established a clear functional link between protein synthesis and long - term memory ( see davis and squire , 1984 for review ) , indicating that proteome remodeling underlies behavioral plasticity . this link between protein synthesis and long - term plasticity is most recently reinforced by studies showing that targeted genetic disruption of signaling molecules that regulate protein translation interfere with long - term synaptic or behavioral memories ( costa - mattioli et al . , 2009 ) . we now know dendrites and axons of neurons represent specialized cellular outposts that can function with a high degree of autonomy at long distances from the soma , as illustrated by the remarkable ability of growing axons to navigate correctly after soma removal ( harris et al . , 1987 ) or isolated synapses to undergo plasticity ( kang and schuman , 1996 ; vickers et al . , 2005 ) . the identification of polyribosomes at the base or in spines ( steward and levy , 1982 ) together with metabolic labeling experiments that provided the first evidence of de novo synthesis of specific proteins in axons and dendrites ( feig and lipton , 1993 ; giuditta et al . , 2011 ) . , 2012 ; perry et al . , 2005 ; increasing evidence indicates that the complex morphology of neurons has created biological compartments that subdivide the neuron into spatially distinct signaling domains important for neuronal function ( hanus and schuman , 2013 ) . receptors , channels , signaling molecules , and scaffolds ) are collected together with a common function for receiving and processing electrical and chemical input . in axons , for example , some membrane proteins are localized to restricted segments of the axon ( fasciclins , tag1/l1 , robo ) ( bastiani et al . selective activation of a single spine on a dendrite , for example , can provide the receiving neuron with information about a specific stimulus ( varga et al . compartments may be overlapping or distinct and range in size depending on the biological function . as such , future experiments aimed at understanding how different compartments emerge and what mechanisms generate such spatially precise intracellular patterning will be very informative . one of the remarkable features of rna is its ability to be spatially localized and , therefore , potentially contribute to neuronal compartmentalization . the proteins encoded by localized mrnas are also concentrated at the site suggesting that mrna localization and the ensuing local translation plays an important role in positioning proteins for cellular functions . mrnas tend to be abundantly localized to the peripheral domains and motile parts of neurons where they are optimally positioned for the arrival of external signals , e.g. this polarized translation leads to a rapid ( 5 min ) polarized increase in -actin protein that helps to drive axon turning towards the gradient source . different growth factors , for example , trigger the transport of a specific repertoire of mrnas in axons ( willis et al . , 1999 ) , and different guidance cues elicit the translation of specific subsets of mrnas ( leung et al . , 2013 ; -actin mrna translation is triggered by netrin-1 but not sema3a , whereas rhoa and cofilin mrna translation is induced by sema3a but not netrin-1 . several aspects of this translation - driven cue - induced turning remain to be understood , such as how receptor activation signals mrna recruitment and , critically , how specific subsets of mrna are translated . although there are several examples of stimulus - induced local translation in axons in vitro ( shigeoka et al . , 2013 ) , it has only recently become possible to investigate translation in neuronal compartments in vivo . , at the midline ) that triggers the region - specific translation of proteins needed for pathfinding ( brittis et al . , 2013 ) . it was not previously technically possible to inhibit translation of a specific transcript in a compartment - specific manner . recently , however , new tools have been developed that allow separate manipulation of specific neuronal compartments in vivo such as targeted delivery of sirnas or antisense morpholinos and conditional targeting of 3utrs ( perry et al . these subcellular - directed approaches are beginning to yield information suggesting that local translation is involved in regulating multiple aspects of axonal and dendritic biology . guidance cues induce immediate steering responses in growth cones via classical signaling pathways that involve receptor activation and phosphorylation of downstream signaling molecules ( bashaw and klein , 2010 ) . moreover , recent high - resolution in situ hybridization data suggest that mrna molecules are distributed in local domains ( cajigas et al . these data imply that there is local sharing of cell biological machineries , including the machinery for protein synthesis and degradation . it remains unclear , however , over what spatial scale local translation can be regulated and stimulated in dendrites . for example , is stimulation of a single spine sufficient to regulate local translation , and , if so , over what spatial domain do the newly synthesized proteins function ? the past view that rna acts primarily as an inert intermediate between genes and proteins has undergone a revolution in recent years with discoveries of both new classes of rnas ( e.g. , noncoding rnas , ( see ulitsky and bartel , 2013 for review ) and new rna - based mechanisms of gene regulation ( e.g. indeed , given the relatively constrained diversity of proteomes across cells and organisms , rna - based mechanisms ( diverse rna species and rna functions ) represent a unique platform to diversify and specialize cells , especially neurons . numerous new roles for rna have been found in recent years , expanding the role of rna to controlling many and diverse cellular processes , including stimulus - induced local translation that underlie adaptive responses in neurons ( e.g. , memory , axon guidance , and maintenance ) . , 2013 ) and even between organisms ( sarkies and miska , 2013 ) , bringing a whole new era of rna function in cellular communication into focus . the demonstrations that local protein translation functions during synaptic development and plasticity led to the hunt for specific mrnas that could be translated in these local compartments . for many years , in situ hybridization was the method of choice , and several individual mrnas were visualized in dendrites , including the mrna for the ca - calmodulin - dependent protein kinase alpha subunit , camkii ( burgin et al . one of the most surprising findings to come out of these studies is the vast number of mrnas that are present in these neuronal compartments . many functional categories are shared between the two compartments , although there are numerous distinct compartment - specific subsets of mrnas , e.g. the use of mrna localization to achieve protein localization may arise from the fact that , at least theoretically , unlimited address information can be built into the 3 and/or 5 utrs of mrna without altering its gene - coding function , whereas there is a tight limit to how much additional coding sequence can be added to a protein without ramifications for function . the family of proteins that bind , transport , localize , and regulate the translation of mrnas are known as rna - binding proteins ( rbps ) ( see darnell , 2013 for review ) . rna - binding proteins complexed with mrna , other rna species , and accessory proteins are thought to be assembled in the cell body and form rna granules ( kiebler and bassell , 2006 ) . , hirokawa , 2006 and czaplinski and singer , 2006 ) , the mrna cargo is thought to be silenced by translational repressors ( krichevsky and kosik , 2001 ) . rna - binding proteins are an important class of regulatory molecule that recognizes specific nucleotide sequences in rna ( ray et al . some rbps , however , appear to be cell - type specific , such as hermes ( rpbms2 ) that is expressed exclusively in retinal ganglion cells in the cns and its knockdown causes severe defects in axon terminal branching ( hrnberg et al . the number of mrna - binding proteins identified by known rna - binding domains is relatively small ( around 270 ) given the increasingly large number of transcripts found in axons and dendrites . recent work using interactome capture in embryonic stem cells has significantly expanded the number of rbps , adding a further 280 proteins to the repertoire , including , remarkably , many enzymes such as e3 ubiquitin ligases with previously unknown rna - binding function ( kwon et al . several rbps have been implicated in neurological disorders , such as fmrp in fragile x syndrome and survival of motor neuron protein ( smn ) in spinal muscular atrophy ( bear et al . , 2013 ; santini et al . in recent years the discovery of noncoding rnas , including mirnas ( which use sequence complementarity to recognize target mrna ) , has revealed unanticipated and enormous potential for the regulation of mrna stability and translation , as well as other functions . given the huge and unanticipated number of mrnas detected in axons and dendrites , it is perhaps not surprising that these noncoding rnas also exist and are even enriched in neuronal compartments . one might even argue the complex morphology and functional specialization of neurons provides a hotbed for mrna regulation that can potentially be mediated by noncoding rnas . , 2012 ) . this includes small - nucleolar rna - derived and transfer rna - derived small rnas , firstly identified as degradation products , and long noncoding rna known as regulators of gene transcription , that may regulate gene expression posttranscriptionally . , 2013 ) . , 1968 ; koenig , 2009 ) , but the possibility that the signal arose from cell - body contamination could not be eliminated due to these technical limitations . localized translation was convincingly demonstrated by surgically severing the soma from its processes ( aakalu et al . , 2001 ; campbell and holt , 2001 ; kang and schuman , 1996 ) and , more recently , by the use of chambers in which the processes ( dendrites or axons ) are fluidically isolated from cell bodies ( eng et al . , other methods for isolating neuronal processes include substrates with limited pore size that allow axons to penetrate but not cell bodies ( torre and steward , 1992 ; zheng et al . these methods combined with the rapid increase in the sensitivity of profiling techniques have enabled genome - wide transcriptome analyses to be performed on axons and dendrites in a variety of neurons ( see below ) . the visualization and identification of newly synthesized proteins has also been a hurdle due to issues of sensitivity ( detecting low levels of newly synthesized proteins ) as well as difficulties in distinguishing between the movement of existing proteins and the synthesis of new proteins . puromycin , a trna analog , can be used together with fluorescent tags ( smith et al . fluorescent reporters , such as photo - switchable kaede , fused to the 3utr regulatory region of mrnas of interest have enabled de novo protein synthesis to be monitored live in neuronal processes ( aakalu et al . , in addition , new methods have been developed to selectively label the pool of newly synthesized proteins , to ascertain a given cell type or cellular compartment as the site of synthesis , and to visualize the newly synthesized proteins . these methods make use of noncanonical amino acids that cross cell membranes and get charged onto trnas by the cell s own trna synthetases and then incorporated into new protein during protein synthesis . , 2006 ) or visualize ( dieterich et al . , 2012 ) , and ncat can be used in combination with 2d difference gel electrophoresis ( dige - ncat ) to compare the proteomes of specific subcellular ( e.g. , 2012 ) . in addition , in some cases the signal for recognition by an rna - binding protein may reside in the secondary structure of the mrna , rather than the nucleotide sequence . adding to the complexity is the recent observation that low - complexity regions of rna - binding proteins are sufficient to create reversible rna granule - like structures ( kato et al . future studies should focus on the dynamics of the rna - protein interactions in cellular contexts . in addition , the possibility that rna might be delivered from extracellular sources ( e.g. unbiased genome - wide analyses have shown that the mrna repertoire is dynamically regulated with the mrna repertoire changing over time ( gumy et al . evidence with ephrinb in rgcs indicates that although the transcripts are present in somas early in development , they do not move into axons until later , suggesting that some kind of specific gating mechanism may exist . currently , little is known about the quantitative aspects of mrna localization and translation in neurons . for example , how many rna molecules are needed to provide a functionally significant amount of protein ? this also raises the intriguing question of whether translation from monosomes , rather than polysomes , may be more common in distal neuronal compartments where there could be demand for a few highly localized proteins . new high - resolution single molecule detection methods ( cajigas et al . with the advent of trap ( translating affinity purification ) technology ( heiman et al . , 2008 ) it will be possible in the future to answer this question in specific neuronal compartments of specific subsets of neurons . for example , cell - type specific cre - driver lines can be crossed with the ribotag mouse ( sanz et al . these can be isolated from mouse brains by immunoprecipitation at different ages and under different conditions ( and diseased ) , and rna - seq analysis can identify the ribosome - protected , and therefore , actively translating transcripts . this will be of huge importance in characterizing and understanding the translatome of neuronal compartments . thus , current technology now offers the exciting possibility of being able to discover differences in the dendritic or axonal translatome of diseased ( e.g. how does the spatial morphology of the dendrite , axon , or spine contribute to or constrain protein synthesis ? this could allow spines to enhance the ability of neurons to detect , uniquely respond to , and store distinct synaptic input patterns ( harnett et al . , 2012 ) . different patterns of synapse activation can lead to protein synthesis - dependent or -independent plasticity ( govindarajan et al . , however , the importance and mechanism of specific protein translation remains to be examined in this cooperativity . thus , while it is clear that protein synthesis occurs in the dendrite and that it is regulated by neuronal activity , the extent to which the activity of single synapses or synaptic regions stimulates protein synthesis , or alters protein localization , remains unknown . in the small cytoplasmic volume of a dendritic spine or growth cone , there is a limit to the amount of protein that can fit into the space before molecular crowding becomes a problem . while it is clear that changes in synaptic transmission involve extensive regulation of the synaptic proteome via the regulated synthesis and degradation of proteins ( fonseca et al . changes in the steady - state level of a protein have to be particularly fast and fine - tuned in neurons , due to the fast nature of synaptic transmission and the rapid induction of plasticity . under physiological conditions the spatial and temporal profile of synaptic and cue stimulation is on a much finer scale and the translational readout is likely limited . indeed , we know that different cues can trigger translation of specific subsets of mrnas in the growth cone ( lin and holt , 2007 ) . the mechanisms by which specific patterns of synaptic signals ( e.g. the use of high - resolution imaging techniques and focal stimulation should provide answers to these questions . in neurons , the mirna function has been explored both individually and on a population level , but a broad conceptual understanding is still lacking . moreover , if mirnas regulate mrna translation and expression in different neuronal compartments , what regulates the expression of mirna themselves ? based on this , it is likely that a real understanding of the complexity of rna function in neurons will require not only investigation of individual molecules but also a systems biology perspective where the entire network of rna molecules and their targets can be considered together ( see pelez and carthew , 2012 ) . , 1998 ; bunge , 1973 ) how they are transported and whether they are sequestered or anchored is not well understood . a mechanism that could provide specificity or docking would be the specialization of ribosomes by accessory proteins or subunits . this could provide proteins for in situ repair of ribosomes , or even more interestingly could provide onsite tuning of translation ( lee et al . 2011 ) consistent with axonal protein synthesis being required for axon maintenance ( hillefors et al . 2012 ) . , recent years have witnessed a transformation in our appreciation of rna function in dendrites / axons on the one hand and of neuronal compartments as spatially distinct signaling / processing units on the other . here we have highlighted the convergence of these two areas and have sought to define some of the many interesting questions and challenges that lie ahead . as technical approaches become increasingly sensitive for unbiased profiling there is the promise of improved understanding of the qualitative concepts that govern the various active rna species and formation and function of compartments as well as quantitative details on the stoichiometries of all of the players positioned within the morphological framework of the neuron and its remarkable dendritic and axonal arbor .

twenty - five years ago , when the first issue of neuron was published , it was well appreciated that the neurons were capable of local information processing , but the potential cellular mechanisms that established and regulated local compartments were not well understood . dendritic spines had been proposed as biochemical and/or electrical compartments ( harris and kater , 1994 ; koch and zador , 1993 ) , and polyribosomes had been identified at the base of spines ( steward and levy , 1982 ) . however , the view that dominated until nearly the end of the twentieth century was that the central dogma ( dna - rna - protein ) was carried out centrally in the nuclei and somata of neurons . in that context , the localization of mrna observed in some cells was thought to represent a specialized mechanism that operated in unique biological systems , such as egg cells , where storage of mrnas is needed for subsequent patterning of the early embryo ( see martin and ephrussi , 2009 for review ) . evidence from a number of studies in the last decade , particularly in neurons , has led to a revolution in our thinking . although the field is still young , it is becoming clear that rna - based mechanisms provide a highly adaptable link between extrinsic signals in the environment and the functional responses of a neuron or parts of a neuron . this is accomplished by the localization of both protein - coding and noncoding rna in neuronal processes and the subsequent regulated local translation of mrna into protein . here we discuss some of the key findings that lead us to the view that mrna localization and rna - regulated and localized translation underlie many fundamental cellular processes that are regulated by extrinsic signals in neurons , such as memory , dendrite and arbor branching , synapse formation , axon steering , survival , and likely proteostasis . over 50 years ago , in vivo experiments ( in a variety of species ) established a clear functional link between protein synthesis and long - term memory ( see davis and squire , 1984 for review ) , indicating that proteome remodeling underlies behavioral plasticity . these observations were paralleled by in vitro studies of synaptic plasticity demonstrating a clear requirement for newly synthesized proteins in the long - term modification of synaptic function ( see sutton and schuman , 2006 for review ; also , tanaka et al . this link between protein synthesis and long - term plasticity is most recently reinforced by studies showing that targeted genetic disruption of signaling molecules that regulate protein translation interfere with long - term synaptic or behavioral memories ( costa - mattioli et al . we now know dendrites and axons of neurons represent specialized cellular outposts that can function with a high degree of autonomy at long distances from the soma , as illustrated by the remarkable ability of growing axons to navigate correctly after soma removal ( harris et al . the identification of polyribosomes at the base or in spines ( steward and levy , 1982 ) together with metabolic labeling experiments that provided the first evidence of de novo synthesis of specific proteins in axons and dendrites ( feig and lipton , 1993 ; giuditta et al . in axons , the idea of local protein synthesis has been slower to find acceptance , no doubt hindered by the classical view of axons as information transmitters rather than receivers ; so , why would local protein synthesis be required ? it is now thought that at least part of the explanation for their apparent paucity lies in their localization close to the plasma membrane in axons ( sotelo - silveira et al . indeed , the first functional evidence for local protein synthesis in axons came from studies that showed that cue - induced directional steering is abolished by inhibitors of protein synthesis , including rapamycin , in surgically isolated axons ( campbell and holt , 2001 ) . , 2006 ) and revealed that local protein synthesis underlies growth - cone adaptation , gradient sensing , and directional turning in growing axons ( leung et al . , 2005 ; in addition , axonal protein synthesis is elicited in response to injury and plays key roles in axon regeneration and maintenance ( jung et al . , 2005 ; increasing evidence indicates that the complex morphology of neurons has created biological compartments that subdivide the neuron into spatially distinct signaling domains important for neuronal function ( hanus and schuman , 2013 ) . receptors , channels , signaling molecules , and scaffolds ) are collected together with a common function for receiving and processing electrical and chemical input . although spines are small ( 1 m ) , they can still be subdivided into further functional compartments ( see chen and sabatini , 2012 for review ) with multiple microdomains , raising the question of how a compartment is defined . , 1987 ; in addition , second - messenger signaling molecules such as calcium and cyclic nucleotides , once thought to signal extensively throughout a cell , are now known to be highly regulated such that increases in concentration can be confined to a small space , creating a signaling compartment . as such , future experiments aimed at understanding how different compartments emerge and what mechanisms generate such spatially precise intracellular patterning will be very informative . compartmentalized signaling presents several challenges to the cell , a prime one being the localization of its component parts . one of the remarkable features of rna is its ability to be spatially localized and , therefore , potentially contribute to neuronal compartmentalization . that localized rna is more often the rule than the exception is spectacularly illustrated by the finding that 71% of the drosophila embryo transcriptome is localized to specific subcellular compartments ( lcuyer et al . the proteins encoded by localized mrnas are also concentrated at the site suggesting that mrna localization and the ensuing local translation plays an important role in positioning proteins for cellular functions . mrnas tend to be abundantly localized to the peripheral domains and motile parts of neurons where they are optimally positioned for the arrival of external signals , e.g. to navigate in retinal growth cones , for example , which are only 5 m in diameter , a polarized external gradient of netrin-1 triggers increases in both the transport and translation of -actin mrna on the gradient near side ( leung et al . this has given rise to the differential translation model suggesting that translation - dependent repulsive and attractive turning in growth cones depends on the differential translation of mrnas involved in assembly or disassembly of the actin cytoskeleton ( lin and holt , 2007 ) . several aspects of this translation - driven cue - induced turning remain to be understood , such as how receptor activation signals mrna recruitment and , critically , how specific subsets of mrna are translated . early studies by flanagan and colleagues showing compartmentalized expression of epha2 , recapitulated by a translation reporter , in the post - midline crossing segment of commissural spinal cord axons introduced the idea that the growing tip of the axon is stimulated by a regionally expressed cue ( e.g. a recent study provides direct evidence for this type of mechanism in the control of robo expression and midline guidance ( colak et al . two robo3 receptor isoforms have opposing roles in guiding axons to and away from the midline , and their expression is compartmentalized in pre - crossing ( robo3.1 ) and postcrossing ( robo3.2 ) axonal segments ( chen et al . the switch to robo3.2 expression at the midline ( the transcript of which contains a premature termination codon ) is controlled by midline - induced axonal protein synthesis coupled with nonsense - mediated mrna decay . recently , however , new tools have been developed that allow separate manipulation of specific neuronal compartments in vivo such as targeted delivery of sirnas or antisense morpholinos and conditional targeting of 3utrs ( perry et al . these subcellular - directed approaches are beginning to yield information suggesting that local translation is involved in regulating multiple aspects of axonal and dendritic biology . guidance cues induce immediate steering responses in growth cones via classical signaling pathways that involve receptor activation and phosphorylation of downstream signaling molecules ( bashaw and klein , 2010 ) . taken together , it seems reasonable to consider a range of spatial domains over which signaling can occur , which would span the scale from subdomains in spines to dendritic branches to the entire neuron . these data can be compared to what we know about the quantitative localization of the protein - synthesis machinery . preliminary estimates of mrna numbers indicate that there may not be sufficient copies of individual mrna species for each synapse to have an exclusive and dedicated molecular toolbox . the past view that rna acts primarily as an inert intermediate between genes and proteins has undergone a revolution in recent years with discoveries of both new classes of rnas ( e.g. , noncoding rnas , ( see ulitsky and bartel , 2013 for review ) and new rna - based mechanisms of gene regulation ( e.g. indeed , given the relatively constrained diversity of proteomes across cells and organisms , rna - based mechanisms ( diverse rna species and rna functions ) represent a unique platform to diversify and specialize cells , especially neurons . numerous new roles for rna have been found in recent years , expanding the role of rna to controlling many and diverse cellular processes , including stimulus - induced local translation that underlie adaptive responses in neurons ( e.g. in addition , rna s role may not be limited to the cells where it is synthesized , as new studies indicate it can be transferred between cells ( via exosomes ) ( sharma et al . the demonstrations that local protein translation functions during synaptic development and plasticity led to the hunt for specific mrnas that could be translated in these local compartments . for many years , in situ hybridization was the method of choice , and several individual mrnas were visualized in dendrites , including the mrna for the ca - calmodulin - dependent protein kinase alpha subunit , camkii ( burgin et al . one of the most surprising findings to come out of these studies is the vast number of mrnas that are present in these neuronal compartments . many functional categories are shared between the two compartments , although there are numerous distinct compartment - specific subsets of mrnas , e.g. the use of mrna localization to achieve protein localization may arise from the fact that , at least theoretically , unlimited address information can be built into the 3 and/or 5 utrs of mrna without altering its gene - coding function , whereas there is a tight limit to how much additional coding sequence can be added to a protein without ramifications for function . the family of proteins that bind , transport , localize , and regulate the translation of mrnas are known as rna - binding proteins ( rbps ) ( see darnell , 2013 for review ) . rna - binding proteins complexed with mrna , other rna species , and accessory proteins are thought to be assembled in the cell body and form rna granules ( kiebler and bassell , 2006 ) . ip - seq analysis has revealed , unexpectedly , that some rbps can bind hundreds of different mrnas ( see darnell , 2013 for review ) . some rbps , however , appear to be cell - type specific , such as hermes ( rpbms2 ) that is expressed exclusively in retinal ganglion cells in the cns and its knockdown causes severe defects in axon terminal branching ( hrnberg et al . the number of mrna - binding proteins identified by known rna - binding domains is relatively small ( around 270 ) given the increasingly large number of transcripts found in axons and dendrites . recent work using interactome capture in embryonic stem cells has significantly expanded the number of rbps , adding a further 280 proteins to the repertoire , including , remarkably , many enzymes such as e3 ubiquitin ligases with previously unknown rna - binding function ( kwon et al . several rbps have been implicated in neurological disorders , such as fmrp in fragile x syndrome and survival of motor neuron protein ( smn ) in spinal muscular atrophy ( bear et al . in recent years the discovery of noncoding rnas , including mirnas ( which use sequence complementarity to recognize target mrna ) , has revealed unanticipated and enormous potential for the regulation of mrna stability and translation , as well as other functions . given the huge and unanticipated number of mrnas detected in axons and dendrites , it is perhaps not surprising that these noncoding rnas also exist and are even enriched in neuronal compartments . one might even argue the complex morphology and functional specialization of neurons provides a hotbed for mrna regulation that can potentially be mediated by noncoding rnas . indeed , an analysis of 100 different mirnas discovered the differential distribution of some mirnas in dendrites versus somata and copy numbers in individual neurons as high as 10,000equivalent to the number of synapses a typical pyramidal neuron possesses ( kye et al . moreover , the enrichment of mirnas in synaptosomes isolated from specific brain regions has also been reported ( pichardo - casas et al . , 2010 ) as well as more recently , the appreciation of other types of noncoding rnas have come into focus , though very little is known about their function in neurons . this includes small - nucleolar rna - derived and transfer rna - derived small rnas , firstly identified as degradation products , and long noncoding rna known as regulators of gene transcription , that may regulate gene expression posttranscriptionally . a recent study demonstrated , for example , that a long noncoding rna that is anti - sense to a k channel subunit ( kcna2 ) is upregulated following peripheral nerve injury , leading to a downregulation of the k channel and a resulting increase in the excitability of drg neurons , increasing neuropathic pain ( zhao et al . in the early years , the study of local translation was hampered by the technical difficulty of obtaining pure and sufficient quantities of dendrites and axons for analysis . these methods combined with the rapid increase in the sensitivity of profiling techniques have enabled genome - wide transcriptome analyses to be performed on axons and dendrites in a variety of neurons ( see below ) . the visualization and identification of newly synthesized proteins has also been a hurdle due to issues of sensitivity ( detecting low levels of newly synthesized proteins ) as well as difficulties in distinguishing between the movement of existing proteins and the synthesis of new proteins . fluorescent reporters , such as photo - switchable kaede , fused to the 3utr regulatory region of mrnas of interest have enabled de novo protein synthesis to be monitored live in neuronal processes ( aakalu et al . , in addition , new methods have been developed to selectively label the pool of newly synthesized proteins , to ascertain a given cell type or cellular compartment as the site of synthesis , and to visualize the newly synthesized proteins . a modification of the ncat method , which in principle enables one to label newly synthesized proteins in specific cell types , has also recently been developed ( ngo et al . , 2012 ) , and ncat can be used in combination with 2d difference gel electrophoresis ( dige - ncat ) to compare the proteomes of specific subcellular ( e.g. in addition , in some cases the signal for recognition by an rna - binding protein may reside in the secondary structure of the mrna , rather than the nucleotide sequence . adding to the complexity is the recent observation that low - complexity regions of rna - binding proteins are sufficient to create reversible rna granule - like structures ( kato et al . evidence with ephrinb in rgcs indicates that although the transcripts are present in somas early in development , they do not move into axons until later , suggesting that some kind of specific gating mechanism may exist . these can be isolated from mouse brains by immunoprecipitation at different ages and under different conditions ( and diseased ) , and rna - seq analysis can identify the ribosome - protected , and therefore , actively translating transcripts . thus , current technology now offers the exciting possibility of being able to discover differences in the dendritic or axonal translatome of diseased ( e.g. these observations suggest that the amplifying and coordinating properties of dendritic spines have an effect on neuronal input processing and may influence information storage by promoting the induction of clustered forms of synaptic and dendritic plasticity among coactive spines . since there are mrnas that are differentially distributed in the length of the dendrites , it is tempting to speculate that there is a role for protein synthesis in regulating the functional compartment in dendrites and spines . thus , while it is clear that protein synthesis occurs in the dendrite and that it is regulated by neuronal activity , the extent to which the activity of single synapses or synaptic regions stimulates protein synthesis , or alters protein localization , remains unknown . while it is clear that changes in synaptic transmission involve extensive regulation of the synaptic proteome via the regulated synthesis and degradation of proteins ( fonseca et al . changes in the steady - state level of a protein have to be particularly fast and fine - tuned in neurons , due to the fast nature of synaptic transmission and the rapid induction of plasticity . indeed , we know that different cues can trigger translation of specific subsets of mrnas in the growth cone ( lin and holt , 2007 ) . in addition , the recent appreciation of the abundance and regulatory potential of other noncoding rnas , mostly in nonneuronal cell types , adds another level of complexity , including the recent demonstration of regulation by circular rnas that may serve as either shuttles , assembly factories , or sponges for mirnas and/or rbps ( hentze and preiss , 2013 ) . based on this , it is likely that a real understanding of the complexity of rna function in neurons will require not only investigation of individual molecules but also a systems biology perspective where the entire network of rna molecules and their targets can be considered together ( see pelez and carthew , 2012 ) . ribocode that suggests heterogeneity in the composition of ribosomes , enabling ribosomes to be tuned to translate specific mrnas via specific ribosomal proteins ( xue and barna , 2012 ) . mouse models of neurodevelopmental disorders such as autism spectrum disorder ( asd ) show significant improvement on treatment with reagents that target the protein - synthesis pathway ( bear et al . indeed , the first effective oral drug treatment that prevents neurodegeneration in a prion disease / alzheimer s mouse model targets a kinase ( perk ) that shuts down protein synthesis as part of the unfolded protein response ( moreno et al . , recent years have witnessed a transformation in our appreciation of rna function in dendrites / axons on the one hand and of neuronal compartments as spatially distinct signaling / processing units on the other . as technical approaches become increasingly sensitive for unbiased profiling there is the promise of improved understanding of the qualitative concepts that govern the various active rna species and formation and function of compartments as well as quantitative details on the stoichiometries of all of the players positioned within the morphological framework of the neuron and its remarkable dendritic and axonal arbor .

more than 90% of the increasing radiative forcing imbalance is reflected as global ocean warming [ alexander et al . , the deceleration of the upper ocean heat storage since the mid2000s [ levitus et al . cheng and zhu , 2015 ] has instigated an active search for the missing heat in the deep ocean [ meehl et al . , 2011 ; trenberth and fasullo , 2010 ; katsman and van oldenborgh , 2011 ; balmaseda et al . , 2013 ; kosaka and xie , 2013 ] . the enhanced downward heat transfer necessary for this vertical heat redistribution has been proposed to be related to the hiatus in global warming , although the hiatus started earlier and lasted for the 15 years period 19982013 [ trenberth et al . two natural mechanisms have been proposed to explain the deceleration of the rise in global mean surface temperature and the efficient transfer of heat to the deep ocean : ( i ) the intensification of zonal winds causing la nialike conditions in the tropical pacific [ meehl et al . , 2011 ; trenberth and fasullo , 2010 ; katsman and van oldenborgh , 2011 ; balmaseda et al . , 2013 ; kosaka and xie , 2013 ] and ( ii ) decadal variations in the strength of deep convection and currents in the north atlantic [ meehl et al . , 2011 ; katsman and van oldenborgh , 2011 ; chen and tung , 2014 ; drijfhout et al . , modeling simulations show that la nia surface cooling in the eastern and central tropical pacific can decrease the rate of increase in atmospheric temperature ( although the similarity of observed and simulated deceleration is still debated [ trenberth , 2015 ; karl et al . , 2015 ] ) and induce an anomalous heat flux into the ocean [ kosaka and xie , 2013 ; england et al . , 2014 ; balmaseda et al . , associated to this anomalous heat flux into the ocean , subsurface heat uptake in the western tropical pacific and indian oceans occurs [ goddard , 2014 ; england et al . , 2014 ; meehl et al . , 2013 ; lee et al . , 2015 ] mostly above 300 m ( see figure s1 in the supporting information ) [ chen and tung , 2014 , figures 1 and 3 ; nieves et al . , 2015 ] , thus not largely involving transfer of heat into the deep ocean . regarding the second mechanism , vertical heat inventories of the north atlantic show notable heat transfer from the upper ( 0300 m ) to intermediate ( 300700 m ) and deep ( > 700 m ) layers since the mid2000s ( see figure s1 and corresponding comments ) [ alexander et al . , 2013 , figure 3.1 ; chen and tung , 2014 , figure 6 ; williams et al . such recent downward heat transfer follows decades of sustained surface warming and increasing ocean heat content in the basin [ drijfhout et al . , 2014 ; however , concomitant with the downward heat export after the mid2000s , the increase in ocean heat content in the upper 2000 m of the north atlantic paused [ chen and tung , 2014 , figures 1 and 3 ; hkkinen et al . , 2015 , figure 1 ] , making the bulk rate of change of ocean heat content per unit area in the basin almost zero . bulk rates of change in ocean heat content per unit area are considered an estimation of the ocean heat uptake ( q , w m ) at ocean basin scales [ e.g. , mauritzen et al . , 2012 ; guemas et al . , 2013 ; chen and tung , 2014 ] . changes in ocean circulation can generate ocean heat content anomalies of opposite signs between different ocean layers and regions ( e.g. , subtropical versus subpolar gyre ) , but the anomalies compensate when integrated at ocean basin scales and decadal timescales [ lozier et al . , 2008 ; mauritzen et al . , therefore , one can conclude that the heat redistribution in the north atlantic since the mid2000s as a whole has apparently occurred without extra heat uptake from the atmosphere . overall , during the past decade , different basins have played contrasting roles on the earth 's atmosphereocean heat budget distribution . in particular , the north atlantic would not have contributed notably as in previous decades to atmospheric heat uptake , in contrast to that observed in the tropical pacific and southern oceans [ roemmich et al . , 2015 ] , but to the quick transfer of heat accumulated during the previous decades in the upper layers to the deep ocean . what are the causes for this shift ? deep convection in the subpolar north atlantic under highsalinity conditions might account for the quick transfer of heat to the deep ocean [ chen and tung , 2014 ] . according to these authors , recent highsalinity conditions may be caused by a strengthening of the atlantic meridional overturning circulation ( amoc ) . agreeing that high salinity in deep convection regions favors heat transfer to the deep ocean [ mauritzen et al . , first , available observational records do not show a strengthening of the amoc [ kanzow et al . second , deep convection in the subpolar north atlantic is restricted to the labrador and irminger seas ( see figure 1 ) , but far from these prominent deep ocean ventilation sites , ocean observations seem to show the largest deep ocean heat uptake at midlatitudes in the eastern north atlantic ( figure 1 ) [ desbruyres et al . , warming pattern in the north atlantic at middepth . temperature differences ( t in color , c ) in the 300700 m layer between the 20072012 and the 19972002 periods ( i.e. , average values are estimated for each time range ) . figure 1 is the zoom into the north atlantic in figure s1 ocean reanalysis system 4 ( oras4 ) ( d ) ( see figure s1 procedure description for details ) . arrows represent ( in red ) warm and salty upper water currents , ( in dark blue ) cold and fresh deep water currents , ( in black , dotted ) the subpolar gyre , and ( in black , dashed ) the subtropical gyre . deep convection sites in the irminger and greenland seas as well as the position of the santander standard section in the bay of biscay are marked by green squares . midlatitudes in the eastern north atlantic ( mlena ) , considered as the oceanic area including the mode water formation area of the eastern north atlantic central water ( enacw ) and the oceanic waters of the bay of biscay and surrounding the iberian peninsula to north africa as in somavilla et al . we reexamine the mechanism responsible for the quick transfer of ocean heat from upper to deeper layers in the north atlantic focusing on the role of ena modal waters . highquality hydrographic data with global coverage are required for providing observational evidence to the mid2000s shift in the north atlantic . these data are obtained from regularly observed hydrographic sections , which are affected by gaps in coverage both in time and space . to overcome this issue , we combined standard hydrographic observations ( salinity and temperature profiles ) at oceanographic sections regularly maintained in the bay of biscay and the greenland sea ( see figure 1 ) with other data sets that offer more global coverage such as argo floats , globalgridded ocean reanalysis ( european centre for mediumrange weather forecasts ocean reanalysis system 4 , oras4 ) and objective analysis ( met office hadley centre monthly objective analysis , en4 , and world ocean database 2001 , woa01 ) . along the work , temperature and salinity time series from oceanographic sections are systematically presented together with those constructed from argo floats and reanalysis data . in this manner , we prove that the changes detected at oceanographic sections are representative of larger oceanic areas ( e.g. , figures 2 and 5 ) , and it serves as an important validation for both argo floats and gridded data sets affected by factors such as changes in observation coverage around 2005 [ roemmich et al . , 2015 ] , contamination from model errors in ocean reanalysis data [ balmaseda et al . , 2013 ] , or the gapfilling strategy in climatological data [ cheng and zhu , 2015 ] . with a similar purpose of increasing the reliability of individual data sets , warming / cooling and saltening / freshening maps obtained from different gridded data are compared ( figures s1 and s2 ) thus , we increase the confidence of the enhanced warming at midlatitudes in the eastern north atlantic as shown in figure 1 , since similar warming distributions showing enhanced heat uptake at midlatitudes in the ena and surrounding the subpolar north atlantic margins are observed in other gridded products ( figure s1 based on en4 ; figure 3c in chen and tung based on ishii data ; figure 4 to 6 in hkkinen et al . based on noaa / national oceanographic data center database ; figure 3.1 on the intergovernmental panel on climate change report [ alexander et al . , 2013 ] constructed from an update of the annual analysis of levitus et al . ) . a complete description of the data sets , methods , and further details are included in the supporting information . strong mixinginduced deep ocean heat injection at mlena . ( a ) mean temperature ( white line ) between the shallower winter mixed layer depth achieved in warm winters and the enacw upper bound ( 100300 m ) and ( black line ) in the depth range of the enacw core ( 300550 m ) in the enacw formation area ( 4348n , 1020w ) obtained from oras4 reanalysis data set . ( b and c ) same as figure 2a but for temperature and salinity at =27.227.3 kg m ( corresponding to the + 0.1 kg m 2005 denser enacw core ) . the blue circles indicate temperature and salinity at =27.227.3 kg m as recorded at station 7 of the santander standard section ( figure 1 ) . the magenta line in figure 2b shows the background average warming trend ( + 0.1c decade ) at the intermediate depth ( 300700 m ) for the global ocean ( table s1 ) . the winter of 2005 was exceptionally cold and dry in southwestern europe [ shein , 2006 ; somavilla et al . , , the ocean surface at midlatitudes in the eastern north atlantic ( mlena , see figure 1 for geographical reference ) experienced the highest densification ( buoyancy loss ) since the 1960s , causing the winter mixed layer to reach very deep levels likely unprecedented for decades [ somavilla et al . the induced deep convective mixing resulted in homogeneization of properties of the water column from the surface to density levels of =27.227.3 kg m , at depth ranges of 350500 m , well beyond the normal airsea interaction extent at the core of enacw ( =27.127.2 kg m ) . this mixing of ( warmer and saltier ) upper water and ( colder and fresher ) modal waters made the upper waters colder ( 0.4c ) , creating a denser ( + 0.1 kg m ) , warmer ( + 0.23c ) , and more saline ( + 0.015 ) type of enacw ( figures 2 and s1 ) [ somavilla et al . , 2009 ] . such temperature increase at the enacw core ( + 0.23c ) resulted in a sudden heat content increase of 2.410 j m for the 350500 m layer ( 3.9 0.610 j m for the 300700 m layer , table s1 ) . this suggests that 70% of the estimated 5.810 j m of heat gained slowly but persistently between 1994 and 2005 in the shallower levels ( 0.7c in the 100300 m layer , table s1 and figure 2a ) was transferred into a denser variety of mode waters through the extreme winter mixing . in addition , considering that such heat injection took place during winter 2005 , the heat uptake ( q ) at intermediate depths would be 30 w m ( 5 w m for the 300700 m layer ) , between 65 and 100 times faster than the average ocean heat uptake at such intermediate depths ( 300700 m ) between 2000 and 2010 ( see table s1 ) . note that the heat uptake at intermediate depths originates from the heat transfer from the upper layers and not from the atmosphere . forced by atmospheric cooling , the convective mixing would have released heat to the atmosphere ( see comments in table s1 for additional information ) . most of the enhanced warming observed at intermediate depths at mlena shown in figure 1 ( + 0.33c gained between 1997 and 2012 ) is explained by the increase in the modal waters during the winter of 2005 ( + 0.23c ) . the estimated overall heat content increase in the region is 1.2 10 j and accounts for 44% of the total heat content increase at intermediate depths in the north atlantic from 36 to 65n from 1997 to 2012 ( table s2 ) . evidence suggests that this extreme winter mixing episode may have contributed to the deep ocean heat injection at higher latitudes by inducing ocean circulation changes that enabled northward propagation of the relatively salty enacw . mlena climatological circulation charts and specific studies indicate slow southward flow west of the bay of biscay and iberia [ pollard et al . , 1996 ; pingree , 1993 ; reid , 1994 ] ( figure 3a ) . the 27.15 isopycnal became shallower from the midatlantic eastward and peaked at about 18w , beyond which it deepened slowly . after winter 2005 , the isopycnals became shallower all the way against the european margin ( figures 3b3d ) . the associated zonal dynamic height sections were thus altered , and the zonal dynamic height slope eastward of 22w changed from almost flat or negative to positive ( figures 3e3 g ) . hence , the associated meridional flow shifts from southward to northward . estimated as the geostrophic meridional current between 22 and 12w ( v g=/f l , where is the difference between the dynamic height at 12w and 22w , f the coriolis parameter , and l the distance between the two longitudes ) , the meridional current after 2005 estimated from the mean filtered dynamic height from argo floats is + 0.7 cm s ( northward ) with respect to 0.4 cm s ( southward ) estimated from woa01 climatological dynamic height ( with reference level at 1500 m both for argo floats and woa01 data ) . oras4 meridional current data show a comparable northward reversal of the flow centered at 18w from 2005 to 2010 ( mean value = + 1 cm s , maximum value = + 3 cm s ) with respect to its southward direction ( mean value = 0.5 cm s , maximum value = 3 cm s ) from 1990 to 2005 ( see figure s3 ) . the inversion of the zonal dynamic height slope is a robust feature ( e.g. , with reference level at 1000 m , the meridional current after 2005 estimated from argo floats is + 0.6 cm s ( northward ) with respect to 0.35 cm s ( southward ) estimated from woa01 climatological dynamic height ) as also observed in oras4 meridional current ( figure s3 ) . a similar flow reversal was predicted by a sensitivity experiment in a global ocean circulation model , and anomalous northward advection of salt was obtained as a result of surface cooling at 45n in the ena [ svellec et al . , ( a ) map of the northeast atlantic showing the location of the nwse , we , and swne sections across the enacw formation area for which the data are shown in figures 3b3 g . ( b d ) location of the = 27.15 at the nwse , ew , and swne sections marked in figure 3a based on woa01 climatological data ( grey ) and available argo floats in the corresponding section between 2005 and 2010 ( cyan ) . the blue line indicates the mean filtered value of all the argo floats each 2 of longitude . ( e g ) idem for the dynamic height between 300 and 1500 m ( see section 2.3 in the supporting information for details ) . black arrows in figure 3a indicate the southward flow west of the bay of biscay based on pollard et al . , inferred in figures 3e3 g from the climatological data ( grey dots ) ; and the red arrow indicates the reversed flow after 2005 , shown in figures 3e3 g by argo floats ( blue line ) . it is worth noting that the maximum water exchange between the subtropical and the subpolar gyres occurs at a density level of =27.227.4 kg m [ brambilla and talley , 2006 ] . thus , the + 0.1 kg m denser core of enacw ( =27.227.3 kg m after 2005 ) might easily get incorporated to the major branch of the north atlantic current flowing northward . the anomalous northward flow of salty enacw is also inferred directly from largescale hydrography ( see figure 4b ) as will be described in detail in the next section . increasing salinities at middepths after 2005 . salinity difference ( s in color ) in the 300700 m layer between ( a ) the 20022005 and the 19972002 periods and ( b ) the 20072012 and the 19972002 periods based on en4 data set ( i.e. , average values are estimated for each time range ) . figure 4 is the zoom into the north atlantic in figure s2 en4 ( c and d ) ( see figure s2 procedure description for details ) . due to the weak circulation and dominance of mesoscale activity in the mlena region , finding direct evidence of a flow reversal as the one inferred from dynamic height and reanalysis current data is difficult . argobased deep displacements [ ollitrault and rannou , 2013 ] are an available source , and luckily , a number of floats drifting at enacw levels were active in the region ( see figure s4 ) . the outcome from the analysis of their lagrangian displacements upper water temperature and salinity in the subpolar gyre and the nordic seas increased since early the 2000s ; this tendency is attributed to the arrival of warmer and saltier western subtropical waters caused by the contraction of the subpolar gyre [ htn et al . , 2005 ; woollings , 2011 ] . though the higher salinities peaking between 2007 and 2010 in these areas ( see figure 4b ) [ holliday et al . , 2008 ; beszczynskamller and dye , 2013 ; http://ocean.ices.dk/iroc/ ] would be consistent with a higher transport of western subtropical waters , that higher transport does not account for the drop in nutrient concentration in the eastern subpolar north atlantic ( rockall trough , figure 1 ) observed from 2007 onward [ johnson et al . , 2013 ] . this drop in nutrient concentration is explained by a higher contribution of enacw in the area [ johnson et al . , 2013 ] , thus adding further evidence in support of the circulation reversal previously described . the enhanced northward flow in the ena would have reinforced the northwestward retreat of the subpolar front [ hkkinen et al . , 2013 ] , broadening the passage for both enacw and western subtropical waters toward the north ( see figure 4b ) . both the eastern and western north atlantic central water types have the potential to increase temperature and salinity at higher latitudes . however , enacw are saltier than western waters arriving at the eastern subpolar north atlantic [ johnson et al . , 2013 ; hkkinen et al . , 2013 ] , providing an alternative explanation for the highestsalinity anomalies observed between 2007 and 2010 in the subpolar north atlantic and nordic seas . assuming mean anomaly propagation speeds of 2 to 3 cm s [ larsen et al . 2008 ] , a lag of 2 to 5 years is expected for the salinity anomaly ( from the mlena ) to reach first the eastern subpolar north atlantic and then the western subpolar gyre and northern nordic seas . in these areas , the arrival of salty southern waters due to the circulation shift in the mlena would have contributed to the highsalinity conditions required for deep ocean heat uptake through density compensated anomalies [ mauritzen et al . , 2012 ; chen and tung , 2014 ] without requiring an increase of the amoc as suggested by chen and tung . such circulation shift and enhanced northward flow in the mlena do not seem to contradict the observed weakening of amoc [ srokosz and bryden , 2015 ] . indeed , a strong northward flow east of 22w and south of 48n in the a priori recirculation area at the ' ovide ' section crossing the north atlantic from portugal to greenland was observed in 2006 , coincident with the lowest amoc measured through the section between 1997 and 2010 [ mercier et al . , consistent with the aforementioned spreading timescale and presence of the saltiest upper waters after 2007 in these areas , the heat and salt uptake below 700 m in the subpolar gyre and the nordic seas increased after 2007 ( figures s1 and s2 ) , coinciding with more intensive deep convection activity in both the subpolar gyre ( irminger and labrador seas , [ yashayaev and loder , 2009 ; kieke and yashayaev , 2015 ; de jong et al . , 2012 ; piron et al . , 2015 ] ) and nordic seas ( greenland sea , [ beszczynskamller and dye , 2013 ] ) . after years of sustained warming and increasing salinity in the upper layers , the first deep convection events at each of these sites resemble the enacw transformation in 2005 . in the deep water formation region of the greenland sea , a strong mixinginduced deep ocean heat injection event occurred through density compensated anomalies in 2008 ( figure 5 ) . upper ocean salinity in this region had been increasing since the mid1990s , and the 2008 winter cooling made the surface waters dense enough to trigger deep convection with temperature and salinity higher than typical values ( figure s5 ) . as a consequence of the overturning , the heat and salt gained slowly over time by the upper layers were directly injected to great depths , with a resulting sudden temperature and salinity increase of 0.16c and 0.01 , respectively ( figures 5c and 5d ) . with a less stratified water column , convection occurred at greater depths in the subsequent years as compared to previous decades in the greenland sea [ beszczynskamller and dye , 2013 ] . coincident with the arrival of highly saline waters in the upper layers of the subpolar gyre , a strong winter mixing occurred in the irminger sea in 2007 [ piron et al . , figures 5a and 5b show sudden increases in temperature ( 0.15c ) and salinity ( 0.01 ) at depth , related to this event . as in the greenland sea , subsequent deep convection events followed in later years ( 2008 , 2009 , and 2012 ) [ de jong et al . , 2012 ; piron et al . , 2015 ] . finally , coinciding with the arrival of highly saline waters in the upper levels , deep convection has reached levels deeper than 1700 m in the labrador sea in 2008 after more than a decade of weak convection , being repeated in 2012 and 2014 [ kieke and yashayaev , 2015 ] . as in the greenland and irminger seas , the first deep convection events appear to be associated with downward transfer of heat and salt [ see yashayaev and loder , 2009 , figure 2a ; kieke and yashayaev , 2015 , figure 4b ] . in general , the steplike temperature increases at depth at deep convection sites caused by the strong mixinginduced heat injection events are equivalent to heat uptake pulses between 16 and 25 w m , which is between 50 and 100 times faster than the average heat uptake below 700 m in the global ocean ( see table s1 ) . as in the mlena , concurrent with the deep ocean heat injection , the deep convective mixing events would have released heat to the atmosphere , favoring deceleration of the north atlantic heat uptake from the atmosphere , as apparently observed since the mid2000s [ chen and tung , 2014 ; hkkinen et al . , deep ( a ) temperature and ( b ) salinity ( 14001600 dbar , =27.7827.8 kg m ) in the irminger sea ( 6062n ; 3540w , green square in figure grey dots indicate vertically averaged argo temperature and salinity values in the pressure range of interest from individual profiles . deep ( c ) temperature and ( d ) salinity ( 15001800 dbar , =28.05828.062 kg m ) in the greenland sea ( 7476n ; 06w , see figure 1 ) . blue circles indicate vertically and spatially averaged conductivitytemperaturedepth ( ctd ) casts in the area and pressure range of interest and grey dots vertically averaged temperature and salinity of individual argo floats . the magenta line in figures 5a and 5c shows the background global ocean average warming trend below 700 m ( + 0.015c decade ) ( table s1 ) . blue lines highlight the years 2007 ( in figures 5a and 5b ) and 2008 ( in figures 5c and 5d ) . the primary cause of the extremely cold winter in 2005 in the mlena was the occurrence of atmospheric blocking anomalies in the ena . in addition to its effects on ocean circulation described in section 2 ( i.e. , regional enhanced mixing altering the largescale density structure ) , blocking anomalies in the ena generate a dipole of temperature anomalies between europe ( cold ) and greenland ( warm ) associated with a strong northward meridional atmospheric flow [ barriopedro et al . , 2008 ] with a potential to reinforce largescale ocean circulation changes . blocking anomalies over the ena occurred previously in the late 1950s and 1960s and extreme winter mixing events ( similar to that in 2005 ) enhanced northward flow of enacw may have occurred and contributed ( along with western subtropical waters ) to the intrusion of warmer and saltier waters into the eastern subpolar north atlantic and nordic seas during the 1960s [ holliday et al . , 2005 ] , as well as enhanced heat uptake by the subpolar gyre through densitycompensated temperature anomalies [ mauritzen et al . , 2012 ; chen and tung , 2014 ; hkkinen et al . , 2013 ; williams et al . , the heat uptake in the 1960s had smaller amplitude compared to the 2005 event , and this may be related to the sustained warming trend in the global upper ocean in the few decades prior to 2005 . the ultimate driver for the occurrence of blocking anomalies at midlatitudes is under debate [ kintisch , 2014 ] . both warmer arctic and tropical pacific have been associated with a wavier jet stream responsible for an increase in blocking events in the north atlantic and higher frequency of extreme weather events in general [ francis and vavrus , 2012 ; trenberth et al . , the blocking pattern developed in winter 2005 in the ena region is not inconsistent with natural climate variability [ barnes et al . however , overall global warming is expected to affect the development of such atmospheric features [ alexander et al . , 2013 ] as well as the subsequent ocean response . estimating the global significance of ocean heat transfer from upper to deeper levels by the mechanism described here requires a better model representation of the frequency and duration of wintertime blocking events , which have been underestimated in most global climate models thus far [ hov et al . , since the mid2000s , in contrast to the enhanced ocean heat uptake observed in other ocean basins , the north atlantic deep ocean heat content has significantly increased without apparently intervening ocean heat uptake from the atmosphere . about 40% of the increase in deep ocean heat content increase below 300 m is centered at midlatitudes in the eastern north atlantic and occurred relatively quickly around the mid2000s far from prominent deep ocean ventilation sites . we suggest that a sequence of connected strong mixinginduced heat injection events originating from this region triggered large transfer of heat from upper to deeper ocean layers , implying a deceleration or halt of the north atlantic heat uptake from the atmosphere . a large transformation of modal water masses in the eastern north atlantic in the winter of 2005 played a major role in the observed quick transfer of heat from upper to deep ocean layers but also aided a reverse of the southward circulation of saltier waters from the eastern side of the temperate atlantic with far reaching consequences ; the northward flow of saltier waters into the subpolar north atlantic and nordic seas favored additional heat injection through deep convection events in later years . thus , we agree that existing highsalinity conditions in the late 2000s in areas of deep convection have aided heat transfer to the deep ocean , but the suggestion that an increase of the amoc is behind such salinity increase is not supported by observations . a greater influence of the saltier enacw and broadened passage for both enacw and western subtropical waters toward the north appears to be a more plausible explanation . anomalous atmospheric patterns such as the one behind this shift are not unique to the last decade , although they may have been exacerbated under global warming .

abstractprior to the 2000s , the north atlantic was the basin showing the greatest warming . however , since the mid2000s during the socalled global warming hiatus , large amounts of heat were transferred in this basin from upper to deeper levels while the dominance in terms of atmospheric heat capture moved into the indopacific . here we show that a large transformation of modal waters in the eastern north atlantic ( ena ) played a crucial role in such contrasting behavior . first , strong winter mixing in 2005 transformed ena modal waters into a much saltier , warmer , and denser variety , transferring upper ocean heat and salt gained slowly over time to deeper layers . the new denser waters also altered the zonal dynamic height gradient reversing the southward regional flow and enhancing the access of saltier southern waters to higher latitudes . then , the excess salinity in northern regions favored additional heat injection through deep convection events in later years .

Introduction Data Sets Transformation of NorthEast Atlantic Modal Waters in 2005 and Implications for BasinScale Circulation Increasing Salinities and Deep Ocean Heat Injection at Higher Latitudes Further Remarks Conclusions Supporting information

, the deceleration of the upper ocean heat storage since the mid2000s [ levitus et al . the enhanced downward heat transfer necessary for this vertical heat redistribution has been proposed to be related to the hiatus in global warming , although the hiatus started earlier and lasted for the 15 years period 19982013 [ trenberth et al . two natural mechanisms have been proposed to explain the deceleration of the rise in global mean surface temperature and the efficient transfer of heat to the deep ocean : ( i ) the intensification of zonal winds causing la nialike conditions in the tropical pacific [ meehl et al . , 2013 ; kosaka and xie , 2013 ] and ( ii ) decadal variations in the strength of deep convection and currents in the north atlantic [ meehl et al . , modeling simulations show that la nia surface cooling in the eastern and central tropical pacific can decrease the rate of increase in atmospheric temperature ( although the similarity of observed and simulated deceleration is still debated [ trenberth , 2015 ; karl et al . , associated to this anomalous heat flux into the ocean , subsurface heat uptake in the western tropical pacific and indian oceans occurs [ goddard , 2014 ; england et al . , 2015 ] , thus not largely involving transfer of heat into the deep ocean . regarding the second mechanism , vertical heat inventories of the north atlantic show notable heat transfer from the upper ( 0300 m ) to intermediate ( 300700 m ) and deep ( > 700 m ) layers since the mid2000s ( see figure s1 and corresponding comments ) [ alexander et al . such recent downward heat transfer follows decades of sustained surface warming and increasing ocean heat content in the basin [ drijfhout et al . , 2014 ; however , concomitant with the downward heat export after the mid2000s , the increase in ocean heat content in the upper 2000 m of the north atlantic paused [ chen and tung , 2014 , figures 1 and 3 ; hkkinen et al . , 2015 , figure 1 ] , making the bulk rate of change of ocean heat content per unit area in the basin almost zero . , therefore , one can conclude that the heat redistribution in the north atlantic since the mid2000s as a whole has apparently occurred without extra heat uptake from the atmosphere . in particular , the north atlantic would not have contributed notably as in previous decades to atmospheric heat uptake , in contrast to that observed in the tropical pacific and southern oceans [ roemmich et al . , 2015 ] , but to the quick transfer of heat accumulated during the previous decades in the upper layers to the deep ocean . deep convection in the subpolar north atlantic under highsalinity conditions might account for the quick transfer of heat to the deep ocean [ chen and tung , 2014 ] . agreeing that high salinity in deep convection regions favors heat transfer to the deep ocean [ mauritzen et al . second , deep convection in the subpolar north atlantic is restricted to the labrador and irminger seas ( see figure 1 ) , but far from these prominent deep ocean ventilation sites , ocean observations seem to show the largest deep ocean heat uptake at midlatitudes in the eastern north atlantic ( figure 1 ) [ desbruyres et al . , warming pattern in the north atlantic at middepth . figure 1 is the zoom into the north atlantic in figure s1 ocean reanalysis system 4 ( oras4 ) ( d ) ( see figure s1 procedure description for details ) . deep convection sites in the irminger and greenland seas as well as the position of the santander standard section in the bay of biscay are marked by green squares . midlatitudes in the eastern north atlantic ( mlena ) , considered as the oceanic area including the mode water formation area of the eastern north atlantic central water ( enacw ) and the oceanic waters of the bay of biscay and surrounding the iberian peninsula to north africa as in somavilla et al . we reexamine the mechanism responsible for the quick transfer of ocean heat from upper to deeper layers in the north atlantic focusing on the role of ena modal waters . highquality hydrographic data with global coverage are required for providing observational evidence to the mid2000s shift in the north atlantic . to overcome this issue , we combined standard hydrographic observations ( salinity and temperature profiles ) at oceanographic sections regularly maintained in the bay of biscay and the greenland sea ( see figure 1 ) with other data sets that offer more global coverage such as argo floats , globalgridded ocean reanalysis ( european centre for mediumrange weather forecasts ocean reanalysis system 4 , oras4 ) and objective analysis ( met office hadley centre monthly objective analysis , en4 , and world ocean database 2001 , woa01 ) . with a similar purpose of increasing the reliability of individual data sets , warming / cooling and saltening / freshening maps obtained from different gridded data are compared ( figures s1 and s2 ) thus , we increase the confidence of the enhanced warming at midlatitudes in the eastern north atlantic as shown in figure 1 , since similar warming distributions showing enhanced heat uptake at midlatitudes in the ena and surrounding the subpolar north atlantic margins are observed in other gridded products ( figure s1 based on en4 ; figure 3c in chen and tung based on ishii data ; figure 4 to 6 in hkkinen et al . a complete description of the data sets , methods , and further details are included in the supporting information . strong mixinginduced deep ocean heat injection at mlena . ( b and c ) same as figure 2a but for temperature and salinity at =27.227.3 kg m ( corresponding to the + 0.1 kg m 2005 denser enacw core ) . , , the ocean surface at midlatitudes in the eastern north atlantic ( mlena , see figure 1 for geographical reference ) experienced the highest densification ( buoyancy loss ) since the 1960s , causing the winter mixed layer to reach very deep levels likely unprecedented for decades [ somavilla et al . this mixing of ( warmer and saltier ) upper water and ( colder and fresher ) modal waters made the upper waters colder ( 0.4c ) , creating a denser ( + 0.1 kg m ) , warmer ( + 0.23c ) , and more saline ( + 0.015 ) type of enacw ( figures 2 and s1 ) [ somavilla et al . this suggests that 70% of the estimated 5.810 j m of heat gained slowly but persistently between 1994 and 2005 in the shallower levels ( 0.7c in the 100300 m layer , table s1 and figure 2a ) was transferred into a denser variety of mode waters through the extreme winter mixing . in addition , considering that such heat injection took place during winter 2005 , the heat uptake ( q ) at intermediate depths would be 30 w m ( 5 w m for the 300700 m layer ) , between 65 and 100 times faster than the average ocean heat uptake at such intermediate depths ( 300700 m ) between 2000 and 2010 ( see table s1 ) . forced by atmospheric cooling , the convective mixing would have released heat to the atmosphere ( see comments in table s1 for additional information ) . most of the enhanced warming observed at intermediate depths at mlena shown in figure 1 ( + 0.33c gained between 1997 and 2012 ) is explained by the increase in the modal waters during the winter of 2005 ( + 0.23c ) . the estimated overall heat content increase in the region is 1.2 10 j and accounts for 44% of the total heat content increase at intermediate depths in the north atlantic from 36 to 65n from 1997 to 2012 ( table s2 ) . evidence suggests that this extreme winter mixing episode may have contributed to the deep ocean heat injection at higher latitudes by inducing ocean circulation changes that enabled northward propagation of the relatively salty enacw . the associated zonal dynamic height sections were thus altered , and the zonal dynamic height slope eastward of 22w changed from almost flat or negative to positive ( figures 3e3 g ) . estimated as the geostrophic meridional current between 22 and 12w ( v g=/f l , where is the difference between the dynamic height at 12w and 22w , f the coriolis parameter , and l the distance between the two longitudes ) , the meridional current after 2005 estimated from the mean filtered dynamic height from argo floats is + 0.7 cm s ( northward ) with respect to 0.4 cm s ( southward ) estimated from woa01 climatological dynamic height ( with reference level at 1500 m both for argo floats and woa01 data ) . the inversion of the zonal dynamic height slope is a robust feature ( e.g. , with reference level at 1000 m , the meridional current after 2005 estimated from argo floats is + 0.6 cm s ( northward ) with respect to 0.35 cm s ( southward ) estimated from woa01 climatological dynamic height ) as also observed in oras4 meridional current ( figure s3 ) . a similar flow reversal was predicted by a sensitivity experiment in a global ocean circulation model , and anomalous northward advection of salt was obtained as a result of surface cooling at 45n in the ena [ svellec et al . , ( a ) map of the northeast atlantic showing the location of the nwse , we , and swne sections across the enacw formation area for which the data are shown in figures 3b3 g . ( b d ) location of the = 27.15 at the nwse , ew , and swne sections marked in figure 3a based on woa01 climatological data ( grey ) and available argo floats in the corresponding section between 2005 and 2010 ( cyan ) . ( e g ) idem for the dynamic height between 300 and 1500 m ( see section 2.3 in the supporting information for details ) . thus , the + 0.1 kg m denser core of enacw ( =27.227.3 kg m after 2005 ) might easily get incorporated to the major branch of the north atlantic current flowing northward . figure 4 is the zoom into the north atlantic in figure s2 en4 ( c and d ) ( see figure s2 procedure description for details ) . due to the weak circulation and dominance of mesoscale activity in the mlena region , finding direct evidence of a flow reversal as the one inferred from dynamic height and reanalysis current data is difficult . argobased deep displacements [ ollitrault and rannou , 2013 ] are an available source , and luckily , a number of floats drifting at enacw levels were active in the region ( see figure s4 ) . the outcome from the analysis of their lagrangian displacements upper water temperature and salinity in the subpolar gyre and the nordic seas increased since early the 2000s ; this tendency is attributed to the arrival of warmer and saltier western subtropical waters caused by the contraction of the subpolar gyre [ htn et al . , 2008 ; beszczynskamller and dye , 2013 ; http://ocean.ices.dk/iroc/ ] would be consistent with a higher transport of western subtropical waters , that higher transport does not account for the drop in nutrient concentration in the eastern subpolar north atlantic ( rockall trough , figure 1 ) observed from 2007 onward [ johnson et al . both the eastern and western north atlantic central water types have the potential to increase temperature and salinity at higher latitudes . however , enacw are saltier than western waters arriving at the eastern subpolar north atlantic [ johnson et al . , 2013 ] , providing an alternative explanation for the highestsalinity anomalies observed between 2007 and 2010 in the subpolar north atlantic and nordic seas . 2008 ] , a lag of 2 to 5 years is expected for the salinity anomaly ( from the mlena ) to reach first the eastern subpolar north atlantic and then the western subpolar gyre and northern nordic seas . in these areas , the arrival of salty southern waters due to the circulation shift in the mlena would have contributed to the highsalinity conditions required for deep ocean heat uptake through density compensated anomalies [ mauritzen et al . indeed , a strong northward flow east of 22w and south of 48n in the a priori recirculation area at the ' ovide ' section crossing the north atlantic from portugal to greenland was observed in 2006 , coincident with the lowest amoc measured through the section between 1997 and 2010 [ mercier et al . , consistent with the aforementioned spreading timescale and presence of the saltiest upper waters after 2007 in these areas , the heat and salt uptake below 700 m in the subpolar gyre and the nordic seas increased after 2007 ( figures s1 and s2 ) , coinciding with more intensive deep convection activity in both the subpolar gyre ( irminger and labrador seas , [ yashayaev and loder , 2009 ; kieke and yashayaev , 2015 ; de jong et al . after years of sustained warming and increasing salinity in the upper layers , the first deep convection events at each of these sites resemble the enacw transformation in 2005 . in the deep water formation region of the greenland sea , a strong mixinginduced deep ocean heat injection event occurred through density compensated anomalies in 2008 ( figure 5 ) . upper ocean salinity in this region had been increasing since the mid1990s , and the 2008 winter cooling made the surface waters dense enough to trigger deep convection with temperature and salinity higher than typical values ( figure s5 ) . as a consequence of the overturning , the heat and salt gained slowly over time by the upper layers were directly injected to great depths , with a resulting sudden temperature and salinity increase of 0.16c and 0.01 , respectively ( figures 5c and 5d ) . coincident with the arrival of highly saline waters in the upper layers of the subpolar gyre , a strong winter mixing occurred in the irminger sea in 2007 [ piron et al . as in the greenland sea , subsequent deep convection events followed in later years ( 2008 , 2009 , and 2012 ) [ de jong et al . finally , coinciding with the arrival of highly saline waters in the upper levels , deep convection has reached levels deeper than 1700 m in the labrador sea in 2008 after more than a decade of weak convection , being repeated in 2012 and 2014 [ kieke and yashayaev , 2015 ] . as in the greenland and irminger seas , the first deep convection events appear to be associated with downward transfer of heat and salt [ see yashayaev and loder , 2009 , figure 2a ; kieke and yashayaev , 2015 , figure 4b ] . in general , the steplike temperature increases at depth at deep convection sites caused by the strong mixinginduced heat injection events are equivalent to heat uptake pulses between 16 and 25 w m , which is between 50 and 100 times faster than the average heat uptake below 700 m in the global ocean ( see table s1 ) . as in the mlena , concurrent with the deep ocean heat injection , the deep convective mixing events would have released heat to the atmosphere , favoring deceleration of the north atlantic heat uptake from the atmosphere , as apparently observed since the mid2000s [ chen and tung , 2014 ; hkkinen et al . , deep ( a ) temperature and ( b ) salinity ( 14001600 dbar , =27.7827.8 kg m ) in the irminger sea ( 6062n ; 3540w , green square in figure grey dots indicate vertically averaged argo temperature and salinity values in the pressure range of interest from individual profiles . the primary cause of the extremely cold winter in 2005 in the mlena was the occurrence of atmospheric blocking anomalies in the ena . blocking anomalies over the ena occurred previously in the late 1950s and 1960s and extreme winter mixing events ( similar to that in 2005 ) enhanced northward flow of enacw may have occurred and contributed ( along with western subtropical waters ) to the intrusion of warmer and saltier waters into the eastern subpolar north atlantic and nordic seas during the 1960s [ holliday et al . , the heat uptake in the 1960s had smaller amplitude compared to the 2005 event , and this may be related to the sustained warming trend in the global upper ocean in the few decades prior to 2005 . both warmer arctic and tropical pacific have been associated with a wavier jet stream responsible for an increase in blocking events in the north atlantic and higher frequency of extreme weather events in general [ francis and vavrus , 2012 ; trenberth et al . , the blocking pattern developed in winter 2005 in the ena region is not inconsistent with natural climate variability [ barnes et al . however , overall global warming is expected to affect the development of such atmospheric features [ alexander et al . estimating the global significance of ocean heat transfer from upper to deeper levels by the mechanism described here requires a better model representation of the frequency and duration of wintertime blocking events , which have been underestimated in most global climate models thus far [ hov et al . , since the mid2000s , in contrast to the enhanced ocean heat uptake observed in other ocean basins , the north atlantic deep ocean heat content has significantly increased without apparently intervening ocean heat uptake from the atmosphere . about 40% of the increase in deep ocean heat content increase below 300 m is centered at midlatitudes in the eastern north atlantic and occurred relatively quickly around the mid2000s far from prominent deep ocean ventilation sites . we suggest that a sequence of connected strong mixinginduced heat injection events originating from this region triggered large transfer of heat from upper to deeper ocean layers , implying a deceleration or halt of the north atlantic heat uptake from the atmosphere . a large transformation of modal water masses in the eastern north atlantic in the winter of 2005 played a major role in the observed quick transfer of heat from upper to deep ocean layers but also aided a reverse of the southward circulation of saltier waters from the eastern side of the temperate atlantic with far reaching consequences ; the northward flow of saltier waters into the subpolar north atlantic and nordic seas favored additional heat injection through deep convection events in later years . thus , we agree that existing highsalinity conditions in the late 2000s in areas of deep convection have aided heat transfer to the deep ocean , but the suggestion that an increase of the amoc is behind such salinity increase is not supported by observations . anomalous atmospheric patterns such as the one behind this shift are not unique to the last decade , although they may have been exacerbated under global warming .

the enhanced downward heat transfer necessary for this vertical heat redistribution has been proposed to be related to the hiatus in global warming , although the hiatus started earlier and lasted for the 15 years period 19982013 [ trenberth et al . two natural mechanisms have been proposed to explain the deceleration of the rise in global mean surface temperature and the efficient transfer of heat to the deep ocean : ( i ) the intensification of zonal winds causing la nialike conditions in the tropical pacific [ meehl et al . , 2013 ; kosaka and xie , 2013 ] and ( ii ) decadal variations in the strength of deep convection and currents in the north atlantic [ meehl et al . , modeling simulations show that la nia surface cooling in the eastern and central tropical pacific can decrease the rate of increase in atmospheric temperature ( although the similarity of observed and simulated deceleration is still debated [ trenberth , 2015 ; karl et al . , associated to this anomalous heat flux into the ocean , subsurface heat uptake in the western tropical pacific and indian oceans occurs [ goddard , 2014 ; england et al . regarding the second mechanism , vertical heat inventories of the north atlantic show notable heat transfer from the upper ( 0300 m ) to intermediate ( 300700 m ) and deep ( > 700 m ) layers since the mid2000s ( see figure s1 and corresponding comments ) [ alexander et al . , 2014 ; however , concomitant with the downward heat export after the mid2000s , the increase in ocean heat content in the upper 2000 m of the north atlantic paused [ chen and tung , 2014 , figures 1 and 3 ; hkkinen et al . , 2015 , figure 1 ] , making the bulk rate of change of ocean heat content per unit area in the basin almost zero . , therefore , one can conclude that the heat redistribution in the north atlantic since the mid2000s as a whole has apparently occurred without extra heat uptake from the atmosphere . in particular , the north atlantic would not have contributed notably as in previous decades to atmospheric heat uptake , in contrast to that observed in the tropical pacific and southern oceans [ roemmich et al . , 2015 ] , but to the quick transfer of heat accumulated during the previous decades in the upper layers to the deep ocean . deep convection in the subpolar north atlantic under highsalinity conditions might account for the quick transfer of heat to the deep ocean [ chen and tung , 2014 ] . second , deep convection in the subpolar north atlantic is restricted to the labrador and irminger seas ( see figure 1 ) , but far from these prominent deep ocean ventilation sites , ocean observations seem to show the largest deep ocean heat uptake at midlatitudes in the eastern north atlantic ( figure 1 ) [ desbruyres et al . temperature differences ( t in color , c ) in the 300700 m layer between the 20072012 and the 19972002 periods ( i.e. figure 1 is the zoom into the north atlantic in figure s1 ocean reanalysis system 4 ( oras4 ) ( d ) ( see figure s1 procedure description for details ) . arrows represent ( in red ) warm and salty upper water currents , ( in dark blue ) cold and fresh deep water currents , ( in black , dotted ) the subpolar gyre , and ( in black , dashed ) the subtropical gyre . deep convection sites in the irminger and greenland seas as well as the position of the santander standard section in the bay of biscay are marked by green squares . midlatitudes in the eastern north atlantic ( mlena ) , considered as the oceanic area including the mode water formation area of the eastern north atlantic central water ( enacw ) and the oceanic waters of the bay of biscay and surrounding the iberian peninsula to north africa as in somavilla et al . we reexamine the mechanism responsible for the quick transfer of ocean heat from upper to deeper layers in the north atlantic focusing on the role of ena modal waters . to overcome this issue , we combined standard hydrographic observations ( salinity and temperature profiles ) at oceanographic sections regularly maintained in the bay of biscay and the greenland sea ( see figure 1 ) with other data sets that offer more global coverage such as argo floats , globalgridded ocean reanalysis ( european centre for mediumrange weather forecasts ocean reanalysis system 4 , oras4 ) and objective analysis ( met office hadley centre monthly objective analysis , en4 , and world ocean database 2001 , woa01 ) . with a similar purpose of increasing the reliability of individual data sets , warming / cooling and saltening / freshening maps obtained from different gridded data are compared ( figures s1 and s2 ) thus , we increase the confidence of the enhanced warming at midlatitudes in the eastern north atlantic as shown in figure 1 , since similar warming distributions showing enhanced heat uptake at midlatitudes in the ena and surrounding the subpolar north atlantic margins are observed in other gridded products ( figure s1 based on en4 ; figure 3c in chen and tung based on ishii data ; figure 4 to 6 in hkkinen et al . ( a ) mean temperature ( white line ) between the shallower winter mixed layer depth achieved in warm winters and the enacw upper bound ( 100300 m ) and ( black line ) in the depth range of the enacw core ( 300550 m ) in the enacw formation area ( 4348n , 1020w ) obtained from oras4 reanalysis data set . ( b and c ) same as figure 2a but for temperature and salinity at =27.227.3 kg m ( corresponding to the + 0.1 kg m 2005 denser enacw core ) . the magenta line in figure 2b shows the background average warming trend ( + 0.1c decade ) at the intermediate depth ( 300700 m ) for the global ocean ( table s1 ) . , , the ocean surface at midlatitudes in the eastern north atlantic ( mlena , see figure 1 for geographical reference ) experienced the highest densification ( buoyancy loss ) since the 1960s , causing the winter mixed layer to reach very deep levels likely unprecedented for decades [ somavilla et al . the induced deep convective mixing resulted in homogeneization of properties of the water column from the surface to density levels of =27.227.3 kg m , at depth ranges of 350500 m , well beyond the normal airsea interaction extent at the core of enacw ( =27.127.2 kg m ) . this mixing of ( warmer and saltier ) upper water and ( colder and fresher ) modal waters made the upper waters colder ( 0.4c ) , creating a denser ( + 0.1 kg m ) , warmer ( + 0.23c ) , and more saline ( + 0.015 ) type of enacw ( figures 2 and s1 ) [ somavilla et al . such temperature increase at the enacw core ( + 0.23c ) resulted in a sudden heat content increase of 2.410 j m for the 350500 m layer ( 3.9 0.610 j m for the 300700 m layer , table s1 ) . this suggests that 70% of the estimated 5.810 j m of heat gained slowly but persistently between 1994 and 2005 in the shallower levels ( 0.7c in the 100300 m layer , table s1 and figure 2a ) was transferred into a denser variety of mode waters through the extreme winter mixing . in addition , considering that such heat injection took place during winter 2005 , the heat uptake ( q ) at intermediate depths would be 30 w m ( 5 w m for the 300700 m layer ) , between 65 and 100 times faster than the average ocean heat uptake at such intermediate depths ( 300700 m ) between 2000 and 2010 ( see table s1 ) . most of the enhanced warming observed at intermediate depths at mlena shown in figure 1 ( + 0.33c gained between 1997 and 2012 ) is explained by the increase in the modal waters during the winter of 2005 ( + 0.23c ) . the estimated overall heat content increase in the region is 1.2 10 j and accounts for 44% of the total heat content increase at intermediate depths in the north atlantic from 36 to 65n from 1997 to 2012 ( table s2 ) . estimated as the geostrophic meridional current between 22 and 12w ( v g=/f l , where is the difference between the dynamic height at 12w and 22w , f the coriolis parameter , and l the distance between the two longitudes ) , the meridional current after 2005 estimated from the mean filtered dynamic height from argo floats is + 0.7 cm s ( northward ) with respect to 0.4 cm s ( southward ) estimated from woa01 climatological dynamic height ( with reference level at 1500 m both for argo floats and woa01 data ) . oras4 meridional current data show a comparable northward reversal of the flow centered at 18w from 2005 to 2010 ( mean value = + 1 cm s , maximum value = + 3 cm s ) with respect to its southward direction ( mean value = 0.5 cm s , maximum value = 3 cm s ) from 1990 to 2005 ( see figure s3 ) . , with reference level at 1000 m , the meridional current after 2005 estimated from argo floats is + 0.6 cm s ( northward ) with respect to 0.35 cm s ( southward ) estimated from woa01 climatological dynamic height ) as also observed in oras4 meridional current ( figure s3 ) . a similar flow reversal was predicted by a sensitivity experiment in a global ocean circulation model , and anomalous northward advection of salt was obtained as a result of surface cooling at 45n in the ena [ svellec et al . , ( a ) map of the northeast atlantic showing the location of the nwse , we , and swne sections across the enacw formation area for which the data are shown in figures 3b3 g . ( b d ) location of the = 27.15 at the nwse , ew , and swne sections marked in figure 3a based on woa01 climatological data ( grey ) and available argo floats in the corresponding section between 2005 and 2010 ( cyan ) . it is worth noting that the maximum water exchange between the subtropical and the subpolar gyres occurs at a density level of =27.227.4 kg m [ brambilla and talley , 2006 ] . thus , the + 0.1 kg m denser core of enacw ( =27.227.3 kg m after 2005 ) might easily get incorporated to the major branch of the north atlantic current flowing northward . salinity difference ( s in color ) in the 300700 m layer between ( a ) the 20022005 and the 19972002 periods and ( b ) the 20072012 and the 19972002 periods based on en4 data set ( i.e. figure 4 is the zoom into the north atlantic in figure s2 en4 ( c and d ) ( see figure s2 procedure description for details ) . due to the weak circulation and dominance of mesoscale activity in the mlena region , finding direct evidence of a flow reversal as the one inferred from dynamic height and reanalysis current data is difficult . the outcome from the analysis of their lagrangian displacements upper water temperature and salinity in the subpolar gyre and the nordic seas increased since early the 2000s ; this tendency is attributed to the arrival of warmer and saltier western subtropical waters caused by the contraction of the subpolar gyre [ htn et al . , 2008 ; beszczynskamller and dye , 2013 ; http://ocean.ices.dk/iroc/ ] would be consistent with a higher transport of western subtropical waters , that higher transport does not account for the drop in nutrient concentration in the eastern subpolar north atlantic ( rockall trough , figure 1 ) observed from 2007 onward [ johnson et al . , 2013 ] , providing an alternative explanation for the highestsalinity anomalies observed between 2007 and 2010 in the subpolar north atlantic and nordic seas . 2008 ] , a lag of 2 to 5 years is expected for the salinity anomaly ( from the mlena ) to reach first the eastern subpolar north atlantic and then the western subpolar gyre and northern nordic seas . in these areas , the arrival of salty southern waters due to the circulation shift in the mlena would have contributed to the highsalinity conditions required for deep ocean heat uptake through density compensated anomalies [ mauritzen et al . indeed , a strong northward flow east of 22w and south of 48n in the a priori recirculation area at the ' ovide ' section crossing the north atlantic from portugal to greenland was observed in 2006 , coincident with the lowest amoc measured through the section between 1997 and 2010 [ mercier et al . , consistent with the aforementioned spreading timescale and presence of the saltiest upper waters after 2007 in these areas , the heat and salt uptake below 700 m in the subpolar gyre and the nordic seas increased after 2007 ( figures s1 and s2 ) , coinciding with more intensive deep convection activity in both the subpolar gyre ( irminger and labrador seas , [ yashayaev and loder , 2009 ; kieke and yashayaev , 2015 ; de jong et al . after years of sustained warming and increasing salinity in the upper layers , the first deep convection events at each of these sites resemble the enacw transformation in 2005 . as a consequence of the overturning , the heat and salt gained slowly over time by the upper layers were directly injected to great depths , with a resulting sudden temperature and salinity increase of 0.16c and 0.01 , respectively ( figures 5c and 5d ) . with a less stratified water column , convection occurred at greater depths in the subsequent years as compared to previous decades in the greenland sea [ beszczynskamller and dye , 2013 ] . coincident with the arrival of highly saline waters in the upper layers of the subpolar gyre , a strong winter mixing occurred in the irminger sea in 2007 [ piron et al . finally , coinciding with the arrival of highly saline waters in the upper levels , deep convection has reached levels deeper than 1700 m in the labrador sea in 2008 after more than a decade of weak convection , being repeated in 2012 and 2014 [ kieke and yashayaev , 2015 ] . as in the greenland and irminger seas , the first deep convection events appear to be associated with downward transfer of heat and salt [ see yashayaev and loder , 2009 , figure 2a ; kieke and yashayaev , 2015 , figure 4b ] . in general , the steplike temperature increases at depth at deep convection sites caused by the strong mixinginduced heat injection events are equivalent to heat uptake pulses between 16 and 25 w m , which is between 50 and 100 times faster than the average heat uptake below 700 m in the global ocean ( see table s1 ) . as in the mlena , concurrent with the deep ocean heat injection , the deep convective mixing events would have released heat to the atmosphere , favoring deceleration of the north atlantic heat uptake from the atmosphere , as apparently observed since the mid2000s [ chen and tung , 2014 ; hkkinen et al . , deep ( a ) temperature and ( b ) salinity ( 14001600 dbar , =27.7827.8 kg m ) in the irminger sea ( 6062n ; 3540w , green square in figure grey dots indicate vertically averaged argo temperature and salinity values in the pressure range of interest from individual profiles . , regional enhanced mixing altering the largescale density structure ) , blocking anomalies in the ena generate a dipole of temperature anomalies between europe ( cold ) and greenland ( warm ) associated with a strong northward meridional atmospheric flow [ barriopedro et al . blocking anomalies over the ena occurred previously in the late 1950s and 1960s and extreme winter mixing events ( similar to that in 2005 ) enhanced northward flow of enacw may have occurred and contributed ( along with western subtropical waters ) to the intrusion of warmer and saltier waters into the eastern subpolar north atlantic and nordic seas during the 1960s [ holliday et al . , the heat uptake in the 1960s had smaller amplitude compared to the 2005 event , and this may be related to the sustained warming trend in the global upper ocean in the few decades prior to 2005 . both warmer arctic and tropical pacific have been associated with a wavier jet stream responsible for an increase in blocking events in the north atlantic and higher frequency of extreme weather events in general [ francis and vavrus , 2012 ; trenberth et al . estimating the global significance of ocean heat transfer from upper to deeper levels by the mechanism described here requires a better model representation of the frequency and duration of wintertime blocking events , which have been underestimated in most global climate models thus far [ hov et al . , since the mid2000s , in contrast to the enhanced ocean heat uptake observed in other ocean basins , the north atlantic deep ocean heat content has significantly increased without apparently intervening ocean heat uptake from the atmosphere . about 40% of the increase in deep ocean heat content increase below 300 m is centered at midlatitudes in the eastern north atlantic and occurred relatively quickly around the mid2000s far from prominent deep ocean ventilation sites . we suggest that a sequence of connected strong mixinginduced heat injection events originating from this region triggered large transfer of heat from upper to deeper ocean layers , implying a deceleration or halt of the north atlantic heat uptake from the atmosphere . a large transformation of modal water masses in the eastern north atlantic in the winter of 2005 played a major role in the observed quick transfer of heat from upper to deep ocean layers but also aided a reverse of the southward circulation of saltier waters from the eastern side of the temperate atlantic with far reaching consequences ; the northward flow of saltier waters into the subpolar north atlantic and nordic seas favored additional heat injection through deep convection events in later years . thus , we agree that existing highsalinity conditions in the late 2000s in areas of deep convection have aided heat transfer to the deep ocean , but the suggestion that an increase of the amoc is behind such salinity increase is not supported by observations .

msc are multipotent cells , capable of differentiating , in vitro , into different lineages , including osteocytes , chondrocytes , adipocytes , muscle cells , cardiomyocytes , and neural precursor . more recently , the capacity of human msc ( hmsc ) to suppress both innate and adaptive immunity has been described [ 2 , 3 ] as well as their poor immunogenicity . as a result , the therapeutic potential of hmsc as immunoregulatory agents is currently being explored in several phase i / ii clinical trials [ 46 ] . a number of recent studies have focused on the influence of hmsc on dc functions [ 2 , 7 , 8 ] . can be generated from cd34 stem / progenitor cells and from cd14 monocytes [ 10 , 11 ] . the in vitro interaction between hmsc and either cd34 or cd14 dc progenitors inhibits the generation of functional dc [ 2 , 7 , 8 ] , skewing their differentiation toward phenotypically abnormal dc , which express lower level of cd1a , cd40 , cd80 , cd86 , and cd83 . different mechanisms are responsible for the effects of hmsc on dc differentiation including the involvement of both soluble factors and cell - to - cell contact interactions [ 12 , 13 ] . taken together , these studies demonstrated the profound immunosuppressive effects of hmsc on dc . however , the topic of whether the immunological properties of hmsc persist after in vitro differentiation into hdoc has been not deeply investigated . indeed , it has only been shown that hdoc suppress t cell proliferation elicited by allogeneic cells [ 14 , 15 ] . in fact , the characterization of the immunological properties of hdoc may be of crucial importance for cell - based tissue - engineering therapeutic strategies . although hmsc have been shown to contribute to repair bone defects in vivo either through infusion or local implantation [ 16 , 17 ] , more recent strategies aim to in vitro differentiate hmsc into hdoc before implantation , in order to optimize bone regeneration [ 18 , 19 ] . therefore , the evaluation of the interactions between hdoc and the host allogeneic immune system , in particular of antigen presenting cells ( apc ) such as dc , may be important in view of clinical trials . since the preparation of hdoc for cell - based tissue - engineering strategies may be different [ 18 , 19 ] , it is interesting to evaluate whether different culture systems may affect the capacity of doc of modulating the immune response . for these reasons , here we characterized the immunological properties of hdoc manipulated in two different ways , which mimic the use of hdoc as cell suspension or as adherent cells , by showing their capacity to modulate the phenotype and the functions of allogeneic dc . human mscs ( hmscs ) were isolated from bm aspirates of healthy donors after obtaining written informed consent . the characteristics of the donors are shown in table s1 ( see supplementary material available online at http://dx.doi.org/10.1155/2015/526195 ) . to induce hdoc , hmscs were seeded at 3.1 10 cells / cm and grown in osteogenic differentiation medium ( lonza ) containing l - glutamine , mcgs , dexamethasone , ascorbate , -glycerophosphate , and penicillin / streptomycin . the osteogenic differentiation was analysed by cytological staining and by the evaluation of runx2 mrna levels ( figure s1 ) . briefly , cells were fixed in 10% pfa in pbs for 15 min at room temperature ( rt ) , rinsed with pbs and distilled water , and then stained with 40 mm alizarin red solution ( sigma aldrich ) ph 4.2 , for 75 min at rt with gentle agitation . after washing , alizarin red was extracted from fixed cells by incubating in 10% ( w / v ) cetylpyridinium chloride ( cpc ) solution ( sigma aldrich ) in 10 mm sodium phosphate for 15 min at rt with gentle agitation . after 2 weeks hmsc and hdoc were lysed and total rna was extracted using rneasy micro kit ( qiagen ) and reverse transcribed using a promega improm ii kit and random examers in 20 l final volume . quantitative real - time pcr was performed using an abi prism 7900 sequence detection system ( applied biosystems ) . the relative level of runx2 mrna was calculated by subtracting ct values of the control gene ( gapdh ) from the ct values of the specific gene ( runx2 ) . primers probe for runx2 , hs00231692_m1 , and gapdh , hs00266705_g1 were purchased from applied biosystems . for immunological assays , hdoc were used either in adhesion on the same plate used for differentiation ( adh - doc ) or after harvesting and replating ( det - doc ) . for harvesting , monolayers of hdoc were washed with washing buffer ( 0.5% bsa , 5 mm edta in pbs ) , incubated with trypsin ( 0.25% trypsin with 0.1% edta , euroclone ) , and collected with a cell scraper . where indicated the cellular component was lysed by incubating the monolayer with 0.1 m nacl , 0.01 m tris , 0.2% edta , and 0.1% tritonx-100 . in all the assays , the same number of adh - doc and det - doc was used . for each experiment , the cell number was evaluated the day before the assays , by counting 3 wells of adh - doc harvested separately . the mean values of these 3 counts were used to plate det - doc . then , adh - doc and det - doc were cultured overnight in complete medium ( rpmi 1640 medium ( lonza ) supplemented with 10% heat - inactivated fbs ( gibco - invitrogen ) , 2 mm l - glutamine , 100 u / ml penicillin , and 100 g / ml streptomycin ( mp biomedicals ) ) at 37c in 5% co2 , before coculturing with dc and cd3 t cells . the experimental strategy is showed in a schema ( figure s2 ) . the same strategy and the same conditions indeed , the number of det - msc to plate was established by counting independently 3 wells of adh - msc . buffy coats were obtained from healthy donors and were used to isolate mononuclear cells ( mnc ) , by gradient centrifugation ( lymphoprep ; 1.077 g / ml ; nycomed pharma ) . after separation , cd14 monocytes and cd3 t cells were purified from total mnc by magnetic separation columns ( miltenyi biotec ) , according to manufacturer 's instructions . monocyte - derived dcs ( mo - dcs ) were generated by a 5-day culture of cd14 cells in complete medium plus 50 ng / ml granulocyte - macrophage colony - stimulation factor ( gm - csf ) and 800 u / ml il-4 ( both by endogen ) , at 37c in 5% co2 . for maturation , day 5 mo - dcs were cultured with gm - csf and il-4 and incubated for 48 hours in presence of a cocktail of cytokine made of 10 ng / ml tnf , 10 ng / ml il-6 , 10 ng / ml il-1 , and 1 g / ml pge2 ( all by endogen ) . to detect lymphocytes proliferation , cd3 t cells were labelled with carboxyfluorescein succinimidyl ester ( cfse , 2.5 m from sigma aldrich ) before plating . to test the capacity of adh - doc and det - doc to modulate the dc allostimulatory capacity , allogeneic mo - dc , either immature or mature , were irradiated ( 3000 cgy ) and plated ( 1 : 1 ) at 37c on either adh - doc or det - doc in presence of third party cfse - labeled cd3 t cells ( 1 : 10 ) . as a positive control , cd3 t cells were cultured with mo - dc and , as a negative control , with medium alone . for a second set of experiments , cd3 t cells were added to the upper chamber of a 0.4 m pore polycarbonate filter in 24-well transwell chambers ( corning costar ) to keep them separated from dc and from either adh - doc or det - doc , which were plated in the lower chamber of the transwell system . after 5 days , the cells in the upper chamber were collected and analyzed using bd facscantoii equipment ( bd biosciences ) . to test the capacity of adh - doc and det - doc to modulate the dc capacity to induce in cd3 t cells the treg cell or the th17 cell phenotype , allogeneic mo - dc , either immature or mature , were plated ( 1 : 1 ) on either adh - doc or det - doc in presence of allogeneic cd3 t cells ( 1 : 20 ) . as a positive control , cd3 t cells were cultured with mo - dc , as a negative control , with medium alone . for a second set of experiments , cd3 t cells were added to the upper chamber of a 0.4 m pore polycarbonate filter in 24-well transwell chambers ( corning costar ) , while dc and either adh - doc or det - doc were added in the lower chamber . cell cultures were incubated at 37c for 5 days ; then the t cells were harvested from the upper chamber and stained for immunophenotype via tricolor immunofluorescence , which was performed using fluorescein isothiocyanate- ( fitc- ) conjugated anti - human cd4 ( clone rpa - t4 ) , phycoerythrin- ( pe- ) conjugated anti - human foxp3 ( clone 206d ) , and allophycocyanin- ( apc- ) conjugated anti - human cd25 ( clone bc96 , biolegend ) . for cell - surface staining , 1 10 cells/100 l were incubated in the dark for 20 min at rt with mabs in phosphate - buffered saline- ( pbs- ) 1% bovine serum albumin . subsequently , for foxp3 intracellular staining , cells were incubated at rt in the dark for 20 min with fix / perm buffer followed by 15 min with perm solution and additional 30 min with the mab . after 2 washes , samples were analyzed using bd facscantoii equipment ( bd biosciences ) . immature and mature mo - dc were incubated for 5 days either alone or with adh - doc or det - doc ( 1 : 1 ) . dual - color immunofluorescence was performed using the following panel of mabs : pe- or fitc - conjugated anti - human hla - dr ( bd pharmingen ; clone l242 ) , cd86 ( biolegend ; clone it2.2 ) , and cd80 ( biolegend ; clone 2d10 ) . immature and mature mo - dc were incubated for 5 days either alone or with adh - doc or det - doc ( 1 : 1 ) . supernatants were collected and tested for the release of tgf-1 ( drg diagnostics , marburg , germany ) il-6 and il-10 ( thermo scientific , erembodegem , belgium ) , according to the manufacturer 's instructions . = p < 0.05 , = p < 0.01 , and = p < 0.001 , bonferroni corrected . we first compared the suppressive capacity of hdoc on dc - mediated t cell proliferation . in particular , we used hdoc either after detachment from culture plates ( det - doc ) or as adherent cells ( adh - doc ) to mimic , in vitro , the conditions in which they are used therapeutically ( i.e. , cultured and then detached to be injected as cellular suspension or grown adherent on a scaffold and implanted without detachment ) . as shown in figures 1(a ) and 1(b ) , both det - doc and adh - doc inhibited the allostimulatory capacity of dc ( kruskal wallis p < 0.0001 ) . however , the inhibition induced by adh - doc is much stronger than det - doc thus , we asked why det - doc partially lost their ability to inhibit dc allostimulation . to investigate the mechanism(s ) involved in such process , we first asked whether det - doc were less viable than adh - doc , since they were harvested after treatment with trypsin and collected with a cell scraper . we could not find a reduction of viability in harvested det - doc ( data not shown ) . then , we asked whether the extracellular matrix secreted by hdoc ( routinely discarded to obtain det - doc ) could itself inhibit the allostimulatory capacity of dc . therefore , we lysed the cellular component of hdoc and recovered the extracellular matrix to perform the same assay . as shown in figure 1(c ) , the extracellular matrix had no inhibitory capacity by itself . then , we asked whether the procedure used to detach cells from the plates influenced by itself the tolerogenic capacity of detached doc . thus , we compared the capacity of undifferentiated hmsc used after detachment from culture plates ( mimicking det - msc ) with that of cells used as adherent cells ( mimicking adh - msc ) . as shown in figure 1(d ) , both det - msc and adh - msc were able to inhibit the dc allostimulation at the same level , suggesting that the detachment procedure , by itself , had no influence on the tolerogenic capacity of the cells . finally we investigated whether the different capacity to inhibit dc allostimulation of det - doc and adh - doc was mediated by soluble factors rather than by a cell - to - cell contact - dependent mechanism(s ) . therefore , we performed the same suppression assay by separating cd3 t cells with a 0.4 m pore transwell chamber , which allows the migration of only soluble factors . as shown in figure 1(e ) , neither det - doc nor adh - doc could inhibit dc allostimulation when dc are cultured without any contact with cd3 t cells . therefore , in this setting , hdoc modified the allostimulatory capacity of dc , mainly by modulating cell - to - cell contact - dependent mechanism(s ) . thus , we wanted to investigate which surface marker(s ) on dc may be affected by the inhibitory activity of hdoc [ 24 , 25 ] . to this end , we cocultured dc with either det - doc or adh - doc , for 5 days and then analyzed dc phenotype ( kruskal - wallis p < 0.0001 for cd80 , p = 0.0012 for cd86 ) . to discriminate dc from hdoc we gated on hla - dr cells . as shown in figure 2(a ) , the coculture of dc with det - doc or adh - doc resulted in the downregulation of cd80 . notably , adh - doc and det - doc could downregulate this marker to a comparable extent . conversely , det - doc were not able to downregulate cd86 , while this molecule was strongly downregulated by adh - doc , as shown in figure 2(b ) . taken together , our results demonstrate that , if hdoc are left in their own extracellular matrix ( adh - doc ) , they acquire a high capacity to inhibit the dc expression of molecules involved in t cell costimulation . on the other hand , if the interactions of hdoc with their extracellular matrix are destroyed ( i.e. , hdoc are harvested from their own extracellular matrix and seeded back in a new culture support , as for det - doc ) , they partly lose this capacity , resulting in lower inhibition of costimulatory molecules on dc and , consequently , of t cell proliferation . dc have a crucial role not only in the activation of t cell response but also in the induction of tolerance by generating cd4cd25foxp3 t cell population . therefore , we asked whether det - doc and adh - doc could modulate this function of dc . to test this hypothesis , we cocultured dc with allogeneic t cells , in the presence or absence of either det - doc or adh - doc ( kruskal - wallis p < 0.0001 ) . coculture of t cells with dc increased the percentage of cd4cd25foxp3 t cells , as compared to cd3 t cells alone ( figures 3(a ) and 3(b ) ) . the addition of det - doc or adh - doc decreased the population of cd4cd25foxp3 t cells induced by dc ( figures 3(a ) and 3(b ) ) , with the adh - doc showing the greatest inhibitory effect ( p < 0.001 , bonferroni corrected ) . then , we asked whether the different capacity to inhibit the dc - mediated cd4cd25foxp3 t cell generation of det - doc and adh - doc was mediated by soluble factors rather than by a cell - to - cell contact - dependent mechanism . therefore , we performed the same assay by separating cd3 t cells with a 0.4 m pore transwell chamber . as shown in figure 3(c ) , det - doc and adh - doc did not inhibit the capacity of dc to induce a cd4cd25foxp3 t cell population and there was no significant difference among different hdoc . therefore , adh - doc had an enhanced inhibitory effect on dc to induce cd4cd25foxp3 t cell population , in comparison to det - doc , by modulating mechanism(s ) involved in cell - to - cell , contact - dependent suppression . as shown in figure 2 , adh - doc were able to downregulate the expression of both cd80 and cd86 , while det - doc decreased only cd80 at a lower extent . . taken together , these data demonstrate that hdoc inhibit the capacity of dc to induce cd4cd25foxp3 t cells . however , such inhibitory capacity is markedly increased when hdoc are left adherent to the same plate used for differentiation ( adh - doc ) , whereas it is reduced when hdoc are detached from culture plates ( det - doc ) . this effect may be mediated by different modulation of cd80 and cd86 costimulatory molecules on dc by the two different preparations of hdoc . since recent reports showed that there is a reciprocal relationship between cd4cd25foxp3 t cells and th17 cells in their development [ 28 , 29 ] , we asked whether adh - doc and det - doc showed differential inhibitory capacity during th17 generation as well as during cd4cd25foxp3 t cell induction . since th17 cells can be identified as cd4cd161cd196 t cells [ 30 , 31 ] , we analyzed the induction of cd4cd161cd196 t cells , after coculture of cd3 t cells with dc alone or incubated either with det - doc or with adh - doc ( kruskal - wallis p < 0.0001 ) . coculture of t cells with dc increased the percentage of cd4cd161cd196 t cells , as compared to cd3 t cells alone ( figures 4(a ) and 4(b ) ) . interestingly , the addition of det - doc enhanced the generation of cd4cd161cd196 t cells induced by dc , while the addition of adh - doc significantly decreased that population ( figures 4(a ) and 4(b ) ) . then , we asked whether the different capacity to modulate the dc - mediated cd4cd161cd196 t cell generation of det - doc and adh - doc was mediated by cytokine secretion rather than by a cell - to - cell contact - dependent mechanism(s ) . therefore , we performed the same assay but after separating cd3 t cells from both dc and doc with a 0.4 m pore transwell chamber . as shown in figure 4(c ) , when added in the upper chamber of the transwell , cd3 t cells differentiated into cd4cd161cd196 t cells similarly to cell - to - cell contact culture conditions . therefore , we conclude that hdoc influence the generation of cd4cd161cd196 t cells by dc , by modulating soluble factor(s ) . to investigate which soluble factor(s ) involved in the differentiation of cd4cd161cd196 t cells was regulated by the presence of hdoc , we analyzed the supernatants of the coculture for the presence of tgf- and il-6 , since it has been previously shown that dc can induce th17 differentiation through the release of il-6 , which acts in concert with tgf- . our results showed that il-6 secretion was enhanced in presence of det - doc , while it was not influenced by the presence of adh - doc ( figure 5(a ) , kruskal - wallis p < 0.0001 ) . however , tgf- was decreased in the supernatants of both the cocultures , compared with the supernatant of the dc cultured alone ( figure 5(b ) , kruskal - wallis p < 0.0001 ) . to evaluate whether det - doc and/or adh - doc could modulate the extracellular milieu towards a tolerogenic environment , il-10 was also tested and no differences were found ( figure s3 ) . taken together , these data demonstrate that hdoc can modulate the dc induction of cd4cd161cd196 t cells via il-6/tgf- regulation . the different capacity of det - doc and of adh - doc to modulate the induction of the cd4cd161cd196 t cell phenotype may be due to the different balance between il-6 and tgf- secreted or induced by these cells , a difference that according to our results is generated by the way hdoc are manipulated . the most efficient tissue source would be the autologous bone , but the donor - site morbidity , the inadequate supply , and the problems about size and shape make this source not always feasible . allografts from cadaveric donors are increasingly used , but the risk of disease transmission and/or immune reaction limit their use [ 33 , 34 ] . indeed , allogeneic peptides may be presented by apc , such as dc , on mhc - i and mhc - ii molecules and can activate cytotoxic cd8 t cells and cd4 helper t cells . this activation can ( i ) mediate allograft rejection , ( ii ) dramatically inhibit bone generation , and ( iii ) lead to gradual , long - term immune response . among the other sources tested to prevent these adverse effects , msc have been shown to be immunoprivileged , that is , nontargeted by mhc - mismatched immune cells , to exert a suppressive effect on the host immune system and to repair bone defects in vivo either through infusion or through local implantation [ 16 , 40 ] . the main limitation for bone repair is the low capacity of engraftment of the in vitro - cultured msc . since the basic functional unit for the repair of bone defects is the differentiated osteogenic cell ( doc ) derived from msc , it has been suggested that osteogenic differentiation of msc before implantation might be useful to optimize bone regeneration . in this way , the interval between implantation and subsequent osteogenesis in situ might be shortened , with a consequent reduction of loss of the implanted cells . however , differentiated osteoprogenitors may be not immunoprivileged and may not have the same suppressive effect on the host immune system as their undifferentiated msc progenitors . here we show for the first time that the immunological properties of hmsc to modulate dc phenotype and functions can persist after in vitro differentiation into hdoc . however , our data demonstrate that the capacity of hdoc of modulating recipient dc function depends on different processing protocols and experimental conditions ( i.e. , the presence or absence of extracellular matrix ) . in fact , det - doc are less potent than adh - doc to inhibit the allostimulation of dc in a contact - dependent manner , and this is due to the partial downregulation of the costimulatory molecules expressed on dc . indeed , det - doc do not modify the expression of cd86 , while they weakly downregulate cd80 . on the other hand , adh - doc strongly downregulate cd80 and cd86 on dc , resulting in a more profound inhibition of t cell proliferation . the different capacity of det - doc and adh - doc to downregulate cd80 and cd86 might also explain why adh - doc are more efficient than det - doc in inhibiting also the induction of cd4cd25foxp3 t cells by dc , in a contact - dependent manner . similarly , adh - doc have an opposite effect on dc in the induction of cd4cd161cd196 t cells in comparison with det - doc . in fact , adh - doc inhibit , while det - doc enhance , the capacity of dc to generate cd4cd161cd196 t cells in a cytokine - dependent manner , depending on their capacity to differently modulate the balance between tgf- and il-6 . the same results were obtained also with mature dc ( data not shown ) , suggesting that the capacity of hdoc to modulate dc function is independent from the maturation state of dc . overall , here we show that adh - doc strongly inhibit some of the main functions of dc , such as the induction of t cell proliferation , the generation of cd4cd25foxp3 , and the generation of cd4cd161cd196 t cells . on the other hand we also show that det - doc weakly inhibit the dc capacity of inducing t cell proliferation and of generating cd4cd25foxp3 t cells , while they enhance the dc capacity of generating cd4cd161cd196 t cells . thus , the coculture of either det - doc or adh - doc with dc results in altered apc functions . in the present work , we showed that hdoc are able to modulate the function of dc and , therefore , the host immune response . moreover , by comparing hdoc as cellular suspension ( i.e. , det - doc ) to cells implanted on a scaffold ( i.e. , adh - doc ) , we showed that different manipulation techniques could result in different immunological properties of hdoc . as a consequence , the culture system of hdoc can produce highly different immunological outcome . . however , our report about different in vitro capacity of det - doc and adh - doc in modulating dc functions offers the rationale to specifically address , at the clinical level , the safety and immunomodulatory capacity of doc . indeed , better definition of the most suitable culture system for hdoc preparation may have relevant clinical implications for their implantation in the context of bone repair cell - based tissue - engineering clinical trials .

