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Evaluation on the Hub 34

the treatment of chronic hepatitis c ( chc ) has evolved in the last 15 years from monotherapy with interferon alpha ( ifnalpha ) to the combination treatment with pegylated ifn ( peg - ifn ) plus ribavirin for 24 - 48 weeks . up to now , several viral , host and drug - related reactions in response to ifnalpha - based therapy have been identified . recent studies suggest that liver inflammation in chc is controlled by several mechanisms , including host regulatory immune responses and viral polypeptides interacting with cells involved in innate and adaptive immunity . it is well known that cutaneous side effects of treatment with ifnalpha alone or ifnalpha plus ribavirin in patients with chc have been widely reported , beyond the fact that the virus itself can cause skin lesions . however , the cutaneous side effects during therapy of chc are of inflammatory type with local erythema , edema and , much less frequently , necrosis at the injection skin sites . by contrast , skin side effects of such drugs have few data available in the literature , although the number of reports has been increasing in the last years , including cosmetic filler site injections . roughly one half of these patients had cutaneous findings , either alone or with systemic involvement . more recently there have been reported dermatological diseases in patients receiving the combination of ifn / ribavirin or ifn / ribavirin / amantadine for the treatment of chc . a 44-year - old man with chc genotype 1a and a viral load of hcv > 800,000 iu / ml , a2-f1 on liver biopsy according to metavir criteria , was treated for the hepatic disease . peg - ifnalpha-2b 1.5 g / kg weekly and 1,000 mg of ribavirin daily was introduced . in the 40th week the patient reported sudden intense pain , pruritus , erythema and skin hypertrophy in the left deltoid area over the seahorse tattoo and in a scar on his face ( fig . 1 ) . a skin biopsy on the scar and tattoo showed granulomatous dermatitis ( fig . chest x - ray was normal and the level of angiotensin enzyme converter was 50 iu / ml ( range 18 - 55 iu / ml ) . we intend to call attention especially to dermatologists to the possibility of a granulomatous tattoo reaction as a side effect during chc treatment with peg - ifnalpha-2b plus ribavirin . until now , eight cases of sarcoidosis as a complication of ifn therapy in chc have been reported in the english language literature . the most relevant topic in this case is the occurrence of disease on a tattoo area . six months later retreatment using peg - ifnalpha-2a 180 g once a week plus ribavirin 1,000 mg daily was tried . in the 6th week , cutaneous symptoms relapsed in an unbearable way . the patient did not accept to continue therapy , since hydrocortisone cream was prescribed again but no satisfactory pruritus and pain improvement was noticed . a prompt regression of skin reaction occurred after stopping therapy . sarcoidosis is a granulomatous disorder of unknown etiology and whose epidemiology suggest a genetic tendency face to infectious agents being supposed to result of immune system deregulation leading to non - caseating granulomas as an immune reaction to an unknown persistent antigenic stimulus . ifn has been linked to pulmonary macrophage activation , a characteristic feature of sarcoidosis which has been assumed an exaggerated t helper 1 ( th-1 ) immune response to a variety of exogenous antigens . it seems very likely that a potent immunoregulatory protein for th-1 response such as ifn may induce the disease . in january 2003 the eighth case of ifn - related sarcoidosis was reported with a review of the literature . chest x - ray revealed hilar lymphadenopathy in three patients , with reticulonodular shadows in another three , while the patient with only cutaneous involvement had a normal chest x - ray . the case we report presented with cutaneous sarcoidosis during chc therapy and spontaneous regression of the lesions was noted with treatment discontinuation . we intend to emphasize , especially to dermatologists , the risks of a granulomatous tattoo and other cutaneous sites during chc treatment with peg - ifnalpha-2b plus ribavirin . thalidomide has been also shown to have specific activity to the inflammatory mediators of sarcoidosis and to be an alternative beneficial therapy .

the treatment of chronic hepatitis c ( chc ) has evolved in the past 15 years and combination of pegylated interferon plus ribavirin is its current standard therapy . however , several side effects are commonly observed and frequently lead to transient or definitive interruption of treatment . although sarcoidosis in its systemic or cutaneous form is a very rare side effect in such circumstances , some cases have been reported even with conventional interferon . this brief review of the literature and description of a case of sarcoidosis occurring in a tattoo and a scar patient 's face , during treatment with pegylated interferon alpha-2b plus ribavirin , is an educative report directed in special to dermatologists . the lesion improved after drug interruption and recurred after retreatment with pegylated interferon alpha-2a . we conclude that this side effect must call the attention of doctors to seek for the diagnosis and therapy as soon as possible in such circumstances . no differences were noticed neither with alpha-2a nor alpha-2b pegylated interferon employment .

necrotising fasciitis ( nf ) is a serious soft tissue and life threatening infection , primarily involving the skin and superficial fascia , characterised by a rapid and extensive necrosis of the subcutaneous tissue . most patients have associated comorbidities , such as diabetes , hiv infection or treatment with immunosuppressive drugs , but it can also affect previously healthy individuals . infection is one of the common causes of morbidity and mortality in patients with systemic lupus erythematosus ( sle ) . it is associated with immunosuppressive agents , renal failure , and increased disease activity . despite the increased propensity of sle patients to develop common and opportunistic infections , nf has rarely been reported . to our knowledge , only 31 cases are described in the literature to date ( see table 1 ) . nf has also been described in other rheumatic diseases including polymyositis , dermatomyositis , systemic sclerosis , rheumatoid arthritis and ankylosing spondylitis , but it appears that this association is more common in sle.1 2 clinical features of nf in systemic lupus erythematosus a , asian ; aa , african american ; aps , antiphospholipid syndrome ; art , arthritis ; avn , avascular necrosis ; aza , azathioprine ; cad , coronary artery disease ; cs , corticosteroid therapy ; cyc , cyclophosphamide ; d , died ; dm , diabetes mellitus ; f , female ; fg , fournier 's gangrene ; h , hispanic ; hcq , hydroxychloroquine ; lym , lymphocytes ; m , male ; mmf , mycophenolate ; mtx , methotrexate ; n , nephritis ; na , not available ; nf , necrotising fasciitis ; pe , pulmonary embolism ; rtx , rituximab ; s , survived ; sa , staphylococcus aureus ; ttp , thrombotic thrombocytopenic purpura ; wbc , white blood cells . we describe the case of a nf caused by pseudomonas aeruginosa , complicated by recurrent sepsis and multiorgan failure in a young patient with a history of sle . a 26-year - old asian woman with a complex history of sle , diagnosed at age 14 years when she presented with malar rash , arthralgia , mouth ulcers , pulmonary vasculitis , strongly positive anti - nuclear antibody and anti - dsdna antibodies . subsequently , she had idiopathic detrusor overactivity with repeated urinary tract infection . linked to her corticosteroid treatment she developed avascular necrosis of her elbow ( at that time , on prednisolone 6 mg daily ) . the patient was also treated with hydroxychloroquine ( hcq ) , azathioprine ( aza ) ( 100 mg from 1999 to 2009 ) and had 14 courses of cyclophosphamide ( cyc ) ( a cumulative dose of 14 g ) and two of rituximab ( rtx ) ( two 1 g intravenous infusions separated by 2 weeks ) , the last being given in march 2012 for a sle flare manifested by severe vasculitic rash . immediately prior to b - cell depletion , her c3 level was 0.33 g / l ( normal : 0.651.65 ) , cluster designation ( cd ) 19 count was 0.146/l ( 0.110.69 ) , and immunoglobulin g level was 23.4 g / l ( 716 ) . in may 2012 , she presented to her local hospital with painful swelling of her left lower limb , she denied a history of trauma , and her inflammatory markers and ultrasonography were normal , and she was discharged . within 12 h she was found at home with a glasgow coma scale of 6 , and was admitted to the intensive care unit ( icu ) . on examination , the patient was in septic shock with hypotension ( systolic blood pressure 60 mm hg ) , tachycardia ( 135/min ) , and respiratory failure ( sao2 70% ) . laboratory results showed erythrocyte sedimentation rate 96 mm / h ( normal : 17 ) , c - reactive protein 281 mg / dl ( 05 ) , white blood cells 15 400/l ( 300010 000 ) ( 97% neutrophils ; 1.9% lymphocytes ) , haemoglobin 9.6 g / dl ( 11.515.5 ) , platelet 50 000/l ( 150 000400 000 ) , na 138 mmol / l ( 135145 ) , creatine kinase 474 iu / l ( 26140 ) , urea 16.8 mmol / l ( 1.78.3 ) , alamine transaminase 384 iu / l ( 1035 ) , total bilirubin 11 mg / dl ( 0.31.9 ) , albumin 17 g / l ( 3450 ) , creatinine 290 mol / l ( 4992 ) and proteinuria 2.30 g / l ( 00,10 ) . her c3 level was 1.0 g / l ( 0.651.65 ) , cd19 count was 0.001/l ( 0.110.69 ) , and immunoglobulin g level was 9.32 g / cultures of muscle tissue only grew p aeruginosa , resistant to piperazillin - tazobactam ; therefore , she was treated with meropenem , teicoplanin and clindamycin . as her clinical condition however , 1 week later , she deteriorated , with thick yellowish sputum , fever , respiratory distress and the chest x - ray showed right consolidation . ciprofloxacin and teicoplanin were empirically started , and the patient 's clinical condition gradually improved . a week after this treatment had finished , the patient became worse with a new left lower lobe consolidation , so the antibiotic therapy was changed to ceftazidime . her lupus flared with a marked malar rash over both cheeks , so her steroids ( prednisolone ) were increased to 20 mg per day and hcq was restarted . she was in the icu from may to august 2012 , but made a remarkable recovery and was discharged home with a steroid - tapering regimen and hcq . later , she developed two more sle flares , which were treated again with cyc and rtx , achieving good response . at 18 months follow - up , there is no evidence of new recurrent or severe ongoing infections . most patients with nf ranged from 38years to 44 years , with a male to female ratio of 23 : 1 , and apparently an increased incidence in african and asian countries.3 the true incidence is not known ( it is estimated to be approximately 0.4 cases per 100 000 ) , with a reported mortality from 20% to as high as 80%.35 the causative agents of nf vary and include two main categories , polymicrobial ( type 1 ) and infection of group a streptococcal ( type 2 ) . patients with sle have an increased risk of infections , due to immunological dysfunction and the use of steroids and immunosuppressive agents.6 other factors , such as the presence of a variant form of the fc receptor are also believed to contribute to the risk of pneumococcal infection.7 nf due to pseudomonas has very rarely been reported.8 9 the clinical presentation of patients with nf may be deceptively benign at onset , and it may not be possible to distinguish it clearly from minor soft tissue infections . our patient was discharged from the original hospital before being admitted subsequently severely ill to university college hospital . the lesion was rapidly progressive , probably due to her sle , and the prolonged steroid treatment and recent administration of cyc and rtx might have increased the risk of infection . during the initial period , exploration of the wound may be necessary even before the diagnosis is clear , particularly in a patient who is clearly toxic . in addition to the present case , the other 31 cases of nf in sle patients that have been reported are summarised in table 1 . the ages ranged from 12 years to 66 years , and 27 ( 84% ) were female . most of the patients reported were asian ( 33% , 7 patients ) , 33% ( 7 patients ) hispanic and 19% ( 4 patients ) african american . the sites of infection included the upper or lower limb ( 60% , 17 patients ) , the face , neck or tongue ( 18% , 5 patients ) , the genital area ( 11% , 3 patients ) and the abdomen ( 11% , 3 patients ) . nf type 1 was identified in 22 patients ( 71% ) and type 2 in 7 patients ( 23% ) , and when reported , the most common isolation was streptococcus ( 50% , 16 patients ) . most of the patients presented with intense pain , poorly demarcated erythematous , swollen lesions over the limbs and systemic inflammatory response ; 28 patients ( 88% ) were receiving corticosteroids and 7 hcq ( 22% ) . additionally , use of other immunosuppressive drugs has been reported ( 5 cyc , 5 aza , 2 rtx , 1 mycophenolate , 1 methotrexate , 1 plasmapheresis ) before or at the time of infection . other associated conditions have been identified , including nephritis ( 54% ; 15 patients of 28 reported ) , either during the current presentation or in the past , 13 ( 42% ) had active disease ( presumably those taking > 20 mg prednisolone or equivalent per day ) , 76% ( 13 of 17 reported ) had lymphopenia , 100% ( 12 out of 12 reported ) low serum albumin levels and 12 ( 37% ) had anaemia . interestingly , before the admission , 9 ( 28% ) reported a previous episode of infection : urinary , bartholin abcess , diarrhoea , respiratory tract infection , carious tooth , colitis , hepatitis c , cutaneous ulcers ( 2 ) , breast abscess and diverticulitis . most patients responded to therapy , but 8 of the 32 patients died ( 25% ) , probably due to the infection or because of complications ( such as sepsis or pulmonary embolism ) . the case presented here and the literature reviewed suggests that active disease , nephropathy , lymphopenia , anaemia , low serum albumin levels , immunosuppressive therapy and significant infections in the past , may constitute risk factors for the development of nf in patients with sle . a high index of suspicion for the diagnosis is required , and surgical exploration should not be delayed , especially during the early stages , to improve the prognosis of this devastating complication .

necrotising fasciitis ( nf ) is a rare infection of the subcutaneous tissue , known to be rapidly progressive and potentially fatal . patients with systemic lupus erythematosus ( sle ) may be predisposed to this condition , and early clinical recognition can be difficult . we report a case of necrotising fasciitis in a 26-year - old woman with sle . she presented with painful swelling of her left leg , then developed clinical features of septic shock . emergency debridement was performed . intraoperative findings revealed nf and cultures grew pseudomonas aeruginosa . the patient survived after a lengthy hospital admission , following several further debridements complicated by recurrent chest sepsis and multiorgan failure . we also review and discuss the published cases of nf in sle patients .

spontaneous perforation of rectum is a rare event ; however evisceration of the small bowel through the perforated site without predisposing factors is extremely rare , complex and worth reporting . the operative findings revealed it to be a case of spontaneous perforation of rectum with evisceration of the small bowel through the perforation . sudden increase in the intra - abdominal pressure leads to the perforation in the chronically deranged rectal wall and pushes the small bowel loops into the pelvis and through the perforated rectum to appear transanally . evisceration of small bowel through a spontaneous perforation in rectum is an extremely rare event . although 55 cases of evisceration of small bowel through anus due to perforation of rectum have been documented in world literature since the first report in 1827 by benjamin brodie , the number of cases without any significant predisposing factor is very less . the article reports a case of spontaneous perforation of rectum with evisceration of small bowel in a normal bowel without an apparent cause which is an extremely rare presentation . a 14 years old male was admitted with the chief complaint of acute lower abdominal pain which started suddenly after he passed stools . the abdomen was tender on deep palpation in the left iliac fossa and bowel sounds were sluggish . however , on per rectal examination gut loops were felt in the anal canal and finger was stained with blood thereby making intussusception the most probable diagnosis . a lump was felt in the left iliac fossa once the patient was anaesthetized for surgery . peroperative findings revealed a distended and a tubular sigmoid colon ( intussusception like appearance ) and twisting of ileal loops around the sigmoid colon simulating ileo - sigmoid knotting . on further mobilization and gentle traction on the ileal loops gangrenous segment of ileum became evident till it finally emerged out from a longitudinal perforation ( approximately15 mm ) in the anterior wall of rectum just above the peritoneal reflection ( fig . rectal tear was closed primarily in single layer after taking the biopsy from the whole edge which revealed non specific inflammation . on repeated questioning in postoperative period patient denied any history suggestive of constipation , rectal prolapse , trauma , weight lifting , rectal instrumentation or homosexual activity ; however intermittent straining at stools was present . figure showing rectal perforation and the gangrenous segment of ileum which had eviscerated through the perforated rectum . urinary bladder ( small square ) , the peritoneal reflection ( thin arrow ) and the rectal perforation ( thick arrow ) are clearly visible . spontaneous rectal perforation usually occurs due to excessive straining on the anterior rectal wall with a pre - existing pathology like , diverticulosis , colitis , ulceration , malignancy , adhesions , irradiation , rectal and uterine prolapse and as a consequence of iatrogenic injuries and blunt trauma abdomen . . chronic strain because of the underlying pathology causes progressive deepening of rectovesical and rectouterine pouches and the rectal wall becomes thin and weak as it is unsupported . contraction of the abdominal muscles increases the intra - abdominal pressure and spontaneous perforation occurs through this thinned out area , mostly at the ant mesenteric border where the blood supply is poorest . similarly , in a constipated patient the changed defecation pattern in some individuals per se causes a preliminary lesion in the intestinal wall which becomes friable due to chronic inflammation of its layers and may perforate . nevertheless intestinal evisceration through the perforated gut without any contamination of the peritoneal cavity is enigmatic and little difficult to explain . in a recent study presumed that two factors predisposed patients to this unusual complication : one was the sudden increase in intra - abdominal pressure and the other was the presence of rectal prolapse . whatever the mechanics involved the sudden increase in the intra - abdominal pressure seems to be the main contributing factor . it pushes the ileal loops into the rectovesical / rectouterine pouch which violently presses upon the anterior rectal wall . as the week rectal wall gives way the ileal loops sneak in . this seems to be the only plausible explanation for the spontaneous act particularly when there are no signs of peritonitis as in our case . in a perforated simple ulcer of rectum there is an area of induration around edges while a spontaneous perforation appears slit like , runs longitudinally ( rarely transversally ) with minimal signs of inflammation . it can occur in all age groups , the youngest one reported was six years old and oldest being ninety six years old . the treatment follows basic surgical principles . if reduced bowel is not viable it should be resected and followed by anastomosis or ileostomy depending upon the condition of the patient and contamination involved . in the follow up period patients should be investigated to detect exact etiology and cause specific management will prevent recurrence and complications . therefore , one must remember that per rectal palpation of bowel loops may be more than just rectal prolapse or intussusception .

context : spontaneous perforation of rectum is a rare event ; however evisceration of the small bowel through the perforated site without predisposing factors is extremely rare , complex and worth reporting.case report : a 14 years old presented to us apparently as a case of intussception . the operative findings revealed it to be a case of spontaneous perforation of rectum with evisceration of the small bowel through the perforation.conclusion:sudden increase in the intra - abdominal pressure leads to the perforation in the chronically deranged rectal wall and pushes the small bowel loops into the pelvis and through the perforated rectum to appear transanally .

the motor system is a dynamic network of cortical and subcortical areas interacting through excitatory and inhibitory circuits , modulated by somatosensory input . modifications of cortical excitability enable recovery and reorganization of the remaining motor areas both in animal models [ 4 , 5 ] and in humans [ 1 , 6 ] . transcranial magnetic stimulation ( tms ) and magnetoencephalography ( meg ) have both been applied in stroke patients to reveal cortical excitability changes . motor threshold ( mt ) , that is , the minimal tms intensity eliciting motor evoked potentials ( meps ) , is related to membrane excitability as it is influenced by drugs affecting neuronal ion channels . paired pulse tms ( pp - tms ) reveals short - interval intracortical inhibition ( sici ) , related to the activation of gaba - a receptors and intracortical facilitation ( icf ) attributed mainly to glutamatergic nmda receptor activation ( for references , see ) . in acute stroke , the mt is increased in the lesioned hemisphere ( lh ) whereas in the nonlesioned ( nh ) hemisphere it is normal or decreased one month after stroke . sici is decreased in both hemispheres early after stroke ; icf is stronger in nh in severe than mild strokes [ 1 , 6 ] . finger movements and median nerve or finger stimulation modify spontaneous meg oscillations over the sensorimotor cortex in the beta band ( 1525 hz ) . they are suppressed at 100300 ms after tactile stimulation ( event - related desynchronization ; erd ) , reflecting increased excitability , and increased at 5001000 ms ( event - related synchronization ; ers ) , reflecting removal of excitation [ 12 , 13 ] or reduced excitability . inhibitory gaba - a agonist diazepam increases meg beta activity [ 15 , 16 ] . combined meg and magnetic resonance spectroscopy showed a linear relationship between the beta ers strength and gaba concentration . beta ers has been shown to be significantly weaker in the lh than nh ; stronger ers amplitude was correlated with a better affected hand function up to 3 months after stroke . reduction of beta ers , however , correlated with clinical improvement after physiotherapy of patients with chronic stroke . movement - related beta erd was significantly reduced in lh of stroke patients . the hand representation in the somatosensory cortex ( s1 ) , estimated by somatosensory evoked fields ( sefs ) , is the largest one month after stroke and its increase was correlated with the affected hand function . in tms mapping , hand motor representation expands in the lh up to 1 month . in animal models , somatosensory reorganization is activated immediately after the lesion by diminished gaba - a - related inhibition and by glutamatergic activation after one month . we hypothesized to see correlations between erd and mt related to the early cortical excitability and between ers and sici , both attributed to gaba - a - related processes . moreover , we expected that icf , reflecting glutamatergic activity , would correlate with the somatosensory modifications in meg , as glutamate is associated with late somatosensory plasticity . meg and ntms were recorded from thirteen patients ( age 67.3 11 years , 8 women ) , with first - ever ischemic stroke in the middle cerebral artery territory one ( t1 ) and three months after stroke ( t2 ) . six patients had a subcortical and seven patients had a subcorticocortical or cortical stroke ( table 1 ) . at t1 , one patient used amitriptyline 10 mg / day and another used citalopram 10 mg / day . one patient used zopiclone 7.5 mg for occasional sleeplessness . at t2 , citalopram 10 mg / day was used by one patient . data from these patients has been presented previously [ 18 , 21 , 23 , 24 ] . as the patients having both tms and meg recordings are a subset of the previous patient groups , we recalculated the group - level electrophysiological parameters to show that the present patient group is sufficiently similar to those reported previously ( see supplementary tables 1 and 2 in supplementary material available online at http://dx.doi.org/10.1155/2015/309546 ) . an eximia navigated magnetic stimulator with a coplanar figure - of - eight coil of 70 mm wing radius ( nexstim ltd . , helsinki , finland ) was used for ntms . patients ' mris , required for navigation , were scanned at t1 and used also at t2 . the site was then stimulated by single tms pulses at 110% of mt and by the pp - tms at 90% for conditioning and 110% of mt for test pulses . fifteen single pulses or pairs of conditioning and test pulses were delivered in each stimulation session . the interval between stimuli was 3.3 s and the intersession interval varied between 1 and 3 min . the isi selected for the paired - pulse stimuli was 2 ms for sici and 12 ms for icf . the peak - to - peak amplitudes of meps elicited by pp - tms were normalized by dividing them by the corresponding single - pulse mep amplitude to simplify subject - to - subject comparisons . meg during rest and tactile stimulation of the thumb ( d1 ) , index ( d2 ) , and little finger ( d5 ) were recorded with a 306-sensor neuromagnetometer ( elekta neuromag , helsinki , finland ) in biomag laboratory , right before the ntms measurement . time - frequency representations ( tfr ) in the 1030 hz band were calculated to define the frequency range of beta modulation , which was quantified by the temporal spectral evolution method ( tse ) from signals of 2 to 4 meg sensors showing the strongest reactivity . only the contralateral beta modifications ( the affected hand stimulation for the lh and the unaffected hand for nh ) were analyzed . onset and offset of the erd and ers were defined as a time point when the signal differed 2 sds from the baseline . the absolute erd and ers strengths were calculated from the peak amplitudes and converted into relative values in relation to the 300 ms prestimulus baseline . for sefs , about 120 responses were averaged for stimulation of d2 ( isi 3005 ms ) , and d1 and d5 ( isi 1005 ms ) in separate sessions . the size of the hand representation in the si was determined by calculating the euclidean distance in xyz - space between the equivalent current dipoles ( ecds ) of the earliest responses to d1 and d5 stimulation . multiple comparison correction was carried out according to the number of tests ( n = 32 ) suggested by the four prior hypotheses ( t1 and t2 were tested separately ; lh and nh variations lead to four tests in each case ; n = 442 = 32 ) . we also present significances of correlation values without multiple comparisons and supply all correlation coefficients and corresponding p values ( cf . [ 1 , 9 , 28 ] for a similar approach and for its statistical aspects ) . the differences between ntms and meg values obtained at t1 and t2 were tested with student 's t - test . in the lh , meps were found in 11 patients both at t1 and at t2 and were present in the nh in all patients . mt was higher for lh than nh in 9 patients at t1 ( p < 0.05 , binomial test ) and in 10 patients at t2 ( p < 0.05 , binomial test ) . the mts in the lh and nh correlated strongly both at t1 ( r = .82 , p < .01 ) and at t2 ( r = .78 , p < .01 ) . pp2ms stimulations of nh did not inhibit meps ( disinhibition ; diminished sici ) in 5 patients at t1 and in 3 patients at t2 . pp12ms stimulation of lh facilitated meps ( icf ) in 10 out of 11 patients at t1 and in all patients at t2 . in nh mep amplitudes elicited by pp12ms stimulations were correlated between the lh and nh at t1 ( r = .62 ; p < .05 ) but not at t2 ( supplementary table 2 ) . erd started 140 10 ms after tactile stimulation and peaked at 250 10 ms . the subsequent ers started at 520 40 ms and peaked at 900 70 ms . at t1 , erd was absent in one patient and ers in two patients in the lh ; ers was missing from the nh in one patient . at t2 , ers was smaller in the lh than nh at t1 ( 46 31% versus 63 32% ; p < .05 ) ; at t2 , the difference was nonsignificant . sefs from both hemispheres were detected in 12 patients at t1 and in all patients at t2 . they were smaller in the lh than nh at t1 ( 25 nam versus 32 nam ; p < .04 ) but not at t2 . the si hand representation area was larger in the lh than nh at t1 ( 12 3 mm versus 10 3 mm p < .003 ) but not at t2 ( supplementary table 3 ) . the plots of the most relevant correlations are depicted in figure 1 to show that they were not driven by outliers . all correlations are displayed in table 2 to enable evaluation of significance of our hypotheses against general effects of the lesions . the mt and erd were correlated in the lh at t1 ( r = .66 , p < .03 ) , indicating that small erd was associated with a high mt ( figure 1(a ) ) . at t2 , this correlation was weaker ( r = .58 , p < .06 ) . however , the mt of the lh correlated with the erd of nh ( r = .62 , p < .04 ) , and the mt of the nh correlated with erd of the lh ( r = .65 , p < .02 ) , suggesting that high mt was associated with a small erd in the opposite hemisphere at t2 ( table 2 ) . sici and the ers did not correlate at t1 or in lh at t2 . in the nh , high ers was associated with a strong sici ( r = .59 , p < .04 ; figure 1(b ) ) . in addition to hypothesized correlations , sici of the nh and erd of the lh at t2 were correlated ( r = .82 , p < .001 ) , indicating that strong erd in the lh was associated with a strong sici in the nh . sici in the lh was correlated also with the si amplitude of the lh ( r = .64 , p < .04 ) , indicating that small si amplitude was associated with a weak sici ( table 2 ) . icf and sef parameters did not correlate at t1 . at t2 , icf in the lh correlated with the s1 amplitude of lh ( r = .65 , p in addition , icf in the nh correlated ( r = .82 , p < .001 ) with the si finger representation area of the lh ; this correlation remained significant also after bonferroni correction ( table 2 ) . moreover , icf in the lh at t1 was correlated ( r = .83 ; p < .002 ) with the si finger representation area of lh at t2 ; high icf at t1 resulted in a small hand representation area at t2 ( figure 1(c ) ) . our study is the first to compare meg and ntms excitability parameters during stroke recovery . navigated tms , not applied previously in longitudinal studies of stroke patients , enabled the precise replication of the stimulus site between separate measurements , adding reliability to the follow - up . we found correlations between cortical excitability estimates derived from ntms and meg . as expected , we found correlations between mt and erd , but in only one of the four possible intrahemispheric and two out of four interhemispheric conditions . the sici and beta ers , both attributed to gabaergic mechanisms , were correlated in one of their four possible intrahemispheric conditions ( in the nh at t2 ) . one reason for this may be that various gaba - a receptor subtypes contribute to sici . nonselective gaba - a receptor activators modify sici whereas the gaba - a1 receptor specific zolpidem did not . nonselective gaba - a agonist zopiclone increased meg beta activity whereas zolpidem suppressed beta activity in the vicinity of stroke lesion . moreover , in healthy subjects , diazepam increased meg erd but did not affect ers when the increase of baseline beta activity was taken into account . this nonspecificity could contribute to the correlations of sici with the erd and mt as well . correlations between ntms parameters and meg erd / ers were stronger at t2 than at t1 . analogously , most tms intracortical excitability measures did not correlate with the hand function acutely but did so 3 months after stroke . recovery of sensorimotor fmri activation to digit stimulation from 1 to 3 months was correlated with final motor function , emphasizing the importance of this time period for stroke recovery . is attributed mainly to glutaminergic mechanisms , glutamate may contribute to stroke - induced plasticity . somewhat surprisingly , high icf at t1 correlated with small si hand area at t2 ( figure 1(c ) ) ; thus , the narrowing towards normal hand representation size may be supported by glutaminergic activity . icf did not correlate with the meg erd / ers , and the si hand area and beta erd / ers were not correlated ; this suggests different mechanisms underlying sici and icf ( see for a detailed discussion ) . for example , high mt was associated with a small erd in the opposite hemisphere , and strong erd in the lh was associated with a strong sici in the nh . increased excitability within the unaffected motor cortex may cause imbalance between the homologous cortical motor areas and worsen also the ipsilesional hand coordination ( for references , see ) . thus , some functional correlations may relate to the modified general excitability properties of the motor system , instead of effects in the immediate vicinity of the stroke . tms results give direct information of the changes in the motor output and the immediate effects of tms are relatively local . however , also subcortical and spinal processes affect the meps used to evaluate the tms effects . meg reveals the activity of the whole cortical mantle and enables mapping of network effects generated by stroke . meg source analysis suggests mainly motor cortex origin of beta ers [ 13 , 33 , 34 ] . however , in electrocorticography , recorded directly from the cortex , beta erd and ers appear outside of pre- and postcentral gyri , in supplementary motor cortex , or broadly from pre- and postcentral gyri , frontolateral and medial cortex [ 37 , 38 ] . the widespread cortical generation of the erd and ers may make them resilient to small cortical strokes . multitude of generators may contribute to considerable variability of source locations of beta erd in stroke patients ( cf . ) . multiple sources underlying meg signals may also explain resilience of auditory evoked fields after small strokes . stronger correlations between icf and si parameters than between mt and sici and ers / erd may , in part , result from spatially more limited source areas of s1 responses than those of erd / ers . it can be expected that meg and ntms produce complementary information about the effects of stroke on cortical networks . moreover , meg parameters in the affected hemisphere and ntms indices in the unaffected hemisphere were correlated with the motor performance of the affected hand ( cf . [ 18 , 21 , 24 ] ) . erd in the 822 hz band may reflect downregulation of intracortical inhibition in the human motor cortex , as tms delivered during erd is associated with increased mep amplitudes and reduced sici . however , 1 hz repetitive tms over the motor cortex reduces meps to subsequent single tms pulses , indicating inhibition , but decreases postmovement beta ers , and intermittent theta burst tms facilitates meps but increases postmovement beta ers . beta ers is reduced in patients with myoclonus epilepsy , indicating increased cortical excitability . in line , sici is decreased in myoclonus epilepsy patients ; however , mt is increased [ 44 , 45 ] , and the silent period after the tms pulse , reflecting motor cortical postsynaptic inhibition , is prolonged , indicating prevailing inhibitory cortical tonus . thus , only some aspects of the cortical excitability may be shared in excitability estimates obtained by tms and meg . our results suggest that some tms and meg excitability measures reflect the activity of the same transmitter systems . however , high mt and absence of erd / ers may also correlate because of severely affected sensorimotor connections between the periphery and the cortex . we detected sefs in 12/13 patients and meps in 11/13 patients already at t1 , indicating that both somatosensory and motor pathways were conveying signals . motor function can be maintained despite significant damage to the corticospinal tract , as estimated from mt of stroke patients . moreover , we observed fewer correlations at t1 , when the sensorimotor pathways probably were more affected , than at t2 . however , such spurious correlations should remain stable or decrease during recovery from t1 to t2 but not increase , as in our data . the limitations of our study include the small size of the patient group as the precise features of structural and functional changes may differ among the patients . cortical excitability is modified differently in cortical and subcortical strokes [ 46 , 47 ] . this , however , should not alter our correlations between the tms and meg , as both were recorded from the same patients . possible effects of medication on excitability should go in parallel for meg and ntms , as the patients were tested sequentially during the same day . the patients were not tested in the acute stage with tms , and meg recordings showed most dramatic erd and ers modifications between the acute phase and t1 . although mt in the lh is correlated with the paretic hand function in acute stroke , this correlation , however , weakens during recovery , and tms intracortical excitability parameters correlated with the clinical performance best at 3 months . the 2 ms or 12 ms isis , selected for our paired - pulse stimuli , produce clear sici and icf in healthy subjects and in stroke patients , but we did not test other parameters , which could have produced stronger correlations between ntms and meg . icf and si response amplitude and area size , mt and the erd of the hemisphere harboring the stroke lesion , and sici and ers of the nonlesioned hemisphere are correlated in stroke patients . numerous correlations of the excitability parameters between the lh and nh emphasize the importance of the hemispheric balance of the excitatory - inhibitory properties of the sensorimotor system in analyzing the stroke - related dysfunction during stroke recovery .

objective . stroke alters cortical excitability both in the lesioned and in the nonlesioned hemisphere . stroke recovery has been studied using transcranial magnetic stimulation ( tms ) . spontaneous brain oscillations and somatosensory evoked fields ( sefs ) measured by magnetoencephalography ( meg ) are modified in stroke patients during recovery . methods . we recorded sefs and spontaneous meg activity and motor threshold ( mt ) short intracortical inhibition ( sici ) and intracortical facilitation ( icf ) with navigated tms ( ntms ) at one and three months after first - ever hemispheric ischemic strokes . changes of meg and ntms parameters attributed to gamma - aminobutyrate and glutamate transmission were compared . results . icf correlated with the strength and extent of sef source areas depicted by meg at three months . the ntms mt and event - related desynchronization ( erd ) of beta - band meg activity and sici and the beta - band meg event - related synchronization ( ers ) were correlated , but less strongly . conclusions . this first report using sequential ntms and meg in stroke recovery found intra- and interhemispheric correlations of ntms and meg estimates of cortical excitability . icf and sef parameters , mt and the erd of the lesioned hemisphere , and sici and ers of the nonlesioned hemisphere were correlated . covarying excitability in the lesioned and nonlesioned hemispheres emphasizes the importance of the hemispheric balance of the excitability of the sensorimotor system .

the u.s . food and drug administration ( fda ) has approved around 30 antidepressant medications for the treatment of major depression . among them are selective serotonin reuptake inhibitors ( ssris ) . these drugs change the balance of serotonin in the brain , such as fluoxetine ( prozac ) , paroxetine ( paxil ) , sertraline ( zoloft ) , escitalopram ( lexapro ) , and citalopram ( celexa ) . another family of medications , selective serotonin and norepinephrine reuptake inhibitors ( snris ) , help increase serotonin and norepinephrine levels in the brain , such as venlafaxine ( effexor ) , duloxetine ( cymbalta ) , and levomilnacipran ( fetzima ) . still others in this family include bupropion ( wellbutrin ) , vortioxetine ( brintellix ) , mirtazapine ( remeron ) vilazodone ( viibryd ) , nefazodone ( serzone ) , and trazodone ( desyrel ) . in addition , tricyclics and monoamine oxidase inhibitors , which are two classes of older antidepressants that work by inhibiting the brain s reuptake of serotonin and norepinephrine , are also approved but tend to cause more side effects than the other classes of antidepressants . but pharmacotherapy is nt the only option ; two other major classes of treatment are also available psychotherapy and somatic nonpharmacological treatments . in randomized , controlled trials , cognitive - behavioral therapy ( cbt ) and interpersonal psychotherapy ( ipt ) repeatedly have been demonstrated to be effective in the treatment of major depressive disorder ( mdd ) . whether other forms of psychotherapy , such as insight - oriented , psychodynamically based therapy , are effective in major depression remains controversial . brain stimulation therapies involve activating or touching the brain directly with electricity , magnets , or implants , and the fda has approved three somatic nonpharmacological treatments for depression : electroconvulsive therapy ( ect ) , vagus nerve stimulation ( vns ) , and repetitive transcranial magnetic stimulation ( rtms ) . ect is generally considered the most effective of all depression treatments , although no head - to - head , randomized , controlled trial has compared it with other interventions . it generally requires inpatient hospitalization , at least initially , and general anesthesia with nine to twelve treatments over a three- to four - week period . vns and rtms are both fda - approved for treatment - resistant depression ; the former requires an invasive surgical procedure . researchers have conducted relatively few controlled studies of these devices compared with the vast number of pharmacotherapy and psychotherapy treatment trials . with a plethora of drugs and psychotherapy approaches available , let us consider the problem psychiatrists encounter on a daily basis . a 50-year - old academic physician suffers from a classic major depressive episode associated with severe work stress . he has difficulty falling asleep , awakens several times during the night , and rises early with severe anxiety . he has reduced appetite , difficulty concentrating , and trouble enjoying any leisure activities , and he feels pessimistic about the future . he admits to passive contemplations about suicide , with recurring thoughts that if a car jumped the median and landed on his car , it would be an end to his suffering . he has no underlying medical disorder that might be contributing to depression , such as hypothyroidism or drug or alcohol abuse i want to recommend the treatment most likely to be successful in producing a complete remission of his depressive syndrome and relieving him of his considerable misery . what are the known and best - validated predictors of response ? our group has previously reviewed the scientific findings in this area.36 the most reliable predictor is past response , but in this case the patient has never been treated for depression . a positive response in first - degree family relatives is also predictive of a beneficial response to antidepressants , but again , this is not applicable to this patient . some evidence suggests that certain subtypes of depression respond best to certain treatments monoamine oxidase inhibitors ( the first type of antidepressants developed ) are believed to be the most effective for patients with so - called atypical depression characterized by hypersomnia , overeating , extreme rejection sensitivity , and feeling better in the morning than later in the day . combinations of antidepressants and antipsychotics or ect are best for patients with major depression with psychotic features . surely patient choice is an important consideration , but it will likely be guided by my discussion with the patient . in 2013 , mayberg , holtzheimer , dunlop , and craighead ) were colleagues of mine for many years , and we continue to collaborate on various projects . mayberg s study sought to identify a biomarker that could predict which type of treatment would benefit a patient based on the individual s brain activity . using regional brain glucose metabolism as measured by positron emission tomography ( pet ) as a proxy for neural activity , her group sought to determine whether baseline resting state activity predicted remission after twelve weeks of treatment with either the selective serotonin reuptake inhibitor escitalopram ( 10 to 20 mg per day ) or sixteen sessions of cognitive - behavioral therapy . the study sample initially comprised eighty - two men and women who were randomized between the two treatments . of these , sixty - five patients completed the study and thirty - eight had clear outcomes and acceptable pet data . the thirty - eight patients who comprise the analyzable data set were distributed as follows : eleven who went into remission with escitalopram ( six non - responders ) and twelve who did so with cbt ( nine nonresponders ) . the major finding were that hypometabolism of glucose in the insula , likely reflecting reduced activity of neurons in this brain region , was associated with remission using cbt , and with poor response to escitalopram . contrariwise , insula hypermetabolism , reflecting increased activity of neurons in this brain region , was associated with remission using escitalopram and with poor response to cbt . the authors conclude that baseline insula metabolism is the first objective marker to guide initial treatment selection in depression first they eliminated from their primary analysis the responders to cbt or to escitalopram who did not go into remission . more specifically , partial responders to escitalopram or cbt were excluded from the analysis . they did so in order to accentuate the differences between the extremes in the depressed population ; the results revealed clear differences in glucose metabolism in six regions : the right anterior insula , right motor cortex , left premotor cortex , right inferior temporal cortex , left amygdala , and precuneus . mean regional activity values for remitters and nonresponders segregated by treatment arm are plotted for the six regions showing a significant treatment outcome analysis of variance interaction effect . regional metabolic activity values are displayed as region / whole - brain metabolism converted to z scores . cbt indicates cognitive - behavioral therapy.7 when all six regions were compared , the right insula exhibited the greatest effect as a discriminator of treatment response , followed by the precuneus . when the whole sample was studied , right insular activity was positively correlated with the depression symptom severity scale , and with the hamilton depression rating scale ( hrsd ) score in the cbt treatment group while right insular activity was negatively correlated with the hrsd in the escitalopram treatment group . if additional research can replicates these results , it suggests that a simple brain imaging test could reliably predict whether a given patient should be treated with psychotherapy or antidepressant medication . it also raises a plethora of additional questions : a wealth of data , now summarized in a research meta - analysis , indicate that mdd patients with a history of child abuse and neglect exhibit a poorer response to pharmacotherapy and psychotherapy and exhibit unique brain imaging differences.8 mayberg s research does not address this critical clinical characteristic in this population.it is somewhat unclear how the six brain regions of interest were identified and why several regions repeatedly identified to be implicated in the pathophysiology of depression either were not selected or exhibited no significant effect , including the hippocampus , subgenual cingulate , and others.it is hard to know what to make of the findings that only the right anterior insula , right motor cortex , left premotor cortex , left amygdala , left precuneus , and right inferior temporal region show dramatic differences in the cbt versus escitalopram induced remission versus nonresponder groups whereas their counterparts , namely the left anterior insula , left motor cortex , right premotor cortex , right amygdala , right precuneus , and left inferior cortex did not . was a composite of the left and right sides of these structures informative?as the authors themselves point out , the study comprises a relatively small number of patients and our field is replete with pilot study findings that , unfortunately , have not been replicated in larger trials.this study utilized pet instead of the more often used functional magnetic resonance imaging ( fmri ) technology . as mayberg and her colleagues appropriately point out in their paper , fmri studies have examined regional brain activity and , more recently , resting state connectivity to identify mdd or mdd subtypes , but neither type of imaging has been used to discriminate response either among antidepressants or between antidepressants and psychotherapy.9 a wealth of data , now summarized in a research meta - analysis , indicate that mdd patients with a history of child abuse and neglect exhibit a poorer response to pharmacotherapy and psychotherapy and exhibit unique brain imaging differences.8 mayberg s research does not address this critical clinical characteristic in this population . it is somewhat unclear how the six brain regions of interest were identified and why several regions repeatedly identified to be implicated in the pathophysiology of depression either were not selected or exhibited no significant effect , including the hippocampus , subgenual cingulate , and others . it is hard to know what to make of the findings that only the right anterior insula , right motor cortex , left premotor cortex , left amygdala , left precuneus , and right inferior temporal region show dramatic differences in the cbt versus escitalopram induced remission versus nonresponder groups whereas their counterparts , namely the left anterior insula , left motor cortex , right premotor cortex , right amygdala , right precuneus , and left inferior cortex did not . was a composite of the left and right sides of these structures informative ? as the authors themselves point out , the study comprises a relatively small number of patients and our field is replete with pilot study findings that , unfortunately , have not been replicated in larger trials . this study utilized pet instead of the more often used functional magnetic resonance imaging ( fmri ) technology . as mayberg and her colleagues appropriately point out in their paper , fmri studies have examined regional brain activity and , more recently , resting state connectivity to identify mdd or mdd subtypes , but neither type of imaging has been used to discriminate response either among antidepressants or between antidepressants and psychotherapy.9 the expanding area of genetics in general , and pharmacogenetics in particular , is also of vital importance . a burgeoning database documents the role of certain genetic variations in vulnerability to mood disorders , and more recently how variations may affect treatment response to different antidepressants . whether genetic material was collected in mayberg s study is unclear , but this focus is crucial , particularly in view of recent findings in imaging genomics . the lack of random assignment of the mdd patients as regards , for example , the vulnerability gene variants of the serotonin transporter or others , now shown to be associated with clear alterations in regional brain activity , could have confounded the results . this research group has always been willing to take great leaps forward , and they should be applauded for it . subsequent studies will reveal if the insula is truly the region that predicts response to cbt versus a selective serotonin reuptake inhibitor such as escitalopram or whether other regions or biomarkers also need to be a component of the ultimate formula . this is part of the ongoing and exciting scientific process that is emblematic of the marriage of neuroscience and psychiatry . ultimately , i believe this work will be judged as crucial in eventually attaining the goal all of us seek : a valid predictor of individual treatment response in depression , still the holy grail in psychiatry research .

editor s note:holy grail is a well - known metaphor for the eternal spiritual pursuit for truth and wisdom . it suggests that in order for us to find what no one has found , we must search where few have looked . in 2013 , a group led by helen mayberg published a groundbreaking paper that sought an answer to one of the most discussed conundrums in psychiatry and neuroscience : can specific patterns of brain activity indicate how a depressed person will respond to treatment with medication or psychotherapy ? our author examines the findings and discusses their potential impact on treatment for a public health problem that affects millions of people worldwide .

primary mitochondrial disorders due to impaired oxidative phosphorylation ( oxphos ) are a well - established cause of severe disability and precocious death in both children and adults ( koopman et al . , 2012 ) . no effective therapies are currently available for these conditions , but encouraging results have recently been obtained by stimulating mitochondrial biogenesis acting on either the ppar system ( wenz et al . , 2008 ) or the amp - kinase ( ampk)/pgc1 axis ( viscomi et al . , 2011 ) . importantly , these approaches can in principle be extended to several mitochondrial diseases with different genetic causes , as they do not point to the correction of a specific defect but are based on a more general strategy aimed at increasing the overall residual activity of the respiratory chain . additional targets able to activate the mitochondriogenic program and boost mitochondrial function are sirtuins 17 ( houtkooper et al . , 2012 ) , the mammalian orthologs of the yeast silent information regulator ( sir ) 2 gene ( imai et al . , 2000 ; sirtuins have different subcellular localization , sirt1 and sirt6 being mainly found in the nucleus , sirt2 in the cytosol , sirt3sirt5 in mitochondria , and sirt7 in the nucleolus . sirtuins are important regulators of several proteins , including key metabolic players , acting as either deacetylases or adp - ribosylases ( houtkooper et al . , 2012 ) . the most - investigated member of the family is sirtuin1 ( sirt1 ) , a nad - dependent type iii nuclear deacetylase that utilizes nad as a cosubstrate to remove acetyl groups from lysine residues of a target protein . known targets of sirt1 are the tumor suppressor p53 , the myocyte - specific enhancer factor 2 ( mef2 ) , the forkhead box o ( foxo ) , and pgc1 , all of which regulate transcriptional programs related to increased mitochondrial function ( andreux et al . , 2013 ) . sirt1 activity is directly regulated by nad availability , by substrate - dependent activation , raising the hypothesis that nad acts as a metabolic sensor . for instance , both nad levels and sirt1 activity increase in mammalian tissues in response to energy / nutrient stresses such as exercise ( cant and auwerx , 2009 , 2010 ) and fasting ( cant and auwerx , 2010 ; chen et al . recent studies have shown that sirt1 activation can prevent diet - induced obesity in mice . this effect was achieved by increasing the content of nad in cells and tissues essentially through ( 1 ) dietary supplementation of suitable nad precursors , such as nicotinamide riboside , nr ( cant et al . , 2012 ) , or ( 2 ) inhibition of nad - consuming enzymes , such as the poly(adp - ribose ) polymerase 1 , parp1 ( bai et al . , 2011 ) . here , we have tested the therapeutic efficacy of these strategies on a genetic mitochondrial disease model , the sco2 knockout / knockin ( sco2 ) mouse ( yang et al . , 2010 ) . sco2 encodes a metallochaperone involved in the formation of the copper redox centers into nascent complex iv ( cytochrome c oxidase , cox ) ( leary et al . , 2009 ) . mutations in this gene lead to infantile fatal encephalocardiomyopathy ( papadopoulou et al . , 1999 ) . most of the patients carry at least one allele encoding the common mutation p.e140k , corresponding to the p.e129k mutation in the knockin murine sco2 allele . the second knockout allele in this animal model is functionally null ( yang et al . , 2010 ) . while homozygous knockout individuals are embryonic lethal , the sco2 mice show a predominantly myopathic phenotype characterized by exercise intolerance and associated with ubiquitous cox deficiency . to test the effects of persistent increase of the nad pool on the sco2 mouse , we first crossed it with a constitutive parp1 mouse ( de murcia et al . , 1997 ) , which shows increased levels of nad in skeletal muscle ( bai et al . , 2011 ) . the sco2-parp1 double mutants showed reduced fasting blood glucose levels , body weight and epididymal white adipose tissue ( wat ) compared to sco2 littermates , whereas no differences were observed between wt and parp1 littermates ( see figure s1 available online ) . the endurance motor performance of sco2-parp1 double mutants , sco2 , parp1 , and wt littermates ( four animals per group ) was monitored weekly by a standard treadmill test for 4 weeks starting at 2 months of age . while sco2 mice showed markedly reduced motor performance compared to wt littermates throughout the observation time , the double mutant individuals performed as well as the wt and parp1 littermates ( figure 1a ) . biochemically , significant reduction of complex iv activity and , to a lesser extent , complex iii as well ( yang et al . , 2010 ) , was measured in skeletal muscle of sco2 mice . in the muscle homogenate of sco2-parp1 double mutants , these activities were comparable to that of parp1 and wt littermates , whereas complex i and ii activities were even higher ( figure 1b ; table s1 ) . accordingly , the intensity of the histochemical staining specific to cox was increased in skeletal muscle of the double mutants , compared to the sco2 mutants ( figure 1c ) . the activities of complex iii and iv ( figure 1d ; table s2 ) were also increased in the brains of double mutants relative to sco2 animals , but remained significantly lower than those measured in parp1 and wt littermates ; the histochemical reaction to cox was concordant with the biochemical data ( figure 1e ) . to test the effects of pharmacological intervention , we first administered nr to sco2 mice and wt littermates ( n = 4/group ) , as a food admix ( 400 mg / kg ) ( cant et al . , 2012 ) for four weeks . metabolic parameters in the treated groups , including reduced blood glucose , plasma fatty acids , and epididymal wat , mirrored those of parp1 mice ( figure s2 ) , confirming that nr , or its derivative nmn , was pharmacologically active in vivo . nr - treated sco2 mice significantly improved their motor performance compared to the vehicle - treated sco2 group , rapidly achieving the levels of motor endurance displayed by treated and untreated wt mice ( figure 2a ) , which showed no difference to each other . these results suggest that nr treatment increases mitochondrial function in the sco2 mice through nad activation of sirt1 ( cant et al . , 2012 ) . accordingly , the nad / nadh ratio was significantly increased ( figure 2b ) , and the ratio between acetylated and total foxo1 , a direct target of sirt1 , was clearly reduced ( figure 2c ) in skeletal muscle of nr - treated versus vehicle - treated sco2 and wt animals . we did not observe differences in mtdna content ( data not shown ) and citrate synthase ( cs ) activity ( figure 2f ) , but mrna levels of several genes related to either fatty acids oxidation ( fao ) , including acox and cd36 , or oxidative phosphorylation ( coxi , coxii , coxiv , coxva ) were significantly increased in nr - treated versus vehicle - treated sco2 but not in wt animals ( figure 2d ) . tfam , a key factor of mtdna transcription , was also increased , and ucp3 and pdk4 , which were downregulated in sco2 mice , returned to control levels upon nr treatment . as expected , we found no change in transcripts specific to pgc1 , which is activated posttranscriptionally by sirt1 , and to two pgc1 partners , nrf1 and nrf2 . accordingly , western blot immunovisualization demonstrated increased levels of several nuclear- and mitochondrial - dna - encoded oxphos - related proteins in nr - treated versus vehicle - treated skeletal muscle samples ( figure 2e ) . in the same specimens , mitochondrial respiratory chain activities were significantly increased ( figure 2f ; table s3 ) in nr - treated versus vehicle - treated sco2 mice , but not in wt animals . the histochemical staining for cox reflected the biochemical results ( figure 2 g ) . in both c. elegans and mammalian cells , nr - dependent sirt1 activation can induce the mitochondrial unfolded protein response ( mtupr ) , a stress - response protective mechanism which can improve mitochondrial function ( durieux et al . we found that the mtupr - specific transcripts clpp , hsp60 , and sod2 were significantly increased in muscle samples of nr - treated sco2 mice , whereas sod3 , which is unrelated to mtupr , was unchanged ( figure 2h ) . no effect of the nr treatment was observed on the mitochondrial respiratory chain activities in the brain of our animals ( table s4 ) . next , we administered a pan - parp inhibitor ( mrlb-45696 , ic50 for parp < 1 nm ; pirinen et al . , 2014 , in this issue of cell metabolism ) at 50 mg / kg as a food admix for 4 weeks . in mrlb-45696-treated sco2 mice , we observed metabolic effects similar to those of nr treatment ( figure s3 ) . weekly treadmill tests showed progressive increase , up to normal , of the motor endurance in mrlb-45696-treated sco2 , whereas no change was seen in wt mice ( figure 3a ) . the nad / nadh ratio was significantly increased in treated versus untreated wt , but not in the sco2 , animals ( figure 3b ) , whereas the acetylated / total foxo1 ratio was reduced in both treated groups ( figure 3c ) , indicating activation of sirt1 by mrlb-45696 . similar to nr , mrlb-45696 increased the mrna expression levels of oxphos- and fao - related genes in both sco2 and wt mice , whereas mtdna content ( data not shown ) and cs activity ( figure 3f ) remained unchanged . in treated sco2 but not in wt animals , western blot analysis showed increased content of mitochondrial respiratory chain subunits ( figures 3d and 3e ) , which was paralleled by significantly increased mitochondrial respiratory chain activities ( figure 3f ; table s3 ) . histochemistry for cox showed increased staining in treated versus vehicle - treated sco2 mice ( figure 3 g ) . again , we found that expression of the mtupr genes hsp60 , clpp , and sod2 was significantly increased , unlike the mtupr - unrelated gene sod3 ( figures 3h and 3i ) . in contrast to the nr treatment , the mrlb-45696 treatment determined significant increase of cox transcripts ( figure 4a ) , and respiratory chain activities in the brain ( figure 4b ; table s4 ) . the intensity of cox staining was increased as well ( figure 4c ) . similar results were obtained in both skeletal muscle and brain by using pj34 , a commercially available pan - parp inhibitor ( figures s3 and s4 ; table s4 ) . the nad pool is set by the balance between de novo and salvage biosynthetic pathways and utilization by nad - consuming enzymes . nad is synthesized de novo from tryptophan , but the main source of nad is from salvage pathways ( houtkooper et al . , 2010 ) . these require the uptake of other nad precursors from the diet , including nr . upon its entry in the cell , nr is phosphorylated by nr kinases into nicotinamide mononucleoside ( nmn ) , which is then converted to nad by nmn adenylyltransferase ( bieganowski and brenner , 2004 ) . nad biosynthesis and cellular levels are also controlled by a circadian clock related to the feeding / fasting cycle . for example , in the mitochondrial compartment nad levels regulate sirtuin 3 , a deacetylase targeting respiratory chain subunits ( peek et al . , 2013 ) , while in the nucleus nad is a substrate of both parp1 and sirt . parp1 , the highest consumer of nad in mammalian tissues , is activated upon binding to damaged or abnormal dna ( durkacz et al . , 1980 ) and catalyzes the formation of poly(adp - ribose ) polymers ( par ) using nad as a substrate , onto different acceptor proteins , including parp1 itself ( adamietz , 1987 ) . ablation of the parp1 gene , supplementation of nr or administration of parp inhibitors ( parpi ) can expand the nad pool and activate sirtuins , particularly sirt1 , a master regulator of mitochondrial homeostasis . these effects can protect mice from high - fat ( hf)-induced metabolic disease ( cant et al . , we have shown here that these treatments can correct the biochemical and clinical phenotype of the sco2 mouse , a model of genetically determined mitochondrial disease . increased transcription of genes related to both oxphos and mtupr was associated with activation of oxidative metabolism , increase of mitochondrial respiratory chain activities , and normalization of the endurance motor deficit , displayed by naive sco2 animals . notably , these effects were hardly seen in wt littermates , suggesting that mitochondrial dysfunction sensitizes muscle and possibly other tissues to activators of mitochondriogenic programs . increased mitochondrial function can be achieved in wt animals only by much longer - term treatments ( > 6 months ) ( pirinen et al . , 2014 ) . in contrast with previous results , we found no change in mtdna copy number and cs activity in nr- or parpi - treated versus untreated animals , possibly because of the shorter timeframe of our experimental protocol ( 4 weeks ) compared to that of other studies ( 12 weeks ) . this observation suggests time - dependent activation of different mitochondriogenic programs , with induction of oxphos- and fao - related genes occurring much earlier than stimulation of mitochondrial proliferation and increase in mtdna content . likewise , prolonged nr supplementation up to 6 months induced mitochondrial biogenesis in the brain and improvement of cognitive dysfunction of an alzheimer disease mouse model ( gong et al . , 2013 ) . while we observed hardly any effect of nr in our 4 week trial , two pan - parp inhibitors did correct the respiratory chain defect in the brains of our sco2 mice . this observation is particularly relevant , as the brain is an exquisite target of mitochondrial dysfunction , and progressive encephalopathy is the most frequent clinical presentation of mitochondrial disease in infancy and childhood . our work supports the idea that the increase of nad levels in critical tissues is an effective therapeutic option for mitochondrial disease . nr is a natural vitamin with no known adverse effects , which could be administered as a dietary supplement , particularly in case of isolated mitochondrial myopathy . our results are concordant with very recent works reporting beneficial effects of nad precursors in mouse models characterized by reduced nad / nadh ratio , such as aging ( gomes et al . , 2013 ) or complex i deficiency ( karamanlidis et al . , 2013 ) initially shown to boost oxidative metabolism in diet - induced models of obesity , parp1 ablation or inhibition has recently been reported to remarkably rescue pharmacological models of liver cirrhosis , partly by correcting the associated mitochondrial impairment ( mukhopadhyay et al . , 2013 ) . several parp inhibitors are currently under clinical trial as anticancer molecules , and seem to be associated with relatively mild side effects ( bundred et al . , 2013 ; tutt et al . , however , more work is needed to evaluate their use in chronic conditions such as primary mitochondrial disorders in view of their potential genotoxic effects . mouse tissues were homogenized in 15 volumes of 10 mm potassium phosphate buffer ( ph 7.5 ) . mitochondrial - enriched fractions were collected after centrifugation at 800 g for 10 min in the presence of protease inhibitors , and frozen and thawed three times in liquid nitrogen . aliquots , 70 g each , were run through a 12% sds - page and electroblotted onto a nitrocellulose membrane , which was then matched with different antibodies . carlo besta neurological institute , in accordance with guidelines of the italian ministry of health . mice were maintained in a temperature- and humidity - controlled animal - care facility , with a 12 hr light / dark cycle and free access to water and food . a standard treadmill apparatus ( columbus instruments , columbus , oh ) was used to measure motor exercise endurance , as described in viscomi et al . series of 8 m thick sections were stained for cox and sdh , as described ( sciacco and bonilla , 1996 ) . muscle quadriceps samples stored in liquid nitrogen were homogenized in 10 mm phosphate buffer ( ph 7.4 ) , and the spectrophotometric activity of ci , cii , ciii , and civ , as well as cs , was measured as described ( bugiani et al . , 2004 ) . note that in all panels the activity of cii has been multiplied by 10 for visualization clarity . nad was extracted using acidic and alkaline extraction methods , respectively ( yang and sauve , 2006 ) . tissue nad was analyzed with mass spectrometry as previously described ( yang and sauve , 2006 ) . mtdna content and transcripts analysis was carried out by sybr green real - time pcr , as described ( viscomi et al . , 2011 ) .

summarymitochondrial disorders are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain . pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling , often fatal , disorders . pathways related to mitochondrial biogenesis are targets of sirtuin1 , a nad+-dependent protein deacetylase . as nad+ boosts the activity of sirtuin1 and other sirtuins , intracellular levels of nad+ play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell . we show here that supplementation with nicotinamide riboside , a natural nad+ precursor , or reduction of nad+ consumption by inhibiting the poly(adp - ribose ) polymerases , leads to marked improvement of the respiratory chain defect and exercise intolerance of the sco2 knockout / knockin mouse , a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis . this strategy is potentially translatable into therapy of mitochondrial disorders in humans .

noroviruses ( novs ) , a group of small , round - structured rna viruses constituting the norovirus genus in the family caliciviridae , infect both humans and animals . human novs cause epidemic acute gastroenteritis , affecting millions of people and claiming over 200,000 lives annually worldwide . at present structurally , novs are nonenveloped , containing an outer protein capsid that encapsulates the single - stranded , positive sense rna genome of 7.7 kb . the nov capsid is made from a single major structural viral protein , vp1 . crystallography of recombinant nov - like particles ( vlps ) reveals a t = 3 icosahedral symmetry consisting of 180 copies of vp1 organized into 90 dimers . vp1 is divided into two major domains , the shell ( s ) and the protruding ( p ) domains . the s domain forms the interior , icosahedral shell ; while the p domain forms the dimeric protrusions extending outward from the shell . the p domain can be further divided into p1 and p2 subdomains , corresponding to the legs and the head of the arch - like protrusion , respectively . the p2 subdomain forms the outermost surface of the capsid with highly variable sequence , responsible for the virus human novs are difficult to study due to the lack of an efficient cell culture system and a small animal model . currently , research into nov host interactions relies on various nov subviral particles . expression of full - length vp1 results in vlps that are structurally similar to an authentic virus . furthermore , expression of various subdomains results in smaller subviral particles or complexes . for example , production of the s domain forms s particles , corresponding to the interior shell of the capsid , while expressions of the p domains with or without modifications can form p dimers , 12-mer small p particles , or 24-mer p particles . in addition , various glutathione s - transferase ( gst)-p domain fusion proteins have been shown to form polyvalent complexes owing to the dimeric and oligomeric features of the gst and the p domain . these vlps , p particles and p complexes retain the basic structures of the capsid or p dimer , recognize host ligands and , thus , have been used as tools or models for the study of nov host interactions . human novs recognize histo - blood group antigens ( hbgas ) as attachment factors or receptors , which play an important role in the host susceptibility of nov infection , as shown by both human challenge studies and outbreak investigations . hbgas are oligosaccharides linked to membrane proteins or lipids as glycoprotein or glycolipid that are distributed extensively on the surfaces of red blood cells and mucosal epithelia . they are also present as free oligosaccharides in biological fluids , such as saliva or milk . human novs interact with hbgas in a strain - specific manner , whereby a number of nov - hbga binding patterns involved in all abo , lewis and secretor / nonsecretor types have been identified . the structural basis of these interactions have been elucidated by x - ray crystallography of nov p dimers in complex with hbga oligosaccharides . however , it has been observed that some human novs , such as va115 ( gi.3 ) , desert shield virus ( gi.3 ) and noda485 ( gii.1 ) , do not bind any hbgas . a human challenge study of snow mountain virus ( smv , gii.2 ) did not reveal a dependence of host susceptibility on hbga type , despite the fact that the smv vlp recognizes only the b antigen . in addition , a recent study showed that nov vlps of ueno 7k ( gii.6 ) and noda485 binds caco-2 cells and human small intestinal epithelium biopsy in a hbga - independent manner . these data suggest that hbgas may not be the only receptors for human novs . for example , using thin - layer chromatography and quartz crystal microbalance with dissipation monitoring , larson et al . reported binding of gii.4 vlps to galactosylceramide and hbga glycosphingolipids that were purified from human meconium samples . takeda and co - workers demonstrated that vlps of gii novs bound heparan sulfate on the cell surface , while belliot and co - workers showed that gii.4 vlps recognized sialic acid - containing carbohydrates , such as sialyl lewis x ( le ) , sialyl - lacto - n - fucopentaose , sialyl - lacto - n - tetraose , and sialyl - lacto - n - neotetraose , with affinities comparable to those of hbga ligands . using saturation transfer difference nuclear magnetic resonance spectroscopy , peters and co - workers detected the interactions between gii.4 vlps and the sialic acid moiety of sialyl le and sialyl le . however , they also found that carbohydrates containing sialic acid , but not fucose , e.g. 3-sialyllactose and 6-sialyllactose , do not exhibit detectable binding with the vlp . the results of these studies , taken together , imply that sialic acid - containing oligosaccharides could also be ligands of human novs . in fact , sialic acid - containing oligosaccharides have been shown to be ligands or receptors for some animal caliciviruses ( cvs ) , including murine nov ( mnv1 ) , feline calicivirus ( fcv ) and a porcine sapovirus ( psav , cowden strain ) . however , solid evidence to establish the ligand status of sialic acid for human cvs ( human novs and human sapoviruses ) is lacking . here , we report the first experimental evidence that human novs recognize sialic acid - containing glycosphingolipids ( gangliosides ) . the catch - and - release electrospray ionization mass spectrometry ( car - esi - ms ) assay was used to screen a library of gangliosides against the p particle of human nov va387 ( gii.4 ) . the affinities of 13 gangliosides for the p dimer of va387 and of a second human nov strain , va115 ( gi.3 ) , were measured using the direct esi - ms assay . using a competitive esi - ms assay , the proxy protein method , the highest affinity ligand , gm3 , was subjected to additional binding measurements and the affinities for both the va387 p particle and vlp were determined . notably , the ganglioside affinities measured for nov va387 are comparable to those of known hbga oligosaccharide receptors . enzyme - linked immunosorbent assays ( elisa ) provided additional evidence that both strains of novs exhibit binding to sialic acid - containing oligosaccharides . the vlps of va387 ( gii.4 ) were produced in insect cells ( sf9 ) through a recombinant baculovirus containing the gene encoding va387 vp1 ( genbank accession number ay038600 , molecular weight ( mw ) of monomer 58,887 da ) as described previously . the resulting vlps were purified by sucrose gradient . va387 p particles ( 24-mer , mw 865,036 da ) , p dimers ( mw 69,312 da ) , and gst - p domain fusion proteins were produced based on the p domain sequences ( residues 222539 ) of vp1 via e. coli as reported in our previous studies . the gst gene fusion system ( ge healthcare life sciences , piscataway , nj ) with plasmid vector pgex-4t-1 was used for the p proteins expression . preparations of va115 ( gi.3 ) vlps and p particles were attempted based on the vp1 sequences ( genbank accession number ay038598 ) and the established procedure described above , but the yields for both particles were found to be very low . the p dimers ( mw 67,712 da ) and the gst - p fusion proteins of va115 were produced in high yield ( > 20 mg l bacteria ) through the same procedure as used for the production of the p proteins of va387 . formations of the 24-mer p particles , p dimers , and the gst - p polymers were analyzed by gel - filtration chromatography via a superdex 200 size exclusion column ( ge healthcare life sciences ) controlled by an akta fast performance liquid chromatography system ( fplc , model 920 , ge healthcare life sciences ) . a single chain fragment ( scfv , mw 26,539 da ) of the monoclonal antibody se1554 was produced using recombinant technology as described elsewhere . a recombinant fragment of the c - terminus of human galectin-3 ( gal-3c , mw 16,330 da ) was generously provided by prof . c. cairo ( university of alberta ) . bovine ubiquitin ( ubq , mw 8565 da ) was purchased from sigma - aldrich canada ( oakville , canada ) . the proteins were concentrated and exchanged into an aqueous 200 mm ammonium acetate ( ph 7 ) using vivaspin 0.5 ml centrifugal filters ( sartorius stedim biotech , gttingen , germany ) with a mw cutoff of 10 kda and stored at 80 c until use . the structures of the oligosaccharides and glycoconjugates used in this study are shown in figure s1 ( supporting information ) . the 17 ganglioside and globoside oligosaccharides ( gm3 , gm2 , gm1a , gm1b , gd3 , gd2 , gd1a , gd1b , gt3 , gt2 , gt1a , gt1c , fucosyl - gm1 , asialo - gm2 , asialo - gm1 , gb3 and gb4 ) were purchased from elicityl sa ( crolles , france ) . h type 3 trisaccharide , a type 3 tetrasaccharide , and b type 3 tetrasaccharide were a gift from prof . each solid compound was dissolved in ultrafiltered milli - q water ( millipore , ma ) to give a 1 mm stock solution . polyacrylamide ( paa)-conjugated neu5ac , 6-sialylacnac , and gm3 trisaccharide were purchased from vector lab ( burlingame , ca ) . all of the esi - ms assays were carried out on a synapt g2s quadrupole - ion mobility separation - time - of - flight ( q - ims - tof ) mass spectrometer ( waters , manchester , u.k . ) the car - esi - ms and direct esi - ms assays were performed in negative ion mode , whereas the proxy protein esi - ms assay was implemented in positive ion mode . , 0.68 mm i.d . ) pulled to 5 m using a p1000 micropipette puller ( sutter instruments , novato , ca ) . a platinum wire was inserted into the nanoesi tip , and a capillary voltage was applied to carry out esi . the source parameters for both negative and positive ion modes were : capillary voltage 0.8 kv ( negative ion mode ) or 1.0 kv ( positive ion mode ) , source temperature 60 c , cone voltage 60 v ( negative ion mode ) or 35 v ( positive ion mode ) , trap voltage 5 v , and transfer voltage 2 v. data acquisition and processing were performed using masslynx software ( version 4.1 ) . the car - esi - ms assay was performed to identify carbohydrate ligands of the nov va387 p particle . ions corresponding to ligand - bound p particle were isolated using the quadrupole mass filter . the quadrupole was set to transmit a broad mass - to - charge ratio ( m / z ) window ( approximately 200 m / z units ) , which allows for the simultaneous passage of free and ligand - bound p particle complexes at a given charge state . protein ligand complexes were subjected to collision - induced dissociation ( cid ) in the trap region of the synapt g2s by increasing the trap voltage from 5 to 200 v. argon ( 1.42 10 mbar ) was used to carry out cid in the trap region . in most instances , the deprotonated ligands released from the complexes could be identified from their mws . where required , ims was used to separate the released isomeric ligands . for ims separation a wave height of 35 v was used , and the wave velocity was ramped from 2000 to 500 m s. in all cases a helium flow rate of 150 ml min and a nitrogen flow rate of 40 ml min were used . the arrival time distributions ( atds ) for the released ligands were compared to reference atds , which were measured for the deprotonated carbohydrates produced directly from solution . the direct esi - ms assay was used to quantify the affinities of the carbohydrate ligands for the nov p dimers of va387 and va115 . at least four different initial ligand concentrations were used for each oligosaccharide tested , and the binding measurements were carried out in triplicate . a complete description of the data analysis method employed to calculate the intrinsic association constants ( ka , int ) can be found elsewhere . briefly , the abundance ratio ( ri ) of the ligand - bound protein ( pli ) , bound to i molecules of l , to free protein ( p ) measured by esi - ms ( after correction for nonspecific ligand - protein binding ) is taken to be equal to the equilibrium concentration ratio in solution , eq 1:1 assuming the protein has h independent and identical binding sites , ka , int can be expressed by eq 2:2where [ p]0 and [ l]0 are the initial concentrations of the protein and ligand , respectively , and f is the fraction of occupied binding sites , eq 3:3 in the case of the p dimer , which has two equivalent binding sites , ka , int can be found using eq 4:4 the proxy protein esi - ms assay was used to quantify the affinities of gm3 trisaccharide for nov va387 p particle and vlp . a complete description of the data analysis method employed to calculate ka , int can be found elsewhere . briefly , a proxy protein ( pproxy ) , which binds to l with a known affinity , is used to monitor the extent of l binding to p. specifically , in the presence of p , the abundance ratio rproxy (= [ pproxyl]/[pproxy ] ) will quantitatively reflect the concentration of l bound to p and ka , int can be evaluated using eq 5:5where the initial concentrations of target protein ( [ p]0 ) , proxy protein ( [ pproxy]0 ) and ligand ( [ l]0 ) as well as the association constant for binding of pproxy to the ligand ( ka , pproxy ) are known ; [ p]m,0 is the initial concentration of binding sites in the target protein , i.e. , [ p]m,0 = h [ p]0 . paa - conjugated neu5ac , 6-sialylacnac and gm3 trisaccharide were dissolved in 1x pbs ( ph 7.4 ) . they were diluted and coated on a 96-well microtiter plate at concentration of 2 g ml and stored at 4 c overnight . after blocking with 5% nonfat dry milk , nov vlp , p particle , or gst - p domain fusion proteins as well as gst ( negative control ) at 50 ng l were added and incubated for 2 h at 37 c . the ligand - bound nov vlp and p proteins were detected by homemade guinea pig hyperimmune serum against va387 vlp and va115 p protein ( 1:3000 ) , respectively , followed by horseradish peroxidase ( hrp)-conjugated goat antiguinea pig immunoglobulin g ( igg , 1:3000 ; icn , aurora , oh ) . the signals were displayed using a tmb kit ( thermo fisher scientific , rockford , il ) . evidence of ganglioside binding to novs was initially revealed through the screening of a small ( 20 components ) carbohydrate library against the p particle ( 24-mer , mw 865,036 da ) of nov va387 ( gii.4 ) using the car - esi - ms assay . the library consisted of the oligosaccharides of 17 glycosphingolipids , gm1a , gm1b , gm2 , gm3 , gd1a , gd1b , gd2 , gd3 , gt1a , gt1c , gt2 , gt3 , fucosyl - gm1 ( referred to as fuc - gm1 ) , asialo gm1 , asialo gm2 , gb3 and gb4 as well as three known hbga oligosaccharide ligands , h type 3 trisaccharide ( referred to as h3 ) , a type 3 tetrasaccharide ( a3 ) , and b type 3 tetrasaccharide ( b3 ) . the intrinsic affinities of the hbga ligands range from 700 to 1500 m. the car - esi - ms assay was carried out by first incubating the p particle with the carbohydrate library , followed by direct esi - ms analysis of the mixture . because of the high mw of the p particle , the identity of the bound ligands could not be established directly from the mass spectrum . instead , using a quadrupole mass filter set to pass a range of mass - to - charge - ratio ( m / z ) ions , all of the ligand - bound p particle ions at a given charge state were isolated and then activated ( heated ) using cid to release the ligands ( as ions ) from the complex . given that carbohydrates have relatively low gas - phase acidities and are able to effectively compete with proteins for negative charge , accurate mass analysis , alone or in combination with ion mobility separation ( ims ) , which separates ions based on size and shape , allowed for positive ligand identification . shown in figure 1a is a representative esi mass spectrum acquired in negative ion mode for an aqueous ammonium acetate ( 200 mm , ph 7 , 25 c ) solution of p particle ( 3 m ) and the carbohydrate library ( 10 m each ) . from the mass spectrum it can be seen that the p particle exists predominately as a 24-mer , with a charge state distribution ranging from 60 to 65 . signal corresponding to an 18-mer is also present , although at lower abundance , with a charge state distribution of 51 to 54 . due to the high mw of the p particle and the formation of adducts during the esi process , it was impossible to resolve the ions corresponding to free p particle and its complexes with one or more oligosaccharide ligands . however , cid , performed using a 200 m / z wide isolation window centered at 14,350 to pass ions corresponding to the 61 charge state of the p particle , led to the appearance of singly deprotonated ions of the three hbga oligosaccharides as well as gm3 ( m / z 632.2 ) , gm2 ( m / z 835.3 ) , gd3 ( m / z 923.3 ) , gm1a and/or gm1b ( m / z 997.3 ) , and fuc - gm1 ( m / z 1143.4 ) ( figure 1b ) . ions corresponding to the singly deprotonated gd2 ( m / z 1126.4 ) and the doubly deprotonated ions of gd1a and/or gd1b ( m / z 644.1 ) were also detected , although at low abundance ( figure 1b ) . abundant multiply charged protein monomer ions , pm at n = 1023 , were also evident ( figure 1b ) . implementation of the car - esi - ms assay using other charge states of the p particle complexes produced similar results ( figure s2 , supporting information ) . ion mobility separation of the released ligands revealed evidence that both gm1a and gm1b are released from the p particle , with gm1a being more abundant ( figure s3a , supporting information ) . the doubly deprotonated ions of gd1a and gd1b could not be differentiated using optimized ims conditions ( figure s3b , supporting information ) , and therefore , it was not possible to establish whether one or both oligosaccharides bind to the p particle directly from these measurements . instead , the car - esi - ms assay was applied to solutions containing p particle ( 3 m ) and 10 m of gd1a or gd1b . these data revealed that only gd1a binds to the p particle under these solution conditions ( figure s4 , supporting information ) . the car - esi - ms results provide compelling evidence that the p particle of va387 exhibits a broad specificity for mono- and disialylated gangliosides . however , there is a clear preference for gm3 , and the addition of saccharides to gal ( e.g. , gm1 or gm2 ) or sia ( e.g. , gd3 , gd2 or gd1b ) decreases binding , compared to gm3 . these data , combined with affinities measured for ganglioside oligosaccharides , vide infra , suggest that the sia - gal - glc moiety represents the dominant recognition epitope for this nov . ( a ) esi mass spectrum acquired in negative ion mode for an aqueous ammonium acetate solution ( 200 mm , ph 7 and 25 c ) of nov va387 p particle ( 3 m ) and a 20-component ( 10 m each ) carbohydrate library consisting of the oligosaccharides of gm1a , gm1b , gm2 , gm3 , gd1a , gd1b , gd2 , gd3 , gt1a , gt1c , gt2 , gt3 , fuc - gm1 , asialo gm1 , asialo gm2 , gb3 , and gb4 as well as the h3 , b3 , and a3 oligosaccharides . ( b ) cid mass spectrum measured for the 61 charge state of the free and ligand - bound p particle . based on the relative abundances of the released oligosaccharide ligands measured by car - esi - ms ( figure 1 ) it would appear that the affinities of the ganglioside ligands are similar to those of the highest affinity hbga oligosaccharides . however , this conclusion is predicated on the assumption that the release efficiency of the bound - ligands is essentially independent of structure . because of the presence of the sialic acid , it is possible that gangliosides ( which are likely deprotonated in the gaseous complexes ) are preferentially released from the p particle due to a lower activation energy resulting from coulombic repulsion . therefore , it was important to measure the affinities directly . in order to do this , the affinities were measured using the direct esi - ms assay , which has been shown to provide reliable ka values for many protein carbohydrate interactions . affinities were measured for the oligosaccharides of 13 gangliosides ( gm3 , gm2 , gm1a , gm1b , gd3 , gd2 , gd1a , gd1b , gt3 , gt2 , gt1a , gt1c , and fuc - gm1 ) for the va387 p dimer ( mw 69,312 da ) . a reference protein ( pref ) was used in all cases to correct the mass spectra for the occurrence of nonspecific carbohydrate a representative esi mass spectrum acquired for an aqueous ammonium acetate solution ( 200 mm , ph 7 , 25 c ) of va387 p dimer ( 12 m ) and gm3 trisaccharide ( 80 m ) is shown in figure 2a as well as the distribution of ligand - bound p dimer after correction for nonspecific binding . from the esi - ms data , ka , int values were calculated for each oligosaccharide ( table 1 ) . affinities were also measured for a3 , b3 , and h3 and shown to agree well with the reported values ( table s1 , supporting information ) . inspection of the ka , int values reveals that , of the tested gangliosides , gm3 exhibits the highest affinity for the va387 p dimer , which is consistent with the results of the car esi - ms measurements , vide supra . moreover , the ka , int ( 1500 m ) is identical , within experimental error , to that of b3 ( 1500 150 m ) . of the 12 other gangliosides investigated , nine bind weakly ( ka , int < 500 m ) and three ( gd1b , gt3 and gt1a ) do not show any detectable binding . notably , the quantitative binding data obtained for the p dimer agree qualitatively with the relative affinities inferred from the car - esi - ms measurements performed on the p particle . moreover , all ligands with affinities > 100 m were detected in the car - esi - ms measurements ( table s2 , supporting information ) . ( a ) esi mass spectrum acquired in negative ion mode for aqueous ammonium acetate solution ( 200 mm , ph 7 and 25 c ) of nov va387 p dimer ( p2 , 12 m ) , gm3 trisaccharide ( 80 m ) and pref ( 4 m ) . another minor form of p dimer ( p2 , mw 74,080 da ) was also detected with lower abundance . inset , normalized distribution of gm3 bound to p2 after correction for nonspecific ligand binding . ( b ) esi mass spectrum acquired in negative ion mode for aqueous ammonium acetate solution ( 200 mm , ph 7 and 25 c ) of nov va115 p dimer ( p2 , 12 m , mw 67,712 da ) , gm3 trisaccharide ( 80 m ) , and pref ( 4 m ) . inset , normalized distribution of gm3 bound to p2 after correction for nonspecific ligand binding . nb = no binding detected . to demonstrate the relevance of the affinity data acquired for the p dimer , affinity measurements were also carried out for gm3 trisaccharide binding to the va387 p particle and vlp ( 180-mer , mw 10.5 mda ) . an adaptation of the proxy protein esi - ms method , which combines direct esi - ms binding measurements and competitive protein binding , was used to evaluate the affinities . a recombinant fragment of the c - terminus of human galectin-3 ( gal-3c , mw 16,330 da ) , which contains a carbohydrate recognition domain and interacts with a -galactoside moiety , served as the proxy protein ( pproxy ) . importantly , gal-3c binds to gm3 trisaccharide with an affinity of ( 1.20 0.02 ) 10 m. the extent of binding of gm3 trisaccharide to gal-3c , as determined by esi - ms , in the presence of known concentrations of the target protein ( p particle or vlp ) allowed for a quantitative measure of gm3 binding to the target . esi - ms measurements were performed on aqueous ammonium acetate solutions ( 160 mm , ph 7 and 25 c ) of pproxy ( 3.0 m ) , pref ( 1.0 m ) , gm3 trisaccharide ( 40 m ) , and either p particle , at concentrations ranging from 0 to 7.2 m ( corresponds to monomer concentration of 0172.8 m ) , or vlp , at concentrations ranging from 0 to 570 nm ( monomer concentration of 0102.6 m ) . representative esi mass spectra acquired in positive ion mode in the absence and presence of nov vlp ( 570 nm ) are shown in figure 3a and 3b , respectively . the distributions of ligand - bound pproxy , following correction for nonspecific ligand binding , are also given . inspection of the distributions reveals a measurable decrease in the extent of gm3 trisaccharide binding to gal-3c upon addition of vlp . the dependence of the extent of gm3 trisaccharide binding to pproxy on vlp concentration is shown in figure 3c . binding measurements performed on solutions containing p particle yielded qualitatively similar results ( figure s5 , supporting information ) . analysis of the pproxy binding data acquired in the presence of vlp or p particle using the procedure outlined in experimental section yields gm3 affinities of 2600 200 and 5500 600 m for the p particle and vlp , respectively . the slight differences in the magnitude of the affinities measured for the binding of a common carbohydrate ligand to the p dimer , p particle , and vlp of a nov ( the first such data set to be reported ) , likely reflect subtle differences in the structure of the carbohydrate binding site presented by these related protein complexes . these differences notwhithstanding , the present results suggest that the p dimer can serve as a surrogate of the vlp for carbohydrate binding studies . representative esi mass spectra measured in positive ion mode for aqueous ammonium acetate solutions ( 160 mm , ph 7 and 25 c ) of pproxy ( gal-3c , 3.0 m ) , pref ( ubq , 1.0 m ) , and gm3 trisaccharide ( 40 m ) without ( a ) or with ( b ) nov va387 vlp ( 570 nm , 180-mer ) . insets show the fraction of free and gm3-bound pproxy , after correction for nonspecific ligand binding . ( c ) plot of the abundance ratio of gm3-bound pproxy to free pproxy ( rproxy ) versus vlp concentration . the solution conditions for each measurement were the same as in ( a ) , but with the addition of vlp . it has been proposed that nov va387 has a binding interface that recognizes hbgas through the -l - fuc epitope as the major binding interaction and either the -d - galnac or -d - gal epitope as a minor binding interaction . however , these core recognition elements are missing in the ganglioside ligands identified in the present study . therefore , it is of interest to establish whether the ganglioside ligands interact with the nov through the hbga binding site or through a distinct ganglioside binding site . it is not possible to answer this question through competitive binding measurements carried out using a ganglioside oligosaccharide ( e.g. , gm3 trisaccharide ) and va387 p dimer in the presence of varying concentrations of a hbga oligosaccharide ligand due to the low affinities of these ligands . instead , future efforts will rely on x - ray crystallography to establish whether va387 nov has distinct binding sites for hbga and ganglioside ligands . the aforementioned binding data reveal that nov va387 binds to mono- and disialylated gangliosides , with affinities comparable to those of the highest affinity hbga oligosaccharide ligands . to demonstrate that this is not an isolated example of a human nov that recognizes gangliosides , the affinities of the 13 ganglioside oligosaccharides for the p dimer of nov va115 ( gi.3 genotype ) , which does not bind to human hbgas , were also measured ( table 1 ) . a representative esi mass spectrum acquired for an aqueous ammonium acetate solution ( 200 mm , ph 7 , 25 c ) of nov va115 p dimer ( 12 m ) and gm3 trisaccharide ( 80 m ) is shown in figure 2b as well as the distribution of ligand - bound p dimer after correction for nonspecific binding . notably , the va115 p dimer binds to all 13 oligosaccharides tested , and overall , the affinities are slightly higher than those for va387 . additional evidence for the recognition of sialic acid by human novs comes from elisa measurements carried out on the va387 vlp , p particle , and gst - p fusion protein as well as va115 gst - p fusion protein , with paa - conjugated neu5ac , 6-sialylacnac , and gm3 trisaccharide . as shown in figure 4 , it is curious that the va387 vlp exhibited weaker binding than that of the p particle to the three glycoconjugates and the cause of the weaker binding is , at this time , unknown . nevertheless , the fact that all three assemblies of nov capsid protein exhibit a similar binding pattern to the three glycoconjugates ( gm3 > 6-sialylacnac > neu5ac ) validate their applications as models for nov ligand interaction . moreover , comparing the binding of gst - p fusion protein of va387 to that of va115 indicates that the sialic acid - containing glycoconjugates have slightly higher affinities for va115 , consistent with the esi - ms data . these results , together with those from esi - ms , suggest both -(2,3)- and -(2,6)- linked sialic acids as critical motifs in va387 and va115 binding , similar to what has been reported for mnv1 and psav . it is important to point out that , although sialic acid - containing oligosaccharides have been identified as receptors for an animal nov ( mnv1 ) and two other animal cvs ( fcv and psav ) , human novs generally recognize gangliosides in addition to hbgas . furthermore , human novs differ greatly from mnvs in many other important aspects , including host tropism ( human vs mouse ) , clinical manifestation ( with vs without diarrhea / vomiting ) , and pathogenesis . binding of nov vlp , p particles , and gst - p fusion protein of va387 as well as gst - p fusion protein of va115 to paa - conjugated gm3 trisaccharide , 6-sialylacnac , and neu5ac in 1x pbs ( ph 7.4 ) . gst , which does not show binding to any of the three glycoconjugates , served as a negative control . taken together , the results of esi - ms and elisa measurements performed on two human novs representing two different genogroups ( gi and gii ) provide the first experimental evidence of interactions between human novs and gangliosides and sialic acid - containing glycoconjugates . notably , the affinities measured for the oligosaccharides of the ganglioside ligands by esi - ms are comparable in magnitude to those reported for the oligosaccharides of known hbga receptors . these experimental data demonstrate sialic acid - containing oligosaccharides as alternative ( to hbgas ) ligands for human novs and suggest a new mechanism of human nov host interaction , one that involves hbga and sialic acid - containing oligosaccharide receptors and co - receptors for attachment and penetration into host cells and opens a new direction in human nov research . further studies to characterize the role of cell surface sialic acids / gangliosides in the early stage of viral infection and its potential coordination with hbgas for viral attachment and/or entry are needed .

human noroviruses ( novs ) are known to recognize histo - blood group antigens ( hbgas ) as attachment factors . we report the first experimental evidence that sialic acid - containing glycosphingolipids ( gangliosides ) are also ligands for human novs . electrospray ionization mass spectrometry - based carbohydrate binding measurements performed on assemblies ( p dimer , p particle , and virus - like particle ) of recombinant viral capsid proteins of two nov strains , va387 ( gii.4 ) and va115 ( gi.3 ) , identified binding to the oligosaccharides of mono- , di- , and trisialylated gangliosides . the intrinsic ( per binding site ) affinities measured for these ligands are similar in magnitude ( 102103 m1 ) to those of human hbgas . binding of nov vlps , p particles , and glutathione s - transferase ( gst)-p domain fusion proteins to sialic acid - containing glycoconjugates , observed in enzyme - linked immunosorbent assays , provided additional confirmation of the nov ganglioside interactions .

hypertension is one of the most common if not the commonest medical complication in pregnancy.19 hyper - tensive disorders in pregnancy are found to be the greatest single cause of maternal mortality.911 it also causes a lot of prenatal mortality.12 most of the complications caused by this problem could be reduced by early detection and proper management.1314 studies on the incidence of hypertensive disease in pregnancy in most developing countries , including saudi arabia , are scarce.15 few of those studies , if any , have looked at the extent of knowledge , attitude or practice of doctors or nurses dealing with hypertensive pregnant mothers . hence , the objective of this study was to determine the status of knowledge , attitude and practice ( kap ) of doctors and nurses in phc centers with regard to hypertension in pregnancy and to analyze factors affecting kap in the al - khobar area of the eastern province of the kingdom of saudi arabia . terms used to describe hypertension and its complications in pregnancy differ according to its presentation , gestational age of discovery and the presence of previous history of hypertension before pregnancy . the one defined by the 1972 committee on terminology of the american obstetricians and gynecologists ( acog)131519 was adopted for the purpose of this kap study . doctors and female nurses working at all 8 phc centers in al - khobar area were enrolled for the study . two self - administered questionnaires structured to examine the essential knowledge , attitude , and practice of doctors and nurses in dealing with hypertensive disorders during pregnancy . the first was intended to gather demographic characteristics and information on the respondent that might affect the kap . these variables included age , sex , nationality , year of graduation , certification , training and experience in obstetrics , duration of work in phc in the country and any in - service training received . the second part of the questionnaire consisted of 49 questions divided into three sections dealing with practice , attitude and knowledge . the practice sections included questions designed to test competence of doctors and nurses in the skills of measuring blood pressure , management of the hypertensive pregnant mother and practices of health education . the knowledge section was composed of 28 questions to test basic medical knowledge regarding blood pressure reading , management of the problem and its complications . both questionnaires were given to three consultant obstetricians and two nurse educators for consent validity and elimination of non - essential questions . both knowledge and practice areas were scored on a zero and one additive scoring system in which each correct answer or practice was given one score and no mark given for a wrong response . the attitude part included questions that measured attitudes of participants towards the seriousness and commonness of hypertension in pregnancy and their motivation to improve their knowledge on the subject . attitude questions were scored using the five - point likert scaling system . in this scale , a high attitude was assigned to the answer if the respondent 's answer was to agree or strongly agree to the question in the scale and a low attitude level for not sure or final scoring of the knowledge and practice section was satisfactory if the participant scored at least 60% of the total marks in these two sections . this was an arbitrary cut - off point based on the pilot study and judgment of the experts . on attitude , the questionnaire was handed over to five postgraduate doctors in general practice and their responses to attitude questions were used as a reference standard . the questionnaire for nurses both versions of the questionnaire were pilot tested on a pilot group of 10 doctors and 22 nurses , selected randomly from phc centers in a nearby city , al - dammam to assess level of difficulty , clarity , suitability and time required for their completion . data were fed into a personal computer , cleaned and analyzed using epi info and spss statistical packages . frequency distribution tables were constructed and appropriate tests including the multiple regression analysis to identify significant independent factors were applied . the total number of phc doctors in al - khobar area was 44 . among those 36 ( 81.4% ) who were present at time of data collection their ages ranged between 25 - 47 with mean of 37.1 years ( sd 6.4 ) and 56% were in the 35 - 44 years group . arab nationals other than saudis including egyptians , palestinian , and jordanian formed the majority of doctors ( 41.7% ) , while saudis constituted 22.2% of the group and the rest were from the indian subcontinent . the mean number of years in practice after graduation was 12.6 years ( sd 7.38 ) . distribution of phc doctors according to years of practice after graduation the mean number of years working in phc was 4.7 years ( sd 3.4 ) and 17 doctors ( 47.2% ) had worked for over 5 years in phc centers . only , 4 doctors ( 11.1% ) had postgraduate qualifications ; out of these only one held a diploma in obstetrics . five doctors ( 13.9% ) had had 3 - 6 months post - internship training in obstetrics but no certification . of the entire group , only 2 doctors ( 5.6% ) had had courses in hypertensive disorders in pregnancy in the course of their work . in describing their general attitude towards managing hypertension in pregnancy , a majority of doctors ( 80% ) stated clearly that they did not feel confident in managing hypertensive pregnant mothers and would prefer to see children or adult male patients in their clinic . nevertheless , the attitude of 34 doctors ( 94.4% ) toward learning more about hypertension in pregnancy was positive . out of the maximum attitude score of 45 marks , the mean score for the attitude questions for the group reached a reasonably high figure of 30.69 marks . doctors attitudes towards hypertension in pregnancy doctors management of hypertension in pregnancy was quite deficient as 16 doctors ( 44.4% ) reported that they had not actually seen any hypertensive pregnant mother during last year . around 53% of the doctors depended on bp measurement taken by a competent staff and did not check it . the doctors scores for practice was generally low , as only 60% of them got the correct answers to the skill of blood pressure measurement , a basic skill necessary for any medical graduate . in addition , almost all doctors wrongly reported that they had advised their pregnant hypertensive patients to lose weight and reduce their salt intake . another major mistake noted among 25% of the doctors was that they said they would start treatment of hypertensive cases immediately after diagnosis in their clinic without considering referral . the scores on practice of 75% of the doctors were below the satisfactory cut - of point . again the doctors score of 58.3% on all areas of knowledge was unsatisfactory since this directly affected the diagnosis and quality of care for hypertensive mothers . some of the responses dealt with normal and abnormal readings of blood pressure and the presenting symptoms of preeclampsia . doctors mean score 12.1 marks out of a total score of 21 on knowledge was moderate . the total number of phc nurses in al - khobar area was 120 . among these , the 91 ( 75.8% ) who were present at the time of data collection answered the questionnaire . their mean age was 29.8 years ( sd 6.3 ) ranging between 20 - 44 years , 74% of them fell within the 20 - 34 age group . indians formed the second largest group ( 37.4% ) followed by filipinos ( 11% ) . according to their qualification , 14 nurses ( 15.4% ) held bachelor degrees and 77 ( 84.6% ) had diplomas . mean years of practice after registration was 8.7 ( sd 5.4 ) and their distribution is shown in table 3 . mean duration of work in phc was 4.1 years ( sd 2.8 ) and most nurses ( 69.2% ) had spent < 5 years working in phc in the country . distribution of phc nurses according to years of practice after registration thirty - seven nurses ( 40.7% ) had spent < 6 months in obstetrics training and 10 nurses ( 11% ) had spent > 1 year . most of the group ( 48.3% ) , mainly the saudis , had minimal training of 3 weeks in obstetrics ward during their internship period as required by the saudi female health institution . a majority of the nurses 73 ( 80.2% ) had not worked in a phc setting before their current positions . out of those who had had previous phc experience , nine of them had had courses in hypertensive disorders in pregnancy during their work in phc . in describing their general attitude towards hypertension in pregnancy , 86% of them reported that it was a common health problem and they were all positive about learning more . they were all keen to talk to pregnant women about their problem and advice them to take bed rest . out of the maximum attitude score of 35 marks , the highest mean score was 30.02 marks . table 4 shows some of the attitude questions and their responses . nurses attitude towards hypertension in pregnancy the nurses were quite good on practice in the area of hypertension in pregnancy , as 95.6% of them scored satisfactory . the mean score of this section on practice was quite high for nurses being 8.3 marks out of a total score of 12 marks . the nurses level of knowledge was very low since only five of them ( 5.5% ) scored satisfactory on the questions in this section . the deficiencies were in areas dealing with diagnosis and quality of care for hypertensive mothers . most of them ( 96.7% ) wrongly considered salt restriction and weight reduction as important in the health education of hypertensive pregnant women . mean scores on knowledge was 9.02 marks out of a total score of 20 marks which was quite low . in the regression analysis of independent factors associated with kap of doctors and nurses , all variables in the first part of the questionnaire such as age , nationality and certification were included in a series of multiple regression equations against each of the dependent variables , namely , scores on knowledge , attitude , and practice for doctors and nurses . there was a positive significant association of the doctors attitude with their sex i.e. , male , nationality , being older and previous enrollment in a course on hypertension ( r square = 0.26 p=0.02 ) . their knowledge was only associated with previous training in obstetrics ( r square = 0.14 , p=0.02 ) , while practices of both doctors and nurses were not associated with any of the factors under study . the attitude of nurses , on the other hand , showed different associations as non - saudi nationals and training in obstetrics had a positive significant effect on their attitude ( r square = 0.08 , p=0.02 ) . their knowledge was only associated significantly with being non - saudi ( r square = 0.19 , p=0.001 ) . hypertension in pregnancy is one of the major causes of prenatal morality and morbidity.20 it is responsible for about 18% of maternal mortality,102122 maternal mortality in hypertensive disorders of pregnancy is primarily due to low standard of care and delay in referral.14 antenatal care ( anc ) is a major part of maternal and child services in phc . one of the most important functions of anc is to detect high - risk pregnancies and to give them the necessary care . findings from this study will help to identify the status kap of workers in phc on hypertensive disorders in pregnancy . it was discovered in this study that the definition of raised blood pressure and its management was not standardized among doctors and nurses in phc . this is not surprising as previous study by bisson in bristol23 in which a large group of general practitioners , hospital doctors , hospital midwives , community midwives and student midwives who were questioned , gave variety of action plans according to their understanding of diagnostic criteria . they considered the reading of 90 mmhg the model value of diastolic bp at which further action would be taken , whether proteinuria was present or absent . edema was considered a useful indicator by 93% of the respondents and 49% would use ankle edema in their assessment . another questionnaire - based survey by hutton24 on the management of hypertension in pregnancy completed by 65 new zealand obstetricians found that 40 ( 61.5% ) doctors considered the diastolic of 80 - 85 mmhg , the lowest abnormal reading , at 28 weeks , and by 18 ( 27.7% ) doctors at 36-week gestation . however , 20 ( 30.8% ) doctors considered the diastolic of 90 mmhg , at 28 weeks and 42 ( 64.6% ) doctors at 36 weeks gestation , the lowest abnormal reading . around 47% of phc doctors had spent more than 5 years in phc service in ksa and only 14% of them had had post - internship training in obstetrics but being males had not been involved in anc activity in phc . only 2 had had courses in hypertensive disorders of pregnancy . about 44% of the doctors had not seen any cases of hypertensive pregnant mothers during the last year . questions asked were concerned with four aspects namely , techniques of blood pressure measurements , history and physical examination , health education practice and action management to be taken by the phc doctors on discovered cases . it is obvious that having such little contact with hypertensive cases and lacking the basic training or refresher courses , their management skills will be inadequate . since hypertension is one of the commonest medical complications in pregnancy , it is important to educate the pregnant mother about it . almost all doctors report that they advise hypertensive mothers on the need for bed rest . this is an important non - pharmacological measure in the management of the problem.25 on the other hand , almost all doctors report that they advise their patients to restrict their salt intake and to go on a weight - reducing diet . salt restriction in hypertensive non - pregnant women can be effective but in pregnancy may aggravate the condition . moreover , diet restriction in pregnancy can lead to delivery of small - for - date fetus.25 similar findings of wrong advice were reported in other studies by trudinger26 and bisson.23 around 85% of phc doctors reported that they would not refer hypertensive cases after diagnosis and 25% of them would start medication immediately after diagnosis . in fact , the cornerstone of management of hypertensive cases starts in the phc center with accurate diagnosis and undelayed referral of these cases from the phc to the specialist or hospital.1621 phc doctors should be aware of their limitation in the management of these cases and not to jeopardize the health of the mother and her fetus . around 31% did not recognize this is a common health problem in pregnancy , while 97% of them stressed the importance of taking blood pressure reading at each anc visit . that 53% of phc doctors depended on blood pressure measurement taken by clinical staff without confirming it , the reading themselves is an alarming negative attitude as well as wrong practice . the diagnosis of hypertension in general or in pregnancy in any patient is not an easy task and proper management and modifications depend on it . it is therefore , important that the person responsible for the management , namely , the doctor , should confirm the reading again . in looking for factors affecting the attitude of doctors , it was found that gender i.e. , male , arab , older and previous training in a course on hypertension were factors associated with higher scores . longer experience in phc work , being male and older and the absence of a language barrier for arabic speakers , including saudis , might explain to some extent their better attitude scores as compared to those of the females and younger doctors . the knowledge of about 58% of doctors on questions that bare on the diagnosis and quality of care of hypertensive mothers as well as nurses provide most of the vital anc services in the phc . around 53% of the phc nurses were non - saudis and non - arabic speakers and the language barrier adversely affects the health education in hypertensive cases . it is obvious from the results that there is a shortage of specialized courses for nurses . none of the saudi nurses had more than a diploma in nursing and there did nt seem to be any arrangements for their further movement at universities . a period of 3 weeks in an obstetrics ward for new graduate nurses as part of an internship period is not enough to give clear , pragmatic information on anc services in a phc setting . about 31% of the nurses have spent > 5 years in the phc centers in the ksa , but only 10% had taken courses in hypertension . this clearly , demonstrates the necessity of a better arrangement for on - job training classes for them . their responses to the question of history and physical examinations were 87% correct and about 90% of them responded correctly to the questions on health education . their erroneous responses were on the items on restriction on salt intake and weight reduction . these good scores indicate that except for their wrong ideas on salt and diet restriction , which should be corrected at refresher courses , the correct procedure have been learnt . although the phc nurses were very good with practice in general , around 95% of them had poor knowledge . knowledge and facts , on the other hand , need to be updated by continuous education . around 86% of them felt that it was a common health problem and reported that they needed to know more about it . the importance of a health education as a vital task for nurses can not be ignored . the presence of these cases in the clinic , therefore , provides a good opportunity for the performance of this task . there was a strong positive association of attitude of nurses to non - saudi nationality and training in obstetrics . while all saudi nurses hold diplomas , about 30% of the non - saudi nurses have bachelors degrees . there is a masked difference in the type , duration and content of curriculum at the pre - graduate level for both groups and non - saudi nurses had higher knowledge scores than saudis . the implication of these findings clearly points out the need to improve the knowledge and attitude of phc nurses through refresher courses . good in attitude questions but had low scores on the practice and knowledge component of the questionnaire . the nurses scored high on practice and attitude but had low scores on the knowledge component . it is recommended that appropriate regular refresher courses on common and serious problems like hypertension be organized for doctors and nurses in the phcs . there should be opportunities for effective training of reasonable duration with clearly defined objectives under proper supervision in good hospitals to improve their knowledge and practice . it would be also appropriate to offer saudi doctors extra incentives for postgraduate study in family medicine to deal with these common problems , and to institute a suitable program of continuing medical education within the health centers for both doctors and nurses . it is also vital to review the curriculum of the female nursing institutions to update both its theoretical and practical content , and extend the duration of training in such common problems as hypertension in obstetrics .

objective : to assess the status of knowledge , attitude and practice ( kap ) of doctors and nurses in primary health care ( phc ) centers with regard to hypertension in pregnancy and to identify factors associated with kap in al - khobar , saudi arabia.methodology:using a self - administered comprehensive questionnaire , all available doctors and nurses in phc centers of the al - khobar area were approached to determine their knowledge , attitude and practice in hypertension during pregnancy . questionnaires were validated and pilot tested . each section of the questionnaire was scored and the mean scores calculated . factors affecting each section were identified by means of multiple regression analysis.results:a total of 36 doctors and 91 nurses were enrolled in the study . saudis formed 22.2% of the doctors and 47.3% of nurses . mean years of practice after graduation were 12.6 and 8.7 years for doctors and nurses , respectively . saudi nurses spend only 3 weeks in the obstetrics ward during the whole period of their internship . all saudi nurses hold only diplomas and not many courses on the hypertensive disorder are offered to both doctors and nurses after graduation . the practice of doctors particularly in the management of patients after reaching a diagnosis and educating them on diet and salt intake was poor . furthermore , their knowledge was also poor . though their level of knowledge was poor , the nurses practice was satisfactory , particularly in taking history and physical examination . the attitude of both doctors and nurses towards hypertensive disorders was in general , positive and satisfactory towards health education . nurses nationality and duration of post - internship training were the factors that influenced their attitude and scores on knowledge.conclusion and recommendation : the study revealed that both doctors and nurses working in the phc lacked training and knowledge in this area of their work . it is therefore necessary to give phc doctors and nurses refresher courses on common and serious problems like hypertension . a longer period of training in action management is needed to improve the knowledge and practice of doctors and nurses working in antenatal clinics in this area .

renal cell carcinoma ( rcc ) is the most common primary malignant neoplasm of the kidney and accounts for about 3% of all adult neoplasms . the incidence of rcc has been increasing during the past two decades in all age groups . the greatest increase has been in patients with localized tumors as a result of incidental detection , which is thought to be the result of more widespread use of abdominal ultrasound and computed tomography . however , about 30% of patients with rcc already have metastatic disease at the time of diagnosis , and 10% to 14% of patients experience local recurrence or distant metastasis after nephrectomy despite a finding of pathologically confined disease at the time of nephrectomy [ 3 - 5 ] . well - known pathological and clinical variables have been identified and sometimes integrated into nomograms to better predict disease recurrence , but with only partial success . with a better understanding of the biology of rcc , laboratory values and molecular markers are increasingly being investigated as potential predictive factors for rcc [ 6 - 8 ] . disease progression appears to depend on a complex interaction between the tumor and host inflammatory responses . it is estimated that underlying infection and inflammatory responses are linked to 15% to 20% of all deaths from cancer worldwide . the serum level of c - reactive protein ( crp ) , a nonspecific inflammatory acute - phase protein , is frequently increased in patients with metastatic rcc and is a predictor of prognosis . however , secondary thrombocytosis has been reported with many malignant diseases ; the survival of such patients has been reported to be significantly shorter . the prognostic value of the platelet count has been studied in patients with localized and metastatic rcc ; patients with thrombocytosis had a poorer survival than did patients with a normal platelet count . the purpose of this study was to investigate the association of preoperative crp elevation and thrombocytosis with the prognosis of patients with non - metastatic rcc . the medical records of 177 patients with non - metastatic rcc who underwent a radical nephrectomy between march 2000 and may 2008 and for whom preoperative crp and platelet levels were available for analysis were retrospectively reviewed . there were 130 men and 43 women , with a mean age of 53.5 years ( range , 28 - 83 years ) . preoperatively , all patients were evaluated with a physical examination , routine hematology and biochemical analysis , and radiology studies , including abdominal computed tomography and chest x - ray . the tumors were staged by using the 2002 tnm classification of the american joint committee on cancer ( ajcc ) and were graded according to fuhrman 's nuclear grading system . all patients were evaluated postoperatively every 3 months for the first 2 years , every 6 months for the next 2 years , and yearly thereafter . crp elevation was defined as a crp level 0.8 mg / dl , and thrombocytosis was defined as a platelet count 380,000/l in men and 370,000/l in women according to the normal reference range in our hospital . disease recurrence was defined as local failure in the tumor bed or regional lymph nodes or distant metastasis . the chi - square test or fisher 's exact test was used to analyze the correlation between preoperative crp elevation or thrombocytosis and the clinical and pathological variables , including age , gender , anemia , histology subtype , tumor size , t stage , nuclear grade , and metastasis during follow - up . univariate and multivariate analyses were performed by using the log - rank test or the cox proportional hazards regression model . for all tests , the clinical and pathological characteristics of the 177 patients with non - metastatic rcc are summarized in table 1 . the mean tumor size was 5.12 cm ( median , 4.50 ; range , 1 - 22 cm ) . regional lymph node dissection was performed in 24 patients who showed suspicious lymph node metastasis on computed tomography . however , none had a pathological diagnosis of lymph node metastasis . there were 38 patients ( 21.5% ) with crp elevations and 11 patients ( 6.2% ) with thrombocytosis . among patients with crp elevation , the mean follow - up period was 48.3 months ( median , 48.0 ; range , 13 - 111 months ) . twenty - three patients ( 13.0% ) developed metastases and six patients died during the follow - up period . preoperative crp elevation was significantly correlated with anemia ( p=0.001 ) , t stage ( p=0.004 ) , grade ( p=0.025 ) , and metastasis ( p<0.001 ) , but not with age , gender , tumor histology subtype , or tumor size ( table 2 ) . preoperative thrombocytosis was significantly correlated with anemia ( p=0.003 ) , t stage ( p=0.002 ) , and metastasis ( p=0.001 ) , but not with age , gender , tumor histology subtype , tumor size , or grade ( table 3 ) . no significant correlation was found between preoperative crp levels and platelet counts when analyzed by using pearson 's correlation coefficient ( r=0.132 , p=0.079 ) . kaplan - meier recurrence - free survival curves according to crp level showed that the survival rate of patients with crp elevation ( crp0.8 mg / dl ) was significantly lower than that of patients with normal crp levels ( p<0.001 ) ( fig . the univariate analysis identified anemia , crp elevation , thrombocytosis , tumor histology subtype , tumor size , t stage , and grade as significant prognostic factors for recurrence - free survival , whereas the multivariate analysis showed that crp elevation ( p=0.033 ) and tumor size ( p=0.007 ) were independent prognostic factors ( table 4 ) . crp is an acute phase reactant that is produced exclusively by hepatocytes in response to cytokines such as interleukin ( il)-6 , which are known mediators of ongoing inflammatory processes . an elevation of the serum concentration of this protein indicates the presence of acute inflammation , which is observed 8 to 12 hours after the onset of infection . the relationship between cancer and crp elevation hepatic production of crp is induced by cytokines such as il-1 , tumor necrosis factor ( tnf ) , and primarily il-6 , which is frequently overproduced by the tumor cells themselves . experimental studies on rcc cell lines and expression studies of renal surgical specimens have shown that at least some renal tumors produce il-6 , which functions as an autocrine growth factor of the rcc . therefore , the presence of a systemic inflammatory response might be associated with aggressive rcc behavior . the prognostic value of crp levels has been reported in many recent studies of cancers , including those of the esophagus , ovaries , and colon [ 21 - 23 ] . however , the mechanism by which a systemic inflammatory response affects cancer - specific survival in patients with rcc is unclear . hwang et al studied the relationship between crp and survival in patients with rcc who underwent a radical nephrectomy . they reported a statistically insignificant difference in the survival rates of patients with and without crp elevation and concluded that crp was not a significant prognostic factor for rcc when compared with tumor stage , grade , tumor size , and cell type . however , komai et al demonstrated that the 5-year and 10-year disease - specific survival rates ( 75% and 30% , respectively ) in patients with high crp levels were significantly worse than the rates in patients with normal crp levels ( both 93% , p<0.001 ) and suggested that the preoperative crp level is associated with poor survival in patients with localized rcc . in addition , they reported that the crp level is a useful preoperative screening tool in patients with localized rcc . blay et al reported that pretreatment il-6 and crp were significantly associated with the effects of cytokine therapy and high il-6 and that the crp level indicated a poor response to cytokine therapy . in the present study , preoperative crp elevation was associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc . this result confirms the importance of the preoperative crp level as an independent prognostic factor for tumor recurrence and is consistent with the findings of earlier reports showing that a high crp level is associated with poor survival in localized rcc . secondary or reactive thrombocytosis has been associated with tumor metastasis and poor survival in cancer patients . the pathophysiologic mechanism of reactive thrombocytosis may be tumor - associated elevation of circulating platelets by tumor - derived humoral factors such as il-6 and macrophage colony - stimulating factor ( m - csf ) that may be responsible for the development of cancer - related thrombocytosis . il-6 is a potent stimulator of megakaryocytopoiesis , and tumor cells have been shown to release il-6 both in vitro and in vivo . these observations suggest that il-6 plays a role in the development of tumor - associated thrombocytosis . karakiewicz et al analyzed 1828 patients with rcc by univariable , multivariable , and predictive accuracy analyses with regard to rcc - specific mortality . in that study , they showed that the addition of thrombocytosis to the base model ( age , tumor size , tnm stage , ecog - performance status , fuhrman grade , and histology subtype ) increased the predictive accuracy by only 0.3% ( from 85.3% to 85.6% ) ; these changes in predictive accuracy were not statistically significant . consequently , they concluded that patients who presented with thrombocytosis did not have poorer prognosis than did their counterparts who did not exhibit these apparently unfavorable characteristics , as long as the effects of the tnm stage , histology , tumor grade , and ecog - performance status were considered . however , many investigators have reported that thrombocytosis is related to a poor prognosis in patients with rcc . , they showed that patients with thrombocytosis had a mean survival of 45.2 months compared with 76.6 months in patients without thrombocytosis ( p=0.0002 ) and concluded that preoperative thrombocytosis is associated with a poorer survival than is a normal platelet count . in the present study , we showed that preoperative thrombocytosis was significantly correlated with anemia , t stage , and metastasis . in addition , thrombocytosis was a significant prognostic factor associated with recurrence - free survival in the univariate analysis . therefore , thrombocytosis was associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc ; these findings are consistent with those of previous studies . ito et al showed that preoperative crp levels and platelet counts had a significant correlation and suggested that this was because the reactive thrombocytosis and crp elevation in rcc were both caused by the production of inflammatory cytokines such as il-6 . as in the study by ito et al , we analyzed the relationship between preoperative crp and platelet count . however , we did not find a statistically significant association between preoperative crp and platelet count ( r=0.132 , p=0.079 ) . therefore , the exact relationship between preoperative crp and platelet count remains to be determined . a limitation of this study is that several variables could not be considered in the current analysis . nonetheless , all available variables were included , and our findings indicate that several known prognostic factors were valuable as prognostic factors the reason for this result was , to some extent , too many patients in stage t1 rather than other t stages and a small number of patients with grade 4 compared with other grades according to the early detection of rcc nowadays . preoperative crp elevation and thrombocytosis were associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc . moreover , however , preoperative thrombocytosis was not an independent prognostic factor for tumor recurrence and prognosis .

purposethe aim of this study was to investigate the association of preoperative c - reactive protein ( crp ) elevation and thrombocytosis with the prognosis of patients with non - metastatic renal cell carcinoma ( rcc).materials and methodsthis was a retrospective review of the medical records of 177 patients ( 130 men and 47 women ) with non - metastatic rcc who underwent a radical nephrectomy between march 2000 and may 2008 and for whom preoperative crp and platelet data were available for analysis . preoperative crp elevation and thrombocytosis were compared with clinical and pathological variables.resultsthere were 38 patients with crp elevation and 11 patients with thrombocytosis . the mean follow - up time was 48.3 months ( median , 48.0 ; range , 13 - 111 months ) . twenty - three patients ( 13.0% ) developed metastases and six patients died during the follow - up period . crp elevation was significantly correlated with anemia ( p=0.001 ) , t stage ( p=0.004 ) , grade ( p=0.025 ) , and metastasis ( p<0.001 ) . thrombocytosis was significantly correlated with anemia ( p=0.003 ) , t stage ( p=0.002 ) , and metastasis ( p=0.001 ) . the univariate analysis identified anemia , crp elevation , thrombocytosis , tumor histology subtype , tumor size , t stage , and grade as significant prognostic factors associated with recurrence - free survival , whereas the multivariate analyses showed that crp elevation ( p=0.033 ) and tumor size ( p=0.007 ) were independent prognostic factors.conclusionspreoperative crp elevation and thrombocytosis were associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc . moreover , preoperative crp elevation appeared to be an independent predictor of tumor recurrence and prognosis . preoperative thrombocytosis , however , was not an independent prognostic factor for tumor recurrence and prognosis .

angiogenesis is a constant hallmark of multiple myeloma ( mm ) progression and has prognostic potential . we have previously isolated endothelial cells ( ec ) from bone marrow of patients with mm ( mmec ) . they show intrinsic angiogenic ability , because they rapidly form a capillary network in vitro , and extrinsic ability , because they generate numerous new vessels in vivo in the chick embryo chorioallantoic membrane ( cam ) . moreover , mmec secrete angiogenic cytokines , such as fibroblast growth factor-2 ( fgf-2 ) and vascular endothelial growth factor ( vegf ) , as well as matrix metalloproteinase-2 and 9 ( mmp-2 and mmp-9 ) and express adhesion molecules facilitating dissemination of these cells . studies by rajkumar et al . , dominici et al . , and laroche et al . the bone marrow microenviromnent plays a crucial role in inducing the angiogenic response in mm [ 6 , 7 ] . reciprocal positive and negative interactions between plasma cells and bone marrow stromal cells , namely haematopoietic stem cells , fibroblasts , ostoblasts / osteoclasts , ec , ec progenitor cell , t lymphocytes , macrophages and mast cells are mediated by an array of cytokines , receptors and adhesion molecules . these cells secrete angiogenic factors , such as interleukin-6 ( il-6 ) , vegf , fgf-2 , hepato - cyte growth factor / scatter factor ( hgf / sf ) , transforming growth factor- ( tgf- ) and tumour necrosis factor ( tnf- ) [ 6 , 7 ] . here , we have attempted a fine characterization of the angiogenic response induced by mmec , as compared with monoclonal gammopathy of undetermined significance ec ( mgusec ) in the cam assay . we investigated the angiogenic response induced by gelatin sponges soaked with the cell suspensions and implantedonthecamsurfacefrom8today12ofincu - bation and we studied the effects of mmec and mgusec on the expression of endogenous levels in the cam of vegf , fgf-2 , angiopoietin-1(ang-1 ) , hypoxia - inducible factor-1 ( hif-1 ) and endostatin by reverse transcriptase - polymerase chain reaction ( rt - pcr ) . mmec were obtained from 10 patients with mm and mgsuec were obtained from 10 patients with mgus , as previously described . fertilized white leghorn chicken eggs were incubated at 37c at constant humidity . on day 3 of incubation a square window was opened in the egg shell after removal of 23 ml of albumen so as to detach the developing cam from the shell . the window was sealed with a glass and the eggs were returned to the incubator . gelatin sponges ( gelfoam , upjohn company , kalamazoo , mi , usa ) were cut to a size of 1 mm3 and placed on top of a growing cam at day 8 incubation under sterile conditions . the sponges were then adsorbed with 2 l of cell suspension ( 18,000 cells per sponge ) of mmec or mgusec or with rpmi-1640 medium alone or supplemented with fgf-2 ( 200 g / ml ) ( r & d systems , abington , uk ) , used as negative and positive control , respectively . the angiogenic response was evaluated 96 hrs after the implants by means of a stereomicroscope connected to an image analyser system ( olympus italia , italy ) . blood vessels entering the sponges within the focal plane of the cam were counted by two observers in a double blind fashion at a magnification of 50x . means 1 standard deviation ( sd ) were evaluated for all the parameters and the statistical significance of the differences between the counts was determined by student 's t - test for unpaired data . total rna was extracted from 12 cam treated with ec obtained from six patients with mgus and six patients with mm and from six control cam using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufactures 's protocol . the concentration and the purity of the isolated rna were determined using a spettrofotometric analysis and the integrity of the rna was verified by means of electroforesis in a formaldehyde agarose gel followed by ethidium bromide staining . the cdna was then synthetized with 5 g of total rna using m - mlv and oligo ( dt)1820 as primer ( invitrogen , carlsbad , ca , usa ) . level of mrna expression of pro - angiogenic molecules vegf , fgf-2 , ang-1 , hif-1 and mrna expression of anti - angiogenic factor endostatin were evaluated by pcr using specific primers and the relative level of the each amplified transcripts were normalized to those of the housekeeping gene -actin . pcr reactions were performed in a final volume of 25 l containing 2 l of rt products , 10 picomoles of each primer and 1x of red - taq polymerase mix ( sigma - aldrich , milano , italy ) . preliminary experiments were performed to determine the appropriate conditions ( ta and specific number of cycles ) for semi - quantitative rt - pcr and transcript were amplified using different cycle numbers:25 cycles for -actin , 30 cycles for vegf , fgf-2 , ang-1 and for endostatin and 35 cycles for hif-1. pcr products were subjected to electrophoresis in a 1.5% agarose gel and ethidium bromide stained , and then photographed under an ultraviolet transilluminator ( amersham biosciences ) . intensity of bands was quantified as arbitrary optical density units ( od ) using the scion image software ( based on nih image ) . six cam treated with ec obtained from three patients with mgus and three patients with mm and three control cam were homogenized in ice cold buffer containing 250 mm sucrose , 10 mm tris - hcl , ph 7.4 and protease inhibitors [ 1g / ml leupeptin , 1g / ml pepstatin and 1 mm phenyl - methylsulfonyl fluoride ( pmsf ) ] . after homogenization in a potter apparatus and centrifugation at 1000 g for 10 min , the protein content of the supernatant was measured using a bradford assay ( bio - rad laboratories , hercules , ca , usa ) . for sds - page , samples ( 60 g / lane ) were solubilized in laemmli buffer , boiled at 90c for 10 min and runned on a 12% polyacrylamide . the proteins were blotted to a pvdf membrane ( millipore corporate , billeira , ma , usa ) and immunoblotting was performed using a monoclonal mouse primary antibody against endostatin ( novus biologicals , littleton , co , usa ) diluted 1:500 in tbst . peroxidase activity was revealed by chemiluminescence ( ecl kit , santa cruz biotechnology , santa cruz , ca , usa ) and blot was immediately exposed to x - ray film . control experiments were performed using a mouse - anti--actin ( abcam , cambridge , ca , usa ) to detect housekeeping protein -actin to normalize the protein load . stained bands were scanned and intensity was quantified using the scion image system ( based on nih image ) . mmec were obtained from 10 patients with mm and mgsuec were obtained from 10 patients with mgus , as previously described . fertilized white leghorn chicken eggs were incubated at 37c at constant humidity . on day 3 of incubation a square window was opened in the egg shell after removal of 23 ml of albumen so as to detach the developing cam from the shell . the window was sealed with a glass and the eggs were returned to the incubator . gelatin sponges ( gelfoam , upjohn company , kalamazoo , mi , usa ) were cut to a size of 1 mm3 and placed on top of a growing cam at day 8 incubation under sterile conditions . the sponges were then adsorbed with 2 l of cell suspension ( 18,000 cells per sponge ) of mmec or mgusec or with rpmi-1640 medium alone or supplemented with fgf-2 ( 200 g / ml ) ( r & d systems , abington , uk ) , used as negative and positive control , respectively . the angiogenic response was evaluated 96 hrs after the implants by means of a stereomicroscope connected to an image analyser system ( olympus italia , italy ) . blood vessels entering the sponges within the focal plane of the cam were counted by two observers in a double blind fashion at a magnification of 50x . means 1 standard deviation ( sd ) were evaluated for all the parameters and the statistical significance of the differences between the counts was determined by student 's t - test for unpaired data . total rna was extracted from 12 cam treated with ec obtained from six patients with mgus and six patients with mm and from six control cam using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufactures 's protocol . the concentration and the purity of the isolated rna were determined using a spettrofotometric analysis and the integrity of the rna was verified by means of electroforesis in a formaldehyde agarose gel followed by ethidium bromide staining . the cdna was then synthetized with 5 g of total rna using m - mlv and oligo ( dt)1820 as primer ( invitrogen , carlsbad , ca , usa ) . level of mrna expression of pro - angiogenic molecules vegf , fgf-2 , ang-1 , hif-1 and mrna expression of anti - angiogenic factor endostatin were evaluated by pcr using specific primers and the relative level of the each amplified transcripts were normalized to those of the housekeeping gene -actin . pcr reactions were performed in a final volume of 25 l containing 2 l of rt products , 10 picomoles of each primer and 1x of red - taq polymerase mix ( sigma - aldrich , milano , italy ) . preliminary experiments were performed to determine the appropriate conditions ( ta and specific number of cycles ) for semi - quantitative rt - pcr and transcript were amplified using different cycle numbers:25 cycles for -actin , 30 cycles for vegf , fgf-2 , ang-1 and for endostatin and 35 cycles for hif-1. pcr products were subjected to electrophoresis in a 1.5% agarose gel and ethidium bromide stained , and then photographed under an ultraviolet transilluminator ( amersham biosciences ) . intensity of bands was quantified as arbitrary optical density units ( od ) using the scion image software ( based on nih image ) . six cam treated with ec obtained from three patients with mgus and three patients with mm and three control cam were homogenized in ice cold buffer containing 250 mm sucrose , 10 mm tris - hcl , ph 7.4 and protease inhibitors [ 1g / ml leupeptin , 1g / ml pepstatin and 1 mm phenyl - methylsulfonyl fluoride ( pmsf ) ] . after homogenization in a potter apparatus and centrifugation at 1000 g for 10 min , the protein content of the supernatant was measured using a bradford assay ( bio - rad laboratories , hercules , ca , usa ) . for sds - page , samples ( 60 g / lane ) were solubilized in laemmli buffer , boiled at 90c for 10 min and runned on a 12% polyacrylamide . the proteins were blotted to a pvdf membrane ( millipore corporate , billeira , ma , usa ) and immunoblotting was performed using a monoclonal mouse primary antibody against endostatin ( novus biologicals , littleton , co , usa ) diluted 1:500 in tbst . peroxidase activity was revealed by chemiluminescence ( ecl kit , santa cruz biotechnology , santa cruz , ca , usa ) and blot was immediately exposed to x - ray film . control experiments were performed using a mouse - anti--actin ( abcam , cambridge , ca , usa ) to detect housekeeping protein -actin to normalize the protein load . stained bands were scanned and intensity was quantified using the scion image system ( based on nih image ) . the ability of mmec and mgusec to induce an angiogenic response in vivo was assessed with the cam - gelatin sponge assay . 1 ) . cam implanted with the medium containing fgf-2 or mmec gave significantly higher vessel counts and numerous allantoic vessels converging like spokes toward the sponges were recognizable ( table 1 and fig . when the sponges were loaded with the medium alone and with mgusec , physiologic angiogenesis was observed in the form of few allantoic vessels partly around and partly converging toward the sponge ( table 1 and fig . angiogenic response assessed by the chick embryo chorioallantoic membrane ( cam)-gelatin sponge assay p<0.001 versus rpmi-1640 and mgusec angiogenic activity in the chorioallantoic membrane ( cam ) assay . gelatin sponges loaded with fgf-2 ( a ) , mmec ( b ) , rpmi-1640 ( c ) and mgusec ( d ) were implanted on top of the cam on day 8 . macroscopic view of the cam on day 12 shows numerous allantoic vessels converging like spokes toward the sponge in a and b , while few allantoic vessels are recognizable in c and d. original magnification : a d , x 50 . in order to evaluate the angiogenic effects of mmec and mgusec , mrna expression of pro - angiogenic factors including vegf , fgf2 , ang-1 , hif-1 and mrna expression of anti - angiogenic molecule endostatin the expression level of each transcript was compared to those detected in control cam . as shown in fig . 2 , treatment of cam with mmec and mgusec did not produce significant difference in the expression of pro - angiogenic factors mrna in comparison with control cam treated with medium alone . moreover , no statistical difference was found in vegf , fgf-2 , ang-1 , hif-1 and endostatin mrna content between mm and mgus treated cam ( p<0.05 ) . in contrast , the expression of endostatin mrna detected in both mgus and mm treated cam was significantly lower respect to control cam ( mgus od:120.113 and mm od:48.319.6 versus ctrl od:205.84 19.6 ; p<0.05 ) . expression levels of mrna coding for vegf , fgf-2 , ang-1 , hif1 and endostatin evaluated by semi - quantitative rt - pcr . transcript levels from the cam assay are referred to ec obtained from 6 mm and 6 mmgus patients and the error band represents the standard deviation of six experiments . western blot analyses were also performed to study whether the expression of endostatin changes in mmec and mgusec treated cam respect to control . as shown in fig . 3 , according to rt - pcr quantification , endostatin was significantly lower also at the protein level in both mgusec and mmec treated cam respect to control cam ( mgus od : 97.1410 and mm od : 52.313.6 versus ctrl od : 158.715.52 ; p<0.05 ) . immunoblotting analysis and quantification after western blot analysis of endostatin expression in cam treated with ec obtained from 3 mm and 3 mgus patients . mm plasma cells secrete vegf , whereas both vegfr-1 and vegfr-2 are markedly elevated in the bone marrow stromal cells , suggesting that a paracrine growth pathway mediated by vegf - activated stromal cells , secretion of il-6 on their part , and subsequent activation of plasma cells may occur . in active mm , we found secretion of the vegf - a isoform by plasma cells , overexpression of vegfr-2 by bone marrow microvessels and isolated ec and of vegfr-1 by the other stromal cells , secretion of vegf - c and vegf - d by stromal cells , and expression of their cognate receptor vegfr-3 by plasma cells , which suggests another paracrine loop of angiogenesis and tumour growth in mm . parallel studies by kumar et al . found secretion of vegf by several mm cell lines and fresh patients ' bone marrow plasma cells , as well as expression of vegfr-1 and vegfr-2 , which suggests multiple vegf - mediated autocrine pathways of tumour growth and paracrine stimulation of angiogenesis . when we studied the angiogenic potential of plasma cell suspension obtained from patients with active mm and implanted them onto the cam , we demonstrated a vasoproliferative response , significantly higher than that induced by cell suspension obtained from patients either with non - active mm or mgus . hgf / sf is yet another angiogenic factor identified in the culture medium of both human mm cell lines and freshly isolated plasma cells . still other factors may be responsible for bone marrow angiogenesis in mm : the plasma cell secretion products , such as tgf- , il-1 , il-6 and il-8 ; granulocyte - colony stimulating factor ( g - csf ) , granulocyte macrophage - colony stimulating factor ( gm - csf ) and tnf- , secreted by bone marrow microenvironment cells recruited and activated by plasma cells . since human mm cell lines produce ang-1 that up - regulates the tie-2 cognate receptor in bone marrow ec , involvement of the ang-1/tie-2 loop in mm neovascularization has been demonstrated . conversely , mmec secrete angiogenic cytokines , such as fgf-2 , vegf and ang-1 as well as matrix metalloproteinase-2 and -9 ( mmp-2 and mmp-9 ) and express adhesion molecules facilitating dissemination of these cells . other studies , however , showed that the expression levels of vegf , fgf-2 , and their receptors were similar among plasma cells from mgus and mm , suggesting that increasing angiogenesis from mgus to mm is , at least in part , explained by increased tumour burden rather than increased expression of vegf / vegfr-2 by plasma cells . another possibility is that the inhibition of angio - genesis in mgus is lost with progression , hence that the switch from mgus to mm may involve a loss of an anti - angiogenic activity . our data are in accord with this statement , because the expression of endostatin mrna detected in both mgus and mm treated cam was significantly lower respect to control cam . the phenotypic switch to angiogenesis in tumour growth involves more than simple up - regulation of angiogenic activity and is thought to be the result of a net balance of positive and negative regulators , and our study suggests that angiogenic switch in mm , investigated by using an in vivo experimental model such as the chick cam , may involve loss of an endoge - nous angiogenesis inhibitor , namely endostatin .

abstractwe have attempted a fine characterization of the angiogenic response induced by multiple myeloma endothelial cells ( mmec ) by using the chick embryo chorioallantoic membrane ( cam ) assay and by reverse transcriptase - polymerase chain reaction ( rt - pcr ) . results showed that in the cam assay mmec induced an angiogenic response comparable to that of a well - known angiogenic cytokine , namely fibroblast growth factor-2 ( fgf-2 ) , while rt - pcr demonstrated that the expression of endostatin mrna detected in mm treated cam was significantly lower respect to control cam . these data suggest that angiogenic switch in mm may involve loss of an endogenous angiogenesis inhibitor , such as endostatin .

six cases were reported from 2007 to 2012 to the oral and maxillofacial unit of annaswamy mudaliar general hospital , bangalore , out of which four patients were males with the lesion occurring between the second decade of life and sixth decade of life [ figures 1a3c ] . in all cases , the mandible was involved with the greatest size of tumor seen being 11 cm 9.6 cm [ figure 1a ] . all cases exhibited enlargement of cortical plates with perforation , pain and tenderness with regional lymph node enlargement , with three cases showing fixed submandibular groups of lymph nodes ( level ib ) and in one case parasthesia of inferior alveolar nerve . five cases showed unilocular radiolucency and one case showed multilocular radiolucency with erosion of bone , including inferior border of mandible in one case [ figure 1c ] . maximum follow - up was for 39 months with one patient being lost to the follow - up [ table 1 ] . all patients had a chest x - ray and an abdomen ultrasound done to avoid missing distant metastasis as we were aware of the potential of this lesion to metastasize . incisional biopsy was done for diagnosis for all cases and was diagnosed as features suggestive of ameloblastoma with areas showing dysplastic changes in the epithelium suggestive of a neoplastic change . ( a ) patient exhibiting large extra oral tumor with draining sinus , ( b ) intra oral view of tumor : ( c ) orthopantomogram showing bicortical involvement of mandible in one patient , wide excision was done as the lesion was small and the patient being young did not wish aggressive measures that may have had postsurgical cosmetic deformity [ figure 2a c ] . he chose the wait and watch policy and there is no evidence of disease after 32 months . ( a ) preoperative view showing tumor involving left mandible and sulcus , ( b ) preoperative orthopantomogram , ( c ) intra oral view of tumor being excised two patients underwent hemimandibulectomy and neck dissection because of increased involvement of mandible with palpable level ib nodes where fine - needle aspiration cytology showed positive for carcinoma , as also one more patient who had underwent a wide excision with hemimandibulectomy and neck dissection due to skin involvement and palpable fixed nodes at ib level [ figure 1a c ] . one patient had a segmental resection of the mandibleone patient had a hemimandibulectomy [ figure 3a c]all the resected specimens were sent for histological examination . one patient had a segmental resection of the mandible one patient had a hemimandibulectomy [ figure 3a c ] all the resected specimens were sent for histological examination . ( a ) preoperative view of tumor involving left mandible , ( b ) intra oral view of tumor , ( c ) orthopantomogram showing involvement of left mandible the largest diameter of the tumors was 10 cm 9 cm [ figure 1a ] showing increased vascularity with numerous cystic spaces and areas of necrosis . typical features found were multiple pieces of tissue with predominant tumor islands within connective tissue seen under scanner . under higher magnification tumor islands appearing epithelial odontogenic in origin showed infiltrative pattern with each island showing peripheral columnar cells with central stellate reticulum exhibiting feature of ameloblastoma , but with atypical features such as bizarre mitosis , altered nuclear chromatin ratio , hyperchromatisim , and mild pleomorphism . individual cell keratinization with keratin pearl formation with stroma showed predominant inflammatory component and endothelial lined blood vessels [ figure 4 ] . immunohistochemical findings showed that ameloblastic carcinomas reacted strongly with antibodies directed against cytokeratin chm and aei and ae3 . histopathological picture showing bizarre mitosis , altered nuclear chromatin ratio , hyperchromatisim , mild pleomorphism , and central stellate reticulum confirming ameloblastic carcinoma no evidence of disease or metastasis was seen postsurgery in any of the patients . it is interesting to note that up to date of completion of this study 71 cases of ameloblastoma had reported to our department between february 2007 and may 2012 . in this series , ameloblastic carcinoma is a lesion with histologic behavior that dictates a more aggressive surgical approach than that of a simple ameloblastoma . mean age for occurrence is 30.5 years , male : female ratio is 1.5:1 , and 80% are located in the mandible posterior region . involvement of the maxilla is less frequent than that of the mandible . the clinical symptoms of ameloblastic carcinoma are more aggressive than conventional ameloblastoma , with swelling , pain , rapid growth , trismus , dysphonia , expansion of the jaws , and frequently perforation of the cortex . these were mirrored in our series also . radiographic appearance of ameloblastic carcinoma is consistent with that of ameloblastoma except for occasional presence of some focal radiopacities , reflecting dystrophic calcification . histologically , ameloblastic carcinoma does not show uniform proliferation , and has pleomorphic and hyperchromatic cells arranged in the form of sheets and chords with reversed polarity . an additional consideration in the differential diagnosis is the squamous cell carcinoma arising in the lining of an odontogenic cyst . histologically , this lesion tends to more closely resemble oral squamous cell carcinoma ameloblastic carcinoma . the squamous odontogenic tumor may also be mistaken for ameloblastic carcinoma , being composed of islands of squamous epithelium that lack stellate reticulum like zones , peripheral palisading , microcystic changes and dystrophic calcifications . however , the epithelium of the squamous odontogenic tumor lacks any cytologic evidence of malignant disease . thus , the term ameloblastic carcinoma can be applied to our series , where all showed evidence of malignant disease including cytologic atypia and mitoses with indisputable features of classic ameloblastoma . whether ameloblastoma may transform biologically and histologically from a classic ameloblastoma to a malignant lesion is controversial . many authors have shown that metastasizing ameloblastomas are histologically indistinguishable from classic ameloblastomas and others have identified malignant features in the tumor , usually after repeated surgical excisions . the high rate of recurrence maybe due to the mode of growth and surgical mismanagement rather that any inherent malignant properties and metastases are exceedingly rare . wide local excision is the treatment of choice as most investigators have recommended , which we had followed in all our cases . regional lymph node dissection should be performed selectively as we did in three of our cases where in the regional lymph node was palpable . the efficacy of adjuvant radiation or chemotherapy as a postsurgical treatment is not clear and seems to have a limited value . lanham treated a patient with doxorubicin , cisplatin , cyclophosphamide , dacarbazine , and 5 fu ; but , the tumor failed to respond . yoon et al . have reported distant metastasis as early as 4 months and as late as 47 months after surgery . none of our patients showed recurrence , but one patient was lost to follow - up . none of our patients were sent for radiotherapy or chemotherapy . in our case series , least follow - up until date is 22 months and maximum follow - up being 39 months . this small series of cases illustrates the malignant portion in the spectrum of ameloblastomas and it is possible that ameloblastoma may show a wide variety of histologic and biologic behavior ranging from benignity to frank malignancy . cases of ameloblastoma should be studied carefully , correlating their histologic pattern with biologic behavior to detect changes in histology that may predict aggressive behavior . when a case is diagnosed as ameloblastic carcinoma , assessment of nodal metastasis and evidence of distant metastasis treatment of ameloblastic carcinoma is wide surgical resection and if evidence of nodal metastasis is present , neck dissection should also be considered .

ameloblastic carcinoma is a rare odontogenic tumor exhibiting not only features of ameloblastoma , but also features of carcinoma . clinical dissemination of this lesion is more aggressive and rapid than that of ameloblastoma and it can metastasize to the lung or regional lymph node . histologically , there are features of both ameloblastoma and carcinoma . < 50 cases have been reported until 2011 . we report a series of six cases with our treatment modalities .

the calcium - sensing receptor ( car ) , a g protein - coupled receptor that signals through gi , gq and sometimes g12/13 pathways , is best known and understood for its role in regulating the secretion and synthesis of parathyroid hormone in response to extracellular ca in the parathyroid glands . like many other g protein - coupled receptors , the car signals through a defined set g proteins , but also interacts with other proteins that probably give it its unique signalling personality. this receptor was first cloned as an extracellular ca sensor using a xeno - pus oocyte expression system . since that time , it has been detected in most epithelial and mesenchymal cell types including renal and gastrointestinal epithe - lium , endothelial cells , keratinocytes , breast tissue , osteoblasts , cardiac myocytes and cells of the central and peripheral nervous systems where it can be expressed in non - polarized cells and polarized cells on either the apical or basolateral membrane . the car appears to have distinct functions in these different cell types , although the functions are not precisely defined . for example , the car can either stimulate cell division ( rat-1 cells ) or inhibit division and promote differentiation ( colonocytes or parathyroid cells ) [ 36 ] . additionally , its biologic effects in tissues such as the kidney or parathyroid glands can not be explained completely on the basis of known second messenger signalling effects [ 1 , 7 ] . the original model of signalling by g protein - dependent receptors was relatively simple . the receptors and g proteins through which the intracellular second messenger systems are activated are attached to the plasma membrane of the cell . these proteins could be relatively free in the membrane or loosely associated with each other . upon activation of a receptor , the protein interactions change , g proteins are activated , which in turn activate effector molecules such as enzymes or ion channels , to generate intracellular signals ( fig . protein interactions have been developed and as more precise analysis of cell signalling has become possible , the inadequacy of the original model has become clear through demonstration that g protein - coupled receptors interact with many intracellular proteins in addition to g proteins . these interacting proteins include rgs proteins ( regulator of g protein signalling ) , scaffolding and structural proteins , ion channels , additional signalling proteins , chaperone and trafficking proteins and others that may not have defined functions yet [ 811 ] . receptors that act through similar sets of g proteins may have different signalling or biologic effects in different regions of a cell and in different cells . this finding suggests that they may have common activities based on the g proteins with which they interact , but that they may also have distinct functions based on the unique sets of other proteins with which they interact as well as their unique locations within a cell . although no one receptor in a native tissue has been characterized completely , enough work has been done with different receptors including the 2- , -adrenergic and metabotropic glutamate receptors in various experimental systems to indicate that such a scenario is not only plausible , but likely ( fig . 1 ) [ 911 ] . four scenarios in which all g protein - coupled receptors interact with g subunits that regulate standard second messenger generation and other common proteins ( grey triangle , e.g. arrestin ) . they also interact with additional different proteins that give them unique signalling personalities . in a the receptor shown in b interacts with an accessory protein ( e.g. a ramp ) as well as another unique protein . the receptor shown in c has a long third intracellular loop that interacts with a unique protein ( octagon ) , and a long c - terminus with a pdz domain that binds a pdz protein that itself brings additional proteins into the complex . in the scenario shown in d , the c - terminus of another membrane protein ( e.g. a channel ) interacts with the c - terminus of the receptor , and the receptor binds an additional protein . most work on the signalling pathways controlled by the car has focused on traditional g protein - coupled pathways , gi , gq , and in some cases g12 or g13[1 , 1216 ] ( fig . , the car inhibits adenylyl cyclase and activates extracellular signal - regulated kinase ( erk ) , through gq it activates phos - pholipase c , increases cai and dag ( diacyl glycerol ) levels , and activates phospholipase a2 , and through g12/13 , it activates rho and phospholipase d . although the full physiologic significance is not understood , activation of the car initiates ca oscillations via a gq - dependent mechanism that when prolonged or forming a plateau , inhibit adenylyl cyclase activity co - ordinately with gi activation . this system serves as an active turn - off system for camp signals and depends on the forms of adenylyl cyclase expressed in different tissues . in the intestine , the car inhibits cholera toxin and e. coli heat stable enterotox - in - stimulated fluid secretion via activation of cyclic nucleotide phosphodiesterases and inhibition of nkcc1 ( sodium , potassium , 2cl transporter-1 ) activity . in keeping with studies of other g protein - coupled receptors , the car transactivates , the epidermal growth factor receptor presumably via a matrix metal - loproteinase [ 19 , the car is expressed on apical or basolateral membranes of epithelial cells where it is likely to come into contact with distinct sets of proteins that should give the car different signalling characteristics and biologic effects . similarly , the car is expressed in many different cell types with different functions , so its signalling and biologic functions could vary from cell type to cell type . a schematic diagram of the principal second messenger signalling pathways that have been described for the car . most of these studies pathways were identified in heterologous expression systems , and may not all exist in all cells where the car is expressed at all times . a good example of the unique signalling and physiologic activities of the car is found in the distal nephron of the kidney where activation of the car results in significant na , k , ca , mg , cl and h2o loss . although the car acts via gi , gq and g12/13 , its biologic effects in the distal nephron can not be explained solely on this basis . a number of g protein - coupled receptors that also act via gi , gq and g12/13 are also expressed in the distal nephron , including those for angiotensin ii ( at1 ) , bradykinin ( b2 ) and endothelin ( etb ) . all of these receptors inhibit camp production , stimulate phospholipase c ( with increases in cai and protein kinase c activity , and decreases in pip2 levels ) , and stimulate phop - spholipase a2 ( with increases in 20-hete and other arachidonic acid metabolites ) . in all studies and experimental systems , activation of the car results in physiologically significant inhibition of na transport in the distal nephron , while activation of the at1 receptor ( presumably at1 ) can stimulate or inhibit transport depending on the concentration , experimental system and study and the effects are minor [ 2131 , 31 ] . these differences between the responses of the distal nephron to ca and aii indicate that the two receptors have distinct effects on cells despite the fact that they stimulate production of the same second messengers . the unique signalling characteristics and biologic effects of the car may be explained by its interactions with proteins in addition to g protein subunits ( table 1 ) . g protein - coupled receptors have four domains that are exposed to the intracellular space and that are available to interact with other proteins , three intracellular loops that connect transmembrane ( tm ) domains 1 and 2 ( intracellular loop 1 or ic1 ) , tm domains 3 and 4 ( ic2 ) , tm domains 5 and 6 ( ic3 ) and the c - terminus . the intracellular loops , particularly ic2 and 3 interact with g proteins as well as other proteins including proteins involved in signalling , such as the arrestins or spinophilin . in g protein - coupled receptors , the size of the loops and c - termini are highly variable . in the car , ic1 and 3 are 14 aa , ic2 is 24 aa , all relatively short and the c - terminus is 220 aa , relatively long . because of its size , the c - terminus is easier to use for techniques to identify interacting protein such as yeast two hybrid cloning . for the car , as well as the better - characterized metabotropic glutamate receptors ( both have a similar structures ) , interacting proteins have generally been identified using yeast two hybrid assays with tissue - specific cdna libraries and the c - terminus as bait . the interactions are verified with co - immunopre - cipitation and the functional interactions are characterized in relatively generic expression systems , such as hek-293 cells . although these car - interacting proteins have not been characterized completely in native tissue , enough data exist to believe that the interactions are real , and that the biologic functions they contribute are also likely to be real . interactions with other proteins have been identified presumptively based on the fact that they are receptor interacting proteins and affect the behaviour of the car . a number of these interacting proteins such as -arrestin or receptor kinases are common to many other g protein - coupled receptors , or are involved in trafficking and degradation , and so are unlikely to be responsible for distinct signalling features of the car . consequently , we will focus on three sets of proteins , receptor activity modifying proteins ( ramps ) , filamin , a potential scaffolding protein , and two inwardly rectifying potassium channels , kir4.1 and kir4.2 that through their interactions with the car , could begin to explain some of its unique signalling characteristics . proteins with which the car interacts . table 1 lists the car interacting proteins that have been described so far alphabetically in the first column . the third column lists the experimental basis for the interaction of a protein with the car , y2h for yeast two hybrid , co - ip for co - immunoprecipitation , gst for gst pull - down assays and functional if expression or mutagenesis studies indicate that the interaction is functionally significant . the fourth column ( car domain ) the fifth column states whether co - localization studies of the car were performed with the interacting protein . native means that studies were performed in native tissue , and het means that studies were performed in heterologous expression systems . the affinity of the car for its agonists or calcimimet - ics ( agents that sensitize the car to activation by agonists ) , appears to depend on the cell type where it is expressed . in the parathyroid gland and parathy - roid cells , the ec50 for ca is approximately 1.0 mm , while for the car expressed in cultured cells , the ec50 for ca is on the order of 3.5 mm [ 32 , 33 ] . in ker - atinocytes , the ec50 for induction of a differentiation marker , involucrin ( presumably a car - mediated event ) , by extracellular ca is approximately 0.1 mm . parathyroid cells are forty times more sensitive to cinacalcet for inhibition of parathyroid hormone secretion than are thyroid c cells to stimulation of calcitonin release . this variability in car responsiveness could be explained by its interaction with different sets of intracellular proteins or accessory proteins such as ramps , a family of proteins that affect trafficking , glycosylation , ligand specificity and second messenger production by the receptors with which they interact . ramp1 was discovered using xenopus oocyte expression cloning to identify an accessory protein for the calcitonin - like receptor ( clr ) that could explain differences in ligand binding and sig - nalling observed in vivo and in different expression systems . ramp1 is a 148 aa protein with a long extracellular domain , a single tm domain and a short cytoplasmic tail that interacts with both the calcitonin receptor ( ctr ) and clr . in these settings , ramp1 acts as a chaperone permitting cell surface expression and alters the affinity as well as the selectivity of these receptors for agonists . ramp2 and ramp3 were subsequently identified using database searches and have similar structures and effects on the ctr and clr . ramp3 has a c - terminal pdz domain , so it may have different functions from ramp2 and 3 . the ramps interact with their target receptors via their extracellular n - termini and tm domains . this family of proteins is relatively new and has not been studied extensively , so their tissue distributions , subcellular localizations and catalogue of receptors with which they interact are not fully defined , although ramps are expressed in all tissues studied to date [ 35 , 36 ] . the level of ramp expression changes in physiologic states and disease models , so receptor activity could be modulated by this mechanism [ 36 , 37 ] . most work has focused on the ctr , the clr and their ligands calci - tonin , adrenomedullin , calcitonin gene - related pep - tides ( cgrp ) 1 and 2 and amylin . adrenomedullin and the cgrp affect vascular tone and blood pressure [ 38 , 39 ] . ramps interact with other class 2 gpcrs , the vasopressin / pituitary adenylate cyclase - activating peptide ( vpac)-1 , parathyroid hormone1 , parathyroid hormone 2 and glucagons receptors , but the functional and physiologic consequences of these interactions are not known . the car , a class 3 g protein - coupled receptor , interacts with ramp1 and ramp3 . the initial observation was that the car could not reach the cell surface in cos7 cells . subsequently , the investigators found that cos7 cells do not express any of the known ramps . interaction of the car with one of these ramps is required for cell surface expression . in the absence of ramp1 or 2 , the car is trapped in the endoplasmic reticulum in an immature core gly - coslyated form . the ramps facilitate its exit from the endoplasmic reticulum , and its transit to the golgi where it is glycosylated and then moves to the cell surface . the functional consequences of interaction with ramp1 or ramp3 were not tested , so it is possible that interaction with the two different ramps leads to different car activation kinetics . the fact that ramp3 has a c - terminal pdz domain means that it could also contribute to localization of the car in the cell and with other signalling proteins . for example , ramp-3 interacts with n - ethylmaleimide - sensitive factor ( nsf ) via its pdz domain . interaction with nsf is required for recycling of the agonist - occupied clr and 2-adrenergic receptor [ 9 , 35 ] . at least two groups identified filamin as a car interacting protein that could serve as a scaffold for other signalling proteins [ 40 , 41 ] . filamin is a homodimer made up of 280 kd proteins that contain n - terminal actin - binding domains , 24 96 aa igg - like repeats , a c - terminal dimerization domain ( repeat 24 ) and two hinge regions [ 42 , 43 ] . filamin was originally described as an actin cross - linking protein that provides mechanical strength to the actin cytoskeleton . subsequently , it was found to interact with a number of tm proteins ( many of them signalling proteins ) and anchor them and the plasma membrane to the cytoskeleton . a partial list of these proteins includes integrins , ca and k channels , a subset of g protein - coupled receptors ( the d2 dopamine receptor , the ctr , some metabotropic glutamate receptors , and the car ) , and the insulin receptor [ 11 , 42 ] . filamin also interacts with intracellular signalling proteins including map kinases , rho gtpases , rho guanine nucleotide exchange factors ( gefs ) , rho kinase , smads and phosphatases [ 42 , 43 ] . filamin appears to be involved in organization of these proteins in the cell and in their trafficking in that under the correct experimental conditions , loss of filamin or interference with its interaction with the protein of interest results in altered membrane expression and lost or reduced function [ 44 , 45 ] . a problem in understanding the function of filamin is that its distribution in various differentiated cells has not been fully determined . despite the fact that filamin is generally considered a cytoskeletal protein , most of it is found in the soluble fraction of hepatocytes , endothelial cells and presumably other cell types [ 4648 ] . this distribution suggests that filamin may have a role in processes , such as protein trafficking in addition to anchoring proteins to the cytoskeleton and plasma membrane . one recent study indicates that in polarized epithelial cells , fil - amin may be preferentially expressed in the apical membrane , suggesting that in polarized cells , fil - amin 's scaffolding function may be more important at the apical surface . the mid - portion of the car c - terminus interacts with the region of filamin that contains repeats 1517 and hinge 1 based in studies utilizing yeast two hybrid , co - immunoprecipitation and gst - fusion protein - binding assays . in the absence of filamin or when the interaction of filamin and the car is blocked , the car does not activate erk or jun n - terminal kinases ( jnk ) appropriately [ 40 , 41 , 50 ] . this interaction is also important for car - mediated rho activation because expression of peptides that interfere with the car - filamin interaction block car - mediated rho activation . the simplest explanation for these observations is that filamin contributes to cell surface expression of the car . this is certainly the case , but even in the absence of filamin some car reaches the cell surface . evidence that fil - amin also provides an important scaffolding function comes from studies of ca or phe - induced car - mediated intracellular ca oscillations . the oscillations stimulated by phe require g12/13 , rho , trpc1 , an intact cytoskeleton and filamin , while the ca - stimulated oscillations persist with disruption of the interactions of filamin and the car [ 12 , 52 ] . although the precise mechanism by which filamin is involved in car - mediated inhibition of parathyroid hormone secretion on the apical surface of parathy - roid cells is not defined , an intact cytoskeleton is required . if filamin is primarily found on the apical surface of epithelial cells , its interaction with the car may be important for those cell types ( e.g. renal proximal tubule , intestinal and parathyroid cells ) where the car is expressed on the apical surface . the c - terminus of the car interacts with and inactivates two inwardly rectifying k channels , kir4.1 and kir4.2 in the kidney that are expressed in the distal nephron ( thick ascending limb of henle and distal convoluted tubule ) as well as other tissues . four kir subfamilies , kir2.x , kir4.x , kir5.x and kir7.x , are expressed on the basolateral membrane of the distal nephron where the car is also expressed [ 5558 ] . a number of channels undoubtedly contribute to the distal nephron basloateral k conductance , but studies indicate that the biophysical properties of a component of it are compatible with homomeric kir4.1 , kir4.2 , or heteromeric kir4-kir5.1 channels [ 55 , 59 , 60 ] . these channels are probably involved in recycling k for na , k - atpases ( and possibly h , k - atpases ) and regulating membrane potential . g protein - dependent signalling systems regulate kir channels by a number of mechanisms that involve protein protein interactions and second messengers including release of g subunits that interact directly with kir3 channels ( girk ) to activate them , and inhibition of kir3 channels by rgs ( regulator of g protein signalling ) proteins [ 6169 ] . the 2 adren - ergic and dopamine d2 and d4 receptors interact directly with heteromeric kir3 channels ( kir3.1/3.4 and kir3.1/3.2 ) , but the physiologic consequences of these interactions are not defined . control of inositol lipid metabolism by plc and pi kinases regulate kir channel activity by determining the level of pip2 , a lipid that is required for channel activity . the car interacts directly and selectively with kir4.1 and kir4.2 as demonstrated with yeast two hybrid assays and co - immunoprecipitation . the channels can be co - immunoprecipitated from kidney cortex , and kir4.2 and the car can be co - immuno - precipitated from heart and liver . the reason for this association may be for regulation of channel activity by direct interaction with the receptor , to localize pip2 metabolism near the channel , ( the car interacts with and regulates pi4-kinase and regulates plc ) or for trafficking to affect the stochiometry of the receptor channel complex [ 54 , 71 ] . kir4.1 and kir4.2 have c - terminal pdz domains , and may be organized in multi - protein complexes on that basis . although frequently overlooked in the category of interacting proteins , g protein - coupled receptors interact with each other to form homodimers and with other receptors to form heterodimers . the car forms homodimers in the er via interactions of cystienes in the extracellular domain and this dimerization is important for cell surface expression [ 7274 ] . interaction of the three intracellular loops of the car with other proteins has not been specifically demonstrated , but can be inferred from functional studies and knowledge of the behaviour of the potential interacting proteins . the car must make contact with the g protein subunits through which it acts , gi , gq and g12/13 . mutagenesis of the second and third intracellular loops and naturally occurring mutants demonstrate that these loops contribute to activation of , and presumably interact directly with gq and gi because mutants are defective in plc activation , a gq - dependent event [ 75 , 76 ] . these loops are usually sites of phosphorylation by g protein receptor kinases ( grks ) in other receptors , and sites for interaction with proteins like arrestin and spinophilin . arrestins then bind to the phosphorylated receptor , block interaction with g proteins and initiate termination of the signal as well as internalization of the receptor [ 9 , 10 ] . arrestins are also capable of activating the erks via src kinase in a g protein - independent manner . based on the fact that over - expression of grk 2 or 3 and -arrestins 1 or 2 reduces car signalling , these proteins probably interact with the car , but the sites of phosphorylation and of interaction were not defined , and interaction was not specifically demonstrated directly . precisely how interactions with grks and arrestins affect car signalling and trafficking have not been studied . in addition to filamin and the k channels , kir4.1 and kir4.2 , a number of other proteins interact with the car c - terminus . the c - terminus is phosphory - lated by , and so must interact with protein kinase c. this phosphorylation at thr 888 results in reduced intracellular ca store release . pkc - mediated car phosphorylation may also be important for mediating the effects of -arrestins because signalling by a mutant car that lacks pkc phospho - rylation sites is not affected by expression of -arrestins . the degradation of the car is mediated by the ubiquitin system through interactions with the e3 ubiquitin ligase , dorfin and amsh ( associated molecule with the sh3 domain of stam ) , a deubiqui - tinating enzyme [ 79 , 80 ] . both interactions were initially identified using yeast two hybrid cloning and co - immunoprecipitation or gst pull - down assays . as expected and consistent with effects of ubiquitination of other g protein - coupled receptors , over - expression of dorfin resulted in reduced levels of car protein while a dominant negative dorfin construct increased car protein . dorfin can mediate degradation of immature car protein from the er . although expression of amsh would be expected to increase car protein expression , it had the opposite effect . the car interacts with several other proteins , but the region of the car responsible for the interaction is not defined . caveolin , a protein that also interacts with filamin , co - immunoprecipitates with , and co - localizes with the car in parathyroid cells [ 81 , 82 ] . caveolae are membrane microdomains enriched in cholesterol and glycosphingolipids that in contrast to lipid rafts also contain caveolin . these structures concentrate the components of g protein signalling systems , and caveolin may be involved in stable membrane expression of these proteins as well as endocytosis and sorting . precisely how its presence in caveolae or its interactions with caveolin affect car function are not defined , but the correlation of reduced caveolin and car expression in parathyroid adenomas suggests that the interaction may be important functionally . this arrangement of proteins could result in a signalling module that regulates the metabolism of inositol lipids in a coordinated manner and in a limited region of the cell to precisely control processes such as channel activity , or it could contribute to protein trafficking that can also depend on inositol lipid metabolism [ 9 , 54 , 69 ] . regulators of g protein signalling ( rgs ) proteins have been implicated in car signalling through expression studies , but interactions of the car or a car - based signalling complex with specific rgs proteins have not been demonstrated [ 16 , 71 ] . g protein - coupled receptors act not only through het - erotrimeric g proteins , but though additional proteins that may interact directly with the receptor or be brought into proximity by scaffolding or structural proteins . a long - standing question has been why so many receptors exist to signal through the same g proteins . part of the answer is that signalling by receptors through subsets of g proteins represents a common characteristic of many receptors , but that the unique signalling and biologic characteristics of receptors are determined by the additional restricted , and possibly unique proteins with which an individual receptor interacts . studies of proteins that interact with the car are less advanced than studies of 2- , -adrenergic or metabotropic glutamate receptors , but some common themes are becoming clear . g protein - coupled receptors interact with each other in the er , an interaction that is important for trafficking and cell - surface expression . these receptors also interact with a restricted set of g proteins , although the location for the initial interaction is not established . signalling by this class of receptors involves rgs proteins that participate in signal termination , can initiate signals ( e.g. rho gef ) , and that can contribute to the specificity of g protein - receptor interactions . g protein - coupled receptors interact with grks and second messenger - dependent kinases ( e.g. protein kinase c and protein kinase a ) , and arrestins as a part of the signal termination process and for receptor traffick - ing . the placement of these receptors in cells is specific and is determined by interactions with other proteins that may have structural , scaffolding , signalling , or mixed functions , and that may contain motifs such as pdz domains . many interacting proteins such as arrestins have multiple functions that include sig - nalling and trafficking , so these aspects of receptor biology may not be separable . based on current information , several interactions may distinguish the car from other gi , gq and g12/13-coupled receptors . filamin appears to serve as a scaffolding protein that also affects car traffick - ing . the scaffolding function is probably important in all cells , but particularly so on the apical surface of polarized cells were filamin is enriched . although the significance of the interaction with ramps1 and 3 is not fully established , these proteins have the potential to affect localization and signalling by the car . the ability of the car to interact directly with ion channels ( kir4.1 and kir4.2 ) in tissues could permit tight control over channel activity through direct protein protein contact or with minimal dispersion of second messengers in the cell . undoubtedly , additional interacting proteins will be discovered that will provide a more complete understanding ofthe function of the car and other similar receptors .

abstractseven membrane - spanning , or g protein - coupled receptors were originally thought to act through het - erotrimeric g proteins that in turn activate intracellular enzymes or ion channels , creating relatively simple , linear signalling pathways . although this basic model remains true in that this family does act via a relatively small number of g proteins , these signalling systems are considerably more complex because the receptors interact with or are located near additional proteins that are often unique to a receptor or subset of receptors . these additional proteins give receptors their unique signalling personalities. the extracellular ca - sensing receptor ( car ) signals via gi , gq and g12/13 , but its effects in vivo demonstrate that the signalling pathways controlled by these subunits are not sufficient to explain all its biologic effects . additional structural or signalling proteins that interact with the car may explain its behaviour more fully . although the car is less well studied in this respect than other receptors , several car - interacting proteins such as filamin , a potential scaffolding protein , receptor activity modifying proteins ( ramps ) and potassium channels may contribute to the unique characteristics of the car . the car also appears to interact with additional proteins common to other g protein - coupled receptors such as arrestins , g protein receptor kinases , protein kinase c , caveolin and proteins in the ubiquitination pathway . these proteins probably represent a few initial members of car - based signalling complex . these and other proteins may not all be associated with the car in all tissues , but they form the basis for understanding the complete nature of car signalling .

throughout life , we are constantly challenged by pathogens , and re - exposure to previously encountered viruses or bacteria happens frequently . homologous re - infections are often effectively controlled by neutralizing antibodies , but pathogens that express altered serological epitopes ( heterosubtypic re - infection ) can bypass antibody - mediated immunity and cause a secondary infection ( corti and lanzavecchia , 2013 ) . right at the beginning of such a re - infection , the t cell repertoire may already contain large numbers of pathogen - specific t cells , and some of these can immediately exert effector function ( zhang and bevan , 2011 ) . moreover , preexisting pathogen - specific but insufficiently neutralizing antibodies could strongly alter infection and replication kinetics ( beura et al . , 2016 ) of a pathogen , and this may significantly alter tissue tropism ( rothman , 2011 ) . it has been shown that cells of the innate arm of the immune system can respond more vigorously ( sun et al . , 2009 ) , and memory cd8 t cells can even contribute to controlling pathogen load in the early phase of infection in a non - cognate , innate - like fashion ( chu et al . , 2013 ) . thus , pathogen spreading , antigen presentation , t cell activation kinetics , and levels of inflammation and tissue destruction can significantly vary between primary and secondary infections . a particular feature of heterosubtypic re - infections is that many antigens are shared in the primary and secondary infection , but the second pathogen expresses epitopes , which are new to the immune system . such a situation of preexisting partial immunity to a pathogen can also occur in a primary infection when a pathogen happens to share an epitope with a previously encountered unrelated pathogen a phenomenon known as heterologous immunity ( welsh et al . , 2010 , che et al . , 2015 ) . though highly relevant for understanding immunity in humans , interferences between past and present infections ( or vaccinations ) are rarely investigated under well - defined conditions . thus , we still have very limited insight into how existing immunity impacts t cell activation , expansion , and conversion into effector and memory t cells . similarly , pre - existing partial immunity can significantly alter the outcome of vaccination ( frahm et al . , 2012 ) . it is also particularly important to consider that pre - existing immunity does not inevitably result in better immune protection . instead , certain re - infections are known to be accompanied by enhanced pathology , as occurs when a dengue - virus - immune individual is exposed to a different virus serovar ( rothman , 2011 ) . similarly , certain vaccination trials are suspected to have augmented the severity of subsequent infections ( blanco et al . , 2010 , moore et al . , 2008 ) . the mechanisms underlying such disease enhancement are not clearly resolved , and it is not known how frequently this may occur . all of these points strongly underline the need to investigate immune responses in immune or partially immune individuals . here , we utilize well - controlled experimental systems to dissect and characterize how different levels of pre - existing immunity influence t cell responses . we observed that t cell activation thresholds differ substantially between primary and secondary infections . we report that only the highest - affinity ligands induce t cell expansion in heterosubtypic re - infections , whereas minor reductions in the levels of tcr stimulation fail to induce t cell expansion . importantly , we show that neither a shortened inflammatory response nor a globally altered antigen presentation pattern are responsible for raising the threshold . instead , we found that even a single shared epitope recognized by previously formed memory cd8 t cells is sufficient to increase the t cell activation threshold of naive t cells . we noted that this elevated threshold is mediated by a non - transferrable endogenous memory t cell population . together our data provide relevant insights into t cell differentiation mechanisms in secondary heterosubtypic infections and for the design of vaccines that are given multiple times . we previously reported that low - affinity t cells differentiate into effector and memory cells in primary infections ( zehn et al . , 2009 ) . we established this using recombinant listeria strains , which express full - length ovalbumin [ ova ] that contains either the original high - affinity , h-2k - restricted ot-1 ligand siinfekl ( ova357364 ) or different altered peptide ligands ( apls ) a2 > y3 > q4 > t4 apl ( listed in decreasing ot-1 stimulatory potency ) . we then became interested in addressing the stimulation requirements in secondary heterosubtypic re - infections . to do so , we infected naive or wild - type listeria ( lm - wt)-experienced mice with lm - ova- or apl - expressing listeria ( see figure 1a ) . with this experimental setup , we mimic a frequently occurring situation where an individual is immune to some but not all antigens in a secondary infection . we observed that the high - affinity ot-1 ligand n4 induces similar expansion in mice with or without a previous lm - wt immunization . in contrast , ot-1 expansion in response to listeria expressing the slightly weaker sainfekl ( a2 ) or siynfekl ( y3 ) apl was decreased in lm - wt immune mice ( figure 1b ) and significantly impaired in response to q4 or t4 . similar exclusions of low - affinity t cells were observed when we transferred ot-1 t cells before the primary lm - wt infection ( figure s1a ) and when memory ot-1 t cells were used instead of naive ot-1 t cells ( figure s1b ) . notably , both n4 and t4 induce similar carboxyfluorescein succinimidyl ester ( cfse ) dilution in primary infections , but only n4 induces robust proliferation in immune mice ( figure 1c ) . some ot-1 t cells proliferate in response to t4 in listeria - immune mice , but this did not increase total ot-1 t cells ( data not shown ) . we therefore conclude that low - affinity stimulation leads to an abortive t cell response in heterosubtypic re - infections . to illustrate the relevance of our observation , we transferred low - affinity h-2k / ova - specific ot-3 tcr transgenic t cells ( enouz et al . , 2012 ) into naive or lm - wt immune rip - mova mice . the lm - wt immune mice were then infected with the same challenge as in figure 1a , whereas the naive rip - mova mice received a priming dose . using this setup , only naive mice showed diabetic blood glucose levels above 300 mg / dl ( figure 1d ) . next , we wanted to define the difference in the affinity range of t cells responding in primary versus heterosubtypic infections . we previously showed that the low - affinity v4 ligand activates ot-1 t cells ( zehn et al . , 2009 ) , even though it has a 1,000-fold lower ec50 than the n4 ligand ( figure 2a ) . interestingly , the d4 apl has an ec50 value that is 10,000-fold below n4 . to test the ot-1 t cell response to this very low - affinity ligand we confirmed that the lm - d4 strain is infectious and comparable to lm - n4 ( figure s2a ) . unexpectedly , even lm - d4 induced rapid initial ot-1 proliferation ( figure 2b ) . d4 activated ot-1 t cells secrete tnf and ifn ( figure 2c ) and produce granzyme b ( figure 2d ) , and they differentiate into memory t cells that can undergo secondary expansion ( figure 2e ) . we illustrated this by transferring ot-1 cells into mice that were either infected with lm - d4 or with lm - wt . four weeks later , we challenged the mice with vesicular stomatits virus expressing ova ( vsv - ova ) . we noticed higher numbers of ot-1 cells in mice primed by d4 in comparison to lm - wt - primed mice that do not stimulate ot-1 t cells ( figure 2e ) . t cells indicated that the signal 1 ( tcr stimulation ) activation requirement in primary infections is very low . in contrast , the difference between the normal n4 and the a2 or y3 apl is rather small ( figure 2a ) . nonetheless , this level already strongly impairs t cell expansion in secondary or cross - reactive infections . we therefore conclude that there is a substantial stimulation threshold difference between primary and secondary infections . to further illustrate the significance of this difference , we marked the known boundaries for ligands which induce positive and negative selection of ot-1 t cells in figure 2a , whereby t4 is the threshold ligand between positive and negative selection ( figure 2a ) ( daniels et al . g4 is known to induce positive selection in fetal thymic organ cultures ( yachi et al . , 2006 ) . given that d4 with a potency much below g4 supports effector t cell differentiation , we wondered whether even the presumed endogenous positive selecting ligands for ot-1 , isfkfdhl from the f - actin capping protein ( cp1 ) and rtytyekl from -catenin ( hogquist et al . 2002 ) would drive ot-1 expansion when expressed by listeria , but this was not the case ( figure 2f ) . we confirmed that the lack of an ot-1 response is not due to a lack of bacterial replication and that an lm-cat infection is , in this respect , comparable to an lm - n4 infection ( figure s2a ) . e1 ova ( eiinfekl ) is the only known apl that supports ot-1-positive selection in vivo ( stefanski et al . , 2001 ) . the lack of a peripheral response to e1 ( figure 2f ) suggests that it more closely resembles the quality of ligands ( cp1 and -catenin ) that are thought to naturally support positive selection . given the activation threshold differences in primary versus heterosubtypic infections , we sought to define the responsible mechanism . depletion of cd4 t cells and nk cells from lm - wt primed mice did not restore low - affinity t cell activation ( data not shown ) . taking a possible role of listeria - specific memory cd8 t cells into consideration , we set up a system that allowed us to control the size of the antigen - specific memory population by sharing only one cd8 t cell epitope between two consecutive infections . we constructed listeria strains that express n4 or t4 plus the lymphocytic choriomeningitis virus ( lcmv)-derived gp33 - 41 epitope . we found that lm - gp33-n4 and lm - gp33-t4 have similar in vivo growth rates ( figure s2a ) and induce similarly sized populations of endogenous gp33-specific t cells ( data not shown and figures s2b and s2c ) . t cells are primed by these listeria strains in naive or lcmv immune hosts ( figure 3a ) , we observed a 2-fold reduction in ot-1 expansion to n4 and an essentially non - detectable response to t4 in lcmv immune mice ( figure 3b ) . this indicates that pre - existing antigen - specific memory cd8 t cells effectively raise the t cell activation threshold and that only one epitope ( or two if one also considers the h-2k - restricted gp34 epitope ) ( hudrisier et al . , 1997 ) we considered that rapid listeria clearance in immune hosts might shorten the duration of antigen - presentation and lower the magnitude of concomitant inflammation ( zehn et al . , 2014 , prlic et al . , 2006 ) . to address this point , we transferred cfse - labeled ot-1 t cells into naive or lm - wt - primed mice and challenged them with lm - n4 . we observed similar cfse dilution in naive and immune hosts excluding major differences in antigen presentation kinetics in the two hosts ( figures 3c and 3d ) . also anti - cd40-mediated dc maturation or administration of recombinant ifn to increase inflammation left the t4 response unchanged ( data not shown ) . next , we established comparable levels of listeria induced inflammation and tissue destruction in lcmv immune and naive mice by co - injecting lm - wt and lm - gp33-t4 into lcmv immune mice . here , only lm - gp33-t4 are recognized by memory t cells , whereas the lm - wt infection propagates normally . this resulted in similar absolute listeria titers in lcmv immune and naive mice , but the response to t4 remained unchanged in immune mice ( figure 3e ) . to control for an impact of the different listeria challenge doses in naive or immune mice ( figures 1 , 3a , and 3b ) , we used acta - deficient listeria . these strains can not spread from cell to cell and are rapidly cleared by the host , allowing c57bl/6 mice to be challenged with a high listeria dose . this creates a setup in which antigen levels are primarily dependent on the amount of the initial bacterial challenge dose , but not the in vivo expansion , of injected bacteria . we observed a lack of low - affinity t cell expansion in immune mice challenged with acta - deficient lm ( figure 3 g ) . this excludes a principle shortage in antigen presentation and a lower inflammatory response as causes of the observed effect . to determine whether the presence of memory t cells is sufficient to raise the activation threshold , we transferred gp33-specific memory t cells into new c57bl/6 hosts along with cd45.1/2 congenic ot-1 t cells . contrasting our prior observation , the transferred memory p14 t cells reduced , but did not completely suppress , the ot-1 response to lm - gp33-t4 . in an lm - gp33-n4 infection , the same p14 dose caused only a minor impact on the ot-1 response ( figure 4a ) . similar outcomes were observed when p14 memory t cells were transferred 28 or 80 days after the lcmv infection ( figure s3a ) . we considered the possibility that the number of transferred p14 t cells was too low to suppress the t4 response effectively and therefore increased the number of transferred p14 to 10 cells . again , the t4 response was incompletely blocked , but surprisingly , this p14 dose already caused a strong decline in the high - affinity ot-1 response ( figure 4b ) . these data suggest that transferring memory t cells is not sufficient to recapitulate the selective and complete exclusion of low - affinity t cells , as observed when endogenous memory t cells respond to a pathogen expressing a shared antigen ( figure 1 ) . to exclude a sole ot-1-dependent phenomenon , we reversed the strategy and used the recently described gp33c6 low - affinity apl for p14 t cells ( utzschneider et al . , 2016 ) . we transferred ot-1 memory and naive p14 t cells into naive b6 mice and infected them with listeria expressing ova and wild - type ( lm - gp33-n4 ) or the low - affinity apl gp33c6 ( lm - gp33c6-n4 ) . again , the memory ot-1 had a minor impact on the p14 response to high - affinity ligands and a much stronger impact on the lower - affinity gp33c6 response ( figure s3b ) . these data also show that a weak or strong response of de novo primed naive ot-1 or p14 t cells does not impact the response of memory p14 ( figure s3a ) or ot-1 t cells ( figure s3b ) . we also examined the phenotype of p14 t cells before ( endogenous situation ) and after the transfer and noticed a larger population of klrg1 t cells among endogenous compared to transferred memory t cells and a higher proportion of cd43cd27 cells ( figures 4c and 4d ) . both populations are thought to have immediate effector function ( olson et al . , 2013 ) . moreover , listeria monocytogenes enters the spleen via the bloodstream and in a dendritic cell dependent fashion ( neuenhahn et al . , 2006 ) . at this stage , they will likely be in contact with effector / memory t cells that are positioned in the spleen in close proximity to the bloodstream . by injecting fluorescently labeled anti - cd8 antibody into mice , we marked the fraction of p14 t cells that are in close contact with blood ( galkina et al . , 2005 ) . interestingly , but in line with an earlier publication ( olson et al . , 2013 ) , we saw that the klrg1 or cd43cd27 t cells ( figure 4e ) , which show a low engraftment efficacy in our memory transfers , were enriched among the p14 cells with access to the blood stream . therefore , we favor the conclusion that a form of tissue - resident t cell population is more effective than transferred t cells in raising the activation threshold in heterosubtypic infections . we show a clear difference in the tcr stimulation threshold between primary and secondary infections . only very high - affinity ligands activate t cells in immune mice , whereas ligands such as a2 and y3 , whose ec50 for inducing a half - maximum ifn response differed only 2- to 4-fold from the wild - type n4 peptide ( figure 2a ) , trigger a much weaker response in immune compared to naive mice . this is in contrast to the situation in naive mice in which even ligands with 10,000-fold lower ec50 activate ot-1 t cells ( figure 2a ) . our observations help to understand t cell responses in heterosubtypic re - infections caused for instance by different influenza strains or dengue virus serotypes . serial infections with the distantly related lymphocytic choriomeningitis virus and pichinde virus were shown to induce a narrowed pathogen - specific t cell repertoire because of antigens that are shared between these infections ( cornberg et al . , 2006 , welsh et al . , 2010 ) . our data indicate that this exclusion is caused by a memory t - cell - mediated elevation of the stimulation threshold and that even minimum cross - reactivity is sufficient to cause this effect . the exclusion of low - affinity t cells by previously established memory t cells has consequences for designing vaccine strategies that rely on a prime - boost regimen and intend to elicit a broad t cell repertoire ( such as hiv vaccine trials ) . several examples underline that optimum protection against pathogens is mediated by highly diverse t cell populations . this ensures cross - reactivity to pathogens expressing mutated epitopes , and it enables individuals to better handle a heterosubtypic infection with a related but non - identical pathogen . it also prevents the selection of escape variants ( price et al . , 2004 , meyer - olson et al . , 2004 , t cell receptor diversity in the population of antigen - specific t cells is largely facilitated by expanding t cells , which suboptimally respond to antigen ( low - affinity t cells ) ( zehn et al . , 2009 ) . the focusing of the t cell repertoire as we observed it may be beneficial for the ongoing secondary infection , but the narrow repertoire may severely diminish the ability to subsequently cross - react to related antigens . similarly , tumor immune responses rely on recruiting t cells of intermediate or low affinity given that high - affinity t cells are often eliminated by tolerance mechanisms ( enouz et al . , 2012 ) . our data imply that vaccination strategies , which involve repetitive injections , will perform suboptimally if the vaccine vector contains previously experienced epitopes . in addition , pre - existing immunity to vaccine vectors were shown to strongly diminish the efficacy of inducing t cell immunity . specifically , adenovirus - seropositive vaccine recipients had lower hiv - specific responses to an adenovirus 5-vectored hiv vaccine in comparison to adenovirus - seronegative recipients ( frahm et al . , 2012 ) . in line with this previous study , our data show that a minimal epitope overlap is sufficient to impact not only the quantity but also the breadth of the recruited t cell repertoire . in contrast , memory t cells do not seem to impact the clonal - like expansion of antigen - specific nk cells ( johnson et al . , 2016 ) . our data suggest that populations of memory t cells , which are difficult to transfer , are more effective than transferrable memory populations in raising the stimulation threshold . these cells may impair the translocation of antigen - loaded dcs from the marginal zone into the t cell zone , and this barrier function may lead to altered antigen presentation and consequently a higher stimulation threshold . this is in line with a recent report that highlights the presence of a tissue - resident t cell population in the spleen ( schenkel et al . , 2014 ) . nonetheless , it remains difficult to precisely distinguish between qualitative and quantitative causes for the observed differences between transferred and endogenous memory t cells . what supports the notion of a qualitative difference is that the transfer of a large number of memory p14 t cells , which already diminished a high - affinity response , did not resemble the effect seen with endogenous memory t cells . c57bl/6 mice were obtained from charles river , and ot-1 , rip - mova , and cd45.1 congenic c57bl/6 mice were obtained from jackson laboratories . low - affinity ot-3 tcr transgenic mice were described previously ( enouz et al . , 2012 ) . mice were bred in specific pathogen - free ( spf ) facilities and infected in spf or conventional facilities at the university of lausanne . mice that were at least 6 weeks old were used for experiments in compliance with the university of lausanne institutional regulations , and the experiments were approved by the veterinarian authorities of the swiss canton vaud . single - cell suspensions were obtained by mashing spleens through a 100 m nylon cell strainer ( bd falcon ) . red blood cells were lysed with hypotonic ammonium - chloride - potassium ( ack ) lysis buffer . if cells were harvested less than 5 days after the infection , then the spleens were before mashing digested with 150 g / ml dnase i ( roche ) and 200 g / ml liberase tl ( roche ) for 40 min at 37c . the mouse cd8 t cell enrichment kit ( miltenyi biotech ) was used for cd8 t cell enrichment . memory p14 cells were isolated by using fluorescein isothiocyanate ( fitc ) , phycoerythrin ( pe ) , or biotin - conjugated cd45.1 antibodies followed by anti - fitc , anti - pe , or anti - biotin microbeads ( miltenyi biotech ) according to the manufacturer s instructions . cells were labeled in serum - free medium with 5 m cfse at 37c for 10 min . mice were infected intraperitoneally ( i.p . ) with 2 10 plaque - forming units ( pfu ) of lymphocytic choriomeningitis virus ( lcmv strain 53b , armstrong ) grown and titrated on vero cells ( utzschneider et al . , 2013 ) . vesicular stomatitis virus expressing siinfekl ( n4 ) ( kim et al . , 1998 ) was grown and titrated on bhk cells . recombinant listeria monocytogenes strains expressing ovalbumin that contain k / ova - derived apl were previously described ( zehn et al . , 2010 ) . 2002 ) , which contains native ovalbumin ( aa134387 ) or ovalbumin with the siitfekl variant ( zehn et al . , 2009 ) , were digested with sapi . oligonucleotides encoding kavynfatc ( gp33 ) or kavyncatc ( gp33c6 ) plus an alanine on both sides were ligated into the sapi restriction site . the constructs were inserted into wild - type or acta - deficient 10403s listeria by e. coli conjugation as previously described ( lauer et al . listeria were grown in brain heart infusion broth ( oxoid , thermo fisher ) to mid - log phase . then , bacterial numbers were determined by measuring the od at 600 nm , and diluted stocks were i.v . naive mice received 1,0003,000 colony - forming units ( cfu ) , mice previously infected with listeria received 5 10 cfu , mice previously infected with lcmv received 2 10 cfu , and , for infection with acta - listeria , 10 cfu were injected . bacterial loads were determined by lysing spleens in pbs supplemented with 0.1% tergitol np-40 ( sigma - aldrich ) . serial dilutions were spread out on brain - heart infusion plates containing streptomycin ( 200 g / ml ) , and colonies were enumerated . a large pool of siinfekl - derived synthetic altered peptide ligands were tested for h-2 kb stabilization in rma - s cells and recognition by ot-1 t cells as previously described ( zehn et al . , 2009 ) . the d4 peptide shows similar surface stabilization as the n4 peptide , but it is a weak agonist for ot-1 t cells ( figure 2a ) . ot-1 cells were stimulated in rpmi ( 10% fcs , 100 iu / ml penicillin , 100 iu / ml streptomycin , 5 m 2-me , 5 mm hepes ; invitrogen ) with anti - cd3/cd28-coated beads ( dynabeads , invitrogen ) and cultured with 50 u / ml human il-2 ( chiron ) in 7% co2 . 6 days after activation , 2 10 ot-1 cells and 1 10 rma cells were mixed in 96-well plates , and titrated concentrations of siinfekl or apl peptides ( emc microcollections ) were added . after 30 min , 7 m brefeldin a ( sigma - aldrich ) was added , and cultures were incubated for another 3.5 hr . up to 4 10 cells were plated in 96-well plates in pbs supplemented with 2% fcs and 0.01% azide . cells were surface stained for 20 min at 4c with fluorescently labeled anti - cd8 ( 536.7 ) , cd127 ( a7r34 ) , klrg1 ( 2f1 ) , cd27 ( lg7.f9 ) , cd43 glyco ( 1b11 ) , cd4 ( gk1.5 ) , cd45.1 ( a20 ) , and cd45.2 ( 104 ) antibodies and fixed in pbs supplemented with 1% formaldehyde , 2% glucose , and 0.03% azide . for intracellular cytokine staining , cells were fixed in pbs 2% formaldehyde and permeabilized in pbs with 0.25% saponin and 0.25% bsa ( perm buffer ) . cells were stained in perm buffer for ifn ( xmg1.2 ) , tnf ( mp6-xt22 ) , or granzyme b ( gb12 ) . fluorescently labeled mhc - i - gp33 multimers were used for detecting gp33-specific t cells ( tc - metrix ) . for in vivo staining experiments , we injected mice i.v . with fluorescently labeled anti - cd8 antibodies . mice were sacrificed 3 min later , and harvested organs were processed as indicated above . flow cytometry data were obtained on a bd facs lsrii machine and analyzed using flowjo software ( tree star ) . graphpad prism was used for graphic representation of data , calculation of ec50 values , and statistical calculations . p values 0.05 were considered significant ( p < 0.05 ; p < 0.01 ; p < 0.001 ) , and p values > 0.05 were considered non - significant ( ns ) .