in vitro differentiation of mesenchymal stromal cells ( msc ) into osteocytes ( human differentiated osteogenic cells , hdoc ) before implantation has been proposed to optimize bone regeneration . however , a deep characterization of the immunological properties of doc , including their effect on dendritic cell ( dc ) function , is not available . doc can be used either as cellular suspension ( detached , det - doc ) or as adherent cells implanted on scaffolds ( adherent , adh - doc ) . by mimicking in vitro these two different routes of administration , we show that both det - doc and adh - doc can modulate dc functions . specifically , the weak downregulation of cd80 and cd86 caused by det - doc on dc surface results in a weak modulation of dc functions , which indeed retain a high capacity to induce t - cell proliferation and to generate cd4+cd25+foxp3 + t cells . moreover , det - doc enhance the dc capacity to differentiate cd4+cd161+cd196 + th17-cells by upregulating il-6 secretion . conversely , adh - doc strongly suppress dc functions by a profound downregulation of cd80 and cd86 on dc as well as by the inhibition of tgf- production . in conclusion , we demonstrate that different types of doc cell preparation may have a different impact on the modulation of the host immune system . this finding may have relevant implications for the design of cell - based tissue - engineering strategies .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

msc are multipotent cells , capable of differentiating , in vitro , into different lineages , including osteocytes , chondrocytes , adipocytes , muscle cells , cardiomyocytes , and neural precursor . more recently , the capacity of human msc ( hmsc ) to suppress both innate and adaptive immunity has been described [ 2 , 3 ] as well as their poor immunogenicity . a number of recent studies have focused on the influence of hmsc on dc functions [ 2 , 7 , 8 ] . the in vitro interaction between hmsc and either cd34 or cd14 dc progenitors inhibits the generation of functional dc [ 2 , 7 , 8 ] , skewing their differentiation toward phenotypically abnormal dc , which express lower level of cd1a , cd40 , cd80 , cd86 , and cd83 . different mechanisms are responsible for the effects of hmsc on dc differentiation including the involvement of both soluble factors and cell - to - cell contact interactions [ 12 , 13 ] . however , the topic of whether the immunological properties of hmsc persist after in vitro differentiation into hdoc has been not deeply investigated . in fact , the characterization of the immunological properties of hdoc may be of crucial importance for cell - based tissue - engineering therapeutic strategies . although hmsc have been shown to contribute to repair bone defects in vivo either through infusion or local implantation [ 16 , 17 ] , more recent strategies aim to in vitro differentiate hmsc into hdoc before implantation , in order to optimize bone regeneration [ 18 , 19 ] . therefore , the evaluation of the interactions between hdoc and the host allogeneic immune system , in particular of antigen presenting cells ( apc ) such as dc , may be important in view of clinical trials . since the preparation of hdoc for cell - based tissue - engineering strategies may be different [ 18 , 19 ] , it is interesting to evaluate whether different culture systems may affect the capacity of doc of modulating the immune response . for these reasons , here we characterized the immunological properties of hdoc manipulated in two different ways , which mimic the use of hdoc as cell suspension or as adherent cells , by showing their capacity to modulate the phenotype and the functions of allogeneic dc . for immunological assays , hdoc were used either in adhesion on the same plate used for differentiation ( adh - doc ) or after harvesting and replating ( det - doc ) . in all the assays , the same number of adh - doc and det - doc was used . for each experiment , the cell number was evaluated the day before the assays , by counting 3 wells of adh - doc harvested separately . the mean values of these 3 counts were used to plate det - doc . then , adh - doc and det - doc were cultured overnight in complete medium ( rpmi 1640 medium ( lonza ) supplemented with 10% heat - inactivated fbs ( gibco - invitrogen ) , 2 mm l - glutamine , 100 u / ml penicillin , and 100 g / ml streptomycin ( mp biomedicals ) ) at 37c in 5% co2 , before coculturing with dc and cd3 t cells . the same strategy and the same conditions indeed , the number of det - msc to plate was established by counting independently 3 wells of adh - msc . to test the capacity of adh - doc and det - doc to modulate the dc allostimulatory capacity , allogeneic mo - dc , either immature or mature , were irradiated ( 3000 cgy ) and plated ( 1 : 1 ) at 37c on either adh - doc or det - doc in presence of third party cfse - labeled cd3 t cells ( 1 : 10 ) . for a second set of experiments , cd3 t cells were added to the upper chamber of a 0.4 m pore polycarbonate filter in 24-well transwell chambers ( corning costar ) to keep them separated from dc and from either adh - doc or det - doc , which were plated in the lower chamber of the transwell system . to test the capacity of adh - doc and det - doc to modulate the dc capacity to induce in cd3 t cells the treg cell or the th17 cell phenotype , allogeneic mo - dc , either immature or mature , were plated ( 1 : 1 ) on either adh - doc or det - doc in presence of allogeneic cd3 t cells ( 1 : 20 ) . for a second set of experiments , cd3 t cells were added to the upper chamber of a 0.4 m pore polycarbonate filter in 24-well transwell chambers ( corning costar ) , while dc and either adh - doc or det - doc were added in the lower chamber . cell cultures were incubated at 37c for 5 days ; then the t cells were harvested from the upper chamber and stained for immunophenotype via tricolor immunofluorescence , which was performed using fluorescein isothiocyanate- ( fitc- ) conjugated anti - human cd4 ( clone rpa - t4 ) , phycoerythrin- ( pe- ) conjugated anti - human foxp3 ( clone 206d ) , and allophycocyanin- ( apc- ) conjugated anti - human cd25 ( clone bc96 , biolegend ) . immature and mature mo - dc were incubated for 5 days either alone or with adh - doc or det - doc ( 1 : 1 ) . immature and mature mo - dc were incubated for 5 days either alone or with adh - doc or det - doc ( 1 : 1 ) . we first compared the suppressive capacity of hdoc on dc - mediated t cell proliferation . in particular , we used hdoc either after detachment from culture plates ( det - doc ) or as adherent cells ( adh - doc ) to mimic , in vitro , the conditions in which they are used therapeutically ( i.e. , cultured and then detached to be injected as cellular suspension or grown adherent on a scaffold and implanted without detachment ) . as shown in figures 1(a ) and 1(b ) , both det - doc and adh - doc inhibited the allostimulatory capacity of dc ( kruskal wallis p < 0.0001 ) . however , the inhibition induced by adh - doc is much stronger than det - doc thus , we asked why det - doc partially lost their ability to inhibit dc allostimulation . to investigate the mechanism(s ) involved in such process , we first asked whether det - doc were less viable than adh - doc , since they were harvested after treatment with trypsin and collected with a cell scraper . we could not find a reduction of viability in harvested det - doc ( data not shown ) . then , we asked whether the extracellular matrix secreted by hdoc ( routinely discarded to obtain det - doc ) could itself inhibit the allostimulatory capacity of dc . thus , we compared the capacity of undifferentiated hmsc used after detachment from culture plates ( mimicking det - msc ) with that of cells used as adherent cells ( mimicking adh - msc ) . as shown in figure 1(d ) , both det - msc and adh - msc were able to inhibit the dc allostimulation at the same level , suggesting that the detachment procedure , by itself , had no influence on the tolerogenic capacity of the cells . finally we investigated whether the different capacity to inhibit dc allostimulation of det - doc and adh - doc was mediated by soluble factors rather than by a cell - to - cell contact - dependent mechanism(s ) . therefore , we performed the same suppression assay by separating cd3 t cells with a 0.4 m pore transwell chamber , which allows the migration of only soluble factors . as shown in figure 1(e ) , neither det - doc nor adh - doc could inhibit dc allostimulation when dc are cultured without any contact with cd3 t cells . therefore , in this setting , hdoc modified the allostimulatory capacity of dc , mainly by modulating cell - to - cell contact - dependent mechanism(s ) . thus , we wanted to investigate which surface marker(s ) on dc may be affected by the inhibitory activity of hdoc [ 24 , 25 ] . to this end , we cocultured dc with either det - doc or adh - doc , for 5 days and then analyzed dc phenotype ( kruskal - wallis p < 0.0001 for cd80 , p = 0.0012 for cd86 ) . as shown in figure 2(a ) , the coculture of dc with det - doc or adh - doc resulted in the downregulation of cd80 . notably , adh - doc and det - doc could downregulate this marker to a comparable extent . conversely , det - doc were not able to downregulate cd86 , while this molecule was strongly downregulated by adh - doc , as shown in figure 2(b ) . taken together , our results demonstrate that , if hdoc are left in their own extracellular matrix ( adh - doc ) , they acquire a high capacity to inhibit the dc expression of molecules involved in t cell costimulation . , hdoc are harvested from their own extracellular matrix and seeded back in a new culture support , as for det - doc ) , they partly lose this capacity , resulting in lower inhibition of costimulatory molecules on dc and , consequently , of t cell proliferation . therefore , we asked whether det - doc and adh - doc could modulate this function of dc . to test this hypothesis , we cocultured dc with allogeneic t cells , in the presence or absence of either det - doc or adh - doc ( kruskal - wallis p < 0.0001 ) . coculture of t cells with dc increased the percentage of cd4cd25foxp3 t cells , as compared to cd3 t cells alone ( figures 3(a ) and 3(b ) ) . the addition of det - doc or adh - doc decreased the population of cd4cd25foxp3 t cells induced by dc ( figures 3(a ) and 3(b ) ) , with the adh - doc showing the greatest inhibitory effect ( p < 0.001 , bonferroni corrected ) . then , we asked whether the different capacity to inhibit the dc - mediated cd4cd25foxp3 t cell generation of det - doc and adh - doc was mediated by soluble factors rather than by a cell - to - cell contact - dependent mechanism . therefore , we performed the same assay by separating cd3 t cells with a 0.4 m pore transwell chamber . as shown in figure 3(c ) , det - doc and adh - doc did not inhibit the capacity of dc to induce a cd4cd25foxp3 t cell population and there was no significant difference among different hdoc . therefore , adh - doc had an enhanced inhibitory effect on dc to induce cd4cd25foxp3 t cell population , in comparison to det - doc , by modulating mechanism(s ) involved in cell - to - cell , contact - dependent suppression . as shown in figure 2 , adh - doc were able to downregulate the expression of both cd80 and cd86 , while det - doc decreased only cd80 at a lower extent . taken together , these data demonstrate that hdoc inhibit the capacity of dc to induce cd4cd25foxp3 t cells . however , such inhibitory capacity is markedly increased when hdoc are left adherent to the same plate used for differentiation ( adh - doc ) , whereas it is reduced when hdoc are detached from culture plates ( det - doc ) . this effect may be mediated by different modulation of cd80 and cd86 costimulatory molecules on dc by the two different preparations of hdoc . since recent reports showed that there is a reciprocal relationship between cd4cd25foxp3 t cells and th17 cells in their development [ 28 , 29 ] , we asked whether adh - doc and det - doc showed differential inhibitory capacity during th17 generation as well as during cd4cd25foxp3 t cell induction . since th17 cells can be identified as cd4cd161cd196 t cells [ 30 , 31 ] , we analyzed the induction of cd4cd161cd196 t cells , after coculture of cd3 t cells with dc alone or incubated either with det - doc or with adh - doc ( kruskal - wallis p < 0.0001 ) . coculture of t cells with dc increased the percentage of cd4cd161cd196 t cells , as compared to cd3 t cells alone ( figures 4(a ) and 4(b ) ) . interestingly , the addition of det - doc enhanced the generation of cd4cd161cd196 t cells induced by dc , while the addition of adh - doc significantly decreased that population ( figures 4(a ) and 4(b ) ) . then , we asked whether the different capacity to modulate the dc - mediated cd4cd161cd196 t cell generation of det - doc and adh - doc was mediated by cytokine secretion rather than by a cell - to - cell contact - dependent mechanism(s ) . therefore , we performed the same assay but after separating cd3 t cells from both dc and doc with a 0.4 m pore transwell chamber . as shown in figure 4(c ) , when added in the upper chamber of the transwell , cd3 t cells differentiated into cd4cd161cd196 t cells similarly to cell - to - cell contact culture conditions . therefore , we conclude that hdoc influence the generation of cd4cd161cd196 t cells by dc , by modulating soluble factor(s ) . to investigate which soluble factor(s ) involved in the differentiation of cd4cd161cd196 t cells was regulated by the presence of hdoc , we analyzed the supernatants of the coculture for the presence of tgf- and il-6 , since it has been previously shown that dc can induce th17 differentiation through the release of il-6 , which acts in concert with tgf- . our results showed that il-6 secretion was enhanced in presence of det - doc , while it was not influenced by the presence of adh - doc ( figure 5(a ) , kruskal - wallis p < 0.0001 ) . however , tgf- was decreased in the supernatants of both the cocultures , compared with the supernatant of the dc cultured alone ( figure 5(b ) , kruskal - wallis p < 0.0001 ) . to evaluate whether det - doc and/or adh - doc could modulate the extracellular milieu towards a tolerogenic environment , il-10 was also tested and no differences were found ( figure s3 ) . taken together , these data demonstrate that hdoc can modulate the dc induction of cd4cd161cd196 t cells via il-6/tgf- regulation . the different capacity of det - doc and of adh - doc to modulate the induction of the cd4cd161cd196 t cell phenotype may be due to the different balance between il-6 and tgf- secreted or induced by these cells , a difference that according to our results is generated by the way hdoc are manipulated . among the other sources tested to prevent these adverse effects , msc have been shown to be immunoprivileged , that is , nontargeted by mhc - mismatched immune cells , to exert a suppressive effect on the host immune system and to repair bone defects in vivo either through infusion or through local implantation [ 16 , 40 ] . since the basic functional unit for the repair of bone defects is the differentiated osteogenic cell ( doc ) derived from msc , it has been suggested that osteogenic differentiation of msc before implantation might be useful to optimize bone regeneration . however , differentiated osteoprogenitors may be not immunoprivileged and may not have the same suppressive effect on the host immune system as their undifferentiated msc progenitors . here we show for the first time that the immunological properties of hmsc to modulate dc phenotype and functions can persist after in vitro differentiation into hdoc . however , our data demonstrate that the capacity of hdoc of modulating recipient dc function depends on different processing protocols and experimental conditions ( i.e. in fact , det - doc are less potent than adh - doc to inhibit the allostimulation of dc in a contact - dependent manner , and this is due to the partial downregulation of the costimulatory molecules expressed on dc . indeed , det - doc do not modify the expression of cd86 , while they weakly downregulate cd80 . on the other hand , adh - doc strongly downregulate cd80 and cd86 on dc , resulting in a more profound inhibition of t cell proliferation . the different capacity of det - doc and adh - doc to downregulate cd80 and cd86 might also explain why adh - doc are more efficient than det - doc in inhibiting also the induction of cd4cd25foxp3 t cells by dc , in a contact - dependent manner . similarly , adh - doc have an opposite effect on dc in the induction of cd4cd161cd196 t cells in comparison with det - doc . in fact , adh - doc inhibit , while det - doc enhance , the capacity of dc to generate cd4cd161cd196 t cells in a cytokine - dependent manner , depending on their capacity to differently modulate the balance between tgf- and il-6 . the same results were obtained also with mature dc ( data not shown ) , suggesting that the capacity of hdoc to modulate dc function is independent from the maturation state of dc . overall , here we show that adh - doc strongly inhibit some of the main functions of dc , such as the induction of t cell proliferation , the generation of cd4cd25foxp3 , and the generation of cd4cd161cd196 t cells . on the other hand we also show that det - doc weakly inhibit the dc capacity of inducing t cell proliferation and of generating cd4cd25foxp3 t cells , while they enhance the dc capacity of generating cd4cd161cd196 t cells . thus , the coculture of either det - doc or adh - doc with dc results in altered apc functions . in the present work , we showed that hdoc are able to modulate the function of dc and , therefore , the host immune response . moreover , by comparing hdoc as cellular suspension ( i.e. , det - doc ) to cells implanted on a scaffold ( i.e. , adh - doc ) , we showed that different manipulation techniques could result in different immunological properties of hdoc . however , our report about different in vitro capacity of det - doc and adh - doc in modulating dc functions offers the rationale to specifically address , at the clinical level , the safety and immunomodulatory capacity of doc . indeed , better definition of the most suitable culture system for hdoc preparation may have relevant clinical implications for their implantation in the context of bone repair cell - based tissue - engineering clinical trials .

msc are multipotent cells , capable of differentiating , in vitro , into different lineages , including osteocytes , chondrocytes , adipocytes , muscle cells , cardiomyocytes , and neural precursor . more recently , the capacity of human msc ( hmsc ) to suppress both innate and adaptive immunity has been described [ 2 , 3 ] as well as their poor immunogenicity . as a result , the therapeutic potential of hmsc as immunoregulatory agents is currently being explored in several phase i / ii clinical trials [ 46 ] . the in vitro interaction between hmsc and either cd34 or cd14 dc progenitors inhibits the generation of functional dc [ 2 , 7 , 8 ] , skewing their differentiation toward phenotypically abnormal dc , which express lower level of cd1a , cd40 , cd80 , cd86 , and cd83 . different mechanisms are responsible for the effects of hmsc on dc differentiation including the involvement of both soluble factors and cell - to - cell contact interactions [ 12 , 13 ] . taken together , these studies demonstrated the profound immunosuppressive effects of hmsc on dc . however , the topic of whether the immunological properties of hmsc persist after in vitro differentiation into hdoc has been not deeply investigated . in fact , the characterization of the immunological properties of hdoc may be of crucial importance for cell - based tissue - engineering therapeutic strategies . although hmsc have been shown to contribute to repair bone defects in vivo either through infusion or local implantation [ 16 , 17 ] , more recent strategies aim to in vitro differentiate hmsc into hdoc before implantation , in order to optimize bone regeneration [ 18 , 19 ] . therefore , the evaluation of the interactions between hdoc and the host allogeneic immune system , in particular of antigen presenting cells ( apc ) such as dc , may be important in view of clinical trials . since the preparation of hdoc for cell - based tissue - engineering strategies may be different [ 18 , 19 ] , it is interesting to evaluate whether different culture systems may affect the capacity of doc of modulating the immune response . for these reasons , here we characterized the immunological properties of hdoc manipulated in two different ways , which mimic the use of hdoc as cell suspension or as adherent cells , by showing their capacity to modulate the phenotype and the functions of allogeneic dc . to induce hdoc , hmscs were seeded at 3.1 10 cells / cm and grown in osteogenic differentiation medium ( lonza ) containing l - glutamine , mcgs , dexamethasone , ascorbate , -glycerophosphate , and penicillin / streptomycin . for each experiment , the cell number was evaluated the day before the assays , by counting 3 wells of adh - doc harvested separately . then , adh - doc and det - doc were cultured overnight in complete medium ( rpmi 1640 medium ( lonza ) supplemented with 10% heat - inactivated fbs ( gibco - invitrogen ) , 2 mm l - glutamine , 100 u / ml penicillin , and 100 g / ml streptomycin ( mp biomedicals ) ) at 37c in 5% co2 , before coculturing with dc and cd3 t cells . the same strategy and the same conditions indeed , the number of det - msc to plate was established by counting independently 3 wells of adh - msc . monocyte - derived dcs ( mo - dcs ) were generated by a 5-day culture of cd14 cells in complete medium plus 50 ng / ml granulocyte - macrophage colony - stimulation factor ( gm - csf ) and 800 u / ml il-4 ( both by endogen ) , at 37c in 5% co2 . for maturation , day 5 mo - dcs were cultured with gm - csf and il-4 and incubated for 48 hours in presence of a cocktail of cytokine made of 10 ng / ml tnf , 10 ng / ml il-6 , 10 ng / ml il-1 , and 1 g / ml pge2 ( all by endogen ) . to test the capacity of adh - doc and det - doc to modulate the dc allostimulatory capacity , allogeneic mo - dc , either immature or mature , were irradiated ( 3000 cgy ) and plated ( 1 : 1 ) at 37c on either adh - doc or det - doc in presence of third party cfse - labeled cd3 t cells ( 1 : 10 ) . as a positive control , cd3 t cells were cultured with mo - dc and , as a negative control , with medium alone . for a second set of experiments , cd3 t cells were added to the upper chamber of a 0.4 m pore polycarbonate filter in 24-well transwell chambers ( corning costar ) to keep them separated from dc and from either adh - doc or det - doc , which were plated in the lower chamber of the transwell system . to test the capacity of adh - doc and det - doc to modulate the dc capacity to induce in cd3 t cells the treg cell or the th17 cell phenotype , allogeneic mo - dc , either immature or mature , were plated ( 1 : 1 ) on either adh - doc or det - doc in presence of allogeneic cd3 t cells ( 1 : 20 ) . for a second set of experiments , cd3 t cells were added to the upper chamber of a 0.4 m pore polycarbonate filter in 24-well transwell chambers ( corning costar ) , while dc and either adh - doc or det - doc were added in the lower chamber . cell cultures were incubated at 37c for 5 days ; then the t cells were harvested from the upper chamber and stained for immunophenotype via tricolor immunofluorescence , which was performed using fluorescein isothiocyanate- ( fitc- ) conjugated anti - human cd4 ( clone rpa - t4 ) , phycoerythrin- ( pe- ) conjugated anti - human foxp3 ( clone 206d ) , and allophycocyanin- ( apc- ) conjugated anti - human cd25 ( clone bc96 , biolegend ) . supernatants were collected and tested for the release of tgf-1 ( drg diagnostics , marburg , germany ) il-6 and il-10 ( thermo scientific , erembodegem , belgium ) , according to the manufacturer 's instructions . = p < 0.05 , = p < 0.01 , and = p < 0.001 , bonferroni corrected . we first compared the suppressive capacity of hdoc on dc - mediated t cell proliferation . in particular , we used hdoc either after detachment from culture plates ( det - doc ) or as adherent cells ( adh - doc ) to mimic , in vitro , the conditions in which they are used therapeutically ( i.e. as shown in figures 1(a ) and 1(b ) , both det - doc and adh - doc inhibited the allostimulatory capacity of dc ( kruskal wallis p < 0.0001 ) . however , the inhibition induced by adh - doc is much stronger than det - doc thus , we asked why det - doc partially lost their ability to inhibit dc allostimulation . to investigate the mechanism(s ) involved in such process , we first asked whether det - doc were less viable than adh - doc , since they were harvested after treatment with trypsin and collected with a cell scraper . therefore , we lysed the cellular component of hdoc and recovered the extracellular matrix to perform the same assay . then , we asked whether the procedure used to detach cells from the plates influenced by itself the tolerogenic capacity of detached doc . thus , we compared the capacity of undifferentiated hmsc used after detachment from culture plates ( mimicking det - msc ) with that of cells used as adherent cells ( mimicking adh - msc ) . as shown in figure 1(d ) , both det - msc and adh - msc were able to inhibit the dc allostimulation at the same level , suggesting that the detachment procedure , by itself , had no influence on the tolerogenic capacity of the cells . finally we investigated whether the different capacity to inhibit dc allostimulation of det - doc and adh - doc was mediated by soluble factors rather than by a cell - to - cell contact - dependent mechanism(s ) . therefore , we performed the same suppression assay by separating cd3 t cells with a 0.4 m pore transwell chamber , which allows the migration of only soluble factors . therefore , in this setting , hdoc modified the allostimulatory capacity of dc , mainly by modulating cell - to - cell contact - dependent mechanism(s ) . thus , we wanted to investigate which surface marker(s ) on dc may be affected by the inhibitory activity of hdoc [ 24 , 25 ] . to this end , we cocultured dc with either det - doc or adh - doc , for 5 days and then analyzed dc phenotype ( kruskal - wallis p < 0.0001 for cd80 , p = 0.0012 for cd86 ) . as shown in figure 2(a ) , the coculture of dc with det - doc or adh - doc resulted in the downregulation of cd80 . taken together , our results demonstrate that , if hdoc are left in their own extracellular matrix ( adh - doc ) , they acquire a high capacity to inhibit the dc expression of molecules involved in t cell costimulation . , hdoc are harvested from their own extracellular matrix and seeded back in a new culture support , as for det - doc ) , they partly lose this capacity , resulting in lower inhibition of costimulatory molecules on dc and , consequently , of t cell proliferation . dc have a crucial role not only in the activation of t cell response but also in the induction of tolerance by generating cd4cd25foxp3 t cell population . therefore , we asked whether det - doc and adh - doc could modulate this function of dc . to test this hypothesis , we cocultured dc with allogeneic t cells , in the presence or absence of either det - doc or adh - doc ( kruskal - wallis p < 0.0001 ) . coculture of t cells with dc increased the percentage of cd4cd25foxp3 t cells , as compared to cd3 t cells alone ( figures 3(a ) and 3(b ) ) . the addition of det - doc or adh - doc decreased the population of cd4cd25foxp3 t cells induced by dc ( figures 3(a ) and 3(b ) ) , with the adh - doc showing the greatest inhibitory effect ( p < 0.001 , bonferroni corrected ) . then , we asked whether the different capacity to inhibit the dc - mediated cd4cd25foxp3 t cell generation of det - doc and adh - doc was mediated by soluble factors rather than by a cell - to - cell contact - dependent mechanism . as shown in figure 3(c ) , det - doc and adh - doc did not inhibit the capacity of dc to induce a cd4cd25foxp3 t cell population and there was no significant difference among different hdoc . therefore , adh - doc had an enhanced inhibitory effect on dc to induce cd4cd25foxp3 t cell population , in comparison to det - doc , by modulating mechanism(s ) involved in cell - to - cell , contact - dependent suppression . as shown in figure 2 , adh - doc were able to downregulate the expression of both cd80 and cd86 , while det - doc decreased only cd80 at a lower extent . taken together , these data demonstrate that hdoc inhibit the capacity of dc to induce cd4cd25foxp3 t cells . since recent reports showed that there is a reciprocal relationship between cd4cd25foxp3 t cells and th17 cells in their development [ 28 , 29 ] , we asked whether adh - doc and det - doc showed differential inhibitory capacity during th17 generation as well as during cd4cd25foxp3 t cell induction . since th17 cells can be identified as cd4cd161cd196 t cells [ 30 , 31 ] , we analyzed the induction of cd4cd161cd196 t cells , after coculture of cd3 t cells with dc alone or incubated either with det - doc or with adh - doc ( kruskal - wallis p < 0.0001 ) . coculture of t cells with dc increased the percentage of cd4cd161cd196 t cells , as compared to cd3 t cells alone ( figures 4(a ) and 4(b ) ) . interestingly , the addition of det - doc enhanced the generation of cd4cd161cd196 t cells induced by dc , while the addition of adh - doc significantly decreased that population ( figures 4(a ) and 4(b ) ) . then , we asked whether the different capacity to modulate the dc - mediated cd4cd161cd196 t cell generation of det - doc and adh - doc was mediated by cytokine secretion rather than by a cell - to - cell contact - dependent mechanism(s ) . therefore , we performed the same assay but after separating cd3 t cells from both dc and doc with a 0.4 m pore transwell chamber . as shown in figure 4(c ) , when added in the upper chamber of the transwell , cd3 t cells differentiated into cd4cd161cd196 t cells similarly to cell - to - cell contact culture conditions . therefore , we conclude that hdoc influence the generation of cd4cd161cd196 t cells by dc , by modulating soluble factor(s ) . to investigate which soluble factor(s ) involved in the differentiation of cd4cd161cd196 t cells was regulated by the presence of hdoc , we analyzed the supernatants of the coculture for the presence of tgf- and il-6 , since it has been previously shown that dc can induce th17 differentiation through the release of il-6 , which acts in concert with tgf- . our results showed that il-6 secretion was enhanced in presence of det - doc , while it was not influenced by the presence of adh - doc ( figure 5(a ) , kruskal - wallis p < 0.0001 ) . however , tgf- was decreased in the supernatants of both the cocultures , compared with the supernatant of the dc cultured alone ( figure 5(b ) , kruskal - wallis p < 0.0001 ) . to evaluate whether det - doc and/or adh - doc could modulate the extracellular milieu towards a tolerogenic environment , il-10 was also tested and no differences were found ( figure s3 ) . taken together , these data demonstrate that hdoc can modulate the dc induction of cd4cd161cd196 t cells via il-6/tgf- regulation . the different capacity of det - doc and of adh - doc to modulate the induction of the cd4cd161cd196 t cell phenotype may be due to the different balance between il-6 and tgf- secreted or induced by these cells , a difference that according to our results is generated by the way hdoc are manipulated . the most efficient tissue source would be the autologous bone , but the donor - site morbidity , the inadequate supply , and the problems about size and shape make this source not always feasible . this activation can ( i ) mediate allograft rejection , ( ii ) dramatically inhibit bone generation , and ( iii ) lead to gradual , long - term immune response . among the other sources tested to prevent these adverse effects , msc have been shown to be immunoprivileged , that is , nontargeted by mhc - mismatched immune cells , to exert a suppressive effect on the host immune system and to repair bone defects in vivo either through infusion or through local implantation [ 16 , 40 ] . the main limitation for bone repair is the low capacity of engraftment of the in vitro - cultured msc . since the basic functional unit for the repair of bone defects is the differentiated osteogenic cell ( doc ) derived from msc , it has been suggested that osteogenic differentiation of msc before implantation might be useful to optimize bone regeneration . in this way , the interval between implantation and subsequent osteogenesis in situ might be shortened , with a consequent reduction of loss of the implanted cells . here we show for the first time that the immunological properties of hmsc to modulate dc phenotype and functions can persist after in vitro differentiation into hdoc . however , our data demonstrate that the capacity of hdoc of modulating recipient dc function depends on different processing protocols and experimental conditions ( i.e. in fact , det - doc are less potent than adh - doc to inhibit the allostimulation of dc in a contact - dependent manner , and this is due to the partial downregulation of the costimulatory molecules expressed on dc . the different capacity of det - doc and adh - doc to downregulate cd80 and cd86 might also explain why adh - doc are more efficient than det - doc in inhibiting also the induction of cd4cd25foxp3 t cells by dc , in a contact - dependent manner . similarly , adh - doc have an opposite effect on dc in the induction of cd4cd161cd196 t cells in comparison with det - doc . in fact , adh - doc inhibit , while det - doc enhance , the capacity of dc to generate cd4cd161cd196 t cells in a cytokine - dependent manner , depending on their capacity to differently modulate the balance between tgf- and il-6 . the same results were obtained also with mature dc ( data not shown ) , suggesting that the capacity of hdoc to modulate dc function is independent from the maturation state of dc . overall , here we show that adh - doc strongly inhibit some of the main functions of dc , such as the induction of t cell proliferation , the generation of cd4cd25foxp3 , and the generation of cd4cd161cd196 t cells . on the other hand we also show that det - doc weakly inhibit the dc capacity of inducing t cell proliferation and of generating cd4cd25foxp3 t cells , while they enhance the dc capacity of generating cd4cd161cd196 t cells . thus , the coculture of either det - doc or adh - doc with dc results in altered apc functions . in the present work , we showed that hdoc are able to modulate the function of dc and , therefore , the host immune response . however , our report about different in vitro capacity of det - doc and adh - doc in modulating dc functions offers the rationale to specifically address , at the clinical level , the safety and immunomodulatory capacity of doc . indeed , better definition of the most suitable culture system for hdoc preparation may have relevant clinical implications for their implantation in the context of bone repair cell - based tissue - engineering clinical trials .

because of their novel mechanisms of antibiotic activity , which generally involves some type of membrane disruption , antimicrobial peptides ( amp ) have the potential to be developed into useful antibiotic therapeutic agents . generally amps are small highly positively charged amphipathic peptides with well - defined hydrophobic and hydrophilic regions [ 24 ] . it is generally accepted that the electrostatic interactions that occur between an amp and the target cell 's membrane are the first step in the binding of an amp to the surface of a cell membrane [ 57 ] . amps exhibit a high net positive charge ( + 3 to + 9 ) while most bacterial cell membranes contain a relatively high percentage of negatively charged phospholipids as compared to mammalian cells . the resulting difference in the electrostatic nature of the two cell membranes explains , in part , the inherent selectivity of amps for bacterial membranes over mammalian membranes . amps have evolved in almost every class of living organism , including humans , amphibians , insects , mammals , birds , fish , and plants , as a host defense mechanism against invading microorganisms including bacteria , fungi , protozoa , and parasites [ 1315 ] ; they are also considered to be key components in the innate immune response system [ 1619 ] . the antibacterial and anticancer activity of the antimicrobial peptides ll-37 [ 20 , 21 ] , human beta - defensin-3 [ 21 , 22 ] , and other amps has been extensively investigated and reviewed in the literature . particularly beneficial has been the application of solid state nmr methods which have been extensively employed to investigate the interactions that occur between peptides and phospholipid suvs and luv phospholipid membrane models [ 2329 ] . some researchers have suggested that antimicrobial activity is not the primary function of mammalian amps such as the defensins . their primary function may involve immunomodulatory processes in controlling the interaction of acquired and innate immunity [ 3033 ] . the research of porcelli and coworkers reported the nmr derived structure of the defensin peptide ll-37 bound to dpc micelles . the results obtained were consistent with previous solid state nmr studies that supported a nonpore forming carpet - like mechanism of action for these amps . the amps developed in our laboratory were designed to be members of the mechanistic class known as membrane - disruptors [ 19 , 35 , 36 ] . in our laboratory we developed a series of amps structurally very different from the defensins by incorporating various unnatural amino acids into the primary amino acid sequence with the intent to introduce specific physicochemical properties that will control membrane binding . it is well documented that the selectivity and potency of an amp against a particular organism are defined in large measure by the complementary nature of the physicochemical surface properties of the amp and of the target membrane [ 10 , 13 , 35 , 3841 ] . unnatural amino acids provide a toolbox of different physicochemical properties that are not available in peptides composed of the 20 naturally occurring rna encoded amino acids [ 4246 ] . toolbox to facilitate the development of peptides with specific physicochemical properties that have the ability to interact with membranes in novel ways [ 47 , 48 ] . the work of gottler and coworkers , who previously reported the application of fluorinated analogs of protegrin-1 and other antimicrobial peptides to investigate the role played by changing hydrophobicity on the physical and biological properties of the interactions with lipid membranes and improve activity , can be used as an example of the application of unnatural amino acids to modify biological and physical properties of antimicrobial peptides . gram - negative bacteria such as pseudomonas aeruginosa [ 5156 ] and klebsiella pneumoniae represent major threats to human health , causing hundreds of thousands of severe infections each year . infections associated with gram - negative bacteria are difficult to treat in part because the cell membranes consist of two distinct lipid bilayers of very different chemical compositions [ 58 , 59 ] . the surface of the outer membrane of gram - negative bacteria is comprised almost exclusively of negatively charged lipopolysaccharides ( lps ) [ 6062 ] . a molecule of lps is subdivided into three major components , the chemical compositions of the two outer components varying by bacterial strain [ 6365 ] . the outermost component consists of a polysaccharide known as the o - antigen , the core oligosaccharide unit constitutes the middle region , and the innermost portion is the highly conserved phospholipid known as lipid a [ 6365 ] . one of the key functions of lps is to control the transport of antibiotics , antimicrobial peptides , and host defense proteins into the cell [ 63 , 6668 ] . because of the reduced transport of antimicrobial peptides across the outer membrane , often higher concentrations of the peptide are required to exhibit antibacterial activity against gram - negative strains than are required to obtain the same level of activity against gram - positive strains . therefore , in the case of gram - negative bacteria , it is critically important to understand the physicochemical interactions that occur between an amp and lps in order to design amps with increased antibacterial activity against gram - negative bacteria [ 58 , 60 , 64 ] . the first step in the binding of an amp to the membranes of gram - negative bacteria involves the insertion of the amp into the outer leaflet [ 6971 ] which causes expansion or loosening of the lipid bilayer resulting in the depolarization of the lps vesicles and allows a transient self - promoted uptake pathway to occur , destabilizing the bilayer [ 72 , 73 ] . this process may be similar to the carpet - like mechanism proposed for the binding of amps to phospholipid membranes [ 7476 ] . the primary amino acid sequence of the amps in this investigation incorporates six tic - oic dipeptide units , as well as four additional residues ( a , b , c , and d ) on either side of the intervening hydrophobic and charged residues as shown in figure 1 . these residues define the overall conformational mobility of the peptide backbone . a fifth residue , e , defines the distance between the polypeptide backbone and the positively charged side chain amine group . we have previously shown using electrostatic surface calculations that the distance between the positively charged amino group and the electronegative carbonyl oxygen of the amide bond determines the resulting positive charge density of the side chain . the amino acid residues used for residues a e are defined in table 1 . the amino acid sequences of the amps used in this investigation are listed in table 2 . we previously reported that increasing the number of tic - oic dipeptide units from three to six without the incorporation of residues a , b , c , and d in amps 70 or 22 resulted in a dramatic loss in activity against all of the gram - negative bacteria tested , compared to the analogues containing three tic - oic dipeptide units ( e.g. , amp 23 : ac - gf - tic - oic - gk - tic - oic - gf - tic - oic - gk - tic - kkkk - conh2 ) . ( please see table 3 for in vitro inhibitory activity of the amps investigated in this study ) . we propose that the observed differences in inhibitory activity of these amps ( table 3 ) against these two strains of gram - negative bacteria largely arise from variations in how these peptides interact with the lps components of the bacteria . to obtain insight into how these amps interact with lps , h nmr and cd investigations were conducted using suvs consisting of the lps isolated from p. aeruginosa and k. pneumonia . peptide synthesis was performed either manually using t - boc chemistry or with an automated peptide synthesizer using fmoc protocols [ 79 , 80 ] as previously reported [ 47 , 81 , 82 ] . all peptides were purified by reverse phase hplc [ 47 , 81 , 82 ] . purified peptides were analyzed again by hplc and mass spectrometry [ 47 , 82 ] . a 4 mg sample of the appropriate lipopolysaccharide was hydrated with 4 ml of buffer ( 40 mm sodium phosphate , ph = 6.8 ) and vortexed extensively . suvs were prepared by sonication of the milky lipid suspension using a titanium tip ultrasonicator for approximately 10 minutes at a temperature of 40c until the solution became transparent . the titanium debris was removed by centrifugation at 8,800 ppm for 10 minutes using a table - top centrifuge . peptide solutions were prepared by dissolving approximately 2 mg of amps 70 , 74 , 75 , 79 , or 80 in 1.0 ml of phosphate buffer . due to solubility issues , the cd spectra of amps 22 , 76 , 77 , and 78 were collected at a concentration approximately 50% lower than the other amps . cd spectra of amps 71 , 72 , and 73 in the presence of either lps could not be obtained due to precipitation of uncharacterized amp - lps complexes . for the lps liposome studies 350 l of stock lps solution was mixed with 50 l of stock peptide solution . cd spectroscopy is a sensitive technique , so it is commonly used to monitor conformational changes in peptides and proteins [ 8385 ] . however , as lps can exhibit strong cd absorption , only after careful subtraction of the lps background signal can meaningful spectra of the amps bound to the lps be obtained [ 64 , 65 , 86 ] . all cd spectra were obtained by acquiring 8 scans using a 0.1 mm cylindrical quartz cell with a spectral range of 260 to 195 nm ( at wavelengths below 195 nm the htv exceeded 400 , and therefore data collection was terminated at 195 nm ) and a scanning rate of 20 nm / min . acquisition parameters were bandwidth 1 nm , data pitch 0.2 nm , response time 2.0 s , and 5 mdeg sensitivity . all analyses of cd spectra were conducted after smoothing with a means - movement function [ 47 , 87 , 88 ] . cd spectra that exhibited ht values of greater than 500 were not used due to excessive light scattering and/or absorption . all h experiments were conducted at 298 k on a bruker avance iii 400 mhz nmr spectrometer equipped with a 5 mm direct observe z - gradient broad - band probe . the spectral width was 4,000 hz , 256 fids were collected per experiment . data were processed using exponential multiplication with a line - broadening function of 5 hz . samples contained 1.0 mg / ml of the lps in the presence of 0.1 mg of the amp in 600 l of a 150 mm perdeuterated sodium acetate buffer at a ph of 5.64 in d2o . the cd spectra of amps 70 , 74 , 75 , 79 , and 80 in the presence of lps isolated from both p. aeruginosa and k. pneumoniae are shown in figure 2 . the cd spectra of amps 22 , 76 , 77 , and 78 in the presence of lps isolated from both organisms are shown in figure 3 . as can be seen in figures 2 and 3 , the cd spectra fall into two different spectral types . the first exhibited a max at approximately 198 nm and double min at approximately 210 and 225 nm . these cd spectra appear similar to those observed for peptides comprised of only the 20 naturally occurring amino acids with predominantly -helical secondary structure . in the case of the peptides under investigation the incorporation of a high percentage of unnatural amino acids means that the traditional methods of characterizing peptide secondary structure by spectral deconvolution are not valid . helical - like . in the presence of lps isolated from p. aeruginosa , the cd spectra of amps 22 , 70 , 74 , 75 , and 77 exhibit helical - like features , while in the presence of lps isolated from k. pneumoniae , the cd spectra of amps 22 , 70 , 75 , and 77 ( but not 74 ) exhibit helical - like features . the second type of cd spectra consisting of amps 76 , 78 , 79 , and 80 exhibits only negative absorptions with double min at approximately 204210 and 225 nm in the presence of the lps isolated from both bacterial strains . in the presence of lps isolated from k. the observation of two different types of cd spectra implies that these amps adopt two very different sets of conformations on binding to lps and further suggests two distinct binding mechanisms for these amps . the different binding conformations and mechanisms may be explained by the amps interacting with different sites or regions of the lps . bhunia and coworkers have reported the nmr - derived three dimensional structures of pardaxins pa1 , pa2 , pa3 , and pa4 bound to lps micelles . in the pardaxin pa4-lps complex , the structure of the peptide was found to be very different from those adopted in the presence of organic solvents and other micelles . these results may provide insight into the structural requirements for selectivity for gram - negative bacteria , but unfortunately two practical issues prevented us from conducting similar experiments using lps suvs with these amps . at the concentrations of the amp required to conduct 2d nmr experiments , the amp - lps mixtures precipitated out of solution , and no nmr signals were detected . in addition , the incorporation of six tic - oic dipeptide units ( which , as secondary amides , lack amide protons ) into the sequence of these peptides , coupled with severe overlap of the side chain protons in the region 2.51.0 ppm , makes the application of standard homonuclear 2d experiments such as the tocsy [ 89 , 90 ] and noesy very problematic . however , one - dimensional h nmr spectra of amp - lps complexes could be employed to monitor changes in the local chemical environments of the lps as a function of amp binding . compared to the h nmr spectrum of the lps alone , a significant reduction in the peak heights of the resonances in the region between 1.5 and 0.5 ppm ( figure 4 ) was observed in the spectra of a 1.0 mg / ml sample of lps isolated from p. aeruginosa as a result of the addition of 0.1 mg of amps 70 , 74 , 75 , 79 , and 80 . ( at this low concentration of amp , no nmr signals corresponding to the amps are observed ) . the region between 1.5 and 0.5 ppm corresponds to the resonances associated with the side chain protons of the lipid a region of lps . the reduction in peak area indicates a strong binding interaction of these amps with this region of lipid a. the region between 4.5 and 3.8 ppm , which corresponds to the polysaccharide resonances of the lps , exhibits a change in peak position but little change in peak intensity . this indicates a weaker interaction between the amp and the polysaccharide region of the lps . the h nmr spectra of a 1.0 mg / ml sample of the lps isolated from k. pneumoniae in the presence of 0.1 mg of amps 70 , 74 , 75 , 79 , and 80 ( figure 5 ) showed a reduction in the signal intensity as well as changes in the observed chemical shifts in the region of 1.8 to 0.7 ppm relative to lps alone . such a decrease in peak area would arise from complexation between amps and the lipid a region of the lps isolated from k. pneumoniae . these data suggest that the present amps exhibit a higher partition coefficient for the lipid a portion than for the polysaccharide or core oligosaccharide of the lps . this is in accord with the second mechanism of amp - lps binding , which involves hydrophobic interactions between the amp and the hydrocarbon chain region of lipid a [ 92 , 93 ] . at lower field , between 4.5 and 3.8 ppm , the polysaccharide resonances of the lps exhibit a change in peak position but little change in peak intensity . this indicates a weaker interaction between the amp and the polysaccharide region of the lps . lps is believed to act as barrier to the transport of material , including drugs , across the outer membrane of gram - negative bacteria via two mechanisms . the first involves hydrophilic interactions between the substrate to be transported and the densely packed negatively charged oligosaccharide core of lps . the second mechanism involves sequestering of lipophilic moieties within the hydrocarbon chains of lipid a [ 92 , 93 ] . the transport of hydrophobic molecules from bulk solvent through the lps bilayer occurs at a rate that is 98 - 99% slower than that observed for the transport of the same molecule across a phospholipid bilayer [ 95 , 96 ] . these two mechanisms indicated that both hydrophobic interactions and electrostatic attractions between an amp and lps are possible , this is because amps are highly amphipathic , presenting a hydrophobic face and a hydrophilic face to lps . other investigations have been conducted attempting to link the interactions of amps with lps and the observed antibacterial activity against gram - negative bacteria . for example , the amp msi-594 and its mutant analog msi-594f5a exhibit very different activity against gram - negative bacteria , with msi-594 exhibiting greater potency , while exhibiting similar activity against gram - positive bacteria . domadia and coworkers reported using nmr that msi-594 and msi-594f5a adopt different helical structures in the presence of lps micelles . msi-594 adopts a hairpin helical structure , while msi-594f5a adopts an amphipathic curved helix without the packing interactions that controlled the lps binding of msi-594 . the differences in the helical conformations adopted by these two amps seem to be related to the 3d spatial orientation of the lys residues . the six lys residues of msi-594f5a are on one amphipathic face and are evenly spaced out at a distance of 25 . while the six lys residues of msi-594 are on one amphipathic face and they are clustered together over a distance of only 17 . domadia and coworkers proposed that , the compact structure and geometrical compatibility of lps / msi-594 , provided by the orientation of the side chain of basic residues , could be related to an efficient permeabilization of an lps membrane of gram - negative bacteria . it has been shown that helical content alone does not account for antibacterial activity against gram - negative bacteria because increasing the helical content of an amp by incorporation of unnatural -amino acids does not necessarily increase antibacterial activity . as we and others have shown it is the three - dimensional character and complementarity of the physicochemical properties , such as charge density and hydrophobicity presented to the cell membranes that define antibacterial activity . in an effort to explain how the two different spectral shapes observed in the cd spectra for these amps relate to inhibitory activity the construction of this model is guided by the findings of domadia and coworkers that the positioning of the lys residues is critical for transport of the amp across lps . a cartoon depiction of a proposed active site that is able to accommodate the helical conformation of the amp is given in figure 6 . multiple regions of a single lps molecule , or multiple lps molecules , may be required to form the scaffolding of the site . five cationic residue groupings are present in the amps under investigation , and it appears that all five must be paired with negatively charged side chains on lps for high - affinity binding . this assumption is based on the observation that the cd spectra of analogues containing only three tic - oic dipeptide units and three cationic residue groupings , such as amp 23 ( ac - gf - tic - oic - gk - tic - oic - gf - tic - oic - gk - tic - kkkk - conh2 ) , do not exhibit helical characteristics in the presence of lps and exhibit greater in vitro inhibitory activity compared to the larger amps under investigation in this study . based on the amino acid residues incorporated into these amps there may be as many as eight hydrophobic microenvironments ( or four localized ones and a single large one ) included within the active site on lps , the number and location of which may vary between bacterial strains . for both strains of bacteria a hydrophobic pocket likely appears at some distance before and after each anionic binding pocket . this model can be used to explain the observed cd spectra and the inhibitory activity for these amps . the cd spectra of amps 22 and 70 exhibit helical characteristics in the presence of lps isolated from both strains of bacteria . k. pneumoniae , amps 22 , and 70 exhibit very poor in vitro inhibitory activity ( 100 g / ml ) against p. aeruginosa and k. pneumoniae . the combined cd and biological activity data suggests , based on the work of domadia and coworkers , that 22 and 70 adopt helical conformations that bind lps very strongly and these amps are not efficiently transported across the lps bilayer . amps 76 , 78 , 79 , and 80 exhibited cd spectra with nonhelical characteristics as well as an increased in vitro inhibitory activity of 50 g / ml . the combined cd and biological activity data suggests that 74 adopts a nonhelical conformation that binds lps very loosely and is efficiently transported across the lps bilayer . the cd spectra of amps 75 and 77 exhibited helical characteristics in the presence of lps isolated from p. aeruginosa ; however this is inconsistent with the observed increased in vitro inhibitory activity of 50 g / ml for these amps . the cd spectra of amps 22 , 70 , and 75 in the presence of lps isolated from k. pneumoniae exhibited helical characteristics of the cd spectra and also exhibited poor in vitro inhibitory activity of 100 g / ml . the combined cd and biological activity data suggests , based on the work of domadia and coworkers , that 22 and 70 adopt helical conformations that bind lps very strongly and these amps are not efficiently transported across the lps bilayer . notably , these two amps do not contain any of the four residues a , b , c , or d. amp 74 features gly residues as residue a , increasing the distance between each lys residue and the following tic residue . for the lps isolated from p. aeruginosa , the hydrophobic pocket for the active site is probably large enough to accommodate this increase in distance since the cd spectrum of amp 74 exhibits helical characteristics . the three additional gly residues will increase the distances between the lys residues and depending on the conformation adopted by the amp dramatically alter the three - dimensional spatial orientation of these residues , thus modifying the type of helical structure adopted by 74 on binding to lps . amp 74 , exhibited very poor in vitro inhibitory activity ( 100 g / ml ) against p. aeruginosa . the combined cd and biological activity data suggests , based on the work of domadia and coworkers , that 74 adopts a helical conformation that binds lps very strongly and this amp is not efficiently transported across the lps bilayer . however , the hydrophobic pocket for the active site for the lps isolated from k. pneumoniae appears to be unable to accommodate the increase in distance since the cd spectrum of amp 74 is not helical in nature . amp 74 exhibits in vitro inhibitory activity of 100 g / ml against k. pneumonia . the combined cd and biological activity data suggests that 74 adopts a nonhelical conformation that binds lps very loosely and is efficiently transported across the lps bilayer . amp 78 , contains gly residues as residue b , resulting in a greater distance between each lys residue and the preceding oic residue . the active site hydrophobic pockets for lps isolated from both p. aeruginosa and k. pneumonia are incompatible with this increase in distance , as the cd spectra of amp 78 do not exhibit helical characteristics . amp 78 exhibits in vitro inhibitory activity of 50 g / ml against both p. aeruginosa and k. pneumonia . the combined cd and biological activity data suggests that 78 adopts a nonhelical conformation that binds lps very loosely and is efficiently transported across the lps bilayer . amps 75 and 77 contain gly residues as residues c and d ; residue c increases the distance from a phe residue to the following tic residue and residue d increases the distance from a phe residue to the preceding oic residue . either the hydrophobic pocket for both bacteria is very large and can accommodate the increased molecular bulk of these two modifications , or the proposed pocket plays no role in the binding active site , since the cd spectra of both amps in the presence of both lpss exhibit helical characteristics . the cd spectra of amps 75 and 77 exhibited helical characteristics in the presence of lps isolated from p. aeruginosa ; however this is inconsistent with the observed increased in vitro inhibitory activity of 50 g / ml for these amps . the two additional gly residues will increase the distances between the lys residues and depending on the conformation adopted by the amp dramatically alter the three - dimensional spatial orientation of these residues , thus modifying the type of helical structures adopted by 75 and 77 on binding to lps . the combined cd and biological activity data suggests , based on the work of domadia and coworkers , that 75 and 77 adopt helical conformations that bind lps very strongly and these amps are not efficiently transported across the lps bilayer . the argument for this proposed hydrophobic pocket playing a role in the active site of the lps isolated from p. aeruginosa is provided by amp 76 , which contains gly residues for residues a and c. individually these residues are accommodated by the active site binding model , as noted above . however , collectively these residues disfavor binding to the active site as indicated by the nonhelical character of the observed cd spectra . amp 76 exhibits in vitro inhibitory activity against p. aeruginosa of 50 g / ml . the combined effect of these two residues is to change the relative position of the anionic lys residues and the hydrophobic phe residues in three - dimensional space preventing binding to the active site . the data indicate that 76 adopts a nonhelical conformation that binds lps very loosely and is efficiently transported across the lps bilayer . amps 79 and 80 also appear not to interact with the active site of either lps since their cd spectra do not exhibit helical characteristics . both amps exhibited in vitro inhibitory activity of 50 g / ml against both bacteria strains . the data indicate that amps 79 and 80 adopt nonhelical conformations that bind lps very loosely and are efficiently transported across the lps bilayer . this investigation has shown that synthetic amps with elongated primary amino acid sequences exhibit helical characteristics in their cd spectra upon binding to suvs comprised of lps isolated from either p. aeruginosa or k. pneumoniae . peptides with cd spectra that exhibit characteristics of helical secondary structure appear to bind to an active site on the lps . in vitro inhibitory data suggest that there are two possible helical conformations resulting from two different amp - lps binding mechanisms . mechanism one involves a helical binding conformation where the amp binds lps very strongly and is not efficiently transported across the lps bilayer resulting in the loss of inhibitory activity . mechanism two involves a helical binding conformation where the amp binds lps very loosely and is efficiently transported across the lps bilayer resulting in an increase in inhibitory activity . mechanism three involves a nonhelical binding conformation where the amp binds lps very loosely and is efficiently transported across the lps bilayer resulting in an increase in inhibitory activity .

circular dichroism and 1h nmr were used to investigate the interactions of a series of synthetic antimicrobial peptides ( amps ) with lipopolysaccharides ( lps ) isolated from pseudomonas aeruginosa and klebsiella pneumoniae . previous cd studies with amps containing only three tic - oic dipeptide units do not exhibit helical characteristics upon interacting with small unilamellar vesicles ( suvs ) consisting of lps . increasing the number of tic - oic dipeptide units to six resulted in five analogues with cd spectra that exhibited helical characteristics on binding to lps suvs . spectroscopic and in vitro inhibitory data suggest that there are two possible helical conformations resulting from two different amp - lps binding mechanisms . mechanism one involves a helical binding conformation where the amp binds lps very strongly and is not efficiently transported across the lps bilayer resulting in the loss of inhibitory activity . mechanism two involves a helical binding conformation where the amp binds lps very loosely and is efficiently transported across the lps bilayer resulting in an increase in inhibitory activity . mechanism three involves a nonhelical binding conformation where the amp binds lps very loosely and is efficiently transported across the lps bilayer resulting in an increase in inhibitory activity .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusions

because of their novel mechanisms of antibiotic activity , which generally involves some type of membrane disruption , antimicrobial peptides ( amp ) have the potential to be developed into useful antibiotic therapeutic agents . it is generally accepted that the electrostatic interactions that occur between an amp and the target cell 's membrane are the first step in the binding of an amp to the surface of a cell membrane [ 57 ] . the antibacterial and anticancer activity of the antimicrobial peptides ll-37 [ 20 , 21 ] , human beta - defensin-3 [ 21 , 22 ] , and other amps has been extensively investigated and reviewed in the literature . particularly beneficial has been the application of solid state nmr methods which have been extensively employed to investigate the interactions that occur between peptides and phospholipid suvs and luv phospholipid membrane models [ 2329 ] . some researchers have suggested that antimicrobial activity is not the primary function of mammalian amps such as the defensins . in our laboratory we developed a series of amps structurally very different from the defensins by incorporating various unnatural amino acids into the primary amino acid sequence with the intent to introduce specific physicochemical properties that will control membrane binding . it is well documented that the selectivity and potency of an amp against a particular organism are defined in large measure by the complementary nature of the physicochemical surface properties of the amp and of the target membrane [ 10 , 13 , 35 , 3841 ] . the work of gottler and coworkers , who previously reported the application of fluorinated analogs of protegrin-1 and other antimicrobial peptides to investigate the role played by changing hydrophobicity on the physical and biological properties of the interactions with lipid membranes and improve activity , can be used as an example of the application of unnatural amino acids to modify biological and physical properties of antimicrobial peptides . gram - negative bacteria such as pseudomonas aeruginosa [ 5156 ] and klebsiella pneumoniae represent major threats to human health , causing hundreds of thousands of severe infections each year . the surface of the outer membrane of gram - negative bacteria is comprised almost exclusively of negatively charged lipopolysaccharides ( lps ) [ 6062 ] . a molecule of lps is subdivided into three major components , the chemical compositions of the two outer components varying by bacterial strain [ 6365 ] . the outermost component consists of a polysaccharide known as the o - antigen , the core oligosaccharide unit constitutes the middle region , and the innermost portion is the highly conserved phospholipid known as lipid a [ 6365 ] . one of the key functions of lps is to control the transport of antibiotics , antimicrobial peptides , and host defense proteins into the cell [ 63 , 6668 ] . because of the reduced transport of antimicrobial peptides across the outer membrane , often higher concentrations of the peptide are required to exhibit antibacterial activity against gram - negative strains than are required to obtain the same level of activity against gram - positive strains . the first step in the binding of an amp to the membranes of gram - negative bacteria involves the insertion of the amp into the outer leaflet [ 6971 ] which causes expansion or loosening of the lipid bilayer resulting in the depolarization of the lps vesicles and allows a transient self - promoted uptake pathway to occur , destabilizing the bilayer [ 72 , 73 ] . the primary amino acid sequence of the amps in this investigation incorporates six tic - oic dipeptide units , as well as four additional residues ( a , b , c , and d ) on either side of the intervening hydrophobic and charged residues as shown in figure 1 . we previously reported that increasing the number of tic - oic dipeptide units from three to six without the incorporation of residues a , b , c , and d in amps 70 or 22 resulted in a dramatic loss in activity against all of the gram - negative bacteria tested , compared to the analogues containing three tic - oic dipeptide units ( e.g. , amp 23 : ac - gf - tic - oic - gk - tic - oic - gf - tic - oic - gk - tic - kkkk - conh2 ) . ( please see table 3 for in vitro inhibitory activity of the amps investigated in this study ) . we propose that the observed differences in inhibitory activity of these amps ( table 3 ) against these two strains of gram - negative bacteria largely arise from variations in how these peptides interact with the lps components of the bacteria . to obtain insight into how these amps interact with lps , h nmr and cd investigations were conducted using suvs consisting of the lps isolated from p. aeruginosa and k. pneumonia . cd spectra of amps 71 , 72 , and 73 in the presence of either lps could not be obtained due to precipitation of uncharacterized amp - lps complexes . cd spectroscopy is a sensitive technique , so it is commonly used to monitor conformational changes in peptides and proteins [ 8385 ] . cd spectra that exhibited ht values of greater than 500 were not used due to excessive light scattering and/or absorption . samples contained 1.0 mg / ml of the lps in the presence of 0.1 mg of the amp in 600 l of a 150 mm perdeuterated sodium acetate buffer at a ph of 5.64 in d2o . the cd spectra of amps 70 , 74 , 75 , 79 , and 80 in the presence of lps isolated from both p. aeruginosa and k. pneumoniae are shown in figure 2 . the cd spectra of amps 22 , 76 , 77 , and 78 in the presence of lps isolated from both organisms are shown in figure 3 . as can be seen in figures 2 and 3 , the cd spectra fall into two different spectral types . these cd spectra appear similar to those observed for peptides comprised of only the 20 naturally occurring amino acids with predominantly -helical secondary structure . in the case of the peptides under investigation the incorporation of a high percentage of unnatural amino acids means that the traditional methods of characterizing peptide secondary structure by spectral deconvolution are not valid . in the presence of lps isolated from p. aeruginosa , the cd spectra of amps 22 , 70 , 74 , 75 , and 77 exhibit helical - like features , while in the presence of lps isolated from k. pneumoniae , the cd spectra of amps 22 , 70 , 75 , and 77 ( but not 74 ) exhibit helical - like features . the second type of cd spectra consisting of amps 76 , 78 , 79 , and 80 exhibits only negative absorptions with double min at approximately 204210 and 225 nm in the presence of the lps isolated from both bacterial strains . in the presence of lps isolated from k. the observation of two different types of cd spectra implies that these amps adopt two very different sets of conformations on binding to lps and further suggests two distinct binding mechanisms for these amps . the different binding conformations and mechanisms may be explained by the amps interacting with different sites or regions of the lps . bhunia and coworkers have reported the nmr - derived three dimensional structures of pardaxins pa1 , pa2 , pa3 , and pa4 bound to lps micelles . in the pardaxin pa4-lps complex , the structure of the peptide was found to be very different from those adopted in the presence of organic solvents and other micelles . at the concentrations of the amp required to conduct 2d nmr experiments , the amp - lps mixtures precipitated out of solution , and no nmr signals were detected . in addition , the incorporation of six tic - oic dipeptide units ( which , as secondary amides , lack amide protons ) into the sequence of these peptides , coupled with severe overlap of the side chain protons in the region 2.51.0 ppm , makes the application of standard homonuclear 2d experiments such as the tocsy [ 89 , 90 ] and noesy very problematic . however , one - dimensional h nmr spectra of amp - lps complexes could be employed to monitor changes in the local chemical environments of the lps as a function of amp binding . compared to the h nmr spectrum of the lps alone , a significant reduction in the peak heights of the resonances in the region between 1.5 and 0.5 ppm ( figure 4 ) was observed in the spectra of a 1.0 mg / ml sample of lps isolated from p. aeruginosa as a result of the addition of 0.1 mg of amps 70 , 74 , 75 , 79 , and 80 . this indicates a weaker interaction between the amp and the polysaccharide region of the lps . the h nmr spectra of a 1.0 mg / ml sample of the lps isolated from k. pneumoniae in the presence of 0.1 mg of amps 70 , 74 , 75 , 79 , and 80 ( figure 5 ) showed a reduction in the signal intensity as well as changes in the observed chemical shifts in the region of 1.8 to 0.7 ppm relative to lps alone . such a decrease in peak area would arise from complexation between amps and the lipid a region of the lps isolated from k. pneumoniae . these data suggest that the present amps exhibit a higher partition coefficient for the lipid a portion than for the polysaccharide or core oligosaccharide of the lps . this is in accord with the second mechanism of amp - lps binding , which involves hydrophobic interactions between the amp and the hydrocarbon chain region of lipid a [ 92 , 93 ] . this indicates a weaker interaction between the amp and the polysaccharide region of the lps . the first involves hydrophilic interactions between the substrate to be transported and the densely packed negatively charged oligosaccharide core of lps . the transport of hydrophobic molecules from bulk solvent through the lps bilayer occurs at a rate that is 98 - 99% slower than that observed for the transport of the same molecule across a phospholipid bilayer [ 95 , 96 ] . other investigations have been conducted attempting to link the interactions of amps with lps and the observed antibacterial activity against gram - negative bacteria . domadia and coworkers reported using nmr that msi-594 and msi-594f5a adopt different helical structures in the presence of lps micelles . msi-594 adopts a hairpin helical structure , while msi-594f5a adopts an amphipathic curved helix without the packing interactions that controlled the lps binding of msi-594 . the differences in the helical conformations adopted by these two amps seem to be related to the 3d spatial orientation of the lys residues . domadia and coworkers proposed that , the compact structure and geometrical compatibility of lps / msi-594 , provided by the orientation of the side chain of basic residues , could be related to an efficient permeabilization of an lps membrane of gram - negative bacteria . it has been shown that helical content alone does not account for antibacterial activity against gram - negative bacteria because increasing the helical content of an amp by incorporation of unnatural -amino acids does not necessarily increase antibacterial activity . in an effort to explain how the two different spectral shapes observed in the cd spectra for these amps relate to inhibitory activity the construction of this model is guided by the findings of domadia and coworkers that the positioning of the lys residues is critical for transport of the amp across lps . a cartoon depiction of a proposed active site that is able to accommodate the helical conformation of the amp is given in figure 6 . this assumption is based on the observation that the cd spectra of analogues containing only three tic - oic dipeptide units and three cationic residue groupings , such as amp 23 ( ac - gf - tic - oic - gk - tic - oic - gf - tic - oic - gk - tic - kkkk - conh2 ) , do not exhibit helical characteristics in the presence of lps and exhibit greater in vitro inhibitory activity compared to the larger amps under investigation in this study . this model can be used to explain the observed cd spectra and the inhibitory activity for these amps . the cd spectra of amps 22 and 70 exhibit helical characteristics in the presence of lps isolated from both strains of bacteria . k. pneumoniae , amps 22 , and 70 exhibit very poor in vitro inhibitory activity ( 100 g / ml ) against p. aeruginosa and k. pneumoniae . the combined cd and biological activity data suggests , based on the work of domadia and coworkers , that 22 and 70 adopt helical conformations that bind lps very strongly and these amps are not efficiently transported across the lps bilayer . amps 76 , 78 , 79 , and 80 exhibited cd spectra with nonhelical characteristics as well as an increased in vitro inhibitory activity of 50 g / ml . the combined cd and biological activity data suggests that 74 adopts a nonhelical conformation that binds lps very loosely and is efficiently transported across the lps bilayer . the cd spectra of amps 75 and 77 exhibited helical characteristics in the presence of lps isolated from p. aeruginosa ; however this is inconsistent with the observed increased in vitro inhibitory activity of 50 g / ml for these amps . the cd spectra of amps 22 , 70 , and 75 in the presence of lps isolated from k. pneumoniae exhibited helical characteristics of the cd spectra and also exhibited poor in vitro inhibitory activity of 100 g / ml . the combined cd and biological activity data suggests , based on the work of domadia and coworkers , that 22 and 70 adopt helical conformations that bind lps very strongly and these amps are not efficiently transported across the lps bilayer . notably , these two amps do not contain any of the four residues a , b , c , or d. amp 74 features gly residues as residue a , increasing the distance between each lys residue and the following tic residue . for the lps isolated from p. aeruginosa , the hydrophobic pocket for the active site is probably large enough to accommodate this increase in distance since the cd spectrum of amp 74 exhibits helical characteristics . the three additional gly residues will increase the distances between the lys residues and depending on the conformation adopted by the amp dramatically alter the three - dimensional spatial orientation of these residues , thus modifying the type of helical structure adopted by 74 on binding to lps . amp 74 , exhibited very poor in vitro inhibitory activity ( 100 g / ml ) against p. aeruginosa . the combined cd and biological activity data suggests , based on the work of domadia and coworkers , that 74 adopts a helical conformation that binds lps very strongly and this amp is not efficiently transported across the lps bilayer . however , the hydrophobic pocket for the active site for the lps isolated from k. pneumoniae appears to be unable to accommodate the increase in distance since the cd spectrum of amp 74 is not helical in nature . amp 74 exhibits in vitro inhibitory activity of 100 g / ml against k. pneumonia . the combined cd and biological activity data suggests that 74 adopts a nonhelical conformation that binds lps very loosely and is efficiently transported across the lps bilayer . amp 78 , contains gly residues as residue b , resulting in a greater distance between each lys residue and the preceding oic residue . the active site hydrophobic pockets for lps isolated from both p. aeruginosa and k. pneumonia are incompatible with this increase in distance , as the cd spectra of amp 78 do not exhibit helical characteristics . amp 78 exhibits in vitro inhibitory activity of 50 g / ml against both p. aeruginosa and k. pneumonia . the combined cd and biological activity data suggests that 78 adopts a nonhelical conformation that binds lps very loosely and is efficiently transported across the lps bilayer . either the hydrophobic pocket for both bacteria is very large and can accommodate the increased molecular bulk of these two modifications , or the proposed pocket plays no role in the binding active site , since the cd spectra of both amps in the presence of both lpss exhibit helical characteristics . the cd spectra of amps 75 and 77 exhibited helical characteristics in the presence of lps isolated from p. aeruginosa ; however this is inconsistent with the observed increased in vitro inhibitory activity of 50 g / ml for these amps . the two additional gly residues will increase the distances between the lys residues and depending on the conformation adopted by the amp dramatically alter the three - dimensional spatial orientation of these residues , thus modifying the type of helical structures adopted by 75 and 77 on binding to lps . the combined cd and biological activity data suggests , based on the work of domadia and coworkers , that 75 and 77 adopt helical conformations that bind lps very strongly and these amps are not efficiently transported across the lps bilayer . the argument for this proposed hydrophobic pocket playing a role in the active site of the lps isolated from p. aeruginosa is provided by amp 76 , which contains gly residues for residues a and c. individually these residues are accommodated by the active site binding model , as noted above . however , collectively these residues disfavor binding to the active site as indicated by the nonhelical character of the observed cd spectra . amp 76 exhibits in vitro inhibitory activity against p. aeruginosa of 50 g / ml . the combined effect of these two residues is to change the relative position of the anionic lys residues and the hydrophobic phe residues in three - dimensional space preventing binding to the active site . the data indicate that 76 adopts a nonhelical conformation that binds lps very loosely and is efficiently transported across the lps bilayer . amps 79 and 80 also appear not to interact with the active site of either lps since their cd spectra do not exhibit helical characteristics . both amps exhibited in vitro inhibitory activity of 50 g / ml against both bacteria strains . the data indicate that amps 79 and 80 adopt nonhelical conformations that bind lps very loosely and are efficiently transported across the lps bilayer . this investigation has shown that synthetic amps with elongated primary amino acid sequences exhibit helical characteristics in their cd spectra upon binding to suvs comprised of lps isolated from either p. aeruginosa or k. pneumoniae . peptides with cd spectra that exhibit characteristics of helical secondary structure appear to bind to an active site on the lps . in vitro inhibitory data suggest that there are two possible helical conformations resulting from two different amp - lps binding mechanisms . mechanism one involves a helical binding conformation where the amp binds lps very strongly and is not efficiently transported across the lps bilayer resulting in the loss of inhibitory activity . mechanism two involves a helical binding conformation where the amp binds lps very loosely and is efficiently transported across the lps bilayer resulting in an increase in inhibitory activity . mechanism three involves a nonhelical binding conformation where the amp binds lps very loosely and is efficiently transported across the lps bilayer resulting in an increase in inhibitory activity .

because of their novel mechanisms of antibiotic activity , which generally involves some type of membrane disruption , antimicrobial peptides ( amp ) have the potential to be developed into useful antibiotic therapeutic agents . it is generally accepted that the electrostatic interactions that occur between an amp and the target cell 's membrane are the first step in the binding of an amp to the surface of a cell membrane [ 57 ] . the resulting difference in the electrostatic nature of the two cell membranes explains , in part , the inherent selectivity of amps for bacterial membranes over mammalian membranes . amps have evolved in almost every class of living organism , including humans , amphibians , insects , mammals , birds , fish , and plants , as a host defense mechanism against invading microorganisms including bacteria , fungi , protozoa , and parasites [ 1315 ] ; they are also considered to be key components in the innate immune response system [ 1619 ] . the antibacterial and anticancer activity of the antimicrobial peptides ll-37 [ 20 , 21 ] , human beta - defensin-3 [ 21 , 22 ] , and other amps has been extensively investigated and reviewed in the literature . particularly beneficial has been the application of solid state nmr methods which have been extensively employed to investigate the interactions that occur between peptides and phospholipid suvs and luv phospholipid membrane models [ 2329 ] . the research of porcelli and coworkers reported the nmr derived structure of the defensin peptide ll-37 bound to dpc micelles . the amps developed in our laboratory were designed to be members of the mechanistic class known as membrane - disruptors [ 19 , 35 , 36 ] . in our laboratory we developed a series of amps structurally very different from the defensins by incorporating various unnatural amino acids into the primary amino acid sequence with the intent to introduce specific physicochemical properties that will control membrane binding . it is well documented that the selectivity and potency of an amp against a particular organism are defined in large measure by the complementary nature of the physicochemical surface properties of the amp and of the target membrane [ 10 , 13 , 35 , 3841 ] . unnatural amino acids provide a toolbox of different physicochemical properties that are not available in peptides composed of the 20 naturally occurring rna encoded amino acids [ 4246 ] . toolbox to facilitate the development of peptides with specific physicochemical properties that have the ability to interact with membranes in novel ways [ 47 , 48 ] . the work of gottler and coworkers , who previously reported the application of fluorinated analogs of protegrin-1 and other antimicrobial peptides to investigate the role played by changing hydrophobicity on the physical and biological properties of the interactions with lipid membranes and improve activity , can be used as an example of the application of unnatural amino acids to modify biological and physical properties of antimicrobial peptides . infections associated with gram - negative bacteria are difficult to treat in part because the cell membranes consist of two distinct lipid bilayers of very different chemical compositions [ 58 , 59 ] . the surface of the outer membrane of gram - negative bacteria is comprised almost exclusively of negatively charged lipopolysaccharides ( lps ) [ 6062 ] . a molecule of lps is subdivided into three major components , the chemical compositions of the two outer components varying by bacterial strain [ 6365 ] . the outermost component consists of a polysaccharide known as the o - antigen , the core oligosaccharide unit constitutes the middle region , and the innermost portion is the highly conserved phospholipid known as lipid a [ 6365 ] . one of the key functions of lps is to control the transport of antibiotics , antimicrobial peptides , and host defense proteins into the cell [ 63 , 6668 ] . because of the reduced transport of antimicrobial peptides across the outer membrane , often higher concentrations of the peptide are required to exhibit antibacterial activity against gram - negative strains than are required to obtain the same level of activity against gram - positive strains . therefore , in the case of gram - negative bacteria , it is critically important to understand the physicochemical interactions that occur between an amp and lps in order to design amps with increased antibacterial activity against gram - negative bacteria [ 58 , 60 , 64 ] . the first step in the binding of an amp to the membranes of gram - negative bacteria involves the insertion of the amp into the outer leaflet [ 6971 ] which causes expansion or loosening of the lipid bilayer resulting in the depolarization of the lps vesicles and allows a transient self - promoted uptake pathway to occur , destabilizing the bilayer [ 72 , 73 ] . this process may be similar to the carpet - like mechanism proposed for the binding of amps to phospholipid membranes [ 7476 ] . the primary amino acid sequence of the amps in this investigation incorporates six tic - oic dipeptide units , as well as four additional residues ( a , b , c , and d ) on either side of the intervening hydrophobic and charged residues as shown in figure 1 . these residues define the overall conformational mobility of the peptide backbone . a fifth residue , e , defines the distance between the polypeptide backbone and the positively charged side chain amine group . we have previously shown using electrostatic surface calculations that the distance between the positively charged amino group and the electronegative carbonyl oxygen of the amide bond determines the resulting positive charge density of the side chain . we previously reported that increasing the number of tic - oic dipeptide units from three to six without the incorporation of residues a , b , c , and d in amps 70 or 22 resulted in a dramatic loss in activity against all of the gram - negative bacteria tested , compared to the analogues containing three tic - oic dipeptide units ( e.g. , amp 23 : ac - gf - tic - oic - gk - tic - oic - gf - tic - oic - gk - tic - kkkk - conh2 ) . ( please see table 3 for in vitro inhibitory activity of the amps investigated in this study ) . we propose that the observed differences in inhibitory activity of these amps ( table 3 ) against these two strains of gram - negative bacteria largely arise from variations in how these peptides interact with the lps components of the bacteria . to obtain insight into how these amps interact with lps , h nmr and cd investigations were conducted using suvs consisting of the lps isolated from p. aeruginosa and k. pneumonia . due to solubility issues , the cd spectra of amps 22 , 76 , 77 , and 78 were collected at a concentration approximately 50% lower than the other amps . cd spectra of amps 71 , 72 , and 73 in the presence of either lps could not be obtained due to precipitation of uncharacterized amp - lps complexes . however , as lps can exhibit strong cd absorption , only after careful subtraction of the lps background signal can meaningful spectra of the amps bound to the lps be obtained [ 64 , 65 , 86 ] . samples contained 1.0 mg / ml of the lps in the presence of 0.1 mg of the amp in 600 l of a 150 mm perdeuterated sodium acetate buffer at a ph of 5.64 in d2o . the cd spectra of amps 70 , 74 , 75 , 79 , and 80 in the presence of lps isolated from both p. aeruginosa and k. pneumoniae are shown in figure 2 . the cd spectra of amps 22 , 76 , 77 , and 78 in the presence of lps isolated from both organisms are shown in figure 3 . these cd spectra appear similar to those observed for peptides comprised of only the 20 naturally occurring amino acids with predominantly -helical secondary structure . in the case of the peptides under investigation the incorporation of a high percentage of unnatural amino acids means that the traditional methods of characterizing peptide secondary structure by spectral deconvolution are not valid . in the presence of lps isolated from p. aeruginosa , the cd spectra of amps 22 , 70 , 74 , 75 , and 77 exhibit helical - like features , while in the presence of lps isolated from k. pneumoniae , the cd spectra of amps 22 , 70 , 75 , and 77 ( but not 74 ) exhibit helical - like features . the second type of cd spectra consisting of amps 76 , 78 , 79 , and 80 exhibits only negative absorptions with double min at approximately 204210 and 225 nm in the presence of the lps isolated from both bacterial strains . in the presence of lps isolated from k. the observation of two different types of cd spectra implies that these amps adopt two very different sets of conformations on binding to lps and further suggests two distinct binding mechanisms for these amps . the different binding conformations and mechanisms may be explained by the amps interacting with different sites or regions of the lps . bhunia and coworkers have reported the nmr - derived three dimensional structures of pardaxins pa1 , pa2 , pa3 , and pa4 bound to lps micelles . in the pardaxin pa4-lps complex , the structure of the peptide was found to be very different from those adopted in the presence of organic solvents and other micelles . these results may provide insight into the structural requirements for selectivity for gram - negative bacteria , but unfortunately two practical issues prevented us from conducting similar experiments using lps suvs with these amps . at the concentrations of the amp required to conduct 2d nmr experiments , the amp - lps mixtures precipitated out of solution , and no nmr signals were detected . in addition , the incorporation of six tic - oic dipeptide units ( which , as secondary amides , lack amide protons ) into the sequence of these peptides , coupled with severe overlap of the side chain protons in the region 2.51.0 ppm , makes the application of standard homonuclear 2d experiments such as the tocsy [ 89 , 90 ] and noesy very problematic . however , one - dimensional h nmr spectra of amp - lps complexes could be employed to monitor changes in the local chemical environments of the lps as a function of amp binding . compared to the h nmr spectrum of the lps alone , a significant reduction in the peak heights of the resonances in the region between 1.5 and 0.5 ppm ( figure 4 ) was observed in the spectra of a 1.0 mg / ml sample of lps isolated from p. aeruginosa as a result of the addition of 0.1 mg of amps 70 , 74 , 75 , 79 , and 80 . the region between 1.5 and 0.5 ppm corresponds to the resonances associated with the side chain protons of the lipid a region of lps . the reduction in peak area indicates a strong binding interaction of these amps with this region of lipid a. the region between 4.5 and 3.8 ppm , which corresponds to the polysaccharide resonances of the lps , exhibits a change in peak position but little change in peak intensity . this indicates a weaker interaction between the amp and the polysaccharide region of the lps . the h nmr spectra of a 1.0 mg / ml sample of the lps isolated from k. pneumoniae in the presence of 0.1 mg of amps 70 , 74 , 75 , 79 , and 80 ( figure 5 ) showed a reduction in the signal intensity as well as changes in the observed chemical shifts in the region of 1.8 to 0.7 ppm relative to lps alone . such a decrease in peak area would arise from complexation between amps and the lipid a region of the lps isolated from k. pneumoniae . these data suggest that the present amps exhibit a higher partition coefficient for the lipid a portion than for the polysaccharide or core oligosaccharide of the lps . this is in accord with the second mechanism of amp - lps binding , which involves hydrophobic interactions between the amp and the hydrocarbon chain region of lipid a [ 92 , 93 ] . at lower field , between 4.5 and 3.8 ppm , the polysaccharide resonances of the lps exhibit a change in peak position but little change in peak intensity . lps is believed to act as barrier to the transport of material , including drugs , across the outer membrane of gram - negative bacteria via two mechanisms . the transport of hydrophobic molecules from bulk solvent through the lps bilayer occurs at a rate that is 98 - 99% slower than that observed for the transport of the same molecule across a phospholipid bilayer [ 95 , 96 ] . these two mechanisms indicated that both hydrophobic interactions and electrostatic attractions between an amp and lps are possible , this is because amps are highly amphipathic , presenting a hydrophobic face and a hydrophilic face to lps . other investigations have been conducted attempting to link the interactions of amps with lps and the observed antibacterial activity against gram - negative bacteria . for example , the amp msi-594 and its mutant analog msi-594f5a exhibit very different activity against gram - negative bacteria , with msi-594 exhibiting greater potency , while exhibiting similar activity against gram - positive bacteria . the differences in the helical conformations adopted by these two amps seem to be related to the 3d spatial orientation of the lys residues . domadia and coworkers proposed that , the compact structure and geometrical compatibility of lps / msi-594 , provided by the orientation of the side chain of basic residues , could be related to an efficient permeabilization of an lps membrane of gram - negative bacteria . it has been shown that helical content alone does not account for antibacterial activity against gram - negative bacteria because increasing the helical content of an amp by incorporation of unnatural -amino acids does not necessarily increase antibacterial activity . as we and others have shown it is the three - dimensional character and complementarity of the physicochemical properties , such as charge density and hydrophobicity presented to the cell membranes that define antibacterial activity . in an effort to explain how the two different spectral shapes observed in the cd spectra for these amps relate to inhibitory activity the construction of this model is guided by the findings of domadia and coworkers that the positioning of the lys residues is critical for transport of the amp across lps . a cartoon depiction of a proposed active site that is able to accommodate the helical conformation of the amp is given in figure 6 . multiple regions of a single lps molecule , or multiple lps molecules , may be required to form the scaffolding of the site . five cationic residue groupings are present in the amps under investigation , and it appears that all five must be paired with negatively charged side chains on lps for high - affinity binding . this assumption is based on the observation that the cd spectra of analogues containing only three tic - oic dipeptide units and three cationic residue groupings , such as amp 23 ( ac - gf - tic - oic - gk - tic - oic - gf - tic - oic - gk - tic - kkkk - conh2 ) , do not exhibit helical characteristics in the presence of lps and exhibit greater in vitro inhibitory activity compared to the larger amps under investigation in this study . based on the amino acid residues incorporated into these amps there may be as many as eight hydrophobic microenvironments ( or four localized ones and a single large one ) included within the active site on lps , the number and location of which may vary between bacterial strains . the combined cd and biological activity data suggests , based on the work of domadia and coworkers , that 22 and 70 adopt helical conformations that bind lps very strongly and these amps are not efficiently transported across the lps bilayer . the cd spectra of amps 75 and 77 exhibited helical characteristics in the presence of lps isolated from p. aeruginosa ; however this is inconsistent with the observed increased in vitro inhibitory activity of 50 g / ml for these amps . the cd spectra of amps 22 , 70 , and 75 in the presence of lps isolated from k. pneumoniae exhibited helical characteristics of the cd spectra and also exhibited poor in vitro inhibitory activity of 100 g / ml . the combined cd and biological activity data suggests , based on the work of domadia and coworkers , that 22 and 70 adopt helical conformations that bind lps very strongly and these amps are not efficiently transported across the lps bilayer . notably , these two amps do not contain any of the four residues a , b , c , or d. amp 74 features gly residues as residue a , increasing the distance between each lys residue and the following tic residue . for the lps isolated from p. aeruginosa , the hydrophobic pocket for the active site is probably large enough to accommodate this increase in distance since the cd spectrum of amp 74 exhibits helical characteristics . the three additional gly residues will increase the distances between the lys residues and depending on the conformation adopted by the amp dramatically alter the three - dimensional spatial orientation of these residues , thus modifying the type of helical structure adopted by 74 on binding to lps . the combined cd and biological activity data suggests , based on the work of domadia and coworkers , that 74 adopts a helical conformation that binds lps very strongly and this amp is not efficiently transported across the lps bilayer . however , the hydrophobic pocket for the active site for the lps isolated from k. pneumoniae appears to be unable to accommodate the increase in distance since the cd spectrum of amp 74 is not helical in nature . the active site hydrophobic pockets for lps isolated from both p. aeruginosa and k. pneumonia are incompatible with this increase in distance , as the cd spectra of amp 78 do not exhibit helical characteristics . either the hydrophobic pocket for both bacteria is very large and can accommodate the increased molecular bulk of these two modifications , or the proposed pocket plays no role in the binding active site , since the cd spectra of both amps in the presence of both lpss exhibit helical characteristics . the cd spectra of amps 75 and 77 exhibited helical characteristics in the presence of lps isolated from p. aeruginosa ; however this is inconsistent with the observed increased in vitro inhibitory activity of 50 g / ml for these amps . the two additional gly residues will increase the distances between the lys residues and depending on the conformation adopted by the amp dramatically alter the three - dimensional spatial orientation of these residues , thus modifying the type of helical structures adopted by 75 and 77 on binding to lps . the combined cd and biological activity data suggests , based on the work of domadia and coworkers , that 75 and 77 adopt helical conformations that bind lps very strongly and these amps are not efficiently transported across the lps bilayer . the argument for this proposed hydrophobic pocket playing a role in the active site of the lps isolated from p. aeruginosa is provided by amp 76 , which contains gly residues for residues a and c. individually these residues are accommodated by the active site binding model , as noted above . however , collectively these residues disfavor binding to the active site as indicated by the nonhelical character of the observed cd spectra . the combined effect of these two residues is to change the relative position of the anionic lys residues and the hydrophobic phe residues in three - dimensional space preventing binding to the active site .