summarymany infections are caused by pathogens that are similar , but not identical , to previously encountered viruses , bacteria , or vaccines . in such re - infections , pathogens introduce known antigens , which are recognized by memory t cells and new antigens that activate naive t cells . how preexisting memory t cells impact the repertoire of t cells responding to new antigens is still largely unknown . we demonstrate that even a minimum epitope overlap between infections strongly increases the activation threshold and narrows the diversity of t cells recruited in response to new antigens . thus , minimal cross - reactivity between infections can significantly impact the outcome of a subsequent immune response . interestingly , we found that non - transferrable memory t cells are most effective in raising the activation threshold . our findings have implications for designing vaccines and suggest that vaccines meant to target low - affinity t cells are less effective when they contain a strong cd8 t cell epitope that has previously been encountered .

under innate immune conditions , the inflammatory responses mediated by macrophages , mast cells , and neutrophils comprise an important barrier against infectious pathogens , such as viruses , fungi , and bacteria , as well as chemical toxins [ 1 , 2 ] . among the cellular components of innate immunity , macrophages are regarded as central inflammatory cells , as they identify external pathogens using special surface receptors ( e.g. , toll - like receptors ( tlrs ) ) and are widely distributed in the human body . the inflammatory responses mediated by macrophages and their role in pathophysiology have been previously studied . activated macrophages induce various intracellular signaling cascades , including src , syk , phosphatidylinositide 3-kinase ( pi3k ) , akt , inhibitor of b ( ib ) kinase ( ikk ) , and ib [ 46 ] . the signaling pathway also stimulates the nuclear translocation of nuclear factor- ( nf- ) b and activator protein ap-1 , triggering the expression of inflammatory genes that lead to secretion of inflammatory mediators ( e.g. , nitric oxide ( no ) , reactive oxygen species ( ros ) , prostaglandin e2 ( pge2 ) , chemokines , and cytokines ( e.g. , tumor necrosis factor- ( tnf- ) ) ) [ 79 ] . recently , ample evidence has suggested that unchecked , prolonged inflammatory responses can cause serious immunological diseases , including diabetes , septic shock , cancer , arthritis , and cardiovascular disease . the understanding of inflammatory responses and exploration of strategies for suppressing inflammation are thus considered appropriate approaches to reducing disease incidence [ 1013 ] . the cordyceps genus including cordyceps sinensis , cordyceps militaris , cordyceps pruinosa , and cordyceps bassiana grow in korea , japan , china , and the congo . the cordyceps genus can be administered through traditional routes and is known to ameliorate various inflammatory diseases , including chronicle bronchitis , asthma , and eczema . the biological and pharmacological activities of cordyceps genus are antioxidative , antiviral , antifibrotic , anti - inflammatory , antinociceptive , antiangiogenic , antiplatelet aggregation , and antidiabetic [ 14 , 15 ] . studies have also demonstrated the anti - inflammatory mechanisms of butanol ( bf ) and hexane ( hf ) fractions of cordyceps bassiana . however , the specific chemical compounds responsible for the plant 's anti - inflammatory properties have not yet been elucidated . recently , we isolated a promising novel compound [ kth-13 : 4-isopropyl-2,6-bis(1-phenylethyl)phenol ] with anticancer activity from cordyceps bassiana . despite the novel chemical structure of this compound , we have established a method for its total synthesis and derivatization to develop more effective molecules . so far , almost 60 compounds were newly synthesized and tested to check their activities by employing no assay and antiproliferative activity . of them , interestingly , kth-13-amine - diastereomer 1 [ 4-isopropyl-2,6-bis(1-phenylethyl)aniline 1 ( kth-13-ad1 ) ] has been reported to have stronger activity than that of the original compound in terms of anticancer activity ( data not shown ) . in this study , therefore , we further aimed to demonstrate the anti - inflammatory potential of kth-13-ad1 , a derivative of kth-13 , and to explore its mechanism of action using activated macrophages . sodium nitroprusside ( snp ) , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , dihydrorhodamine 123 ( dhr123 ) , fluorescein isothiocyanate- ( fitc- ) dextran , ascorbic acid , and lps ( e. coli 0111:b4 ) were purchased from sigma chemical co. ( st . louis , mo , usa ) . fetal bovine serum and rpmi 1640 the murine macrophage cell line raw264.7 and human embryonic kidney ( hek ) 293 cells were purchased from the american type culture collection ( rockville , md , usa ) . luciferase constructs containing binding promoters for nf-b and ap-1 were gifts from professors chung , hae young ( pusan national university , pusan , korea ) and rhee , man hee ( kyungpook national university , daegu , korea ) . phospho- and total protein - specific antibodies to p65 , p50 , c - fos , c - jun , ib , ikk , akt , p85 , src , syk , lamin a / c , and -actin were obtained from cell signaling technology ( beverly , ma , usa ) . primers ( table 1 ) designed in our laboratory were synthesized by bioneer ( daejeon , korea ) . to synthesize kth-13-ad1 ( figure 1(a ) ) , a solution of 4-isopropylaniline ( 4.00 g , 29.6 mmol ) in xylene ( 14 ml ) was mixed with styrene ( 9.48 g , 91.1 mmol ) and cf3so3h ( 1.0 ml , 11.4 mmol ) . the reaction mixture was allowed to heat to 160c and stirred for 24 h. at that time , the reaction was allowed to cool to room temperature and the volatiles were removed under vacuo . the resulting residue was purified by silica gel column chromatography ( hexanes : etoac = 9 : 1 ) to afford the desired kth-13-ad1 ( 2.60 g , 7.57 mmol , and 1 : 1 diastereomers ) in 26% yield as a brown oil . infrared ( ir ) spectra of kth-13-ad1 were recorded on a bruker vertex 70 spectrophotometer , max in cm . bands are characterized as strong ( s ) , medium ( m ) , and weak ( w ) . h nuclear magnetic resonance ( nmr ) spectra of this compound were recorded on a jeol jnm - al400 ( 400 mhz ) spectrometer . chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance as the internal standard ( cdcl3 : 7.27 ppm ) . data are reported as follows : chemical shift , multiplicity ( s = singlet , d = doublet , t = triplet , q = quartet , and m = multiplet ) , coupling constants ( hz ) , and integration . c nmr spectra were recorded on a jeol jnm - al400 ( 100 mhz ) spectrometer with complete proton decoupling . chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance as the internal standard ( cdcl3 : 77.16 ppm ) . low - resolution mass spectrometry was performed on an agilent 6890n gc ( hewlett - packard co. , palo alto , california , usa ) . h nmr ( 400 mhz , cdcl3 , diastereomer 1 ) : 7.407.12 ( m , 12h ) , 4.07 ( q , j = 6.9 hz , 2h ) , 3.29 ( br s , 2h ) , 3.00 ( sept , j = 6.9 hz , 1h ) , 1.67 ( d , j = 7.1 hz , 6h ) , 1.37 ( d , j = 7.1 hz , 6h ) ; c nmr ( 100 mhz , cdcl3 ) : 145.8 , 139.4 , 138.9 , 130.2 , 128.7 , 127.5 , 126.3 , 123.5 , 40.2 , 33.7 , 24.4 , 22,2 ; ir ( neat ) : 3471 ( s ) , 3384 ( s ) , 3060 ( m ) , 3024 ( m ) , 2982 ( s ) , 2869 ( s ) , 2293 ( w ) , 1947 ( w ) , 1878 ( w ) , 1803 ( w ) , 1623 ( s ) , 1600 ( s ) , 1471 ( s ) , 1450 ( s ) , 1373 ( m ) , 1318 ( m ) , 1257 ( m ) , 1172 ( m ) , 1027 ( m ) , 881 ( s ) , 761 ( s ) , 700 ( s ) cm ; lr - ms ( esi ) : m / z calculated for c25h30n ( [ m + h ] ) 344.2 , and found 344.2 . raw264.7 and hek293 cells were cultured with rpmi1640 medium supplemented with 10% heat - inactivated fbs , glutamine , and antibiotics ( penicillin and streptomycin ) at 37c in a 5% co2 atmosphere . in each experiment , cells were detached with a scraper . examination of cell densities at 2 10 cells / ml revealed that the proportion of dead cells was consistently < 1% according to trypan blue dye exclusion as the criterion for viability . raw264.7 macrophage cells ( 1 10 cells / ml ) were cultured for 18 h , pretreated with kth-13-ad1 ( 0 to 200 m ) for 30 min , and further incubated with lps ( 1 g / ml ) for 24 h. the inhibitory effect of kth-13-ad1 on lps - induced no production was determined by analyzing no level using griess reagent , as previously described [ 18 , 19 ] . the od at 550 nm ( od550 ) was measured using a spectramax 250 microplate reader ( molecular devices , sunnyvale , ca , usa ) . the level of intracellular ros was determined by recording the change in fluorescence resulting from the oxidation of the fluorescent probe dhr123 . briefly , 5 10 raw264.7 cells were exposed to kth-13-ad1 ( 0 to 150 m ) for 30 min and then incubated with snp ( 0.25 mm ) at 37c for 20 min to induce ros production . the cells were further incubated with 20 m of the fluorescent probe dhr123 for 30 min at 37c . the degree of fluorescence , which corresponded to the level of intracellular ros , was determined using a facscan flow cytometer ( becton - dickinson ) , as reported previously . to measure the phagocytic activity of raw264.7 cells , we modified a previously reported method . raw264.7 cells ( 5 10 ) were pretreated with kth-13-ad1 ( 0 to 150 m ) for 1 h , resuspended in 100 l phosphate buffered saline ( pbs ) containing 1% human ab serum , and then incubated with fitc - dextran ( 1 mg / ml ) at 37c for 20 min . the reactions were stopped by adding 2 ml ice - cold pbs containing 1% human serum and 0.02% sodium azide . the cells were then washed three times with cold pbs - azide and analyzed on a facscan flow cytometer ( becton - dickinson , san jose , ca , usa ) , as reported previously . the level of fitc - dextran or ros in raw264.7 cells was determined through flow cytometric analysis [ 22 , 23 ] . 10 cells / ml ) treated with kth-13-ad1 in the presence or absence of fitc - dextran ( 1 mg / ml ) or dhr123 ( 20 m ) were washed with staining buffer containing 2% rabbit serum and 1% sodium azide in pbs and then incubated with direct - labeled antibodies for an additional 45 min on ice . after washing three times with staining buffer , stained cells were analyzed on a facscan flow cytometer ( becton - dickinson ) . raw264.7 cells ( 1 10 cells / ml ) were cultured for 18 h , after which kth-13-ad1 ( 0 to 150 m ) was added for the final 24 or 8 h of culture . the cytotoxic effects of ats - e3 kth-13-ad1 were then evaluated using a conventional mtt assay , as reported previously [ 24 , 25 ] . for the final 3 h of culture , 10 l mtt solution ( 10 mg / ml in pbs , ph 7.4 ) was added to each well . the incubation was stopped by the addition of 15% sodium dodecyl sulfate ( sds ) to each well , which solubilized the formazan . the absorbance at 570 nm ( od570630 ) was measured using a spectramax 250 microplate reader ( biotek , bad friedrichshall , germany ) . raw264.7 cells ( 1 10 cells / ml ) were cultured for 18 h , pretreated with kth-13-ad1 ( 0 to 150 m ) for 30 min , and further cultured with lps ( 1 g / ml ) for 6 h. the inhibitory effect of kth-13-ad1 on the expression of inos and tnf- was determined using semiquantitative rt - pcr and real - time quantitative reverse transcription - polymerase chain reaction ( qrt - pcr ) , as reported previously [ 18 , 27 ] . the primers ( bioneer , daejeon , korea ) used in these reactions are listed in table 1 . hek293 cells ( 1 10 cells / ml ) were transfected with 1 g plasmids to drive the expression of -galactosidase and either nf-b - luc or ap-1-luc in the presence or absence of an inducing molecule ( myd88 , trif , or ha - src ) . transfections were performed using the pei method in 12-well plates , as previously outlined [ 28 , 29 ] . luciferase assays were performed using the luciferase assay system ( promega , madison , wi , usa ) , as previously reported . raw264.7 cells ( 5 10 cells / ml ) were washed three times in cold pbs supplemented with 1 mm sodium orthovanadate , resuspended in lysis buffer ( 20 mm tris - hcl , ph 7.4 , 2 mm edta , 2 mm ethyleneglycotetraacetic acid , 50 mm -glycerophosphate , 1 mm sodium orthovanadate , 1 mm dithiothreitol , 1% triton x-100 , 10% glycerol , 10 g / ml aprotinin , 10 g / ml pepstatin , 1 mm benzamide , and 2 mm pmsf ) , lysed by sonication , and rotated for 30 min at 4c . the lysates were clarified by centrifugation at 16,000 g for 10 min at 4c and stored at 20c until use . the soluble fractions of the cell lysates were immunoblotted , and total and phosphoprotein levels of c - fos , c - jun , p50 , p65 , ib , ikk , akt , p85 , src , syk , lamin a / c , and -actin were visualized , as previously reported . data are expressed as the mean standard deviation ( sd ) , as calculated from one ( n = 6 ) of two independent experiments . other data are representative of three different experiments with similar results . for statistical comparisons , the results were analyzed using analysis of variance / scheffe 's post hoc test and the kruskal - wallis / mann - whitney test . a p value < 0.05 was considered to be statistically significant . all statistical tests were conducted using spss ( spss inc . , chicago , il , usa ) . in previous studies , we and other investigators have reported on the anti - inflammatory , anticancer , and immunomodulatory activities of cordyceps bassiana [ 3235 ] . unlike other cordyceps species , such as cordyceps militaris and cordyceps sinensis , recently , we identified a promising new compound called 4-isopropyl-2,6-bis(1-phenylethyl)phenol with anticancer activity against several cancer cell lines , such as c6 glioma and mda - mb-231 . we further established a method for total synthesis of this compound in order to facilitate its mass production and derivatization . in this study , we tested one ( kth-13-ad1 ( figure 1(a ) ) ) of its derivatives , specifically to see whether this compound is able to suppress macrophage - mediated inflammatory responses . as shown in figure 1(b ) , left panel , the production of no was reduced by up to 70% in a dose - dependent manner by kth-13-ad1 in lps - stimulated macrophage - like raw264.6 cells at 150 m , though it did not downregulate no released from snp , a drug that directly releases no in vitro ( figure 1(b ) , right panel ) . these results indicate that kth-13-ad1 does not act as a chemically directed neutralizing agent but rather as a modulator of no biosynthesis during tlr4 stimulation following lps treatment . in similar fashion , the radical scavenging activity of kth-13-ad1 was observed only at 100 and 150 m when applied to snp - treated cells ( figure 1(c ) , left panel ) but not in the dpph assay ( figure 1(c ) , right panel ) . since kth-13-ad1 did not suppress the viability of raw264.7 cells ( figure 1(d ) ) , the results demonstrate that though this compound does not have direct antioxidative activity , it displays anti - inflammatory properties that indirectly suppress the production of inflammatory mediators , such as no , similarly to ginsenosides that have only anti - inflammatory properties [ 37 , 38 ] . however , unlike these compounds , flavonoids ( e.g. , quercetin , luteolin , and kaempferol ) and anthraquinones ( e.g. , rhodoptilometrin ) , are active components of numerous medicinal plants and have been reported to demonstrate both antioxidative and anti - inflammatory activities [ 3942 ] . to determine the mechanism by which kth-13-ad1 suppresses no production , we investigated tlr4-mediated transcriptional activation levels in lps - treated raw264.7 cells pretreated with kth-13-ad1 . first , we evaluated mrna levels in proinflammatory genes , including inos and tnf- , using quantitative real - time and semiquantitative pcr . as shown in figures 2(a ) and 2(b ) , kth-13-ad1 ( 150 m ) significantly decreased the mrna levels of inos and tnf- , suggesting that kth-13-ad1-driven suppression of inflammatory responses occur at the transcriptional level . since nf-b and ap-1 are representative of transcription factors regulating inflammatory responses [ 43 , 44 ] , we determined the ability of this compound to suppress the activation of nf-b and ap-1 using a luciferase reporter gene assay with promoter sites for nf-b and ap-1 . to determine how kth-13-ad1 regulates promoter activity in inflammatory genes , we pretreated the kth-13-ad1 for 30 min before activating hek293 cells with pma , an activator of pkc that induces the inflammatory signaling pathway . as shown in figure 2(c ) , luciferase activity mediated by nf-b and ap-1 was greatly enhanced by pma , up to 500- and 40-fold , respectively . interestingly , kth-13-ad1 ( 100 and 150 m ) strongly suppressed nf-b - mediated luciferase activity ( figure 2(c ) , left panel ) but not ap-1 ( figure 2(c ) , right panel ) . it has also been reported that myd88 and trif , adaptor molecules regulating the tlr4-mediated intracellular signaling pathway , are good inducers of luciferase activity in hek293 cells [ 47 , 48 ] . the results in figure 2(d ) support this conclusion , as cotransfection of these molecules increased both nf-b- and ap-1-mediated luciferase activity up to 4,000- and 250-fold , respectively , as seen in previous studies . comparing these two conditions , myd88-induced nf-b activation was strongly suppressed by kth-13-ad1 , with 100 m of the compound inhibiting activation by 45% ( figure 2(d ) ) . in addition , kth-13-ad1 reduced the nuclear translocation of the nf-b subunits p65 and p50 at 30 and 60 min , although there was no inhibition at 15 min ( figure 2(e ) ) . these results suggest that the nf-b signaling pathway affecting the activation of nf-b dimerization at 30 to 60 min might be a potential target for kth-13-ad1 . these inhibitory patterns observed by kth-13-ad1 treatment seem to also imply that nf-b activation signals might be timely regulated by some upstream signaling units . to prove such raised possibility , we subsequently focused on identifying the nf-b regulatory pathways that are recognized by this compound . first , we determined the phosphorylation level of ib , an essential step in the nuclear translocation of nf-b . the phosphorylation of ib was blocked by kth-13-ad1 at 15 , 30 , and 60 min , while the total form of ib was greatly increased at 30 and 60 min , compared to lps treatment alone ( figure 3(a ) ) , implying that this compound is able to suppress both phosphorylation and degradation of ib between 30 min and 1 h. in addition , kth-13-ad1 was found to reduce the phosphorylation of ikk/ , upstream of ib , at 5 min ( figure 3(a ) ) . similarly , the phosphorylation of akt and p85/pi3k , upstream enzymes for ikk activation , was blocked by kth-13-ad1 at 3 to 5 min ( figure 3(b ) ) . additionally , src phosphorylation occurring at 1 and 3 min was also suppressed by this compound ( figure 3(b ) ) . to confirm our results , we evaluated the dose - responsive activity of kth-13-ad1 on the phosphorylation levels of putative target proteins , p85/pi3k and src . as seen in figure 3(c ) , the phosphorylation of p85 and src was continuously diminished by kth-13-ad1 ( 100 and 150 m ) under the same conditions . as these results strongly suggest that the putative target of kth-13-ad1 is src , we next analyzed whether this compound is able to regulate the autophosphorylation of src , using an overexpression strategy , as reported previously [ 50 , 51 ] . as shown in figure 3(d ) , kth-13-ad1 ( 100 and 150 m ) clearly decreased the level of src phosphorylation , implying that src is directly modulated by this compound . this is further supported by the fact that the promoter activity of nf-b , as stimulated by src overexpression , was also blocked by kth-13-ad1 ( figure 3(e ) ) . based on these results , we propose that src serves as a potential target protein in kth-13-ad1-mediated anti - inflammatory actions . many anti - inflammatory compounds , both naturally occurring and chemically synthesized , have been found to possess direct inhibitory activity against src kinase [ 5052 ] . additionally , previous studies have reported on src activity under nf-b activation and inflammatory gene expression [ 51 , 53 , 54 ] , suggesting an important role of src in inflammatory responses and in oncogenic activity . finally , to demonstrate whether kth-13-ad1 modulates macrophage phagocytic uptake , fitc - dextran was applied to raw264.7 cells during exposure to this compound . as shown in figure 4 , there was no inhibition of phagocytosis by kth-13-ad1 , as measured by fitc - derived fluorescence [ 55 , 56 ] ; however , significant enhancement was observed at 100 and 150 m concentrations , implying that the compound stimulates macrophage phagocytosis , an important step in the innate immune response . this result suggests that innate immune responses might be regulated by kth-13-ad1 via different cellular mechanisms . in summary , kth-13-ad1 demonstrated impressive suppression of inflammatory mediator production , including ros and no , without inducing cytotoxicity , while this compound strongly enhanced the phagocytotic uptake , suggesting that kth-13-ad1 is able to regulate cellular responses related to innate immunity . in addition , the transcriptional activation of nf-b and its upstream signaling pathway , composed of src , p85/pi3k , and akt , was blocked by kth-13-ad1 , as summarized in figure 5 . thus , the present study proposes that kth-13-ad1 , a chemical analog of kth-13 extracted from cordyceps bassiana , has clinical utility as an anti - inflammatory drug .

the cordyceps species has been a good source of compounds with anticancer and anti - inflammatory activities . recently , we reported a novel compound ( 4-isopropyl-2,6-bis(1-phenylethyl)phenol , kth-13 ) with anticancer activity isolated from cordyceps bassiana and created several derivatives to increase its pharmacological activity . in this study , we tested one of the kth-013 derivatives , 4-isopropyl-2,6-bis(1-phenylethyl)aniline 1 ( kth-13-ad1 ) , with regard to anti - inflammatory activity under macrophage - mediated inflammatory conditions . kth-13-ad1 clearly suppressed the production of nitric oxide ( no ) and reactive oxygen species ( ros ) in lipopolysaccharide ( lps ) and sodium nitroprusside- ( snp- ) treated macrophage - like cells ( raw264.7 cells ) . similarly , this compound also reduced mrna expression of inducible no synthase ( inos ) and tumor necrosis factor- ( tnf- ) , as analyzed by rt - pcr and real - time pcr . interestingly , kth-13-ad1 strongly diminished nf-b - mediated luciferase activities and nuclear translocation of nf-b family proteins . in accordance , kth-13-ad1 suppressed the upstream signaling pathway of nf-b activation , including ib , ikk/ , akt , p85/pi3k , and src in a time- and dose - dependent manner . the autophosphorylation of src and nf-b observed during the overexpression of src was also suppressed by kth-13-ad1 . these results strongly suggest that kth-13-ad1 has strong anti - inflammatory features mediated by suppression of the src / nf-b regulatory loop .

neuroendocrine tumors ( nets ) originate from neural crest cells which belong to the amine precursor uptake and decarboxylation ( apud ) lineage and have both neural and endocrine cell features . these tumors are generally seen in the gastroenteropancreatic tract and lungs and rarely in ovary . these tumors can be imaged using metaiodobenzyl guanidine ( mibg ) tagged to 131-iodine ; which enters by a specific energy - dependent uptake mechanism competing with norepinephrine and majority of it is trapped in the intracellular granule fraction . this tracer has shown better sensitivity in sympathoadrenomedullary tumors as compared to the other net though the uptake is heterogeneous . over - expression of somatostatin receptors ( sstr ) is noted in these tumors and this patho - physiology is exploited in radioimmunoscintigraphy ( ris ) . sstr imaging in net is indicated for detection of the primary , staging , monitoring response to therapeutic somatostatin and treatment planning for sstr directed radionuclide therapy . all the subtypes of sstr expressed by net have affinity for the native peptide but vary in their affinity for the somatostatin analogues ; hence , the sensitivity of the study depends on the density of the sstr in the tumor and the type of analogue used in the study . indium 111 ( in-111 ) tagged somatostatin analogues were the commonly used tracers and majority of the literature related to somatostatin receptor scintigraphy ( srs ) had been done using this tracer . studies have revealed the sensitivity of in-111 labeledsrsto be in the range of 80 - 90% . it has shown superiority to other diagnostic imaging methods ( such ascomputed tomography [ ct ] and magnetic resonance ) in identifying and assessing the staging of net , except for insulinoma ( density of sstr is very low ) . the disadvantages of long half - life , physiological uptake in abdominal organs , and a higher energy of in-111 warranted research in use of a technitium-99 m ( 99mtc ) labeled agent for somatostatin receptor imaging , which is better suited for single photon emission computed tomography ( spect ) imaging . tc labeled tyrosine-3 octreotide ( toc ) has been identified as a suitable tracer which uses hydrazinonicotinic acid ( hynic ) as a complexing ligand . the pharmacokinetic properties of tc - hynic toc were found to be better than those of in - octreotide . higher target - to - non - target ratios and higher absolute tumor uptake values were observed for tc - hynic toc and the optimal acquisition time for imaging was identified as 4 h after injection . srs has low sensitivity for lesions that are present in organs having physiological tracer concentration like the liver and lesions smaller in size due to the limitation of the mechanics and tracers used in spect . imaging with pet ( positron emission tomography ) has higher resolution of the lesions , an inherent property of the modality . initial data showed the tracer gallium 68 ( ga 68 ) dota toc to have a good pharmacokinetic and imaging characteristic as compared to conventional nuclear medicine procedures . a large prospective study also demonstrates a higher accuracy of ga 68 dota toc in comparison to the anatomical imaging modality , ct , and conventional srs . however , the pet / ct modality is not often available and spect imaging is still the feasible option for imaging of net . our pictorial will try to demonstrate the utility of sstr imaging using tc hynic toc in various clinical settings and project its role in prognostication when done in conjunction with 18f flouro- deoxy glucose ( fdg)_pet / ct . patients receiving cold somatostatin therapy were asked to refrain from the therapy for 4 weeks , whereas those patients who had undergone a surgery had their imaging done after the 3 post - operative week . patients were injected with 20 mci ( 740 mbq ) of the tracer ; a whole body planar image was obtained at 30 min post - injection ( p.i ) on a dual head gamma camera ( infinia hawkeye , ge , milwaukee ) . a repeat whole body planar image and spect of the abdomen and regions with abnormal tracer uptake were performed 2 hafter injection in majority of the cases ; a pilot study of 15 cases revealed the 2-h images to be as sensitive as a 4-h image ( as suggested in literature ) . in cases with a doubtful lesion in the2-hp.i image , a delayed image at 4 h p.i . fdg pet / ct was done 60 - 90 min after intravenous injection of 18 fdg , with the patient in a fasting state within a week of the srs . acquisition was done as per the snm guidelines , from base of skull to mid - thigh on a dedicated pet / ct scanner ( discovery st , ge , milwaukee ) . the normal distribution of the tc hynic toc tracer is seen in thegall bladder , kidneys , liver , spleen [ figure 1 ] , and sometimes in the pituitary and thyroid . this image ( ( a ) anterior and ( b ) posterior ) depicts the normal distribution of the radiotracer , 99 m tc hynic toc . note the uptake in the thyroid ( small arrow ) , the liver and spleen . the kidney and urinary bladder are seen due to the part excretion through this system staging of histologically proven neuroendocrine malignancies.the management of net depends on the stage of the disease , i.e. , whether it is localized or metastatic . surgery is offered as an option to patients who have a non non - metastatic primary mass lesion . patients with locally advanced disease generally undergo a debulking surgery with the residual disease being treated with targeted therapies . cytoreduction followed by targeted therapies or specific local therapies like radioablation is the treatment option for a local disease with a solitary metastatic site . a disseminated disease is tried to control with targeted therapies.the conventional staging for net is done with a cect of the suspected local site with ct of abdominopelvic and thorax regions . ris is now incorporated in the staging of net as it helps trace the extent of the primary disease and also the spread of the malignancy in a single setting as seen in figure 2.netsshow unusual site of metastases less frequently though not uncommon . ris helps locate the odd sites of disease as seen in figure 3.initial detection and localization of suspected net and potential metastases in presence of a clinical or biochemical suspicion or to locate primary in a case identified to have a solitary metastatic lesion on conventional imaging.patients with netsmore often present with symptoms due to high endocrine secretion rather than the pressure effect caused by the primary mass . conventional imaging modalities are able to map the metastatic sites but tracing the primary site is difficult at times . figures 4 and 5 depict the utility of srs in this indication.treatment response assessment of net : patients with metastatic disease are treated with medical line of treatment and the treatment response assessment is generally done with biochemical markers and clinically . reduction of the symptoms with a decline in tumor markers is noted with responsive tumors . imaging studies are used to document treatment response ; however , it is difficult to differentiate between functional and non - functioning residual tissue . the ability to identify residual functioning tissue by a non - invasive procedure is useful to plan continuation of therapy . pre- and post - therapy srsis a helpful tool in this respect as shown in figures 6 and 7 . the management of net depends on the stage of the disease , i.e. , whether it is localized or metastatic . surgery is offered as an option to patients who have a non non - metastatic primary mass lesion . patients with locally advanced disease generally undergo a debulking surgery with the residual disease being treated with targeted therapies . cytoreduction followed by targeted therapies or specific local therapies like radioablation is the treatment option for a local disease with a solitary metastatic site . the conventional staging for net is done with a cect of the suspected local site with ct of abdominopelvic and thorax regions . ris is now incorporated in the staging of net as it helps trace the extent of the primary disease and also the spread of the malignancy in a single setting as seen in figure 2 . initial detection and localization of suspected net and potential metastases in presence of a clinical or biochemical suspicion or to locate primary in a case identified to have a solitary metastatic lesion on conventional imaging . patients with netsmore often present with symptoms due to high endocrine secretion rather than the pressure effect caused by the primary mass . identifying conventional imaging modalities are able to map the metastatic sites but tracing the primary site is difficult at times . treatment response assessment of net : patients with metastatic disease are treated with medical line of treatment and the treatment response assessment is generally done with biochemical markers and clinically . reduction of the symptoms with a decline in tumor markers is noted with responsive tumors . imaging studies are used to document treatment response ; however , it is difficult to differentiate between functional and non - functioning residual tissue . the ability to identify residual functioning tissue by a non - invasive procedure is useful to plan continuation of therapy . pre- and post - therapy srsis a helpful tool in this respect as shown in figures 6 and 7 . whole body planar images ( a ) of a 99 m tc hynic toc study of a recently detected case of pancreatic net reveals the uptake in the primary ( arrow ) and the metastatic lesions which are localized on the transaxial spect / ct images to correlate with the lesion in the skull ( b ) , a enlarged prevascular node ( c ) and a tiny pleural based pulmonary nodule ( d ) which is identified on the correlative ct image ( triangulated in e ) . another pulmonary nodule is seen as a focus of uptake in the left hemithorax on the whole body planar image ( a ) a diagnosed case of small cell carcinoma of the lung referred for staging using sstr imaging revealed a large uptake in the upper abdomen on the whole body planar images ( a ) , better visualized in the anterior aspect ( arrow ) . a spect / ct of the abdomen shows a large peritoneal mass ( b and c ) with focal tracer uptake in the primary in the left lung mass ( d ) and an unusual subcutaneous metastases in the posterior chest wall in paravertebral region ( e ) . no fdg pet / ct study was done for this patient patient with diarrhea evaluated for net , conventional ct imaging revealed hepatic metastases and was referred for somatostatin receptor scintigraphy . planar wb images show avid tracer concentration in the known sites of hepatic metastasis ( bold arrow in a and b ) . a small focus of uptake to the right of the midline in the abdomen ( small arrow in a ) corresponded to site of the primary in the duodenum well depicted on the spect / ct images ( triangulated in c ) patient who presented with metastatic left supraclavicular node from an unknown net with multiple hepatic metastases on cect of the abdomen was investigated with srs . the wb image in lesser intensity showed multiple hepatic lesions ( b ) , while the darker intensity images ( a ) showed focal uptake in the left supraclavicular node ( thin arrow a ) and an uptake in the left aspect of the upper abdomen ( thick arrow a ) which correlates on the transaxial spect ct image to the stomach wall ( arrow head in c ) suggesting gastrinoma as a possible primary , later confirmed histologically responder : a metastatic case of small bowel net ; the baseline planar whole body image ( a ) shows uptake in the primary ( thin arrow ) and the multiple hepatic metastases ( thick arrow ) . whole body planar sstr imaging ( b ) after 3 cycles of somatostatin therapy shows complete regression of the tracer uptake at the primary and the metastatic liver lesions depicting the suppression of somatostatin receptors due to the therapy . he was documented to be df on his last follow up , 1.5 year post last radioimmunoscintigraphy non responders : a case of net of the duodenum with hepatic metastases , the pre treatment wb planar images show multiple abnormal uptakes in the liver with no obvious focal uptake at the primary site ( a ) , the hepatic lesions showed partial regression in the post therapy scan ( b ) . a small focus of tracer in the right aspect of the abdomen corresponding to the primary ( arrow b and d ) of the post therapy scan which was not appreciated in the pre therapy scan ( c ) probably due to masking . the combined studies suggest suboptimal suppression of the somatostatin receptor pathway change in biology of the tumors is a known phenomenon and is attributed to either a change in the tumor receptor density or expression of a new receptor . delineating these patients on follow - up with clinical or biochemical suspicion of a recurrence evaluated with ris with poor to absent sstr expression raise the probability of altering receptor status . net is a well - differentiated pathology and does not express glucose transporter ( glut ) receptors and hence a fdg pet / ct study is not utilized in the work up . dedifferentiating tumors show an increase in the glut receptor expression with a decline in the somatostatin receptor density ; hence , a fdg pet / ct study would be efficacious in locating sites of tumor spread . combination receptor imaging will help in staging the disease as per the who classification which is based on the histology type 1a : well - differentiated benign , type 1b : welldifferentiated with low - grade malignancy , and type 2 poorly differentiated . the prognosis of the tumor is dependent on the differentiation of the tumor , poorly differentiated having a bad prognosis . netscan be categorized depending on the pattern of somatostatin and glut receptors expression with type i at one end of the spectrum suggestive of a well - differentiated tumor and type iv which depicts a dedifferentiated tumor with poor to absent sstr at the other end [ table 1 ] . categorisation of neuroendocrine tumors based on somatostatin receptor scintigraphy and flouro deoxy - glucose uptakes . figures 8 - 13 illustrate the various combinations of ris and fdg scans depicting the varied biologies of nets confirming the utility of conjugate receptor imaging . type i uptake pattern : the wb planar images of 99 m tc hynic toc study show multiple hepatic metastases ( a ) . the transaxial spect / ct images show focal concentration in the primary in head of pancreas ( b ) and the fused image of the hepatic metastases ( c ) . the mip image of the fdg pet / ct study ( d ) of this patient does not show abnormal focal fdg concentration either in the primary or the hepatic metastases . the combined srs and fdg images in this patient portray a type i pattern type ii uptake pattern : the anterior and posterior whole body planar image of ris ( a ) with avid uptakes seen in the liver metastases and right aspect of the midline region , the transaxial spect / ct images show the focal uptake in the right of the midline correlating with the primary in the body of the pancreas ( b ) and the multiple hepatic metastases ( c ) type ii uptake pattern ( contd ) : the fdg pet / ct study of the patient in fig 9 revealed an area of minimal increased tracer uptake in the right lobe of the liver on the mip image ( arrow a ) which on transaxial images corresponds to the largest hepatic lesion in the right lobe ( triangulated in b ) with no glut expression in the primary lesion at the junction of the body and head of the pancreas ( arrow c ) or the other larger hepatic lesions ( d ) , these tumors may have a propensity for alteration of tumor biology type iii uptake pattern : tumors with poor differentiation show reduction in density of sstr with increase in density of glut receptors ; these are visualized as fdg and srs avid lesions categorized as type iii pattern . this is a case of recurrent net of the tail of the pancreas evaluated with ris , the planar and the correlative fused transaxial spect / ct images show somatostatin avid recurrent lesion in tail of pancreas ( thin arrow a and triangulation in ( b ) . note the intense focal uptake in the gall bladder ( arrow head a ) , confirmed in the spect / ct image ( c ) type iii uptake pattern ( contd ) : the recurrent pancreatic tail lesion seen in fig 11 showed avid fdg uptake seen on mip image of the 18 f fdg pet / ct ( arrow head a ) and the transaxial image ( b ) suggestive of dense glut receptor expression . a low grade fdg uptake is seen in a hepatic lesion on the fdg pet / ct study ( arrow a and c ) with no hynic toc concentration in the liver on correlative transaxial spect image of the ris ( d ) . the metastatic lesions demonstrate altered biology , no somatostatin receptor expression but expression of glut receptor type iv uptake pattern : dedifferentiated tumors show poor somatostatin receptor expression with high expression of glut receptor which shows a type iv pattern of uptake -fdg avid and poor sstr lesions . a net of the lung with mediastinal nodal metastases shows poor somatostatin expression as seen by low uptakes on the whole body planar images of 99 m tc hynic toc study ( a ) with intense fdg concentration in the primary and the nodal disease seen on the mip image ( b ) and the transaxial images of the thorax ( c and d ) of the 18 f fdg pet / ct it would be appropriate to suggest that in combined srs and fdg pet / ct studies , an increasing fdg uptake with declining sr uptake would convey loss of tumor differentiation and predict a poor prognosis . peptide receptor radionuclide therapy ( prrnt ) is emerging as a promising therapeutic option in view of the specific targeting of tumor receptors . the consensus report of the net clinical trials planning meeting mentions the need for a randomized phase iii trial with use of prrnt in one arm which is based on the somatostatin receptor expression . sstr would be useful in this setting to identify the differentiation of the tumor , its spread , and will also be used for tailored dosimetry . it would be worthy to note the advantage of ris in that it provides all the necessary treatment planning information in a single study . change in biology of the tumors is a known phenomenon and is attributed to either a change in the tumor receptor density or expression of a new receptor . delineating these receptor changes assists in prognosticating the disease and alter management . patients on follow - up with clinical or biochemical suspicion of a recurrence evaluated with ris with poor to absent sstr expression raise the probability of altering receptor status . net is a well - differentiated pathology and does not express glucose transporter ( glut ) receptors and hence a fdg pet / ct study is not utilized in the work up . dedifferentiating tumors show an increase in the glut receptor expression with a decline in the somatostatin receptor density ; hence , a fdg pet / ct study would be efficacious in locating sites of tumor spread . combination receptor imaging will help in staging the disease as per the who classification which is based on the histology type 1a : well - differentiated benign , type 1b : welldifferentiated with low - grade malignancy , and type 2 poorly differentiated . the prognosis of the tumor is dependent on the differentiation of the tumor , poorly differentiated having a bad prognosis . netscan be categorized depending on the pattern of somatostatin and glut receptors expression with type i at one end of the spectrum suggestive of a well - differentiated tumor and type iv which depicts a dedifferentiated tumor with poor to absent sstr at the other end [ table 1 ] . categorisation of neuroendocrine tumors based on somatostatin receptor scintigraphy and flouro deoxy - glucose uptakes . figures 8 - 13 illustrate the various combinations of ris and fdg scans depicting the varied biologies of nets confirming the utility of conjugate receptor imaging . type i uptake pattern : the wb planar images of 99 m tc hynic toc study show multiple hepatic metastases ( a ) . the transaxial spect / ct images show focal concentration in the primary in head of pancreas ( b ) and the fused image of the hepatic metastases ( c ) . the mip image of the fdg pet / ct study ( d ) of this patient does not show abnormal focal fdg concentration either in the primary or the hepatic metastases . the combined srs and fdg images in this patient portray a type i pattern type ii uptake pattern : the anterior and posterior whole body planar image of ris ( a ) with avid uptakes seen in the liver metastases and right aspect of the midline region , the transaxial spect / ct images show the focal uptake in the right of the midline correlating with the primary in the body of the pancreas ( b ) and the multiple hepatic metastases ( c ) type ii uptake pattern ( contd ) : the fdg pet / ct study of the patient in fig 9 revealed an area of minimal increased tracer uptake in the right lobe of the liver on the mip image ( arrow a ) which on transaxial images corresponds to the largest hepatic lesion in the right lobe ( triangulated in b ) with no glut expression in the primary lesion at the junction of the body and head of the pancreas ( arrow c ) or the other larger hepatic lesions ( d ) , these tumors may have a propensity for alteration of tumor biology type iii uptake pattern : tumors with poor differentiation show reduction in density of sstr with increase in density of glut receptors ; these are visualized as fdg and srs avid lesions categorized as type iii pattern . this is a case of recurrent net of the tail of the pancreas evaluated with ris , the planar and the correlative fused transaxial spect / ct images show somatostatin avid recurrent lesion in tail of pancreas ( thin arrow a and triangulation in ( b ) . note the intense focal uptake in the gall bladder ( arrow head a ) , confirmed in the spect / ct image ( c ) type iii uptake pattern ( contd ) : the recurrent pancreatic tail lesion seen in fig 11 showed avid fdg uptake seen on mip image of the 18 f fdg pet / ct ( arrow head a ) and the transaxial image ( b ) suggestive of dense glut receptor expression . a low grade fdg uptake is seen in a hepatic lesion on the fdg pet / ct study ( arrow a and c ) with no hynic toc concentration in the liver on correlative transaxial spect image of the ris ( d ) . the metastatic lesions demonstrate altered biology , no somatostatin receptor expression but expression of glut receptor type iv uptake pattern : dedifferentiated tumors show poor somatostatin receptor expression with high expression of glut receptor which shows a type iv pattern of uptake -fdg avid and poor sstr lesions . a net of the lung with mediastinal nodal metastases shows poor somatostatin expression as seen by low uptakes on the whole body planar images of 99 m tc hynic toc study ( a ) with intense fdg concentration in the primary and the nodal disease seen on the mip image ( b ) and the transaxial images of the thorax ( c and d ) of the 18 f fdg pet / ct it would be appropriate to suggest that in combined srs and fdg pet / ct studies , an increasing fdg uptake with declining sr uptake would convey loss of tumor differentiation and predict a poor prognosis . peptide receptor radionuclide therapy ( prrnt ) is emerging as a promising therapeutic option in view of the specific targeting of tumor receptors . the consensus report of the net clinical trials planning meeting mentions the need for a randomized phase iii trial with use of prrnt in one arm which is based on the somatostatin receptor expression . sstr would be useful in this setting to identify the differentiation of the tumor , its spread , and will also be used for tailored dosimetry . it would be worthy to note the advantage of ris in that it provides all the necessary treatment planning information in a single study . the ability of the modality to delineate the somatostatin receptor expression gives explicit information of the biology of the net , both at primary and metastatic site and helps in treatment planning . srs in conjunction with glut receptor imaging helps locate change in tumor receptor expression and thus helps in prognostication of the disease . this can stratify patients who would benefit from somatostatin analogue or peptide therapy , which is the emerging treatment option for net . ris will be an effective method to monitor response to radioimmunotherapy , which will identify a non - responder early and help alter treatment in such patients .

somatostatin receptor scintigraphy is considered as a comprehensive imaging modality for many neuroendocrine tumors . multiple radiotracers using combinations of gamma or positron emitting radionuclides and tracers are now available . newer radiopharmaceuticals using 99mtc labeled with toc , tate , noc are good alternatives to the 68 - gallium radiotracers where the pet facility is not available . the pictoral depicts the role of srs using 99 m tc hynic toc radiotracers in staging and treatment planning of nets . characterization of the tumor biology using combined srs and fdg pet / ct is also demonstrated with a proposed categorization method . the emerging role of srs in tailored targeted radionuclide therapy is outlined in brief .

recently , a single center study conducted by oiva and coworkers and published in critical care demonstrated that phospho - specific whole blood flow cytometry could be used to assess activated signaling path - ways in leukocytes isolated from pancreatitis patients . pancreatitis is an inflammatory process in which pancreatic enzymes are activated leading to autodigestion of the pancreas and adjacent tissues . during pernicious cases , the initial inflammatory response is excessive and can result in tissue injury , organ failure , and death . to counter this inflammation , these steps can include leukocyte apoptosis , anti - inflammatory cytokine production , and anergy . when patients become leukopenic and immunosuppressed , they often become susceptible to infections with organisms to which a healthy patient would normally be resistant . although therapy aimed at blunting the initial over - exuberant inflammation may be beneficial if applied early , it may also be detrimental by impairing the host 's ability to fight infection that is a common cause of death in these patients . to complicate the potential use of immune - modulating therapy , inflammatory and anti - inflammatory processes can occur along different time - lines for the varied leukocyte subsets . for example , during immune response , t lymphocytes might be undergoing apoptosis while neutrophils are primed to produce reactive oxygen species . thus , during prolonged pancreatitis , leukocyte subsets may be concurrently exhibiting both pro- and anti - inflammatory processes . as noted above , a potential promising therapy of pancreatitis is modulation of the immune response . this is a difficult undertaking as the host response will be affected by a number of variables , including genetic background , co - morbidities , age , gender , and so on . in addition , the patient 's immune response will vary during the course of the disease . thus , what is needed for immunotherapy to be practical is a rapid , robust measure of the host immune system . in the manuscript published by oiva and co - workers , the authors determined the signaling profiles of circulating leukocytes isolated from pancreatitis patients using phospho - specific whole blood flow cytometry . this is a powerful new technology that allows for simultaneous single - cell determination of leukocyte subsets using cell surface markers as well as intracellular protein phosphorylation . this work represents a continuation of work published previously . in this earlier work , oiva and co - workers demonstrated that stimulated monocytes isolated from patients with acute pancreatitis had decreased phosphorylated erk 1/2 , nf-b , and stat1 and 3 . the authors concluded that these changes could lead to impaired monocyte recruitment as well as increased susceptibility to infections . here , changes in activated t lymphocyte p38 , nf-b , stat1 and stat6 activity was observed that could be interpreted as being pro- or anti - inflammatory . interestingly , they determined that patients ' lymphocytes exhibited decreased phosphorylated nf-b and stat1 and increased phosphorylation of p38 and stat6 , suggesting a transition from a th1 to th2 phenotype . additionally , this minimally invasive methodology could be used to generate immune status within 6 hours under ideal conditions . thus , this methodology represents an essential step prior to targeting the underlying immune response to pancreatitis . although beyond the scope of the report , a potentially necessary step after immune status determination , and prior to treatment with inflammatory altering treatments , would be to first treat the whole blood cells isolated from the patients with the potential therapeutic agent and evaluate the immune effector cell response . phospho - specific flow cytometry could also be used to determine if the leukocytes responded in a way that would be beneficial . thus , the minimally invasive , relatively quick methodology developed in this paper could be utilized to determine immune status as well as provide a method to test potential therapies . one unavoidable limitation to the report is that the signaling processes were determined using only peripheral leukocytes . a possibility exists that peripheral leukocytes may not respond similarly to ex vivo stimuli as leukocytes isolated from the inflammatory site(s ) . when feasible , future studies need to be undertaken to compare the response of peripheral leukocytes to these tissue leukocytes . rapid results from whole blood phospho - specific flow cytometry during pancreatitis will allow for immune status determination , likely improve early diagnosis and provide a rational basis for immune targeting therapies . this work was supported in part by national institutes of health grants gm44118 and gm55194 ( to rsh ) , gm72760 ( to ccc ) , and the alan a and edith l wolff foundation .

recently , a single center study conducted by oiva and coworkers and published in critical care demonstrated that phospho - specific whole blood flow cytometry could be used to assess activated signaling pathways in leukocytes isolated from pancreatitis patients . the authors demonstrated that this methodology had the potential to determine the current status of a patient 's immune state . although the experimental cohort was clinically homogeneous , the observed data were heterogeneous . altogether , these results suggest that prior to administering immune - modulatory therapies in inflammatory diseases , it will be beneficial to first determine immune status . rapid results from whole blood phospho - specific flow cytometry may allow for determination of immune status , improve early diagnosis , and provide a rational basis for immunomodulatory therapies .

planning , evaluating , and budget allocation for prevention programs are contingent upon estimating the size of their target groups . population size estimation of the target groups in some context , such as hiv / aids , always entail numerous challenges in countries where such diseases are concentrated in specific sub - populations with stigmatized behaviors & characteristics ( e.g. : injecting drug users ) ( 15 ) . traditional sampling methods that are used to estimate the size of these high risk populations , including multiplier , capture - recapture , etc . , are very complicated , if not impossible , since locating people with high risk behaviors is difficult , and approaching them directly can greatly reduce response reliability ( 510 ) . estimating social network size of a representative sample , through a general population survey , and asking participants questions about specific high - risk behaviors among their acquaintances ( 1113 ) is one of the best means of gathering information on the sizes of such populations , also called hidden or hard - to - count populations due to the fact that it is not possible to calculate their sizes through regular , direct methods ( 14 ) one such method that has held the interest of researchers for the past decade is network scale - up ( nsu ) ( 6 ) . in this method , the average social network size of the respondents ( shown by c ) is used as a prerequisite for estimating the size of a high risk population in the society ( shown by e ) based on the mean number of persons with high risk behavior known by respondents ( shown by m ) ( 15 ) . in the past two decades , several studies in different countries including the united states , ukraine , brazil , moldova , rwanda , japan , china , and thailand have used this method to determine social network sizes as a pre - requisite for estimating high risk population sizes . this difference points to the need for local studies to determine this value and its determinants ( 1421 ) . in iran , the estimated social network size on a national scale is between 308 and 380 . however , tehran , as a megacity and the capital of iran , has unique demographic , cultural , and social features , special inter - individual relationship patterns , and different subpopulation proportions . in addition , due to the migration of different ethnic groups to this city , various ethnic networks have been formed ( 22 ) . therefore to observe these different characteristics and the technical considerations which are recommended in the nsu method , the present study was designed to examine the social network size of the tehran province residents and the demographic factors affecting it in order to prepare the grounds for estimating the size of hidden populations . the social network size was estimated using an indirect approach , that is , respondents were asked how many persons they knew in a certain sub - population within the society , and estimations were made based on the respondents answers . the term knowing a person has a specific definition in this study , and involves a certain time span and space ( 23 , 24 ) ; therefore by saying a knows b , it is understood that a knows b by name and face ; a can visit , call or email b whenever he / she wants , and vice versa . moreover , a has contacted b by phone , email or in person at least once within the past 2 years , and b is a resident of tehran province ( 2527 ) . for the total sample size 1029 , we selected 829 persons from tehran and 200 from robat karim , the capital of robat karim county in tehran provincein 2012 . the city of tehran was divided into 5 geographical zones : north , south , east , west , and center and the sample size were divided proportional to the population size of these five zones . sampling was done in crowded areas , such as streets and parks ; individuals were selected through convenience sampling , and questions were asked in the form of street interviews . all participants were 18 or older , and had been residents of tehran province for the past five years . upon entering the survey , participants were briefed on the study objectives , and their informed consent was obtained . in order to determine the social network size , participants were asked questions regarding the number of people in their personal networks using the indirect method , that is , they were asked how many people they each knew in specific subpopulations . after coordination with the ministry of health and the statistical center of iran , twenty - three known populations with clear and available provincial level statistics were selected to be used in this study . in order to improve estimation accuracy , and in view of the findings of similar studies ( 20 , 28 ) , several considerations were taken into account in preparing the final list of the known populations to be used in the present study ; for instance , the size of all selected populations was between 0.1% and 4% of the total population of tehran province . moreover , we excluded populations that could potentially induce a transmission error in the estimations for which there was no practical correction method . popularity of names in the past three decades was observed in assigning names , and more popular names were used at an almost even distribution . out of the twenty - three initial subpopulations , thirteen were eventually used in the study : first graders , high school graduates , university applicants , university students , married people , divorced people , those who had had normal vaginal deliveries ( nvd ) , those who had had cesarean sections ( c / s ) , primary school staff , people with the first names hamed , abulfazl , sara and marjan . in this study , social network size was estimated using the maximum likelihood estimation ( mle ) method and the equation below : c^i = j mijj ej.t where c^i is the social network size of the respondent i , mij is the number of people that i knows in the known population j , ej is the real size of the known population j , the statistical information for which is available through previous censuses or surveys , and t is the total size of the general population in the survey area ( 11 , 13 ) . to compute the 95% confidence intervals of the social network size , standard error c was calculated through the formula below : ( 29 ) se ci = tcijej this simple model will only work if the following strong assumptions exist ( 11 , 24 ) : all respondents have equal opportunity to know each member of the subpopulations under study.all respondents are fully informed about their own social network.all respondents can recall the number of their acquaintances in the subpopulations under study quickly and clearly . all respondents can recall the number of their acquaintances in the subpopulations under study quickly and clearly . violation of each of these assumptions brings about errors in the results achieved through the network scale - up method . one thing that can violate assumption a is the barrier effect , which pertains to barriers causing some respondents to know the subpopulations in the study with stronger or weaker possibility . transmission error , on the other hand , can violate assumption b , and that is when all people in a given person s network are similarly likely not to be aware of him / her belonging in a subpopulation . estimation effect is another factor that can violate assumption c , and it is due to a lack of precise knowledge of certain details about the people in a high risk social network . in order to control various effects and errors that can bias estimations , the following methods were employed to investigate the sources for these potential errors : 1 ) back estimation of each of the known populations to determine the best groups for estimating the ultimate social network size , and 2 ) variable stratification of the study samples , carrying out all analyses independently in each subgroup , and comparing results with that for the total sample . moreover , acquaintances who were among the respondents social network but were not residents of tehran province were eliminated from all estimations . in order to perform the first correction , a preliminary calculation of the social network size was done , the size of each known population was assumed unknown ( e ) , and then the size of each subpopulation was estimated through the formula below : at this point , the estimate / real ratio ( e / r ratio ) for each population was calculated by dividing the estimated population sizes by the real sizes throughout the province . the next step was to eliminate the first known population in which this ratio was not between 0.5 and 2 . after removing the population in which this ratio was the farthest away from the above - mentioned range of 0.5 and 2 , the whole process was repeated one more time . the procedure was repeated for each of the populations , starting with the farthest outlier , until none of the calculated ratios was outside of this range . the final social network size was computed from the average social network size calculated for people in this stage ( ci ) ( 14 , 24 , 30 ) . in the end , the effect of various demographic and background factors on the social network size of the residents of tehran province was analyzed using a linear regression model . a total of 1029 people were interviewed in this study ; 46.7% participants were male . the majority ( 42.3% ) was in the 18 25 year age group ; the last educational degree of most participants was high school graduate or bachelor s degree , and they were mostly university students ( table 1 ) . demographics of study participants using the thirteen known populations , the social network size was estimated at 200.4 ( ci95% : 188.3 , 212.5 ) , which was used for the back estimation of the known populations in this study . the pearson correlation coefficient between the real and estimated sizes of these known populations was 0.44 ( p value = 0.12 ) . the e / r ratio was approximately 1 in all but three of the subpopulations ; exceptions were first graders , and nvd , c / s where e / r ratios were 0.40 , 0.43 , and 0.3 respectively , and therefore the social network size of tehran province was estimated by eliminating the subpopulations c / s , first graders and nvds in that order . table 2 represents the estimated social network size after excluding each of the above subpopulations , as well as the e / r ratio for populations whose sizes have been calculated based on the estimated network sizes that continue to be out of the desired range . having eliminated c / s , primary school and nvd groups , the estimated cs were 220.8 , 243.4 and 259.1 respectively . in addition , the correlation between the real and estimated sizes of remaining groupwas improved significantly in every steps ( r= 0.32 based on groups , 0.51 , 0.72 and 0.82 by dropping c / s , primary school and nvd groups respectively ) . estimate / real ( e / r ) ratio & estimate - real correlation changes after stepwise excluding the known populations with out of range e / r ratio table 3 demonstrates results of applying the back estimation method to subpopulations with estimated sizes within the acceptable range that were used in the final network size estimations . back estimations and e / r ratios of all populations are presented in table 4 . known populations , source of data , back estimation of their population sizes , and the e / r ratio e / r ratio in separate estimations of social network size for men and women ( before and after excluding the three out of range e / r ratio known groups ) figure 1 represents the scatter plot of the real against estimated sizes of the thirteen known populations , and the same measures after excluding the three abovementioned subpopulations . the final network size estimated by using the remaining ten subpopulations was 259.1 ( ci95% : 242.2 , 276 ) . real estimate scatter plot of known population sizes ( before and after excluding the three out of range e / r ratio known groups ) in the univariate analysis , social network size was significantly associated with gender ( p=0.01 ) , age ( p<0.001 ) , and occupation ( p<0.001 ) . men had larger networks comparing to women ( 291.8 versus 230.4 ) , and c in younger people was larger than that in older ones ( 328.5 versus 201.1 ) . multivariate analysis showed that variables of gender and age impacted social network size ( table 5 ) . age was also proved to have a significant impact on the respondents knowing the studied subpopulations ( p < 0.001 ) . comparison of social network size in different sub groups based on different background variables the possibility of knowing a person who had graduated from high school , participated in the university entrance exam , or been admitted into university in the past year was higher in the younger age group ( 56.6% , 63.4% and 39.3% respectively ) ( table 6 ) . based on the findings of this study and by using the mle method , the social network size of the residents of tehran province was estimated at 259.1 which were calculated using 10 known populations with an e / r ratio between 0.5 and 2 . age and gender were determinants of peoples network sizes . estimated network sizes were different in male and female respondents , and appropriate known populations used for c estimation in these two groups were not equal . our experience showed that all subpopulations with known size were not appropriate to be used in the estimation of c. we dropped three sub - populations to improve the internal validity of our size estimations , which also used in other comparable studies ( 11 , 14 , 20 , 22 ) . by eliminating these 3 subpopulations , the social network size the national study was primarily biased in a similar manner as well ( 22 ) . therefore , estimation of c based on the size of some known subpopulations is a stepwise technique and has to be applied with some considerations ; otherwise the estimated c might have big bias . the estimated sizes for known subpopulations c / s , nvd , and first graders were less than half the real sizes , and for the subpopulation of people with the first name marjan the estimated size was more than twice the actual size . after all three subpopulations were eventually eliminated , the correlation coefficient was more than double ( 0.82 as opposed to 0.32 ) , and became statistically significant . moreover , the estimations for the subpopulation marjan a persian female first name- fit within the acceptable range , and was closer to 1 . this indicated that the social network size estimated by using the ten remaining known populations would yield a more reliable estimate of hidden subpopulations too . similar studies confirm likewise that elimination of populations with estimations outside of the range 0.5 1.5increases the correlation coefficient between back estimations and real sizes . conducted a research in ukraine to estimate the number of idu s fsw s and msm . of the 22 known populations used in this study , 13 fell within the acceptable e / r ratio range , and were used in the final network scale - up calculations . the estimated sizes of known populations had a high correlation coefficient with the real sizes using the back estimation technique ( r = 0.912 ) , and this number reached 0.94 after nine of the known populations were eliminated ( 14 ) . in another study conducted on 1554 individuals in the u.s . after elimination of two populations with a discrepancy between their real and estimated sizes , the correlation rose to 0.94 ( 11 ) . in the present study , the average e / r ratio using thirteen known populations was calculated to be 1.17 . removing each of the outliers brought this ratio closer to 1 , and after eliminating the 3 sub - populations mentioned earlier , the e / r ratio reached 1.09 . this figure indicated an average overestimation of about 9% in the calculations . in the ukrainian study , the e / r ratio was 1.65 , which denotes an overestimation of 65% in population size estimations ( 14 ) . overestimation seems to be one of the common limitations of the network scale - up method with small subpopulations ( 11 , 30 ) . in the present study , this problem occurred with the subpopulation of people with the first name marjan , which was the smallest subpopulation with only 0.12% of the total population of tehran province . since nicknames are common in iran , this overestimation may be because the respondents network includes people called marjan who are registered in the census with a different name ; this can be true with several names . after eliminating the c / s subpopulation , the e / r ratio for the subpopulation marjan fell within the acceptable range ( e / r = 2 ) , and after the two subpopulations nvd and first graders were removed , this ratio decreased even further ( e / r = 1.7 ) . another common calculation error in the network scale - up method is underestimation of the size of large subpopulations ( 11 , 30 ) . the largest subpopulations used in this study were c / s , first graders , married people , and university applicants . the first two were underestimated as expected , while the other two had more accurate estimations , probably on account of a more satisfactory transmission and the respondents being more informed on these properties due to the significance that iranians attribute to marriage and university education . study on se - roprevalence in the united states , it is difficult for respondents to estimate the number of acquaintances they have in large subpopulations ( 11 ) . in addition to the estimation error in the c / s subpopulation , another explanation for this group having the lowest e / r ratio ( 0.34 ) and an e / r ratio lower than 0.5 for the nvd group is transmission error . the similar natures of these two subpopulations as well as their connection to gender suggested that respondents gender impacts their awareness of nvd or c / s occurring within their social networks . in order to assess the validity of this theory , network size estimations and back estimations for populations were performed based on the respondents gender . based on our findings , the e / r ratios for known populations were different in male and female res pondents ( table 4 ) . the e / r ratios were out of range for the subpopulations c / s , nvd , and first graders when estimated through the networks of male respondents , but this limitation did not exist in case of female respondents . the difference seems to be understandable considering the nature of these subpopulations and the iranian culture : an iranian male respondent would be less likely to be aware of childbirth conditions or first graders in his social network compared to a female respondent in a similar situation . in the ukrainian study , similar circumstances were encountered when estimating the number of militiamen , since there was a higher likelihood for male respondents to know someone in this subpopulation on account of their cultural status and social relations compared to women ( 77% vs. 68% ) . therefore , using the militiamen subpopulation to estimate the social network size leads to different results in male and female respondents ( 14 ) . barrier effect is another significant factor that can compromise the estimation of population sizes in the network scale - up method . one common barrier , which could be a concern in the present study , is the respondents age . those in the 18 25 year group were more likely than others to know someone in the subpopulations high school graduates , university applicants , and university students , considering how these categories are age - related . based on the results shown in table6 however , barrier effect did not affect the results of this study . this is probably because the 18 - 25 year old group is relatively larger in the general population , and our 18 - 25 year old group was proportionately larger too . in the ukrainian study , the two subpopulations polish and moldavian were removed due to the risk of barrier effect and consequent estimation errors . in the final stage of the present study , the remaining ten subpopulations were used to estimate the social network size at 259.1 , and this was used as the base for estimating the known population sizes with the most suitable e / r ratio and the highest correlation with their true sizes . this number was smaller compared to similar study in the country , carried out by shokoohi et al . in kerman province in 2010 ( c = 303 ) . their study was performed on 500 men between 18 and 45 years of age , and the data were collected through interviews . the difference between the social network sizes of the two provinces of tehran and kerman seems to be due to the lifestyle specific to metropolitan areas and capital cities that somehow limits social relationships ; one other reason may be that the kerman study was restricted to male samples , who are expected to have more social contacts and consequently larger networks than females in a city like kerman ( 27 ) . two estimates were computed by entering 23 known groups in the study using regression - based ( c=308 ) and ratio - based ( c=380 ) methods . according to their results , the ratio - based method , which is also used in the present study , is the recommended approach because of higher internal validity and prediction validity ( 22 ) . the ratio of the population size to the total tehran population was beyond the range of 0.1% to 4.0% for 10 of the 23 known groups in the national study ; differences in population compositions and their effect on results can partly explain the different estimates in these two studies . moreover , unique social , cultural , political , and economic processes in tehran have created a different pattern of social interactions that restricts inter - individual relationships and limits the social network size . social science studies also confirm that in recent years , the nature of urban development in megaci - ties such as tehran has entailed loosening in social ties . this is due to concentration of population in metropolitan areas and absence of alternative social practices ( 31 ) . in the ukrainian study , the social network size was estimated at 202 using a network scale - up approach and the mle method , which was also employed by the present study ( 27 ) . in a 2012 study conducted in japan by ezoe et al . with the purpose of estimating the msm population , the network size was estimated to be 363.5 regardless of gender , with 174 being male . out of the initial ten known populations used in this study , only three were eventually used to estimate the social network size in the pilot stage : male fire fighter , policemen , and military personnel . of the seven eliminated populations , five were removed on account of estimation error , that is , their estimated sizes were not consistent with their real sizes , and two were removed due to transmission error ( 20 ) . the social network size was estimated at approximately 291 in the united states , as shown in the 2001 study by mccarty et al . , using four separate national sets of samples . the study used the network scale - up method on 29 known populations , three of which were similar to the subpopulations in the present study and those were : first names , people who gave birth within the last year , and victims of car accidents ( 24 ) . the social network size estimated by killworth et al . was 286 , which is larger than the present study , and this may be due to the numerous cultural and social differences between iran and the united states ( 11 ) . although in the kerman study , there was no significant relationship between the social network size and any of the demographic factors ( age , education , marital status , and occupation ) ( 27 ) ; the results of the present study showed that men comparing to women and younger people comparing older ones had a significantly larger social network , which seems understandable on account of cultural and social considerations . in the national study and the chinese study , the social network sizes of men and younger people was larger compared to those of women and other age groups , and this is due to men and young people having wider social circles ( 21 , 22 ) . married people had smaller social networks compared to single people in the two studies mentioned above ; this difference was found to exist in the present study as well , although the difference between these two groups was statistically not significant in tehran province ( 248.8 versus 270.1 ) . in this study , there were limitations associated with the network scale - up method . firstly , it is unrealistic to expect accurate and flawless estimations , because although there is a precise and specific definition for the term knowing a person , data collected on social networks is self - reported . in minimize this limitation , the present study attempted to use known populations that were easily recognizable by all respondents , and had clear and uniform definitions for all members of the society . furthermore , the time span for the questions was the past year so that respondents could remember the details with fewer errors . another issue , which is quite unavoidable , is lack of definite boundaries separating many subpopulations in the respondents points of view . researchers are limited to subpopulations for which official statistics exist , and these subpopulations have been assigned specific definitions that are not necessarily consistent with those of various members of the society . we intended to select a representative sample of general population in order to minimize different types of selection and information biases . although it was not a fully random sample of whole community and there are some methodological considerations , based on the existing experiences in iran and the result of a methodological study that was performed for comparing three popular sampling method in this type of studies ( street - based , telephone - based and home - based interviews ) in this regard , it seems street - based sampling from deferent geographical zones would be a feasible sampling scheme which was used in the national study as well . based on the above explanation , it seems that the social network size of tehran s residents is different not only with similar studies in other countries but also with the results of national survey in iran , which might be mainly because of the special social and cultural pattern of communications among different communities . we also showed that the c varies considerably in males and females , in young and old people . therefore , local estimations for c in different sub - populations are needed to improve the accuracy of nsu estimations . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or fal sification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

abstractbackgroundnetwork scale - up is an indirect method for estimating the size of hidden , hard - to - count or high risk populations . social network size estimation is the first step in this method . the present study was conducted with the purpose of estimating the social network size of the tehran province residents and its determinants.methodsmaximum likelihood estimation was applied to estimate people s network sizes by using populations of known sizes and the scale - up method . respondents were selected from tehran province through convenience sampling in 2012 . out of thirteen selected subpopulations with known size , ten had minimum accuracy which used in our analysis.resultsof the 1029 respondents in this study , 46.7% were male . the social network size of tehran province residents was estimated to be 259.1 ( ci95% : 242.2 , 276 ) based on the ten known populations remained in this study . this size was 291.8 in men and 230.4 in women . younger people ( 1825 years old ) had larger network sizes compared to the other age groups ( p<0.001).conclusionour estimation for social network size of tehran inhabitants was smaller than that previously estimated size for the whole country ( c=380 ) . in addition , we found that the social network of subpopulations was different . this difference means that we need local estimations for sub - populations to improve the accuracy of population size estimation using network scale up method .

experimental animals : male c57bl/6ncrslc mice ( 8 weeks old ) were purchased from japan slc ( hamamatsu , japan ) . all mice were maintained in 40.5 20.5 18.5 cm individual ventilated cages ( sealsafe plus mouse ; tecniplast , buguggiate , italy ) under controlled temperature ( 23 2c ) and humidity ( 50 10% ) on a 12-hr light / dark cycle at the kobe university life - science laboratory with ad libitum access to a pellet diet ( dc-8 ; clea japan , tokyo , japan ) and filtered water . this study was approved by the institutional animal care and use committee ( permission # 24 - 10 - 03 ) and carried out according to the kobe university animal experimental regulation . ctd purification and hplc analysis : water - soluble dantotsu ( involving 16% of ctd ; sumitomo chemical co. , tokyo , japan ) , donated by sado city ( niigata , japan ) , was washed with 10 times the amount of distilled water to remove the surfactant activating and granulating agents . after being left to stand for at least 48 hr , this step was repeated five times , and then , the white precipitate was collected and air - dried naturally for a week . the content rate of ctd in the white precipitate was measured by a lachrom high - performance liquid chromatography ( hplc ) system ( interface l-7000 , pump l-7100 , auto sampler l-7200 , column oven l-7350 and uv - vis detector l-7420 ; hitachi , tokyo , japan ) using a capcell pak c18 ug120 column ( 5 m particles , 4.6 250 mm ; shiseido , tokyo , japan ) . ctd standard ( > 99.8% ; wako chemical , osaka , japan ) and the obtained white powder were completely dissolved in dimethyl sulfoxide ( dmso ) and then serial - diluted with a mobile phase consisting of 55% acetonitrile in 50 mm potassium phosphate buffer ( ph 3.0 ) , followed by filtration with a 0.20 m syringe driven filter unit ( millex - lg ; millipore , billerica , ma , u.s.a . ) . the column maintained at 40c was eluted with the mobile phase at a flow rate of 1.0 ml / min . after the column was equilibrated , 10 l of the samples was injected into the hplc system . we monitored the resultant chromatograph at the wavelength of 260 nm and then calculated the ctd content of the obtained white powder from the calibration curve created by the peak areas and heights of the ctd standard . a single peak was observed in the samples from the white precipitate with the same retention time as that of the ctd standard . a linear calibration curve ( r=0.999 ) created from samples serial - diluted with the ctd standard showed that our purification makes the content rate of ctd increased from 1416% to 9397% by weight . ctd administration and stress exposure : all mice were allowed to acclimate to their home cages for a week prior to the initiation of experiments . we divided the mice into eight groups ( n=5 mice in each cage ) : ctd-0 ( control ) , ctd-10 ( 10 mg / kg / day ) , ctd-50 ( 50 mg / kg / day ) and ctd-250 ( 250 mg / kg / day ) with the presence or absence of stress exposure . as a substitute for filtered water for the mice , we used the medigel sucrarose 2 oz cup ( clearh2o , portland , me , u.s.a . ) , which is a flavored thermoreversible hydration gel matrix . the amounts of the purified ctd for the respective administration groups were calculated from the ctd purity ( 95% ) , daily gel intake ( 5 g / day / mouse ) , total gel weight ( 60 g ; excluding the package weight ) and average mouse weight ( 24 g ; weighed at initiation of experiments ) . these amounts of ctd were completely dissolved in 600 l dmso ( 1% volume of a gel ) and injected into the gels and then double - boiled at 60c followed by shaking to ensure that the ctd was diffused well . for the ctd-0 group , the same volume of dmso without the purified ctd all gels were strapped with cable ties under the grate of the cage lid to prevent contamination with the beddings and the excretions . all gels were weighed daily to estimate the amounts of the putative ctd exposure . in the four stress groups , the mice were subjected to an unpredictable chronic stress procedure as described in our earlier report with some modifications . briefly , the following six stressors were used : 5 min forced swimming in water at room temperature ( rt ) , 24 hr food and water deprivation , continuous overnight illumination , 30 min horizontal cage shaking ( 80 rpm ) , 24 hr switching of cagemates ( being housed with another mouse ) and 24 hr wet bedding . to maximize the unpredictability of this paradigm , the mice were randomly exposed to two mild stressors per day at varying times for 4 weeks . behavioral analysis : on the last day of the 4 week experimental period , an open field test was conducted during the light phase to evaluate the locomotor activity and the anxiety - like behavior of the mice . briefly , the mouse was placed on the corner of an open field ( 60 60 30 cm ) with led illumination . all of the mouse s activities were recorded by a video camera for the subsequent 10 min , and we then analyzed the total distance traveled and the time spent in the center zone ( 30 30 cm ) using smart video tracking software v3.0 ( san diego instruments , san diego , ca , u.s.a . ) . tissue preparation : on the day after the completion of the 4 weeks of combined exposure to ctd and stress , all mice were deeply anesthetized with diethyl ether and transcardially perfused with 0.9% normal saline , followed by perfusion with ice - cold 4% paraformaldehyde in phosphate buffer . the testes were excised , weighed and postfixed with the same fixative overnight at 4c . the testes were dehydrated through a graded series of ethanol followed by xylene and embedded in paraffin . serial sections of testes were then cut at 4 m thickness on a sliding microtome ( sm2000r ; leica microsystems , wetzlar , germany ) and mounted on slide glasses ( platinum pro ; matsunami glass ind . histological and immunohistochemical analyses : for the general histological analysis , testis sections were stained with hematoxylin and eosin ( he ; merck kgaa , darmstadt , germany ) after their deparaffinization and hydration , following the manufacturer s instructions . to detect antioxidant enzymes in the testes the sections were immersed in absolute methanol and 0.5% h2o2 for 30 min , respectively , at rt to quench the endogenous peroxidase activity . kyoto , japan ) for 1 hr at rt for protein blocking and then incubated with the rabbit polyclonal anti - gpx4 antibody ( item no . 10005258 ; cayman chemicals , ann arbor , mi , u.s.a . ) diluted 1:8,000 in phosphate buffered saline with 0.05% tween-20 ( pbst ; ph 7.4 ) for 18 hr at 4c . after being washed with pbst , the sections were reacted with goat anti - rabbit immunoglobulins conjugated to peroxidase - labeled dextran polymer in tris ( hydroxymethyl ) aminomethane - hcl buffer ( envision+ ; dako , glostrup , denmark ) for 1 hr at rt . immunoreactivity was then detected by incubation with 3,3-diaminobenzidine solution ( envision+ kit / hrp[dab ] , dako ) . the sections were then rinsed with distilled water and counterstained lightly with hematoxylin solution for 1 min . next , the sections were placed in a graded series of ethanol , dehydrated with absolute ethanol , cleared by xylene and coverslipped with eukitt ( o. kindler gmbh , freiburg , germany ) . statistical analysis : statistical analyses were performed with excel statistics 2012 ( ssri version 1.00 , tokyo , japan ) . in the behavioral analyses , outliers more distant than 1.5 interquartile ranges from the upper or lower quartile were omitted . all data were analyzed by two - way anova ( ctd stress ) followed by the tukey - kramer s post hoc test . the results were considered significant when the p - value was less than 0.05 . gel intake , body and testis weights : the daily gel consumption , body weights and testes weights are shown in fig . 1fig . 1.effects of combined exposure on the daily gel intake ( a ) , body weights ( b ) and testis weights ( c ) in the non - stressed ( open column ) and stressed groups ( closed column ) . the two - way anova showed significant main effects for the ctd and stress ( p<0.01 ) , but the interaction effects were not significant in gel intake and body weights . ( a ) ctd dose - dependently inhibited the gel intake , and the inhibition was significant in all ctd - administration groups compared to the ctd-0 groups . the stress procedure significantly inhibited the gel intake in each ctd - administration group . ( b ) the body weights of the ctd-250 stress and non - stressed groups were significantly lower than those of the other dose groups , in addition to the significant difference between the ctd-0 and ctd-10 groups . the post hoc analyses also revealed that there were significant differences between the ctd-0 and ctd-250 stress and non - stressed groups . ( c ) the two - way anova showed a significant main effect for stress ( p<0.01 ) , but the interaction effect was not significant in the absolute weights of testes . the stress procedure significantly suppressed the absolute testes weights in all stress groups , except the ctd-10 group . ctd significantly suppressed the daily gel intake in all ctd administration groups compared to the non - ctd administration groups ( fig . although the daily intake was significantly inhibited in the stress groups , there was little difference if the values in the days of food deprivation in our stress protocol were omitted ( table 1table 1.body weight , gel intake , putative ctd exposure and testis weightsgroupsctd-0ctd-0 + stressctd-10ctd-10 + stressctd-50ctd-50 + stressctd-250ctd-250 + stressbody weight ( g)28.62 1.6626.48 0.7226.98 0.9925.36 1.2227.50 0.8225.24 0.7825.82 0.9923.76 0.40gel intake / day ( g)6.13 1.375.64 2.804.41 0.842.85 1.774.60 1.593.55 2.093.64 0.642.38 1.42 [ 6.66 2.56 ] [ 3.73 1.59 ] [ 4.65 1.79 ] [ 3.16 1.09 ] putative exposure ( mg / kg / day)008.82 1.685.70 3.5446.0 15.935.5 20.9182 32119 71 [ 7.46 3.18 ] [ 46.5 17.9 ] [ 158 54.5 ] testis weight ( mg)99.6 5.588.2 11.895.7 9.690.5 9.7100.3 9.881.1 8.297.5 7.887.5 the values in square brackets are the averages after the removal of values in the deprivation days from the calculation . ) . as a result , the body weights of mice reflected the same tendency of the gel intake . the body weights were suppressed in all stress groups relative to the non - stressed groups at the same dose of ctd ( fig . the body weights of the ctd-250 groups were significantly lower than those of the other three dose groups . on the other hand , the absolute weights of the testes were decreased by the stress procedure in the ctd-0 , ctd-50 and ctd-250 groups ( fig . effects of combined exposure on the daily gel intake ( a ) , body weights ( b ) and testis weights ( c ) in the non - stressed ( open column ) and stressed groups ( closed column ) . the two - way anova showed significant main effects for the ctd and stress ( p<0.01 ) , but the interaction effects were not significant in gel intake and body weights . ( a ) ctd dose - dependently inhibited the gel intake , and the inhibition was significant in all ctd - administration groups compared to the ctd-0 groups . the stress procedure significantly inhibited the gel intake in each ctd - administration group . ( b ) the body weights of the ctd-250 stress and non - stressed groups were significantly lower than those of the other dose groups , in addition to the significant difference between the ctd-0 and ctd-10 groups . the post hoc analyses also revealed that there were significant differences between the ctd-0 and ctd-250 stress and non - stressed groups . ( c ) the two - way anova showed a significant main effect for stress ( p<0.01 ) , but the interaction effect was not significant in the absolute weights of testes . the stress procedure significantly suppressed the absolute testes weights in all stress groups , except the ctd-10 group . the values in square brackets are the averages after the removal of values in the deprivation days from the calculation . 2.behavioral effect of combined exposure of ctd and stress in the open field activity in the non - stressed and stressed groups . ( a ) a representative trajectory map of the mice as illustrated by the video tracking software . the exploratory behaviors in the center zone ( 30 30 cm ) of the open field ( 60 60 cm ) were dose - dependently suppressed by ctd compared to the non - administration groups . ( b ) total distances traveled in the open field of the non - stressed ( open columns ) and stressed groups ( closed columns ) . no marked difference was detected by two - way anova in the total distances traveled . ( c ) time spent in the center zone in the open field of the non - stressed ( open columns ) and stressed groups ( closed columns ) . two - way anova showed significant main effects for ctd ( p<0.05 ) and stress ( p<0.01 ) , but the interaction effect was not significant . ctd significantly inhibited the times spent in the center zone in the ctd-250 groups compared to the non - ctd administration groups . a significant anxiogenic effect of the stress procedure was observed between the ctd-0 groups . in the non - stressed condition , there were significant differences in the ctd-10 and ctd-50 groups compared to the ctd-0 group . * p<0.05 , * * p<0.01 . ) revealed that the mice with the combined ctd exposure and stress procedure tended to walk alongside the walls . ctd and the stress procedure had insignificant impacts on the locomotor activities measured by the total distances traveled ( fig . by contrast , the anxiety - like behaviors that avoid being in the center zone were elevated by the stress procedure and were significantly different between the ctd-0 groups ( fig . ctd also enhanced the anxiety - like behaviors in a dose - dependent manner , and in the non - stressed condition , the difference was significant in the ctd-10 and ctd-250 groups compared to the ctd-0 group . behavioral effect of combined exposure of ctd and stress in the open field activity in the non - stressed and stressed groups . ( a ) a representative trajectory map of the mice as illustrated by the video tracking software . the exploratory behaviors in the center zone ( 30 30 cm ) of the open field ( 60 60 cm ) were dose - dependently suppressed by ctd compared to the non - administration groups . ( b ) total distances traveled in the open field of the non - stressed ( open columns ) and stressed groups ( closed columns ) . no marked difference was detected by two - way anova in the total distances traveled . ( c ) time spent in the center zone in the open field of the non - stressed ( open columns ) and stressed groups ( closed columns ) . two - way anova showed significant main effects for ctd ( p<0.05 ) and stress ( p<0.01 ) , but the interaction effect was not significant . ctd significantly inhibited the times spent in the center zone in the ctd-250 groups compared to the non - ctd administration groups . a significant anxiogenic effect of the stress procedure was observed between the ctd-0 groups . in the non - stressed condition , there were significant differences in the ctd-10 and ctd-50 groups compared to the ctd-0 group . histological and immunohistochemical findings : the general histological analyses of the testes by he staining are shown in fig the seminiferous tubules of the control group showed robust spermatogenesis with densely stacked germ cells . multinucleated giant cells ( arrow ) were occasionally observed in the testes of the ctd-0+stress groups . in the ctd groups , dose - dependent vacuoled degeneration ( arrowhead ) of seminiferous tubules was observed . degenerated seminiferous tubules only composed of sertoli cells were present in the ctd-250 groups . bar=100 m .. although there were seminiferous tubules densely arranged with germ cells and sperms in the testes of the control group , several testicular signs of toxicity were observed in combined exposure groups . multinucleated giant cells were occasionally observed in the seminiferous tubules of the mice in the stress groups . in addition , variably sized vacuolizations were scattered at the bottom of seminiferous tubules of the ctd - administered groups . in particular , seriously degenerated seminiferous tubules , from which almost all germ cells had dropped off , were found in the ctd-250 groups . there was no marked histological difference between the leydig cells in the testicular interstitial tissues of any of the experimental groups . the seminiferous tubules of the control group showed robust spermatogenesis with densely stacked germ cells . multinucleated giant cells ( arrow ) were occasionally observed in the testes of the ctd-0+stress groups . in the ctd groups , dose - dependent vacuoled degeneration ( arrowhead ) of seminiferous tubules was observed . degenerated seminiferous tubules only composed of sertoli cells were present in the ctd-250 groups . bar=100 m . the results of the immunohistochemical analyses of the testes visualizing gpx4 are shown in fig . 4.representative immunohistochemistry for gpx4 of the testis in the non - stressed and stressed groups . gpx4 immunoreactivities were detected in sperms and spermatids in the testes of the control group . in the testes of the stressed groups , reduced immunoreactivity of gpx4 in sperm was observed . in the ctd groups , spermatids showed weakened immunoreactivity of gpx4 . sertoli cells ectopically expressed gpx4 in degenerated seminiferous tubules of the ctd-50+stress group and the ctd-250 groups . bar=100 m .. in the control group , strong gpx4 immunoreactivity was detected in the sperm , and diffuse gpx4 immunoreactivity was seen in the cytoplasm of spermatids . the intensity of gpx4 immunoreactivity in the sperm was decreased by stress , and the cytoplasmic immunoreactivity in the spermatids was dose - dependently decreased by ctd . in addition , abnormal immunoreactivity of gpx4 in sertoli cells was detected in the combined exposure groups , and it became strong in degenerated seminiferous tubules in the ctd-50+stress group and the ctd-250 groups . representative immunohistochemistry for gpx4 of the testis in the non - stressed and stressed groups . gpx4 immunoreactivities were detected in sperms and spermatids in the testes of the control group . in the testes of the stressed groups , reduced immunoreactivity of gpx4 in sperm was observed . in the ctd groups sertoli cells ectopically expressed gpx4 in degenerated seminiferous tubules of the ctd-50+stress group and the ctd-250 groups . we verified the combined effects of ctd and stress on the reproductive and behavioral function in mature male mice by a non - invasive administration method . there are several advantages of the gels we used ; compared to baking pellets , the gels can uniformly deliver the test articles to animals without exposing them to high temperature . the gels could not completely cloak the bad palatability of ctd as reported earlier using similar gels mixed with an aromatase inhibitor , anastrozole ; however , the gel consumption observed in combined exposure groups was enough for maintaining the health of the mice . in our preliminary experiments , we initially suspected that the ctd avoidance effect was attributable to any accessory constituents of the commercial pesticide containing ctd , but ctd itself appeared to have some repellent effects on mice . in agricultural fields , ctd - containing pesticides are used at dilutions from 1:20 to 1:4,000 for treating seeds and plants . our purification protocol and the water solubility of ctd imply that the white precipitate resulting from the recommended concentration of ctd products is particularly dangerous for pesticide users . in the present study , the unpredictable stress model , which is often used in the assessment of antidepressant medications , evidently raised the anxiety level in the mice . ctd dose - dependently induced anxiety alone , but there was little difference in this measure among the combined exposure groups . to the best of our knowledge , there are only a few reports about the behavioral effects of neonicotinoids on mammals . several parameters of behavioral developmental tests that evaluated the sense of equilibrium , coordinated movement and muscular power of offspring were changed by maternal exposure to low - dose ctd , with a sex difference [ 34 , 35 ] . thiamethoxam ( tmx , 100 mg / kg / day ) significantly reduced the locomotor activity of rats in an open field test and induced anxiety in an elevated plus - maze test while suppressing acetylcholinesterase ( ache ) activity in three brain regions . considering that tmx is metabolized to ctd in mammals , our findings regarding the anxiogenic effects of ctd sufficiently agree with the findings of these prior studies . although the mechanisms of the behavioral effects of neonicotinoids remain unclear , cholinergic systems and downstream neurotransmitters are undoubtedly involved . an in vivo study revealed that ctd directly injected into the rat brain evoked a striatal dopamine surge through nachrs . early types of neonicotinoids caused a subsequent desensitization of nachrs as well as immediate neuronal excitation . the behavioral consequences of chronic nicotine intake are also attributed to both the upregulation and desensitization of nachrs . the anxiety - like behaviors in several behavioral tests are part of the phenotype of nachrs subunit 4 knockout mice , implying that the anxiogenic effect of ctd observed in the present study was presumably triggered by the chronic desensitization of nachrs . the chrna7 coding 7 subunit of nachrs contains a glucocorticoid response element , which may explain the decreasing nachrs 7 expression by chronic immobilization stress . these observations may support the hypothesis that the behavioral effects of ctd and stress partly share a common mechanism mediating weakened cholinergic activities , such as the desensitization of nachrs , the suppressed level of ache and the disturbance of the release of downstream neurotransmitters . in the ctd-250+stress groups , the stress procedure and the ctd - induced feeding suppression presumably fully suppressed the cholinergic activities , which made the anxiogenic effect of ctd invisible . on the other hand , we found that the chronic exposure to ctd and the stress influenced the testes in different ways . the stress procedure significantly decreased the testicular weight in addition to causing the emergence of the multinucleated giant cells , and both of these are common toxicant observations in testes produced by an abnormal meiotic division of germ cells . ctd did not show an impact on the testicular weight , whereas it dose - dependently degenerated the seminiferous epithelia . in particular , the sertoli cell - only seminiferous tubules found in the ctd-250 groups were unexplainable by ctd - induced chronic nutritional deprivation . in the clinical field , sertoli cell - only syndrome is known to result from partial deletions of the azoospermia factor region on the y - chromosome , which is one of the markers of male infertility . from the toxicological perspective , germ cells are vulnerable to damage from exogenous chemicals , and they are easily phagocytized by sertoli cells . several endocrine - disrupting compounds ( edcs ) including diethylstilbestrol and flutamide induce such a destruction of seminiferous tubules [ 1 , 11 , 40 ] . an increasing number of vacuolizations in seminiferous epithelia are also caused by the absence of germ cells , suggesting that they are the first target of ctd in the testes through the blood - testis barrier . this corresponds to the ctd dose - dependent increase of single - stranded dna immunopositive germ cells observed in the testes of quails [ 15 , 36 ] . gpx4 ( also called phospholipid - hydroperoxide glutathione peroxidase as a member of the glutathione peroxidase family ) is an antioxidant enzyme expressed in every mammalian organ . there are three types of transcripts from the same gpx4 gene : mitochondrial , non - mitochondrial and nuclear types were identified . the mitochondrial and non - mitochondrial types in particular are expressed in spermatocytes , and gpx4 is the principal antioxidant enzymes in the testes with 30 times greater expression in the testes compared to other organs . notably , in sperm , gpx4 is responsible for the abilities to scavenge approx . testis - specific gpx4 knockout mice showed a phenotype of infertility with degenerated testicular tissue and morphological aberration of sperm . the antigen of the polyclonal antibody used in this study is a common sequence among the three types of mouse gpx4 , and thus , we could not classify the types of gpx4 immunoreactivity . the weakened gpx4 immunoreactivity in the lumen of seminiferous tubules presumably resulted from the decreases in the number and quality of the sperm . neonicotinoids are known to have strong oxidizing properties that affect the liver and the reproductive and central nervous systems [ 7 , 19 ] . the present report is the first to describe the localized decrease of antioxidant enzymes by ctd in the testes of a mammal . similarly , oxidative effects of psychological stress via stress hormones have been reported [ 23 , 42 ] . although a constant level of reactive oxygen species is necessary for the proliferation of spermatogonial stem cells , the present results also showed that oxidative mechanisms partly explain how stress impacts the reproductive function cooperatively with ctd . although there are no reports of plentiful expressions of gpx4 in sertoli cells , strong ectopic expression of antioxidant enzymes was observed in sertoli cells in degenerated seminiferous tubules in the ctd-250 groups . because gpx4 mrna was not expressed in sertoli cell in rat , investigated whether gpx4 mrna in the testes was altered by several edcs , implying the measurements of oxidative conditions provide an indication of the testicular toxicity by environmental factors . in the present study , although no remarkable interaction effect was detected , we investigated whether ctd and stress affected the behavioral and reproductive functions additively rather than synergistically in adult mice . the endogenous nachrs agonist lynx1 was recently reported to serve as a molecular break system for brain plasticity . the developmental effects of neonicotinoids in juvenile and fetal periods remain to be determined . neonicotinoids and their metabolites are detected at high frequency in urine samples from japanese people . human exposure to ctd at a high concentration as high as that of the present ctd-250 groups does not occur naturally , but the precipitates that result from the low water solubility of ctd are very dangerous . in addition , a low concentration of ctd could become harmful under a stressed condition , such as fasting . the finding that one of the metabolites of imidacloprid ( imi ) seriously disturbs mapk / erk signaling compared to imi itself suggests that the safety coefficient ( 100 ) for calculating the acceptable daily intake is not satisfactory . taking the results of the present study as an example , it is clear that further research is needed to identify the toxicant mechanisms of environmental chemicals in order to protect humans and animals with high sensitivities under stressed conditions .

neonicotinoids , some of the most widely used pesticides in the world , act as agonists to the nicotinic acetylcholine receptors ( nachrs ) of insects , resulting in death from abnormal excitability . neonicotinoids unexpectedly became a major topic as a compelling cause of honeybee colony collapse disorder , which is damaging crop production that requires pollination worldwide . mammal nachrs appear to have a certain affinity for neonicotinoids with lower levels than those of insects ; there is thus rising concern about unpredictable adverse effects of neonicotinoids on vertebrates . we hypothesized that the effects of neonicotinoids would be enhanced under a chronic stressed condition , which is known to alter the expression of targets of neonicotinoids , i.e. , neuronal nachrs . we performed immunohistochemical and behavioral analyses in male mice actively administered a neonicotinoid , clothianidin ( ctd ; 0 , 10 , 50 and 250 mg / kg / day ) , for 4 weeks under an unpredictable chronic stress procedure . vacuolated seminiferous epithelia and a decrease in the immunoreactivity of the antioxidant enzyme glutathione peroxidase 4 were observed in the testes of the ctd+stress mice . in an open field test , although the locomotor activities were not affected , the anxiety - like behaviors of the mice were elevated by both ctd and stress . the present study demonstrates that the behavioral and reproductive effects of ctd become more serious in combination with environmental stress , which may reflect our actual situation of multiple exposure .

women have among the highest rates of new hiv infections globally and the rate of new infections has not lessened in recent years [ 14 ] . african americans account for 66% of new hiv infections among all us women over 5 times their proportion representation ( 12% ) in the population . one in 30 african - american women can expect to acquire hiv infection during their lifetime ; the vast majority of these are through heterosexual intercourse . crack - using african - american women are a distinct , hard - to - reach population for whom empowerment to reduce hiv / sti risk takes on additional complexities due to the entangled dependencies of drugs , partnerships with men ( who are often drug involved ) for material support for women and their children , and constant threat of violent reprisal [ 4 , 79 ] . concurrent infection with sti is considered to be endemic among african - american , drug - using , hiv+ women . women 's biological inequality in vulnerability to sexually transmitted infection ( sti ) can be addressed with greater availability and use of male and female condoms ( fc ) . in addition to challenges in the diffusion of available protection technologies , however , gender inequity in sexual relationships , limited bargaining power of women complicated by threats of violence , and insufficient resource commitment to health issues among minorities and the poor all contribute to women 's continuing entrenchment of hiv / aids in the united states . the fc is the only female - initiated protection method that protects against hiv / sti and unwanted pregnancy at a level equivalent to a male condom ( mc ; ) . full integration of the fc has lagged far behind in hiv prevention efforts [ 1214 ] . interventions for us populations at high risk infrequently ( 4 of 14 studies ) included information and counseling on the fc , although most studies were targeted to heterosexual risk groups . formal cdc documents on voluntary counseling and testing ( vct ) do not specifically refer to fc as integral to the counseling approach . effective interventions targeted to drug - using women to reduce sexual risk are few in number and , as pointed out by wechsberg et al . , have rarely involved follow - up intervals of longer than 6 months . studies with short - term followup evaluating effective intervention approaches have included two woman - focused interventions : the co - op model as well as the nida standard model tested by sterk and colleagues , as against two enhanced models . the womens ' co - op intervention is a brief 4-session intervention with mixed individual and group sessions , based on empowerment and feminist theory , encouraging women to understand their hiv risks in the context of substance abuse and effects on personal power and vulnerability to victimization . numerous adaptations have been conducted since the first trial of the intervention , which resulted in its recognition as a best evidence intervention , based on results from 6-month followup . long - term followup ( 4 years ) of 60% of the original study population , however , found evidence of differential attrition favoring the retention of the high - risk participants and lack of long - term sustained salutary effects observed at 6 months . sterk and colleagues followed 333 mainly crack - using women who were randomly assigned to three different conditions . with 96% retention at 6 months , positive change was demonstrated in drug use behavior and sexual risk behavior , as well as drug - related sexual risk behavior such as sex trade . however , no differences among the nida standard and woman - focused interventions were found . the enhanced interventions were based on five behavioral theories with a gender- and culture - specific focus . all conditions involved access to prevention methods and included practice of negotiation and protection techniques and technologies . the behavioral intervention tested here ( bestbet ) integrated elements from three existing theories the theory of gender and power , community empowerment theory , and risk reduction / harm reduction theory as well as an original theory of body empowerment . the latter draws heavily from feminist health principles espoused widely in the 1970s in such works as our bodies , ourselves . the bestbet intervention was initially developed as a multiple session , clinic - based intervention among hiv+ women , and has evolved through a series of studies on diverse populations of high - risk women [ 2325 ] . increased body knowledge appeared to facilitate use of women 's barrier methods in these studies because risk behavior declined . we believed that active crack / polydrug users would attend a relatively long , multisession intervention with women - focused content despite the inherent physical and mental challenges involved . we posited that the woman - specific intervention themes would have cross - cutting relevance and produce positive behavioral change among this population at very high sexual risk for hiv / sti . in this paper , we describe a randomized controlled trial of an intervention ( bestbet ) from a sample of 198 drug - using women with high - risk sexual behavior who were followed for 12 months . the feminist health model as applied to hiv underscores the need for holistic education about reproductive organs and genitals , rather than a narrow focus on hiv . our intervention included session content on normal female anatomy , the menstrual cycle , pregnancy , menopause , sti signs and symptoms , female cancers , benefits of screening ( mammography and pap smear ) , and common surgeries . the body empowerment approach posits that increased knowledge , sense of ownership , and pride in the body ( body knowledge ) are an independent pathway to self - esteem and to self - efficacy for protection behavior use of female - initiated barriers , mc negotiation with partners , and refusal of unsafe sex . thus , we introduced to intervention women the entire range of female methods thought possibly to reduce risk of sti / hiv female condom , diaphragm , cervical cap , and spermicides ( the study was initiated prior to the findings concerning vaginal irritation with nonoxynol-9 ; we counseled women to abandon use of spermicides for disease protection following publication of the findings)arranged hierarchically according to what was known about protection level in a harm reduction approach to increasing protection behavior ( i.e. , something is better than nothing ) . we also focused on the need for female solidarity and mutual support . these four pillars body knowledge , access to female initiated methods , emphasis on harm reduction , and a setting promoting female solidarity provided the intervention core components . we integrated content on female and male barriers in each intervention session , focusing on practical applications of the fc with diverse partners , including paid sexual transactions ( e.g. , cheeking or oral application of mc without partner initiation ) concomitant with drug use ( e.g. , inserting an fc before getting high ) , and situations of potentially violent partners ( inserting cervical barrier with spermicide for discreet risk reduction ) . women tried the female barriers at home and shared their experiences at the following group session , where troubleshooting for problems encountered in the initial adoption period was facilitated by a trained counselor . the impact of basic body education coupled with access to a full range of female barrier options on decreasing sexual risk behavior has not , to our knowledge , been evaluated in active substance users . for this trial , we enhanced certain elements and adapted the study to target out - of - treatment drug users . we added a session dedicated to intimate partner violence ( for paying , nonprimary , and primary partners ) including a module on basic self - defense techniques and exit strategies and use of protection methods in the context of forced sex . the intervention was held at a university - operated , community storefront site with a long history of provision of hiv counseling and testing in a poor , inner city neighborhood . the elements of the intervention were ( 1 ) small groups with interactive counseling ; ( 2 ) trained and certified near - peer community counselors with a history of substance abuse and in recovery for at least 2 years ; ( 3 ) role play and rehearsal , ( 4 ) multimedia educational approaches , such as videos , audio tapes , brochures , posters , and anatomical models ; ( 5 ) active referrals to local service organizations ; ( 6 ) access to hiv testing ; and ( 7 ) training of cbo staff for community capacity building . audio tapes of diverse risk vignettes based on interviews with the target population and accompanying illustrated handouts were prepared by an outside nonprofit health organization . we collaborated with a nearby branch of a national family planning organization to facilitate diaphragm and cervical cap fittings and offered patient advocate support to accompany study participants to clinic appointments . to be eligible for the bestbet study , we required women to ( 1 ) be 18 years of age or older , ( 2 ) be hiv seronegative , ( 3 ) report 30% or more unprotected vaginal or anal sex acts over the preceding three months ( averaged over all partners ) , ( 4 ) report not currently being in drug user treatment other than with methadone , and ( 5 ) report that heroin or cocaine were either injected , snorted , or smoked at least 12 times during the same three - month interval . women were excluded for psychiatric problems or if they had been in drug treatment for more than 6 months . because changing drug use behavior was not an explicit study objective , we did not exclude women from attending sessions if they were high except if such use disrupted group dynamics . eligible women were recruited in philadelphia between november 2001 and august 2003 , with the use of a mobile outreach van staffed with trained interviewers and harm reduction counselors . the van was parked in designated , high - risk areas known for crack - selling and smoking activity . interested women gave the first written consent to be administered a 20-minute , confidential prescreening assessment interview that included demographic and behavioral risk items as well as to provide locator information . the second screening assessment was conducted at a community storefront site that served as an information and referral center for drug users . recruitment and study methods have , in part , been presented in a prior publication . this study was approved by and conducted in compliance with the institutional review board of the university of pennsylvania . participants completed a baseline risk assessment instrument delivered via audio , computer - assisted self - interview ( a - casi ) targeting sexual and drug user behavior over prior 6 months . additional baseline data were collected in a face - to - face interview including the following : demographic and reproductive health history , history of sti , health care insurance , and types and location of health care services used in past 6 months . following baseline data collection , all women received enhanced hiv and sti harm - reduction counseling that exceeds standard guidelines for hiv voluntary counseling and testing ( vct ) ; both the mc and fc were taught . we counseled women with sti on treatment locations and required these women to bring written confirmation of treatment for formal enrollment . we provided hiv - positive women with referrals for further counseling and care but they were excluded from this study . women randomized to the intervention arm were invited back for five , 3-hour , weekly group sessions within 2 months of enrollment and were contacted for 6- and 12-month reunion sessions at community sites . control participants received limited case management and the offer of free mcs and fcs , but no other proactive study intervention . at 6- and 12-month following enrollment , all assessments were repeated at the community sites or other locations when necessary . masters ' level interviewers were trained and certified in vct and in standardized interviewing techniques . intensive retention efforts ( phone , mailed correspondence , and in person ) were used to follow participants considering their mobility , including community outreach , visits to known crack houses and other hang - outs , and hospital and prison outreach . retention challenges were discussed weekly in study meetings . using multiple methods and extensive efforts to locate hard - to - reach participants , the retention rate was 95% at 12 months . to test for differences between the control and intervention groups at baseline , chi - square for dichotomous variables and independent sample t - test for continuous variables were conducted for demographic variables and background characteristics . univariate analyses including frequencies and percentages were completed to describe the total sample and its subsets at baseline and 12-month follow - up . our main outcome variable , frequency of unprotected vaginal intercourse ( monthly ) , is the focus of this paper . data for this analysis were derived from a series of questions delivered via a - casi , asking first , the total frequency of acts during the interval ( vaginal , anal , and oral ) followed by the frequency of acts protected by mc or fc , diaphragm , cervical cap , or spermicide ( these latter three related only to vaginal sex ) . the remainder was defined as unprotected vaginal sex acts . protected and unprotected vaginal sexual acts were calculated at baseline and at 12-month followup for two types of partners : primary partners and hiv - negative nonprimary partners . the number of hiv - positive nonprimary partners was prohibitively small to include for analysis . a primary ( male ) partner was described as someone you have lived with or have seen a lot , and to whom you have felt a special emotional commitment . if a woman answered that she did not have vaginal sex in the prior interval ( this was only possible at followup ) or did not have that type of partner , she was considered as having zero frequency of unprotected acts . for this study we used an intent - to - treat analysis with the inclusion of women who may not have been sexually active during followup and/or may not have had a primary or nonprimary sexual partner . pairwise analysis was performed on the outcome variable separately for primary and nonprimary partners from baseline to followup , for each intervention arm , which included generating a paired sampled t - test ( p < 0.05 was considered significant ) . for comparison of intervention versus control arms , first a change either parametric ( student 's t - test ) or nonparametric tests ( mann - whitney u test ) were used to assess statistical significance , depending on the normality of distribution . all analyses were conducted using spss version 19.0 ( spss , inc . , chicago , illinois ) . a total of 1134 women were prescreened at the van . of the 616 eligible women , 304 ( 49% ) successfully completed the baseline interviews and specimen collection and were invited to participate in the bestbet study . among eligible women completing baseline interviews ( n = 304 ) , of these women , 227 ( 75% ) returned for enrollment procedures and were randomized ( 113 as intervention subjects and 114 as controls ) . twenty - nine women were later excluded based on contradictory information related to eligibility criteria ( see figure 1 ) . a slightly greater percentage of eligible women who completed baseline interviews , as compared to the final study group , reported drug injection ( 49% versus 42% ) and somewhat fewer had health insurance ( 65% versus 70% ) . the majority of the study participants were african - american ( see table 1 ) . a majority of participants were unemployed with nearly two - thirds reporting use of food stamps . most women had a history of drug treatment , with crack / rock cocaine and marijuana as the drugs of choice . most participants ( c 79.8% , i 85.9% ) had a primary male sex partner , and more than half ( c 56.6% , i 60.6% ) had both a recent nonprimary male sex partner(s ) and a primary partner . women with primary partners reported substantial levels of recent ( past 6 months ) intimate partner violence : 21% had partners who had made threats on their life , 34% had been forced to have vaginal or anal sex without a condom , 25% had been punched or hit with something that hurt , and 11% had consulted medical care because of a fight ( data not shown ) . no significant differences across arms were found . at baseline , the overall mean frequency of unprotected vaginal sex acts ( all enrolled women ) with primary partners in the past 6 months was 34.3 ( median = 12 ) , and with nonprimary , hiv - negative partners was 22.7 acts ( median 0 ) . at 12-month followup , the overall mean frequency of unprotected vaginal sex acts ( all enrolled women ) with primary partners was 15 . 6 ( median 0 ) ; with nonprimary hiv - negative partners the follow - up mean frequency was 2.9 acts ( median 0 ) . a total of 189 women completed assessments at 12-month followup ( retention rate : 95.4% ) . for the paired baseline - to - followup comparisons , among controls , for primary partner , baseline frequency was 32.7 acts ; at 12 months this was reduced to 19.3 acts ( paired t - test , p = 0.008 ; see table 2 ) . among intervention women , mean of unprotected vaginal acts at baseline for primary partner was 35.9 and at follow - up was 12.3 ( paired t - test , p = 0.000 ) . for control women with nonprimary hiv partners , mean baseline frequency of unprotected vaginal acts was 21.6 acts ; at 12-month followup frequency was 2.5 ( paired t - test , p = 0.000 ) . among intervention women , frequency of unprotected vaginal acts with non- primary hiv - partners at baseline was 23.8 , and at 12-month followup was 3.5 ( paired t - test , p = 0.002 ) . a statistical test of the change over time ( i.e. , reduction in unprotected acts ) , per partner type , compared across arms , indicated borderline significance for primary partners ( p = 0.075 ) , favoring the intervention arm . for nonprimary partners , no statistical difference could be detected between the change observed across the two arms from baseline to followup ( p = 0.80 ) . proportions of women reporting mc use were greater in both arms in cross - sectional analyses comparing baseline to followup . mc use with primary partners was lower than with nonprimary partners across both arms , at both baseline and followup ( table 3 ) . intervention women reported mc use with primary partners more frequently at followup but the difference across arms was not statistically significant for either primary or nonprimary partners . mc use with nonprimary partner was reported by two - thirds ( i ) and three - quarters ( c ) of women ; the higher rates in i women at follow - up were achieved despite lower usage at baseline with nonprimary partners ( i versus c ) . use of the fc was also substantially higher at followup than at baseline in both arms . the lowest proportions of women reporting fc use at followup were c women with nonprimary partners ( 7% ) . use of spermicide and cervical barriers was also greater at followup compared with baseline for all women except c women with their nonprimary partners . very few protected acts involved spermicide use alone and only 1 woman reported use of a cervical barrier unaccompanied by either mc or fc use . thus , followup method use as compared with baseline method use appeared to increase the least when considering the subgroup control women - nonprimary partners . for i women , by contrast , there were consistent and substantial differences when comparing baseline to followup , with both partner types and across all methods . the sample of active , drug - using women in this study demonstrated large changes in risk behavior over 12-month follow - up . frequency of unprotected sex acts dropped significantly for both study arms , across both partner types . the difference in frequency of unprotected sex for both study arms was modified by partner type . for primary partners , statistical significance would likely have been realized with greater numbers due to the large variability in the outcome measure . for nonprimary partners , there was no observed difference across the study arms ; women in both arms reported substantial increases in protection . our findings agree with those of others indicating that use of mc is less frequent with a primary partner than with nonprimary partners ; nevertheless , the results with primary partner for intervention women are encouraging as these partners are often considered to pose a greater risk of sti / hiv transmission to drug - using women . although both types of counseling reduced unprotected acts , the value - added impact of the group - based intervention appeared to reside mainly with primary partners . in our study , fc use also increased in both arms , with greater and more uniform increases among i women . in particular , at followup , fewer c women used fc with nonprimary partners , with proportions that were significantly different from i women . we have previously shown that introduction to the fc and other female protection methods promoted mc negotiation and possibly a greater rejection of unprotected sex . these findings taken together suggest that our group - based intervention treatment was superior in encouraging behavior change . prior studies among drug - using women have often suffered from short follow - up periods . study participants who used fc found the method sufficiently practical to use with primary and nonprimary partners . there has been a tendency to discount female barrier methods as impractical and unacceptable though no wide - scale evidence supports this contention and indeed there is evidence to the contrary [ 13 , 14 ] . women have potentially more control over fc ( versus mc ) use but its impact will only be realized with strong promotional programs and quality counseling including outreach activities to men wherever possible and easy access to continuing supplies , to ensure adoption and maintenance of the behavior . the fc should be a regular component of vct , formalized into easily accessible counseling guidelines and promoted widely . a separate analysis of knowledge changes with this intervention suggests that a key component of fc adoption is empowerment of women through better knowledge of their bodies . such education should be incorporated routinely into future interventions and programs for drug - using women including , importantly , woman - focused drug treatment programs , still few in number and often lacking a reproductive health approach . our body empowerment approach and counselor training supplied women with key elements to boost the use of female - initiated methods , including cervical barriers and devices that are important and relevant to drug - using women but poorly promoted and underresearched as a dual protection method . study participants were selected on the basis of specific criteria drawn up to concentrate a high level of drug - related and sexual risk . they agreed and were expected to attend five , relatively long , intervention sessions and return for follow - up assessments . other drug - using populations with a lower risk profile or less availability for study procedures might have demonstrated different results . nevertheless , we used a - casi , an established technique to increase the validity of reporting of sensitive behavior in numerous populations . additionally , there was high variability in frequency of reported unprotected acts at both time points . these considerations tend to mitigate concerns about reporting bias , though there is no way of eliminating this possibility . comparisons of fc and mc use proportions were cross - sectional due to high levels of changes in partner and sexual activity . drug - using women continue to be at extremely high risk for hiv / sti , with large demonstrated racial disparities , despite successful inroads made for other at - risk populations in the interim , such as non - injection - using men who have sex with men ; additional approaches are still urgently needed . arm , as we designed two alternative counseling approaches that could be used in the field ; thus changes over time can not be attributed to the intervention procedures with complete confidence and may be due in part to the additional time duration spent with counselors . of concern , we may well have underestimated the impact of our intervention as compared to mc counseling only , which is practiced widely . also , our analysis was based on an intention - to - treat approach ; women assigned to the intervention arm did not necessarily complete all five sessions . the sample size may have been too small to detect some changes that were statistically significant for example , the differences within main partner across intervention and control arms . finally , contamination of the message across study arms was certainly possible , given the relatively small geographic recruitment area and overlapping social networks ; this too would have acted to minimize observed differences across the study arms . the hiv primary prevention landscape is changing , with oral preexposure prophylaxis ( prep ) as well as postexposure prophylaxis ( pep ) now documented to be valuable tools in the prevention mix and potentially applicable to drug - using populations . the potential for reducing sexual risk with vaginal prep for women is still unclear , with conflicting and recently disappointing results regarding the efficacy of vaginal tenofovir [ 37 , 38 ] . a daily , voluntary application of an antiviral vaginal compound may suffer from poor adherence ; long - term intact options may be considerably more effective . as additional topical microbicidal compounds complete testing , including microbicidally impregnated vaginal devices , the counseling on the use of such methods , especially among women at high risk such as idu and nidu women , will become more routine . our results suggest that active drug - using women at high risk of sexually transmitted hiv / sti will partake of such counseling and capitalize on expanded access to these technologies .

background . we tested an original , woman - focused intervention , based on body empowerment , and female - initiated barrier methods , including the female condom ( fc ) and cervical barriers . methods . eligible women were > = 18 years of age , hiv seronegative , and active drug users , reporting 30% or greater unprotected sex acts . both controls ( c ) and intervention ( i ) participants received enhanced hiv / sti harm reduction counseling . i participants underwent 5 additional weekly group sessions . we compared change in frequency of unprotected vaginal intercourse across arms at 12 months . results . among 198 enrolled women , over 95% completed followup . two - thirds were african - american ; most of them used crack , had a primary partner , and reported sex exchange . in paired t - tests from baseline to followup , the frequency of unprotected vaginal sex dropped significantly for i ( primary p < 0.00 , nonprimary p < 0.002 ) and c ( primary p < 0.008 , nonprimary p < 0.000 ) arms with all partners . the difference in change across arms was of borderline significance for primary partner ( p = 0.075 ) ; no difference was seen for nonprimary partner ( p = 0.8 ) . use of male condom and fc increased with both partner types over time , but more consistently among i women . conclusion : the value - added impact of the intervention was observed mainly with primary partners . body knowledge with routine fc counseling should be incorporated into interventions for drug - using women .

vitamin d is a fat - soluble vitamin obtained by the human body in two possible ways . there can be a dietary intake , mainly through fatty fish , eggs and fortified food as well as endogenous production , where transformation of 7-dehydrocholesterol into vitamin d in the skin occurs after exposure to ultraviolet b radiation . in the liver , vitamin d is hydroxylated to 25-hydroxyvitamin d. subsequent hydroxylation in the kidney forms the active metabolite , 1,25-oh vitamin d. in pregnancy , there is a 2-fold higher concentration of 1,25-oh vitamin d in maternal serum due to activity of placental 1--hydroxylase ( novakovic et al . , 2009 ) . vitamin d not only influences bone mineralisation but has implications on maternal and fetal well - being . 25-oh vitamin d deficiency is associated with an increased risk of developing preeclampsia ( bodnar et al . , 2007 ; haugen et al . , 2009 ) , multiple sclerosis ( hayes et al . , 1997 ; pierrot - deseilligny , 2009 ) and schizophrenia ( mcgrath , 1999 ; mcgrath et al . , there is also an association with type 1 diabetes ( stene et al . , 2000 ) and asthma ( brehm et al . , 2009 ; erkkola et al . , 2009 ; willers et al . , 2007 ) after deficiency in utero or in early life . neonates from deficient mothers have lower birthweight ( mannion et al . , 2006 ; sabour et al . , 2006 ; scholl and chen , 2009 ) and at age 9 still demonstrate a lower bone mineral content ( javaid et al . , 2006 ) . in vitro studies have suggested a protective effect of 25-oh vitamin d against malignancies such as breastcancer and cancer of the colon . risk factors for developing deficiency have been identified ; living in northern latitudes , limited sun exposure , dark skin , poor social circumstances and extensive clothing are amongst the most common ones ( baile et al . , 1979 ; , 2009 ; mulligan et al . , 2009 ; sachan et al . , 2005 ; , 2009 ; taha et al . , 1984 ; waiters et al . , 1999 ; this makes immigrants in northern countries , especially when dark skinned and/or covered , extremely vulnerable ( bowyer et al . , 2009 ; , 2006 ; wielders et al . , 2006 ) ( clemens et al . , 1982 ) . results of vitamin d status in the pregnant flemish population have never been published before . this pilot study aims at determining the prevalence of vitamin d deficiency in the antwerp population . the pilot study ran from august 1 , 2009 until november 30 , 2009 after approval of the local ethical commitee was obtained . in each blood sample that was taken from pregnant women consulting the antenatal clinic , we determined 25-oh vitamin d. the nurse taking the blood samples put every patient in a category according to their sun exposure . there were three possible categories : not covered , covering of only the head and leaving arms exposed to sun or complete covering with no sun exposure except for the face . because part of the period studied coincided with the month ramadan , we also asked the women whether they were fasting or not . our laboratory uses the elecsys 25-oh d3 immunoassay ( leino et al . , 2008 ) for determination of 25-oh vitamin d3 levels . this immunoassay is performed on a modular analytics e170 apparatus ( roche diagnostics , manheimm , germany ) . the measuring range is 4 - 100 ng / ml ( 10 - 250 nmol / l ) , the reference range for hypovitaminosis in our laboratory is < 30 ng / ml , corresponding the health based reference values . vitamin d levels between 16 - 20 ng / ml are seen as mild deficiency , 6 - 16 ng / ml as moderate deficiency and values lower than 6 ng / ml as severe deficiency . for the statistical analysis , we used spss statistics 17.0 ( spss inc . , chicago , illinois ) . normality was tested with the kolmogorov - smirnov test , the means of vitamin d between all three groups were compared with anova - analysis . the means of each group according to exposure were compared with an independent samples t - test . to compare the groups of women who used a prenatal vitamin and those who did not we also used the independent samples t - test . linear regression was used to analyse wether vitamin d level is influenced by gestational age , maternal age , parity , gravidity , sun exposure , intake of supplements and/or date of blood sample . for all tests significance was accepted at p < 0.05 . the odd s ratio between covered and non - covered women with vitamin d levels lower than 30 ng / ml was calculated , and 95% confidence intercal group were also compared using the chi squared test . we determined 25-oh vitamin d3 in 171 women ( n = 171 ) . the mean age of this population was 29,1 4,6 ( standard deviation or sd ) years . the median gestational age was 24 weeks ( range : 4 - 37 ) , most women were primiparae ( 72/171 , 42,1% ) . the number of fasting women was too small ( 4/171 , 2,3% ) to draw any conclusions . 17% ( n = 29 ) of the women was taking a multivitamin preparation . they all used the same brand containing 10 microgram ( 400 iu ) of vitamin d. in the entire population the mean 25-oh vitamin d value was 28 12,4 ( sd ) ng / ml . there was no effect of age , gravidity , parity , intake of supplements nor gestational age on the vitamin d level . there was however a statistically significant influence of date of blood sampling and sun exposure ( p = 0,001 ) . the mean vitamin d value of women taking supplements was 28,5 12,5 ( sd ) ng / ml , the group without supplements had a mean of 27,9 12,5 ( sd ) ng / ml . the difference in means between these two groups was not significantly different ( p = 0,8 ) . of all women 86% ( n = 147 ) was not covered , 10,5% ( n = 18 ) wore head covers but had other body parts exposed to the sun , 3,5% ( n = 6 ) was completely covered , leaving only the face open for sun exposure . there was a significant effect of degree of body covering on vitamin d level ( linear regression , p < 0,001 ) . the mean 25-oh vitamin d value in the non - covered group was 29,5 12,2 ( sd ) ng / ml . there was a significant difference in vitamin d level between the three groups ( anova , p = 0,001 ) ( fig . 1 ) . the mean of the partially veiled group , 17,2 7,2 ( sd ) ng / ml , was significantly lower than the mean of the non - veiled group ( t - test , p = 0,001 ) . the mean of the completely veiled group , being 22,5 12,9 ( sd ) ng / ml was not significantly higher than that of the partially veiled group and not significantly lower than the non - veiled group ( t - test , p > 0,05 ) . when we only compare covered versus non - covered groups , there are significantly more covered than non - covered women with a concentration of vitamin d lower than 30 ng / ml ( odds ratio 6,2 ; 95% ci : 1,8 - 21,7 ; p < 0,05 ) . the mean vitamin d value of our population , 28 ng / ml , was below the reference range used in our laboratory . this suggests a widespread shortage of vitamin d in our population , 1,75% was suffering from severe deficiency , 9,9% had moderate deficiency and 18,1% had mild deficiency . the values used are based on concentrations that avoid development of rickets and osteomalacia ( vieth et al . , 2007 ) . with new date on the role of vitamin d besides bone metabolism , some authors are pleading for higher cut - off values to determine deficiency ( norman et al . heaney ( 2005 ) states that 32 ng / ml should be considered as a minimum for normal physiology , bischoff - ferrari ( 2008 ) defines values of 36 - 40 ng / ml as optimal serum concentrations . we found no effect of maternal age , gestational age , parity , gravidity and intake of supplements on the vitamin d level . no consensus exists on the recommended intake and supplementation of 25-oh vitamin d during pregnancy . , 2007 ; madelenat et al . , 2001 ; saadi et al . , 2007 ; , 2009 ; yu et al . , 2009 ) and several authors agree that the current recommended intake of 200 - 600 iu ( or 5 - 15 g ) is too low , daily requirements may be closer to 1000 iu ( 25 g ) or higher ( bisschof - ferrari et al . the most commonly prescribed multivitamin preparation in belgium , only contains 10 g ( 400 iu ) of vitamin d , a value that will not suffice to maintain or build sufficient levels . this fact is confirmed in our pilot study : no significant difference in vitamin d levels between women taking supplements and those who did not . there clearly was an effect of sun exposure and date of sampling , probably because august has more sun hours than november . in the population studied , the non - covered women had higher mean serum concentrations of vitamin d compared to partially covered women . the higher mean values of the completely covered group were probably due to small group size and were not statistically significant compared with other groups . however , we did not classify the participants according to their nationality nor their skin tone and as most women wearing head / body cover also have a darker skin , this is a fact that we should have taken into account . furthermore , most of the covered women are immigrants who often live in poorer social circumstances . hence , their lower vitamin d levels might not be only attributable to wearing head / body cover but also to poor dietary intake . although most of the vitamin d production comes from skin conversion , the dietary intake of vitamin d is something we ll have to examine in further studies , as well as darkness of skin and nationality . as this was a pilot study , we only measured 25-oh vitamin d. in the future , we will start including other relevant parameters such as medical history , serum levels of calcium and parathyroid hormone . low vitamin d levels are frequently found in pregnancy but the optimal serum concentration remains unknown . , we found that low vitamin d levels in the antwerp population are frequent and that there is an effect of the seasonal moment of the year and degree of sun exposure . a large scale study is needed to come to clinical guidelines and recommendation for obstetricians .

introduction : vitamin d deficiency in utero or early neonatal life may have a major impact on children s health . little is known on vitamin d deficiency in pregnant women in belgium , non on the impact of wearing head and/or body cover.objectives : this was a preliminary exploration of the vitamin d status in pregnant women visiting the antenatal clinic in the antwerp university hospital.method : from august 1 2009 until november 30 2009 we systematically determined 25-hydroxy vitamin d ( 25-oh vitamin d ) in each blood sample taken from pregnant women visiting the antenatal clinic . we also registered the degree of head / body cover and inquired for intake of vitamin supplements.results : our population consisted of 171 women , mostly primiparous , of which 86% were not covered . the mean value of 25-oh vitamin d was 28 ng / ml . non - covered women had a mean of 29,5 12,2 ( sd ) ng / ml , the partially covered group had a mean of 17,2 7,2 ( sd ) ng / ml and the completely covered group had a mean of 22,5 12,9 ( sd ) ng / ml . the difference in serum concentrations between the 3 groups was statistically significant ( anova , p < 0,00001 ) . there were significantly more covered than non - covered women with a vitamin d concentration lower than 30 ng / ml ( or6.2 ; 95% ci : 1,8 - 21,7 ; p < 0,05).there was no effect of gestational age , maternal age , gravidity , parity and intake of supplements on vitamin d levels . there was a siginificant seasonal effect from summer to fall , with vitamine d levels lowering from august to november ( linear regression , p < 0,05).conclusion : low vitamin d levels seem to be frequent and covered woman are at a higher risk of deficiency .

a 7-year - old spayed domestic longhair cat from perth , western australia , presented with left - sided head tilt , dysphonia , head shaking , inappetence and weight loss . otitis media with extension into the external ear canal was suspected and investigated using video - otoscopy and computed tomography examination . invasive disease with extension from the middle ear to the base of the skull , and intracranial extension into the caudal fossa and cranial cervical vertebral canal was detected . cytology of external ear canal exudate showed capsulated budding yeasts and cryptococcus gattii vgii was cultured . treatment with amphotericin b infusions and oral fluconazole was prescribed , with nutritional support via oesophagostomy tube . this case report describes the successful medical treatment of otogenic meningoencephalomyelitis due to c gattii ( vgii ) infection in a cat . cryptococcosis is a common systemic mycosis affecting cats worldwide and can affect cats of any age and sex . the two most common species causing disease in animals are cryptococcus neoformans and cryptococcus gattii . historically , cryptococcus was classified into five serotypes ( a , b , c , d and ad ) on the basis of antigenic differences in capsular polysaccharides . using a modern molecular typing scheme , cryptococcal strains are now divided into eight multilocus sequence types ( vni , vnii , vniii , vniv , vgi , vgii , vgiii , vgiv ) , which are likely to be cryptic species . c gattii has been associated with tropical and subtropical climates , with an outbreak of disease affecting both humans and animals in the pacific northwest of the usa , emanating from an initial focus of infection on vancouver island , british columbia , canada . c gattii primarily infects immunocompetent hosts and the organism can cause disease in unusual host species ( eg , horses , ferrets , goats , dolphins ) . vgii has a high environmental presence in perth , western australia , and areas of the northern territory , in contrast to eastern australia where vgi isolates predominate . the genetic diversity of vgii environmental and clinical isolates obtained from the perth environs suggests there is a sexually recombining population structure in this geographical niche . while c gattii vgi is strongly associated with detritus in mature eucalyptus tree hollows , the ecological niche of vgii is largely unknown . in vancouver island , where an outbreak of vgiia was diagnosed in humans and animals , c gattii vgii colonises coniferous , evergreen and deciduous trees in the douglas fir tree bioclimatic zone , with transient isolation from soil , saltwater , freshwater and air . infection most likely occurs subsequent to inhalation of airborne infectious propagules such as basidiospores or desiccated yeast cells with clinical signs of sneezing , stertor , nasal deformity and discharge reflecting nasal cavity infection . it is common for the infection to spread , both locally to contiguous adjacent structures and haematogenously to other sites . we present an unusual case of otogenic infection with intracranial extension attributable to c gattii vgiib in a cat for which signs resolved with aggressive medical therapy . a 7-year - old spayed domestic longhair cat was presented to murdoch university veterinary hospital for evaluation of left head tilt , dysphonia , head shaking , inappetance and weight loss of 2 weeks duration . initial investigations by the referring veterinarian identified an aural mass in the left horizontal ear canal , which had been removed . sabouraud s dextrose agar culture of an ear swab had resulted in heavy growth of c gattii . serum latex cryptococcal antigen agglutination test ( lcat ) was positive , with a titre of 1:256 . the cat was treated with enrofloxacin ( 5 mg / kg po q24h [ baytril ; bayer animal health ] ) , itraconazole ( 10 mg / kg po q24h [ sporanox ; janssen pharmaceutica ] ) , meloxicam ( 0.05 mg / kg po q24h [ meloxicam ; troy laboratories australia ] ) and a topical compounded enrofloxacin / dexamethasone otic preparation . the cat was thin ( body condition score [ bcs ] 3/9 ; weight 4.9 kg ) and had a left - sided head tilt . the remainder of the clinical examination , including full ophthalmic and neurological examinations , was unremarkable . the presence of a head tilt without ipsilateral postural reaction deficits was consistent with left peripheral vestibular disease . hand - held otoscopic examination demonstrated a mass occluding the lumen of the left horizontal ear canal , and ceruminous discharge . the cat was premedicated with buprenorphine ( 0.018 mg / kg i m [ temgesic ; symbion pharmacy services ] ) and acepromazine ( 0.04 mg / kg i m [ acp ; westralian holdings ] ) , anaesthetised with propofol and maintained with isoflurane in 100% oxygen . a ct ( siemens emotion duo 2 slice ct scanner ; ge healthcare australia ) examination of the head was performed using soft tissue and bone spatial reconstruction algorithm and 1 mm slice thickness pre- and postintravenous contrast ( iohexol ; 10 ml [ 300 mmol / l ] iv ) ( figure 1 ) , followed by video - otoscopic examination . ( a ) the medial portion of the left external ear canal is filled with strongly and homogeneously contrast - enhancing soft tissue attenuating material ( 1 ) . the left tympanic bulla is filled with mildly and homogeneously contrast - enhancing material ( 3 ) . ( b ) the material seen ventromedial to the tympanic bulla continues caudally and forms a 1.6 cm ( width ) 1.3 cm ( height ) 2.5 cm ( length ) soft tissue structure , poorly enhancing centrally but strongly enhancing peripherally ( 1 ) . this mass ( * ) distorts local tissue architecture such that the nasopharyngeal lumen is narrowed > 50% , and the hyoid bones are displaced laterally . additionally , at this level , there is a rim of contrast enhancement seen in the ventral aspect of the left temporal lobe of the cerebrum ( 2 ) . ( c ) at the level of the caudal aspect of the left tympanic bulla , the soft tissue lesion can still be seen ventromedial to the bulla ( 1 ) . additionally , there is a rim of contrast enhancement outlining a hypoattenuating area in the left lateral cerebellum ( 2 ) . there is poorly contrast - enhancing soft tissue attenuating material in the caudal fossa that is displacing the brainstem dorsally and to the right ( 3 ) . additionally , there is strong meningeal contrast enhancement in this region . ( d ) the poorly contrast - enhancing material in the cranial cavity can be followed further caudally , to the level of the atlas . this image , at the level of the foramen magnum , demonstrates abnormal tissue ( arrow ) displacing and compressing the cervical spinal cord ( * ) dorsally and to the right . ( e ) bone window computed tomography image at the most caudal aspect of the bulla . note the discontinuity of the temporal bone ( black arrows ) , indicative of bony lysis ct showed moderate contrast enhancement of the left ear canal lining . the medial portion of the left external ear canal and left tympanic bulla were filled with homogenous , contrast - enhancing soft tissue attenuating material ( figure 1a ) . ventromedial and rostral to the tympanic bulla , a region ( 1.6 cm width 1.3 cm height 2.5 cm length ) of poorly contrast - enhancing tissue , outlined by a rim of strong contrast enhancement ( figure 1b ) causing a mass effect , was seen , which compressed the nasopharyngeal lumen by > 50% and displaced the hyoid bones laterally ( figure 1b ) . ventromedial and caudal to the bulla , a similarly enhancing region was seen ( 0.6 cm height 0.3 cm width 0.6 cm length ) . in the caudal left bulla , there was bony lysis of the temporal bone ( figure 1e ) . lateral to the oval foramen and dorsal to the larger parapharyngeal lesion , an intracranial rim of contrast enhancement was seen in the ventral aspect of the left temporal lobe . adjacent to the lytic region of the temporal bone , contrast enhancement outlining a hypoattenuating area with broad dural base was seen ( figure 1c ) . poorly enhancing material extended caudally along the left ventral portion of the brainstem to mid first cervical vertebra , displacing and compressing the brainstem and cranial spinal cord , accompanied by strong meningeal contrast enhancement ( figure 1d ) . video otoscopy ( medrx video otoscope ) identified a large pink fleshy mass obscuring the lumen of the horizontal ear canal . the mass traversed the tympanic membrane , and was contiguous the tympanic bulla ( figure 2 ) , in agreement with ct findings . video - otoscopic photograph of the left tympanic membrane , which appears to have been breached by abnormal inflammatory tissue ( arrows ) , which extends into the tympanic bulla impression smears of the mass revealed large numbers of round yeast , surrounded by a prominent clear halo and narrow - necked budding consistent with cryptococcal infection . a heavy pure growth of a cryptococcus species resulted , which was identified as c gattii by conventional phenotypic mycology testing . further analysis by sequencing of the ribosomal internal transcribed spacer region identified c gattii vgii by comparing the sequence with published signature sequences ; however , the isolate was not available for further molecular typing . the isolate was susceptible to fluconazole , itraconazole , posaconazole , flucytosine and amphotericin b , but resistant to caspofungin . the minimum inhibitory concentration ( mic ) for fluconazole was 8 mg / l ( table 1 ) . antifungal susceptibility data for the cryptococcus gattii vgii isolate cultured from the cat s = susceptible ; r = resistant ; mic = minimum inhibitory concentration antifungal therapy was commenced with fluconazole ( 10 mg / kg po q12h [ symbion pharmacy services ] ) and amphotericin deoxycholate ( 0.5 mg / kg ; subcutaneous infusion in 350 ml 0.45% nacl and 2.5% dextrose three times weekly ) . four weeks after treatment commenced , the cat was brighter , with complete resolution of the head tilt , although inappetence persisted . , the cat had received a cumulative amphotericin b dose of 12.5 mg / kg , and was starting to eat and gain weight . owing to finances , the frequency of amphotericin b infusion was reduced to twice weekly ( dose 0.7 mg / kg / infusion ) . the frequency of amphotericin b infusion was reduced to once weekly ( 0.7 mg / kg / infusion ) . four months after diagnosis , lcat was 1:256 and the plasma concentration of fluconazole was 45 mg / l . seven months after diagnosis , the lcat was 1:64 and was 1:32 a further 3 months later , with a corresponding plasma concentration of fluconazole of 47 mg / l . after 12 months , the weekly amphotericin b infusions ( 0.7 mg / kg ) were discontinued and the cat continued to be clinically well , receiving only fluconazole ( 50 mg q12h ) . at the time of writing , 21 months after starting therapy , the lcat was 1:8 and the cat continued to be clinically well . otitis media due to c neoformans var grubii infection was managed by a total ear canal ablation and lateral bulla osteotomy , followed by oral itraconazole . in the third cat ventral bulla osteotomy and twice weekly amphotericin b subcutaneous infusions , with oral flucytosine and fluconazole , were used . in these latter two cats , diagnosis was made on cytological and histological evaluation of material removed via bulla osteotomy , culture and serum antigen testing . the cat presented initially with unilateral peripheral vestibular disease . as a mass in the left external ear canal was detected during preliminary examination , an aural polyp arising from the middle ear was initially suspected . presence of additional signs , including dysphonia , inappetence and weight loss , may have suggested a more sinister underlying aetiology . cytology from the external ear canal resulted in a prompt diagnosis of cryptococcosis ; however , failure to improve clinically to azole monotherapy prompted referral . video otoscopy and ct of the head demonstrated extensive and invasive disease of the ear , skull , cervical spinal cord and ipsilateral intracranial dural space . inhalation of air - borne basidiospores or desiccated yeast cells into the nasal cavity is considered the primary route of infection in cats . colonisation of the caudal nasal cavity , with subsequent epithelial invasion , possibly in the vicinity of the nasopharynx , may have been the primary route of infection , with extension via the auditory tube to the middle ear . possible pathways from the inner ear into the brainstem include erosion through the medial aspect of the petrous temporal bone ( although this was not apparent in the ct scans ) , along the nerves and vessels of the internal acoustic meatus , or via haematogenous spread . inoculation of cryptococcal organisms directly into the external ear canal , with subsequent spread to the middle ear by penetration of the tympanic membrane is considered unlikely and usually results in only superficial lesions . a grass seed heavily contaminated with cryptococcal cells may provide an explanation for how this might have occurred , although these are less commonly found in the ear canal of cats than dogs . this case is quite distinct from a recent feline case of bilateral cryptococcal otitis interna , which was thought to have arisen haematogenously as there was no evidence of rhinosinusitis disease or otitis media , and a small cryptococcal lesion was also present in the thalamus ; this patient was infected with c neoformans var grubii . the pathogenesis of cryptococcus in this cat is unusual and supports the contention by sykes and malik that vgii and vgiii infections are more invasive than c gattii vgi and c neoformans var grubii infections . all clinical signs resolved after aggressive medical therapy and initial de - bulking within the ear canal . while recent papers concerning c gattii vgi infections of the nasal cavity and contiguous tissues in koalas have emphasised the need for surgical intervention and/or intralesional therapy because antifungal agents may not penetrate well into poorly perfused tissues , the requirement for this probably should be made on a case - by - case basis . c gattii vgii can be divided into vgiia , vgiib and vgiic strains , with vgiia being the major genotype in the vancouver island outbreak . determining the specific biotype is critical because c gattii vgii and vgiii isolates tend to demonstrate heteroresistance to fluconazole . the epidemiological cut - off values for fluconazole in vgii and vgiii infections are therefore higher than for vgi and c neoformans var grubii isolates . in this instance the causal vgii isolate was susceptible to fluconazole in vitro , with a mic of 8 mg / l . given the pharmacokinetics of fluconazole in the cat , a dose of 10 mg / kg po q12h should produce peak blood concentrations of fluconazole at steady state in the order of 2080 mg / l . in eastern australia , about 20% of cryptococcosis in cats and dogs is caused by c gattii ( predominantly vgi ) . in western australia , vgiib ( mating type ) is the predominant species of c gattii , although vgi and vgii ( mating type a ) also occur . studies on the c gattii vgiia isolates from the vancouver island outbreak and vgiib isolates from australia have demonstrated that such strains are highly virulent in experimental infections of mice and rats . although central nervous system ( cns ) involvement is an important negative prognostic indicator in feline cryptococcosis , this cat improved markedly with combination therapy using amphotericin b and fluconazole . cross - sectional imaging was not often undertaken when cases of cryptococcosis were first recorded in the veterinary literature . in this case , clinical findings and plain radiographs would not have provided indication of the extensiveness of the disease , and the success of medical therapy in this cat testifies that good outcomes may occur despite extensive invasive disease . treatment for cns cryptococcosis is prolonged and should be continued until the lcat titre reaches zero , which may take 12 years . in recovered cats , the titre can be monitored every 36 months to allow early detection of recurrences , which is more common than re - infection with a new strain or antifungal resistance . this case report describes the successful medical treatment of otogenic meningoencephalomyelitis due to c gattii ( vgii ) infection in a cat .

case summarya 7-year - old spayed domestic longhair cat from perth , western australia , presented with left - sided head tilt , dysphonia , head shaking , inappetence and weight loss . a polypoid lesion had previously been removed from the external ear canal . otitis media with extension into the external ear canal was suspected and investigated using video - otoscopy and computed tomography examination . invasive disease with extension from the middle ear to the base of the skull , and intracranial extension into the caudal fossa and cranial cervical vertebral canal was detected . cytology of external ear canal exudate showed capsulated budding yeasts and cryptococcus gattii vgii was cultured . treatment with amphotericin b infusions and oral fluconazole was prescribed , with nutritional support via oesophagostomy tube . the cat clinically recovered 12 months after treatment commenced.relevance and novel informationthis case report describes the successful medical treatment of otogenic meningoencephalomyelitis due to c gattii ( vgii ) infection in a cat .

neuroretinitis ( nr ) is considered to be an inflammatory condition which is characterized by optic disc edema and , as a result , formation of a macular star figure . nr is an atypical presentation of toxoplasmosis infection , and such cases are quite rare . a 13-year - old girl presented with painless subacute visual loss in her right eye for a week at khatam - al - anbia eye hospital in mashhad , iran . although toxoplasmosis nr is rare , it should be considered in the differential diagnoses of nr . neuroretinitis ( nr ) is considered an inflammatory condition which is characterized by optic disc edema and , as a result , formation of a macular star figure ( 1 ) . this disorder is possibly caused by an infectious process affecting the disc ; in other instances , a post - viral or autoimmune mechanism seems to be a more probable cause ( 1 ) . furthermore , some instances of nr have been reported to be accompanied by a wide spectrum of infectious pathogens ( 1 ) . the most common case reported so far is the result of cat scratch disease ( csd ) , accounting for two - thirds of cases in one study ( 2 ) . however , there are other infectious etiologies of neuroretinitis , including rubeola , toxoplasmosis , herpes simplex , varicella , tuberculosis , lyme disease , leptospirosis , syphilis , various fungi , and multiple viral illnesses ( 1 ) . additionally , sporadic cases of nr may occur owing to noninfectious forms of uveitis , such as sarcoidosis and periarteritis nodosa ( 3 , 4 ) . optic disc edema with a macular star may also occur as a result of other factors such as diabetic papillopathy , hypertensive neuropathy , and anterior ischemic optic neuropathy papilledema ( 1 ) . to determine the relevant history of patients with nr , the practitioner needs to concentrate on plausible risk factors for specific infectious parameters , such as travelling to areas where lyme disease or tuberculosis ( for example ) are endemic , exposure to waste material ( e.g. , leptospirosis ) , animal exposure ( especially cats ) , and sexual contact that may have resulted in the contraction of syphilis . the clinician should investigate patients for systemic symptoms such as lymphadenopathy , headache , fever , and skin rash . laboratory tests ought to be customized for the individuals on the basis of information from both their reported history and the examination . serologic tests for most cases may include the fluorescent treponemal antibody absorption test ( fta - abs ) , cat scratch titers ( bartonella species ) , and a tuberculosis skin test ( purified protein derivative , or ppd ) ( 1 ) . in this study , we present a case of unilateral neuroretinitis in which the serology result was apparently negative for acute infection of toxoplasmosis ( negative igm titer for toxoplasmosis , but positive for igg ) . based on anti - toxoplasma treatment that was administered for the positive igg levels , the condition responded well with near optimal visual recovery . a 13-year - old girl was referred to the ophthalmic emergency department of khatam - al - anbia eye hospital , mashhad , iran , which is affiliated with mashhad university of medical sciences ( mums ) , in may of 2013 with painless subacute visual loss in her right eye for one week . she denied any focal neurologic condition , pain on eye movement or ophthalmodynia , or any other systemic symptom . she also stated that she had not engaged in outdoor camping or any other related activity . furthermore , she had no remarkable past medical and ophthalmologic history , and she had no contact or proximity to pets , especially cats . upon examination , the best corrected visual acuity ( bcva ) was 20/250 in the right eye and 20/20 in the left eye . moreover , the patient had a grade 2 + relative afferent papillary defect ( rapd ) in the right eye . an examination of the anterior segment demonstrated 2 + cells in the anterior chamber ( a / c ) and 1 + vitreous reaction with normal intraocular pressure ( iop ) in the right eye . the investigation of the right fundus revealed optic disc swelling , macular star lipid ( hard exudates ) deposition , and peripapillary vascular sheathing ( figure 1 ) . many tests were performed for the patient , including the erythrocyte sedimentation rate ( esr ) , complete blood count differential ( cbc diff . ) , c - reactive protein ( crp ) , toxoplasmosis serology , purified protein derivative ( ppd ) , angiotensin converting enzyme ( ace ) , fta - abs , brain and orbital mri , chest x - ray , and infectious and rheumatologic consultation . bartonella henselae serology was not available in our ophthalmology center , nor in any other nearby neighborhood . based on our clinical results , neuroretinitis etiology revealed csd as the most common infectious etiology , along with the negative systemic and ocular history , and empirical treatment commenced including azithromycin 500 mg daily , trimethoprim / sulfamethoxazole , and the application of a topical steroid . after 72 hours , all the above - mentioned laboratory indices returned to the normal level , except for the toxoplasmosis serology . furthermore , the serologic test for toxoplasmosis was negative for igm antibody whereas the igg antibody titer was > 100 iu / ml . relying on the positive igg toxoplasmosis serology and the absence of any response to initial treatment , we embarked on shifting to classic anti - toxoplasmosis treatment , i.e. , sulfadiazine 500 mg qid ( four times a day ) , pyrimethamine 50 mg / day , and folinic acid . after 72 hours had passed , prednisolone 1mg / kg / day was added and thereafter tapered during the treatment . the disc edema diminished over the course of a week . also , visual acuity gradually improved and the a / c inflammatory reaction faded away . after the decrease in disc swelling , a very small hyperpigmented focus appeared in the juxtapapillary position . gradually , over the next two months , the patient s visual acuity improved to 9/10 and the optic disc swelling and macular exudates resolved ( figure 2 ) . the clinical syndrome of idiopathic stellate maculopathy accompanied by optic nerve edema was first identified by leber in 1916 ( 5 ) . later on , this syndrome was renamed as leber s stellate neuroretinitis . as is now known , the most common form of infectious nr results from csd ( 1 ) . the engagement of the optic nerve suggests the need for urgent intervention . in spite of the lack of evidence either for animal exposure or any other systemic condition for this case , empirical csd treatment was instituted as lab tests were pending and given that cat scratch serology was not accessible . toxoplasmosic retinochoroiditis is rendered as one of the most frequent causes of posterior uveitis , specifically in young patients ( 6 ) . however , toxoplasmic optic neuropathy is an infrequent condition and is often characterized by subacute visual loss and optic nerve swelling , and is sometimes associated with a macular star ( neuroretinitis ) ( 7 ) . the engagement of the optic nerve most commonly encountered in ocular toxoplasmosis is optic nerve edema with a simultaneous distant active lesion . other types of optic nerve involvement include pure papillitis presented as optic disc swelling , associated with peripapillary vascular sheathing close to the healed lesion , and neuroretinitis , i.e. , optic disc swelling with macular hard exudate deposition . additionally , monocular involvement has been observed in most cases with favorable visual prognosis ( 8) . considering the positive toxoplasmosis serology in this patient , toxoplasmosis neuroretinitis was suspected , hence prompting the specified treatment . this case disclosed optic disc involvement and neuroretinitis owing to juxtapapillary chorioretinitis reactivation . in some cases , the juxtapapillary chorioretinal scar can not be found in the acute stage of disease due to disc and peripapillary edema . although toxoplasmosis neuroretinitis is rare , it should be still considered in any suspected case of neuroretinitis . this case re - emphasizes the necessity of considering toxoplasmosis in the differential diagnosis of neuroretinitis . awareness of this potential cause and prompt treatment after positive testing may therefore result in a good visual outcome .

introductionneuroretinitis ( nr ) is considered to be an inflammatory condition which is characterized by optic disc edema and , as a result , formation of a macular star figure . nr is an atypical presentation of toxoplasmosis infection , and such cases are quite rare.case presentationa 13-year - old girl presented with painless subacute visual loss in her right eye for a week at khatam - al - anbia eye hospital in mashhad , iran . following comprehensive evaluation , a diagnosis of toxoplasmic nr was made . the nr favorably responded to classic anti - toxoplasmosis treatment . a juxtapapillary retinochoroidal scar appeared after disc swelling resolution.conclusionsalthough toxoplasmosis nr is rare , it should be considered in the differential diagnoses of nr .

three types of calibrated microspheres have recently been approved in japan as embolic agents for hypervascular tumors and arteriovenous malformations . to date , two types of embolic microspheres capable of being loaded with drug have been introduced : dc bead ( biocompatibles , farnham , uk ) and hepasphere ( biosphere medical , roissy cdg cedex , france ) . in a phase ii randomized controlled study of transarterial chemoembolization ( tace ) using a drug - eluting bead ( deb - tace ) for hepatocellular carcinoma ( hcc ) , the precision - v study , the response rate after 6 months was 52% in the deb - tace group and 44% in the tace group using a lipiodol ( laboratoire guerbet , aulnay - sous - bois , france)/anticancer drug mixture solution ( lip - tace ) , showing no significant difference or superiority of deb - tace . however , complication by postembolization syndrome , such as abdominal pain , fever , nausea , vomiting and liver enzyme elevation , developed in 2080% after lip - tace [ 3 , 4 ] , whereas symptoms developed in only 1837% after deb - tace [ 5 , 6 ] , showing that the complications were milder than those after lip - tace . on the other hand , deb - tace induced liver abscesses at a relatively high frequency , which could be fatal [ 5 , 7 , 8 , 9 ] . adverse events after deb - tace have been reported occasionally [ 5 , 7 , 8 , 9 , 10 , 11 ] , but as it has just been introduced in japan , many points regarding adverse events are unclear with respect to the developmental mechanism and risk factors . we encountered a hcc patient who underwent deb - tace and experienced delayed intratumoral hemorrhage . an 81-year - old male regularly visited his physician for treatment of hepatitis c virus - related cirrhosis . on routine abdominal ultrasonography ( us ) , on esophagogastroduodenoscopy , a type 0-iia early gastric carcinoma with a diameter of 25 mm was detected in the lesser curvature of the middle body . the patient was referred to our hospital for further examination and treatment . during the first examination , the liver function was classified as child - pugh class a , and the tumor markers were normal ( afp level : 3.4 ng / ml , afp - l3 fraction level < 0.5% ) , but the pivka ii had a level of 118 mau / ml , showing a mild elevation . it was capsulated and enhanced in the arterial phase on dynamic computed tomography ( ct ) ( fig . 1 ) , and showed a mosaic pattern accompanied by a marginal hypoechoic zone on gray - scale us . the mass was enhanced in the vascular phase ( 040 s ) on contrast - enhanced us ( ceus ) using sonazoid ( daiichi sankyo , tokyo , japan ) , and showed a hypoechoic area in the postvascular phase ( 10 min after an intravenous injection of sonazoid ) . based on the tumor marker and imaging findings , the patient was diagnosed with hcc . deb - tace was performed because the patient declined to undergo surgical resection of hcc . access for tace was performed under sterile conditions and under local anesthesia , via the right femoral artery using a 3-fr sheath ( medikit , tokyo , japan ) and in a retrograde fashion . when a microcatheter was advanced to the anterior segmental artery of the right hepatic artery and contrast imaging was applied , an intensely stained tumor was observed , with a5 as the feeding artery . a solution impregnated with 100300 m dc bead and 50 mg epirubicin hydrochloride , with a dc bead volume of 0.35 ml , was administered via the feeding artery to perform deb - tace . disappearance of the intensely stained tumor image was confirmed by right hepatic arterial angiography , and the treatment was completed . the course after deb - tace was favorable , and no adverse events above grade 3 according to the common terminology criteria for adverse events ( ctcae ) version 4.0 were observed . endoscopic submucosal dissection ( esd ) of the early gastric carcinoma was performed about 1 month after deb - tace . right hypochondriac pain suddenly developed 3 days after esd , but no adverse events were assumed to be caused by esd ( i.e. , free air , noted on dynamic ct ) . however , the tumor diameter had increased from that before deb - tace , and the tumor showed a high - intensity area on unenhanced ct ( fig . 2a ) , which was not enhanced in the arterial phase on dynamic ct , suggesting intratumoral hemorrhage . the hemodynamics of the tumor were followed using ceus over the time before and after deb - tace . on ceus carried out on the day after deb - tace , the whole intratumoral enhancement decreased , and nonenhanced patchy regions , assumed to be necrosis , were noted in the tumor in the vascular phase . on ceus performed 4 weeks after deb - tace , i.e. , immediately before esd , the tumor size was 33 31 mm , and the enhancement area was increased in the vascular phase ( fig . however , the tumor had enlarged to 41 36 mm on ceus when the right hypochondriac pain developed ( 5 weeks after deb - tace ) , and changes in the echogenicity on gray - scale us and nonenhancement of almost the entire tumor in the vascular phase on ceus were noted ( fig . based on the above findings , the cause of right hypochondriac pain may have been deb - tace - associated intratumoral hemorrhage . since the hemorrhage was limited to inside the tumor , it stopped spontaneously with rest . the advantages of deb - tace are as follows : the infusion dose of the chemotherapeutic agent can be modified by adjusting the chemotherapeutic agent carrier , while altering the depth of embolization with beads , the duration of tumor exposure to chemotherapeutic agent can be fine - tuned through sustained release of the chemotherapeutic agent , and systemic adverse reactions of chemotherapeutic agent can be attenuated by continuous release of high - dose chemotherapeutic agents into the tumors . deb has proven to be extremely potent and safe by allowing large amounts of the chemotherapeutic agent to concentrate within the tumor over time , thereby maximizing the cytotoxic effect , which also makes it attractive as an effective treatment for large lesions . favorable therapeutic effects have been reported occasionally [ 8 , 9 ] and have increased the expectations of hcc treatment with deb - tace ; however , the beads exhibit characteristic physical properties and behavior that are different from those of gelatin particles . it is important to fully investigate these characteristics to avoid inadequacy of the therapeutic effect and to prevent the onset of adverse events . previous studies have reported that the incidence of mild adverse events , such as postembolization syndrome , is lower in the deb - tace group compared with the lip - tace group [ 3 , 4 , 5 , 6 ] . meanwhile , liver abscesses were induced at a relatively high frequency ( about 1.67.4% ) [ 5 , 7 , 8 , 9 ] . malagari et al . reported that the 30-day mortality was 1.26% among 237 hcc patients treated with deb - tace with dc bead loaded with doxorubicin , and that the cause was liver abscess in all cases . regarding other adverse events , acute pancreatitis and cholecystitis developed with incidences of 15 and 16.3% , respectively . previous studies have shown that rare adverse events included liver failure and infection , bile duct injury , upper gastrointestinal bleeding , pulmonary embolism , splenic infarction , or spinal embolization [ 1 , 7 , 10 , 11 ] . unlike lip - tace , many adverse events occurred due to ischemic changes induced by the inflow of beads into organs other than the hcc lesion , which is characteristic of deb - tace . tumor hemorrhage has also been reported after deb - tace [ 1 , 7 , 10 , 11 , 13 ] with large tumors , with a diameter of 516 cm , and the tumor locations were subcapsular in all previous reports . to our knowledge , there has been no case of a tumor with a diameter < 5 cm distinct from the subcapsular , as was observed in our patient . there has been no report of a mechanism underlying the deb - tace - specific tumor hemorrhage . in general , the mechanism of tumor rupture after tace might be related to increased intratumoral pressure as a result of rapid edematic expansion due to tumor necrosis or vascular injury , secondary to embolization . the risk is particularly high in subcapsular lesions , and we must be careful because the spread of hemorrhage in the abdominal cavity can not only lead to hemorrhagic shock , but also peritoneal metastasis . unlike the reported cases of tumor hemorrhage after deb - tace , in the present case , the tumor diameter was not large and the tumor was distinct from the subcapsular region . however , intratumoral hemorrhage occurred about 1 month after deb - tace . local pooling of the contrast agent within the target tumor could occur during deb - tace , and seki et al . reported that it occurred in 25.2% of 135 hcc patients treated with deb - tace ( epirubicin - loaded hepasphere ) . while the cause of this local pooling remains unclear , it is similar to extravasation or blood sinus , suggesting that it was a local hemorrhage due to intratumor vascular collapse as a consequence of the treatment . when local pooling of the contrast agent was noted , seki et al . added a small amount of gelatin sponge particles , with a diameter ranging from 1 to 2 mm , to the feeding artery until the pooling disappeared . based on the above findings , the observation of local pooling of the contrast agent within the target tumor during treatment suggests that intratumoral pressure has already increased , which was unlikely in our patient . treatment - induced intratumoral hemorrhage continued immediately after deb - tace because no pooling was noted during treatment or on confirmation angiography following treatment . however , it is possible that the amount of dc bead was insufficient and embolization was incomplete . it was assumed that incomplete embolization subsequently caused intratumoral vascular collapse , i.e. , a phenomenon similar to pooling , and that the hemorrhage expanded to the necrotic intratumoral area by sustained release of the chemotherapeutic agent and embolization , leading to complication of intratumoral hemorrhage despite the low risk of tumor hemorrhage . many points remain unclear , i.e. , the appropriate bead sizes for each case and the level of embolization , but it may be important to apply sufficient embolization because insufficient embolization is likely to lead to tumor hemorrhage . deb - tace is considered to rarely cause adverse events , but fatal complications have been reported [ 5 , 7 , 8 , 9 ] , and reports of adverse events may increase with augmentation of the numbers of deb - tace - treated patients . if reports of tumor hemorrhage after deb - tace increase , the mechanism of intratumoral hemorrhage in cases similar to our patient may be elucidated . intratumoral hemorrhage was suspected due to the presence of a high - intensity area in the tumor on unenhanced ct and a rapid increase in the tumor diameter and accompanying enlargement of the unenhanced area on ceus performed before and after deb - tace . based on these findings it was difficult to assess the therapeutic effect of deb - tace by ct , based on lipiodol accumulation , unlike that after conventional lip - tace , and evaluation of the intratumoral hemodynamics could be important . ceus is superior in evaluating intratumoral hemodynamics in real time , and the utility of ceus to evaluate the therapeutic effect after deb - tace was suggested . we encountered a patient who experienced intratumoral hemorrhage about 1 month after deb - tace . deb - tace for hcc is a new treatment method in japan , and reports of adverse events may increase in the future . to obtain the therapeutic effect of deb - tace while preventing the adverse events , it may be important to understand the characteristics of the beads themselves and to apply the appropriate embolization to each individual case . it was also suggested that ceus is useful to evaluate the therapeutic effect after deb - tace .

transarterial chemoembolization ( tace ) using a drug - eluting bead ( deb - tace ) for hepatocellular carcinoma ( hcc ) is a new treatment method . we report on a case of delayed intratumoral hemorrhage after deb - tace . an 81-year - old male with hepatitis c virus - related cirrhosis was diagnosed with a hcc of 35 mm in diameter in s5 detected by dynamic computed tomography ( ct ) and contrast - enhanced ultrasonography ( ceus ) . deb - tace with dc bead and epirubicin hydrochloride was performed because the patient declined to undergo surgical resection . the treatment was completed , and the course after deb - tace was favorable . however , right hypochondriac pain suddenly developed about 1 month after deb - tace . unenhanced ct showed an increase of the tumor diameter and intratumoral high - intensity area , which was not enhanced in the arterial phase . ceus performed at the time of right hypochondriac pain ( 5 weeks after deb - tace ) showed nonenhancement of almost the entire tumor in the vascular phase . the cause of the symptom may have been deb - tace - associated intratumoral hemorrhage . tumor hemorrhage has been reported after deb - tace with tumors > 5 cm in diameter , and the tumor locations were subcapsular in all previous reports . there has been no case of a tumor with a diameter < 5 cm distinct from the subcapsular , as was observed in our patient . incomplete embolization might be the cause of the intratumoral hemorrhage experienced by this case presenting a few risks . to obtain the therapeutic effect of deb - tace while preventing the adverse events , it may be important to understand the characteristics of the beads and to apply the appropriate embolization to each individual case .

natriuretic peptides are a family of hormones , sharing similar chemical structure ( a characteristic 17-amino acid ring structure , stabilized by a cysteine bridge , which contains several invariant amino acids and variable c- and n - terminal tails ) and biological function , with relevant effects in cardiovascular physiology and pathology . the study of natriuretic peptides began 50 years ago , when electron microscopy revealed the presence of secretory granules in cardiac atrial cells containing atrial natriuretic peptide ( anp ) as demonstrated by de bold and coworkers in the early 80s . from 1988 to 1990 , two more members of the natriuretic peptide family , the brain natriuretic peptide ( bnp ) and c - type natriuretic peptide ( cnp ) were identified in porcine brain [ 2 , 3 ] . more recently a new peptide that shares structural and functional characteristics with the previous natriuretic peptides was identified in the venom of the green mamba ( dendroaspis angusticeps ) and received the denomination of dendroaspis natriuretic peptide ( dnp ) . in addition , other peptides with similar cardiovascular effects have been identified in mammalians , including urodilatin , a peptide derived from alternative cleavage of pro - anp in the distal tubules of the kidney , where it exerts its natriuretic actions , or the intestinal epithelium derived peptides , guanylin and uroguanylin , which participate in water absorption . the classic physiological role of natriuretic peptides includes promotion of renal excretion of sodium ( natriuresis ) and water ( diuresis ) . natriuretic peptides also exert autocrine and paracrine effects within circulation , such as vasodilatation by relaxing vascular smooth muscle cells , regulation of renin , progesterone , endothelin and vasopressin secretion . anp and bnp are 28- and 32-amino acid peptides , respectively , mainly synthesized and released by atrial ( in the case of anp ) and ventricular cardiomyocytes ( for bnp ) in response to blood pressure and volume loading [ 8 , 9 ] . in addition , anp and bnp secretion from the ventricles increases associated to a number of ventricular dysfunctions , and the extent of anp and bnp release under these circumstances is in relation to the severity of the pathology , that has led to the use of both natriuretic peptides as diagnostic tests for heart failure [ 10 , 11 ] . anp and bnp are synthesized as larger molecules that are subsequently cleaved to yield the active peptide hormone and the biologically inactive n - terminal peptide fragment [ 12 , 13 ] . both natriuretic peptides circulate in the blood reducing vascular tone and promoting diuresis / natriuresis to lower blood volume and pressure . anp and bnp are removed from the circulation by two different mechanisms : receptor - mediated internalization and proteolytic degradation by neutral endopeptidase , that has been shown to take place in the kidneys , vascular endothelium , lungs and heart . circulating half - life of anp is approximately 35 min , whereas the half - life of bnp is significantly greater , about 23 min . , and even greater is that of the inactive terminal fragment of bnp , nt - probnp , from 60 to 120 min , which is relevant to their value as diagnostic tests . cnp , the third natriuretic peptide identified , is mainly expressed in the nervous system and vascular endothelial cells [ 14 , 15 ] , where cnp exerts autocrine and paracrine actions on vascular tone and muscle cell growth [ 15 , 16 ] . the cardiovascular effects of cnp are more likely mediated by its vascular local effects or by central actions on vasopressin and adrenocorticotropine release than to its natriuretic and diuretic effects , that are weaker than that of anp and bnp [ 16 , 17 ] . cnp gene expression is stimulated by several vasoactive mediators , such as interleukin 1 , vascular endothelial growth factor , transforming growth factor , tumour necrosis factor - a and insulin [ 15 , 1820 ] . dnp is a recently isolated 38-amino acid peptide that shows structural and functional properties of the previously identified members of the natriuretic peptide family . although dnp purification from human blood has not yet been achieved , dnp immunoreactivity has been reported in human plasma . dnplike immunoreactivity has been found in rat aorta , carotid artery and kidney [ 22 , 23 ] , where it has been found to induce vasorelaxation and inhibition of vascular smooth muscle cell proliferation . natriuretic peptides regulate cardiovascular homeostasis by the occupation of three membrane receptors ; two are guanylyl cyclase - coupled receptors , known as natriuretic peptide receptor ( npr)-a and npr - b , while npr - c lacks enzymatic activity and among other functions acts as a clearance receptor [ 24 , 25 ] . npr - a is activated by anp , bnp and dnp [ 25 , 26 ] , npr - b shows high affinity and is activated by cnp and , finally , the npr - c binds all natriuretic peptides . npr - a and npr - b are single - transmembrane receptors of approximately 120 kd with a similar basic structure that consist of a variable extracellular natriuretic peptide - binding region , a conserved intracellular kinase homology domain and a guanylyl cyclase domain with enzyme activity . occupation of npr - a and npr - b induces cellular responses through the elevation of intracellular cgmp levels , which , in turn , leads to the activation of a cgmpdependent protein kinase that phosphorylates a target protein in serine of threonine residues and mediates the specific physiological function . signal transduction activated by these receptors is terminated by cgmp phosphodiesterases that modulate the intracellular concentrations of cgmp and the duration and magnitude of the responses . npr - c is a transmembrane receptor with an extracellular domain containing the natriuretic peptide - binding region , a transmembrane domain and a cytosolic domain . two different subtypes of npr - c , of approximately 67 and 77 kd in size , have been identified [ 28 , 29 ] . npr - c has been involved in peptide clearance , removing natriuretic peptides from the circulation and in the mediation of natriuretic peptide - induced inhibition of camp synthesis , an effect that requires the involvement of a heterotrimeric g protein [ 7 , 31 , 32 ] . we have provided evidence that the 77 kd npr - c - like protein is involved in peptide internalization whereas the 67 kd npr - c - like protein is likely involved in adenylyl cyclase inhibition . in the past decade , much attention has been given to the investigation of natriuretic peptides testing in the evaluation of patients with different pathologies , such as congestive heart failure ( chf ) . studies in patients with chf showed that both anp and bnp secretion from ventricular myocytes increases in relation to the rigor of dysfunction . these findings led to investigate whether the plasma levels of anp and bnp may assist in the diagnosis of patients with heart failure . since half - life of bnp is greater than that of anp comparison of the diagnostic value of anp and bnp as reported above , the inactive n - terminal fragment of bnp , ntprobnp , has an even greater half - life than bnp ; thus , most of the clinical investigations concerning natriuretic peptides as biochemical markers for chf has focused on bnp or nt - probnp . basal plasma levels of bnp have been established between 5 and 50 pg / ml , while the corresponding values for nt - probnp are from 7 to 160 pg / ml . the approved cut - off levels to consider an abnormal range are 100 pg / ml for bnp and 125 pg / ml for ntprobnp ( 450 pg / ml for individuals older than 75 years old ) , although they vary according to age , sex and estimated glomerular filtration rate . levels of both bnp and nt - probnp are elevated under a number of pathological situations including cardiac dysfunctions , such as chf , diastolic dysfunction , valvular heart disease , atrial fibrillation and non - cardiac pathologies , such as acute pulmonary embolism , pulmonary hypertension , sepsis or hyperthyroidism [ 11 , 3436 ] . four major applications of bnp and nt - probnp testing in patients with chf have been presented in the last few years , including diagnosis , screening , prognosis and monitoring of therapy . different studies have reported the efficacy of bnp and ntprobnp tests increasing the diagnostic accuracy of heart failure [ 37 , 38 ] . the sensitivity and specificity of the test is especially high at a cut - off of 76 pg / ml bnp , and nt - probnp has been proved to be particularly important in decreasing the overdiagnosis of heart failure that occurs in primary care . for ntprobnp , levels over 450 pg / ml ( over 900 pg / ml for patients older than 50 years old ) are sensitive and specific for heart failure ; however , if the value is below 300 pg / ml , heart failure is highly unlikely with a negative predictive value of 99% . in addition , recent studies have reported that bnp is a significant prognostic indicator in patients diagnosed with heart failure and in asymptomatic patients . nt - probnp has been found to be a stronger risk biomarker for cardiovascular disease and death than c - reactive protein . high - risk subject with cardiovascular risk , which would be a great advance prompting more aggressive primary prevention . however , a number of limitations have been reported about the prognostic value of bnp and nt - probnp . in patients with advanced chf , atrial fibrillation bnp or nt - probnp can also be used as a guide for therapy in heart failure . for this use , it should be taken into account that therapies that reduce clinical disorders in heart failure also reduce bnp levels and those that improve heart failure conditions act primarily through mechanisms that are linked with changes in natriuretic peptide levels . in addition , bnp and nt - probnp levels do not decrease as rapidly in response to therapy as might be expected from their short half - lives , suggesting that the natriuretic peptide system need some time to autoregulate . considering this , bnp and nt - probnp levels might guide the clinician to adjust the treatment in order to achieve a plasmatic level of these agents below a critical value . sepsis and septic shock is associated with an elevation of cardiac troponin levels that has been shown to indicate left ventricular dysfunction and a poor prognosis . whereas cardiac troponins are a good biomarker for myocardial dysfunction in patients with severe sepsis or septic shock the use of natriuretic peptides , including bnp , as an indicator of prognosis remains controversial . renal insufficiency has also been shown to affect the plasmatic levels of both bnp and nt - probnp both in children and adult patients . the levels of bnp are correlated with renal function , so that an increase reaching about 200 pg / ml have been reported in patients with reduced creatinine clearance ( below 60 ml similar observations have been reported with nt - probnp and a recommended reference value of 1200 pg / ml has been shown for patients with reduced creatinine clearance . it has been shown that peritoneal dialysis does not alter plasma nt - bnp or bnp levels in patients with renal failure , whereas both blood urea nitrogen and creatinine levels declined as expected , which should be taken into account if plasmatic levels of these hormones are used as a guide to the management of patients with renal failure . nt - pro - bnp and bnp have been recently presented as useful biomarkers for coronary artery disease and left ventricular hypertrophy in patients with chronic kidney disease , where detection of coronary artery disease and left ventricular hypertrophy in these patients has remained elusive despite the greater prevalence in patients with chronic kidney disease . natriuretic peptides , especially anp , have long been reported to have the potential to restore renal function after ischaemic injury , and have been shown to counteract renal sympathetic nerve activity in renal function . anp overexpression has been shown to exhibit protection against gentamycin - induced nephrotoxicity , as demonstrated by daily subcutaneous administration of gentamycin , for 10 days , in sprague dawley rats either treated with an intravenous injection of adenovirus ( ad.rsv-anp ) , carrying the human anp gene , the first day of gentamycin administration , or not treated ( control ) for comparison . this finding raises the possibility of using anp gene therapy for the treatment of drug - induced renal failure . in addition , anp has been reported to exert a protective effect on the outcome of acute renal failure in animals when infused over short periods of time , while it has been shown that prolonged infusion of anp does not alter the course of acute renal failure . on the other hand , infusion of wistar rats with anp exerted little renoprotective effect against endotoxin - induced acute renal failure , since anp infusion did not improve the hyponatriuresis and oliguria induced by endotoxin administration . recent studies have pointed out that continuous infusion of synthetic human anp is effective for preventing acute renal failure , which is a major problem occurring immediately after liver transplantation and requiring haemodialysis . this is the case of a model of transgenic mice overexpressing bnp that show reduced glomerular injury than control mice during the development of diabetes mellitus . glomerular hyperfiltration is an early haemodynamic alteration and one of the key mechanisms of the pathogenesis of diabetic nephropathy . this observation suggests that renoprotective effects of natriuretic peptides may prevent the progression of diabetic nephropathy , although further studies are necessary to establish therapeutic strategies to palliate renal complications associated to diabetes mellitus . further evidences for the renoprotective role of natriuretic peptides come from studies where the peptide hydrolysis is inhibited . neutral endopeptidase is an endothelial cell surface zinc metallopeptidase and the major pathway involved in the degradation of the natriuretic peptides . inhibition of neutral endopeptidase increases levels of anp , bnp and cnp , and has been shown to offer a therapeutic advantage in the treatment of hypertension , heart failure and endothelial dysfunction . endopeptidase inhibitors reduce vasoconstriction and improve sodium / water balance ; as a result these inhibitors decrease peripheral vascular resistance and blood pressure and improve local blood flow . endopeptidase inhibitors also reduce the activity of the angiotensin - converting enzyme ( ace ) , leading to a reduction of vasoconstrictor and proliferative mediators , such as angiotensin ii and increase local levels of bradykinin . recent studies have reported that anp reduces angiotensin ii - induced renomedullary interstitial cells proliferation and extracellular matrix synthesis in diabetic subjects more efficiently in neutral endopeptidase - deficient mice , which provide evidence for the beneficial effect of inhibition of neutral endopeptidase in attenuating abnormal cell growth associated with diabetic nephropathy . in addition to the renoprotective effect , neutral endopeptidase inhibition , in combination with inhibition of the ace activity , has been shown to reduce blood pressure in spontaneously hypertensive rats . this is the case of omapatrilat , a potent vasopeptidase inhibitor that exerts anti - hypertensive effects by inhibition of the neutral endopeptidase and ace at the tissue level , which results in beneficial effects on the cardiovascular structure . the dual metalloprotease inhibitors of ace and neutral endopeptidases , called vasopeptidase inhibitors , provide an advance over individual ace or neutral endopeptidase inhibitors , and might represent a new and attractive therapeutic strategy for the treatment of cardiovascular disease . nesiritide is a recombinant form of human bnp and its amino acid sequence is identical to that of endogenous human bnp . administration of nesiritide results in venous , arterial and coronary vasodilatation , reducing cardiac the pre - load and after - load , which increase cardiac output without direct inotropic effects [ 6062 ] . in addition , nesiritide increases glomerular filtration rate and filtration fraction , suppresses the renin - angiotensin - aldosterone axis , and enhances diuresis and natriuresis . nesiritide is currently used in the treatment of acute decompensated heart failure , where it has been shown to decrease pulmonary capillary wedge pressure , pulmonary artery pressure , right atrial pressure and systemic vascular resistance , as well as increase cardiac index and stroke volume index [ 6466 ] . in comparison with nitroglycerin , treatment with nesiritide did not induce the appearance of tachyphylaxis and other adverse effects . unlike inotropes , the beneficial haemodynamic effects produced by nesiritide do not cause an increase in myocardial oxygen consumption , an important consideration for patients with acutely decompensated heart failure . since nesiritide is not an inotrope , it does not affect myocardial contractility , as does the agonists of -adrenergic receptors or the inhibitors of phosphodiesterase iii . as a result studies aimed to determine the impact of early initiation of acute decompensated heart failure therapy with nesiritide on subsequent outcomes have confirmed its beneficial effects reducing the severity of the associated complications , reducing the mean total hospital length of stay and the requirement of transfer to the intensive care unit . in comparison with dobutamine and milrinone , nesiritide therapy was associated with a lower in - hospital mortality rate and shorter length of stay . in addition , total health care costs with nesiritide were decreased compared with the other drugs , since although the acquisition cost of nesiritide was higher than that of milrinone and dobutamine , nesiritide has been shown as a more cost - effective treatment option for patients with acute decompensated heart failure [ 69 , 70 ] . despite its beneficial effects on the cardiovascular system , recent studies have raised the question of safety with nesiritide therapy . in three randomized controlled trials sackner - bernstein and coworkers expressed concern of possible short - term ( 30-day ) risk of death after nesiritide use for the treatment of acute decompensated heart failure . as mentioned by the authors , first , the three randomized controlled trials used the nesiritide study group efficacy trial ( nsget ) , the vasodilation in the management of acute congestive heart failure ( vmac ) , and the prospective randomized outcomes study of acutely decompensated chf treated initially in outpatients with natrecor ( proaction ) were not designed to determine whether nesiritide is associated with risk of death . in addition , there was not complete information concerning the use of additional medications or procedures through the 30-day period of the study . the same group has reported that nesiritide significantly increases the risk of worsening renal function in patients with acute decompensated heart failure , although whether renal dysfunction reflects haemodynamic effect or renal injury is unclear . concerning this issue , more recent studies have reported that the effect of nesiritide on renal function strongly depends on the infusion times , so that nesiritide infusion time 24 hrs is associated with elevated markers of worsening renal function in patients with acutely decompensated heart failure compare with infusion of less than 24 hrs . several studies have reported that an increase in serum creatinine levels , such as that observed with nesiritide , predicts a higher risk of death even when that increase is transient [ 74 , 75 ] . unlike these previous studies , a more recent meta - analysis has reported that nesiritide is not associated with a higher 30- or 180-day mortality . given the limitations of the meta - analyses , without randomized controlled trials powered to evaluate mortality , arora and coworkers suggest that it is premature to abandon the use of nesiritide . a summary of the current information concerning the benefits and deleterious effects of the use of nesiritide is provided in table 1 . future clinical trials are necessary to address the concerns raised and provide a better understanding of the actions of nesiritide in the management of acute decompensated heart failure . in addition , recent studies have reported that npr - b , not npr - a , which is the receptor of nesiritide , is the predominant npr in the failing heart , suggesting that drugs directed towards both nprs might provide a greater benefit than nesiritide per se . beneficial and deleterious effects of nesiritide in cardiovascular and renal systems the clinical role of natriuretic peptides either as biomarkers for diagnosis , prognosis or monitoring of therapy , or as therapeutic strategies for cardiovascular and renal disorders has gained acceptance over the last decade . despite specific basic studies and clinical trials necessary to better understand the possibilities of natriuretic peptides in therapeutic interventions , the use of these peptides to treat cardiovascular dysfunction seems to be most promising .