this article investigates the correlates of the timing of transition into motherhood among adolescent girls in two informal settlements or slums korogocho and viwandani in nairobi city , kenya . other markers include leaving school , entering the labour force , leaving one 's natal home and getting married . however , adolescent fertility has been viewed as both a social and policy challenge in many countries in sub - saharan africa ( world health organization , 2011 ; united nations children 's fund , 2012a ; national research council & institute of medicine , 2005 ) . to effectively address this challenge , there is a need to understand the factors driving adolescent childbearing , especially in resource - poor settings such as informal settlements . in many sub - saharan african countries , the contribution of adolescents ' fertility to total fertility rates remains higher than in other parts of the world . recent data indicate that 18% of adolescent girls aged 1519 in eastern / southern africa and 21% in western / central africa had initiated childbearing ( national research council & institute of medicine , 2005 ) . other estimates show that , on average , more than 25% of women aged 2024 in sub - saharan africa have given birth before age 18 , with great disparities between countries in the region ( united nations children 's fund , 2012b ) . in fact , much of the high fertility in sub - saharan africa can be attributed to first birth occurring at very young ages . the timing of the first birth is usually an indicator of future fertility patterns , largely because it determines the length of a woman 's childbearing window . in the absence of contraception , one of the long - term demographic effects of adolescent fertility ( wulf & singh , 1991 ) . although childbearing is a key marker of the transition to adulthood and carries parental responsibilities , giving birth during adolescence is likely to lead to poor health outcomes for the young mother and her child . for instance , the risk of maternal and child morbidity and mortality is known to be higher among adolescent mothers . furthermore , younger mothers have a higher likelihood of having clandestine induced abortions and acquiring hiv and other sexually transmitted infections ( mensch et al . , 2005 ) . patton and colleagues ( 2009 ) show that maternal mortality accounts for nearly 26% of the increased number of deaths in females aged 1024 years in the african region . the incidence of low birth weight , prematurity , stillbirth and neonatal mortality are known to be higher among children whose mothers are adolescents . early childbearing is also known to negatively impact on educational and employment prospects , and hence lead to a greater likelihood of poverty ( singh , 1998 ; gupta & mahy , 2003b ) . in many instances , pregnant females are forced to drop out of school , while for those who are able to continue schooling , the very possible conflict between school and child care responsibilities , which women primarily bear , results in poor academic performance . other consequences of early parenthood include marital instability , single parenthood , social ostracism and an increased risk of getting into poverty . accordingly , adolescents ' fertility has become a source of social and policy concern for many governments and non - governmental organizations in sub - saharan africa ( national research council & institute of medicine , 2005 ; world health organization , 2011 ; united nations children 's fund , 2012a ) . indeed , the 1994 international conference on population and development ( icpd ) paved the way for highlighting the concerns of adolescents ' fertility by prioritizing adolescents ' reproductive health issues . since then , there have been an increasing number of studies addressing reproductive health and behaviour challenges among adolescents in sub - saharan africa . although sexual and reproductive health was excluded from the millennium development goals ( mdgs ) , universal access to reproductive health by 2015 was later set as a goal by governments at the world summit in 2005 since it was clear that achievement of mdgs largely depended on addressing sexual and reproductive health challenges , especially in high - fertility regions ( glasier et al . , 2006 ) . beyond the achievement of the objective of providing reproductive health services for the youth , a better understanding of adolescents ' childbearing is of great importance for policymakers involved in helping the continent to achieve its desirable fertility levels . in kenya , where the fertility decline is stalling , the proportion of teenagers initiating childbearing was 18% in 2008 , with teenagers from poorer households ( 24% ) being more likely to have begun childbearing than those from wealthier households ( 16% ) ( kenya national bureau of statistics ( knbs ) & icf macro , 2010 ) . these figures also show considerable differences across educational level ( with education versus no education ) , residence ( urban versus rural ) and regional divides . recent research also shows growing gaps in the disparities between the poor versus non - poor within urban areas . however , little attention has been paid to understanding the dimensions and factors driving the fertility patterns among adolescents living in resource - poor urban settings and in particular in informal settlements or slums . in fact , despite the significant proportion of adolescents making up the slum population , little attention has been paid to the challenges they face during their transition to adulthood . adolescents living in nairobi slums grow up in a very poor environment characterized by lack of basic social infrastructure and services , high rates of unemployment , crime and substance abuse , poor schooling facilities and lack of recreational facilities , and so on ( african population and health research center , 2002 ; mugisha & zulu , 2004 ; fotso et al . also , they are known to engage in riskier sexual behaviours , including early sexual debut , transactional sex and multiple sexual partnerships than those in the non - slums parts of the city ( african population and health research center , 2002 ; zulu et al . , 2002 , 2007 ; 2010 ) . actually , early entry into childbearing among young slum dwellers is a sign of early involvement in unprotected sexual intercourse , which exposes these youth to sexually transmitted infections , including hiv , given the relatively high prevalence of hiv / aids in slum settlements . they live in an environment where knowledge about contraception is inadequate , and access to contraceptive methods is limited ( ezeh et al . , 2010 ) . in these conditions , early sexual debut automatically lengthens the period of exposure to the risk of pregnancy and childbearing . these constitute social barriers that may impinge on adolescents ' ability to have control over their reproductive life or decide on the timing of childbearing , and thus have key implications for the well - being and welfare of adolescents . indeed , previous studies show that 16% of female adolescents in slums become parents by age 18 , meaning that a considerable number are exposed to the risks of teenage pregnancy and delivery because they have not reached physical maturity or physiological development ( beguy et al . , 2009b ) . consequently , early parenthood becomes a health threat , not only for the young women but also for their children . these challenges are further compounded by the lack of basic health facilities and the high cost of care , which impede access to obstetric services within slum settings ( izugbara et al . , 2009 ) . it is paramount to help prevent early childbearing , which is often unintended , and its adverse health consequences . in addition , averting early childbearing among adolescent girls living in urban slums could also contribute to slowing rates of urban growth , which is substantially brought about by natural increase in sub - saharan africa ( chen et al . the mushrooming of informal settlements in urban areas in sub - saharan africa is one of the tangible manifestations of the rapid urban growth in this region over the past 20 years ( un - habitat , 2008a , 2008b , 2010 ) . in kenya , un - habitat estimates suggest that between 60 and 70% of the nairobi city residents live in slum settlements or slum - like conditions ( african population and health research center , 2002 ; un - habitat , 2008a ) . understanding and identifying ways to address the unique reproductive and health outcomes among slum adolescents , who constitute a substantial part of urban residents , is central to the attainment of development goals such as the mdgs . it is apparent that the well - being of the urban poor will increasingly drive national development indicators , including health and poverty indicators , in kenya . the findings could help understand the circumstances and factors surrounding early childbearing among young people in order to design and implement appropriate intervention programmes . this study examines predictors of entry into motherhood among adolescents living in the two slum settlements using the problem behaviour theory ( pbt ) framework ( jessor et al . , 1998 , 2003 ; this was developed in the us by jessor and colleagues to explain adolescent problem behaviours such as early sexual initiation , substance abuse and deviant behaviour ( jessor et al . , this stems from the fact that many event markers of the transition to adulthood that are age - graded are considered as problem behaviours if they are experienced at a socially proscribed age ( e.g. at earlier age ) . the pbt framework posits that problem behaviours are driven by common psychosocial root causes , including contextual attributes and individual characteristics conceptualized as three protective ( models protection , controls protection and support protection ) and three risk factors ( models risk , opportunity risk and vulnerability risk ) . models protection refers to models for positive or prosocial behaviour from adults , peers , etc . ; controls protection is related to social regulations against problem behaviour and sanctions for social contraventions ; and support protection refers to a supportive environment to promote conventional behaviour . with respect to controls protection , a distinction could be made between social controls protection , which is captured at the social environment level , and individual controls protection , measured at the individual level . models risk relates to models for unconventional behaviour ; opportunity risk operates through exposure to contextual elements or situations that could lead to engaging in antisocial behaviour ; and vulnerability has to do with individual attributes that increase the risks of engaging in socially proscribed behaviour . protective factors are negatively associated with problem behaviour while risk factors increase the likelihood of engaging in problem behaviour ( jessor , 1998 , 2002 , 2003 ; jessor , 2008 ) . the pbt framework has been used in various contexts in and outside the us to explain various problem behaviours ( vazsonyi et al . , 2008 ; 2010 ) . in this paper , the pbt framework is used to explain early childbearing among adolescent girls aged 1519 in two informal settlements in nairobi city . early childbearing is considered a problem behaviour because of its attendant adverse health , social and economic consequences for the adolescent girls living in such resource - constrained settings . an adolescent 's entry into motherhood is therefore expected to be negatively associated with protective factors , and positively associated with risk factors . in addition to psychosocial factors , socio - demographic factors are known to be associated with adolescent fertility in different regions of the world . in her global review of adolescent childbearing , singh ( 1998 ) points out that factors associated with adolescent fertility include , among others , cultural practices of early marriage , especially in africa and asia , as well as cultural tolerance of premarital sexual relationships to prove one 's fertility before marriage . further , socioeconomic factors such as level of urbanization and level of education have been shown to have an effect on adolescent childbearing in many countries . adolescents residing in urban areas and those with higher levels of education are likely to delay their childbearing due to known conflicts between schooling , work and childrearing ( singh , 1998 ; gupta & mahy , 2003a ) . however , some studies show that education can also be a risk factor for childbearing through its weakening of parental controls , especially where young people spend considerable periods of time in school ( zabin & kiragu , 1998 ) . parental education is also an important determinant of adolescent childbearing , with daughters of more educated parents being more likely to delay their entry into motherhood ( tambashe & shapiro , 1996 ; lee , 2001 ) . family structure is an important determinant of adolescent fertility , with adolescents raised in polygamous households being more likely to initiate sexual activity early compared with those in monogamous households ( tambashe & shapiro , 1996 ) . parental survival status is also associated with early childbearing , with orphans being more likely to begin childbearing early ( tambashe & shapiro , 1996 ; cavazos - rehg et al . , 2010 ) . child communication is an important protective factor against early sex and pregnancy . in a study conducted in kenya , magadi et al . ( 2009b ) found that adolescents who discussed sexual issues with their boyfriends as opposed to their parents or peers began childbearing earlier . they also noted that adolescents who made autonomous reproductive health decisions had a higher likelihood of childbearing during adolescence , as were those who had strong gender bias . in general , major factors associated with early adolescent childbearing in developing countries include low education level , unstable family structure , absence of a father figure , low self - esteem , low socioeconomic status , early sexual debut and experience of physical abuse ( baumgartner et al . this paper investigates whether these factors are also correlated with adolescent entry into motherhood in two slum settlements in nairobi , kenya . this paper draws on data collected under the transition to adulthood ( tta ) component of the urbanization , poverty and health dynamics ( uphd ) project that the african population and health research center ( aphrc ) conducted between 2007 and 2010 in two informal settlements , korogocho and viwandani , in nairobi city . the uphd is nested in the nairobi urban health and demographic surveillance system ( nuhdss ) , which has been implemented by aphrc in the two slums since 2002 . nearly 72,000 people in about 28,000 households are annually covered in the two slum settlements . every 4 months , fieldworkers visit all households within the demographic surveillance area ( dsa ) to collect information on births , deaths , migrations , health , childhood vaccination , health - seeking behaviour , education , household possessions and amenities , livelihood sources , and so on . the tta is a 3-year prospective study that aims to identify protective and risk factors in the lives of adolescents growing up in the two informal settlements , and examine how these factors influence key markers of the transition to adulthood ( transitions to secondary school , sexual intercourse , marriage , parenthood , independent housing ) . the specific aims are to : ( a ) identify sexual , reproductive health , livelihood , educational and other key concerns and aspirations of young people as they grow up in urban informal settlements ; ( b ) determine both protective and risk factors ( including coercion ) that influence young people 's transition to secondary school , employment , independent housing , sexual and marital partnerships and parenthood and the sequencing of these transitions ; and ( c ) investigate the implications of childbearing aspirations for hiv / sti prevention and vice versa , with particular focus on dual - protection strategies . existing instruments developed and used in various settings were adapted to develop the tta questionnaire ( e.g. national study of youth and religion , 2002 ; jessor et al . , 2002 ) , which was piloted and tested in areas outside the dsa . the questionnaire was translated and administered in swahili , kenya 's national language . however , to ensure comparability , the english and swahili versions of the questionnaires were reviewed . during fieldwork activities , proper and close supervision of fieldworkers was ensured by the field supervisors and some of the senior researchers within the team . spot checks and quality controls during data entry and cleaning were done by the team . interviewers underwent rigorous training to ensure that all had a uniform understanding of the questions . the nuhdss database of residents for the year 2007 was used to randomly select adolescents aged 1222 years in the study areas ( beguy et al . during the first wave ( november 2007june 2008 ) , 4058 randomly selected young people aged 1222 years were interviewed at their homes ( 50% males ) . refusal rate was less than 5% of young people who were requested to be respondents in the study . although age definitions of adolescence vary from one culture to another , it is defined in this study as the age group 1519 years . individuals aged 2022 years are considered to be in their early adulthood . to generate the analytical sample of 15- to 19-year - old girls for this paper , 114 observations were first omitted from the 4058 successfully interviewed youth because of inconsistent or missing data on age , date of birth and/or age at first event ( child , sex , marriage , housing ) , leaving 3944 ( 1957 females ) individuals with valid data . finally , all males and females aged 1214 and 2022 years were excluded from the sample . adolescents aged 1214 years are considered to be in their early adolescence and were not included in the analysis on entry into motherhood . ethical approval for the study was granted by the kenya medical research institute 's ethical review board . the outcome variable was based on answers to the following three questions : have you ever given birth? and if so , in what month and year did you have your first child , how old were you when you had your first child? the outcome variable was the age at which a person makes the transition to first motherhood ( uncensored individuals ) or age at interview if the event had not occurred as at the time of the survey ( censored individuals ) . a dichotomous variable ( coded 1 if the first birth occurred and 0 if not ) was used to define the censoring status . differences in the timing of entry into motherhood may be explained by socio - demographic and psychosocial factors , and other markers of the transition to adulthood , whose measures were included as explanatory factors in the regression models . socio - demographic characteristics ( table 2 ) used in the multivariate analysis included study site ( korogocho vs viwandani ) , age in years , educational level , ethnic group , religion , place of birth ( born in slum vs born outside ) , mother 's survival status ( mother alive or not ) , father 's survival status ( father alive or not ) , schooling status ( in school vs out of school ) . educational attainment was divided into three categories : no education , primary education , secondary education or higher . also , a variable indicating whether the respondent has ever used contraception was included in the analysis ( yes vs no ) . two key markers of the transition to adulthood , namely marital status and independent housing , were also included as part of the explanatory variables . marital status was coded 1 if the individual was married or living together with a partner and 0 otherwise . similarly , independent housing was coded 1 if an individual owned or rented her house and 0 otherwise . an index computed based on responses to six items measuring attitudes towards contraceptives was also used as an explanatory variable . protective and risk factors , as categorized by jessor in the pbt framework , were generated using composite scores derived from standardized values of individual items . cronbach 's alpha was used to assess internal consistency of scores for each composite measure ( crocker & algina , 1986 ) . the alpha reliabilities of the composite measures were acceptable ( cronbach 's alpha > 0.6 ) ( see table 1 ) . table 1.description of items used to measure protective and risk factorsquestionsitemresponse codesocial controls protection ( cronbach 's alpha=0.83)19how much would you say your parents / guardians really know about the following things about you:1 ( never know ) to3 ( always know)where you spend time in the evenings on weekdays ? who you spend time with in the evenings on week days ? where you spend time on weekends ? who you spend time with on weekends ? who your friends are?1011how often does your parent / guardian scold or reprimand you when you do something wrong ; for example , if you come home late , do n't do your chores , watch too much tv ? when you do something wrong , how often does your parent / guardian spank or slap you?1 ( never ) to5 ( every time)12if you are currently in school , how important is it to your friends that you do well in school?1 ( not too important ) to3 ( very important)1314how do most of your friends feel about someone your age drinking alcohol ? how do most of your friends feel about someone your age using marijuana or other drugs?1 ( strongly disapprove)to 4 ( strongly approve)individual controls protection ( cronbach 's alpha=0.76)16how important is it to you to rely on religious teaching when you have a problem ? how important is it to you to believe in god ? how important is it to you to rely on religious beliefs as a guide for day - to - day living ? how important is it to be able to turn to prayer when you are facing a personal problem ? how important are the following things to you : finishing secondary school ? going to university?1 ( not important ) to4 ( very important)support protection ( cronbach 's alpha=0.83)14since the beginning of this school year how often has your ( father / father figure ) checked your homework or asked you to make sure you had done it ? since the beginning of this school year , how often have you talked to your ( father / father figure ) about any progress or problems you were having at school?1 ( never ) to5 ( almost every day)since the beginning of this school year how often has your ( mother / mother figure ) checked your homework or asked you to make sure you had done it ? since the beginning of this school year , how often have you talked to your ( mother / mother figure ) about any progress or problems you were having at school?510how often does your father / father figure teach you things you did n't know ? how often do you share secrets or private feelings with your father / father figure ? how often does your father / father figure try to help you when you need something ? how often does your mother / mother figure teach you things you did n't know ? how often do you share secrets or private feelings with your mother / mother figure ? how often does your mother / mother figure try to help you when you need something?1 ( never ) to5 ( all the time)1116please indicate whether you agree or disagree with the following statements:1 ( strongly agree)4 ( strongly disagree)you feel very close to your girlfriend / boyfriend . your girlfriend / boyfriend always takes the time to talk over your problems with you . when you are with your girlfriend / boyfriend you feel completely able to relax and be yourself . no matter what happens , you know that your girlfriend / boyfriend will always be there for you . you know that your girlfriend /boyfriend has confidence in you . your girlfriend / boyfriend / partner often lets you know that he / she thinks you are a worthwhile person.models risk ( cronbach 's alpha=0.77)17have any of your brothers or sisters ever had to drop out of school for any reason ? have any of your brothers or sisters ever had premarital sex ? have any of your brothers or sisters ever smoked or do any currently smoke cigarettes ? have any of your brothers or sisters ever drunk or do any currently drink alcohol ? do you know of any close friends who have had sexual intercourse?1 ( yes ) , 2 ( no)vulnerability risk ( cronbach 's alpha=0.79)15 in the last month , has your family / household ever not had enough food to feed everyone ? in the past three months has your family / household suffered because your parent(s)/guardian(s ) were out of a job ? did your parents ever divorce or separate ? sometimes parents or other adults hurt children . has a parent or other adult living in your home ever hit you hard enough to cause injury?1 ( yes ) , 2 ( no)67how well do you get along with others your age ? how well do you live up to what other people expect of you?1 ( very well ) to4 ( not well at all)8what about your ability to do well in school ( even if you are not in school currently)?1 ( very able ) to4 ( not able at all)9how attractive do you think you are?1 ( very attractive ) to4 ( not attractive at all)10on the whole , how satisfied are you with yourself?1 ( very satisfied ) to4 ( not satisfied at all)11how much peer pressure is there on people your age to have sex?1 ( none ) to 4 ( a lot)1223what are the chances that : you will finish primary school ? you will join secondary school ? you will be respected in your community?1 ( high ) 3 ( low)multiple problem behaviour index ( mpbi ) ( cronbach 's alpha=0.69)14delinquent behaviors : how many times have you done any of the following things in the last 4 months:0 ( never ) , 1 ( once),2 ( more than once)you stayed away from home at least one night without your parent 's permission ? you started a fight with your peers ? you took or tried to take something that belonged to someone else , without their knowledge ? you hit or threatened to hit a peer or adult?multiple pro - social behaviour index ( mpsbi ) ( cronbach 's alpha=0.67)18civic participation : do you belong to a [ group ] ? ( groups : religious group , foot / netball club , drama group / dance group / choir , anti - aids club , anti - drugs club , girl guides / boy scouts , wildlife society , self - help group)1 ( yes ) , 2 ( no)attitudes towards contraception ( cronbach 's alpha=0.74)16it 's smart to use birth control to prevent an unplanned pregnancy . using birth control is just too much of a hassle . it is a good idea to use condoms to protect against getting aids . teenagers who use birth control show they care about themselves and their future.1 ( agreed ) 2 ( disagree ) description of items used to measure protective and risk factors in this paper , protective factors included social controls protection , individual controls protection and support protection . the social controls protection index was measured using fourteen items related to parental monitoring and perceived peer attitudes towards antisocial behaviours . the individual controls protection included six items related to religiosity , positive attitude towards schooling , resistance to peer pressure and traditional attitudes towards sexual behaviour . support protection refers to the presence of a supportive environment and was measured using sixteen items . the multiple pro - social behaviour index ( mpsbi ) is a composite eight - item index constructed measuring involvement in community activities ( eight items ) . the models risk index was measured using seven items measuring models for risk behaviour in family , peers and school context . on the other hand , 23 items related to low self - esteem , low perceived life chances , adverse life experiences and perceived peer pressure to engage in sex were used to measure vulnerability risk . the multiple problem behaviour index ( mpbi ) is a composite index constructed measuring engagement in delinquent behaviours ( four items ) . event history analysis ( eha ) techniques were employed for analysis , with kaplan meier estimates being used to examine the timing of first motherhood and cox regression models to investigate the influence of various factors on entry into motherhood ( allison , 1991 ; cleves et al . , 2008 ) . to allow age at first motherhood to be censored , individuals were considered to be at risk from birth until they first become mothers or censored at the time of the survey for those who were still childless . for girls who had yet to reach a particular age and who had not experienced first motherhood , the risk of having a first child at that age was assumed to be the same as that for individuals who had reached that age . cox 's proportional hazards models were used for the multivariate analysis , since they do not need specification of the form of the distribution of the baseline hazard rate ( cox , 1972 ; cox & oakes , 1984 ; blossfeld et al . , 1989 ) . these models also allow for use of time - varying covariates : that is , characteristics whose status may change over time . when the hazard ratio is greater than one , it means a higher risk of first child in the corresponding category , as compared with the reference category . conversely , the risk of having first child is lower when the hazard ratio is less than one . the hazard rate in the cox model is computed as : , where the regression coefficients are to be estimated from the data . the term h0(t ) is the baseline hazard function ( the hazard when z=0 ) , zj(t ) is the individual covariates vector and j is a vector of the regression parameters that indicates the effects of these covariates , some of them varying with t ( hence the term time - varying covariate ) . variations in the timing of entry into motherhood were examined in two age cohorts : 1517 years ( younger cohort ) and 1819 years ( older cohort ) . actually , being an early mother among 15- to 17-year - olds could be a different phenomenon than being an . indeed , the explanatory factors are likely to play a very different role in the younger cohort than in the older cohort . in other words , what are the correlates of earliness of entry into motherhood among the 15- to 17-year - olds as against those of the 18- to 19-year - olds ? descriptive characteristics for the 897 female participants in the analytic sample are displayed in table 2 . about 15% of the 1519 year female adolescents reported having had a child , with older adolescents being more likely than the younger ones to have done so ( 27% vs 5% ) . about 47% of adolescents were living in korogocho and 53% in viwandani ; the same distribution is observed within the two age groups . fifty - eight per cent of all adolescents were born in the slum areas ; this percentage is higher among the younger cohort ( 65% vs 49% ) . most of the adolescents reported having their parents alive : 93% for mother and 78% for father . overall , only 1% never attended school ( 1% for younger cohort and 2% for older cohort ) , 55% of the respondents have attained primary level of education ( 59% for younger cohort vs 50% for older cohort ) and 44% have at least secondary level education ( 40% for younger cohort and 48% for older cohort ) . at the time of the survey , 52% of the 1519 year female adolescents were attending school , with those from the younger cohort being more likely to do so ( 72% vs 27% ) . about 13% of the participants reported having ever been married ( 4% of younger adolescents vs 24% of older ones ) and 9% had ever owned or rented their residential unit ( 2% of younger adolescents vs 16% of older ones ) . overall , most of the individual adolescent characteristics are significantly associated with first motherhood , as shown in table 2 . the proportion of adolescents who are primiparous is higher among adolescents born outside of the slums ( p<0.01 ) , not enrolled in school ( p<0.01 ) , ever married ( p<0.01 ) , ever been residentially independent ( p<0.01 ) , without religion ( p<0.01 ) and those who had ever used contraception ( p<0.01 ) . the proportion of adolescents who had a child was lowest among those with at least secondary education level ( p<0.01 ) . in the younger cohort , all these associations are also statistically significant , except for that between mother 's survival , father 's survival and first child . in the older cohort , it is only the association between contraceptive use and first child that is not significant . among the older cohort , contrary to what was observed in the overall sample , the proportion of primiparous adolescents is higher among those who have never used contraception , although the association is not statistically significant . table 2.descriptive characteristics of the sample of 15- to 19-year - old female adolescents by age cohort , nairobi informal settlements , 20071517 years(n=498)1819 years(n=399)1519 years(n=897)socio - demographiccharacteristic%distribution%first child%distribution%first child%distribution%first childtotal100.04.6100.027.3100.014.7slum of residencekorogocho53.04.552.428.252.715.0viwandani47.04.747.626.347.314.4born in the slum?no35.36.8 * 50.633.7***42.121.2***yes64.73.449.420.857.910.0mother aliveno5.610.78.845.7**7.030.2***yes94.44.391.225.593.013.5father aliveno20.94.823.638.3***22.120.7***yes79.14.676.423.977.913.0ever used contraceptionno95.03.6***84.527.990.313.7***yes5.024.015.524.29.724.1currently in schoolno28.116.4***73.237.3***48.230.6***yes71.90.026.80.051.80.0education levelnever attended school0.825.0***1.842.9***1.236.4***primary58.67.249.642.954.621.6secondary40.20.547.910.543.65.4other0.40.00.833.30.620.0ever married / lived togetherno96.22.5***76.211.8***87.36.1***yes3.857.923.876.812.773.7ever rented / owned a houseno97.83.7***83.720.4***91.510.5***yes2.245.516.363.18.560.5*p<0.1 ; * * p<0.05 ; * * * p<0.001 . descriptive characteristics of the sample of 15- to 19-year - old female adolescents by age cohort , nairobi informal settlements , 2007 * p<0.1 ; * * p<0.05 ; * * * p<0.001 . in the following section , the results of the event history analysis enable the timing of entry into motherhood to be examined , as well as its associations with possible predictors , while controlling for the effects of other important factors , using kaplan meier estimates and cox regression models . the median age can not be calculated for the whole sample as it had not yet been attained . a quarter of adolescents had their first child by age 18.9 . by age 15 , 1% of adolescents aged 1517 and 3% of those aged 1819 had had their first child . by age 18 , 21% of the whole sample and 34% of those aged 1819 were already mothers . table 3.descriptive statistics of age at first birth by age cohort , female adolescents , nairobi informal settlements , 20071517 yearcohort1819 yearcohorttotalproportion having a first birth by age ( years):120%0%0%130%1%0%141%1%1%151%3%2%162%4%3%178%16%13%1824%21%1931%29%age at : first quartilena18.919.0mediannananathird quartilenananaperson - years at risk8164.47365.115,529.5n498399897events23109132na = not attained . descriptive statistics of age at first birth by age cohort , female adolescents , nairobi informal settlements , 2007 results from the cox models are presented in table 4 , by age cohort . models 1 and 4 include only the socio - demographic variables . in models 2 and 5 , the two other key markers of transition to adulthood are controlled for , in addition to the socio - demographic variables . table 4.hazard ratios of having first birth among female adolescents by birth cohort ( cox model ) , nairobi informal settlements , 2007hazard ratios ( 95% ci)15- to 17-year - old18- to 19-year - oldvariablesmodel 1model 2model 3model 4model 5model 6socio - demographic variablesage ( years)1.22 ( 0.562.67)1.20 ( 0.453.20)1.12 ( 0.216.08)0.85 ( 0.551.31)0.73 ( 0.461.14)0.81 ( 0.511.29)slum residence ( ref . koch)viwandani0.81 ( 0.272.44)0.24 * * ( 0.060.94)0.31 ( 0.042.41)0.89 ( 0.531.49)0.92 ( 0.571.48)1.06 ( 0.671.70)education ( ref . primary)never attended4.95 ( 0.6041.04)2.00 ( 0.429.42)4.80 ( 0.6038.67)2.06 ( 0.498.67)1.88 ( 0.615.79)2.74 ( 0.779.80)secondary or higher0.13 * ( 0.021.05)0.29 ( 0.042.35)0.20 ( 0.013.61)0.41 * * * ( 0.240.70)0.57 * * ( 0.350.94)0.67 ( 0.371.18)other0.30 ( 0.042.05)0.05 * * * ( 0.010.33)0.21 ( 0.014.61)0.73 ( 0.105.17)0.33 ( 0.034.45)0.42 ( 0.035.12)currently in school0.00 * * * ( 0.000.00)0.00 * * * ( 0.000.00)0.00 * * * ( 0.000.00)0.00 * * * ( 0.000.00)0.00 * * * ( 0.000.00)0.00 * * * ( 0.000.00)born in slums0.64 ( 0.271.53)0.71 ( 0.251.97)0.47 ( 0.141.66)0.68 ( 0.401.13)0.98 ( 0.581.67)0.95 ( 0.571.59)mother alive0.56 ( 0.074.38)0.67 ( 0.133.40)0.43 ( 0.062.88)0.67 ( 0.341.34)0.79 ( 0.391.58)0.60 ( 0.291.23)father alive1.77 ( 0.437.23)1.87 ( 0.487.27)2.80 ( 0.3920.16)0.87 ( 0.551.38)0.98 ( 0.591.62)0.96 ( 0.581.59)ever used contraception0.98 ( 0.137.64)0.73 ( 0.391.37)transition markersmarried18.46 * * * ( 6.6751.12)13.72 * * * ( 1.9994.40)8.16 * * * ( 5.1512.95)8.22 * * * ( 5.2412.90)owned or rented a house1.44 ( 0.405.19)1.12 ( 0.158.28)1.14 ( 0.711.83)1.09 ( 0.651.82)psychosocial variablesmpbi1.53 ( 0.862.72)1.10 ( 0.831.46)mpsbi0.64 ( 0.104.21)0.93 ( 0.461.88)social controls protection0.61 ( 0.261.46)0.76 * * ( 0.591.00)individual controls protection1.29 ( 0.533.15)0.73 * * ( 0.540.98)social support protection1.11 ( 0.393.14)1.09 ( 0.801.50)model risk2.27 * * ( 1.144.52)1.03 ( 0.781.35)vulnerability risk1.15 ( 0.274.92)1.39 ( 0.812.42)positive attitudes towardscontraception1.27 ( 0.413.87)0.70 ( 0.421.18)wald 11,001.622***19,727.065***10,400.520***18,609.371***29,892.313***33,661.686***2log likelihood188.96162.54150.161122.661030.71013.24subjects ( events)498 ( 23)498 ( 23)498 ( 23)399 ( 109)399 ( 109)399 ( 109)time at risk8164.3598164.3598164.3597365.0947365.0947365.094*p<0.1 ; * * p<0.05 ; * * * p<0.01 . models controlled for ethnicity , religion.amultiple problem behaviour index.bmultiple pro - social behaviour index . hazard ratios of having first birth among female adolescents by birth cohort ( cox model ) , nairobi informal settlements , 2007 * p<0.1 ; * * p<0.05 ; * * * p<0.01 . multiple pro - social behaviour index . among adolescents aged 1517 years , none of the socio - demographic characteristics , with the exception of current school attendance , significantly affected the timing of entry into motherhood after controlling for other factors . hazard ratios ( hrs ) from the full model 3 indicate that adolescents currently attending school are significantly more likely ( the hr is almost equal to zero ) to delay childbearing . adolescents without education enter into motherhood earlier , while those with at least secondary level of education do so at a later age , although the ratios are not significant at the 5% level . the significant and negative effective of secondary education only disappears when marriage and residential independence are controlled for in the models . there is no significant difference in the timing of childbearing based on adolescents ' lifetime contraceptive use , although the findings show that adolescents aged 1517 years who have ever used contraception were more likely to delay childbearing . results from model 3 suggest that married adolescents are more than 13 times more likely to have their first child earlier that the non - married adolescents . although those who are residentially independent are also more likely to give birth earlier , the hr is not statistically significant . these effects were also observed in model 2 , which includes only those two variables and the socio - demographic characteristics . as for protective factors , the findings from model 3 indicate that although all the hrs corresponding to the protective factors are in the expected direction , i.e. less than 1 , none of them is significantly associated with the timing of making the transition into motherhood . in a model where only the psychosocial variables were controlled for ( results not shown here ) , mpsbi and support protection are significantly associated with entry into motherhood , with both higher scores on these two indices associated with a delay in the timing of first birth . with regard to risk factors , the results suggest that having negative models is associated with the outcome of interest ; adolescents with a high level of model risk are more likely to enter into motherhood earlier . although only marginally significant at 10% , in a model that only controls for psychosocial variables , increasing vulnerability risk is associated with earlier entry into motherhood . there is no significant difference in the timing of entry into motherhood based on attitudes towards contraception . for adolescents aged 1819 years , the findings indicate similar associations when it comes to socio - demographic characteristics and transition markers . as with the 15- to 17-years - olds , older adolescents with at least a secondary level of education delay their entry into motherhood , although the corresponding hazard ratio is not significant at the 5% level . the negative effect of secondary education became insignificant after the introduction of psychosocial variables in the full model ( they were highly significant in previous models ) . no significant difference was observed with regard to the other socio - demographic characteristics . among the protective factors , social and individual controls protection adolescents with high levels of these two factors are likely to delay their entry into motherhood ( chances are reduced by 24% and 27% for social controls and individual controls , respectively ) . none of the risk factors is significantly associated with timing of entry into motherhood in the older cohort , although in a model that only controls for psychosocial variables , adolescents with high vulnerability risk are likely to enter into motherhood earlier . as with the younger cohort , there is no significant difference in the timing of childbearing based on attitudes towards contraception . this paper is based on data collected from 15- to 19-year - old adolescents in two resource - poor settings in nairobi to investigate patterns and determinants of the timing of entry into motherhood . the focus is on the association between the timing of childbearing and other key transitions to adult roles , socio - demographic characteristics and psychosocial variables . this study adds to the growing body of evidence on transition to adult roles among young people in urban informal settlements in sub - saharan africa . the findings suggest that some female adolescents become mothers at very young ages ; 21% of the whole sample and 34% of those aged 1819 are already mothers by age 18 . given their incomplete physical maturity , these young females are likely to face special risks during pregnancy and delivery , especially in such resource - poor settings characterized by lack of basic health facilities and relatively high cost of health care ( izugbara et al . , 2009 ) . in addition , in a context where educational and employment opportunities are scarce , younger mothers are more likely to drop out of school and hence have reduced opportunities for gainful employment . previous evidence has shown that living in resource - constrained settings predisposes adolescents to increased risk of negative social outcomes , including risky sexual behaviour , substance use , delinquency and violence ( blum et al . , 2000 ; mugisha et al . , 2003 ; ngom et al . , 2003 ; dodoo et al . , 2007 ) . consequently , programmes aiming to reduce risky sexual behaviours that could lead to childbearing among adolescents should be made in tandem with initiatives to enhance livelihood prospects for those in lower income brackets . further , these programmes must be introduced very early , and before the onset of sexual activity . as expected , marriage has a strong relationship with the timing of first birth for both younger and older cohorts , with adolescents who are married or living together with their partners entering into motherhood significantly earlier than those who are not . this is consistent with previous evidence that shows that marriage is a key driver of fertility in sub - saharan africa , although out - of - wedlock childbearing is becoming increasingly common in the region ( tambashe & shapiro , 1996 ) . consequently , efforts to reduce early childbearing must include initiatives to discourage girls from marrying early . early childbearing , which often stems from early marriage , entails potential health risks for the young mother and the child ( zabin & kiragu , 1998 ) . early marriage may also limit educational opportunities and may occur when the young person is not fully prepared to take over marital and parental responsibilities ( singh & samara , 1996 ) . in many cases , early marriage is associated with higher chances of divorce or separation , which may leave the young mother bearing the sole responsibility of raising the child , without any social or family support , either emotionally or financially . specifically , being in school was associated with a delay in the timing of childbearing . although the observed association between school enrolment or educational level may be misleading because some girls who are not in school at the time of the survey could have dropped out of school because of pregnancy - related reasons , previous studies do show that in - school youth tend to delay sexual activity ( ajayi et al . , 1991 ; this suggests that even within impoverished communities like slum settlements , education is an important pathway for reducing early childbearing and associated negative reproductive health and socioeconomic outcomes . this needs to be further enlightened through , for instance , exploration of the association between school curricula , including sexual and reproductive health education , and reproductive outcomes among girls living in such poor settings . other studies have also found that adolescents with at least secondary education are less likely to give birth or get pregnant than those with no or little formal education ( mboup & saha , 1998 ; quamrun & hosik , 2008 ; magadi & agwanda , 2009a ) . in their analysis of dhs data in eight countries in sub - saharan africa , gupta & mahy ( 2003a ) found a strong negative influence of education on the probability of childbearing in all countries . in particular , they concluded that ensuring that adolescent girls receive at least a secondary level of education is the optimal way of delaying childbearing . our analyses somehow support this argument : adolescents with at least secondary education level enter into motherhood later . it may be not only important to enrol girls in school , but also necessary to retain them in the education system until they complete secondary school . consequently , policies and programmes such as universal primary education should be extended to ensure increased access to , and completion of , secondary education . however , this may not be done in isolation as the poor job prospects in poor settings such as slums may lower the motivation among adolescent girls to complete secondary school . although the hazard ratios are not statistically significant , being residentially independent appears to hasten entry into motherhood for both age cohorts . given the space constraints that typify the slum settlements in nairobi , young adults are almost forced to leave their parental home , leaving them highly exposed to risky sexual behaviours that lead to increased risks of early childbearing , especially in an environment where contraceptive use is low ( amuyunzu - nyamongo & magadi , 2006 ; dodoo et al . , 2007 ) . further investigations are needed to understand the consequences of home - leaving among adolescents living is such poor settings . models ( both protection and risk ) , controls ( both individual and social ) , supports and vulnerability in the pbt framework . among 15- to 17-year - old adolescents , models risk ( the presence of models for problem or anti - social behaviours , including early sexual activity ) is significantly associated with entry into motherhood , with those having high models risk being more likely to have a first child earlier . this indicates the vulnerability of younger girls in an environment where indulgence in problem behaviours is common practice . in a recent study , ndugwa and others ( 2010 ) found that models risk is a strong predictor of involvement in problem behaviour among 12- to 19-year - old adolescents living in korogocho and viwandani slum settlements in nairobi . in contexts typified by early childbearing , young parenthood may become normative , meaning that adolescent girls become pregnant because many of their peers and family members also begin childbearing early . social controls protection , such as parental monitoring and perceived peer disapproval of antisocial behaviours , reduces the likelihood of early entry into motherhood for 18- to 19-year - old female adolescents living in slum settlements . this is consistent with other evidence that shows that parental monitoring often prevents young people from engaging in risky sexual behaviours that could trigger early childbearing ( babalola et al . , 2005 ; kumi - kyereme et al . , 2007 ) previous findings from nairobi slums show that it is the father 's presence , in particular , that is associated with lower risk of engaging in sexual activity and , hence , lower risk of pregnancy ( ngom et al . , 2003 ) . also , adolescents often identify themselves with peer groups while growing up , allowing the latter to serve as models for appropriate behaviour . adolescents ' sexual behaviour has been found to be associated with peers ' attitudes and behaviours in slum and non - slum communities in nairobi ( kabiru et al . , 2010 ) . in addition , 18- to 19-year - old adolescent girls with strong individual controls protection , such high religiosity and positive orientation towards school , are likely to delay their first birth . it is possible that highly religious adolescent girls are frequently exposed to moral messages against early sexual debut and pregnancy ; they are also likely to commit to stand by such teachings given their stronger religious engagement . moreover , they may often interact within a network of religious peers who serve as models for conventional behaviours . this finding also suggests that adolescents with high educational expectations might choose to delay sexual experience and childbearing to meet those expectations . first , the analysis is based on self - reported information and thus is subject to self - report bias . for example , there is possible under - reporting of children who died shortly after birth . also , retrospective recording of timing of first birth , marriage and independent housing may affect the accuracy of reporting . this means that the true magnitude of adolescent childbearing in the sample could be higher than that observed . second , most of the independent variables are time - invariant , thus limiting the possibility of inferring causality . as a result , most of the findings do not infer causation , apart from those related to marriage and independent housing . further , another marker of the transition to adulthood , i.e. entry into income - generating activity , can not be used in analysis as the related data were not collected during wave 1 of the transition to adulthood ( tta ) questionnaire used in this paper . also , qualitative information was not available to enlighten the quantitative findings . these limitations notwithstanding , the findings highlight some key factors that are likely to be important drivers of early entry into motherhood among the two age groups of adolescents living in urban nairobi slums . these findings support the need for programmes or policies that enhance controls protection , buffer or moderate the exposure to models that support unconventional behaviour , keep girls in school and improve livelihood opportunities among adolescents at high risk of early childbearing .

summarythe contribution of adolescents ' childbearing to total fertility rates in many sub - saharan african countries is higher than in other parts of the world . in this paper , data collected from 897 female adolescents aged 1519 years are analysed to investigate patterns and determinants of entry into motherhood in two informal settlements in nairobi , kenya , using kaplan meier estimates and cox regression models . about 15% of these adolescents have had a child . the findings show that marriage , being out of school and having negative models in peer , family and school contexts are associated with early childbearing among females aged 1517 years . for adolescents aged 1819 years , school attendance considerably delays entry into motherhood while marriage hastens its timing . furthermore , older adolescents with high levels of social controls ( parental monitoring or perceived peer orientation to or approval of prosocial behaviours ) and individual controls ( high religiosity and positive orientation to schooling ) are likely to delay childbearing . programmes aiming to reduce risky sexual behaviours that could lead to childbearing among adolescents should be introduced very early , and before the onset of sexual activity . also , the findings underscore the need to identify and address the risky factors and reinforce the protective ones in order to improve sexual and reproductive health outcomes of adolescent girls in nairobi slum settlements .

Introduction Methods Results Discussion

this article investigates the correlates of the timing of transition into motherhood among adolescent girls in two informal settlements or slums korogocho and viwandani in nairobi city , kenya . however , adolescent fertility has been viewed as both a social and policy challenge in many countries in sub - saharan africa ( world health organization , 2011 ; united nations children 's fund , 2012a ; national research council & institute of medicine , 2005 ) . in many sub - saharan african countries , the contribution of adolescents ' fertility to total fertility rates remains higher than in other parts of the world . recent data indicate that 18% of adolescent girls aged 1519 in eastern / southern africa and 21% in western / central africa had initiated childbearing ( national research council & institute of medicine , 2005 ) . in fact , much of the high fertility in sub - saharan africa can be attributed to first birth occurring at very young ages . although childbearing is a key marker of the transition to adulthood and carries parental responsibilities , giving birth during adolescence is likely to lead to poor health outcomes for the young mother and her child . patton and colleagues ( 2009 ) show that maternal mortality accounts for nearly 26% of the increased number of deaths in females aged 1024 years in the african region . early childbearing is also known to negatively impact on educational and employment prospects , and hence lead to a greater likelihood of poverty ( singh , 1998 ; gupta & mahy , 2003b ) . in many instances , pregnant females are forced to drop out of school , while for those who are able to continue schooling , the very possible conflict between school and child care responsibilities , which women primarily bear , results in poor academic performance . accordingly , adolescents ' fertility has become a source of social and policy concern for many governments and non - governmental organizations in sub - saharan africa ( national research council & institute of medicine , 2005 ; world health organization , 2011 ; united nations children 's fund , 2012a ) . indeed , the 1994 international conference on population and development ( icpd ) paved the way for highlighting the concerns of adolescents ' fertility by prioritizing adolescents ' reproductive health issues . since then , there have been an increasing number of studies addressing reproductive health and behaviour challenges among adolescents in sub - saharan africa . although sexual and reproductive health was excluded from the millennium development goals ( mdgs ) , universal access to reproductive health by 2015 was later set as a goal by governments at the world summit in 2005 since it was clear that achievement of mdgs largely depended on addressing sexual and reproductive health challenges , especially in high - fertility regions ( glasier et al . beyond the achievement of the objective of providing reproductive health services for the youth , a better understanding of adolescents ' childbearing is of great importance for policymakers involved in helping the continent to achieve its desirable fertility levels . in kenya , where the fertility decline is stalling , the proportion of teenagers initiating childbearing was 18% in 2008 , with teenagers from poorer households ( 24% ) being more likely to have begun childbearing than those from wealthier households ( 16% ) ( kenya national bureau of statistics ( knbs ) & icf macro , 2010 ) . also , they are known to engage in riskier sexual behaviours , including early sexual debut , transactional sex and multiple sexual partnerships than those in the non - slums parts of the city ( african population and health research center , 2002 ; zulu et al . actually , early entry into childbearing among young slum dwellers is a sign of early involvement in unprotected sexual intercourse , which exposes these youth to sexually transmitted infections , including hiv , given the relatively high prevalence of hiv / aids in slum settlements . these constitute social barriers that may impinge on adolescents ' ability to have control over their reproductive life or decide on the timing of childbearing , and thus have key implications for the well - being and welfare of adolescents . in addition , averting early childbearing among adolescent girls living in urban slums could also contribute to slowing rates of urban growth , which is substantially brought about by natural increase in sub - saharan africa ( chen et al . the mushrooming of informal settlements in urban areas in sub - saharan africa is one of the tangible manifestations of the rapid urban growth in this region over the past 20 years ( un - habitat , 2008a , 2008b , 2010 ) . in kenya , un - habitat estimates suggest that between 60 and 70% of the nairobi city residents live in slum settlements or slum - like conditions ( african population and health research center , 2002 ; un - habitat , 2008a ) . the findings could help understand the circumstances and factors surrounding early childbearing among young people in order to design and implement appropriate intervention programmes . this study examines predictors of entry into motherhood among adolescents living in the two slum settlements using the problem behaviour theory ( pbt ) framework ( jessor et al . with respect to controls protection , a distinction could be made between social controls protection , which is captured at the social environment level , and individual controls protection , measured at the individual level . models risk relates to models for unconventional behaviour ; opportunity risk operates through exposure to contextual elements or situations that could lead to engaging in antisocial behaviour ; and vulnerability has to do with individual attributes that increase the risks of engaging in socially proscribed behaviour . in this paper , the pbt framework is used to explain early childbearing among adolescent girls aged 1519 in two informal settlements in nairobi city . an adolescent 's entry into motherhood is therefore expected to be negatively associated with protective factors , and positively associated with risk factors . in addition to psychosocial factors , socio - demographic factors are known to be associated with adolescent fertility in different regions of the world . in her global review of adolescent childbearing , singh ( 1998 ) points out that factors associated with adolescent fertility include , among others , cultural practices of early marriage , especially in africa and asia , as well as cultural tolerance of premarital sexual relationships to prove one 's fertility before marriage . adolescents residing in urban areas and those with higher levels of education are likely to delay their childbearing due to known conflicts between schooling , work and childrearing ( singh , 1998 ; gupta & mahy , 2003a ) . parental education is also an important determinant of adolescent childbearing , with daughters of more educated parents being more likely to delay their entry into motherhood ( tambashe & shapiro , 1996 ; lee , 2001 ) . family structure is an important determinant of adolescent fertility , with adolescents raised in polygamous households being more likely to initiate sexual activity early compared with those in monogamous households ( tambashe & shapiro , 1996 ) . parental survival status is also associated with early childbearing , with orphans being more likely to begin childbearing early ( tambashe & shapiro , 1996 ; cavazos - rehg et al . this paper investigates whether these factors are also correlated with adolescent entry into motherhood in two slum settlements in nairobi , kenya . this paper draws on data collected under the transition to adulthood ( tta ) component of the urbanization , poverty and health dynamics ( uphd ) project that the african population and health research center ( aphrc ) conducted between 2007 and 2010 in two informal settlements , korogocho and viwandani , in nairobi city . the tta is a 3-year prospective study that aims to identify protective and risk factors in the lives of adolescents growing up in the two informal settlements , and examine how these factors influence key markers of the transition to adulthood ( transitions to secondary school , sexual intercourse , marriage , parenthood , independent housing ) . the specific aims are to : ( a ) identify sexual , reproductive health , livelihood , educational and other key concerns and aspirations of young people as they grow up in urban informal settlements ; ( b ) determine both protective and risk factors ( including coercion ) that influence young people 's transition to secondary school , employment , independent housing , sexual and marital partnerships and parenthood and the sequencing of these transitions ; and ( c ) investigate the implications of childbearing aspirations for hiv / sti prevention and vice versa , with particular focus on dual - protection strategies . to generate the analytical sample of 15- to 19-year - old girls for this paper , 114 observations were first omitted from the 4058 successfully interviewed youth because of inconsistent or missing data on age , date of birth and/or age at first event ( child , sex , marriage , housing ) , leaving 3944 ( 1957 females ) individuals with valid data . adolescents aged 1214 years are considered to be in their early adolescence and were not included in the analysis on entry into motherhood . differences in the timing of entry into motherhood may be explained by socio - demographic and psychosocial factors , and other markers of the transition to adulthood , whose measures were included as explanatory factors in the regression models . socio - demographic characteristics ( table 2 ) used in the multivariate analysis included study site ( korogocho vs viwandani ) , age in years , educational level , ethnic group , religion , place of birth ( born in slum vs born outside ) , mother 's survival status ( mother alive or not ) , father 's survival status ( father alive or not ) , schooling status ( in school vs out of school ) . teenagers who use birth control show they care about themselves and their future.1 ( agreed ) 2 ( disagree ) description of items used to measure protective and risk factors in this paper , protective factors included social controls protection , individual controls protection and support protection . the social controls protection index was measured using fourteen items related to parental monitoring and perceived peer attitudes towards antisocial behaviours . event history analysis ( eha ) techniques were employed for analysis , with kaplan meier estimates being used to examine the timing of first motherhood and cox regression models to investigate the influence of various factors on entry into motherhood ( allison , 1991 ; cleves et al . variations in the timing of entry into motherhood were examined in two age cohorts : 1517 years ( younger cohort ) and 1819 years ( older cohort ) . indeed , the explanatory factors are likely to play a very different role in the younger cohort than in the older cohort . in other words , what are the correlates of earliness of entry into motherhood among the 15- to 17-year - olds as against those of the 18- to 19-year - olds ? about 15% of the 1519 year female adolescents reported having had a child , with older adolescents being more likely than the younger ones to have done so ( 27% vs 5% ) . at the time of the survey , 52% of the 1519 year female adolescents were attending school , with those from the younger cohort being more likely to do so ( 72% vs 27% ) . the proportion of adolescents who are primiparous is higher among adolescents born outside of the slums ( p<0.01 ) , not enrolled in school ( p<0.01 ) , ever married ( p<0.01 ) , ever been residentially independent ( p<0.01 ) , without religion ( p<0.01 ) and those who had ever used contraception ( p<0.01 ) . the proportion of adolescents who had a child was lowest among those with at least secondary education level ( p<0.01 ) . table 2.descriptive characteristics of the sample of 15- to 19-year - old female adolescents by age cohort , nairobi informal settlements , 20071517 years(n=498)1819 years(n=399)1519 years(n=897)socio - demographiccharacteristic%distribution%first child%distribution%first child%distribution%first childtotal100.04.6100.027.3100.014.7slum of residencekorogocho53.04.552.428.252.715.0viwandani47.04.747.626.347.314.4born in the slum?no35.36.8 * 50.633.7***42.121.2***yes64.73.449.420.857.910.0mother aliveno5.610.78.845.7**7.030.2***yes94.44.391.225.593.013.5father aliveno20.94.823.638.3***22.120.7***yes79.14.676.423.977.913.0ever used contraceptionno95.03.6***84.527.990.313.7***yes5.024.015.524.29.724.1currently in schoolno28.116.4***73.237.3***48.230.6***yes71.90.026.80.051.80.0education levelnever attended school0.825.0***1.842.9***1.236.4***primary58.67.249.642.954.621.6secondary40.20.547.910.543.65.4other0.40.00.833.30.620.0ever married / lived togetherno96.22.5***76.211.8***87.36.1***yes3.857.923.876.812.773.7ever rented / owned a houseno97.83.7***83.720.4***91.510.5***yes2.245.516.363.18.560.5*p<0.1 ; * * p<0.05 ; * * * p<0.001 . descriptive characteristics of the sample of 15- to 19-year - old female adolescents by age cohort , nairobi informal settlements , 2007 * p<0.1 ; * * p<0.05 ; * * * p<0.001 . in the following section , the results of the event history analysis enable the timing of entry into motherhood to be examined , as well as its associations with possible predictors , while controlling for the effects of other important factors , using kaplan meier estimates and cox regression models . by age 15 , 1% of adolescents aged 1517 and 3% of those aged 1819 had had their first child . among adolescents aged 1517 years , none of the socio - demographic characteristics , with the exception of current school attendance , significantly affected the timing of entry into motherhood after controlling for other factors . there is no significant difference in the timing of childbearing based on adolescents ' lifetime contraceptive use , although the findings show that adolescents aged 1517 years who have ever used contraception were more likely to delay childbearing . as for protective factors , the findings from model 3 indicate that although all the hrs corresponding to the protective factors are in the expected direction , i.e. in a model where only the psychosocial variables were controlled for ( results not shown here ) , mpsbi and support protection are significantly associated with entry into motherhood , with both higher scores on these two indices associated with a delay in the timing of first birth . with regard to risk factors , the results suggest that having negative models is associated with the outcome of interest ; adolescents with a high level of model risk are more likely to enter into motherhood earlier . although only marginally significant at 10% , in a model that only controls for psychosocial variables , increasing vulnerability risk is associated with earlier entry into motherhood . there is no significant difference in the timing of entry into motherhood based on attitudes towards contraception . for adolescents aged 1819 years , the findings indicate similar associations when it comes to socio - demographic characteristics and transition markers . as with the 15- to 17-years - olds , older adolescents with at least a secondary level of education delay their entry into motherhood , although the corresponding hazard ratio is not significant at the 5% level . among the protective factors , social and individual controls protection adolescents with high levels of these two factors are likely to delay their entry into motherhood ( chances are reduced by 24% and 27% for social controls and individual controls , respectively ) . none of the risk factors is significantly associated with timing of entry into motherhood in the older cohort , although in a model that only controls for psychosocial variables , adolescents with high vulnerability risk are likely to enter into motherhood earlier . this paper is based on data collected from 15- to 19-year - old adolescents in two resource - poor settings in nairobi to investigate patterns and determinants of the timing of entry into motherhood . this study adds to the growing body of evidence on transition to adult roles among young people in urban informal settlements in sub - saharan africa . the findings suggest that some female adolescents become mothers at very young ages ; 21% of the whole sample and 34% of those aged 1819 are already mothers by age 18 . in addition , in a context where educational and employment opportunities are scarce , younger mothers are more likely to drop out of school and hence have reduced opportunities for gainful employment . consequently , programmes aiming to reduce risky sexual behaviours that could lead to childbearing among adolescents should be made in tandem with initiatives to enhance livelihood prospects for those in lower income brackets . further , these programmes must be introduced very early , and before the onset of sexual activity . this is consistent with previous evidence that shows that marriage is a key driver of fertility in sub - saharan africa , although out - of - wedlock childbearing is becoming increasingly common in the region ( tambashe & shapiro , 1996 ) . although the observed association between school enrolment or educational level may be misleading because some girls who are not in school at the time of the survey could have dropped out of school because of pregnancy - related reasons , previous studies do show that in - school youth tend to delay sexual activity ( ajayi et al . , 1991 ; this suggests that even within impoverished communities like slum settlements , education is an important pathway for reducing early childbearing and associated negative reproductive health and socioeconomic outcomes . this needs to be further enlightened through , for instance , exploration of the association between school curricula , including sexual and reproductive health education , and reproductive outcomes among girls living in such poor settings . although the hazard ratios are not statistically significant , being residentially independent appears to hasten entry into motherhood for both age cohorts . given the space constraints that typify the slum settlements in nairobi , young adults are almost forced to leave their parental home , leaving them highly exposed to risky sexual behaviours that lead to increased risks of early childbearing , especially in an environment where contraceptive use is low ( amuyunzu - nyamongo & magadi , 2006 ; dodoo et al . among 15- to 17-year - old adolescents , models risk ( the presence of models for problem or anti - social behaviours , including early sexual activity ) is significantly associated with entry into motherhood , with those having high models risk being more likely to have a first child earlier . in a recent study , ndugwa and others ( 2010 ) found that models risk is a strong predictor of involvement in problem behaviour among 12- to 19-year - old adolescents living in korogocho and viwandani slum settlements in nairobi . social controls protection , such as parental monitoring and perceived peer disapproval of antisocial behaviours , reduces the likelihood of early entry into motherhood for 18- to 19-year - old female adolescents living in slum settlements . this is consistent with other evidence that shows that parental monitoring often prevents young people from engaging in risky sexual behaviours that could trigger early childbearing ( babalola et al . , 2007 ) previous findings from nairobi slums show that it is the father 's presence , in particular , that is associated with lower risk of engaging in sexual activity and , hence , lower risk of pregnancy ( ngom et al . adolescents ' sexual behaviour has been found to be associated with peers ' attitudes and behaviours in slum and non - slum communities in nairobi ( kabiru et al . in addition , 18- to 19-year - old adolescent girls with strong individual controls protection , such high religiosity and positive orientation towards school , are likely to delay their first birth . this finding also suggests that adolescents with high educational expectations might choose to delay sexual experience and childbearing to meet those expectations . entry into income - generating activity , can not be used in analysis as the related data were not collected during wave 1 of the transition to adulthood ( tta ) questionnaire used in this paper . these limitations notwithstanding , the findings highlight some key factors that are likely to be important drivers of early entry into motherhood among the two age groups of adolescents living in urban nairobi slums . these findings support the need for programmes or policies that enhance controls protection , buffer or moderate the exposure to models that support unconventional behaviour , keep girls in school and improve livelihood opportunities among adolescents at high risk of early childbearing .

this article investigates the correlates of the timing of transition into motherhood among adolescent girls in two informal settlements or slums korogocho and viwandani in nairobi city , kenya . however , adolescent fertility has been viewed as both a social and policy challenge in many countries in sub - saharan africa ( world health organization , 2011 ; united nations children 's fund , 2012a ; national research council & institute of medicine , 2005 ) . to effectively address this challenge , there is a need to understand the factors driving adolescent childbearing , especially in resource - poor settings such as informal settlements . recent data indicate that 18% of adolescent girls aged 1519 in eastern / southern africa and 21% in western / central africa had initiated childbearing ( national research council & institute of medicine , 2005 ) . other estimates show that , on average , more than 25% of women aged 2024 in sub - saharan africa have given birth before age 18 , with great disparities between countries in the region ( united nations children 's fund , 2012b ) . in the absence of contraception , one of the long - term demographic effects of adolescent fertility ( wulf & singh , 1991 ) . indeed , the 1994 international conference on population and development ( icpd ) paved the way for highlighting the concerns of adolescents ' fertility by prioritizing adolescents ' reproductive health issues . although sexual and reproductive health was excluded from the millennium development goals ( mdgs ) , universal access to reproductive health by 2015 was later set as a goal by governments at the world summit in 2005 since it was clear that achievement of mdgs largely depended on addressing sexual and reproductive health challenges , especially in high - fertility regions ( glasier et al . beyond the achievement of the objective of providing reproductive health services for the youth , a better understanding of adolescents ' childbearing is of great importance for policymakers involved in helping the continent to achieve its desirable fertility levels . in kenya , where the fertility decline is stalling , the proportion of teenagers initiating childbearing was 18% in 2008 , with teenagers from poorer households ( 24% ) being more likely to have begun childbearing than those from wealthier households ( 16% ) ( kenya national bureau of statistics ( knbs ) & icf macro , 2010 ) . however , little attention has been paid to understanding the dimensions and factors driving the fertility patterns among adolescents living in resource - poor urban settings and in particular in informal settlements or slums . in fact , despite the significant proportion of adolescents making up the slum population , little attention has been paid to the challenges they face during their transition to adulthood . adolescents living in nairobi slums grow up in a very poor environment characterized by lack of basic social infrastructure and services , high rates of unemployment , crime and substance abuse , poor schooling facilities and lack of recreational facilities , and so on ( african population and health research center , 2002 ; mugisha & zulu , 2004 ; fotso et al . also , they are known to engage in riskier sexual behaviours , including early sexual debut , transactional sex and multiple sexual partnerships than those in the non - slums parts of the city ( african population and health research center , 2002 ; zulu et al . actually , early entry into childbearing among young slum dwellers is a sign of early involvement in unprotected sexual intercourse , which exposes these youth to sexually transmitted infections , including hiv , given the relatively high prevalence of hiv / aids in slum settlements . indeed , previous studies show that 16% of female adolescents in slums become parents by age 18 , meaning that a considerable number are exposed to the risks of teenage pregnancy and delivery because they have not reached physical maturity or physiological development ( beguy et al . in addition , averting early childbearing among adolescent girls living in urban slums could also contribute to slowing rates of urban growth , which is substantially brought about by natural increase in sub - saharan africa ( chen et al . the mushrooming of informal settlements in urban areas in sub - saharan africa is one of the tangible manifestations of the rapid urban growth in this region over the past 20 years ( un - habitat , 2008a , 2008b , 2010 ) . in kenya , un - habitat estimates suggest that between 60 and 70% of the nairobi city residents live in slum settlements or slum - like conditions ( african population and health research center , 2002 ; un - habitat , 2008a ) . understanding and identifying ways to address the unique reproductive and health outcomes among slum adolescents , who constitute a substantial part of urban residents , is central to the attainment of development goals such as the mdgs . in this paper , the pbt framework is used to explain early childbearing among adolescent girls aged 1519 in two informal settlements in nairobi city . in her global review of adolescent childbearing , singh ( 1998 ) points out that factors associated with adolescent fertility include , among others , cultural practices of early marriage , especially in africa and asia , as well as cultural tolerance of premarital sexual relationships to prove one 's fertility before marriage . in general , major factors associated with early adolescent childbearing in developing countries include low education level , unstable family structure , absence of a father figure , low self - esteem , low socioeconomic status , early sexual debut and experience of physical abuse ( baumgartner et al . this paper draws on data collected under the transition to adulthood ( tta ) component of the urbanization , poverty and health dynamics ( uphd ) project that the african population and health research center ( aphrc ) conducted between 2007 and 2010 in two informal settlements , korogocho and viwandani , in nairobi city . every 4 months , fieldworkers visit all households within the demographic surveillance area ( dsa ) to collect information on births , deaths , migrations , health , childhood vaccination , health - seeking behaviour , education , household possessions and amenities , livelihood sources , and so on . the tta is a 3-year prospective study that aims to identify protective and risk factors in the lives of adolescents growing up in the two informal settlements , and examine how these factors influence key markers of the transition to adulthood ( transitions to secondary school , sexual intercourse , marriage , parenthood , independent housing ) . the specific aims are to : ( a ) identify sexual , reproductive health , livelihood , educational and other key concerns and aspirations of young people as they grow up in urban informal settlements ; ( b ) determine both protective and risk factors ( including coercion ) that influence young people 's transition to secondary school , employment , independent housing , sexual and marital partnerships and parenthood and the sequencing of these transitions ; and ( c ) investigate the implications of childbearing aspirations for hiv / sti prevention and vice versa , with particular focus on dual - protection strategies . to generate the analytical sample of 15- to 19-year - old girls for this paper , 114 observations were first omitted from the 4058 successfully interviewed youth because of inconsistent or missing data on age , date of birth and/or age at first event ( child , sex , marriage , housing ) , leaving 3944 ( 1957 females ) individuals with valid data . socio - demographic characteristics ( table 2 ) used in the multivariate analysis included study site ( korogocho vs viwandani ) , age in years , educational level , ethnic group , religion , place of birth ( born in slum vs born outside ) , mother 's survival status ( mother alive or not ) , father 's survival status ( father alive or not ) , schooling status ( in school vs out of school ) . how well do you live up to what other people expect of you?1 ( very well ) to4 ( not well at all)8what about your ability to do well in school ( even if you are not in school currently)?1 ( very able ) to4 ( not able at all)9how attractive do you think you are?1 ( very attractive ) to4 ( not attractive at all)10on the whole , how satisfied are you with yourself?1 ( very satisfied ) to4 ( not satisfied at all)11how much peer pressure is there on people your age to have sex?1 ( none ) to 4 ( a lot)1223what are the chances that : you will finish primary school ? you will be respected in your community?1 ( high ) 3 ( low)multiple problem behaviour index ( mpbi ) ( cronbach 's alpha=0.69)14delinquent behaviors : how many times have you done any of the following things in the last 4 months:0 ( never ) , 1 ( once),2 ( more than once)you stayed away from home at least one night without your parent 's permission ? ( groups : religious group , foot / netball club , drama group / dance group / choir , anti - aids club , anti - drugs club , girl guides / boy scouts , wildlife society , self - help group)1 ( yes ) , 2 ( no)attitudes towards contraception ( cronbach 's alpha=0.74)16it 's smart to use birth control to prevent an unplanned pregnancy . teenagers who use birth control show they care about themselves and their future.1 ( agreed ) 2 ( disagree ) description of items used to measure protective and risk factors in this paper , protective factors included social controls protection , individual controls protection and support protection . event history analysis ( eha ) techniques were employed for analysis , with kaplan meier estimates being used to examine the timing of first motherhood and cox regression models to investigate the influence of various factors on entry into motherhood ( allison , 1991 ; cleves et al . cox 's proportional hazards models were used for the multivariate analysis , since they do not need specification of the form of the distribution of the baseline hazard rate ( cox , 1972 ; cox & oakes , 1984 ; blossfeld et al . the term h0(t ) is the baseline hazard function ( the hazard when z=0 ) , zj(t ) is the individual covariates vector and j is a vector of the regression parameters that indicates the effects of these covariates , some of them varying with t ( hence the term time - varying covariate ) . overall , only 1% never attended school ( 1% for younger cohort and 2% for older cohort ) , 55% of the respondents have attained primary level of education ( 59% for younger cohort vs 50% for older cohort ) and 44% have at least secondary level education ( 40% for younger cohort and 48% for older cohort ) . at the time of the survey , 52% of the 1519 year female adolescents were attending school , with those from the younger cohort being more likely to do so ( 72% vs 27% ) . about 13% of the participants reported having ever been married ( 4% of younger adolescents vs 24% of older ones ) and 9% had ever owned or rented their residential unit ( 2% of younger adolescents vs 16% of older ones ) . the proportion of adolescents who are primiparous is higher among adolescents born outside of the slums ( p<0.01 ) , not enrolled in school ( p<0.01 ) , ever married ( p<0.01 ) , ever been residentially independent ( p<0.01 ) , without religion ( p<0.01 ) and those who had ever used contraception ( p<0.01 ) . among the older cohort , contrary to what was observed in the overall sample , the proportion of primiparous adolescents is higher among those who have never used contraception , although the association is not statistically significant . table 2.descriptive characteristics of the sample of 15- to 19-year - old female adolescents by age cohort , nairobi informal settlements , 20071517 years(n=498)1819 years(n=399)1519 years(n=897)socio - demographiccharacteristic%distribution%first child%distribution%first child%distribution%first childtotal100.04.6100.027.3100.014.7slum of residencekorogocho53.04.552.428.252.715.0viwandani47.04.747.626.347.314.4born in the slum?no35.36.8 * 50.633.7***42.121.2***yes64.73.449.420.857.910.0mother aliveno5.610.78.845.7**7.030.2***yes94.44.391.225.593.013.5father aliveno20.94.823.638.3***22.120.7***yes79.14.676.423.977.913.0ever used contraceptionno95.03.6***84.527.990.313.7***yes5.024.015.524.29.724.1currently in schoolno28.116.4***73.237.3***48.230.6***yes71.90.026.80.051.80.0education levelnever attended school0.825.0***1.842.9***1.236.4***primary58.67.249.642.954.621.6secondary40.20.547.910.543.65.4other0.40.00.833.30.620.0ever married / lived togetherno96.22.5***76.211.8***87.36.1***yes3.857.923.876.812.773.7ever rented / owned a houseno97.83.7***83.720.4***91.510.5***yes2.245.516.363.18.560.5*p<0.1 ; * * p<0.05 ; * * * p<0.001 . descriptive characteristics of the sample of 15- to 19-year - old female adolescents by age cohort , nairobi informal settlements , 2007 * p<0.1 ; * * p<0.05 ; * * * p<0.001 . in the following section , the results of the event history analysis enable the timing of entry into motherhood to be examined , as well as its associations with possible predictors , while controlling for the effects of other important factors , using kaplan meier estimates and cox regression models . table 3.descriptive statistics of age at first birth by age cohort , female adolescents , nairobi informal settlements , 20071517 yearcohort1819 yearcohorttotalproportion having a first birth by age ( years):120%0%0%130%1%0%141%1%1%151%3%2%162%4%3%178%16%13%1824%21%1931%29%age at : first quartilena18.919.0mediannananathird quartilenananaperson - years at risk8164.47365.115,529.5n498399897events23109132na = not attained . table 4.hazard ratios of having first birth among female adolescents by birth cohort ( cox model ) , nairobi informal settlements , 2007hazard ratios ( 95% ci)15- to 17-year - old18- to 19-year - oldvariablesmodel 1model 2model 3model 4model 5model 6socio - demographic variablesage ( years)1.22 ( 0.562.67)1.20 ( 0.453.20)1.12 ( 0.216.08)0.85 ( 0.551.31)0.73 ( 0.461.14)0.81 ( 0.511.29)slum residence ( ref . hazard ratios of having first birth among female adolescents by birth cohort ( cox model ) , nairobi informal settlements , 2007 * p<0.1 ; * * p<0.05 ; * * * p<0.01 . among adolescents aged 1517 years , none of the socio - demographic characteristics , with the exception of current school attendance , significantly affected the timing of entry into motherhood after controlling for other factors . there is no significant difference in the timing of childbearing based on adolescents ' lifetime contraceptive use , although the findings show that adolescents aged 1517 years who have ever used contraception were more likely to delay childbearing . in a model where only the psychosocial variables were controlled for ( results not shown here ) , mpsbi and support protection are significantly associated with entry into motherhood , with both higher scores on these two indices associated with a delay in the timing of first birth . with regard to risk factors , the results suggest that having negative models is associated with the outcome of interest ; adolescents with a high level of model risk are more likely to enter into motherhood earlier . among the protective factors , social and individual controls protection adolescents with high levels of these two factors are likely to delay their entry into motherhood ( chances are reduced by 24% and 27% for social controls and individual controls , respectively ) . none of the risk factors is significantly associated with timing of entry into motherhood in the older cohort , although in a model that only controls for psychosocial variables , adolescents with high vulnerability risk are likely to enter into motherhood earlier . this paper is based on data collected from 15- to 19-year - old adolescents in two resource - poor settings in nairobi to investigate patterns and determinants of the timing of entry into motherhood . the focus is on the association between the timing of childbearing and other key transitions to adult roles , socio - demographic characteristics and psychosocial variables . as expected , marriage has a strong relationship with the timing of first birth for both younger and older cohorts , with adolescents who are married or living together with their partners entering into motherhood significantly earlier than those who are not . this is consistent with previous evidence that shows that marriage is a key driver of fertility in sub - saharan africa , although out - of - wedlock childbearing is becoming increasingly common in the region ( tambashe & shapiro , 1996 ) . although the observed association between school enrolment or educational level may be misleading because some girls who are not in school at the time of the survey could have dropped out of school because of pregnancy - related reasons , previous studies do show that in - school youth tend to delay sexual activity ( ajayi et al . given the space constraints that typify the slum settlements in nairobi , young adults are almost forced to leave their parental home , leaving them highly exposed to risky sexual behaviours that lead to increased risks of early childbearing , especially in an environment where contraceptive use is low ( amuyunzu - nyamongo & magadi , 2006 ; dodoo et al . among 15- to 17-year - old adolescents , models risk ( the presence of models for problem or anti - social behaviours , including early sexual activity ) is significantly associated with entry into motherhood , with those having high models risk being more likely to have a first child earlier . in a recent study , ndugwa and others ( 2010 ) found that models risk is a strong predictor of involvement in problem behaviour among 12- to 19-year - old adolescents living in korogocho and viwandani slum settlements in nairobi . , 2007 ) previous findings from nairobi slums show that it is the father 's presence , in particular , that is associated with lower risk of engaging in sexual activity and , hence , lower risk of pregnancy ( ngom et al .

structural brain abnormalities have been consistently described in schizophrenic patients compared to healthy controls [ 19 , 27 ] . in a recent meta - analysis , volumetric deficits at diagnosis were seen in total brain volume , in the hippocampus , in cortical grey matter , in heschl s gyrus , in the planum temporal and in temporal grey matter , and in longitudinal studies continued volumetric loss over time could be demonstrated in these structures . in addition , the lateral ventricles were significantly larger than normal at onset of the illness and the ventricular volume tended to increase significantly in the disease course . only few studies focused on the predictive value of brain morphology for the course of schizophrenia in first - episode patients and reported inconsistent results [ 4 , 5 , 13 , 1618 , 23 ] . it was observed that hippocampal volume was associated with a higher risk of relapse , greater ventricular expansion was related to an unremitting course and poor outcome [ 4 , 5 , 11 ] , smaller temporal gray matter volume was associated with persistence of hallucinations , and more normal cerebral asymmetry was associated with adequate social / vocational functioning and full recovery . two other investigations could not demonstrate a correlation between volumetric measurement and treatment response or 2-year outcome . the aim of the presented study was to investigate if there is a difference in brain morphology focusing on structures suggested in previous studies to be relevant for the clinical course ( hippocampus , lateral ventricles ) in a sample consisting of first - episode patients well characterized for the outcome over 1 year . based on previous findings of reduced internal capsule volume and cross sectional area in families affected with schizophrenia and in first - episode patients , we decided to include volumetric measurement of the anterior limb of the internal capsule ( alic ) . the first - episode study program of the german research network on schizophrenia ( grns ) consisted of an 8-week acute treatment phase and a consecutive 2-year long - term treatment phase ( registered in clinicaltrials.gov : nct00159081 ) . between 2000 and 2004 , 158 first - episode patients , aged 1855 years and treated in inpatients departments of the participating centres , were randomly assigned to double - blind , low - dose haloperidol or risperidone . drugs could be increased ( up to 8 mg / day ) or lowered ( minimum 1 mg / day ) depending on symptoms or side effects as indicated by the respective clinical global impressions ( cgi ) scales . concomitant medications were permitted throughout the trial , except for additional antipsychotics or mood stabilizers . patients were diagnosed with schizophrenia according to dsm - iv by experienced clinical psychiatrists using a standardized clinical interview ( scid ; german version ) . before entering the long - term treatment study the diagnosis of schizophrenia was confirmed by independent experienced clinical psychiatrists . a total of 75 patients of this long - term treatment phase first - episode sample participated in the mr - imaging study and were recruited from the psychiatry departments of the universities of bonn , cologne , dsseldorf , duisburg - essen , jena , mainz , munich , and tbingen , as well as from the central institute of mental health in mannheim . out of this sample only 45 patients received standardized rating of psychopathology with the positive and negative syndrome scale ( panss ) at baseline and during follow - up , and underwent magnetic resonance imaging ( mri ) at baseline due to a defined protocol fulfilling the quality - criteria . exclusion criteria for the first - episode long - term study and the mri study part were any psychiatric comorbidity ( dsm - iv ) , neurological diseases , contraindication for antipsychotic treatment , suicidal behavior in previous history , mental retardation , pregnancy , substance dependence , disorders which affect cerebral metabolism , in addition to the usual exclusion criteria for mri ( e.g. metal implants , cardiac pace makers ) . after a complete description of the study , written informed consent was obtained from each patient . the local ethics committees approved the protocol , which is in accordance with the declaration of helsinki . from the mri study sample we extracted 39 first - episode patients , who received follow - up visits covering a period of at least 3 months up to 1 year ( mean 46.6 10.7 weeks ) . in this mr sample volumetric measurement of all regions of interest we divided this patient sample in three subgroups according to panss rating during the 1-year follow - up : 12 patients with stable psychopathology ( good outcome ; increase of panss total score below 10% of baseline score , approximately 5 points ) , 9 patients with intermediate outcome ( moderate worsening ) , and 11 patients with clinically relevant deterioration ( bad outcome ; increase of panss total score above 40% of baseline score , approximately 20 points ) . the subgroup with good outcome did not show any clinically relevant worsening of symptoms , presenting a stable course ( change of mean panss total score less than 4 points ) , while the subgroup with bad outcome demonstrated a clinically relevant increase of symptoms at least in one visit during the 1-year course , predominantly in negative symptoms and general psychopathology , ( change of mean panss total score more than 40 points ) ( see table 1 ) . table 1clinical and sociodemographic data in the first - episode subgroupsfe - sz stable ( n = 12)fe - sz bad outcome ( n = 11)anovamsdmsdfpage ( years)30.409.4233.1812.870.310.58education ( years)11.603.0312.112.620.150.70education of parents ( years)12.673.4613.294.500.100.76baseline panss positive score10.833.938.822.522.100.16baseline panss negative core15.336.1111.093.184.240.052baseline panss gen . score0.501.0025.8211.1162.07<0.0005fe - sz stablefe - sz bad outcomepgender ( male / female ) , n6/66/5 0.05 0.83fe - sz first - episode schizophrenic patients , diff . general psychopathology , n sample size , m mean , sd standard deviation , anova analysis of variance , ff - statistics , p probability , chi - square - test clinical and sociodemographic data in the first - episode subgroups fe - sz first - episode schizophrenic patients , diff . general psychopathology , n sample size , m mean , sd standard deviation , anova analysis of variance , ff - statistics , p probability , chi - square - test we compared the subgroups of good and bad outcome with regard to volumetric measurement of three distinct brain regions : hippocampus , lateral ventricles and the alic . magnetic resonance imaging ( mri ) was performed on siemens and phillips 1.5 tesla mr scanners with t1-weighted 3d data ( mprage ) sequences providing a spatial resolution of 1 1 1 mm ( repetition time = 11.4 ms , echo time = 4.4 ms , flip angle = 15 ) . manual area measurements for lateral ventricles , hippocampus and alic were obtained using the region of interest volumes of rois were calculated by multiplying the outlined areas with slice thickness , and relative volumes were calculated to adjust for differences in total brain volumes . the total brain volume , gray and white matter volumes were determined using an automatic algorithm programmed in matlab and spm99 ( statistical parametric mapping ) . the areas of the lateral ventricles were traced for each side separately in coronal slices , and hippocampal contours were drawn in the sagittal view following the borders described in the literature . the measurement of alic was performed as described previously dividing the internal capsule into an anterior and posterior part by a line bisecting the angle of internal capsule on horizontal slices . the ventral boundary was determined by a line connecting the lateral edge of ventral part of the caudate and the medial edge of the ventral part of the lentiform nucleus . the first slice included was the most inferior one comprising a detectable amount of white matter belonging to the internal capsule . the last slice was the one inferior to the slice which comprised the merging of internal capsule and the lamina pallidi externa . the method of manual delineation required individual optimization of contrast and brightness in each slice . alic identification was controlled for each slice in coronal and sagittal orientation . additionally , alic length and maximal cross sectional area were measured , length as the sum of slices containing alic , maximal cross section as maximal area of one particular slice among all slices ( see fig . 1 ) . in all cases the slice with maximal cross sectional area was identical to the most superior slice directly beneath the delta of merging internal capsule and lamina pallidi externa . the internal capsule is divided into an anterior ( alic ) and posterior part ( plic ) by a line bisecting the angle of internal capsule on horizontal slices . the right anterior limb of the internal capsule ( alic ) is marked tracing of alic t1-weighted mr - image . the internal capsule is divided into an anterior ( alic ) and posterior part ( plic ) by a line bisecting the angle of internal capsule on horizontal slices . the right anterior limb of the internal capsule ( alic ) is marked statistical analyses were performed with spss 10 for windows . dependent variables were bilateral volumes of the hippocampus and the ventricular system , and bilateral volumes , maximal cross sectional area and length of the alic . since there were no significant deviations from the normality assumption , the general linear model ( glm ) was performed using a multivariate approach for left and right side measurements separately for the blocks of dependent variables . independent factors outcome ( good / bad ) and gender and covariate age were included into the model . the first - episode study program of the german research network on schizophrenia ( grns ) consisted of an 8-week acute treatment phase and a consecutive 2-year long - term treatment phase ( registered in clinicaltrials.gov : nct00159081 ) . between 2000 and 2004 , 158 first - episode patients , aged 1855 years and treated in inpatients departments of the participating centres , were randomly assigned to double - blind , low - dose haloperidol or risperidone . drugs could be increased ( up to 8 mg / day ) or lowered ( minimum 1 mg / day ) depending on symptoms or side effects as indicated by the respective clinical global impressions ( cgi ) scales . concomitant medications were permitted throughout the trial , except for additional antipsychotics or mood stabilizers . patients were diagnosed with schizophrenia according to dsm - iv by experienced clinical psychiatrists using a standardized clinical interview ( scid ; german version ) . before entering the long - term treatment study the diagnosis of schizophrenia was confirmed by independent experienced clinical psychiatrists . a total of 75 patients of this long - term treatment phase first - episode sample participated in the mr - imaging study and were recruited from the psychiatry departments of the universities of bonn , cologne , dsseldorf , duisburg - essen , jena , mainz , munich , and tbingen , as well as from the central institute of mental health in mannheim . out of this sample only 45 patients received standardized rating of psychopathology with the positive and negative syndrome scale ( panss ) at baseline and during follow - up , and underwent magnetic resonance imaging ( mri ) at baseline due to a defined protocol fulfilling the quality - criteria . exclusion criteria for the first - episode long - term study and the mri study part were any psychiatric comorbidity ( dsm - iv ) , neurological diseases , contraindication for antipsychotic treatment , suicidal behavior in previous history , mental retardation , pregnancy , substance dependence , disorders which affect cerebral metabolism , in addition to the usual exclusion criteria for mri ( e.g. metal implants , cardiac pace makers ) . after a complete description of the study , written informed consent was obtained from each patient . the local ethics committees approved the protocol , which is in accordance with the declaration of helsinki . from the mri study sample we extracted 39 first - episode patients , who received follow - up visits covering a period of at least 3 months up to 1 year ( mean 46.6 10.7 weeks ) . in this mr sample volumetric measurement of all regions of interest we divided this patient sample in three subgroups according to panss rating during the 1-year follow - up : 12 patients with stable psychopathology ( good outcome ; increase of panss total score below 10% of baseline score , approximately 5 points ) , 9 patients with intermediate outcome ( moderate worsening ) , and 11 patients with clinically relevant deterioration ( bad outcome ; increase of panss total score above 40% of baseline score , approximately 20 points ) . the subgroup with good outcome did not show any clinically relevant worsening of symptoms , presenting a stable course ( change of mean panss total score less than 4 points ) , while the subgroup with bad outcome demonstrated a clinically relevant increase of symptoms at least in one visit during the 1-year course , predominantly in negative symptoms and general psychopathology , ( change of mean panss total score more than 40 points ) ( see table 1 ) . table 1clinical and sociodemographic data in the first - episode subgroupsfe - sz stable ( n = 12)fe - sz bad outcome ( n = 11)anovamsdmsdfpage ( years)30.409.4233.1812.870.310.58education ( years)11.603.0312.112.620.150.70education of parents ( years)12.673.4613.294.500.100.76baseline panss positive score10.833.938.822.522.100.16baseline panss negative core15.336.1111.093.184.240.052baseline panss gen . score0.501.0025.8211.1162.07<0.0005fe - sz stablefe - sz bad outcomepgender ( male / female ) , n6/66/5 0.05 0.83fe - sz first - episode schizophrenic patients , diff . general psychopathology , n sample size , m mean , sd standard deviation , anova analysis of variance , ff - statistics , p probability , chi - square - test clinical and sociodemographic data in the first - episode subgroups fe - sz first - episode schizophrenic patients , diff . general psychopathology , n sample size , m mean , sd standard deviation , anova analysis of variance , ff - statistics , p probability , chi - square - test we compared the subgroups of good and bad outcome with regard to volumetric measurement of three distinct brain regions : hippocampus , lateral ventricles and the alic . magnetic resonance imaging ( mri ) was performed on siemens and phillips 1.5 tesla mr scanners with t1-weighted 3d data ( mprage ) sequences providing a spatial resolution of 1 1 1 mm ( repetition time = 11.4 ms , echo time = 4.4 ms , flip angle = 15 ) . manual area measurements for lateral ventricles , hippocampus and alic were obtained using the region of interest volumes of rois were calculated by multiplying the outlined areas with slice thickness , and relative volumes were calculated to adjust for differences in total brain volumes . the total brain volume , gray and white matter volumes were determined using an automatic algorithm programmed in matlab and spm99 ( statistical parametric mapping ) . the areas of the lateral ventricles were traced for each side separately in coronal slices , and hippocampal contours were drawn in the sagittal view following the borders described in the literature . the measurement of alic was performed as described previously dividing the internal capsule into an anterior and posterior part by a line bisecting the angle of internal capsule on horizontal slices . the ventral boundary was determined by a line connecting the lateral edge of ventral part of the caudate and the medial edge of the ventral part of the lentiform nucleus . the first slice included was the most inferior one comprising a detectable amount of white matter belonging to the internal capsule . the last slice was the one inferior to the slice which comprised the merging of internal capsule and the lamina pallidi externa . the method of manual delineation required individual optimization of contrast and brightness in each slice . alic identification was controlled for each slice in coronal and sagittal orientation . additionally , alic length and maximal cross sectional area were measured , length as the sum of slices containing alic , maximal cross section as maximal area of one particular slice among all slices ( see fig . 1 ) . in all cases the slice with maximal cross sectional area was identical to the most superior slice directly beneath the delta of merging internal capsule and lamina pallidi externa . the internal capsule is divided into an anterior ( alic ) and posterior part ( plic ) by a line bisecting the angle of internal capsule on horizontal slices . the right anterior limb of the internal capsule ( alic ) is marked tracing of alic t1-weighted mr - image . the internal capsule is divided into an anterior ( alic ) and posterior part ( plic ) by a line bisecting the angle of internal capsule on horizontal slices . dependent variables were bilateral volumes of the hippocampus and the ventricular system , and bilateral volumes , maximal cross sectional area and length of the alic . independent variable was the outcome of the patients . only the extreme groups with good or bad outcome since there were no significant deviations from the normality assumption , the general linear model ( glm ) was performed using a multivariate approach for left and right side measurements separately for the blocks of dependent variables . independent factors outcome ( good / bad ) and gender and covariate age were included into the model . the patient subgroups with good and poor outcome did not differ in sociodemographic data ( see table 1 ) . first - episode patients who subsequently worsened during the first year of the illness revealed a significantly smaller maximal cross sectional area of the alic compared to those patients with stable psychopathology ( glm ; multivariate : f = 4.5 , df = 2 , 17 , p = 0.028 ) . subsequent univariate tests showed that this reduction was significant on the left side ( 19% , f = 8.2 , df = 1 , 18 , p = 0.010 ; see fig . 2 ) . other measurements of alic , as well as hippocampal and lateral ventricular volumes were not significantly reduced in patients with poor outcome compared to the stable group ( see table 2 ) . alic , anterior limb of the internal capsule ; fe - sz , first - episode schizophrenia patients ; ci , confidence interval ; max . cs , maximal cross sectional ; rel . , relativetable 2results of volumetric measurementsregion of interestfe - sz stable ( n = 12)fe - sz bad outcome ( n = 11)diff ( % ) mancovamsdmsdfdfphippocampus volume left side ( rel.)0.003280.000440.003130.000344.40.41 , 170.23hippocampus volume right side ( rel.)0.003150.000550.003070.000372.50.41 , 170.45lateral ventricle volume left side ( rel.)0.011390.004670.009770.0052814.20.81 , 180.38lateral ventricle volume right side ( rel.)0.010380.004660.009460.005078.80.21 , 180.64alic volume left side(rel.)0.000560.000110.000550.000101.70.01 , 181.00alic volume right side(rel.)0.000590.000100.000510.0001012.32.21 , 180.15alic max . area right side ( rel.)0.00850.00150.00790.00136.90.81 , 180.38alic length left side ( rel.)0.09230.01280.09450.01162.50.61 , 180.44alic length right side ( rel.)0.09300.01080.09550.01202.70.81 , 180.39total brain volume ( absolute , cm)1136.5116.61133.1103.21.40.11 , 180.73fe - sz first - episode schizophrenic patients , diff . difference , n sample size , m mean , sd standard deviation , ff - statistics , df degrees of freedom , p probability , rel . maximal cross sectional , mancova multivariate analysis of covariance , f , p for factor subgroup relative left alic maximal cross sectional area in first - episode schizophrenia patients . alic , anterior limb of the internal capsule ; fe - sz , first - episode schizophrenia patients ; ci , confidence interval ; max . cs , maximal cross sectional ; rel . , relative results of volumetric measurements fe - sz first - episode schizophrenic patients , diff . difference , n sample size , m mean , sd standard deviation , ff - statistics , df degrees of freedom , p probability , rel . relative , max . cs . maximal cross sectional , mancova multivariate analysis of covariance , f , p for factor subgroup we found no significant correlations between the cumulative doses of risperidone or haloperidol and the alic maximal cross sectional area in the total group of patients and in the subgroups of schizophrenic patients with stable psychopathology or clinically relevant deterioration in the 1-year course . the cumulative doses of risperidone and haloperidol were not significantly different in both subgroups ( df = 1 , f = 0.001 ; p = 0.98 , oneway - anova ) , and the daily doses of risperidone and haloperidol in the subgroups were comparable ( fe - sz stable psychopathology : risperidone 4.3 2.0 mg / day , haloperidol 3.0 1.5 mg / day ; fe - sz poor outcome : risperidone 3.8 1.3 mg / day , haloperidol 3.1 1.6 mg / day ) . while the educational level may have influenced out results we repeated the analysis with education as covariate . the results were similar , first - episode patients with deterioration during the first year of the illness still presented with a significantly smaller maximal cross sectional area of the alic compared to those patients with stable psychopathology ( mancova ; p = 0.020 ) . the main result of our study assessing the relationship of brain morphology and clinical outcome in first - episode schizophrenia is the significant reduced maximal cross sectional area of the anterior limb of the internal capsule ( alic ) in the subgroup of patients with clinical deterioration during the 1-year course compared to patients with stable psychopathology . there was no volumetric difference between the two subgroups with regard to the other selected regions of interest , hippocampus and lateral ventricles . while most prominent replicable brain abnormalities in schizophrenia are volume changes of the grey matter ( e.g. 19 , 22 , 27 ) , some studies using voxel - based morphometry revealed evidence for white matter abnormalities , e.g. bilaterally in the frontal lobe or in the left temporal and frontal lobe of schizophrenic patients with predominant negative symptoms . in addition , significant decreases in white matter density were detected in connecting structures like the genu and truncus of the corpus callosum , the right alic and the right anterior commissure compared to healthy subjects . only a few studies assessed white matter changes in schizophrenia that were hypothesis - driven using the region of interest ( roi ) approach . in the present study we morphometrically assessed the hippocampus and the lateral ventricles , whose volumetric alterations are most consistently associated with the course of the illness , as well as the anterior limb of the internal capsule , a connecting white matter structure , which has been found to exhibit decreased volumes both in schizophrenia and in unaffected first - degree relatives of schizophrenic patients . out of the three mentioned structures , in our study only the left maximal cross sectional area of the anterior limb of the internal capsule was associated with the clinical course in first - episode schizophrenia . in this sample of first - episode patients those with poor outcome ( high increase of positive symptoms ) showed a reduced maximal cross sectional area of the alic on the left side compared to those with good outcome . however , when left and right alic maximal cross sectional areas of both subgroups are compared , there is an inverse relationship . in the good outcome group the mean left alic maximal cross sectional area is larger than the right one , and in the bad outcome group it is the other way round . this inverse relationship may be at least in part responsible for the significant difference between the subgroups . this may point towards a different asymmetry or shape of this structure in the subgroups . nevertheless we can not rule out the possibility that the left alic maximal cross sectional area in the patients with good outcome group may be disproportionately large in our sample . the alic is a very important connecting structure linking the frontal cortex to subcortical structures through the medial and the basolateral limbic circuit . abnormalities in the alic can thus be taken as measure for disturbances in the frontothalamic connectivity that is discussed to be relevant for the pathophysiology of schizophrenia . interestingly , a recent study shows that a reduced area of the alic in dorsal mri slices was associated with poor outcome based on longitudinal analysis of self - care deficits in chronic schizophrenia . the authors suggest disruption of internal capsule fibers in poor - outcome patients with schizophrenia , which is supported by the results of our study . the reduced maximal cross sectional area measured in our investigation corresponds to the reduced alic area in dorsal mri slices of the cited study . additionally , our results indicate that reduced white fiber structures in this area might increase the risk not only for a poor outcome in chronic schizophrenic patients , but also increase the vulnerability for the onset of schizophrenic psychosis and an unfavourable course in first - episode schizophrenia . to our knowledge this study is the first one investigating the relevance of white matter structures , especially the alic , for the course of first - episode schizophrenia . we were not able to control for the influence of possible confounding factors determining poor outcome , e.g. psychosocial circumstances or neurocognitive function . secondly , the follow - up observational period without any intermediate anchor point may be too long to document the disease course adequately . while the cumulative and daily doses of risperidone and haloperidol were low and similar distributed in both subgroups we do not assume a relevant influence of antipsychotic medication on our volumetric results or on clinical outcome , for instance by the induction of secondary negative symptoms . in conclusion our finding of reduced internal capsule size in patients with bad outcome supports the dysconnectivity concept of schizophrenia and points towards disturbances in the striato - thalamo - cortical neuronal circuit and abnormalities in related structures .

subtle structural brain abnormalities are an established finding in first - episode psychosis . nevertheless their relationship to the clinical course of schizophrenia is controversially discussed . in a multicentre study 45 first - episode schizophrenia patients ( fe - sz ) underwent standardized mri scanning and were followed up to 1 year . in 32 fe - sz volumetric measurement of three regions of interests ( rois ) potentially associated with disease course , hippocampus , lateral ventricle and the anterior limb of the internal capsule ( alic ) could be performed . the subgroups of fe - sz with good ( 12 patients ) and poor outcome ( 11 patients ) , defined by a clinically relevant change of the panss score , were compared with regard to these volumetric measures . multivariate analysis of covariance revealed a significant reduced maximal cross sectional area of the left alic in fe - sz with clinically relevant deterioration compared to those with stable psychopathology . there were no differences in the other selected rois between the two subgroups . in conclusion , reduced maximal area of alic , which can be interpreted as a disturbance of fronto - thalamic connectivity , is associated with poor outcome during the 1 year course of first - episode schizophrenia .

Introduction Method Subjects Magnetic resonance imaging and measurements Statistics Results Discussion

structural brain abnormalities have been consistently described in schizophrenic patients compared to healthy controls [ 19 , 27 ] . in a recent meta - analysis , volumetric deficits at diagnosis were seen in total brain volume , in the hippocampus , in cortical grey matter , in heschl s gyrus , in the planum temporal and in temporal grey matter , and in longitudinal studies continued volumetric loss over time could be demonstrated in these structures . in addition , the lateral ventricles were significantly larger than normal at onset of the illness and the ventricular volume tended to increase significantly in the disease course . only few studies focused on the predictive value of brain morphology for the course of schizophrenia in first - episode patients and reported inconsistent results [ 4 , 5 , 13 , 1618 , 23 ] . it was observed that hippocampal volume was associated with a higher risk of relapse , greater ventricular expansion was related to an unremitting course and poor outcome [ 4 , 5 , 11 ] , smaller temporal gray matter volume was associated with persistence of hallucinations , and more normal cerebral asymmetry was associated with adequate social / vocational functioning and full recovery . the aim of the presented study was to investigate if there is a difference in brain morphology focusing on structures suggested in previous studies to be relevant for the clinical course ( hippocampus , lateral ventricles ) in a sample consisting of first - episode patients well characterized for the outcome over 1 year . based on previous findings of reduced internal capsule volume and cross sectional area in families affected with schizophrenia and in first - episode patients , we decided to include volumetric measurement of the anterior limb of the internal capsule ( alic ) . the first - episode study program of the german research network on schizophrenia ( grns ) consisted of an 8-week acute treatment phase and a consecutive 2-year long - term treatment phase ( registered in clinicaltrials.gov : nct00159081 ) . between 2000 and 2004 , 158 first - episode patients , aged 1855 years and treated in inpatients departments of the participating centres , were randomly assigned to double - blind , low - dose haloperidol or risperidone . a total of 75 patients of this long - term treatment phase first - episode sample participated in the mr - imaging study and were recruited from the psychiatry departments of the universities of bonn , cologne , dsseldorf , duisburg - essen , jena , mainz , munich , and tbingen , as well as from the central institute of mental health in mannheim . exclusion criteria for the first - episode long - term study and the mri study part were any psychiatric comorbidity ( dsm - iv ) , neurological diseases , contraindication for antipsychotic treatment , suicidal behavior in previous history , mental retardation , pregnancy , substance dependence , disorders which affect cerebral metabolism , in addition to the usual exclusion criteria for mri ( e.g. from the mri study sample we extracted 39 first - episode patients , who received follow - up visits covering a period of at least 3 months up to 1 year ( mean 46.6 10.7 weeks ) . in this mr sample volumetric measurement of all regions of interest we divided this patient sample in three subgroups according to panss rating during the 1-year follow - up : 12 patients with stable psychopathology ( good outcome ; increase of panss total score below 10% of baseline score , approximately 5 points ) , 9 patients with intermediate outcome ( moderate worsening ) , and 11 patients with clinically relevant deterioration ( bad outcome ; increase of panss total score above 40% of baseline score , approximately 20 points ) . the subgroup with good outcome did not show any clinically relevant worsening of symptoms , presenting a stable course ( change of mean panss total score less than 4 points ) , while the subgroup with bad outcome demonstrated a clinically relevant increase of symptoms at least in one visit during the 1-year course , predominantly in negative symptoms and general psychopathology , ( change of mean panss total score more than 40 points ) ( see table 1 ) . table 1clinical and sociodemographic data in the first - episode subgroupsfe - sz stable ( n = 12)fe - sz bad outcome ( n = 11)anovamsdmsdfpage ( years)30.409.4233.1812.870.310.58education ( years)11.603.0312.112.620.150.70education of parents ( years)12.673.4613.294.500.100.76baseline panss positive score10.833.938.822.522.100.16baseline panss negative core15.336.1111.093.184.240.052baseline panss gen . score0.501.0025.8211.1162.07<0.0005fe - sz stablefe - sz bad outcomepgender ( male / female ) , n6/66/5 0.05 0.83fe - sz first - episode schizophrenic patients , diff . general psychopathology , n sample size , m mean , sd standard deviation , anova analysis of variance , ff - statistics , p probability , chi - square - test clinical and sociodemographic data in the first - episode subgroups fe - sz first - episode schizophrenic patients , diff . general psychopathology , n sample size , m mean , sd standard deviation , anova analysis of variance , ff - statistics , p probability , chi - square - test we compared the subgroups of good and bad outcome with regard to volumetric measurement of three distinct brain regions : hippocampus , lateral ventricles and the alic . the measurement of alic was performed as described previously dividing the internal capsule into an anterior and posterior part by a line bisecting the angle of internal capsule on horizontal slices . the ventral boundary was determined by a line connecting the lateral edge of ventral part of the caudate and the medial edge of the ventral part of the lentiform nucleus . the first slice included was the most inferior one comprising a detectable amount of white matter belonging to the internal capsule . the last slice was the one inferior to the slice which comprised the merging of internal capsule and the lamina pallidi externa . additionally , alic length and maximal cross sectional area were measured , length as the sum of slices containing alic , maximal cross section as maximal area of one particular slice among all slices ( see fig . in all cases the slice with maximal cross sectional area was identical to the most superior slice directly beneath the delta of merging internal capsule and lamina pallidi externa . the internal capsule is divided into an anterior ( alic ) and posterior part ( plic ) by a line bisecting the angle of internal capsule on horizontal slices . the right anterior limb of the internal capsule ( alic ) is marked tracing of alic t1-weighted mr - image . the internal capsule is divided into an anterior ( alic ) and posterior part ( plic ) by a line bisecting the angle of internal capsule on horizontal slices . the right anterior limb of the internal capsule ( alic ) is marked statistical analyses were performed with spss 10 for windows . dependent variables were bilateral volumes of the hippocampus and the ventricular system , and bilateral volumes , maximal cross sectional area and length of the alic . since there were no significant deviations from the normality assumption , the general linear model ( glm ) was performed using a multivariate approach for left and right side measurements separately for the blocks of dependent variables . the first - episode study program of the german research network on schizophrenia ( grns ) consisted of an 8-week acute treatment phase and a consecutive 2-year long - term treatment phase ( registered in clinicaltrials.gov : nct00159081 ) . between 2000 and 2004 , 158 first - episode patients , aged 1855 years and treated in inpatients departments of the participating centres , were randomly assigned to double - blind , low - dose haloperidol or risperidone . a total of 75 patients of this long - term treatment phase first - episode sample participated in the mr - imaging study and were recruited from the psychiatry departments of the universities of bonn , cologne , dsseldorf , duisburg - essen , jena , mainz , munich , and tbingen , as well as from the central institute of mental health in mannheim . exclusion criteria for the first - episode long - term study and the mri study part were any psychiatric comorbidity ( dsm - iv ) , neurological diseases , contraindication for antipsychotic treatment , suicidal behavior in previous history , mental retardation , pregnancy , substance dependence , disorders which affect cerebral metabolism , in addition to the usual exclusion criteria for mri ( e.g. from the mri study sample we extracted 39 first - episode patients , who received follow - up visits covering a period of at least 3 months up to 1 year ( mean 46.6 10.7 weeks ) . in this mr sample volumetric measurement of all regions of interest we divided this patient sample in three subgroups according to panss rating during the 1-year follow - up : 12 patients with stable psychopathology ( good outcome ; increase of panss total score below 10% of baseline score , approximately 5 points ) , 9 patients with intermediate outcome ( moderate worsening ) , and 11 patients with clinically relevant deterioration ( bad outcome ; increase of panss total score above 40% of baseline score , approximately 20 points ) . the subgroup with good outcome did not show any clinically relevant worsening of symptoms , presenting a stable course ( change of mean panss total score less than 4 points ) , while the subgroup with bad outcome demonstrated a clinically relevant increase of symptoms at least in one visit during the 1-year course , predominantly in negative symptoms and general psychopathology , ( change of mean panss total score more than 40 points ) ( see table 1 ) . table 1clinical and sociodemographic data in the first - episode subgroupsfe - sz stable ( n = 12)fe - sz bad outcome ( n = 11)anovamsdmsdfpage ( years)30.409.4233.1812.870.310.58education ( years)11.603.0312.112.620.150.70education of parents ( years)12.673.4613.294.500.100.76baseline panss positive score10.833.938.822.522.100.16baseline panss negative core15.336.1111.093.184.240.052baseline panss gen . score0.501.0025.8211.1162.07<0.0005fe - sz stablefe - sz bad outcomepgender ( male / female ) , n6/66/5 0.05 0.83fe - sz first - episode schizophrenic patients , diff . general psychopathology , n sample size , m mean , sd standard deviation , anova analysis of variance , ff - statistics , p probability , chi - square - test clinical and sociodemographic data in the first - episode subgroups fe - sz first - episode schizophrenic patients , diff . general psychopathology , n sample size , m mean , sd standard deviation , anova analysis of variance , ff - statistics , p probability , chi - square - test we compared the subgroups of good and bad outcome with regard to volumetric measurement of three distinct brain regions : hippocampus , lateral ventricles and the alic . the measurement of alic was performed as described previously dividing the internal capsule into an anterior and posterior part by a line bisecting the angle of internal capsule on horizontal slices . the ventral boundary was determined by a line connecting the lateral edge of ventral part of the caudate and the medial edge of the ventral part of the lentiform nucleus . the first slice included was the most inferior one comprising a detectable amount of white matter belonging to the internal capsule . the last slice was the one inferior to the slice which comprised the merging of internal capsule and the lamina pallidi externa . additionally , alic length and maximal cross sectional area were measured , length as the sum of slices containing alic , maximal cross section as maximal area of one particular slice among all slices ( see fig . in all cases the slice with maximal cross sectional area was identical to the most superior slice directly beneath the delta of merging internal capsule and lamina pallidi externa . the internal capsule is divided into an anterior ( alic ) and posterior part ( plic ) by a line bisecting the angle of internal capsule on horizontal slices . the right anterior limb of the internal capsule ( alic ) is marked tracing of alic t1-weighted mr - image . the internal capsule is divided into an anterior ( alic ) and posterior part ( plic ) by a line bisecting the angle of internal capsule on horizontal slices . dependent variables were bilateral volumes of the hippocampus and the ventricular system , and bilateral volumes , maximal cross sectional area and length of the alic . only the extreme groups with good or bad outcome since there were no significant deviations from the normality assumption , the general linear model ( glm ) was performed using a multivariate approach for left and right side measurements separately for the blocks of dependent variables . the patient subgroups with good and poor outcome did not differ in sociodemographic data ( see table 1 ) . first - episode patients who subsequently worsened during the first year of the illness revealed a significantly smaller maximal cross sectional area of the alic compared to those patients with stable psychopathology ( glm ; multivariate : f = 4.5 , df = 2 , 17 , p = 0.028 ) . other measurements of alic , as well as hippocampal and lateral ventricular volumes were not significantly reduced in patients with poor outcome compared to the stable group ( see table 2 ) . alic , anterior limb of the internal capsule ; fe - sz , first - episode schizophrenia patients ; ci , confidence interval ; max . , relativetable 2results of volumetric measurementsregion of interestfe - sz stable ( n = 12)fe - sz bad outcome ( n = 11)diff ( % ) mancovamsdmsdfdfphippocampus volume left side ( rel. )0.09300.01080.09550.01202.70.81 , 180.39total brain volume ( absolute , cm)1136.5116.61133.1103.21.40.11 , 180.73fe - sz first - episode schizophrenic patients , diff . maximal cross sectional , mancova multivariate analysis of covariance , f , p for factor subgroup relative left alic maximal cross sectional area in first - episode schizophrenia patients . alic , anterior limb of the internal capsule ; fe - sz , first - episode schizophrenia patients ; ci , confidence interval ; max . , relative results of volumetric measurements fe - sz first - episode schizophrenic patients , diff . maximal cross sectional , mancova multivariate analysis of covariance , f , p for factor subgroup we found no significant correlations between the cumulative doses of risperidone or haloperidol and the alic maximal cross sectional area in the total group of patients and in the subgroups of schizophrenic patients with stable psychopathology or clinically relevant deterioration in the 1-year course . the cumulative doses of risperidone and haloperidol were not significantly different in both subgroups ( df = 1 , f = 0.001 ; p = 0.98 , oneway - anova ) , and the daily doses of risperidone and haloperidol in the subgroups were comparable ( fe - sz stable psychopathology : risperidone 4.3 2.0 mg / day , haloperidol 3.0 1.5 mg / day ; fe - sz poor outcome : risperidone 3.8 1.3 mg / day , haloperidol 3.1 1.6 mg / day ) . the results were similar , first - episode patients with deterioration during the first year of the illness still presented with a significantly smaller maximal cross sectional area of the alic compared to those patients with stable psychopathology ( mancova ; p = 0.020 ) . the main result of our study assessing the relationship of brain morphology and clinical outcome in first - episode schizophrenia is the significant reduced maximal cross sectional area of the anterior limb of the internal capsule ( alic ) in the subgroup of patients with clinical deterioration during the 1-year course compared to patients with stable psychopathology . there was no volumetric difference between the two subgroups with regard to the other selected regions of interest , hippocampus and lateral ventricles . in addition , significant decreases in white matter density were detected in connecting structures like the genu and truncus of the corpus callosum , the right alic and the right anterior commissure compared to healthy subjects . in the present study we morphometrically assessed the hippocampus and the lateral ventricles , whose volumetric alterations are most consistently associated with the course of the illness , as well as the anterior limb of the internal capsule , a connecting white matter structure , which has been found to exhibit decreased volumes both in schizophrenia and in unaffected first - degree relatives of schizophrenic patients . out of the three mentioned structures , in our study only the left maximal cross sectional area of the anterior limb of the internal capsule was associated with the clinical course in first - episode schizophrenia . in this sample of first - episode patients those with poor outcome ( high increase of positive symptoms ) showed a reduced maximal cross sectional area of the alic on the left side compared to those with good outcome . in the good outcome group the mean left alic maximal cross sectional area is larger than the right one , and in the bad outcome group it is the other way round . this inverse relationship may be at least in part responsible for the significant difference between the subgroups . nevertheless we can not rule out the possibility that the left alic maximal cross sectional area in the patients with good outcome group may be disproportionately large in our sample . interestingly , a recent study shows that a reduced area of the alic in dorsal mri slices was associated with poor outcome based on longitudinal analysis of self - care deficits in chronic schizophrenia . the authors suggest disruption of internal capsule fibers in poor - outcome patients with schizophrenia , which is supported by the results of our study . the reduced maximal cross sectional area measured in our investigation corresponds to the reduced alic area in dorsal mri slices of the cited study . additionally , our results indicate that reduced white fiber structures in this area might increase the risk not only for a poor outcome in chronic schizophrenic patients , but also increase the vulnerability for the onset of schizophrenic psychosis and an unfavourable course in first - episode schizophrenia . to our knowledge this study is the first one investigating the relevance of white matter structures , especially the alic , for the course of first - episode schizophrenia . in conclusion our finding of reduced internal capsule size in patients with bad outcome supports the dysconnectivity concept of schizophrenia and points towards disturbances in the striato - thalamo - cortical neuronal circuit and abnormalities in related structures .