abstractthe natriuretic peptides are a family of related hormones that play a crucial role in cardiovascular and renal homeostasis . they have recently emerged as potentially important clinical biomarkers in heart failure . natriuretic peptides , particularly brain natriuretic peptide ( bnp ) and the inactive n - terminal fragment of bnp , nt - probnp , that has an even greater half - life than bnp , are elevated in heart failure and therefore considered to be excellent predictors of disease outcome . nesiritide , a recombinant human bnp , has been shown to provide symptomatic and haemodynamic improvement in acute decompensated heart failure , although recent reports have suggested an increased short - term risk of death with nesiritide use . this review article describes : the current use of bnp and its inactive precursor nt - probnp in diagnosis , screening , prognosis and monitoring of therapy for congestive heart failure , the renoprotective actions of natriuretic peptides after renal failure and the controversy around the therapeutic use of the recombinant human bnp nesiritide .

in cases of paraesophageal hiatal hernia with gastric volvulus , which is referred to as upside - down stomach , the first choice is surgical treatment . in high - surgical - risk patients , endoscopic repositioning and gastropexy is useful to ensure a good quality of life . however , there are no detailed reports regarding recurrent cases of upside - down stomach after endoscopic treatment ; therefore , the risks of recurrence are unknown . we had a case of recurrent upside - down stomach after endoscopic repositioning and gastropexy that indicated the limits of endoscopic treatment and the risk of recurrence , as reported here . an 88-year - old woman with periodic vomiting and inability to eat was admitted to our hospital on three separate occasions where she received conservative treatment for a few days . because of her advanced age , complications ( bedridden state , left hemiplegia after cerebral infarction , heart failure ) and poor general condition , the minimally invasive method of endoscopic repositioning and gastropexy was performed . the stomach was reduced to the normal anatomic position , and the anterior stomach wall was fixed to the abdominal wall in three places as widely as possible ( fig . chest and abdominal computed tomography showed the gastric fornix in the abdominal cavity , but the gastric corpus was above the diaphragm with organoaxial volvulus ( fig . she was then diagnosed with a recurrent upside - down stomach after endoscopic repositioning and gastropexy . she was initially managed conservatively and had a nasogastric tube inserted , but her condition did not improve . although her anterior stomach wall had been fixed to the abdominal wall in three places as widely as possible in the last hospitalization , she again presented with upside - down stomach . we concluded that it was impossible to prevent the recurrence of an upside - down stomach by endoscopic therapy . therefore , we treated the condition with a laparoscopic repair of hernias and gastropexy . using a laparoscope , we observed that the anterior stomach wall was adhered to the abdominal wall in three places from the antrum to the lower corpus of the stomach ( fig . the upper corpus of the stomach was rotated and herniated into the esophageal hiatus ( fig . however , it quickly returned into the esophageal hiatus with rotation because of the adhesion between the omentum and the esophageal hiatus ( fig . this adhesion was peeled off and the crura were closed by a primary suture ( fig . the cardial part of the stomach wall was fixed to the crura , and the anterior stomach wall was fixed in three places to the abdominal wall widely from the upper corpus to the antrum ( fig . the suture used for fixing the stomach wall was removed 44 days after the operation . a paraesophageal hiatal hernia is a herniation of the gastric fundus anterior to a normally positioned esophagogastric junction . in large paraesophageal hernias , the entire stomach can be herniated with volvulus , which we refer to as upside - down stomach . surgical intervention comprising reduction of the stomach in the gastric lodge and calibration of the hiatus adding a gastropexy or antireflux procedure is the first choice of treatment for this condition . although publications describe the results of retrospective studies , the hospital stay after laparoscopic repair of paraesophageal hiatal hernias was shorter than that after conventional surgery . the morbidity reported in patients treated by conventional surgery exceeded the morbidity reported in laparoscopically operated patients , and follow - up was longer for conventional surgery . the recurrence rate after laparoscopic repair for large hiatal hernias was higher than after conventional repair . reoperative findings were intraluminal mesh erosion , esophageal stenosis , dense fibrosis , esophageal perforation and intragastric migration [ 5 , 6 ] . considering the recurrence and reoperation rates , the differences between mesh repair and primary repair were small and perhaps clinically inconsequential . in our case , the esophageal hiatus was not so large and could be closed easily ; therefore , we did not use mesh . the addition of a fundoplication in the laparoscopic repair of paraesophageal hiatal hernia minimized postoperative reflux symptoms . when no objective evidence of reflux was seen preoperatively , an antireflux procedure was not necessary . in our case , a fundoplication , therefore , was not performed and reflux symptoms were not observed following the procedure . for upside - down stomach , preventing recurrence of volvulus , a gastropexy is added to repair the paraesophageal hiatal hernia . in our case , the anterior stomach wall was fixed in three places to the abdominal wall widely from the upper corpus to the antrum . many patients with upside - down stomach are not candidates for surgical therapy because of advanced age or comorbid conditions . for these patients , endoscopic repositioning and fixation by percutaneous endoscopic gastrostomy ( peg ) can be performed [ 10 , 11 ] . in our case , one reason is the narrow range where the stomach wall can be fixed endoscopically , although the fixation was performed as widely as possible . therefore , performing endoscopic gastropexy fixation of the stomach by 2 pegs is recommended [ 12 , 13 ] . in our case , the anterior stomach wall was fixed to the abdominal wall in three places as widely as possible . however , as observed with a laparoscope , the anterior stomach wall was adhered only from the antrum to the lower corpus of the stomach , and the upper corpus of the stomach was rotated and herniated into the esophageal hiatus . in endoscopic gastropexy , the stomach wall is fixed by penetrating the peg tube or stomach wall fixture through the stomach wall and the abdominal wall . to avoid penetrating the chest wall and the lungs , the range of abdominal wall which can be fixed to the stomach wall since the stomach wall can not be pulled caudally , unlike in the surgical approach , the range of stomach wall which can be fixed to the abdominal wall is limited from the antrum to the lower corpus of the stomach . as a result the other reason for recurrence is the adhesion between the omentum and the esophageal hiatus . therefore , a repeated case has risks of recurrence of upside - down stomach after endoscopic treatment . in conclusion , although endoscopic repositioning and gastropexy for upside - down stomach can be a useful alternative method in high - risk patients , some cases may recur . repeated upside - down stomach caused by adhesions has risks of recurrence after endoscopic treatment . to use this therapy more effectively , we hope that more cases will be reported and the risks of recurrences revealed .

patients with an upside - down stomach usually receive surgical treatment . in high - risk patients , endoscopic repositioning and gastropexy can be performed . however , the risk of recurrence after endoscopic treatment is not known . we treated a case of recurrent upside - down stomach after endoscopic therapy that indicated the limits of endoscopic treatment and risk of recurrence . an 88-year - old woman was treated three times for vomiting in the past . she presented to our hospital with periodic vomiting and an inability to eat , and a diagnosis of upside - down stomach was made . endoscopic repositioning and gastropexy were performed . the anterior stomach wall was fixed to the abdominal wall in three places as widely as possible . following treatment , she became symptom - free . three months later , she was hospitalized again because of a recurrent upside - down stomach . laparoscopic repair of hernias and gastropexy was performed . using a laparoscope , two causes of recurrence were found . one cause was that the range of adherence between the stomach and the abdominal wall was narrow ( from the antrum only to the lower corpus of stomach ) , so the upper corpus of stomach was rotated and herniated into the esophageal hiatus . the other cause was adhesion between the omentum and the esophageal hiatus which caused the stomach to rotate and repeatedly become herniated . although endoscopic treatment for upside - down stomach can be a useful alternative method in high - risk patients , its ability to prevent recurrence is limited . moreover , a repeated case caused by adhesions has risks of recurrence .

the general public is exposed to multiple sources of information on medicine , either through traditional mass media ( e.g. , television , journals , and radio ) or through the internet ( e.g. , blogs , twitter , facebook , and the many features of web 2.0 ) . the media 's potential to reach large audiences gives it an important role in providing health - related information , shaping public health - related beliefs , and influencing health behaviors . however , medical information published in newspapers can often be based on scant evidence because of a lack of specialized knowledge of medical journalists , criticisms of medical information in newspapers , and a lack of available time to secure strong evidence prior to copy deadlines . in addition , information from newspapers , which should be understood by the general population , is sometimes an exaggeration or overstatement . erectile dysfunction is defined as a consistent or recurrent inability of a man to obtain and/or maintain a penile erection sufficient for sexual activity . a duration of symptoms of at least 3 months is considered acceptable to establish a diagnosis of erectile dysfunction , except in some instances of trauma or surgery . erectile dysfunction affected about 150 million men worldwide in 2000 , and this number is expected to double by 2025 as a result of improved life expectancy and the age - related nature of erectile dysfunction . this disorder is also associated with lower overall life satisfaction scores , mental health quality of life ( qol ) scores , and vitality qol scores . many patients look for information on erectile dysfunction from various media outlets , and commercials on treatment for erectile dysfunction have been developed . recently , sugita and miyakawa reported that the size of the japanese market for counterfeit phosphodiesterase-5 inhibitors was estimated to be about 2.5 times larger than that for genuine phosphodiesterase-5 inhibitors . the aim of the present study was to evaluate the accuracy of news information on erectile dysfunction from 10 nationwide newspapers . our present study will add to the growing body of research documenting the nature and influence of news information on erectile dysfunction in korea . articles on erectile dysfunction from 10 nationwide daily newspapers in korea from january 2011 through december 2011 were evaluated in this study . the newspapers included were the kyunghyang shinmun , the kukminilbo , the dong - a ilbo , the munhwa ilbo , the seoul shinmun , the segye ilbo , the hankyoreh , the hankok ilbo , the chosun ilbo , and the korea joongang daily . newspaper articles or columns that reported results of clinical or experimental studies were included ; however , articles that discussed disease occurrence , accidents , crime , policies , social phenomena related to medicine , questionnaire surveys , educational information , esoteric medical information from unreliable sources , and advertisements were excluded . news articles that summarized scientific congresses or journal articles that did not include direct quotations were also excluded . because a public set of data was used that did not include personal data , the present study was exempted from institutional review board review . the websites of both the chosun ilbo and korea joongang daily ( http://www.chosun.com and http://joongang.joinsmsn.com ) newspapers were used to search for relevant articles , whereas the website of the korean press foundation ( http://www.kinds.or.kr ) was used to search for articles from the other newspapers identified above . , we identified 20 articles in a pilot test to confirm the evaluation process for interpretation . all newspaper articles were separately extracted by two researchers ( y.s.h . and j.y.l . ) . differences in the results of the extraction and interpretation were discussed , and agreement was sought from a senior physician specializing in andrology ( k.s.c . ) . the articles were first categorized into articles with accurate statements and articles with inaccurate or misleading statements . on the basis of a previous study , articles with inaccurate or misleading statements were subclassified into four groups : 1 ) using inappropriate surrogate outcomes as clinical endpoints , 2 ) extrapolating nonhuman results to humans , 3 ) exaggerating the significance of results , and 4 ) using incorrect words . surrogate outcomes are defined as physiological or anatomical results ; however , clinical endpoints are defined as patient - related or functional outcomes , such as symptomatic improvement , recovery of normal activity , and survival . traditionally , surrogate outcomes have been interpreted as clinical endpoints , although the two should not be considered equivalent . for instance , an article on post - prostatectomy - induced erectile dysfunction that could be cured by mesenchymal stem cells demonstrated a typical example of confusing surrogate outcomes with end outcomes . results from animal experimental research or the use of cell lines may be inappropriately interpreted as equivalent to human data . although experimental , physiological , and animal studies are valuable and useful , they do not directly influence clinical decisions . finally , the use of incorrect words suggests that subjective and apocryphal terminologies may have been used . data extraction , sharing , and cooperation with our researchers were performed by using google drive ( http://drive.google.com ) and google docs ( google inc . , mountain view , ca , usa ) . a pearson chi - squared test and fisher exact test were used to compare distributions of categorical values , and statistical analyses were performed by using r ( r ver . 2.15.3 , r foundation for statistical computing , vienna , austria ; http://www.r-project.org ) . articles on erectile dysfunction from 10 nationwide daily newspapers in korea from january 2011 through december 2011 were evaluated in this study . the newspapers included were the kyunghyang shinmun , the kukminilbo , the dong - a ilbo , the munhwa ilbo , the seoul shinmun , the segye ilbo , the hankyoreh , the hankok ilbo , the chosun ilbo , and the korea joongang daily . newspaper articles or columns that reported results of clinical or experimental studies were included ; however , articles that discussed disease occurrence , accidents , crime , policies , social phenomena related to medicine , questionnaire surveys , educational information , esoteric medical information from unreliable sources , and advertisements were excluded . news articles that summarized scientific congresses or journal articles that did not include direct quotations were also excluded . because a public set of data was used that did not include personal data , the present study was exempted from institutional review board review . the websites of both the chosun ilbo and korea joongang daily ( http://www.chosun.com and http://joongang.joinsmsn.com ) newspapers were used to search for relevant articles , whereas the website of the korean press foundation ( http://www.kinds.or.kr ) was used to search for articles from the other newspapers identified above . searching was not performed by using the real newspaper . however , the korean press foundation supplied the nearest article in the printed version . , we identified 20 articles in a pilot test to confirm the evaluation process for interpretation . all newspaper articles were separately extracted by two researchers ( y.s.h . and j.y.l . ) . differences in the results of the extraction and interpretation were discussed , and agreement was sought from a senior physician specializing in andrology ( k.s.c . ) . the articles were first categorized into articles with accurate statements and articles with inaccurate or misleading statements . on the basis of a previous study , articles with inaccurate or misleading statements were subclassified into four groups : 1 ) using inappropriate surrogate outcomes as clinical endpoints , 2 ) extrapolating nonhuman results to humans , 3 ) exaggerating the significance of results , and 4 ) using incorrect words . surrogate outcomes are defined as physiological or anatomical results ; however , clinical endpoints are defined as patient - related or functional outcomes , such as symptomatic improvement , recovery of normal activity , and survival . traditionally , surrogate outcomes have been interpreted as clinical endpoints , although the two should not be considered equivalent . for instance , an article on post - prostatectomy - induced erectile dysfunction that could be cured by mesenchymal stem cells demonstrated a typical example of confusing surrogate outcomes with end outcomes . results from animal experimental research or the use of cell lines may be inappropriately interpreted as equivalent to human data . although experimental , physiological , and animal studies are valuable and useful , they do not directly influence clinical decisions . finally , the use of incorrect words suggests that subjective and apocryphal terminologies may have been used . data extraction , sharing , and cooperation with our researchers were performed by using google drive ( http://drive.google.com ) and google docs ( google inc . , mountain view , ca , usa ) . a pearson chi - squared test and fisher exact test were used to compare distributions of categorical values , and statistical analyses were performed by using r ( r ver . 2.15.3 , r foundation for statistical computing , vienna , austria ; http://www.r-project.org ) . after website searches , a total of 188 news articles on erectile dysfunction from 10 newspapers in 2011 were reviewed . 1 ) ; excluded articles were those discussing disease occurrence ( 16 ) , accidents ( 11 ) , crime and policy ( 7 ) , social phenomena related to medicine ( 5 ) , questionnaire surveys ( 4 ) , educational information ( 4 ) , esoteric medical information from unreliable sources ( 63 ) , and advertisements ( 57 ) . in addition , 21 articles that summarized scientific congresses or journal articles were also excluded . our exclusion criteria were based on the concept that simple reporting of an article without discussion of the outcomes of the research was not new health information . of the 47 articles that met our inclusion criteria , 27 ( 57.4% ) contained inaccurate or misleading statements on the basis of an evidence - based medicine evaluation . these included using inappropriate surrogate outcomes as clinical endpoints ( three cases , 6.4% ) , extrapolating nonhuman results to humans ( two cases , 4.3% ) , exaggerating the significance of results ( eight cases , 17.0% ) , and using incorrect words ( 14 cases , 29.8% ; fig . the rate of error was higher in information from korean sources than in information from international sources ( 22 cases vs. 5 cases ) . however , there were no significant differences between korean and foreign sources in any category ( table 1 ) . there were also no differences between articles written by general journalists and those written by medical journalists or specialists ( 13 cases by general journalists vs. 14 cases by medical journalists ) . after website searches , a total of 188 news articles on erectile dysfunction from 10 newspapers in 2011 were reviewed . 1 ) ; excluded articles were those discussing disease occurrence ( 16 ) , accidents ( 11 ) , crime and policy ( 7 ) , social phenomena related to medicine ( 5 ) , questionnaire surveys ( 4 ) , educational information ( 4 ) , esoteric medical information from unreliable sources ( 63 ) , and advertisements ( 57 ) . in addition , 21 articles that summarized scientific congresses or journal articles were also excluded . our exclusion criteria were based on the concept that simple reporting of an article without discussion of the outcomes of the research was not new health information . of the 47 articles that met our inclusion criteria , 27 ( 57.4% ) contained inaccurate or misleading statements on the basis of an evidence - based medicine evaluation . these included using inappropriate surrogate outcomes as clinical endpoints ( three cases , 6.4% ) , extrapolating nonhuman results to humans ( two cases , 4.3% ) , exaggerating the significance of results ( eight cases , 17.0% ) , and using incorrect words ( 14 cases , 29.8% ; fig . the rate of error was higher in information from korean sources than in information from international sources ( 22 cases vs. 5 cases ) . however , there were no significant differences between korean and foreign sources in any category ( table 1 ) . there were also no differences between articles written by general journalists and those written by medical journalists or specialists ( 13 cases by general journalists vs. 14 cases by medical journalists ) . good health is a prime global concern , and various factors including economic development play a significant role in shaping and defining a population 's perception of health . in particular , men 's health , including erectile dysfunction and andropause , are important matters in developing and developed countries . in asia , the men 's attitudes to life events and sexuality ( males ) study , which studies the prevalence and treatment of erectile dysfunction in china , japan , korea , malaysia , and taiwan , reported that 6.4% of 10,934 men between 20 and 75 years of age self - reported erectile dysfunction , and 83.5% found suitable treatments in the media . tannenbaum , in a survey on men 's health of 2,325 canadians between 55 and 97 years of age , reported that 35.7% of men surveyed , especially those of a younger age , showed an interest in erectile dysfunction . in an epidemiological study on erectile dysfunction in korean men , the rate of self - reported erectile dysfunction was 13.4% in 1,570 men aged 40 - 75 years , and the percentage of men scoring fewer than 17 points on the international index of erectile function scale was 32.4% , demonstrating a high prevalence of erectile dysfunction among korean men . thus , the prevalence of erectile dysfunction is high , particularly in developed countries like korea . it might be difficult for men with erectile dysfunction who are keen to receive treatment to find a urologist , schedule an interview , and finally receive medical treatment . reported on the numbers of prescriptions written for phosphodiesterase-5 inhibitors from each department within three medical institutions in korea . in their study , the mean rate of prescriptions for phosphodiesterase-5 inhibitors was approximately 46.4% from the urology departments , compared with 33.0% from endocrinology , 5.1% from neurology , 4.4% from cardiology , and 3.9% from family medicine , which indirectly suggests that the medical diagnosis and treatment of erectile dysfunction was relatively low in the urology departments of the three hospitals . mass media such as newspapers and television broadcasts are alternative sources of medical and health information . with the recent introduction of medical journalists , many medical news articles have appeared in newspapers and television broadcasts and are subsequently redistributed via the internet . unfortunately , as shown in our present study , the accuracy of these medicine - related articles can be unreliable . hwang et al . performed an evidence - based medical evaluation of health information from the television news . in that study , over an 8-month period , there were 85 such reports on the evening news , and 34 of them ( 40.0% ) were found to be inaccurate or misleading . in particular , using inappropriate surrogate outcomes as clinical endpoints was the most frequent error . this could be attributed to the misinterpretation of results owing to inappropriate study designs , or medical researchers assuming that anatomical or physiological endpoints in their studies could be translated directly to represent clinical outcomes in patients , and the ignorance of news reporters on the medical issues they were reporting . the reason for this might be that the researchers published exaggerated results , and the news media chose to focus on this misinformation of popular interest . the frequency of errors was higher in articles from korean sources than in articles from foreign sources . news articles from the foreign presses were re - reported by the korean media mainly because they were easily accessible to the journalists . on the contrary , domestic sources were often more interested in publicizing study researchers and their associated hospitals , often exaggerating claims although the level of korean media has been increasing , it may report biased content not based on exact evidence . because an increasing number of medical commentaries initially reported on television news and newspapers are now available online in media such as twitter , facebook , and youtube , the accuracy of primary sources ( i.e. , television news and newspapers ) has become more crucial . presently , the korean urological association and the korean society for sexual medicine and andrology do not use twitter or facebook aggressively , so as to reduce " retweeting " or " liking " misleading information and comments made by the television news or newspapers . to curtail these errors , evidence - based medical methodology must be agreed upon and practiced by all medical journalists and the news media . doctors must first embrace evidence - based medicine during the course of their training and clinical work and publish their research findings in relevant peer - reviewed journals before making their results public . additionally , medical journalists should spend more time studying and digesting medical - related information and receive training in evidence - based medicine . last but not least , both the korean urological association and the korean society for sexual medicine and andrology should constantly monitor misleading medical articles and request revisions whenever necessary . it is only through these measures that the general public can be spared the negative effects of medical misinformation . approximately 57% of all articles on erectile dysfunction from 10 nationwide daily newspapers were found to be inaccurate on the basis of an evidence - based medicine evaluation . to this effect , urologists , the korean urological association , and the korean society for sexual medicine and andrology play a paramount role in identifying articles containing inaccurate information . in addition , medical journalists should be trained to write articles based on evidence rather than sensation .

purposea rapid growth in the socioeconomic status of koreans has triggered an unprecedented explosion of health information for the general population . despite its obvious benefits , this increase in information could also result in potentially harmful effects for both consumers and professionals who do not use it appropriately . thus , this study was conducted to evaluate the quality and accuracy of health information on erectile dysfunction from 10 nationwide daily newspapers.materials and methodsthis study analyzed health information from 10 nationwide daily newspapers in korea from january 2011 through december 2011 . we reviewed the health information for quality by using evidence - based medicine tools and evaluated the accuracy of the information provided . articles that simply summarized scientific congresses or journal articles and that did not include direct quotations were excluded , as were advertisements.resultsa total of 47 articles were gathered . among them , 27 ( 57.4% ) contained inaccurate or misleading statements on the basis of an evidence - based medicine evaluation . these statements included using inappropriate surrogate outcomes as clinical endpoints ( three cases , 6.4% ) , extrapolating nonhuman results to humans ( two cases , 4.3% ) , exaggerating the significance of results ( eight cases , 17.0% ) , and using incorrect words ( 14 cases , 29.8% ) . the rate of error was higher in the information from korean sources than in that from international sources ( 22 cases vs. 5 cases).conclusionsapproximately 57% of all articles on erectile dysfunction from 10 nationwide daily newspapers were found to contain inaccuracies .

for the invasive treatment of symptomatic aortic stenosis several attractive options became available beyond conventional cardiosurgical implantation of stented bioprostheses or mechanical valves . besides stentless prostheses , in particular transcatheter procedures are of growing importance . nevertheless the number of implanted stented bioprostheses in germany expanded or at least remained stable over the last years . thus , 9.704 bioprostheses were implanted in germany in 2010 , according to 84% of all implanted prostheses in aortic position . implantation of bioprostheses was associated with a 30-day - mortality of 3,3 % , which is higher than for mechanical valves ( 1,5% ) due to the fact that patients for bioprostheses are significantly older or suffer from more complex co - morbidities . the purpose of this review is to summarize the features of the currently available bioprostheses in the light of competing procedures . there has been a continuous development to improve the design of stented bioprostheses with respect to hemodynamic profile , biological durability and ease of implantation . various designs are available , allowing a valve selection according to individual patient factors or anatomical criteria including techniques for intra - annular and supra - annular positioning [ 3 , 4 ] . it would have the same biological and hemodynamic properties as a normal valve and would not undergo degeneration . stented bioprostheses have proven to be effective even in small aortic roots regarding hemodynamic improvement , left ventricular mass reduction and improvement of the patient s quality of life . in vitro examinations showed pericardial valves to be slightly superior to porcine valves with regard to gradient and orifice area . associated with the scalloped designs of stented bioprostheses a tension - free implantation with avoidance of paravalvular leaks can be expected . implantation techniques evolved over the last decades which resulted in short cross - clamp times in normal findings . surgeons learned that meticulous care in decalcifying the aortic annulus and sizing helps to prevent paravalvular leaks . additionally , accurate sizing prevents a patients - prosthesis - mismatch which could result in poor postoperative clinical performance . avoiding the use of running suture for stented valve implantation the evolvement of low profile valves reduces the risk of coronary occlusion in patients with a small distance between annulus and coronary ostia . the currently available and established bioprostheses show rates of degeneration at 20 years around 15% in patients aged 65 or higher . the freedom from repeat aortic valve replacement reaches over 85% after 20 years in patients older than 60 years and 65% in all age groups . along with these convincing results of durability and freedom of re - implantation stented bioprostheses became also attractive for younger patients . if a repeated valve replacement is necessary due to functional deterioration the operative mortality is acceptable with 4 to 6% and is mainly due to active endocarditis and comorbidities . lately transcatheter aortic valve implantation ( tavi ) with valve - in - valve implantation into the degenerated xenograft showed to be an additional option in high risk patients with the need for redo valve replacement . stentless porcine valves were initially believed to have superior hemodynamic properties resulting in more effective reduction of left ventricular mass and better clinical performance according to nyha class . these findings could nt be yet approved by long - term follow up investigations [ 15 , 16 ] . bearing this in mind calls the more demanding implantation technique of stentless prostheses in question . stented bioprostheses are still used more commonly than stentless ones because of their relative ease of implantation , their extensively documented long - term results , and the low risk associated with reoperation . another biological approach is the use of an autograft like the ross procedure , where the patient s pulmonic valve is transferred to the aortic position . these pulmonic autografts have excellent hemodynamic properties as well as low rates of thrombosis , degeneration , and endocarditis . the ross procedure is suitable for children and young adults because it is compatible with further growth of the aortic root . nevertheless since the pulmonary valve has to been replaced by a bioprosthesis it is a demanding two valve procedure with prolonged cross clamping times . aside from this , special means are necessary for pulmonary autograft stabilization to prevent its degeneration . there are only a few current randomized trials comparing the long - term results of biological and mechanical valves . a large - scale review revealed no difference in survival rates at 10 years and a slightly higher survival rate at 15 years for patients with mechanical prostheses . the reoperation rate for mechanical valves in the aortic position is less than 5% at 10 years and less than 10% at 15 years , while the corresponding figures for bioprostheses are 10% and 30% , respectively . hemorrhagic complications are significantly more common in patients with mechanical valves because of anticoagulation . chronic atrial fibrillation is no longer a contraindication to bioprostheses since permanent oral anticoagulation can be avoided if concomitant ablative surgery results in permanent conversion to sinus rhythm . end stage renal failure was also defined a contraindication to bioprostheses since a rapid degeneration was feared due to altered metabolism in these patients . it has been found that life expectancy is already curtailed to such an extent that bioprosthesis degeneration often does not occur in the remaining lifetime . the supposed advantage of the longer durability of a mechanical valve is also offset by the potential complications of oral anticoagulation , especially because anticoagulation is more difficult to manage in dialysis patients than in others . nevertheless aortic valve replacement using a tissue valve remains controversial for patients younger than 60 years since there are studies reporting reduced mid - term results . some younger patients are averse to oral anticoagulation and therefore prefer a biological valvular prosthesis . thus , younger patients opting for a bioprosthesis can enjoy a normal quality of life without anticoagulation for many years but may need to undergo a second valve replacement procedure with an acceptable degree of risk . implantation of conventional prostheses can be performed through a limited direct access like a hemi - sternotomy or a lateral access . reported large series show that aortic valve operations can be safely performed in experienced centers . substantial progress in valve technology led to the development of self - expanding valves . since a few years a number of bioprostheses are available for suture - less implantation which is usually combined with a limited surgical access . . future will show the benefits of this procedure characterized by short cross clamping times and whether it can coexist with tavi procedures . after its clinical inauguration by cribier in 2002 the transcatheter aortic valve implantation gained widespread use in high risk patients with aortic stenosis . in germany a massive increase of its application can be observed ; in 2010 nearly every fourth aortic valve replacement was performed as a tavi procedure . the partner trial was a randomized trial comparing tavi with standard - of - care therapies in high risk patients . a 2-year follow - up of patients in the partner trial supports lately tavi as an alternative to surgery in high - risk patients . the two treatments were similar with respect to mortality , reduction in symptoms , and improved valve hemodynamics , but paravalvular regurgitation was more frequent after tavi and was associated with increased late mortality . there are a growing number of publications reporting promising short term results in high risk patients . nevertheless there is still a lack of evidence that tavi is superior to open valvular replacement as the gold standard regarding long - term results . current guidelines recommend the use of tavi restricted to patients with contraindications for open surgery or inacceptable perioperative risks . current studies reveal that bioprostheses of the most recent generation last longer than earlier types . because life expectancies in general have risen , more and more elderly patients are presenting for valve replacement and for these patients a bioprosthesis is usually chosen . in addition the cost effectiveness of stented bioprostheses appears unbeatable and the surgical ease of implantation allows for cross - clamp times between 30 and 60 minutes . overall , even in face of more innovative biological alternatives the implantation of stented bioprostheses is still a very interesting option and represents actually the most frequent valve implantation technique for aortic stenosis . especially in the light of growing use of interventional valve replacement there is the urgent need for complete nationwide registry with adequate long term follow up , quality of life information and relevant subgroup analysis to define new standards in the treatment of patients with aortic stenosis .

introductionbiological stented prostheses are currently the main type of prosthetic valve used for aortic valve replacement . the ratio of bioprotheses to mechanical prostheses has switched in the last 15 years ; the percentage of biological prostheses implanted has risen from 30 % to 85 % . moreover the total number of implanted stented bioprostheses remained stable over the last years despite competing procedures like stentless prostheses or transcatheter aortic valve implantation.methodsa literature search of all published aortic valve replacement studies was performed from january 2000 through may 2012.resultsthe recommendations guiding the type of heart valve replacement have been revised in recent years . of particular interest are the new generation of biological prostheses with extended durability , a decrease in mortality of reoperation and an increase in life expectancy . comorbidities such as chronic renal insufficiency or chronic atrial fibrillation are no longer contraindications to bioprostheses.conclusionoverall , even in face of more innovative biological alternatives the implantation of stented bioprostheses is still a very interesting option and represents actually the most frequent valve implantation technique for aortic stenosis .

lumbosacral radicular pain is a common and disabling condition in adults with pathology of the bones in the distal spine , and it represents a significant quality - of - life issue.1 lower back pain is not always associated with radicular symptoms . clinical findings of neuropathy may vary significantly , depending on the pathogenesis of the pain symptoms . neuropathic pain is still a dark zone for neuroscientists and treatment remains challenging for clinicians . it has been described as a direct consequence of involvement of the somatosensory system , with a population prevalence between 7 and 10% , thus stressing the importance of a standardized approach for its recognition.2 pharmacological therapies have often proved ineffective , surgery has poorly characterized indications , and the use of interventional neuromodulation ( eg , pulsed radiofrequency and spinal cord stimulation ) is still debated or not always applicable.3 some older studies reported that opioids have poor efficacy for neuropathic pain , although this conclusion has been progressively questioned because of the lack of compelling evidence , and several reports arguing that a substantial proportion of patients with neuropathic pain might significantly respond to opioids.4 some investigators have evaluated the additional efficacy of local anesthetics , stressing evidence for the development of synergistic effects after coadministration with opioids , and found that bupivacaine and morphine together showed better results than either alone , in terms of patient satisfaction , medication intake , and pain clinic admissions.5 different routes of administration have been tested , and epidural and intrathecal drug delivery confers benefits in comparison with other routes . for instance , these routes avoid first - pass metabolism and the blood brain barrier and allow the use of lower doses at comparable efficacy , given that the medications are deposited directly at the spinal cord.68 considering that epidural administration can be maintained for longer periods with lower risks relative to the intrathecal route , it may be possible to utilize other analgesics to obtain adequate analgesia for prolonged periods . to the best of our knowledge , no previous study has assessed how epidural therapy with combined opioids and local anesthetics might provide long - lasting pain modulation . the aim of this study was to investigate the effectiveness of epidural morphine and bupivacaine in patients with chronic neuropathic pain associated with lumbosacral radiculopathy . the research protocol was approved by the institutional review board committee of the santa maria maddalena hospital , occhiobello ( ro ) , italy . informed consent was obtained from each patient , and the patients enrolled in the research were allowed to withdraw from the study at any point if they wished . prior to the enrollment , every patient was evaluated to confirm probable or definite neuropathic involvement.9 the following criteria were adopted as inclusion criteria for the study subjects : severe pain intensity assessed with a numeric rating scale ( nrs ) score of 7/10.definite or probable neuropathic involvement as assessed by clinical examination and grading system.chronic lower extremity radicular pain lasting for > 6 months.clinical examination suggestive of radicular pain with 1 positive provocation tests ( eg , straight leg raise test).poor response to pharmacological therapy , physical therapy , or epidural injection of anti - inflammatories ( ie , pain reduction in nrs 30% during the 6 months before enrollment).magnetic resonance imaging and/or electromyographic test suggestive of radiculopathy . severe pain intensity assessed with a numeric rating scale ( nrs ) score of 7/10 . definite or probable neuropathic involvement as assessed by clinical examination and grading system . clinical examination suggestive of radicular pain with 1 positive provocation tests ( eg , straight leg raise test ) . poor response to pharmacological therapy , physical therapy , or epidural injection of anti - inflammatories ( ie , pain reduction in nrs 30% during the 6 months before enrollment ) . magnetic resonance imaging and/or electromyographic test suggestive of radiculopathy . the following exclusion criteria were applied : possible or unlikely neuropathic pain.positive response to previous treatments.comorbid neurological or psychiatric disorders.significant motor deficits . . significant motor deficits . the procedure was performed in a suitable operating room , with disinfection and sterilization of all devices . a sterile operative field was maintained while each patient was lying on a fluoroscopy table in the prone position for needle introduction . after injection of local anesthetic to numb the operative zone , a 16-g hollow needle was inserted through the paramedian tissues at a 40 angle , with the skin and epidural space located by standard loss of resistance to air techniques . an epidural catheter was inserted at the t12l1 intervertebral space , with the correct placement verified by fluoroscopy . the position of the catheter tip was confirmed by injection of x - ray contrast medium ( iopamiro 300 ; bracco imaging , s.p.a . , the catheter was tunneled for 5 cm subcutaneously and then fixed at 10 cm in the abdominal skin , and the patient was transported to the recovery room ( figure 1 ) . on the following day , each patient received an epidural analgesic injection followed by a 2 ml flush with normal saline . the injection solution was composed of morphine chlorhydrate ( 0.1 mg / ml ) and bupivacaine ( 1 mg / ml ) , and the dose was progressively increased to 3 ml three times a day ( total dosage per day : morphine 0.9 mg and bupivacaine 9 mg ) . a variable ratio of the opioid and local anesthetic agents was used , such that the dosages of the two drugs were adjusted in order to obtain the optimal analgesia with the fewest side effects in comparison to the postoperative pain treatment ( ie , morphine 24 mg / day and bupivacaine 0.125%).10 the medication was administered in the hospital for the first week and self - administered by the patient in the outpatient setting for the following 3 weeks . proper medication intake and catheter functioning were evaluated every week by an expert nurse and a pain physician . on the 30th day , the catheter was removed and an appointment for clinical follow - up in 1 month was scheduled . each patient was evaluated for pain levels by two independent reviewers not involved with catheter placement and drug administration , before catheter placement and at 1 month after removal . pain intensity was assessed with the 010 nrs and by the italian version of the mcgill pain questionnaire ( quid ) , which has 42 descriptors divided into four main pain rating index ranks ( sensory , affective , evaluative , and mixed ) . the total prir ( prir - t ) value , given by the sum of all the rank values , describes and quantifies the pain.11,12 response to treatment was considered successful with a pain reduction in nrs > 30% at follow - up . the wilcoxon s rank sum test was applied to evaluate changes in nrs and prir - t , comparing baseline and follow - up levels . moreover , differences between the four prir coefficients describing sensory ( prirc - s ) , affective ( prirc - a ) , evaluative ( prirc - e ) , and mixed ( prirc - m ) aspects of pain were compared in the responders and nonresponders . the association between pain variation in nrs and quid at the follow - up was calculated using the spearman s test . the kolmogorov the sample size was chosen in order to obtain a pain reduction > 30% at follow - up in patients with a pretreatment nrs pain intensity 7/10 . considering an alpha level of < 5% , a study group of 20 patients allowed achievement of a statistical significance of > 95% with statistical power of > 80% . the research protocol was approved by the institutional review board committee of the santa maria maddalena hospital , occhiobello ( ro ) , italy . informed consent was obtained from each patient , and the patients enrolled in the research were allowed to withdraw from the study at any point if they wished . prior to the enrollment , every patient was evaluated to confirm probable or definite neuropathic involvement.9 the following criteria were adopted as inclusion criteria for the study subjects : severe pain intensity assessed with a numeric rating scale ( nrs ) score of 7/10.definite or probable neuropathic involvement as assessed by clinical examination and grading system.chronic lower extremity radicular pain lasting for > 6 months.clinical examination suggestive of radicular pain with 1 positive provocation tests ( eg , straight leg raise test).poor response to pharmacological therapy , physical therapy , or epidural injection of anti - inflammatories ( ie , pain reduction in nrs 30% during the 6 months before enrollment).magnetic resonance imaging and/or electromyographic test suggestive of radiculopathy . severe pain intensity assessed with a numeric rating scale ( nrs ) score of 7/10 . definite or probable neuropathic involvement as assessed by clinical examination and grading system . clinical examination suggestive of radicular pain with 1 positive provocation tests ( eg , straight leg raise test ) . poor response to pharmacological therapy , physical therapy , or epidural injection of anti - inflammatories ( ie , pain reduction in nrs 30% during the 6 months before enrollment ) . magnetic resonance imaging and/or electromyographic test suggestive of radiculopathy . the following exclusion criteria were applied : possible or unlikely neuropathic pain.positive response to previous treatments.comorbid neurological or psychiatric disorders.significant motor deficits . . significant motor deficits . the procedure was performed in a suitable operating room , with disinfection and sterilization of all devices . a sterile operative field was maintained while each patient was lying on a fluoroscopy table in the prone position for needle introduction . after injection of local anesthetic to numb the operative zone , a 16-g hollow needle was inserted through the paramedian tissues at a 40 angle , with the skin and epidural space located by standard loss of resistance to air techniques . an epidural catheter was inserted at the t12l1 intervertebral space , with the correct placement verified by fluoroscopy . the position of the catheter tip was confirmed by injection of x - ray contrast medium ( iopamiro 300 ; bracco imaging , s.p.a . , the catheter was tunneled for 5 cm subcutaneously and then fixed at 10 cm in the abdominal skin , and the patient was transported to the recovery room ( figure 1 ) . on the following day , each patient received an epidural analgesic injection followed by a 2 ml flush with normal saline . the injection solution was composed of morphine chlorhydrate ( 0.1 mg / ml ) and bupivacaine ( 1 mg / ml ) , and the dose was progressively increased to 3 ml three times a day ( total dosage per day : morphine 0.9 mg and bupivacaine 9 mg ) . a variable ratio of the opioid and local anesthetic agents was used , such that the dosages of the two drugs were adjusted in order to obtain the optimal analgesia with the fewest side effects in comparison to the postoperative pain treatment ( ie , morphine 24 mg / day and bupivacaine 0.125%).10 the medication was administered in the hospital for the first week and self - administered by the patient in the outpatient setting for the following 3 weeks . proper medication intake and catheter functioning were evaluated every week by an expert nurse and a pain physician . on the 30th day , the catheter was removed and an appointment for clinical follow - up in 1 month was scheduled . each patient was evaluated for pain levels by two independent reviewers not involved with catheter placement and drug administration , before catheter placement and at 1 month after removal . pain intensity was assessed with the 010 nrs and by the italian version of the mcgill pain questionnaire ( quid ) , which has 42 descriptors divided into four main pain rating index ranks ( sensory , affective , evaluative , and mixed ) . the total prir ( prir - t ) value , given by the sum of all the rank values , describes and quantifies the pain.11,12 response to treatment was considered successful with a pain reduction in nrs > 30% at follow - up . the wilcoxon s rank sum test was applied to evaluate changes in nrs and prir - t , comparing baseline and follow - up levels . moreover , differences between the four prir coefficients describing sensory ( prirc - s ) , affective ( prirc - a ) , evaluative ( prirc - e ) , and mixed ( prirc - m ) aspects of pain were compared in the responders and nonresponders . the association between pain variation in nrs and quid at the follow - up was calculated using the spearman s test . the sample size was chosen in order to obtain a pain reduction > 30% at follow - up in patients with a pretreatment nrs pain intensity 7/10 . considering an alpha level of < 5% , a study group of 20 patients allowed achievement of a statistical significance of > 95% with statistical power of > 80% . eighty - three consecutive patients , diagnosed with radicular lumbosacral pain between january 2014 and december 2015 , were evaluated in the pain medicine unit of santa maria maddalena hospital . oral medications that were prescribed for therapy before the enrollment included non - steroidal anti - inflammatory drugs , antidepressants , anticonvulsants , and opioids , and the pre - enrollment physical therapy was based on a progressive specific exercise program involving the training of the deep abdominal and lumbar muscles . radicular epidural injection with triamcinolone 40 mg / ml and lidocaine 2% 0.3 ml was performed with ultrasound guidance.13,14 fifty - three patients did not meet inclusion criteria and were excluded from the study . two patients were excluded during the treatment period after malfunction and subsequent removal of the epidural catheter , and six patients reported no improvement in pain level at the fourth week and were removed from follow - up . ultimately , 22 patients remained enrolled in the study , and most ( 73% ) of these patients reached significant pain relief during treatment . the study profile and the epidural treatment effect on pain scores are summarized in figure 2 and table 2 , respectively . nrs and prir - t were significantly reduced at follow - up ( p=0.001 and p=0.03 , respectively ) . these data were confirmed by the parallel evolution in the two scores ( r=0.75 , p<0.001 ) , strengthening marked pain relief lasting up to 1 month after treatment cessation ( figure 3 ) . if the four pain rating coefficients were analyzed , none of the single items was significantly modified compared to the others either in responders or in nonresponders . the oswestry disability index was applied for clinical purposes and showed an overall improvement at follow - up . nevertheless , considering the frequent association with low back pain , disability caused by axial pain therefore , we chose not to include this index in the study due to the complex nature of lumbosacral radicular pain . a successful clinical outcome was observed in eleven of the 22 patients at the follow - up , with 50.4% of effect size and 97.2% of statistical power . in this study , we evaluated pain improvement after the cessation of epidural treatment with morphine and bupivacaine . at follow - up , half of the patients suffering from moderate - to - severe pain at baseline reported a significant improvement , which was confirmed with two validated scores , affecting the sensory and evaluative as much as affective aspects of pain . to our knowledge , this is the first study to report the effectiveness of epidural treatment with combined opioids and anesthetics persisting after catheter removal . if pain improvement had occurred , it was maintained for 1 month after therapy cessation , thereby suggesting a potential role for modifying the natural history of the disease . although the protocol was complex , involving hospital admission , placement of epidural catheter in the operating room , and the administration of medications for 4 weeks , all the patients included in the study had severe chronic pain that was refractory to several standard treatments . opioid infusion has increasingly been used as a treatment in noncancer pain and more recently has been assessed in patients with chronic pain . pain levels improve significantly with higher doses , but these levels can promote the risk of abuse and often burden the patient with adverse effects . one of the main pitfalls in opioid therapy lies in the progressive development of tolerance , which is responsible for decreasing efficacy.15 epidural delivery of anesthetics may be associated with prolonged analgesia and reduced mechanical allodynia in rats with nerve injury , whereas similar effects have also been described in humans with neuropathic pain after lidocaine administration.16 in other trials , the combination of intrathecal bupivacaine and morphine showed good efficacy in most cancer and nonmalignant pain patients with neuropathic or mixed pain . in addition to enhancing analgesia , bupivacaine combinations seem to be responsible for opioid - sparing regimens with diminished adverse effects.17 opioids are usually claimed to target dorsal horn cell receptors and cause pre- and postsynaptic modulation of pain , although peripheral effects have also been taken into account due to the identification of opioid receptors on peripheral sensory neuron processes.18 a previous study has also shown that microinjection of opioids into the brainstem may elicit powerful antinociceptive effect in animals and humans , likely involving the periaqueductal gray ( pag ) and the rostro - ventromedial medulla ( rvm).19 although the cellular mechanisms and sites of action are still debated , many local anesthetics block ion channels at the dorsal entry root zone and antagonize pain transmission via ad and c fibers.20 moreover , bupivacaine may inhibit spinal glutamatergic transmission , and its analgesic effect might also be subsequent to the modulation of n - methyl - d - aspartate receptors in the superficial dorsal horn.21 compelling evidence suggests that ectopic neural discharge may represent an underlying mechanism of neuropathic pain . these abnormal impulses may arise at the site of nerve injury or far from it , in apparently intact axons or structures , driven by abnormal discharges in ab or c fibers.22 in this regard , the role of the dorsal root ganglion ( drg ) has been widely investigated , due to its location as a border area between spinal cord and peripheral nerves . abnormal discharges in the drg tend to be more sensitive to lidocaine injection than those at the site of nerve injury . several studies highlighted that hyperexcitable drg neurons begin to show ongoing activity that lasts many days or weeks after nerve injury . in turn , this pathological activity might induce reversible central sensitization.23 in experimental models , systemic lidocaine silences the drg ectopic discharges without blocking nerve conduction , whereas local anesthetic agents applied intrathecally and to the drg surface in patients with phantom limb pain produce transient improvement of painful and nonpainful sensations in the majority of subjects , strengthening the conclusion that lidocaine can antagonize spontaneous bursting action potential discharge and sensitization.24 despite the distance from the catheter tip , sensitized drg might therefore have responded to low - dose bupivacaine and , because of a lack of efficient neurovascular barriers , it could be considered as a potential target in our study . in addition , ion channels are widely expressed in the central nervous system as much as in the periphery . the number of sodium channels ( eg , nav 1.3 ) has been found to be increased in dorsal horn and thalamic neurons after traumatic spinal cord injury , producing central pain . blocking the expression of such channels may reverse the excitability changes in either the central nervous system or the peripheral nervous system , whereas injection of lidocaine into the rvm blocks tactile and thermal hyperesthesia.25 increasing evidence is also documenting the anti - inflammatory properties of anesthetics , resulting from their modulating action on the immune system . manchikanti et al26 showed comparable long - lasting efficacy of epidural steroids and anesthetics in patients suffering from chronic lumbar disk herniation . this result might explain the improvement in low - back pain with radicular involvement , which often combines mixed features of nociceptive and neuropathic effects . how can we explain the maintenance of pain control efficacy in our results , lasting up to 1 month after the cessation of treatment and catheter removal ? several experiments showed that administration of systemic or local anesthetics may improve symptoms related to neuropathic pain for variable amounts of time after the end of therapy.27 some data stress the important role of bupivacaine in pain control : 1 ) the common tendency to increase the dose of opioids to optimize pain control , whereas in our study , it was maintained at a constant dose , and 2 ) the reported pain improvement after the addition of intrathecal bupivacaine in patients unresponsive to opioid alone.28 bupivacaine may enhance the analgesic effect of opioids through its action on voltage - gated calcium channels , changing opioid pharmacokinetics and receptor conformation.29 moreover , chronic pain has been associated with structural and functional changes in the human cortex and with dysfunctional descending pain modulation , which may be affected by medications and pain resolution , suggesting cortical plasticity processes.30 epidural addition of bupivacaine to opioids might help in restoring physiological antinociceptive mechanisms , therefore preventing or antagonizing chronic neuropathic pain.31 because our patients were suffering from refractory neuropathic pain , we considered a reduction of pain > 30% ( at follow - up ) as utilized in clinical trials , as significant.32 the analysis of quid coefficients failed to disclose significant pain variation among prirc - s , prirc - a , prirc - e , and prirc - m . the treatment improved sensory pain as much as affective quality of pain in responders , whereas none of these items changed compared to the others in poor responders . nevertheless , the use of pain coefficients is not sufficient to rule out placebo effects , which may play an important role in the subjective response to treatments.33 the heterogeneous and small number of patients may be another issue , given that neuropathic pain may develop from different disorders and etiologies , making tighter criteria too restrictive . nevertheless , we believe that the statistical power in this study was strong enough to support reliable results . finally , the short duration of follow - up in this study does not allow us to comment on the potential for long - term pain relief . in animal models , intravenous infusion of lidocaine was able to eliminate mechanical allodynia for at least 3 weeks after treatment , but the maximal duration in humans needs to be elucidated in further studies . the present results showed significant efficacy of combined epidural morphine and bupivacaine in the treatment of radicular neuropathic pain , which was maintained for up to 1 month after the end of administration . as a result of multiple pathophysiological events , central and peripheral neural changes may produce clinical symptoms and lead to chronic pain . maintaining significant pain relief with low doses of epidural bupivacaine / opioid may allow the physician to minimize adverse effects , neurotoxicity , and drug dosages , or to adopt more conservative approaches ( eg , physical therapy ) in pain management . therefore , further studies are needed to evaluate the safety and reliability of this regimen over long periods , to promote the use of epidural treatment in selected cases , or ultimately to develop new implantable pump systems engineered to provide infusions for extended pain relief .

objectivethe aim of this study was to investigate the therapeutic effectiveness of epidural morphine and bupivacaine in patients with chronic lumbosacral radicular neuropathic pain after the cessation of treatment.methodstwenty-two patients with chronic lumbosacral pain with neuropathic features were enrolled . an indwelling catheter was placed into the epidural space , and each patient received an epidural injection of morphine chlorhydrate and bupivacaine up to three times a day . the medication was administered for 4 weeks . the pain intensity score on a 010 numeric rating scale ( nrs ) , the total pain rating index rank ( prir - t ) , and its coefficients were evaluated before treatment and 1 month after catheter removal . p - value < 0.05 was considered statistically significant.resultsnrs and prir - t were significantly reduced at follow - up ( p=0.001 and p=0.03 , respectively ) , whereas the parallel evolution of the two scores ( r=0.75 and p<0.001 , respectively ) confirmed significant pain relief lasting up to 1 month after treatment cessation . none of the four pain rating coefficients was significantly modified compared to the others in either responders or nonresponders . successful clinical outcome ( pain reduction > 30% in nrs ) was reached and maintained in half of the patients at follow-up.conclusioncombined epidural morphine and bupivacaine seems to be effective in the treatment of neuropathic pain .

the need for categorization of anomalies and congenital aberrancies formed due to developmental vascular defects produce identifiable birthmarks of the skin and mucosa and a variable degree of underlying soft tissue abnormalities . presently a surge in the knowledge of criterias to classify these various anomalies has put forth classifications purely with respect to histopathological features of the disease . these lesions predominantly occur within the head and neck and affect approximately 1 in 22 children . involvement of the oral cavity is common but frequently requires unconventional treatment strategies for its management . though previously termed angiomas or vascular birthmarks , vascular anomalies are divided into two main categories : vascular tumors and vascular malformations . infantile hemangiomas comprise the majority of vascular anomalies and are considered the predominant vascular tumor type composed of rapidly proliferating endothelial cells . blood vessel architecture is incomplete and surrounded by hyperplastic cells in hemangiomas and other vascular tumors . in contrast , vascular malformations do not contain hyperplastic cells but consist of progressively enlarging aberrant and ectatic vessels composed of a particular vascular architecture such as veins , lymphatic vessels , venules , capillaries , arteries or mixed vessel type . the latter comprises lymphangiomas or lymphatic malformations which are congenital collections of ectatic lymph vessels that form endothelial lined cystic spaces . the pathogenesis of these tumors could be of importance in thoroughly understanding the mode of these varying histopathological presentations . they represent about 6% of the total number of benign tumors of the soft tissue in patients aged less than 20 years . regarding gender distribution of lymphangioma , it is equally divided between males and females , with about 50% of the lesions being noted at birth and 90% developing by 2 years of age . oral lesions may occur at various sites but they form most frequently on the anterior two thirds of tongue . they may increase in size , producing macroglossia which may lead to difficulties in mastication , deglutition , and speech ; and displacement of the teeth , with a resulting malocclusion . they may interfere with normal breathing , particularly during sleep , produce sleep apnea , and in certain instances , produce a life - threatening upper airway compromise . they can also be present in the palate , buccal mucosa , gingiva and lip . the tumor is superficial in location and demonstrates a white pebbly surface that resembles a cluster of translucent vesicles . the deeper lesions could mimic various soft tissue tumors since the color which is classically used for diagnosing such tumors would seem to be irrelevant . they appear as a nodule or masses without significant change in surface texture or color . a 9-year - old female patient reported to the department of oral & maxillofacial pathology , i.t.s cdsr , with a complaint of a painless growth with respect to left side of tongue . patient had given a history of trauma due to tongue bite around 3 months back and was enlarging slowly in size the swelling was initially small , peanut sized , which increased to the present size . on examination of the swelling a growth of 2 1 cm on the anterior part of the dorsal surface of tongue . the lesion was pale pink in color and oval in shape with well - defined margins [ figure 1 ] . intraoral photograph showing nodular swelling resembling cluster of vesicles on the left side of dorsum of tongue an incisional biopsy was performed and the tissue was histopathologically diagnosed as lymphangioma , since large lymphatic vessels lined by flattened endothelial cells pushing into the overlying epithelium were seen [ figure 2 ] . patient was recalled after 4 days and a total excision of the lesion was performed under local anesthesia ( la ) . photomicrograph of incisional biopsy showing large lymphatic vessels ( h and e , 10 ) examination of gross macroscopic appearance revealed the excised tissue to be oval shaped , measuring 1.5 1 cm in size and was creamish brown in color with a pebbly surface . microscopic examination of the excised lesion showed numerous large , dilated lymphatic channels of irregular shape , lined by flattened endothelial cells , of which some of the vessels were filled with lymph . numerous large- to medium - sized channels with thin endothelial lining , engorged with rbcs , were also present in the deeper area of the connective tissue [ figure 3 , inset ] . lymphangioma was first described by virchow in 1854 , and in 1872 , krester hypothesized that hygromas were derived from lymphatic tissue . the origin of lesion is considered to be congenital abnormality of lymphatic system rather than true neoplasm . a portion of the jugular lymphatic sac is thought to sequestrate from the primary sacs during fetal development with failure to establish communications with other lymphatic system . the fact that most lymphangiomas manifest clinically during early childhood and develop in areas where the primitive lymph sacs occur ( neck , axilla ) provides presumptive evidence for this hypothesis . on the contrary , it is argued that instead of being a congenital malformation , lymphangioma is a true neoplasm resulting from transformed lymphatic endothelial cells and/or stromal cells . the classic sequence of events in embryology and development of vasculogenesis falls into three stages : the undifferentiated capillary network stage , the retiform developmental stage and the final developmental stage . the first theory is that the lymphatic system develops from five primitive sacs arising from venous system . concerning the head and neck , endothelial outpouchings from the jugular sacs spread centrifugally to form the lymphatic systems . another theory proposes that the lymphatic system develops from mesenchymal clefts in the venous plexus reticulum and spreads centripetally toward the jugular sacs . several studies have been published regarding possible lymphangiogenic growth factor involvement in the etiology of lymphatic malformations . these factors include vascular endothelial growth factor ( vegf)-c , vascular endothelial growth factor receptor 3 ( vegfr-3 ) , and transcription factor prox-1 . vegf - c and vegfr-3 have been shown to be upregulated in lymphatic malformed tissue , and both are involved in lymphatic tissue proliferation . the tumor may be localized in a small area of tongue or floor of mouth or it may diffusely infiltrate these areas . if the tumor is located in a deeper area , it may present as submucosal mass . cervical lesions in a child cause dysphagia and airway obstruction which is rare in adults . the misunderstanding on the nosologic distinction between oral hemangiomas and vascular malformations leads to diagnostic mistakes . hemangiomas are differentiated from vascular malformations by their clinical appearance , histopathological features , and biologic features . the natural history of hemangiomas involves rapid proliferations for the first several months of life with subsequent spontaneous regression . vascular malformations are often recognized at birth and grow proportionately with the child , with many becoming more prominent at puberty . histologically , hemangiomas in the proliferating phase show endothelial hyperplasia and large number of mast cells . in contrast , vascular malformations show normal number of mast cells , and consist of mature , often combined , capillary , arterial , venous , and lymphatic elements . lymphatic malformations / lymphangiomas are classified microscopically into four categories : lymphangioma simplex ( lymphangioma circumscriptum ) composed of small , thin - walled lymphatics ; cavernous lymphangioma comprising dilated lymphatic vessels with surrounding adventitia ; cystic lymphangioma ( cystic hygroma ) consisting of huge , macroscopic lymphatic spaces with surrounding fibrovascular tissues and smooth muscle ; and benign lymphangioendothelioma ( acquired progressive lymphangioma ) , in which lymphatic channel dissects through dense collagen bundles . occasionally , channels may be filled with blood , a mixed hemangiolymphangioma , an uncommon developmental anomaly with a propensity to invade underlying tissues and to recur locally , distinguishing it from the simple lymphangioma or hemangioma . although histologically it is a benign disorder , local invasion into the muscle , bone , and underlying tissue can lead to severe deformity . in the present case , numerous large - sized lymphatic channels along with medium- to large - sized channels entrapped with rbcs , lined by endothelium , were seen and hence it was subcategorized as hemangiolymphangioma . we reviewed the archival cases of lymphatic malformations in our department , the demographical information , location and histopathological features of which are shown in table 1 . the only significant difference in the three archival cases and the present case was in the histopathological features of lymphangioma and hemangiolymphangioma . the demographic information , location and histopathological features these anomalies present the necessity for sound discretion with regards to their approach therapeutically.although spontaneous regression of lesions is rarely encountered , the treatment seems to weigh heavily on individual assessments of the observer . sclerosing agents are ineffective , probably as a result of the discontinuous basement membrane of the lymphatic vessels . nd - yag laser surgery has been widely preferred because of its advantages of less bleeding and edema . due to a rate of recurrence of nearly 21% , long - term follow - up is essential of these tumorigenic anomalies . the vascular lesions consist of both blood vessels and lymphatic vessels . whether these can be termed as hemangiolymphangioma or just vascular malformation is still confusing . thus through the present article we would like to highlight the complexities which can arise from the terminal categorization of the large group of tumors called vascular neoplasms when based on their histopathological representation . further detailed analysis of a larger case series would be imperative in the correct classification and diagnosis which could enormously help to accurately ascertain prognosis and direct treatment .

malformations of vascular nature originate as anomalies caused due to errors in vasculogenesis . these tumors are generally broadly classified into vascular tumors ( hemangiomas ) and vascular malformations ( venous malformations , arteriovenous malformations , lymphatic malformations ) . these descriptive tumors and malformations have been categorized based on the architectural assembly of vessels . lymphangiomas are further subclassified microscopically into capillary , cavernous , cystic and lymphangioendothelioma , depending upon their histopathological features . lymphatic malformations or lymphangiomas are uncommon congenital malformations of the lymphatic system , usually occurring in the head and neck region , characterized by collections of ectatic lymph vessels that form endothelial lined cystic spaces . advancements in the knowledge of pathogenesis of such vascular malformations are continuously changing their treatment protocols . early recognition is of utmost importance for initiation of proper treatment and avoiding serious complications . hemangiolymphangioma is a variant of lymphangioma showing vascular component . herewith , we present a case of vascular malformation diagnosed as hemangiolymphangioma histopathologically in a 9-year - old girl , along with a review of literature regarding its categorization .

growing evidence has shown that early life experiences can have lasting effects on adult health ( 13 ) . low birth weight ( < 2,500 g ) , an indicator of a compromised fetal growth , has been associated with a higher risk of developing type 2 diabetes ( 4,5 ) . however , to our knowledge , there are no studies assessing the relation of low birth weight to type 2 diabetes in african american women , a population disproportionately affected by low birth weight ( 6 ) and type 2 diabetes ( 7 ) . in addition , few studies have examined very low birth weight ( < 1,500 g ) , which might confer an even higher risk of type 2 diabetes as adult ( 8) . two major hypotheses have been proposed to explain the observed association between low birth weight and type 2 diabetes : the thrifty phenotype , or fetal programming hypothesis ( 9,10 ) , and the fetal insulin hypothesis ( 11 ) . the thrifty phenotype hypothesis states that as a consequence of intrauterine malnutrition , the individual s metabolism is reprogrammed to become nutritionally thrifty . according to this hypothesis , the thrifty phenotype would confer a survival advantage under conditions of nutritional deprivation , but the individual would be more prone to developing diabetes and other metabolic defects as an adult under improved nutritional conditions ( 9,10 ) . children born small for gestational age tend to have high serum leptin concentrations during catch - up growth ( 12 ) , which in turn has been associated with fat accumulation and higher insulin levels in adult life ( 13,14 ) . the fetal insulin hypothesis states that low birth weight and diabetes are different phenotypes of the same genotype ; genetic variants affecting fetal pancreas development would result in both reduced fetal growth , due to deficient insulin secretion , and higher risk of type 2 diabetes later in life because of the same underlying problem of compromised -cell mass ( 11 ) . recent results provide support to the fetal insulin hypothesis , as three genetic loci associated with type 2 diabetes ( adcy5 , cdkal1 , and hhex - ide ) were also associated with low birth weight ( 1517 ) , and at least two of the shared loci ( cdkal1 and hhex - ide ) are involved in -cell dysfunction ( 18,19 ) . because the underlying defect would be a deficiency in pancreas development , we would not expect , based on the fetal insulin hypothesis , that increased adiposity is a mediator between low birth weight and type 2 diabetes later in life . in the current study , we assessed the relation of self - reported birth weight to adult risk of type 2 diabetes in the black women s health study ( bwhs ) , a prospective cohort study . in particular , we evaluated whether extremes of the birth weight distribution are associated with an increased risk of incident type 2 diabetes . we also examined whether increased adiposity , as measured by higher prevalence of adult obesity ( bmi 30 kg / m ) , is a potential mediator of the relationship . the bwhs is an ongoing prospective follow - up study of african american women in the u.s . the study began in 1995 when 59,000 women aged 2169 years enrolled through completing health questionnaires . participants were approximately equally distributed in the northeast , south , midwest , and west . we collected information on demographics , medical and reproductive history , body weight , height , diet , smoking , physical activity , and other factors through the baseline questionnaire . participants have been followed through biennial questionnaires to collect information on incident diseases and update information on risk factors . questions about birth weight were asked on the 1997 questionnaire ( see below ) , and thus the present analyses are based on follow - up beginning in 1997 . among the 24,085 women who provided adequate data on birth weight , we excluded those with diabetes , cancer , myocardial infarction , stroke , or coronary artery bypass graft surgery at baseline or incident diabetes diagnosed before age 30 years , which resulted in a final analytic sample of 21,624 women ( fig . final analytic sample after exclusions . among the 24,085 women who provided adequate data on birth weight , we excluded those with diabetes ( n = 1,319 ) , cancer ( n = 677 ) , myocardial infarction ( n = 234 ) , stroke ( n = 171 ) , or coronary artery bypass graft surgery ( n = 25 ) at baseline or incident diabetes diagnosed before 30 years of age ( n = 35 ) , which resulted in a final analytic sample of 21,624 women . on the 1997 follow - up questionnaire , women were asked their birth weight in categories ( < 4 lb ; 4 lb to 5 lb , 8 oz ; > 5 lb , 8 oz ; do not know ) and their exact birth weight in pounds and ounces , if known . we used information from both questions to create four categories of birth weight ( very low , < 1,500 g ; low , 1,5002,499 g ; normal , 2,5003,999 g ; and high , 4,000 g ) . we carried out a validation study among 637 bwhs participants born in massachusetts using birth registry data from the massachusetts department of public health to corroborate self - reported data on birth weight . the coefficient of agreement for the categorical data was 0.80 , and there were no significant differences across categories of adult bmi at the time of reporting in 1997 ( p = 0.57 ) . for exact self - reported birth weight , the pearson correlation coefficient was 0.88 , and there were no significant differences across bmi categories ( p = 0.38 ) . these results are in agreement with previous studies ( 21,22 ) that have shown the validity of retrospectively collected self - reported birth weight information . we also assessed reproducibility of self - reported birth weight in a subset of 776 bwhs participants who completed the 1997 questionnaire two times . the coefficient was 0.86 for categorical birth weight , and the pearson correlation coefficient was 0.96 for exact birth weight . on each of the biennial questionnaires the accuracy of self - reported diabetes was assessed in a sample of 229 women who reported being diagnosed with diabetes , who consented to the release of medical records from their physicians , and whose providers replied to the request . we found the diagnosis of type 2 diabetes to be confirmed in 220 ( 96% ) of the women . of the nine remaining participants , two had type 1 diabetes , one had metabolic syndrome with no diabetes , one had steroid - induced diabetes , two had gestational diabetes mellitus , and three did not have diabetes . weight information was updated on each biennial follow - up questionnaire , and it was used to calculate current bmi ( weight in kilograms divided by the square of height in meters ) using height in 1995 . in validation studies of anthropometric measures conducted among 115 bwhs participants , spearman correlations for self - reported versus technician - measured weight , as well as height , were 0.97 , as well as 0.93 , respectively ( 23,24 ) . information on whether the participant was born preterm was obtained from the 1997 questionnaire through the question , were you born 3 or more weeks early ? ( yes , no , do nt know ) . we observed high reproducibility ( = 0.86 ) of self - reported preterm birth based on data from 776 bwhs participants who returned duplicate questionnaires in 1997 . data on vigorous physical activity ( hours / week ) were obtained from the 1995 questionnaire and updated in follow - up questionnaires . information on energy intake ( calories per day ) was estimated from 1995 and 2001 food frequency questionnaires ( 25,26 ) using the diet*calc software , version 1.4.1 , from the national cancer institute ( 27 ) . for assessment of individual socioeconomic status ( ses ) , years of education were ascertained in 1995 and 2003 . briefly , participants current addresses were linked through geocoding ( mapping analytics , rochester , ny ) to 2000 u.s . factor analysis of block group census variables identified six variables ( median household income ; median housing value ; percentage of households receiving interest , dividend , or net rental income ; percentage of adults aged 25 years or older who have completed college ; percentage of employed persons age 16 years or older who are in occupations classified as managerial , executive , or professional ; and percentage of families with children that are not headed by a single female ) that were used to calculate an index of neighborhood ses . we compared age - adjusted baseline characteristics across birth weight categories by computing means of continuous risk factors and proportions of categorical variables in each group . we calculated incidence rate ratios ( irrs ) and 95% cis using age- and period - stratified cox proportional hazards models . we calculated person - years of follow - up as the number of years from 1997 ( i.e. , baseline of the current study ) to first diagnosis of diabetes , death , loss of follow - up , or end of follow - up ( 2013)whichever came first . multivariable models included terms for first - degree family history of diabetes ( yes or no ) , preterm birth ( yes , no , or do not know ) , dietary caloric intake ( quintiles of kilocalories per day ) , vigorous physical activity ( none , < 1 h / week , 14 h / week , or 5 h / week ) , years of education ( 12 , 1315 , 16 , or 17 years ) , and quintiles of the index of neighborhood ses . in secondary analysis , we restricted our models to women not born preterm to make sure any observed association between very low and low birth weight and diabetes is due to fetal growth restriction rather than being born preterm . we used several approaches to assess whether birth weight affects risk of type 2 diabetes through an effect on bmi . first , we compared analyses without and with adjustment for bmi ( < 25 , 2529 , 3034 , 3539 , or 40 kg / m ) . second , we performed mediation analysis to estimate the proportion of the association between birth weight and type 2 diabetes that is explained by bmi . we estimated mediation proportion , defined as , and 95% ci using the partial likelihood function ( 30 ) of cox models with and without bmi as implemented in the sas mediate macro ( 31 ) . third , we conducted bmi - stratified analyses ( nonobese women , bmi < 30 kg / m , and obese women , bmi 30 kg / m ) . finally , we assessed the association of birth weight with incident obesity ( bmi 30 kg / m ) by estimating irrs adjusted for age , questionnaire cycle , being born preterm , energy intake , vigorous physical activity , years of education ( 12 , 1315 , 16 , or 17 years ) , and quintiles of the index of neighborhood ses . the bwhs is an ongoing prospective follow - up study of african american women in the u.s . the study began in 1995 when 59,000 women aged 2169 years enrolled through completing health questionnaires . participants were approximately equally distributed in the northeast , south , midwest , and west . we collected information on demographics , medical and reproductive history , body weight , height , diet , smoking , physical activity , and other factors through the baseline questionnaire . participants have been followed through biennial questionnaires to collect information on incident diseases and update information on risk factors . questions about birth weight were asked on the 1997 questionnaire ( see below ) , and thus the present analyses are based on follow - up beginning in 1997 . among the 24,085 women who provided adequate data on birth weight , we excluded those with diabetes , cancer , myocardial infarction , stroke , or coronary artery bypass graft surgery at baseline or incident diabetes diagnosed before age 30 years , which resulted in a final analytic sample of 21,624 women ( fig . final analytic sample after exclusions . among the 24,085 women who provided adequate data on birth weight , we excluded those with diabetes ( n = 1,319 ) , cancer ( n = 677 ) , myocardial infarction ( n = 234 ) , stroke ( n = 171 ) , or coronary artery bypass graft surgery ( n = 25 ) at baseline or incident diabetes diagnosed before 30 years of age ( n = 35 ) , which resulted in a final analytic sample of 21,624 women . on the 1997 follow - up questionnaire , women were asked their birth weight in categories ( < 4 lb ; 4 lb to 5 lb , 8 oz ; > 5 lb , 8 oz ; do not know ) and their exact birth weight in pounds and ounces , if known . we used information from both questions to create four categories of birth weight ( very low , < 1,500 g ; low , 1,5002,499 g ; normal , 2,5003,999 g ; and high , 4,000 g ) . we carried out a validation study among 637 bwhs participants born in massachusetts using birth registry data from the massachusetts department of public health to corroborate self - reported data on birth weight . the coefficient of agreement for the categorical data was 0.80 , and there were no significant differences across categories of adult bmi at the time of reporting in 1997 ( p = 0.57 ) . for exact self - reported birth weight , the pearson correlation coefficient was 0.88 , and there were no significant differences across bmi categories ( p = 0.38 ) . these results are in agreement with previous studies ( 21,22 ) that have shown the validity of retrospectively collected self - reported birth weight information . we also assessed reproducibility of self - reported birth weight in a subset of 776 bwhs participants who completed the 1997 questionnaire two times . the coefficient was 0.86 for categorical birth weight , and the pearson correlation coefficient was 0.96 for exact birth weight . on each of the biennial questionnaires , we asked about a diagnosis of diabetes in the previous 2 years . the accuracy of self - reported diabetes was assessed in a sample of 229 women who reported being diagnosed with diabetes , who consented to the release of medical records from their physicians , and whose providers replied to the request . we found the diagnosis of type 2 diabetes to be confirmed in 220 ( 96% ) of the women . of the nine remaining participants , two had type 1 diabetes , one had metabolic syndrome with no diabetes , one had steroid - induced diabetes , two had gestational diabetes mellitus , and three did not have diabetes . weight information was updated on each biennial follow - up questionnaire , and it was used to calculate current bmi ( weight in kilograms divided by the square of height in meters ) using height in 1995 . in validation studies of anthropometric measures conducted among 115 bwhs participants , spearman correlations for self - reported versus technician - measured weight , as well as height , were 0.97 , as well as 0.93 , respectively ( 23,24 ) . information on whether the participant was born preterm was obtained from the 1997 questionnaire through the question , were you born 3 or more weeks early ? we observed high reproducibility ( = 0.86 ) of self - reported preterm birth based on data from 776 bwhs participants who returned duplicate questionnaires in 1997 . data on vigorous physical activity ( hours / week ) were obtained from the 1995 questionnaire and updated in follow - up questionnaires . information on energy intake ( calories per day ) was estimated from 1995 and 2001 food frequency questionnaires ( 25,26 ) using the diet*calc software , version 1.4.1 , from the national cancer institute ( 27 ) . for assessment of individual socioeconomic status ( ses ) , years of education were ascertained in 1995 and 2003 . briefly , participants current addresses were linked through geocoding ( mapping analytics , rochester , ny ) to 2000 u.s . factor analysis of block group census variables identified six variables ( median household income ; median housing value ; percentage of households receiving interest , dividend , or net rental income ; percentage of adults aged 25 years or older who have completed college ; percentage of employed persons age 16 years or older who are in occupations classified as managerial , executive , or professional ; and percentage of families with children that are not headed by a single female ) that were used to calculate an index of neighborhood ses . we compared age - adjusted baseline characteristics across birth weight categories by computing means of continuous risk factors and proportions of categorical variables in each group . we calculated incidence rate ratios ( irrs ) and 95% cis using age- and period - stratified cox proportional hazards models . we calculated person - years of follow - up as the number of years from 1997 ( i.e. , baseline of the current study ) to first diagnosis of diabetes , death , loss of follow - up , or end of follow - up ( 2013)whichever came first . multivariable models included terms for first - degree family history of diabetes ( yes or no ) , preterm birth ( yes , no , or do not know ) , dietary caloric intake ( quintiles of kilocalories per day ) , vigorous physical activity ( none , < 1 h / week , 14 h / week , or 5 h / week ) , years of education ( 12 , 1315 , 16 , or 17 years ) , and quintiles of the index of neighborhood ses . in secondary analysis , we restricted our models to women not born preterm to make sure any observed association between very low and low birth weight and diabetes is due to fetal growth restriction rather than being born preterm . we used several approaches to assess whether birth weight affects risk of type 2 diabetes through an effect on bmi . first , we compared analyses without and with adjustment for bmi ( < 25 , 2529 , 3034 , 3539 , or 40 kg / m ) . second , we performed mediation analysis to estimate the proportion of the association between birth weight and type 2 diabetes that is explained by bmi . we estimated mediation proportion , defined as , and 95% ci using the partial likelihood function ( 30 ) of cox models with and without bmi as implemented in the sas mediate macro ( 31 ) . third , we conducted bmi - stratified analyses ( nonobese women , bmi < 30 kg / m , and obese women , bmi 30 kg / m ) . finally , we assessed the association of birth weight with incident obesity ( bmi 30 kg / m ) by estimating irrs adjusted for age , questionnaire cycle , being born preterm , energy intake , vigorous physical activity , years of education ( 12 , 1315 , 16 , or 17 years ) , and quintiles of the index of neighborhood ses . table 1 shows baseline characteristics of participants by birth weight categories . at baseline , of the 21,624 women included in the study , 2.3% had a very low birth weight , 23.9% had low birth weight , 66.0% had normal birth weight , and 7.8% had high birth weight . age - adjusted baseline ( 1997 ) characteristics by birth weight categories over 16 years of follow - up and a total of 263,980 person - years , there were 2,388 incident diabetes cases ( table 2 ) . in the multivariate model , very low and low birth weight were associated with an increased risk of type 2 diabetes relative to normal birth weight . irrs were 1.40 ( 95% ci 1.081.82 ) for very low birth weight and 1.13 ( 1.021.25 ) for low birth weight . mediation analysis showed that bmi was not a significant mediator of the association of birth weight with type 2 diabetes . in an analysis restricted to women who were not born preterm , an association of low birth weight with risk of type 2 diabetes was observed similar to that in the overall sample : irr 1.19 ( 1.041.35 ) . however , there was almost complete overlap between being born preterm and having a very low birth weight , preventing analysis of very low birth weight among women born full term . irr ( 95% ci ) for diabetes according to birth weight categories in the bwhs : 19972013 * multivariate model : adjusted for age , questionnaire cycle , first - degree family history of diabetes , being born preterm ( yes , no , or do not know ) , activity levels ( none , < 1 h / week , 14 h / week , or 5 h / week ) , energy intake ( quintiles of kcal / day ) , neighborhood ses quintiles , and education level ( 12 , 1315 , 16 , or 17 years ) . bmi ( < 25 , 2529 , 3034 , 3539 , or 40 adjusted for age , questionnaire cycle , first - degree family history of diabetes , activity levels , energy intake , neighborhood ses , subject s education level , and bmi ( categories ) . adjusted for age , questionnaire cycle , first - degree family history of diabetes , being born preterm , activity levels , energy intake , neighborhood ses , subject s education level , and bmi ( continuous ) . the same patterns of risk were found within strata of bmi ( bmi < 30 vs. 30 kg / m ; p for interaction = 0.25 ) . in addition , no significant differences on risk were observed across the five bmi categories ( < 25 , 2539 , 3034 , 3539 , and 40 kg / m ; p for interaction = 0.33 ) ( data not shown ) . to further explore the hypothesis that very low and low birth weight may affect risk of type 2 diabetes through a higher risk of obesity , we assessed the relation of birth weight with incident obesity ( bmi 30 kg / m ) in a multivariate model adjusting for age , questionnaire cycle , being born preterm , energy intake , vigorous physical activity , years of education , and neighborhood ses . relative to women with normal birth weight , neither women with very low birth weight , irr 1.01 ( 95% ci 0.811.26 ) , nor women with low birth weight , 0.91 ( 0.840.99 ) , had an increased risk of incident obesity ; women with high birth weight had a borderline higher risk of incident obesity , 1.12 ( 1.001.26 ) ( data not shown ) . black women , we found that very low and low birth weights were associated with higher risk of incident type 2 diabetes . although the association with low birth weight was independent of being born preterm , we could not distinguish between very low birth weight and being born preterm , since virtually all with very low birth weight were also born preterm . the association of very low and low birth weight with incidence of type 2 diabetes was not mediated by bmi , as the association remained almost unchanged after adjustment for bmi , mediation analysis did not show significant mediation by bmi , and an association was also present among nonobese women . the observed association also was not explained by variation in ses . with regard to high birth weight , we did not observe an increased risk of incident type 2 diabetes , despite the fact that women with high birth weight tended to have a higher risk of incident obesity relative to women with normal birth weight . although the relation of birth weight to risk of type 2 diabetes has been assessed in several previous studies ( 3236 ) , to our knowledge ours is the first report on this relation in african american women , a population with high frequency of low birth weight ( 6 ) and high incidence of type 2 diabetes ( 7 ) . a meta - analysis of 14 studies reported a u - shaped relation of birth weight with risk of type 2 diabetes , with both low birth weight and high birth weight associated with higher risk of type 2 diabetes relative to normal birth weight ( 4 ) . a more recent and bigger meta - analysis of 31 studies reported an overall inverse relation of birth weight with risk of type 2 diabetes ( 5 ) , and exclusion of macrosomic infants ( > 4,000 g birth weight ) had little effect on the overall inverse association ( 5 ) . however , there was substantial heterogeneity between populations , with few groups , particularly native americans , showing a u - shaped relation of birth weight and risk of type 2 diabetes ( 5 ) . our results are consistent with both meta - analyses in showing that low birth weight is associated with higher risk of type 2 diabetes , and we show that this increased risk extends to very low birth weight . however , because of the almost complete overlap between very low birth weight and being born preterm , we could not assess an independent effect of very low birth weight , and it is unclear how much of the observed association is due to being born preterm . we did not find an increased risk of type 2 diabetes for women who had a high birth weight even though they had a higher risk of incident obesity relative to women who had normal birth weight . to date , the relation of high birth weight to type 2 diabetes is unclear , as two meta - analyses provide conflicting findings ( 4,5 ) . our results are also consistent with a recent report using data from the national health and nutrition examination survey ( nhanes ) cycles 20012010 , which found that low birth weight but not high birth weight was associated with type 2 diabetes risk factors such as fasting glucose , fasting insulin , and homa in 10,758 u.s . although the evidence is still limited , it may be that higher adult bmi in persons who had a high birth weight reflects more lean tissue than fat mass ( 38,39 ) , explaining the apparently paradoxical observation that individuals who had high birth weight had higher bmi as an adult but not higher risk of type 2 diabetes relative to persons who had normal birth weight . mediation analysis in our study suggested that adult bmi did not play a major role as a mediator of the relation between very low and low birth weight and development of type 2 diabetes in adulthood . this conclusion is supported by other results such as the following : 1 ) adjustment for bmi did not attenuate the association of very low and low birth weight with risk of type 2 diabetes , 2 ) very low and low birth weight were associated with increased risk of type 2 diabetes even among nonobese women , and 3 ) women who had very low and low birth weight did not have an increased risk of incident obesity relative to women who had a normal birth weight . results from animal models and human studies suggest a multifactorial etiology that includes neuroendocrine alterations ( 4042 ) , deregulation of lipid metabolism ( 4345 ) , and pancreatic dysfunction ( 4648 ) among others . recent evidence suggests that low birth weight and type 2 diabetes may share a genetic basis ( 1517 ) , and at least two of the shared loci ( cdkal1 and hhex - ide ) have been implicated in -cell dysfunction ( 18,19 ) . these observations lend support to the fetal insulin hypothesis ( 49 ) , which states that low birth weight and type 2 diabetes later in life are manifestations of the same genotype , and they are due to impairment of -cell development that is genetically programmed ( 15 ) . it is noteworthy that regardless of the potential mechanisms , the impact of low birth weight on the risk of type 2 diabetes may be stronger among african americans , as a recent study found a more substantial association between low birth weight and components of the insulin resistance syndrome among african american children than among white children ( 45 ) . the current study has several strengths including its large size , high rate of follow - up , and ability to control for important confounding variables . information on birth weight was self - reported many years after the fact , raising the possibility of exposure misclassification . however , our validation study showed high correlations between self - reported birth weight and birth registry data , and there were no differences across bmi categories . thus , although we can not exclude a certain degree of misclassification , this was most likely at random and generally would result in attenuation of our findings . while information about type 2 diabetes was also self - reported , a validation study found self - report to have high sensitivity ( 96% ) for diabetes diagnosis . in addition , because the prevalence of undiagnosed diabetes among african american women is ~4% ( 7 ) we do not expect a major effect of undiagnosed diabetes on our estimates of risk . with respect to anthropometric measurements , our validation study showed very high correlations between self - reported and technician - measured weight and height . we can not rule out the presence of residual confounding due to unmeasured variables such as maternal metabolic status during pregnancy . for example , we had no information about maternal gestational diabetes mellitus , which is a risk factor for development of type 2 diabetes in the offspring later in life ( 50).the current prevalence of gestational diabetes mellitus among african american women is ~4% ( 51 ) , but based on temporal trends this prevalence was most likely < 2% during the time most of the study s participants were born ( 51 ) . it is unlikely that unmeasured gestational diabetes mellitus , given its low prevalence , had a major impact on our results . finally , although we can not establish a causal link between low birth weight and type 2 diabetes in adulthood , taken together our results , most previous observational studies , and results from animal models do suggest a causal role of compromised fetal growth in the development of type 2 diabetes . in summary , this large prospective study of african american women suggests that both very low and low birth weight are associated with a higher risk of incident type 2 diabetes . this relation was not mediated by bmi , suggesting that mechanisms independent of bmi are responsible for the observed association . the prevalence of low birth weight is especially high in african american populations , and this may explain in part the higher occurrence of type 2 diabetes .

objectiveto assess the association of birth weight with incident type 2 diabetes , and the possible mediating influence of obesity , in a large cohort of u.s . black women.research design and methodsthe black women s health study is an ongoing prospective study . we used cox proportional hazards models to estimate incidence rate ratios ( irrs ) and 95% ci for categories of birth weight ( very low birth weight [ < 1,500 g ] , low birth weight [ 1,5002,499 g ] , and high birth weight [ 4,000 g ] ) in reference to normal birth weight ( 2,5003,999 g ) . models were adjusted for age , questionnaire cycle , family history of diabetes , caloric intake , preterm birth , physical activity , years of education , and neighborhood socioeconomic status with and without inclusion of terms for adult bmi.resultswe followed 21,624 women over 16 years of follow - up . there were 2,388 cases of incident diabetes . women with very low birth weight had a 40% higher risk of disease ( irr 1.40 [ 95% ci 1.081.82 ] ) than women with normal birth weight ; women with low birth weight had a 13% higher risk ( irr 1.13 [ 95% ci 1.021.25 ] ) . adjustment for bmi did not appreciably change the estimates.conclusionsvery low birth weight and low birth weight appear to be associated with increased risk of type 2 diabetes in african american women , and the association does not seem to be mediated through bmi . the prevalence of low birth weight is especially high in african american populations , and this may explain in part the higher occurrence of type 2 diabetes .