structural brain abnormalities have been consistently described in schizophrenic patients compared to healthy controls [ 19 , 27 ] . in a recent meta - analysis , volumetric deficits at diagnosis were seen in total brain volume , in the hippocampus , in cortical grey matter , in heschl s gyrus , in the planum temporal and in temporal grey matter , and in longitudinal studies continued volumetric loss over time could be demonstrated in these structures . in addition , the lateral ventricles were significantly larger than normal at onset of the illness and the ventricular volume tended to increase significantly in the disease course . only few studies focused on the predictive value of brain morphology for the course of schizophrenia in first - episode patients and reported inconsistent results [ 4 , 5 , 13 , 1618 , 23 ] . it was observed that hippocampal volume was associated with a higher risk of relapse , greater ventricular expansion was related to an unremitting course and poor outcome [ 4 , 5 , 11 ] , smaller temporal gray matter volume was associated with persistence of hallucinations , and more normal cerebral asymmetry was associated with adequate social / vocational functioning and full recovery . the aim of the presented study was to investigate if there is a difference in brain morphology focusing on structures suggested in previous studies to be relevant for the clinical course ( hippocampus , lateral ventricles ) in a sample consisting of first - episode patients well characterized for the outcome over 1 year . based on previous findings of reduced internal capsule volume and cross sectional area in families affected with schizophrenia and in first - episode patients , we decided to include volumetric measurement of the anterior limb of the internal capsule ( alic ) . the first - episode study program of the german research network on schizophrenia ( grns ) consisted of an 8-week acute treatment phase and a consecutive 2-year long - term treatment phase ( registered in clinicaltrials.gov : nct00159081 ) . between 2000 and 2004 , 158 first - episode patients , aged 1855 years and treated in inpatients departments of the participating centres , were randomly assigned to double - blind , low - dose haloperidol or risperidone . drugs could be increased ( up to 8 mg / day ) or lowered ( minimum 1 mg / day ) depending on symptoms or side effects as indicated by the respective clinical global impressions ( cgi ) scales . a total of 75 patients of this long - term treatment phase first - episode sample participated in the mr - imaging study and were recruited from the psychiatry departments of the universities of bonn , cologne , dsseldorf , duisburg - essen , jena , mainz , munich , and tbingen , as well as from the central institute of mental health in mannheim . out of this sample only 45 patients received standardized rating of psychopathology with the positive and negative syndrome scale ( panss ) at baseline and during follow - up , and underwent magnetic resonance imaging ( mri ) at baseline due to a defined protocol fulfilling the quality - criteria . exclusion criteria for the first - episode long - term study and the mri study part were any psychiatric comorbidity ( dsm - iv ) , neurological diseases , contraindication for antipsychotic treatment , suicidal behavior in previous history , mental retardation , pregnancy , substance dependence , disorders which affect cerebral metabolism , in addition to the usual exclusion criteria for mri ( e.g. from the mri study sample we extracted 39 first - episode patients , who received follow - up visits covering a period of at least 3 months up to 1 year ( mean 46.6 10.7 weeks ) . in this mr sample volumetric measurement of all regions of interest we divided this patient sample in three subgroups according to panss rating during the 1-year follow - up : 12 patients with stable psychopathology ( good outcome ; increase of panss total score below 10% of baseline score , approximately 5 points ) , 9 patients with intermediate outcome ( moderate worsening ) , and 11 patients with clinically relevant deterioration ( bad outcome ; increase of panss total score above 40% of baseline score , approximately 20 points ) . the subgroup with good outcome did not show any clinically relevant worsening of symptoms , presenting a stable course ( change of mean panss total score less than 4 points ) , while the subgroup with bad outcome demonstrated a clinically relevant increase of symptoms at least in one visit during the 1-year course , predominantly in negative symptoms and general psychopathology , ( change of mean panss total score more than 40 points ) ( see table 1 ) . table 1clinical and sociodemographic data in the first - episode subgroupsfe - sz stable ( n = 12)fe - sz bad outcome ( n = 11)anovamsdmsdfpage ( years)30.409.4233.1812.870.310.58education ( years)11.603.0312.112.620.150.70education of parents ( years)12.673.4613.294.500.100.76baseline panss positive score10.833.938.822.522.100.16baseline panss negative core15.336.1111.093.184.240.052baseline panss gen . score0.501.0025.8211.1162.07<0.0005fe - sz stablefe - sz bad outcomepgender ( male / female ) , n6/66/5 0.05 0.83fe - sz first - episode schizophrenic patients , diff . general psychopathology , n sample size , m mean , sd standard deviation , anova analysis of variance , ff - statistics , p probability , chi - square - test clinical and sociodemographic data in the first - episode subgroups fe - sz first - episode schizophrenic patients , diff . general psychopathology , n sample size , m mean , sd standard deviation , anova analysis of variance , ff - statistics , p probability , chi - square - test we compared the subgroups of good and bad outcome with regard to volumetric measurement of three distinct brain regions : hippocampus , lateral ventricles and the alic . magnetic resonance imaging ( mri ) was performed on siemens and phillips 1.5 tesla mr scanners with t1-weighted 3d data ( mprage ) sequences providing a spatial resolution of 1 1 1 mm ( repetition time = 11.4 ms , echo time = 4.4 ms , flip angle = 15 ) . manual area measurements for lateral ventricles , hippocampus and alic were obtained using the region of interest volumes of rois were calculated by multiplying the outlined areas with slice thickness , and relative volumes were calculated to adjust for differences in total brain volumes . the areas of the lateral ventricles were traced for each side separately in coronal slices , and hippocampal contours were drawn in the sagittal view following the borders described in the literature . the ventral boundary was determined by a line connecting the lateral edge of ventral part of the caudate and the medial edge of the ventral part of the lentiform nucleus . the right anterior limb of the internal capsule ( alic ) is marked tracing of alic t1-weighted mr - image . dependent variables were bilateral volumes of the hippocampus and the ventricular system , and bilateral volumes , maximal cross sectional area and length of the alic . since there were no significant deviations from the normality assumption , the general linear model ( glm ) was performed using a multivariate approach for left and right side measurements separately for the blocks of dependent variables . the first - episode study program of the german research network on schizophrenia ( grns ) consisted of an 8-week acute treatment phase and a consecutive 2-year long - term treatment phase ( registered in clinicaltrials.gov : nct00159081 ) . between 2000 and 2004 , 158 first - episode patients , aged 1855 years and treated in inpatients departments of the participating centres , were randomly assigned to double - blind , low - dose haloperidol or risperidone . drugs could be increased ( up to 8 mg / day ) or lowered ( minimum 1 mg / day ) depending on symptoms or side effects as indicated by the respective clinical global impressions ( cgi ) scales . a total of 75 patients of this long - term treatment phase first - episode sample participated in the mr - imaging study and were recruited from the psychiatry departments of the universities of bonn , cologne , dsseldorf , duisburg - essen , jena , mainz , munich , and tbingen , as well as from the central institute of mental health in mannheim . out of this sample only 45 patients received standardized rating of psychopathology with the positive and negative syndrome scale ( panss ) at baseline and during follow - up , and underwent magnetic resonance imaging ( mri ) at baseline due to a defined protocol fulfilling the quality - criteria . exclusion criteria for the first - episode long - term study and the mri study part were any psychiatric comorbidity ( dsm - iv ) , neurological diseases , contraindication for antipsychotic treatment , suicidal behavior in previous history , mental retardation , pregnancy , substance dependence , disorders which affect cerebral metabolism , in addition to the usual exclusion criteria for mri ( e.g. from the mri study sample we extracted 39 first - episode patients , who received follow - up visits covering a period of at least 3 months up to 1 year ( mean 46.6 10.7 weeks ) . in this mr sample volumetric measurement of all regions of interest we divided this patient sample in three subgroups according to panss rating during the 1-year follow - up : 12 patients with stable psychopathology ( good outcome ; increase of panss total score below 10% of baseline score , approximately 5 points ) , 9 patients with intermediate outcome ( moderate worsening ) , and 11 patients with clinically relevant deterioration ( bad outcome ; increase of panss total score above 40% of baseline score , approximately 20 points ) . the subgroup with good outcome did not show any clinically relevant worsening of symptoms , presenting a stable course ( change of mean panss total score less than 4 points ) , while the subgroup with bad outcome demonstrated a clinically relevant increase of symptoms at least in one visit during the 1-year course , predominantly in negative symptoms and general psychopathology , ( change of mean panss total score more than 40 points ) ( see table 1 ) . table 1clinical and sociodemographic data in the first - episode subgroupsfe - sz stable ( n = 12)fe - sz bad outcome ( n = 11)anovamsdmsdfpage ( years)30.409.4233.1812.870.310.58education ( years)11.603.0312.112.620.150.70education of parents ( years)12.673.4613.294.500.100.76baseline panss positive score10.833.938.822.522.100.16baseline panss negative core15.336.1111.093.184.240.052baseline panss gen . score0.501.0025.8211.1162.07<0.0005fe - sz stablefe - sz bad outcomepgender ( male / female ) , n6/66/5 0.05 0.83fe - sz first - episode schizophrenic patients , diff . general psychopathology , n sample size , m mean , sd standard deviation , anova analysis of variance , ff - statistics , p probability , chi - square - test clinical and sociodemographic data in the first - episode subgroups fe - sz first - episode schizophrenic patients , diff . general psychopathology , n sample size , m mean , sd standard deviation , anova analysis of variance , ff - statistics , p probability , chi - square - test we compared the subgroups of good and bad outcome with regard to volumetric measurement of three distinct brain regions : hippocampus , lateral ventricles and the alic . magnetic resonance imaging ( mri ) was performed on siemens and phillips 1.5 tesla mr scanners with t1-weighted 3d data ( mprage ) sequences providing a spatial resolution of 1 1 1 mm ( repetition time = 11.4 ms , echo time = 4.4 ms , flip angle = 15 ) . manual area measurements for lateral ventricles , hippocampus and alic were obtained using the region of interest volumes of rois were calculated by multiplying the outlined areas with slice thickness , and relative volumes were calculated to adjust for differences in total brain volumes . the areas of the lateral ventricles were traced for each side separately in coronal slices , and hippocampal contours were drawn in the sagittal view following the borders described in the literature . the ventral boundary was determined by a line connecting the lateral edge of ventral part of the caudate and the medial edge of the ventral part of the lentiform nucleus . dependent variables were bilateral volumes of the hippocampus and the ventricular system , and bilateral volumes , maximal cross sectional area and length of the alic . only the extreme groups with good or bad outcome since there were no significant deviations from the normality assumption , the general linear model ( glm ) was performed using a multivariate approach for left and right side measurements separately for the blocks of dependent variables . first - episode patients who subsequently worsened during the first year of the illness revealed a significantly smaller maximal cross sectional area of the alic compared to those patients with stable psychopathology ( glm ; multivariate : f = 4.5 , df = 2 , 17 , p = 0.028 ) . subsequent univariate tests showed that this reduction was significant on the left side ( 19% , f = 8.2 , df = 1 , 18 , p = 0.010 ; see fig . other measurements of alic , as well as hippocampal and lateral ventricular volumes were not significantly reduced in patients with poor outcome compared to the stable group ( see table 2 ) . alic , anterior limb of the internal capsule ; fe - sz , first - episode schizophrenia patients ; ci , confidence interval ; max . , relativetable 2results of volumetric measurementsregion of interestfe - sz stable ( n = 12)fe - sz bad outcome ( n = 11)diff ( % ) mancovamsdmsdfdfphippocampus volume left side ( rel. maximal cross sectional , mancova multivariate analysis of covariance , f , p for factor subgroup relative left alic maximal cross sectional area in first - episode schizophrenia patients . alic , anterior limb of the internal capsule ; fe - sz , first - episode schizophrenia patients ; ci , confidence interval ; max . maximal cross sectional , mancova multivariate analysis of covariance , f , p for factor subgroup we found no significant correlations between the cumulative doses of risperidone or haloperidol and the alic maximal cross sectional area in the total group of patients and in the subgroups of schizophrenic patients with stable psychopathology or clinically relevant deterioration in the 1-year course . the cumulative doses of risperidone and haloperidol were not significantly different in both subgroups ( df = 1 , f = 0.001 ; p = 0.98 , oneway - anova ) , and the daily doses of risperidone and haloperidol in the subgroups were comparable ( fe - sz stable psychopathology : risperidone 4.3 2.0 mg / day , haloperidol 3.0 1.5 mg / day ; fe - sz poor outcome : risperidone 3.8 1.3 mg / day , haloperidol 3.1 1.6 mg / day ) . the results were similar , first - episode patients with deterioration during the first year of the illness still presented with a significantly smaller maximal cross sectional area of the alic compared to those patients with stable psychopathology ( mancova ; p = 0.020 ) . the main result of our study assessing the relationship of brain morphology and clinical outcome in first - episode schizophrenia is the significant reduced maximal cross sectional area of the anterior limb of the internal capsule ( alic ) in the subgroup of patients with clinical deterioration during the 1-year course compared to patients with stable psychopathology . there was no volumetric difference between the two subgroups with regard to the other selected regions of interest , hippocampus and lateral ventricles . in addition , significant decreases in white matter density were detected in connecting structures like the genu and truncus of the corpus callosum , the right alic and the right anterior commissure compared to healthy subjects . in the present study we morphometrically assessed the hippocampus and the lateral ventricles , whose volumetric alterations are most consistently associated with the course of the illness , as well as the anterior limb of the internal capsule , a connecting white matter structure , which has been found to exhibit decreased volumes both in schizophrenia and in unaffected first - degree relatives of schizophrenic patients . out of the three mentioned structures , in our study only the left maximal cross sectional area of the anterior limb of the internal capsule was associated with the clinical course in first - episode schizophrenia . in this sample of first - episode patients those with poor outcome ( high increase of positive symptoms ) showed a reduced maximal cross sectional area of the alic on the left side compared to those with good outcome . in the good outcome group the mean left alic maximal cross sectional area is larger than the right one , and in the bad outcome group it is the other way round . nevertheless we can not rule out the possibility that the left alic maximal cross sectional area in the patients with good outcome group may be disproportionately large in our sample . abnormalities in the alic can thus be taken as measure for disturbances in the frontothalamic connectivity that is discussed to be relevant for the pathophysiology of schizophrenia . interestingly , a recent study shows that a reduced area of the alic in dorsal mri slices was associated with poor outcome based on longitudinal analysis of self - care deficits in chronic schizophrenia . additionally , our results indicate that reduced white fiber structures in this area might increase the risk not only for a poor outcome in chronic schizophrenic patients , but also increase the vulnerability for the onset of schizophrenic psychosis and an unfavourable course in first - episode schizophrenia . to our knowledge this study is the first one investigating the relevance of white matter structures , especially the alic , for the course of first - episode schizophrenia . while the cumulative and daily doses of risperidone and haloperidol were low and similar distributed in both subgroups we do not assume a relevant influence of antipsychotic medication on our volumetric results or on clinical outcome , for instance by the induction of secondary negative symptoms . in conclusion our finding of reduced internal capsule size in patients with bad outcome supports the dysconnectivity concept of schizophrenia and points towards disturbances in the striato - thalamo - cortical neuronal circuit and abnormalities in related structures .

accurate and automatic assessment of retinal images has been considered as a powerful tool for the diagnosis of retinal disorders such as diabetic retinopathy , hypertension , and arteriosclerosis . blood vessels have varying contrast due to which the darker vessels ( thick vessels ) can be extracted easily using standard techniques mentioned in the literature while it is difficult to extract the vessels having poor contrast ( thin vessels ) . segmentation of blood vessels in retinal images is a field of interest for scientists since last two decades [ 14 ] . various kinds of eye abnormalities are indicated by changes in vessel tree structure [ 5 , 6 ] . a true vessel tree structure should contain information about precise thickness of blood vessels in the retinal images . central retinal artery occlusion produces dilated tortuous veins , age related macular degeneration and diabetes can generate new blood vessels ( neovascularization ) , and the study of retinopathy of prematurity in premature infants is not possible without the knowledge of vessel tree structure . progression of such eye diseases can only be tracked by noticing the changes in the vessel tree structure with the passage of time . normal images are those containing high contrast vessels and uniform background illumination and which do not contain eye abnormalities such as drusen , exudates , lesions , and microaneurysms . extraction of vessel tree for normal images is not much useful in comparison with the abnormal retinal images which convey useful information about the progression of different eye abnormalities . a retinal image has blood vessels with varying thicknesses ( 36 micron to 180 micron ) and varying foreground illumination . the contrast of the blood vessels also varies : higher for thick vessels and lower for thin vessels . in the presence of anomalies in the retinal images , extraction of tree structure some of them are kernel based methods such as edge detection filters and matched filters [ 9 , 10 ] . in matched filter methods if a larger kernel is selected , thick vessels are obtained precisely whereas thin vessels with increased thickness are obtained . the use of smaller kernels can help to precisely select the thin vessels with higher correlation , but the thick vessels are obtained with reduced thicknesses . a conventional matched filtering technique thus requires a large number of different sized kernels with different orientations . local and region based properties to segment blood vessels have been reported in using a probing technique . the method in uses matched filtering , thresholding , thinning and linear classifier algorithm to obtain vessel tree . a classification rate of 91% of blood vessel segmentation and 95% of the vessel network was reported . some relatively new vessel segmentation techniques which work well for normal images have been reported in [ 2131 ] . the readers may be interested in recent work on localisation and segmentation of optic disc in retinal images [ 33 , 34 ] . in this paper the first one processes the given retinal image by extracting horizontal line cross - sections which are thresholded according to local statistical properties of the line data and the binary lines are combined to obtain binary vessel tree . we are referring this technique as binary vessel extraction ( bve ) using image line cross - sections ( ilcs ) . we are referring the second bve technique as edge enhancement and edge detection ( eeed ) . in this technique a minimum of the two images : original retinal image and its blurred version , is generated which is further blurred by using a log filter which gives an enhanced version of blood vessels . the image is thresholded using otsu and the noise is removed from the binary image . the third technique that we are reporting is the modified matched filtering ( mmf ) technique . in mmf preprocessing of the original retinal image includes application of homomorphic filtering for contrast enhancement , assignment of thresholded value ( obtained using otsu ) to all image pixels with threshold value while retaining lower image values for enhancement of blood vessels . the image is then subjected to conventional matched filtering and is thresholded by otsu for vessel tree extraction . the fourth technique is based on an algorithm which we are referring to as continuation algorithm . in ca two binary images : one containing thick vessels ( reference image ) and the other containing thin vessels plus the thick vessels ( test image ) , are processed . the vessel tree in the reference image is extended with the help of vessel tree in the test image . when further extension is halted , the reference image contains a complete blood vessel tree . the images presented in this paper were either taken from stare or from alexandra eye pavilion hospital , edinburgh , uk . from the available images only 30 images were chosen for bve by these four methods which were abnormal and were hard to process with the techniques given in the references [ 131 ] . out of the thirty blurred retinal images , we are reporting the images with highest abnormalities . one image as shown in figure 1(a ) was processed with all the four techniques for intercomparison . the three techniques ilcs , eeed , and ca are novel and the fourth technique mmf is an improved and modified version of the existing technique called matched filtering . matched filtering technique is one of the most cited techniques in literature and has been included to make comparison with the rest of the three techniques . we are describing these four methods for bve one by one in the following sections . in this paper , our main emphasis is on the extraction of blood vessels from abnormal retinal images . we have also tested these four techniques on 105 normal retinal images with higher success rates , but we are not reporting them as they form trivial cases which can be dealt successfully with most of the techniques given in the references [ 132 ] . retinal images have inhomogeneous illumination pattern due to built - in shape of retina . to remove inhomogeneity in illumination the retinal images a typical poor quality highly abnormal retinal image is shown in figure 1(a ) . the image in figure 1(a ) was processed with homomorphic filter which is shown in figure 1(b ) . horizontal cross - sections of the image in figure 1(b ) are extracted one by one for further processing . a typical horizontal cross - section from the image in figure 1(b ) the data shown in figure 2(a ) is convolved with kern1 = { 0 , 0.1 , 0 } , and the resulting data is shown in figure 2(b ) . the application of the convolution process with kern1 has resulted in the inversion of the data . the data shown in figure 2(b ) is then convolved with kern2 = { 0.35 , 1 , 0.35 } and the resulting data is shown in figure 2(c ) . the application of the convolution process with kern2 has produced vessels with improved signal to noise ratio . the horizontal axis shown on the plot of figure 2(c ) corresponds to the threshold level given by ( 1 ) . the kern1 and kern2 were determined on the basis of a set of experiments yielding best results in terms of signal to noise ratio for blood vessels(1)threshold = x+standard deviationrms2 , where ( 2)standard deviation=i(xix)2n1,rms=ixi2n1,n is the of number data points in a given line cross section , xi represents the ith data point , and x- represents the mean value of the data in a line cross section . is a constant equal to a value of 3.3 10 , and this value has been found to give very satisfactory threshold values for all the cross sections in 96% retinal images tested . some retinal images with very poor contrast required a small variation in the value of to give an excellent tree structure . once all the line cross sections of a retinal image are processed , they are combined to form a 2d binary vessel tree as shown in figure 3(a ) . this method has been implemented on 30 highly abnormal retinal images , and 78% efficiency was achieved for high contrast thick blood vessels and for low contrast blood vessels the efficiency varied from 74 to 78% depending upon the contrast of the blood vessels against background . thin blood vessels with contrast as small as 0.01 were extracted with 77% efficiency using the proposed method . however , blood vessels contrast can be further improved by using histogram equalization method in overlapping windows with unit step and then applying line cross - section method on this contrast enhanced image . the image in figure 3(a ) after length filtering at 10 pixels is shown in figure 3(b ) . length filtering is an algorithm by which small isolated structures most probably nonvessels are removed from the image . the original thickness of all the blood vessels has been exactly reproduced without any artifacts by ilcs . some retinal images contain a lot of noise and when such images are processed for bve using ilcs the resulting vessel tree also contains a lot of noise . an example of such a retinal image is shown in figure 4(a ) , and vessel tree extracted by ilcs is shown in figure 4(b ) . the thin vessels in the image shown in figure 4(a ) have very poor contrast which matches with noise spread over the image background . an application of length filtering technique at this stage for noise removal also removes a significant portion of thin blood vessels . to make ilcs more effective , we suggest a slight blurring of such retinal images with a small sized gaussian kernel of standard deviation = 0.824 and then suggest the application of ilcs on resulting blurred retinal images . a vessel tree obtained in this way is shown in figure 5(a ) and after application of length filtering at 10 pixels is shown in figure 5(b ) . figure 6 shows the results obtained with two different matched filters ( of different lengths and widths ) when applied to the image shown in figure 4(a ) . the vessel trees obtained with matched filtering technique shown in figure 6 can be compared with the result obtained with ilcs shown in figure 5(b ) . the result shown in figure 6(a ) corresponds to a matched filter kernel of length 2 and width 4 , whereas the result shown in figure 6(b ) corresponds to matched filter of length 4 and width 6 . the drawback of the matched filtering method , as can be easily seen from these results , is that the shape of tortuous blood vessels has been greatly exaggerated near the optic disc region in figure 6(b ) . the reason for the failure is that each matched filter kernel is designed for a specific environment related to the thickness and linearity of the vessels . since in normal retinal images blood vessels are not very tortuous , therefore matched filtering can work well , but in case of retinal images with tortuous vessels ( an indication of hypertension ) , it has not worked due to mismatching of filter parameters with the nature of vessels . so in general terms we can conclude that the matched filtering technique is not appropriate option especially when the images are abnormal containing drusen or tortuous vessels . the working principle of the proposed technique has been explained through the flow chart diagram given in figure 7 . some important vessel segmentation techniques reported so far in the literature [ 131 ] are based on some sort of thresholding criterion . the techniques either use threshold selection based on the whole image data ( called global thresholding ) or data from the patches of the image ( called adaptive thresholding ) . the thresholding in ilcs is based on the horizontal line cross - section of the image under process and the selection of threshold is based on ( 1 ) . the way the ilcs extracts vessel tree from retinal images is entirely a new concept . in this section , we address the issue of extraction of blood vessels in retinal images using a novel edge enhancement and edge detection technique . most of the techniques mentioned in [ 131 ] work well for normal retinal images containing no abnormalities . as the retinal image becomes abnormal due to the inclusion of drusen or due to lower image contrast , the performance of the techniques in [ 131 ] falls and the vessel tree structures obtained with those techniques do not represent the actual blood vessels tree . to demonstrate the capability of eeed we have chosen an image shown in figure 8(a ) which is low in contrast and at the same time contains a large number of drusen spread over the entire retinal image . the techniques mentioned in [ 131 ] do not give satisfactory results particularly for images like the one shown in figure 8(a ) . the main objectives of the eeed technique are to enhance the contrast of blood vessels and at the same time diffuse the other abnormal features present in the retinal image . application of laplacian of gaussian ( log ) filter further enhances the vessel contrast and suppresses the other abnormal image features . the image can then simply be thresholded using any standard technique , for example , well known otsu thresholding technique can be applied to obtain binary vessel tree . for the extraction of blood vessels with eeed , a retinal image is first convolved with a large gaussian blurring kernel . the blurred image will lose all the details contained in the retinal image and will contain only the illumination pattern . once a gaussian blurred image is obtained from a retinal image , another image is formed out of the two images . the blood vessels in a retinal image normally have intensities lying in the lower range . the minimum image enhances this fact ; that is , the blood vessels have the lowest values with enhanced edges in comparison with background . the background in the minimum image is completely diffused suppressing information about the drusen and the optic disc . the minimum image is then blurred a little bit , normally with a gaussian blurring technique with a small kernel . the blurred - minimum image is then convolved with a log filter with a kernel size of ( 9 , 9 ) . the resultant image is contrast enhanced and contrast reversed with more prominent vessel trees and more uniform background . if the retinal image contains noise comparable with the contrast of vessel trees , then the noise will also be enhanced . due to contrast reversal , the vessels now look bright with boundaries having dark edges . this feature is similar to the use of a homomorphic filter on a retinal image . due to uniform background intensity and the vessel tree having higher intensities as compared with background , the image is simply thresholded using any optimum thresholding technique . we used the otsu algorithm to obtain optimum thresholding for the image obtained with the log filter . noise in the binary images can be eliminated using a length filtering technique or a noise removal technique . the algorithm works in windows of sizes ( x , x ) where x may vary from 4 to 16 . in this algorithm if all the boundary pixels have zero values , that is , the sum of boundary pixel values is zero , then all the pixels within the window are deleted . this method is fast and does not involve human intervention at any stage . to illustrate the eeed technique for vessel tree extraction this image was convolved with a gaussian blurring kernel of = 24 . the blurred image so obtained the minimum of the two images , ( image in figure 8(a ) and image in figure 8(b ) ) was computed and the resultant image is shown in figure 8(c ) . the image in figure 8(c ) is a vessel enhanced image on a nonuniform background . the image in figure 8(c ) was then convolved with a gaussian blurring kernel of = 1 . this process helps to develop continuity in the vessel tree structure ; otherwise broken or missing pixels in the binary vessel tree will be observed . the image obtained in this process is shown in figure 8(d ) . an optimum threshold computed on the image in figure 8(d ) does not give an appropriate vessel tree because the illumination of the background is still nonuniform . to eliminate this nonuniform background problem , we convolved the image in figure 8(d ) with a log kernel of size ( 9 , 9 ) . an optimum threshold was computed for the image in figure 8(e ) and the binary image so obtained is shown in figure 8(f ) . the image in figure 8(f ) has binary noise which is not a part of the vessel tree structure . the noise in the image shown in figure 8(f ) can be removed using a number of techniques . we applied the length - filtering technique to remove the binary noise which is either not a part of vessel tree or a small broken isolated part of a vessel tree . length filtering algorithm has been explained earlier . a window of size ( 8 , 8) was chosen and the noise removal algorithm was applied to the image in figure 8(f ) . the image in figure 8(g ) has much of the noise removed , but there is some noise which can still be removed . to remove the rest of the noise in image in figure 8(g ) we applied the noise removal algorithm with a window size of ( 16 , 16 ) and obtained a completely noise free image , which is shown in figure 8(h ) . the image in figure 8(h ) is the final step in our novel eeed technique for vessel tree extraction . the novel technique for vessel extraction presented in this section has been tested on 30 retinal images of varying illumination and contrast , and the success rate without human intervention was ( 88 - 89% ) and with human intervention was ( 90 to 91% ) . one of the 30 images required human intervention for filtering the image to produce the best result . eeed gave improved results and the success rate varied in the range ( 96 to 98% ) when tested on 105 retinal images . in this section matched filtering method for extraction of blood vessels is used with preprocessing in order to get improved quality of the extracted blood vessels . the important feature in our method is that it produces good quality totally automatic bve , which can be useful for the eye care professionals for patients screening , treatment , evaluation , and clinical study . in this approach the image background at corners since we are interested in enhancing the signal of the blood vessels which lie in the range below the threshold value , the image is automatically thresholded using the otsu algorithm keeping the information in the image below the threshold value and assigning the rest of the image the same value as the threshold . we have done this because our required data lies towards the lower end of the image data spectrum . then this image is subjected to the matched filtering technique and thresholded automatically by otsu algorithm . 80 to 85% was the success rate in obtaining correct vessel trees by mmf without human intervention . this is because by assigning threshold value to higher intensity spectrum leads to a decrease in within - class variance and an increase in between - class variance . then matched filtering technique is applied to that enhanced image and resultant image is thresholded automatically . the work presented in this section is more relevant to the work reported in [ 1 , 10 , 18 , 19 ] . a matched filter technique has been presented for the purpose of enhancement of blood vessels in retinal images . this technique is fully automated for thresholding purposes in order to get a binary tree of blood vessels . the method used for automatic threshold selection is known as the otsu method of threshold determination . three properties of the blood vessels in retinal images may be noted . in a 2d retinal image , blood vessels may have three possible orientations : one orientation is when the blood vessels are along the horizontal axis of the image ( say x - axis ) , blood vessels may be oriented along the vertical axis ( say y - axis ) , or the blood vessels may be at some angle with the x - axis . blood vessels can have small curvatures with antiparallel curvature edges and these anti - parallel pairs may also be approximated by piecewise linear segments . blood vessels are darker relative to the surrounding region in a retinal image due to lower reflectance relative to the surrounding regions . blood vessels seem to follow an inverted gaussian profile with the width of the vessels varying from 2 pixels to 10 pixels for a standard image . this range may correspond to 36 to 180 microns . to introduce the concept of matched filtering in retinal images , let us consider ( 3 ) which is used in the theory of communication [ 22 , 32 ] ( 3)so(t)=h(f){s(f)+(f)}ei2ftdf , where so(t ) is the output , s(t ) is the input with an additive gaussian noise n(t ) , such that s(f ) = {s(t ) } and (f ) = {n(t ) } , where is the fourier transform operator and f is the variable in the frequency domain corresponding to the variable t in the time domain . h(f ) = {h(t ) } is the transfer function of the system with impulse response h(t ) . it has been shown that the filter h(f ) that maximizes the signal to noise ratio for so(t ) is given by hopt(f ) = {s(t ) } = s(f ) , where hopt(f ) is known as the matched filter with impulse response hopt(t ) = s(t ) = s(t ) , when s(t ) is an even function . in a typical communication system , if there are n different inputs si(t ) , i = 1,2 , , n received , then these inputs are passed through a stack of n matched filters and the output with the maximum value is selected . for the 2d retinal images , s(t ) needs to be defined at the vessels and may be approximated by an inverted gaussian function of appropriate width with gaussian centred at the centre of the vessels . as the vessels move away from the optic disc the width of the vessels narrows down . for the inverted gaussian to act as a perfect matched filter for the vessels , as the vessels change their orientation the gaussian function needs to be rotated as well . we have suggested an inverted gaussian to act as a matched filter for the enhancement of blood vessels in retinal images . ideally the matched filter must have all possible orientations and varying thickness to enhance vessels with different orientations and thicknesses . assuming blood vessels are the signal to be enhanced , s(t ) may take the form given by ( 4)f(x , y)=a{1kexp(d222 ) } , where d is the perpendicular distance between the point ( x , y ) on the vessel and the straight line passing through the centre of the blood vessel in a direction along its length . corresponds to the spread of the gaussian intensity profile of the vessel , a is the grey - level intensity of the local background , and k is a measure of the reflectance of the blood vessel relative to its neighbourhood . the optimal filter to act as a matched filter for the vessels may be written as given by . the negative sign implies that the vessels are darker than the background . for simulation purposes , ( 5 ) for the matched filter in its 2d form ( 6)f(x , y)=exp(x222 ) |y|l2 . this will act as an ideal matched filter for a segment of the vessels lying along the y - axis and l is the length of the vessel segment for which the vessel is assumed to have a fixed orientation . the matched filter above is valid only for a segment of the vessels of certain gaussian width . this filter must have different values of to act as a matched filter for different widths of the vessels lying along the y - axis . for vessels other than along the y - axis , the filter above needs to be rotated to act as a matched filter for vessels with different orientations . in our simulation , we used only one value of but rotated the filter from 0 to 180 with a step size of 9. the application of matched filtering with these orientations generates 21 images . these images are then combined retaining the maximum value of the pixels among the 21 images . it has been observed that by changing the width of the matched filter , there is a considerable effect on the thickness of the extracted blood vessels . if the filter is very wide , then the blood vessels extracted will be thicker in size as compared with their original width in the image and vice versa . kernel with width ranging from w = 6 to w = 8 is the most accurate range for vessel extraction in comparison with the original thickness of the blood vessels . this image was processed with a matched filter at w = 6 and the response of the matched filter is shown in figure 9(b ) . figure 10 shows the intermediate stage in which the matched filter response for w = 6 is shown at angles of 0 , 45 , 90 , and 135 for the image shown in figure 9(a ) . it shows how blood vessels at different locations in an image are enhanced with the rotation of the matched filter kernel . the results described in figure 9 are for the suitable range of width of matched filter kernel , but if we increase or decrease the width of the matched filter kernel , then the results obtained are not as good as in the previous case . the width of the gaussian is determined by and the sampling is determined by w. appropriate values of both and w are required to obtain optimum matched filter results . the overall response for the matched filter at w = 2 , 4 , 10 , it was also found that by changing the length of the kernel , there was an adverse effect on the quality of image obtained after the matched filtering technique . figure 12(a ) is the maximum matched filter response with w = 6 , but this time length along the y - axis has been changed from ( w , + w ) to ( 2w , + 2w ) ; that is , the length of kernel has been doubled while the length along the x - axis remained the same as ( w , + w ) . figure 12(b ) shows the result when the length along the y - axis has been halved from ( w , + w ) to ( 0.5w , + 0.5w ) . in order to get the coordinates of the blood vessels we need to threshold the image obtained after the matched filtering . the selection of an adequate threshold of the grey - level for extracting objects of interest from background is very important . most of the methods used to threshold the image automatically were based on the image histogram . in the ideal case the histogram for the image should be bimodal . ideally there is a deep and sharp valley between these two peaks , but practically , for most grey - level images , it is difficult to detect the exact valley bottom precisely , especially when two peaks are unequal in height producing no trace of a valley . if the image histogram is unimodal , such methods do not work . let there be l grey - levels in a retinal image [ 1,2 , , let ni be the number of pixels at level i. the total number of pixels in the image is then given by n = n1 + n2 + +nl . the probability of the ith grey - level is given by pi = ni / n , where i=1pi = 1 . let us assume that we want to divide the grey - levels in the retinal image into two classes c1 and c2 to separate the background from the foreground , where c1 denotes pixels with levels [ 1,2 , , k ] and c2 denotes pixels with levels [ k + 1 , k + 2 , , l ] , where k is the grey - level value that thresholds the background from the foreground . in retinal images information about the blood vessels falls in the category of lower grey - levels . the probability of class occurrence and class mean levels are given , respectively , by the following equations : ( 7)1=i=1kpi,2=i = k+1lpi,1=i=1kipi1,2=i = k+1lipi2 , where 1 , 2 is the probability of class occurrences of c1 and c2 , respectively , and 1 , 2 are the class means for c1 and c2 , respectively . t = i=1i pi is the total mean level of the original image . it is easy to establish 1 1 + 2 2 = t and 1 + 2 = 1 . the class variances may be found from the following equations : ( 8)12=i=1k(i1)2pi122=i = k+1l(i2)2pi2 . to determine the goodness of the threshold at level k , we can use the following discriminant criterion measures or the measures of class separability used in the discriminant analysis : ( 9)=b2t2 , where ( 10)b2=i=12i(it)2 . the relationship between b and t is given by ( 11)b2+w2=t2 , where w is given by ( 12)w2=112+222.b and t are the between - class variance and the total variance of levels , respectively , where w is defined as within - class variance . it should be noted that w and b are functions of the threshold k where w is based on second order statistics while b is based on the first order statistics and t is independent of k. thus the problem is to find the optimum value of k labelled as k that maximises or equivalently b once the value of k is determined , the retinal image is split into two levels at the value of k. this method of finding the threshold has also been tested for multilevel thresholding in order to calculate a suitable threshold for the replacement of black regions at corners by a suitable grey - level as shown in figure 9(a ) . the algorithm worked well by splitting the image into three classes but was unable to classify that darker region into a single separate class . in order to extract the coordinates of blood vessels in the matched filtered image we have applied the algorithm described earlier also known as otsu for thresholding . results have suggested that if we use an image in which the surrounding dark part ( corners ) is very black , then the threshold selected by the algorithm is not very good . however , if we assign those black regions by a value which is close to image background , then much better results are obtained . the image in figure 13(a ) is modified when the black surrounding corners are replaced with the image mean value . this information is gathered from the image histogram which gives a distinct peak for the background . figures 13(c ) and 13(d ) show the results of automatic thresholding on matched filtered output of the images in figures 13(a ) and 13(b ) , respectively . if one wants to reveal further detail in the image , for example , if the blood vessels have very poor contrast against the background , the threshold decided by the algorithm can be further reduced manually and one can see the results , but in most cases this will increase the noise in the image . it has been observed that a better threshold decision takes account of the data of the surrounding corners as well . if the data at these edges is just like the image data , then even better results can be achieved . the reason for better results with modified surroundings can be explained on the basis of ( 11 ) which shows that the sum of squares of variances between the classes and within - class is a constant and is independent of the threshold k. in discriminant analysis , within - class variance shows the scatter of samples around their class expected value . thus by replacing the small value pixels from the surroundings by a higher value the variance within the class will be reduced . in an unmodified image the variance within the class is high while after modification with higher values at these pixels the variance within the class becomes lower . since the sum of two variances ( within - class , between the classes ) is constant if the within - class variance is high then the between - class variance will be small and vice versa . thus by performing the above action of intensity modification at the surrounding pixels we have actually reduced within - class variance and consequently between - class variance increases . from the results of figure 13(d ) we can see that we can extract the coordinates of the main blood vessels in retinal images successfully . the method for the extraction of blood vessels is further modified to improve the quality of the extracted blood vessel tree . in this approach , the image background at corners is modified and then a homomorphic filter is applied to obtain smooth image . since we are interested in enhancing the signal of the blood vessels which is lying in the range below the threshold value , the image is automatically thresholded using the otsu algorithm keeping the information below the threshold value and giving the rest of the image the same value as the threshold . figure 14(c ) shows the image obtained as a result of application of homomorphic filtering , and figure 14(d ) shows the image obtained by automatic thresholding of the image in figure 14(c ) by the otsu method keeping the data of the image below the decided threshold unchanged and the data above the threshold being replaced by the threshold value . we have done this because our required data lies towards the lower end of the image data spectrum . then this image is subjected to the matched filtering technique and thresholded automatically by otsu . 100% correct results have been obtained for thresholding the image by this method , and there is no need to play around with the threshold value for better results as we needed to do with the conventional matched filtering method because now there is a big difference between background and signal ; that is , between - class variance is increased and at the same time within - class variance has been decreased . figure 15(a ) shows the result obtained with the conventional matched filtering method , and figure 15(b ) shows the result obtained with the modified matched filter method . it has been observed that along with the enhancement of blood vessels in the abnormal images some other features are also sometimes enhanced and it is essential to eliminate them . the method used to filter them out is called length filtering . in length filtering a single identity is assigned to a single piece of connected objects and the number of pixels in each connected objects is determined and so they can be filtered easily . the effect of length filtering is shown in part ( c ) and ( d ) of figure 15 . we have tried to prevent information ( other than blood vessels ) being enhanced with the use of matched filters by making an effort to enhance the contrast of the blood vessels and at the same time to reduce the contrast of background features . in some cases , when the lines are small and disjoint , it is possible to filter them out completely but when they combine to form bigger structures then it is difficult to eliminate them . in figure 15(b ) the small lines in the image can easily be removed by the length filtering technique , but it is very difficult to clear the lines in the image in figure 15(a ) as they combine together to form structures with large numbers of pixels and any effort to clear those structures may lead to the loss of blood vessel information . figure 15(c ) shows the image in figure 15(a ) after length filtering at 136 pixels , and figure 15(d ) shows the image in figure 15(b ) after length filtering at the same number of pixels . it has been found that if an image contains thin blood vessels , then by using a kernel of small width these blood vessels can be enhanced . one can try kernels of different widths , and a detailed tree of blood vessels can be extracted by combining the responses at each pixel from the images obtained by using kernels of varying widths . figure 16(b ) shows the blood vessel tree using single kernel of length 4 and width 10 , and figure 16(c ) shows the blood vessel tree obtained using 4 different kernels of different widths and lengths . figure 16(d ) shows the tree extracted with kernel of length 2 and width 4 ( without length filtering ) overlapped with the unprocessed image . at certain points where the width of the blood vessels is more than the width measured by the matched filtering method , one can see black lines along the blood vessels , and where the width is less in the original scene than the width measured by the matched filter , one can see a black line in the central region with as outer region of low intensity which shows the enhanced width due to the matched filtering method . at the points where the matched filter correlates exactly with the width of vessels one can observe an exact overlap . it has also been observed that if the image is first processed with a high pass filter followed by matched filter method , then superior results are achieved . this is because the high pass filter can greatly enhance the edges of the vessels increasing the contrast of the vessels against background . one of the most frequently cited technique , for bve is a matched filtering technique . moreover , extraction of thick and thin and intermediate sized vessels requires different sized kernels in the matched filtering technique . different binary images obtained with different sized and different orientation kernels in the matched filtering technique are combined to give one binary vessel tree image , but in addition to this some noise filtering techniques are always required to filter out the segments which are not part of a vessel tree . the full automation of matched filtering technique requires careful selection of kernels of different sizes and orientations and of course a noise filtering technique as well . one of the fundamental drawbacks is a discontinuity in the vessel tree especially where the vessels are very thin . this is because of the smaller signal to noise ratio for weaker or thinner vessels . due to this discontinuity the application of any noise ( broken segments of vessels ) removal or length filtering technique would result in a vessel tree image which is not complete especially on the sides where thin vessels end . sometimes thin vessels provide more important information to an ophthalmologist about the progression of a disease . the other vessel extraction techniques as mentioned in [ 131 ] suffer from more or less similar defects especially when concerned with thin vessels . the broken thin vessels in the binary vessel tree if joined could give us a complete vessel tree , and then the other broken parts which are not the part of blood vessels can easily be removed using any noise filtering technique , for example , a length filtering technique . we felt the need to develop an algorithm that could join the broken parts of the vessel tree segments to develop continuity and at the same time identifying the noisy bits in the image for their removal if necessary . in this section we present a novel technique for the development of continuity between the broken thin vessel segments in a binary vessel tree image avoiding the joining of broken segments which are not part of the actual vessel tree . in the proposed technique called continuation algorithm ( ca ) we generate two binary tree images of the given retinal image and process them to obtain a noise free continued segments vessel tree . the two binary images can be generated using matched filtering techniques [ 1 , 10 , 18 , 19 ] . we have used the matched filtering technique for the extraction of binary tree as it provides appropriate control to obtain two pre - requisite images for our proposed continuation algorithm . other techniques for blood vessel extraction as cited in the reference list may be used provided two conditions are satisfied by the binary tree images : ( 1 ) the first binary image ( refer to it as reference binary image ) should contain thick blood vessel tree structure which is obtained with matched kernel of relatively larger size . the reference image would contain only thick vessels and no noise , that is , no broken segments . if there are any broken segments length filtering or any other noise filtering technique could be used . ( 2 ) the second binary image ( refer to it as test image or image2 ) should contain thick as well as thin vessel tree structure as it is obtained with matched filtering the retinal image with a smaller kernel size . as the image2 contains thin vessels , it also contains a lot of noise . a typical low quality abnormal retinal image chosen for simulation using ca the image chosen for the simulation is abnormal in the sense that it consists of drusen , it is a poor quality image in terms of contrast , the contrast of thin vessels is extremely poor , and it is hard to visualise the ending points of thin vessels being submerged in the background noise . two binary vessel trees are extracted from a retinal image using the matched filter technique . one binary tree image called the reference image consists of a thick vessel tree and can be made free of noise by the application of a length - filtering algorithm if required . the second binary tree image ( called image2 ) required by the algorithm consists of thick as well as thin vessels . a lot of noise appears in the image as a result of the application of matched filtering . the noise in this image can not be filtered out using a length - filtering technique as most of the thin vessel structures will also be filtered out . matched filters with different kernel sizes and with different orientations were used to obtain the two binary vessel trees . the main drawback of the matched filtering technique is that it is poor at extracting noise - free thin vessels . the pixel values in the image are either 0 or 100 . for each pixel ( i , j ) in the reference image where the value is zero that is , refimg ( i , j ) = 0 , and for the same pixel ( i , j ) in the second binary image where the value is 1 , that is , img2 ( i , j ) = 1 , a sum of pixel values is calculated as ( 14)sum = refimg(i , j+1)+refimg(i+1,j+2)+refimg(i+1,j+3)+refimg(i+1,j+1)+refimg(i+2,j+3)+refimg(i+2,j+1)+refimg(i+3,j+2)+refimg(i+3,j+1)+refimg(i+1,j ) . if the sum 100 , then the pixel value in the reference image is set to 100 , that is , refimg ( i , j ) = 100 . this process is repeated indefinitely unless all the zero value pixels in the refimg find no unity value pixel in the img2 for all ( i , j ) in the refimg . first it extends the tree in the reference image from top to bottom using the information from the second binary image . the tree extension obtained in the first step is shown in figure 18(a ) . the vertically flipped version of the image shown in figure 18(a ) the image after the second application of the algorithm is shown in figure 18(b ) . the image in figure 18(b ) is then flipped horizontally and the algorithm is applied . the resultant image is shown in figure 18(c ) . the image in figure 18(c ) is then flipped vertically and after the fourth application of the algorithm the resulting image is filliped horizontally and is shown in figure 18(d ) . the comparison of the two images ( image in figure 17(b ) and image in figure 18(d ) ) reveals that our proposed continuation algorithm gives reasonably accurate vessel tree structure of a poor quality retinal image . the algorithm has been tested on 30 images for vessel tree extraction and the performance has been found to be satisfactory . each technique was tested on the same set of 30 highly abnormal poor quality retinal images . most of the techniques in the literature can work well on normal images but fail to perform well on abnormal retinal images . in this paper , we have focussed mainly on the abnormal retinal images . a set of four typical abnormal images are shown in figures 1(a ) , 4(a ) , 9(a ) , and 14(a ) . the image in figure 1(a ) was processed by all the four techniques and their results have been reproduced for comparison in figure 19 . images in figures 1(a ) and 4(a ) were processed with ilcs , image in figure 1(a ) was processed by eeed , images in figures 1(a ) , 9(a ) , and 14(a ) were processed by mmf technique and the image in figure 1(a ) was processed with ca . we have chosen to present the image in figure 1(a ) as a typical abnormal retinal image for comparison . the four techniques in the order of increased simplicity are mmf , ilcs , eeed , and ca . mmf being relatively more complex has been discussed in greater detail and three retinal images have been presented to support the technique . ilcs which is comparatively less complex has been demonstrated with two retinal images and the rest of the two techniques eeed and ca being much simpler have been supported by one image each . the images obtained with eeed and ca shown , respectively , in figures 19(b ) and 19(d ) are superior in quality to the images obtained with ilcs and mmf shown , respectively , in figures 19(a ) and 19(c ) . the vessel tree structure obtained with ilcs ( figure 19(a ) ) contains more isolated segments or broken vessels whereas the broken vessels in the image figure 19(c ) by mmf are relatively less . the image in figure 19(b ) by eeed contains a few broken pieces of vessels and the image in figure 19(d ) by ca contains almost no broken vessel part . the four techniques ( ilcs , eeed , mmf , and ca ) were run for the image in figure 1(a ) on the machine : pentium(r ) d cpu 3.4 ghz , 2 gb of ram , microsoft windows xp professional service pack 2 version 2002 and mathematica 4.1 in a stand - alone mode . 14 seconds 200 milliseconds for ilcs , 1 min . 1 second 99 milliseconds for eeed , 1 min . the four techniques can be arranged in the order of increasing time consumption as ca , eeed , ilcs , and mmf . for rest of the 29 images the four images shown in figure 19 were presented to three expert human graders for evaluation purposes . their averaged ratings for the images in figures 19(a ) , 19(b ) , 19(c ) and 19(d ) , respectively , were 76% , 88% , 82% , and 97% . for the rest of the images , ca was always evaluated in the range ( 95 to 99% ) , eeed was always evaluated in the range ( 88 to 91% ) , mmf was always evaluated in the range ( 80 to 85% ) , and ilcs was evaluated in the range ( 74 to 78% ) . these results were found when the relevant codes were run in fully automatic mode requiring no human intervention during the processing time . it has also been observed that the techniques ilcs and mmf can be improved by 1 to 2% if human intervention is involved for some images . the techniques ca and eeed were found to be fully automatic and even a human intervention could not improve the results obtained with them . the four techniques in the order of improved automation may be listed as mmf , ilcs , eeed , and ca . these techniques when analysed on normal retinal images gave improved results : ( 99% to 100% ) for ca , ( 96% to 98% ) for eeed , ( 94% to 96% ) for mmf , and ( 88% to 93% ) for ilcs . it is worth noticing that even the results of the expert human graders varied by 2.5% and we averaged their results for comparison with our results . most of the techniques mentioned in [ 131 ] extract binary vessel trees of retinal images embedded in noise . such techniques extract the vessel trees along with noise like the one shown in figure 17(b ) . some types of noise filters are then used to remove noise but at the cost of losing parts of the vessels which are very thin . the reason is that the very thin parts of the vessel trees are usually treated as discontinued segments by the existing noise removal techniques . the trade - off in removing the noise is to lose parts of the vessel trees which are very thin and the resulting vessel tree structures do not represent the true picture . the existing vessel tree extraction techniques either give up the extraction of thinnest part of vessel trees like the image shown in figure 17(a ) or produce images with a lot of noise as shown in figure 17(b ) . it retrieves noise free vessel tree images and at the same time preserves the thinner vessel segments in the vessel trees . some of the existing vessel tree extraction techniques [ 132 ] produce reasonable tree images for only those retinal images which are good in quality , in contrast and in appearance and in which the vessel tree can be perceived easily with naked eye . the existing techniques do not seem to give reasonable solution when applied to the poor quality images like the ones as shown in figure 1 . we deliberately chose an image with poor quality , with poor contrast , and with poor appearance in which the thinner parts of the vessel tree structure are hardly visible with naked eye . the image ( figure 1(a ) ) also contains a lot of drusen which act as embedded noise . in the presence of these abnormalities in the processed image , the proposed method based on continuation algorithm seems to give accurate results in the extraction of full tree structure retaining the thinner vessel parts and at the same time removing the noise as its inherent capability . the technique does not require the application of any noise removal technique during its processing steps if the matched filter is carefully chosen in obtaining the reference image used in the proposed technique . the proposed continuation algorithm ensures continuity in the extracted vessel trees till the end of the thinner parts of the vessel segments . the existing vessel extraction techniques find it hard to ensure this characteristic and a discontinuity is observed especially with poor quality and noisy images . the built - in feature of the continuation algorithm is such that it removes the noise during its processing steps and does not require any postprocessing which is its second main characteristic . this type of characteristic is lacking in existing techniques and some existing techniques may add noise in the vessel tree images during processing and require some postprocessing for noise removal . if retinal images are available from the same fundus camera , the matched filtering kernel used in obtaining the reference image will be fixed and the continuation algorithm can work without any human intervention . that is , ca does not require any pre- or postprocessing in the sense required by other vessel extraction techniques . filtering of noise ( removal of nonvessel segments ) is an essential part of many bve methods for clean outputs . the other three methods ( ilcs , eeed , mmf ) and most of the methods given in the references require some sort of filtering to remove the broken vessel pieces , pieces of leftovers of other removed segments which were not vessels and the background noise . if the success rate for a given technique is , for example , 95% , then it means that only 5 pixels out of 100 pixels differed from the averaged ground truth image constructed by the three expert human graders . the success rate of 95% does not mean that a particular technique worked only for 95 images out of 100 . all the four techniques worked for all the test images including 30 abnormal images and 105 normal retinal images but with different accuracies . results of the three expert human graders in the form of ground truth images were found to vary by a maximum of 2.5% . the interpretation of our success rate is different from the meaning of the success rate used in the literature . two noise removal techniques were developed to remove noise from the final binary vessel trees . one was the length - filtering technique and the other was a noise removal algorithm . the appropriate value for the threshold has been found to vary from 10 to 50 depending upon the noise level in the input retinal image . from automation point of view a value of 50 for the threshold in the length filtering algorithm the isolated structures in the binary vessel tree are grouped and the number of pixels in each group is computed . a square window of size ( x , x ) pixels is centred at each pixel position in the binary vessel tree . if all the boundary pixels are zero , then the data within the window is all set to zero . the appropriate window sizes vary from ( 8 , 8) to ( 16 , 16 ) . this algorithm was found to give satisfactory results , and the validity has been established on 30 abnormal retinal images and 105 normal retinal images . we have suggested a modification to the existing method that gives better results as compared with the results obtained using the ordinary or conventional matched filtering technique . our method of matched filtering includes some preprocessing of the retinal images . in the ordinary or conventional method , a matched filter is directly convolved with a retinal image , which also enhances nonvessel structure in the retinal image and gives rise to noise in the binary vessel tree structure . in our method , a threshold value is computed for the image using the otsu threshold method and all the data in the image greater than the threshold is replaced by the threshold value . the resulting image may have uniform background illumination with vessel structure more enhanced as compared with the preprocessed retinal image . a threshold value is then computed using the otsu method and a binary vessel tree is produced with reduced nonvessel structure in the image . the modifications we have suggested have greatly improved the vessel tree structure as compared with that obtained using only the conventional matched filter technique . drawbacks of using only one matched filter to recover the vessel tree have been discussed . we have also introduced a multimatched filtering technique in which a preprocessed retinal image is convolved with matched filters of different kernel sizes and corresponding binary vessel tree images are combined at the end . the results reveal that the modified matched filtering technique could be a promising technique for bve in retinal images . kernels of different lengths and widths with all possible orientations ( with a minimum step of 9 ) should act as matched filters for a given retinal image . preprocessing of retinal images and post processing of uncleaned binary trees are the important ingredients of vessel tree extraction process by mmf method . in the continuation method one of the binary vessel tree images contains only thick vessels and is noise free which we named as the reference image . the second binary vessel tree image contains thick vessels , thin vessels , and a lot of noise . the proposed method extends the vessel tree in the reference binary image with the help of the other binary vessel tree structure . this process is repeated four times for different orientation of the images generated from the reference image with the application of continuation method . this process extends the vessel tree in the reference image according to the vessel tree in the second binary image except where the nonvessel structure ( noise ) occurs . the final vessel tree obtained from the reference image with successive applications of the continuation algorithm is an exact copy of the second binary vessel tree image except that the nonvessel tree structure is absent . this technique does not require noise filtering at any stage as required by other vessel tree extraction methods . we have tested our continuation method on 30 poor quality abnormal retinal images and on 105 normal ( good quality ) retinal images and found the technique to work satisfactorily . the success rate of the ca method was 95% to 99% when tested over 30 abnormal retinal images which approach to 99 - 100% for normal retinal when tested over 105 normal images . the technique is superior to existing vessel tree extraction techniques and the other three techniques ( ilcs , eeed , and mmf ) . we conclude that the four techniques for vessel tree extraction from retinal images are good additions to the existing vessel segmentation techniques and the two techniques : eeed and ca , are especially important as they satisfied the human graders who recommended these two techniques for bve from retinal images on commercial grounds .

a variety of blood vessel extraction ( bve ) techniques exist in the literature , but they do not always lead to acceptable solutions especially in the presence of anomalies where the reported work is limited . four techniques are presented for bve : ( 1 ) bve using image line cross - sections ( ilcs ) , ( 2 ) bve using edge enhancement and edge detection ( eeed ) , ( 3 ) bve using modified matched filtering ( mmf ) , and ( 4 ) bve using continuation algorithm ( ca ) . these four techniques have been designed especially for abnormal retinal images containing low vessel contrasts , drusen , exudates , and other artifacts . the four techniques were applied to 30 abnormal retinal images , and the success rate was found to be ( 95 to 99% ) for ca , ( 8891% ) for eeed , ( 8085% ) for mmf , and ( 7478% ) for ilcs . application of these four techniques to 105 normal retinal images gave improved results : ( 99 - 100% ) for ca , ( 9698% ) for eeed , ( 94 - 95% ) for mmf , and ( 8893% ) for ilcs . investigations revealed that the four techniques in the order of increasing performance could be arranged as ilcs , mmf , eeed , and ca . here we demonstrate these four techniques for abnormal retinal images only . ilcs , eeed , and ca are novel additions whereas mmf is an improved and modified version of an existing matched filtering technique . ca is a promising technique .

1. Introduction 2. Blood Vessel Extraction (BVE) with Image Line Cross-Section (ILCS) 3. Blood Vessel Extraction (BVE) Using Edge Enhancement and Edge Detection (EEED) 4. Blood Vessel Extraction (BVE) Using Modified Matched Filtering (MMF) 5. Blood Vessel Extraction (BVE) with Continuation Algorithm (CA) 6. Discussion and Comparison of Proposed Vessel Extraction Techniques 7. Conclusion

accurate and automatic assessment of retinal images has been considered as a powerful tool for the diagnosis of retinal disorders such as diabetic retinopathy , hypertension , and arteriosclerosis . segmentation of blood vessels in retinal images is a field of interest for scientists since last two decades [ 14 ] . a true vessel tree structure should contain information about precise thickness of blood vessels in the retinal images . central retinal artery occlusion produces dilated tortuous veins , age related macular degeneration and diabetes can generate new blood vessels ( neovascularization ) , and the study of retinopathy of prematurity in premature infants is not possible without the knowledge of vessel tree structure . normal images are those containing high contrast vessels and uniform background illumination and which do not contain eye abnormalities such as drusen , exudates , lesions , and microaneurysms . in the presence of anomalies in the retinal images , extraction of tree structure some of them are kernel based methods such as edge detection filters and matched filters [ 9 , 10 ] . a classification rate of 91% of blood vessel segmentation and 95% of the vessel network was reported . in this paper the first one processes the given retinal image by extracting horizontal line cross - sections which are thresholded according to local statistical properties of the line data and the binary lines are combined to obtain binary vessel tree . we are referring this technique as binary vessel extraction ( bve ) using image line cross - sections ( ilcs ) . we are referring the second bve technique as edge enhancement and edge detection ( eeed ) . the third technique that we are reporting is the modified matched filtering ( mmf ) technique . in mmf preprocessing of the original retinal image includes application of homomorphic filtering for contrast enhancement , assignment of thresholded value ( obtained using otsu ) to all image pixels with threshold value while retaining lower image values for enhancement of blood vessels . from the available images only 30 images were chosen for bve by these four methods which were abnormal and were hard to process with the techniques given in the references [ 131 ] . out of the thirty blurred retinal images , we are reporting the images with highest abnormalities . one image as shown in figure 1(a ) was processed with all the four techniques for intercomparison . the three techniques ilcs , eeed , and ca are novel and the fourth technique mmf is an improved and modified version of the existing technique called matched filtering . matched filtering technique is one of the most cited techniques in literature and has been included to make comparison with the rest of the three techniques . we are describing these four methods for bve one by one in the following sections . in this paper , our main emphasis is on the extraction of blood vessels from abnormal retinal images . we have also tested these four techniques on 105 normal retinal images with higher success rates , but we are not reporting them as they form trivial cases which can be dealt successfully with most of the techniques given in the references [ 132 ] . to remove inhomogeneity in illumination the retinal images a typical poor quality highly abnormal retinal image is shown in figure 1(a ) . a typical horizontal cross - section from the image in figure 1(b ) the data shown in figure 2(a ) is convolved with kern1 = { 0 , 0.1 , 0 } , and the resulting data is shown in figure 2(b ) . the application of the convolution process with kern1 has resulted in the inversion of the data . is a constant equal to a value of 3.3 10 , and this value has been found to give very satisfactory threshold values for all the cross sections in 96% retinal images tested . some retinal images with very poor contrast required a small variation in the value of to give an excellent tree structure . this method has been implemented on 30 highly abnormal retinal images , and 78% efficiency was achieved for high contrast thick blood vessels and for low contrast blood vessels the efficiency varied from 74 to 78% depending upon the contrast of the blood vessels against background . some retinal images contain a lot of noise and when such images are processed for bve using ilcs the resulting vessel tree also contains a lot of noise . an example of such a retinal image is shown in figure 4(a ) , and vessel tree extracted by ilcs is shown in figure 4(b ) . an application of length filtering technique at this stage for noise removal also removes a significant portion of thin blood vessels . the vessel trees obtained with matched filtering technique shown in figure 6 can be compared with the result obtained with ilcs shown in figure 5(b ) . since in normal retinal images blood vessels are not very tortuous , therefore matched filtering can work well , but in case of retinal images with tortuous vessels ( an indication of hypertension ) , it has not worked due to mismatching of filter parameters with the nature of vessels . so in general terms we can conclude that the matched filtering technique is not appropriate option especially when the images are abnormal containing drusen or tortuous vessels . the thresholding in ilcs is based on the horizontal line cross - section of the image under process and the selection of threshold is based on ( 1 ) . in this section , we address the issue of extraction of blood vessels in retinal images using a novel edge enhancement and edge detection technique . most of the techniques mentioned in [ 131 ] work well for normal retinal images containing no abnormalities . as the retinal image becomes abnormal due to the inclusion of drusen or due to lower image contrast , the performance of the techniques in [ 131 ] falls and the vessel tree structures obtained with those techniques do not represent the actual blood vessels tree . the background in the minimum image is completely diffused suppressing information about the drusen and the optic disc . noise in the binary images can be eliminated using a length filtering technique or a noise removal technique . the blurred image so obtained the minimum of the two images , ( image in figure 8(a ) and image in figure 8(b ) ) was computed and the resultant image is shown in figure 8(c ) . a window of size ( 8 , 8) was chosen and the noise removal algorithm was applied to the image in figure 8(f ) . the novel technique for vessel extraction presented in this section has been tested on 30 retinal images of varying illumination and contrast , and the success rate without human intervention was ( 88 - 89% ) and with human intervention was ( 90 to 91% ) . eeed gave improved results and the success rate varied in the range ( 96 to 98% ) when tested on 105 retinal images . in this section matched filtering method for extraction of blood vessels is used with preprocessing in order to get improved quality of the extracted blood vessels . then matched filtering technique is applied to that enhanced image and resultant image is thresholded automatically . a matched filter technique has been presented for the purpose of enhancement of blood vessels in retinal images . to introduce the concept of matched filtering in retinal images , let us consider ( 3 ) which is used in the theory of communication [ 22 , 32 ] ( 3)so(t)=h(f){s(f)+(f)}ei2ftdf , where so(t ) is the output , s(t ) is the input with an additive gaussian noise n(t ) , such that s(f ) = {s(t ) } and (f ) = {n(t ) } , where is the fourier transform operator and f is the variable in the frequency domain corresponding to the variable t in the time domain . it has been shown that the filter h(f ) that maximizes the signal to noise ratio for so(t ) is given by hopt(f ) = {s(t ) } = s(f ) , where hopt(f ) is known as the matched filter with impulse response hopt(t ) = s(t ) = s(t ) , when s(t ) is an even function . for the 2d retinal images , s(t ) needs to be defined at the vessels and may be approximated by an inverted gaussian function of appropriate width with gaussian centred at the centre of the vessels . assuming blood vessels are the signal to be enhanced , s(t ) may take the form given by ( 4)f(x , y)=a{1kexp(d222 ) } , where d is the perpendicular distance between the point ( x , y ) on the vessel and the straight line passing through the centre of the blood vessel in a direction along its length . corresponds to the spread of the gaussian intensity profile of the vessel , a is the grey - level intensity of the local background , and k is a measure of the reflectance of the blood vessel relative to its neighbourhood . ideally there is a deep and sharp valley between these two peaks , but practically , for most grey - level images , it is difficult to detect the exact valley bottom precisely , especially when two peaks are unequal in height producing no trace of a valley . to determine the goodness of the threshold at level k , we can use the following discriminant criterion measures or the measures of class separability used in the discriminant analysis : ( 9)=b2t2 , where ( 10)b2=i=12i(it)2 . if one wants to reveal further detail in the image , for example , if the blood vessels have very poor contrast against the background , the threshold decided by the algorithm can be further reduced manually and one can see the results , but in most cases this will increase the noise in the image . figure 14(c ) shows the image obtained as a result of application of homomorphic filtering , and figure 14(d ) shows the image obtained by automatic thresholding of the image in figure 14(c ) by the otsu method keeping the data of the image below the decided threshold unchanged and the data above the threshold being replaced by the threshold value . then this image is subjected to the matched filtering technique and thresholded automatically by otsu . 100% correct results have been obtained for thresholding the image by this method , and there is no need to play around with the threshold value for better results as we needed to do with the conventional matched filtering method because now there is a big difference between background and signal ; that is , between - class variance is increased and at the same time within - class variance has been decreased . figure 15(a ) shows the result obtained with the conventional matched filtering method , and figure 15(b ) shows the result obtained with the modified matched filter method . in figure 15(b ) the small lines in the image can easily be removed by the length filtering technique , but it is very difficult to clear the lines in the image in figure 15(a ) as they combine together to form structures with large numbers of pixels and any effort to clear those structures may lead to the loss of blood vessel information . one can try kernels of different widths , and a detailed tree of blood vessels can be extracted by combining the responses at each pixel from the images obtained by using kernels of varying widths . at certain points where the width of the blood vessels is more than the width measured by the matched filtering method , one can see black lines along the blood vessels , and where the width is less in the original scene than the width measured by the matched filter , one can see a black line in the central region with as outer region of low intensity which shows the enhanced width due to the matched filtering method . one of the most frequently cited technique , for bve is a matched filtering technique . moreover , extraction of thick and thin and intermediate sized vessels requires different sized kernels in the matched filtering technique . different binary images obtained with different sized and different orientation kernels in the matched filtering technique are combined to give one binary vessel tree image , but in addition to this some noise filtering techniques are always required to filter out the segments which are not part of a vessel tree . the full automation of matched filtering technique requires careful selection of kernels of different sizes and orientations and of course a noise filtering technique as well . one of the fundamental drawbacks is a discontinuity in the vessel tree especially where the vessels are very thin . the broken thin vessels in the binary vessel tree if joined could give us a complete vessel tree , and then the other broken parts which are not the part of blood vessels can easily be removed using any noise filtering technique , for example , a length filtering technique . in the proposed technique called continuation algorithm ( ca ) we generate two binary tree images of the given retinal image and process them to obtain a noise free continued segments vessel tree . we have used the matched filtering technique for the extraction of binary tree as it provides appropriate control to obtain two pre - requisite images for our proposed continuation algorithm . other techniques for blood vessel extraction as cited in the reference list may be used provided two conditions are satisfied by the binary tree images : ( 1 ) the first binary image ( refer to it as reference binary image ) should contain thick blood vessel tree structure which is obtained with matched kernel of relatively larger size . ( 2 ) the second binary image ( refer to it as test image or image2 ) should contain thick as well as thin vessel tree structure as it is obtained with matched filtering the retinal image with a smaller kernel size . a typical low quality abnormal retinal image chosen for simulation using ca the image chosen for the simulation is abnormal in the sense that it consists of drusen , it is a poor quality image in terms of contrast , the contrast of thin vessels is extremely poor , and it is hard to visualise the ending points of thin vessels being submerged in the background noise . a lot of noise appears in the image as a result of the application of matched filtering . for each pixel ( i , j ) in the reference image where the value is zero that is , refimg ( i , j ) = 0 , and for the same pixel ( i , j ) in the second binary image where the value is 1 , that is , img2 ( i , j ) = 1 , a sum of pixel values is calculated as ( 14)sum = refimg(i , j+1)+refimg(i+1,j+2)+refimg(i+1,j+3)+refimg(i+1,j+1)+refimg(i+2,j+3)+refimg(i+2,j+1)+refimg(i+3,j+2)+refimg(i+3,j+1)+refimg(i+1,j ) . the algorithm has been tested on 30 images for vessel tree extraction and the performance has been found to be satisfactory . most of the techniques in the literature can work well on normal images but fail to perform well on abnormal retinal images . in this paper , we have focussed mainly on the abnormal retinal images . a set of four typical abnormal images are shown in figures 1(a ) , 4(a ) , 9(a ) , and 14(a ) . the image in figure 1(a ) was processed by all the four techniques and their results have been reproduced for comparison in figure 19 . images in figures 1(a ) and 4(a ) were processed with ilcs , image in figure 1(a ) was processed by eeed , images in figures 1(a ) , 9(a ) , and 14(a ) were processed by mmf technique and the image in figure 1(a ) was processed with ca . the four techniques in the order of increased simplicity are mmf , ilcs , eeed , and ca . ilcs which is comparatively less complex has been demonstrated with two retinal images and the rest of the two techniques eeed and ca being much simpler have been supported by one image each . the four techniques ( ilcs , eeed , mmf , and ca ) were run for the image in figure 1(a ) on the machine : pentium(r ) d cpu 3.4 ghz , 2 gb of ram , microsoft windows xp professional service pack 2 version 2002 and mathematica 4.1 in a stand - alone mode . the four techniques can be arranged in the order of increasing time consumption as ca , eeed , ilcs , and mmf . for the rest of the images , ca was always evaluated in the range ( 95 to 99% ) , eeed was always evaluated in the range ( 88 to 91% ) , mmf was always evaluated in the range ( 80 to 85% ) , and ilcs was evaluated in the range ( 74 to 78% ) . the four techniques in the order of improved automation may be listed as mmf , ilcs , eeed , and ca . these techniques when analysed on normal retinal images gave improved results : ( 99% to 100% ) for ca , ( 96% to 98% ) for eeed , ( 94% to 96% ) for mmf , and ( 88% to 93% ) for ilcs . the trade - off in removing the noise is to lose parts of the vessel trees which are very thin and the resulting vessel tree structures do not represent the true picture . the existing techniques do not seem to give reasonable solution when applied to the poor quality images like the ones as shown in figure 1 . in the presence of these abnormalities in the processed image , the proposed method based on continuation algorithm seems to give accurate results in the extraction of full tree structure retaining the thinner vessel parts and at the same time removing the noise as its inherent capability . if retinal images are available from the same fundus camera , the matched filtering kernel used in obtaining the reference image will be fixed and the continuation algorithm can work without any human intervention . the other three methods ( ilcs , eeed , mmf ) and most of the methods given in the references require some sort of filtering to remove the broken vessel pieces , pieces of leftovers of other removed segments which were not vessels and the background noise . all the four techniques worked for all the test images including 30 abnormal images and 105 normal retinal images but with different accuracies . the interpretation of our success rate is different from the meaning of the success rate used in the literature . one was the length - filtering technique and the other was a noise removal algorithm . from automation point of view a value of 50 for the threshold in the length filtering algorithm the isolated structures in the binary vessel tree are grouped and the number of pixels in each group is computed . this algorithm was found to give satisfactory results , and the validity has been established on 30 abnormal retinal images and 105 normal retinal images . we have suggested a modification to the existing method that gives better results as compared with the results obtained using the ordinary or conventional matched filtering technique . the results reveal that the modified matched filtering technique could be a promising technique for bve in retinal images . the final vessel tree obtained from the reference image with successive applications of the continuation algorithm is an exact copy of the second binary vessel tree image except that the nonvessel tree structure is absent . we have tested our continuation method on 30 poor quality abnormal retinal images and on 105 normal ( good quality ) retinal images and found the technique to work satisfactorily . the success rate of the ca method was 95% to 99% when tested over 30 abnormal retinal images which approach to 99 - 100% for normal retinal when tested over 105 normal images . the technique is superior to existing vessel tree extraction techniques and the other three techniques ( ilcs , eeed , and mmf ) . we conclude that the four techniques for vessel tree extraction from retinal images are good additions to the existing vessel segmentation techniques and the two techniques : eeed and ca , are especially important as they satisfied the human graders who recommended these two techniques for bve from retinal images on commercial grounds .

accurate and automatic assessment of retinal images has been considered as a powerful tool for the diagnosis of retinal disorders such as diabetic retinopathy , hypertension , and arteriosclerosis . central retinal artery occlusion produces dilated tortuous veins , age related macular degeneration and diabetes can generate new blood vessels ( neovascularization ) , and the study of retinopathy of prematurity in premature infants is not possible without the knowledge of vessel tree structure . progression of such eye diseases can only be tracked by noticing the changes in the vessel tree structure with the passage of time . extraction of vessel tree for normal images is not much useful in comparison with the abnormal retinal images which convey useful information about the progression of different eye abnormalities . in the presence of anomalies in the retinal images , extraction of tree structure some of them are kernel based methods such as edge detection filters and matched filters [ 9 , 10 ] . in this paper the first one processes the given retinal image by extracting horizontal line cross - sections which are thresholded according to local statistical properties of the line data and the binary lines are combined to obtain binary vessel tree . in mmf preprocessing of the original retinal image includes application of homomorphic filtering for contrast enhancement , assignment of thresholded value ( obtained using otsu ) to all image pixels with threshold value while retaining lower image values for enhancement of blood vessels . the three techniques ilcs , eeed , and ca are novel and the fourth technique mmf is an improved and modified version of the existing technique called matched filtering . matched filtering technique is one of the most cited techniques in literature and has been included to make comparison with the rest of the three techniques . we have also tested these four techniques on 105 normal retinal images with higher success rates , but we are not reporting them as they form trivial cases which can be dealt successfully with most of the techniques given in the references [ 132 ] . a typical horizontal cross - section from the image in figure 1(b ) the data shown in figure 2(a ) is convolved with kern1 = { 0 , 0.1 , 0 } , and the resulting data is shown in figure 2(b ) . the kern1 and kern2 were determined on the basis of a set of experiments yielding best results in terms of signal to noise ratio for blood vessels(1)threshold = x+standard deviationrms2 , where ( 2)standard deviation=i(xix)2n1,rms=ixi2n1,n is the of number data points in a given line cross section , xi represents the ith data point , and x- represents the mean value of the data in a line cross section . this method has been implemented on 30 highly abnormal retinal images , and 78% efficiency was achieved for high contrast thick blood vessels and for low contrast blood vessels the efficiency varied from 74 to 78% depending upon the contrast of the blood vessels against background . to make ilcs more effective , we suggest a slight blurring of such retinal images with a small sized gaussian kernel of standard deviation = 0.824 and then suggest the application of ilcs on resulting blurred retinal images . the drawback of the matched filtering method , as can be easily seen from these results , is that the shape of tortuous blood vessels has been greatly exaggerated near the optic disc region in figure 6(b ) . since in normal retinal images blood vessels are not very tortuous , therefore matched filtering can work well , but in case of retinal images with tortuous vessels ( an indication of hypertension ) , it has not worked due to mismatching of filter parameters with the nature of vessels . so in general terms we can conclude that the matched filtering technique is not appropriate option especially when the images are abnormal containing drusen or tortuous vessels . the thresholding in ilcs is based on the horizontal line cross - section of the image under process and the selection of threshold is based on ( 1 ) . in this section , we address the issue of extraction of blood vessels in retinal images using a novel edge enhancement and edge detection technique . as the retinal image becomes abnormal due to the inclusion of drusen or due to lower image contrast , the performance of the techniques in [ 131 ] falls and the vessel tree structures obtained with those techniques do not represent the actual blood vessels tree . to demonstrate the capability of eeed we have chosen an image shown in figure 8(a ) which is low in contrast and at the same time contains a large number of drusen spread over the entire retinal image . the main objectives of the eeed technique are to enhance the contrast of blood vessels and at the same time diffuse the other abnormal features present in the retinal image . we applied the length - filtering technique to remove the binary noise which is either not a part of vessel tree or a small broken isolated part of a vessel tree . to remove the rest of the noise in image in figure 8(g ) we applied the noise removal algorithm with a window size of ( 16 , 16 ) and obtained a completely noise free image , which is shown in figure 8(h ) . the novel technique for vessel extraction presented in this section has been tested on 30 retinal images of varying illumination and contrast , and the success rate without human intervention was ( 88 - 89% ) and with human intervention was ( 90 to 91% ) . the important feature in our method is that it produces good quality totally automatic bve , which can be useful for the eye care professionals for patients screening , treatment , evaluation , and clinical study . in this approach the image background at corners since we are interested in enhancing the signal of the blood vessels which lie in the range below the threshold value , the image is automatically thresholded using the otsu algorithm keeping the information in the image below the threshold value and assigning the rest of the image the same value as the threshold . in a 2d retinal image , blood vessels may have three possible orientations : one orientation is when the blood vessels are along the horizontal axis of the image ( say x - axis ) , blood vessels may be oriented along the vertical axis ( say y - axis ) , or the blood vessels may be at some angle with the x - axis . to introduce the concept of matched filtering in retinal images , let us consider ( 3 ) which is used in the theory of communication [ 22 , 32 ] ( 3)so(t)=h(f){s(f)+(f)}ei2ftdf , where so(t ) is the output , s(t ) is the input with an additive gaussian noise n(t ) , such that s(f ) = {s(t ) } and (f ) = {n(t ) } , where is the fourier transform operator and f is the variable in the frequency domain corresponding to the variable t in the time domain . in a typical communication system , if there are n different inputs si(t ) , i = 1,2 , , n received , then these inputs are passed through a stack of n matched filters and the output with the maximum value is selected . assuming blood vessels are the signal to be enhanced , s(t ) may take the form given by ( 4)f(x , y)=a{1kexp(d222 ) } , where d is the perpendicular distance between the point ( x , y ) on the vessel and the straight line passing through the centre of the blood vessel in a direction along its length . corresponds to the spread of the gaussian intensity profile of the vessel , a is the grey - level intensity of the local background , and k is a measure of the reflectance of the blood vessel relative to its neighbourhood . figure 10 shows the intermediate stage in which the matched filter response for w = 6 is shown at angles of 0 , 45 , 90 , and 135 for the image shown in figure 9(a ) . the results described in figure 9 are for the suitable range of width of matched filter kernel , but if we increase or decrease the width of the matched filter kernel , then the results obtained are not as good as in the previous case . the overall response for the matched filter at w = 2 , 4 , 10 , it was also found that by changing the length of the kernel , there was an adverse effect on the quality of image obtained after the matched filtering technique . figure 12(a ) is the maximum matched filter response with w = 6 , but this time length along the y - axis has been changed from ( w , + w ) to ( 2w , + 2w ) ; that is , the length of kernel has been doubled while the length along the x - axis remained the same as ( w , + w ) . figure 12(b ) shows the result when the length along the y - axis has been halved from ( w , + w ) to ( 0.5w , + 0.5w ) . let there be l grey - levels in a retinal image [ 1,2 , , let ni be the number of pixels at level i. the total number of pixels in the image is then given by n = n1 + n2 + +nl . let us assume that we want to divide the grey - levels in the retinal image into two classes c1 and c2 to separate the background from the foreground , where c1 denotes pixels with levels [ 1,2 , , k ] and c2 denotes pixels with levels [ k + 1 , k + 2 , , l ] , where k is the grey - level value that thresholds the background from the foreground . the probability of class occurrence and class mean levels are given , respectively , by the following equations : ( 7)1=i=1kpi,2=i = k+1lpi,1=i=1kipi1,2=i = k+1lipi2 , where 1 , 2 is the probability of class occurrences of c1 and c2 , respectively , and 1 , 2 are the class means for c1 and c2 , respectively . to determine the goodness of the threshold at level k , we can use the following discriminant criterion measures or the measures of class separability used in the discriminant analysis : ( 9)=b2t2 , where ( 10)b2=i=12i(it)2 . the relationship between b and t is given by ( 11)b2+w2=t2 , where w is given by ( 12)w2=112+222.b and t are the between - class variance and the total variance of levels , respectively , where w is defined as within - class variance . it should be noted that w and b are functions of the threshold k where w is based on second order statistics while b is based on the first order statistics and t is independent of k. thus the problem is to find the optimum value of k labelled as k that maximises or equivalently b once the value of k is determined , the retinal image is split into two levels at the value of k. this method of finding the threshold has also been tested for multilevel thresholding in order to calculate a suitable threshold for the replacement of black regions at corners by a suitable grey - level as shown in figure 9(a ) . in order to extract the coordinates of blood vessels in the matched filtered image we have applied the algorithm described earlier also known as otsu for thresholding . figures 13(c ) and 13(d ) show the results of automatic thresholding on matched filtered output of the images in figures 13(a ) and 13(b ) , respectively . if one wants to reveal further detail in the image , for example , if the blood vessels have very poor contrast against the background , the threshold decided by the algorithm can be further reduced manually and one can see the results , but in most cases this will increase the noise in the image . the reason for better results with modified surroundings can be explained on the basis of ( 11 ) which shows that the sum of squares of variances between the classes and within - class is a constant and is independent of the threshold k. in discriminant analysis , within - class variance shows the scatter of samples around their class expected value . since the sum of two variances ( within - class , between the classes ) is constant if the within - class variance is high then the between - class variance will be small and vice versa . from the results of figure 13(d ) we can see that we can extract the coordinates of the main blood vessels in retinal images successfully . since we are interested in enhancing the signal of the blood vessels which is lying in the range below the threshold value , the image is automatically thresholded using the otsu algorithm keeping the information below the threshold value and giving the rest of the image the same value as the threshold . figure 14(c ) shows the image obtained as a result of application of homomorphic filtering , and figure 14(d ) shows the image obtained by automatic thresholding of the image in figure 14(c ) by the otsu method keeping the data of the image below the decided threshold unchanged and the data above the threshold being replaced by the threshold value . 100% correct results have been obtained for thresholding the image by this method , and there is no need to play around with the threshold value for better results as we needed to do with the conventional matched filtering method because now there is a big difference between background and signal ; that is , between - class variance is increased and at the same time within - class variance has been decreased . figure 15(a ) shows the result obtained with the conventional matched filtering method , and figure 15(b ) shows the result obtained with the modified matched filter method . in figure 15(b ) the small lines in the image can easily be removed by the length filtering technique , but it is very difficult to clear the lines in the image in figure 15(a ) as they combine together to form structures with large numbers of pixels and any effort to clear those structures may lead to the loss of blood vessel information . figure 15(c ) shows the image in figure 15(a ) after length filtering at 136 pixels , and figure 15(d ) shows the image in figure 15(b ) after length filtering at the same number of pixels . at certain points where the width of the blood vessels is more than the width measured by the matched filtering method , one can see black lines along the blood vessels , and where the width is less in the original scene than the width measured by the matched filter , one can see a black line in the central region with as outer region of low intensity which shows the enhanced width due to the matched filtering method . different binary images obtained with different sized and different orientation kernels in the matched filtering technique are combined to give one binary vessel tree image , but in addition to this some noise filtering techniques are always required to filter out the segments which are not part of a vessel tree . we felt the need to develop an algorithm that could join the broken parts of the vessel tree segments to develop continuity and at the same time identifying the noisy bits in the image for their removal if necessary . in this section we present a novel technique for the development of continuity between the broken thin vessel segments in a binary vessel tree image avoiding the joining of broken segments which are not part of the actual vessel tree . in the proposed technique called continuation algorithm ( ca ) we generate two binary tree images of the given retinal image and process them to obtain a noise free continued segments vessel tree . we have used the matched filtering technique for the extraction of binary tree as it provides appropriate control to obtain two pre - requisite images for our proposed continuation algorithm . other techniques for blood vessel extraction as cited in the reference list may be used provided two conditions are satisfied by the binary tree images : ( 1 ) the first binary image ( refer to it as reference binary image ) should contain thick blood vessel tree structure which is obtained with matched kernel of relatively larger size . a typical low quality abnormal retinal image chosen for simulation using ca the image chosen for the simulation is abnormal in the sense that it consists of drusen , it is a poor quality image in terms of contrast , the contrast of thin vessels is extremely poor , and it is hard to visualise the ending points of thin vessels being submerged in the background noise . for each pixel ( i , j ) in the reference image where the value is zero that is , refimg ( i , j ) = 0 , and for the same pixel ( i , j ) in the second binary image where the value is 1 , that is , img2 ( i , j ) = 1 , a sum of pixel values is calculated as ( 14)sum = refimg(i , j+1)+refimg(i+1,j+2)+refimg(i+1,j+3)+refimg(i+1,j+1)+refimg(i+2,j+3)+refimg(i+2,j+1)+refimg(i+3,j+2)+refimg(i+3,j+1)+refimg(i+1,j ) . the comparison of the two images ( image in figure 17(b ) and image in figure 18(d ) ) reveals that our proposed continuation algorithm gives reasonably accurate vessel tree structure of a poor quality retinal image . the images obtained with eeed and ca shown , respectively , in figures 19(b ) and 19(d ) are superior in quality to the images obtained with ilcs and mmf shown , respectively , in figures 19(a ) and 19(c ) . the four techniques ( ilcs , eeed , mmf , and ca ) were run for the image in figure 1(a ) on the machine : pentium(r ) d cpu 3.4 ghz , 2 gb of ram , microsoft windows xp professional service pack 2 version 2002 and mathematica 4.1 in a stand - alone mode . for the rest of the images , ca was always evaluated in the range ( 95 to 99% ) , eeed was always evaluated in the range ( 88 to 91% ) , mmf was always evaluated in the range ( 80 to 85% ) , and ilcs was evaluated in the range ( 74 to 78% ) . these techniques when analysed on normal retinal images gave improved results : ( 99% to 100% ) for ca , ( 96% to 98% ) for eeed , ( 94% to 96% ) for mmf , and ( 88% to 93% ) for ilcs . some of the existing vessel tree extraction techniques [ 132 ] produce reasonable tree images for only those retinal images which are good in quality , in contrast and in appearance and in which the vessel tree can be perceived easily with naked eye . in the presence of these abnormalities in the processed image , the proposed method based on continuation algorithm seems to give accurate results in the extraction of full tree structure retaining the thinner vessel parts and at the same time removing the noise as its inherent capability . the other three methods ( ilcs , eeed , mmf ) and most of the methods given in the references require some sort of filtering to remove the broken vessel pieces , pieces of leftovers of other removed segments which were not vessels and the background noise . from automation point of view a value of 50 for the threshold in the length filtering algorithm the isolated structures in the binary vessel tree are grouped and the number of pixels in each group is computed . we conclude that the four techniques for vessel tree extraction from retinal images are good additions to the existing vessel segmentation techniques and the two techniques : eeed and ca , are especially important as they satisfied the human graders who recommended these two techniques for bve from retinal images on commercial grounds .

unlike the more stereotypical development of the body s main blood vessels , the formation of the capillary networks responsible for tissue perfusion is an adaptive process primarily governed by the metabolic needs of the target tissues ( fraisl et al . , 2009 ; the plastic nature of this adaptive angiogenesis is further highlighted by the dramatic changes in vascularization observed in tumors or in obese adipose tissue : changes that contribute to the progression of pathologies such as cancer and obesity and are becoming increasingly central to their treatment ( cao , 2010 ; kerbel , 2008 ; lijnen , 2008 ) . although environmental factors such as diet are widely believed to affect the development and progression of these pathologies , exploration of the link between nutrition and angiogenesis has largely been confined to correlative studies . these include descriptions of the effects of gestational nutrition on the placental vasculature ( belkacemi et al . , 2010 ; rutland et al . , 2007 ) or the pro / anti - angiogenic actions of nutrients and metabolites with a potential modulatory role in cancer ( adolphe et al . , 2010 ; kumar et al . , a tantalizing new study has shown that increasing adipose tissue vascularization can ameliorate the deleterious metabolic effects of a high - fat diet , pointing to a central metabolic role for these vascular changes ( sung et al . . however , whether modulation of angiogenesis is associated with metabolic benefits remains a controversial topic , partly because it is not trivial to genetically target the blood vessels of specific organs to recapitulate the changes associated with certain dietary interventions without affecting other cell types or vascular pools ( cao , 2010 ; lijnen , 2008 ; sun et al . regardless of its metabolic consequences , adaptive angiogenesis is widely believed to be mechanistically driven by target - derived signals ( cao , 2007 ; fraisl et al . , a close spatial association between mammalian nerves and vessels was observed as long ago as 1543 ( vesalius , 1543 ) , an association that has subsequently been shown to result from mutual guidance or common pathfinding mechanisms during the formation of the neural and vascular networks ( carmeliet and tessier - lavigne , 2005 ; mukouyama et al . notably , interplay of innervation and vascularisation of internal organs has also been described ( davies , 2009 ) . a functional role for these neurovascular interactions was suggested following the discovery that vessel abnormalities precede a number of neurodevelopmental and neurodegenerative disorders : an observation that points to angiogenesis as a therapeutically relevant process ( quaegebeur et al . , 2011 ; the question remains whether , in a reciprocal process , neuronal activity may affect adaptive angiogenesis . in spite of some intriguing associations ( asano et al . , 1997 ; tonello et al . , 1999 ) , no neuronal populations have been identified that effect long - lasting changes in angiogenesis in response to environmental factors . drosophila melanogaster has an open circulation , but its tracheal system has a role analogous to that of the vertebrate vasculature in supplying tissues and internal organs with oxygen ( fraisl et al . , 2009 during embryogenesis , developmental mechanisms akin to those discovered in the vertebrate lung and vasculature make use of signaling pathways such as fibroblast growth factor ( fgf ) signaling to sculpt this complex tracheal network of interconnected tubes ( ghabrial et al . , 2002 ; metzger et al . , 2008 ; uv et al . , 2003 ) . these embryonic proliferative and morphogenetic stages are superseded by a larval period of extensive , but mechanistically less understood , cellular growth . growth is particularly prominent in the tracheal terminal cells : the cells at the end of each airway that make contact with target tissues and through which gas exchange takes place ( ghabrial et al . , 2003 ; drosophila tracheal terminal cells branch profusely in response to low oxygen using conserved fgf and hypoxia - inducible factor ( hif ) signaling pathways ( centanin et al . , 2008 this hypoxic remodeling has been assumed to be the only source of tracheal plasticity and , in normal conditions , the tracheal system is generally believed to grow in proportion to the whole organism . in this study , we use a combination of genetic approaches , metabolic profiling , and in vivo imaging to uncover previously unrecognized nutritional plasticity in the fly tracheal system . in contrast to the known target - derived mechanisms of adaptive remodeling , we find this plasticity to be regulated by a mechanism , previously undescribed in either flies or vertebrates , involving nutrient - responsive neurons effecting long - lasting and metabolically significant changes in tracheal architecture . while subjecting wild - type drosophila larvae to different dietary conditions , we observed that a severe reduction in dietary yeast ( the main source of lipid and amino acids in the larval diet ) was accompanied by an almost ubiquitous reduction in tracheal terminal cell branching , even when controlling for overall developmental delay by allowing nutrient - restricted larvae to develop to a comparable stage ( figures 1a1c , 1g1i and figures s1a , s1c , s1d , s1f , s1 g , s1i , s1j , s1l , s1 m , and s1o available online ) . the single exception was the tracheal branches of the central nervous system ( cns ) , which were refractory to this dietary manipulation ( figures 1a and 1 g ) . by contrast , a mild reduction in dietary yeast neither affected developmental timing nor led to major changes in the size of organs or that of most tracheal terminal cells ( figures 1d and 1e and data not shown ) but did lead to a severe reduction in tracheal coverage throughout the digestive tract ( figures 1f , s1b , s1e , s1h , s1k , and s1n ) . reduced tracheal coverage was not caused by cell death ( figures s2a and s2b ) and could not be solely accounted for by defects in gas filling ( figures s2q and s2r ) . s2 m , and s2q s2r ) . to investigate the reversibility of the tracheal changes described above , we reared larvae under the mild nutrient restriction conditions shown to reduce intestinal tracheation and transferred them to more nutritious food immediately after eclosion . even after 7 days on a nutritious diet , the intestinal tracheae of these adults flies were significantly less branched than those of control adult flies always reared on a nutritious diet ( figures 1j and 1k ) , indicating that a defined period of nutrient restriction has long - term effects on the tracheal scaffold . dietary plasticity could be a feature unique to larval tracheae , given that their branches are undergoing extensive growth . to investigate whether adult tracheae are also responsive to diet , we allowed wild - type flies to develop under our standard nutritional conditions and then exposed them to nutritionally poor or imbalanced diets as adult flies . as figure 1l shows , a 7 day nutritional imbalance ( 9% sucrose ) led to increased intestinal tracheation of the mid - midgut , confirming the dietary plasticity of the tracheal system also in adult flies . collectively , these data uncover previously unrecognized nutritional plasticity of the insect tracheal system , shaped by both previous and current nutritional states . the tracheae of different organs exhibit different degrees of nutritional plasticity ; intestinal tracheal terminal cells are particularly sensitive to a reduction in yeast availability , while cns tracheae are preferentially spared . we then focused on the larval phenotypes to investigate the molecular mechanisms of nutritional plasticity . hypoxia , the only known regulator of tracheal plasticity , has been shown to promote tracheal branching by inducing fgf ligand in target tissues and receptor upregulation in tracheal cells ( centanin et al . although downregulation of the fgf receptor gene breathless ( btl ) did lead to reduced tracheation in most scored tissues , consistent with the known fgf requirement for the establishment of the tracheal scaffold during earlier developmental stages ( ghabrial et al . , 2003 ; uv et al . , 2003 ) , further attempts to manipulate fgf signaling or to detect fgf ligand expression and differential pathway activation under different nutritional conditions all failed to support a role for fgf signaling in coupling nutrition with larval tracheal growth ( data not shown ) . these included expression of btl , constitutively active btl , and its ligand branchless ( bnl ) in tracheal terminal cells and analysis of bnl and stumps ( a downstream signaling component ) expression under different nutritional conditions . we then turned our attention to the insulin signaling pathway : the major coordinator of nutrient intake and tissue size in all animals including drosophila ( andersen et al . , 2013 ) . we first suppressed the intracellular insulin signal transducer phosphoinositide 3-kinase ( pi3k ) by expressing the dominant - negative dp110 ( referred to as pi3k - dn ; leevers et al . , 1996 ) in tracheal terminal cells using dsrf - gal4 ( gervais and casanova , 2011 ) . this led to reduced tracheal terminal cell branching both in the periphery and throughout the digestive tract , but not in the cns ( figures 2a2f , s2s , s2 t , s3a s3d and data not shown ) : a reduction qualitatively and quantitatively comparable to that observed in these tracheal terminal cells following severe nutrient restriction ( figures 1g1i ) . as in the case of diet , the intestinal branches appeared to be more severely affected by this manipulation . because the selective intestinal phenotype was not caused by stronger gal4 expression in intestinal tracheae ( data not shown ) , we tested whether it resulted from increased sensitivity to insulin signaling . to this end , we made use of an rnai construct against the insulin receptor ( inr ) known to lead to incomplete receptor downregulation and a milder reduction in insulin signaling ( slaidina et al . 2011 ) . driving this rnai transgene in all tracheal terminal cells led to a significant reduction in intestinal , but not body wall or cns , tracheal coverage ( figures 2g2i , s3e , and s3f and data not shown ) . as in the case of dietary or pi3k manipulations , reduced coverage resulted from reduced tracheal terminal cell branching ( figures s2c , s2d , s2h s2j , and s2n s2p ) . together , these results confirm the cell - autonomous role for the insulin signaling pathway in the regulation of tracheal terminal cell growth and suggest that the enhanced nutritional plasticity of the gut tracheae is a consequence of their higher sensitivity to insulin signaling . in drosophila larvae , nutrient restriction leads to growth inhibition , caused by the reduced release of several insulin - like peptides ( ilps ) from brain insulin - producing cells ( the so - called median neurosecretory cells , mnscs , represented schematically in figure 3j ) into the hemolymph ( gminard et al . , 2009 ) . a triple mutation of the three main mnsc ilps ( ilp2 , ilp3 , and ilp5 ; grnke et al . , 2010 ) largely recapitulated the phenotype resulting from expression of pi3k - dn in tracheal terminal cells . indeed , reduced growth was observed in both body wall ( figures 3a and 3d ) and intestinal tracheal terminal cells ( figures 3b , 3e , and s4a s4d ) . however , we found the posterior hindgut tracheal branches to be spared in these larvae ( figures 3c and 3f ) . immunohistochemical and ultrastructural analyses of this intestinal portion revealed that these posterior tracheal branches were adjacent to the two hindgut nerves that run along both sides of the hindgut ( figures 3k , 3l , and 7b ) . we have previously shown that axons emanating from a different population of cns ilp - producing neurons , the ilp7 neurons , contribute to this innervation ( figure 3j ; miguel - aliaga et al . , 2008 ) and thus could provide a local peptide supply to this portion of the gut . functional inactivation of the ilp7 neurons by expression of the inward - rectifying potassium channel kir2.1 or by expression of tetanus toxin light chain did not affect most tracheae but led to reduced tracheal coverage of two portions of the hindgut ( figures 3g3i , s4e , and s4f and data not shown ) : the posterior hindgut ( figures 3c and 3i ) , where the ilp7 axons are adjacent to the posterior visceral tracheal branches ( figures 3k and 7b ) , but also the mid - hindgut ( figure s4f ) , which we had also found to be regulated by systemic mnsc - derived ilps ( figures s4b and s4d ) . in this latter region , the visceral tracheal branches emanate from the segmentally repeated main lateral branches ( figures 7a and 7b ) and do not abut the ilp7 axons , suggesting paracrine growth regulation . we then characterized the peptidergic profile of the central neurons contributing to the hindgut nerves using immunohistochemistry . four of the eight ilp7-expressing neurons coexpress pigment dispersing factor ( pdf ) ( figures 4a and 4b ) : a neuropeptide that shares functional and signaling similarities with vertebrate vasoactive intestinal polypeptide ( vip ) ( taghert and nitabach , 2012 ) . four other central hindgut - innervating neurons also express pdf and bundle together with the ilp7 hindgut nerves ( figure 4b ; talsma et al . , 2012 ) . together , both neuronal populations deliver pdf and ilp7 to the hindgut in a regionalized fashion : ilp7 is apparent only in the posterior hindgut , whereas pdf is present in both posterior and mid - hindgut terminals ( figures 4c and 7b ) . mutation of these peptides , alone or in combination , revealed complex control of different intestinal tracheal subsets by local ilp7 and pdf peptides in combination with the systemic ilp2 , ilp3 and ilp5 peptides ( figures 4d4u , s5a s5l , and 7b ) : in the posterior hindgut , neither loss of ilp7 alone nor ilp2 , ilp3 , and ilp5 together affected tracheal branching ( figures 4f , 4i , s5c , 3c , and 3f ) , but loss of all four peptides resulted in reduced tracheal terminal cell growth ( figures 4l and s5c ) , indicating partially redundant control of tracheal terminal growth . loss of ilp7 or pdf alone , or tracheal - specific downregulation of the pdf receptor ( dsrf - gal4 , uas - pdfr - rnai ) , resulted in reduced tracheal growth only in the mid - hindgut ( figures 4g4i , 4m4r and s5a s5i ) : a region also affected by the lack of systemic ilps ( figures s4b and s4d ) and not directly exposed to ilp7 peptide ( figure 7b ) . finally , mutants lacking both ilp7 and pdf displayed reduced tracheal growth in both the mid - hindgut and posterior hindgut ( figures 4 t , 4u , s5k , and s5l ) , indicating that ilp7 and pdf act redundantly in the posterior hindgut . collectively , neuropeptide mutation and tracheal receptor downregulation experiments indicate that growth of tracheal terminal cells is directly regulated by the nervous system . the systemically secreted ilps act as virtually pan - tracheal regulators , but in some intestinal portions they synergize in a combinatorial and sometimes partially redundant manner with locally delivered ilp and pdf neuropeptides . both mnscs and ilp7 neurons have been shown to modulate feeding responses to nutrient scarcity in adult flies ( cognigni et al . , 2011 ) . however , the dietary dependency of ilp release has only been investigated in mnscs using immunohistochemistry ( gminard et al . , , we expressed the genetically encoded green fluorescent ca indicator gcamp3 ( tian et al . , 2009 ) in ilp7 neurons , together with a red fluorescent protein to visualize the cell bodies . ilp7 cell bodies displayed some transient activity in the absence of a stimulus , which rapidly increased following yeast presentation ( figures 5a , 5c , s6a , and movie s1 ) . in most neurons , the frequency and amplitude of the transient ca peaks increased and then adapted after about one minute , possibly a consequence of persistent exposure to yeast . this response was yeast - specific because exposure to sucrose did not elicit any responses in these neurons ( data not shown ) , consistent with the yeast dependency of tracheal growth . it was also specific to ilp7 neurons , given that gcamp3 fluorescence intensity was unaffected by yeast in the capa - expressing va neurons , used as a control population of six unrelated peptidergic efferent neurons ( suska et al . , 2011 ) the only well - characterized environmental trigger of tracheal branching is hypoxia ( centanin et al . we therefore monitored oxygen - evoked ca responses in these two neuronal populations and found that hypoxia led to a fast and very robust response in the ilp7but not in the va neurons ( figures 5b , 5d , and s6b , and movie s2 ) . indeed , it was predominantly tonic , although some animals mainly showed transient peaks of increased amplitude , and lasted throughout the hypoxic period , decreasing slightly over time . interestingly , the return to normoxia almost completely abrogated the basal transient firing of ilp7 neurons , suggesting hyperpolarization . this effect was not a consequence of excessive firing and cellular exhaustion because repeated hypoxic stimulation continued to activate the ilp7 neurons ( figure s6c ) . together , these findings indicate that the activity of the ilp7- and pdf - producing neurons is increased in vivo by both nutritional cues and reductions in oxygen availability . together with previous ilp / pdf loss - of - function experiments , the above experiments suggested that nutritional modulation of ilp neuronal activity underlies the nutritional plasticity of tracheae . to test this idea , we used thermogenetics to achieve persistent , low - level activation of the ilp7 neurons throughout larval life by expressing the heat - sensitive channel trpa1 from ilp7-gal4 in larvae reared at 25c . this promoted tracheal branching in a paracrine fashion ; it increased branching of the adjacent visceral tracheal branch of the posterior hindgut and the tracheal terminal cells of the neighboring mid - hindgut , but did not redirect those of the anterior hindgut or other regions ( figures 5e and 5f , and data not shown ) . hence , in addition to being necessary , ilp7 neurons are sufficient to sustain tracheal growth in the hindgut . the finding that tracheal branching is directly regulated by nutrient - responsive neurons suggests that tracheal terminal cells may be used by the nervous system as effectors of metabolic adaptations to nutrient availability . to investigate this possibility , we recapitulated the differential effects of nutrient restriction on tracheae by either reducing tracheal terminal cell growth in all tissues ( except for the cns tracheae , using dsrf > btl - rnai ) , or specifically in the gut tracheae ( using dsrf > inr - rnai ) . reduced tracheation of all tissues did not affect larval development ( figure 6a ) or carbohydrate metabolism ( figures s7a s7c ) but resulted in leaner larvae ( figure 6c ) with reduced lipid stores ( figure 6d ) and increased hemolymph glycerol ( a metabolite derived from the hydrolysis of triglycerides ) ( figure 6e ) , consistent with reduced lipid storage capacity in the fat body . these larvae did manage to eclose as adults but were sick and short - lived even in the presence of nutritious food ( figure 6b and data not shown ) . by contrast , when reduced tracheation was confined to the gut , no developmental or metabolic phenotypes were apparent in larvae ( figures s7d s7h ) , and there was no difference in adult lifespan between the experimental flies and controls on nutritious food ( figure 6f ) . we then hypothesized that the specific effect of nutrient restriction on gut tracheae may fulfil an adaptive role to allow flies to deal with poor nutritional conditions . to test this idea , we exposed the dsrf > inr - rnai flies with reduced gut tracheation to a low - calorie diet throughout their adult lifetime , and found them to be significantly more resistant to nutrient scarcity than control flies : a tracheal phenotype that was confirmed using the recently published tracheal driver 14d03-gal4 ( guo et al . , 2013 ) metabolic profiling of these adult flies revealed no differences in carbohydrate metabolism but showed a reduction in lipid stores in poor nutritional conditions ( figures 6h , 6i , and s7i s7l ) . in summary , manipulations that recapitulate the effects of nutrient restriction and reduced insulin signaling specifically in tracheal terminal cells show that these cells are important metabolic mediators . our work has uncovered a new mechanism coupling nutrition and metabolism . in response to specific nutritional cues , small subsets of neurons are activated to regulate tracheal branching in an organ - specific and metabolically significant fashion . at least one of the two yeast - responsive neuronal subsets also responds to reduced oxygen the other environmental modulator of tracheal branching in flies so it will be interesting to determine the contribution of these neurons to the previously reported tracheal adaptations to hypoxia . importantly , our identification of a shared neuronal substrate for both nutritional and hypoxic stimuli is , to our knowledge , the first of its kind in invertebrates and one remarkably similar to the mammalian carotid body : a cluster of chemoreceptors that monitors arterial oxygen concentration and nutrient levels to regulate breathing and cardiovascular tone ( pardal and lpez - barneo , 2002 ; prabhakar , 2000 ) . future work will aim to establish whether these drosophila neurons are able to sense oxygen and/or nutrients directly and whether they do so using mechanisms akin to those described in the carotid body . this would lend further support to the existence of an evolutionarily conserved link between oxygen and nutrient neuronal sensing . molecularly , the neuronal control of different tracheal subsets involves both local and systemic actions of insulin- and vip - like neuropeptides : neuronal mechanisms that are particularly complex and combinatorial along the digestive tract ( figure 7 ) and that differ from the known adaptive target - derived signals that sculpt tissue - specific angiogenesis ( cao , 2007 ; fraisl et al . , 2009 ) . in this metabolic motor neurons ; as the nervous system modulates motor neuron activity to regulate muscle contraction , it also modulates the branching of tracheal terminal cells to control the metabolic state of cells such as those of the fat body or the gut epithelium . it will be of interest to investigate whether similar mechanisms are deployed in vertebrates to effect long - lasting , tissue - specific and metabolically significant changes in angiogenesis in response to nutrition , in a manner distinct from ( but reminiscent of ) the acute changes in blood supply effected by neurons by acting on blood vessel musculature ( see , for example , matheson et al . , 2000 ) . in drosophila , previous gain- and loss - of - function experiments had failed to reveal unique functions for most of the eight known ilps ( brogiolo et al . , 2001 ; grnke et al . , 2010 ) . the regional regulation of tracheal subsets hence provides one possible explanation for the apparent redundancy of the ilp gene family in drosophila : while all these ilps may indeed have the same function ( in this case , to modulate tracheal growth in response to nutrition ) , they may carry it out in different places for example , in the posterior hindgut in the case of ilp7 and in other parts of the digestive tract for ilp2 , ilp3 , and ilp5 . this regional control of tracheal growth may extend to other regions : gut visceral musculature and cns glia are known to activate ilp3 and ilp2/ilp6 gene expression respectively in a nutrient - dependent fashion ( chell and brand , 2010 ; obrien et al . the recent discovery that intestinal tracheae can regulate stem cell proliferation ( li et al . , 2013 ) , it is possible that local regulation of tracheal branching by ilps contributes to their reported action on intestinal or neuronal stem cell proliferation ( chell and brand , 2010 ; obrien et al . effects of insulin and vip on blood vessels have been described in vertebrates ( chaudhuri et al . indeed , although the effect of pdf on intestinal tracheal branching is unexpected in drosophila ( where this peptide is known for its central role in clock neurons ; taghert and nitabach , 2012 ) , neurally derived vip has a vasodilatory effect on the arterioles of small intestine and colon ( holzer , 2006 ) . however , the physiological significance of these ( largely ex vivo ) observations has not been entirely elucidated ( matheson et al . , 2000 ) . in contrast to this mode of regulation , involving acute modulation of endothelial muscle tone , the evidence for longer - lasting effects of these peptides on angiogenesis which would be more akin to their action on the drosophila tracheal system is more tenuous and often contradictory ( see , for example , ogasawara et al . , 1999 ; ribatti et al . , 2007 ) . our findings suggest that their effects may have been underestimated because they act in partially redundant fashion and in response to specific nutritional cues . mechanistically , it has been proposed that the vertebrate peptides regulate proangiogenic target - derived signals . by contrast , our tracheae - specific receptor downregulation experiments clearly indicate that these peptides can act directly on the tracheal cells , so it will be of interest to establish whether both modes of action contribute to their effects on vertebrate angiogenesis . in drosophila , whole - organism manipulations of insulin signaling such as ablation of insulin - producing cells or ilp mutation result in both slower development and diabetic phenotypes , highlighting their dual insulin / igf - like role ( grnke et al . , 2010 ; strikingly , downregulation of insulin signaling only in one cellular target the tracheal terminal cells uncouples the developmental from the metabolic phenotypes of these peptides , thus identifying the tracheal system as an important and previously unrecognized metabolic target of insulin signaling in the fly . hence , the tracheal involvement in previously reported insulin - modulated phenotypes , such as lifespan or resistance to oxidative stress ( grnke et al . , 2010 ) , deserves further investigation . interestingly , a pan - tracheal reduction in insulin signaling results in normal carbohydrate metabolism but leads to reduced adiposity . this is suggestive of abnormal lipid metabolism in the fat body and is consistent with the recent finding that reduced fat tissue vascularity leads to fat mass reduction without affecting glucose homeostasis in young mice ( sung et al . , 2013)although in both mice and flies this phenotype may eventually prove to be deleterious ( sung et al . reduced adiposity is a phenotype that , although also consistent with one of the classic symptoms of type i diabetes in humans , had not previously been observed in flies with a ubiquitous reduction in insulin signaling or lacking the systemic ilp peptides ( puzzlingly , these flies were actually found to accumulate triglyceride ; bhni et al . , 1999 ; grnke et al . , 2010 ) . we suggest that this increased adiposity may have been secondary to the igf - like effects of ilps on developmental time , and only by uncoupling these developmental from the metabolic effects of ilps , as we have done with the tracheal - specific reduction of insulin signaling , can some of the true insulin - like phenotypes of ilps be unmasked . we have also found that subtle changes in insulin signaling or in the nutritional content of the fly s diet ( some of which are within the range of those normally found in diets used for fly rearing in different labs ) have a striking effect on an unexpected tracheal population : that of the digestive tract . it will be of interest to explore the cellular mechanisms underlying their differential sensitivity . these might result from differences in receptor levels or composition the ret - like receptor tyrosine kinase stitcher , recently shown to synergize with inr in mitotic tissues , is a possible candidate ( ofarrell et al . , 2013 ) . alternatively , it could be caused by differences in downstream signaling components such as foxo , which has been shown to account for some organ - specific responses ( tang et al . , 2011 ) . functionally , by uncovering gut - specific effects of tracheation on adult survival and lipid mobilization upon nutrient scarcity , we have identified the tracheal system as a possible anatomical substrate for the previously reported effects of nutrient acquisition during developmental and growth periods on a variety of adult features ( foley and luckinbill , 2001 ; zwaan et al . , 1991 ) . enterocytes would appear to be the obvious cellular mediators of these effects ; changes in oxygen supply may modulate the metabolic state of these absorptive cells , and long - term adaptations to nutrient scarcity may result from differential nutrient absorption and/or utilization . however , enterocytes need not be the only intestinal targets of the nutrient - driven tracheal changes : the tracheal regulation of stem cell proliferation described above ( li et al . , 2013 ) consistent with this idea , there is correlative as well as ( more limited ) functional data implicating neuronal factors in the regulation of angiogenesis in tumor environments ( jang et al . , 2000 ; madden et al . , 2011 ; toda et al . , furthermore , oxygen need not be the sole mediator of the gut tracheae - driven adaptations : li et al . ( 2013 ) also found that tracheae produce dpp , an important tgf-like signaling molecule . in future , it will be of interest to explore not only these intestinal targets , but also whether the intestinal tracheal plasticity is more widely regulated by other environmental stimuli such as gut epithelial infection or damage . from a more translational perspective , most studies of adaptive angiogenesis in vertebrates have focused on the adipose vasculature ( cao , 2010 ; lijnen , 2008 ) . in light of our drosophila findings , it will be of interest to explore the nutritional plasticity of the gastrointestinal vasculature , as well as its contribution to pathologies such as obesity or to the metabolic improvements following gastric bypass interventions . tracheae were imaged and blindly scored using dic optics ( see extended experimental procedures for details ) . quantifications were performed as follows : the stereotypical endings of the third dorsal branch , directly posterior to the large tracheal commissure on the third segment , were counted as described in ( centanin et al . , 2008 ) . tracheal coverage was quantified in the vnc a relatively flat tissue with well - defined anatomical boundaries as the ratio between the total length of tracheal arbours ( which are complex and nonstereotypical ) divided by total vnc area ( m/m ) . tracheal length was measured using a custom - written imagej macro ( schneider et al . , 2012 ) . after median filtering ( radius = 3 pixels ) to reduce image noise , a polygonal region of interest ( roi ) was manually drawn to mark the tissue area . following background subtraction to enhance the visibility of tracheae , the image was segmented and the tracheal area within the roi was measured . in the mid - hindgut , where the tissue surface and three - dimensional properties allowed semiautomated quantification , the same procedure as for the vnc was used , but the segmented image was subsequently skeletonized . parts of gut tissue wrongly identified as tracheae or segments of the tracheal tree missed by the program were manually edited before counting the total number of pixels in the skeletonized tracheal tree . in other intestinal portions , where the ruggedness and/or bends and twists of the target tissue made semiautomated quantification impractical , tracheal coverage was blindly scored using likert - type scales ranging from no difference to wild - type ( 3 ) to strongly increased ( 5 ) or strongly reduced ( 1 ) ( see figure 1 legend for color coding of displays ) . the validity of this scoring system was confirmed in the body wall and mid - hindgut , where likert - quantified scores were comparable to those obtained by counting or by semiautomated quantification respectively ( data not shown ) . likert rank data were displayed as the mean ( circled ) on diverging stacked bar charts , with the percentage of samples assigned to each likert rank reflected in the length of each differently colored segment . we refer to extended experimental procedures for details of statistical analyses , fly stocks , diets , and more standard methods ( immunohistochemistry , transmission electron microscopy , metabolic assays , survival assays , developmental rate and size quantifications , and in vivo recordings of neuronal activity ) . extended experimental proceduresfly stocks and rearingthe following fly stocks were used : dsrf - gal4 ( gervais and casanova , 2011 ) , uas - pi3k - dn ( uas - dp110 ; leevers et al . , 1996 ) , uas - inr - rnai ( vdrc stock 992 ) , uas - btl - rnai ( vdrc stock 110277 ) , ilp7 , ilp2,3,5 and ilp2,3,5,7 mutants ( grnke et al . , 2010 ) , btl - gal4 ( shiga et al . , 1996 ) , uas - cd8-gfp ( lee and luo , 1999 ) , ilp7-gal4 ( yang et al . , 2008 ) , uas - kir2.1 ( baines et al . , 2001 ) , pdf mutants ( renn et al . , 1999 ) , uas - pdfr - rnai ( trip stock hms01815 ) , va - gal4 ( suska et al . , 2011 ) , uas - gcamp3 ( tian et al . , 2009 ) , uas - myr - mrfp ( hop b2 , following re - mobilization of the original hop contributed to the bloomington stock center by h. chang ) , va - gal4 ( allan et al . , 2003 ) and uas - trpa1 ( hamada et al . , 2008 ) . oregon r ( orer ) were used as wild - type flies . in genetic experiments involving the gal4-uas system , two parental controls were used : the gal4 driver and the uas drivers separately crossed to w flies . an isogenic w stock was used as control for the experiments involving the pdf mutant , and all ilp mutants were backcrossed for at least 8 generations to a large population of w flies , which was used as a control strain in all the ilp mutant experiments . all crosses and experiments were kept at 25c , including those designed to achieve persistent , low - level activation of the ilp7 neurons by expression of trpa1.to obtain larvae of the desired developmental stage , a small number of flies ( less than 10 virgin females ) were housed in embryo collection cages with apple juice plates topped with abundant yeast ( see section below ) . following frequent plate changes , eggs / larvae were allowed to develop until the desired stage depending on the experiment , but they were typically screened at 72h after egg laying ( ael ) to control for developmental delay and discard larvae that had not reached 3-instar stage . at this point , larvae were transferred to fresh plates in controlled numbers ( 25 per plate ) to avoid nutrient restriction resulting from overcrowding.for the experiments requiring adult flies , an equal volume of eggs ( 29 l , corresponding to ca . 200 eggs ) was squirted into bottles to control for the density of developing larvae and synchronize the emergence of adults . adult flies were maintained in vials at low densities ( 1015 flies per vial ) and were transferred to fresh food vials every 23 days.dietsour stocks are normally reared on a standard cornmeal / agar diet ( 5.5% cornmeal , 6% dextrose , 1.3% yeast , 0 . 55% agar supplemented with 0.18% nipagin and 2.9 ml / l propionic acid ) . for most larval experiments , larvae were raised and staged under nutrient - rich conditions using apple juice plates ( 2.3% agar , 2.3% dextrose , 25% apple juice in dh2o ) with abundant baker s yeast ( prepared fresh every time using 5 g of yeast and 10 ml dh2o ) . for the experiments assessing the effect of yeast concentration on larval tracheal growth ( figure 1 ) , the standard cornmeal / agar diet above was used with the following three yeast concentrations : 8% ( nutritious ) , 2% ( mild nutrient restriction ) or 0.8% ( severe nutrient restriction ) . these same diets were used to assess the effect of larval diet on adult tracheation ; control flies ( figure 1j ) were raised and maintained in the nutritious cornmeal / agar diet with 8% yeast , whereas flies that were nutrient restricted as larvae ( figure 1k ) were raised in the cornmeal / agar diet with 0.8% yeast , and then were switched to the nutritious cornmeal / agar diet with 8% yeast after eclosion.for the adult survival or metabolic experiments , flies of the different genotypes were reared and aged for 1 day in standard food , and were then transferred to a more defined diet with the same sugar / yeast ratio ( 60%/40% ) but differing nutritional content : nutritious ( 5.4% sucrose and 3.6% yeast in 1% agar , 0.18% nipagin ) , or restricted ( 0.9% sucrose and 0.6% yeast in 1% agar , 0.18% nipagin ) . metabolic assays were conducted as described below 7 days after feeding on either diet.visualization and scoring of tracheal growthfor visualization of tracheae using dic optics , larval or adult tissues were dissected in 100% glycerol and were immediately mounted and used to quantify / image phenotypes using a zeiss axioplan equipped with a leica dfc425c camera . tracheal coverage was measured in specific regions of the gut , brain or body wall . the criterion for defining larval gut regions was typically their proximity to an obvious landmark : e.g. , the gastric caeca ( anterior midgut ) or the malpighian tubules ( posterior midgut ) . gut and brain tracheae were scored in early 3-instar larvae ( 72 hr ael ) , whereas body wall tracheae were scored in mid-3instar larvae as previously done by ( centanin et al . , 2008 ) . in the cases where it was necessary to correct for developmental delay ( ilp2,3,5 mutants and severe nutrient restriction ) , the developmental delay was first monitored and found to be ca . egg collections and transfers to fresh plates were thus carried out 24 hr earlier , so as to obtain 3-instar larvae at the same time as controls . when comparing genotypes , mutants and controls were mounted and processed on the same slides . in cns images , anterior is to the top . in body wall images , anterior is to the top and the right dorsal terminal branch is always shown . in all gut images , anterior ( oral ) is to the left.tracheal scoring was performed as described in the experimental procedures . tracheal coverage data were tested for normality using the shapiro - wilk test . because most distributions were found to be nonnormal , coverage data were analyzed using nonparametric statistics . the wilcoxon rank - sum test was used for pairwise comparisons , and was preceded by a kruskal - wallis test if two parental controls were analyzed . multiple comparisons were performed with the kruskal - wallis test and the wilcoxon test with the benjamini and hochberg / yekutieli procedure for controlling false discovery rate . in all figures , significance levels are indicated with asterisks as follows : p < 0.05 , p < 0.01 , p < 0.001 .developmental rate and size quantificationsto assess developmental rate , 4 hr lays were collected and aged as described in the fly rearing section . by 72 hr ael , the percentage of larvae reaching the 3-instar stage ( as assessed by the anatomy of their mouth hooks and anterior spiracles ) was comparable across genotypes . these 3-instar larvae were transferred to fresh apple juice plates with yeast in groups of 10 , and the emergence of pupae was scored hourly . statistical analysis was performed using survival statistics ( harrington and fleming , 1982).the size of mid-3-instar larvae ( 96 hr ael ) larvae was determined following their incubation in a 60c water bath for 20 min , which relaxed all muscles . the dead larvae were immediately imaged in groups of three with a leica mz16f equipped with a dfc420c camera . size measurements were done semi - automatically using imagej.survival assaysupon eclosion , experimental flies reared in noncrowding conditions were collected daily and allowed to mate for a further 24 hr to avoid multiple matings . the flies were then separated into males and females and were kept as described in the fly rearing section ( see extended experimental procedures ) . male and female survival data were analyzed separately as described in ( harrington and fleming , 1982 ) . since no differences between males and females were found , only the data for male flies is shown in figure 3.metabolic assaysfor whole - animal measurements , metabolic assays were performed on fresh ( not previously frozen ) 96 hr ael larvae or adult males ( previously frozen in liquid nitrogen ) . groups of 10 adult males or larvae ( previously washed in dh20 , then briefly dried on tissue paper ) were put in a 2 ml eppendorf tube containing a steel bead and 500 l ( or 200 l for adult tag assays ) grinding buffer ( pbs for carbohydrate assays and pbs with 0.05% tween for the tag assays ) . grinding was performed twice for 1 min at 30hz using a qiagen tissuelyserii , after which the samples were immediately incubated at 70c for 5 min . for the carbohydrate assays , the samples were centrifuged for 3 min at 13,000 g. for each sample , 25 l of supernatant was added to 25 l pbs , pbs with amyloglucosidase or pbs with trehalase for carbohydrate digestion in 96-well plates . 10 l were then transferred to 100 l of glucose assay reagent ( sigma ) or protein quantification kit rapid ( sigma ) . for tag assays , 25 l of each sample were added to 25 l pbs or triglyceride reagent ( sigma ) . following a 30 min incubation at 37c , samples were centrifuged for 3 min at 13,000 g , and 10 l of each sample were transferred to individual wells in 96-well plates containing 100 l of free glycerol reagent ( sigma ) or protein quantification kit absorbance was read according to manufacturer s description using a perkin elmer wallac victor 1420 and a bio - rad model 680 microplate reader . concentration values were calculated using standard curves , and the protein values were used to normalize the tag and sugar assays.hemolymph measurements were performed in a similar fashion . groups of 10 cleaned and dried larvae were torn open with forceps on a clean glass slide . to measure trehalose , 2 l of hemolymph were diluted in 38 l of pbs and the mix was immediately heated to 70c . following a 10 min centrifugation step at 13,000 g , 10 l were added to 30 l of pbs or pbs with trehalase . the remaining steps were performed as described above for the whole - animal assays without the protein normalization . to measure hemolymph free glycerol , hemolymph was extracted as described for trehalose and 2 l were diluted in 10 l pbs . samples were then heated and centrifugated in an identical fashion , and 10 l of each sample were added to single wells in a 96-well plate containing 100 l of free glycerol reagent ( sigma).all metabolic data were tested for normality using the shapiro - wilk test . because most distributions were found to be nonnormal , data were analyzed using nonparametric statistics . the wilcoxon rank - sum test was used for pairwise comparisons , and was preceded by a kruskal - wallis test if two parental controls were analyzed . in all figures , significance levels are indicated with asterisks as follows : p < 0.05 , p < 0.01 , p < 0.001 .immunohistochemistrylarval guts were dissected in pbs and fixed in 4% paraformaldehyde for 20 min . subsequent washes and incubations were done in pbs with 0.2% triton . tissues were incubated overnight with primary antibody at 4c , followed by a 2 hr incubation with secondary antibodies at room temperature the next day . the following antibodies were used : mouse -futsch ( 22c10 , developmental studies hybridoma bank ; 1:10 ) , goat -green fluorescent protein ( gfp ) ( abcam , 1:2,000 ) , rabbit -ilp7 ( miguel - aliaga et al . , 2008 ; 1:5,000 ) , mouse anti - pdf propeptide ( pdf c7 , developmental studies hybridoma bank ; 1:50 ) and mouse anti - dsrf antibody ( active motif , 1:10 ) . fitc- , cy3- , and cy5-conjugated secondary antibodies were obtained from jackson immunolabs and used at 1:200 ( 1:100 for the cy5-conjugated antibody ) . fluorescent preparations were mounted in vectashield with dapi ( vector labs ) to visualize all nuclei . images were acquired using a leica sp5 confocal microscope.transmission electron microscopytransmission electron microscopy was performed using standard techniques . briefly , guts were dissected and fixed overnight at 4c in 2.5% glutaraldehyde in 0.1 m phosphate buffer . the next day the guts were postfixed for 30 min in 1% osmium tetroxide in 2% glutaraldehyde in 0.1 m phosphate buffer , and for a further 1 hr in 2% osmium tetroxide . guts were then bulk - stained in 2% uranylacetate and embedded in araldite after dehydration steps . ultrathin sections were cut using a leica uc6 ultramicrotome ( leica microsystems ) mounted on 100-mesh copper grids . these sections were double - stained for 10 min with 2% uranylacetate in 70% methanol , followed by 5 min lead citrate in water . imaging was performed using a fei - tecnai 12 biotwin ( 100 kv , tungsten filament ) and a f214a digital camera.in vivo recordings of neuronal activityimmobilized 72 hr 3rd - instar larvae expressing uas - gcamp3 , uas - myr - mrfp from either ilp7-gal4 or va - gal4 reared on apple juice plates with yeast were used for these experiments . ca imaging of nutrient and oxygen responses was performed essentially as described for c. elegans worms ( busch et al . , 2012 ) . briefly , larvae were immobilized on agar pads ( 2% in h2o ) with dermabond tissue adhesive ( ethicon ) , and were individually housed by placing a 6x6 mm wide and 0.2 mm deep polydimethylsiloxane microfluidics chamber over them . for the hypoxia experiments , humidified gas mixtures ( boc ) of 21% or 1% o2 ( balance n2 ) were continuously delivered to the microfluidics device at 2.5 ml min-1 from a syringe pump . teflon valves ( automate scientific ) were used to rapidly switch between the two gas streams every 120 s , as described in ( busch et al . , 2012 ) . for the nutrition experiments , circa 2 min after the start of each recording , larvae were presented with a suspension of liquid yeast ( 1 g:3 ml in dh2o ) or 9% sucrose in dh20 , pumped in through pe50 polyethylene tubing ( bd intramedic ) from a syringe . the time when the yeast reached the chamber was manually recorded and was used to realign traces to compare and average the responses of different larvae . ilp7 or va neurons were recorded with a 100 ms exposure time in 0.5 s intervals on an inverted compound microscope ( axiovert , zeiss ) , using a 2.0 neutral density filter , a gfp / dsred emission filter set ( chroma ) , a 40x c - apochromat lens ( zeiss ) , a dual - view beamsplitter with gfp / dsred emission filters ( photometrics ) and a cascade ii 512 emccd camera ( photometrics ) . the fluorescence intensity of individual neurons was tracked over time using a region of interest in both the gcamp and rfp channels with custom - written software in matlab ( mathworks ) . from each region of interest , the 20 brightest pixels were selected , median pixel intensity was subtracted for each channel separately , and the gcamp / rfp fluorescence ratio was then determined to normalize the gcamp signal for z axis drift . the following fly stocks were used : dsrf - gal4 ( gervais and casanova , 2011 ) , uas - pi3k - dn ( uas - dp110 ; leevers et al . , 1996 ) , uas - inr - rnai ( vdrc stock 992 ) , uas - btl - rnai ( vdrc stock 110277 ) , ilp7 , ilp2,3,5 and ilp2,3,5,7 mutants ( grnke et al . , 2010 ) , btl - gal4 ( shiga et al . , 1996 ) , uas - cd8-gfp ( lee and luo , 1999 ) , ilp7-gal4 ( yang et al . , 2008 ) , uas - kir2.1 ( baines et al . , 2001 ) , pdf mutants ( renn et al . , 1999 ) , uas - pdfr - rnai ( trip stock hms01815 ) , va - gal4 ( suska et al . , 2011 ) , uas - gcamp3 ( tian et al . , 2009 ) , uas - myr - mrfp ( hop b2 , following re - mobilization of the original hop contributed to the bloomington stock center by h. chang ) , va - gal4 ( allan et al . , 2003 ) and uas - trpa1 ( hamada et al . , 2008 ) . oregon r ( orer ) were used as wild - type flies . in genetic experiments involving the gal4-uas system , two parental controls were used : the gal4 driver and the uas drivers separately crossed to w flies . an isogenic w stock was used as control for the experiments involving the pdf mutant , and all ilp mutants were backcrossed for at least 8 generations to a large population of w flies , which was used as a control strain in all the ilp mutant experiments . all crosses and experiments were kept at 25c , including those designed to achieve persistent , low - level activation of the ilp7 neurons by expression of trpa1 . to obtain larvae of the desired developmental stage , a small number of flies ( less than 10 virgin females ) were housed in embryo collection cages with apple juice plates topped with abundant yeast ( see section below ) . following frequent plate changes , eggs / larvae were allowed to develop until the desired stage depending on the experiment , but they were typically screened at 72h after egg laying ( ael ) to control for developmental delay and discard larvae that had not reached 3-instar stage . at this point , larvae were transferred to fresh plates in controlled numbers ( 25 per plate ) to avoid nutrient restriction resulting from overcrowding . for the experiments requiring adult flies , an equal volume of eggs ( 29 l , corresponding to ca . 200 eggs ) was squirted into bottles to control for the density of developing larvae and synchronize the emergence of adults . adult flies were maintained in vials at low densities ( 1015 flies per vial ) and were transferred to fresh food vials every 23 days . our stocks are normally reared on a standard cornmeal / agar diet ( 5.5% cornmeal , 6% dextrose , 1.3% yeast , 0 . 55% agar supplemented with 0.18% nipagin and 2.9 ml / l propionic acid ) . for most larval experiments , larvae were raised and staged under nutrient - rich conditions using apple juice plates ( 2.3% agar , 2.3% dextrose , 25% apple juice in dh2o ) with abundant baker s yeast ( prepared fresh every time using 5 g of yeast and 10 ml dh2o ) . for the experiments assessing the effect of yeast concentration on larval tracheal growth ( figure 1 ) , the standard cornmeal / agar diet above was used with the following three yeast concentrations : 8% ( nutritious ) , 2% ( mild nutrient restriction ) or 0.8% ( severe nutrient restriction ) . these same diets were used to assess the effect of larval diet on adult tracheation ; control flies ( figure 1j ) were raised and maintained in the nutritious cornmeal / agar diet with 8% yeast , whereas flies that were nutrient restricted as larvae ( figure 1k ) were raised in the cornmeal / agar diet with 0.8% yeast , and then were switched to the nutritious cornmeal / agar diet with 8% yeast after eclosion . for the adult survival or metabolic experiments , flies of the different genotypes were reared and aged for 1 day in standard food , and were then transferred to a more defined diet with the same sugar / yeast ratio ( 60%/40% ) but differing nutritional content : nutritious ( 5.4% sucrose and 3.6% yeast in 1% agar , 0.18% nipagin ) , or restricted ( 0.9% sucrose and 0.6% yeast in 1% agar , 0.18% nipagin ) . metabolic assays were conducted as described below 7 days after feeding on either diet . for visualization of tracheae using dic optics , larval or adult tissues were dissected in 100% glycerol and were immediately mounted and used to quantify / image phenotypes using a zeiss axioplan equipped with a leica dfc425c camera . tracheal coverage was measured in specific regions of the gut , brain or body wall . the criterion for defining larval gut regions was typically their proximity to an obvious landmark : e.g. , the gastric caeca ( anterior midgut ) or the malpighian tubules ( posterior midgut ) . gut and brain tracheae were scored in early 3-instar larvae ( 72 hr ael ) , whereas body wall tracheae were scored in mid-3instar larvae as previously done by ( centanin et al . , 2008 ) . in the cases where it was necessary to correct for developmental delay ( ilp2,3,5 mutants and severe nutrient restriction ) , the developmental delay was first monitored and found to be ca . egg collections and transfers to fresh plates were thus carried out 24 hr earlier , so as to obtain 3-instar larvae at the same time as controls . when comparing genotypes , mutants and controls were mounted and processed on the same slides . in cns images , anterior is to the top . in body wall images , anterior is to the top and the right dorsal terminal branch is always shown . in all gut images , anterior ( oral ) is to the left . tracheal coverage data were tested for normality using the shapiro - wilk test . because most distributions were found to be nonnormal , coverage data were analyzed using nonparametric statistics . the wilcoxon rank - sum test was used for pairwise comparisons , and was preceded by a kruskal - wallis test if two parental controls were analyzed . multiple comparisons were performed with the kruskal - wallis test and the wilcoxon test with the benjamini and hochberg / yekutieli procedure for controlling false discovery rate . in all figures , significance levels are indicated with asterisks as follows : p < 0.05 , p < 0.01 , p < 0.001 . to assess developmental rate , 4 hr lays were collected and aged as described in the fly rearing section . by 72 hr ael , the percentage of larvae reaching the 3-instar stage ( as assessed by the anatomy of their mouth hooks and anterior spiracles ) was comparable across genotypes . these 3-instar larvae were transferred to fresh apple juice plates with yeast in groups of 10 , and the emergence of pupae was scored hourly . the size of mid-3-instar larvae ( 96 hr ael ) larvae was determined following their incubation in a 60c water bath for 20 min , which relaxed all muscles . the dead larvae were immediately imaged in groups of three with a leica mz16f equipped with a dfc420c camera . experimental flies reared in noncrowding conditions were collected daily and allowed to mate for a further 24 hr to avoid multiple matings . the flies were then separated into males and females and were kept as described in the fly rearing section ( see extended experimental procedures ) . male and female survival data were analyzed separately as described in ( harrington and fleming , 1982 ) . since no differences between males and females were found , only the data for male flies is shown in figure 3 . for whole - animal measurements , metabolic assays were performed on fresh ( not previously frozen ) 96 hr ael larvae or adult males ( previously frozen in liquid nitrogen ) . groups of 10 adult males or larvae ( previously washed in dh20 , then briefly dried on tissue paper ) were put in a 2 ml eppendorf tube containing a steel bead and 500 l ( or 200 l for adult tag assays ) grinding buffer ( pbs for carbohydrate assays and pbs with 0.05% tween for the tag assays ) . grinding was performed twice for 1 min at 30hz using a qiagen tissuelyserii , after which the samples were immediately incubated at 70c for 5 min . for the carbohydrate assays , the samples were centrifuged for 3 min at 13,000 g. for each sample , 25 l of supernatant was added to 25 l pbs , pbs with amyloglucosidase or pbs with trehalase for carbohydrate digestion in 96-well plates . 10 l were then transferred to 100 l of glucose assay reagent ( sigma ) or protein quantification kit rapid ( sigma ) . for tag assays , 25 l of each sample were added to 25 l pbs or triglyceride reagent ( sigma ) . following a 30 min incubation at 37c , samples were centrifuged for 3 min at 13,000 g , and 10 l of each sample were transferred to individual wells in 96-well plates containing 100 l of free glycerol reagent ( sigma ) or protein quantification kit rapid ( sigma ) . absorbance was read according to manufacturer s description using a perkin elmer wallac victor 1420 and a bio - rad model 680 microplate reader . concentration values were calculated using standard curves , and the protein values were used to normalize the tag and sugar assays . groups of 10 cleaned and dried larvae were torn open with forceps on a clean glass slide . to measure trehalose , 2 l of hemolymph were diluted in 38 l of pbs and the mix was immediately heated to 70c . following a 10 min centrifugation step at 13,000 g , 10 l were added to 30 l of pbs or pbs with trehalase . the remaining steps were performed as described above for the whole - animal assays without the protein normalization . to measure hemolymph free glycerol , hemolymph was extracted as described for trehalose and 2 l were diluted in 10 l pbs . samples were then heated and centrifugated in an identical fashion , and 10 l of each sample were added to single wells in a 96-well plate containing 100 l of free glycerol reagent ( sigma ) . all metabolic data were tested for normality using the shapiro - wilk test . because most distributions were found to be nonnormal , data were analyzed using nonparametric statistics . the wilcoxon rank - sum test was used for pairwise comparisons , and was preceded by a kruskal - wallis test if two parental controls were analyzed . in all figures , significance levels are indicated with asterisks as follows : p < 0.05 , p < 0.01 , p < 0.001 . larval guts were dissected in pbs and fixed in 4% paraformaldehyde for 20 min . tissues were incubated overnight with primary antibody at 4c , followed by a 2 hr incubation with secondary antibodies at room temperature the next day . the following antibodies were used : mouse -futsch ( 22c10 , developmental studies hybridoma bank ; 1:10 ) , goat -green fluorescent protein ( gfp ) ( abcam , 1:2,000 ) , rabbit -ilp7 ( miguel - aliaga et al . , 2008 ; 1:5,000 ) , mouse anti - pdf propeptide ( pdf c7 , developmental studies hybridoma bank ; 1:50 ) and mouse anti - dsrf antibody ( active motif , 1:10 ) . fitc- , cy3- , and cy5-conjugated secondary antibodies were obtained from jackson immunolabs and used at 1:200 ( 1:100 for the cy5-conjugated antibody ) . fluorescent preparations were mounted in vectashield with dapi ( vector labs ) to visualize all nuclei . briefly , guts were dissected and fixed overnight at 4c in 2.5% glutaraldehyde in 0.1 m phosphate buffer . the next day the guts were postfixed for 30 min in 1% osmium tetroxide in 2% glutaraldehyde in 0.1 m phosphate buffer , and for a further 1 hr in 2% osmium tetroxide . guts were then bulk - stained in 2% uranylacetate and embedded in araldite after dehydration steps . ultrathin sections were cut using a leica uc6 ultramicrotome ( leica microsystems ) mounted on 100-mesh copper grids . these sections were double - stained for 10 min with 2% uranylacetate in 70% methanol , followed by 5 min lead citrate in water . imaging was performed using a fei - tecnai 12 biotwin ( 100 kv , tungsten filament ) and a f214a digital camera . immobilized 72 hr 3rd - instar larvae expressing uas - gcamp3 , uas - myr - mrfp from either ilp7-gal4 or va - gal4 reared on apple juice plates with yeast were used for these experiments . ca imaging of nutrient and oxygen responses was performed essentially as described for c. elegans worms ( busch et al . , 2012 ) . briefly , larvae were immobilized on agar pads ( 2% in h2o ) with dermabond tissue adhesive ( ethicon ) , and were individually housed by placing a 6x6 mm wide and 0.2 mm deep polydimethylsiloxane microfluidics chamber over them . for the hypoxia experiments , humidified gas mixtures ( boc ) of 21% or 1% o2 ( balance n2 ) were continuously delivered to the microfluidics device at 2.5 ml min-1 from a syringe pump . teflon valves ( automate scientific ) were used to rapidly switch between the two gas streams every 120 s , as described in ( busch et al . , 2012 ) . for the nutrition experiments , circa 2 min after the start of each recording , larvae were presented with a suspension of liquid yeast ( 1 g:3 ml in dh2o ) or 9% sucrose in dh20 , pumped in through pe50 polyethylene tubing ( bd intramedic ) from a syringe . the time when the yeast reached the chamber was manually recorded and was used to realign traces to compare and average the responses of different larvae . ilp7 or va neurons were recorded with a 100 ms exposure time in 0.5 s intervals on an inverted compound microscope ( axiovert , zeiss ) , using a 2.0 neutral density filter , a gfp / dsred emission filter set ( chroma ) , a 40x c - apochromat lens ( zeiss ) , a dual - view beamsplitter with gfp / dsred emission filters ( photometrics ) and a cascade ii 512 emccd camera ( photometrics ) . the fluorescence intensity of individual neurons was tracked over time using a region of interest in both the gcamp and rfp channels with custom - written software in matlab ( mathworks ) . from each region of interest , the 20 brightest pixels were selected , median pixel intensity was subtracted for each channel separately , and the gcamp / rfp fluorescence ratio was then determined to normalize the gcamp signal for z axis drift .

summaryduring adaptive angiogenesis , a key process in the etiology and treatment of cancer and obesity , the vasculature changes to meet the metabolic needs of its target tissues . although the cues governing vascular remodeling are not fully understood , target - derived signals are generally believed to underlie this process . here , we identify an alternative mechanism by characterizing the previously unrecognized nutrient - dependent plasticity of the drosophila tracheal system : a network of oxygen - delivering tubules developmentally akin to mammalian blood vessels . we find that this plasticity , particularly prominent in the intestine , drives rather than responds to metabolic change . mechanistically , it is regulated by distinct populations of nutrient- and oxygen - responsive neurons that , through delivery of both local and systemic insulin- and vip - like neuropeptides , sculpt the growth of specific tracheal subsets . thus , we describe a novel mechanism by which nutritional cues modulate neuronal activity to give rise to organ - specific , long - lasting changes in vascular architecture .

Introduction Results Discussion Experimental Procedures

unlike the more stereotypical development of the body s main blood vessels , the formation of the capillary networks responsible for tissue perfusion is an adaptive process primarily governed by the metabolic needs of the target tissues ( fraisl et al . , 2009 ; the plastic nature of this adaptive angiogenesis is further highlighted by the dramatic changes in vascularization observed in tumors or in obese adipose tissue : changes that contribute to the progression of pathologies such as cancer and obesity and are becoming increasingly central to their treatment ( cao , 2010 ; kerbel , 2008 ; lijnen , 2008 ) . although environmental factors such as diet are widely believed to affect the development and progression of these pathologies , exploration of the link between nutrition and angiogenesis has largely been confined to correlative studies . however , whether modulation of angiogenesis is associated with metabolic benefits remains a controversial topic , partly because it is not trivial to genetically target the blood vessels of specific organs to recapitulate the changes associated with certain dietary interventions without affecting other cell types or vascular pools ( cao , 2010 ; lijnen , 2008 ; sun et al . regardless of its metabolic consequences , adaptive angiogenesis is widely believed to be mechanistically driven by target - derived signals ( cao , 2007 ; fraisl et al . , a close spatial association between mammalian nerves and vessels was observed as long ago as 1543 ( vesalius , 1543 ) , an association that has subsequently been shown to result from mutual guidance or common pathfinding mechanisms during the formation of the neural and vascular networks ( carmeliet and tessier - lavigne , 2005 ; mukouyama et al . , 2011 ; the question remains whether , in a reciprocal process , neuronal activity may affect adaptive angiogenesis . , 1999 ) , no neuronal populations have been identified that effect long - lasting changes in angiogenesis in response to environmental factors . drosophila melanogaster has an open circulation , but its tracheal system has a role analogous to that of the vertebrate vasculature in supplying tissues and internal organs with oxygen ( fraisl et al . , 2009 during embryogenesis , developmental mechanisms akin to those discovered in the vertebrate lung and vasculature make use of signaling pathways such as fibroblast growth factor ( fgf ) signaling to sculpt this complex tracheal network of interconnected tubes ( ghabrial et al . growth is particularly prominent in the tracheal terminal cells : the cells at the end of each airway that make contact with target tissues and through which gas exchange takes place ( ghabrial et al . , 2008 this hypoxic remodeling has been assumed to be the only source of tracheal plasticity and , in normal conditions , the tracheal system is generally believed to grow in proportion to the whole organism . in this study , we use a combination of genetic approaches , metabolic profiling , and in vivo imaging to uncover previously unrecognized nutritional plasticity in the fly tracheal system . in contrast to the known target - derived mechanisms of adaptive remodeling , we find this plasticity to be regulated by a mechanism , previously undescribed in either flies or vertebrates , involving nutrient - responsive neurons effecting long - lasting and metabolically significant changes in tracheal architecture . while subjecting wild - type drosophila larvae to different dietary conditions , we observed that a severe reduction in dietary yeast ( the main source of lipid and amino acids in the larval diet ) was accompanied by an almost ubiquitous reduction in tracheal terminal cell branching , even when controlling for overall developmental delay by allowing nutrient - restricted larvae to develop to a comparable stage ( figures 1a1c , 1g1i and figures s1a , s1c , s1d , s1f , s1 g , s1i , s1j , s1l , s1 m , and s1o available online ) . by contrast , a mild reduction in dietary yeast neither affected developmental timing nor led to major changes in the size of organs or that of most tracheal terminal cells ( figures 1d and 1e and data not shown ) but did lead to a severe reduction in tracheal coverage throughout the digestive tract ( figures 1f , s1b , s1e , s1h , s1k , and s1n ) . to investigate the reversibility of the tracheal changes described above , we reared larvae under the mild nutrient restriction conditions shown to reduce intestinal tracheation and transferred them to more nutritious food immediately after eclosion . even after 7 days on a nutritious diet , the intestinal tracheae of these adults flies were significantly less branched than those of control adult flies always reared on a nutritious diet ( figures 1j and 1k ) , indicating that a defined period of nutrient restriction has long - term effects on the tracheal scaffold . as figure 1l shows , a 7 day nutritional imbalance ( 9% sucrose ) led to increased intestinal tracheation of the mid - midgut , confirming the dietary plasticity of the tracheal system also in adult flies . collectively , these data uncover previously unrecognized nutritional plasticity of the insect tracheal system , shaped by both previous and current nutritional states . hypoxia , the only known regulator of tracheal plasticity , has been shown to promote tracheal branching by inducing fgf ligand in target tissues and receptor upregulation in tracheal cells ( centanin et al . this led to reduced tracheal terminal cell branching both in the periphery and throughout the digestive tract , but not in the cns ( figures 2a2f , s2s , s2 t , s3a s3d and data not shown ) : a reduction qualitatively and quantitatively comparable to that observed in these tracheal terminal cells following severe nutrient restriction ( figures 1g1i ) . as in the case of diet , the intestinal branches appeared to be more severely affected by this manipulation . together , these results confirm the cell - autonomous role for the insulin signaling pathway in the regulation of tracheal terminal cell growth and suggest that the enhanced nutritional plasticity of the gut tracheae is a consequence of their higher sensitivity to insulin signaling . we have previously shown that axons emanating from a different population of cns ilp - producing neurons , the ilp7 neurons , contribute to this innervation ( figure 3j ; miguel - aliaga et al . functional inactivation of the ilp7 neurons by expression of the inward - rectifying potassium channel kir2.1 or by expression of tetanus toxin light chain did not affect most tracheae but led to reduced tracheal coverage of two portions of the hindgut ( figures 3g3i , s4e , and s4f and data not shown ) : the posterior hindgut ( figures 3c and 3i ) , where the ilp7 axons are adjacent to the posterior visceral tracheal branches ( figures 3k and 7b ) , but also the mid - hindgut ( figure s4f ) , which we had also found to be regulated by systemic mnsc - derived ilps ( figures s4b and s4d ) . four of the eight ilp7-expressing neurons coexpress pigment dispersing factor ( pdf ) ( figures 4a and 4b ) : a neuropeptide that shares functional and signaling similarities with vertebrate vasoactive intestinal polypeptide ( vip ) ( taghert and nitabach , 2012 ) . mutation of these peptides , alone or in combination , revealed complex control of different intestinal tracheal subsets by local ilp7 and pdf peptides in combination with the systemic ilp2 , ilp3 and ilp5 peptides ( figures 4d4u , s5a s5l , and 7b ) : in the posterior hindgut , neither loss of ilp7 alone nor ilp2 , ilp3 , and ilp5 together affected tracheal branching ( figures 4f , 4i , s5c , 3c , and 3f ) , but loss of all four peptides resulted in reduced tracheal terminal cell growth ( figures 4l and s5c ) , indicating partially redundant control of tracheal terminal growth . loss of ilp7 or pdf alone , or tracheal - specific downregulation of the pdf receptor ( dsrf - gal4 , uas - pdfr - rnai ) , resulted in reduced tracheal growth only in the mid - hindgut ( figures 4g4i , 4m4r and s5a s5i ) : a region also affected by the lack of systemic ilps ( figures s4b and s4d ) and not directly exposed to ilp7 peptide ( figure 7b ) . collectively , neuropeptide mutation and tracheal receptor downregulation experiments indicate that growth of tracheal terminal cells is directly regulated by the nervous system . in most neurons , the frequency and amplitude of the transient ca peaks increased and then adapted after about one minute , possibly a consequence of persistent exposure to yeast . we therefore monitored oxygen - evoked ca responses in these two neuronal populations and found that hypoxia led to a fast and very robust response in the ilp7but not in the va neurons ( figures 5b , 5d , and s6b , and movie s2 ) . together , these findings indicate that the activity of the ilp7- and pdf - producing neurons is increased in vivo by both nutritional cues and reductions in oxygen availability . together with previous ilp / pdf loss - of - function experiments , the above experiments suggested that nutritional modulation of ilp neuronal activity underlies the nutritional plasticity of tracheae . to test this idea , we used thermogenetics to achieve persistent , low - level activation of the ilp7 neurons throughout larval life by expressing the heat - sensitive channel trpa1 from ilp7-gal4 in larvae reared at 25c . the finding that tracheal branching is directly regulated by nutrient - responsive neurons suggests that tracheal terminal cells may be used by the nervous system as effectors of metabolic adaptations to nutrient availability . to investigate this possibility , we recapitulated the differential effects of nutrient restriction on tracheae by either reducing tracheal terminal cell growth in all tissues ( except for the cns tracheae , using dsrf > btl - rnai ) , or specifically in the gut tracheae ( using dsrf > inr - rnai ) . to test this idea , we exposed the dsrf > inr - rnai flies with reduced gut tracheation to a low - calorie diet throughout their adult lifetime , and found them to be significantly more resistant to nutrient scarcity than control flies : a tracheal phenotype that was confirmed using the recently published tracheal driver 14d03-gal4 ( guo et al . in response to specific nutritional cues , small subsets of neurons are activated to regulate tracheal branching in an organ - specific and metabolically significant fashion . at least one of the two yeast - responsive neuronal subsets also responds to reduced oxygen the other environmental modulator of tracheal branching in flies so it will be interesting to determine the contribution of these neurons to the previously reported tracheal adaptations to hypoxia . importantly , our identification of a shared neuronal substrate for both nutritional and hypoxic stimuli is , to our knowledge , the first of its kind in invertebrates and one remarkably similar to the mammalian carotid body : a cluster of chemoreceptors that monitors arterial oxygen concentration and nutrient levels to regulate breathing and cardiovascular tone ( pardal and lpez - barneo , 2002 ; prabhakar , 2000 ) . future work will aim to establish whether these drosophila neurons are able to sense oxygen and/or nutrients directly and whether they do so using mechanisms akin to those described in the carotid body . molecularly , the neuronal control of different tracheal subsets involves both local and systemic actions of insulin- and vip - like neuropeptides : neuronal mechanisms that are particularly complex and combinatorial along the digestive tract ( figure 7 ) and that differ from the known adaptive target - derived signals that sculpt tissue - specific angiogenesis ( cao , 2007 ; fraisl et al . in this metabolic motor neurons ; as the nervous system modulates motor neuron activity to regulate muscle contraction , it also modulates the branching of tracheal terminal cells to control the metabolic state of cells such as those of the fat body or the gut epithelium . it will be of interest to investigate whether similar mechanisms are deployed in vertebrates to effect long - lasting , tissue - specific and metabolically significant changes in angiogenesis in response to nutrition , in a manner distinct from ( but reminiscent of ) the acute changes in blood supply effected by neurons by acting on blood vessel musculature ( see , for example , matheson et al . the regional regulation of tracheal subsets hence provides one possible explanation for the apparent redundancy of the ilp gene family in drosophila : while all these ilps may indeed have the same function ( in this case , to modulate tracheal growth in response to nutrition ) , they may carry it out in different places for example , in the posterior hindgut in the case of ilp7 and in other parts of the digestive tract for ilp2 , ilp3 , and ilp5 . this regional control of tracheal growth may extend to other regions : gut visceral musculature and cns glia are known to activate ilp3 and ilp2/ilp6 gene expression respectively in a nutrient - dependent fashion ( chell and brand , 2010 ; obrien et al . , 2013 ) , it is possible that local regulation of tracheal branching by ilps contributes to their reported action on intestinal or neuronal stem cell proliferation ( chell and brand , 2010 ; obrien et al . effects of insulin and vip on blood vessels have been described in vertebrates ( chaudhuri et al . in contrast to this mode of regulation , involving acute modulation of endothelial muscle tone , the evidence for longer - lasting effects of these peptides on angiogenesis which would be more akin to their action on the drosophila tracheal system is more tenuous and often contradictory ( see , for example , ogasawara et al . mechanistically , it has been proposed that the vertebrate peptides regulate proangiogenic target - derived signals . , 2010 ; strikingly , downregulation of insulin signaling only in one cellular target the tracheal terminal cells uncouples the developmental from the metabolic phenotypes of these peptides , thus identifying the tracheal system as an important and previously unrecognized metabolic target of insulin signaling in the fly . reduced adiposity is a phenotype that , although also consistent with one of the classic symptoms of type i diabetes in humans , had not previously been observed in flies with a ubiquitous reduction in insulin signaling or lacking the systemic ilp peptides ( puzzlingly , these flies were actually found to accumulate triglyceride ; bhni et al . we suggest that this increased adiposity may have been secondary to the igf - like effects of ilps on developmental time , and only by uncoupling these developmental from the metabolic effects of ilps , as we have done with the tracheal - specific reduction of insulin signaling , can some of the true insulin - like phenotypes of ilps be unmasked . we have also found that subtle changes in insulin signaling or in the nutritional content of the fly s diet ( some of which are within the range of those normally found in diets used for fly rearing in different labs ) have a striking effect on an unexpected tracheal population : that of the digestive tract . alternatively , it could be caused by differences in downstream signaling components such as foxo , which has been shown to account for some organ - specific responses ( tang et al . functionally , by uncovering gut - specific effects of tracheation on adult survival and lipid mobilization upon nutrient scarcity , we have identified the tracheal system as a possible anatomical substrate for the previously reported effects of nutrient acquisition during developmental and growth periods on a variety of adult features ( foley and luckinbill , 2001 ; zwaan et al . enterocytes would appear to be the obvious cellular mediators of these effects ; changes in oxygen supply may modulate the metabolic state of these absorptive cells , and long - term adaptations to nutrient scarcity may result from differential nutrient absorption and/or utilization . however , enterocytes need not be the only intestinal targets of the nutrient - driven tracheal changes : the tracheal regulation of stem cell proliferation described above ( li et al . in future , it will be of interest to explore not only these intestinal targets , but also whether the intestinal tracheal plasticity is more widely regulated by other environmental stimuli such as gut epithelial infection or damage . in light of our drosophila findings , it will be of interest to explore the nutritional plasticity of the gastrointestinal vasculature , as well as its contribution to pathologies such as obesity or to the metabolic improvements following gastric bypass interventions . in the mid - hindgut , where the tissue surface and three - dimensional properties allowed semiautomated quantification , the same procedure as for the vnc was used , but the segmented image was subsequently skeletonized . parts of gut tissue wrongly identified as tracheae or segments of the tracheal tree missed by the program were manually edited before counting the total number of pixels in the skeletonized tracheal tree . all crosses and experiments were kept at 25c , including those designed to achieve persistent , low - level activation of the ilp7 neurons by expression of trpa1.to obtain larvae of the desired developmental stage , a small number of flies ( less than 10 virgin females ) were housed in embryo collection cages with apple juice plates topped with abundant yeast ( see section below ) . these same diets were used to assess the effect of larval diet on adult tracheation ; control flies ( figure 1j ) were raised and maintained in the nutritious cornmeal / agar diet with 8% yeast , whereas flies that were nutrient restricted as larvae ( figure 1k ) were raised in the cornmeal / agar diet with 0.8% yeast , and then were switched to the nutritious cornmeal / agar diet with 8% yeast after eclosion.for the adult survival or metabolic experiments , flies of the different genotypes were reared and aged for 1 day in standard food , and were then transferred to a more defined diet with the same sugar / yeast ratio ( 60%/40% ) but differing nutritional content : nutritious ( 5.4% sucrose and 3.6% yeast in 1% agar , 0.18% nipagin ) , or restricted ( 0.9% sucrose and 0.6% yeast in 1% agar , 0.18% nipagin ) . in the cases where it was necessary to correct for developmental delay ( ilp2,3,5 mutants and severe nutrient restriction ) , the developmental delay was first monitored and found to be ca . to obtain larvae of the desired developmental stage , a small number of flies ( less than 10 virgin females ) were housed in embryo collection cages with apple juice plates topped with abundant yeast ( see section below ) . in the cases where it was necessary to correct for developmental delay ( ilp2,3,5 mutants and severe nutrient restriction ) , the developmental delay was first monitored and found to be ca .

unlike the more stereotypical development of the body s main blood vessels , the formation of the capillary networks responsible for tissue perfusion is an adaptive process primarily governed by the metabolic needs of the target tissues ( fraisl et al . , 2009 ; the plastic nature of this adaptive angiogenesis is further highlighted by the dramatic changes in vascularization observed in tumors or in obese adipose tissue : changes that contribute to the progression of pathologies such as cancer and obesity and are becoming increasingly central to their treatment ( cao , 2010 ; kerbel , 2008 ; lijnen , 2008 ) . however , whether modulation of angiogenesis is associated with metabolic benefits remains a controversial topic , partly because it is not trivial to genetically target the blood vessels of specific organs to recapitulate the changes associated with certain dietary interventions without affecting other cell types or vascular pools ( cao , 2010 ; lijnen , 2008 ; sun et al . , a close spatial association between mammalian nerves and vessels was observed as long ago as 1543 ( vesalius , 1543 ) , an association that has subsequently been shown to result from mutual guidance or common pathfinding mechanisms during the formation of the neural and vascular networks ( carmeliet and tessier - lavigne , 2005 ; mukouyama et al . a functional role for these neurovascular interactions was suggested following the discovery that vessel abnormalities precede a number of neurodevelopmental and neurodegenerative disorders : an observation that points to angiogenesis as a therapeutically relevant process ( quaegebeur et al . , 2009 during embryogenesis , developmental mechanisms akin to those discovered in the vertebrate lung and vasculature make use of signaling pathways such as fibroblast growth factor ( fgf ) signaling to sculpt this complex tracheal network of interconnected tubes ( ghabrial et al . in this study , we use a combination of genetic approaches , metabolic profiling , and in vivo imaging to uncover previously unrecognized nutritional plasticity in the fly tracheal system . in contrast to the known target - derived mechanisms of adaptive remodeling , we find this plasticity to be regulated by a mechanism , previously undescribed in either flies or vertebrates , involving nutrient - responsive neurons effecting long - lasting and metabolically significant changes in tracheal architecture . while subjecting wild - type drosophila larvae to different dietary conditions , we observed that a severe reduction in dietary yeast ( the main source of lipid and amino acids in the larval diet ) was accompanied by an almost ubiquitous reduction in tracheal terminal cell branching , even when controlling for overall developmental delay by allowing nutrient - restricted larvae to develop to a comparable stage ( figures 1a1c , 1g1i and figures s1a , s1c , s1d , s1f , s1 g , s1i , s1j , s1l , s1 m , and s1o available online ) . by contrast , a mild reduction in dietary yeast neither affected developmental timing nor led to major changes in the size of organs or that of most tracheal terminal cells ( figures 1d and 1e and data not shown ) but did lead to a severe reduction in tracheal coverage throughout the digestive tract ( figures 1f , s1b , s1e , s1h , s1k , and s1n ) . to investigate the reversibility of the tracheal changes described above , we reared larvae under the mild nutrient restriction conditions shown to reduce intestinal tracheation and transferred them to more nutritious food immediately after eclosion . even after 7 days on a nutritious diet , the intestinal tracheae of these adults flies were significantly less branched than those of control adult flies always reared on a nutritious diet ( figures 1j and 1k ) , indicating that a defined period of nutrient restriction has long - term effects on the tracheal scaffold . hypoxia , the only known regulator of tracheal plasticity , has been shown to promote tracheal branching by inducing fgf ligand in target tissues and receptor upregulation in tracheal cells ( centanin et al . although downregulation of the fgf receptor gene breathless ( btl ) did lead to reduced tracheation in most scored tissues , consistent with the known fgf requirement for the establishment of the tracheal scaffold during earlier developmental stages ( ghabrial et al . , 2003 ) , further attempts to manipulate fgf signaling or to detect fgf ligand expression and differential pathway activation under different nutritional conditions all failed to support a role for fgf signaling in coupling nutrition with larval tracheal growth ( data not shown ) . this led to reduced tracheal terminal cell branching both in the periphery and throughout the digestive tract , but not in the cns ( figures 2a2f , s2s , s2 t , s3a s3d and data not shown ) : a reduction qualitatively and quantitatively comparable to that observed in these tracheal terminal cells following severe nutrient restriction ( figures 1g1i ) . together , these results confirm the cell - autonomous role for the insulin signaling pathway in the regulation of tracheal terminal cell growth and suggest that the enhanced nutritional plasticity of the gut tracheae is a consequence of their higher sensitivity to insulin signaling . in drosophila larvae , nutrient restriction leads to growth inhibition , caused by the reduced release of several insulin - like peptides ( ilps ) from brain insulin - producing cells ( the so - called median neurosecretory cells , mnscs , represented schematically in figure 3j ) into the hemolymph ( gminard et al . functional inactivation of the ilp7 neurons by expression of the inward - rectifying potassium channel kir2.1 or by expression of tetanus toxin light chain did not affect most tracheae but led to reduced tracheal coverage of two portions of the hindgut ( figures 3g3i , s4e , and s4f and data not shown ) : the posterior hindgut ( figures 3c and 3i ) , where the ilp7 axons are adjacent to the posterior visceral tracheal branches ( figures 3k and 7b ) , but also the mid - hindgut ( figure s4f ) , which we had also found to be regulated by systemic mnsc - derived ilps ( figures s4b and s4d ) . four of the eight ilp7-expressing neurons coexpress pigment dispersing factor ( pdf ) ( figures 4a and 4b ) : a neuropeptide that shares functional and signaling similarities with vertebrate vasoactive intestinal polypeptide ( vip ) ( taghert and nitabach , 2012 ) . together , both neuronal populations deliver pdf and ilp7 to the hindgut in a regionalized fashion : ilp7 is apparent only in the posterior hindgut , whereas pdf is present in both posterior and mid - hindgut terminals ( figures 4c and 7b ) . mutation of these peptides , alone or in combination , revealed complex control of different intestinal tracheal subsets by local ilp7 and pdf peptides in combination with the systemic ilp2 , ilp3 and ilp5 peptides ( figures 4d4u , s5a s5l , and 7b ) : in the posterior hindgut , neither loss of ilp7 alone nor ilp2 , ilp3 , and ilp5 together affected tracheal branching ( figures 4f , 4i , s5c , 3c , and 3f ) , but loss of all four peptides resulted in reduced tracheal terminal cell growth ( figures 4l and s5c ) , indicating partially redundant control of tracheal terminal growth . loss of ilp7 or pdf alone , or tracheal - specific downregulation of the pdf receptor ( dsrf - gal4 , uas - pdfr - rnai ) , resulted in reduced tracheal growth only in the mid - hindgut ( figures 4g4i , 4m4r and s5a s5i ) : a region also affected by the lack of systemic ilps ( figures s4b and s4d ) and not directly exposed to ilp7 peptide ( figure 7b ) . finally , mutants lacking both ilp7 and pdf displayed reduced tracheal growth in both the mid - hindgut and posterior hindgut ( figures 4 t , 4u , s5k , and s5l ) , indicating that ilp7 and pdf act redundantly in the posterior hindgut . we therefore monitored oxygen - evoked ca responses in these two neuronal populations and found that hypoxia led to a fast and very robust response in the ilp7but not in the va neurons ( figures 5b , 5d , and s6b , and movie s2 ) . to test this idea , we used thermogenetics to achieve persistent , low - level activation of the ilp7 neurons throughout larval life by expressing the heat - sensitive channel trpa1 from ilp7-gal4 in larvae reared at 25c . this promoted tracheal branching in a paracrine fashion ; it increased branching of the adjacent visceral tracheal branch of the posterior hindgut and the tracheal terminal cells of the neighboring mid - hindgut , but did not redirect those of the anterior hindgut or other regions ( figures 5e and 5f , and data not shown ) . to investigate this possibility , we recapitulated the differential effects of nutrient restriction on tracheae by either reducing tracheal terminal cell growth in all tissues ( except for the cns tracheae , using dsrf > btl - rnai ) , or specifically in the gut tracheae ( using dsrf > inr - rnai ) . reduced tracheation of all tissues did not affect larval development ( figure 6a ) or carbohydrate metabolism ( figures s7a s7c ) but resulted in leaner larvae ( figure 6c ) with reduced lipid stores ( figure 6d ) and increased hemolymph glycerol ( a metabolite derived from the hydrolysis of triglycerides ) ( figure 6e ) , consistent with reduced lipid storage capacity in the fat body . by contrast , when reduced tracheation was confined to the gut , no developmental or metabolic phenotypes were apparent in larvae ( figures s7d s7h ) , and there was no difference in adult lifespan between the experimental flies and controls on nutritious food ( figure 6f ) . to test this idea , we exposed the dsrf > inr - rnai flies with reduced gut tracheation to a low - calorie diet throughout their adult lifetime , and found them to be significantly more resistant to nutrient scarcity than control flies : a tracheal phenotype that was confirmed using the recently published tracheal driver 14d03-gal4 ( guo et al . at least one of the two yeast - responsive neuronal subsets also responds to reduced oxygen the other environmental modulator of tracheal branching in flies so it will be interesting to determine the contribution of these neurons to the previously reported tracheal adaptations to hypoxia . importantly , our identification of a shared neuronal substrate for both nutritional and hypoxic stimuli is , to our knowledge , the first of its kind in invertebrates and one remarkably similar to the mammalian carotid body : a cluster of chemoreceptors that monitors arterial oxygen concentration and nutrient levels to regulate breathing and cardiovascular tone ( pardal and lpez - barneo , 2002 ; prabhakar , 2000 ) . molecularly , the neuronal control of different tracheal subsets involves both local and systemic actions of insulin- and vip - like neuropeptides : neuronal mechanisms that are particularly complex and combinatorial along the digestive tract ( figure 7 ) and that differ from the known adaptive target - derived signals that sculpt tissue - specific angiogenesis ( cao , 2007 ; fraisl et al . in this metabolic motor neurons ; as the nervous system modulates motor neuron activity to regulate muscle contraction , it also modulates the branching of tracheal terminal cells to control the metabolic state of cells such as those of the fat body or the gut epithelium . it will be of interest to investigate whether similar mechanisms are deployed in vertebrates to effect long - lasting , tissue - specific and metabolically significant changes in angiogenesis in response to nutrition , in a manner distinct from ( but reminiscent of ) the acute changes in blood supply effected by neurons by acting on blood vessel musculature ( see , for example , matheson et al . the regional regulation of tracheal subsets hence provides one possible explanation for the apparent redundancy of the ilp gene family in drosophila : while all these ilps may indeed have the same function ( in this case , to modulate tracheal growth in response to nutrition ) , they may carry it out in different places for example , in the posterior hindgut in the case of ilp7 and in other parts of the digestive tract for ilp2 , ilp3 , and ilp5 . indeed , although the effect of pdf on intestinal tracheal branching is unexpected in drosophila ( where this peptide is known for its central role in clock neurons ; taghert and nitabach , 2012 ) , neurally derived vip has a vasodilatory effect on the arterioles of small intestine and colon ( holzer , 2006 ) . in contrast to this mode of regulation , involving acute modulation of endothelial muscle tone , the evidence for longer - lasting effects of these peptides on angiogenesis which would be more akin to their action on the drosophila tracheal system is more tenuous and often contradictory ( see , for example , ogasawara et al . in drosophila , whole - organism manipulations of insulin signaling such as ablation of insulin - producing cells or ilp mutation result in both slower development and diabetic phenotypes , highlighting their dual insulin / igf - like role ( grnke et al . , 2010 ; strikingly , downregulation of insulin signaling only in one cellular target the tracheal terminal cells uncouples the developmental from the metabolic phenotypes of these peptides , thus identifying the tracheal system as an important and previously unrecognized metabolic target of insulin signaling in the fly . reduced adiposity is a phenotype that , although also consistent with one of the classic symptoms of type i diabetes in humans , had not previously been observed in flies with a ubiquitous reduction in insulin signaling or lacking the systemic ilp peptides ( puzzlingly , these flies were actually found to accumulate triglyceride ; bhni et al . we suggest that this increased adiposity may have been secondary to the igf - like effects of ilps on developmental time , and only by uncoupling these developmental from the metabolic effects of ilps , as we have done with the tracheal - specific reduction of insulin signaling , can some of the true insulin - like phenotypes of ilps be unmasked . we have also found that subtle changes in insulin signaling or in the nutritional content of the fly s diet ( some of which are within the range of those normally found in diets used for fly rearing in different labs ) have a striking effect on an unexpected tracheal population : that of the digestive tract . functionally , by uncovering gut - specific effects of tracheation on adult survival and lipid mobilization upon nutrient scarcity , we have identified the tracheal system as a possible anatomical substrate for the previously reported effects of nutrient acquisition during developmental and growth periods on a variety of adult features ( foley and luckinbill , 2001 ; zwaan et al . in other intestinal portions , where the ruggedness and/or bends and twists of the target tissue made semiautomated quantification impractical , tracheal coverage was blindly scored using likert - type scales ranging from no difference to wild - type ( 3 ) to strongly increased ( 5 ) or strongly reduced ( 1 ) ( see figure 1 legend for color coding of displays ) . , 1996 ) , uas - inr - rnai ( vdrc stock 992 ) , uas - btl - rnai ( vdrc stock 110277 ) , ilp7 , ilp2,3,5 and ilp2,3,5,7 mutants ( grnke et al . all crosses and experiments were kept at 25c , including those designed to achieve persistent , low - level activation of the ilp7 neurons by expression of trpa1.to obtain larvae of the desired developmental stage , a small number of flies ( less than 10 virgin females ) were housed in embryo collection cages with apple juice plates topped with abundant yeast ( see section below ) . these same diets were used to assess the effect of larval diet on adult tracheation ; control flies ( figure 1j ) were raised and maintained in the nutritious cornmeal / agar diet with 8% yeast , whereas flies that were nutrient restricted as larvae ( figure 1k ) were raised in the cornmeal / agar diet with 0.8% yeast , and then were switched to the nutritious cornmeal / agar diet with 8% yeast after eclosion.for the adult survival or metabolic experiments , flies of the different genotypes were reared and aged for 1 day in standard food , and were then transferred to a more defined diet with the same sugar / yeast ratio ( 60%/40% ) but differing nutritional content : nutritious ( 5.4% sucrose and 3.6% yeast in 1% agar , 0.18% nipagin ) , or restricted ( 0.9% sucrose and 0.6% yeast in 1% agar , 0.18% nipagin ) . for the experiments assessing the effect of yeast concentration on larval tracheal growth ( figure 1 ) , the standard cornmeal / agar diet above was used with the following three yeast concentrations : 8% ( nutritious ) , 2% ( mild nutrient restriction ) or 0.8% ( severe nutrient restriction ) . these same diets were used to assess the effect of larval diet on adult tracheation ; control flies ( figure 1j ) were raised and maintained in the nutritious cornmeal / agar diet with 8% yeast , whereas flies that were nutrient restricted as larvae ( figure 1k ) were raised in the cornmeal / agar diet with 0.8% yeast , and then were switched to the nutritious cornmeal / agar diet with 8% yeast after eclosion . for the adult survival or metabolic experiments , flies of the different genotypes were reared and aged for 1 day in standard food , and were then transferred to a more defined diet with the same sugar / yeast ratio ( 60%/40% ) but differing nutritional content : nutritious ( 5.4% sucrose and 3.6% yeast in 1% agar , 0.18% nipagin ) , or restricted ( 0.9% sucrose and 0.6% yeast in 1% agar , 0.18% nipagin ) . ilp7 or va neurons were recorded with a 100 ms exposure time in 0.5 s intervals on an inverted compound microscope ( axiovert , zeiss ) , using a 2.0 neutral density filter , a gfp / dsred emission filter set ( chroma ) , a 40x c - apochromat lens ( zeiss ) , a dual - view beamsplitter with gfp / dsred emission filters ( photometrics ) and a cascade ii 512 emccd camera ( photometrics ) .

it is estimated that 68,470 new cases of primary brain and central nervous system tumors are expected to be diagnosed in the united states in 2015 , and gliomas account for the majority of primary malignant brain tumors in adults ( ostrom et al . , 2014 ) . although low - grade lesions have a better prognosis , they often undergo transformation to a more malignant , higher grade at the time of progression . both primary and recurrent gliomas infiltrate into adjacent brain tissue , making it difficult to define tumor margins . proper diagnosis and grading , correct localization , and assessment of response to therapy are of great importance for all phases of treatment planning and selection . conventional t1- and t2-weighted mri are applied in conjunction with an injection of a gadolinium - based contrast agent to delineate structural abnormalities in the brain and assess regions where the brain blood barrier has been compromised . however , these conventional anatomic sequences have failed to reliably distinguish between complex tissue components within and around brain tumors such as interstitial edema , gliosis , inflammation , cysts , necrosis and active tumor . the specificity of conventional mri is further compromised by treatment with radiation , temozolomide and anti - angiogenic agents ( wen et al . , 2010 ) . diffusion weighted imaging ( dwi ) has been a rapidly expanding field in mri and has been found valuable in evaluating many diseases in the brain ( lerner et al . , 2014 ; rovaris et al . , 2005 ; van everdingen et al . , signal to the random motion of water molecule protons at a microscopic level ( of the order of 520 m ) , it is able to probe tissue microstructures in the brain such as axons , dendrites , glial cells , and extra - cellular spaces ( yablonskiy and sukstanskii , 2010 ) , in a manner that may provide valuable insights into tumor physiology . a simple and most widely used model to describe changes in signal intensity is diffusion tensor imaging ( dti ) , where the directional motion of water behavior is characterized by a 3 dimensional tensor . the apparent diffusion coefficient ( adc ) derived from tensor modeling is a sensitive yet non - specific metric when evaluated in the highly heterogeneous tumor environment . on serial mri studies , reduction in adc may be the result of decreasing vasogenic edema or an increase in cell density . increased adc may reflect tissue necrosis , an increase in interstitial fluid , or a decreasing cell density due to effective treatment . the ability to distinguish between the mechanisms that lead to changes in adc would be very important for treatment planning and monitoring patients . one approach to addressing this problem is to use a more sophisticated diffusion model that can distinguish between the different tissue compartments . neurite orientation dispersion and density imaging ( noddi ) is a recently proposed diffusion model that allows the quantification of specific microstructural features directly related to neuronal morphology ( zhang et al . , 2012 ) . in particular , the noddi model ascribes the signal from water protons in neuronal tissue to one of three different pools : i ) free water in areas such as the ventricles that contain csf and exhibit isotropic diffusion ; ii ) restricted water within linear structures that represent dendrites and axons ; and iii ) water that is anisotropically hindered , representing diffusion within glial cells , neuronal cell bodies and extracellular environment . an orientation dispersion index ( od ) is produced to describe the coherence of neurite directions , with a lower value indicating more coherent organization . the optimized 30-minute protocol limited the noddi model to a 2-shell 90-direction dwi acquisition , which has been shown to produce reliable noddi maps ( zhang et al . , 2012 ) . recent applications of noddi that have provided encouraging results are from patients with multiple sclerosis ( magnollay , 2014 ; schneider et al . , 2014 ) , focal cortical dysplasia ( billiet et al . , 2014 ; winston et al . , 2014 ) , neurofibromatosis ( billiet et al . , 2014 ) , healthy newborn brain ( kunz et al . , 2014 ) , as well as in the spinal cord ( grussu et al . , 2015 ) . these results are all at 3 t , and the method has not yet been investigated at 7 t , or in patients with brain tumors . the technique is enticing for the evaluation of patients with glioma , in that we hypothesize that changes in vasogenic edema would be reflected in the free water compartment ( stummer , 2007 ) , and changes in cell density would be associated with changes in the hindered water compartment . the availability of improved hardware and fast acquisition techniques make it possible to obtain 90-directional multi - shell dwi within a clinically feasible time . conventional parallel imaging ( blaimer et al . 2012 ) results in a significant loss in snr because the number of phase encoding steps that are acquired is reduced . for 2d multi - slice imaging , significant improvements can be achieved by exciting several slices at the same time using multiband radio frequency ( rf ) pulses . since each slice is excited and sampled identically without skipping or missing k - space data , there is no loss of snr due to reduced acquisition time as is encountered with parallel imaging . this technique has been successfully implemented in the highly publicized human connectome project for accelerated diffusion imaging acquisition at 3 t ( setsompop et al . , 2013 ; sotiropoulos et al . , 2013 ) . at higher field strengths , reduced t2 compromises the gain in snr from the increased static magnetization , making the benefit of diffusion imaging at ultra - high field unclear . other challenges , such as increased b0 and b1 field inhomogeneity , can also influence the quality of diffusion data at high field ( uurbil et al . , 2013 ) . ( wu et al . , 2013 ) have attempted to compensate for the b1 inhomogeneity through rf shimming of multiband pulses in both single and parallel transmit settings . another group has also implemented the multiband spoke technique to both improve the b1 homogeneity across slices and reduce peak rf power ( sharma et al . , 2015 ) . with the efforts put into the optimization for acquisition of diffusion data at 7 t , it has been anticipated that the diffusion data set acquired with a more powerful gradient set at 7 t will be comparable to 3 t data ( uurbil et al . , 2013 ) . despite these advances , the applications of ultra - high field diffusion weighted imaging have been limited , and there has not yet been a rigorous direct comparison of the snr for diffusion imaging between 3 t and 7 t. the goal of this study was to use multiband dwi at 7 t in order to obtain 90-directional multi - shell data within a clinically feasible acquisition time for patients with glioma . this study included an snr comparison between 3 t and 7 t , and the application of b1 mapping and distortion correction procedures for reducing the impact of variations in b0 and b1 . the optimized multiband sequence was applied to generate both dti and noddi maps and to compare the values in tumor and normal appearing white matter ( nawm ) . multiband epi was conducted with a quadrature transmit and 32-channel receive head coil ( nova medical , wilmington , ma ) on ge scanners . three slices ( 40 mm apart ) were simultaneously excited ( multiband factor of 3 , or mb = 3 ) with a three - band rf excitation ( no inter - slice shift ) and axial spin - echo echo planer ( se - epi ) readout with phase encoding ( pe ) in the anterior posterior ( ap ) direction , resulting in 60 slices for whole brain coverage with isotropic voxels of 2 2 2 mm over a field of view of 256 256 mm . 75% partial fourier k - space sampling was employed to reduce te and an in - plane acceleration factor of 3 ( r = 3 ) was used , which we found to be the best trade - off between te and distortion . with a 50 mt / m amplitude gradient system at b = 2000 s / mm , te was 71.6 ms and tr was 3200 ms . calibration images were acquired at the beginning of the sequence with the same three - band excitation pulses but with different phase offsets applied between the bands so that they could be separated through a fourier transform ( ft ) . this used the same se - epi readouts but was interleaved three times to fully sample k - space while having the same k - space traversal speed as the actual accelerated acquisition . a b1 mapping procedure was performed prior to the multiband acquisition to determine the optimal transmit gain ( tg ) ( d. kelley , 2013 ) that can account for the b1 inhomogeneity at high field . b1 maps were generated using a gradient echo acquisition sequence ( tr / te = 250/8 ms ) with a 1 ms adiabatic bloch siegert pulse and the transmitter gain was automatically adjusted based on the median b1 of the image volume . to correct for the susceptibility - induced distortion , one additional b = 0 image this resulted in a pair of b = 0 images with distortions going in opposite directions and allowed the off - resonance field to be estimated with a method similar to that described in andersson et al . ( 2003 ) and implemented in fsl ( smith et al . , 2004 ) . 5 volunteers were scanned with both ge 3 t mr 750 and ge 7 t mr950 scanners ( ge healthcare , waukesha , wi ) using 32-channel receive - only head coils ( nova medical , wilmington , ma ) and the same gradient system ( 50 mt / m amplitude and 200 t / m / s slew rate ) . ten b = 0 ( t2 weighted ) images were repetitively acquired with both standard epi ( mb = 1 ) and mb = 3 using the acquisition parameters described above . with the same tr , only the central slab of the brain was acquired for mb = 1 . a homogeneous spherical water phantom was used to evaluate the geometry factor ( g - factor ) map which quantifies the fractional loss in snr caused by the non - orthogonality of the array coil sensitivities ( pruessmann et al . , 1999 ) . two data sets were acquired in each scanner using a gradient - recalled echo ( gre ) imaging sequence ( fov = 256 256 mm , matrix size = 64 64 , slice thickness = 2 mm ) . the first data set was used to assess the noise covariance matrix and was obtained with all rf pulses suppressed . the second data set served to determine coil sensitivity maps for each coil element and was obtained with regular rf excitation . twenty patients with glioma ( 13 males and 7 females , median age = 54 years ) were referred by physicians from the neuro - oncology service at our institution and recruited to this study . tumors had been graded by histological examination of tissue samples obtained during biopsy or surgical resection : 10 had grade ii , 3 had grade iii and 7 had grade iv . all patients were recurrent and 4 patients were showing progressive disease at the time of the scan . a two - shell diffusion imaging protocol was acquired on the 7 t scanner with mb = 3 . this protocol included 7 b = 0 images ( and one additional b = 0 image with reversed phase - encoding gradient for distortion correction ) , 30 directions at b = 1000 s / mm , and 60 directions at b = 2000 s / mm with a total acquisition time of 5 42. anatomical images of these patients were acquired at the 3 t scanner , including sagittal t1-weighted spin echo , axial fluid attenuated inversion recovery ( flair ) , contrast - enhanced 3d spoiled gradient - recalled acquisition in the steady state ( spgr ) t1-weighted and t1-weighted post - contrast spin echo images ( t1-gad ) . in 14 patients , 24-directional dwi with a standard se - epi sequence was also acquired ( repetition time ( tr)/echo time ( te ) = 10 s/99 ms , voxel size = 22 2 mm , in - plane r = 2 , b = 1000 s / mm ) . table 2 summarized the data acquisition with phantom , volunteers and patients . the image was aliased in both si and ap directions , and was unfolded using the sense / grappa procedure as described in blaimer et al . , ( 2004 ) . the calibration data were first concatenated and fourier transformed ( ft ) to generate the fully sampled k - space . once the kernel has been generated , aliased images went through the reconstruction pipeline as illustrated in fig . 1 . the undersampled k - space was first zero filled in the direction that no acceleration was performed ( left right , fig . 1b ) and a standard grappa / arc procedure was applied to reconstruct the k - space data ( fig . after an inverse fourier transform ( ifft ) , the unaliased images ( fig . 1f ) through a partial k - space reconstruction method using projection onto convex sets ( pocs ) ( yudilevich and stark , 1989 ) . a 2d fermi filter was then applied to reduce gibbs ringing , and single coil images ( fig . 1 g ) were combined with a sum of squares coil combination ( fig . 1h ) . repeated acquisitions of b = 0 ( t2 weighted ) images from mb1 and mb3 of all 5 volunteers were used to calculate snr according to dietrich o et al . ( 1 ) for each voxel within the brain.(1)snr = mean(voxel)std(voxel ) to calculate g - factor maps , phantom data were used to estimate relative coil sensitivity maps using the eigenvector method described by walsh et al . g - factor maps were then estimated from the sensitivity maps with the equation shown in eq . ( 2 ) ( pruessmann et al . , 1999 ) for different under - sampling schemes ( mb1 , r = 3 and mb3 , r = 3).(2)gj = cr*-1crj , jcr*-1cr-1j , jcr is the coil sensitivity encoding matrix corresponding to an r fold accelerated acquisition . is the noise covariance matrix of the 32-channel coils , which was estimated by calculating the covariate matrix of the noise data acquired with rf excitation disabled . after the multiband data were reconstructed , a susceptibility distortion correction was applied using the topup method available in fsl ( andersson et al . , 2003 ; smith et al . , 2004 ) . eddy current correction was followed by applying affine registration to a reference volume ( eddy_correct , fsl ) . the noddi matlab toolbox ( http://www.nitrc.org/projects/noddi_toolbox/ ) was employed for noddi . in order to fit the noddi model , the two diffusivities representing the diffusion coefficient of the isotropic compartment ( diso ) and the intrinsic diffusivity of the intra - neurite compartments ( d// ) were fixed as in the original model ( zhang et al . , 2012 ) to diso = 3.00 m / s and d// = 1.70 m / s , which are the values commonly employed in literature for the free diffusivity of water particles in csf and neural tissue in vivo at body temperature . the dti fitting program in fsl was employed to fit dti with weighted least squares tensor fitting ( dtifit , fsl ) . both models were fitted to the whole double - shell data set , and the dti model was also applied separately to the shell acquired at b = 1000 s / mm and a shell acquired at b = 2000 s / mm . the following voxel - wise maps were obtained . for noddi : the isotropic volume fraction ( viso ) , the intra - neurite ( restricted ) volume fraction ( vic ) , the extra - neurite ( hindered ) volume fraction ( vec ) and the orientation dispersion index ( od ) . in this study , we calculated the effective volume fraction for vic and vec so that viso + vic + vec = 1 . 3 t anatomical images were rigidly aligned ( flirt , fsl ) to 7 t diffusion images in order to generate region of interests ( rois ) for the evaluation of noddi parameters . rois included voxels within the region of t2 hyperintensity ( t2l ) on the flair images and contrast - enhancing lesions ( cel ) on the t1-gad images . nawm , gray matter ( gm ) and cerebrospinal fluid ( csf ) were defined from the 3d spgr brain images . nonparametric wilcoxon rank sum tests were applied to assess differences in diffusion maps between tumor grades , or between different regions of interests ( rois ) , including t2l , cel , nawm , gm and csf . adjustment for multiple comparisons was not applied due to the exploratory nature of this study . fig . 2a showed the center slice and the snr map in a volunteer acquired with mb1 and mb3 at 3 t and 7 t. it can be noted that snr is less homogeneous at 7 t due to b1 inhomogeneity , and the reduction in snr from mb1 to mb3 is smaller at 7 t compared to 3 t. the median snrs of all slices were compared by a scatter plot for all 5 volunteers in fig . the median snr was 68.4 and 43.9 for mb1 and mb3 at 3 t , and 50.2 and 46.1 for mb1 and mb3 at 7 t. g - factor maps ( center slice and slices 40 mm above and below ) at 3 t and 7 t for different undersampling schemes were shown in fig . median 1/g was 0.97 and 0.57 for 3 t mb1 and mb3 and 0.98 and 0.71 for 7 t mb1 and mb3 . the noise correlation matrix of the 3 t 32-channel coil and 7 t 32-channel coil was shown in fig . 14 out of 20 patients had 3 t dti data available acquired with a standard sequence . 3 t and 7 t dti data were compared for this population in their native space , which included : ( 1 ) 3 t standard dti with b = 1000 , 24dir ; ( 2 ) 7 t multiband dti with b = 1000 , 30dir ; ( 3 ) 7 t multiband dti with b = 2000 , 60dir and ( 4 ) 7 t multiband double shell , 90dir . the median values of adc and fa within nawm and gm for 14 patients from different data sets are shown with bar plots in fig . the adc was not significantly different between 3 t and 7 t within the nawm , and fa was significantly higher at 7 t ( p < 0.0001 ) . as expected , the adc obtained at b = 2000 was significantly lower than adc at b = 1000 ( p < 0.0001 ) due to the non - gaussianity of water diffusion in a restricted environment , and the values fitted from the double shell were in between the values fitted from each shell . 4c for a patient data set that was acquired at 7 t. it can be seen that distortions were in the opposite direction between reversed and regular phase encoding blips , and with topup the distortion was well corrected . the mean and standard deviation of noddi and dti parameters in the nawm , gm , csf , t2l and cel were summarized in table 3 , as a function of tumor grade . within both the t2l and cel , the adc was significantly higher and fa was lower than in nawm ( p < 0.0001 ) . both viso and vec were significantly elevated ( p < 0.0001 ) compared to nawm and vic was significantly decreased ( p < 0.0001 ) . adc was significantly lower in the cel than the t2l , while od and vic were significantly higher ( p < 0.01 , p < 0.001 ) , viso was significantly lower ( p < 0.05 ) and vec was not significantly different between the two lesions . these metrics were not found to be different between tumor grades ( p > 0.1 ) . noddi maps of three patients who had lesions with different grades are shown in fig . 5 , together with adc and fa maps fitted from the same double - shell data , and 3 t t1-gad and flair images . all three patients had received radiation therapy ( rt ) and were stable at the time of scan . within in the t2l , variations were seen in viso , vic and vec , that reflected different water mobility characteristics . contrast enhancing lesions were present in all patients ( blue arrow ) and all demonstrated elevated od and vic compared to surrounding tissues . the white arrow indicates two interesting regions at the edge of t2l of the grade ii and grade iii patients . these regions could easily be missed on anatomical images and dti maps , as their intensities were very close to nawm , but were highlighted in noddi maps due to elevated vec . the use of sophisticated diffusion models to provide improved characterization of tissue composition is a promising technique for evaluating lesion heterogeneity in patients with glioma . the application of these models has typically been limited by the need to acquire a large number of diffusion directions at high b - values which is not feasible in clinical settings ( assaf and basser , 2005 ; panagiotaki et al . , 2014 ) . in this study , we demonstrated the feasibility of acquiring multishell diffusion weighted data in the whole brain using the multiband technique within the same acquisition time that has been used to acquire routine dti . our results showed that the noddi maps were able to provide unique contrast within the t2l and are likely to provide information that is complementary to fa and adc . major concerns for obtaining diffusion data using ultra high field strength mr scanners are increased variation in b0 and b1 . in this study , we used 3-fold in - plane acceleration to shorten te , as well as to increase the bandwidth in the phase - encoding direction in order to reduce the extent of distortion . to further correct for the susceptibility distortion , we used the topup correction by adding an additional b = 0 image with reversed phase encoding blips into the sequence ( andersson et al . b1 inhomogeneity caused spin - echo imperfections over the entire brain , resulting in non - uniform signal intensity . to ameliorate this situation , we optimized the transmitter gain based on the median b1 of the image volume by incorporating the measured b1 field strength . it has been previously demonstrated that this technique can improve the situation by providing 82% of the available spin echo signal ( d. kelley , 2013 ) . the performance of the epi sequence at ultra - high field has been difficult to assess because the t2 values of brain tissue are difficult to measure accurately at 7 t. we carried out a straightforward snr comparison between 3 t and 7 t with the same hardware settings ( gradient , coils ) and acquisition parameters ( tr , te , k - space coverage and voxel size ) . our results showed that without multiband , the snr was higher at 3 t ( snr 68.4 ) than 7 t ( snr 50.2 ) , indicating that the shortened t2 as well as increased b0 and b1 inhomogeneity at 7 t outweighed the increased static magnetization and the total signal was reduced at the same echo time . however , when 3-fold acceleration was applied with multiband , the snr was comparable between 3 t ( snr 43.9 ) and 7 t ( snr 46.1 ) . the snr loss after simultaneous multi - slice acquisition is caused by the spatially dependent amplification of noise , known as the geometry factor , or g - factor. our estimation of g - factor maps showed that with mb = 3 and in - plane r = 3 , the g - factor was lower at 7 t ( g = 1.4 ) than 3 t ( g = 1.75 ) . this explained why snr is no longer superior at 3 t with a multiband factor of 3 . the improved g - factor map at 7 t compared to 3 t that was observed in our study is in great agreement with the literature ( wiesinger et al . , 2004b ) . , 2008 ; ohliger et al . , 2003 ; wiesinger et al . , 2004a ) and in experiments ( kelley et al . , 2008 ; wiesinger et al , 2004b ) that the g - factor is dependent upon b0 field strength at high field ( b0 > 1.5 t ) , and improves as the field increases , because the coil sensitivities grow progressively asymmetric ( collins et al . , 2002 ) and become increasingly structured due to the shortening rf wavelength and related interference effects ( yang et al . , it has also been demonstrated that improvement in the g - factor at high field becomes more obvious as acceleration factor increases ( wiesinger et al . , 2004b ) . our data demonstrated comparable snr between 7 t and 3 t with 9-fold acceleration , and it can be predicted that a higher acceleration factor can be achieved at 7 t with less reduction in snr compared to 3 t. the multiband diffusion sequence was applied to the characterization of glioma using a 2-shell , 90-direction protocol that is straightforward to implement and sufficiently economical for clinical applications , yet sophisticated enough to distinguish three types of water diffusion in the brain . the adc and fa maps fitted from this 7 t protocol were first compared to the data acquired at 3 t for patients with both data sets available ( n = 14 ) . our results showed that adc values at b = 1000 mm / s within nawm and gm were close between 3 t and 7 t , with adc higher in gm than nawm , which is consistent with the trend reported in the literature ( helenius et al . fa values were higher at 7 t in both nawm and gm , which could be caused by the difference in snr between the two acquisitions , with a lower snr at 7 t ( mb3 , snr 46.1/33 db ) than 3 t ( standard dti , snr > 68.4/37 db ) . increasing fa with decreasing snr has been reported in both experiments and computer simulations . 1996 ) showed that , for lower snr , the longitudinal principal eigenvalue 1 was overestimated , whereas the eigenvalues 2/3 are underestimated , leading to overestimation of fa . ( landman et al . , 2007 ) developed a theoretical framework to model the influence of noise in dti and showed that for snr < 40 db in b = 0 s / mm images , mean fa increased with decreasing snr , and adc was less affected than fa . the noddi model distinguishes three types of water diffusion behavior and each was quantified with a compartment fraction . when applied to tumor , it is natural to speculate that vasogenic edema would belong to viso given its isotropically fast diffusing movement . invading tumor cells along fiber tracts should be categorized as vec , as they co - exist with glial cells in the space around the neurites . our data were supportive of these assumptions by showing increased viso and decreased vic within t2l and cel lesions compared to nawm , consistent with increased edema and loss of neurons . these assumptions should be validated with tissue samples , which were not available in the current study . patients recruited into this study were at different stages of disease and had received varying treatments , which may explain why these matrices were not found to be significantly different between tumor grades . the noddi maps provided unique contrast across the tumor and highlighted interesting regions that could not be seen in adc or fa maps . the different types of contrast observed may be reflective of progressive disease or of treatment effects that are worth noting for radiologists . obtaining follow - up data at later time points would be valuable for tracking changes in these regions and may be helpful in interpreting them . definitive verification and interpretation would require the use of image guided tissue samples and will be considered for future studies . 5 was that vic values were higher in cel compared to the t2l . in the noddi model , vic represents neuron density , which conflicts with this finding as we know that the cel contains less neurons than t2l and reflects either the most aggressive tumor packed with dense tumor cells or non - specific changes related to treatment effect . this suggests that when applied to tumor , vic can no longer be interpreted as neuron density . rather , the elevated vic can be explained by the presence of restrictive structures with restrictions in all direction , which is consistent with the similarly elevated od . this warned us that we should be very cautious when interpreting noddi results in tumor , as this model was not directly built on tumor and some parameters were prefixed based on values in normal brain such as the intrinsic free diffusivity in vic . the 3-fold acceleration in the slice direction was the highest that could be obtained with our current multiband technique , as the separation of aliased slices ( 40 mm ) is close to the coil size and snr lost would be exacerbated if a higher acceleration factor was used in that direction . another technique , termed blipped - caipirinha can be implemented to both increase the snr and achieve a higher acceleration factor ( setsompop et al . , 2012 ) . this technique can reduce the g - factor in simultaneous multi - slice acquisitions by introducing interslice image shifts and thus increase the distance between aliased voxels . le roux ( slr ) optimized rf pulses where the high rf energy transmission limited the optimal tr that can be achieved at high field due to specific absorption rate ( sar ) . advanced rf pulse design techniques have been reported to lower the peak rf power at ultra high field ( eichner et al . , 2014 ; sharma et al . , 2015 ) . these will be considered for future studies . in conclusion , we have demonstrated the feasibility of using multiband diffusion weighted imaging at 7 t within 6 min in order to apply the noddi model to characterizing glioma . the 7 t diffusion data quality was generally comparable to data acquired at 3 t and quantitative diffusion measurements ( adc and fa ) were similar . anatomical imaging at 7 t benefits from the higher snr , and the ability to consistently obtain high quality diffusion data at 7 t will contribute towards the implementation of a comprehensive brain mri examination at ultra - high field . we have shown that noddi maps provided unique contrast within the t2l lesion that was not seen in anatomical images or dti maps . such contrast may reflect the complexity of tissue compositions associated with disease progression and treatment effects . changes in viso , vic and vec in tumor lesions compared with nawm were consistent with the alternations in tissue components within tumor . histological analysis of image - guided tissue samples is needed in future studies to better understand these variations .

recent technological progress in the multiband echo planer imaging ( mb epi ) technique enables accelerated mr diffusion weighted imaging ( dwi ) and allows whole brain , multi - b - value diffusion imaging to be acquired within a clinically feasible time . however , its applications at 7 t have been limited due to b1 field inhomogeneity and increased susceptibility artifact . it is an ongoing debate whether dwi at 7 t can be performed properly in patients , and a systematic snr comparison for multiband spin - echo epi between 3 t and 7 t has not been methodically studied . the goal of this study was to use mb epi at 7 t in order to obtain 90-directional multi - shell dwi within a clinically feasible acquisition time for patients with glioma . this study included an snr comparison between 3 t and 7 t , and the application of b1 mapping and distortion correction procedures for reducing the impact of variations in b0 and b1 . the optimized multiband sequence was applied in 20 patients with glioma to generate both dti and noddi maps for comparison of values in tumor and normal appearing white matter ( nawm ) . our snr analysis showed that mb epi at 7 t was comparable to that at 3 t , and the data quality acquired in patients was clinically acceptable . noddi maps provided unique contrast within the t2 lesion that was not seen in anatomical images or dti maps . such contrast may reflect the complexity of tissue compositions associated with disease progression and treatment effects . the ability to consistently obtain high quality diffusion data at 7 t will contribute towards the implementation of a comprehensive brain mri examination at ultra - high field .