incremental cost of pim use in the elderly is estimated to be $ 7.2 billion , pim use in ambulatory elderly patients has been found to be 50 per 1,000 person years . the elderly population is especially susceptible to use of pim as up to 94% take at least one pim . pim is defined as use of a drug with risk of an adverse event that outweighs its clinical benefit , particularly when there is a safer or more effective alternative therapy for the same condition . beers criteria to assess and reduce the risk of pim in the elderly by identifying pim has been developed . utilization of this criteria has shown pims to be associated with a 25% increase in the risk of hospitalizations in elderly nursing home residents . in addition , the incidence of pim use has been reported to range from 22% to 66% during hospitalization , and 44% upon discharge . geriatric population is very fragile and prone to medication side effects in view of changing physiological capacities of the body . the decline is much more rapid when co morbidities , especially diabetes and hypertension set in . there is a corresponding decline in other body systems , including : cardiac , respiratory , gastrointestinal , and neurovascular . this results in accentuated drug side effects and toxicities secondary to slowed pharmacokinetics and pharmacodynamics . this is the subset of population where more and more use of polypharmacy and pim use is observed . however , there are very few studies available evaluating the association of pims with hospital readmission . mansur and colleagues evaluated several objectives in a cohort of hospitalized elderly patients , including the relationship of pims with readmission and mortality , 3 months post discharge . they found no significant difference in pim use among patients who were readmitted or died as compared to those who were not readmitted or survived . however , this trial had several limitations , including a small sample size of 212 patients . the present study was designed to see if polypharmacy and/or pim use was related to readmission rates to the hospital irrespective of the admission diagnosis . the objective of this study was to evaluate the association of pim use with hospital readmission within 30 days regardless of the admitting diagnosis . this nested case - control study included all patients readmitted within 30 days of discharge to a 250 bed community hospital located in scranton , pennsylvania usa . all patients 65 years of age or older admitted between january 2008 and december 2009 were eligible for study enrollment . day 0 was defined as the day of discharge and day1 was defined as the day - after surgery . the present study included all the consecutive readmissions and no controls were used for comparison . the modified beer 's criteria is the most widely followed drug list that is used to screen potentially inappropriate drug use in the geriatric population [ table 1 ] . modified beer criteria showing list of the potential associated with inappropriate usage of medicines this was a retrospective case record review of 414 patients who were hospitalized within 30 days of discharge from the hospital between january 2008 and december 2009 . there was no bias in the selection of patients on the basis of age , sex , co morbidities , height , weight , marital status , ethnicity , and socio - economic status . the institutional review board of hospital approved the study and waived the requirement for informed consent for the retrospective review of medical records . the goal of the study was to identify the patient characteristics associated with readmission to the hospital within 30 days of discharge from the hospital . day 0 was defined as readmission on the same day before midnight and any readmission after midnight was defined as 1 day . rest of the readmissions were defined as within 30 days of discharge but after day 1 . demographic and laboratory data was retrieved from an electronic hospital database . after merging data from the different sources , automated and manual data verification was performed . statistical analysis was carried out using a two - way anova on the data to see if polypharmacy and/or pim use was related to readmission within 30 days of discharge irrespective of admission diagnosis . this was a retrospective case record review of 414 patients who were hospitalized within 30 days of discharge from the hospital between january 2008 and december 2009 . there was no bias in the selection of patients on the basis of age , sex , co morbidities , height , weight , marital status , ethnicity , and socio - economic status . the institutional review board of hospital approved the study and waived the requirement for informed consent for the retrospective review of medical records . the goal of the study was to identify the patient characteristics associated with readmission to the hospital within 30 days of discharge from the hospital . day 0 was defined as readmission on the same day before midnight and any readmission after midnight was defined as 1 day . rest of the readmissions were defined as within 30 days of discharge but after day 1 . demographic and laboratory data was retrieved from an electronic hospital database . after merging data from the different sources , automated and manual data verification was performed . statistical analysis was carried out using a two - way anova on the data to see if polypharmacy and/or pim use was related to readmission within 30 days of discharge irrespective of admission diagnosis . a total of 312 patients were on polypharmacy ( five or more medications at the time of admission ) figure 2 . gender profile of readmissions polypharmacy and its relationship to readmission to the hospital on the same day for readmission and readmission the immediate day after discharge was statistically significant . the impact of polypharmacy and pim combined on hospital readmission was statistically significant only when the readmission occurred on the same day or immediate next day . no polypharmacy and no inappropriate drugs were statistically significant as compared to polypharmacy and pim . at day 1 and day 0 , data for all the readmissions was compiled and statistical analysis was carried out . the statistical comparison is expressed as significant ( p < 0.05 ) and highly significant ( p < 0.001 ) with regard to polypharmacy and pim . no polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.001)polypharmacy + no inappropriate drugs versus no polypharmacy + inappropriate drugs : ( p < 0.05)poypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.05)no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.001)no polypharmacy versus polypharmacy : ( p < 0.001 ) . no polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.001 ) polypharmacy + no inappropriate drugs versus no polypharmacy + inappropriate drugs : ( p < 0.05 ) poypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.05 ) no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.001 ) no polypharmacy versus polypharmacy : ( p < 0.001 ) . no polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.05)no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.05)no polypharmacy versus polypharmacy : ( p < 0.01 ) . no polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.05 ) no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.05 ) no polypharmacy versus polypharmacy : ( p < 0.01 ) . no polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.001)polypharmacy + no inappropriate drugs versus no polypharmacy + inappropriate drugs : ( p < 0.05)poypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.05)no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.001)no polypharmacy versus polypharmacy : ( p < 0.001 ) . no polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.001 ) polypharmacy + no inappropriate drugs versus no polypharmacy + inappropriate drugs : ( p < 0.05 ) poypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.05 ) no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.001 ) no polypharmacy versus polypharmacy : ( p < 0.001 ) . no polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.05)no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.05)no polypharmacy versus polypharmacy : ( p < 0.01 ) . no polypharmacy + no inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.05 ) no polypharmacy + inappropriate drugs versus polypharmacy + inappropriate drugs : ( p < 0.05 ) no polypharmacy versus polypharmacy : ( p < 0.01 ) . there is a trend to pay for performance and hospitals are not being reimbursed for so - called never events . the list of the never events has been gradually increasing over the last couple of years . the expansion of never events and hospital acquired conditions by center of medicare and medicaid ( cms ) would have a substantial financial impact on tertiary care facilities . this is in sharp contrast to earlier trends when hospitals were reimbursed irrespective of the quality of the services offered . costs have continued to rise at double - digit rates , and quality is far from optimal . in the 21 century , we see aging of the population ; the fastest growing population being people over 85 years of age . adverse drug events , which can be especially problematic in older adults , often can be prevented by detecting potential risk factors . many primary care doctors possess a poor knowledge of pim and are unaware of prescribing guidelines such as the beers criteria . also there is poor communication of drugs potential side effects profile between the physician and the patient . hospital readmission within 30 days of discharge from the hospital is a multifaceted dragon which has to be dealt with a multipronged approach . a substantial amount of economic resources are spent on readmissions , especially in terms of finances . the most common cause of readmission by admitting diagnosis is congestive heart failure followed by psychosis [ figure 4 ] . the usual approach is to blame it on the disease process without addressing the patients co - morbidities . at the hospital , where this study was conducted , this study was designed to quantify the use of inappropriate medications among older adult outpatients . patients have a more difficult time trying to remember their drug regimen daily and can infringe on quality of life . not to forget to mention the cost of the polypharmacy . healthcare spending is also increased with more follow - up visits to doctor 's offices . healthcare spending can also involve the side effects caused by polypharmacy that can include acute drug reactions and readmissions . tracking the number of patients who experience unplanned readmissions to a hospital after a previous hospital stay is another category of data used to judge the quality of hospital care . in usa , cms services began to look into the issue after a national push by the obama administration . in usa , the president 's health - care reform has identified readmission rates as a key target for medical care savings . the hospital compare site allows patients to compare hospitals based on the how the rate of readmission for three treatments heart attack , heart failure , and pneumonia compare with the national average . the rates take into account how sick patients were before they were admitted to the hospital . the modified beer 's criteria is the most widely followed drug list that is used to screen potentially inappropriate drug use in the geriatric population . for a complet e list of medications included in the beer 's criteria although it remains a useful screening tool to screen for pim , its practical application in acute - care settings remains obscure . critics of the beer 's criteria opine that it is too big to be implemented in its entirety in the acute - care settings . in fact adrs could contribute to accelerated functional decline in elderly hospitalized patients independently of the use of beers listed pims . for example , pregabalin is very poorly tolerated by geriatric population and is not included in the beer 's criteria . furthermore , potentially any medication could be inappropriate in the geriatric population if it is not titrated by the physiological decline , especially renal decline . in this subset of population , , all anticholinergics , opiods , and benzodiazepams should raise a red flag , especially at the time of medication reconciliation . consideration should be placed on benefit to risk ratio when a new medication is added . on each hospitalization there should be a diligent review of the drug profile and every effort should be made to minimize medications . screening tool of older persons pim ( stopp ) have recently found favor as a tool for screening pim . in the hospital setting , proton pump inhibitors ( ppi ) are very commonly prescribed and are the most over prescribed medications . this not only compounds the cost of healthcare delivery but is detrimental to the patients medical management by increasing morbidity . along with the over prescribed ppi 's , a significant portion of the geriatric population is on clopidogrel because of coexistent coronary artery disease . ppi added in conjunction with plavix has a 64% higher risk of re hospitalization for myocardial infraction mi or coronary stent placement than patients receiving plavix alone . there is also an increase in stent thrombosis in patients who are on both plavix and ppis . healthcare professionals should be vigilant patient advocates when it comes to polypharmacy or at least be cautious of all the side effects . this is to drive home the point that pim use be at the back of the mind with a larger goal to minimize polypharmacy . antidepressants are another class of medications , which is widely used in the geriatric population but does not figure into the beer 's criteria . considering the benefit to risk ratio the widespread use of antidepressants need to be minimized . if we look at the natural course of depression , 60% of the cases are self - resolving if we consider the side effects profile to antidepressants , there is an extensive list and of course like most other medication , side effects are more pronounced in the elderly . most patients are on antidepressants forever because most physicians are hesitant to taper them off . a good discharge plan should include a meticulous medication reconciliation especially weighing the benefit to risk ratio . we would also recommend a mandated continued medical education on pim on renewal of state license . a good surgeon knows when not to operate and a good physician knows when not to give medicines . polypharmacy is a big contributor to increasing healthcare cost and increasing morbidity as well as mortality in the elderly population . there should be focused attention on all patients whether in hospital , or in physician 's follow - up clinic towards minimizing polypharmacy . if clinicians practice polypharmacy , they are bound to lead to use of pim . in our opinion , internship year should be geared towards orienting the physicians to focus on the geriatric aspect of medicine and improve medication use in elderly population . this is important because india is seeing rapid surge of elderly with infrastructure and health care system ill equipped to handle it . we would need a larger prospective multicenter randomized controlled intervention study to assess the effect of optimization strategies on the appropriateness of prescribing in elderly people .

background and aim : readmission to the hospital within 30 days of discharge from the hospital is a common occurrence . congestive heart failure is the most common cause of readmissions in the hospital . we hypothesized that irrespective of the admission diagnosis polypharmacy and potentially inappropriate use of medications ( pim ) leads to readmissions within 30 days of discharge from the hospital.materials and methods : a retrospective study was carried out by reviewing the hospital records of 414 patients who were readmitted to the hospital within 30 days of discharge from the hospital between january 2008 and december 2009 . the data was stratified to see which patients were on polypharmacy and/or on pim . polypharmacy was defined as use of more than 5 medications . pim was defined as per the modified beers criteria . day 0 was defined as the day of discharge and day1 was defined as the day - after admission to the hospital . statistical analysis was carried out using a two - way analysis of variance ( anova ) on the data to see if polypharmacy and/or pim was related to readmission within 30 days of discharge irrespective of admission diagnosis.results:polypharmacy was related to hospital readmission at day 1 and day 0 , however inappropriate drug use was found to be not related at any day . polypharmacy and pim combined had a positive correlation to readmission only on days 1 and 0 and it was statistically significant . the use of minimal and appropriate use of drugs was statistically significant compared to polypharmacy and pim use.conclusions:polypharmacy and pim are under recognized cause of readmissions to the hospital .

the northern house mosquito , culex pipiens l. ( diptera : culicidae ) , is a member of a species complex that includes vectors of lymphatic filariasis and several arboviral diseases , such as st . louis encephalitis ( sle ) , rift valley fever ( rfv ) , and west nile virus ( wnv ) . in the northeastern quadrant of the united states , c. pipiens recently has become important because of its role in the transmission of wnv . the virus was introduced into new york city in 1999 and is currently the most widespread arboviral disease in the u.s . ( cdc , 2010 ) . the blood - feeding pattern of c. pipiens ( shifting from avian to human feeding within the transmission season ) ( edman and taylor , 1968 ; kilpatrick et al . , 2006 ) , coupled with its high vector competence ( turell et al . , 2005 ) and ability to carry wnv through the winter , has allowed the virus to spread from its point of origin and maintain transmission throughout the mosquito s range . thus , c. pipiens is one of the major targets for mosquito vector control and surveillance in the u.s . current methods of monitoring by abatement districts and health departments include centers for disease control ( cdc ) light traps and co2-baited traps , which mainly trap blood - seeking females , along with gravid traps , which target gravid females . plant - derived attractants have the potential to act as surveillance - trap lures and , when combined with poison - laced sugar solutions ( mller et al . , mosquitoes are attracted to various plant species , from which they obtain nectar and other juices . sugar feeding by mosquitoes has a significant influence on dispersal ( hocking , 1953 ; magnarelli , 1977 ) and vectorial capacity ( gary and foster , 2001 ; gu et al . , 2011 ) , and , contrary to long - held conjecture , is required by both males and females throughout the adult stage ( downes , 1958 ; yuval , 1992 ; foster , 1995 ) . both sexes typically first visit plants soon after emergence . males then require sugar at frequent intervals to maintain their energy reserves in order to join nightly mating swarms ( yuval et al . , 1992 ) . females take sugar between blood meals , when they are digesting blood , or when they are gravid ( clements , 1999 ; foster , 2008 ) . furthermore , females of most temperate - climate culex and anopheles species enter reproductive diapause in late summer , after which they no longer are attracted to blood hosts , but engage heavily in plant - sugar feeding ( bowen , 1992a ) . the potential for plant volatiles to lure mosquitoes has been demonstrated in several laboratory behavioral assays that have used stimuli such as natural plant extracts ( vargo and foster , 1982 ; jepson and healy , 1988 ; mauer and rowley , 1999 ) or single floral compounds ( jhumur et al . , 2006 ) . in field studies , bates ( 1949 ) reported the successful trapping of anopheline mosquitoes with plant and fruit baits ; fruit also proved to be an effective attractant in cdc traps for c. tarsalis ( reisen et al . , 1986 ) . sandholm and price ( 1962 ) observed that mosquitoes in the field were attracted to light - colored flowers with distinct fragrances ; odor appears to be primarily responsible for long - range attraction , with visual cues playing a role at shorter range ( thorsteinson and brust , 1962 ; healy and jepson , 1988 ; jepson and healy , 1988 ) . orientation to commercially obtained floral extracts and honey has been demonstrated for various mosquito species ( thorsteinson and brust , 1962 ; hancock and foster , 1997 ; foster and takken , 2004 ) . schlein and mller ( 2008 ) and mller et al . ( 2008 , 2010 ) reported dramatic population reductions of c. pipiens and other mosquito species by spraying a fruit - based sugar bait containing insecticide on vegetation surrounding larval habitats . light - less cdc traps baited with the blossoms of tamarix jordanis were highly effective in trapping c. pipiens , and populations were reduced where these blossoms were treated with spinosad insecticide ( schlein and mller , 2008 ) . flower species that elicit the highest attraction probably vary by region . during mid - summer in the u.s . , mosquitoes have been observed probing blossoms of common milkweed , asclepias syriaca , at rates disproportionate to their abundance relative to other flowering plants , with one study reporting 54 species occurring in the study area that did not serve as nectar sources ( grimstad and defoliart , 1974 ) . another study collected 25 aedes vexans from one milkweed cluster in just 15 min ( sandholm and price , 1962 ) . the observed preference of insects for milkweed flowers , both day and night , possibly is due to its strong and distinctive fragrance , lighter - colored flowers , and greater nectar production ( sandholm and price , 1962 ) . common milkweed is indigenous to eastern and midwestern north america , where its principal native pollinators are mainly large hymenoptera and lepidoptera ( e.g. , jennersten and morse , 1991 ) . mosquitoes are incapable of transferring milkweed pollinia and therefore function as nectar thieves , appearing to co - opt the attractive properties used by the pollinators to locate the flowers ( foster , 1995 ) . solvent extracts of common milkweed flowers also are attractive to mosquitoes ( vargo and foster , 1982 ; mauer and rowley , 1999 ) , confirming that volatile chemicals are at least partly responsible for that attraction . mauer and rowley ( 1999 ) determined the headspace profile of common milkweed flower to be predominantly 2-phenylethanol and benzyl alcohol ; however , they found that a synthetic blend of these two compounds was not attractive to c. pipiens . due to the multiple demonstrations of c. pipiens attraction to milkweed , both in the laboratory and the field , we used a solvent extract of the flower as a potential model for synthetic mosquito lures . experiments were conducted with c. pipiens from a colony established in 2009 from larvae collected near columbus , oh , usa . larvae were identified at l4 by siphonal hair tufts ( vinogradova , 2000 ) . adults were maintained in 41-l clear acrylic cages on a diet of 10 % sucrose , water , and weekly blood meals from the legs of a rooster ( ilacuc permit no . oviposition water was prepared by soaking grass clippings in aged tap water and allowing fermentation over a 3-d period . three days after each blood meal , oviposition cups were placed with caged adults , and eggs were collected the following day . two hundred first - instar larvae were placed into 22.8 33.0 cm aluminum pans with 450 ml of aged tap water . the larvae were provided finely ground tetramin flakes , increasing the quantity daily from 50 mg for first instars to 500 mg for final instars until pupae appeared on the 8th and 9th day post - hatching . pupae then were counted and transferred to plastic cups and placed in a 41-l cage supplied with water wicks . emerging adults were given ad libitum access to water , but were deprived of sugar . the mosquito rearing and maintenance conditions were 27 1 c , 85 5 % rh , and 16:8 ( l : d ) , with 30-min gradual crepuscular transitions between photophase and scotophase . phenylacetaldehyde ( > 90 % ) , benzaldehyde ( 99.5 % , purified by redistillation ) , nonanal ( 95 % ) , ( e)-2-nonenal ( 97 % ) , and an alkane - standards mixture ( c8 - c20 ) were purchased from sigma - aldrich ( st . louis , mo , usa ) . ( phillipsburg , nj , usa ) , and -ocimene [ ( 75 % ( e)--ocimene ) ] was synthesized by chemos gmbh ( regenstauf , germany ) . synthetics were diluted with hplc - grade n - pentane ( fisher scientific , pittsburgh , pa , usa ) . flowers of common milkweed , a. syriaca , were collected in early summer from the ohio state university campus in columbus ( 400018.95 " n , 830247.11 " w ) , placed in an ice cooler , and transported 10-min to the laboratory . single florets were plucked from the main milkweed umbel and separated from the calyx and other green parts of the flower , weighed , and placed into a 500 ml narrow - mouth glass erlenmeyer flask . milkweed florets were submerged in hplc - grade n - pentane ( fisher scientific ) in a 1:8 ratio ( w / v ) for a total solvent volume of 480 ml and held for 24 h at room temperature , at which time the extract was decanted into 21-ml borosilicate glass vials , capped with teflon - lined screw caps and stored at -20 c . the volatile profile of milkweed flower was characterized by placing a single floret into a 21-ml borosilicate glass vial with a teflon - lined rubber septum . volatiles were allowed to equilibrate in the vial headspace at 30 c for 10 min before collections were made . volatiles were collected by using a divinylbenzene / carboxen / polydimethylsiloxane ( dvb / car / pdms ) ( 50/30 m ) solid phase micro - extraction ( spme ) fiber ( supelco , bellefonte , pa , usa ) . this mixed - chemistry fiber provides affinity for chemicals with a far broader range of polarity and volatility than pdms alone . the fiber was introduced to the vial through the septum and exposed for 30 min . the collected volatiles were analyzed immediately by using an agilent technologies 6890 series gas chromatograph interfaced to an agilent technologies 5973 quadrupole mass selective detector ( gc - ms ) . the spme fiber was desorbed at 275 c for 3 min with a splitless injection onto a zebron zb-1 ms column ( 30 m 0.25 mm , 0.25 m film thickness ) ( phenomenex , torrance , ca , usa ) . the oven temperature was held at 25 c for 3 min , then ramped at 15 c / min to 250 c , where it was held for 2 min . the carrier gas was helium at a flow rate of 1 ml / min . the mass selective detector was operated in ei mode at 70 ev , scanning 19 - 350 m / z with a quadrupole temperature of 180 c and source temperature of 240 c . to guide the construction of a synthetic floral blend , pentane extracts of milkweed florets were analyzed by gc - ms on a zebron zb-50 column ( 15 m 0.25 mm , 0.25 m film thickness ) ( phenomenex ) , with a 1-l splitless injection at 210 c . the temperature program began at 25 c for 3 min and increased at 13 c / min to 200 c , followed by a 25 c / min increase to 280 c to remove high - boiling lipids . peak identities were validated by re - injection on a finnegan trace gc / ms ( thermo fisher scientific inc . , waltham , ma , usa ) , with a 1:10 split at 180 c and a rtx-5ms column ( 30 m 0.25 mm , 0.25 m film thickness ) ( restek , bellefonte , pa , usa ) . the helium flow was 1 ml / min , and temperature program was 40 c for 1 min , ramped at 15 c / min to 275 c , with a 2-min final hold time . retention indices ( ri ) of compounds were determined on both columns relative to n - alkanes ( c8c20 ) . components of floral headspace and extract were identified by matching their mass spectra to nist / epa / nih mass spectral library 2005 and comparison of retention indices to published values ( adams , 1995 ) . identity of major components was confirmed by comparison of spectra and retention times to authentic standards . peak purity was determined by using amdis deconvolution software ( nist , gaithersburg , md , usa ) to assure the absence of any co - eluting components . a synthetic mimic of the extract was constructed by blending authentic standards of the major components identified in the extract . quantities of each component were individually adjusted until the overall concentration and component proportions closely approximated those of the floral extract ( fig . 1total ion chromatograms ( a ) of florets of milkweed , asclepias syriaca , pentane extract ( broken line ) , and a synthetic blend ( solid line ) used to assay upwind attraction of culex pipiens ( peak # 1 . phenylethanol ; column = zebron zb-50 ) and total ion chromatogram ( b ) of the headspace profile of a single a. syraica floret captured on a divinylbenzene / carboxen / polydimethylsiloxane spme fiber ( peak # 1 . ( e , e)-alloocimene ; column = zebron zb-1 ) total ion chromatograms ( a ) of florets of milkweed , asclepias syriaca , pentane extract ( broken line ) , and a synthetic blend ( solid line ) used to assay upwind attraction of culex pipiens ( peak # 1 . phenylethanol ; column = zebron zb-50 ) and total ion chromatogram ( b ) of the headspace profile of a single a. syraica floret captured on a divinylbenzene / carboxen / polydimethylsiloxane spme fiber ( peak # 1 . ( e , e)-alloocimene ; column = zebron zb-1 ) all behavioral assays were conducted in a clear acrylic dual - port flight olfactometer , modified from hancock and foster ( 1993 ) , with three main parts : an introduction / release chamber , flight chamber , and trapping ports . the introduction / release chamber was located at the downwind end , measuring 30 40 cm wide and 30 cm long . a sliding gate separated the release chamber from the main flight chamber ( 30 40 cm wide , 90 cm long ) , which had two cylindrical glass jar trapping ports ( 15 cm long by 7 cm diameter ) on its upwind end . ports were fitted with borosilicate glass funnels , with the wide end opening into the flight chamber and the narrower ( 3 mm diam ) end pointing into a glass jar . thus , mosquitoes entering the wide end of the funnel from the flight chamber were channeled into the jar , where they were retained . trapping ports were located 11 cm above the flight chamber floor and were separated by 21 cm . the funnel and glass jar were held together by parafilm sealing film ( pechiney plastic company , menasha , wi , usa ) , which was replaced between treatments . we recognized the pitfalls associated with the use of parafilm in solvent - based behavioral assays ( millar and haynes , 1998 ) , and we ensured that this sealant would not come in contact with any chemicals in the olfactometer . a 7.5-mm diam hole in the upwind end of each jar allowed the introduction of purified / humidified air after it had passed from an oil - free air pump through an activated carbon canister , and then through a water column . air flow was maintained at 50 ml / s into each port , providing a velocity of 72 mm / s in the center of the flight chamber . a black cotton cloth , covering a dampened layer of cotton wool , covered the entire floor of the flight chamber , to maintain a level of humidity similar to that within the choice ports . data loggers ( hobo , onset computer corporation , bourne , ma , usa ) recorded temperature and humidity in the olfactometer , which was 25.0 - 27.5 c and 75 - 95 % rh . an exhaust duct , connected at the downwind end of the release chamber , directed the effluent air out of the building through an exhaust hood . to test for positional bias of the ports , mosquitoes were released into the chamber after either baiting both ports with honey or leaving both empty . mosquitoes were divided equally between left and right ports when baited with honey and were found rarely in ports when both were empty . response to whole milkweed flowers was determined in the flight olfactometer with 2 g of florets placed in an aluminum weighing boat in one of the trapping jars , and compared with an empty control jar . extracts and synthetic chemicals were applied to 15-cm long cotton wicks ( tidi products , neenah , wi , usa ) and compared to a pentane control . the positions of the treatment and control ports were alternated between bioassays as a further safeguard against positional bias . the olfactometer parts were cleaned with 70 % ethanol followed by water after each experiment ; gloves were used at all times to avoid contamination with human - related kairomones . the 16:8 ( l : d ) light cycle used for mosquito rearing was maintained in the bioassay room . at 2 h prior to scotophase , approximately 200 - 300 mosquitoes of both sexes in similar numbers were released through a sleeve connection , directly from an acrylic plastic cage ( 30 30 30 cm ) into the holding / release chamber , where they were held for 15 min to acclimatize before the release gate was opened . after 12 h , the numbers of mosquitoes in the treatment port , the control port , and remaining in the flight chamber were recorded . use of a 12-h period captured the bimodal nocturnal - crepuscular sugar - feeding rhythm of c. pipiens , which includes some sugar feeding throughout the night ( yee and foster , 1992 ) . subtractive bioassays were conducted on the synthetic blend to determine the relative activity of the identified components . the full six - component blend was presented in one of the paired olfactometer ports alongside a reducted five - compound blend , in which one of the components was removed . after establishing the significance of each chemical on mosquito attraction , we compared the minimal attractive blend against the full six - component blend . this was followed by a dose response study of the minimal blend to determine the optimal concentration for response . doses were presented in a randomized block design ( n = 5 ) , with percentage response calculated as the number in the synthetic blend port minus the number in the control port and expressed relative to the number of mosquitoes released . mosquito flight behavior was recorded by using three infrared rca closed - circuit video cameras ( lancaster , pa , usa ) onto which was mounted a tc1824b wide - angle es 25 mm 1:1.4 lens for flight - chamber recordings or a tc1874c es 75 mm 1:1.8 lens for each of the choice ports . ( lancaster , pa , usa ) eight - channel switcher , with video output to two rca tc1109 video monitors . video output was recorded with an emerson ewv404 vcr ( parsippany , nj , usa ) onto vhs tape in time - lapse mode , 20 s each min . mosquito response in the flight olfactometer was analyzed by comparing numbers caught in the two ports by a goodness - of - fit chi - squared test with spss v. 17 ( spss inc , chicago , il , usa ) . response experiment , a regression model of mosquito response to log - transformed blend dose was determined by using the fitted line plot module of minitab v. 16 ( minitab inc . six compounds comprised > 90 % of the relative abundance of the milkweed floret components in the pentane extract : ( e)--ocimene , benzaldehyde , nonanal , benzyl alcohol , phenylacetaldehyde , and ( e)-2-nonenal ( fig . 1a , table 1 ) . the volatile profile collected by dvb / car / pdms spme directly from fresh florets was dominated by three of these compounds : ( e)--ocimene , benzaldehyde , and phenylacetaldehyde ( > 75 % of the total ) , but there were also substantial differences compared to the pentane extract ( fig . most notably , ( e , z)- and ( e , e)-alloocimene were collected from the flower headspace , but were almost completely absent from the extract , whereas nonanal , a major constituent of floret extract , was missing in the headspace sample . these disparities can be explained partly by variation in volatility among the components , but also likely reflect differences between the chemical composition in flower tissue and what is released . the extract also contained higher - boiling hydrocarbons and fatty acids , but due to their low volatility , they were not included in behavioral studies . based on the quantities of synthetic standards used to mimic the extract gc profile ( table 1 ) , the total floral concentration of the six major volatiles was estimated at 32 g / g floret fresh weight.table 1major volatile constituents of a pentane extract of the common milkweed , asclepias syriaca , as determined by gas chromatography mass spectrometry , and quantities used in a synthetic mimiccompoundrelative retention indexdiagnostic ei - ms fragment ions ( % intensity)synthetic blend ( g / ml)(e)--ocimene112477(36 ) , 79(42 ) , 91(53 ) , 93(100)6.14benzaldehyde115551(54 ) , 77(100 ) , 105(88 ) , 106(88)3.27nonanal122029(66 ) , 41(100 ) , 56(61 ) , 57(94)4.14benzyl alcohol123677(85 ) , 79(100 ) , 107(59 ) , 108(75)4.58phenylacetaldehyde125765(22 ) , 91(100 ) , 92(24 ) , 120(16)11.25(e)-2-nonenal130429(56 ) , 41(100 ) , 55(80 ) , 70(58)2.12identity established by comparison to authentic standard of each compoundgc retention times relative to n - alkane standards on a phenomenex zb-50 column phasesynthetic -ocimene contained a 3:1 ( e : z ) isomer mixture major volatile constituents of a pentane extract of the common milkweed , asclepias syriaca , as determined by gas chromatography mass spectrometry , and quantities used in a synthetic mimic identity established by comparison to authentic standard of each compound gc retention times relative to n - alkane standards on a phenomenex zb-50 column phase synthetic -ocimene contained a 3:1 ( e : z ) isomer mixture infrared video recordings showed c. pipiens in the flight chamber engaged in a zigzagging flight that was directed upwind towards ports baited with florets , extracts , or synthetic blends . the angle of the turns progressively decreased as the mosquitoes approached the target . in most cases , mosquitoes flew into the ports , but in a few cases they landed on the entrance of the port and walked in . there was no observed oriented flight towards the control port . in the first behavioral assay , mosquitoes showed a significant response to whole milkweed florets , where 67 % of released mosquitoes were captured in the floret - baited port , compared to only 6 % in the control port ( fig . 2a ; = 515.02 df = 1 , p < 0.001 ) . despite the chemical differences measured between the headspace and extract profiles of milkweed florets , mosquitoes showed a similarly strong response to a pentane extract of milkweed florets ( 52 % ) compared to pentane alone ( 11 % ) ( fig . 2b ; = 61.44 , df = 1 , p < 0.001 ) . moreover , mosquitoes were observed in video recordings to probe the extract - treated cotton wick during the early scotophase and early photophase , suggesting that the extract also stimulated a feeding response . 2percentage of culex pipiens flying upwind in a dual - port flight olfactometer to : a whole milkweed , asclepias syriaca , florets ; b pentane extract of a. syriaca florets ; and ( c ) synthetic a. syriaca floret blend compared against a water a or solvent ( b , c ) control . * * * p < 0.001 by chi - squared test percentage of culex pipiens flying upwind in a dual - port flight olfactometer to : a whole milkweed , asclepias syriaca , florets ; b pentane extract of a. syriaca florets ; and ( c ) synthetic a. syriaca floret blend compared against a water a or solvent ( b , c ) control . * * * p < 0.001 by chi - squared test based on the positive response to the milkweed floret extract , a six - component synthetic blend was formulated to simulate the concentrations and relative proportions of major constituents of the pentane extract ( fig . one discrepancy in the gc profiles was caused by the presence of the ( z)-isomer in synthetic ( e)--ocimene , resulting in a synthetic blend that exceeded the levels of this isomer in the floret extract . in the olfactometer , the synthetic floret blend performed similarly to the natural extract , with 48 % of released mosquitoes trapped , compared to 16 % for the control ( fig . 2c ; = 120.6 , df = 1 , p < 0.001 ) . again , video - recordings showed a directed upwind flight response to ports containing the synthetic blend , accompanied by vigorous probing of the treated wicks , but not to the controls . males and females responded to milkweed floret odors in similar numbers in all three experiments as the sex ratio of mosquitoes captured in either the treatment or control ports did not deviate significantly from 1:1 . the subtractive bioassay of the synthetic blend indicated a significant role for three compounds in c. pipiens response to milkweed : benzaldehyde ( = 13.22 , df = 1 , p < 0.001 ) , phenylacetaldehyde ( = 8.25 , df = 1 , p = 0.004 ) , and ( e)-2-nonenal ( = 3.62 , df = 1 , p = 0.05 ) ( fig . 3 ) . when any of these compounds was removed , mosquitoes showed a significant preference for the full blend over the reducted blend . a nearly significant preference was seen for blends missing either -ocimene ( p = 0.078 ) or benzyl alcohol ( p = 0.082 ) compared to the full blend . the activity of benzaldehyde , phenylacetaldehyde , and ( e)-2-nonenal was confirmed when the three were combined : the three - component blend captured 31 % of mosquitoes compared to only 7 % for the control ( fig . 4a ; = 196.56 , df = 1 , p < 0.001 ) and was as active as the full blend ( fig . 4b , 4 ; = 3.02 , df = 1 , p = 0.082).fig . 3choice response of culex pipiens in a dual - port flight olfactometer to a six - component ( full ) synthetic blend of milkweed , asclepias syriaca , floret volatiles compared to blends in which each component has been removed individually ( reducted ) . total response indicates the percentage of c. pipiens released that were captured by one of the blends ( no significant differences by 1-way anova ) . relative response shows the proportional response to the reducted ( gray bars ) and full ( black bars ) blends . numbers in bars indicate the mean number of c. pipiens responding ( n = 46 ) . asterisks denote significant differences in mean response to reducted versus full blend by chi - squared test ( * p < 0.05 , * * p < 0.005)fig . 4a percentage of culex pipiens flying upwind in a dual - choice flight olfactometer in response to a three - component blend , consisting of benzaldehyde , ( e)-2-nonenal , and phenylacetaldehyde ; * * * p < 0.001 by chi - squared test . b percentage of c. pipiens flying upwind in response to the full ( six - component ) and minimal ( three - component ) synthetic blends of a. syriaca floral odor . ns = no significant difference choice response of culex pipiens in a dual - port flight olfactometer to a six - component ( full ) synthetic blend of milkweed , asclepias syriaca , floret volatiles compared to blends in which each component has been removed individually ( reducted ) . total response indicates the percentage of c. pipiens released that were captured by one of the blends ( no significant differences by 1-way anova ) . relative response shows the proportional response to the reducted ( gray bars ) and full ( black bars ) blends . numbers in bars indicate the mean number of c. pipiens responding ( n = 46 ) . asterisks denote significant differences in mean response to reducted versus full blend by chi - squared test ( * p < 0.05 , * * p < 0.005 ) a percentage of culex pipiens flying upwind in a dual - choice flight olfactometer in response to a three - component blend , consisting of benzaldehyde , ( e)-2-nonenal , and phenylacetaldehyde ; * * * p < 0.001 by chi - squared test . b percentage of c. pipiens flying upwind in response to the full ( six - component ) and minimal ( three - component ) synthetic blends of a. syriaca floral odor . ns = no significant difference because quantity as well as quality can determine the intensity of chemically mediated behavior , c. pipiens response was measured in the flight olfactometer to different doses of the three - component blend compared to a solvent control . there was a significant positive quadratic response [ log(dose ) ( y = 20.527x + 73.476x43.709 ; r = 57.6 % ] , with a response maximum predicted at 62 g ( fig . 5 ) . the response flattened out at both the lowest and highest doses ; removal of the two most extreme doses produced a stronger - fitting regression model ( r = 93.3 % ) , but with a similar predicted optimal dose of 65 g.fig . 5percentage of culex pipiens flying upwind in a dual - choice flight olfactometer in response to different concentrations of a three - component kairomone blend ( benzaldehyde , ( e)-2-nonenal , and phenylacetaldehyde ) after subtracting the response to a paired solvent ( pentane ) control percentage of culex pipiens flying upwind in a dual - choice flight olfactometer in response to different concentrations of a three - component kairomone blend ( benzaldehyde , ( e)-2-nonenal , and phenylacetaldehyde ) after subtracting the response to a paired solvent ( pentane ) control a positive upwind orientation by c. pipiens to floret volatiles of a. syriaca was demonstrated in this study , suggesting a new potential source of attractants for use in the field as lures . males and females showed significant response to milkweed florets , a pentane extract , and a synthetic blend of the extract s six major constituents : benzaldehyde , ( e)--ocimene , benzyl alcohol , phenylacetaldehyde , nonanal , and ( e)-2-nonenal . the first three of these compounds are among the most common floral odor constituents in plants ( knudsen et al . , 2006 ) . subsequent subtractive bioassays of the synthetic blend demonstrated that only three of its components contributed to attraction : benzaldehyde , phenylacetaldehyde , and ( e)-2-nonenal . the attraction of mosquitoes to plant odors has been demonstrated in numerous field and laboratory studies , but few studies have shown significant attraction to synthetics . mauer and rowley ( 1999 ) observed orientation of c. pipiens to a methylene chloride extract of a. syriaca in a dual port olfactometer . however , a synthetic blend of the two dominant compounds in the extract headspace , benzyl alcohol and 2-phenylethanol , failed to attract the mosquitoes . we did not test 2-phenylethanol , but benzyl alcohol was not active in our flight olfactometer bioassays . ( 2007 ) also found benzyl alcohol to not be very attractive to c. pipiens var . flowers of tanacetum vulgare also attract culex species in the field ( andersson and jaenson , 1987 ) , and bowen ( 1992b ) discovered a high proportion of both broadly- and narrowly - tuned antennal receptor neurons of c. pipiens sensitive to thujone , the primary constituent of t. vulgare fragrance . however , thujone only elicited a close - range dose - dependent probing response in c. pipiens ; it did not stimulate upwind flight in a wind - tunnel olfactometer ( bowen , 1992b ) . the only previous flower - based synthetic blend causing mosquito orientation was developed from silene otites , whose flowers attract mosquitoes in the field ( brantjes and leemans , 1976 ) . the synthetic blend mimic consisted of phenylacetaldehyde , veratrole , and 2-methoxyphenol , the first of which elicited the strongest attraction in c. pipiens var . phenylacetaldehyde also was a prominent component of our a. syriaca headspace and solvent extract , and it was essential for maximum attraction to the synthetic blend . although nonanal was a major component of a. syriaca floret extract , we saw no evidence that it played a role in attraction in our bioassays . in contrast , from single - cell recordings , syed and leal ( 2009 ) determined that ca . 40 % of all antennal olfactory receptors of c. quinquefasciatus were acutely sensitive to nonanal , and it produced a synergistic effect on trap catch when presented with co2 . nonanal is a major skin odorant of birds and mammals , including humans , resulting from the oxidation of sebaceous fatty acids ( haze et al . , 2001 ) this fact , along with the significant behavioral interaction with co2 , suggests that nonanal may play an important role in mediating culex host - finding , but does not attract sugar - seeking mosquitoes . we found notable differences between profiles of the pentane extract and the spme - collected headspace of a. syriaca florets . pentane extracts were dominated by phenylacetaldehyde , ( e)--ocimene , and nonanal , whereas the headspace profile contained primarily benzaldehyde , ocimene isomers , and phenylacetaldehyde . disparities between extracts and chemicals collected in the headspace might be explained by differences in the physico - chemical properties of the constituents , deep penetration by solvents to extract compounds that are not normally released by the tissue , and/or selective trapping of chemicals by spme . in this study , benzaldehyde , -ocimene , and alloocimene have the highest vapor pressures of all the chemicals identified from milkweed , and they make up most of the volatile profile . the vapor pressure of benzaldehyde is almost twice that of nonanal , almost four times that of ( e)-2-nonenal , and more than six times that of benzyl alcohol . the latter three compounds were either absent or found in very low levels in the headspace analysis . phenylacetaldehyde is intermediate in its volatility , but was also the component found in the highest levels in the pentane extract . volatile collection analyses can produce misleading results , as they may not accurately reflect the actual proportionality of constituents in the headspace . we do not believe that this was a major factor for explaining the differences in the headspace and extract profiles of milkweed due to our choice of spme phase . although pdms has been the most widely used spme phase , it is actually a poor choice for characterizing plant volatile profiles that contain constituents with a range of volatilities and functional groups . in preliminary studies ( not shown ) , we found a broader array of volatile compounds was trapped from various flowers by dvb / car / pdms compared to pdms alone . this mixed - bed fiber not only employs a broader range of phase polarity , but also incorporates both adsorption and partitioning as mechanisms of collection ( koziel and novak , 2002 ) . a number of recent studies have demonstrated the higher recovery efficiency and linearity by dvb / car / pdms compared to other spme phases for all of the compound classes that we identified from milkweed ( cui et al . , 2009 ; ferreira et al . , 2009 ; zhang et al . , 2009 ) . 2009 ) found that volatile profiles of longan fruit extracted by a 50/30 m dvb / car / pdms fiber were 3x higher in terpenes , 5x higher in alcohols , and 14x higher in esters than those produced by a 100 m pdms fiber , and revealed volatile carbonyls and acids , which were completely absent from pdms profiles . this study points to floral odors as a potential new model for chemical lures useful for mosquito sampling or control . if similar attraction can be demonstrated at the field level , as has already been demonstrated with human kairomone blends ( mukabana et al . , 2012 ) , synthetic floral blends could potentially be used in trapping devices to sample adult populations . relative to animal - derived odors ( mukabana et al . , 2012 ) , floral odors have the advantage of attracting both sexes of mosquitoes in proportional numbers , and females in all gonotrophic states and in reproductive diapause . given that c. pipiens visits a variety of flowers for nectar - feeding , the three compounds identified here are likely not the only floral components attractive to them , and more effective blends may remain to be discovered . future research should seek additional attractants and determine optimal blend release rates , delivery systems , and trap designs for maximizing capture in the field .

a pentane extract of flowers of common milkweed , asclepias syriaca ( asclepiadaceae ) , elicited significant orientation from both male and female culex pipiens in a dual - port flight olfactometer . analysis of the extract by gas chromatography mass spectrometry revealed six major constituents in order of relative abundance : benzaldehyde , ( e)--ocimene , phenylacetaldehyde , benzyl alcohol , nonanal , and ( e)-2-nonenal . although not all were collected from the headspace profile of live flowers , a synthetic blend of these six compounds , when presented to mosquitoes in the same levels and proportions that occur in the extract , elicited a response comparable to the extract . subtractive behavioral bioassays demonstrated that a three - component blend consisting of benzaldehyde , phenylacetaldehyde , and ( e)-2-nonenal was as attractive as the full blend . these findings suggest the potential use of synthetic floral - odor blends for monitoring or control of both male and female disease - vectoring mosquitoes .

the paradigm of intercellular cell signaling mechanisms has until recently been assumed to be limited to direct cell cell contact and via local soluble factor release . however , there has been increasing interest in the role of plasma membrane - derived small lipid / protein vesicles in cell cell communication in the last decade . microparticles ( mp ) are one such type of vesicles gaining more recognition as their biological roles are being explored . although there is no standardized terminology for these small extracellular vesicles , the term , microparticle , usually refers to a subset of extracellular , cell membrane - derived vesicles ranging from 0.1 to 1 m in diameter , shed directly from the plasma membrane through the process of ectocytosis ( gasser and schifferli , 2004 ; cocucci et al . , 2009 although mp are shed from steady - state cells , elevated levels are observed in actively proliferating cells and cells activated by a wide range of stimuli , from mitogens and inflammatory mediators , to antigenically foreign compounds , such as pathogen components , as well as various other stress - inducing challenges in both in vitro and in vivo models ( ratajczak et al . , 2006 ; in vivo , mp are most commonly found in blood , however , they are also observed in other bodily fluids , such as synovial fluid ( berckmans et al . , 2005 ; distler et al . , 2005b ) and urine ( smalley et al . , 2008 ) . microparticles are often compared with exosomes and apoptotic bodies ( table 1 ) , however , while superficial structural similarities exist between them , both the mechanism of mp formation and their subsequent composition are fundamentally different ( thry et al . , 2009 ; mause and weber , 2010 ; gyrgy et al . , 2011 ) . mp shedding is a multi - enzyme - regulated process . under normal conditions , the asymmetric composition of the plasma membrane is maintained by three principal translocase enzyme families of flippases , floppases , and scramblases , which actively maintain phosphatidylcholine ( pc ) and sphingomyelin ( sm ) on the external leaf , while phosphatidylserine ( ps ) and phosphatidylethanolamine ( pe ) are kept on the internal leaf . on cellular activation , these enzymes are inhibited following an influx of ca into the cytoplasm , and consequently the normal phospholipid asymmetry is disrupted ( coltel et al . , 2006 ; piccin et al . , 2007 ; pap et al . , this is accompanied by the loss of actin spectrin anchorages and the degradation of actin filaments by calpain . these protrusions are pinched off as individual mp in a rho - associated kinase i ( rock - i)-dependent manner from the cell membrane , with ps and pe exposed on the outer surfaces . in contrast , exosomes fall into the 50100 nm range and are formed by inward budding into the endosomal lumen of small intra - luminal vesicles ( ilv ) that aggregate to become multivesicular bodies ( mvb ) . mvb subsequently fuse with the plasma membrane to release the ilv into the extracellular space as exosomes ( thry et al . , exosomes are enriched in cd63 , and major histocompatibility complex ( mhc ) i and ii molecules , but tend to have low levels of ps and pro - coagulation factors on the surface ( denzer et al . , 2000 ; thry et al . , 2009 ; mause and weber , 2010 ; gyrgy et al . , 2011 ) . apoptotic bodies , yet another type of subcellular vesicle , are much larger ( 14 m ) and are essentially fragments of cells produced in late stage apoptosis ( table 1 ) . the process by which mp are produced results in their possessing many of the characteristics of the cell from which they originated . this includes many of the surface molecules , such as integrins , receptor ligands , co - stimulatory molecules , as well as cytoplasmic contents , such as activated signaling molecules and even genetic materials e.g. , mrna ( baj - krzyworzeka et al . , 2006 ; aliotta et al . , 2010 ) and mirna ( yuan et al . , 2009 ) . mp either attach and fuse to their target cell ( del conde et al . , 2005 ) , or are internalized and processed by pinocytic or phagocytic mechanisms ( dasgupta et al . since mp carry biological molecules from their cells of origin , they may induce associated responses in their target cells , depending on the source of mp , the cell type on which they act and the route by which they are incorporated . given that mp are produced in response to many stress conditions and are involved in several immunological processes , it seems likely that mp are involved in the regulation of the immune response against invading pathogens , however , their potential role in infectious diseases has not been extensively studied . this review aims to consolidate current evidence on their biological role and venture opinion on their potential impact in infectious disease . one of the hallmarks of bacterial , viral , fungal , or parasitic infection is the formation of an inflammatory milieu around infected or affected cells . pathogen - associated molecular pattern ( pamp ) recognition receptors ( prr ) , such as toll - like receptors , certain classes of rna helicases , like the retinoic acid inducible gene - i ( rig - i ) , and nod - like receptors ( nlr ) , specifically detect components of the invading pathogen and trigger a cascade of intracellular signaling events that lead to the activation of transcription to initiate the first wave of innate immune responses ( creagh and oneill , 2006 ; seth et al . , 2006 ) . however , endotoxins , produced by bacteria during infection , or the sheer load of replicating intracellular virus would undoubtedly cause stress in affected cells . indeed , generation of mp may be closely associated with the initiation of apoptosis and necrosis ( ardoin et al . , 2007 ) . whether stress - inducible proteins , such as hypoxia - inducible factor 1 ( hif-1 ) , heat - shock factor ( hsf-1 ) , heat - shock protein ( hsp ) family , nuclear factor b ( nf-b ) , jun nh2-terminal kinase ( jnk ) , and p53 ( ohiro et al . , 2003 ; razorenova et al . , 2005 ; thompson and locarnini , 2007 ; zinkernagel et al . , 2007 ; de maio , 2011 ; li et al . , 2011 ; rawat and mitra , 2011 ; zhang et al . , 2011 ) and mhc class i polypeptide - related sequence a and b ( mica , micb ; stern - ginossar and mandelboim , 2009 ) are involved , remains to be determined . nevertheless , a broad array of pro - inflammatory cytokines , such as tumor necrosis factor ( tnf ) , interleukin-1 ( il-1 ) , il-6 , and type 1 interferons ( ifn - i ) are produced in response to infection ( medzhitov and horng , 2009 ) , and many of these induce mp shedding in the absence of infection ( combes et al . , 1999 ; sheremata et al . 2008 ) . not surprisingly , therefore , both stimulation in vitro with lipopolysaccharide ( lps ; aharon et al . , 2008 ; bernimoulin et al . , 2009 ) and intravenous injection of bacterial endotoxin in vivo ( zubairova et al . , 2006 ; wang et al . , furthermore , elevated blood mp levels are also observed in septic patients ( nieuwland et al . , 2000 ; mostefai et al . , 2008 ) , in human immunodeficiency virus ( hiv ) patients ( aupeix et al . , 1997 ) and ebola virus - infected macaques ( geisbert et al . , 2003 ) , as well as malaria patients and plasmodium - inoculated laboratory animals ( combes et al . , 2004 , 2005 ; penet et al . , 2008 ; couper et al . , 2010 ; pankoui mfonkeu et al . , many activated immune cells not necessarily infected by pathogens , such as monocytes , macrophages , dendritic cells ( dc ) , and lymphocytes , produce mp when activated by relevant stimuli . mp have been shown to be produced by monocytes and macrophages stimulated with a23187 ( cerri et al . , , 2006 ; aharon et al . , 2008 ) , and starvation ( aharon et al . , 2008 ) , by dc stimulated with lps ( obregon et al . , 2006 , 2009 ) , by t cells stimulated with agonistic cd3 antibodies , fas ligand , heat , actinomycin - d , staurosporine , pha , pma ( scanu et al . , 2008 ) , and by neutrophils stimulated with fmlp and c5a ( gasser and schifferli , 2004 ) . in addition , mp are produced by cells isolated from patients with autoimmune disease , such as diabetes ( martin et al . , 2004 ) , as well as vascular disease ( tesse et al . , 2005 ) . the wide variety of possible stimuli producing mp in the absence of infection makes it difficult to predict pathways leading to mp production in infectious disease , or predict their function in this context and few experimental models utilize infectious pathogens . nevertheless , these studies provide valuable clues to what possible responses might occur in infection . as mentioned above , elevated blood mp levels have been reported in models of sepsis , viral infection , and cerebral malaria . because of the generalized inflammatory conditions that such events generally create , it seems reasonable to propose this would be the case for most , if not all infections accompanied by significant hematogenous spread , i.e. , during parasitemia , bacteremia , viremia or fungemia . furthermore , due to the propensity of mp to carry contents derived from their cells of origin , it is likely that they contain antigens , either present or induced in infected cells . plasmodium parasite antigens were detected in the mp purified from the plasma of infected animals ( couper et al . , 2010 ) . this is unlikely to be unique to parasitic infections , as infecting viruses inevitably become an integral part of the cell , inducing a range of virus - specified proteins crucial for their replication and these could become incorporated into the mp contents , either specifically or non - specifically . thus , mp could remotely convey viral antigens via the blood stream to professional antigen - presenting cells ( apc ) , such as macrophages and dc residing centrally in the spleen and liver without the necessity for their direct interaction with infected cells . both organs normally have high throughputs from the systemic circulation , increasing the likelihood that mp will interact with apc . from a host point of view , this maintains a desirable separation between the peripheral site of infection and the site of immune induction . additionally , tnf - stimulated , endothelial cell - derived mp have the capacity to induce activation and maturation of plasmacytoid dc , although not conventional dc ( angelot et al . , 2009 ) . together , such remote interactions could increase the probability of detecting infection early , enabling prompt initiation of innate and adaptive responses by apc responding to mp in the blood . indeed , it has been shown that mp produced by mycobacterium - infected macrophages were able to activate and present antigen to naive t cells ( ramachandra et al . , 2010 ) , thus obviating the necessity for direct apc and t cell contact to generate a t cell response . in these experiments , mp were better at inducing t cell proliferation than exosomes . ( 2010 ) also demonstrated the activation of bone marrow - derived macrophages , with upregulation of cd40 and tnf expression . this raises the further possibility that mp may facilitate the mobilization of subsets recruited from the bone marrow during infection ( king et al . , 2007 ; getts et al . , 2008 ) . as well as accepting mp by fusion , dc also produce mp following activation . lps - induced mp from dc can transfer antigen to recipient dc , which can present this to t cells ( obregon et al . . other cellular membrane function can be transferred by mp from activated to nave t cells via the tetraspanin , cd81 , important for adhesion and lymphocyte maturation ( fritzsching et al . , 2002 ; quast et al . , 2011 ) . furthermore , cd154 platelet - derived mp introduced into cd154 mice triggers cd4 t cell - dependent production of antigen - specific igg and germinal center formation , suggesting that with the right surface molecules mp can fully activate the adaptive immune response ( sprague et al . , 2008 ) . indeed , mp transfer could explain the rapid exchange of t cell receptors ( tcr ) observed in the early stages of t cell activation to expand antigen - specific t cell clones ( chaudhri et al . , 2009 ) . irrespective , it seems likely that mp containing pathogen antigens along with the necessary co - stimulatory molecules can activate t cells , bypassing cell cell contact , thus enabling a wider , more efficient initiation , amplification and/or modulation of early adaptive immune responses . microparticles may also contain pro - inflammatory cytokines , such as il-1 , providing an alternative or additional pathway for cytokine secretion into the extracellular space to modulate local cytokine / chemokine signaling ( mackenzie et al . depending on their mode of manufacture and cellular release , it seems highly likely that other cytokines / chemokines , may make use of this pathway ( garcia et al . , 2005 ; dean et al . , 2009 ) . certainly , t cell - derived mp have been shown to activate monocytes ( distler et al . , 2005a ; huber et al . , 2007 ; scanu et al . , 2008 ) and monocytes activated with mp from pha- or pma - stimulated blood t cells produce tnf , il-1 , and soluble interleukin-1 receptor antagonist ( sil-1ra ; scanu et al . , 2008 ) . mp derived from t cells and monocytes also induce nf-b - dependent expression of matrix metalloproteinases ( mmp ; distler et al . , 2005b ) , as well as some pro - inflammatory cytokines and chemokines such as il-6 , il-8 , macrophage chemoattractant protein ( mcp)-1 , mcp-2 , and regulated upon activation , normal t cell expressed and secreted ( rantes ) in synovial fibroblasts ( berckmans et al . , 2005 ; distler et al . , 2005b ) . monocyte / macrophage - derived mp also upregulate inflammatory mediator synthesis by epithelial cells ( cerri et al . , 2006 ) . not surprisingly , under certain conditions mp can also induce anti - inflammatory il-10 expression in monocytes ( kppler et al . , 2006 ) and transforming growth factor 1 ( tgf-1 ) in macrophages ( sadallah et al . , mp can even be pro - apoptotic , especially in macrophages , which can undergo apoptosis upon uptake ( distler et al . for example , neutrophil - derived mp specifically upregulate the expression of tgf-1 , which helps to suppress the inflammatory response ( distler et al . the mp used in these models induce macrophage apoptosis , possibly because the mp were generated from apoptotic cells , which might have transferred the apoptotic signals to the macrophages ( bing et al . , 2008 ) . this is similar to the behavior of macrophages to dampen inflammation after ingesting apoptotic bodies , to prevent over - reaction ( savill et al . , 2002 ) . however , these studies did not use pathogens and macrophages activated by plasmodium were in contrast highly pro - inflammatory ( couper et al . , , mp might contain factors induced or expressed by the infecting organism that could impede or abrogate the early induction of an effective immune response . for example , influenza a ns1 protein expressed intracellularly has the capacity to suppress ifn - i production and inhibit apoptosis to enable better viral replication ( pauli et al . , 2008 ; wang et al . , several flavivirus subunits interfere with ifn - signaling at various levels and in different ways ( reviewed in daffis et al . such molecules , carried into cells yet to be infected could arguably give the infecting organism a survival advantage , enabling greater levels of replication in the host for longer periods of time , thus increasing the probability of transmission to vectors and/or to the next host . in the case of arboviruses , considering the relatively short times that organisms spend in the blood stream , even small increases in concentration and time spent in the blood stream could potentially subtend a significant survival advantage . the inflammatory conditions created by mp could in principle play important roles in controlling infection and pathogen clearance . how crucial these roles are in the immune system remain unclear , but as one of many factors that regulate the immune response against invading pathogens , mp could significantly influence the outcome for the host , both during the initial knockdown phase of controlling pathogen and in the ongoing adaptive response to it . pro - inflammatory cytokines induced by mp would act to manage and contain the infection , while chemoattractant proteins would send signals to recruit leukocytes to the site of infection to eliminate the pathogen . indeed , a further interesting characteristic of mp is their ability to upregulate adhesion molecules such as intercellular adhesion molecule-1 ( icam - i ; berckmans et al . , 2005 ) in target cells and promote monocyte recruitment ( mause et al . , 2005 ) . along with ability of mp to induce mmp expression and tnf release , the endothelium becomes more permeable for immune active proteins and leukocytes to access the site of infection . although this is an important step to enable pathogen clearance , it can be a double - edged sword in certain infectious diseases . in cases of lethal flavivirus encephalitis , in which immunopathology is one of the prominent features of pathogenesis , excessive monocyte infiltration of the central nervous system ( cns ) , and the accompanying pro - inflammatory cytokine spike are a major cause of mortality ( king et al . , 2007 ; getts et al . , 2008 ) . we hypothesize that during viremia preceding encephalitis , flavivirus infecting endothelium , and blood monocytes induce mp , both as a consequence of infection and via tnf released by infected cells ( cheng et al . , brain barrier , potentially enabling virus access to the cns ( wang et al . , 2004 ) . mp from tnf - activated endothelium could further activate uninfected monocytes and endothelium , while mp from infected cells could carry viral antigen , as well as activation signals to the liver and spleen . early antigen presentation would likely advantage the host in generating anti - viral immune responses . however , wnv - infected cns neurons recruit activated ( ccr2 ) monocytes from the bone marrow to the brain via ccl2 ( getts et al . , 2008 ) . here , tnf- and mp - mediated activation of cns endothelium would facilitate macrophage margination , adhesion , and diapedesis in response to the chemokine gradient , thus potentially increasing numbers of monocytes recruited to the brain and exacerbating immunopathology ( figure 1 ) . putative role of mp in immunopathogenesis of flavivirus encephalitis . after local replication of virus at the site of infection , the ensuing viremia results in endothelial and monocyte infection . mp produced as a result of infection and via the consequent tnf secretion , are transported in the blood stream . on the one hand , mp from tnf - activated endothelium could further activate uninfected monocytes and endothelium at remote sites , such as in the brain , while mp from infected cells could carry viral antigen and activation signals to liver and spleen . early antigen presentation would likely advantage the host by generating anti - viral immune responses . on the other hand , endothelial activation may enable increased migration of monocytes into the brain in response to ccl2 secreted from infected neurons . while this immigration may initially enable early viral clearance , over - exuberant immigration of activated monocytes that differentiate into macrophages may be immunopathological for neurons via toxic soluble factors produced . this does not exclude a possible trojan horse scenario , in which virus may also further gain access to the brain via the cerebral endothelium and/or via infected immigrant monocytes during encephalitis . brain barrier in cerebral malaria via monocyte , tnf , and mp activities ( combes et al . , moreover , in mice deficient in the floppase , atp - binding cassette transporter a1 , which modulates ps distribution on the outer plasma membrane leaflet , mp production is reduced in cerebral malaria , abrogating neurological signs in this model , and presenting the possibility for pharmacological modulation of mp to ameliorate immunopathology ( combes et al . , 2005 ; penet et al . , 2008 ) . immunological aspects aside , mp could potentially also be used directly as transport by viruses , including flavivirus , influenza , and herpes simplex virus ( hsv ) that propagate via a process of ectocytosis from the plasma membrane of an infected cell . in a model of hiv-1 , the glycome profile and the budding signal of the virion were similar to that of the mp secreted in conjunction , suggesting that the mechanism for viral propagation was similar to that of the formation of mp ( krishnamoorthy et al . even if mp formation selectively excludes budding virions , the mp may still potentially contain unpackaged viral genome and other viral proteins necessary for replication in the target cell . in either scenario , mp may be used by viruses as trojan horses to spread to other cells . indeed , hiv-1 induces mp that express cd45 , cd80 , cd86 , as well as mhc - i and ii ( esser et al . , 2001 ) , thus , potentially masking themselves as being of host origin . in addition , hiv also induces ccr5-containing mp and can transfer this receptor to other peripheral blood mononuclear cells ( pbmc ) deficient of endogenous ccr5 , enabling infection of these cells ( mack et al . , 2000 ) . however , the success of allogeneic transplantation of 32/32 ccr5 stem cells in keeping the patient hiv free , indicates that much more remains to be understood about these processes ( htter et al . , 2009 ) . as with all emerging areas , a number of technical issues remain to be fully resolved . for example , the accurate determination of mp size and phenotype , their clear distinction from and relationship to , other membrane vesicles , and their pure separation for functional experimental purposes , remain a significant challenge ( gyrgy et al . , 2011 ) . while mp clearly have likely roles in homeostasis and inflammation , few studies have to date directly addressed their involvement in infectious disease . thus , many questions remain to be answered , perhaps most important among which are , whether the production of mp as a programmatic response to infection confers an overall species survival advantage . at the mechanistic level , also , how cellular decision - making occurs to produce mp , what they should contain , and whether production of the full range of membranous vesicles is required and/or produced kinetically to accommodate host requirements from initial infection to disease resolution , remains to be elucidated . as with most defenses against invading pathogens , on the one hand , the transfer of phenotype and function by mp may be of considerable benefit to the host , in the early warning and initiating of immune defenses at both innate and adaptive levels . on the other , however , this may exacerbate immune responses leading to immune pathology , or be commandeered by the invading pathogen for its own survival . 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 .

despite their clear relationship to immunology , few existing studies have examined the potential role of microparticles ( mp ) in infectious disease . mp have a different size range from exosomes and apoptotic bodies , with which they are often grouped and arise by different mechanisms in association with inflammatory cytokine action or stress on the source cell . infection with pathogens usually leads to the expression of a range of inflammatory cytokines and chemokines , as well as significant stress in both infected and uninfected cells . it is thus reasonable to infer that infection - associated inflammation also leads to mp production . mp are produced by most of the major cell types in the immune system , and appear to be involved at both innate and adaptive levels , potentially serving different functions in each . thus , they do not appear to have a universal function ; instead their functions are source- or stimulus - dependent , although likely to be primarily either pro- or anti - inflammatory . we argue that in infectious diseases , mp may be able to deliver antigen , derived from the biological cargo acquired from their cells of origin , to antigen - presenting cells . another potential benefit of mp would be to transfer and/or disseminate phenotype and function to target cells . however , mp may also potentially be manipulated , particularly by intracellular pathogens , for survival advantage .