Introduction Data acquisition Results Discussion

it is estimated that 68,470 new cases of primary brain and central nervous system tumors are expected to be diagnosed in the united states in 2015 , and gliomas account for the majority of primary malignant brain tumors in adults ( ostrom et al . conventional t1- and t2-weighted mri are applied in conjunction with an injection of a gadolinium - based contrast agent to delineate structural abnormalities in the brain and assess regions where the brain blood barrier has been compromised . diffusion weighted imaging ( dwi ) has been a rapidly expanding field in mri and has been found valuable in evaluating many diseases in the brain ( lerner et al . , signal to the random motion of water molecule protons at a microscopic level ( of the order of 520 m ) , it is able to probe tissue microstructures in the brain such as axons , dendrites , glial cells , and extra - cellular spaces ( yablonskiy and sukstanskii , 2010 ) , in a manner that may provide valuable insights into tumor physiology . a simple and most widely used model to describe changes in signal intensity is diffusion tensor imaging ( dti ) , where the directional motion of water behavior is characterized by a 3 dimensional tensor . increased adc may reflect tissue necrosis , an increase in interstitial fluid , or a decreasing cell density due to effective treatment . the ability to distinguish between the mechanisms that lead to changes in adc would be very important for treatment planning and monitoring patients . the optimized 30-minute protocol limited the noddi model to a 2-shell 90-direction dwi acquisition , which has been shown to produce reliable noddi maps ( zhang et al . these results are all at 3 t , and the method has not yet been investigated at 7 t , or in patients with brain tumors . the technique is enticing for the evaluation of patients with glioma , in that we hypothesize that changes in vasogenic edema would be reflected in the free water compartment ( stummer , 2007 ) , and changes in cell density would be associated with changes in the hindered water compartment . the availability of improved hardware and fast acquisition techniques make it possible to obtain 90-directional multi - shell dwi within a clinically feasible time . conventional parallel imaging ( blaimer et al . for 2d multi - slice imaging , significant improvements can be achieved by exciting several slices at the same time using multiband radio frequency ( rf ) pulses . since each slice is excited and sampled identically without skipping or missing k - space data , there is no loss of snr due to reduced acquisition time as is encountered with parallel imaging . this technique has been successfully implemented in the highly publicized human connectome project for accelerated diffusion imaging acquisition at 3 t ( setsompop et al . at higher field strengths , reduced t2 compromises the gain in snr from the increased static magnetization , making the benefit of diffusion imaging at ultra - high field unclear . other challenges , such as increased b0 and b1 field inhomogeneity , can also influence the quality of diffusion data at high field ( uurbil et al . with the efforts put into the optimization for acquisition of diffusion data at 7 t , it has been anticipated that the diffusion data set acquired with a more powerful gradient set at 7 t will be comparable to 3 t data ( uurbil et al . despite these advances , the applications of ultra - high field diffusion weighted imaging have been limited , and there has not yet been a rigorous direct comparison of the snr for diffusion imaging between 3 t and 7 t. the goal of this study was to use multiband dwi at 7 t in order to obtain 90-directional multi - shell data within a clinically feasible acquisition time for patients with glioma . this study included an snr comparison between 3 t and 7 t , and the application of b1 mapping and distortion correction procedures for reducing the impact of variations in b0 and b1 . the optimized multiband sequence was applied to generate both dti and noddi maps and to compare the values in tumor and normal appearing white matter ( nawm ) . three slices ( 40 mm apart ) were simultaneously excited ( multiband factor of 3 , or mb = 3 ) with a three - band rf excitation ( no inter - slice shift ) and axial spin - echo echo planer ( se - epi ) readout with phase encoding ( pe ) in the anterior posterior ( ap ) direction , resulting in 60 slices for whole brain coverage with isotropic voxels of 2 2 2 mm over a field of view of 256 256 mm . 75% partial fourier k - space sampling was employed to reduce te and an in - plane acceleration factor of 3 ( r = 3 ) was used , which we found to be the best trade - off between te and distortion . a b1 mapping procedure was performed prior to the multiband acquisition to determine the optimal transmit gain ( tg ) ( d. kelley , 2013 ) that can account for the b1 inhomogeneity at high field . to correct for the susceptibility - induced distortion , one additional b = 0 image this resulted in a pair of b = 0 images with distortions going in opposite directions and allowed the off - resonance field to be estimated with a method similar to that described in andersson et al . 5 volunteers were scanned with both ge 3 t mr 750 and ge 7 t mr950 scanners ( ge healthcare , waukesha , wi ) using 32-channel receive - only head coils ( nova medical , wilmington , ma ) and the same gradient system ( 50 mt / m amplitude and 200 t / m / s slew rate ) . ten b = 0 ( t2 weighted ) images were repetitively acquired with both standard epi ( mb = 1 ) and mb = 3 using the acquisition parameters described above . twenty patients with glioma ( 13 males and 7 females , median age = 54 years ) were referred by physicians from the neuro - oncology service at our institution and recruited to this study . tumors had been graded by histological examination of tissue samples obtained during biopsy or surgical resection : 10 had grade ii , 3 had grade iii and 7 had grade iv . a two - shell diffusion imaging protocol was acquired on the 7 t scanner with mb = 3 . this protocol included 7 b = 0 images ( and one additional b = 0 image with reversed phase - encoding gradient for distortion correction ) , 30 directions at b = 1000 s / mm , and 60 directions at b = 2000 s / mm with a total acquisition time of 5 42. anatomical images of these patients were acquired at the 3 t scanner , including sagittal t1-weighted spin echo , axial fluid attenuated inversion recovery ( flair ) , contrast - enhanced 3d spoiled gradient - recalled acquisition in the steady state ( spgr ) t1-weighted and t1-weighted post - contrast spin echo images ( t1-gad ) . in 14 patients , 24-directional dwi with a standard se - epi sequence was also acquired ( repetition time ( tr)/echo time ( te ) = 10 s/99 ms , voxel size = 22 2 mm , in - plane r = 2 , b = 1000 s / mm ) . table 2 summarized the data acquisition with phantom , volunteers and patients . 1b ) and a standard grappa / arc procedure was applied to reconstruct the k - space data ( fig . after the multiband data were reconstructed , a susceptibility distortion correction was applied using the topup method available in fsl ( andersson et al . in order to fit the noddi model , the two diffusivities representing the diffusion coefficient of the isotropic compartment ( diso ) and the intrinsic diffusivity of the intra - neurite compartments ( d// ) were fixed as in the original model ( zhang et al . both models were fitted to the whole double - shell data set , and the dti model was also applied separately to the shell acquired at b = 1000 s / mm and a shell acquired at b = 2000 s / mm . for noddi : the isotropic volume fraction ( viso ) , the intra - neurite ( restricted ) volume fraction ( vic ) , the extra - neurite ( hindered ) volume fraction ( vec ) and the orientation dispersion index ( od ) . in this study , we calculated the effective volume fraction for vic and vec so that viso + vic + vec = 1 . 3 t anatomical images were rigidly aligned ( flirt , fsl ) to 7 t diffusion images in order to generate region of interests ( rois ) for the evaluation of noddi parameters . rois included voxels within the region of t2 hyperintensity ( t2l ) on the flair images and contrast - enhancing lesions ( cel ) on the t1-gad images . nawm , gray matter ( gm ) and cerebrospinal fluid ( csf ) were defined from the 3d spgr brain images . adjustment for multiple comparisons was not applied due to the exploratory nature of this study . 2a showed the center slice and the snr map in a volunteer acquired with mb1 and mb3 at 3 t and 7 t. it can be noted that snr is less homogeneous at 7 t due to b1 inhomogeneity , and the reduction in snr from mb1 to mb3 is smaller at 7 t compared to 3 t. the median snrs of all slices were compared by a scatter plot for all 5 volunteers in fig . the median snr was 68.4 and 43.9 for mb1 and mb3 at 3 t , and 50.2 and 46.1 for mb1 and mb3 at 7 t. g - factor maps ( center slice and slices 40 mm above and below ) at 3 t and 7 t for different undersampling schemes were shown in fig . median 1/g was 0.97 and 0.57 for 3 t mb1 and mb3 and 0.98 and 0.71 for 7 t mb1 and mb3 . the noise correlation matrix of the 3 t 32-channel coil and 7 t 32-channel coil was shown in fig . 14 out of 20 patients had 3 t dti data available acquired with a standard sequence . 3 t and 7 t dti data were compared for this population in their native space , which included : ( 1 ) 3 t standard dti with b = 1000 , 24dir ; ( 2 ) 7 t multiband dti with b = 1000 , 30dir ; ( 3 ) 7 t multiband dti with b = 2000 , 60dir and ( 4 ) 7 t multiband double shell , 90dir . the adc was not significantly different between 3 t and 7 t within the nawm , and fa was significantly higher at 7 t ( p < 0.0001 ) . as expected , the adc obtained at b = 2000 was significantly lower than adc at b = 1000 ( p < 0.0001 ) due to the non - gaussianity of water diffusion in a restricted environment , and the values fitted from the double shell were in between the values fitted from each shell . 4c for a patient data set that was acquired at 7 t. it can be seen that distortions were in the opposite direction between reversed and regular phase encoding blips , and with topup the distortion was well corrected . adc was significantly lower in the cel than the t2l , while od and vic were significantly higher ( p < 0.01 , p < 0.001 ) , viso was significantly lower ( p < 0.05 ) and vec was not significantly different between the two lesions . 5 , together with adc and fa maps fitted from the same double - shell data , and 3 t t1-gad and flair images . within in the t2l , variations were seen in viso , vic and vec , that reflected different water mobility characteristics . these regions could easily be missed on anatomical images and dti maps , as their intensities were very close to nawm , but were highlighted in noddi maps due to elevated vec . the use of sophisticated diffusion models to provide improved characterization of tissue composition is a promising technique for evaluating lesion heterogeneity in patients with glioma . the application of these models has typically been limited by the need to acquire a large number of diffusion directions at high b - values which is not feasible in clinical settings ( assaf and basser , 2005 ; panagiotaki et al . in this study , we demonstrated the feasibility of acquiring multishell diffusion weighted data in the whole brain using the multiband technique within the same acquisition time that has been used to acquire routine dti . our results showed that the noddi maps were able to provide unique contrast within the t2l and are likely to provide information that is complementary to fa and adc . major concerns for obtaining diffusion data using ultra high field strength mr scanners are increased variation in b0 and b1 . in this study , we used 3-fold in - plane acceleration to shorten te , as well as to increase the bandwidth in the phase - encoding direction in order to reduce the extent of distortion . b1 inhomogeneity caused spin - echo imperfections over the entire brain , resulting in non - uniform signal intensity . the performance of the epi sequence at ultra - high field has been difficult to assess because the t2 values of brain tissue are difficult to measure accurately at 7 t. we carried out a straightforward snr comparison between 3 t and 7 t with the same hardware settings ( gradient , coils ) and acquisition parameters ( tr , te , k - space coverage and voxel size ) . our results showed that without multiband , the snr was higher at 3 t ( snr 68.4 ) than 7 t ( snr 50.2 ) , indicating that the shortened t2 as well as increased b0 and b1 inhomogeneity at 7 t outweighed the increased static magnetization and the total signal was reduced at the same echo time . however , when 3-fold acceleration was applied with multiband , the snr was comparable between 3 t ( snr 43.9 ) and 7 t ( snr 46.1 ) . our estimation of g - factor maps showed that with mb = 3 and in - plane r = 3 , the g - factor was lower at 7 t ( g = 1.4 ) than 3 t ( g = 1.75 ) . this explained why snr is no longer superior at 3 t with a multiband factor of 3 . the improved g - factor map at 7 t compared to 3 t that was observed in our study is in great agreement with the literature ( wiesinger et al . , 2004a ) and in experiments ( kelley et al . , 2008 ; wiesinger et al , 2004b ) that the g - factor is dependent upon b0 field strength at high field ( b0 > 1.5 t ) , and improves as the field increases , because the coil sensitivities grow progressively asymmetric ( collins et al . , 2002 ) and become increasingly structured due to the shortening rf wavelength and related interference effects ( yang et al . , it has also been demonstrated that improvement in the g - factor at high field becomes more obvious as acceleration factor increases ( wiesinger et al . our data demonstrated comparable snr between 7 t and 3 t with 9-fold acceleration , and it can be predicted that a higher acceleration factor can be achieved at 7 t with less reduction in snr compared to 3 t. the multiband diffusion sequence was applied to the characterization of glioma using a 2-shell , 90-direction protocol that is straightforward to implement and sufficiently economical for clinical applications , yet sophisticated enough to distinguish three types of water diffusion in the brain . the adc and fa maps fitted from this 7 t protocol were first compared to the data acquired at 3 t for patients with both data sets available ( n = 14 ) . our results showed that adc values at b = 1000 mm / s within nawm and gm were close between 3 t and 7 t , with adc higher in gm than nawm , which is consistent with the trend reported in the literature ( helenius et al . fa values were higher at 7 t in both nawm and gm , which could be caused by the difference in snr between the two acquisitions , with a lower snr at 7 t ( mb3 , snr 46.1/33 db ) than 3 t ( standard dti , snr > 68.4/37 db ) . 1996 ) showed that , for lower snr , the longitudinal principal eigenvalue 1 was overestimated , whereas the eigenvalues 2/3 are underestimated , leading to overestimation of fa . , 2007 ) developed a theoretical framework to model the influence of noise in dti and showed that for snr < 40 db in b = 0 s / mm images , mean fa increased with decreasing snr , and adc was less affected than fa . patients recruited into this study were at different stages of disease and had received varying treatments , which may explain why these matrices were not found to be significantly different between tumor grades . the noddi maps provided unique contrast across the tumor and highlighted interesting regions that could not be seen in adc or fa maps . obtaining follow - up data at later time points would be valuable for tracking changes in these regions and may be helpful in interpreting them . this warned us that we should be very cautious when interpreting noddi results in tumor , as this model was not directly built on tumor and some parameters were prefixed based on values in normal brain such as the intrinsic free diffusivity in vic . this technique can reduce the g - factor in simultaneous multi - slice acquisitions by introducing interslice image shifts and thus increase the distance between aliased voxels . le roux ( slr ) optimized rf pulses where the high rf energy transmission limited the optimal tr that can be achieved at high field due to specific absorption rate ( sar ) . advanced rf pulse design techniques have been reported to lower the peak rf power at ultra high field ( eichner et al . in conclusion , we have demonstrated the feasibility of using multiband diffusion weighted imaging at 7 t within 6 min in order to apply the noddi model to characterizing glioma . the 7 t diffusion data quality was generally comparable to data acquired at 3 t and quantitative diffusion measurements ( adc and fa ) were similar . anatomical imaging at 7 t benefits from the higher snr , and the ability to consistently obtain high quality diffusion data at 7 t will contribute towards the implementation of a comprehensive brain mri examination at ultra - high field . we have shown that noddi maps provided unique contrast within the t2l lesion that was not seen in anatomical images or dti maps . such contrast may reflect the complexity of tissue compositions associated with disease progression and treatment effects .

it is estimated that 68,470 new cases of primary brain and central nervous system tumors are expected to be diagnosed in the united states in 2015 , and gliomas account for the majority of primary malignant brain tumors in adults ( ostrom et al . although low - grade lesions have a better prognosis , they often undergo transformation to a more malignant , higher grade at the time of progression . proper diagnosis and grading , correct localization , and assessment of response to therapy are of great importance for all phases of treatment planning and selection . conventional t1- and t2-weighted mri are applied in conjunction with an injection of a gadolinium - based contrast agent to delineate structural abnormalities in the brain and assess regions where the brain blood barrier has been compromised . however , these conventional anatomic sequences have failed to reliably distinguish between complex tissue components within and around brain tumors such as interstitial edema , gliosis , inflammation , cysts , necrosis and active tumor . diffusion weighted imaging ( dwi ) has been a rapidly expanding field in mri and has been found valuable in evaluating many diseases in the brain ( lerner et al . , signal to the random motion of water molecule protons at a microscopic level ( of the order of 520 m ) , it is able to probe tissue microstructures in the brain such as axons , dendrites , glial cells , and extra - cellular spaces ( yablonskiy and sukstanskii , 2010 ) , in a manner that may provide valuable insights into tumor physiology . a simple and most widely used model to describe changes in signal intensity is diffusion tensor imaging ( dti ) , where the directional motion of water behavior is characterized by a 3 dimensional tensor . the apparent diffusion coefficient ( adc ) derived from tensor modeling is a sensitive yet non - specific metric when evaluated in the highly heterogeneous tumor environment . on serial mri studies , reduction in adc may be the result of decreasing vasogenic edema or an increase in cell density . the ability to distinguish between the mechanisms that lead to changes in adc would be very important for treatment planning and monitoring patients . one approach to addressing this problem is to use a more sophisticated diffusion model that can distinguish between the different tissue compartments . neurite orientation dispersion and density imaging ( noddi ) is a recently proposed diffusion model that allows the quantification of specific microstructural features directly related to neuronal morphology ( zhang et al . in particular , the noddi model ascribes the signal from water protons in neuronal tissue to one of three different pools : i ) free water in areas such as the ventricles that contain csf and exhibit isotropic diffusion ; ii ) restricted water within linear structures that represent dendrites and axons ; and iii ) water that is anisotropically hindered , representing diffusion within glial cells , neuronal cell bodies and extracellular environment . an orientation dispersion index ( od ) is produced to describe the coherence of neurite directions , with a lower value indicating more coherent organization . these results are all at 3 t , and the method has not yet been investigated at 7 t , or in patients with brain tumors . the technique is enticing for the evaluation of patients with glioma , in that we hypothesize that changes in vasogenic edema would be reflected in the free water compartment ( stummer , 2007 ) , and changes in cell density would be associated with changes in the hindered water compartment . 2012 ) results in a significant loss in snr because the number of phase encoding steps that are acquired is reduced . for 2d multi - slice imaging , significant improvements can be achieved by exciting several slices at the same time using multiband radio frequency ( rf ) pulses . this technique has been successfully implemented in the highly publicized human connectome project for accelerated diffusion imaging acquisition at 3 t ( setsompop et al . at higher field strengths , reduced t2 compromises the gain in snr from the increased static magnetization , making the benefit of diffusion imaging at ultra - high field unclear . other challenges , such as increased b0 and b1 field inhomogeneity , can also influence the quality of diffusion data at high field ( uurbil et al . , 2013 ) have attempted to compensate for the b1 inhomogeneity through rf shimming of multiband pulses in both single and parallel transmit settings . with the efforts put into the optimization for acquisition of diffusion data at 7 t , it has been anticipated that the diffusion data set acquired with a more powerful gradient set at 7 t will be comparable to 3 t data ( uurbil et al . despite these advances , the applications of ultra - high field diffusion weighted imaging have been limited , and there has not yet been a rigorous direct comparison of the snr for diffusion imaging between 3 t and 7 t. the goal of this study was to use multiband dwi at 7 t in order to obtain 90-directional multi - shell data within a clinically feasible acquisition time for patients with glioma . this study included an snr comparison between 3 t and 7 t , and the application of b1 mapping and distortion correction procedures for reducing the impact of variations in b0 and b1 . the optimized multiband sequence was applied to generate both dti and noddi maps and to compare the values in tumor and normal appearing white matter ( nawm ) . three slices ( 40 mm apart ) were simultaneously excited ( multiband factor of 3 , or mb = 3 ) with a three - band rf excitation ( no inter - slice shift ) and axial spin - echo echo planer ( se - epi ) readout with phase encoding ( pe ) in the anterior posterior ( ap ) direction , resulting in 60 slices for whole brain coverage with isotropic voxels of 2 2 2 mm over a field of view of 256 256 mm . 75% partial fourier k - space sampling was employed to reduce te and an in - plane acceleration factor of 3 ( r = 3 ) was used , which we found to be the best trade - off between te and distortion . calibration images were acquired at the beginning of the sequence with the same three - band excitation pulses but with different phase offsets applied between the bands so that they could be separated through a fourier transform ( ft ) . a b1 mapping procedure was performed prior to the multiband acquisition to determine the optimal transmit gain ( tg ) ( d. kelley , 2013 ) that can account for the b1 inhomogeneity at high field . b1 maps were generated using a gradient echo acquisition sequence ( tr / te = 250/8 ms ) with a 1 ms adiabatic bloch siegert pulse and the transmitter gain was automatically adjusted based on the median b1 of the image volume . to correct for the susceptibility - induced distortion , one additional b = 0 image this resulted in a pair of b = 0 images with distortions going in opposite directions and allowed the off - resonance field to be estimated with a method similar to that described in andersson et al . 5 volunteers were scanned with both ge 3 t mr 750 and ge 7 t mr950 scanners ( ge healthcare , waukesha , wi ) using 32-channel receive - only head coils ( nova medical , wilmington , ma ) and the same gradient system ( 50 mt / m amplitude and 200 t / m / s slew rate ) . a homogeneous spherical water phantom was used to evaluate the geometry factor ( g - factor ) map which quantifies the fractional loss in snr caused by the non - orthogonality of the array coil sensitivities ( pruessmann et al . twenty patients with glioma ( 13 males and 7 females , median age = 54 years ) were referred by physicians from the neuro - oncology service at our institution and recruited to this study . this protocol included 7 b = 0 images ( and one additional b = 0 image with reversed phase - encoding gradient for distortion correction ) , 30 directions at b = 1000 s / mm , and 60 directions at b = 2000 s / mm with a total acquisition time of 5 42. anatomical images of these patients were acquired at the 3 t scanner , including sagittal t1-weighted spin echo , axial fluid attenuated inversion recovery ( flair ) , contrast - enhanced 3d spoiled gradient - recalled acquisition in the steady state ( spgr ) t1-weighted and t1-weighted post - contrast spin echo images ( t1-gad ) . in 14 patients , 24-directional dwi with a standard se - epi sequence was also acquired ( repetition time ( tr)/echo time ( te ) = 10 s/99 ms , voxel size = 22 2 mm , in - plane r = 2 , b = 1000 s / mm ) . (1)snr = mean(voxel)std(voxel ) to calculate g - factor maps , phantom data were used to estimate relative coil sensitivity maps using the eigenvector method described by walsh et al . in order to fit the noddi model , the two diffusivities representing the diffusion coefficient of the isotropic compartment ( diso ) and the intrinsic diffusivity of the intra - neurite compartments ( d// ) were fixed as in the original model ( zhang et al . , 2012 ) to diso = 3.00 m / s and d// = 1.70 m / s , which are the values commonly employed in literature for the free diffusivity of water particles in csf and neural tissue in vivo at body temperature . both models were fitted to the whole double - shell data set , and the dti model was also applied separately to the shell acquired at b = 1000 s / mm and a shell acquired at b = 2000 s / mm . for noddi : the isotropic volume fraction ( viso ) , the intra - neurite ( restricted ) volume fraction ( vic ) , the extra - neurite ( hindered ) volume fraction ( vec ) and the orientation dispersion index ( od ) . in this study , we calculated the effective volume fraction for vic and vec so that viso + vic + vec = 1 . rois included voxels within the region of t2 hyperintensity ( t2l ) on the flair images and contrast - enhancing lesions ( cel ) on the t1-gad images . 2a showed the center slice and the snr map in a volunteer acquired with mb1 and mb3 at 3 t and 7 t. it can be noted that snr is less homogeneous at 7 t due to b1 inhomogeneity , and the reduction in snr from mb1 to mb3 is smaller at 7 t compared to 3 t. the median snrs of all slices were compared by a scatter plot for all 5 volunteers in fig . the median snr was 68.4 and 43.9 for mb1 and mb3 at 3 t , and 50.2 and 46.1 for mb1 and mb3 at 7 t. g - factor maps ( center slice and slices 40 mm above and below ) at 3 t and 7 t for different undersampling schemes were shown in fig . median 1/g was 0.97 and 0.57 for 3 t mb1 and mb3 and 0.98 and 0.71 for 7 t mb1 and mb3 . 3 t and 7 t dti data were compared for this population in their native space , which included : ( 1 ) 3 t standard dti with b = 1000 , 24dir ; ( 2 ) 7 t multiband dti with b = 1000 , 30dir ; ( 3 ) 7 t multiband dti with b = 2000 , 60dir and ( 4 ) 7 t multiband double shell , 90dir . the median values of adc and fa within nawm and gm for 14 patients from different data sets are shown with bar plots in fig . the adc was not significantly different between 3 t and 7 t within the nawm , and fa was significantly higher at 7 t ( p < 0.0001 ) . as expected , the adc obtained at b = 2000 was significantly lower than adc at b = 1000 ( p < 0.0001 ) due to the non - gaussianity of water diffusion in a restricted environment , and the values fitted from the double shell were in between the values fitted from each shell . the mean and standard deviation of noddi and dti parameters in the nawm , gm , csf , t2l and cel were summarized in table 3 , as a function of tumor grade . within both the t2l and cel , the adc was significantly higher and fa was lower than in nawm ( p < 0.0001 ) . both viso and vec were significantly elevated ( p < 0.0001 ) compared to nawm and vic was significantly decreased ( p < 0.0001 ) . adc was significantly lower in the cel than the t2l , while od and vic were significantly higher ( p < 0.01 , p < 0.001 ) , viso was significantly lower ( p < 0.05 ) and vec was not significantly different between the two lesions . the white arrow indicates two interesting regions at the edge of t2l of the grade ii and grade iii patients . the use of sophisticated diffusion models to provide improved characterization of tissue composition is a promising technique for evaluating lesion heterogeneity in patients with glioma . the application of these models has typically been limited by the need to acquire a large number of diffusion directions at high b - values which is not feasible in clinical settings ( assaf and basser , 2005 ; panagiotaki et al . in this study , we demonstrated the feasibility of acquiring multishell diffusion weighted data in the whole brain using the multiband technique within the same acquisition time that has been used to acquire routine dti . our results showed that the noddi maps were able to provide unique contrast within the t2l and are likely to provide information that is complementary to fa and adc . in this study , we used 3-fold in - plane acceleration to shorten te , as well as to increase the bandwidth in the phase - encoding direction in order to reduce the extent of distortion . to ameliorate this situation , we optimized the transmitter gain based on the median b1 of the image volume by incorporating the measured b1 field strength . the performance of the epi sequence at ultra - high field has been difficult to assess because the t2 values of brain tissue are difficult to measure accurately at 7 t. we carried out a straightforward snr comparison between 3 t and 7 t with the same hardware settings ( gradient , coils ) and acquisition parameters ( tr , te , k - space coverage and voxel size ) . our results showed that without multiband , the snr was higher at 3 t ( snr 68.4 ) than 7 t ( snr 50.2 ) , indicating that the shortened t2 as well as increased b0 and b1 inhomogeneity at 7 t outweighed the increased static magnetization and the total signal was reduced at the same echo time . however , when 3-fold acceleration was applied with multiband , the snr was comparable between 3 t ( snr 43.9 ) and 7 t ( snr 46.1 ) . the snr loss after simultaneous multi - slice acquisition is caused by the spatially dependent amplification of noise , known as the geometry factor , or g - factor. our estimation of g - factor maps showed that with mb = 3 and in - plane r = 3 , the g - factor was lower at 7 t ( g = 1.4 ) than 3 t ( g = 1.75 ) . the improved g - factor map at 7 t compared to 3 t that was observed in our study is in great agreement with the literature ( wiesinger et al . , 2008 ; wiesinger et al , 2004b ) that the g - factor is dependent upon b0 field strength at high field ( b0 > 1.5 t ) , and improves as the field increases , because the coil sensitivities grow progressively asymmetric ( collins et al . our data demonstrated comparable snr between 7 t and 3 t with 9-fold acceleration , and it can be predicted that a higher acceleration factor can be achieved at 7 t with less reduction in snr compared to 3 t. the multiband diffusion sequence was applied to the characterization of glioma using a 2-shell , 90-direction protocol that is straightforward to implement and sufficiently economical for clinical applications , yet sophisticated enough to distinguish three types of water diffusion in the brain . the adc and fa maps fitted from this 7 t protocol were first compared to the data acquired at 3 t for patients with both data sets available ( n = 14 ) . our results showed that adc values at b = 1000 mm / s within nawm and gm were close between 3 t and 7 t , with adc higher in gm than nawm , which is consistent with the trend reported in the literature ( helenius et al . fa values were higher at 7 t in both nawm and gm , which could be caused by the difference in snr between the two acquisitions , with a lower snr at 7 t ( mb3 , snr 46.1/33 db ) than 3 t ( standard dti , snr > 68.4/37 db ) . , 2007 ) developed a theoretical framework to model the influence of noise in dti and showed that for snr < 40 db in b = 0 s / mm images , mean fa increased with decreasing snr , and adc was less affected than fa . our data were supportive of these assumptions by showing increased viso and decreased vic within t2l and cel lesions compared to nawm , consistent with increased edema and loss of neurons . in the noddi model , vic represents neuron density , which conflicts with this finding as we know that the cel contains less neurons than t2l and reflects either the most aggressive tumor packed with dense tumor cells or non - specific changes related to treatment effect . this warned us that we should be very cautious when interpreting noddi results in tumor , as this model was not directly built on tumor and some parameters were prefixed based on values in normal brain such as the intrinsic free diffusivity in vic . the 3-fold acceleration in the slice direction was the highest that could be obtained with our current multiband technique , as the separation of aliased slices ( 40 mm ) is close to the coil size and snr lost would be exacerbated if a higher acceleration factor was used in that direction . in conclusion , we have demonstrated the feasibility of using multiband diffusion weighted imaging at 7 t within 6 min in order to apply the noddi model to characterizing glioma . anatomical imaging at 7 t benefits from the higher snr , and the ability to consistently obtain high quality diffusion data at 7 t will contribute towards the implementation of a comprehensive brain mri examination at ultra - high field .

breast cancer is the commonest neoplasm and the second cause of cancer death in women worldwide . it is estimated that in the world more than one million women are diagnosed with breast cancer every year , and more than 410,000 will die from the disease , representing approximately 14% of female cancer deaths . human leukocyte antigen ( hla ) class i molecules have a central role in the cell - mediated immune system , especially as antigen - presenting molecules for cytotoxic t lymphocytes ( ctls ) , which can recognize tumor antigenic bound peptides , presented on the cell surface with hla class i molecules , and kill the target cell [ 2 , 3 ] . i expression seems to be lost or downregulated on the tumor cell surface and this might represent a mechanism for neoplastic cells to escape from being killed by ctls , allowing tumor dissemination and metastasis . hla class ii molecules ( hla - dr and hla - dq ) are essential for peptide presentation to t - helper lymphocytes , and their expression may be responsible for triggering the immune response . thus , the presence of these antigens may make the tumor more immunogenic , which could lead to a favorable prognosis . however , it has been proposed that hla - dr molecules offer protection against nk cell cytotoxicity , as has been described for hla class i antigens . the class i human major histocompatibility complex ( mhc ) genes encode both the classical ( extremely polymorphic ) hla - ia ( hla - a , -b and -c ) molecules and the nonclassical hla - ib ( -e , -f and -g ) molecules , characterized by low allelic polymorphism , limited tissue distribution , and the presence of membrane - bound and soluble isoforms . hla - g and hla - e expression at the tumor cell surface might allow it to escape t and natural killer ( nk ) cell immune surveillance . surface hla - e appears to confer protection from the nk cell - mediated lysis via the cd94/nkg2a receptor . indeed , the role of hla - g may be to interact with nk cell inhibitory receptors , such as ilt2 or ilt4 . the purpose of the present study was to analyze the relation between hla - ia , hla - ib , and classical hla - ii ( -dq , -dr ) expression in idc of the breast and breast cancer aggressiveness and metastatic tumor behavior . tumor tissue specimens were taken at the institute of clinical pathology ( ipc ) of araraquara , state of so paulo , brazil . fifty - two idc of the breast biopsies were analyzed by hematoxylin / eosin staining methods and stratified according to histological differentiation ( well , moderately , and poorly differentiated ) and the presence of metastasis in axillary lymph nodes . a total of 52 formalin - fixed and paraffin - embedded biopsies of idc of the breast were collected from 52 patients and stratified according to lesion grade . in the case of breast carcinoma with metastasis ( 16 patients ) , one biopsy of the respective axillary lymph node immunohistochemical tests with the streptavidin - biotin system ( ep - usa/500 , signet , usa ) were carried out , to detect the hla - ia , hla - dq , hla - dr , hla - g , and hla - e antigens . tissue specimens were dewaxed in xylene , rehydrated in graded alcohol , and rinsed in water . for antigen detection , endogenous peroxidase was blocked by immersion in a hydrogen peroxide bath in absolute methanol ( 15 minutes each change ) and nonspecific binding was performed with 3% low - fat dried milk diluted 1 : 100 in phosphate - buffered saline ( pbs ) . slides were incubated with the primary monoclonal antibodies ( mabs ) for hla - ia ( ab70328 , diluted 1 : 50 ; abcam , cambridge , england ) , hla - dq ( ab55158 , diluted 1 : 50 ; abcam , cambridge , england ) , hla - dr ( ab175085 , diluted 1 : 50 ; abcam , cambridge , england ) , hla - g ( 5a6g7 , diluted 1 : 50 ; exbio , prague , czech republic ) , and for hla - e ( mem - e/02 , diluted 1 : 50 ; exbio , prague , czech republic ) in a humidified chamber at 4c overnight and then incubated with the streptavidin - peroxidase complex at 37c for 30 min . the sections were then incubated in a solution containing 5 mg of diaminobenzidine ( gibbico , gaithersburg , maryland , usa ) , dissolved in 5 ml of pbs , and 100 l of fresh peroxidase solution ( 450 l pbs and 50 l hydrogen peroxide ) for 10 min , lightly counterstained with carrazzi 's hematoxylin without acid for 60 sec , exhaustively washed with tap water , air - dried , and mounted with permount mounting medium ( merck ; darmstadt , germany ) . on each section , the mean number of positive - staining membranes was counted for both hla - g and hla - e immunostaining and classified as negative ( absence of immunolabelling to 25% positivity ) and positive ( 25% to 100% positivity ) . for hla - ia , hla - dq , and hla - dr , immunostaining was classified as negative ( absence of immunolabeling to 10% positivity ) and positive ( 10% to 100% positivity ) ( figure 1 ) . image analysis of tissues subjected to immunohistochemistry to determine the expression of hla - ia , hla - dr , hla - dq , hla - g , and hla - e was performed in the image analyzer image - pro plus ( md , usa ) . this equipment consists of a microscope ( olympus bx50 ) coupled to a color camera ( olympus dp10 ) and a dedicated computer containing the software , responsible for the mathematical determination of labeled cells . in each blade , an average of 10 fields was selected and digitized images were obtained in those fields in which it was established that the number of positive cells ( marking in the cytoplasmic membrane ) is 1000 cells / biopsy . such quantification that could be interactively controlled by targeting through an rgb filter exists in software , allowing automatic retrieval of the number of positive cells . thus , the average number labeled cells / biopsy of the 52 biopsies was determined and established the average percentage of positivity . to validate the anti - hla - ia , anti - hla - dq , and anti - hla - dr mabs and the immunohistochemical method , we systematically analyzed a paraffin - embedded section of human tonsil ( positive control ) . a negative control was prepared by omitting the primary antibody from the same tissue . to validate the anti - hla - g and anti - hla - e mabs and the immunohistochemical method , a paraffin - embedded section of trophoblastic tissue from a third - trimester human placenta ( positive control ) data were analyzed statistically with the program instat mac 2.01 ( graphpad , san diego , ca , usa ) . to analyze the quantitative expression of hla molecules , the nonparametric distribution kruskal - wallis test was used , followed by the dunn test for multiple comparison between pairs among the groups . this test was utilized since the data did not exhibit gaussian distribution in the normality test . the test and , when appropriate , fisher 's exact test were used to compare the quantitative immunohistochemistry results for hla molecules , tumor size , histological grade , nuclear grade , and presence or otherwise of metastases in axillary lymph nodes . based on the tolerogenic functions of breast tumors , we investigated possible associations between the expression of hla class ia , class ib ( hla - g and hla - e ) , and class ii ( hla - dq and hla - dr ) in breast tissue ( figure 1 ) . the quantitative immunohistochemical assay results for hla types , obtained by image analysis and expressed as mean proportion of positive - staining cells and standard deviation , are shown in figure 2 . a correlation was detected between low expression of hla - ia and high expression of hla - g ( p < 0.001 ) , likewise between hla - g high expression and hla - e low expression ( p < 0.01 ) . no relation was found between hla - ia and hla - e expression ( p > 0.05 ) . hla - dr was significantly less expressed in idc lesions than hla - g and hla - dq molecules ( p < 0.01 ) but at the same level as hla class ia and hla - e . when hla - dq was compared with the other hla molecules , it was found that its expression was raised in the invasive tumors a little more than the level of hla - g . there was a significant difference between expression of hla - dr and hla - dq ( p < 0.001 ) that could indicate an association between high levels of hla - dq and low levels of hla - dr expression . the relationships between hla - ia , hla - ii , hla - g , and hla - e immunohistochemical expression and axillary lymph node metastasis are demonstrated in figure 3(a ) . no association was found between hla - ia ( p = 0.2830 ; p > 0.05 ) , hla - g high expression ( p = 0.9512 ; p > 0.05 ) , hla - e ( p = 0.3963 ; p > 0.05 ) , hla - dr ( p = 0.3010 ; p > 0.05 ) , and hla - dq ( p = 0.6894 ; p > 0.05 ) and metastases in axillary lymph nodes of idc patients . the relation between the hla immunohistochemical profile and tumor size is demonstrated in figure 3(b ) . a correlation ( p = 0.00683 ; p < 0.05 ) between tumor size and hla - i expression was observed . tumors larger than 2 cm showed low hla - i expression in 53.8% of the cases ; on the other hand , tumors classified as positive for hla - i were observed in 3.8% of patients with a tumor < 2 cm . thus , the results suggest that bigger lesions , possibly with higher proliferative activity , showed lower hla - i expression . smaller than 2 cm and 30.7% of larger lesions . on the other hand , low expression was observed in 17.3% of patients with lesions under 2 cm and 26.9% with larger lesions . for the hla - e molecules , hla - e low expression was observed in 32.7% of the smaller lesions and in 42.3% of the larger . however , no association was observed between hla - g ( p = 0.5801 ) and hla - e ( p = 0.1826 ) expression and tumor size . in the hla class ii results , no association was found between levels of expression ( hla - dr ; p = 1.000 and hla - dq ; p = 0.4420 ; p > 0.05 ) and tumor size , suggesting that there is no modification of the hla class ii levels with increasing tumor mass , an indirect indicator of tumor proliferation . the results for hla class i , class ii , hla - g and hla - e , and histological grade ( hg ) are demonstrated in figure 3(c ) . a significant correlation was observed between higher histological grades and hla - i low expression ( p = 0.0062 ; p < 0.05 ) . among patients with low expression of hla - i , greater histological disorganization ( hg2 + hg3 ) was observed in 75.0% , compared to 3.8% patients with more organized histological lesions ( hg1 ) . thus , a relationship can be seen between hla - i low expression and less histological differentiation . higher hla - g expression and greater histological disorganization ( hg2 + hg3 ) were observed in 55.7% , while hla - e high expression and lower histological differentiation ( hg2 + hg3 ) were observed in 25.0% of idc patients . on the other hand , low hla - g and hla - e expression was observed in the less differentiated lesions in , respectively , 40.4% and 71.2% of patients . however , no significant correlation was observed between lower histological differentiation and hla - g regulation ( p = 0.2695 ) or hla - e downregulation ( p = 0.7070 ) . also , no association was found between hla class ii ( dr and dq ) expression ( resp . the hla class i , class ii ( dr and dq ) , hla - g and hla - e immunohistochemical results , and cellular atypia levels of idc of the breast , classified as nuclear grade , are shown in figure 3(d ) . lower expression of hla - i ( p = 0.0071 , p < 0.05 ) was observed in the higher nuclear atypia lesions ( ng2 + ng3 ) in 78.8% patients . only 21.1% of the patients did not show hla - i downregulation , classified in this study as a positive response , and of these patients 7.7% presented lower nuclear atypia ( ng1 ) . hence , it may be suggested that in the less differentiated tumors , with a greater number of cellular atypias , hla - i downregulation could be expected . hla - g and hla - e high expression was observed in higher nuclear atypia lesions ( ng2 + ng3 ) in , respectively , 53.8% and 21% patients . on the other hand , hla - g and hla - e low expression was seen in , respectively , 38.5% and 71.2% of the idc analyzed . in the same way , no relation was found between lesser cellular differentiation and hla - g downregulation ( p = 0.4086 ) and hla - e downregulation ( p = 0.0661 ) . no significant association between hla class ii dr ( p = 0.5621 ) and dq ( p = 1.000 ) expression and the cellular atypia of idc of the breast was observed , apparently indicating that the expression of neither molecule was altered by cellular atypia . to assess the similarities or differences in the expression of all hla molecules investigated in the breast tumors and in their metastases in axillary lymph nodes , hla class i , class ii ( dr and dq ) , hla - g , and hla - e expression was assayed in the 16 lymph nodes of the patients that presented metastases ( figure 4 ) . from these results , it can be seen that hla class i , class ii ( dr and dq ) and hla - e low expression and hla - g ( p = 0.0091 , p < 0.05 ) high expression , in metastases in the lymph nodes , showed similar expression to that in the breast lesions . the results suggest a reduction in the expression of classical hla molecules and an increase in hla - g expression , related to the capacity for metastasis of this breast invasive tumor in axillary lymph nodes . the competent immune system has the capacity to recognize tumor cells and destroy them , preventing the seeding and growth of a series of tumors . notwithstanding this , the same types of tumor cell may develop the ability to evade such immune control , by means of the loss or low expression of hla class i molecules . decreased or absent hla - i molecules expression has frequently been observed in a range of malignant neoplasms , unlike what occurs in normal breast tissue , in which the expression of hla - ia occurs [ 79 ] . however , the relationship between hla - i expression and the process of carcinogenesis and metastatic progression of breast cancer has yet to be established with any certainty . the few studies carried out on this question have produced controversial results with regard to the deregulation of the expression of hla - ia and the potential for metastasis of the invasive ductal carcinoma of the breast . it is well documented that tumor antigens are present in the lymphatic nodes nearest to the region of the neoplasm , either through the migration of malignant cells to the lymph nodes or by means of crossed presentation of molecules from the tumor on the antigen - presenting cells ( apc ) in the node . this mechanism triggers the immune effector function but also generates some degree of tolerance to the development of the tumor . during tumor expansion , the presentation of tumor - specific antigen to the cd8 + t - cells by the apcs in the lymph nodes seems to lead to the proliferation of transitory effector cells , which is insufficient to trigger the functional response of the cytotoxic t - cells . on the other hand , some studies demonstrate that changes in the expression of hla class i and ii molecules are related to early events in breast carcinogenesis and play an important part in the metastatic progression of breast idc . the deregulation of classical hla - i is strongly associated with human breast cancer metastasis . the occurrence of metastasis in lymph nodes is generally taken to indicate a poor outcome for the breast cancer and heterogeneous hla - i expression could be an additional sign of the preexistence and dissemination of the tumor . the heterogeneous expression of class mhc molecules , frequently involving reduced levels or the complete absence of hla - a , -b , and -c molecules , could reflect a major dissemination of tumor cells leading to metastasis of bone marrow and breast tumors . in the present study , however , no significant association could be demonstrated between deregulation of hla - i expression and metastasis in the axillary lymph nodes of patients suffering from idc of the breast . few studies have assessed the relation between hla expression and tumor size . in this study , an association was found between diminished expression of hla - i and increase in tumor size . larger breast tumors tend to be associated with worse patient outcomes and , when it is assessed , tumor size can be of use in predicting the evolution of the disease . although previous work has not revealed an association between immunohistochemical expression of the hla antigens and tumor size [ 5 , 10 ] , the size of the neoplasm can demonstrate that a tumor has acquired the capacity to evade destruction by the immune system . there is evidence that the modulation of the immune effector function is substantially weakened by the abnormal expression of growth factors or the loss of expression of antigens by the tumor . also , tumor cells are wrapped in stroma , whose extracellular matrix permits the anchoring of inflammatory cells , such as macrophages , granulocytes , and dendritic cells . the immune cells found in the stroma can produce tumorigenic factors , as well as assisting in the evasion of the immune response by preventing the dendritic cells from maturing . in the vicinity of a tumor , the t lymphocytes sometimes fail to migrate to the region because of the microenvironment created by the tumor itself , which strongly hinders t - cell migration . in addition to losing the anchoring of t lymphocytes , the stroma adjacent to neoplastic tissue may help the tumor cells to multiply faster by promoting angiogenesis . from the results reported here , it may be suggested that , in idcs with a large tumor mass , there is a diminution of hla - i expression , indicating the possible evasion of the immune response and thence a grave biological outcome . histological and nuclear grade , are histological parameters used to assess tumor differentiation , malignancy , prognosis and life - expectancy . their correlation with a panel of clinical and pathological parameters may indicate the less differentiated character of breast tumors and the low expression of hla - i has been closely associated with poorly differentiated tumors . the histological aggressiveness and staging of lesions are vital data for the prognosis of breast cancer . moreover , there is a consensus that nuclear grade , scored by a pathologist on the basis of the size and shape of the nucleus , can be used to predict the aggressiveness and potential for metastasis of malignant breast neoplasms . in the present study , an association was found between the reduced immunohistochemical expression of hla - i and the histological grade of the tumors [ 4 , 5 , 10 ] . cell atypia is strongly indicative of the cell disarray found in malignant tumors , while abnormalities in the expression of classical hla - i molecules have been described as a negative influence on the clinical course of the disease . thus , the present results demonstrate that larger lesions which show less histological differentiation exhibit anomalous hla - i expression , possibly allowing the tumor cells evade the immune system and promoting the development of breast idc . in this study , quantitative analysis of the expression of classical and nonclassical hla molecules revealed a negative correlation between hla - i and hla - g expression in idc tissue , where hla - i was found to be underexpressed and hla - g overexpressed . an association between raised levels of hla - g molecules and human carcinogenesis has already been noted and their expression by certain types of tumor is well documented , though some results are controversial [ 2031 ] . it is well established that hla - g molecules are expressed in trophoblast cells , thus warding off the host immune response so as to protect the fetus from attack by cytolytic cells . deregulation of hla - g expression seems to be a common occurrence , associated with downregulation of hla - i , and this may represent a pattern pointing to malignant transformation [ 33 , 34 ] . a target showing resistance to the hla - i mediated immune response in a tumor could exhibit a raised hla - g response and it has been postulated that abnormal expression of nonclassical hla molecules may be required to inhibit the signal to natural killer ( nk ) cells , making the neoplastic cells resist lysis and enabling them to avoid detection by the immune system . tumors can follow a number of paths to escape from nk cells and thus both of these molecules could work together to keep the tumor cells alive within their microenvironment , favoring their proliferation and malignant progression . an unexpected pattern of hla - ib expression was observed in this study , namely , low levels of hla - e molecules in idc lesions , combined with high levels of hla - g . generally , hla - g is not expressed on normal cells ( absence of malignance ) . due to this factor , it was possible to check in our study that memg-9 ab did stain in a significant number of tumor tissues . normally , hla - e surface expression is dependent on the availability of hla class i signal sequence - derived peptides . accordingly , hla - e surface expression is generally considered to be coexpressed with classical hla class i , which is expressed in the majority of healthy tissues [ 9 , 33 , 34 ] . few lesions were found to express hla - e and those that showed positive immunostaining did so at low intensity . the expression of hla - e had been expected to rise alongside that of hla - g , as had occurred previously in cells transformed by cytomegalovirus . hla - e is normally transcribed in various tissues , but is expressed weakly on the surface of the cells , where its stability depends on the coexpression of hla - c , hla - g , and hla - a molecules . the normal expression of class i hla molecules allows the hla - e complex to become stable and thus be expressed more strongly . it was evident in our study that the fall in hla - i expression , followed by overexpression of hla - g , resulted in the inhibition of hla - e expression . earlier research has demonstrated that there is competition between hla - e and class ia molecules for the 2 m ( light ) chains . malignant transformation frequently changes the heavy : light chain ratio and could thus modify the level of hla - e detected on the cell surface . in this situation , there is concomitantly a total loss , selective loss , or reduced expression of class i mhc molecules . in this study , when the expression of hla - g and hla - e was compared with the presence of anatomopathological features , no association was found between deregulation of these hlas and increase in tumor size , lower differentiation of the lesion , or axillary lymph node metastasis . it is possible that the higher expression of hla - g and lower expression of hla - e are not directly related to the aggressiveness or metastatic potential of the idc . to our knowledge , no other study has investigated a possible correlation between these clinical features and the expression of the nonclassical hla - g and hla - e molecules . regarding the expression of class ii molecules ( hla - dr and -dq ) , the present results demonstrate an association between low hla - dr and high hla - dq expression . an earlier study of hla - dr , -dq , and -dp expression in langerhans cells in cervical neoplasia reported that hla - dr expression was lower than that of hla - dq . hla - dr and -dq molecules have different functions in the induction of the immune response to tumors . hla - dr has a role in the proliferation of t - helper / inducer lymphocytes , while -dq mainly modulates the cytotoxic , suppressor , or both activities of t lymphocytes . contrary to the results of our study , the -dr and -dq responses have most frequently been seen as interdependent , meaning that the function of hla - dr is positively correlated with those of hla - dq and the levels of protein expression are similar . in the papers published to date on hla status in breast cancer , only hla - dr was assayed ( in class ii molecules ) and it was found to be expressed in low amounts , as was class i hla [ 5 , 40 ] , corroborating the present results . moreover , those authors pointed out that no association was observed between the anatomopathological features and hla - dr expression , as in this study . the expression of hla class ii molecules ( hla - dr and -dq ) in the present study demonstrated an association between low hla - dr and high hla - dq expression . since hla - dr has a role in the proliferation of t - helper / inducer lymphocytes and hla - dq mainly modulates the cytotoxic , suppressor , or both activities of t lymphocytes , it would be expected if there was , in addition to decreased expression of hla - dr , decreased expression of hla - dq , due to its ability to stimulate cytotoxicity in t lymphocyte . therefore , the low expression of hla - ia and hla - dr may suggest a mechanism of escape for idc of the breast from the t helper - mediated immune response . in addition , the rise in hla - dq levels could stimulate the suppressor t - cell response , assisting the tumor in evading immune recognition . currently it is established that her-2 ( human epidermal growth factor receptor-2 ) , estrogen receptor ( er ) , and progesterone receptor ( pr ) are the most commonly used biomarkers and therapeutic targets in breast cancer patients . however , these biomarkers are not expressed in 1730% of women with breast cancer which restricts the use of existing therapies . the triple negative breast cancer phenotype , which means tumors that are negative for her-2 , er , and pr , is even more aggressive and resistant [ 41 , 42 ] . current evidence has plainly established the heterogeneity of cancer and consequently demonstrates the role of other molecules involved in the behavior of breast cancer . assessment of the expression of hla - ia , ib ( -g and -e ) , and ii ( -dr and -dq ) molecules in axillary lymph node metastases has very rarely been carried out in previous studies of various kinds of tumor . in this study , a similar pattern of response was observed in the metastases and primary tumors , indicating that the low expression of hla - ia ( -a , -b , and -c ) and hla - ii ( -dr ) molecules and the high levels of hla - g should be related to both primary tumor carcinogenesis and the metastatic capacity of breast idc . in conclusion , the results reported here suggest that the metastatic capacity of this tumor is associated with deregulation of classical and nonclassical hla molecules , which results in its evasion of the host immune system , enabling the tumor to form metastases in the axillary lymph nodes .

considering that downregulation of hla expression could represent a potential mechanism for breast carcinogenesis and metastasis , the aim of the present study was to use immunohistochemical methods to analyze the expression of hla - ia , hla - dr , hla - dq , hla - e , and hla - g in invasive ductal carcinoma ( idc ) of the breast and to relate this hla profile to anatomopathological parameters . fifty - two idc from breast biopsies were stratified according to histological differentiation ( well , moderately , and poorly differentiated ) and to the presence of metastases in axillary lymph nodes . the expression of hla molecules was assessed by immunohistochemistry , using a computer - assisted system . overall , 31 ( 59.6% ) out of the 52 idc breast biopsies exhibited high expression of hla - g , but only 14 ( 26.9% ) showed high expression of hla - e . a large number ( 41 , 78.8% ) of the biopsies showed low expression of hla - ia , while 45 ( 86.5% ) showed high expression of hla - dq and 36 ( 69.2% ) underexpressed hla - dr . moreover , 24 ( 41.2% ) of 52 biopsies had both low hla - ia expression and high hla - g expression , while 11 ( 21.2% ) had low hla - ia expression and high hla - e expression . these results suggest that , by different mechanisms , the downregulation of hla - ia , hla - e , and hla - dr and the upregulation of hla - g and hla - dq are associated with immune response evasion and breast cancer aggressiveness .

1. Introduction 2. Material and Methods 3. Results 4. Discussion

hla class ii molecules ( hla - dr and hla - dq ) are essential for peptide presentation to t - helper lymphocytes , and their expression may be responsible for triggering the immune response . the class i human major histocompatibility complex ( mhc ) genes encode both the classical ( extremely polymorphic ) hla - ia ( hla - a , -b and -c ) molecules and the nonclassical hla - ib ( -e , -f and -g ) molecules , characterized by low allelic polymorphism , limited tissue distribution , and the presence of membrane - bound and soluble isoforms . hla - g and hla - e expression at the tumor cell surface might allow it to escape t and natural killer ( nk ) cell immune surveillance . the purpose of the present study was to analyze the relation between hla - ia , hla - ib , and classical hla - ii ( -dq , -dr ) expression in idc of the breast and breast cancer aggressiveness and metastatic tumor behavior . fifty - two idc of the breast biopsies were analyzed by hematoxylin / eosin staining methods and stratified according to histological differentiation ( well , moderately , and poorly differentiated ) and the presence of metastasis in axillary lymph nodes . a total of 52 formalin - fixed and paraffin - embedded biopsies of idc of the breast were collected from 52 patients and stratified according to lesion grade . in the case of breast carcinoma with metastasis ( 16 patients ) , one biopsy of the respective axillary lymph node immunohistochemical tests with the streptavidin - biotin system ( ep - usa/500 , signet , usa ) were carried out , to detect the hla - ia , hla - dq , hla - dr , hla - g , and hla - e antigens . slides were incubated with the primary monoclonal antibodies ( mabs ) for hla - ia ( ab70328 , diluted 1 : 50 ; abcam , cambridge , england ) , hla - dq ( ab55158 , diluted 1 : 50 ; abcam , cambridge , england ) , hla - dr ( ab175085 , diluted 1 : 50 ; abcam , cambridge , england ) , hla - g ( 5a6g7 , diluted 1 : 50 ; exbio , prague , czech republic ) , and for hla - e ( mem - e/02 , diluted 1 : 50 ; exbio , prague , czech republic ) in a humidified chamber at 4c overnight and then incubated with the streptavidin - peroxidase complex at 37c for 30 min . on each section , the mean number of positive - staining membranes was counted for both hla - g and hla - e immunostaining and classified as negative ( absence of immunolabelling to 25% positivity ) and positive ( 25% to 100% positivity ) . for hla - ia , hla - dq , and hla - dr , immunostaining was classified as negative ( absence of immunolabeling to 10% positivity ) and positive ( 10% to 100% positivity ) ( figure 1 ) . image analysis of tissues subjected to immunohistochemistry to determine the expression of hla - ia , hla - dr , hla - dq , hla - g , and hla - e was performed in the image analyzer image - pro plus ( md , usa ) . thus , the average number labeled cells / biopsy of the 52 biopsies was determined and established the average percentage of positivity . to validate the anti - hla - ia , anti - hla - dq , and anti - hla - dr mabs and the immunohistochemical method , we systematically analyzed a paraffin - embedded section of human tonsil ( positive control ) . to validate the anti - hla - g and anti - hla - e mabs and the immunohistochemical method , a paraffin - embedded section of trophoblastic tissue from a third - trimester human placenta ( positive control ) data were analyzed statistically with the program instat mac 2.01 ( graphpad , san diego , ca , usa ) . to analyze the quantitative expression of hla molecules , the nonparametric distribution kruskal - wallis test was used , followed by the dunn test for multiple comparison between pairs among the groups . the test and , when appropriate , fisher 's exact test were used to compare the quantitative immunohistochemistry results for hla molecules , tumor size , histological grade , nuclear grade , and presence or otherwise of metastases in axillary lymph nodes . based on the tolerogenic functions of breast tumors , we investigated possible associations between the expression of hla class ia , class ib ( hla - g and hla - e ) , and class ii ( hla - dq and hla - dr ) in breast tissue ( figure 1 ) . a correlation was detected between low expression of hla - ia and high expression of hla - g ( p < 0.001 ) , likewise between hla - g high expression and hla - e low expression ( p < 0.01 ) . no relation was found between hla - ia and hla - e expression ( p > 0.05 ) . hla - dr was significantly less expressed in idc lesions than hla - g and hla - dq molecules ( p < 0.01 ) but at the same level as hla class ia and hla - e . when hla - dq was compared with the other hla molecules , it was found that its expression was raised in the invasive tumors a little more than the level of hla - g . there was a significant difference between expression of hla - dr and hla - dq ( p < 0.001 ) that could indicate an association between high levels of hla - dq and low levels of hla - dr expression . the relationships between hla - ia , hla - ii , hla - g , and hla - e immunohistochemical expression and axillary lymph node metastasis are demonstrated in figure 3(a ) . no association was found between hla - ia ( p = 0.2830 ; p > 0.05 ) , hla - g high expression ( p = 0.9512 ; p > 0.05 ) , hla - e ( p = 0.3963 ; p > 0.05 ) , hla - dr ( p = 0.3010 ; p > 0.05 ) , and hla - dq ( p = 0.6894 ; p > 0.05 ) and metastases in axillary lymph nodes of idc patients . tumors larger than 2 cm showed low hla - i expression in 53.8% of the cases ; on the other hand , tumors classified as positive for hla - i were observed in 3.8% of patients with a tumor < 2 cm . thus , the results suggest that bigger lesions , possibly with higher proliferative activity , showed lower hla - i expression . for the hla - e molecules , hla - e low expression was observed in 32.7% of the smaller lesions and in 42.3% of the larger . however , no association was observed between hla - g ( p = 0.5801 ) and hla - e ( p = 0.1826 ) expression and tumor size . in the hla class ii results , no association was found between levels of expression ( hla - dr ; p = 1.000 and hla - dq ; p = 0.4420 ; p > 0.05 ) and tumor size , suggesting that there is no modification of the hla class ii levels with increasing tumor mass , an indirect indicator of tumor proliferation . the results for hla class i , class ii , hla - g and hla - e , and histological grade ( hg ) are demonstrated in figure 3(c ) . among patients with low expression of hla - i , greater histological disorganization ( hg2 + hg3 ) was observed in 75.0% , compared to 3.8% patients with more organized histological lesions ( hg1 ) . thus , a relationship can be seen between hla - i low expression and less histological differentiation . higher hla - g expression and greater histological disorganization ( hg2 + hg3 ) were observed in 55.7% , while hla - e high expression and lower histological differentiation ( hg2 + hg3 ) were observed in 25.0% of idc patients . on the other hand , low hla - g and hla - e expression was observed in the less differentiated lesions in , respectively , 40.4% and 71.2% of patients . however , no significant correlation was observed between lower histological differentiation and hla - g regulation ( p = 0.2695 ) or hla - e downregulation ( p = 0.7070 ) . the hla class i , class ii ( dr and dq ) , hla - g and hla - e immunohistochemical results , and cellular atypia levels of idc of the breast , classified as nuclear grade , are shown in figure 3(d ) . lower expression of hla - i ( p = 0.0071 , p < 0.05 ) was observed in the higher nuclear atypia lesions ( ng2 + ng3 ) in 78.8% patients . hla - g and hla - e high expression was observed in higher nuclear atypia lesions ( ng2 + ng3 ) in , respectively , 53.8% and 21% patients . on the other hand , hla - g and hla - e low expression was seen in , respectively , 38.5% and 71.2% of the idc analyzed . in the same way , no relation was found between lesser cellular differentiation and hla - g downregulation ( p = 0.4086 ) and hla - e downregulation ( p = 0.0661 ) . no significant association between hla class ii dr ( p = 0.5621 ) and dq ( p = 1.000 ) expression and the cellular atypia of idc of the breast was observed , apparently indicating that the expression of neither molecule was altered by cellular atypia . to assess the similarities or differences in the expression of all hla molecules investigated in the breast tumors and in their metastases in axillary lymph nodes , hla class i , class ii ( dr and dq ) , hla - g , and hla - e expression was assayed in the 16 lymph nodes of the patients that presented metastases ( figure 4 ) . from these results , it can be seen that hla class i , class ii ( dr and dq ) and hla - e low expression and hla - g ( p = 0.0091 , p < 0.05 ) high expression , in metastases in the lymph nodes , showed similar expression to that in the breast lesions . the results suggest a reduction in the expression of classical hla molecules and an increase in hla - g expression , related to the capacity for metastasis of this breast invasive tumor in axillary lymph nodes . notwithstanding this , the same types of tumor cell may develop the ability to evade such immune control , by means of the loss or low expression of hla class i molecules . decreased or absent hla - i molecules expression has frequently been observed in a range of malignant neoplasms , unlike what occurs in normal breast tissue , in which the expression of hla - ia occurs [ 79 ] . however , the relationship between hla - i expression and the process of carcinogenesis and metastatic progression of breast cancer has yet to be established with any certainty . the few studies carried out on this question have produced controversial results with regard to the deregulation of the expression of hla - ia and the potential for metastasis of the invasive ductal carcinoma of the breast . during tumor expansion , the presentation of tumor - specific antigen to the cd8 + t - cells by the apcs in the lymph nodes seems to lead to the proliferation of transitory effector cells , which is insufficient to trigger the functional response of the cytotoxic t - cells . on the other hand , some studies demonstrate that changes in the expression of hla class i and ii molecules are related to early events in breast carcinogenesis and play an important part in the metastatic progression of breast idc . the occurrence of metastasis in lymph nodes is generally taken to indicate a poor outcome for the breast cancer and heterogeneous hla - i expression could be an additional sign of the preexistence and dissemination of the tumor . the heterogeneous expression of class mhc molecules , frequently involving reduced levels or the complete absence of hla - a , -b , and -c molecules , could reflect a major dissemination of tumor cells leading to metastasis of bone marrow and breast tumors . in the present study , however , no significant association could be demonstrated between deregulation of hla - i expression and metastasis in the axillary lymph nodes of patients suffering from idc of the breast . from the results reported here , it may be suggested that , in idcs with a large tumor mass , there is a diminution of hla - i expression , indicating the possible evasion of the immune response and thence a grave biological outcome . their correlation with a panel of clinical and pathological parameters may indicate the less differentiated character of breast tumors and the low expression of hla - i has been closely associated with poorly differentiated tumors . in the present study , an association was found between the reduced immunohistochemical expression of hla - i and the histological grade of the tumors [ 4 , 5 , 10 ] . cell atypia is strongly indicative of the cell disarray found in malignant tumors , while abnormalities in the expression of classical hla - i molecules have been described as a negative influence on the clinical course of the disease . thus , the present results demonstrate that larger lesions which show less histological differentiation exhibit anomalous hla - i expression , possibly allowing the tumor cells evade the immune system and promoting the development of breast idc . in this study , quantitative analysis of the expression of classical and nonclassical hla molecules revealed a negative correlation between hla - i and hla - g expression in idc tissue , where hla - i was found to be underexpressed and hla - g overexpressed . deregulation of hla - g expression seems to be a common occurrence , associated with downregulation of hla - i , and this may represent a pattern pointing to malignant transformation [ 33 , 34 ] . a target showing resistance to the hla - i mediated immune response in a tumor could exhibit a raised hla - g response and it has been postulated that abnormal expression of nonclassical hla molecules may be required to inhibit the signal to natural killer ( nk ) cells , making the neoplastic cells resist lysis and enabling them to avoid detection by the immune system . normally , hla - e surface expression is dependent on the availability of hla class i signal sequence - derived peptides . the expression of hla - e had been expected to rise alongside that of hla - g , as had occurred previously in cells transformed by cytomegalovirus . hla - e is normally transcribed in various tissues , but is expressed weakly on the surface of the cells , where its stability depends on the coexpression of hla - c , hla - g , and hla - a molecules . the normal expression of class i hla molecules allows the hla - e complex to become stable and thus be expressed more strongly . it was evident in our study that the fall in hla - i expression , followed by overexpression of hla - g , resulted in the inhibition of hla - e expression . in this study , when the expression of hla - g and hla - e was compared with the presence of anatomopathological features , no association was found between deregulation of these hlas and increase in tumor size , lower differentiation of the lesion , or axillary lymph node metastasis . it is possible that the higher expression of hla - g and lower expression of hla - e are not directly related to the aggressiveness or metastatic potential of the idc . to our knowledge , no other study has investigated a possible correlation between these clinical features and the expression of the nonclassical hla - g and hla - e molecules . regarding the expression of class ii molecules ( hla - dr and -dq ) , the present results demonstrate an association between low hla - dr and high hla - dq expression . an earlier study of hla - dr , -dq , and -dp expression in langerhans cells in cervical neoplasia reported that hla - dr expression was lower than that of hla - dq . hla - dr and -dq molecules have different functions in the induction of the immune response to tumors . contrary to the results of our study , the -dr and -dq responses have most frequently been seen as interdependent , meaning that the function of hla - dr is positively correlated with those of hla - dq and the levels of protein expression are similar . in the papers published to date on hla status in breast cancer , only hla - dr was assayed ( in class ii molecules ) and it was found to be expressed in low amounts , as was class i hla [ 5 , 40 ] , corroborating the present results . moreover , those authors pointed out that no association was observed between the anatomopathological features and hla - dr expression , as in this study . the expression of hla class ii molecules ( hla - dr and -dq ) in the present study demonstrated an association between low hla - dr and high hla - dq expression . since hla - dr has a role in the proliferation of t - helper / inducer lymphocytes and hla - dq mainly modulates the cytotoxic , suppressor , or both activities of t lymphocytes , it would be expected if there was , in addition to decreased expression of hla - dr , decreased expression of hla - dq , due to its ability to stimulate cytotoxicity in t lymphocyte . therefore , the low expression of hla - ia and hla - dr may suggest a mechanism of escape for idc of the breast from the t helper - mediated immune response . assessment of the expression of hla - ia , ib ( -g and -e ) , and ii ( -dr and -dq ) molecules in axillary lymph node metastases has very rarely been carried out in previous studies of various kinds of tumor . in this study , a similar pattern of response was observed in the metastases and primary tumors , indicating that the low expression of hla - ia ( -a , -b , and -c ) and hla - ii ( -dr ) molecules and the high levels of hla - g should be related to both primary tumor carcinogenesis and the metastatic capacity of breast idc . in conclusion , the results reported here suggest that the metastatic capacity of this tumor is associated with deregulation of classical and nonclassical hla molecules , which results in its evasion of the host immune system , enabling the tumor to form metastases in the axillary lymph nodes .

human leukocyte antigen ( hla ) class i molecules have a central role in the cell - mediated immune system , especially as antigen - presenting molecules for cytotoxic t lymphocytes ( ctls ) , which can recognize tumor antigenic bound peptides , presented on the cell surface with hla class i molecules , and kill the target cell [ 2 , 3 ] . i expression seems to be lost or downregulated on the tumor cell surface and this might represent a mechanism for neoplastic cells to escape from being killed by ctls , allowing tumor dissemination and metastasis . hla class ii molecules ( hla - dr and hla - dq ) are essential for peptide presentation to t - helper lymphocytes , and their expression may be responsible for triggering the immune response . however , it has been proposed that hla - dr molecules offer protection against nk cell cytotoxicity , as has been described for hla class i antigens . the class i human major histocompatibility complex ( mhc ) genes encode both the classical ( extremely polymorphic ) hla - ia ( hla - a , -b and -c ) molecules and the nonclassical hla - ib ( -e , -f and -g ) molecules , characterized by low allelic polymorphism , limited tissue distribution , and the presence of membrane - bound and soluble isoforms . hla - g and hla - e expression at the tumor cell surface might allow it to escape t and natural killer ( nk ) cell immune surveillance . the purpose of the present study was to analyze the relation between hla - ia , hla - ib , and classical hla - ii ( -dq , -dr ) expression in idc of the breast and breast cancer aggressiveness and metastatic tumor behavior . fifty - two idc of the breast biopsies were analyzed by hematoxylin / eosin staining methods and stratified according to histological differentiation ( well , moderately , and poorly differentiated ) and the presence of metastasis in axillary lymph nodes . in the case of breast carcinoma with metastasis ( 16 patients ) , one biopsy of the respective axillary lymph node immunohistochemical tests with the streptavidin - biotin system ( ep - usa/500 , signet , usa ) were carried out , to detect the hla - ia , hla - dq , hla - dr , hla - g , and hla - e antigens . on each section , the mean number of positive - staining membranes was counted for both hla - g and hla - e immunostaining and classified as negative ( absence of immunolabelling to 25% positivity ) and positive ( 25% to 100% positivity ) . for hla - ia , hla - dq , and hla - dr , immunostaining was classified as negative ( absence of immunolabeling to 10% positivity ) and positive ( 10% to 100% positivity ) ( figure 1 ) . image analysis of tissues subjected to immunohistochemistry to determine the expression of hla - ia , hla - dr , hla - dq , hla - g , and hla - e was performed in the image analyzer image - pro plus ( md , usa ) . to validate the anti - hla - ia , anti - hla - dq , and anti - hla - dr mabs and the immunohistochemical method , we systematically analyzed a paraffin - embedded section of human tonsil ( positive control ) . to validate the anti - hla - g and anti - hla - e mabs and the immunohistochemical method , a paraffin - embedded section of trophoblastic tissue from a third - trimester human placenta ( positive control ) data were analyzed statistically with the program instat mac 2.01 ( graphpad , san diego , ca , usa ) . to analyze the quantitative expression of hla molecules , the nonparametric distribution kruskal - wallis test was used , followed by the dunn test for multiple comparison between pairs among the groups . based on the tolerogenic functions of breast tumors , we investigated possible associations between the expression of hla class ia , class ib ( hla - g and hla - e ) , and class ii ( hla - dq and hla - dr ) in breast tissue ( figure 1 ) . a correlation was detected between low expression of hla - ia and high expression of hla - g ( p < 0.001 ) , likewise between hla - g high expression and hla - e low expression ( p < 0.01 ) . hla - dr was significantly less expressed in idc lesions than hla - g and hla - dq molecules ( p < 0.01 ) but at the same level as hla class ia and hla - e . when hla - dq was compared with the other hla molecules , it was found that its expression was raised in the invasive tumors a little more than the level of hla - g . there was a significant difference between expression of hla - dr and hla - dq ( p < 0.001 ) that could indicate an association between high levels of hla - dq and low levels of hla - dr expression . the relationships between hla - ia , hla - ii , hla - g , and hla - e immunohistochemical expression and axillary lymph node metastasis are demonstrated in figure 3(a ) . no association was found between hla - ia ( p = 0.2830 ; p > 0.05 ) , hla - g high expression ( p = 0.9512 ; p > 0.05 ) , hla - e ( p = 0.3963 ; p > 0.05 ) , hla - dr ( p = 0.3010 ; p > 0.05 ) , and hla - dq ( p = 0.6894 ; p > 0.05 ) and metastases in axillary lymph nodes of idc patients . tumors larger than 2 cm showed low hla - i expression in 53.8% of the cases ; on the other hand , tumors classified as positive for hla - i were observed in 3.8% of patients with a tumor < 2 cm . for the hla - e molecules , hla - e low expression was observed in 32.7% of the smaller lesions and in 42.3% of the larger . however , no association was observed between hla - g ( p = 0.5801 ) and hla - e ( p = 0.1826 ) expression and tumor size . in the hla class ii results , no association was found between levels of expression ( hla - dr ; p = 1.000 and hla - dq ; p = 0.4420 ; p > 0.05 ) and tumor size , suggesting that there is no modification of the hla class ii levels with increasing tumor mass , an indirect indicator of tumor proliferation . the results for hla class i , class ii , hla - g and hla - e , and histological grade ( hg ) are demonstrated in figure 3(c ) . among patients with low expression of hla - i , greater histological disorganization ( hg2 + hg3 ) was observed in 75.0% , compared to 3.8% patients with more organized histological lesions ( hg1 ) . higher hla - g expression and greater histological disorganization ( hg2 + hg3 ) were observed in 55.7% , while hla - e high expression and lower histological differentiation ( hg2 + hg3 ) were observed in 25.0% of idc patients . on the other hand , low hla - g and hla - e expression was observed in the less differentiated lesions in , respectively , 40.4% and 71.2% of patients . however , no significant correlation was observed between lower histological differentiation and hla - g regulation ( p = 0.2695 ) or hla - e downregulation ( p = 0.7070 ) . the hla class i , class ii ( dr and dq ) , hla - g and hla - e immunohistochemical results , and cellular atypia levels of idc of the breast , classified as nuclear grade , are shown in figure 3(d ) . lower expression of hla - i ( p = 0.0071 , p < 0.05 ) was observed in the higher nuclear atypia lesions ( ng2 + ng3 ) in 78.8% patients . only 21.1% of the patients did not show hla - i downregulation , classified in this study as a positive response , and of these patients 7.7% presented lower nuclear atypia ( ng1 ) . on the other hand , hla - g and hla - e low expression was seen in , respectively , 38.5% and 71.2% of the idc analyzed . no significant association between hla class ii dr ( p = 0.5621 ) and dq ( p = 1.000 ) expression and the cellular atypia of idc of the breast was observed , apparently indicating that the expression of neither molecule was altered by cellular atypia . to assess the similarities or differences in the expression of all hla molecules investigated in the breast tumors and in their metastases in axillary lymph nodes , hla class i , class ii ( dr and dq ) , hla - g , and hla - e expression was assayed in the 16 lymph nodes of the patients that presented metastases ( figure 4 ) . from these results , it can be seen that hla class i , class ii ( dr and dq ) and hla - e low expression and hla - g ( p = 0.0091 , p < 0.05 ) high expression , in metastases in the lymph nodes , showed similar expression to that in the breast lesions . the results suggest a reduction in the expression of classical hla molecules and an increase in hla - g expression , related to the capacity for metastasis of this breast invasive tumor in axillary lymph nodes . notwithstanding this , the same types of tumor cell may develop the ability to evade such immune control , by means of the loss or low expression of hla class i molecules . decreased or absent hla - i molecules expression has frequently been observed in a range of malignant neoplasms , unlike what occurs in normal breast tissue , in which the expression of hla - ia occurs [ 79 ] . however , the relationship between hla - i expression and the process of carcinogenesis and metastatic progression of breast cancer has yet to be established with any certainty . the few studies carried out on this question have produced controversial results with regard to the deregulation of the expression of hla - ia and the potential for metastasis of the invasive ductal carcinoma of the breast . it is well documented that tumor antigens are present in the lymphatic nodes nearest to the region of the neoplasm , either through the migration of malignant cells to the lymph nodes or by means of crossed presentation of molecules from the tumor on the antigen - presenting cells ( apc ) in the node . during tumor expansion , the presentation of tumor - specific antigen to the cd8 + t - cells by the apcs in the lymph nodes seems to lead to the proliferation of transitory effector cells , which is insufficient to trigger the functional response of the cytotoxic t - cells . on the other hand , some studies demonstrate that changes in the expression of hla class i and ii molecules are related to early events in breast carcinogenesis and play an important part in the metastatic progression of breast idc . the occurrence of metastasis in lymph nodes is generally taken to indicate a poor outcome for the breast cancer and heterogeneous hla - i expression could be an additional sign of the preexistence and dissemination of the tumor . the heterogeneous expression of class mhc molecules , frequently involving reduced levels or the complete absence of hla - a , -b , and -c molecules , could reflect a major dissemination of tumor cells leading to metastasis of bone marrow and breast tumors . in the present study , however , no significant association could be demonstrated between deregulation of hla - i expression and metastasis in the axillary lymph nodes of patients suffering from idc of the breast . although previous work has not revealed an association between immunohistochemical expression of the hla antigens and tumor size [ 5 , 10 ] , the size of the neoplasm can demonstrate that a tumor has acquired the capacity to evade destruction by the immune system . there is evidence that the modulation of the immune effector function is substantially weakened by the abnormal expression of growth factors or the loss of expression of antigens by the tumor . the immune cells found in the stroma can produce tumorigenic factors , as well as assisting in the evasion of the immune response by preventing the dendritic cells from maturing . in the vicinity of a tumor , the t lymphocytes sometimes fail to migrate to the region because of the microenvironment created by the tumor itself , which strongly hinders t - cell migration . from the results reported here , it may be suggested that , in idcs with a large tumor mass , there is a diminution of hla - i expression , indicating the possible evasion of the immune response and thence a grave biological outcome . their correlation with a panel of clinical and pathological parameters may indicate the less differentiated character of breast tumors and the low expression of hla - i has been closely associated with poorly differentiated tumors . moreover , there is a consensus that nuclear grade , scored by a pathologist on the basis of the size and shape of the nucleus , can be used to predict the aggressiveness and potential for metastasis of malignant breast neoplasms . in the present study , an association was found between the reduced immunohistochemical expression of hla - i and the histological grade of the tumors [ 4 , 5 , 10 ] . cell atypia is strongly indicative of the cell disarray found in malignant tumors , while abnormalities in the expression of classical hla - i molecules have been described as a negative influence on the clinical course of the disease . thus , the present results demonstrate that larger lesions which show less histological differentiation exhibit anomalous hla - i expression , possibly allowing the tumor cells evade the immune system and promoting the development of breast idc . in this study , quantitative analysis of the expression of classical and nonclassical hla molecules revealed a negative correlation between hla - i and hla - g expression in idc tissue , where hla - i was found to be underexpressed and hla - g overexpressed . an association between raised levels of hla - g molecules and human carcinogenesis has already been noted and their expression by certain types of tumor is well documented , though some results are controversial [ 2031 ] . deregulation of hla - g expression seems to be a common occurrence , associated with downregulation of hla - i , and this may represent a pattern pointing to malignant transformation [ 33 , 34 ] . a target showing resistance to the hla - i mediated immune response in a tumor could exhibit a raised hla - g response and it has been postulated that abnormal expression of nonclassical hla molecules may be required to inhibit the signal to natural killer ( nk ) cells , making the neoplastic cells resist lysis and enabling them to avoid detection by the immune system . an unexpected pattern of hla - ib expression was observed in this study , namely , low levels of hla - e molecules in idc lesions , combined with high levels of hla - g . hla - e is normally transcribed in various tissues , but is expressed weakly on the surface of the cells , where its stability depends on the coexpression of hla - c , hla - g , and hla - a molecules . it was evident in our study that the fall in hla - i expression , followed by overexpression of hla - g , resulted in the inhibition of hla - e expression . in this study , when the expression of hla - g and hla - e was compared with the presence of anatomopathological features , no association was found between deregulation of these hlas and increase in tumor size , lower differentiation of the lesion , or axillary lymph node metastasis . it is possible that the higher expression of hla - g and lower expression of hla - e are not directly related to the aggressiveness or metastatic potential of the idc . to our knowledge , no other study has investigated a possible correlation between these clinical features and the expression of the nonclassical hla - g and hla - e molecules . regarding the expression of class ii molecules ( hla - dr and -dq ) , the present results demonstrate an association between low hla - dr and high hla - dq expression . an earlier study of hla - dr , -dq , and -dp expression in langerhans cells in cervical neoplasia reported that hla - dr expression was lower than that of hla - dq . hla - dr has a role in the proliferation of t - helper / inducer lymphocytes , while -dq mainly modulates the cytotoxic , suppressor , or both activities of t lymphocytes . contrary to the results of our study , the -dr and -dq responses have most frequently been seen as interdependent , meaning that the function of hla - dr is positively correlated with those of hla - dq and the levels of protein expression are similar . in the papers published to date on hla status in breast cancer , only hla - dr was assayed ( in class ii molecules ) and it was found to be expressed in low amounts , as was class i hla [ 5 , 40 ] , corroborating the present results . the expression of hla class ii molecules ( hla - dr and -dq ) in the present study demonstrated an association between low hla - dr and high hla - dq expression . since hla - dr has a role in the proliferation of t - helper / inducer lymphocytes and hla - dq mainly modulates the cytotoxic , suppressor , or both activities of t lymphocytes , it would be expected if there was , in addition to decreased expression of hla - dr , decreased expression of hla - dq , due to its ability to stimulate cytotoxicity in t lymphocyte . therefore , the low expression of hla - ia and hla - dr may suggest a mechanism of escape for idc of the breast from the t helper - mediated immune response . assessment of the expression of hla - ia , ib ( -g and -e ) , and ii ( -dr and -dq ) molecules in axillary lymph node metastases has very rarely been carried out in previous studies of various kinds of tumor . in this study , a similar pattern of response was observed in the metastases and primary tumors , indicating that the low expression of hla - ia ( -a , -b , and -c ) and hla - ii ( -dr ) molecules and the high levels of hla - g should be related to both primary tumor carcinogenesis and the metastatic capacity of breast idc . in conclusion , the results reported here suggest that the metastatic capacity of this tumor is associated with deregulation of classical and nonclassical hla molecules , which results in its evasion of the host immune system , enabling the tumor to form metastases in the axillary lymph nodes .

drug and alcohol addiction is a major problem in western society where substance abuse is associated with considerable social , medical , and financial costs ( collins and lapsley , 2002 ) . it has been estimated that annually around $ 7.5 billion is associated with alcohol and alcohol - related problems in australia and about 5% of the total annual mortality is due to this ( collins and lapsley , 2002 ) . the australian institute of health and welfare estimated that in 2004 , more than 56,000 people had used opiates in the previous 12 months and around 1% of the population had used cocaine ( aihw 2005 ) . due to the impact on society and limited success of current therapies to treat addiction , extensive research into the pathophysiology underlying alcoholism and drug abuse is ongoing . in particular , research efforts are focused toward understanding the persistent vulnerability to relapse displayed by drug - addicted individuals despite months or even years of abstinence ( gossop et al . , 1989 ; dejong , 1994 ; jupp and lawrence , 2009 ) . current preclinical research into potential treatments for addiction is focused on gaining a greater understanding of the neurobiological mechanisms underpinning both relapse vulnerability and the transition from casual to compulsive drug use . at present there is a paucity of pharmacotherapies on the market for the treatment of addictions ; none of which are particularly effective and involve multiple off - target effects ( jupp and lawrence , 2009 ) . for this reason it is vital that new therapeutic targets are identified that have potential in this regard . complexes containing the metabotropic glutamate receptor type 5 ( mglu5 ) , and/or functional interactions between mglu5 and other g protein - coupled receptors ( gpcrs ) , represent a novel therapeutic option worthy of investigation due to the possibility of limiting off - target effects . mglu5 has been implicated in numerous central processes , thus targeting mglu5-containing complexes / functional receptor interactions that are restricted to addiction relevant brain areas provide an avenue to selectively target those mglu5 involved in reward and drug - seeking behavior ( bird and lawrence , 2009b ) . a variety of gpcrs have been shown to play critical roles in the reinforcing and motivational properties of drugs , drug - induced plasticity and drug - seeking behavior ( traynor , 2010 ) . a substantial body of evidence exists which implicates metabotropic glutamate receptors in many of these facets of drug addiction ( bird and lawrence , 2009a ; olive , 2009 ) and among them mglu5 seems to play a significant role . mglu5 belongs to a distinct family of eight gpcrs that are activated by l - glutamate , the major fast excitatory neurotransmitter in the central nervous system ( cns ) . at the signaling level , mglu5 is known to be associated with phosphoinositide hydrolysis and the activation of phospholipase c , stimulation of adenylate cyclase and inhibition of voltage - operated calcium channels ( dhami and ferguson , 2006 ) . signaling via mglu5 is thought to play a role in synaptic plasticity , ultimately affecting learning and memory , neuronal development and neurodegeneration ( lu et al . , 1997 ) . it is also thought to have a role in a number of behaviors including stress responses , anxiety - like , and depressive - like behavior ( tatarczynska et al . , 2001 ) . mglu5 is highly expressed in the olfactory bulb and tubercle , dorsal striatum , nucleus accumbens , cerebral cortex , hippocampal formation , and amygdala ( shigemoto et al . , 1993 ; romano et al . , 1995 ) . in the striatum , mglu5 is located primarily in the perisynaptic ring and at the postsynaptic density ( kennedy , 2000 ; smith et al . , 2000 ) , including striatal projection neurons containing enkephalin ( testa et al . , evidence of presynaptic localization of mglu5 on cortical inputs to the striatum also exists ( romano et al . , 1995 ; rodrigues et al . , 2005 ) . the mglu5 receptor is structurally linked to the nmda receptor via a protein scaffold ( naisbitt et al . , 1999 ) and functional interactions have been demonstrated between the two receptors ( attucci et al . , 2001 ) . thus , mglu5 receptors are perfectly positioned to influence the mesolimbic , corticotegmental , and corticostriatal pathways known to be critical for the actions of drugs of abuse and drug - induced plasticity . the first evidence to suggest a role for mglu5 in the rewarding properties of drugs of abuse ( 2001 ) who reported that mice lacking mglu5 failed to intravenously self - administer a range of doses of cocaine and failed to show any hyperactivity after acute administration of the drug . antagonism of mglu5 can reduce the self - administration of multiple drugs of abuse as well as reinstatement of drug - seeking , indicating a facilitatory role for this receptor in these behaviors ( olive , 2009 ) . for example , the mglu5 antagonists 2-methyl-6-(phenylethynyl)-pyridine ( mpep ) and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine ( mtep ) reduce self - administration of alcohol , cocaine , and nicotine in rats ( kenny et al . 2005 ) as well as alcohol and morphine in mice ( cowen et al . , 2007 ; brown et al . 2004 ) , cocaine- ( backstrom and hyytia , 2006 ) , nicotine- ( bespalov et al . , 2005 ) , opiate- ( brown et al . , 2011b ) , and methamphetamine - seeking ( gass et al . , 2009 ) in animal models of relapse , indicating a clear role for mglu5 in drug - seeking behavior across a broad range of reinforcers . genetic mouse models have also implicated mglu5 in the reinforcing and motivational properties of drugs of abuse . in addition to the original work by chiamulera and colleagues , deletion of the mglu5 gene results in altered central reward processing in the case of ethanol ( bird et al . , 2008 ) and an absence of drug - induced synaptic plasticity in the ventral tegmental area in the case of cocaine ( bird et al . , 2010 ) . thus , the putative involvement of mglu5 in addictive behavior suggests that selective inhibition of this receptor may provide an exciting new approach for treatment of addictive disorders . more recently evidence has been forthcoming which implicates mglu5 in associative reward learning processes more generally . published reports suggest systemic administration of the selective mglu5 antagonist mtep prior to conditioning disrupts the expression of conditioned reinforcement by food delivery ( oconnor et al . , 2010 ) . this suggests a critical role for mglu5 in the acquisition of incentive properties by a conditioned stimulus ( oconnor et al . , 2010 ) ; an observation which is supported by the finding that mice with knockdown of mglu5 in striatal d1-expressing neurons exhibit diminished cue - induced cocaine - seeking , as well as impaired incentive learning ( novak et al . , 2010 ) . hence mglu5 may play a central role in associative reward learning processes ( novak et al . , this possible role in incentive learning is not inconsistent with the involvement of mglu5 in cue - induced drug - seeking behavior ( backstrom et al . , 2004 ; backstrom and hyytia , 2006 ) . indeed , in the operant conditioning paradigm , the discrete cue is a conditioned stimulus and as such needs to acquire incentive motivational properties in order to elicit drug - seeking upon re - presentation . collectively , these studies provide further insight into the potentially complex role of mglu5 in both reward and reward learning . a role for mglu5 in extinction learning relevant to drug use and drug - associated cues has also been established by recent research using positive allosteric modulators of mglu5 ( cleva and olive , 2011 ) . the first mglu5 positive allosteric modulators to be characterized were 3-cyano - n-(1,3-diphenyl-1h - pyrazol-5-yl)benzamide ( cdppb ; lindsley et al . , 2004 ) and s-(4-fluoro - phenyl)[3-[3-(4-fluoro - phenyl)-1,2,4-oxadiazol-5-yl]piperidin-1-yl]methanone ( adx47273 ; liu et al . , 2008 ) and were suggested to be beneficial in alleviating the cognitive deficits associated with chronic drug abuse ( reichel et al . , 2011 ) . studies using animal models of addiction have also shown that cdppb facilitates extinction of both cocaine - conditioned place preference ( gass and olive , 2009 ) and operant responding for cocaine ( cleva et al . , this is supported by observations from our laboratory , which demonstrate deficits in extinction learning in mice lacking mglu5 ( lawrence laboratory , unpublished observations ) . therefore , positive allosteric modulators of mglu5 have potential use as pharmacological adjuncts to cue exposure therapy in the treatment of drug addiction , with the potential benefit of reversing cognitive deficits associated with chronic drug use ( cleva and olive , 2011 ) . recently , a range of compounds that selectively inhibit mglu5 function have been published and tested in various neurological disorder models and some of them exhibit good tolerability , long - term action , and oral bioavailability ( keywood et al . , 2009 ; zerbib et al . , 2010 ; lindemann et al . , 2011 ) . consequently , there is a promising future for the development of new treatments for addiction based on both positive and negative allosteric modulation of mglu5 . in order to maintain the speed and subtly of neuronal signal transmission , intramembrane protein protein interactions are likely to play a role ( vilardaga et al . , 2008 ) . consequently , researchers in the gpcr field are now focusing their efforts on understanding gpcr signaling and function in the context of higher - order structures and not just monomers . textbooks describing gpcr signaling as a linear cascade , where agonist activation results in the receptor coupling to one g protein and therefore the activation of one signaling pathway , are now being updated to include receptor homomers ( macromolecular complexes made up of two or more identical functional receptor units ) and receptor heteromers ( macromolecular complexes composed of at least two different functional receptor units with biochemical properties that are demonstrably different from those of their individual components ; ferre et al . , 2009 ) . heteromerization not only provides a potential mechanism for increased signaling diversity , but also specificity by , for example , allowing the development of drugs to target in a tissue selective manner and thereby minimize off - target effects ( mustafa et al . , 2010 ) . indeed , there are now numerous examples of physiologically relevant gpcr heteromers ( dalrymple et al . , 2008 ) , particularly in the cns ( albizu et al . , 2010 ) , and new methodologies are being developed to investigate such complexes ( mustafa et al . , 2010 ) . dopaminergic and glutamatergic inputs converge in the dendritic spines of -aminobutyric acid ( gaba)ergic medium spiny neurons ( totterdell and smith , 1989 ; sesack and pickel , 1992 ) which constitute more than 90% of the neuronal population of the striatum ( smith and bolam , 1990 ) . dopamine release in the striatum activates two classes of gpcrs that are expressed by distinct subpopulations of medium spiny neurons . dopamine d1 receptors ( d1r ) are localized on the striatonigral neurons of the direct pathway whereas dopamine d2 receptors ( d2r ) are expressed by the striatopallidal neurons of the indirect pathway ( gerfen , 1992 ; le moine and bloch , 1995 ) . d2rs are coupled to gi , and activation of these receptors triggers a cascade of signaling pathways , reducing camp production and protein kinase a ( pka ) activity and activating phospholipase c ( plc)-dependent processes as well as the release of ca from internal stores ( missale et al . , 1998 ) . d2r has been linked to a number of behavioral disorders , including parkinson s disease , obsessive compulsive disorder , addiction , and schizophrenia ( bonci and hopf , 2005 ; kreitzer and malenka , 2008 ) . treatments targeting dopaminergic signaling have utilized dopamine receptor agonists to alleviate symptoms of hypokinesia associated with parkinson s disease , and dopamine receptor antagonists to treat hallucinations and delusions in schizophrenic patients ( missale et al . , however , side effects have been associated with both therapies , presumably due to the difficulty in re - establishing the balance in dopamine signaling required for appropriate physiological function . adenosine is often described as a neuromodulator , which acts on a family of four adenosine receptors : a1 , a2a , a2b , and a3 receptors ( fredholm et al . , 1994 , 2001b ) . of the four known adenosine receptors , a1 and a2a receptors are primarily responsible for the central effects of adenosine ( fredholm et al . , 2001a ) . a1 receptors are widely distributed in the brain ( fastbom et al . , 1987 ; fredholm et al . , 2001a ) whereas a2a receptors are highly enriched in the terminal regions of ascending dopamine projections , concentrated in the dendritic spines of striatopallidal neurons , particularly in the vicinity of glutamatergic synapses ( fink et al . , 1992 ; svenningsson et al . , 1997 ; hettinger et al . , 2001 ; rosin et al . , 2003 ) . adenosine a2a receptors are also found presynaptically in glutamatergic , but not in dopaminergic , terminals although with lower density ( hettinger et al . , 2001 ; rosin et al . , 2003 therefore , it has been suggested that the a2a receptor plays a role in the fine - tuning modulation of glutamatergic neurotransmission onto striatal gabaergic neurons both at the postsynaptic and presynaptic level ( hettinger et al . , 2001 ; ciruela et al . recently , presynaptic a2a receptors were shown to be preferentially localized in cortical glutamatergic terminals that contact medium spiny neurons of the direct pathway ( quiroz et al . , 2009 ) , while their postsynaptic counterparts are located on striatopallidal neurons of the indirect pathway . thus adenosine acting at a2a receptors has the capacity to modulate neurotransmission of both output pathways of the basal ganglia . signaling via the a2a receptor has been implicated in the reinforcing properties of multiple drugs of abuse ( brown and short , 2008 ) . for example , in mice lacking the a2a receptor , a reduction in self - administration of morphine has been reported ( brown et al . , 2009 ) . similarly , a2ar antagonists have also been reported to reduce alcohol - seeking and opiate - seeking behavior in rats ( arolfo et al . , 2004 ; yao et al . , 2006 ) . a remarkable feature of the two striatal output pathways is the segregation of adenosine and dopamine receptors . gabaergic / enkephalinergic neurons predominantly express adenosine a2a ( stimulatory , coupled to gs / golf ) and dopamine d2 ( inhibitory , coupled to gi ) receptors , while gabaergic / dynorphinergic neurons predominantly express adenosine a1 ( inhibitory , coupled to gi ) and dopamine d1 ( stimulatory , coupled to gs ) receptors ( schiffmann et al . , 2007 ) it is not surprising therefore , that evidence has accumulated documenting interactions between these gpcrs in relation to striatal gabaergic efferent neuron function ( ferre et al . these interactions have been reported to include direct receptor receptor interactions as well as interactions at the second messenger and behavioral levels , all of which have been reviewed elsewhere ( ferre et al . , 1994 , 2004 ; fredholm and svenningsson , 2003 ; fuxe et al . , 2003 ) . d2 interaction is predominantly found to be antagonistic as activation of a2ar reduces d2r binding , coupling , signaling as well as behavioral effects ; thus d2r exerts a tonic inhibition on striatopallidal neurons which a2ar activation antagonizes ( see reviews above ) . evidence for the existence of heteromers containing a2a and d2 receptors in expression systems has been provided by means of co - immunoprecipitation and resonance energy transfer ( ret ) techniques such as bioluminescence - ret ( bret ) and fluorescence - ret ( fret ; canals et al . ultrastructural and confocal analysis indicate this to be mainly on dendritic spines in the perisynaptic zones of dopamine terminals and glutamatergic synapses in striatopallidal neurons ( fuxe et al . , 2010 ) . in addition , interactions involving the a2ar have been described for other receptors , including adenosine a1 , cannabinoid cb1 and , most relevant to this review , mglu5 ( ferre et al . in the striatum mglu5 is expressed postsynaptically on both types of striatal gabaergic efferent neuron and potentially presynaptically on glutamatergic terminals ( romano et al . , 1995 ; tallaksen - greene et al . , 1998 ; similar to a2ar , mglu5 is predominantly found postsynaptically and perisynaptically to asymmetric synapses ( smith et al . , 2000 ) . ( 2002 ) to investigate the possibility of the existence of heteromers containing these two receptors . thus , co - localization of a2ar and mglu5 at the membrane level was demonstrated by confocal laser microscopy in co - transfected human embryonic kidney ( hek)293 cells . in addition , western blotting has provided evidence for complexes containing a2ar and mglu5 in membrane preparations from both co - transfected hek293 cells and rat striatal tissue ( ferre et al . , 2002 ) . binding of the a2a receptor antagonist [ i]zm241385 in mouse striatum is reduced as a result of pretreatment with mglu5 antagonist mtep ( brown et al . , 2011a ) , providing a neurochemical correlate for a possible allosteric interaction between these receptors . potential for cross - talk also exists between the receptors signal transduction pathways ( agnati et al . , 2003 ) , and there is functional evidence of a relationship between mglu5 and a2ar to this effect . firstly , an apparent interdependence exists between the actions of a2ar and mglu5 in the phosphorylation of dopamine and camp - regulated phosphoprotein of 32,000 kda ( darpp-32 ) at thr-34 ( nishi et al . , 2003 ) . thus , a2ar antagonism attenuates the ability of mglu5 activation to phosphorylate darpp-32 at thr-34 in mouse striatal slices and vice versa . in addition , co - activation of mglu5 and a2ar synergistically increases phosphorylation of darpp-32 at thr-34 , apparently via an extracellular signal - regulated kinase ( erk)1/2-dependent mechanism involving increases in camp formation ( nishi et al . , 2003 ) . these findings are consistent with an erk1/2-dependent synergistic interaction between a2ar and mglu5 with respect to c - fos expression in hek293 cells ( ferre et al . , 2002 ) . collectively , these data have led some researchers in the field to suggest that the existence of this a2ar mglu5 heteromer may provide a point of temporal and spatial convergence of gs and gq signaling onto the mapk cascade , thereby providing the capacity to influence striatal plasticity ( agnati et al . , 2010a ) , thus raising the possibility of the involvement of this heteromer in plasticity resulting from repeated exposure to drugs of abuse . of interest is the observation that both a2ar and mglu5 have been shown to play a role in corticostriatal plasticity ( dalcantara et al . in vivo microdialysis experiments have shown that perfusion with the a2ar agonist 2-[4-(2-carboxy ethyl)phenethylamine-50-n - ethylcarboxamidoadenosine ( cgs21680 ) potentiates the facilitation of gaba release from striatopallidal neurons by the mglu5 agonist ( rs)-2-chloro-5-hydroxyphenylglycine ( chpg ; diaz - cabiale et al . , 2002 ) . in addition , a2ar and mglu5 have been shown to interact synergistically to modulate glutamate release from corticostriatal afferents ( rodrigues et al . , 2005 ) . submaximal concentrations of the a2ar agonist cgs21680 and mglu5 agonist chpg have been shown to synergistically facilitate glutamate release , with the cgs21680-facilitated release prevented by mpep . conversely , facilitation of glutamate release by chpg is prevented by the a2ar antagonist sch58261 ( rodrigues et al . , 2005 ) . these results demonstrate that in addition to modulating striatal output , mglu5a2ar interactions also play a role in regulating cortical input into the striatum , providing an additional avenue to influence striatal function and ultimately behavior . indeed , behavioral interactions between a2ar and mglu5 have been reported , particularly with respect to motor behaviors due to the strong interest in these receptors for their potential in the treatment of parkinson s disease . for example , central co - administration of cgs21680 and chpg has been found to reduce the motor activity induced by phencyclidine at doses that have no effect alone ( ferre et al . , 2002 ) . furthermore , in both normal and reserpinized mice , locomotion is enhanced in a synergistic manner with combined treatment of mpep and the a2ar antagonist kw-6002 ( kachroo et al . , 2005 ) . similarly , akinetic symptoms of 6-hydroxydopamine - lesioned rats are improved with combinations of sub - threshold doses of mglu5 and a2ar antagonists ( coccurello et al . , 2004 ) . mglu5 interaction is also relevant to reward and drug - seeking behavior ( discussed in subsequent section ) . recently , co - immunoprecipitation experiments in native striatal tissue provided evidence for the existence of a higher - order oligomeric complex containing mglu5 , d2r , and a2ar , localized to the dendritic spines of striatopallidal neurons ( cabello et al . , 2009 ) . early studies reported synergy between a2ar and mglu5 in reducing dopamine d2r - mediated behaviors such as phencyclidine - induced hyperactivity ( ferre et al . , 2002 ) and dopamine signaling through darpp-32 ( nishi et al . , 2003 ) . in rat striatal membranes , the a2ar agonist cgs21680 potentiates the effect of mglu5 agonist chpg in reducing the affinity of d2 receptors for the agonist ( popoli et al . , 2001 ) . it therefore appears that a2ar and mglu5 work together to antagonize d2r - mediated signaling in the striatum . under baseline conditions , there would be strong tonic activation of d2r in the striatum which would impair the ability of the a2ar to signal through adenylate cyclase / camp / pka . co - stimulation of mglu5 thus allows a2ar stimulation to override the inhibitory tone imposed by endogenous dopamine acting at d2 receptors ( ferre et al . , this is supported by behavioral data as activation of a2ar and mglu5 causes attenuation of d2r agonist quinpirole - induced contralateral turning in 6-hydroxydopamine - lesioned rats ( popoli et al . studies performed in heterologous expression systems utilizing a range of biophysical and biochemical techniques have provided evidence for the existence of this unique biochemical fingerprint as a result of the specific receptor interaction ( agnati et al . , 2010b ) . for example , in the case of a2ar d2r and mglu5d2r complexes , heteromerization appears to result in an antagonistic allosteric effect where a2ar and mglu5 agonists , respectively , reduced the affinity of the d2 agonist - binding sites ( ferre et al . , 1999 ; popoli et al . , 2001 ; torvinen et al . , in the case of the mglu5a2ar heteromer however , co - stimulation of both receptors appears to result in a synergistic effect on downstream signaling ( ferre et al . , 2002 ) . mglu5 and a2ar antagonists have been reported to reduce alcohol - seeking behavior in rats ; interestingly , co - administration of antagonists for these receptors has a synergistic effect in rats , supporting the existence and functional importance of the heteromer in addiction - associated behaviors ( adams et al . , 2008 ) . we have consequently suggested exploiting the limited overlapping expression of mglu5 and a2ar for developing potential therapies , which target a sub - population of receptors ( bird and lawrence , 2009b ) . by developing putative heteromer - selective drugs , the problematic issue of modulating the widely distributed and multifunctional mglu5 receptor the idea of receptor mosaics suggests that proteins can form even more complex and dynamic networks , with respect to time and protein make up , which has the potential to make significant contributions to the diversity and specificity of gpcr signaling , particularly in neuropharmacology , where a few key receptors have been implicated in multiple neurological and psychiatric disorders ( agnati et al . , 2009 ) . agnati and fuxe first introduced this concept of receptor mosaics in 1982 in the context of learning and memory ( agnati et al . , 1982 ) . ( 2008 ) this receptor mosaic term not only allows the interactions between the different receptors ( stoichiometry ) to be taken into account but also their spatial organization ( topography ) and their order of activation . it has been proposed that by changing the order of activation , functionally distinct receptor mosaics can transduce signals resulting in distinct biological responses . this may be of particular importance for neuropharmacology where different agonist concentrations direct activation of one receptor mosaic activity over another . this can be exemplified by considering a receptor mosaic which consists of three different receptors ; a , b , and c and therefore can potentially result in six functional outcomes ( fuxe et al . , agnati et al . , 2010a , b ; fuxe and kenakin , 2010 ) . furthermore , it has been suggested that receptor mosaics are composed of at least one so called hub receptor a receptor , capable of forming multiple connections , which can result in differing pharmacology depending on these connections ( agnati et al . , 2010a ) . hub receptors have been classified into three distinct groups ; passive hubs which allow the flow of information ; intelligent hubs modify the information as it flows through ; and switching hubs , which direct the information to the correct destination ( agnati et al . , 2010a ) . it has been postulated that the d2 receptor acts as a hub receptor , with evidence in the literature for its interaction with the a2a and mglu5 receptors ( cabello et al . , 2009 ) as well as a2a and cb1 ( agnati et al . , 2009 ) to form trimeric receptor mosaics . ( 2009 ) have reported the existence of these receptor mosaics in the perisynaptic and extrasynaptic parts of the neuronal plasma membrane . as mentioned above , it is thought that this trimeric receptor mosaic plays a role on the extrasynaptic striatopallidal gaba neurons and possibly in corticostriatal glutamate terminals . as only a small population of these receptors are expected to form complexes at any given time or location , this may allow even more selectivity when designing drug therapies . modulation of specific interactions and pathways has the potential to reduce side effects , of paramount importance in the treatment of neurological disorders , where lack of specificity could impair vital brain function . given the specific localization of apparent mglu5 receptor complexes in the striatum , as well as the established involvement of plasticity in glutamatergic corticostriatal neurons in the reinstatement of drug - seeking in animal models of relapse ( kalivas , 2009 ) , one may hypothesize that interactions between mglu5 and a2ar have the potential to regulate drug - seeking or the conditioned effects of drugs of abuse . firstly , as mentioned previously , functional interactions between mglu5 and a2a receptors were shown to regulate operant self - administration of alcohol and cue - induced reinstatement of alcohol - seeking ( adams et al . , 2008 ) . sub - threshold doses of sch58261 , a selective a2a receptor antagonist and mtep , an mglu5 antagonist , were administrated in combination and were found to act synergistically to decrease operant self - administration of alcohol and cue - induced reinstatement of alcohol - seeking ( adams et al . , 2008 ) . in addition , no support was found for interactions between adenosine a1 and a2ar , or a1 and mglu5 , suggesting this reduction in alcohol self - administration and alcohol - seeking behavior was specific to a functional interaction between a2a and mglu5 receptors ( adams et al . , 2008 , 2010 ) . it is now recognized that reinstatement of drug - seeking involves corticostriatal glutamatergic input into the striatum ( kalivas and mcfarland , 2003 ; kalivas et al . , 2005 ) . prior co - administration of sch58261 and mtep may have prevented cue - elicited release of glutamate in this context , thus preventing drug - seeking behavior . this fits nicely with the role discussed earlier for a2ar and mglu5 in regulating glutamate release from corticostriatal afferent neurons ( rodrigues et al . , 2005 ) . in rats , stimulation of corticostriatal afferents has been shown to result in phosphorylation of erk which can be prevented by a2ar antagonism ( quiroz et al . , 2009 ) , thus providing one possible mechanism for this observation . though no direct evidence exists as yet , it is also possible that specialized receptor complexes containing multiple gpcrs are involved in this behavior , and that plasticity may occur with repeated drug use which alters the arrangement of or signaling through this receptor complex . though the exact nature of the interactions between mglu5 and a2a receptors are yet to be determined and involvement of potential hub receptors such as the d2r remains to be explored , it is clear that a biologically relevant functional interaction between these two gpcrs can act to regulate drug - seeking behavior providing evidence of the therapeutic potential of targeting mglu5 and a2ar in combination . a similar functional interaction was found between mglu5 and a2ar in relation to cocaine - driven behaviors ( brown et al . , 2011a ) providing further evidence of the potential of this possible drug target . despite preventing a conditioned place preference to cocaine in wildtype mice , mtep treatment had no impact on the ability of a2ar knockout mice to develop a robust conditioned place preference to cocaine , suggesting a functional a2ar is required for mglu5 to regulate the acquisition of incentive properties in a cocaine - paired context . this finding was mirrored by conditioned hyperactivity data , whereby mtep completely prevented conditioned hyperactivity in wildtype mice but had no impact in a2ar knockout mice . in contrast , mtep was able to block the locomotor - activating properties of cocaine in both wildtype and a2ar knockout mice . it should be noted that conditioned hyperactivity was assessed during the test session when all mice were in a drug - free state . conditioned hyperactivity therefore , as with conditioned place preference , is driven purely by contextual associations and is hence likely to be underpinned by similar neural processes . hence , it appears that a functional a2ar may be critical for the actions of mtep in regulating both the conditioned reinforcing and conditioned locomotor - activating properties of cocaine , but not the direct locomotor effects of acute cocaine . these data are in line with recent evidence supporting a role for mglu5 in incentive learning processes ( novak et al . , 2010 ; oconnor et al . , 2010 ) . as the ventral striatum is a key component of the neural circuitry underlying pavlovian conditioning ( robbins et al . , 2008 ) , it seems probable that it is a likely site of action for this interaction . however it should be noted that both a2ar and mglu5 were recently shown to be co - localized in the hippocampus ( tebano et al . , 2005 ) and have been implicated in learning and memory processes ( simonyi et al . , 2005 ; for example , a2ar have been shown to play a key role in hippocampal - dependent associative learning in a classical eye - blinking paradigm ( fontinha et al . , 2009 ) and given that conditioned place preference is a paradigm that relies on contextual learning , functional interactions between mglu5 and a2ar in the hippocampus can not be ruled out . this review has outlined the evidence supporting a role for functional interactions between mglu5 and other gpcrs , particularly the adenosine a2ar , in reward , drug - seeking , and drug - induced plasticity . though much evidence now exists describing the individual contribution that mglu5 , d2r , and a2ar make to various facets of addictive behavior , only a small number of studies thus far have investigated interactions between mglu5 and these other gpcrs in this context . initial studies suggest that mglu5 receptor interactions may play a role in the rewarding and incentive motivational properties of drugs of abuse , drug - seeking , as well as the conditioned effects of drugs of abuse . this is consistent with the substantial influence that interactions between mglu5 and these other receptors appear to have on striatal function , regulating both gabaergic output of striatopallidal neurons and glutamatergic input from corticostriatal afferents . the specific nature of these interactions has not yet been determined and though it is possible that striatal mglu5-containing complexes are responsible for regulating drug - seeking , future studies should aim to determine whether or not this is the case as well as determine the exact site of action as mglu5 has recently been shown to co - localize with a2ar in the hippocampus ( tebano et al . , 2005 ) . furthermore , in order to overcome the problematic issue of therapeutically modulating the widely distributed and multifunctional mglu5 , the limited overlapping expression of mglu5 , d2 , and a2ar may be exploited when designing drug therapies aimed at exhibiting little or no off - target effects . on the other hand , if such complexes do not prove to be relevant in vivo in humans , exploitation of specific functional interactions that have biological significance ( e.g. , downstream mglu5a2a receptor functional interactions which are well - established ) could provide an equally attractive proposition . indeed , the therapeutic targeting of receptor mosaics / functional interactions in a tissue specific or temporal manner ( for example , a sub - population of receptors in a pathological state ) has the potential to reduce detrimental side effects that may otherwise impair vital brain function . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

the idea of receptor mosaics is that proteins may form complex and dynamic networks with respect to time and composition . these have the potential to markedly expand the diversity and specificity of g protein - coupled receptors ( gpcr ) signaling , particularly in neural cells , where a few key receptors have been implicated in many neurological and psychiatric disorders , including addiction . metabotropic glutamate type 5 receptors ( mglu5 ) can form complexes with other gpcrs , including adenosine a2a and dopamine d2 receptors . mglu5-containing complexes have been reported in the striatum , a brain region critical for mediating the rewarding and incentive motivational properties of drugs of abuse . mglu5-containing complexes and/or downstream interactions between divergent receptors may play roles in addiction relevant behaviors . interactions between mglu5 receptors and other gpcrs can regulate the rewarding and conditioned effects of drugs as well as drug - seeking behaviors . mglu5 complexes may influence striatal function , including gabaergic output of striatopallidal neurons and glutamatergic input from corticostriatal afferents . given their discrete localization , mglu5-[non - mglu5 ] receptor interactions and/or mglu5-containing complexes may minimize off - target effects and thus provide a novel avenue for drug discovery . the therapeutic targeting of receptor receptor functional interactions and/or receptor mosaics in a tissue specific or temporal manner ( for example , a sub - population of receptors in a pathological state ) might reduce detrimental side effects that may otherwise impair vital brain functions .