since their discovery in 1967 , radio pulsars have provided insights into physics on length scales covering the range from 1 m ( giant pulses from the crab pulsar ) to 10 km ( neutron star ) to kpc ( galactic ) to hundreds of mpc ( cosmological ) . pulsars present an extreme stellar environment , with matter at nuclear densities , magnetic fields of 10 g to nearly 10 g , and spin periods ranging from 1.5 ms to 8.5 s. the regular pulses received from a pulsar each correspond to a single rotation of the neutron star . it is by measuring the deviations from perfect observed regularity that information can be derived about the neutron star itself , the interstellar medium between it and the earth , and effects due to gravitational interaction with binary companion stars . in particular , pulsars have proved to be remarkably successful laboratories for tests of the predictions of general relativity ( gr ) . the tests of gr that are possible through pulsar timing fall into two broad categories : setting limits on the magnitudes of parameters that describe violation of equivalence principles , often using an ensemble of pulsars , and verifying that the measured post - keplerian timing parameters of a given binary system match the predictions of strong - field gr better than those of other theories . long - term millisecond pulsar timing can also be used to set limits on the stochastic gravitational - wave background ( see , e.g. , [ 73 , 86 , 65 ] ) , as can limits on orbital variability in binary pulsars for even lower wave frequencies ( see , e.g. , [ 20 , 78 ] ) . however , these are not tests of the same type of precise prediction of gr and will not be discussed here . this review will present a brief overview of the properties of pulsars and the mechanics of deriving timing models , and will then proceed to describe the various types of tests of gr made possible by both single and binary pulsars . the properties and demographics of pulsars , as well as pulsar search and timing techniques , are thoroughly covered in the article by lorimer in this series . this section will present only an overview of the topics most important to understanding the application of pulsar observations to tests of gr . radio pulsars were firmly established to be neutron stars by the discovery of the pulsar in the crab nebula ; its 33-ms period was too fast for a pulsating or rotating white dwarf , leaving a rotating neutron star as the only surviving model [ 108 , 53 ] . the 1982 discovery of a 1.5-ms pulsar , psr b1937 + 21 , led to the realization that , in addition to the young crab - like pulsars born in recent supernovae , there exists a separate class of older millisecond or recycled pulsars , which have been spun up to faster periods by accretion of matter and angular momentum from an evolving companion star . ( see , for example , and for reviews of the evolution of such binary systems . ) it is precisely these recycled pulsars that form the most valuable resource for tests of gr . the exact mechanism by which a pulsar radiates the energy observed as radio pulses is still a subject of vigorous debate . the basic picture of a misaligned magnetic dipole , with coherent radiation from charged particles accelerated along the open field lines above the polar cap [ 55 , 128 ] , will serve adequately for the purposes of this article , in which pulsars are treated as a tool to probe other physics . while individual pulses fluctuate severely in both intensity and shape ( see figure 1 ) , a profile integrated over several hundred or thousand pulses ( i.e. , a few minutes ) yields a shape a standard profile ( there is generally some evolution of pulse profiles with frequency , but this can usually be taken into account . ) it is the reproducibility of time - averaged profiles that permits high - precision timing . figure 1 top : 100 single pulses from the 253-ms pulsar b0950 + 08 , demonstrating pulse - to - pulse variability in shape and intensity . bottom : cumulative profile for this pulsar over 5 minutes ( about 1200 pulses ) ; this approaches the reproducible standard profile . top : 100 single pulses from the 253-ms pulsar b0950 + 08 , demonstrating pulse - to - pulse variability in shape and intensity . bottom : cumulative profile for this pulsar over 5 minutes ( about 1200 pulses ) ; this approaches the reproducible standard profile . ( stairs , unpublished . ) of some importance later in this article will be models of the pulse beam shape , the envelope function that forms the standard profile . the collection of pulse profile shapes and polarization properties have been used to formulate phenomenological descriptions of the pulse emission regions . at the simplest level ( see , e.g. , and other papers in that series ) , the classifications can be broken down into gaussian - shaped regions with little linear polarization and some circular polarization , and double - peaked cone regions with stronger linear polarization and s - shaped position angle swings in accordance with the rotating vector model while these models prove helpful for evaluating observed changes in the profiles of pulsars undergoing geodetic precession , there are ongoing disputes in the literature as to whether the core / cone split is physically meaningful , or whether both types of emission are simply due to the patchy strength of a single emission region ( see , e.g. , ) . a short description of pulsar observing techniques is in order . as pulsars have quite steep radio spectra ( see , e.g. , ) , they are strongest at frequencies f0 of a few hundred mhz . at these frequencies , the propagation of the radio wave through the ionized interstellar medium ( ism ) can have quite serious effects on the observed pulse . multipath scattering will cause the profile to be convolved with an exponential tail , blurring the sharp profile edges needed for the best timing . figure 2 shows an example of scattering ; the effect decreases with sky frequency as roughly f0 - 4 ( see , e.g. , ) , and thus affects timing precision less at higher observing frequencies . a related effect is scintillation : interference between the rays traveling along the different paths causes time - and frequency - dependent peaks and valleys in the pulsar s signal strength . the decorrelation bandwidth , across which the signal is observed to have roughly equal strength , is related to the scattering time and scales as f04 ( see , e.g. , ) . there is little any instrument can do to compensate for these effects ; wide observing bandwidths at relatively high frequencies and generous observing time allocations are the only ways to combat these problems . figure 2pulse profile shapes for psr j1740 - 3052 at multiple frequencies , aligned by pulse timing . all observations taken with the green bank telescope ( stairs , unpublished ) , except for the 660-mhz profile which was acquired at the parkes telescope [ 9 , 122 ] . pulse profile shapes for psr j1740 - 3052 at multiple frequencies , aligned by pulse timing . all observations taken with the green bank telescope ( stairs , unpublished ) , except for the 660-mhz profile which was acquired at the parkes telescope [ 9 , 122 ] . another important effect induced by the ism is the dispersion of the traveling pulses . acting as a tenuous electron plasma , the ism causes the wavenumber of a propagating wave to become frequency - dependent . by calculating the group velocity of each frequency component , it is easy to show ( see , e.g. , ) that lower frequencies will arrive at the telescope later in time than the higher - frequency components , following a 1/f law . the magnitude of the delay is completely characterized by the dispersion measure ( dm ) , the integrated electron content along the line of sight between the pulsar and the earth . all low - frequency pulsar observing instrumentation is required to address this dispersion problem if the goal is to obtain profiles suitable for timing . one standard approach is to split the observing bandpass into a multichannel filterbank , to detect the signal in each channel , and then to realign the channels following the 1/f law when integrating the pulse . however , when the ratio of the pulse period to its dm becomes small , much sharper profiles can be obtained by sampling the voltage signals from the telescope prior to detection , then convolving the resulting time series with the inverse of the easily calculated frequency - dependent filter imposed by the ism . as a result , the pulse profile is perfectly aligned in frequency , without any residual dispersive smearing caused by finite channel bandwidths . in addition , full - stokes information can be obtained without significant increase in analysis time , allowing accurate polarization plots to be easily derived . this coherent dedispersion technique is now in widespread use across normal observing bandwidths of several tens of mhz , thanks to the availability of inexpensive fast computing power ( see , e.g. , [ 10 , 66 , 123 ] ) . some of the highest - precision experiments described below have used this approach to collect their data . figure 3pulse profile of the fastest rotating pulsar , psr b1937 + 21 , observed with the 76-m lovell telescope at jodrell bank observatory . the top panel shows the total - intensity profile derived from a filterbank observation ( see text ) ; the true profile shape is convolved with the response of the channel filters . the lower panel shows the full - stokes observation with a coherent dedispersion instrument [ 126 , 123 ] . total intensity is indicated by black lines , and linear and circular power by red and blue lines , respectively . the coherent dedispersion observation results in a much sharper and more detailed pulse profile , less contaminated by instrumental effects and more closely resembling the pulse emitted by the rotating neutron star . pulse profile of the fastest rotating pulsar , psr b1937 + 21 , observed with the 76-m lovell telescope at jodrell bank observatory . the top panel shows the total - intensity profile derived from a filterbank observation ( see text ) ; the true profile shape is convolved with the response of the channel filters . the lower panel shows the full - stokes observation with a coherent dedispersion instrument [ 126 , 123 ] . total intensity is indicated by black lines , and linear and circular power by red and blue lines , respectively . the coherent dedispersion observation results in a much sharper and more detailed pulse profile , less contaminated by instrumental effects and more closely resembling the pulse emitted by the rotating neutron star . much better timing precision can be obtained with these sharper pulses . once dispersion has been removed , the resultant time series is typically folded modulo the expected pulse period , in order to build up the signal strength over several minutes and to obtain a stable time - averaged profile . the pulse period may not be very easily predicted from the discovery period , especially if the pulsar happens to be in a binary system . the goal of pulsar timing is to develop a model of the pulse phase as a function of time , so that all future pulse arrival times can be predicted with a good degree of accuracy . the profile accumulated over several minutes is compared by cross - correlation with the standard profile for the pulsar at that observing frequency . a particularly efficient version of the cross - correlation algorithm compares the two profiles in the frequency domain . once the phase shift of the observed profile relative to the standard profile is known , that offset is added to the start time of the observation in order to yield a time of arrival ( toa ) that is representative of that few - minute integration . in practice , observers frequently use a time- and phase - stamp near the middle of the integration in order to minimize systematic errors due to a poorly known pulse period . as a rule , pulse timing precision is best for bright pulsars with short spin periods , narrow profiles with steep edges , and little if any profile corruption due to interstellar scattering . with a collection of toas in hand , it becomes possible to fit a model of the pulsar s timing behaviour , accounting for every rotation of the neutron star . based on the magnetic dipole model [ 108 , 53 ] , the pulsar is expected to lose rotational energy and thus spin down . the primary component of the timing model is therefore a taylor expansion of the pulse phase with time t : ( 1)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \phi = { \phi _ 0 } + \nu ( t - { t_0 } ) + \frac{1}{2}\dot \nu { ( t - { t_0})^2 } + \ldots , $ $ \end{document } where 0 and t0 are a reference phase and time , respectively , and the pulse frequency is the time derivative of the pulse phase . note that the fitted parameters and and the magnetic dipole model can be used to derive an estimate of the surface magnetic field b sin : ( 2)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ b\sin \alpha = { \left ( { \frac { { - 3i\dot \nu { c^3}}}{{8{\pi ^2}{r^6}{\nu ^3 } } } } \right)^{1/2 } } \approx 3.2 \times { 10^{19}}{\left ( { \frac { { - \dot \nu } } { { { \nu ^3 } } } } \right)^{1/2}}{\rm{g , } } $ $ \end{document } where is the inclination angle between the pulsar spin axis and the magnetic dipole axis , r is the radius of the neutron star ( about 10 cm ) , and the moment of inertia is i10 g cm . in turn , integration of the energy loss , along with the assumption that the pulsar was born with infinite spin frequency , yields a characteristic age c for the pulsar : ( 3)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \tau _ c } = - \frac{\nu } { { 2\dot \nu } } . $ $ \end{document } equation ( 1 ) refers to pulse frequencies and times in a reference frame that is inertial relative to the pulsar . toas derived in the rest frame of a telescope on the earth must therefore be translated to such a reference frame before equation ( 1 ) can be applied . the best approximation available for an inertial reference frame is that of the solar system barycentre ( ssb ) . even this is not perfect ; many of the tests of gr described below require correcting for the small relative accelerations of the ssb and the centre - of - mass frames of binary pulsar systems . but certainly for the majority of pulsars it is adequate . the required transformation between a toa at the telescope and the emission time t from the pulsar is ( 4)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ t = \tau - d/{f^2 } + { \delta _ { { \rm{r } } \odot } } + { \delta _ { { \rm{e } } \odot } } - { \delta _ { { \rm{s } } \odot } } - { \delta _ { \rm{r } } } - { \delta _ { \rm{e } } } - { \delta _ { \rm{s}}}. $ $ \end{document } here d / f accounts for the dispersive delay in seconds of the observed pulse relative to infinite frequency ; the parameter d is derived from the pulsar s dispersion measure by d = dm/2.4110 hz , with dm in units of pc cm and the observing frequency f in mhz . the roemer term r takes out the travel time across the solar system based on the relative positions of the pulsar and the telescope , including , if needed , the proper motion and parallax of the pulsar . the einstein delay e accounts for the time dilation and gravitational redshift due to the sun and other masses in the solar system , while the shapiro delay s expresses the excess delay to the pulsar signal as it travels through the gravitational well of the sun a maximum delay of about 120 s at the limb of the sun ; see for a fuller discussion of these terms . the terms r , e , and s in equation ( 4 ) account for similar delays within the pulsar binary system , if needed , and will be discussed in section 2.3.2 below . most observers accomplish the model fitting , accounting for these delay terms , using the program @@page11tempo . the correction of toas to the reference frame of the ssb requires an accurate ephemeris for the solar system . it is also clear that accurate time - keeping is of primary importance in pulsar modeling . general practice is to derive the time - stamp on each observation from the observatory s local time standard typically a hydrogen maser and to apply , retroactively , corrections to well - maintained time standards such as utc(bipm ) , universal coordinated time as maintained by the bureau international des poids et mesures in paris . the terms r , e , and s in equation ( 4 ) , describe the roemer , the majority of binary pulsar orbits are adequately described by five keplerian parameters : the orbital period pb , the projected semi - major axis , the eccentricity e , and the longitude and epoch t0 of periastron . the angle is measured from the line of nodes where the pulsar orbit intersects the plane of the sky . in many cases , one or more relativistic corrections to the keplerian parameters early relativistic timing models , developed in the first years after the discovery of psr b1913 + 16 , either did not provide a full description of the orbit ( see , e.g. , ) , or else did not define the timing parameters , in a way that allowed deviations from gr to be easily identified ( see , e.g. , [ 49 , 58 ] ) . the best modern timing model [ 33 , 133 , 43 ] incorporates a number of post - keplerian timing parameters which are included in the description of the three delay terms , and which can be fit in a completely phenomenological manner . the delays are defined primarily in terms of the phase of the orbit , defined by the eccentric anomaly u and true anomaly ae(u ) , as well as , pb , and their possible time derivatives . these are related by ( 5)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ u - e\sin u = 2\pi \left [ { \left ( { \frac{{t - { t_0}}}{{{p_{\rm{b } } } } } } \right ) - \frac{{{{\dot p}_{\rm{b}}}}}{2}{{\left ( { \frac{{t - { t_0}}}{{{p_{\rm{b } } } } } } \right)}^2 } } \right ] , $ $ \end{document } ( 6)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { a_{\rm{e}}}(u ) = 2\arctan \left [ { { { \left ( { \frac{{1 + e}}{{1 - e } } } \right)}^{1/2}}\tan \frac{u}{2 } } \right ] , $ $ \end{document } ( 7)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \omega = { \omega _ 0 } + \left ( { \frac{{{p_{\rm{b}}}\dot \omega } } { { 2\pi } } } \right){a_{\rm{e}}}(u ) , $ $ \end{document } where 0 is the reference value of at time t0 . the delay terms then become : ( 8)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \delta _ { \rm{r } } } = x\sin \omega ( \cos u - e(1 + { \delta _ r } ) ) + x{(1 - { e^2}{(1 + { \delta _ \theta } ) ^2})^{1/2}}\cos \omega \sin u , $ $ \end{document } ( 9)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \delta _ { \rm{e } } } = \gamma \sin u , $ $ \end{document } ( 10)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \delta _ { \rm{s } } } = - 2r\ln \left\ { { 1 - e\cos u - s\left [ { \sin \omega ( \cos u - e ) + { { ( 1 - { e^2})}^{1/2}}\cos \omega \sin u } \right ] } \right\}. $ $ \end{document } here represents the combined time dilation and gravitational redshift due to the pulsar s orbit , and r and s are , respectively , the range and shape of the shapiro delay . together with the orbital period derivative b and the advance of periastron , they make up the post - keplerian timing parameters that can be fit for various pulsar binaries . a fuller description of the timing model also includes the aberration parameters r and , but these parameters are not in general separately measurable . the interpretation of the measured post - keplerian timing parameters will be discussed in the context of double - neutron - star tests of gr in section 4 . equivalence principles are fundamental to gravitational theory ; for full descriptions , see , e.g. , or . newton formulated what may be considered the earliest such principle , now called the weak equivalence principle it states that in an external gravitational field , objects of different compositions and masses will experience the same acceleration . the einstein equivalence principle ( eep ) includes this concept as well as those of lorentz invariance ( non - existence of preferred reference frames ) and positional invariance ( non - existence of preferred locations ) for nongravitational experiments . this principle leads directly to the conclusion that non - gravitational experiments will have the same outcomes in inertial and in freely - falling reference frames . the strong equivalence principle ( sep ) adds lorentz and positional invariance for gravitational experiments , thus including experiments on objects with strong self - gravitation . as gr incorporates the sep , and other theories of gravity may violate all or parts of it , it is useful to define a formalism that allows immediate identifications of such violations . the parametrized post - newtonian ( ppn ) formalism was developed to provide a uniform description of the weak - gravitational - field limit , and to facilitate comparisons of rival theories in this limit . this formalism requires 10 parameters ( ppn , , , 1 , 2 , 3 , 1 , 2 , 3 , and 4 ) , which are fully described in the article by will in this series , and whose physical meanings are nicely summarized in table 2 of that article . ( note that ppn is not the same as the post - keplerian pulsar timing parameter ) damour and esposito - farse [ 38 , 36 ] extended this formalism to include strong - field effects for generalized tensor - multiscalar gravitational theories . this allows a better understanding of limits imposed by systems including pulsars and white dwarfs , for which the amounts of self - gravitation are very different . here , for instance , 1 becomes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat \alpha _ 1 } = { \alpha _ 1 } + { \alpha ' _ 1}({c_1 } + { c_2 } ) + \ldots , $ $ \end{document } , where ci describes the compactness of mass mi . the compactness can be written ( 11)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { c_i } = - 2\frac{{\partial \ln { m_i}}}{{\partial \ln g } } \simeq - { \left ( { \frac{{2{e^{{\rm{grav}}}}}}{{m{c^2 } } } } \right)_i } , $ $ \end{document } where g is newton s constant and eigrav is the gravitational self - energy of mass mi , about -0.2 for a neutron star ( ns ) and -10 for a white dwarf ( wd ) . pulsar timing has the ability to set limits on 1 , which tests for the existence of preferred - frame effects ( violations of lorentz invariance ) ; 3 , which , in addition to testing for preferred - frame effects , also implies non - conservation of momentum if non - zero ; and 2 , which is also a non - conservative parameter . pulsars can also be used to set limits on other sep - violation effects that constrain combinations of the ppn parameters : the nordtvedt ( gravitational stark ) effect , dipolar gravitational radiation , and variation of newton s constant . the current pulsar timing limits on each of these will be discussed in the next sections . table 1 summarizes the ppn and other testable parameters , giving the best pulsar and solar - system limits . table 1ppn and other testable parameters , with the best solar - system and binary pulsar tests . physical meanings and most of the solar - system references are taken from the compilations by will . references : ppn , solar system : ; , solar system : ; , solar system : ; 1 , solar system : , pulsar : ; 2 , solar system : [ 105 , 152 ] ; 3 , solar system : , pulsar : ; 2 , pulsar : ; 3 , solar system : [ 15 , 152 ] ; , net , solar system : , pulsar : ; \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left ( { { \alpha _ { { c_1 } } } - { \alpha _ 0 } } \right)^2}$$\end{document } , pulsar : ; / g , solar system : [ 45 , 115 , 59 ] , pulsar : .parameterphysical meaningsolar - system testlimitpulsar testlimit ppn space curvature produced by unit rest massvlbi , light deflection ; measures |ppn1|310 non - linearity in superposition law for gravityperihelion shift of mercury ; measures |1|310 preferred - location effectssolar alignment with ecliptic410 1 preferred - frame effectslunar laser ranging10ensemble of binary pulsars1.410 2 preferred - frame effectssolar alignment with ecliptic410 3 preferred - frame effects and nonconservation of momentumperihelion shift of earth and mercury210ensemble of binary pulsars1.510 1 non - conservation of momentumcombined ppn limits210 2 non - conservation of momentumlimit on p for psr b1913 + 16410 3 non - conservation of momentumlunar acceleration10 4 non - conservation of momentumnot independent , netgravitational stark effectlunar laser ranging10ensemble of binary pulsars910 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left ( { { \alpha _ { { c_1 } } } - { \alpha _ 0 } } \right)^2}$$\end{document } pulsar coupling to scalar fielddipolar gravitational radiation for psr b0655 + 642.710 / g variation of newton s constantlaser ranging to the moon and mars610 yrchanges in chandrasekhar mass4.810 yr ppn and other testable parameters , with the best solar - system and binary pulsar tests . physical meanings and most of the solar - system references references : ppn , solar system : ; , solar system : ; , solar system : ; 1 , solar system : , pulsar : ; 2 , solar system : [ 105 , 152 ] ; 3 , solar system : , pulsar : ; 2 , pulsar : ; 3 , solar system : [ 15 , 152 ] ; , net , solar system : , pulsar : ; \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left ( { { \alpha _ { { c_1 } } } - { \alpha _ 0 } } \right)^2}$$\end{document } , pulsar : ; / g , solar system : [ 45 , 115 , 59 ] , pulsar : . the possibility of direct tests of the sep through lunar laser ranging ( llr ) experiments was first pointed out by nordtvedt . as the masses of earth and the moon contain different fractional contributions from self - gravitation , a violation of the sep would cause them to fall differently in the sun s gravitational field . this would result in a polarization of the orbit in the direction of the sun . llr tests have set a limit of ||<0.001 ( see , e.g. , [ 45 , 147 ] ) , where is a combination of ppn parameters : ( 12)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \eta = 4\beta - \gamma - 3 - \frac{{10}}{3}\xi - { \alpha _ 1 } + \frac{2}{3}{\alpha _ 2 } - \frac{2}{3}{\zeta _ 1 } - \frac{1}{3}{\zeta _ 2}. $ $ \end{document } the strong - field formalism instead uses the parameter i , which for object i may be written as ( 13)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{array}{*{20}{l } } { { { \left ( { \frac{{{m_{{\rm{grav}}}}}}{{{m_{{\rm{inertial } } } } } } } \right)}_i } = 1 + { \delta _ { \rm{i}}}}\\ { \ \ \ \ \ \ \ \ \ \ \ \ \ \ = 1 + \eta { { \left ( { \frac{{{e^{{\rm{grav}}}}}}{{m{c^2 } } } } \right)}_i } + \eta ' \left ( { \frac{{{e^{{\rm{grav}}}}}}{{m{c^2 } } } } \right)_i^2 + \ldots . } \end{array } $ $ \end{document } pulsar - white - dwarf systems then constrain net=pulsarcompanion . if the sep is violated , the equations of motion for such a system will contain an extra acceleration netg , where g is the gravitational field of the galaxy . as the pulsar and the white dwarf fall differently in this field , this netg term will influence the evolution of the orbit of the system . for low - eccentricity orbits , by far the largest effect will be a long - term forcing of the eccentricity toward alignment with the projection of g onto the orbital plane of the system . thus , the time evolution of the eccentricity vector will not only depend on the usual gr - predicted relativistic advance of periastron ( ) , but will also include a constant term . damour and schfer write the time - dependent eccentricity vector as ( 14)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \rm{\mathbf{e}(}}t{\rm { ) = } } { { \rm{\mathbf{e}}}_{\rm{f } } } + { { \rm{\mathbf{e}}}_{\rm{r}}}(t ) , $ $ \end{document } where er(t ) is the -induced rotating eccentricity vector , and ef is the forced component . in terms of net , the magnitude of ef may be written as [ 41 , 145 ] ( 15)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left| { { { \rm{\mathbf{e}}}_{\rm{f } } } } \right| = \frac{3}{2}\frac{{{\delta _ { { \rm{net}}}}{{\rm{\mathbf{g } } } _ \bot } } } { { \dot \omega a(2\pi /{p_{\rm{b } } } ) } } , $ $ \end{document } where g is the projection of the gravitational field onto the orbital plane , and a = x / sin i is the semi - major axis of the orbit . for small - eccentricity systems , this reduces to ( 16)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left| { { { \bf{e}}_{\rm{f } } } } \right| = \frac{1}{2}\frac{{{\delta _ { { \rm{net}}}}{{\bf{g } } _ \bot } { c^2}}}{{fgm{{(2\pi /{p_{\rm{b}}})}^2 } } } , $ $ \end{document } where m is the total mass of the system , and , in gr , f=1 and g is newton s constant . clearly , the primary criterion for selecting pulsars to test the sep is for the orbital system to have a large value of pb2/e , greater than or equal to 10 days . however , as pointed out by damour and schfer and wex , two age - related restrictions are also needed . first of all , the pulsar must be sufficiently old that the -induced rotation of e has completed many turns and er(t ) can be assumed to be randomly oriented . this requires that the characteristic age c be 2/ , and thus young pulsars can not be used . secondly , itself must be larger than the rate of galactic rotation , so that the projection of g onto the orbit can be assumed to be constant . according to wex , this holds true for pulsars with orbital periods of less than about 1000 days . converting equation ( 16 ) to a limit on net requires some statistical arguments to deal with the unknowns in the problem . first is the actual component of the observed eccentricity vector ( or upper limit ) along a given direction . damour and schfer assume the worst case of possible cancellation between the two components of e , namely that |ef||er| . with an angle between g and er ( see figure 4 ) , they write |ef|e/(2sin(/2 ) ) . wex [ 145 , 146 ] corrects this slightly and uses the inequality ( 17)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{array}{*{20}{c } } { \left| { { { \rm{\mathbf{e}}}_{\rm{f } } } } \right| \le e{\xi _ 1}\left ( \theta \right),}&\ \ \ \ \ { { \xi _ 1}\left ( \theta \right ) = \left\ { { \begin{array}{*{20}{l } } { 1/\sin \theta } & { { \rm{for\ } } \theta \in \left [ { 0,\pi /2 } \right),}\\ 1&{{\rm{for\ } } \theta \in \left [ { \pi /2,3\pi /2 } \right],}\\ { - 1/\sin \theta } & { { \rm{for\ } } \theta \in \left ( { 3\pi /2,2\pi } \right ) , } \end{array } } \right . } , is assumed to have a uniform probability distribution between 0 and 2. figure 4polarization of a nearly circular binary orbit under the influence of a forcing vector g , showing the relation between the forced eccentricity ef , the eccentricity evolving under the general - relativistic advance of periastron er(t ) , and the angle . ( after . ) polarization of a nearly circular binary orbit under the influence of a forcing vector g , showing the relation between the forced eccentricity ef , the eccentricity evolving under the general - relativistic advance of periastron er(t ) , and the angle . ( after . ) next comes the task of estimating the projection of g onto the orbital plane . the projection can be written as ( 18)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left| { { \mathbf{g } _ \bot } } \right| = \left| \mathbf{g } \right|{\left [ { 1 - { { \left ( { \cos i\cos \lambda + \sin i\sin \lambda \sin \omega } \right)}^2 } } \right]^{1/2 } } , $ $ \end{document } where i is the inclination angle of the orbital plane relative to the line of sight , is the line of nodes , and is the angle between the line of sight to the pulsar and g . the values of and |g| can be determined from models of the galactic potential ( see , e.g. , [ 83 , 1 ] ) . the inclination angle i can be estimated if even crude estimates of the neutron star and companion masses are available , from statistics of ns masses ( see , e.g. , ) and/or a relation between the size of the orbit and the wd companion mass ( see , e.g. , ) . however , the angle is also usually unknown and also must be assumed to be uniformly distributed between 0 and 2. damour and schfer use the psr b1953 + 29 system and integrate over the angles and to determine a 90% confidence upper limit of net<1.110 . wex uses an ensemble of pulsars , calculating for each system the probability ( fractional area in - space ) that net is less than a given value , and then deriving a cumulative probability for each value of net . in this way he derives net<510 at 95% confidence however , this method may be vulnerable to selection effects ; perhaps the observed systems are not representative of the true population . given a value of net , an upper limit on || is obtained from equation ( 17 ) . a monte carlo simulation of the expected pulsar population ( assuming a range of masses based on evolutionary models and a random orientation of ( ) then yields a certain fraction of the population that agree with this limit on || . this is slightly weaker than wex s previous limit but derived in a more rigorous manner . prospects for improving the limits come from the discovery of new suitable pulsars , and from better limits on eccentricity from long - term timing of the current set of pulsars . in principle , measurement of the full orbital orientation ( i.e. , and i ) for certain systems could reduce the dependence on statistical arguments thus , even though the required angles have in fact been measured for the millisecond pulsar j0437 - 4715 , its comparatively large observed eccentricity of 210 and short orbital period mean it will not significantly affect the current limits . a non - zero 1 implies that the velocity w of a binary pulsar system ( relative to a universal background reference frame given by the cosmic microwave background , or cmb ) will affect its orbital evolution . in a manner similar to the effects of a non - zero net , the time evolution of the eccentricity will depend on both and a term that tries to force the semi - major axis of the orbit to align with the projection of the system velocity onto the orbital plane . the analysis proceeds in a similar fashion to that for net , except that the magnitude of ef is now written as [ 34 , 18 ] ( 19)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left| { { { \rm{\mathbf{e}}}_{\rm{f } } } } \right| = \frac{1}{{12}}{\hat \alpha _ 1}\left| { \frac{{{m_1 } - { m_2}}}{{{m_1 } + { m_2 } } } } \right|\frac{{\left| { { w _ \bot } } \right|}}{{{{\left [ { g({m_1 } + { m_2})(2\pi /{p_{\rm{b } } } ) } \right]}^{1/3 } } } } , $ $ \end{document } where w is the projection of the system velocity onto the orbital plane . the angle , used in determining this projection in a manner similar to that of equation ( 18 ) , is now the angle between the line of sight to the pulsar and the absolute velocity of the binary system . the figure of merit for systems used to test 1 is pb1/3/e . as for the net test , the systems must be old , so that c2/ , and must be larger than the rate of galactic rotation . examples of suitable systems are psr j2317 + 1439 [ 27 , 18 ] with a last published value of e<1.210 in 1996 , and psr j1012 + 5307 , with e<810 . this latter system is especially valuable because observations of its white - dwarf component yield a radial velocity measurement , eliminating the need to find a lower limit on an unknown quantity . this is comparable in magnitude to the weak - field results from lunar laser ranging , but incorporates strong field effects as well . tests of 3 can be derived from both binary and single pulsars , using slightly different techniques . a non - zero 3 , which implies both a violation of local lorentz invariance and non - conservation of momentum , will cause a rotating body to experience a self - acceleration aself in a direction orthogonal to both its spin s and its absolute velocity w : ( 20)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { \rm{\mathbf{a}}}_{{\rm{self } } } } = - \frac{1}{3}{\hat \alpha _ 3}\frac{{{e^{{\rm{grav}}}}}}{{(m{c^2})}}\mathbf{w } \times { \mathbf{\omega } _ { \rm{s}}}. $ $ \end{document } thus , the self - acceleration depends strongly on the compactness of the object , as discussed in section 3 above . an ensemble of single ( isolated ) pulsars can be used to set a limit on 3 in the following manner . for any given pulsar , it is likely that some fraction of the self - acceleration will be directed along the line of sight to the earth . such an acceleration will contribute to the observed period derivative via the doppler effect , by an amount ( 21)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { \dot p}_{{{\hat \alpha } _ 3 } } } = \frac{p}{c}\bf{\hat n } \cdot { \bf{a}_{{\rm{self } } } } , $ $ \end{document } where n is a unit vector in the direction from the pulsar to the earth . the analysis of will assumes random orientations of both the pulsar spin axes and velocities , and finds that , on average , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left| { { { \dot p}_{{{\hat \alpha } _ 3 } } } } \right| \simeq 5 \times { 10^ { - 5}}\left| { { { \hat \alpha } _ 3 } } \right|$$\end{document } , independent of the pulse period . the sign of the 3 contribution to , however , may be positive or negative for any individual pulsar ; thus , if there were a large contribution on average , one would expect to observe pulsars with both positive and negative total period derivatives . young pulsars in the field of the galaxy ( pulsars in globular clusters suffer from unknown accelerations from the cluster gravitational potential and do not count toward this analysis ) all show positive period derivatives , typically around 10 s / s . thus , the maximum possible contribution from 3 must also be considered to be of this size , and the limit is given by |3|<210 . bell applies this test to a set of millisecond pulsars ; these have much smaller period derivatives , on the order of 10s / s . here , it is also necessary to account for the shklovskii effect in which a similar doppler - shift addition to the period derivative results from the transverse motion of the pulsar on the sky : ( 22)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \dot p_{{\rm{pm } } } } = p{\mu ^2}\frac{d}{c } , $ $ \end{document } where is the proper motion of the pulsar and d is the distance between the earth and the pulsar . the distance is usually poorly determined , with uncertainties of typically 30% resulting from models of the dispersive free electron density in the galaxy [ 132 , 30 ] . nevertheless , once this correction ( which is always positive ) is applied to the observed period derivatives for isolated millisecond pulsars , a limit on |3| on the order of 10 results [ 16 , 19 ] . in the case of a binary - pulsar white - dwarf system , the combined accelerations affect both the velocity of the centre of mass of the system ( an effect which may not be readily observable ) and the relative motion of the two bodies . the relativemotion effects break down into a term involving the coupling of the spins to the absolute motion of the centre of mass , and a second term which couples the spins to the orbital velocities of the stars . the second term induces only a very small , unobservable correction to pb and . the first term , however , can lead to a significant test of 3 . both the compactness and the spin of the pulsar will completely dominate those of the white dwarf , making the net acceleration of the two bodies effectively ( 23)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { \rm{\mathbf{a}}}_{{\rm{self } } } } = \frac{1}{6}{\hat \alpha _ 3}{c_{\rm{p}}}{\rm{\mathbf{w } } } \times { \mathbf{\omega } _ { { \rm{sp } } } } , $ $ \end{document } where cp and sp denote the compactness and spin angular frequency of the pulsar , respectively , and w is the velocity of the system . for evolutionary reasons ( see , e.g. , ) , the spin axis of the pulsar may be assumed to be aligned with the orbital angular momentum of the system , hence the net effect of the acceleration will be to induce a polarization of the eccentricity vector within the orbital plane . the forced eccentricity term may be written as ( 24)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left| { { { \rm{\mathbf{e}}}_{\rm{f } } } } \right| = { \hat \alpha _ 3}\frac{{{c_{\rm{p}}}\left| \mathbf{w } \right|}}{{24\pi } } \frac{{p_{\rm{b}}^2}}{p}\frac{{{c^2}}}{{g({m_1 } + { m_2})}}\sin \beta , $ $ \end{document } where is the ( unknown ) angle between w and sp , and p is , as usual , the spin period of the pulsar : p=2/sp . the figure of merit for systems used to test 3 is pb2/(ep ) . the additional requirements of c2/ and being larger than the rate of galactic rotation also hold . the 95% confidence limit derived by wex for an ensemble of binary pulsars is 3<1.510 , much more stringent than for the single - pulsar case . it will contribute , along with 3 , to an acceleration of the centre of mass of a binary system [ 149 , 152 ] ( 25)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { \rm{\mathbf{a}}}_{{\rm{cm } } } } = \left ( { { \alpha _ 3 } + { \zeta _ 2 } } \right)\frac{{\pi { m_1}{m_2}\left ( { { m_1 } - { m_2 } } \right)}}{{{p_{\rm{b}}}{{\left [ { \left ( { { m_1 } + { m_2 } } \right)a\left ( { 1 - { e^2 } } \right ) } \right]}^{3/2}}}}e\ { { \rm{\mathbf{n}}}_{\rm{p } } } , $ $ \end{document } where np is a unit vector from the centre of mass to the periastron of m1 . this acceleration produces the same type of doppler - effect contribution to a binary pulsar s as described in section 3.2.2 . in a small - eccentricity system , this contribution would not be separable from the intrinsic to the pulsar . however , in a highly eccentric binary such as psr b1913 + 16 , the longitude of periastron advances significantly for psr b1913 + 16 , it has advanced nearly 120 since the pulsar s discovery . in this case , the projection of acm along the line of sight to the earth will change considerably over the long term , producing an effective second derivative of the pulse period . this p is given by [ 149 , 152 ] ( 26)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \ddot p = \frac{p}{2}\left ( { { \alpha _ 3 } + { \zeta _ 2 } } \right){m_2}\sin i{\left ( { \frac{{2\pi } } { { { p_{\rm{b } } } } } } \right)^2}\frac{{x\left ( { 1 - x } \right)}}{{{{\left ( { 1 + x } \right)}^2}}}\frac{{e\dot \omega \cos \omega } } { { { { \left ( { 1 - { e^2 } } \right)}^{3/2 } } } } , $ $ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x = { m_1}/{m_2}$$\end{document } is the mass ratio of the two stars and an average value of cos is chosen . as of 1992 , the 95% confidence upper limit on p was 410 s [ 133 , 149 ] . as 3 is orders of magnitude smaller than this ( see section 3.2.2 ) , this can be interpreted as a limit on 2 alone . although psr b1913 + 16 is of course still observed , the infrequent campaign nature of the observations makes it difficult to set a much better limit on p ( j. taylor , private communication , as cited in ) . the other well - studied double - neutron - star binary , psr b1534 + 12 , yields a weaker test due to its orbital parameters and very similar component masses . a complication for this test is that an observed p could also be interpreted as timing noise ( sometimes seen in recycled pulsars ) or else a manifestation of profile changes due to geodetic precession [ 79 , 75 ] . general relativity predicts gravitational radiation from the time - varying mass quadrupole of a binary pulsar system . the spectacular confirmation of this prediction will be discussed in section 4 below . gr does not , however , predict dipolar gravitational radiation , though many theories that violate the sep do . in these theories , dipolar gravitational radiation results from the difference in gravitational binding energy of the two components of a binary . for this reason , neutron - star white - dwarf binaries are the ideal laboratories to test the strength of such dipolar emission . the expected rate of change of the period of a circular orbit due to dipolar emission can be written as [ 152 , 35 ] ( 27)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \dot p_{{\rm{b \ dipole } } } } = - \frac{{4{\pi ^2}{g_*}}}{{{c^3}{p_{\rm{b}}}}}\frac{{{m_1}{m_2}}}{{{m_1 } + { m_2}}}{\left ( { { \alpha _ { { c_1 } } } - { \alpha _ { { c_2 } } } } \right)^2 } , $ $ \end{document } where g*=g in gr , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\alpha _ { { c_i}}}$$\end{document } is the coupling strength of body ( similar expressions can be derived when casting b dipole in terms of the parameters of specific tensor - scalar theories , such as brans - dicke theory . equation ( 27 ) , however , tests a more general class of theories . ) of course , the best test systems here are pulsar white - dwarf binaries with short orbital periods , such as psr b0655 + 64 and psr j1012 + 5307 , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\alpha _ { { c_1 } } } \gg { \alpha _ { { c_2}}}$$\end{document } so that a strong limit can be set on the coupling of the pulsar itself . for psr b0655 + 64 , damour and esposito - farse used the observed limit of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \dot p_{\rm{b } } } = \left ( { 1 \pm 4 } \right ) \times { 10^ { - 13 } } $ $ \end{document } to derive \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left ( { { \alpha _ { { c_1 } } } - { \alpha _ 0 } } \right)^2 } < 3 \times { 10^ { - 4}}\left ( { 1 - \sigma } \right)$$\end{document } , where 0 is a reference value of the coupling at infinity . more recently , arzoumanian has set a somewhat tighter 2- upper limit of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left| { { { \dot p}_{\rm{b}}}/{p_{\rm{b } } } } \right| < 1 \times { 10^ { - 10}}\ { \rm{y}}{{\rm{r}}^ { - 1}}$$\end{document } , or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left| { { { \dot p}_{\rm{b } } } } \right| < 2.7 \times { 10^ { - 13}}$$\end{document } , which yields \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left ( { { \alpha _ { { c_1 } } } - { \alpha _ 0 } } \right)^2 } < 2.7 \times { 10^ { - 4}}$$\end{document}. for psr j1012 + 5307 , a shklovskii correction ( see and section 3.2.2 ) for the transverse motion of the system and a correction for the ( small ) predicted amount of quadrupolar radiation must first be subtracted from the observed upper limit to arrive at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\dot p_{\rm{b } } } = \left ( { - 0.6 \pm 1.1 } \right ) \times { 10^ { - 13}}$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left ( { { \alpha _ { { c_1 } } } - { \alpha _ 0 } } \right)^2 } < 4 \times { 10^ { - 4}}$$\end{document } at 95% confidence . it should be noted that both these limits depend on estimates of the masses of the two stars and do not address the ( unknown ) equation of state of the neutron stars . limits may also be derived from double - neutron - star systems ( see , e.g. , [ 148 , 151 ] ) , although here the difference in the coupling constants is small and so the expected amount of dipolar radiation is also small compared to the quadrupole emission . however , certain alternative gravitational theories in which the quadrupolar radiation predicts a positive orbital period derivative independently of the strength of the dipolar term ( see , e.g. , [ 117 , 99 , 85 ] ) are ruled out by the observed decreasing orbital period in these systems . other pulsar white - dwarf systems with short orbital periods are mostly found in globular clusters , where the cluster potential will also contribute to the observed b , or in interacting systems , where tidal effects or magnetic braking may affect the orbital evolution ( see , e.g. , [ 4 , 50 , 100 ] ) . however , one system that offers interesting prospects is the recently discovered psr j1141 - 6545 , which is a young pulsar with white - dwarf companion in a 4.75-hour orbit . in this case , though , the pulsar was formed after the white dwarf , instead of being recycled by the white - dwarf progenitor , and so the orbit is still highly eccentric . this system is therefore expected both to emit sizable amounts of quadrupolar radiation b could be measurable as soon as 2004 and to be a good test candidate for dipolar emission . theories that violate the sep by allowing for preferred locations ( in time as well as space ) may permit newton s constant g to vary . in general , variations in g are expected to occur on the timescale of the age of the universe , such that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot g / g \sim { h_0 } \sim 0.7 \times { 10^ { - 10}}\ { \rm{y}}{{\rm{r}}^ { - 1}}$$\end{document } , where h0 is the hubble constant . three different pulsar - derived tests can be applied to these predictions , as a sep - violating time - variable g would be expected to alter the properties of neutron stars and white dwarfs , and to affect binary orbits . by affecting the gravitational binding of neutron stars , a non - zero would reasonably be expected to alter the moment of inertia of the star and hence change its spin on the same timescale . goldman writes ( 28)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \left ( { \frac{{\dot p}}{p } } \right)_{\dot g } } = { \left ( { \frac{{\partial \ln i}}{{\partial \ln g } } } \right)_n}\frac{{\dot g}}{g } , $ $ \end{document } where i is the moment of inertia of the neutron star , about 10 g cm , and n is the ( conserved ) total number of baryons in the star . by assuming that this represents the only contribution to the observed of psr b0655 + 64 , in a manner reminiscent of the test of 3 described above , goldman then derives an upper limit of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left| { \dot g / g } \right| \le \left ( { 2.2 - 5.5 } \right ) \times { 10^ { - 11}}\;{\rm{y}}{{\rm{r}}^ { - 1}}$$\end{document } , depending on the stiffness of the neutron star equation of state . arzoumanian applies similar reasoning to psr j2019 + 2425 , which has a characteristic age of 27 gyr once the shklovskii correction is applied . again , depending on the equation of state , the upper limits from this pulsar are \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left| { \dot g / g } \right| \le \left ( { 1.4 - 3.2 } \right ) \times { 10^ { - 11}}\;{\rm{y}}{{\rm{r}}^ { - 1}}$$\end{document } . these values are similar to those obtained by solar - system experiments such as laser ranging to the viking lander on mars ( see , e.g. , [ 115 , 59 ] ) . shklovskii and galactic - acceleration corrections are taken into account , have similarly large characteristic ages ( see , e.g. , [ 28 , 137 ] ) . the effects on the orbital period of a binary system of a varying g were first considered by damour , gibbons , and taylor , who expected ( 29)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \left ( { \frac{{{{\dot p}_{\rm{b}}}}}{{{p_{\rm{b } } } } } } \right)_{\dot g } } = - 2\frac{{\dot g}}{g}. $ $ \end{document } applying this equation to the limit on the deviation from gr of the b for psr 1913 + 16 , they found a value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot g / g = ( 1.0 \pm 2.3 ) \times { 10^ { - 11}}\;{\rm{y}}{{\rm{r}}^ { - 1}}$$\end{document}. nordtvedt took into account the effects of on neutron - star structure , realizing that the total mass and angular momentum of the binary system would also change . the corrected expression for b incorporates the compactness parameter ci and is ( 30)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \left ( { \frac{{{{\dot p}_{\rm{b}}}}}{{{p_{\rm{b } } } } } } \right)_{\dot g } } = - \left [ { 2 - \left ( { \frac{{{m_1}{c_1 } + { m_2}{c_2}}}{{{m_1 } + { m_2 } } } } \right ) - \frac{3}{2}\left ( { \frac{{{m_1}{c_2 } + { m_2}{c_1}}}{{{m_1 } + { m_2 } } } } \right ) } \right]\frac{{\dot g}}{g}. $ $ \end{document } ( note that there is a difference of a factor of -2 in nordtvedt s definition of ci versus the damour definition used throughout this article . ) nordtvedt s corrected limit for psr b1913 + 16 is therefore slightly weaker . a better limit actually comes from the neutron - star white - dwarf system psr b1855 + 09 , with a measured limit on b of ( 0:61.2)10 . using equation ( 29 ) , this leads to a bound of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot g / g = ( - 9 \pm 18 ) \times { 10^ { - 12}}\;{\rm{y}}{{\rm{r}}^ { - 1}}$$\end{document } , which arzoumanian corrects using equation ( 30 ) and an estimate of ns compactness to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot g / g = ( - 1.3 \pm 2.7 ) \times { 10^ { - 11}}\;{\rm{y}}{{\rm{r}}^ { - 1}}$$\end{document}. prospects for improvement come directly from improvements to the limit on b . even though psr j1012 + 5307 has a tighter limit on b , its shorter orbital period means that the limit it sets is a factor of 2 weaker than obtained with psr b1855 + 09 . the chandrasekhar mass , mch , is the maximum mass possible for a white dwarf supported against gravitational collapse by electron degeneracy pressure . its value about 1.4m comes directly from newton s constant : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{{\rm{ch } } } } \sim { \left ( { \hbar c / g } \right)^{3/2}}/m_{\rm{n}}^2$$\end{document } , where is planck s constant and mn is the neutron mass . all measured and constrained pulsar masses are consistent with a narrow distribution centred very close to mch : 1.350.04m . thus , it is reasonable to assume that mch sets the typical neutron star mass , and to check for any changes in the average neutron star mass over the lifetime of the universe . thorsett compiles a list of measured and average masses from 5 double - neutron - star binaries with ages ranging from 0.1 gyr to 12 or 13 gyr in the case of the globular - cluster binary b2127 + 11c . using a bayesian analysis , he finds a limit of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot g / g = ( - 0.6 \pm 4.2 ) \times { 10^ { - 12}}\;{\rm{y}}{{\rm{r}}^ { - 1}}$$\end{document } at the 95% confidence level , the strongest limit on record . the solid lines show predicted changes in the average neutron star mass corresponding to hypothetical variations in g , where -12=10 implies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot g / g = 10 \times { 10^ { - 12}}\;{\rm{y}}{{\rm{r}}^ { - 1}}$$\end{document}. ( from , used by permission . ) measured neutron star masses as a function of age . the solid lines show predicted changes in the average neutron star mass corresponding to hypothetical variations in g , where -12=10 implies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot g / g = 10 \times { 10^ { - 12}}\;{\rm{y}}{{\rm{r}}^ { - 1}}$$\end{document}. ( from , used by permission . ) while some cancellation of observed mass changes might be expected from the changes in neutron - star binding energy ( cf . section 3.4.2 above ) , these will be smaller than the mch changes by a factor of order the compactness and can be neglected . also , the claimed variations of the fine structure constant of order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \alpha /\alpha \simeq - 0.72 \pm 0.18 \times { 10^ { - 5}}$$\end{document } over the redshift range 0.5<z<3.5 could introduce a maximum derivative of 1/(c)d(c)/dt of about 510 yr and hence can not influencee the chandrasekhar mass at the same level as the hypothesized changes in g. one of the five systems used by thorsett has since been shown to have a white - dwarf companion , but as this is one of the youngest systems , this will not change the results appreciably . the recently discovered psr j1811 - 1736 , a double - neutron - star binary , has a characteristic age of only c1 gyr and , therefore , will also not significantly strengthen the limit . ongoing searches for pulsars in globular clusters stand the best chance of discovering old double - neutron - star binaries for which the component masses can eventually be measured . the best - known uses of pulsars for testing the predictions of gravitational theories are those in which the predicted strong - field effects are compared directly against observations . as essentially point - like objects in strong gravitational fields , neutron stars in binary systems provide extraordinarily clean tests of these predictions . this section will cover the relation between the post - keplerian timing parameters and strong - field effects , and then discuss the three binary systems that yield complementary high - precision tests . in any given theory of gravity , the post - keplerian ( pk ) parameters can be written as functions of the pulsar and companion star masses and the keplerian parameters . as the two stellar masses are the only unknowns in the description of the orbit , it follows that measurement of any two pk parameters will yield the two masses , and that measurement of three or more pk parameters will over - determine the problem and allow for self - consistency checks . it is this test for internal consistency among the pk parameters that forms the basis of the classic tests of strong - field gravity . it should be noted that the basic keplerian orbital parameters are well - measured and can effectively be treated as constants here . in general relativity , the equations describing the pk parameters in terms of the stellar masses are ( see [ 33 , 133 , 43 ] ) : ( 31)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \dot \omega = 3{\left ( { \frac{{{p_{\rm{b}}}}}{{2\pi } } } \right)^ { - 5/3}}{\left ( { { t _ \odot } m } \right)^{2/3}}{\left ( { 1 - { e^2 } } \right)^ { - 1 } } , $ $ \end{document } ( 32)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \gamma = e{\left ( { \frac{{{p_{\rm{b}}}}}{{2\pi } } } \right)^{1/3}}t _ \odot ^{2/3}{m^ { - 4/3}}{m_2}\left ( { { m_1 } + 2{m_2 } } \right ) , $ $ \end{document } ( 33)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { \dot p}_{\rm{b } } } = - \frac{{192\pi } } { 5}{\left ( { \frac{{{p_{\rm{b}}}}}{{2\pi } } } \right)^ { - 5/3}}\left ( { 1 + \frac{{73}}{{24}}{e^2 } + \frac{{37}}{{96}}{e^4 } } \right){\left ( { 1 - { e^2 } } \right)^ { - 7/2}}t _ \odot ^{5/3}{m_1}{m_2}{m^ { - 1/3 } } , $ $ \end{document } ( 34)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r = { t _ \odot } { m_2 } , $ $ \end{document } ( 35)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s = x{\left ( { \frac{{{p_{\rm{b}}}}}{{2\pi } } } \right)^ { - 2/3}}t _ \odot ^ { - 1/3}{m^{2/3}}m_2^ { - 1}. $ $ \end{document } where ssin i , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m = { m_1 } + { m_2}$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t _ \odot } \equiv g{m _ \odot } /{c^3 } = 4.925490947\;\mu { \rm{s}}$$\end{document}. other theories of gravity , such as those with one or more scalar parameters in addition to a tensor component , will have somewhat different mass dependencies for these parameters . the prototypical double - neutron - star binary , psr b1913 + 16 , was discovered at the arecibo observatory in 1974 . over nearly 30 years of timing , its system parameters have shown a remarkable agreement with the predictions of gr , and in 1993 hulse and taylor received the nobel prize in physics for its discovery [ 61 , 131 ] . in the highly eccentric 7.75-hour orbit , the two neutron stars are separated by only 3.3 light - seconds and have velocities up to 400 km / s . this provides an ideal laboratory for investigating strong - field gravity . for psr b1913 + 16 , three pk parameters are well measured : the combined gravitational red - shift and time dilation parameter the advance of periastron , and the derivative of the orbital period , b . the orbital parameters for this pulsar , measured in the theory - independent dd system , are listed in table 2 [ 133 , 144 ] . table 2 orbital parameters for psr b1913 + 16 in the dd framework , taken from [ 144 ] . parametervalueorbital period pb ( d)0.322997462727(5)projected semi - major axis x ( s)2.341774(1)eccentricity e0.6171338(4)longitude of periastron ( deg)226.57518(4)epoch of periastron t0 ( mjd)46443.99588317(3)advance of periastron ( deg yr)4.226607(7)gravitational redshift ( ms)4.294(1)orbital period derivative ( b ) ( 10)-2.4211(14 ) orbital parameters for psr b1913 + 16 in the dd framework , taken from [ 144 ] . a second useful timing formalism is ddgr [ 33 , 43 ] , which assumes gr to be the true theory of gravity and fits for the total and companion masses in the system , using these quantities to calculate theoretical thus , one can make a direct comparison between the measured dd pk parameters and the values predicted by ddgr using the same data set ; the parameters for psr b1913 + 16 agree with their predicted values to better than 0.5% . the classic demonstration of this agreement is shown in figure 6 , in which the observed accumulated shift of periastron is compared to the predicted amount . figure 6 the parabola indicates the predicted accumulated shift in the time of periastron for psr b1913 + 16 , caused by the decay of the orbit . the parabola indicates the predicted accumulated shift in the time of periastron for psr b1913 + 16 , caused by the decay of the orbit . ( from [ 144 ] , courtesy joel weisberg . ) in order to check the self - consistency of the overdetermined set of equations relating the pk parameters to the neutron star masses , it is helpful to plot the allowed m1m2 curves for each parameter and to verify that they intersect at a common point . figure 7 displays the and curves for psr b1913 + 16 ; it is clear that the curves do intersect , at the point derived from the ddgr mass predictions . figure 7mass - mass diagram for the psr b1913 + 16 system , using the and parameters listed in table 2 . the lines intersect at the point given by the masses derived under the ddgr formalism . mass - mass diagram for the psr b1913 + 16 system , using the and parameters listed in table 2 . the lines intersect at the point given by the masses derived under the ddgr formalism . clearly , any theory of gravity that does not pass such a self - consistency test can be ruled out . however , it is possible to construct alternate theories of gravity that , while producing very different curves in the m1m2 plane , do pass the psr b1913 + 16 test and possibly weak - field tests as well . such theories are best dealt with by combining data from multiple pulsars as well as solar - system experiments ( see section 4.4 ) . the first is that the unknown radial velocity of the binary system relative to the ssb will necessarily induce a doppler shift in the orbital and neutron - star spin periods . this will change the observed stellar masses by a small fraction but will cancel out of the calculations of the pk parameters . the second is that the measured value of the orbital period derivative b is contaminated by several external contributions . damour and taylor consider the full range of possible contributions to b and calculate values for the two most important : the acceleration of the pulsar binary centre - of - mass relative to the ssb in the galactic potential , and the shklovskii v / r effect due to the transverse proper motion of the pulsar ( cf . section 3.2.2 ) . both of these contributions have been subtracted from the measured value of b before it is compared with the gr prediction . it is our current imperfect knowledge of the galactic potential and the resulting models of galactic acceleration ( see , e.g. , [ 83 , 1 ] ) which now limits the precision of the test of gr resulting from this system . a second double - neutron - star binary , psr b1534 + 12 , was discovered during a drift - scan survey at arecibo observatory in 1990 . this system is quite similar to psr b1913 + 16 : it also has a short ( 10.1-hour ) orbit , though it is slightly wider and less eccentric . psr b1534 + 12 does possess some notable advantages relative to its more famous cousin : it is closer to the earth and therefore brighter ; its pulse period is shorter and its profile narrower , permitting better timing precision ; and , most importantly , it is inclined nearly edge - on to the line of sight from the earth , allowing the measurement of shapiro delay as well as the 3 pk parameters measurable for psr b1913 + 16 . table 3 orbital parameters for psr b1534 + 12 in the dd framework , taken from [ 125 ] . parametervalueorbital period pb ( d)0.420737299122(10)projected semi - major axis x ( s)3.729464(2)eccentricity e0.2736775(3)longitude of periastron ( deg)274.57679(5)epoch of periastron t0 ( mjd)50260.92493075(4)advance of periastron ( deg yr)1.755789(9)gravitational redshift ( ms)2.070(2)orbital period derivative ( b ) ( 10)-0.137(3)shape of shapiro delay s0.975(7)range of shapiro delay r ( s)6.7(1.0 ) orbital parameters for psr b1534 + 12 in the dd framework , taken from [ 125 ] . as for psr b1913 + 16 , a graphical version of the internal consistency test is a helpful way to understand the agreement of the measured pk parameters with the predictions of gr . it is obvious that the allowed - mass region derived from the observed value of b does not in fact intersect those from the other pk parameters . this is a consequence of the proximity of the binary to the earth , which makes the shklovskii contribution to the observed b much larger than for psr b1913 + 16 . the magnitude of this contribution depends directly on the poorly known distance to the pulsar . at present , the best independent measurement of the distance comes from the pulsar s dispersion measure and a model of the free electron content of the galaxy , which together yield a value of 0.70.2 kpc . if gr is the correct theory of gravity , then the correction derived from this distance is inadequate , and the true distance can be found by inverting the problem [ 17 , 121 ] . ne2001 galactic model incorporates the gr - derived distance to this pulsar and hence can not be used in this case . ) it is possible that , in the long term , a timing or interferometric parallax may be found for this pulsar ; this would alleviate the b discrepancy . the gr - derived distance is in itself interesting , as it has led to revisions of the predicted merger rate of double - neutron - star systems visible to gravitational - wave detectors such as ligo ( see , e.g. , [ 121 , 7 , 71 ] ) although recent calculations of merger rates determine the most likely merger rates for particular population models and hence are less vulnerable to distance uncertainties in any one system . figure 8mass - mass diagram for the psr b1534 + 12 system . labeled curves illustrate 68% confidence ranges of the dd parameters listed in table 3 . the filled circle indicates the component masses according to the ddgr solution . the kinematic correction for assumed distance \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d = { \rm{0}}{\rm{.7 } } \pm { \rm{0}}{\rm{.2}}\;{\rm{kpc}}$$\end{document } has been subtracted from the observed value of b ; the uncertainty on this kinematic correction dominates the uncertainty of this curve . a slightly larger distance removes the small apparent discrepancy between the observed and predicted values of this parameter . ( after . ) mass - mass diagram for the psr b1534 + 12 system . labeled curves illustrate 68% confidence ranges of the dd parameters listed in table 3 . the filled circle indicates the component masses according to the ddgr solution . the kinematic correction for assumed distance \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d = { \rm{0}}{\rm{.7 } } \pm { \rm{0}}{\rm{.2}}\;{\rm{kpc}}$$\end{document } has been subtracted from the observed value of b ; the uncertainty on this kinematic correction dominates the uncertainty of this curve . a slightly larger distance removes the small apparent discrepancy between the observed and predicted values of this parameter . ( after . ) despite the problematic correction to b , the other pk parameters for psr b1534 + 12 are in excellent agreement with each other and with the values predicted from the ddgr - derived masses . an important point is that the three parameters , , and s ( shape of shapiro delay ) together yield a test of gr to better than 1% , and that this particular test incorporates only quasi - static strong - field effects . this provides a valuable complement to the mixed quasi - static and radiative test derived from psr b1913 + 16 , as it separates the two sectors of the theory . there are three other confirmed double - neutron - star binaries at the time of writing . psr b2127 + 11c [ 2 , 3 ] is in the globular cluster m15 . while its orbital period derivative has been measured , this parameter is affected by acceleration in the cluster potential , and the system has not yet proved very useful for tests of gr , though long - term observations may demonstrate otherwise . the two binaries psrs j1518 + 4904 and j1811 - 1736 have such wide orbits that , although is measured in each case , prospects for measuring further pk parameters are dim . in several circular pulsar white - dwarf binaries , one or two pk parameters have been measured typically or the shapiro delay parameters but these do not over - constrain the unknown masses . the existing system that provides the most optimistic outlook is again the pulsar white - dwarf binary psr j1141 - 6545 , for which multiple pk parameters should be measurable within a few years although one may need to consider the possibility of classical contributions to the measured from a mass quadrupole of the companion . because of their different orbital parameters and inclinations , the double - neutron - star systems psrs b1913 + 16 and b1534 + 12 provide somewhat different constraints on alternative theories of gravity . taken together with some of the limits on sep violation discussed above , and with solar - system experiments , they can be used to disallow certain regions of the parameter space of these alternate theories . this approach was pioneered by taylor et al . , who combined pk - parameter information from psrs b1913 + 16 and b1534 + 12 and the damour and schfer result on sep violation by psr b1855 + 09 to set limits on the parameters and of a class of tensor - biscalar theories discussed in ( figure 9 ) . in this class of theories , gravity is mediated by two scalar fields as well as the standard tensor , but the theories can satisfy the weak - field solar - system tests . strong - field deviations from gr would be expected for non - zero values of and , but the theories approach the limit of gr as the parameters and approach zero . figure 9portions of the tensor - biscalar - plane permitted by timing observations of psrs b1913 + 16 , b1534 + 12 , and b1855 + 09 up to 1992 . values lying above the curve labeled a are incompatible with the measured and parameters for psr b1913 + 16 . the curves labeled b and d give the allowed ranges of and for psrs b1913 + 16 and b1534 + 12 , respectively , fitting for the two neutron - star masses as well as and , using data available up to 1992 . the vertical lines labeled c represent limits on from the sep - violation test using psr b1855 + 09 . ( reprinted by permission from nature , 1992 , macmillan publishers ltd . ) portions of the tensor - biscalar - plane permitted by timing observations of psrs b1913 + 16 , b1534 + 12 , and b1855 + 09 up to 1992 . values lying above the curve labeled a are incompatible with the measured and parameters for psr b1913 + 16 . the curves labeled b and d give the allowed ranges of and for psrs b1913 + 16 and b1534 + 12 , respectively , fitting for the two neutron - star masses as well as and , using data available up to 1992 . the vertical lines labeled c represent limits on from the sep - violation test using psr b1855 + 09 . ( reprinted by permission from nature , 1992 , macmillan publishers ltd . ) a different class of theories , allowing a non - linear coupling between matter and a scalar field , was later studied by damour and esposito - farse [ 35 , 37 ] . the function coupling the scalar field to matter is given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a\left ( \phi \right ) = \exp \left ( { { \tfrac{1}{2}}{\beta _ 0}{\phi ^2 } } \right)$$\end{document } , and the theories are described by the parameters 0 and 0=00 , where 0 is the value that approaches at spatial infinity ( cf . these theories allow significant strong - field effects when 0 is negative , even if the weak - field limit is small . they are best tested by combining results from psrs b1913 + 16 , b1534 + 12 ( which contributes little to this test ) , b0655 + 64 ( limits on dipolar gravitational radiation ) , and solar - system experiments ( lunar laser ranging , shapiro delay measured by viking , and the perihelion advance of mercury ) . currently , for most neutron - star equations of state , the solar - system tests set a limit on 0 ( 02<10 ) that is a few times more stringent than those set by psrs b1913 + 16 and b0655 + 64 , although the pulsar observations do eliminate large negative values of 0 . with the limits from the pulsar observations improving only very slowly with time , it appears that solar - system tests will continue to set the strongest limits on 0 in this class of theories , unless a pulsar black - hole system is discovered . if such a system were found with a 10-m black hole and an orbital period similar to that of psr b1913 + 16 ( 8 hours ) , the limit on 0 derived from this system would be about 50 times tighter than that set by current solar - system tests , and 10 times better than is likely to be achieved by the gravity probe b experiment . figure 10the parameter space in the non - linear 0 , 0 gravitational theory , for neutron stars described by a polytrope equation of state . the regions below the various curves are allowed by various pulsar timing limits , by solar - system tests ( 1pn ) , and by projected ligo / virgo observations of ns - ns and ns - bh inspiral events . ( from ; used by permission . ) the parameter space in the non - linear 0 , 0 gravitational theory , for neutron stars described by a polytrope equation of state . the regions below the various curves are allowed by various pulsar timing limits , by solar - system tests ( 1pn ) , and by projected ligo / virgo observations of ns - ns and ns - bh inspiral events . the shaded region is allowed by all tests . the plane and limits are symmetric about 0=0 . a different and complementary test of gr has recently been permitted by the millisecond pulsar psr j0437 - 4715 . at a distance of only 140 pc , it is the closest millisecond pulsar to the earth , and is also extremely bright , allowing root - mean - square timing residuals of 35 ns with the 64-m parkes telescope , comparable to or better than the best millisecond pulsars observed with current instruments at the 300-m arecibo telescope . the proximity of this system means that the orbital motion of the earth changes the apparent inclination angle i of the pulsar orbit on the sky , an effect known as the annual - orbital parallax . this results in a periodic change of the projected semi - major axis x of the pulsar s orbit , written as ( 36)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ x\left ( t \right ) = { x_0}\left [ { 1 + \frac{{\cot i}}{d}{{\rm{\mathbf{r } } } _ \oplus } \left ( t \right ) \cdot \mathbf{\omega } ' } \right ] , $ $ \end{document } where r(t ) is the time - dependent vector from the centre of the earth to the ssb , and is a vector on the plane of the sky perpendicular to the line of nodes . a second contribution to the observed i and hence x comes from the pulsar system s transverse motion in the plane of the sky : ( 37)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { \dot x}_{{\rm{pm } } } } = - x\cot i\mu \cdot \mathbf{\omega } ' , $ $ \end{document } where is the proper motion vector . by including both these effects in the model of the pulse arrival times , both the inclination angle i and the longitude of the ascending node can be determined . as sin i is equivalent to the shape of the shapiro delay in gr ( pk parameter s ) , the effect of the shapiro delay on the timing residuals can then easily be computed for a range of possible companion masses ( equivalent to the pk parameter r in gr ) . the variations in the timing residuals are well explained by a companion mass of 0.2360.017m ( figure 11 ) . the measured value of , together with i , also provide an estimate of the companion mass , 0.230.14m , which is consistent with the shapiro - delay value . figure 11solid line : predicted value of the shapiro delay in psr j0437 - 4715 as a function of orbital phase , based on the observed inclination angle of 429. for such low - eccentricity binaries , much of the shapiro delay can be absorbed into the orbital roemer delay ; what remains is the pb/3 periodicity shown . the points represent the timing residuals for the pulsar , binned in orbital phase , and in clear agreement with the shape predicted from the inclination angle . solid line : predicted value of the shapiro delay in psr j0437 - 4715 as a function of orbital phase , based on the observed inclination angle of 429. for such low - eccentricity binaries , much of the shapiro delay can be absorbed into the orbital roemer delay ; what remains is the pb/3 periodicity shown . the points represent the timing residuals for the pulsar , binned in orbital phase , and in clear agreement with the shape predicted from the inclination angle . ( reprinted by permission from nature , 2001 , macmillan publishers ltd . ) while this result does not include a true self - consistency check in the manner of the double - neutron - star tests , it is nevertheless important , as it represents the only case in which an independent , purely geometric determination of the inclination angle of a binary orbit predicts the shape of the shapiro delay . it can thus be considered to provide an independent test of the predictions of gr . a complete discussion of gr effects in pulsar observations must mention geodetic precession , though these results are somewhat qualitative and do not ( yet ) provide a model - free test of gr . in standard evolutionary scenarios for double - neutron - star binaries ( see , e.g. , [ 21 , 109 ] ) , both stellar spins are expected to be aligned with the orbital angular momentum just before the second supernova explosion . after this event , however , the observed pulsar s spin is likely to be misaligned with the orbital angular momentum , by an angle of the order of 20 . a similar misalignment may be expected when the observed pulsar is the second - formed degenerate object , as in psr j1141 - 6545 . as a result , both vectors will precess about the total angular momentum of the system ( in practice the total angular momentum is completely dominated by the orbital angular momentum ) . the evolution of the pulsar spin axis s1 can be written as [ 40 , 14 ] ( 38)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{d{{\rm{\mathbf{s}}}_1}}}{{dt } } = \mathbf{\omega } _ 1^{{\rm{spin } } } \times { { \rm{\mathbf{s}}}_1 } , $ $ \end{document } where the vector 1spin is aligned with the orbital angular momentum . its magnitude is given by ( 39)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \omega _ 1^{{\rm{spin } } } = \frac{1}{2}{\left ( { \frac{{{p_{\rm{b}}}}}{{2\pi } } } \right)^ { - 5/3}}\frac{{{m_2}(4{m_1 } + 3{m_2})}}{{(1 - { e^2}){{({m_1 } + { m_2})}^{4/3}}}}t _ \odot ^{2/3 } , $ $ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t _ \odot } \equiv g{m _ \odot } /{c^3 } = 4.905490947\;\mu { \rm{s}}$$\end{document } , as in section 4.1 . this predicted rate of precession is small ; the three systems with the highest 1spin values are : psr j1141 - 6545 at 1.35 yr , psr b1913 + 16 at 1.21 yr , psr b1534 + 12 at 0.52 yr . the primary manifestation of this precession is expected to be a slow change in the shape of the pulse profile , as different regions of the pulse emission beam move into the observable region . psr j1141 - 6545 at 1.35 yr , psr b1913 + 16 at 1.21 yr , psr b1534 + 12 at 0.52 yr . evidence for long - term profile shape changes is in fact seen in psrs b1913 + 16 and b1534 + 12 . for psr b1913 + 16 , profile shape changes were first reported in the 1980s , with a clear change in the relative heights of the two profile peaks over several years ( figure 12 ) . interestingly , although a simple picture of a cone - shaped beam might lead to an expectation of a change in the separation of the peaks with time , no evidence for this was seen until the late 1990s , at the effelsberg 100-m telescope , by which point the two peaks had begun to move closer together at a rather fast rate . kramer used this changing peak separation , along with the predicted precession rate and a simple conal model of the pulse beam , to estimate a spin - orbit misalignment angle of about 22 and to predict that the pulsar will disappear from view in about 2025 ( see figure 13 ) , in good agreement with an earlier prediction by istomin made before the peak separation began to change . recent results from arecibo confirm the gist of kramer s results , with a misalignment angle of about 21. both sets of authors find there are four degenerate solutions that can fit the profile separation data ; two can be discarded as they predict an unreasonably large misalignment angle of 180-22=158 , and a third is eliminated because it predicts the wrong direction of the position angle swing under the rotating vector model . the main area of dispute is the actual shape of the emission region ; while weisberg and taylor find an hourglass - shaped beam ( see figure 14 ) , kramer maintains that a nearly circular cone plus an offset core is adequate ( see figure 15 ) . in any event , it is clear that the interpretation of the profile changes requires some kind of model of the beam shape . kramer [ 81 , 82 ] lets the rate of precession vary as another free parameter in the pulse - shape fit , and finds a value of 1.20.2. this is consistent with the gr prediction but still depends on the beam - shape model and is therefore not a true test of the precession rate . figure 12 changes in the observed pulse profile of psr b1913 + 16 throughout the 1980s , due to a changing line - of - sight cut through the emission region of the pulsar . figure 13 top : change in peak separation of the relativistic double - neutron - star binary psr b1913 + 16 , as observed with the arecibo ( solid points , [ 141 ] ) and effelsberg ( open circles , [ 80 ] ) telescopes . figure 14 hourglass - shaped beam for psr b1913 + 16 derived from the symmetric - component analysis of [ 143 ] . figure 15 alternate proposed beam shape for psr b1913 + 16 , consisting of a symmetric cone plus an offset core . the red lines indicate an example cut through the emission region , as well as the predicted pulse peak ratio and separation as functions of time . ( after [ 81 ] , courtesy michael kramer . ) changes in the observed pulse profile of psr b1913 + 16 throughout the 1980s , due to a changing line - of - sight cut through the emission region of the pulsar . top : change in peak separation of the relativistic double - neutron - star binary psr b1913 + 16 , as observed with the arecibo ( solid points , [ 141 ] ) and effelsberg ( open circles , [ 80 ] ) telescopes . hourglass - shaped beam for psr b1913 + 16 derived from the symmetric - component analysis of [ 143 ] . ( taken from [ 143 ] ; used by permission . ) alternate proposed beam shape for psr b1913 + 16 , consisting of a symmetric cone plus an offset core . the red lines indicate an example cut through the emission region , as well as the predicted pulse peak ratio and separation as functions of time . psr b1534 + 12 , despite the disadvantages of a more recent discovery and a much longer precession period , also provides clear evidence of long - term profile shape changes . these were first noticed at 1400 mhz by arzoumanian [ 5 , 8 ] and have become more obvious at this frequency and at 430 mhz in the post - upgrade period at arecibo . the principal effect is a change in the low - level emission near to the main pulse ( figure 16 ) , though related changes in polarization are now also seen . as this pulsar shows polarized emission through most of its pulse period , it should be possible to form a better picture of the overall geometry than for psr b1913 + 16 ; this may make it easier to derive an accurate model of the pulse beam shape . figure 16 evolution of the low - level emission surrounding the main pulse of psr b1534 + 12 , over a period of nearly 10 years , as measured with the arecibo telescope [ 96 ] . evolution of the low - level emission surrounding the main pulse of psr b1534 + 12 , over a period of nearly 10 years , as measured with the arecibo telescope [ 96 ] . ( stairs et al . , unpublished . ) as for other tests of gr , the pulsar white - dwarf binary psr j1141 - 6545 promises interesting results . as noted by the discoverers , the region of sky containing this pulsar had been observed at the same frequency in an earlier survey , but the pulsar was not seen , even though it is now very strong . it is possible that interference corrupted that original survey pointing , or that a software error prevented its detection , but it is also plausible that the observed pulsar beam is evolving so rapidly that the visible beam precessed into view during the 1990s . the tremendous success to date of pulsars in testing different aspects of gravitational theory leads naturally to the question of what can be expected in the future . improvements to the equivalence - principle violation tests will come from both refining the timing parameters of known pulsars ( in particular , limits on eccentricities and orbital period derivatives ) and the discovery of further pulsar white - dwarf systems . potentially coalescing pulsar white - dwarf binaries , such as psrs j1141 - 6545 , j0751 + 1807 , and 1757 - 5322 , bear watching from the point of view of limits on dipolar gravitational radiation . another worthy , though difficult , goal is to attempt to derive the full orbital geometry for ultra - low - eccentricity systems , as has been done for psr j0437 - 4715 ; this would quickly lead to significant improvements in the eccentricity - dependent tests . the orbital - period - derivative measurements of double - neutron - star binaries are already limited more by systematics ( galactic acceleration models for psr b1913 + 16 , and poorly known distance for psr b1534 + 12 ) than by pulsar timing precision . however , with improved galactic modeling and a realistic expectation of an interferometric ( vlbi ) parallax for psr b1534 + 12 , there is still hope for testing more carefully the prediction of quadrupolar gravitational radiation from these systems . the other timing parameters , equally important for tests of the quasi - static regime , can be expected to improve with time and better instrumentation , such as the wider - bandwidth coherent dedispersion systems now being installed at many observatories ( see , e.g. , [ 68 , 129 ] ) . especially exciting would be a measurement of the elusive shapiro delay in psr b1913 + 16 ; the longitude of periastron is now precessing into an angular range where it may facilitate such a measurement . in the last few years , surveys of the galactic plane and anking regions , using the 64-m parkes telescope in australia , have discovered several hundred new pulsars ( see , e.g. , [ 91 , 48 ] ) , including several new circular - orbit pulsar white - dwarf systems [ 46 , 47 , 26 ] and the eccentric pulsar a complete reprocessing of the galactic plane survey with improved interference filtering is in progress ; thus there is still hope that a truly new system such as a pulsar black - hole binary may emerge from this large survey . several ongoing smaller surveys of small regions and globular clusters ( see , e.g. , [ 25 , 113 ] ) are also finding a number of new and exotic binaries , some of which may eventually turn out to be useful for tests of gr . the possible recent appearance of psr j1141 - 6545 and the predicted disappearance of psr b1913 + 16 due to geodetic precession make it worthwhile to periodically revisit previously surveyed parts of the sky in order to check for newly - visible exotic binaries . over the next several years , large - scale surveys are planned at arecibo and the new 100-m green bank telescope , offering the promise of over 1000 new pulsars including interesting binary systems . the sensitivity of these surveys will of course be dwarfed by the potential of the proposed square kilometre array radio telescope , which will be sensitive to pulsars clear through our galaxy and into neighbouring galaxies such as m31 . the next 10 or 20 years promise to be exciting times for pulsar searchers and for those looking to set ever - more - stringent limits on deviations from general relativity .

pulsars of very different types , including isolated objects and binaries ( with short- and long - period orbits , and white - dwarf and neutron - star companions ) provide the means to test both the predictions of general relativity and the viability of alternate theories of gravity . this article presents an overview of pulsars , then discusses the current status of and future prospects for tests of equivalence - principle violations and strong - field gravitational experiments .

recent results from the national institutes of health ( nih ) frequent hemodialysis network ( fhn ) daily trial and the following rehabilitation , economics and everyday - dialysis outcome measurements ( freedom ) study have provided confirmation of the clinical and quality of life ( qol ) benefits of short daily hemodialysis ( sdhd ) when compared to conventional thrice weekly in - center hemodialysis ( chd ) . the fhn study was conducted in - center ; however , sdhd in the usa is more commonly performed as a home therapy and is not offered as a standard treatment modality in most dialysis clinics . although it has been nearly 50 years since the early pioneers such as belding scribner and stanley shaldon began dialyzing patients in their homes , peaking at 40% of all us dialysis patients in 1973 , the popularity of home hemodialysis ( hd ) has been limited in the usa for a number of reasons , including lack of suitable equipment and increased cost . the last few years , however , have seen a revival in home hd , resulting in renewed interest in the various forms of more frequent therapy , including sdhd . the revival of home dialysis was led in the mid 1990s by robert uldall , andreas pierratos and others in canada , gradually spreading throughout the usa . this re - emergence has been made possible by recent advances in technology , including portable and more user - friendly machines designed specifically for use in the home environment . in 2005 , the fda cleared the first hd machine for home use in the usa , the nxstage system one. fda clearance was based on an open - label , prospective , two - treatment two - period crossover study on 32 patients to compare sdhd performed in - center ( prescribed six times / week ) versus sdhd performed at home . the study was conducted at six centers in the usa under an investigational device exemption . this study showed sdhd was delivered as efficiently in the home environment as in - center , with 98.5% of treatments performed successfully in - center versus 97.3% at home . notably , the composite rate of intra - dialytic and inter - dialytic adverse events was significantly higher during the in - center phase when compared with the home phase ( 5.3 versus 2.1 adverse events/100 treatments ; p = 0.007 ) , suggesting hd therapy is at least as safe at home as in - center . when comparing clinical parameters from the period immediately preceding the study when patients were treated with conventional thrice weekly center hd , home sdhd was associated with reductions in blood pressure , antihypertensive medications and interdialytic weight gain . since the clearance of the nxstage system one in the usa , the number of patients now performing home dialysis is estimated to be 5000 , with the majority of these patients performing sdhd . although this represents a considerable increase over the past 5 years , it is still only 1% of the total end - stage renal disease ( esrd ) population . sdhd offers the promise of improved clinical outcomes in patients with kidney failure , and given the positive results recently released from both the fhn and freedom studies outlined below , sdhd has the potential to become a more viable and popular choice amongst many esrd patients . results recently published from the fhn daily trial , funded by the nih , showed that in - center sdhd ( prescribed six times / week ) , provided significant benefits in both composite co - primary outcomes of death or 12-month change in left ventricular mass ( lvm ) and death or 12-month change in the rand-36 physical health composite ( phc ) score . specifically , the fhn daily trial showed patients randomized to sdhd reduced their lvm by 16.3 35.3 g ( p < 0.001 ) and improved their phc score by 3.3 8.9 points ( p = 0.004 ) , after 12 months . however , patients on sdhd were more likely to undergo interventions related to vascular access , a result which will require further elucidation with the ongoing analysis of the trial . the fhn trial will be a tremendous source of data to further dissect the advantages seen with daily therapy in the coming years . the freedom study is an ongoing prospective cohort study investigating the clinical and economic benefits of sdhd . the objectives of the freedom study are to compare a cohort of patients starting sdhd using the nxstage system one to a matched cohort of patients receiving chd for all - cause hospitalizations and non - treatment - related medical expenditures . using a 10-to-1 ratio , totaling 5000 patients , the matched thrice weekly in - center hd cohort will be obtained from the us renal data system ( usrds ) database . in addition , changes in qol measures , urea kinetics , management of anemia , bone and mineral metabolism , nutrition , vascular access interventions and use of medications will be examined . the freedom study will involve up to 70 clinical sites and 500 participants , with a minimum 1-year follow - up . study participants complete qol surveys at the time of study enrollment , at 4 and 12 months and every 6 months thereafter . interim data recently released from the freedom study has shown that sdhd , performed in the home environment , is associated with significant improvements in several important clinical and qol measures when compared to conventional thrice weekly in - center hd . interim results from the freedom study investigating the effect of sdhd on depressive symptoms were recently published . depressive symptoms were examined in 248 participants at enrollment and at 4 and 12 months by administering the validated beck depression inventory ( bdi)-ii survey . the study protocol requires that the site investigator be notified immediately of a bdi score > 10 , with mild and moderate to severe depressive symptoms defined as bdi scores of 1115 and > 15 , respectively . in summary , sdhd was associated with a significant improvement in mean bdi score over 12 months [ 11.2 , 95% confidence interval ( ci ) 9.612.9 , versus 7.8 ( 95% ci 6.59.1 ) ; p < 0.001 ] , in the per - protocol ( pp ) cohort . similar results were found in the intent - to - treat ( itt ) cohort . for participants with moderate to severe depressive symptoms , the bdi scores almost halved over 12 months [ 24.4 ( 95% ci 19.429.4 ) versus 12.6 ( 95% ci 8.017.2 ) ; p < 0.001 ] . it should be noted that a bdi score > 15 previously has been shown to be highly predictive of a diagnosis of clinical depression . the percentage of participants with depressive symptoms ( bdi score > 10 ) significantly decreased during 12 months ( 41 versus 27% ; p < 0.03 ) . similarly in the fhn study , patients participating in the daily arm did show a trend toward improvement after 12 months when compared to baseline ( 12.6 8.7 versus 10.4 8.5 , p = 0.1 ) . although this trend was not statistically significant , one might speculate that undergoing daily dialysis therapy at home may contribute to this improvement . considering the practical feasibility of daily dialysis both from a logistical and a patient comfort perspective , one might hypothesize that the home setting may further enhance the benefits of sdhd experienced in - center . the long - term effect of sdhd on post - dialysis recovery time was also assessed in the freedom study by administering the following previously validated question : how long does it take you to recover from a dialysis session and resume your normal , usual activities? interim results were recently published and confirmed that sdhd is associated with a significant decrease in post - dialysis recovery time [ 476 min ( 95% ci 359594 ) versus 63 min ( 95% ci 3295 ) ; p < 0.001 ] . the percentage of participants experiencing prolonged post - dialysis recovery time ( > 60 min ) also significantly decreased over the 12-month period ( 81 versus 35% ; p < 0.001 ) . the fhn daily trial showed significant improvements in control of hyperphosphatemia for patients on sdhd . a significant reduction in mean serum phosphate was also reported in an interim report from the freedom study , along with significant reductions in the calcium phosphate product , serum creatinine , serum potassium and a trend toward a reduction in blood urea nitrogen . both the fhn daily trial and an interim analysis from the freedom study showed no change in serum albumin levels . well - known uremic symptoms , including restless legs symptoms ( rls ) and poor sleep quality are common in the hd population . poor sleep quality and rls have both been linked to increased risk of death for these patients [ 9 , 10 ] . a recent interim report from the freedom study demonstrated initiation of sdhd at home is associated with significant improvement in rls and sleep disturbances . results from 235 patients found 40% suffered from rls at baseline , which was associated with poorer sleep quality and respiratory disturbances . among patients with rls , the mean irls ( international restless legs severity scale ) score improved significantly at month 12 , after adjustment for use of rls - related medications ( 18 versus 11 , p < 0.0001 ) . among patients with moderate - to - severe rls ( irls score 15 ) , there was an even greater improvement in the irls score ( 23 versus 13 , p < 0.001 ) . over the 12-month period , there was decline in the percentage of patients reporting rls ( 35 versus 26% , p = 0.05 ) and those reporting moderate - to - severe restless legs symptoms ( 59 versus 43% , p = 0.06 ) . there was a similar and sustained improvement in several scales of the medical outcomes study sleep survey over 12 months , after adjustment for presence of rls and use of anxiolytics and hypnotics . recent results from both the fhn daily trial and freedom study have shown that sdhd , performed either in - center or at home , is associated with several important clinical and qol benefits when compared to conventional thrice weekly hd . the improvements in left ventricular mass , control of hypertension and improvement in hyperphosphatemia demonstrated in the fhn trial , in conjunction with the positive interim findings from the freedom study , including improvements in depressive symptoms , post - dialysis fatigue , various laboratory parameters , restless leg syndrome and sleep disorders , confirm the anecdotal benefits of sdhd seen and reported by many practicing nephrologists and their patients . until now , a complex variety of reasons has hindered the more widespread use of sdhd in the usa and globally . however , the results of these studies may provide a new impetus for esrd patients and nephrologists to reconsider the paradigm of thrice weekly center hd as the default therapy. such a paradigm shift will likely increase the possibility for improved clinical outcomes and overall patient care for those affected by esrd .

thrice weekly in - center hemodialysis is the standard of care for dialysis patients with end - stage renal disease ( esrd ) . however , there is ongoing debate as to whether more frequent hemodialysis , with its readier management of both toxin and fluid removal , benefits patients . new evidence from recent studies , both in center dialysis and in home haemodialysis patients , adds further confirmation of improved cardiovascular outcome and quality of life in patients undergoing short daily hemodialysis . a paradigm shift in esrd care delivery may be facilitated due to new technology enabling daily therapy at home .

when cells migrate , they extend dynamic membrane - bound actin - rich tubular protrusions known as filopodia . formins are a family of large multi - domain proteins that nucleate and polymerize actin to form linear actin filaments like those found within filopodia . in this review formins function as dimers and nucleate actin by means of a formin homology 2 ( fh2 ) domain that binds globular actin monomers . interaction of the adjacent formin homology 1 ( fh1 ) domain with profilin effectively recruits actin monomers to the formin dimer , facilitating the polymerization process . a subset of formins , known as diaphanous - related formins ( drfs ) , bind to and drfs are rendered inactive by interaction of a c - terminal diaphanous autoregulatory domain ( dad ) with an n - terminal diaphanous inhibitory domain ( did ) ( fig . 1 ) . the binding of a rhogtpase to the n - terminal gtpase binding domain ( gbd ) contributes to disruption of this autoinhibitory interaction , which results in the activation of the drf . in the case of the drf fhod1 , phosphorylation of c - terminal serine and threonine residues by rock other domains found in drfs include a a dimerization domain ( dd ) and a coiled coil ( cc ) region , and some groups believe that the dd and did together constitute a loosely defined formin homology 3 ( fh3 ) domain . an n - terminal phospholipid - binding basic domain ( bd ) mdia1 has three stretches of basic amino acids in this domain , which allows it to localize to the plasma membrane , while mdia2 has two . in contrast , the drf daam1 has only one stretch of basic amino acids in its n - terminal , and the distribution of constitutively active daam1 is restricted to the cytoplasm . this might explain why only mdia1 and mdia2 have been implicated in the formation of membrane - bound cell surface protrusions like filopodia and lamellipodia , while mdia3lacking this bd has only been reported to generate cytoplasmic actin structures . several rhogtpases and other proteins have been reported to bind to mdia1 - 3 . these are summarized in figure 1 . the shortest fragment(s ) known to bind the respective mdia isoforms is shown for each interacting protein . excluded from this diagram are ywk - ii , which binds a 223 aa fragment of hdia1 that shares 96% aa sequence identity with mdia1 ( 903 - 1125 aa ) , and inf2 , which binds to aa 1181 - 1262 and aa 1051 - 1193 of what might be longer splice variants of mdia1 and mdia3 respectively . mdia1 - 3 form several types of actin - based cellular structures . within the cytoplasm , mdia1 gives rise to stress fibers , and mdia2 drives the actin dynamics that power vesicle movement and creates the actin scaffold for constriction of the contractile ring during cytokinesis . at the plasma membrane , both mdia1 and mdia2 a role for formins in lamellipodial protrusion is not surprising once thought to be structures comprised of dendritic networks of branched microfilaments , lamellipodia have recently been reported to contain linear actin filaments as well . we have found mdia3 to be capable of inducing filopodia in n1e115 neuroblastoma cells , despite not being able to detect the presence of endogenous mdia3 protein in this particular cell line . all three mdia isoforms have also been linked to invadopodia protrusion , while mdia2 alone plays a role in forming the filopodial precursors of dendritic spines . actin dynamics leading to the formation of the phagocytic cup in macrophages are believed to involve mdia1 and mdia2 too . previous studies have pointed to a role for mdia2 in mammalian filopodia . a decrease in filopodial protrusion was seen in mouse fibroblasts overexpressing constitutively active cdc42 and microinjected with anti - mdia2 antibodies , as well as cells cotransfected with activated cdc42 and a non - functional mdia2 mutant . in nih3t3 fibroblasts , constitutively active mdia2 , when overexpressed alone in b16f1 melanoma cells , also accumulated at filopodial tips . furthermore , knockdown of mdia2 protein in mouse hippocampal neurons reduced the formation of the filopodial precursors of dendritic spines . in more recent work , mdia1 - 3 have been shown to induce filopodia in neuronal cells when overexpressed on their own . however , only mdia1 was seen within filopodia , as observed by time lapse imaging of live cells . the lack of mdia2 and mdia3 in neuronal filopodia implies that these drfs might be involved in the initiation of filopodium formation but not the elongation of the structures . one possibility is that mdia2 and mdia3 generate short microfilaments that are subsequently elongated by mdia1 to form mature filopodia . this would tie in with the suggestion that mdia2 is a relatively strong nucleator but poor elongator of microfilaments , based on observations that it elongates microfilaments at a slower rate than mdia1 . the filopodia induced by full - length wildtype mdia2 appeared cylindrical and of even thickness along their length , and so did the filopodia formed by fh1fh2-mdia2 , a fragment of mdia2 that consists of only the fh1 and fh2 domains . this is unlike the club - shaped filopodia obtained by transfecting b16f1 cells with constitutively active mdia2 , where the structures are packed with shorter microfilaments at their distal ends but contain relatively few long microfilaments that extend toward the base of the protrusions . the high overexpression of constitutively active mdia2 could have resulted in endogenous mdia1 protein becoming a limiting factor on the process of filopodial microfilament elongation , giving rise to the club - shaped morphology of the filopodia . in addition , both full - length mdia2 and fh1fh2-mdia2 localized mostly to the cytoplasm in these experiments it is likely that less of the protein was present in filopodia , and there was enough endogenous mdia1 to elongate the smaller number of short microfilaments generated , thus resulting in filopodia of even thickness along their shafts . these interpretations of the findings would further implicate mdia1 as the key mdia isoform that elongates microfilaments to form mature filopodia . it would be interesting to see if knocking out mdia1 affects mdia2-driven filopodial protrusion will the resulting mdia2-induced filopodia be shorter in length ? mdia1 was seen throughout the shafts of filopodia when overexpressed alone or together with irsp53 or constitutively active rif in neuronal cells . this is in contrast to the tip nucleation model of filopodium formation , where formins are expected to be found only at the tips of the protrusions . one possible explanation is that mdia1 dimers play a dual role in filopodium formation and are involved in not just polymerising the actin filaments but bundling them together as well . another possibility is that filopodia consist of short , discontinuous actin filaments that do not span the entire length of the filopodial shaft . this has been shown by cryo - electron tomography studies to be the nature of the actin filaments that constitute dictyostelium filopodia . superresolution microscopy studies would be able to reveal detail at the nanometre scale and help elucidate the specific locations of mdia1 and mdia2 , as well as other proteins associated with filopodial protrusion , within the structures with much greater accuracy . this would help in establishing a better understanding of the roles of these proteins in the various stages of filopodium formation . cdc42 works through irsp53 , which recruits to the plasma membrane the following proteins that modulate actin dynamics : n - wasp , mena , wave2 and eps8 . the rif pathway to filopodia does not require irsp53 , n - wasp , mena or wave2 . as for the mdia proteins , mdia1 appears to be the only isoform common to both pathways . in the filopodia of neuronal cells overexpressing irsp53 , mdia1 but not mdia2 was present , and was observed to interact with irsp53 within the structures . while both mdia1 and mdia2 were present in rif filopodia , only mdia1 interacted with the rhogtpase . in addition , knockdown of either of these two isoforms resulted in a decrease in rif - driven filopodium formation , while irsp53 filopodia were affected only by the silencing of mdia1 expression . taken together , it appears that mdia2 is not required for irsp53 to form filopodia , and we have found that coexpressing mdia2 with irsp53 leads to a loss of filopodia instead . it remains to be seen as to why cells need two or even moreyetundiscovered pathways to form filopodia , and why irsp53 requires only mdia1 when rif appears to require both mdia1 and mdia2 . also , rif has been shown to bind the gbd of mdia3 , however the significance of this interaction has yet to be investigated . mdia1 and possibly mdia2 are involved in stress fiber formation in addition to filopodial protrusion . these two types of actin - based cellular structures appear to be linked in fish fibroblasts , microfilaments in filopodia can become incorporated into stress fibers , and it has been suggested that the reverse might occur in rat embryonic fibroblasts , with the actin freed up by the dissolution of stress fibers facilitating the protrusion of filopodia . rif interacts with both mdia1 and mdia2 and is able to trigger the formation of both filopodia and stress fibers . the dual role of these three proteins might point to a major role for them in controlling the balance between these two types of actin structures in cell migration . mdia2 appears to be specific for the rif - mediated pathway whereas mdia1 is required for the pathways controlled by cdc42 and rif . it remains to be seen how exactly rif utilizes two different formins , mdia1 and mdia2 , to form filopodia . how rif couples membrane deformation with actin dynamics to give rise to these structures has also yet to be resolved . apart from mdia1 and mdia2 , are there other proteins specific to the rif pathway to filopodium formation ? what potential roles do irsp53 family proteins ( irtks , mim , abba and pinkbar ) play in filopodial protrusion ? these are some of the important questions to address in future studies on mammalian filopodia .

mdia proteins are members of the formin family of actin nucleating proteins that polymerize linear actin filaments . such filaments form the core of thin , tubular , membrane - bound cell surface protrusions known as filopodia , which are a major feature of mammalian cell morphology . filopodia are dynamic structures that help cells sense environmental cues , and play a role in cell migration , axon guidance , angiogenesis and other processes . rhogtpases bind to and control the activity of mdia proteins , and several other binding partners of the three mdia1 isoforms mdia1 , mdia2 and mdia3have been documented . two independent pathways controlling mammalian filopodium formation have emerged , with one driven by the rhogtpase cdc42 , and the other by rif . while mdia2 has been the main formin implicated in forming filopodia , mdia1 has recently surfaced as the key formin utilized by both the cdc42 and rif pathways to drive filopodial protrusion .

mammals are the only animals that secrete a complex fluid from an elaborated skin gland to provide both innate protection and nourishment for their newborn . there are more than 4,000 species of mammals with striking similarities in the structure and function of their mammary glands as well as in their unique milk components such as the caseins , -lactalbumin , lactoferrin , lactose , and milk fat . nevertheless , variations are exhibited in the arrangement and numbers of mammary gland , milk composition , and suckling strategies . mammary gland development begins at puberty and is maintained throughout pregnancy until lactation . during these last stages , development compromises numerous overlapping programs such as branching morphogenesis , inductive stromal - epithelial interactions , programmed cell death , extracellular matrix remodeling , and hormone action . current knowledge of the molecular regulation of mammary development and lactation has largely been derived from the dissection of signaling networks in cell culture systems and phenotypic characterization of genetically altered mice as well as genomewide approaches such as microarrays . nonetheless , to date , regulation of mammary gland development during pregnancy and lactation is incompletely understood . lactation is regarded as one of the most remarkable products of evolution whose processes include the development of mammary tissue as well as the synthesis and secretion of milk . consequently , despite the fact that the development of mammary tissue and the synthesis and secretion of milk are considered as complex dynamic physiological processes , both must preserve overall common characteristics among mammals . considering the underlying assumption that important biological functions are often conserved across species , genes expressed across multiple species are likely to have conserved functions . given the completion of the dna sequence of the human , mouse , and rat genomes , genes identified in microarray studies can be readily compared across species with respect to orthologous genes . therefore , a cross - species hybridization ( csh ) experiment could provide significant information concerning probable conserved gene networks among mammals . in a csh experiment , there is hybridization of rna from one or more ( target ) species to a microarray that contains dna ( cdna or oligomers ) from another ( reference ) species and represents a valuable tool for the identification of orthologous genes . thus , a csh microarray analysis offers the possibility of furthering our understanding of cross - species commonalities and differences that could lead to more effective use of animal models to understand the regulation of mammary gland development at the molecular level . dissection of unique patterns of expression of orthologous clusters of genes among species throughout distinct physiological time points along pregnancy and lactation could prove useful in the integrative analysis of the information available for discerning the molecular events underlying the regulation of mammary gland development and function that lead to milk synthesis . in this study , these data resulted from heterologous microarrays of target rna samples derived from rat mammary gland during distinct stages of pregnancy and lactation in order to extrapolate and enhance the understanding on transcriptional module networks or coregulated functional gene groups conserved in rodents and in the development of the mammary gland in humans . fifteen female sprague dawley rats were obtained from the animal care facility of centro mdico nacional siglo xxi of the mexican institute of social security ( imss ) in mexico city . animals were housed at 22 2c with a 12 h light / dark cycle with free access to water , and a purified diet was administered ad libitum during pregnancy and lactation . , rats were randomly assigned to five groups representing distinct time points in mammary gland development : virgin ( v ) , day 5 ( p5 ) and , day 14 ( p14 ) of pregnancy and day 1 ( l1 ) , and day 12 ( l12 ) of lactation . the day on which sperm was identified in vaginal smears was designated as day 1 of pregnancy and the day of parturition was designated as day 1 of lactation . rats were euthanized , and whole mammary tissue was removed from v , p5 , p14 , l1 , and l12 rats . tissue was immediately frozen in liquid nitrogen and stored at 70c for subsequent total rna isolation or histological analysis . total rna was isolated from tissue ( 0.1 - 0.2 g ) using trizol ( invitrogen , carlsbad , ca , usa ) following the method of chomczynski and sacchi . total rna from mammary tissue was isolated from three different animals of each physiological period ( v , p5 , p14 , l1 , and l12 ) , pooled , and kept in aliquots for later determination of purity and integrity . four microarray datasets generated using the custom - designed mus musculus oligonucleotide array containing 65-mer probe sets representing 22,000 transcripts ( microarray unit , cellular physiology institute , unam , mexico city ) were analyzed . each dataset represented distinct time points in mammary gland development such as p5 , p14 , l1 , and l12 . histologically , the mammary proliferative stage is represented by p5 , the secretory differentiation stage by p14 , early lactation by l1 , and full lactation by l12 . design of the microarray experiments is presented in table s1 in supplementary materials available online at http://dx.doi.org/10.1155/2013/624681 . ten g of total pooled rna was reverse transcribed into cdna incorporating dutp - cy3 or dutp - cy5 and using the cyscribe first - strand cdna labeling kit ( amersham biosciences , piscataway , nj , usa ) . sunnyvale , ca , usa ) , equal quantities of labeled cdna were hybridized to the m22 k_01 microarray for 14 h at 42c . four hybridization assays were carried out as follows : ( a ) the fluorophore used was dutp - cy3 for control nonpregnant virgin rats ( v ) and dutp - cy5 for p5 , ( b ) dutp - cy3 for v and dutp - cy5 for p14 , ( c ) dutp - cy3 for v and dutp - cy5 for l1 , and ( d ) dutp - cy3 for v and dutp - cy5 for l12 . data acquisition and analysis of array images were performed in scanarray 4000 with its accompanying software scanarray 4000 from packard biochips . microarray data analysis was performed with free software genarise , which was developed in the computing unit of the cellular physiology institute of the unam ( http://www.ifc.unam.mx/genarise/ ) . the goal of genarise is to identify which genes show good evidence of being differentially expressed . the software identifies differentially expressed genes by calculating an intensity - dependent z - score . elements with a z - score > 1.5 standard deviations are considered to be significantly and differentially expressed genes . the complete set of raw excel data files have been deposited at gene expression omnibus ( geo ) and are available on the geo website ( i d geo gse22545 ) . gene lists were generated by a set of multiple comparisons among the distinct developmental stages and intersection in venn diagrams . two - way hierarchical clustering with average linkage and a range of 5 to 15 k - means classifications were used to group our time series data using open source software cluster v3.0 . java treeview was used to display the clustering results as dendogram or heat map representations . we adopted the procedure as described in to code the mean expression of a cluster at each stage as flat , decreased , and increased and converted it to numerical representation . putative orthologous genes in rat , mouse , and human were identified from a genome comparative search with roundup ( http://rodeo.med.harvard.edu/tools/roundup ) . roundup is an ortholog and phylogenetic profile retrieval tool backed by a massive repository of orthologous and associated evolutionary distances that were built using the reciprocal smallest distance algorithm . the search was done with a stringent blast e - value threshold of 1.0 10 and a divergence threshold of 0.2 . the david 2.0 functional annotation tool ( http://david.abcc.ncifcrf.gov/summary.jsp ) was used to sort and arrange the similar , redundant , and heterogeneous annotation contents from a set of genes into defined functional groups . in the case of insufficient gene ontology information , published pathway mapping was accomplished using the kyoto encyclopedia of genes and genomes ( kegg ) database of biological systems that integrates genomic , chemical , and systemic functional information ( http://www.genome.jp/kegg/kegg2.html ) . gene lists were converted to human swissprot ids using tables from the ensembl database , release 42 . for each list of human swissprot ids , interactions between those gene products were obtained from online predicted human interaction database ( ophid ) and postprocessed using custom scripts to determine all linkages in the network and to generate a network file . this network file was then explored using navigator v2.0.15 , a program for large network analysis ( http://ophid.utoronto.ca/navigator/index.html ) . transcription - factor - binding site ( tfbs ) prediction was accomplished using core_tf ( conserved and over - represented transcription factor binding sites ) , a web - based tool that identifies overrepresented tfbs in promoters from coexpressed genes aided by the evaluation of cross - species conservation . we measured the relative transcript levels of 14 target genes , and five genes commonly used as references such as glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) , beta - actin ( actb ) , and ribosomal large protein p0 ( rlp0 ) were used as high abundance internal controls as well as splicing factor arginine / serine - rich 1 ( sfrs1 ) and hypoxanthine guanine phosphoribosyl transferase 1 ( hprt1 ) as medium- and low - abundance internal controls , respectively ( table s2 ) . quantitative real - time pcr was performed in a 20 l reaction with 5.0 l from 1/4 reverse transcription dilution using the lightcycler probes master mix ( roche diagnostics , mannheim , germany ) containing 0.2 m of mrna - specific primers and 0.1 m corresponding upl probe into lightcycler microplate wells under reduced light conditions . the results are expressed as mean sem of at least three individual experimental observations . statistically significant differences among experimental groups ( between the mean values of each group ) were determined by an unpaired students t - test ( z - test ) , anova , or a modified fisher 's exact test . the rat mammary gland undergoes a series of dramatic phenotypic changes during pregnancy and lactation . in order to determine the integrity of the dissected inguinal mammary glands , a gross histological evaluation of the characteristic cytomorphological features four time points ( pregnancy days 5 and 14 ; lactation days 1 and 12 ) were selected to represent distinct periods in mammary gland development . histologically , the mammary proliferative stage is represented by p5 , the secretory differentiation stage by p14 , early lactation by l1 , and full lactation by l12 . as reported elsewhere , initial changes observed during pregnancy include an increase in ductal branching and the formation of alveolar buds ( figures 1(c ) and 1(d ) ) . the latter half of pregnancy is characterized by the expansion of alveolar buds to form clusters of lobuloalveolar units followed by the differentiation of these structures into presecretory structures . by day 14 of pregnancy , there is a readily apparent increase in the size of the epithelial compartment ( ep ) ( figures 1(e ) and 1(f ) ) , and expansion of the epithelium ( whereas the adipose compartment decreases ) continues until the epithelial compartment predominates by onset of lactation ( figures 1(g ) and 1(h ) ) . by day 12 of as expected , examination of the histology of the mammary gland at this stage reveals prominent luminal structures ( l ) and ducts and few adipocytes visible at this time ( figures 1(i ) and 1(j ) ) . in this study , we analyzed expression profiles of 22,000 transcripts using murine microarrays and rna samples of mg from virgin , pregnant , and lactating rats by cross - species hybridization . we first identified the total number of genes differentially expressed throughout distinct time points in mammary gland development such as p5 , p14 , l1 , and l12 . a total of 807 oligonucleotide probe sets representing 521 annotated genes showed differential expression in at least one of four physiological time points evaluated , taking into consideration a mean z - score cutoff value of 1.50 standard deviations using genarise . during early pregnancy ( day 5 ) , most of these transcripts ( 123 , 77.8% ) were upregulated , suggesting a feasible tendency in the direction of gain of function versus the virgin stage ( v ) . likewise , in mid - pregnancy ( day 14 ) , as opposed to the virgin stage , the number of transcripts with an altered expression maintained a similar value ( 133 transcripts ; 89.26% upregulated ) . during early and mid - lactation ( days 1 and 12 ) , 342 and 461 transcripts were differentially expressed , corresponding to a percentage of 64.0 and 38.4 overexpressed , respectively ( figure 2(a ) ) . to further illustrate the differences and commonalities among the four physiological time points , changes in gene expression were also interpreted with a venn diagram . as shown in figure 2(b ) , the descriptive table of the venn diagram denotes the number of genes showing upregulation ( ) or downregulation ( ) uniquely at pregnancy ( day 5 or 14 ) or lactation ( day 1 or 12 ) and differential expression at a combination of stages . venn diagram analysis indicated that 47.2% ( 381/807 ) of all the differentially expressed transcripts presented an average significant z - score fold change ( z > 1.5 ) exclusively during either or both time points of lactation . interestingly , among the 381 altered gene transcripts during lactation , 64.8% ( 247/381 ) were found downregulated , implying as previously stated by lemay et al . ( 2007 ) that mammary epithelial cells become biofactories not by gain of function but by a broad suppression of function to effectively push all cell resources towards a very few important tasks . all the gene sets that shared spatial and temporal distributions ( overlapping changes in expression ) are listed in additional data files ( table s3 ) . to determine global alterations in gene expression across developmental stages of the mammary gland from early pregnancy to mid - lactation , we performed a complete - linkage hierarchical clustering with an euclidean distance similarity metric on the expression profiles of the differentially expressed genes ( annotated and est ) across all four time points . the expression profiles of the 807 genetic elements resulted in six predominant clusters on a dendogram ( designated clusters 16 in figure 3 ) . this major trend is a decline in gene expression during mid - pregnancy that remains low during lactation . cluster 2 ( c2 ) describes 25.77% of the total data population and is characterized by a linear decrement in gene expression towards mid - lactation . however , positive z - score values are retained with respect to the reference stage ( virgin ) . the remainder of the clusters ( c3c6 ) appears to explain between 3.09 and 17.84% of the data variation . in c3 and c4 , gene expression rises exponentially from early pregnancy , reaching a plateau during mid - lactation . however , the slope of the curve is even steeper in c3 in comparison to c4 . in cluster 5 ( c5 ) , 65 elements matched the profile outline ( inverted sigmoid form ) of major trend c1 although the reduction tendency was less marked . in cluster 6 ( c6 ) , expression was roughly unchanged during pregnancy and lactation . even so , the relative abundance of transcripts remained in a higher proportion than the reference virgin stage as described for c2 . this transcriptional profile , involved in the mammary development program identified in rat , could be conserved in others mammals like mouse . consequently , in order to delineate potential groups of coregulated genes , final cluster membership was determined by a k - means analysis based on the preestimated number ( six ) of gene clusters . k - means clustering revealed six distinct clusters ( k16 ) that distinguished up from down , early from middle , and transient from sustained changes in expression ( figure 4 ; table s5 ) . each of the six clusters was designated with its unique trajectory expression profile signature across stages ( pregnancy days 5 and 14 , lactation days 1 and 12 ) as presented in figure 4 . there were two major groups of 245 under- and 175 overexpressed tags during lactation only ( table s5 , k1 : 1,1,0,0 and k3 : 1,1,2,2 according to the procedure of rudolph et al . , to code the mean expression , see section 2 ) . among the typical upregulated genes of lactation stage are the milk protein ( casein alpha ( csn1s1 , csn1s2a ) , casein beta ( csn2 ) , and whey acidic protein ( wap ) ) and biogenesis genes that mainly concern glucose and lipid metabolism ( akr1c6 , aldob , ugt2b1 , plb1 , apoe , and sult2b1 ) and transcriptional regulation ( stat5a , pou2af1 ) . among those genes found significantly downregulated only during lactation , several play an important role in the regulation of apoptosis , mediation of metastatic behavior ( epithelial - mesenchymal transition ) , or ubiquitin - mediated protein catabolism ( lysosome degradation ) in the mammary gland including igfbp5 , mmp2 , and ube2r2 [ 14 , 15 ] . one hundred forty - nine genes were upregulated exclusively during early pregnancy ( k2 : 2,1,1,1 ) such as esr1 , esr2 , tshr , and oxt . these participate in the transduction of hormonal status [ 2 , 16 ] involved in the modulation of important physiological processes such as carbohydrate metabolism ( creb3l4 , hk1 , and coasy ) ; glutathione metabolism ( ggt1 , mgst1 , and gstm6 ) ; cell differentiation ( foxa1 , mtap7 , gdf1 , twist2 , hey1 , dll4 , and pcaf ) [ 1923 ] , stromal - epithelial communication ( cell - cell junctions ) ( cldn10 , mpp5 , and epb4 ) , and cell adhesion ( matn1 , krt71 , mpzl2 , and dscaml1 ) . the smallest group of 24 genes were significantly upregulated exclusively at the onset of lactation ( k4 : 1,1,2,1 ) such as lpo , cd8a , and irs1 , important for lactogenesis , particularly in milk production capabilities and related immunotropic constituents ( antigen - specific cd8 + t cells ) found in colostrum [ 26 , 27 ] . for example , acta1 , flnc , and pax7 , which are either restricted to muscular tissues or involved in myogenic development and cellular differentiation [ 28 , 29 ] , are included in this group . according to the trajectory profile signature , 95 additional genes were found upregulated at all stages evaluated ( k6 : 2,2,2,2 ) . interestingly , most of the overexpressed genes in this group include general transcription and translation ( including spliceosome and protein folding ) machinery factors ( eif4a2 , eif2ak1 , etf1 , taf1 , ercc2 , sart1 , ppih , and dbr1 ) [ 30 , 31 ] as well as structural ( itga5 , actg1 , add2 , cldnd2 , rptn , and triobp ) and basal metabolic genes ( pank4 , agpat5 , cyp24a1 , and phyh ) . once the gene clusters were properly defined , identification of orthologous gene transcripts among the time course differentially expressed gene list subsets was critical for reliable comparison of gene function and subsequent determination of probably conserved transcriptional modules implicated in biological processes during the development of mammary tissue . according to genome comparative roundup orthologous database of a total of 448 transcripts upregulated and 371 downregulated , 213 ( upregulated ) and 183 ( downregulated ) genes were identified as orthologous to rat and/or human . the remainder of the genes was discarded or removed from subsequent analysis due to lack of similarity , insufficient information , or unknown identifiers . a complete list of orthologous genes from each dataset was compiled ( table s6 ) . among the upregulated orthologous genes to rat and/or human are those encoding to milk proteins , carbohydrate and lipid metabolism , transcriptional factors [ 17 , 34 ] , transduction of hormones , glutathione metabolism , and cell differentiation [ 19 , 20 ] . others are associated with stromal - epithelial communication , cell adhesion , lactogenesis , and general transcription and translation machinery factors as well as structural and basal metabolic genes [ 3335 ] . among those genes found significantly downregulated , several play an important role in the regulation of apoptosis , mediation of metastatic behavior ( epithelial - mesenchymal transition ) , or ubiquitin - mediated protein catabolism ( lysosome degradation ) [ 14 , 15 ] . also , genes restricted to muscular tissues or involved in myogenic development and cellular differentiation [ 28 , 36 ] are downregulated . taking into consideration their temporal expression profile signature and the fact that they represent different k - means cluster , 14 genes were selected for real - time pcr analysis ( table s2 ) . results show that the expression trends were consistent with the results from the microarray analysis . correlation analysis showed good agreement between real - time rt - pcr and microarray analysis . microarray results for all 14 genes tested were confirmed by real - time rt - pcr with regard to direction and significance of change ( figure 5 ) . conservation of functional blocks of genes is likely to be more important in a cross - species comparison . we found distinct blocks of significantly differentially expressed genes within different cytogenetic regions of the rat with homologous chromosomal segments in human and mouse . however , human , mouse , and rat have different chromosomal arrangements . genes in these blocks appear in contiguous cytogenetic regions , irrespective of species and chromosomal location . this finding is not surprising considering the close evolutionary distance between the species where 278 orthologous segments are reported to be shared between human and rat , and 280 segments are reported to be shared between human and mouse . it is proposed that these gene blocks may be significant for mammary gland development and maintenance and progression of lactation across human , rat , and mouse . for example , genes in the blocks may be coordinately expressed to share transcription programs as stated in previous studies . the argument may be made against the feasibility of using rodent data to draw inferences to human mammary gland gene expression . however , our objective in this study was to utilize the best available sources of information such as rat gene expression data during mammary development and mapping data to develop hypotheses on putative functional gene blocks conserved across species , underlying similarities despite reported differences in the architecture and hormonal control of mammary glands between rats and other rodents and between rats and humans [ 38 , 39 ] . in an effort to further characterize potential highly coregulated gene blocks , we combined transcription - factor binding - site prediction along the promoters of each gene member with the detection of expression profiles of annotated altered transcripts categorized as nucleic acid binding protein . several families of transcription factors were identified ( table s7 ) . for the most part , the presence of cis - elements found with core_tf ( http://www.lgtc.nl/core_tf ) in the promoters of the genes slc44a4 , ppt2 , and b3galt4 that compromises the conserved block d15 ( table s8 ) along with the cotranscription of mrnas that encoded for trans - regulator elements suggests that they are most likely modulated by transcription factors runx2 , creb3l4 , pou3f2 , and pou2af1 . correspondingly , the gene members of block u1 may possibly be coregulated by transcription factors stat5a , foxa1 , creb3l4 , and pou2af1 . in the same manner , other gene blocks ( u3 , u8 , u9 , d1 , d9 , and d14 , table s8 ) were found most likely co - regulated by a minor number of transcription factors ( foxa1 , creb3l4 , pou2af1 , or egr2 ) . hence , identification of conserved overrepresented upstream motifs unravels putative regulatory elements for transcription ( figure 6 ) in at least half of the gene block members reported in this study . consequently , these results strongly substantiate the maintenance of comparable transcriptional regulation programs among the predicted coexpressed modules . because cotranscription of genes in conserved blocks may allow concerted expression of gene products involved in the same response or pathway , integration of this type of analysis thus , the co - regulated clusters we proposed may indeed be conserved transcriptional modules through evolution , at least between rodents and primates . heterologous hybridization experiments on any microarray are of limited use for genes that have undergone rapid evolutionary change in coding regions , large rearrangements , and duplication . long oligonucleotide - based microarray platform may be more suitable for cross - species gene expression studies than a short oligonucleotide - based system . this comparative approach is based on the assumption that similar gene sequences in closely related species allow a reasonably reliable detection of many orthologous genes . for instance , according to several independent and unrelated studies carried out on comparable 50 to 60-mer oligonucleotide microarrays , cross - hybridization was observed only with genes with 50%75% overall sequence identity , respectively [ 44 , 45 ] . considering that orthologous genes between human and mouse and between human and rat both have a mean of ~85% sequence identity , validity of the results obtained in this study despite the problems encountered by csh in comparison to ssh seems upholding . in fact , the nucleotide sequence alignment confirmed an > 75.3% homology at least for the transcript members of the distinct gene blocks described , depending on the sequence evaluated among primates and rodents reinforcing the notion of attaining valid biological results . in addition , similar expression trends for distinct probe sets for one corresponding gene ( data not shown ) seem to largely substantiate the certainty and reproducibility of hybridization results obtained in this study . because of the challenges inherent to csh data , their confirmation by other techniques is essential . in addition to qrt - pcr , orthologous gene expression profiles with syntenic regions of rat , mouse , and human chromosomes reinforce another confirmation method that potentially substantiates the csh results obtained in this study . nonetheless , further validation of the results must be carried out by using csh of human rna to mouse oligonucleotide arrays . this study provides access to a prevalidated platform for analyzing transcriptional changes in rat mammary gland . this paper will hopefully spur an increase of mammary gland csh transcriptome analysis , thus adding to our knowledge base of this interesting evolutionary feature among mammals . however , although we acknowledge the multitude of aspects that can be elucidated by traditional ssh transcriptome analysis , we believe the biggest potential of the presented microarray lies in the multispecies - type studies described . we demonstrated that data analysis strategies such as the combination of orthologous gene expression profiles and chromosome mapping in conjunction with directed promoter transcription - factor binding - site prediction presented here can add strength to conclusions and help identify systems and responses that are conserved across the mammal taxa . the possibility of studying the evolutionary depth of transcriptional regulation adds a new dimension to comparative transcriptomic , particularly identification of differentially co - regulated gene blocks mapped to highly conserved syntenic chromosomal regions , which is important in mammary gland development using csh experiments among mammal species .