Drug Addiction mGlu5 and Addiction GPCR Heteromerization and A Concepts of Receptor Mosaic and Hub Receptor mGlu5 Receptor Functional Interactions: Evidence for a Role in Addiction Conclusion Conflict of Interest Statement

at present there is a paucity of pharmacotherapies on the market for the treatment of addictions ; none of which are particularly effective and involve multiple off - target effects ( jupp and lawrence , 2009 ) . complexes containing the metabotropic glutamate receptor type 5 ( mglu5 ) , and/or functional interactions between mglu5 and other g protein - coupled receptors ( gpcrs ) , represent a novel therapeutic option worthy of investigation due to the possibility of limiting off - target effects . mglu5 has been implicated in numerous central processes , thus targeting mglu5-containing complexes / functional receptor interactions that are restricted to addiction relevant brain areas provide an avenue to selectively target those mglu5 involved in reward and drug - seeking behavior ( bird and lawrence , 2009b ) . a variety of gpcrs have been shown to play critical roles in the reinforcing and motivational properties of drugs , drug - induced plasticity and drug - seeking behavior ( traynor , 2010 ) . a substantial body of evidence exists which implicates metabotropic glutamate receptors in many of these facets of drug addiction ( bird and lawrence , 2009a ; olive , 2009 ) and among them mglu5 seems to play a significant role . mglu5 is highly expressed in the olfactory bulb and tubercle , dorsal striatum , nucleus accumbens , cerebral cortex , hippocampal formation , and amygdala ( shigemoto et al . in the striatum , mglu5 is located primarily in the perisynaptic ring and at the postsynaptic density ( kennedy , 2000 ; smith et al . , 1999 ) and functional interactions have been demonstrated between the two receptors ( attucci et al . thus , mglu5 receptors are perfectly positioned to influence the mesolimbic , corticotegmental , and corticostriatal pathways known to be critical for the actions of drugs of abuse and drug - induced plasticity . the first evidence to suggest a role for mglu5 in the rewarding properties of drugs of abuse ( 2001 ) who reported that mice lacking mglu5 failed to intravenously self - administer a range of doses of cocaine and failed to show any hyperactivity after acute administration of the drug . antagonism of mglu5 can reduce the self - administration of multiple drugs of abuse as well as reinstatement of drug - seeking , indicating a facilitatory role for this receptor in these behaviors ( olive , 2009 ) . for example , the mglu5 antagonists 2-methyl-6-(phenylethynyl)-pyridine ( mpep ) and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine ( mtep ) reduce self - administration of alcohol , cocaine , and nicotine in rats ( kenny et al . 2005 ) as well as alcohol and morphine in mice ( cowen et al . , 2009 ) in animal models of relapse , indicating a clear role for mglu5 in drug - seeking behavior across a broad range of reinforcers . genetic mouse models have also implicated mglu5 in the reinforcing and motivational properties of drugs of abuse . , 2008 ) and an absence of drug - induced synaptic plasticity in the ventral tegmental area in the case of cocaine ( bird et al . , 2010 ) ; an observation which is supported by the finding that mice with knockdown of mglu5 in striatal d1-expressing neurons exhibit diminished cue - induced cocaine - seeking , as well as impaired incentive learning ( novak et al . , this possible role in incentive learning is not inconsistent with the involvement of mglu5 in cue - induced drug - seeking behavior ( backstrom et al . indeed , in the operant conditioning paradigm , the discrete cue is a conditioned stimulus and as such needs to acquire incentive motivational properties in order to elicit drug - seeking upon re - presentation . therefore , positive allosteric modulators of mglu5 have potential use as pharmacological adjuncts to cue exposure therapy in the treatment of drug addiction , with the potential benefit of reversing cognitive deficits associated with chronic drug use ( cleva and olive , 2011 ) . recently , a range of compounds that selectively inhibit mglu5 function have been published and tested in various neurological disorder models and some of them exhibit good tolerability , long - term action , and oral bioavailability ( keywood et al . textbooks describing gpcr signaling as a linear cascade , where agonist activation results in the receptor coupling to one g protein and therefore the activation of one signaling pathway , are now being updated to include receptor homomers ( macromolecular complexes made up of two or more identical functional receptor units ) and receptor heteromers ( macromolecular complexes composed of at least two different functional receptor units with biochemical properties that are demonstrably different from those of their individual components ; ferre et al . heteromerization not only provides a potential mechanism for increased signaling diversity , but also specificity by , for example , allowing the development of drugs to target in a tissue selective manner and thereby minimize off - target effects ( mustafa et al . , 2008 ) , particularly in the cns ( albizu et al . dopaminergic and glutamatergic inputs converge in the dendritic spines of -aminobutyric acid ( gaba)ergic medium spiny neurons ( totterdell and smith , 1989 ; sesack and pickel , 1992 ) which constitute more than 90% of the neuronal population of the striatum ( smith and bolam , 1990 ) . dopamine release in the striatum activates two classes of gpcrs that are expressed by distinct subpopulations of medium spiny neurons . dopamine d1 receptors ( d1r ) are localized on the striatonigral neurons of the direct pathway whereas dopamine d2 receptors ( d2r ) are expressed by the striatopallidal neurons of the indirect pathway ( gerfen , 1992 ; le moine and bloch , 1995 ) . d2rs are coupled to gi , and activation of these receptors triggers a cascade of signaling pathways , reducing camp production and protein kinase a ( pka ) activity and activating phospholipase c ( plc)-dependent processes as well as the release of ca from internal stores ( missale et al . d2r has been linked to a number of behavioral disorders , including parkinson s disease , obsessive compulsive disorder , addiction , and schizophrenia ( bonci and hopf , 2005 ; kreitzer and malenka , 2008 ) . , however , side effects have been associated with both therapies , presumably due to the difficulty in re - establishing the balance in dopamine signaling required for appropriate physiological function . , 2001a ) whereas a2a receptors are highly enriched in the terminal regions of ascending dopamine projections , concentrated in the dendritic spines of striatopallidal neurons , particularly in the vicinity of glutamatergic synapses ( fink et al . signaling via the a2a receptor has been implicated in the reinforcing properties of multiple drugs of abuse ( brown and short , 2008 ) . for example , in mice lacking the a2a receptor , a reduction in self - administration of morphine has been reported ( brown et al . similarly , a2ar antagonists have also been reported to reduce alcohol - seeking and opiate - seeking behavior in rats ( arolfo et al . gabaergic / enkephalinergic neurons predominantly express adenosine a2a ( stimulatory , coupled to gs / golf ) and dopamine d2 ( inhibitory , coupled to gi ) receptors , while gabaergic / dynorphinergic neurons predominantly express adenosine a1 ( inhibitory , coupled to gi ) and dopamine d1 ( stimulatory , coupled to gs ) receptors ( schiffmann et al . these interactions have been reported to include direct receptor receptor interactions as well as interactions at the second messenger and behavioral levels , all of which have been reviewed elsewhere ( ferre et al . d2 interaction is predominantly found to be antagonistic as activation of a2ar reduces d2r binding , coupling , signaling as well as behavioral effects ; thus d2r exerts a tonic inhibition on striatopallidal neurons which a2ar activation antagonizes ( see reviews above ) . evidence for the existence of heteromers containing a2a and d2 receptors in expression systems has been provided by means of co - immunoprecipitation and resonance energy transfer ( ret ) techniques such as bioluminescence - ret ( bret ) and fluorescence - ret ( fret ; canals et al . ultrastructural and confocal analysis indicate this to be mainly on dendritic spines in the perisynaptic zones of dopamine terminals and glutamatergic synapses in striatopallidal neurons ( fuxe et al . in addition , interactions involving the a2ar have been described for other receptors , including adenosine a1 , cannabinoid cb1 and , most relevant to this review , mglu5 ( ferre et al . in the striatum mglu5 is expressed postsynaptically on both types of striatal gabaergic efferent neuron and potentially presynaptically on glutamatergic terminals ( romano et al . these findings are consistent with an erk1/2-dependent synergistic interaction between a2ar and mglu5 with respect to c - fos expression in hek293 cells ( ferre et al . collectively , these data have led some researchers in the field to suggest that the existence of this a2ar mglu5 heteromer may provide a point of temporal and spatial convergence of gs and gq signaling onto the mapk cascade , thereby providing the capacity to influence striatal plasticity ( agnati et al . , 2010a ) , thus raising the possibility of the involvement of this heteromer in plasticity resulting from repeated exposure to drugs of abuse . in addition , a2ar and mglu5 have been shown to interact synergistically to modulate glutamate release from corticostriatal afferents ( rodrigues et al . these results demonstrate that in addition to modulating striatal output , mglu5a2ar interactions also play a role in regulating cortical input into the striatum , providing an additional avenue to influence striatal function and ultimately behavior . indeed , behavioral interactions between a2ar and mglu5 have been reported , particularly with respect to motor behaviors due to the strong interest in these receptors for their potential in the treatment of parkinson s disease . for example , central co - administration of cgs21680 and chpg has been found to reduce the motor activity induced by phencyclidine at doses that have no effect alone ( ferre et al . furthermore , in both normal and reserpinized mice , locomotion is enhanced in a synergistic manner with combined treatment of mpep and the a2ar antagonist kw-6002 ( kachroo et al . mglu5 interaction is also relevant to reward and drug - seeking behavior ( discussed in subsequent section ) . recently , co - immunoprecipitation experiments in native striatal tissue provided evidence for the existence of a higher - order oligomeric complex containing mglu5 , d2r , and a2ar , localized to the dendritic spines of striatopallidal neurons ( cabello et al . it therefore appears that a2ar and mglu5 work together to antagonize d2r - mediated signaling in the striatum . under baseline conditions , there would be strong tonic activation of d2r in the striatum which would impair the ability of the a2ar to signal through adenylate cyclase / camp / pka . co - stimulation of mglu5 thus allows a2ar stimulation to override the inhibitory tone imposed by endogenous dopamine acting at d2 receptors ( ferre et al . for example , in the case of a2ar d2r and mglu5d2r complexes , heteromerization appears to result in an antagonistic allosteric effect where a2ar and mglu5 agonists , respectively , reduced the affinity of the d2 agonist - binding sites ( ferre et al . , in the case of the mglu5a2ar heteromer however , co - stimulation of both receptors appears to result in a synergistic effect on downstream signaling ( ferre et al . mglu5 and a2ar antagonists have been reported to reduce alcohol - seeking behavior in rats ; interestingly , co - administration of antagonists for these receptors has a synergistic effect in rats , supporting the existence and functional importance of the heteromer in addiction - associated behaviors ( adams et al . we have consequently suggested exploiting the limited overlapping expression of mglu5 and a2ar for developing potential therapies , which target a sub - population of receptors ( bird and lawrence , 2009b ) . by developing putative heteromer - selective drugs , the problematic issue of modulating the widely distributed and multifunctional mglu5 receptor the idea of receptor mosaics suggests that proteins can form even more complex and dynamic networks , with respect to time and protein make up , which has the potential to make significant contributions to the diversity and specificity of gpcr signaling , particularly in neuropharmacology , where a few key receptors have been implicated in multiple neurological and psychiatric disorders ( agnati et al . agnati and fuxe first introduced this concept of receptor mosaics in 1982 in the context of learning and memory ( agnati et al . ( 2008 ) this receptor mosaic term not only allows the interactions between the different receptors ( stoichiometry ) to be taken into account but also their spatial organization ( topography ) and their order of activation . hub receptors have been classified into three distinct groups ; passive hubs which allow the flow of information ; intelligent hubs modify the information as it flows through ; and switching hubs , which direct the information to the correct destination ( agnati et al . it has been postulated that the d2 receptor acts as a hub receptor , with evidence in the literature for its interaction with the a2a and mglu5 receptors ( cabello et al . , 2009 ) as well as a2a and cb1 ( agnati et al . ( 2009 ) have reported the existence of these receptor mosaics in the perisynaptic and extrasynaptic parts of the neuronal plasma membrane . as only a small population of these receptors are expected to form complexes at any given time or location , this may allow even more selectivity when designing drug therapies . modulation of specific interactions and pathways has the potential to reduce side effects , of paramount importance in the treatment of neurological disorders , where lack of specificity could impair vital brain function . given the specific localization of apparent mglu5 receptor complexes in the striatum , as well as the established involvement of plasticity in glutamatergic corticostriatal neurons in the reinstatement of drug - seeking in animal models of relapse ( kalivas , 2009 ) , one may hypothesize that interactions between mglu5 and a2ar have the potential to regulate drug - seeking or the conditioned effects of drugs of abuse . firstly , as mentioned previously , functional interactions between mglu5 and a2a receptors were shown to regulate operant self - administration of alcohol and cue - induced reinstatement of alcohol - seeking ( adams et al . sub - threshold doses of sch58261 , a selective a2a receptor antagonist and mtep , an mglu5 antagonist , were administrated in combination and were found to act synergistically to decrease operant self - administration of alcohol and cue - induced reinstatement of alcohol - seeking ( adams et al . in addition , no support was found for interactions between adenosine a1 and a2ar , or a1 and mglu5 , suggesting this reduction in alcohol self - administration and alcohol - seeking behavior was specific to a functional interaction between a2a and mglu5 receptors ( adams et al . it is now recognized that reinstatement of drug - seeking involves corticostriatal glutamatergic input into the striatum ( kalivas and mcfarland , 2003 ; kalivas et al . prior co - administration of sch58261 and mtep may have prevented cue - elicited release of glutamate in this context , thus preventing drug - seeking behavior . though the exact nature of the interactions between mglu5 and a2a receptors are yet to be determined and involvement of potential hub receptors such as the d2r remains to be explored , it is clear that a biologically relevant functional interaction between these two gpcrs can act to regulate drug - seeking behavior providing evidence of the therapeutic potential of targeting mglu5 and a2ar in combination . despite preventing a conditioned place preference to cocaine in wildtype mice , mtep treatment had no impact on the ability of a2ar knockout mice to develop a robust conditioned place preference to cocaine , suggesting a functional a2ar is required for mglu5 to regulate the acquisition of incentive properties in a cocaine - paired context . it should be noted that conditioned hyperactivity was assessed during the test session when all mice were in a drug - free state . hence , it appears that a functional a2ar may be critical for the actions of mtep in regulating both the conditioned reinforcing and conditioned locomotor - activating properties of cocaine , but not the direct locomotor effects of acute cocaine . , 2005 ) and have been implicated in learning and memory processes ( simonyi et al . , 2005 ; for example , a2ar have been shown to play a key role in hippocampal - dependent associative learning in a classical eye - blinking paradigm ( fontinha et al . , 2009 ) and given that conditioned place preference is a paradigm that relies on contextual learning , functional interactions between mglu5 and a2ar in the hippocampus can not be ruled out . this review has outlined the evidence supporting a role for functional interactions between mglu5 and other gpcrs , particularly the adenosine a2ar , in reward , drug - seeking , and drug - induced plasticity . though much evidence now exists describing the individual contribution that mglu5 , d2r , and a2ar make to various facets of addictive behavior , only a small number of studies thus far have investigated interactions between mglu5 and these other gpcrs in this context . initial studies suggest that mglu5 receptor interactions may play a role in the rewarding and incentive motivational properties of drugs of abuse , drug - seeking , as well as the conditioned effects of drugs of abuse . this is consistent with the substantial influence that interactions between mglu5 and these other receptors appear to have on striatal function , regulating both gabaergic output of striatopallidal neurons and glutamatergic input from corticostriatal afferents . the specific nature of these interactions has not yet been determined and though it is possible that striatal mglu5-containing complexes are responsible for regulating drug - seeking , future studies should aim to determine whether or not this is the case as well as determine the exact site of action as mglu5 has recently been shown to co - localize with a2ar in the hippocampus ( tebano et al . furthermore , in order to overcome the problematic issue of therapeutically modulating the widely distributed and multifunctional mglu5 , the limited overlapping expression of mglu5 , d2 , and a2ar may be exploited when designing drug therapies aimed at exhibiting little or no off - target effects . , downstream mglu5a2a receptor functional interactions which are well - established ) could provide an equally attractive proposition . indeed , the therapeutic targeting of receptor mosaics / functional interactions in a tissue specific or temporal manner ( for example , a sub - population of receptors in a pathological state ) has the potential to reduce detrimental side effects that may otherwise impair vital brain function .

it has been estimated that annually around $ 7.5 billion is associated with alcohol and alcohol - related problems in australia and about 5% of the total annual mortality is due to this ( collins and lapsley , 2002 ) . current preclinical research into potential treatments for addiction is focused on gaining a greater understanding of the neurobiological mechanisms underpinning both relapse vulnerability and the transition from casual to compulsive drug use . at present there is a paucity of pharmacotherapies on the market for the treatment of addictions ; none of which are particularly effective and involve multiple off - target effects ( jupp and lawrence , 2009 ) . complexes containing the metabotropic glutamate receptor type 5 ( mglu5 ) , and/or functional interactions between mglu5 and other g protein - coupled receptors ( gpcrs ) , represent a novel therapeutic option worthy of investigation due to the possibility of limiting off - target effects . mglu5 has been implicated in numerous central processes , thus targeting mglu5-containing complexes / functional receptor interactions that are restricted to addiction relevant brain areas provide an avenue to selectively target those mglu5 involved in reward and drug - seeking behavior ( bird and lawrence , 2009b ) . a variety of gpcrs have been shown to play critical roles in the reinforcing and motivational properties of drugs , drug - induced plasticity and drug - seeking behavior ( traynor , 2010 ) . a substantial body of evidence exists which implicates metabotropic glutamate receptors in many of these facets of drug addiction ( bird and lawrence , 2009a ; olive , 2009 ) and among them mglu5 seems to play a significant role . mglu5 belongs to a distinct family of eight gpcrs that are activated by l - glutamate , the major fast excitatory neurotransmitter in the central nervous system ( cns ) . at the signaling level , mglu5 is known to be associated with phosphoinositide hydrolysis and the activation of phospholipase c , stimulation of adenylate cyclase and inhibition of voltage - operated calcium channels ( dhami and ferguson , 2006 ) . signaling via mglu5 is thought to play a role in synaptic plasticity , ultimately affecting learning and memory , neuronal development and neurodegeneration ( lu et al . it is also thought to have a role in a number of behaviors including stress responses , anxiety - like , and depressive - like behavior ( tatarczynska et al . mglu5 is highly expressed in the olfactory bulb and tubercle , dorsal striatum , nucleus accumbens , cerebral cortex , hippocampal formation , and amygdala ( shigemoto et al . thus , mglu5 receptors are perfectly positioned to influence the mesolimbic , corticotegmental , and corticostriatal pathways known to be critical for the actions of drugs of abuse and drug - induced plasticity . the first evidence to suggest a role for mglu5 in the rewarding properties of drugs of abuse ( 2001 ) who reported that mice lacking mglu5 failed to intravenously self - administer a range of doses of cocaine and failed to show any hyperactivity after acute administration of the drug . antagonism of mglu5 can reduce the self - administration of multiple drugs of abuse as well as reinstatement of drug - seeking , indicating a facilitatory role for this receptor in these behaviors ( olive , 2009 ) . for example , the mglu5 antagonists 2-methyl-6-(phenylethynyl)-pyridine ( mpep ) and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine ( mtep ) reduce self - administration of alcohol , cocaine , and nicotine in rats ( kenny et al . , 2009 ) in animal models of relapse , indicating a clear role for mglu5 in drug - seeking behavior across a broad range of reinforcers . , 2008 ) and an absence of drug - induced synaptic plasticity in the ventral tegmental area in the case of cocaine ( bird et al . thus , the putative involvement of mglu5 in addictive behavior suggests that selective inhibition of this receptor may provide an exciting new approach for treatment of addictive disorders . published reports suggest systemic administration of the selective mglu5 antagonist mtep prior to conditioning disrupts the expression of conditioned reinforcement by food delivery ( oconnor et al . , 2010 ) ; an observation which is supported by the finding that mice with knockdown of mglu5 in striatal d1-expressing neurons exhibit diminished cue - induced cocaine - seeking , as well as impaired incentive learning ( novak et al . , this possible role in incentive learning is not inconsistent with the involvement of mglu5 in cue - induced drug - seeking behavior ( backstrom et al . a role for mglu5 in extinction learning relevant to drug use and drug - associated cues has also been established by recent research using positive allosteric modulators of mglu5 ( cleva and olive , 2011 ) . studies using animal models of addiction have also shown that cdppb facilitates extinction of both cocaine - conditioned place preference ( gass and olive , 2009 ) and operant responding for cocaine ( cleva et al . therefore , positive allosteric modulators of mglu5 have potential use as pharmacological adjuncts to cue exposure therapy in the treatment of drug addiction , with the potential benefit of reversing cognitive deficits associated with chronic drug use ( cleva and olive , 2011 ) . recently , a range of compounds that selectively inhibit mglu5 function have been published and tested in various neurological disorder models and some of them exhibit good tolerability , long - term action , and oral bioavailability ( keywood et al . consequently , there is a promising future for the development of new treatments for addiction based on both positive and negative allosteric modulation of mglu5 . consequently , researchers in the gpcr field are now focusing their efforts on understanding gpcr signaling and function in the context of higher - order structures and not just monomers . textbooks describing gpcr signaling as a linear cascade , where agonist activation results in the receptor coupling to one g protein and therefore the activation of one signaling pathway , are now being updated to include receptor homomers ( macromolecular complexes made up of two or more identical functional receptor units ) and receptor heteromers ( macromolecular complexes composed of at least two different functional receptor units with biochemical properties that are demonstrably different from those of their individual components ; ferre et al . heteromerization not only provides a potential mechanism for increased signaling diversity , but also specificity by , for example , allowing the development of drugs to target in a tissue selective manner and thereby minimize off - target effects ( mustafa et al . dopaminergic and glutamatergic inputs converge in the dendritic spines of -aminobutyric acid ( gaba)ergic medium spiny neurons ( totterdell and smith , 1989 ; sesack and pickel , 1992 ) which constitute more than 90% of the neuronal population of the striatum ( smith and bolam , 1990 ) . dopamine d1 receptors ( d1r ) are localized on the striatonigral neurons of the direct pathway whereas dopamine d2 receptors ( d2r ) are expressed by the striatopallidal neurons of the indirect pathway ( gerfen , 1992 ; le moine and bloch , 1995 ) . d2rs are coupled to gi , and activation of these receptors triggers a cascade of signaling pathways , reducing camp production and protein kinase a ( pka ) activity and activating phospholipase c ( plc)-dependent processes as well as the release of ca from internal stores ( missale et al . d2r has been linked to a number of behavioral disorders , including parkinson s disease , obsessive compulsive disorder , addiction , and schizophrenia ( bonci and hopf , 2005 ; kreitzer and malenka , 2008 ) . treatments targeting dopaminergic signaling have utilized dopamine receptor agonists to alleviate symptoms of hypokinesia associated with parkinson s disease , and dopamine receptor antagonists to treat hallucinations and delusions in schizophrenic patients ( missale et al . , however , side effects have been associated with both therapies , presumably due to the difficulty in re - establishing the balance in dopamine signaling required for appropriate physiological function . , 2001a ) whereas a2a receptors are highly enriched in the terminal regions of ascending dopamine projections , concentrated in the dendritic spines of striatopallidal neurons , particularly in the vicinity of glutamatergic synapses ( fink et al . , 2003 therefore , it has been suggested that the a2a receptor plays a role in the fine - tuning modulation of glutamatergic neurotransmission onto striatal gabaergic neurons both at the postsynaptic and presynaptic level ( hettinger et al . gabaergic / enkephalinergic neurons predominantly express adenosine a2a ( stimulatory , coupled to gs / golf ) and dopamine d2 ( inhibitory , coupled to gi ) receptors , while gabaergic / dynorphinergic neurons predominantly express adenosine a1 ( inhibitory , coupled to gi ) and dopamine d1 ( stimulatory , coupled to gs ) receptors ( schiffmann et al . , 2007 ) it is not surprising therefore , that evidence has accumulated documenting interactions between these gpcrs in relation to striatal gabaergic efferent neuron function ( ferre et al . d2 interaction is predominantly found to be antagonistic as activation of a2ar reduces d2r binding , coupling , signaling as well as behavioral effects ; thus d2r exerts a tonic inhibition on striatopallidal neurons which a2ar activation antagonizes ( see reviews above ) . evidence for the existence of heteromers containing a2a and d2 receptors in expression systems has been provided by means of co - immunoprecipitation and resonance energy transfer ( ret ) techniques such as bioluminescence - ret ( bret ) and fluorescence - ret ( fret ; canals et al . ultrastructural and confocal analysis indicate this to be mainly on dendritic spines in the perisynaptic zones of dopamine terminals and glutamatergic synapses in striatopallidal neurons ( fuxe et al . in addition , interactions involving the a2ar have been described for other receptors , including adenosine a1 , cannabinoid cb1 and , most relevant to this review , mglu5 ( ferre et al . thus , co - localization of a2ar and mglu5 at the membrane level was demonstrated by confocal laser microscopy in co - transfected human embryonic kidney ( hek)293 cells . firstly , an apparent interdependence exists between the actions of a2ar and mglu5 in the phosphorylation of dopamine and camp - regulated phosphoprotein of 32,000 kda ( darpp-32 ) at thr-34 ( nishi et al . in addition , co - activation of mglu5 and a2ar synergistically increases phosphorylation of darpp-32 at thr-34 , apparently via an extracellular signal - regulated kinase ( erk)1/2-dependent mechanism involving increases in camp formation ( nishi et al . collectively , these data have led some researchers in the field to suggest that the existence of this a2ar mglu5 heteromer may provide a point of temporal and spatial convergence of gs and gq signaling onto the mapk cascade , thereby providing the capacity to influence striatal plasticity ( agnati et al . in vivo microdialysis experiments have shown that perfusion with the a2ar agonist 2-[4-(2-carboxy ethyl)phenethylamine-50-n - ethylcarboxamidoadenosine ( cgs21680 ) potentiates the facilitation of gaba release from striatopallidal neurons by the mglu5 agonist ( rs)-2-chloro-5-hydroxyphenylglycine ( chpg ; diaz - cabiale et al . these results demonstrate that in addition to modulating striatal output , mglu5a2ar interactions also play a role in regulating cortical input into the striatum , providing an additional avenue to influence striatal function and ultimately behavior . indeed , behavioral interactions between a2ar and mglu5 have been reported , particularly with respect to motor behaviors due to the strong interest in these receptors for their potential in the treatment of parkinson s disease . furthermore , in both normal and reserpinized mice , locomotion is enhanced in a synergistic manner with combined treatment of mpep and the a2ar antagonist kw-6002 ( kachroo et al . recently , co - immunoprecipitation experiments in native striatal tissue provided evidence for the existence of a higher - order oligomeric complex containing mglu5 , d2r , and a2ar , localized to the dendritic spines of striatopallidal neurons ( cabello et al . in rat striatal membranes , the a2ar agonist cgs21680 potentiates the effect of mglu5 agonist chpg in reducing the affinity of d2 receptors for the agonist ( popoli et al . under baseline conditions , there would be strong tonic activation of d2r in the striatum which would impair the ability of the a2ar to signal through adenylate cyclase / camp / pka . studies performed in heterologous expression systems utilizing a range of biophysical and biochemical techniques have provided evidence for the existence of this unique biochemical fingerprint as a result of the specific receptor interaction ( agnati et al . for example , in the case of a2ar d2r and mglu5d2r complexes , heteromerization appears to result in an antagonistic allosteric effect where a2ar and mglu5 agonists , respectively , reduced the affinity of the d2 agonist - binding sites ( ferre et al . , in the case of the mglu5a2ar heteromer however , co - stimulation of both receptors appears to result in a synergistic effect on downstream signaling ( ferre et al . mglu5 and a2ar antagonists have been reported to reduce alcohol - seeking behavior in rats ; interestingly , co - administration of antagonists for these receptors has a synergistic effect in rats , supporting the existence and functional importance of the heteromer in addiction - associated behaviors ( adams et al . we have consequently suggested exploiting the limited overlapping expression of mglu5 and a2ar for developing potential therapies , which target a sub - population of receptors ( bird and lawrence , 2009b ) . by developing putative heteromer - selective drugs , the problematic issue of modulating the widely distributed and multifunctional mglu5 receptor the idea of receptor mosaics suggests that proteins can form even more complex and dynamic networks , with respect to time and protein make up , which has the potential to make significant contributions to the diversity and specificity of gpcr signaling , particularly in neuropharmacology , where a few key receptors have been implicated in multiple neurological and psychiatric disorders ( agnati et al . it has been postulated that the d2 receptor acts as a hub receptor , with evidence in the literature for its interaction with the a2a and mglu5 receptors ( cabello et al . modulation of specific interactions and pathways has the potential to reduce side effects , of paramount importance in the treatment of neurological disorders , where lack of specificity could impair vital brain function . given the specific localization of apparent mglu5 receptor complexes in the striatum , as well as the established involvement of plasticity in glutamatergic corticostriatal neurons in the reinstatement of drug - seeking in animal models of relapse ( kalivas , 2009 ) , one may hypothesize that interactions between mglu5 and a2ar have the potential to regulate drug - seeking or the conditioned effects of drugs of abuse . firstly , as mentioned previously , functional interactions between mglu5 and a2a receptors were shown to regulate operant self - administration of alcohol and cue - induced reinstatement of alcohol - seeking ( adams et al . sub - threshold doses of sch58261 , a selective a2a receptor antagonist and mtep , an mglu5 antagonist , were administrated in combination and were found to act synergistically to decrease operant self - administration of alcohol and cue - induced reinstatement of alcohol - seeking ( adams et al . in addition , no support was found for interactions between adenosine a1 and a2ar , or a1 and mglu5 , suggesting this reduction in alcohol self - administration and alcohol - seeking behavior was specific to a functional interaction between a2a and mglu5 receptors ( adams et al . though the exact nature of the interactions between mglu5 and a2a receptors are yet to be determined and involvement of potential hub receptors such as the d2r remains to be explored , it is clear that a biologically relevant functional interaction between these two gpcrs can act to regulate drug - seeking behavior providing evidence of the therapeutic potential of targeting mglu5 and a2ar in combination . despite preventing a conditioned place preference to cocaine in wildtype mice , mtep treatment had no impact on the ability of a2ar knockout mice to develop a robust conditioned place preference to cocaine , suggesting a functional a2ar is required for mglu5 to regulate the acquisition of incentive properties in a cocaine - paired context . hence , it appears that a functional a2ar may be critical for the actions of mtep in regulating both the conditioned reinforcing and conditioned locomotor - activating properties of cocaine , but not the direct locomotor effects of acute cocaine . this review has outlined the evidence supporting a role for functional interactions between mglu5 and other gpcrs , particularly the adenosine a2ar , in reward , drug - seeking , and drug - induced plasticity . though much evidence now exists describing the individual contribution that mglu5 , d2r , and a2ar make to various facets of addictive behavior , only a small number of studies thus far have investigated interactions between mglu5 and these other gpcrs in this context . initial studies suggest that mglu5 receptor interactions may play a role in the rewarding and incentive motivational properties of drugs of abuse , drug - seeking , as well as the conditioned effects of drugs of abuse . this is consistent with the substantial influence that interactions between mglu5 and these other receptors appear to have on striatal function , regulating both gabaergic output of striatopallidal neurons and glutamatergic input from corticostriatal afferents . the specific nature of these interactions has not yet been determined and though it is possible that striatal mglu5-containing complexes are responsible for regulating drug - seeking , future studies should aim to determine whether or not this is the case as well as determine the exact site of action as mglu5 has recently been shown to co - localize with a2ar in the hippocampus ( tebano et al . furthermore , in order to overcome the problematic issue of therapeutically modulating the widely distributed and multifunctional mglu5 , the limited overlapping expression of mglu5 , d2 , and a2ar may be exploited when designing drug therapies aimed at exhibiting little or no off - target effects . indeed , the therapeutic targeting of receptor mosaics / functional interactions in a tissue specific or temporal manner ( for example , a sub - population of receptors in a pathological state ) has the potential to reduce detrimental side effects that may otherwise impair vital brain function .

several external and internal factors influence an individual s ability to balance , including genetics , the state of vestibular apparatus , age , the area of support , center of mass positioning , emotional state , strength , coordination , flexibility , frequency of participation in motor activities , and training status . independent of these factors , static and dynamic balance continues to be an indispensable motor skill because it is at the center of all human movement ( 30 ) . not only is falling risk a result of poor balance , the ability to maintain balance is necessary to complete activities of daily living both safely and correctly ( 25 ) as well as to excel in sport - specific activities . the definition of balance is most often related to the goals of the present investigation . because balance is required during movement as well as during stance , several laboratory measures have defined two major types of balance . dynamic balance is the preservation of an upright body position throughout locomotion ( 30 ) , whereas static balance is the process of maintaining the center of mass vertically over the base of support with minimal movement ( 22 ) while maintaining specific poses for an extended period of time ( 30 ) . our study focused exclusively on static balance since the literature has shown that collegiate females ( gymnasts and soccer players ) do not differ in terms of static and dynamic balance for single and double leg stance on stiff and compliant surfaces ( 8) . further , although the definition of a stable body position includes the ability to maintain and return to the proper positioning of body segments during the execution of a task or following a perturbation ( 30 ) , our study only included the components of static balance that demonstrated minimal movement during a specified pose . center of pressure ( cop ) measurements have been shown to be a reproducible measure of static balance ( 17 ) . the ability to minimize displacement of the cop while maintaining an upright stance during proper orientation or desired locomotion is controlled by the central nervous system . further , because the central nervous system processes afferent input from visual , vestibular , and somatosensory systems the utilization of multiple senses provides for greater balance ability . when one of the sensory inputs is reduced or eliminated other systems compensate for the loss . of the three systems though , it is common for the somatosensory input to dominate the balancing task ( 12 ) . dance training strengthens the accuracy of the somatosensory system and effectively shifts the vision - dominated , sensorimotor control of balance to an internal - based system of reference ( 12 , 19 ) . therefore , the way dancers adjust in static and dynamic circumstances and self - correct body positioning is a function of strength , responsiveness of their proprioceptive system ( 18 ) , and advanced spatial skills ( 4 , 12 , 16 ) . due to enhanced balance abilities seen among dancers the purpose of this study was to determine if dancers demonstrate better single - leg static balance ability compared with non - dancers . specifically , we hypothesized that dancers would show better static single - leg balance over the prescribed 30 second time interval more efficiently than non - dancers , would have lower excursion of the cop and would keep their ground reaction force ( grf ) balance more lateral and anterior indicating better balance over the four conditions . the four conditions were static balance on the ( 1 ) dominant leg ( dl ) , ( 2 ) non - dominant leg ( ndl ) , ( 3 ) shod ( s ) and ( 4 ) barefoot ( bf ) . two questionnaires and a consent form were completed by those who volunteered as participants . in order to be included in the study participants were required to complete a pre - participation survey as well as score a 70 percent or greater on the standardized lower extremity functional scale ( lefs ) . the pre - participation survey helped identify whether a participant had any known neurological conditions or symptoms that would interfere with the demonstration of static balance . questions that were included in the survey that identified medications that could alter balance or cause dizziness as well as visual deficits that could interfere with balance . because the survey was given immediately prior to the testing period , we further asked if adequate amounts of food had been consumed since this could potentially alter balance performance and negatively affect concentration and focus . the lefs determined who was qualified to participate in the study since decreased functioning of the lower limb may affect balance . the score for the survey was calculated using the equation below : the lower the score , the greater the disability . we recruited seven female dancers ( d ) from the college dance department and seven female non - dancers ( nd ) from the school of education , health and human performance at the college . inclusion criteria for d included seven years or more of dance experience as well as participation on the collegiate level dance team . individuals in the nd control group had less than one year of dance experience or no experience at all . single - leg stance required participants to keep their hands on their hips , their eyes facing forward , and the foot of the non - testing leg held at knee level . their gaze had to remain fixed on an x that was taped two meters away at the individual s eye level . cop was defined as the resulting position of the force vector for all vertical grfs measured by the force plate . copap and copml represented the excursion of the cop measured in the antero - posterior direction ( ap ) and medio - lateral directions ( ml ) , respectively . total balance time represented the average time of the three trials that the subject maintained single - leg stance . maximum grfs included the grf in the ap direction ( fy ) , the grf in the ml direction ( fx ) , and the grf in the vertical direction ( fz ) . the grf coordinate system for the force plate was positive ( + ) for anterior and negative ( ) for the posterior direction . positive ( + ) was the sign for the medial direction and negative ( ) for the lateral direction . maximum grf variables for the three planes were normalized by dividing the resultant force vectors by subject body weight ( grf / subject weight in newtons ) . the dimensions of the force plate measured 600 mm by 1200 mm . in each corner , a piezoelectrical transducer measured the reaction forces occurring in the ap , ml , and vertical planes . data from the transducer were filtered with a low - pass 12 hz filter using a fourth order zero - lag butterworth frequency and processed by an analogue - to - digital converter , thereby eliminating high - frequency noise . these data were then transferred to the microcomputer and saved to the data collection software . single - leg stance was demonstrated to each of the 14 participants immediately prior to testing . participants were evaluated in four testing conditions : single - leg stance on the dl with and without an athletic shoe and single - leg stance on the ndl with and without an athletic shoe . subject shoe familiarity provided for no learning effect of different and unfamiliar shoe types among subjects , thereby optimizing shod balance . each subject performed single - leg stance with her supporting leg on the center of the force plate . the force plate was on an elevated , flat , and stable surface to reduce the amount of vibrations from the ground and surroundings . for each of the four conditions , participants balanced for 30 seconds . in order to obtain a valid measurement , each subject had three trials for each condition . however , if a subject could not maintain the single - leg stance for this period , termination of the trial occurred and the balancing time was recorded . the criteria for termination was defined as the removal of the foot from the opposite knee , the removal of the hands from the hips , the placement of the foot on the ground ( or toe - tapping ) , or the demonstration of a forward movement by the supporting leg . we controlled for the effect that external and environmental conditions could have on our measurements . specifically , we limited the number of individuals in the room to only the experimenters and the subject because of the disruption that surrounding movements could have on the subject s demonstration of balance . also , the room was consistently well - lit and of the same temperature and humidity . data analysis was conducted through ibm spss version 19 for windows ( chicago , il ) . prior to statistical analysis , because the data were normally distributed , independent t - tests were performed on the directional grf , cop , and total balance times for all four conditions . a bonferroni correction was applied to the original alpha level of ( p0.05 ) due to the increased risk of type i statistical error from the performance of multiple independent paired comparisons ; the adjusted alpha level was 0.05/5= 0.01 . the independent t - tests included comparisons for dancer bf dl versus ndl , dancer s dl versus ndl , non - dancer bf dl versus ndl , non - dancer s dl versus ndl , dancer versus non - dancer bf dl , dancer versus non - dancer bf ndl , dancer versus non - dancer s dl , and dancer versus non - dancer s ndl . two questionnaires and a consent form were completed by those who volunteered as participants . in order to be included in the study participants were required to complete a pre - participation survey as well as score a 70 percent or greater on the standardized lower extremity functional scale ( lefs ) . the pre - participation survey helped identify whether a participant had any known neurological conditions or symptoms that would interfere with the demonstration of static balance . questions that were included in the survey that identified medications that could alter balance or cause dizziness as well as visual deficits that could interfere with balance . because the survey was given immediately prior to the testing period , we further asked if adequate amounts of food had been consumed since this could potentially alter balance performance and negatively affect concentration and focus . the lefs determined who was qualified to participate in the study since decreased functioning of the lower limb may affect balance . the score for the survey was calculated using the equation below : the lower the score , the greater the disability . we recruited seven female dancers ( d ) from the college dance department and seven female non - dancers ( nd ) from the school of education , health and human performance at the college . inclusion criteria for d included seven years or more of dance experience as well as participation on the collegiate level dance team . individuals in the nd control group had less than one year of dance experience or no experience at all . single - leg stance required participants to keep their hands on their hips , their eyes facing forward , and the foot of the non - testing leg held at knee level . their gaze had to remain fixed on an x that was taped two meters away at the individual s eye level . cop was defined as the resulting position of the force vector for all vertical grfs measured by the force plate . copap and copml represented the excursion of the cop measured in the antero - posterior direction ( ap ) and medio - lateral directions ( ml ) , respectively . total balance time represented the average time of the three trials that the subject maintained single - leg stance . maximum grfs included the grf in the ap direction ( fy ) , the grf in the ml direction ( fx ) , and the grf in the vertical direction ( fz ) . the grf coordinate system for the force plate was positive ( + ) for anterior and negative ( ) for the posterior direction . positive ( + ) was the sign for the medial direction and negative ( ) for the lateral direction . maximum grf variables for the three planes were normalized by dividing the resultant force vectors by subject body weight ( grf / subject weight in newtons ) . the dimensions of the force plate measured 600 mm by 1200 mm . in each corner , a piezoelectrical transducer measured the reaction forces occurring in the ap , ml , and vertical planes . data from the transducer were filtered with a low - pass 12 hz filter using a fourth order zero - lag butterworth frequency and processed by an analogue - to - digital converter , thereby eliminating high - frequency noise . these data were then transferred to the microcomputer and saved to the data collection software . single - leg stance was demonstrated to each of the 14 participants immediately prior to testing . participants were evaluated in four testing conditions : single - leg stance on the dl with and without an athletic shoe and single - leg stance on the ndl with and without an athletic shoe . subject shoe familiarity provided for no learning effect of different and unfamiliar shoe types among subjects , thereby optimizing shod balance . each subject performed single - leg stance with her supporting leg on the center of the force plate . the force plate was on an elevated , flat , and stable surface to reduce the amount of vibrations from the ground and surroundings . for each of the four conditions , participants balanced for 30 seconds . in order to obtain a valid measurement , each subject had three trials for each condition . however , if a subject could not maintain the single - leg stance for this period , termination of the trial occurred and the balancing time was recorded . the criteria for termination was defined as the removal of the foot from the opposite knee , the removal of the hands from the hips , the placement of the foot on the ground ( or toe - tapping ) , or the demonstration of a forward movement by the supporting leg . we controlled for the effect that external and environmental conditions could have on our measurements . specifically , we limited the number of individuals in the room to only the experimenters and the subject because of the disruption that surrounding movements could have on the subject s demonstration of balance . also , the room was consistently well - lit and of the same temperature and humidity . data analysis was conducted through ibm spss version 19 for windows ( chicago , il ) . prior to statistical analysis , because the data were normally distributed , independent t - tests were performed on the directional grf , cop , and total balance times for all four conditions . a bonferroni correction was applied to the original alpha level of ( p0.05 ) due to the increased risk of type i statistical error from the performance of multiple independent paired comparisons ; the adjusted alpha level was 0.05/5= 0.01 . the independent t - tests included comparisons for dancer bf dl versus ndl , dancer s dl versus ndl , non - dancer bf dl versus ndl , non - dancer s dl versus ndl , dancer versus non - dancer bf dl , dancer versus non - dancer bf ndl , dancer versus non - dancer s dl , and dancer versus non - dancer s ndl . in table 1 , the demographics of the seven dancers and seven controls are shown . significant differences existed between groups for balance time ( d 30.00.0s ; nd 28.55.9s p0.03 ) . in 6 of the 84 trials for the non - dancer group , single - leg stance statistically significant differences also existed for : ap grf in bf dl ( d 0.0090.057 n ; nd 0.1490.067 n p0.001 ) and ndl ( d 0.0690.062 n ; nd 0.1880.105 n p0.001 ) ; ml grf in bf ndl ( d 0.0120.015 n ; nd 0.0130.025 n p0.001 ) ; ap grf in s dl ( d 0.0110.061 n ; nd 0.1620.041 n p0.001 ) ; ml grf in s dl ( d 0.0030.015 n ; nd 0.0180.006 n p0.001 ) ; ap grf in s ndl ( d 0.0800.041n ; nd 0.1680.097 n p0.001 ) ; and , ml grf in s ndl ( d 0.0080.012 n ; nd 0.0120.013 n p0.001 ) as shown in tables 2 and 3 . within group analyses for dancer and non - dancers can be found in tables 4 and 5 . statistically significant differences existed among dancers for : ap grf in bf ( dl 0.0090.057 ; ndl 0.0690.062 p0.001 ) and s ( dl 0.0100.062 ; ndl 0.0800.041 p0.001 ) ; and , copap in bf ( dl 0.000010.00003 ; ndl 0.000040.00006 p0.001 ) and s ( dl 0.000020.00004 ; ndl 0.000050.00005 p0.002 ) . statistically significant differences existed among non - dancers for : copml in bf ( dl 0.000020.00003 ; ndl 0.0000020.00003 p0.013 ) and s ( dl 0.000040.00002 ; ndl 0.0000020.00003 p0.001 ) . for the dl , a significant difference was found in the ap directional grf between dancers and non - dancers . for the ndl , significantly different grf was found in both the ap and ml directions . specifically , non - dancers demonstrated greater posterior - directed grf compared with dancers as well as more grf directed medially compared to the dancer group , which exhibited lateral grf . between group analyses for the s condition can be found in table 3 . for both the dl and ndl , significant grf differences were found in both the ap and ml directions . these significant differences were the result of non - dancers demonstrating greater posterior grf compared with the dancers . also , while dancers demonstrated a lateral grf in the f ml plane , a medially grf was seen among non - dancers . more posterior grf in both bf and s conditions indicates a greater loss of balance in the non - dancer group since , when in a static stance . sensory awareness in movement has received increased interest in sports and clinical literature as a feed - forward and feedback mechanism which may by extension contribute to static balance ( 6 ) . the greater medial grf for non - dancers in the bf and s conditions may also indicate more global instability . further , for the dl , a statistical trend ( p=0.019 ) was observed for the cop ml . non - dancers had a more medial cop , which we believe is indicative of greater perturbation , and therefore instability . it is also important to note that in the shod ndl condition , cop ap distances were different from each other , but did not reach significance ( p = 0.053 ) . in this condition , dancers showed more posterior grf , which is difficult to explain . since strength and proprioception play an essential role in injury prevention ( 3 ) , current athletic shoe construction may hinder balance ability without shoes . athletic shoes are often designed with features that enhance stability of the foot and body which is important for prevention and protection against injury ( 31 ) . there is evidence however that the rigid constraints of a traditional athletic shoe contribute to a lack of muscle development and proprioception ( 23 ) . because dancers perform movements primarily without shoes , it is possible that poor balance in the shod condition among dancers may be due to loss of sensory information that is available only when barefoot . in the within group analyses there were more statistical differences within the dancer group , indicating more variability in dl and ndl , as seen in tables 4 and 5 . dancers were statistically different in the ap grf in both the bf and s conditions . specifically , the results found that dancers show more posterior grf in the ndl , which would support the concept of greater instability on the non - dominant side and more stability on the dominant side . the copap was also statistically different between dl and ndl in bf and s conditions among dancers . both indicated more posterior deviation on the ndl , which supports the notion that the ndl shows greater instability when shod and barefoot . lastly , the bf ndl ml grf showed a statistical trend ( p = 0.016 ) . although dancers had more lateral grf on the non - dominant side , both measures support the position that a dancer s balance is directed more laterally . among the non - dancers the only statistically significant differences were found in the copml for both bf and s conditions . specifically , a more lateral deviation was observed in the copml for the ndl when barefoot . this finding seems unusual considering dancers seem to direct their grf and cop more lateral to maintain balance , yet in the non - dancers , the bf condition shows more laterality for the ndl , which would indicate greater stability for this side . in the s condition , there was less medial deviation in the ndl than in the dl , which again indicates greater stability for the ndl than the dl . the higher number of statistically significant differences between the dl and ndl within the dancer group may lead to the conclusion that a dancer s balance is not equally distributed between dl and ndl . it is important to recognize , though , that the less variability in these measures for non - dancers may be because they have poor balance in both legs , and therefore , differences can not be demonstrated . the results from two different studies demonstrate the impact that dance activity and associated movements have on enhancing static balance . ricotti and ravaschio measured static balance three times over a course of six months in three homogenous groups of 9-year - old soccer athletes ( 29 ) . for single - leg stance , there were no significant improvements in the dominant limb s cop area values neither for the group involved in soccer activity only nor for the group involved in soccer and swimming activity over the 6-month period . the group involved in the soccer and break dance activity , however , demonstrated a significant improvement in cop area values at month 0 and month 6 for the dominant limb . further , for the non - dominant limb , the combination of soccer and break dance activity yielded a greater number of significant differences in cop area values between month 6 and month 2 as well as between month 4 and month 2 ( 29 ) . stankovi m and radenkovi showed enhanced balance among 39 male and female school age children among those involved in a dance program ( 30 ) . in our study , it is evident that the seven or more years of dance experience increased balance ability when performing single - leg stance . although there was not a defined pattern of cop measurements in the ml and ap directions between dancers and non - dancers throughout all four conditions , dancers consistently demonstrated less deviation in the ap and ml grf during bf and s conditions compared to non - dancers . ambeganokar and colleagues also showed that dancers demonstrated significantly greater balance than non - dancers , especially statically ( 1 ) . our results of a subject s ability to maintain single - leg static balance for a 30-second duration is closely related to the results shown by ambeganokar et al . in our study dancers were able to maintain single - leg stance on the dominant and non - dominant limb for the entire 30-second duration while non - dancers were unable to do so in 6 of the 84 trials . many authors support the notion that balance plays an important role in sport - specific movements and injury reduction both of which are needed for dance success ( 15 , 22 ) . between dancers and soccer players , dancers performed significantly better on tasks measuring sway index during single - leg stance on stable and unstable surfaces ( 15 , 17 , 22 ) . postural control , balance , coordination , proprioception and consistency in movement patterns are successful dance characteristics and are also linked to reduced risk of ankle injury , re - injury and acl tears ( 5 , 15 , 16 , 20 , 3234 ) . diminished or exaggerated proprioceptive responses may be the direct result of musculoskeletal injury and surgical interventions ( 5 , 16 , 27 ) . a deficit in proprioception , independent of its extent , will compromise postural control , and therefore , offset balance and increase injury risk ( 11 , 13 , 26 ) . failure to restore proprioception in the lower extremity following surgery and rehabilitation , even if the athlete regains complete strength , will increase the risk for re - injury because of the joint instability that is perceived by the individual ( 16 , 21 ) . further , peripheral and central alterations in function , including joint laxity and instability , misalignment , localized weakness , diminished muscle reaction time , and altered center motor programming ( 4 , 22 , 24 ) may result if proprioceptive deficits are left untrained . thus , extending proprioceptive training and balance protocols used in dance for treatment of lower - extremity injuries may decrease the risk of injury since proprioceptive training is believed to be beneficial in preventing injuries in the lower extremity , protecting against re - injury , and reducing recovery time ( 4 , 22 , 35 ) . in athletes with recurrent ankle sprains , proprioceptive testing has shown that some with recurrent ankle sprains have lower ankle joint position sense compared with individuals without such injuries ( 7 , 28 ) . following knee and ankle injury in athletes , proprioceptive training improves postural stability ( 14 ) , flexibility ( 13 ) , joint position sense , and faster muscle reaction time ( 24 ) . it is important to note , though , that the ability to reduce the risk of specific injuries in specific sports through proprioceptive balance training remains unclear due to the inclusions of strength and agility throughout the training programs ( 3 , 9 ) . for example , ambegaonkar and colleagues sought to understand why female dancers have lower acl injury rates compared with physically active females ( 1 ) . prior to the study , the authors believed that better balance by experienced dancers contributed to decreased musculoskeletal injury risk . the authors concluded that the examination of balance between dancers and non - dancers does not provide a complete explanation regarding why physically active females experience higher acl injury rates compared with dancers . acl injury risk is multifaceted , with balance and proprioception being important components , however joint structure , including the direction and magnitude of the destabilizing force , the rate at which loads are applied , and joint position all contribute to risk . therefore , balance appears to be one of several components that contributes to acl injury . static testing conditions that use a fixed base of support may not translate into the functionally dynamic movements that occur in sport . thus , an athlete may not demonstrate a deficiency in balance ability during static conditions when in fact one exists throughout his movement patterns ( 15 ) . selective destabilization of a joint through the incorporation of perturbations in balance training should also be included in the protocol for rehabilitation ( 10 ) . the application of graded , controlled forces across injured joints is effective in activating higher neural centers that evoke postural synergies , reducing injury potential ( 2 , 5 ) , allowing the center of mass to remain over the base of support .. following this study , we hoped to better understand how dance impacted balance and whether or not these results could be applied to lower - extremity rehabilitation . our study defined balance as the ability to maintain a specified pose for a duration of 30 seconds as well as the ability to demonstrate minimal movement throughout this time interval . more specifically minimal movement was understood as a decrease in posterior and medial grf and deviation in the cop . the duration that an individual maintained single - leg stance also highlighted static balance ability . rehabilitation should provide balance training as a major component in both rehabilitation as well as pre - season injury prevention programs for their athletes .

the purpose of this study was to compare kinetic differences of static balance between female dancers ( d ) with at least seven years of dance experience and female non - dancers ( nd ) who were typical college students . participants were tested in single - leg stance . both the dominant leg ( dl ) and non - dominant leg ( ndl ) were tested with the participants shod ( s ) and barefoot ( bf ) . kinetic variables ( vertical , medio - lateral [ ml ] , antero - posterior [ ap ] maximum ground reaction forces ( grf ) , and center of pressure ( cop ) ml and ap ) were measured by a bertec force platform at 1000 hz with participants s and bf . each subject s stance was measured over 3 30-second intervals . no significant differences ( p0.05 ) existed between groups for height , body mass , or age . significant differences existed between groups for balance time , ap grf in both bf and s conditions for both dl and ndl , and ml grf in bf ndl and s dl and ndl conditions . d and nd in bf and s conditions with dl and ndl static stance demonstrate different ap and ml grf when balancing over a 30-second time interval . data may suggest that nd are more prone to lose their balance . further investigation is warranted to understand whether individuals in the rehabilitative field and athletic populations can use dance therapy for injury prevention and rehabilitation .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

several external and internal factors influence an individual s ability to balance , including genetics , the state of vestibular apparatus , age , the area of support , center of mass positioning , emotional state , strength , coordination , flexibility , frequency of participation in motor activities , and training status . dynamic balance is the preservation of an upright body position throughout locomotion ( 30 ) , whereas static balance is the process of maintaining the center of mass vertically over the base of support with minimal movement ( 22 ) while maintaining specific poses for an extended period of time ( 30 ) . our study focused exclusively on static balance since the literature has shown that collegiate females ( gymnasts and soccer players ) do not differ in terms of static and dynamic balance for single and double leg stance on stiff and compliant surfaces ( 8) . further , although the definition of a stable body position includes the ability to maintain and return to the proper positioning of body segments during the execution of a task or following a perturbation ( 30 ) , our study only included the components of static balance that demonstrated minimal movement during a specified pose . center of pressure ( cop ) measurements have been shown to be a reproducible measure of static balance ( 17 ) . further , because the central nervous system processes afferent input from visual , vestibular , and somatosensory systems the utilization of multiple senses provides for greater balance ability . therefore , the way dancers adjust in static and dynamic circumstances and self - correct body positioning is a function of strength , responsiveness of their proprioceptive system ( 18 ) , and advanced spatial skills ( 4 , 12 , 16 ) . due to enhanced balance abilities seen among dancers the purpose of this study was to determine if dancers demonstrate better single - leg static balance ability compared with non - dancers . specifically , we hypothesized that dancers would show better static single - leg balance over the prescribed 30 second time interval more efficiently than non - dancers , would have lower excursion of the cop and would keep their ground reaction force ( grf ) balance more lateral and anterior indicating better balance over the four conditions . the four conditions were static balance on the ( 1 ) dominant leg ( dl ) , ( 2 ) non - dominant leg ( ndl ) , ( 3 ) shod ( s ) and ( 4 ) barefoot ( bf ) . in order to be included in the study participants were required to complete a pre - participation survey as well as score a 70 percent or greater on the standardized lower extremity functional scale ( lefs ) . the pre - participation survey helped identify whether a participant had any known neurological conditions or symptoms that would interfere with the demonstration of static balance . questions that were included in the survey that identified medications that could alter balance or cause dizziness as well as visual deficits that could interfere with balance . we recruited seven female dancers ( d ) from the college dance department and seven female non - dancers ( nd ) from the school of education , health and human performance at the college . inclusion criteria for d included seven years or more of dance experience as well as participation on the collegiate level dance team . individuals in the nd control group had less than one year of dance experience or no experience at all . single - leg stance required participants to keep their hands on their hips , their eyes facing forward , and the foot of the non - testing leg held at knee level . cop was defined as the resulting position of the force vector for all vertical grfs measured by the force plate . copap and copml represented the excursion of the cop measured in the antero - posterior direction ( ap ) and medio - lateral directions ( ml ) , respectively . total balance time represented the average time of the three trials that the subject maintained single - leg stance . maximum grfs included the grf in the ap direction ( fy ) , the grf in the ml direction ( fx ) , and the grf in the vertical direction ( fz ) . maximum grf variables for the three planes were normalized by dividing the resultant force vectors by subject body weight ( grf / subject weight in newtons ) . in each corner , a piezoelectrical transducer measured the reaction forces occurring in the ap , ml , and vertical planes . single - leg stance was demonstrated to each of the 14 participants immediately prior to testing . participants were evaluated in four testing conditions : single - leg stance on the dl with and without an athletic shoe and single - leg stance on the ndl with and without an athletic shoe . each subject performed single - leg stance with her supporting leg on the center of the force plate . however , if a subject could not maintain the single - leg stance for this period , termination of the trial occurred and the balancing time was recorded . the criteria for termination was defined as the removal of the foot from the opposite knee , the removal of the hands from the hips , the placement of the foot on the ground ( or toe - tapping ) , or the demonstration of a forward movement by the supporting leg . specifically , we limited the number of individuals in the room to only the experimenters and the subject because of the disruption that surrounding movements could have on the subject s demonstration of balance . prior to statistical analysis , because the data were normally distributed , independent t - tests were performed on the directional grf , cop , and total balance times for all four conditions . a bonferroni correction was applied to the original alpha level of ( p0.05 ) due to the increased risk of type i statistical error from the performance of multiple independent paired comparisons ; the adjusted alpha level was 0.05/5= 0.01 . the independent t - tests included comparisons for dancer bf dl versus ndl , dancer s dl versus ndl , non - dancer bf dl versus ndl , non - dancer s dl versus ndl , dancer versus non - dancer bf dl , dancer versus non - dancer bf ndl , dancer versus non - dancer s dl , and dancer versus non - dancer s ndl . in order to be included in the study participants were required to complete a pre - participation survey as well as score a 70 percent or greater on the standardized lower extremity functional scale ( lefs ) . the pre - participation survey helped identify whether a participant had any known neurological conditions or symptoms that would interfere with the demonstration of static balance . we recruited seven female dancers ( d ) from the college dance department and seven female non - dancers ( nd ) from the school of education , health and human performance at the college . inclusion criteria for d included seven years or more of dance experience as well as participation on the collegiate level dance team . individuals in the nd control group had less than one year of dance experience or no experience at all . single - leg stance required participants to keep their hands on their hips , their eyes facing forward , and the foot of the non - testing leg held at knee level . cop was defined as the resulting position of the force vector for all vertical grfs measured by the force plate . copap and copml represented the excursion of the cop measured in the antero - posterior direction ( ap ) and medio - lateral directions ( ml ) , respectively . total balance time represented the average time of the three trials that the subject maintained single - leg stance . maximum grfs included the grf in the ap direction ( fy ) , the grf in the ml direction ( fx ) , and the grf in the vertical direction ( fz ) . maximum grf variables for the three planes were normalized by dividing the resultant force vectors by subject body weight ( grf / subject weight in newtons ) . in each corner , a piezoelectrical transducer measured the reaction forces occurring in the ap , ml , and vertical planes . single - leg stance was demonstrated to each of the 14 participants immediately prior to testing . participants were evaluated in four testing conditions : single - leg stance on the dl with and without an athletic shoe and single - leg stance on the ndl with and without an athletic shoe . each subject performed single - leg stance with her supporting leg on the center of the force plate . however , if a subject could not maintain the single - leg stance for this period , termination of the trial occurred and the balancing time was recorded . the criteria for termination was defined as the removal of the foot from the opposite knee , the removal of the hands from the hips , the placement of the foot on the ground ( or toe - tapping ) , or the demonstration of a forward movement by the supporting leg . specifically , we limited the number of individuals in the room to only the experimenters and the subject because of the disruption that surrounding movements could have on the subject s demonstration of balance . prior to statistical analysis , because the data were normally distributed , independent t - tests were performed on the directional grf , cop , and total balance times for all four conditions . a bonferroni correction was applied to the original alpha level of ( p0.05 ) due to the increased risk of type i statistical error from the performance of multiple independent paired comparisons ; the adjusted alpha level was 0.05/5= 0.01 . the independent t - tests included comparisons for dancer bf dl versus ndl , dancer s dl versus ndl , non - dancer bf dl versus ndl , non - dancer s dl versus ndl , dancer versus non - dancer bf dl , dancer versus non - dancer bf ndl , dancer versus non - dancer s dl , and dancer versus non - dancer s ndl . significant differences existed between groups for balance time ( d 30.00.0s ; nd 28.55.9s p0.03 ) . in 6 of the 84 trials for the non - dancer group , single - leg stance statistically significant differences also existed for : ap grf in bf dl ( d 0.0090.057 n ; nd 0.1490.067 n p0.001 ) and ndl ( d 0.0690.062 n ; nd 0.1880.105 n p0.001 ) ; ml grf in bf ndl ( d 0.0120.015 n ; nd 0.0130.025 n p0.001 ) ; ap grf in s dl ( d 0.0110.061 n ; nd 0.1620.041 n p0.001 ) ; ml grf in s dl ( d 0.0030.015 n ; nd 0.0180.006 n p0.001 ) ; ap grf in s ndl ( d 0.0800.041n ; nd 0.1680.097 n p0.001 ) ; and , ml grf in s ndl ( d 0.0080.012 n ; nd 0.0120.013 n p0.001 ) as shown in tables 2 and 3 . within group analyses for dancer and non - dancers can be found in tables 4 and 5 . statistically significant differences existed among dancers for : ap grf in bf ( dl 0.0090.057 ; ndl 0.0690.062 p0.001 ) and s ( dl 0.0100.062 ; ndl 0.0800.041 p0.001 ) ; and , copap in bf ( dl 0.000010.00003 ; ndl 0.000040.00006 p0.001 ) and s ( dl 0.000020.00004 ; ndl 0.000050.00005 p0.002 ) . statistically significant differences existed among non - dancers for : copml in bf ( dl 0.000020.00003 ; ndl 0.0000020.00003 p0.013 ) and s ( dl 0.000040.00002 ; ndl 0.0000020.00003 p0.001 ) . for the dl , a significant difference was found in the ap directional grf between dancers and non - dancers . for the ndl , significantly different grf was found in both the ap and ml directions . specifically , non - dancers demonstrated greater posterior - directed grf compared with dancers as well as more grf directed medially compared to the dancer group , which exhibited lateral grf . for both the dl and ndl , significant grf differences were found in both the ap and ml directions . these significant differences were the result of non - dancers demonstrating greater posterior grf compared with the dancers . also , while dancers demonstrated a lateral grf in the f ml plane , a medially grf was seen among non - dancers . more posterior grf in both bf and s conditions indicates a greater loss of balance in the non - dancer group since , when in a static stance . the greater medial grf for non - dancers in the bf and s conditions may also indicate more global instability . non - dancers had a more medial cop , which we believe is indicative of greater perturbation , and therefore instability . it is also important to note that in the shod ndl condition , cop ap distances were different from each other , but did not reach significance ( p = 0.053 ) . since strength and proprioception play an essential role in injury prevention ( 3 ) , current athletic shoe construction may hinder balance ability without shoes . in the within group analyses there were more statistical differences within the dancer group , indicating more variability in dl and ndl , as seen in tables 4 and 5 . dancers were statistically different in the ap grf in both the bf and s conditions . specifically , the results found that dancers show more posterior grf in the ndl , which would support the concept of greater instability on the non - dominant side and more stability on the dominant side . the copap was also statistically different between dl and ndl in bf and s conditions among dancers . both indicated more posterior deviation on the ndl , which supports the notion that the ndl shows greater instability when shod and barefoot . lastly , the bf ndl ml grf showed a statistical trend ( p = 0.016 ) . although dancers had more lateral grf on the non - dominant side , both measures support the position that a dancer s balance is directed more laterally . among the non - dancers the only statistically significant differences were found in the copml for both bf and s conditions . this finding seems unusual considering dancers seem to direct their grf and cop more lateral to maintain balance , yet in the non - dancers , the bf condition shows more laterality for the ndl , which would indicate greater stability for this side . in the s condition , there was less medial deviation in the ndl than in the dl , which again indicates greater stability for the ndl than the dl . the higher number of statistically significant differences between the dl and ndl within the dancer group may lead to the conclusion that a dancer s balance is not equally distributed between dl and ndl . it is important to recognize , though , that the less variability in these measures for non - dancers may be because they have poor balance in both legs , and therefore , differences can not be demonstrated . the results from two different studies demonstrate the impact that dance activity and associated movements have on enhancing static balance . ricotti and ravaschio measured static balance three times over a course of six months in three homogenous groups of 9-year - old soccer athletes ( 29 ) . for single - leg stance , there were no significant improvements in the dominant limb s cop area values neither for the group involved in soccer activity only nor for the group involved in soccer and swimming activity over the 6-month period . the group involved in the soccer and break dance activity , however , demonstrated a significant improvement in cop area values at month 0 and month 6 for the dominant limb . further , for the non - dominant limb , the combination of soccer and break dance activity yielded a greater number of significant differences in cop area values between month 6 and month 2 as well as between month 4 and month 2 ( 29 ) . stankovi m and radenkovi showed enhanced balance among 39 male and female school age children among those involved in a dance program ( 30 ) . in our study , it is evident that the seven or more years of dance experience increased balance ability when performing single - leg stance . although there was not a defined pattern of cop measurements in the ml and ap directions between dancers and non - dancers throughout all four conditions , dancers consistently demonstrated less deviation in the ap and ml grf during bf and s conditions compared to non - dancers . ambeganokar and colleagues also showed that dancers demonstrated significantly greater balance than non - dancers , especially statically ( 1 ) . our results of a subject s ability to maintain single - leg static balance for a 30-second duration is closely related to the results shown by ambeganokar et al . in our study dancers were able to maintain single - leg stance on the dominant and non - dominant limb for the entire 30-second duration while non - dancers were unable to do so in 6 of the 84 trials . between dancers and soccer players , dancers performed significantly better on tasks measuring sway index during single - leg stance on stable and unstable surfaces ( 15 , 17 , 22 ) . a deficit in proprioception , independent of its extent , will compromise postural control , and therefore , offset balance and increase injury risk ( 11 , 13 , 26 ) . failure to restore proprioception in the lower extremity following surgery and rehabilitation , even if the athlete regains complete strength , will increase the risk for re - injury because of the joint instability that is perceived by the individual ( 16 , 21 ) . further , peripheral and central alterations in function , including joint laxity and instability , misalignment , localized weakness , diminished muscle reaction time , and altered center motor programming ( 4 , 22 , 24 ) may result if proprioceptive deficits are left untrained . thus , extending proprioceptive training and balance protocols used in dance for treatment of lower - extremity injuries may decrease the risk of injury since proprioceptive training is believed to be beneficial in preventing injuries in the lower extremity , protecting against re - injury , and reducing recovery time ( 4 , 22 , 35 ) . following knee and ankle injury in athletes , proprioceptive training improves postural stability ( 14 ) , flexibility ( 13 ) , joint position sense , and faster muscle reaction time ( 24 ) . for example , ambegaonkar and colleagues sought to understand why female dancers have lower acl injury rates compared with physically active females ( 1 ) . the authors concluded that the examination of balance between dancers and non - dancers does not provide a complete explanation regarding why physically active females experience higher acl injury rates compared with dancers . acl injury risk is multifaceted , with balance and proprioception being important components , however joint structure , including the direction and magnitude of the destabilizing force , the rate at which loads are applied , and joint position all contribute to risk . selective destabilization of a joint through the incorporation of perturbations in balance training should also be included in the protocol for rehabilitation ( 10 ) . the application of graded , controlled forces across injured joints is effective in activating higher neural centers that evoke postural synergies , reducing injury potential ( 2 , 5 ) , allowing the center of mass to remain over the base of support .. following this study , we hoped to better understand how dance impacted balance and whether or not these results could be applied to lower - extremity rehabilitation . our study defined balance as the ability to maintain a specified pose for a duration of 30 seconds as well as the ability to demonstrate minimal movement throughout this time interval . the duration that an individual maintained single - leg stance also highlighted static balance ability . rehabilitation should provide balance training as a major component in both rehabilitation as well as pre - season injury prevention programs for their athletes .

several external and internal factors influence an individual s ability to balance , including genetics , the state of vestibular apparatus , age , the area of support , center of mass positioning , emotional state , strength , coordination , flexibility , frequency of participation in motor activities , and training status . independent of these factors , static and dynamic balance continues to be an indispensable motor skill because it is at the center of all human movement ( 30 ) . not only is falling risk a result of poor balance , the ability to maintain balance is necessary to complete activities of daily living both safely and correctly ( 25 ) as well as to excel in sport - specific activities . dynamic balance is the preservation of an upright body position throughout locomotion ( 30 ) , whereas static balance is the process of maintaining the center of mass vertically over the base of support with minimal movement ( 22 ) while maintaining specific poses for an extended period of time ( 30 ) . our study focused exclusively on static balance since the literature has shown that collegiate females ( gymnasts and soccer players ) do not differ in terms of static and dynamic balance for single and double leg stance on stiff and compliant surfaces ( 8) . further , although the definition of a stable body position includes the ability to maintain and return to the proper positioning of body segments during the execution of a task or following a perturbation ( 30 ) , our study only included the components of static balance that demonstrated minimal movement during a specified pose . center of pressure ( cop ) measurements have been shown to be a reproducible measure of static balance ( 17 ) . further , because the central nervous system processes afferent input from visual , vestibular , and somatosensory systems the utilization of multiple senses provides for greater balance ability . of the three systems though , it is common for the somatosensory input to dominate the balancing task ( 12 ) . dance training strengthens the accuracy of the somatosensory system and effectively shifts the vision - dominated , sensorimotor control of balance to an internal - based system of reference ( 12 , 19 ) . therefore , the way dancers adjust in static and dynamic circumstances and self - correct body positioning is a function of strength , responsiveness of their proprioceptive system ( 18 ) , and advanced spatial skills ( 4 , 12 , 16 ) . due to enhanced balance abilities seen among dancers the purpose of this study was to determine if dancers demonstrate better single - leg static balance ability compared with non - dancers . specifically , we hypothesized that dancers would show better static single - leg balance over the prescribed 30 second time interval more efficiently than non - dancers , would have lower excursion of the cop and would keep their ground reaction force ( grf ) balance more lateral and anterior indicating better balance over the four conditions . the four conditions were static balance on the ( 1 ) dominant leg ( dl ) , ( 2 ) non - dominant leg ( ndl ) , ( 3 ) shod ( s ) and ( 4 ) barefoot ( bf ) . in order to be included in the study participants were required to complete a pre - participation survey as well as score a 70 percent or greater on the standardized lower extremity functional scale ( lefs ) . because the survey was given immediately prior to the testing period , we further asked if adequate amounts of food had been consumed since this could potentially alter balance performance and negatively affect concentration and focus . the lefs determined who was qualified to participate in the study since decreased functioning of the lower limb may affect balance . the score for the survey was calculated using the equation below : the lower the score , the greater the disability . we recruited seven female dancers ( d ) from the college dance department and seven female non - dancers ( nd ) from the school of education , health and human performance at the college . individuals in the nd control group had less than one year of dance experience or no experience at all . single - leg stance required participants to keep their hands on their hips , their eyes facing forward , and the foot of the non - testing leg held at knee level . copap and copml represented the excursion of the cop measured in the antero - posterior direction ( ap ) and medio - lateral directions ( ml ) , respectively . total balance time represented the average time of the three trials that the subject maintained single - leg stance . maximum grfs included the grf in the ap direction ( fy ) , the grf in the ml direction ( fx ) , and the grf in the vertical direction ( fz ) . the grf coordinate system for the force plate was positive ( + ) for anterior and negative ( ) for the posterior direction . positive ( + ) was the sign for the medial direction and negative ( ) for the lateral direction . participants were evaluated in four testing conditions : single - leg stance on the dl with and without an athletic shoe and single - leg stance on the ndl with and without an athletic shoe . subject shoe familiarity provided for no learning effect of different and unfamiliar shoe types among subjects , thereby optimizing shod balance . the force plate was on an elevated , flat , and stable surface to reduce the amount of vibrations from the ground and surroundings . however , if a subject could not maintain the single - leg stance for this period , termination of the trial occurred and the balancing time was recorded . the criteria for termination was defined as the removal of the foot from the opposite knee , the removal of the hands from the hips , the placement of the foot on the ground ( or toe - tapping ) , or the demonstration of a forward movement by the supporting leg . specifically , we limited the number of individuals in the room to only the experimenters and the subject because of the disruption that surrounding movements could have on the subject s demonstration of balance . also , the room was consistently well - lit and of the same temperature and humidity . prior to statistical analysis , because the data were normally distributed , independent t - tests were performed on the directional grf , cop , and total balance times for all four conditions . a bonferroni correction was applied to the original alpha level of ( p0.05 ) due to the increased risk of type i statistical error from the performance of multiple independent paired comparisons ; the adjusted alpha level was 0.05/5= 0.01 . the independent t - tests included comparisons for dancer bf dl versus ndl , dancer s dl versus ndl , non - dancer bf dl versus ndl , non - dancer s dl versus ndl , dancer versus non - dancer bf dl , dancer versus non - dancer bf ndl , dancer versus non - dancer s dl , and dancer versus non - dancer s ndl . in order to be included in the study participants were required to complete a pre - participation survey as well as score a 70 percent or greater on the standardized lower extremity functional scale ( lefs ) . because the survey was given immediately prior to the testing period , we further asked if adequate amounts of food had been consumed since this could potentially alter balance performance and negatively affect concentration and focus . the lefs determined who was qualified to participate in the study since decreased functioning of the lower limb may affect balance . the score for the survey was calculated using the equation below : the lower the score , the greater the disability . we recruited seven female dancers ( d ) from the college dance department and seven female non - dancers ( nd ) from the school of education , health and human performance at the college . individuals in the nd control group had less than one year of dance experience or no experience at all . single - leg stance required participants to keep their hands on their hips , their eyes facing forward , and the foot of the non - testing leg held at knee level . copap and copml represented the excursion of the cop measured in the antero - posterior direction ( ap ) and medio - lateral directions ( ml ) , respectively . total balance time represented the average time of the three trials that the subject maintained single - leg stance . maximum grfs included the grf in the ap direction ( fy ) , the grf in the ml direction ( fx ) , and the grf in the vertical direction ( fz ) . the grf coordinate system for the force plate was positive ( + ) for anterior and negative ( ) for the posterior direction . positive ( + ) was the sign for the medial direction and negative ( ) for the lateral direction . participants were evaluated in four testing conditions : single - leg stance on the dl with and without an athletic shoe and single - leg stance on the ndl with and without an athletic shoe . subject shoe familiarity provided for no learning effect of different and unfamiliar shoe types among subjects , thereby optimizing shod balance . the force plate was on an elevated , flat , and stable surface to reduce the amount of vibrations from the ground and surroundings . however , if a subject could not maintain the single - leg stance for this period , termination of the trial occurred and the balancing time was recorded . the criteria for termination was defined as the removal of the foot from the opposite knee , the removal of the hands from the hips , the placement of the foot on the ground ( or toe - tapping ) , or the demonstration of a forward movement by the supporting leg . specifically , we limited the number of individuals in the room to only the experimenters and the subject because of the disruption that surrounding movements could have on the subject s demonstration of balance . prior to statistical analysis , because the data were normally distributed , independent t - tests were performed on the directional grf , cop , and total balance times for all four conditions . a bonferroni correction was applied to the original alpha level of ( p0.05 ) due to the increased risk of type i statistical error from the performance of multiple independent paired comparisons ; the adjusted alpha level was 0.05/5= 0.01 . the independent t - tests included comparisons for dancer bf dl versus ndl , dancer s dl versus ndl , non - dancer bf dl versus ndl , non - dancer s dl versus ndl , dancer versus non - dancer bf dl , dancer versus non - dancer bf ndl , dancer versus non - dancer s dl , and dancer versus non - dancer s ndl . in table 1 , the demographics of the seven dancers and seven controls are shown . in 6 of the 84 trials for the non - dancer group , single - leg stance statistically significant differences also existed for : ap grf in bf dl ( d 0.0090.057 n ; nd 0.1490.067 n p0.001 ) and ndl ( d 0.0690.062 n ; nd 0.1880.105 n p0.001 ) ; ml grf in bf ndl ( d 0.0120.015 n ; nd 0.0130.025 n p0.001 ) ; ap grf in s dl ( d 0.0110.061 n ; nd 0.1620.041 n p0.001 ) ; ml grf in s dl ( d 0.0030.015 n ; nd 0.0180.006 n p0.001 ) ; ap grf in s ndl ( d 0.0800.041n ; nd 0.1680.097 n p0.001 ) ; and , ml grf in s ndl ( d 0.0080.012 n ; nd 0.0120.013 n p0.001 ) as shown in tables 2 and 3 . statistically significant differences existed among dancers for : ap grf in bf ( dl 0.0090.057 ; ndl 0.0690.062 p0.001 ) and s ( dl 0.0100.062 ; ndl 0.0800.041 p0.001 ) ; and , copap in bf ( dl 0.000010.00003 ; ndl 0.000040.00006 p0.001 ) and s ( dl 0.000020.00004 ; ndl 0.000050.00005 p0.002 ) . statistically significant differences existed among non - dancers for : copml in bf ( dl 0.000020.00003 ; ndl 0.0000020.00003 p0.013 ) and s ( dl 0.000040.00002 ; ndl 0.0000020.00003 p0.001 ) . for the dl , a significant difference was found in the ap directional grf between dancers and non - dancers . for the ndl , significantly different grf was found in both the ap and ml directions . specifically , non - dancers demonstrated greater posterior - directed grf compared with dancers as well as more grf directed medially compared to the dancer group , which exhibited lateral grf . for both the dl and ndl , significant grf differences were found in both the ap and ml directions . these significant differences were the result of non - dancers demonstrating greater posterior grf compared with the dancers . more posterior grf in both bf and s conditions indicates a greater loss of balance in the non - dancer group since , when in a static stance . sensory awareness in movement has received increased interest in sports and clinical literature as a feed - forward and feedback mechanism which may by extension contribute to static balance ( 6 ) . further , for the dl , a statistical trend ( p=0.019 ) was observed for the cop ml . since strength and proprioception play an essential role in injury prevention ( 3 ) , current athletic shoe construction may hinder balance ability without shoes . athletic shoes are often designed with features that enhance stability of the foot and body which is important for prevention and protection against injury ( 31 ) . there is evidence however that the rigid constraints of a traditional athletic shoe contribute to a lack of muscle development and proprioception ( 23 ) . in the within group analyses there were more statistical differences within the dancer group , indicating more variability in dl and ndl , as seen in tables 4 and 5 . dancers were statistically different in the ap grf in both the bf and s conditions . specifically , the results found that dancers show more posterior grf in the ndl , which would support the concept of greater instability on the non - dominant side and more stability on the dominant side . the copap was also statistically different between dl and ndl in bf and s conditions among dancers . both indicated more posterior deviation on the ndl , which supports the notion that the ndl shows greater instability when shod and barefoot . although dancers had more lateral grf on the non - dominant side , both measures support the position that a dancer s balance is directed more laterally . among the non - dancers the only statistically significant differences were found in the copml for both bf and s conditions . this finding seems unusual considering dancers seem to direct their grf and cop more lateral to maintain balance , yet in the non - dancers , the bf condition shows more laterality for the ndl , which would indicate greater stability for this side . in the s condition , there was less medial deviation in the ndl than in the dl , which again indicates greater stability for the ndl than the dl . the higher number of statistically significant differences between the dl and ndl within the dancer group may lead to the conclusion that a dancer s balance is not equally distributed between dl and ndl . it is important to recognize , though , that the less variability in these measures for non - dancers may be because they have poor balance in both legs , and therefore , differences can not be demonstrated . the results from two different studies demonstrate the impact that dance activity and associated movements have on enhancing static balance . for single - leg stance , there were no significant improvements in the dominant limb s cop area values neither for the group involved in soccer activity only nor for the group involved in soccer and swimming activity over the 6-month period . the group involved in the soccer and break dance activity , however , demonstrated a significant improvement in cop area values at month 0 and month 6 for the dominant limb . further , for the non - dominant limb , the combination of soccer and break dance activity yielded a greater number of significant differences in cop area values between month 6 and month 2 as well as between month 4 and month 2 ( 29 ) . stankovi m and radenkovi showed enhanced balance among 39 male and female school age children among those involved in a dance program ( 30 ) . although there was not a defined pattern of cop measurements in the ml and ap directions between dancers and non - dancers throughout all four conditions , dancers consistently demonstrated less deviation in the ap and ml grf during bf and s conditions compared to non - dancers . in our study dancers were able to maintain single - leg stance on the dominant and non - dominant limb for the entire 30-second duration while non - dancers were unable to do so in 6 of the 84 trials . many authors support the notion that balance plays an important role in sport - specific movements and injury reduction both of which are needed for dance success ( 15 , 22 ) . between dancers and soccer players , dancers performed significantly better on tasks measuring sway index during single - leg stance on stable and unstable surfaces ( 15 , 17 , 22 ) . postural control , balance , coordination , proprioception and consistency in movement patterns are successful dance characteristics and are also linked to reduced risk of ankle injury , re - injury and acl tears ( 5 , 15 , 16 , 20 , 3234 ) . a deficit in proprioception , independent of its extent , will compromise postural control , and therefore , offset balance and increase injury risk ( 11 , 13 , 26 ) . failure to restore proprioception in the lower extremity following surgery and rehabilitation , even if the athlete regains complete strength , will increase the risk for re - injury because of the joint instability that is perceived by the individual ( 16 , 21 ) . further , peripheral and central alterations in function , including joint laxity and instability , misalignment , localized weakness , diminished muscle reaction time , and altered center motor programming ( 4 , 22 , 24 ) may result if proprioceptive deficits are left untrained . thus , extending proprioceptive training and balance protocols used in dance for treatment of lower - extremity injuries may decrease the risk of injury since proprioceptive training is believed to be beneficial in preventing injuries in the lower extremity , protecting against re - injury , and reducing recovery time ( 4 , 22 , 35 ) . following knee and ankle injury in athletes , proprioceptive training improves postural stability ( 14 ) , flexibility ( 13 ) , joint position sense , and faster muscle reaction time ( 24 ) . it is important to note , though , that the ability to reduce the risk of specific injuries in specific sports through proprioceptive balance training remains unclear due to the inclusions of strength and agility throughout the training programs ( 3 , 9 ) . the authors concluded that the examination of balance between dancers and non - dancers does not provide a complete explanation regarding why physically active females experience higher acl injury rates compared with dancers . acl injury risk is multifaceted , with balance and proprioception being important components , however joint structure , including the direction and magnitude of the destabilizing force , the rate at which loads are applied , and joint position all contribute to risk . the application of graded , controlled forces across injured joints is effective in activating higher neural centers that evoke postural synergies , reducing injury potential ( 2 , 5 ) , allowing the center of mass to remain over the base of support .. following this study , we hoped to better understand how dance impacted balance and whether or not these results could be applied to lower - extremity rehabilitation . our study defined balance as the ability to maintain a specified pose for a duration of 30 seconds as well as the ability to demonstrate minimal movement throughout this time interval .

mood disorders , such as major depressive disorder ( mdd ) and bipolar disorder ( bd ) , are highly prevalent and debilitating conditions , associated with high suicidality rates , elevated treatment costs , and heavy social and economic burden . the treatment of depressive episodes is associated with a 3040% rate of nonresponse . electroconvulsive therapy ( ect ) is regarded as the most effective treatment for depression , in both bd and mdd , with remission rates ranging from 50% to 75% [ 46 ] . ect is also effective for manic episodes , with reported remission rates of up to 85% [ 711 ] . despite its high effectiveness , the use of ect is limited by its side effects , especially the development of cognitive deficits and , in particular , memory impairment [ 1216 ] . such side effects can range from mild or nonexistent in some patients up to severe and distressing in others [ 14 , 1719 ] . of those , memory loss is the single most often cognitive complaint reported by patients undergoing ect . the cognitive effects of ect have been shown to be dependent upon numerous ect parameters , namely , electrode placement , frequency of sessions , electric dose , and pulse width [ 14 , 17 , 20 ] . the search for improved ect techniques , which could be able to reduce or minimize memory - related side effects whilst maintaining efficacy , has been the subject of a considerable amount of investigation . however , the use of an electrical stimulus to induce seizures is a fundamental limitation in refining convulsive therapy and limiting its side effects profile . control over the spatial distribution and magnitude of intracerebral current density is limited by high skull impedance , which shunts most of the electrical stimulus through the scalp and cerebrospinal fluid ( csf ) and away from the brain . the substantial impedance of the scalp and skull means that the bulk of the electrical stimulus is shunted away from the brain , resulting in widespread stimulation of cortical and subcortical regions . there are also individual differences in skull anatomy that result in uncontrolled variation in intracerebral current density . measurements in humans of shunting across the scalp and skull range from 80% up to 97% . recent studies with computer models further support the notion that current dissipation in ect is considerable and that the electrode placement associated with more severe cognitive side effects , bitemporal ( bl - ect ) , is also the placement with higher shunting and with deeper brain stimulation . the possibility of noninvasive stimulation of specific areas in the cerebral cortex through magnetic stimulation was first demonstrated in 1985 . the use of transcranial magnetic stimulation ( tms ) has since been studied as a treatment for depression , and a number of meta - analyses and sham - controlled trials have confirmed that it is associated with statistically significant antidepressant effect . tms employs a rapidly changing magnetic field to induce an electrical current in a targeted brain region , most often the left dorsolateral prefrontal cortex ( dlpfc ) . magnetic stimulation is more focal than electrical stimulation because it avoids the impedance of the scalp and skull and results in an induced electric field confined to superficial cortex . thus , magnetic stimulation has allowed more control over current paths and current density within cortical tissue [ 22 , 28 ] and grants tms a safer , well - tolerated side effects profile . a recent meta - analysis has shown that repetitive transcranial magnetic stimulation ( rtms ) , despite significantly reducing depressive symptoms , is not as effective as ect for the treatment of refractory depression , leading to a mean reduction of 9.3 points on the hamilton depression rating scale ( hdrs ) , while ect leads to a reduction of 15.42 points . such phenomenon was initially considered to be accidental and regarded as a complication of method [ 2931 ] . however , the possibility of intentionally inducing seizures by magnetic pulses has been formally proposed as early as 1994 by sackeim . once rtms had been found to have significant antidepressant effects at subconvulsive levels , and considering the superior antidepressant potency of ect , it has been hypothesized that , under controlled conditions in a patient under anesthesia , increasing the magnetic stimulus into the convulsive range the resultant seizure could confer robust antidepressant properties as seen with ect . it was also hypothesized that the more accurate and focal seizures triggered by magnetic stimulation could lead to less adverse effects than seizures triggered by ect , while retaining its therapeutic capacity . the first published report of seizures successfully and deliberately induced by rtms dates back to 2001 , described by lisanby et al . . two exemplars of macaca mulatta were chosen for the experiments because of brain - to - coil size ratio that was closer to humans when pediatric sized coils were used and for their ability to perform cognitive tasks with which to access the potential cognitive side effects of the method . the animals were subjected to rtms sessions under general anesthesia , using both a commercially available tms device ( magstim super rapid , magstim company ltd . , whitland , wales ) and a customized magstim device , with eight booster modules instead of the usual four modules , capable of broader pulse width and yielding 40% more power per pulse . the two trials conducted with the commercial magstim device failed to trigger seizures in the primates . the custom device , however , was capable of inducing seizures in both subjects in all trials , therefore illustrating the need for more powerful stimulation than a standard tms device could deliver in order to elicit seizures . further studies comparing electroconvulsive shock ( ecs ) , the animal equivalent of ect , with mst and sham on non - human primates were conducted in order to assess the safety of the procedure . results pointed that mst has significantly less impact than ecs on functions such as spatial memory , time to task completion , and anterograde memory . in fact , there was no difference between mst and sham , even when with the use of higher - intensity stimulus , with pulse frequency of up to 100 hz [ 3537 ] . postmortem studies conducted on the experimentation animals did not reveal morphologic changes or histological lesions [ 38 , 39 ] . soon after the first description of mst in animals , initial human reports were published . in 2001 , lisanby et al . described the first use of mst in a 20-year - old patient with treatment - resistant episode of mdd , who had undergone for the previous three years several pharmacological trials , with different classes of antidepressant drugs , without success , being referred to ect . generalized tonic - clonic seizures were successfully elicited in the four mst treatments , with hamilton depression rating scale scores decreasing from 20 at baseline to 13 after the fourth session . no severe adverse effects were reported and mini exam of mental state scores remained unaltered throughout the trial . in 2003 , kosel et al . this time , a 66-year - old patient was submitted to a full trial of mst , with 12 sessions , until remission was achieved . hamilton depression rating scale ( hdrs ) scores dropped from 33 to 6 and beck depression inventory ( bdi ) dropped from 40 to 11 . a more comprehensive cognitive assessment battery was employed , and the patient did not show any significant cognitive deficits following mst . actually , there was an improvement on some cognitive tests following treatment . since the mechanism of action of mst involves the induction of seizures , the use of general anesthesia with muscle relaxation and clinical support is necessary , much like in ect . the use of a bite block , which is mandatory for patients receiving ect , on the other hand , is not necessary since there is no direct stimulation of the masseter muscle by shunted electric current . however , the loud clicking noise causing the coil might require the use of earplugs by both patients and staff . different coils have been tested , with the nonfocal round coil and the double cone coil reported to be reliable in seizure induction , while the more focal figure of 8 coil was considered inefficient in inducing seizure . another concern related to the coils is the heating of the equipment , which is more pronounced than in tms coils and required previous cooling and the use of heat - resistant materials . magnetic pulses , such as those employed by rtms and mst , are capable of focusing the stimulation to a specific area , since they pass unhindered into the brain without resistance and are not shunted through scalp , skull , and crl like ect 's electric stimulus . on the other hand , magnetic pulses generated by most commercially available coils only penetrate a few centimeters deep , while electric current can reach deeper structures more easily . therefore , a major difference between mst and ect is the former 's capacity to focus stimulation , with mst - induced seizures originating on superficial regions of the cortex , unlike ect , where electrical current passes deep through the brain . consequently , it is possible that mst may produce similar therapeutic benefits to ect without inducing memory - related side effects , as there is no direct electrical stimulation of medial temporal lobe structures , such as the hippocampus , which are implicated in ect - related memory impairment . our objective was to review current clinical evidence on magnetic seizure therapy , its effectiveness on unipolar and bipolar depression , and its side effects profile , with special emphasis on the cognitive aspects . whenever possible , comparisons with ect were drawn in order to establish similarities and differences between the methods . in order to systematically review the current literature on the use of magnetic seizure therapy in the treatment of mood disorders in accordance with the prisma guidelines , the authors performed searches in the ovid and medline databases using the following terms : ( 1 ) magnetic seizure therapy or ( 2 ) magnetic seizure therapy and depression or ( 3 ) magnetic seizure therapy and bipolar . articles dating from 1985 , the year the first papers on transcranial magnetic stimulation were published , to august 2014 were selected . the results were managed with mendeley desktop ( version 1.10.1.0 , mendeley , ltd . ) software . the inclusion and exclusion criteria for studies were as follows : inclusion : ( a ) manuscript in english ; ( b ) clinical trials , either open - labeled or blinded studies ; ( c ) studies on human subjects and exclusion : ( a ) virtual models ; ( b ) animal studies ; ( c ) case reports . our initial search yielded 75 references , 64 of which were initially excluded , based on eligibility criteria , leaving 11 articles . in a subsequent analysis , 3 references were excluded after the abstracts were reviewed and did not meet eligibility criteria . the following variables were extracted per a structured checklist that we developed : ( a ) overview : study design , authors , year of publication , technique , summary , and other relevant data ; ( b ) demographics : total sample ( number ) and intervention groups ; ( c ) assessment of mood disorder : method of diagnosis ( clinical or structured interviews ) ; and ( d ) outcomes : description of each study 's results . to assess the methodological heterogeneity between studies , each report was evaluated with regard to quality , focusing on 2 critical methodological issues : ( a ) internal validity : for clinical studies , whenever possible the authors followed the cochrane guidelines to determine the risk of bias in randomization ( selection bias ) , blinding and control comparison ( performance bias ) , and outcome assessment and reporting ( attrition , measurement , and reporting biases ) ; however , since a small number of double - blind randomized clinical trials were found , open - label studies were included as well ; ( b ) construct validity : we determined whether the operational criteria for mood disorders were appropriate , that is , whether each study fulfilled the following criteria : ( i ) clinical studies that focused on mst and ( ii ) articles on mood disorders . the first clinical trial assessing mst 's effectiveness compared to ect in reducing depressive symptoms was reported by white et al . . depressive symptoms were evaluated with the hdrs before and after the series of 10 to 12 treatments . researchers reported that both methods resulted in a decrease in depressive scores , and ect reduced mean hdrs from 30 at baseline to 6 after treatment , while on the mst group mean hdrs at baseline was 32 , dropping to 14 at the end of the trial . results suggested that while mst demonstrated a safer cognitive profile , its effectiveness was still below ect . only 53% of the patients treated with mst achieved a 50% or greater improvement on the depression rating scores . considering that ect responses have been previously associated with electric dose in relation to the seizure threshold , a possible explanation for mst 's lower efficacy could be the fact that treatments were delivered only on average 1.3 times above magnetic seizure threshold . the authors suggested that better response rates could have been achieved with higher stimulation intensity ; however , mst devices available at the time could only produce short trains of 8 seconds and 50 hz at maximum output . in an attempt to improve effectiveness of mst , it has been proposed that more intense stimuli could be necessary , which could be achieved by employing higher frequency , up to 100 hz , and longer trains . the first comparison between effectiveness of hd - mst and ect was published in 2011 by kayser et al . in patients on a treatment - resistant depressive episode of bd and mdd . primary outcome measure was response , defined by 50% reduction of montgomery and asberg depression rating scale ( madrs ) or remission , defined by madrs score of less than 10 . other clinical measures included hdrs scores , hamilton anxiety scale ( hama ) , bdi , and the 90-item symptom checklist ( scl-90 ) . out of ten patients on the mst group , six were responders , three of which achieved remission . in the ect group it is noteworthy that ect procedures employed by the researchers differed from the usual practice inasmuch as low down rul - ect was used , which reportedly could be less effective than high - dose rul - ect . further investigation of hd - mst and its impact on depressive symptoms was reported by fitzgerald et al . in 2013 . clinical assessment was done with the madrs , the primary outcome variable , as well as the 17-item ham - d , bdi , bprs , and the core rating of melancholia . scores were rated at baseline , after six sessions of mst and after the end of treatment . out of 13 patients , average madrs scores dropped from 39.0 to 26.5 , hamd decreased from 26.7 to 19.2 , bdi went from 35.8 to 26.7 , and bprs dropped from 20.7 to 13.6 . the authors note that the response to mst in this sample was significantly inferior to the reported effectiveness of ect , which generally yields remission rates of up to 80% . the first investigation of regional glucose brain metabolism in mst - treated patients was reported by hoy et al . in 2013 . patients received high - dose mst three times a week , and clinical measures were performed at baseline , after every 6 treatments and at the end of the mst treatment . the primary clinical outcome variable for response was madrs score , and response was defined as 50% reduction on the score , while reduction of 2050% was considered partial response and less 20% nonresponse . fluorine-18-labeled deoxyglucose ( fdg ) pet / ct was performed in the week preceding the first mst treatment and a few days ( average 3.8 ) after completion of the mst treatment course . significant relative glucose metabolism increases were seen in the globus pallidus , substantia nigra , putamen , and the orbital frontal cortex . other findings , though not at the same level of significance , include increases in the glucose metabolism of the medial frontal cortex and the dorsolateral prefrontal cortex . differences between responders and nonresponders were only significant at a trend level and no relation was found between changes in relative glucose metabolism and changes in depression scores . authors ponder that such findings could be due to the small sample . in 2003 , lisanby et al . a total of 10 patients suffering from mdd and referred to an ect course were selected for a randomized , within - subject trial . the first two sessions of each patient were used to determine seizure threshold for both mst and ect , followed by two sessions of suprathreshold stimulation with mst and ect , and finally ect sessions for the remaining treatments . due to the high - frequency clicking noise intrinsic to magnetic stimulation , earplugs were worn by patients during both mst and ect sessions . a bite - block was inserted immediately prior to seizure elicitation to protect the teeth , even if mst did not produce the marked jaw contraction as typically seen with ect , since no current passes through the masseter . mst was administered with a modified magstim stimulator , with a pulse width of 0.5 ms and 60 hz at 100% output . all suprathreshold mst sessions were all given at maximal stimulator output ( 400 pulses ) . tonic - clonic seizures similar to those seen with conventional ect were elicited on all mst sessions , albeit seizure duration was shorter on average for mst ( 40.9 s on threshold and 49.5 s on suprathreshold stimulation ) compared to ect ( 101.0 s on threshold and 74.4 s on suprathreshold stimulation ) . a neuropsychological battery sampling multiple cognitive domain was administered by a blind rater at baseline and immediately before and after each of the four test sessions , evaluating cognitive functions such as memory , orientation , and attention , as well as the squire memory test ( sentences ) , the buschke selective reminding test , and a complex figure test . patients had fewer subjective side effects and recovered orientation more quickly with mst than ect . mst was also superior to ect on measures of attention , retrograde amnesia , and category fluency . to further investigate the cognitive side effects of mst compared to ect , white et al . conducted a study evaluating the amount of time patients took to recover orientation after each procedure , as well as reduction of depressive symptoms scores . ten patients with mdd were submitted to mst and results compared to other 10 case - matched patients undergoing ect treatment in an open - parallel study design . time to recover orientation was assessed by a blinded observer in the postanesthesia recovery area who asked simple questions such as name , place , and day of the week after each session . orientation recovery time was considerably shorter on the mst group , averaging 4 minutes , while patients submitted to ect took an average of 18 minutes to correctly answer the questions . the first report of hd - mst on humans was published in 2008 by kirov et al . . eleven patients suffering from tr mdd were enrolled for a within - subject open study comparing orientation recovery time of a single ect and a single hd - mst session . recovery of orientation after treatments was assessed by asking the patient for their name , date of birth , age , place , and day of the week . the point of orientation recovery was defined as the time when a patient was able to recall four of these five items . the mean time to recovery after successful seizures elicited by mst was 7 min 12 s , against 26 min 35 s for ect . the authors did not report effectiveness data on this study , since this was not the study focus and only one mst session was performed on each patient . another study , this time comparing the effectiveness , electrophysiological characteristics , and cognitive side effects of hd - mst and ect , was published in 2011 by kayser et al . . twenty patients in a treatment - resistant depressive episode were enrolled and blindly randomized in two groups of ten , treated with either ect or mst . it is noteworthy that for the first time patients suffering from bipolar disorder were included in a mst trial , two patients with type ii bd receiving ect and one type i and one type ii patients on the mst group . a neuropsychological battery was employed before and after treatment to evaluate such cognitive domains as memory , learning , executive function , language , and processing speed . recovery was defined as the time when patients opened their eyes and breathed independently , while reorientation time was assessed by asking the patient for her / his names , date of birth , age , place , and day of the week . recovery and reorientation were faster in the mst group , at 1 min 42 s and 2 m 16 s respectively , compared to the ect group , with recovery at 4 min 3 s and reorientation at 8 min 21 s. except for that , no significant differences in cognitive side effects were found between groups . eeg activity was similar in both groups , consisting of high - amplitude synchronized theta activity and equal postictal suppression . however , on the mst group seizure duration was briefer and some patients showed delayed ictal eeg activity and duration of motor and ictal activity in mst - treated patients had about the same length . it is important to notice that the ect procedures in this paper are quite different from other studies . the authors used right unilateral electrodes ( rul - ect ) but chose to apply stimulus three times above seizure threshold , which is reported to be less effective than high - dose rul - ect . on the other hand , lower electrical doses are associated with less cognitive impairment than high - dose ect , which could explain the absence of significant cognitive side effects on the ect group . kayser et al . further explored seizure characteristics and cognitive aspects of hd - mst compared with ect . seven patients in a treatment - resistant depressive episode were enrolled , six suffering from mdd and one from bipolar disorder type ii . such patients had failed to respond to a 12-session mst course and were then referred to ect 12 ect sessions in an open - label , within - subject trial . reorientation time was , once again , shorter , shorter after mst than after ect . except for the fact that seizures lasted longer on patients receiving ect , the authors found no significant differences in visible motor seizures and eeg activities between the two procedures , including postictal suppression , a measure reported to predict response to ect . in 2013 , fitzgerald et al . reported an open - label trial in which 13 patients with tr - mdd received mst , assessing its effectiveness and side effects . mst was applied three times per week , each patient receiving up to 18 treatments . a comprehensive neuropsychological battery was employed , and patients were evaluated at baseline , after six sessions and lastly after the end of treatments . out of 13 patients , 12 completed the trial ; the remaining subject chose to abandon the study due to poor response and desire to receive ect . in fact , the fast recovery from the procedure might have created an unexpected side effect : four patients awoke from the procedure while still under effect of muscle relaxation . neuropsychological tests showed no cognitive impairment after mst ; in fact nonsignificant improvements were detected in several domains . unlike previous reports [ 44 , 48 ] , the authors reported eeg patterns unlike those expected on ect patients . not only were the motor seizures shorter , but also ictal activity was of less amplitude and there was typically much less postictal suppression . in some patients , ictal activity was not always apparent on the eeg despite a clear motor seizure . the authors also note that the response to mst in this sample was significantly inferior to the reported effectiveness of ect , which generally yields remission rates of up to 80% . the cognitive side effects of mst were recently explored by polster et al . , who compared its impact on acute memory retrieval to ect . twenty patients suffering from treatment - resistant mdd were randomly assigned to two groups , one treated with mst and the other with ect . memory assessment consisted of a customized test . on each of the 2 treatment control days and 2 treatment - free control days , the patients were given 3 consecutive learning trials in the morning to learn 40 words . words were clustered into pairs and assigned to a hypernymic category for additional differentiation between storage or retrieval disruption of memory . after treatment , patients were initially asked to remember all 40 of the words by themselves ( delayed recall ) . subsequently , they were provided with the name of each hypernymic category to enable them to recall all 40 words independently from delayed recall , again . the authors point that if patients extraordinarily benefit from these cues , this is indicative of a retrieval - based rather than a storage - focused memory disruption . by comparing memory performance on treatment days to control days , treatment - induced memory disruption was evaluated . to enable evaluation of treatment caused effects on a particular subject , the patients were treated with mst or ect on 2 of the 4 testing days , whereas the other 2 days served as control . after ect , delayed recall was disturbed , whereas after mst , it was not . however , this difference in performance was no longer apparent upon cue application . the results of this review show that mst is as effective as ect in inducing generalized tonic - clonic seizures , both in animals and in humans . however , there are considerable differences in the mechanism of induction , since magnetic pulses are not shunted by the scalp and skull , unlike ect 's electric current , allowing for a more focused and superficial stimulation , which on its turn might have implications for its electrophysiological aspects , effectiveness , and side effects . mst - induced seizures are shorter lasting . in most animal studies and some clinical studies mst also led to less postictal suppression and less eeg amplitude compared to ect - induced seizures , although two studies reported similar eeg characteristics for both groups [ 44 , 48 ] . such results could possibly be related to the use of rul - ect and low electric dose , which are associated with less cognitive loss but less effectiveness as well . the clinical studies so far reported all support that mst is an effective treatment for depressive episodes , with response rates ranging from 40% to 60% and remission rates ranging from 15% to 30% . it is noteworthy that most trials were conducted on patients suffering from trd , who had failed previous therapeutic strategies and therefore had a worse prognosis . on the other hand , such results are still far from those expected from ect , which is reported to lead to remission rates of 50% to 70% on the same conditions . the results could be related to the parameters of mst , such as frequency of pulses and total pulse count . high - dose mst ( hd - mst ) is an attempt to bridge this gap , and further studies are being conducted in order to improve its effectiveness . both the neurocognitive effects and the effectiveness of ect are related in part to the stimulus parameters with higher doses conferring increased adverse effects but perhaps better response rates on the other hand . for example , 6 seizure thresholds rul - ect can achieve results similar to bl - ect [ 5053 ] . following that rationale , the effectiveness of mst could , theoretically , be improved by increasing the stimulus ' dose , which can be achieved by administering a larger number of pulses in the mst train . early mst equipment employed pulse frequencies of 40 hz to 50 hz and train lengths of up to 8 seconds , for a maximum of 400 pulses . specific equipment , capable of delivering stimulus at 100 hz and for up to 10 seconds ( 1000 pulses ) , was developed in order to test such hypothesis . preclinical studies suggested that hd - mst delivered under such parameters could be safe from a cognitive point of view , with no histological damage , either compared to low - dose mst , sham , or ecs [ 36 , 37 , 39 ] . mst shows a considerable advantage over ect on its side effects profile and induced cognitive loss . such difference was evident from the early preclinical studies comparing it to ecs , subjects showing significantly better cognitive scores after mst than ecs . on human subjects , reorientation time after mst ranges from 2 minutes to 8 minutes , while it takes from 18 minutes to 26 minutes after ect , a considerable improvement . other cognitive functions , such as retrograde and anterograde memory , language , and praxis , seem to be unaffected by mst . ect , on the other hand , is notorious for inducing cognitive loss , which might be its most significant drawback . all clinical studies revised here focused on depressive episodes and the majority of enlisted patients suffered from mdd , with very few cases of bd . considering the challenges of treating depressive episodes in bipolar disorder and the low number of approved effective treatments , mst warrants further investigation as an alternative treatment for such cases . furthermore , all studies which included bd patients included them on a sample majorly composed of mdd patients ; no study so far has focused on bipolar depression specifically . also , there are no reports of mst for manic episodes of bd . considering ect 's record of effectiveness on manic states , the use of mst on such cases could be a focus for future research . being such a novel and still experimental technique , mst studies suffer from small samples and low statistical power . also there is considerable variance in the mst technique employed by the researchers , which is unsurprising , considering that commercially available mst devices have only recently become available . however , there is little standardization of anesthetic methods , rating scales , cognitive assessments , and the protocol for ect control . there is currently only one double - blind randomized clinical trial comparing the effectiveness and side effects of mst compared to ect , and all the others are either case reports , open - label studies , or crossover studies . future research , with larger samples , of double - blind design , and more consistent methods will allow for more statistic power and better understanding of the technique . the findings reported on this review suggest that mst might be an effective and safe alternative for the treatment of mood disorders , specifically on treatment - resistant depression , with a safer and more tolerable side effects profile than the current first choice , ect . however , further studies are necessary to improve the assessment of its potential effectiveness and expand current understanding of its mechanisms .

objective . magnetic seizure therapy ( mst ) is a novel , experimental therapeutic intervention , which combines therapeutic aspects of electroconvulsive therapy ( ect ) and transcranial magnetic stimulation , in order to achieve the efficacy of the former with the safety of the latter . mst might prove to be a valuable tool in the treatment of mood disorders , such as major depressive disorder ( mdd ) and bipolar disorder . our aim is to review current literature on mst . methods . ovid and medline databases were used to systematically search for clinical studies on mst . the terms magnetic seizure therapy , depression , and bipolar were employed . results . out of 74 studies , 8 met eligibility criteria . there was considerable variability in the methods employed and samples sizes were small , limiting the generalization of the results . all studies focused on depressive episodes , but few included patients with bipolar disorder . the studies found reported significant antidepressant effects , with remission rates ranging from 30% to 40% . no significant cognitive side effects related to mst were found , with a better cognitive profile when compared to ect . conclusion . mst was effective in reducing depressive symptoms in mood disorders , with generally less side effects than ect . no study focused on comparing mst to ect on bipolar depression specifically .

1. Introduction 2. Aims 3. Methods 4. Results 5. Discussion

mood disorders , such as major depressive disorder ( mdd ) and bipolar disorder ( bd ) , are highly prevalent and debilitating conditions , associated with high suicidality rates , elevated treatment costs , and heavy social and economic burden . the treatment of depressive episodes is associated with a 3040% rate of nonresponse . electroconvulsive therapy ( ect ) is regarded as the most effective treatment for depression , in both bd and mdd , with remission rates ranging from 50% to 75% [ 46 ] . ect is also effective for manic episodes , with reported remission rates of up to 85% [ 711 ] . despite its high effectiveness , the use of ect is limited by its side effects , especially the development of cognitive deficits and , in particular , memory impairment [ 1216 ] . the cognitive effects of ect have been shown to be dependent upon numerous ect parameters , namely , electrode placement , frequency of sessions , electric dose , and pulse width [ 14 , 17 , 20 ] . the search for improved ect techniques , which could be able to reduce or minimize memory - related side effects whilst maintaining efficacy , has been the subject of a considerable amount of investigation . however , the use of an electrical stimulus to induce seizures is a fundamental limitation in refining convulsive therapy and limiting its side effects profile . control over the spatial distribution and magnitude of intracerebral current density is limited by high skull impedance , which shunts most of the electrical stimulus through the scalp and cerebrospinal fluid ( csf ) and away from the brain . recent studies with computer models further support the notion that current dissipation in ect is considerable and that the electrode placement associated with more severe cognitive side effects , bitemporal ( bl - ect ) , is also the placement with higher shunting and with deeper brain stimulation . the possibility of noninvasive stimulation of specific areas in the cerebral cortex through magnetic stimulation was first demonstrated in 1985 . the use of transcranial magnetic stimulation ( tms ) has since been studied as a treatment for depression , and a number of meta - analyses and sham - controlled trials have confirmed that it is associated with statistically significant antidepressant effect . magnetic stimulation is more focal than electrical stimulation because it avoids the impedance of the scalp and skull and results in an induced electric field confined to superficial cortex . thus , magnetic stimulation has allowed more control over current paths and current density within cortical tissue [ 22 , 28 ] and grants tms a safer , well - tolerated side effects profile . a recent meta - analysis has shown that repetitive transcranial magnetic stimulation ( rtms ) , despite significantly reducing depressive symptoms , is not as effective as ect for the treatment of refractory depression , leading to a mean reduction of 9.3 points on the hamilton depression rating scale ( hdrs ) , while ect leads to a reduction of 15.42 points . such phenomenon was initially considered to be accidental and regarded as a complication of method [ 2931 ] . once rtms had been found to have significant antidepressant effects at subconvulsive levels , and considering the superior antidepressant potency of ect , it has been hypothesized that , under controlled conditions in a patient under anesthesia , increasing the magnetic stimulus into the convulsive range the resultant seizure could confer robust antidepressant properties as seen with ect . it was also hypothesized that the more accurate and focal seizures triggered by magnetic stimulation could lead to less adverse effects than seizures triggered by ect , while retaining its therapeutic capacity . two exemplars of macaca mulatta were chosen for the experiments because of brain - to - coil size ratio that was closer to humans when pediatric sized coils were used and for their ability to perform cognitive tasks with which to access the potential cognitive side effects of the method . , whitland , wales ) and a customized magstim device , with eight booster modules instead of the usual four modules , capable of broader pulse width and yielding 40% more power per pulse . the two trials conducted with the commercial magstim device failed to trigger seizures in the primates . the custom device , however , was capable of inducing seizures in both subjects in all trials , therefore illustrating the need for more powerful stimulation than a standard tms device could deliver in order to elicit seizures . further studies comparing electroconvulsive shock ( ecs ) , the animal equivalent of ect , with mst and sham on non - human primates were conducted in order to assess the safety of the procedure . results pointed that mst has significantly less impact than ecs on functions such as spatial memory , time to task completion , and anterograde memory . in fact , there was no difference between mst and sham , even when with the use of higher - intensity stimulus , with pulse frequency of up to 100 hz [ 3537 ] . described the first use of mst in a 20-year - old patient with treatment - resistant episode of mdd , who had undergone for the previous three years several pharmacological trials , with different classes of antidepressant drugs , without success , being referred to ect . generalized tonic - clonic seizures were successfully elicited in the four mst treatments , with hamilton depression rating scale scores decreasing from 20 at baseline to 13 after the fourth session . this time , a 66-year - old patient was submitted to a full trial of mst , with 12 sessions , until remission was achieved . a more comprehensive cognitive assessment battery was employed , and the patient did not show any significant cognitive deficits following mst . the use of a bite block , which is mandatory for patients receiving ect , on the other hand , is not necessary since there is no direct stimulation of the masseter muscle by shunted electric current . different coils have been tested , with the nonfocal round coil and the double cone coil reported to be reliable in seizure induction , while the more focal figure of 8 coil was considered inefficient in inducing seizure . another concern related to the coils is the heating of the equipment , which is more pronounced than in tms coils and required previous cooling and the use of heat - resistant materials . magnetic pulses , such as those employed by rtms and mst , are capable of focusing the stimulation to a specific area , since they pass unhindered into the brain without resistance and are not shunted through scalp , skull , and crl like ect 's electric stimulus . therefore , a major difference between mst and ect is the former 's capacity to focus stimulation , with mst - induced seizures originating on superficial regions of the cortex , unlike ect , where electrical current passes deep through the brain . consequently , it is possible that mst may produce similar therapeutic benefits to ect without inducing memory - related side effects , as there is no direct electrical stimulation of medial temporal lobe structures , such as the hippocampus , which are implicated in ect - related memory impairment . our objective was to review current clinical evidence on magnetic seizure therapy , its effectiveness on unipolar and bipolar depression , and its side effects profile , with special emphasis on the cognitive aspects . whenever possible , comparisons with ect were drawn in order to establish similarities and differences between the methods . in order to systematically review the current literature on the use of magnetic seizure therapy in the treatment of mood disorders in accordance with the prisma guidelines , the authors performed searches in the ovid and medline databases using the following terms : ( 1 ) magnetic seizure therapy or ( 2 ) magnetic seizure therapy and depression or ( 3 ) magnetic seizure therapy and bipolar . articles dating from 1985 , the year the first papers on transcranial magnetic stimulation were published , to august 2014 were selected . the inclusion and exclusion criteria for studies were as follows : inclusion : ( a ) manuscript in english ; ( b ) clinical trials , either open - labeled or blinded studies ; ( c ) studies on human subjects and exclusion : ( a ) virtual models ; ( b ) animal studies ; ( c ) case reports . in a subsequent analysis , 3 references were excluded after the abstracts were reviewed and did not meet eligibility criteria . the following variables were extracted per a structured checklist that we developed : ( a ) overview : study design , authors , year of publication , technique , summary , and other relevant data ; ( b ) demographics : total sample ( number ) and intervention groups ; ( c ) assessment of mood disorder : method of diagnosis ( clinical or structured interviews ) ; and ( d ) outcomes : description of each study 's results . to assess the methodological heterogeneity between studies , each report was evaluated with regard to quality , focusing on 2 critical methodological issues : ( a ) internal validity : for clinical studies , whenever possible the authors followed the cochrane guidelines to determine the risk of bias in randomization ( selection bias ) , blinding and control comparison ( performance bias ) , and outcome assessment and reporting ( attrition , measurement , and reporting biases ) ; however , since a small number of double - blind randomized clinical trials were found , open - label studies were included as well ; ( b ) construct validity : we determined whether the operational criteria for mood disorders were appropriate , that is , whether each study fulfilled the following criteria : ( i ) clinical studies that focused on mst and ( ii ) articles on mood disorders . the first clinical trial assessing mst 's effectiveness compared to ect in reducing depressive symptoms was reported by white et al . depressive symptoms were evaluated with the hdrs before and after the series of 10 to 12 treatments . researchers reported that both methods resulted in a decrease in depressive scores , and ect reduced mean hdrs from 30 at baseline to 6 after treatment , while on the mst group mean hdrs at baseline was 32 , dropping to 14 at the end of the trial . in an attempt to improve effectiveness of mst , it has been proposed that more intense stimuli could be necessary , which could be achieved by employing higher frequency , up to 100 hz , and longer trains . in the ect group it is noteworthy that ect procedures employed by the researchers differed from the usual practice inasmuch as low down rul - ect was used , which reportedly could be less effective than high - dose rul - ect . further investigation of hd - mst and its impact on depressive symptoms was reported by fitzgerald et al . clinical assessment was done with the madrs , the primary outcome variable , as well as the 17-item ham - d , bdi , bprs , and the core rating of melancholia . out of 13 patients , average madrs scores dropped from 39.0 to 26.5 , hamd decreased from 26.7 to 19.2 , bdi went from 35.8 to 26.7 , and bprs dropped from 20.7 to 13.6 . the authors note that the response to mst in this sample was significantly inferior to the reported effectiveness of ect , which generally yields remission rates of up to 80% . patients received high - dose mst three times a week , and clinical measures were performed at baseline , after every 6 treatments and at the end of the mst treatment . fluorine-18-labeled deoxyglucose ( fdg ) pet / ct was performed in the week preceding the first mst treatment and a few days ( average 3.8 ) after completion of the mst treatment course . significant relative glucose metabolism increases were seen in the globus pallidus , substantia nigra , putamen , and the orbital frontal cortex . other findings , though not at the same level of significance , include increases in the glucose metabolism of the medial frontal cortex and the dorsolateral prefrontal cortex . the first two sessions of each patient were used to determine seizure threshold for both mst and ect , followed by two sessions of suprathreshold stimulation with mst and ect , and finally ect sessions for the remaining treatments . due to the high - frequency clicking noise intrinsic to magnetic stimulation , earplugs were worn by patients during both mst and ect sessions . mst was administered with a modified magstim stimulator , with a pulse width of 0.5 ms and 60 hz at 100% output . tonic - clonic seizures similar to those seen with conventional ect were elicited on all mst sessions , albeit seizure duration was shorter on average for mst ( 40.9 s on threshold and 49.5 s on suprathreshold stimulation ) compared to ect ( 101.0 s on threshold and 74.4 s on suprathreshold stimulation ) . a neuropsychological battery sampling multiple cognitive domain was administered by a blind rater at baseline and immediately before and after each of the four test sessions , evaluating cognitive functions such as memory , orientation , and attention , as well as the squire memory test ( sentences ) , the buschke selective reminding test , and a complex figure test . patients had fewer subjective side effects and recovered orientation more quickly with mst than ect . mst was also superior to ect on measures of attention , retrograde amnesia , and category fluency . to further investigate the cognitive side effects of mst compared to ect , white et al . ten patients with mdd were submitted to mst and results compared to other 10 case - matched patients undergoing ect treatment in an open - parallel study design . time to recover orientation was assessed by a blinded observer in the postanesthesia recovery area who asked simple questions such as name , place , and day of the week after each session . recovery of orientation after treatments was assessed by asking the patient for their name , date of birth , age , place , and day of the week . another study , this time comparing the effectiveness , electrophysiological characteristics , and cognitive side effects of hd - mst and ect , was published in 2011 by kayser et al . it is noteworthy that for the first time patients suffering from bipolar disorder were included in a mst trial , two patients with type ii bd receiving ect and one type i and one type ii patients on the mst group . recovery was defined as the time when patients opened their eyes and breathed independently , while reorientation time was assessed by asking the patient for her / his names , date of birth , age , place , and day of the week . recovery and reorientation were faster in the mst group , at 1 min 42 s and 2 m 16 s respectively , compared to the ect group , with recovery at 4 min 3 s and reorientation at 8 min 21 s. except for that , no significant differences in cognitive side effects were found between groups . the authors used right unilateral electrodes ( rul - ect ) but chose to apply stimulus three times above seizure threshold , which is reported to be less effective than high - dose rul - ect . on the other hand , lower electrical doses are associated with less cognitive impairment than high - dose ect , which could explain the absence of significant cognitive side effects on the ect group . further explored seizure characteristics and cognitive aspects of hd - mst compared with ect . seven patients in a treatment - resistant depressive episode were enrolled , six suffering from mdd and one from bipolar disorder type ii . such patients had failed to respond to a 12-session mst course and were then referred to ect 12 ect sessions in an open - label , within - subject trial . except for the fact that seizures lasted longer on patients receiving ect , the authors found no significant differences in visible motor seizures and eeg activities between the two procedures , including postictal suppression , a measure reported to predict response to ect . reported an open - label trial in which 13 patients with tr - mdd received mst , assessing its effectiveness and side effects . mst was applied three times per week , each patient receiving up to 18 treatments . not only were the motor seizures shorter , but also ictal activity was of less amplitude and there was typically much less postictal suppression . the authors also note that the response to mst in this sample was significantly inferior to the reported effectiveness of ect , which generally yields remission rates of up to 80% . the cognitive side effects of mst were recently explored by polster et al . on each of the 2 treatment control days and 2 treatment - free control days , the patients were given 3 consecutive learning trials in the morning to learn 40 words . to enable evaluation of treatment caused effects on a particular subject , the patients were treated with mst or ect on 2 of the 4 testing days , whereas the other 2 days served as control . the results of this review show that mst is as effective as ect in inducing generalized tonic - clonic seizures , both in animals and in humans . however , there are considerable differences in the mechanism of induction , since magnetic pulses are not shunted by the scalp and skull , unlike ect 's electric current , allowing for a more focused and superficial stimulation , which on its turn might have implications for its electrophysiological aspects , effectiveness , and side effects . in most animal studies and some clinical studies mst also led to less postictal suppression and less eeg amplitude compared to ect - induced seizures , although two studies reported similar eeg characteristics for both groups [ 44 , 48 ] . such results could possibly be related to the use of rul - ect and low electric dose , which are associated with less cognitive loss but less effectiveness as well . the clinical studies so far reported all support that mst is an effective treatment for depressive episodes , with response rates ranging from 40% to 60% and remission rates ranging from 15% to 30% . on the other hand , such results are still far from those expected from ect , which is reported to lead to remission rates of 50% to 70% on the same conditions . the results could be related to the parameters of mst , such as frequency of pulses and total pulse count . high - dose mst ( hd - mst ) is an attempt to bridge this gap , and further studies are being conducted in order to improve its effectiveness . following that rationale , the effectiveness of mst could , theoretically , be improved by increasing the stimulus ' dose , which can be achieved by administering a larger number of pulses in the mst train . specific equipment , capable of delivering stimulus at 100 hz and for up to 10 seconds ( 1000 pulses ) , was developed in order to test such hypothesis . preclinical studies suggested that hd - mst delivered under such parameters could be safe from a cognitive point of view , with no histological damage , either compared to low - dose mst , sham , or ecs [ 36 , 37 , 39 ] . mst shows a considerable advantage over ect on its side effects profile and induced cognitive loss . other cognitive functions , such as retrograde and anterograde memory , language , and praxis , seem to be unaffected by mst . all clinical studies revised here focused on depressive episodes and the majority of enlisted patients suffered from mdd , with very few cases of bd . considering the challenges of treating depressive episodes in bipolar disorder and the low number of approved effective treatments , mst warrants further investigation as an alternative treatment for such cases . furthermore , all studies which included bd patients included them on a sample majorly composed of mdd patients ; no study so far has focused on bipolar depression specifically . being such a novel and still experimental technique , mst studies suffer from small samples and low statistical power . also there is considerable variance in the mst technique employed by the researchers , which is unsurprising , considering that commercially available mst devices have only recently become available . there is currently only one double - blind randomized clinical trial comparing the effectiveness and side effects of mst compared to ect , and all the others are either case reports , open - label studies , or crossover studies . future research , with larger samples , of double - blind design , and more consistent methods will allow for more statistic power and better understanding of the technique . the findings reported on this review suggest that mst might be an effective and safe alternative for the treatment of mood disorders , specifically on treatment - resistant depression , with a safer and more tolerable side effects profile than the current first choice , ect .

mood disorders , such as major depressive disorder ( mdd ) and bipolar disorder ( bd ) , are highly prevalent and debilitating conditions , associated with high suicidality rates , elevated treatment costs , and heavy social and economic burden . electroconvulsive therapy ( ect ) is regarded as the most effective treatment for depression , in both bd and mdd , with remission rates ranging from 50% to 75% [ 46 ] . despite its high effectiveness , the use of ect is limited by its side effects , especially the development of cognitive deficits and , in particular , memory impairment [ 1216 ] . the cognitive effects of ect have been shown to be dependent upon numerous ect parameters , namely , electrode placement , frequency of sessions , electric dose , and pulse width [ 14 , 17 , 20 ] . the search for improved ect techniques , which could be able to reduce or minimize memory - related side effects whilst maintaining efficacy , has been the subject of a considerable amount of investigation . however , the use of an electrical stimulus to induce seizures is a fundamental limitation in refining convulsive therapy and limiting its side effects profile . control over the spatial distribution and magnitude of intracerebral current density is limited by high skull impedance , which shunts most of the electrical stimulus through the scalp and cerebrospinal fluid ( csf ) and away from the brain . the substantial impedance of the scalp and skull means that the bulk of the electrical stimulus is shunted away from the brain , resulting in widespread stimulation of cortical and subcortical regions . recent studies with computer models further support the notion that current dissipation in ect is considerable and that the electrode placement associated with more severe cognitive side effects , bitemporal ( bl - ect ) , is also the placement with higher shunting and with deeper brain stimulation . the possibility of noninvasive stimulation of specific areas in the cerebral cortex through magnetic stimulation was first demonstrated in 1985 . the use of transcranial magnetic stimulation ( tms ) has since been studied as a treatment for depression , and a number of meta - analyses and sham - controlled trials have confirmed that it is associated with statistically significant antidepressant effect . tms employs a rapidly changing magnetic field to induce an electrical current in a targeted brain region , most often the left dorsolateral prefrontal cortex ( dlpfc ) . magnetic stimulation is more focal than electrical stimulation because it avoids the impedance of the scalp and skull and results in an induced electric field confined to superficial cortex . a recent meta - analysis has shown that repetitive transcranial magnetic stimulation ( rtms ) , despite significantly reducing depressive symptoms , is not as effective as ect for the treatment of refractory depression , leading to a mean reduction of 9.3 points on the hamilton depression rating scale ( hdrs ) , while ect leads to a reduction of 15.42 points . once rtms had been found to have significant antidepressant effects at subconvulsive levels , and considering the superior antidepressant potency of ect , it has been hypothesized that , under controlled conditions in a patient under anesthesia , increasing the magnetic stimulus into the convulsive range the resultant seizure could confer robust antidepressant properties as seen with ect . it was also hypothesized that the more accurate and focal seizures triggered by magnetic stimulation could lead to less adverse effects than seizures triggered by ect , while retaining its therapeutic capacity . two exemplars of macaca mulatta were chosen for the experiments because of brain - to - coil size ratio that was closer to humans when pediatric sized coils were used and for their ability to perform cognitive tasks with which to access the potential cognitive side effects of the method . the custom device , however , was capable of inducing seizures in both subjects in all trials , therefore illustrating the need for more powerful stimulation than a standard tms device could deliver in order to elicit seizures . further studies comparing electroconvulsive shock ( ecs ) , the animal equivalent of ect , with mst and sham on non - human primates were conducted in order to assess the safety of the procedure . results pointed that mst has significantly less impact than ecs on functions such as spatial memory , time to task completion , and anterograde memory . in fact , there was no difference between mst and sham , even when with the use of higher - intensity stimulus , with pulse frequency of up to 100 hz [ 3537 ] . soon after the first description of mst in animals , initial human reports were published . described the first use of mst in a 20-year - old patient with treatment - resistant episode of mdd , who had undergone for the previous three years several pharmacological trials , with different classes of antidepressant drugs , without success , being referred to ect . generalized tonic - clonic seizures were successfully elicited in the four mst treatments , with hamilton depression rating scale scores decreasing from 20 at baseline to 13 after the fourth session . no severe adverse effects were reported and mini exam of mental state scores remained unaltered throughout the trial . a more comprehensive cognitive assessment battery was employed , and the patient did not show any significant cognitive deficits following mst . since the mechanism of action of mst involves the induction of seizures , the use of general anesthesia with muscle relaxation and clinical support is necessary , much like in ect . the use of a bite block , which is mandatory for patients receiving ect , on the other hand , is not necessary since there is no direct stimulation of the masseter muscle by shunted electric current . however , the loud clicking noise causing the coil might require the use of earplugs by both patients and staff . different coils have been tested , with the nonfocal round coil and the double cone coil reported to be reliable in seizure induction , while the more focal figure of 8 coil was considered inefficient in inducing seizure . another concern related to the coils is the heating of the equipment , which is more pronounced than in tms coils and required previous cooling and the use of heat - resistant materials . magnetic pulses , such as those employed by rtms and mst , are capable of focusing the stimulation to a specific area , since they pass unhindered into the brain without resistance and are not shunted through scalp , skull , and crl like ect 's electric stimulus . therefore , a major difference between mst and ect is the former 's capacity to focus stimulation , with mst - induced seizures originating on superficial regions of the cortex , unlike ect , where electrical current passes deep through the brain . consequently , it is possible that mst may produce similar therapeutic benefits to ect without inducing memory - related side effects , as there is no direct electrical stimulation of medial temporal lobe structures , such as the hippocampus , which are implicated in ect - related memory impairment . our objective was to review current clinical evidence on magnetic seizure therapy , its effectiveness on unipolar and bipolar depression , and its side effects profile , with special emphasis on the cognitive aspects . in order to systematically review the current literature on the use of magnetic seizure therapy in the treatment of mood disorders in accordance with the prisma guidelines , the authors performed searches in the ovid and medline databases using the following terms : ( 1 ) magnetic seizure therapy or ( 2 ) magnetic seizure therapy and depression or ( 3 ) magnetic seizure therapy and bipolar . articles dating from 1985 , the year the first papers on transcranial magnetic stimulation were published , to august 2014 were selected . the inclusion and exclusion criteria for studies were as follows : inclusion : ( a ) manuscript in english ; ( b ) clinical trials , either open - labeled or blinded studies ; ( c ) studies on human subjects and exclusion : ( a ) virtual models ; ( b ) animal studies ; ( c ) case reports . the following variables were extracted per a structured checklist that we developed : ( a ) overview : study design , authors , year of publication , technique , summary , and other relevant data ; ( b ) demographics : total sample ( number ) and intervention groups ; ( c ) assessment of mood disorder : method of diagnosis ( clinical or structured interviews ) ; and ( d ) outcomes : description of each study 's results . to assess the methodological heterogeneity between studies , each report was evaluated with regard to quality , focusing on 2 critical methodological issues : ( a ) internal validity : for clinical studies , whenever possible the authors followed the cochrane guidelines to determine the risk of bias in randomization ( selection bias ) , blinding and control comparison ( performance bias ) , and outcome assessment and reporting ( attrition , measurement , and reporting biases ) ; however , since a small number of double - blind randomized clinical trials were found , open - label studies were included as well ; ( b ) construct validity : we determined whether the operational criteria for mood disorders were appropriate , that is , whether each study fulfilled the following criteria : ( i ) clinical studies that focused on mst and ( ii ) articles on mood disorders . the first clinical trial assessing mst 's effectiveness compared to ect in reducing depressive symptoms was reported by white et al . depressive symptoms were evaluated with the hdrs before and after the series of 10 to 12 treatments . researchers reported that both methods resulted in a decrease in depressive scores , and ect reduced mean hdrs from 30 at baseline to 6 after treatment , while on the mst group mean hdrs at baseline was 32 , dropping to 14 at the end of the trial . only 53% of the patients treated with mst achieved a 50% or greater improvement on the depression rating scores . considering that ect responses have been previously associated with electric dose in relation to the seizure threshold , a possible explanation for mst 's lower efficacy could be the fact that treatments were delivered only on average 1.3 times above magnetic seizure threshold . in an attempt to improve effectiveness of mst , it has been proposed that more intense stimuli could be necessary , which could be achieved by employing higher frequency , up to 100 hz , and longer trains . the first comparison between effectiveness of hd - mst and ect was published in 2011 by kayser et al . in patients on a treatment - resistant depressive episode of bd and mdd . other clinical measures included hdrs scores , hamilton anxiety scale ( hama ) , bdi , and the 90-item symptom checklist ( scl-90 ) . out of ten patients on the mst group , six were responders , three of which achieved remission . in the ect group it is noteworthy that ect procedures employed by the researchers differed from the usual practice inasmuch as low down rul - ect was used , which reportedly could be less effective than high - dose rul - ect . clinical assessment was done with the madrs , the primary outcome variable , as well as the 17-item ham - d , bdi , bprs , and the core rating of melancholia . out of 13 patients , average madrs scores dropped from 39.0 to 26.5 , hamd decreased from 26.7 to 19.2 , bdi went from 35.8 to 26.7 , and bprs dropped from 20.7 to 13.6 . the authors note that the response to mst in this sample was significantly inferior to the reported effectiveness of ect , which generally yields remission rates of up to 80% . the first investigation of regional glucose brain metabolism in mst - treated patients was reported by hoy et al . patients received high - dose mst three times a week , and clinical measures were performed at baseline , after every 6 treatments and at the end of the mst treatment . the primary clinical outcome variable for response was madrs score , and response was defined as 50% reduction on the score , while reduction of 2050% was considered partial response and less 20% nonresponse . fluorine-18-labeled deoxyglucose ( fdg ) pet / ct was performed in the week preceding the first mst treatment and a few days ( average 3.8 ) after completion of the mst treatment course . significant relative glucose metabolism increases were seen in the globus pallidus , substantia nigra , putamen , and the orbital frontal cortex . other findings , though not at the same level of significance , include increases in the glucose metabolism of the medial frontal cortex and the dorsolateral prefrontal cortex . differences between responders and nonresponders were only significant at a trend level and no relation was found between changes in relative glucose metabolism and changes in depression scores . the first two sessions of each patient were used to determine seizure threshold for both mst and ect , followed by two sessions of suprathreshold stimulation with mst and ect , and finally ect sessions for the remaining treatments . due to the high - frequency clicking noise intrinsic to magnetic stimulation , earplugs were worn by patients during both mst and ect sessions . tonic - clonic seizures similar to those seen with conventional ect were elicited on all mst sessions , albeit seizure duration was shorter on average for mst ( 40.9 s on threshold and 49.5 s on suprathreshold stimulation ) compared to ect ( 101.0 s on threshold and 74.4 s on suprathreshold stimulation ) . a neuropsychological battery sampling multiple cognitive domain was administered by a blind rater at baseline and immediately before and after each of the four test sessions , evaluating cognitive functions such as memory , orientation , and attention , as well as the squire memory test ( sentences ) , the buschke selective reminding test , and a complex figure test . to further investigate the cognitive side effects of mst compared to ect , white et al . ten patients with mdd were submitted to mst and results compared to other 10 case - matched patients undergoing ect treatment in an open - parallel study design . orientation recovery time was considerably shorter on the mst group , averaging 4 minutes , while patients submitted to ect took an average of 18 minutes to correctly answer the questions . eleven patients suffering from tr mdd were enrolled for a within - subject open study comparing orientation recovery time of a single ect and a single hd - mst session . recovery of orientation after treatments was assessed by asking the patient for their name , date of birth , age , place , and day of the week . another study , this time comparing the effectiveness , electrophysiological characteristics , and cognitive side effects of hd - mst and ect , was published in 2011 by kayser et al . twenty patients in a treatment - resistant depressive episode were enrolled and blindly randomized in two groups of ten , treated with either ect or mst . it is noteworthy that for the first time patients suffering from bipolar disorder were included in a mst trial , two patients with type ii bd receiving ect and one type i and one type ii patients on the mst group . a neuropsychological battery was employed before and after treatment to evaluate such cognitive domains as memory , learning , executive function , language , and processing speed . recovery was defined as the time when patients opened their eyes and breathed independently , while reorientation time was assessed by asking the patient for her / his names , date of birth , age , place , and day of the week . recovery and reorientation were faster in the mst group , at 1 min 42 s and 2 m 16 s respectively , compared to the ect group , with recovery at 4 min 3 s and reorientation at 8 min 21 s. except for that , no significant differences in cognitive side effects were found between groups . eeg activity was similar in both groups , consisting of high - amplitude synchronized theta activity and equal postictal suppression . however , on the mst group seizure duration was briefer and some patients showed delayed ictal eeg activity and duration of motor and ictal activity in mst - treated patients had about the same length . on the other hand , lower electrical doses are associated with less cognitive impairment than high - dose ect , which could explain the absence of significant cognitive side effects on the ect group . further explored seizure characteristics and cognitive aspects of hd - mst compared with ect . seven patients in a treatment - resistant depressive episode were enrolled , six suffering from mdd and one from bipolar disorder type ii . except for the fact that seizures lasted longer on patients receiving ect , the authors found no significant differences in visible motor seizures and eeg activities between the two procedures , including postictal suppression , a measure reported to predict response to ect . reported an open - label trial in which 13 patients with tr - mdd received mst , assessing its effectiveness and side effects . a comprehensive neuropsychological battery was employed , and patients were evaluated at baseline , after six sessions and lastly after the end of treatments . in fact , the fast recovery from the procedure might have created an unexpected side effect : four patients awoke from the procedure while still under effect of muscle relaxation . the authors also note that the response to mst in this sample was significantly inferior to the reported effectiveness of ect , which generally yields remission rates of up to 80% . twenty patients suffering from treatment - resistant mdd were randomly assigned to two groups , one treated with mst and the other with ect . on each of the 2 treatment control days and 2 treatment - free control days , the patients were given 3 consecutive learning trials in the morning to learn 40 words . by comparing memory performance on treatment days to control days , treatment - induced memory disruption was evaluated . to enable evaluation of treatment caused effects on a particular subject , the patients were treated with mst or ect on 2 of the 4 testing days , whereas the other 2 days served as control . the results of this review show that mst is as effective as ect in inducing generalized tonic - clonic seizures , both in animals and in humans . however , there are considerable differences in the mechanism of induction , since magnetic pulses are not shunted by the scalp and skull , unlike ect 's electric current , allowing for a more focused and superficial stimulation , which on its turn might have implications for its electrophysiological aspects , effectiveness , and side effects . in most animal studies and some clinical studies mst also led to less postictal suppression and less eeg amplitude compared to ect - induced seizures , although two studies reported similar eeg characteristics for both groups [ 44 , 48 ] . such results could possibly be related to the use of rul - ect and low electric dose , which are associated with less cognitive loss but less effectiveness as well . high - dose mst ( hd - mst ) is an attempt to bridge this gap , and further studies are being conducted in order to improve its effectiveness . both the neurocognitive effects and the effectiveness of ect are related in part to the stimulus parameters with higher doses conferring increased adverse effects but perhaps better response rates on the other hand . following that rationale , the effectiveness of mst could , theoretically , be improved by increasing the stimulus ' dose , which can be achieved by administering a larger number of pulses in the mst train . preclinical studies suggested that hd - mst delivered under such parameters could be safe from a cognitive point of view , with no histological damage , either compared to low - dose mst , sham , or ecs [ 36 , 37 , 39 ] . such difference was evident from the early preclinical studies comparing it to ecs , subjects showing significantly better cognitive scores after mst than ecs . all clinical studies revised here focused on depressive episodes and the majority of enlisted patients suffered from mdd , with very few cases of bd . considering the challenges of treating depressive episodes in bipolar disorder and the low number of approved effective treatments , mst warrants further investigation as an alternative treatment for such cases . there is currently only one double - blind randomized clinical trial comparing the effectiveness and side effects of mst compared to ect , and all the others are either case reports , open - label studies , or crossover studies . the findings reported on this review suggest that mst might be an effective and safe alternative for the treatment of mood disorders , specifically on treatment - resistant depression , with a safer and more tolerable side effects profile than the current first choice , ect .

a number of clinical conditions cause acute hemorrhagic anemia , including massive gastrointestinal tract hemorrhage ( caused by traumatic surgery , trauma , rupture of gastroesophageal varices , and duodenal and gastric ulcer ) , pulmonary or bronchial hemoptysis , the sudden bleeding resulting from tumor erosion into the blood vessel wall , and hemostatic defect - related diseases ( such as hemophilia , von willebrand s disease , and platelet dysfunction ) [ 13 ] . each of these diseases can decrease blood flow and oxygen supply to the brain , and affect the physiological function of the brain , which often manifests as a wide variety of neurological symptoms , including dizziness , tinnitus , and even stroke and paralysis , and high disability and fatality rates , especially for children . in addition , anemia concurrent with brain trauma and a ruptured aneurysm also easily induces delayed cerebral infarction , thereby affecting treatment effect . therefore , the detection of changes in brain function in patients with acute hemorrhagic anemia is helpful in preventing neurological complications and evaluating therapeutic effect . however , the clinical changes of the nervous system in patients with anemia have not received much research attention . one important reason is that the current means can only indirectly detect changes in the brain function following anemia and lag behind the real condition of the brain function . some methods , such as transcranial doppler ultrasound , can detect the changes in cerebral blood flow velocity resulting from compensatory response of deficient oxygen delivery in anemia , including the increase in the state of anemia and returning to normal level when anemia is improved , but can not reflect the actual situation of the oxygen supply and metabolism in the brain or organic brain changes such as stroke . the other means , such as conventional computed tomography ( ct ) and magnetic resonance imaging ( mri ) , can detect early cerebral infarction and thus facilitate early treatment , but can not monitor the physiological changes in the brain from the occurrence of anemia . a non - invasive method , near - infrared spectroscopy ( nirs ) , is able to measure total levels of blood oxygen in the brain [ 68 ] , but is unable to provide fine details concerning the alterations in different cerebral tissues . susceptibility - weighted imaging ( swi ) is a newly developed , contrast - enhanced mri technique that has been used widely in the clinical diagnosis and research of many nervous system diseases , including cerebral hemorrhage and brain tumors [ 912 ] . swi is known as high - resolution blood oxygenation level - dependent ( bold ) venographic imaging , and can reflect oxygen metabolism - related blood protein concentrations . this technology can produce clear images of cerebral venules , and may be used to detect strokes and microbleeds [ 11,1416 ] . swi is potentially a novel , non - invasive method for sensing changes in cerebral oxygen levels and may provide more detailed information on cerebral blood flow in patients with hemorrhage . however , there is a lack of studies in animal models assessing the utility of swi for monitoring pathophysiologic changes in the brain following acute hemorrhagic anemia . in the present study , we have used swi to detect cerebral changes in an animal model of acute hemorrhagic anemia . we found that the swi images and signals showed changes following hemorrhage , and the extent of the change in the swi signal following acute hemorrhagic anemia differed between brain parenchyma and white matter regions . this indicates that the partial pressure of oxygen and carbon dioxide , and the concentration of deoxygenated hemoglobin in cerebral blood , are altered following acute hemorrhagic anemia . our results suggest that swi may be used to assess various regions of the brain after acute post - hemorrhagic anemia , and that it provides valuable information concerning the pathophysiologic changes in the brain . ten male new zealand white rabbits ( weight , 2.12.4 kg ; age , 34 months ) were obtained from the experimental animal center of guangdong medical college , guangzhou , china . the study was approved by the animal care and ethics committee of the first affiliated hospital , shenzhen university , guangzhou , china . construction of the rabbit model of acute hemorrhagic anemia was based on the method used by morimoto et al . . after a 12-h fast , the rabbits were anesthetized by intramuscular injection of 0.2 ml / kg xylazine hydrochloride ( su - mian - xin , veterinary institute , academy of military medicine science , changchun , china ) . once the respiratory rate had stabilized at 1218 breaths / min , the skin of the groin was incised along the groin fold . a catheter was then inserted into the femoral artery , for bloodletting and blood sampling . the blood samples obtained were used for whole blood tests and blood gas analysis ( for determination of paco2 , paco2 , lactic acid , and ph ) ; in addition , the mean arterial pressure ( map ) was measured . an additional catheter was inserted through the femoral vein to the atrium , for infusion of fluid and assessment of red blood cell count ( rbc ) , hemoglobin concentration ( hgb ) , hematocrit ( hct% ) , and central venous pressure ( cvp ) . before the first mri scan was performed , blood samples were drawn for blood gas analysis and whole blood tests . after the first mri scan had been carried out , a 40-ml blood sample was drawn through the arterial catheter . to compensate for the effects of simple blood volume loss , the same volume of a 6% hydroxyethyl starch in 0.9% sodium chloride solution a blood sample was drawn again for blood gas analysis and whole blood tests , and the head of the rabbit was re - scanned by mri . the bloodletting , fluid infusion , and scanning processes were repeated continuously 5 times before the animal recovered from anesthesia ; on the fifth occasion , the bloodletting volume and fluid infusion volume were both 50 ml . the rabbits were anesthetized , fixed to a special board in the supine position , and scanned by a siemens magnetom avanto 1.5 t mri scanner ( siemens ) , using a body coil ( excitation ) and wrap - around surface coil ( reception ) . t2 dual - echo fast spin - echo with fat - suppression ( fse - t2wi / pd ) and swi 3d sequences were used . the scan extended downward from a plane passing through the superior orbital margin to the medulla oblongata of the rabbit . fse - t2wi / pd acquisition was conducted using the following parameters : repetition time ( tr)=2800 ms ; echo time ( te)=33/78 ms ; field of view ( fov)=1212 cm ; matrix size = 256256 ; and acquisition time = 3.09 min . swi acquisition was performed with a 3d gradient echo sequence , as follows : tr=49 ms ; te=40 ms ; flip angle ( fa)=15 ; fov=1515 cm ; bandwidth = 80 khz ; and ipat factor = 2 . the bilateral frontal cortex , frontal white matter , temporal lobe , and thalamus were selected manually as regions of interest ( roi ) . the signal intensity in each of these regions ( area fixed at 0.080.01 cm ) was measured , and the average was calculated . the roi was positioned so as to avoid blood vessels and the skull . brain and cerebellum were harvested , immediately fixed in 4% formalin , and embedded in paraffin . sliced sections ( 35 m ) were stained with hematoxylin and eosin ( he ) and examined under an olympus bx41 microscope . in addition , a physician with five years of experience of mri interpretation was invited to evaluate the cerebral white - gray contrast and vein structure of the swi minimum intensity projection ( mip ) images , without knowledge of the sequence in which the images were acquired . ten male new zealand white rabbits ( weight , 2.12.4 kg ; age , 34 months ) were obtained from the experimental animal center of guangdong medical college , guangzhou , china . the study was approved by the animal care and ethics committee of the first affiliated hospital , shenzhen university , guangzhou , china . construction of the rabbit model of acute hemorrhagic anemia was based on the method used by morimoto et al . . after a 12-h fast , the rabbits were anesthetized by intramuscular injection of 0.2 ml / kg xylazine hydrochloride ( su - mian - xin , veterinary institute , academy of military medicine science , changchun , china ) . once the respiratory rate had stabilized at 1218 breaths / min , the skin of the groin was incised along the groin fold . a catheter was then inserted into the femoral artery , for bloodletting and blood sampling . the blood samples obtained were used for whole blood tests and blood gas analysis ( for determination of paco2 , paco2 , lactic acid , and ph ) ; in addition , the mean arterial pressure ( map ) was measured . an additional catheter was inserted through the femoral vein to the atrium , for infusion of fluid and assessment of red blood cell count ( rbc ) , hemoglobin concentration ( hgb ) , hematocrit ( hct% ) , and central venous pressure ( cvp ) . before the first mri scan was performed , blood samples were drawn for blood gas analysis and whole blood tests . after the first mri scan had been carried out , a 40-ml blood sample was drawn through the arterial catheter . to compensate for the effects of simple blood volume loss , the same volume of a 6% hydroxyethyl starch in 0.9% sodium chloride solution a blood sample was drawn again for blood gas analysis and whole blood tests , and the head of the rabbit was re - scanned by mri . the bloodletting , fluid infusion , and scanning processes were repeated continuously 5 times before the animal recovered from anesthesia ; on the fifth occasion , the bloodletting volume and fluid infusion volume were both 50 ml . the rabbits were anesthetized , fixed to a special board in the supine position , and scanned by a siemens magnetom avanto 1.5 t mri scanner ( siemens ) , using a body coil ( excitation ) and wrap - around surface coil ( reception ) . t2 dual - echo fast spin - echo with fat - suppression ( fse - t2wi / pd ) and swi 3d sequences were used . the scan extended downward from a plane passing through the superior orbital margin to the medulla oblongata of the rabbit . fse - t2wi / pd acquisition was conducted using the following parameters : repetition time ( tr)=2800 ms ; echo time ( te)=33/78 ms ; field of view ( fov)=1212 cm ; matrix size = 256256 ; and acquisition time = 3.09 min . swi acquisition was performed with a 3d gradient echo sequence , as follows : tr=49 ms ; te=40 ms ; flip angle ( fa)=15 ; fov=1515 cm ; bandwidth = 80 khz ; and ipat factor = 2 . additional processing was carried out on the phase - corrected swi sequences . the third ventricle and the olfactory bulb parallel to the corpus callosum were measured . the bilateral frontal cortex , frontal white matter , temporal lobe , and thalamus were selected manually as regions of interest ( roi ) . the signal intensity in each of these regions ( area fixed at 0.080.01 cm ) brain and cerebellum were harvested , immediately fixed in 4% formalin , and embedded in paraffin . sliced sections ( 35 m ) were stained with hematoxylin and eosin ( he ) and examined under an olympus bx41 microscope . for all analyses , a value of p<0.05 was considered to indicate statistical significance . in addition , a physician with five years of experience of mri interpretation was invited to evaluate the cerebral white - gray contrast and vein structure of the swi minimum intensity projection ( mip ) images , without knowledge of the sequence in which the images were acquired . comparisons of the blood test results before and after bloodletting are shown in figure 1 . there was an approximate halving of the rbc , hgb and hct% values after the first bloodletting , with further progressive decreases in the values of these parameters following each of the 4 subsequent bloodletting procedures ( figure 1a ) . the rbc , hgb , and hct% values after the fifth bloodletting ( 4.720.4310/l , 98.2010.22 g / l and 32.543.88% , respectively ) were significantly lower than the corresponding control ( pre - bleed ) values ( 0.270.1110/l , 6.012.31 g / l and 1.971.02% , respectively ) . the bloodletting procedures were associated with substantial increases in lactic acid concentration as well as a small , but statistically significant , change in blood ph ( figure 1b ) . after the fifth bloodletting , the lactic acid concentration rose to 14.476.30 compared to 3.602.48 at pre - bleed . pao2 increased and paco2 decreased progressively with each bloodletting procedure ( figure 1c ) , such that following the fifth bloodletting , pao2 was significantly elevated ( 150.409.78 vs. 80.5319.24 mmhg ) and paco2 was significantly reduced ( 22.007.35 vs. 42.654.13 mmhg ) compared with the corresponding pre - bleed values . bloodletting was not associated with any changes in cvp or map ( figure 1d ) . figure 2 shows representative swi and corresponding t2-weighted images of the brain of a rabbit , obtained at the superior aspect of the olfactory bulb , the border of the olfactory bulb , the thalamus , and the cerebellum ; also evident are examples of the rois chosen for analyses of the swi images ( see materials and methods ) . the mean swi signal intensities ( arbitrary units ) of the frontal cortex , frontal white matter , temporal lobe , and thalamus , before and after bloodletting , are presented in figure 3 . the control ( pre - bleed ) swi signal intensity of the frontal white matter was significantly lower than that of the frontal cortex ( 52.5020.29 vs. 63.1022.82 ; p<0.05 ) . bloodletting was not associated with any significant changes in the swi signal of the frontal white matter . in contrast , there were significant ( p<0.05 ) decreases in the swi signals of the frontal cortex , temporal lobe , and thalamus after the second , third , fourth , and fifth bloodletting procedures , compared with the corresponding control ( pre - bleed ) values . following the fifth bloodletting , the swi signal intensities of the frontal cortex , temporal lobe , and thalamus were 37.704.32 , 42.605.54 , and 39.403.47 , compared with corresponding control ( pre - bleed ) values of 63.1022.82 , 52.5020.29 , and 60.4020.29 , respectively ( p<0.05 ) . the value of the roi only reflects the signal intensity of a localized region of the brain , and may be influenced by the volume effect . therefore , we also evaluated the overall cerebral white - gray contrast and vein structure by inviting a physician with 5 years of experience to examine the swi mip images recorded before and after bloodletting ( figure 4 ) . the interpretation of the physician was that , compared with the control ( pre - bleed ) images , the contrast between the cerebral gray and white matter was higher after bloodletting , particularly after the fourth and fifth bloodletting procedures , and that venous structure was more abundant and clearer after bloodletting . histological sections of brain tissues after bloodletting revealed that degeneration and necrosis of neurons and glial cells were not evident ( figure 5 ) . however , spaces had formed around the blood vessels and cells , consistent with the occurrence of cerebral edema . comparisons of the blood test results before and after bloodletting are shown in figure 1 . there was an approximate halving of the rbc , hgb and hct% values after the first bloodletting , with further progressive decreases in the values of these parameters following each of the 4 subsequent bloodletting procedures ( figure 1a ) . the rbc , hgb , and hct% values after the fifth bloodletting ( 4.720.4310/l , 98.2010.22 g / l and 32.543.88% , respectively ) were significantly lower than the corresponding control ( pre - bleed ) values ( 0.270.1110/l , 6.012.31 g / l and 1.971.02% , respectively ) . the bloodletting procedures were associated with substantial increases in lactic acid concentration as well as a small , but statistically significant , change in blood ph ( figure 1b ) . after the fifth bloodletting , the lactic acid concentration rose to 14.476.30 compared to 3.602.48 at pre - bleed . pao2 increased and paco2 decreased progressively with each bloodletting procedure ( figure 1c ) , such that following the fifth bloodletting , pao2 was significantly elevated ( 150.409.78 vs. 80.5319.24 mmhg ) and paco2 was significantly reduced ( 22.007.35 vs. 42.654.13 mmhg ) compared with the corresponding pre - bleed values . bloodletting was not associated with any changes in cvp or map ( figure 1d ) . figure 2 shows representative swi and corresponding t2-weighted images of the brain of a rabbit , obtained at the superior aspect of the olfactory bulb , the border of the olfactory bulb , the thalamus , and the cerebellum ; also evident are examples of the rois chosen for analyses of the swi images ( see materials and methods ) . the mean swi signal intensities ( arbitrary units ) of the frontal cortex , frontal white matter , temporal lobe , and thalamus , before and after bloodletting , are presented in figure 3 . the control ( pre - bleed ) swi signal intensity of the frontal white matter was significantly lower than that of the frontal cortex ( 52.5020.29 vs. 63.1022.82 ; p<0.05 ) . bloodletting was not associated with any significant changes in the swi signal of the frontal white matter . in contrast , there were significant ( p<0.05 ) decreases in the swi signals of the frontal cortex , temporal lobe , and thalamus after the second , third , fourth , and fifth bloodletting procedures , compared with the corresponding control ( pre - bleed ) values . following the fifth bloodletting , the swi signal intensities of the frontal cortex , temporal lobe , and thalamus were 37.704.32 , 42.605.54 , and 39.403.47 , compared with corresponding control ( pre - bleed ) values of 63.1022.82 , 52.5020.29 , and 60.4020.29 , respectively ( p<0.05 ) . the value of the roi only reflects the signal intensity of a localized region of the brain , and may be influenced by the volume effect . therefore , we also evaluated the overall cerebral white - gray contrast and vein structure by inviting a physician with 5 years of experience to examine the swi mip images recorded before and after bloodletting ( figure 4 ) . the interpretation of the physician was that , compared with the control ( pre - bleed ) images , the contrast between the cerebral gray and white matter was higher after bloodletting , particularly after the fourth and fifth bloodletting procedures , and that venous structure was more abundant and clearer after bloodletting . histological sections of brain tissues after bloodletting revealed that degeneration and necrosis of neurons and glial cells were not evident ( figure 5 ) . however , spaces had formed around the blood vessels and cells , consistent with the occurrence of cerebral edema . recent studies have demonstrated that the cerebral venous contrast of swi images and the swi signal intensity of brain tissues change when certain drugs ( such as narcotics , caffeine , and contrast agents ) are applied or certain pathophysiologic conditions are present . in the present study , swi was used to investigate the association between the swi signal intensities of various regions of the rabbit brain and the partial pressures of blood oxygen and carbon dioxide in these regions , after acute hemorrhagic anemia . the rabbits in our study were anesthetized during mri scanning . to remove the possible influence of the anesthetic on the partial pressures of oxygen and carbon dioxide in the blood , and thereby on the bold signal intensity , we compared the swi signal intensities of different brain regions of the anesthetized rabbits before and after bloodletting . our results showed that following the hemorrhage , the rbc count , hemoglobin , and hematocrit of the rabbits decreased , indicative of a state of hemorrhagic shock . after bloodletting , the rabbits were injected with hydroxyethyl starch in sodium chloride solution to maintain blood volume and venous return in the short term and hence improve organ perfusion , resulting in little or no change in the cvp and map values of the rabbits . this supplement of fluid is often used as emergent treatment of patients in hemorrhagic or extensive burn situations . however , although the hydroxyethyl starch / sodium chloride solution was able to increase blood volume , it did not replenish the red blood cells that carry oxygen , and hence it could not correct the problem of inadequate tissue oxygenation . as a result , the blood lactate levels initially increased , then decreased to some extent , and finally increased again . a possible explanation for this is that at the early stage of blood loss , a rapid decrease in blood volume led to tissue hypoperfusion and an increase in anaerobic metabolism ; subsequently , the injection of hydroxyethyl starch / sodium chloride solution caused an increase in blood volume that partially replenished the blood oxygen , thereby reducing lactic acid levels ; and finally , as more hydroxyethyl starch / sodium chloride solution was injected into the rabbits , the acidic substances deposited in the tissues were able to enter the bloodstream and cause an elevation of the lactic acid level . the bold signal is closely related to intravenous oxygen concentration and can indirectly reflect changes in cerebral blood flow ( cbf ) , thereby allowing the monitoring of oxygen saturation . swi calculates the oxygen saturation based on the differences in the magnetic sensitivities of oxyhemoglobin and deoxyhemoglobin . the swi images of venous structures depend on the t2 * time , which may be shortened by deoxyhemoglobin - induced non - uniformity of the magnetic field , and phase differences between the surrounding tissue and blood vessels ; therefore , changes in cbf can also cause changes in the swi signals . the r2 * parameter of bold may be affected by blood volume , the red blood cell volume ratio , oxygen consumption , and small arteries , as well as by other factors such as subject age . since swi is an imaging technique with full flow compensation , it has a higher sensitivity than bold mri when the magnetic field is uneven , and therefore can minimize the interference of small arteries in the measurement and can accurately detect deoxygenated hemoglobin . the results presented here demonstrate that after repeated bloodletting in rabbits , the swi signal intensities of gray matter structures in the bilateral frontal cortex , temporal lobe , and thalamus were significantly decreased . however , the swi signal intensity of the frontal white matter was not significantly affected by bloodletting . the reductions in the swi signal intensities of the bilateral frontal cortex , temporal lobe , and thalamus after bloodletting may have been the result of decompensation induced by the repeated loss of blood . bloodletting may have resulted in a fall in blood pressure , an elevation of heart rate , and hyperventilation , further increasing the emission of carbon dioxide and thus decreasing its partial pressure , as was evident from our observations . the resulting hypocapnia may have led to cerebral artery contractions and reductions in cbf . to meet the oxygen needs of the brain , the proportion of oxygen extracted from the blood must increase , thereby decreasing cerebral venous oxygen , elevating levels of intravenous deoxyhemoglobin , and reducing the swi signal intensity . previous studies have revealed that the overall metabolic rate , the number of capillaries in gray matter , and the flow rate of the cerebral cortex are 4 times that of the white matter . our study also demonstrated that before bloodletting the swi signal intensity of the frontal cortex of rabbits was significantly higher than that of the frontal white matter , suggesting higher perfusion and more perfusion - induced oxyhemoglobin in the gray matter than in the white matter . furthermore , the results presented here imply that the gray matter was more sensitive to changes in pao2 and paco2 caused by acute hemorrhage , consistent with a previous report using arterial spin - labeling mr imaging . in our study , blood loss led to a decrease in the swi signal intensities of the gray matter structures of the cortex and thalamus , but little or no change in the white matter structures , suggesting that in the hypocapnic state associated with acute hemorrhage , the reductions in cerebral perfusion in the cortex and thalamic nuclei were greater than that in the white matter . in contrast to our findings , rostrup et al . reported that in conscious volunteers scanned using functional mri , elevations in blood carbonic acid content were associated with increases in the bold signal intensity of only the cortical gray matter , with no rise detected in the gray matter nuclei and white matter . a possible explanation for this discrepancy is that the use of anesthetics in our study may have inhibited gray matter structures ; in addition , swi is more sensitive and accurate at measuring alterations in bold signal intensities , allowing detection of changes in the gray matter nuclei that may not have been detectable in the rostrup et al . the gray matter of the brain contains more veins than the white matter , which may explain our observation that the swi signal of the white matter was not significantly decreased . during the hemorrhage , cerebral venous structures became clearer , and the cerebral white - gray contrast was markedly improved . this study has revealed that swi is an effective tool for detecting pao2- and paco2-induced changes in the cerebral oxygenation of different brain regions after acute hemorrhagic anemia . therefore , swi may be a useful technique for monitoring the pathophysiological changes and related complications associated with acute anemia .

backgroundthe aim of this study was to investigate susceptibility - weighted imaging ( swi ) signal changes in different brain regions in a rabbit model of acute hemorrhagic anemia.material/methodsten new zealand white rabbits were used for construction of the model of acute hemorrhagic anemia . signal intensities of swi images of the bilateral frontal cortex , frontal white matter , temporal lobe , and thalamic nuclei were measured . in addition , the cerebral gray - white contrast and venous structures of the swi images were evaluated by an experienced physician.resultsrepeated bloodletting was associated with significant reductions in red blood cell count , hemoglobin concentration , hematocrit , ph , and paco2 , and elevations of blood lactate and pao2 . in normal status , the swi signal intensity was significantly higher in the frontal cortex than in the frontal white matter ( 63.1022.82 vs. 52.5020.29 ; p<0.05 ) . repeated bloodletting ( 5 occasions ) caused significant ( p<0.05 ) decreases in the swi signals of the frontal cortex ( from 63.1022.82 to 37.704.32 ) , temporal lobe ( from 52.5020.29 to 42.605.54 ) , and thalamus ( from 60.4020.29 to 39.403.47 ) , but was without effect in the frontal white matter . the cerebral white - gray contrast and venous structures were clearer after bloodletting than before bloodletting.conclusionsthe effect of hemorrhage on the brain is reflected by swi signal changes in the cerebral cortex and gray matter nuclei .

Background Material and Methods New Zealand white rabbits Construction of a rabbit model of acute hemorrhagic anemia Blood sampling MRI scanning SWI image processing Histology Statistical analysis Results Blood test results SWI signals and images Histology Discussion Conclusions

a number of clinical conditions cause acute hemorrhagic anemia , including massive gastrointestinal tract hemorrhage ( caused by traumatic surgery , trauma , rupture of gastroesophageal varices , and duodenal and gastric ulcer ) , pulmonary or bronchial hemoptysis , the sudden bleeding resulting from tumor erosion into the blood vessel wall , and hemostatic defect - related diseases ( such as hemophilia , von willebrand s disease , and platelet dysfunction ) [ 13 ] . each of these diseases can decrease blood flow and oxygen supply to the brain , and affect the physiological function of the brain , which often manifests as a wide variety of neurological symptoms , including dizziness , tinnitus , and even stroke and paralysis , and high disability and fatality rates , especially for children . therefore , the detection of changes in brain function in patients with acute hemorrhagic anemia is helpful in preventing neurological complications and evaluating therapeutic effect . however , the clinical changes of the nervous system in patients with anemia have not received much research attention . one important reason is that the current means can only indirectly detect changes in the brain function following anemia and lag behind the real condition of the brain function . some methods , such as transcranial doppler ultrasound , can detect the changes in cerebral blood flow velocity resulting from compensatory response of deficient oxygen delivery in anemia , including the increase in the state of anemia and returning to normal level when anemia is improved , but can not reflect the actual situation of the oxygen supply and metabolism in the brain or organic brain changes such as stroke . the other means , such as conventional computed tomography ( ct ) and magnetic resonance imaging ( mri ) , can detect early cerebral infarction and thus facilitate early treatment , but can not monitor the physiological changes in the brain from the occurrence of anemia . a non - invasive method , near - infrared spectroscopy ( nirs ) , is able to measure total levels of blood oxygen in the brain [ 68 ] , but is unable to provide fine details concerning the alterations in different cerebral tissues . susceptibility - weighted imaging ( swi ) is a newly developed , contrast - enhanced mri technique that has been used widely in the clinical diagnosis and research of many nervous system diseases , including cerebral hemorrhage and brain tumors [ 912 ] . however , there is a lack of studies in animal models assessing the utility of swi for monitoring pathophysiologic changes in the brain following acute hemorrhagic anemia . in the present study , we have used swi to detect cerebral changes in an animal model of acute hemorrhagic anemia . we found that the swi images and signals showed changes following hemorrhage , and the extent of the change in the swi signal following acute hemorrhagic anemia differed between brain parenchyma and white matter regions . this indicates that the partial pressure of oxygen and carbon dioxide , and the concentration of deoxygenated hemoglobin in cerebral blood , are altered following acute hemorrhagic anemia . our results suggest that swi may be used to assess various regions of the brain after acute post - hemorrhagic anemia , and that it provides valuable information concerning the pathophysiologic changes in the brain . ten male new zealand white rabbits ( weight , 2.12.4 kg ; age , 34 months ) were obtained from the experimental animal center of guangdong medical college , guangzhou , china . the study was approved by the animal care and ethics committee of the first affiliated hospital , shenzhen university , guangzhou , china . construction of the rabbit model of acute hemorrhagic anemia was based on the method used by morimoto et al . the blood samples obtained were used for whole blood tests and blood gas analysis ( for determination of paco2 , paco2 , lactic acid , and ph ) ; in addition , the mean arterial pressure ( map ) was measured . an additional catheter was inserted through the femoral vein to the atrium , for infusion of fluid and assessment of red blood cell count ( rbc ) , hemoglobin concentration ( hgb ) , hematocrit ( hct% ) , and central venous pressure ( cvp ) . to compensate for the effects of simple blood volume loss , the same volume of a 6% hydroxyethyl starch in 0.9% sodium chloride solution a blood sample was drawn again for blood gas analysis and whole blood tests , and the head of the rabbit was re - scanned by mri . the bloodletting , fluid infusion , and scanning processes were repeated continuously 5 times before the animal recovered from anesthesia ; on the fifth occasion , the bloodletting volume and fluid infusion volume were both 50 ml . the rabbits were anesthetized , fixed to a special board in the supine position , and scanned by a siemens magnetom avanto 1.5 t mri scanner ( siemens ) , using a body coil ( excitation ) and wrap - around surface coil ( reception ) . the bilateral frontal cortex , frontal white matter , temporal lobe , and thalamus were selected manually as regions of interest ( roi ) . in addition , a physician with five years of experience of mri interpretation was invited to evaluate the cerebral white - gray contrast and vein structure of the swi minimum intensity projection ( mip ) images , without knowledge of the sequence in which the images were acquired . ten male new zealand white rabbits ( weight , 2.12.4 kg ; age , 34 months ) were obtained from the experimental animal center of guangdong medical college , guangzhou , china . the study was approved by the animal care and ethics committee of the first affiliated hospital , shenzhen university , guangzhou , china . construction of the rabbit model of acute hemorrhagic anemia was based on the method used by morimoto et al . once the respiratory rate had stabilized at 1218 breaths / min , the skin of the groin was incised along the groin fold . the blood samples obtained were used for whole blood tests and blood gas analysis ( for determination of paco2 , paco2 , lactic acid , and ph ) ; in addition , the mean arterial pressure ( map ) was measured . an additional catheter was inserted through the femoral vein to the atrium , for infusion of fluid and assessment of red blood cell count ( rbc ) , hemoglobin concentration ( hgb ) , hematocrit ( hct% ) , and central venous pressure ( cvp ) . to compensate for the effects of simple blood volume loss , the same volume of a 6% hydroxyethyl starch in 0.9% sodium chloride solution a blood sample was drawn again for blood gas analysis and whole blood tests , and the head of the rabbit was re - scanned by mri . the bloodletting , fluid infusion , and scanning processes were repeated continuously 5 times before the animal recovered from anesthesia ; on the fifth occasion , the bloodletting volume and fluid infusion volume were both 50 ml . the rabbits were anesthetized , fixed to a special board in the supine position , and scanned by a siemens magnetom avanto 1.5 t mri scanner ( siemens ) , using a body coil ( excitation ) and wrap - around surface coil ( reception ) . the bilateral frontal cortex , frontal white matter , temporal lobe , and thalamus were selected manually as regions of interest ( roi ) . in addition , a physician with five years of experience of mri interpretation was invited to evaluate the cerebral white - gray contrast and vein structure of the swi minimum intensity projection ( mip ) images , without knowledge of the sequence in which the images were acquired . comparisons of the blood test results before and after bloodletting are shown in figure 1 . there was an approximate halving of the rbc , hgb and hct% values after the first bloodletting , with further progressive decreases in the values of these parameters following each of the 4 subsequent bloodletting procedures ( figure 1a ) . pao2 increased and paco2 decreased progressively with each bloodletting procedure ( figure 1c ) , such that following the fifth bloodletting , pao2 was significantly elevated ( 150.409.78 vs. 80.5319.24 mmhg ) and paco2 was significantly reduced ( 22.007.35 vs. 42.654.13 mmhg ) compared with the corresponding pre - bleed values . bloodletting was not associated with any changes in cvp or map ( figure 1d ) . figure 2 shows representative swi and corresponding t2-weighted images of the brain of a rabbit , obtained at the superior aspect of the olfactory bulb , the border of the olfactory bulb , the thalamus , and the cerebellum ; also evident are examples of the rois chosen for analyses of the swi images ( see materials and methods ) . the mean swi signal intensities ( arbitrary units ) of the frontal cortex , frontal white matter , temporal lobe , and thalamus , before and after bloodletting , are presented in figure 3 . the control ( pre - bleed ) swi signal intensity of the frontal white matter was significantly lower than that of the frontal cortex ( 52.5020.29 vs. 63.1022.82 ; p<0.05 ) . bloodletting was not associated with any significant changes in the swi signal of the frontal white matter . in contrast , there were significant ( p<0.05 ) decreases in the swi signals of the frontal cortex , temporal lobe , and thalamus after the second , third , fourth , and fifth bloodletting procedures , compared with the corresponding control ( pre - bleed ) values . following the fifth bloodletting , the swi signal intensities of the frontal cortex , temporal lobe , and thalamus were 37.704.32 , 42.605.54 , and 39.403.47 , compared with corresponding control ( pre - bleed ) values of 63.1022.82 , 52.5020.29 , and 60.4020.29 , respectively ( p<0.05 ) . the value of the roi only reflects the signal intensity of a localized region of the brain , and may be influenced by the volume effect . therefore , we also evaluated the overall cerebral white - gray contrast and vein structure by inviting a physician with 5 years of experience to examine the swi mip images recorded before and after bloodletting ( figure 4 ) . the interpretation of the physician was that , compared with the control ( pre - bleed ) images , the contrast between the cerebral gray and white matter was higher after bloodletting , particularly after the fourth and fifth bloodletting procedures , and that venous structure was more abundant and clearer after bloodletting . there was an approximate halving of the rbc , hgb and hct% values after the first bloodletting , with further progressive decreases in the values of these parameters following each of the 4 subsequent bloodletting procedures ( figure 1a ) . the rbc , hgb , and hct% values after the fifth bloodletting ( 4.720.4310/l , 98.2010.22 g / l and 32.543.88% , respectively ) were significantly lower than the corresponding control ( pre - bleed ) values ( 0.270.1110/l , 6.012.31 g / l and 1.971.02% , respectively ) . the bloodletting procedures were associated with substantial increases in lactic acid concentration as well as a small , but statistically significant , change in blood ph ( figure 1b ) . pao2 increased and paco2 decreased progressively with each bloodletting procedure ( figure 1c ) , such that following the fifth bloodletting , pao2 was significantly elevated ( 150.409.78 vs. 80.5319.24 mmhg ) and paco2 was significantly reduced ( 22.007.35 vs. 42.654.13 mmhg ) compared with the corresponding pre - bleed values . bloodletting was not associated with any changes in cvp or map ( figure 1d ) . figure 2 shows representative swi and corresponding t2-weighted images of the brain of a rabbit , obtained at the superior aspect of the olfactory bulb , the border of the olfactory bulb , the thalamus , and the cerebellum ; also evident are examples of the rois chosen for analyses of the swi images ( see materials and methods ) . the mean swi signal intensities ( arbitrary units ) of the frontal cortex , frontal white matter , temporal lobe , and thalamus , before and after bloodletting , are presented in figure 3 . the control ( pre - bleed ) swi signal intensity of the frontal white matter was significantly lower than that of the frontal cortex ( 52.5020.29 vs. 63.1022.82 ; p<0.05 ) . bloodletting was not associated with any significant changes in the swi signal of the frontal white matter . in contrast , there were significant ( p<0.05 ) decreases in the swi signals of the frontal cortex , temporal lobe , and thalamus after the second , third , fourth , and fifth bloodletting procedures , compared with the corresponding control ( pre - bleed ) values . following the fifth bloodletting , the swi signal intensities of the frontal cortex , temporal lobe , and thalamus were 37.704.32 , 42.605.54 , and 39.403.47 , compared with corresponding control ( pre - bleed ) values of 63.1022.82 , 52.5020.29 , and 60.4020.29 , respectively ( p<0.05 ) . the value of the roi only reflects the signal intensity of a localized region of the brain , and may be influenced by the volume effect . therefore , we also evaluated the overall cerebral white - gray contrast and vein structure by inviting a physician with 5 years of experience to examine the swi mip images recorded before and after bloodletting ( figure 4 ) . the interpretation of the physician was that , compared with the control ( pre - bleed ) images , the contrast between the cerebral gray and white matter was higher after bloodletting , particularly after the fourth and fifth bloodletting procedures , and that venous structure was more abundant and clearer after bloodletting . recent studies have demonstrated that the cerebral venous contrast of swi images and the swi signal intensity of brain tissues change when certain drugs ( such as narcotics , caffeine , and contrast agents ) are applied or certain pathophysiologic conditions are present . in the present study , swi was used to investigate the association between the swi signal intensities of various regions of the rabbit brain and the partial pressures of blood oxygen and carbon dioxide in these regions , after acute hemorrhagic anemia . to remove the possible influence of the anesthetic on the partial pressures of oxygen and carbon dioxide in the blood , and thereby on the bold signal intensity , we compared the swi signal intensities of different brain regions of the anesthetized rabbits before and after bloodletting . our results showed that following the hemorrhage , the rbc count , hemoglobin , and hematocrit of the rabbits decreased , indicative of a state of hemorrhagic shock . after bloodletting , the rabbits were injected with hydroxyethyl starch in sodium chloride solution to maintain blood volume and venous return in the short term and hence improve organ perfusion , resulting in little or no change in the cvp and map values of the rabbits . as a result , the blood lactate levels initially increased , then decreased to some extent , and finally increased again . a possible explanation for this is that at the early stage of blood loss , a rapid decrease in blood volume led to tissue hypoperfusion and an increase in anaerobic metabolism ; subsequently , the injection of hydroxyethyl starch / sodium chloride solution caused an increase in blood volume that partially replenished the blood oxygen , thereby reducing lactic acid levels ; and finally , as more hydroxyethyl starch / sodium chloride solution was injected into the rabbits , the acidic substances deposited in the tissues were able to enter the bloodstream and cause an elevation of the lactic acid level . swi calculates the oxygen saturation based on the differences in the magnetic sensitivities of oxyhemoglobin and deoxyhemoglobin . the swi images of venous structures depend on the t2 * time , which may be shortened by deoxyhemoglobin - induced non - uniformity of the magnetic field , and phase differences between the surrounding tissue and blood vessels ; therefore , changes in cbf can also cause changes in the swi signals . the r2 * parameter of bold may be affected by blood volume , the red blood cell volume ratio , oxygen consumption , and small arteries , as well as by other factors such as subject age . the results presented here demonstrate that after repeated bloodletting in rabbits , the swi signal intensities of gray matter structures in the bilateral frontal cortex , temporal lobe , and thalamus were significantly decreased . however , the swi signal intensity of the frontal white matter was not significantly affected by bloodletting . the reductions in the swi signal intensities of the bilateral frontal cortex , temporal lobe , and thalamus after bloodletting may have been the result of decompensation induced by the repeated loss of blood . bloodletting may have resulted in a fall in blood pressure , an elevation of heart rate , and hyperventilation , further increasing the emission of carbon dioxide and thus decreasing its partial pressure , as was evident from our observations . to meet the oxygen needs of the brain , the proportion of oxygen extracted from the blood must increase , thereby decreasing cerebral venous oxygen , elevating levels of intravenous deoxyhemoglobin , and reducing the swi signal intensity . previous studies have revealed that the overall metabolic rate , the number of capillaries in gray matter , and the flow rate of the cerebral cortex are 4 times that of the white matter . our study also demonstrated that before bloodletting the swi signal intensity of the frontal cortex of rabbits was significantly higher than that of the frontal white matter , suggesting higher perfusion and more perfusion - induced oxyhemoglobin in the gray matter than in the white matter . furthermore , the results presented here imply that the gray matter was more sensitive to changes in pao2 and paco2 caused by acute hemorrhage , consistent with a previous report using arterial spin - labeling mr imaging . in our study , blood loss led to a decrease in the swi signal intensities of the gray matter structures of the cortex and thalamus , but little or no change in the white matter structures , suggesting that in the hypocapnic state associated with acute hemorrhage , the reductions in cerebral perfusion in the cortex and thalamic nuclei were greater than that in the white matter . reported that in conscious volunteers scanned using functional mri , elevations in blood carbonic acid content were associated with increases in the bold signal intensity of only the cortical gray matter , with no rise detected in the gray matter nuclei and white matter . a possible explanation for this discrepancy is that the use of anesthetics in our study may have inhibited gray matter structures ; in addition , swi is more sensitive and accurate at measuring alterations in bold signal intensities , allowing detection of changes in the gray matter nuclei that may not have been detectable in the rostrup et al . the gray matter of the brain contains more veins than the white matter , which may explain our observation that the swi signal of the white matter was not significantly decreased . during the hemorrhage , cerebral venous structures became clearer , and the cerebral white - gray contrast was markedly improved . this study has revealed that swi is an effective tool for detecting pao2- and paco2-induced changes in the cerebral oxygenation of different brain regions after acute hemorrhagic anemia .

a number of clinical conditions cause acute hemorrhagic anemia , including massive gastrointestinal tract hemorrhage ( caused by traumatic surgery , trauma , rupture of gastroesophageal varices , and duodenal and gastric ulcer ) , pulmonary or bronchial hemoptysis , the sudden bleeding resulting from tumor erosion into the blood vessel wall , and hemostatic defect - related diseases ( such as hemophilia , von willebrand s disease , and platelet dysfunction ) [ 13 ] . each of these diseases can decrease blood flow and oxygen supply to the brain , and affect the physiological function of the brain , which often manifests as a wide variety of neurological symptoms , including dizziness , tinnitus , and even stroke and paralysis , and high disability and fatality rates , especially for children . therefore , the detection of changes in brain function in patients with acute hemorrhagic anemia is helpful in preventing neurological complications and evaluating therapeutic effect . however , the clinical changes of the nervous system in patients with anemia have not received much research attention . one important reason is that the current means can only indirectly detect changes in the brain function following anemia and lag behind the real condition of the brain function . some methods , such as transcranial doppler ultrasound , can detect the changes in cerebral blood flow velocity resulting from compensatory response of deficient oxygen delivery in anemia , including the increase in the state of anemia and returning to normal level when anemia is improved , but can not reflect the actual situation of the oxygen supply and metabolism in the brain or organic brain changes such as stroke . the other means , such as conventional computed tomography ( ct ) and magnetic resonance imaging ( mri ) , can detect early cerebral infarction and thus facilitate early treatment , but can not monitor the physiological changes in the brain from the occurrence of anemia . a non - invasive method , near - infrared spectroscopy ( nirs ) , is able to measure total levels of blood oxygen in the brain [ 68 ] , but is unable to provide fine details concerning the alterations in different cerebral tissues . susceptibility - weighted imaging ( swi ) is a newly developed , contrast - enhanced mri technique that has been used widely in the clinical diagnosis and research of many nervous system diseases , including cerebral hemorrhage and brain tumors [ 912 ] . swi is known as high - resolution blood oxygenation level - dependent ( bold ) venographic imaging , and can reflect oxygen metabolism - related blood protein concentrations . swi is potentially a novel , non - invasive method for sensing changes in cerebral oxygen levels and may provide more detailed information on cerebral blood flow in patients with hemorrhage . however , there is a lack of studies in animal models assessing the utility of swi for monitoring pathophysiologic changes in the brain following acute hemorrhagic anemia . in the present study , we have used swi to detect cerebral changes in an animal model of acute hemorrhagic anemia . we found that the swi images and signals showed changes following hemorrhage , and the extent of the change in the swi signal following acute hemorrhagic anemia differed between brain parenchyma and white matter regions . this indicates that the partial pressure of oxygen and carbon dioxide , and the concentration of deoxygenated hemoglobin in cerebral blood , are altered following acute hemorrhagic anemia . our results suggest that swi may be used to assess various regions of the brain after acute post - hemorrhagic anemia , and that it provides valuable information concerning the pathophysiologic changes in the brain . ten male new zealand white rabbits ( weight , 2.12.4 kg ; age , 34 months ) were obtained from the experimental animal center of guangdong medical college , guangzhou , china . after a 12-h fast , the rabbits were anesthetized by intramuscular injection of 0.2 ml / kg xylazine hydrochloride ( su - mian - xin , veterinary institute , academy of military medicine science , changchun , china ) . once the respiratory rate had stabilized at 1218 breaths / min , the skin of the groin was incised along the groin fold . the blood samples obtained were used for whole blood tests and blood gas analysis ( for determination of paco2 , paco2 , lactic acid , and ph ) ; in addition , the mean arterial pressure ( map ) was measured . an additional catheter was inserted through the femoral vein to the atrium , for infusion of fluid and assessment of red blood cell count ( rbc ) , hemoglobin concentration ( hgb ) , hematocrit ( hct% ) , and central venous pressure ( cvp ) . to compensate for the effects of simple blood volume loss , the same volume of a 6% hydroxyethyl starch in 0.9% sodium chloride solution a blood sample was drawn again for blood gas analysis and whole blood tests , and the head of the rabbit was re - scanned by mri . the rabbits were anesthetized , fixed to a special board in the supine position , and scanned by a siemens magnetom avanto 1.5 t mri scanner ( siemens ) , using a body coil ( excitation ) and wrap - around surface coil ( reception ) . t2 dual - echo fast spin - echo with fat - suppression ( fse - t2wi / pd ) and swi 3d sequences were used . the bilateral frontal cortex , frontal white matter , temporal lobe , and thalamus were selected manually as regions of interest ( roi ) . in addition , a physician with five years of experience of mri interpretation was invited to evaluate the cerebral white - gray contrast and vein structure of the swi minimum intensity projection ( mip ) images , without knowledge of the sequence in which the images were acquired . ten male new zealand white rabbits ( weight , 2.12.4 kg ; age , 34 months ) were obtained from the experimental animal center of guangdong medical college , guangzhou , china . after a 12-h fast , the rabbits were anesthetized by intramuscular injection of 0.2 ml / kg xylazine hydrochloride ( su - mian - xin , veterinary institute , academy of military medicine science , changchun , china ) . once the respiratory rate had stabilized at 1218 breaths / min , the skin of the groin was incised along the groin fold . the blood samples obtained were used for whole blood tests and blood gas analysis ( for determination of paco2 , paco2 , lactic acid , and ph ) ; in addition , the mean arterial pressure ( map ) was measured . an additional catheter was inserted through the femoral vein to the atrium , for infusion of fluid and assessment of red blood cell count ( rbc ) , hemoglobin concentration ( hgb ) , hematocrit ( hct% ) , and central venous pressure ( cvp ) . to compensate for the effects of simple blood volume loss , the same volume of a 6% hydroxyethyl starch in 0.9% sodium chloride solution a blood sample was drawn again for blood gas analysis and whole blood tests , and the head of the rabbit was re - scanned by mri . the rabbits were anesthetized , fixed to a special board in the supine position , and scanned by a siemens magnetom avanto 1.5 t mri scanner ( siemens ) , using a body coil ( excitation ) and wrap - around surface coil ( reception ) . t2 dual - echo fast spin - echo with fat - suppression ( fse - t2wi / pd ) and swi 3d sequences were used . the bilateral frontal cortex , frontal white matter , temporal lobe , and thalamus were selected manually as regions of interest ( roi ) . in addition , a physician with five years of experience of mri interpretation was invited to evaluate the cerebral white - gray contrast and vein structure of the swi minimum intensity projection ( mip ) images , without knowledge of the sequence in which the images were acquired . there was an approximate halving of the rbc , hgb and hct% values after the first bloodletting , with further progressive decreases in the values of these parameters following each of the 4 subsequent bloodletting procedures ( figure 1a ) . the rbc , hgb , and hct% values after the fifth bloodletting ( 4.720.4310/l , 98.2010.22 g / l and 32.543.88% , respectively ) were significantly lower than the corresponding control ( pre - bleed ) values ( 0.270.1110/l , 6.012.31 g / l and 1.971.02% , respectively ) . the bloodletting procedures were associated with substantial increases in lactic acid concentration as well as a small , but statistically significant , change in blood ph ( figure 1b ) . pao2 increased and paco2 decreased progressively with each bloodletting procedure ( figure 1c ) , such that following the fifth bloodletting , pao2 was significantly elevated ( 150.409.78 vs. 80.5319.24 mmhg ) and paco2 was significantly reduced ( 22.007.35 vs. 42.654.13 mmhg ) compared with the corresponding pre - bleed values . figure 2 shows representative swi and corresponding t2-weighted images of the brain of a rabbit , obtained at the superior aspect of the olfactory bulb , the border of the olfactory bulb , the thalamus , and the cerebellum ; also evident are examples of the rois chosen for analyses of the swi images ( see materials and methods ) . the mean swi signal intensities ( arbitrary units ) of the frontal cortex , frontal white matter , temporal lobe , and thalamus , before and after bloodletting , are presented in figure 3 . the control ( pre - bleed ) swi signal intensity of the frontal white matter was significantly lower than that of the frontal cortex ( 52.5020.29 vs. 63.1022.82 ; p<0.05 ) . in contrast , there were significant ( p<0.05 ) decreases in the swi signals of the frontal cortex , temporal lobe , and thalamus after the second , third , fourth , and fifth bloodletting procedures , compared with the corresponding control ( pre - bleed ) values . following the fifth bloodletting , the swi signal intensities of the frontal cortex , temporal lobe , and thalamus were 37.704.32 , 42.605.54 , and 39.403.47 , compared with corresponding control ( pre - bleed ) values of 63.1022.82 , 52.5020.29 , and 60.4020.29 , respectively ( p<0.05 ) . the value of the roi only reflects the signal intensity of a localized region of the brain , and may be influenced by the volume effect . therefore , we also evaluated the overall cerebral white - gray contrast and vein structure by inviting a physician with 5 years of experience to examine the swi mip images recorded before and after bloodletting ( figure 4 ) . the interpretation of the physician was that , compared with the control ( pre - bleed ) images , the contrast between the cerebral gray and white matter was higher after bloodletting , particularly after the fourth and fifth bloodletting procedures , and that venous structure was more abundant and clearer after bloodletting . histological sections of brain tissues after bloodletting revealed that degeneration and necrosis of neurons and glial cells were not evident ( figure 5 ) . there was an approximate halving of the rbc , hgb and hct% values after the first bloodletting , with further progressive decreases in the values of these parameters following each of the 4 subsequent bloodletting procedures ( figure 1a ) . the rbc , hgb , and hct% values after the fifth bloodletting ( 4.720.4310/l , 98.2010.22 g / l and 32.543.88% , respectively ) were significantly lower than the corresponding control ( pre - bleed ) values ( 0.270.1110/l , 6.012.31 g / l and 1.971.02% , respectively ) . the bloodletting procedures were associated with substantial increases in lactic acid concentration as well as a small , but statistically significant , change in blood ph ( figure 1b ) . pao2 increased and paco2 decreased progressively with each bloodletting procedure ( figure 1c ) , such that following the fifth bloodletting , pao2 was significantly elevated ( 150.409.78 vs. 80.5319.24 mmhg ) and paco2 was significantly reduced ( 22.007.35 vs. 42.654.13 mmhg ) compared with the corresponding pre - bleed values . figure 2 shows representative swi and corresponding t2-weighted images of the brain of a rabbit , obtained at the superior aspect of the olfactory bulb , the border of the olfactory bulb , the thalamus , and the cerebellum ; also evident are examples of the rois chosen for analyses of the swi images ( see materials and methods ) . the mean swi signal intensities ( arbitrary units ) of the frontal cortex , frontal white matter , temporal lobe , and thalamus , before and after bloodletting , are presented in figure 3 . the control ( pre - bleed ) swi signal intensity of the frontal white matter was significantly lower than that of the frontal cortex ( 52.5020.29 vs. 63.1022.82 ; p<0.05 ) . in contrast , there were significant ( p<0.05 ) decreases in the swi signals of the frontal cortex , temporal lobe , and thalamus after the second , third , fourth , and fifth bloodletting procedures , compared with the corresponding control ( pre - bleed ) values . following the fifth bloodletting , the swi signal intensities of the frontal cortex , temporal lobe , and thalamus were 37.704.32 , 42.605.54 , and 39.403.47 , compared with corresponding control ( pre - bleed ) values of 63.1022.82 , 52.5020.29 , and 60.4020.29 , respectively ( p<0.05 ) . the value of the roi only reflects the signal intensity of a localized region of the brain , and may be influenced by the volume effect . therefore , we also evaluated the overall cerebral white - gray contrast and vein structure by inviting a physician with 5 years of experience to examine the swi mip images recorded before and after bloodletting ( figure 4 ) . the interpretation of the physician was that , compared with the control ( pre - bleed ) images , the contrast between the cerebral gray and white matter was higher after bloodletting , particularly after the fourth and fifth bloodletting procedures , and that venous structure was more abundant and clearer after bloodletting . histological sections of brain tissues after bloodletting revealed that degeneration and necrosis of neurons and glial cells were not evident ( figure 5 ) . in the present study , swi was used to investigate the association between the swi signal intensities of various regions of the rabbit brain and the partial pressures of blood oxygen and carbon dioxide in these regions , after acute hemorrhagic anemia . to remove the possible influence of the anesthetic on the partial pressures of oxygen and carbon dioxide in the blood , and thereby on the bold signal intensity , we compared the swi signal intensities of different brain regions of the anesthetized rabbits before and after bloodletting . our results showed that following the hemorrhage , the rbc count , hemoglobin , and hematocrit of the rabbits decreased , indicative of a state of hemorrhagic shock . after bloodletting , the rabbits were injected with hydroxyethyl starch in sodium chloride solution to maintain blood volume and venous return in the short term and hence improve organ perfusion , resulting in little or no change in the cvp and map values of the rabbits . a possible explanation for this is that at the early stage of blood loss , a rapid decrease in blood volume led to tissue hypoperfusion and an increase in anaerobic metabolism ; subsequently , the injection of hydroxyethyl starch / sodium chloride solution caused an increase in blood volume that partially replenished the blood oxygen , thereby reducing lactic acid levels ; and finally , as more hydroxyethyl starch / sodium chloride solution was injected into the rabbits , the acidic substances deposited in the tissues were able to enter the bloodstream and cause an elevation of the lactic acid level . the swi images of venous structures depend on the t2 * time , which may be shortened by deoxyhemoglobin - induced non - uniformity of the magnetic field , and phase differences between the surrounding tissue and blood vessels ; therefore , changes in cbf can also cause changes in the swi signals . since swi is an imaging technique with full flow compensation , it has a higher sensitivity than bold mri when the magnetic field is uneven , and therefore can minimize the interference of small arteries in the measurement and can accurately detect deoxygenated hemoglobin . the results presented here demonstrate that after repeated bloodletting in rabbits , the swi signal intensities of gray matter structures in the bilateral frontal cortex , temporal lobe , and thalamus were significantly decreased . the reductions in the swi signal intensities of the bilateral frontal cortex , temporal lobe , and thalamus after bloodletting may have been the result of decompensation induced by the repeated loss of blood . bloodletting may have resulted in a fall in blood pressure , an elevation of heart rate , and hyperventilation , further increasing the emission of carbon dioxide and thus decreasing its partial pressure , as was evident from our observations . to meet the oxygen needs of the brain , the proportion of oxygen extracted from the blood must increase , thereby decreasing cerebral venous oxygen , elevating levels of intravenous deoxyhemoglobin , and reducing the swi signal intensity . previous studies have revealed that the overall metabolic rate , the number of capillaries in gray matter , and the flow rate of the cerebral cortex are 4 times that of the white matter . our study also demonstrated that before bloodletting the swi signal intensity of the frontal cortex of rabbits was significantly higher than that of the frontal white matter , suggesting higher perfusion and more perfusion - induced oxyhemoglobin in the gray matter than in the white matter . furthermore , the results presented here imply that the gray matter was more sensitive to changes in pao2 and paco2 caused by acute hemorrhage , consistent with a previous report using arterial spin - labeling mr imaging . in our study , blood loss led to a decrease in the swi signal intensities of the gray matter structures of the cortex and thalamus , but little or no change in the white matter structures , suggesting that in the hypocapnic state associated with acute hemorrhage , the reductions in cerebral perfusion in the cortex and thalamic nuclei were greater than that in the white matter . reported that in conscious volunteers scanned using functional mri , elevations in blood carbonic acid content were associated with increases in the bold signal intensity of only the cortical gray matter , with no rise detected in the gray matter nuclei and white matter . a possible explanation for this discrepancy is that the use of anesthetics in our study may have inhibited gray matter structures ; in addition , swi is more sensitive and accurate at measuring alterations in bold signal intensities , allowing detection of changes in the gray matter nuclei that may not have been detectable in the rostrup et al . the gray matter of the brain contains more veins than the white matter , which may explain our observation that the swi signal of the white matter was not significantly decreased .