the mammary gland ( mg ) undergoes functional and metabolic changes during the transition from pregnancy to lactation , possibly by regulation of conserved genes . the objective was to elucidate orthologous genes , chromosome clusters and putative conserved transcriptional modules during mg development . we analyzed expression of 22,000 transcripts using murine microarrays and rna samples of mg from virgin , pregnant , and lactating rats by cross - species hybridization . we identified 521 transcripts differentially expressed ; upregulated in early ( 78% ) and midpregnancy ( 89% ) and early lactation ( 64% ) , but downregulated in mid - lactation ( 61% ) . putative orthologous genes were identified . we mapped the altered genes to orthologous chromosomal locations in human and mouse . eighteen sets of conserved genes associated with key cellular functions were revealed and conserved transcription factor binding site search entailed possible coregulation among all eight block sets of genes . this study demonstrates that the use of heterologous array hybridization for screening of orthologous gene expression from rat revealed sets of conserved genes arranged in chromosomal order implicated in signaling pathways and functional ontology . results demonstrate the utilization power of comparative genomics and prove the feasibility of using rodent microarrays to identification of putative coexpressed orthologous genes involved in the control of human mammary gland development .

methylmalonic acidemia ( mma ) encompasses a heterogeneous group of disorders that is characterized by impaired metabolism of methylmalonic acid that is generated during the metabolism of certain amino acids ( isoleucine , methionine , threonine , or valine ) . the incidence rate of mma is 1 in 50,00080,000 newborns but it is more common in countries with high amount of consanguinity and countries with no systematic newborn screening , like developing countries . patients typically presents at the age of 1-month to 1-year with varied presentations of symptoms ranging from poor feeding , vomiting , dehydration , shock , hypoglycemia , hyperammonemia and hyperglycemias with high anion gap ( ag ) metabolic acidosis if left untreated can lead coma or even death . mma may present suddenly in older infants without initial apparent symptoms , which may mimic septic shock and diabetic ketoacidosis ( dka ) and without early recognition can lead fatal consequences . here we are reporting two cases of mma in infants presented with severe high ag metabolic acidosis mimicking as dka in one case and septic shock in an another case even without any initial apparent symptoms . we report an 8-month - old male child presented in an emergency department with hypotensive shock , respiratory failure and disseminated intravascular coagulation with a short history of nonspecific low grade fever associated with upper respiratory infection and vomiting since 1-day . his initial investigation showed : hemoglobin ( hb)-6.0 g / dl , total leukocyte count ( tlc)-4500 with 55% neutrophil , platelet counts 65/nl , c - reactive protein ( crp)-10 mg / dl , protein thiolation index / activated partial thromboplastin time - no clot , international normalized ratio-9 , serum glutamic - oxaloacetic transaminase / prothrombin time - 65/42 and arterial blood gas was ph-7.0 , hco3 - 2.4 , lactate-2.2 , ag-28 , blood sugar-122 mg / dl , urine analysis 4 + ketones . blood culture was sterile and cerebrospinal fluid ( csf ) examination done after stabilization came normal . ketoacidosis remain persisted even after the 24 h of reversal of shock requiring sodium bicarbonate infusion . the child was conservatively managed with fluids , inotropes , fresh frozen plasma , pack cell transfusion , sodium bicarbonate , antibiotics , low protein diet , methylcobalamin , and carnitine . the child responded well to treatment and was extubated after 72 h of admission was shifted out of pediatric intensive care unit ( picu ) on 5 day . the child was discharged on 10 day on oral b12 supplementation and low protein diet . we report a 2 case of previously healthy 1-year - old male child with altered sensorium with a short history of vomiting and low grade fever with no history of seizures . on examination child was comatose with glasgow coma scale score 9 , kussmaul breathing with signs of dehydration . his initial investigation showed hb-8.4 g / dl , tlc-15,200/cmm with 70% neutrophils , platelet counts - 720/nl , crp - 12 mg / dl , blood gas ph - 7.119 , hco3 - 3.7 , ag - 44 , lactate 1.04 , blood sugar - 485 mg / dl , urine 4 + ketones . an initial diagnosis of dka was made , and stranded treatment with fluids and insulin infusion was started . other causes of poor response to insulin e.g. ; dose , administration , and infection were ruled out . persistence of severe high ag metabolic acidosis after 24 h of admission with hemoglobin a1c 4.9% , underlying metabolic disorder was suspected and was investigated to rule out organic acidemia and had high levels of methyl malonic acid . insulin was discontinued and started on b12 , carnitine , low protein diet and sodium bicarbonate and the child responded well to treatment , acidosis was corrected after 24 h and he was shifted out of picu on 5 day . we report an 8-month - old male child presented in an emergency department with hypotensive shock , respiratory failure and disseminated intravascular coagulation with a short history of nonspecific low grade fever associated with upper respiratory infection and vomiting since 1-day . his initial investigation showed : hemoglobin ( hb)-6.0 g / dl , total leukocyte count ( tlc)-4500 with 55% neutrophil , platelet counts 65/nl , c - reactive protein ( crp)-10 mg / dl , protein thiolation index / activated partial thromboplastin time - no clot , international normalized ratio-9 , serum glutamic - oxaloacetic transaminase / prothrombin time - 65/42 and arterial blood gas was ph-7.0 , hco3 - 2.4 , lactate-2.2 , ag-28 , blood sugar-122 mg / dl , urine analysis 4 + ketones . blood culture was sterile and cerebrospinal fluid ( csf ) examination done after stabilization came normal . ketoacidosis remain persisted even after the 24 h of reversal of shock requiring sodium bicarbonate infusion . the child was conservatively managed with fluids , inotropes , fresh frozen plasma , pack cell transfusion , sodium bicarbonate , antibiotics , low protein diet , methylcobalamin , and carnitine . the child responded well to treatment and was extubated after 72 h of admission was shifted out of pediatric intensive care unit ( picu ) on 5 day . the child was discharged on 10 day on oral b12 supplementation and low protein diet . we report a 2 case of previously healthy 1-year - old male child with altered sensorium with a short history of vomiting and low grade fever with no history of seizures . on examination child was comatose with glasgow coma scale score 9 , kussmaul breathing with signs of dehydration . his initial investigation showed hb-8.4 g / dl , tlc-15,200/cmm with 70% neutrophils , platelet counts - 720/nl , crp - 12 mg / dl , blood gas ph - 7.119 , hco3 - 3.7 , ag - 44 , lactate 1.04 , blood sugar - 485 mg / dl , urine 4 + ketones . an initial diagnosis of dka was made , and stranded treatment with fluids and insulin infusion was started . other causes of poor response to insulin e.g. ; dose , administration , and infection were ruled out . persistence of severe high ag metabolic acidosis after 24 h of admission with hemoglobin a1c 4.9% , underlying metabolic disorder was suspected and was investigated to rule out organic acidemia and had high levels of methyl malonic acid . insulin was discontinued and started on b12 , carnitine , low protein diet and sodium bicarbonate and the child responded well to treatment , acidosis was corrected after 24 h and he was shifted out of picu on 5 day . we report an 8-month - old male child presented in an emergency department with hypotensive shock , respiratory failure and disseminated intravascular coagulation with a short history of nonspecific low grade fever associated with upper respiratory infection and vomiting since 1-day . his initial investigation showed : hemoglobin ( hb)-6.0 g / dl , total leukocyte count ( tlc)-4500 with 55% neutrophil , platelet counts 65/nl , c - reactive protein ( crp)-10 mg / dl , protein thiolation index / activated partial thromboplastin time - no clot , international normalized ratio-9 , serum glutamic - oxaloacetic transaminase / prothrombin time - 65/42 and arterial blood gas was ph-7.0 , hco3 - 2.4 , lactate-2.2 , ag-28 , blood sugar-122 mg / dl , urine analysis 4 + ketones . blood culture was sterile and cerebrospinal fluid ( csf ) examination done after stabilization came normal . ketoacidosis remain persisted even after the 24 h of reversal of shock requiring sodium bicarbonate infusion . the child was conservatively managed with fluids , inotropes , fresh frozen plasma , pack cell transfusion , sodium bicarbonate , antibiotics , low protein diet , methylcobalamin , and carnitine . the child responded well to treatment and was extubated after 72 h of admission was shifted out of pediatric intensive care unit ( picu ) on 5 day . the child was discharged on 10 day on oral b12 supplementation and low protein diet . we report a 2 case of previously healthy 1-year - old male child with altered sensorium with a short history of vomiting and low grade fever with no history of seizures . on examination child was comatose with glasgow coma scale score 9 , kussmaul breathing with signs of dehydration . his initial investigation showed hb-8.4 g / dl , tlc-15,200/cmm with 70% neutrophils , platelet counts - 720/nl , crp - 12 mg / dl , blood gas ph - 7.119 , hco3 - 3.7 , ag - 44 , lactate 1.04 , blood sugar - 485 mg / dl , urine 4 + ketones . an initial diagnosis of dka was made , and stranded treatment with fluids and insulin infusion was started . other causes of poor response to insulin e.g. ; dose , administration , and infection were ruled out . persistence of severe high ag metabolic acidosis after 24 h of admission with hemoglobin a1c 4.9% , underlying metabolic disorder was suspected and was investigated to rule out organic acidemia and had high levels of methyl malonic acid . insulin was discontinued and started on b12 , carnitine , low protein diet and sodium bicarbonate and the child responded well to treatment , acidosis was corrected after 24 h and he was shifted out of picu on 5 day . methylmalonic acidemia is a rare autosomal recessive disease in which there is a deficiency in conversion of methylmalonic coenzyme a ( coa ) to succinyl coa . mma appears to be more common than other organic academia perhaps because it has several underlying causes . affected infants present in the first few days of life with vomiting , respiratory distress , feeding intolerance , lethargy , and severe ketoacidosis , which , if not aggressively treated , often progresses rapidly to coma and death . it occurs in older children who usually have low levels of methyl malonic acid in blood and urine and have normal growth and development . these children present intermittently with acidotic crises and are otherwise normal during crisis - free periods . increased levels of organic acids including metabolic disease must always be considered as possible diagnosis when an infant presents with a severe metabolic acidosis accompanied by an increased ag and other causes of increased ag metabolic acidosis with increased osmolar gap , e.g. drug ingestion should be ruled out . we report two cases of infants with mma with sudden decompensation associated with high ag severe metabolic acidosis without any initial signs and symptoms . in first case infant with presented as mimic septic shock responded well to fluid management , but ketoacidosis was persisted even after the shock was corrected . an underlying metabolic disorder was suspected and had very high levels of methylmalonic acids in urine . in another case infant presented with hyperglycemic ketoacidosis with poor response to insulin . because of persistence of ketoacidosis an underlying metabolic disorder was considered . although hyperglycemia is an unusual presentation for mma , boeckx and hicks , guven et al . and kumar and suthar reported cases with severe and persistent metabolic acidosis and hyperglycemia , despite large doses of insulin . dka is most common cause of ketoacidosis , but it shows excellent response to standard treatment ; therefore other etiologies of acidosis / hyperglycemia should be investigated in poor responders . organic acidurias ( oas ) should be included in the differential diagnosis especially in countries where national newborn screening is not implemented . determining the etiology of hyperglycemic ketoacidosis is important and can lead to a good outcome . the unusual presentation of our patients , mimicking dka and septic shock reminds us of the wide spectrum of clinical signs of organic acidemia . in very young patients with severe acidosis and metabolic decompensation , or with atypical clinical course should lead a suspicion of a less common diagnosis such as organic academia to prevent severe morbidities and even death .

methylmalonic acidemia ( mma ) is most common inherited type of organic acidemia . it has diverse presentation in older infants without any initial apparent symptoms . mma sometimes present with sudden metabolic decompensation , which may mimics common emergencies like septic shock and diabetic ketoacidosis ( dka ) without early recognition can be fatal . in born error of metabolism especially organic acidemia should be suspected in any infant presented with severe high anion gap metabolic acidosis . we report two cases of mma in infants presented acutely mimicking dka and septic shock .

the ultrastructural localization of a molecule requires that a certain degree of electron - density , resulting in adequate electron scattering , be present in sufficient amount . binding of heavy atoms can be accomplished easily with regards to proteins , nucleic acids and other cellular components , in some cases with a high degree of specificity . however , the sub - cellular localization of ions is a challenge , since in many cases ions are diffusible and not easily kept in place after the procedures of sample preparation for electron microscopy ( em ) . many ions can offer enough scattering in spite of not being heavy metals , such as manganese , while others necessitate of blocking or precipitating techniques to be visualized . in particular , calcium ions are easily diffusible inside the cell , and even when protein - bound are not easily detectable . the pioneering paper by tandler et al . demonstrated the presence of calcium ions by em via pyroantimonate precipitation , with a good yield of the final product . the end product of the reaction , however , is probably overrated , since the technique is capable of stabilizing and precipitating calcium and other bivalent cations , such as magnesium , as proved by electron spectroscopic imaging ( esi ) analysis . since their introduction by tsien and coworkers , calcium studies have used specific ca - sensitive fluorochromes , such as fura-2 , indo-1 and fluo-4 , coupled to wide - field digitized video fluorescence microfluorimetry and confocal microscopy to image acute elevations in intracellular ca concentration ( [ ca]i ) with high spatio - temporal resolution . cytosolic and nuclear ca signals regulate a multitude of vital cellular processes , such as gene transcription , cell cycle progression , cytoskeletal and chromatin remodelling , and migration . however , proper ca handling within the endoplasmic reticulum ( er ) , the most abundant intracellular ca reservoir , is also crucial for cell - fate decisions . the steady - state er ca level ( [ ca]er ) does not only control synthesis , folding , export and trafficking of proteins , but it also fuels mitochondrial bioenergetics thereby suppressing autophagy . low affinity ca indicators , such as mag - fura 2 , and genetically - encoded probes , such as the bioluminescent protein aequorin and the gfp - based fluorophores camgaroos , pericams , and cameleons , have been devised to assess er ca levels .. nevertheless , these sophisticated tools do not provide any reliable information on ca distribution within the heterogeneous structure of the er . we propose in this paper a tool for a rapid high - resolution detection of calcium ions which can be used in parallel with other techniques . maranto and sandell and masland have proposed the possibility of photoconverting fluorochromes into an electrondense end product . the technique utilizes the photo - oxidation of diaminobenzidine ( dab ) in presence of the emission of an excited fluorochrome , and it has been shown to be efficient on different dyes and conditions . this method combines the specificity of available fluorophores to the high spatial resolution offered by transmission em to detect fluorescing molecules even when present in low amounts in membrane - bounded organelles . the present manuscript sought to exploit dab photo - oxidation of mag - fura 2 for visualizing er ca distribution in hela cells , which are among the most widespread model cell lines used for studying intracellular ca signaling . hela cells have been cultured in iscove s modified dulbecco s medium ( imdm , lonza , basel , switzerland ) supplemented with 10% fetal bovine serum , 2% l - glutamine , 1% penicillin and streptomycin , at 37c air atmosphere with 5% co2 . after reaching 90% confluence , cells have been plated in 6 wells cell culture plate ( 1x10 cells per well ) , or in chamber slides for staining and microscopic examination ( 8x10 cells per well ) . the day after seeding , when cells reached about 90% confluence , the medium has been removed from the wells and replaced with three solutions , one for each experimental condition . the solutions , different in ca concentration , are the following : physiological salt solution ( pss ) , with the following composition : 150 mm nacl , 6 mm kcl , 1.5 mm cacl2 , 1 mm mgcl2 , 10 mm glucose , and 10 mm hepes ; ca - free solution ( 0 ca ) , where ca has been substituted by 2 mm nacl , and 0.5 mm egta was added , whereas the solution with ca more concentrated is similar to pss in composition except for 120 mm nacl and 20 mm cacl2 . all the solutions have been brought to ph 7.4 with an osmolality of 338 mmol / kg , measured with an osmometer ( wescor 5500 ; wescor , inc . , logan , ut , usa ) . intracellular ca deposits have been marked by fluorescent dye - based calcium indicator mag - fura-2 , acetoxymethyl ( am ) ester uv light - excitable ( life tchnologies , carslbad , ca , usa ) , added to the three calcium solutions to obtain a final concentration of 5 m . cells have thus been incubated at 37c in the dark , for 1 h. cells cultured on chamber slides , and treated as hereto described , have been analyzed at fluorescence microscopy . an olympus bx51 microscope ( olympus italia srl , segrate , italy ) was used under the following conditions : 100-w hg lamp as the excitation source , 330- to 385-nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) ; 100 olympus uplanfl oil - immersion objective lens ( na 1.25 ) . images were recorded with an olympus magnifier digital camera system , and stored by the olympus cellf software . instead , cells in plates have been fixed at room temperature for 20 minutes with a fixative solution ( 2% paraformaldehyde and 1% glutaraldehyde in pbs ) . meanwhile , a solution with dab has been freshly prepared as follows : 20 mg of dab ( sigma aldrich , st . louis , mo , usa ) were dissolved in 1 ml dmso , briefly vortexed and then brought to 30 ml with pbs . thereafter , cells have been washed twice with pbs and incubated for 3 h under a germicidal uv lamp ( sankyo denki , kanagawa , japan ; g30t8 , uv - c ) with 2 ml dab solution which is replaced with fresh solution every half an hour . in parallel , some cells were incubated in the absence of mag - fura 2 , treated with dab and processed as above . moreover , to exclude a possible interference by aldehyde groups , some slides were fixed and then treated with 0.5 m nh4cl in pbs for 30 min at 4c , then incubated with dab and processed as above . fixed and photoconverted cells have been washed twice with distilled water and postfixed with 2% osmium tetroxide in water for 1 h , at room temperature . once the osmium was removed and the cells rinsed several times with distilled water , cells have been scraped , centrifuged discarding supernatant , dehydrated in a graded series of ethanol and embedded in lrwhite resin ( sigma aldrich ) . ninety nm ultrathin sections have been cut by a reichert ultramicrotome , transferred to grids without membrane and visualized by a zeiss em900 transmission electron microscope operating at 80kv . hela cells have been cultured in iscove s modified dulbecco s medium ( imdm , lonza , basel , switzerland ) supplemented with 10% fetal bovine serum , 2% l - glutamine , 1% penicillin and streptomycin , at 37c air atmosphere with 5% co2 . after reaching 90% confluence , cells have been plated in 6 wells cell culture plate ( 1x10 cells per well ) , or in chamber slides for staining and microscopic examination ( 8x10 cells per well ) . the day after seeding , when cells reached about 90% confluence , the medium has been removed from the wells and replaced with three solutions , one for each experimental condition . the solutions , different in ca concentration , are the following : physiological salt solution ( pss ) , with the following composition : 150 mm nacl , 6 mm kcl , 1.5 mm cacl2 , 1 mm mgcl2 , 10 mm glucose , and 10 mm hepes ; ca - free solution ( 0 ca ) , where ca has been substituted by 2 mm nacl , and 0.5 mm egta was added , whereas the solution with ca more concentrated is similar to pss in composition except for 120 mm nacl and 20 mm cacl2 . all the solutions have been brought to ph 7.4 with an osmolality of 338 mmol / kg , measured with an osmometer ( wescor 5500 ; wescor , inc . , logan , ut , usa ) . intracellular ca deposits have been marked by fluorescent dye - based calcium indicator mag - fura-2 , acetoxymethyl ( am ) ester uv light - excitable ( life tchnologies , carslbad , ca , usa ) , added to the three calcium solutions to obtain a final concentration of 5 m . cells have thus been incubated at 37c in the dark , for 1 h. cells cultured on chamber slides , and treated as hereto described , have been analyzed at fluorescence microscopy . an olympus bx51 microscope ( olympus italia srl , segrate , italy ) was used under the following conditions : 100-w hg lamp as the excitation source , 330- to 385-nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) ; 100 olympus uplanfl oil - immersion objective lens ( na 1.25 ) . images were recorded with an olympus magnifier digital camera system , and stored by the olympus cellf software . instead , cells in plates have been fixed at room temperature for 20 minutes with a fixative solution ( 2% paraformaldehyde and 1% glutaraldehyde in pbs ) . meanwhile , a solution with dab has been freshly prepared as follows : 20 mg of dab ( sigma aldrich , st . louis , mo , usa ) were dissolved in 1 ml dmso , briefly vortexed and then brought to 30 ml with pbs . thereafter , cells have been washed twice with pbs and incubated for 3 h under a germicidal uv lamp ( sankyo denki , kanagawa , japan ; g30t8 , uv - c ) with 2 ml dab solution which is replaced with fresh solution every half an hour . in parallel , some cells were incubated in the absence of mag - fura 2 , treated with dab and processed as above . moreover , to exclude a possible interference by aldehyde groups , some slides were fixed and then treated with 0.5 m nh4cl in pbs for 30 min at 4c , then incubated with dab and processed as above . fixed and photoconverted cells have been washed twice with distilled water and postfixed with 2% osmium tetroxide in water for 1 h , at room temperature . once the osmium was removed and the cells rinsed several times with distilled water , cells have been scraped , centrifuged discarding supernatant , dehydrated in a graded series of ethanol and embedded in lrwhite resin ( sigma aldrich ) . ninety nm ultrathin sections have been cut by a reichert ultramicrotome , transferred to grids without membrane and visualized by a zeiss em900 transmission electron microscope operating at 80kv . calcium labeling with mag - fura 2 results in highly fluorescent staining mainly localized in the cytoplasm . after incubation of the cells with 20 mm ca for 1 h , the signal is present in large vesicles in the cytoplasm and around the cell nucleus , as well as in elongated structures probably related to mitochondria ( figure 1a ) . the faint signal visible in the nucleus is very probably due to the superposition of the cytoplasmic layer surrounding it . when the cells are incubated in pss , the signal is lower ( figure 1b ) and mostly present in the perinuclear vesicles , while the cytoplasmic signal far from the nucleus somehow disappears . in the last condition , without calcium and in the presence of egta , some labeling is still visible , although to a much reduced level ( figure 1c ) . this continuous presence of ca represents very probably the last ions the cell can not allow to lose , a sort of surviving threshold concentrations . supplementary figure 1 confirms that altering extracellular ca concentrations results in a consequent change in er ca homeostasis , which may have important consequences also on cytosolic ca dynamics . at em level , the controls did not show any electron dense precipitates , both in the absence of the fluorochrome or after aldehyde blockade ( not shown ) ; consequently , the end product obtained in the mag - fura 2 stained cells is due to the fluorochrome photoconversion . the dab photoconversion is an efficient way to stabilize the ions and to visualize them . it must be underlined here that we observe a thin section , 60 - 80 nm thick , corresponding , consequently , only to a fraction of the fluorescent signal . after pss incubation , the cells show a distinctive positivity ( figure 2a ) . in some cases it is possible to see the presence of precipitates without the direct association with smooth or rough er ( ser or rer , respectively ) membranes , likely related to unbound ca ions ( figure 2b ) , and electron dense material inside the vesicles is also positive ( figure 2c ) . a possible suggestion of ca internalization or extrusion is shown in figure 2 d , e , where the deposits are present on the outer membrane . when the cells are incubated without calcium and in the presence of egta , the deposits are extremely reduced , although still present on the outer cell membrane , suggesting the presence of an extruding mechanism ( figure 2f ) . on the contrary , the diffuse , low density end product is almost absent from the cytoplasm . at higher ca concentration in the medium ( figure 3a ) , the dark precipitate is detectable on the inner membrane of cytoplasmic vesicles possibly pertaining to ser , and in discrete foci with no apparent association with vesicles or membranes . the deposits are present within electron dense vesicles ( figure 3b ) and electron lucent structure , in some cases associated , and suggesting the idea of passage of ions from one compartment to another . in other cases , dab - positive material is present on the membranes of complex systems such as multivesicular bodies ( figure 3c ) or on the outer membranes of cytosolic vesicles ( figure 3d ) . interestingly , calcium deposits are detectable on the outer cell membrane ( figure 3e ) in spots , localized and separated , suggesting the presence of calcium channels . in figure 3f , the end product of the dab photoconversion is a speckled , dense precipitate of varying size . however , another abundant end product is present as small sized dots , showing a markedly lower electrondensity ( figure 3f , inset ) and a widespread distribution . we show in this paper that mag - fura 2 photoconversion via dab oxidation is an efficient way for localizing ca ions at em level . moreover , this could be considered also as a means of stabilizing their presence , thus limiting the loss of ions . as for the method , photoconversion is easily carried out and reproducible , can be obtained on a good amount of cells , since the exposition in our conditions is not limited to the direct irradiation of the sample via an objective but obtained with a germicide lamp . the end product is sufficiently electron dense to be detected clearly when present in sufficient amount within a membrane boundary . in these conditions , the blackness of the product stands out from the background . in our conditions , in fact , ca ions are immediately detectable within the vesicles and the tubules of the ser , while it is much more difficult to see their presence when in the cytosol , due to their low electrondensity . this is a clear advantage over the conventional imaging of mag - fura 2 fluorescence that must be removed by the cytosol in order to study truly er signals . this is achieved by permeabilizing the plasma membrane with brief applications of antibiotics or detergents , but this procedure may destroy intracellular signaling pathways , dilute cytosolic modulators of er ca release / sequestration , and disassemble the cytoskeletal elements that maintain the contact between cell membrane and er . alternatively , the whole - cell configuration of the patch - clamp technique could be exploited to remove the dye , but this experimental approach is rather expensive and challenging , requires highly skilled electrophysiologists and has a quite low throughput . the final yield of photoconverted mag - fura 2 is in direct relation with the amount of ca present in the sample considered . in our condition , the ca - enriched medium gave a higher end product staining than the normal physiological conditions or than ca deprivation . it is interesting that in several cases , dense precipitates could be found in areas suggesting the local accumulation of ca nearby the mouth of plasmalemmal ca channels . accordingly , such precipitates are evident when extracellular ca concentration is increased , which is consistent with the notion that constitutive ca inflow from the extracellular milieu is enhanced . this unexpected outcome of dab photoxidation is extremely relevant in light of the master role served by local ca microdomains generated by extracellular ca influx , which regulate cell functions as diverse as cycle progression , proliferation , migration , nitric oxide production , and adenylyl cyclase activity . one thing that should be underlined here is that we could not visualize the ser tubule filled with the end product . this could be due to three different causes : i ) ca can be dispersed ( lost ) in the conditions used here ; ii ) local intraluminal ca concentration is less than expected ( which is less probable as it is unlikely to be lower than 100 m ) ; or iii ) dab photoconversion is not efficient enough for mag - fura . by comparing fluorescence images with em micrographs , it is clear the difference in the signal coming from vesicles and the elongated filamentous structures , the latter being much less fluorescent . the relative low yield when comparing fluorescence images is definitely related to the extremely lower amount of end product which can be found within a 60 - 80 nm thin section in relation to the fluorescence originating from the whole cell . in addition , albeit [ ca]er fluctuates between 100 and 500 m , ca accumulates in specific regions of the er that may be missed in our sections . moreover , releasable ca is actually trapped by oligomers of the ca - binding proteins calnexin and calreticulin : these form a concealed ca source that is not in rapid equilibrium with the [ ca]er , but is easily accessible to ca - releasing channels upon extracellular stimulation . thus , the lack of ser tubules or cisternae filled with the end product of the photo - conversion , i.e. , ca , is not surprising . finally , the photoconversion process seems to be efficient , and gives an end product more reasonable than the precipitation technique utilized , for instance , by tandler et al . in the latter case , an important amount of precipitates were found in the cell and especially in the nucleus ; our method gives a much finer end product , and this allows a superior resolution and localization . in conclusion , this method is a reliable and efficient technique for stabilizing and visualizing ca ions at em . it gives a high resolution end product thus allowing the fine structural ca localization . in principle , this approach could provide important qualitative information about the amount and sub - cellular distribution of calcium ions both in cultured cells and , more importantly , in ex vivo samples , which are far less amenable to high resolution ca imaging of er ca levels .

we propose a tool for a rapid high - resolution detection of calcium ions which can be used in parallel with other techniques . we have applied a new approach by photo - oxidation of diaminobenzidine in presence of the emission of an excited fluorochrome specific for calcium detection . this method combines the selectivity of available fluorophores to the high spatial resolution offered by transmission electron microscopy to detect fluorescing molecules even when present in low amounts in membrane - bounded organelles . we show in this paper that mag - fura 2 photoconversion via diaminobenzidine oxidation is an efficient way for localizing ca2 + ions at electron microscopy level , is easily carried out and reproducible , and can be obtained on a good amount of cells , since the exposure in our conditions is not limited to the direct irradiation of the sample via an objective but obtained with a germicide lamp . the end product is sufficiently electron dense to be detected clearly when present in sufficient amount within a membrane boundary .

presence of associated anomalies suggests a developmental origin of the arachnoid cysts . we present a case of a 6-year - old boy who presented with paraparesis and a brief review of the etiology and pathogenesis in relevance to clinical decision making is also presented . a 6-year - old boy presented to us with complaints of recent onset difficulty in walking and dribbling of urine . on examination he had grade 3/5 power in both lower limbs and absent reflexes . an mri spine was performed which revealed a clearly defined intradural cystic lesion extending from l2 to s2 which was hypointense on t1-weighted images [ figure 1 ] and hyperintense on t2-weighted images [ figure 2 ] with tethering of cord . a tentative diagnosis of lumbosacral arachnoid cyst was made and the patient was taken up for surgery . multiple level laminectomies were performed and on opening the dura a tense cyst was found [ figure 4 ] along with short and thickened filum terminale . there were no other associated lesions . postoperatively the child 's motor power began to improve though his bladder was kept catheterized . postoperative mri was done which showed complete resolution of cyst [ figure 5 ] . at follow - up of 2 months patient t1-weighted mri showing hypodense intradural cystic lesion extending from l2 to s2 t2-weighted image showing hyperintense lesion syrinx in the cervicodorsal spine intra operative photograph showing tense cyst on opening the dura postoperative mri showing complete decompression of cyst arachnoid cysts of the spine are collections of cerebrospinal fluid ( csf ) within protrusions of arachnoid that can occur in a perineural , extradural , intradural , or intra - extradural site . these lesions are most often located posterior to the spinal cord but have also been identified anteriorly . the thoracic spine is the most common location followed by lumbosacral and cervical spine . spinal arachnoid cysts are equally prevalent in men and women usually presenting in the fourth and fifth decades of life . rarely they may present in children with other anomalies thus lending support to congenital etiology of spinal arachnoid cysts . idiopathic arachnoid cysts in children are associated with neural tube defects and in adults with spinal deformities . type 1 included extradural cysts , type 2 included cysts involving nerve root also known as tarlov perineural cyst and type 3 consisted of intradural cysts . intradural cysts are outpouchings of arachnoid that , regardless of size , lie entirely within the dural space . lumbosacral arachnoid cysts are believed to occur as a disorder of secondary neurulation . while patients with arachnoid cysts / meningoceles are usually neurologically normal , 90% of patients with meningocele have associated occult spinal lesions such as tight filum terminale , split cord malformation , and epidermoids . alterations in the arachnoid trabeculae that overlie the spinal cord are probably the root cause . these alterations may be a congenital or may be caused by inflammation from previous trauma , surgery , or subarachnoid hemorrhage . in some cases no cause can be established , it has been hypothesized that these lesions develop from the septum posticum of schwalbe which is an arachnoid membrane dividing the midline posterior cervical and thoracic subarachnoid space . but such a hypothesis does not explain cysts occurring anterior to the cord . according to the hydrodynamic theory normal pulsations of the csf dilate weak areas of arachnoid which then progressively enlarge and form a cyst . a ball valve effect at the neck of the diverticulum is probably responsible for the progressive enlargement of the cyst . these pockets of fluid lead to further disruption of normal pulsatile csf flow and are capable of expanding and developing into cysts . most spinal arachnoid cysts are asymptomatic and are discovered incidentally on magnetic resonance ( mri ) . mri is the most sensitive and specific study for detecting a spinal arachnoid cyst and for assessing the extent of the cyst wall . the signal intensity of arachnoid cyst fluid is the same as csf on t1 and t2 images . sometimes higher signal intensity is seen on t2 which maybe related to increased protein content of sequestered fluid or absence of motion effects . diffusion - weighted mri not only helps differentiate the arachnoid cyst from epidermoid cyst , abscess , or tumors but also evaluates spinal cord atrophy and inflammatory changes . although ct myelography has been used to establish or refute communication between the intraspinal subarachnoid space and the arachnoid cyst , it is more sensitive in determining whether a communication exists rather than locating the communication . 12 kinematic mri ( cine - mri ) helps locate the communication which appear as pulsating turbulent flow voids . the presentation of sacral arachnoid cysts varies from long standing low back pain to neurological deficits such absent deep tendon reflexes or bowel bladder abnormalities . radicular pain if present is relieved by lying down which pushes fluid out of the cyst . the treatment decision needs consideration of the extent of the cyst , the point at which the spinal cord is maximally compressed and presence or absence of communication between cyst and subarachnoid space . aspiration under mri guidance is advised for small cysts which have no communication with subarachnoid space . for moderate - sized cysts complete excision is advised and multiseptated cysts extending over several segments , cyst fenestration can be done to avoid extended laminectomy . percutaneous cyst drainage can be tried as a temporary measure to decrease symptoms or as a test of success of operative management . the success of the treatment modality employed ultimately depends on the degree of correlation of the mri findings with the patient 's symptoms . on histopathology , the walls of arachnoid cysts are usually seen as fibrous and lined by meningothelial cells . these cells are usually negative for gfap , s-100 , transthyretin ( prealbumin ) , and cea staining thus differentiating them from epithelial cysts . surgery typically results in excellent outcomes in terms of resolution of symptoms , and is effective across a large range of cyst sizes . arachnoid cysts of the spine may occasionally be encountered in the work up of neurological symptoms or incidentally . it is important to be aware of the possibilities of other congenital anomalies of the spine and investigate for the same . for good surgical outcome

arachnoid cysts are cerebrospinal fluid collections in the spine that can present with neurological symptoms or be discovered accidentally . intradural location of such cysts especially in the lumbosacral region is relatively rare . the association of such cysts with other congenital anomalies such as tethered cord lends evidence to the developmental origin of arachnoid cysts . we report a case of lumbosacral arachnoid cyst with tethered cord in a 6-year - old male child and discuss the etiopathogenesis and management options .

a total of 130 consecutive p. aeruginosa isolates recovered from different clinical specimens at sanjay gandhi postgraduate institute of medical sciences in lucknow , uttar pradesh , india , during november 2013april 2014 were included in the study ; all specimens were collected from within the state ( figure 1 ) . aeruginosa isolates were identified by standard microbiological techniques ( 6 ) and further confirmed by phoenix automated identification and sensitivity systems ( bd biosciences , san jose , ca , usa ) . the drug susceptibility profile was interpreted by using clinical and laboratory standards institute breakpoints ( 7 ) . these isolates were subjected to pcr by using blandmspecific primers ( 8) followed by amplicon sequencing . the isolates were further screened for high - level aminoglycoside resistance by their ability to grow on muller hinton agar containing amikacin and gentamicin 256 mg / l each as a marker for 16s rmtase ( 3 ) . a total of 33 ( 86.84% ) isolates were positive for high - level aminoglycoside resistance . each of these isolates were further subjected to pcr for detection of 16s rmtases ( arma and rmta rmth ) by using primers and conditions described previously ( 25 ) ; 17 ( 51% ) isolates were positive for 16s rmtases . their distributions were as follows : arma in 6 ( 18% ) ; rmtb in 4 ( 12% ) ; arma + rmtb in 4 ( 12% ) ; rmtc in 2 ( knpa1a and knpa1c ) ( 6% ) ; and rmtc + rmtf in 1 ( knpa1b ) ( 3% ) . knpa1a , knpa1b , and knpa1c were further characterized ; sequence analysis of amplicons confirmed rmtc with 100% nucleotide identity originally described in proteus mirabilis strain ars68 from japan ( 9 ) and assigned embl / genbank nucleotide accession nos . mics of the 3 isolates for different aminoglycosides , -lactams , -lactam/-lactamase inhibitor combinations , carbapenems , and colistin are provided in the table . location of uttar pradesh state , india , showing geographic location of patients infected with 16s rrna methyl transferase positive pseudomonas aeruginosa ( gray shading ) and rmtc - positive isolates knpa1a , knpa1b , and knpa1c ( black dots ) ; knpa1b was also positive for rmtf . knpa1a was isolated from surgical drainage from a woman , 59 years of age , who had hypertension and underwent an abdominal hysterectomy for cervical carcinoma , followed by external beam radiotherapy . her condition stabilized , and she was discharged with advice for repair of the fistula , but she did not return for further treatment . during hospitalization , she received multiple antimicrobial drugs . knpa1b was isolated from endoscopic nasobiliary drainage ( enbd ) collected from a man , 57 years of age , who had extrahepatic biliary obstruction as a complication of hilar cholangiocarcinoma . stent block and fever occurred , necessitating a repeat of the procedure and drainage of fluid . he was discharged with the drainage tube in situ and was advised to return for surgery . knpa1c was isolated from a man , 68 years of age , who had diabetes mellitus and hypertension . he had stricture of the urethra and meatal narrowing after having a transurethral prostate resection . a urinary tract infection was diagnosed , and p. aeruginosa ( knpa1c ) was recovered from urine . the patient received piperacillin / tazobactam and colistin combination therapy ; urine culture was sterile on day 3 posttreatment . resistance genes such as metallo--lactamases ( e.g. , imp , vim , sim , gim , spm ) and extended - spectrum -lactamases such as tem , shv , ctx - m , and ampc were detected by using pcr ( 8) ( table ) . to study genetic relatedness among the 3 isolates , genomic dna in agarose blocks was separated on 1.0% agarose gels in 0.5 tris - borate - edta buffer with the chef ii d - mapper xa pulsed - field gel electrophoresis system ( bio - rad , hercules , ca , usa ) following standard conditions ( 10 ) . multilocus sequence typing ( mlst ) was done according to protocols described in the pseudomonas aeruginosa mlst database ( http://pubmlst.org/paeruginosa ) . seven chromosomal genes were pcr amplified and sequenced ; the sequences were compared with those on the mlst database to determine allele numbers and sequence types ( sts ) . knpa1a , knpa1b , and knpa1c belonged to st764 , st902 , and st880 , respectively . * caz , ceftazidime ; ctx , cefotaxime ; fep , cefepime ; atm , aztreonam ; cps , cefoperazone / sulbactam ; tzp , piperacillin / tazobactam ; ipm , imipenem ; mer , meropenem ; col , colistin ; ak , amikacin ; g , gentamicin . lane 1 , ladder ; 2 , knpa1a ; 3 , knpa1b ; 4 , knpa1c . b ) chromosomal location of rmtc , rmtf , and blandm-1 genes by i - ceui - digested genomic dna of p. aeruginosa isolates . lane 5 , knpa1a ; 6 , knpa1b ; 7 , knpa1c ; smears show southern blot analysis of genomic dna with probes specific to 16s rrna , rmtc , rmtf , and ndm-1 genes . isecp1 was previously shown to promote both expression and transposition of rmtc ( 11 ) ; hence , to assess association of isecp1 with rmtc , pcr was performed on the 3 isolates with primer pairs isecpir - f and rmtc - down and isecp15 and rmtc - r , as described ( 12 ) . sequence analysis of amplicons revealed association of an intact isecp1 element with rmtc in knpa1a ; however , complete isecp1 could not be amplified in knpa1b and knpa1c , corroborating earlier observations of either partial deletion of this element or role of a different isecp1-like element in the spread of rmtc in gram - negative bacteria ( 13 ) . attempts to transfer rmtc from all 3 isolates to rifampin - resistant escherichia coli 20r764 and ciprofloxacin - resistant p. repeated attempts to obtain amikacin - resistant ( mic 16 g / ml ) transformants of e. coli dh5 and p. aeruginosa pa01 by electroporation with plasmid preparation by using the kado and liu method ( 14 ) were also unsuccessful , despite successful transfer of control plasmids . to determine the location of rmtc , rmtf , and blandm-1 , genomic dna from the 3 isolates was digested separately with restriction enzyme i - ceu-1 ( new england biolabs , beverly , ma , usa ) , separated by pfge , and subsequently assayed with 16s rrna , rmtc , rmtf , and blandm-1 probes ( 13 ) . all these probes were hybridized with chromosomal dna ( figure 2 ) and not with plasmid extract . this result shows that rmtc , rmtf , and blandm-1 were located and stabilized on the chromosome of p. aeruginosa . we describe an occurrence of 16s rmtases rmtc and rmtf in clinical isolates of p. aeruginosa co - producing blandm-1 . the rmtc and rmtf genes might have been acquired from plasmids as part of mobile genetic elements and finally integrated and stabilized on the chromosome , but the underlying mechanism of transmission needs to be elucidated . further , spread of multidrug - resistant p. aeruginosa strains expressing rmtc with and without an intact isecp1 element and ndm-1 is of major clinical concern and calls for further studies to limit the spread of such strains .

we investigated 16s rrna methyltransferases in 38 blandm-1positive pseudomonas aeruginosa isolates and found rmtc in 3 isolates , 1 of which also harbored rmtf . the isolates were clonally unrelated ; rmtc and rmtf genes were located on a chromosome with the blandm-1 gene . strategies are needed to limit the spread of such isolates .

statins ( 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors ) were introduced in denmark in 1990 and are currently recommended as a preventive treatment for men and women of all ages at high risk of cardiovascular events.1 during the past two decades , statins have become one of the most commonly used groups of drugs worldwide . between 1995 and 2012 , 842,484 danish individuals ( the current population of denmark is 5.6 million ) filled at least one statin prescription at a community pharmacy , according to data from the danish national board of health.2 in the 1990s , there was a four- to fivefold increase in numbers of new patients who received statins.3 the prevalence of statin use in denmark was predicted to increase by over 300% between 2006 and 2012,4 based on a continuous rise in new users after 2005 . compliance with statin treatment was rated high in denmark in the 1990s,5 but others have raised concerns regarding persistence of use , with reports of a large number of dropouts in short - term studies.6 while clinical studies often focus on middle- and low - risk populations , population - based studies may focus on real - life data , including high - risk populations.710 clinical trials have demonstrated that the most widely prescribed statins ( ie , simvastatin , atorvastatin , rosuvastatin , and pravastatin ) for both primary and secondary prevention purposes reduce total plasma cholesterol and low - density lipoprotein ( ldl ) cholesterol ; reductions of 0.52.26 mmol / l for total and ldl cholesterol levels , respectively , were reported in a meta - analysis of 181 randomized controlled trials of statin.11 on the other hand , the cholesterol level target attainment in routine clinical care is less well described , with somewhat inconsistent results . among 2,227 elderly medicare advantage members in the us , 67%94% reached the ldl cholesterol goal , depending on statin type prescribed.7 in their study of 2,303 patients starting statins in the netherlands , heintjes et al8 stratified cholesterol target attainment by patients risk for cardiovascular disease ; among high - risk patients , target attainment varied from 40% for patients on pravastatin to 81% for patients on rosuvastatin . in a population - based cohort study in spain , 74% of patients did not attain their ldl cholesterol treatment goal after 3 years of follow - up.9 in contrast , among 1,103 patients from community - based practices in canada who filled prescriptions for statins , 73% achieved the target ldl cholesterol level.10 in the current population - based study , we examined the rate of patients initiating statin use and the prevalence of use in northern denmark during 200410 , a setting in which national guidelines recommend using simvastatin as first - line drug . in addition , we aim to examine changes in lipid values over time after statin initiation and determined the percentage of patients attaining treatment goals for cholesterol levels , stratified by risk groups , and whether statin type was switched . denmark provides tax - funded universal access to primary and secondary health care , with no substantial out - of - pocket expenses and with partial reimbursement for most prescribed medications , including statins . individual - level data from all danish registries can be linked via the unique personal identifier ( cpr number ) assigned at birth by the danish civil registration system ( crs).12 in denmark , statins are available to outpatients only by prescriptions filled at community pharmacies . we retrieved all entries for redemption of a first prescription ( index date ) of a statin ( atc classification system : c10aa statins , c10ba statin + ezetimibe ) in the aarhus university prescription database . this is a research database of all reimbursed prescriptions filled at community pharmacies in northern denmark since 1998.13 the study included all patients initiated on statins in 200410 , excluding patients with a statin treatment 15 months prior to 2004 , who were aged between 18 and 86 years and who filled a first statin prescription between january 2004 and december 2010 at any pharmacy in northern denmark . we excluded patients with a previous cancer diagnosis ( international classification of diseases [ icd ] , eighth edition : 140208 , excluding 172173 and icd-10 : c00c99 ) through linkage with the danish national registry of patients . this registry contains discharge diagnoses from all inpatient admissions to danish hospitals since 1977 and hospital outpatient clinic diagnoses since 1995.14 we obtained information about doses , such as the defined daily dose ( ddd ) , and adherence to statin treatment , including medication possession ratio ( mpr ) , switches among statin types , and persistence . ddd is given to each atc code and is the assumed daily average maintenance dose when used for a main indication . nonpersistence was defined as no reimbursements for new statin prescriptions during the time corresponding to all ddds in the last prescribed package plus the following 6 months ( 180 days ) . the crs contains electronic records of age , sex , vital status , and place of residence ( address ) for the entire danish population since 1968 , updated daily.12 from the crs , we retrieved information on sex , age , place of residence ( urban / rural ) , marital status ( married , never married , widowed / divorced ) , and vital status ( alive , dead , emigrated ) . from the danish national registry of patients we obtained information on each patient s comorbidities , assessing the presence of major conditions based on the complete hospital contact history before start of statin use . from the aarhus university prescription database we obtained information on comedications , including oral glucose - lowering drugs , antithrombotics , and oral anticoagulants . the clinical laboratory information system ( labka ) research database contains information on blood samples obtained by primary care and hospital physicians and analyzed at hospitals in northern denmark since 2000.15 from the labka database we collected information on total cholesterol , ldl cholesterol , high - density lipoprotein ( hdl ) cholesterol , sample dates , and results during 200410 . in total , 76% ( 123,743/161,646 ) of patients had one or more cholesterol measurements before and one after statin initiation . based on international and national guidelines1619 we defined three risk groups , based on availability of information in the hospital contact and prescription registries , and determined the achievement of target cholesterol levels in each group . the first group contained a very high - risk group of patients with a hospital - based diagnosis of cardiovascular disease , whose targets were < 1.8 mmol / l for ldl cholesterol and < 4 mmol / l for total cholesterol . the second group consisted of a high - risk group of patients with diabetes ( receiving antidiabetic medications or with a history of hospitalization or outpatient care for diabetes ) , hypertension , or a diagnosis of familial dyslipidemia , whose targets were < 2.5 mmol / l for ldl cholesterol and < 4.5 mmol / l for total cholesterol . the third group contained the remaining patients with low to moderate risk , whose targets were <3 mmol / l for ldl cholesterol and < 5 mmol / l for total cholesterol . this study was approved by the danish data protection agency ( record number 2009 - 41 - 3866 ) . we examined the rate and prevalence of statin use annually during 200410 and overall ; annual person - years at risk in northern denmark for individuals aged 1886 years ( denominator ) were obtained from statistics denmark . we examined the starting dose ( mg ) among new statin users by calendar year and type of statin . to measure adherence , we calculated average daily use in ddds and mpr . we calculated the mpr as the ratio of days statin pills are supplied to days in the time interval to the next statin prescription ( eg , 30 pills prescribed/40 days mpr of 0.75)20 and the persistence of use ( see previous definition of nonpersistence ) . we calculated the proportion of patients with different demographic and clinical characteristics at the time they filled their first statin prescription ( index date ) . we created similar contingency tables for the subgroup of patients who switched to another statin type , at the time of their switch . we also compared the characteristics of statin initiators with and without cholesterol measurements recorded in the aarhus university prescription database . for the 123,743 patients with at least one cholesterol measurement recorded prior to and at least one after their first statin prescription , we examined cholesterol values before and after start of statin treatment , graphically examined cholesterol values over time , and calculated percentage reduction . we examined median and quartiles of total , ldl , and hdl cholesterol at each measurement , focusing particularly on changes between the last cholesterol measurement before commencement of statin treatment and the first measurement after treatment initiation , and on cholesterol level treatment goals . these analyses were conducted for all new statin users , stratified by switching ( yes / no ) . we also examined the proportion of patients reaching their target levels for ldl and total cholesterol at any time within the study period , stratified by level of risk for cardiovascular events ( very high , high , and low to moderate ) . a sensitivity analysis was conducted to examine cholesterol target attainment at the very first measurement after the date of statin initiation . denmark provides tax - funded universal access to primary and secondary health care , with no substantial out - of - pocket expenses and with partial reimbursement for most prescribed medications , including statins . individual - level data from all danish registries can be linked via the unique personal identifier ( cpr number ) assigned at birth by the danish civil registration system ( crs).12 in denmark , statins are available to outpatients only by prescriptions filled at community pharmacies . we retrieved all entries for redemption of a first prescription ( index date ) of a statin ( atc classification system : c10aa statins , c10ba statin + ezetimibe ) in the aarhus university prescription database . this is a research database of all reimbursed prescriptions filled at community pharmacies in northern denmark since 1998.13 the study included all patients initiated on statins in 200410 , excluding patients with a statin treatment 15 months prior to 2004 , who were aged between 18 and 86 years and who filled a first statin prescription between january 2004 and december 2010 at any pharmacy in northern denmark . we excluded patients with a previous cancer diagnosis ( international classification of diseases [ icd ] , eighth edition : 140208 , excluding 172173 and icd-10 : c00c99 ) through linkage with the danish national registry of patients . this registry contains discharge diagnoses from all inpatient admissions to danish hospitals since 1977 and hospital outpatient clinic diagnoses since 1995.14 we obtained information about doses , such as the defined daily dose ( ddd ) , and adherence to statin treatment , including medication possession ratio ( mpr ) , switches among statin types , and persistence . ddd is given to each atc code and is the assumed daily average maintenance dose when used for a main indication . nonpersistence was defined as no reimbursements for new statin prescriptions during the time corresponding to all ddds in the last prescribed package plus the following 6 months ( 180 days ) . the crs contains electronic records of age , sex , vital status , and place of residence ( address ) for the entire danish population since 1968 , updated daily.12 from the crs , we retrieved information on sex , age , place of residence ( urban / rural ) , marital status ( married , never married , widowed / divorced ) , and vital status ( alive , dead , emigrated ) . from the danish national registry of patients we obtained information on each patient s comorbidities , assessing the presence of major conditions based on the complete hospital contact history before start of statin use . from the aarhus university prescription database we obtained information on comedications , including oral glucose - lowering drugs , antithrombotics , and oral anticoagulants . the clinical laboratory information system ( labka ) research database contains information on blood samples obtained by primary care and hospital physicians and analyzed at hospitals in northern denmark since 2000.15 from the labka database we collected information on total cholesterol , ldl cholesterol , high - density lipoprotein ( hdl ) cholesterol , sample dates , and results during 200410 . in total , 76% ( 123,743/161,646 ) of patients had one or more cholesterol measurements before and one after statin initiation . based on international and national guidelines1619 we defined three risk groups , based on availability of information in the hospital contact and prescription registries , and determined the achievement of target cholesterol levels in each group . the first group contained a very high - risk group of patients with a hospital - based diagnosis of cardiovascular disease , whose targets were < 1.8 mmol / l for ldl cholesterol and < 4 mmol / l for total cholesterol . the second group consisted of a high - risk group of patients with diabetes ( receiving antidiabetic medications or with a history of hospitalization or outpatient care for diabetes ) , hypertension , or a diagnosis of familial dyslipidemia , whose targets were < 2.5 the third group contained the remaining patients with low to moderate risk , whose targets were <3 mmol / l for ldl cholesterol and < 5 mmol / l for total cholesterol . this study was approved by the danish data protection agency ( record number 2009 - 41 - 3866 ) . we examined the rate and prevalence of statin use annually during 200410 and overall ; annual person - years at risk in northern denmark for individuals aged 1886 years ( denominator ) were obtained from statistics denmark . we examined the starting dose ( mg ) among new statin users by calendar year and type of statin . to measure adherence , we calculated average daily use in ddds and mpr . we calculated the mpr as the ratio of days statin pills are supplied to days in the time interval to the next statin prescription ( eg , 30 pills prescribed/40 days mpr of 0.75)20 and the persistence of use ( see previous definition of nonpersistence ) . we calculated the proportion of patients with different demographic and clinical characteristics at the time they filled their first statin prescription ( index date ) . we created similar contingency tables for the subgroup of patients who switched to another statin type , at the time of their switch . we also compared the characteristics of statin initiators with and without cholesterol measurements recorded in the aarhus university prescription database . for the 123,743 patients with at least one cholesterol measurement recorded prior to and at least one after their first statin prescription , we examined cholesterol values before and after start of statin treatment , graphically examined cholesterol values over time , and calculated percentage reduction . we examined median and quartiles of total , ldl , and hdl cholesterol at each measurement , focusing particularly on changes between the last cholesterol measurement before commencement of statin treatment and the first measurement after treatment initiation , and on cholesterol level treatment goals . these analyses were conducted for all new statin users , stratified by switching ( yes / no ) . we also examined the proportion of patients reaching their target levels for ldl and total cholesterol at any time within the study period , stratified by level of risk for cardiovascular events ( very high , high , and low to moderate ) . a sensitivity analysis was conducted to examine cholesterol target attainment at the very first measurement after the date of statin initiation . in total , there were 161,646 statin patients initiated on statins in northern denmark between 2004 and 2010 . in 2004 , there were 16.3 new users per 1,000 person - years ( figure 1a ) . the annual number of patients initiated on statins increased until 2008 ( 19.9 per 1,000 person - years ) and then decreased until 2010 ( 14.1 per 1,000 person - years ) . figure 1b shows the resulting cumulative prevalence of statin use up to 2010 . in 2010 , there were 133,979 adults , corresponding to 93.8 per 1,000 adults ( aged between 18 and 86 years ) , in northern denmark who still used statins and who had initiated their statin use between 2004 and 2010 . at statin initiation among the 161,646 patients , 98% started with simvastatin . demographic characteristics varied little between users of different statin types . sixty percent of patients were aged between 55 and 75 years at statin initiation , and 51% were men ( table 1 ) . as expected , many patients had previous hospital - based diagnoses of heart disease and other chronic conditions ( table 2 ) . in general , patients initiated on statins during 200410 were on statin treatment for a median of 1,075 days , or close to 3 years . in this period , for example , the proportion of simvastatin users who started with 40 mg tablets increased from 40% in 2004 to 75% in 2010 , and the proportion of rosuvastatin users who started with high doses ( 20 mg or 40 mg ) increased from 12% in 2004 to 21% in 2010 . overall , the average mpr for the entire period was 0.93 ( interquartile range [ iqr ] 0.771.0 ) . of the statin initiators in this sample , 88% ( 142,807/161,646 ) did not switch statin type . of these 142,807 nonswitchers , only 18% ( 24,998/142,807 ) altered their statin dose during the study period . of the 18,839 ( 12% ) persons who did switch one or more times , 13,138 ( 70% ) switched only once . by far the most common initial switches were from simvastatin to atorvastatin ( 48% ; 9,104/18,839 ) , to rosuvastatin ( 35% ; 6,644/18,839 ) , or to pravastatin ( 9% ; 1,603/18,839 ) . for example , 21% versus 11% had chronic ischemic heart disease and 11% versus 8% had a previous myocardial infarction . in contrast , the prevalence of other important comorbidities ( eg , chronic obstructive pulmonary disease ) was similar to that in nonswitchers . the sex and age distributions among statin switchers and nonswitchers were similar ( 51% males among nonswitchers and 48% among switchers ; 40% aged over 65 years among nonswitchers and 39% among switchers ) . the mpr for the nonswitchers was 0.93 ( iqr 0.781.0 ) , while the mpr for the patients who switched statin type was 0.90 ( iqr 0.751.0 ) preswitch and 0.93 ( iqr 0.701.0 ) postswitch . among the statin initiators , 26,314 persons ( 16% ) completely stopped their statin treatment : ie , were nonpersistent . compared with all patients who started statins , nonpersistent patients were more likely to be very young ( aged < 45 years ) or very old ( aged > 75 years ) , more likely to live in small municipalities in rural areas , more likely to be divorced , and importantly had a slightly higher prevalence of almost all examined diagnoses of comorbidity , including cardiovascular disease ( data not shown ) . among the 133,856 patients with at least one cholesterol measurement recorded prior to their first statin prescription , the most recent cholesterol measurement was , on average , 9 days ( quartiles 528 days ) before redemption of a statin prescription . furthermore , 123,743 patients had at least one cholesterol surveillance measurement ( median 80 days ) after starting to use statins . the demographic and clinical characteristics of patients with and without cholesterol measurements recorded in the database were very similar , except that more patients without measurement lived in large cities ( 29% vs 8% ) , and more patients without measurement ( 49% vs 38% ) started statin treatment early in the period ( 200406 ) , when the laboratory database may have been less complete . median total cholesterol in new statin users decreased from 6.3 ( quartiles 5.57.0 ) mmol / l at the most recent measurement before statin initiation to 4.5 ( quartiles 3.95.1 ) mmol / l at the first measurement after initiation , and remained remarkably stable thereafter ( figure 2a ) , corresponding to a reduction of 28% . median ldl cholesterol decreased rapidly from 4.0 ( quartiles 3.34.6 ) mmol / l to 2.3 ( quartiles 1.92.8 ) mmol / l , corresponding to a reduction of 43% . median hdl cholesterol remained stable at 1.4 mmol / l before and after statin initiation . among patients who later switched statin type , there was a smaller initial decrease in cholesterol values compared with nonswitchers , and some increase in cholesterol levels after the first surveillance measurement ( figure 2b ) . among patients who switched statin type , we observed a modest reduction in cholesterol level after their switch ( figure 2c ) . table 3 shows attainment of target cholesterol levels by risk group . among all statin users , 66% of patients in the very high - risk group reached their target value for total cholesterol , while corresponding figures were 74% and 80% for patients in the high - risk and low- to moderate - risk groups , respectively . when stratified by switching of statin type ( yes / no ) , results for statin nonswitchers were comparable with the overall results . among patients who switched statin type , the percentages attaining target cholesterol levels after the first switch and before the second were 46% , 56% , and 64% for the three risk groups , respectively . when focusing on target attainment at the very first measurement after statin initiation ( averaging 3 months after statin start ) , 43% in the very high - risk , 49% in the high - risk , and 55% in the low- to moderate - risk groups reached total cholesterol target . in total , there were 161,646 statin patients initiated on statins in northern denmark between 2004 and 2010 . in 2004 , there were 16.3 new users per 1,000 person - years ( figure 1a ) . the annual number of patients initiated on statins increased until 2008 ( 19.9 per 1,000 person - years ) and then decreased until 2010 ( 14.1 per 1,000 person - years ) . figure 1b shows the resulting cumulative prevalence of statin use up to 2010 . in 2010 , there were 133,979 adults , corresponding to 93.8 per 1,000 adults ( aged between 18 and 86 years ) , in northern denmark who still used statins and who had initiated their statin use between 2004 and 2010 . demographic characteristics varied little between users of different statin types . sixty percent of patients were aged between 55 and 75 years at statin initiation , and 51% were men ( table 1 ) . as expected , many patients had previous hospital - based diagnoses of heart disease and other chronic conditions ( table 2 ) . in general , initial use of statins other than simvastatin was associated with higher baseline comorbidity . patients initiated on statins during 200410 were on statin treatment for a median of 1,075 days , or close to 3 years . in this period , for example , the proportion of simvastatin users who started with 40 mg tablets increased from 40% in 2004 to 75% in 2010 , and the proportion of rosuvastatin users who started with high doses ( 20 mg or 40 mg ) increased from 12% in 2004 to 21% in 2010 . overall , the average mpr for the entire period was 0.93 ( interquartile range [ iqr ] 0.771.0 ) . of the statin initiators in this sample , 88% ( 142,807/161,646 ) did not switch statin type . of these 142,807 nonswitchers , only 18% ( 24,998/142,807 ) altered their statin dose during the study period . of the 18,839 ( 12% ) persons who did switch one or more times , 13,138 ( 70% ) switched only once . by far the most common initial switches were from simvastatin to atorvastatin ( 48% ; 9,104/18,839 ) , to rosuvastatin ( 35% ; 6,644/18,839 ) , or to pravastatin ( 9% ; 1,603/18,839 ) . for example , 21% versus 11% had chronic ischemic heart disease and 11% versus 8% had a previous myocardial infarction . in contrast , the prevalence of other important comorbidities ( eg , chronic obstructive pulmonary disease ) was similar to that in nonswitchers . the sex and age distributions among statin switchers and nonswitchers were similar ( 51% males among nonswitchers and 48% among switchers ; 40% aged over 65 years among nonswitchers and 39% among switchers ) . the mpr for the nonswitchers was 0.93 ( iqr 0.781.0 ) , while the mpr for the patients who switched statin type was 0.90 ( iqr 0.751.0 ) preswitch and 0.93 ( iqr 0.701.0 ) postswitch . among the statin initiators , 26,314 persons ( 16% ) completely stopped their statin treatment : ie , were nonpersistent . compared with all patients who started statins , nonpersistent patients were more likely to be very young ( aged < 45 years ) or very old ( aged > 75 years ) , more likely to live in small municipalities in rural areas , more likely to be divorced , and importantly had a slightly higher prevalence of almost all examined diagnoses of comorbidity , including cardiovascular disease ( data not shown ) . among the 133,856 patients with at least one cholesterol measurement recorded prior to their first statin prescription , the most recent cholesterol measurement was , on average , 9 days ( quartiles 528 days ) before redemption of a statin prescription . furthermore , 123,743 patients had at least one cholesterol surveillance measurement ( median 80 days ) after starting to use statins . the demographic and clinical characteristics of patients with and without cholesterol measurements recorded in the database were very similar , except that more patients without measurement lived in large cities ( 29% vs 8% ) , and more patients without measurement ( 49% vs 38% ) started statin treatment early in the period ( 200406 ) , when the laboratory database may have been less complete . median total cholesterol in new statin users decreased from 6.3 ( quartiles 5.57.0 ) mmol / l at the most recent measurement before statin initiation to 4.5 ( quartiles 3.95.1 ) mmol / l at the first measurement after initiation , and remained remarkably stable thereafter ( figure 2a ) , corresponding to a reduction of 28% . median ldl cholesterol decreased rapidly from 4.0 ( quartiles 3.34.6 ) mmol / l to 2.3 ( quartiles 1.92.8 ) mmol / l , corresponding to a reduction of 43% . median hdl cholesterol remained stable at 1.4 mmol / l before and after statin initiation . among patients who later switched statin type , there was a smaller initial decrease in cholesterol values compared with nonswitchers , and some increase in cholesterol levels after the first surveillance measurement ( figure 2b ) . among patients who switched statin type , we observed a modest reduction in cholesterol level after their switch ( figure 2c ) . table 3 shows attainment of target cholesterol levels by risk group . among all statin users , 66% of patients in the very high - risk group reached their target value for total cholesterol , while corresponding figures were 74% and 80% for patients in the high - risk and low- to moderate - risk groups , respectively . when stratified by switching of statin type ( yes / no ) , results for statin nonswitchers were comparable with the overall results . among patients who switched statin type , the percentages attaining target cholesterol levels after the first switch and before the second were 46% , 56% , and 64% for the three risk groups , respectively . when focusing on target attainment at the very first measurement after statin initiation ( averaging 3 months after statin start ) , 43% in the very high - risk , 49% in the high - risk , and 55% in the low- to moderate - risk groups reached total cholesterol target . our population - based data show that the rate of new statin use in northern denmark seemed to reach a plateau and begin to decline starting in 2008 , and the initiators were adherent in their use . the reduction in median total cholesterol and ldl cholesterol was similar to that observed in clinical trials . following european guideline lipid goals , 66% in the very high - risk group reached target levels for overall cholesterol and 54% for ldl cholesterol . corresponding percentages were about 80% among the high - risk and low- to moderate - risk groups . previous reports have shown a large increase in population - wide statin use in the 2000s . raymond et al21 reported a 1.7-fold increase in incident statin use based on prescription drug claims in british colombia during 19992004 . our finding that new statin use levelled off starting in the late 2000s may be due to statin treatment saturation within a large pool of previously untreated individuals , or possibly more restrictive use after reports of potential adverse long - term effects of statins . as expected , many patients initiated on statins had previous hospital - based diagnoses of cardiovascular diseases and other comorbidities . the higher proportions of documented cardiovascular disease among patients initiated on the more potent statins may be related to guidelines recommending their use in patients with acute cardiovascular disease.19 furthermore , the increase in starting doses of statins during the observation period is also in accordance with newer treatment guidelines;22 further examination of the impact on the changed starting doses over time is warranted , as dose is related to efficacy in lowering cholesterol.11 we chose to examine attainment of cholesterol levels overall for the statins , since almost all new users start with simvastatin in denmark , in accordance with danish guidelines . our finding that attainment of target cholesterol levels differs among patient risk groups is in line with previous real - world findings from the netherlands.8 additionally , the cholesterol target level attainment in this routine clinical care setting was similar to findings from randomized controlled trials.11 while danish and european guidelines contain lipid goals stratified on risk,17,18 the newest us guidelines have removed the use of lipid goals , and suggestions have been voiced that they ought to be refined rather than removed.23 given that the main desired outcome of increased statin therapy is a decrease in cardiovascular events , it is interesting to note that the incidence of new myocardial infarction has continously decreased over the past 20 years in denmark , as reported by schmidt et al24 based on population - based data . they only included follow - up until 2008 ; thus , further observation is warranted as to whether the continuing increase in statin use and the plateau from 2008 have been accompanied by a continuing decrease in cardiovascular events in denmark . adherence was high in this study ; both a high medication possession ( mpr ) and high treatment persistence were observed during our study period . whereas others have reported clinically important declines in persistence over time,6 only 16% among our study participants appeared to quit statin use totally . compared with previous studies of statin initiators , our study had the advantage of a rather long follow - up time and allowed for shorter gaps of treatment . the generally high adherence observed in this study is positive news , and further examinations of predictors of adherence are warranted . danish guidelines state that simvastatin should be the first - line drug of choice , and it is apparent from our findings that danish physicians adhere to these guidelines.17 twelve percent of patients initiated on statins switched statin type , most often from simvastatin to a more potent statin . in clinical practice , reasons for switching may include adverse side effects and/or failure to attain target cholesterol levels . accordingly , we observed that patients who later shifted statin type experienced less initial reduction of cholesterol levels . unfortunately , our data did not allow us to determine whether insufficient cholesterol reduction was related to poor drug effectiveness , reduced compliance ( eg , due to side effects ) , or choice of dose with the first statin prescribed , but we know that adherence ( expressed as mpr ) is comparable . patients who later shifted statin type also had substantially more heart disease than all initiators , which may explain this group s greater difficulty in reaching their cholesterol targets . strengths of this study include its large size , population - based design , and access to high - quality danish medical databases providing a complete hospital contact , prescription , and laboratory history . our findings are based on data from a wide range of unselected patients in the community and may be transferrable to other population - based settings . some statin - treated patients apparently did not present for surveillance measurements , some may have emigrated outside our data catchment area , some died , and some started statins late in the study period and were followed for only a short time . the patients without cholesterol measurement did not differ in demographic or clinical characteristics compared with the patients with cholesterol measurements . we focused on new statin users from 2004 onward , and patients who had already received statins before the start of the study were left - truncated . this may have excluded from our study early statin users in the background population with a strong medical indication for statins , and subjects who had earlier started and then entirely discontinued statins due to adverse events or lack of effect . on the other hand , having a short look - back period of only 15 months before 2004 may have artificially inflated new statin user rates in the earliest study years because of the inclusion of some previous statin users who restarted statin use during the first study years . in addition , filling a statin prescription is only a proxy for statin utilization . however , the calculated medication possession ratio in our patients was close to 1 , suggesting a surprisingly high adherence to the medication in our region . furthermore , diagnoses made in the primary care setting by patients general practitioners were not included in our data , and our estimates of comorbidity prevalence are likely to be underestimates , as can be identified by the discrepancy between diseases and marker drugs . another concern is that lifestyle changes such as a healthier diet and increased physical exercise are the recommended first interventions to decrease high lipid levels . unfortunately , we had no information on such and other ( eg , smoking cessation ) behavioral changes . finally , our patient risk group classification had to be modified according to data availability . in particular , we lacked data on individual risk factors necessary to evaluate the indication for statin treatment in the low- to moderate - risk group . in conclusion , our population - based data from routine clinical care show that statin use is very prevalent in northern denmark . cholesterol level target attainment in the community setting is similar to findings from clinical trials ; however , a substantial proportion of patients , particularly those at high risk , did not reach target cholesterol levels .

purposeto examine the annual rate and cumulative prevalence of statin use in denmark 200410 , including adherence of use and attainment of cholesterol targets.methodswe included all individuals aged 1886 years with a first statin prescription in northern denmark in 200410 . we calculated the annual rate and cumulative prevalence of statin use . we examined cholesterol values before and after start of statins and the proportion reaching targets according to european guidelines and cardiovascular risk group.resultsthe study population consisted of 161,646 new statin users ( 51% men ; median age 62 years ) . the peak rate of new statin initiators occurred in 2008 , and a cumulative prevalence of 94 users per 1,000 population was reached in 2010 . in total , 98% of new users started with simvastatin . eighty - eight percent ( 142,897 ) did not switch statin type during follow - up . overall persistence was 84% . the reduction in median total cholesterol in new statin users was 28% ( from 6.3 mmol / l to 4.5 mmol / l ) , while it was 43% ( from 4.0 mmol / l to 2.3 mmol / l ) for low - density lipoprotein cholesterol . among patients with very high cardiovascular risk , 66% attained the recommended total cholesterol target ; corresponding figures were 74% among high - risk patients and 80% among low- to moderate - risk patients . corresponding figures for low - density lipoprotein cholesterol were 54% , 82% , and 88% , respectively.conclusionsstatin use has become very prevalent in danish adults , with high adherence . cholesterol reduction after statin initiation is similar to that found in clinical trials , yet a substantial proportion of patients does not reach target cholesterol levels .

all study subjects were from the ongoing , prospective , population - based korean genome and epidemiology study ( koges ) cohort . the original study was designed to establish a representative adult cohort in an urban area , the city of ansan , and to identify the epidemiologic characteristics , frequency , and determinants of chronic diseases in koreans . initially , 5,015 participants ( 2,521 men and 2,494 women 4069 years old ) who participated in a comprehensive health examination and onsite interviews at korea university ansan hospital formed a longitudinal cohort from june 2001 to january 2003 . , subjects signed an informed consent form , which was approved by the human subjects review committee at the korea university ansan hospital . the fifth biennial examination was conducted from march 2009 to february 2011 and the sixth examination from march 2011 to february 2013 . polysomnography ( psg ) was included randomly in the study protocol in september 2009 in about one - half of the koges participants . although psg will be administered to the entire study population during the 4-year period , the present study includes only the subset of the sample with psg data acquired between september 2009 and november 2011 . after excluding the subjects who had missing data and extreme outliers of glucose concentrations , 1,344 subjects ( 706 men and 638 women ) further details from the protocol and design of the koges are described elsewhere ( 11 ) . all participants responded to an interviewer - administered questionnaire and underwent a comprehensive physical examination . lifestyle characteristics were smoking status and alcohol consumption categorized as never , former , and current . level of exercise was categorized as never , lightly ( <3 times / week , 30 min / session ) , or regularly ( 3 times / week , 30 min / session ) during the previous month . the presence of chronic illnesses , including diabetes , hypertension , dyslipidemia , and cardiovascular disease ( cvd ) , was noted as were prescribed medications . subjects with documented events or medical records of myocardial infarction , angina , heart failure , stroke , or peripheral artery disease were considered to have cvd . diabetes was defined by american diabetes association criteria , using a 75-g oral glucose tolerance test ( fasting plasma glucose [ fpg ] 7.0 mmol / l ) , and medical history ( 12 ) . for subjects without diabetes , glucose tolerance status was assessed by american diabetes association criteria ( 12 ) as follows : ifg only ( 5.6 fpg mmol / l ) , and ifg + igt ( 5.6 fpg < 7.0 mmol / l and 7.8 2hpg < 11.1 hypertension was defined as systolic blood pressure 140 mmhg , diastolic blood pressure 90 mmhg , or medical history ( 13 ) . height and body weight were measured to the nearest 0.1 cm and 0.1 kg , respectively . wc was measured at the midpoint between the lower rib margin and the iliac crest in the standing position . obesity was defined as bmi 25 kg / m according to the asian - specific bmi cutoffs from the world health organization report ( 14 ) . plasma glucose , serum triglycerides , hdl cholesterol , and ldl cholesterol levels were measured with an autoanalyzer ( advia 1650 ; siemens , tarrytown , ny ) . insulin was measured with an immunoradiometric assay kit ( ins - irma kit ; biosource , nivelles , belgium ) using a packard counter system . insulin resistance was estimated with the homeostasis model of assessment for insulin resistance ( homa - ir ) and calculated as fasting glucose ( mmol / l ) fasting insulin ( u / ml ) / 22.5 . homa -cell function ( homa- ) ( % ) was calculated as 20 fasting insulin ( u / ml ) / fasting glucose ( mmol / l ) 3.5 ( 15 ) . for subjects who participated in the fifth biennial examination ( n = 820 ) , single - slice computed tomography ( ct ) scanning ( brilliance 64 ; philips , cleveland , oh ) the scans were conducted at 120 kv with a slice thickness of 5 mm at the level of the l4l5 vertebral interspace . the total area of intra - abdominal fat was delineated by manual tracing within the muscle wall , and the visceral fat area ( vfa ) was defined as an area with an attenuation range between 190 and 30 hounsfield units . overnight sleep study was performed at home with a portable device ( embletta x100 ; embla systems , san carlos , ca ) . two trained sleep technologists visited each subject s home in the evening , applied sensors , and instructed the subject on how to start and stop the recording . subjects were required to record the lights - off and -on times and to report them the next morning . recording channels were one for electroencephalography ( c4a1 ) , one for electrooculography ( right upper outer canthus to left lower outer canthus ) , one for chin electromyography , one for modified lead ii electrocardiography , one for airflow from nasal airflow pressure transducer , two for respiratory effort from chest and abdominal respiratory inductance plethysmography , one pulse oximeter , and one position sensor . data were scored by two well - trained technicians who had 5 years of experience with psg monitoring and scoring according to standard guidelines ( 16,17 ) . internal consistency for scoring the apnea - hypopnea index ( ahi ) was high ( cronbach = 0.996 and 1.00 for each rater ) , and interrater reliability was strong ( cronbach = 0.998 ) . although we did not perform a validity study to compare psg recordings obtained in the home and laboratory settings , the sleep heart health study clearly demonstrated that the median respiratory disturbance index was similar in the unattended home and attended laboratory settings , with differences of a small magnitude in some sleep parameters ( 18 ) . obstructive apnea was defined when airflow dropped by 90% of the baseline with ongoing chest and abdominal movement , and hypopnea was defined as a reduction in airflow by 30% associated with at least a 4% oxygen desaturation . the duration threshold for the respiratory events was 10 s. ahi was calculated , and osa was defined as an ahi 5 ( 1,16,19 ) . daytime sleepiness was assessed with the epworth sleepiness scale ( ess ) ( 20 ) , a well - validated and frequently used subjective eight - item , self - administered questionnaire . subjects were asked to score the likelihood of falling asleep in eight different situations with different levels of stimulation . the higher the ess score , the greater propensity for sleepiness implied . in the current study , eds was defined as ess scores > 10 ( 20 ) . subject characteristics at baseline were compared among groups stratified by the presence or absence of osa and obesity by student t test for continuous variables and test for categorical variables . nonnormally distributed variables , such as hba1c , homa - ir , homa- , ahi , and triglyceride level , are presented as the median and interquartile range for each group , and the differences were tested after logarithmic transformation . variables associated with glucose metabolism were compared by ancova after adjusting for age , sex , and bmi among the groups stratified by osa and obesity . for ancova , we confirmed the homogeneity of the slope between the covariates and the dependent variables in subjects with or without osa . the interaction term for osa and obesity on homa- was calculated to determine whether the association between osa and homa- was modified by obesity status . to exclude the potential confounding effect of medication for diabetes or dyslipidemia on glucose metabolism , the same analyses were repeated after excluding subjects who were taking these medications . to evaluate the impact of osa on ifg only , igt only , ifg + igt , and diabetes according to the presence or absence of obesity , the following five models were fit for each outcome : model 1 was adjusted for age and sex ; model 2 was adjusted for age , sex , alcohol consumption , smoking status , exercise , presence of hypertension or cvd , and medication for dyslipidemia ; model 3 was the same as model 2 and adjusted for bmi ; model 4 was the same as model 2 and adjusted for wc ; and model 5 was the same as model 2 and adjusted for vfa in subjects who underwent abdominal ct scan . to exclude the potential confounding effect of medication for diabetes or dyslipidemia on glucose metabolism odds ratios ( ors ) for ifg only , igt only , ifg + igt , and diabetes were calculated according to the tertile of ahi to demonstrate the dose - response relationship . in addition , to examine the additive effect of eds and osa on glucose metabolism , ors for ifg only , igt only , ifg + igt , and diabetes were evaluated in subjects with osa according to the presence or absence of eds compared with those without osa . for logistic regression these models showed no evidence of lack of fit according to the hosmer - lemeshow statistic . all statistical analyses were performed with sas version 9.1 for windows software ( sas institute inc . , cary , nc ) . all participants responded to an interviewer - administered questionnaire and underwent a comprehensive physical examination . lifestyle characteristics were smoking status and alcohol consumption categorized as never , former , and current . level of exercise was categorized as never , lightly ( <3 times / week , 30 min / session ) , or regularly ( 3 times / week , 30 min / session ) during the previous month . the presence of chronic illnesses , including diabetes , hypertension , dyslipidemia , and cardiovascular disease ( cvd ) , was noted as were prescribed medications . subjects with documented events or medical records of myocardial infarction , angina , heart failure , stroke , or peripheral artery disease were considered to have cvd . diabetes was defined by american diabetes association criteria , using a 75-g oral glucose tolerance test ( fasting plasma glucose [ fpg ] 7.0 mmol / l or 2-h plasma glucose [ 2hpg ] 11.1 mmol / l ) , and medical history ( 12 ) . for subjects without diabetes , glucose tolerance status was assessed by american diabetes association criteria ( 12 ) as follows : ifg only ( 5.6 fpg mmol / l ) , igt only ( fpg < 5.6 mmol / l and 7.8 2hpg < 11.1 mmol / l ) , and ifg + igt ( 5.6 fpg < 7.0 mmol / l and 7.8 2hpg < 11.1 hypertension was defined as systolic blood pressure 140 mmhg , diastolic blood pressure 90 mmhg , or medical history ( 13 ) . height and body weight were measured to the nearest 0.1 cm and 0.1 kg , respectively . wc was measured at the midpoint between the lower rib margin and the iliac crest in the standing position . obesity was defined as bmi 25 kg / m according to the asian - specific bmi cutoffs from the world health organization report ( 14 ) . plasma glucose , serum triglycerides , hdl cholesterol , and ldl cholesterol levels were measured with an autoanalyzer ( advia 1650 ; siemens , tarrytown , ny ) . insulin was measured with an immunoradiometric assay kit ( ins - irma kit ; biosource , nivelles , belgium ) using a packard counter system . insulin resistance was estimated with the homeostasis model of assessment for insulin resistance ( homa - ir ) and calculated as fasting glucose ( mmol / l ) fasting insulin ( u / ml ) / 22.5 . homa -cell function ( homa- ) ( % ) was calculated as 20 fasting insulin ( u / ml ) / fasting glucose ( mmol / l ) 3.5 ( 15 ) . for subjects who participated in the fifth biennial examination ( n = 820 ) , single - slice computed tomography ( ct ) scanning ( brilliance 64 ; philips , cleveland , oh ) was used to quantify intra - abdominal adipose tissue . the scans were conducted at 120 kv with a slice thickness of 5 mm at the level of the l4l5 vertebral interspace . the total area of intra - abdominal fat was delineated by manual tracing within the muscle wall , and the visceral fat area ( vfa ) was defined as an area with an attenuation range between 190 and 30 hounsfield units . overnight sleep study was performed at home with a portable device ( embletta x100 ; embla systems , san carlos , ca ) . two trained sleep technologists visited each subject s home in the evening , applied sensors , and instructed the subject on how to start and stop the recording . subjects were required to record the lights - off and -on times and to report them the next morning . recording channels were one for electroencephalography ( c4a1 ) , one for electrooculography ( right upper outer canthus to left lower outer canthus ) , one for chin electromyography , one for modified lead ii electrocardiography , one for airflow from nasal airflow pressure transducer , two for respiratory effort from chest and abdominal respiratory inductance plethysmography , one pulse oximeter , and one position sensor . data were scored by two well - trained technicians who had 5 years of experience with psg monitoring and scoring according to standard guidelines ( 16,17 ) . internal consistency for scoring the apnea - hypopnea index ( ahi ) was high ( cronbach = 0.996 and 1.00 for each rater ) , and interrater reliability was strong ( cronbach = 0.998 ) . although we did not perform a validity study to compare psg recordings obtained in the home and laboratory settings , the sleep heart health study clearly demonstrated that the median respiratory disturbance index was similar in the unattended home and attended laboratory settings , with differences of a small magnitude in some sleep parameters ( 18 ) . obstructive apnea was defined when airflow dropped by 90% of the baseline with ongoing chest and abdominal movement , and hypopnea was defined as a reduction in airflow by 30% associated with at least a 4% oxygen desaturation . the duration threshold for the respiratory events was 10 s. ahi was calculated , and osa was defined as an ahi 5 ( 1,16,19 ) . daytime sleepiness was assessed with the epworth sleepiness scale ( ess ) ( 20 ) , a well - validated and frequently used subjective eight - item , self - administered questionnaire . subjects were asked to score the likelihood of falling asleep in eight different situations with different levels of stimulation . the higher the ess score , the greater propensity for sleepiness implied . in the current study subject characteristics at baseline were compared among groups stratified by the presence or absence of osa and obesity by student t test for continuous variables and test for categorical variables . nonnormally distributed variables , such as hba1c , homa - ir , homa- , ahi , and triglyceride level , are presented as the median and interquartile range for each group , and the differences were tested after logarithmic transformation . variables associated with glucose metabolism were compared by ancova after adjusting for age , sex , and bmi among the groups stratified by osa and obesity . for ancova , we confirmed the homogeneity of the slope between the covariates and the dependent variables in subjects with or without osa . the interaction term for osa and obesity on homa- was calculated to determine whether the association between osa and homa- was modified by obesity status . to exclude the potential confounding effect of medication for diabetes or dyslipidemia on glucose metabolism , the same analyses were repeated after excluding subjects who were taking these medications . to evaluate the impact of osa on ifg only , igt only , ifg + igt , and diabetes according to the presence or absence of obesity , multivariate logistic regression analyses were conducted . in the analysis , the following five models were fit for each outcome : model 1 was adjusted for age and sex ; model 2 was adjusted for age , sex , alcohol consumption , smoking status , exercise , presence of hypertension or cvd , and medication for dyslipidemia ; model 3 was the same as model 2 and adjusted for bmi ; model 4 was the same as model 2 and adjusted for wc ; and model 5 was the same as model 2 and adjusted for vfa in subjects who underwent abdominal ct scan . to exclude the potential confounding effect of medication for diabetes or dyslipidemia on glucose metabolism odds ratios ( ors ) for ifg only , igt only , ifg + igt , and diabetes were calculated according to the tertile of ahi to demonstrate the dose - response relationship . in addition , to examine the additive effect of eds and osa on glucose metabolism , ors for ifg only , igt only , ifg + igt , and diabetes were evaluated in subjects with osa according to the presence or absence of eds compared with those without osa . for logistic regression , we calculated adjusted r and hosmer - lemeshow statistics to assess model adequacy . these models showed no evidence of lack of fit according to the hosmer - lemeshow statistic . all statistical analyses were performed with sas version 9.1 for windows software ( sas institute inc . , cary , nc ) . table 1 shows the characteristics of the participants stratified by the presence or absence of osa and obesity . about 36.6% of nonobese and 58.2% of obese subjects had osa . however , the severity of osa was mostly mild and moderate ( 27 , 8.8 , and 0.8% of nonobese and 38.4 , 14.6 , and 5.2% obese subjects had mild , moderate , and severe osa , respectively ) . among 396 subjects with diabetes , 351 had a history of diabetes of whom 142 were treated with oral hypoglycemic agents ( n = 140 ) or insulin ( n = 4 ) . subjects with osa were older and had a higher 2hpg , hba1c , bmi , wc , vfa , and homa - ir than those without osa , regardless of obesity . in the nonobese group , subjects with osa had higher fpg and triglyceride levels and lower homa- and hdl cholesterol levels than those without osa . the proportion of ifg + igt and diabetes was higher according to osa status in nonobese subjects , whereas only the prevalence of diabetes was higher in obese subjects with osa . characteristics of subjects according to the presence or absence of obesity and osa table 2 presents the age- , sex- , and bmi - adjusted values for glucose metabolism according to osa and obesity status . in the nonobese group , fpg , 2hpg , and hba1c levels were significantly higher , and homa - ir was modestly higher in those with osa than in those without osa . however , in the obese group , only insulin and homa - ir levels were associated with osa . although not statistically significant , homa- was lower in nonobese subjects with osa than in those without osa but higher in obese subjects with osa than in those without osa . therefore , a significant interaction between osa and obesity was observed for homa- ( p = 0.025 ) . after excluding subjects who were taking medication for diabetes or dyslipidemia , in the nonobese group , fpg and 2hpg were still higher in those with osa than in those without . on the contrary , in the obese group , although glucose concentrations were not different , insulin , homa - ir , homa- were higher in those with osa than in those without ( supplementary table 1 ) . variables associated with glucose metabolism according to obesity and osa status after adjusting for age , sex , and bmi multivariate logistic regression analyses were conducted to investigate the effects of osa on glucose metabolism according to obesity status ( table 3 ) . in subjects without obesity , the presence of osa showed higher ors for ifg + igt and diabetes , even after adjusting for several confounding variables , including bmi or wc . osa was significantly associated with diabetes even after adjusting for vfa in nonobese subjects . in an analysis to show the dose - response relationship , nonobese subjects with the highest ahi tertile had the highest ors for ifg + igt or diabetes ( supplementary table 2 ) . in contrast , in those with obesity , none of the abnormal glucose categories was associated with osa . these findings were consistent even after excluding subjects who were taking medication for diabetes or dyslipidemia ( supplementary table 3 ) . association between osa and abnormal glucose metabolism according to obesity status supplementary table 4 shows the joint effect of osa and eds on glucose tolerance categories . in the nonobese group , compared with subjects without osa , nonsleepy subjects with osa had higher ors for diabetes , but sleepy subjects with osa had the highest ors for diabetes ( hazard ratio 5.78 [ 95% ci 2.0516.3 ] ) . in this large community - based cohort study , we demonstrated the differential association between osa and impaired glucose metabolism according to the presence of obesity . of note , osa was significantly associated with abnormal glucose metabolism in nonobese subjects , even after adjusting for several confounding variables , including generalized or visceral adiposity . to our knowledge , this study is the first to show that the impact of osa on glucose metabolism is more evident in the nonobese than in the obese population . we also clearly demonstrated that in the nonobese group , fpg , 2hpg , and hba1c were significantly higher and that homa - ir was modestly higher in subjects with osa than in those without . the only previous study to analyze the effect of osa according to obesity status was the sleep heart health study , where the association between osa and abnormal glucose metabolism was not different between the nonoverweight and overweight / obese groups ( 3 ) . we can hypothesize that osa may have a lesser impact on glucose metabolism in obese than in nonobese individuals because of preexisting derangements in those who are obese . in accordance with this theory , the improvement of insulin sensitivity in patients with osa receiving cpap therapy was minimal in those with a bmi of > 30 kg / m , whereas it was more prominent in those with less obesity ( 5 ) . ( 21 ) demonstrated that the acute intermittent hypoxia caused insulin resistance and increased secretion of leptin and tumor necrosis factor- in lean mice . however , obesity was associated with striking increases in leptin and tumor necrosis factor- levels , which overwhelmed the effects of hypoxia . on the contrary , drager et al . ( 22 ) showed that the effect of chronic intermittent hypoxia on glucose intolerance , inflammation , and oxidative stress was prominent in obese mice but not in lean mice . the causes of these contradictory findings are not clear ; however , the metabolic and proinflammatory responses to acute and chronic intermittent hypoxia may be different . an alternative hypothesis may be a presence or absence of eds in subjects with osa . eds is reportedly associated with hyperglycemia and insulin resistance in subjects with osa ( 23,24 ) . intermittent hypoxemia has been shown to result in disturbed sleep architecture or to damage neuronal structures that promote wakefulness in animal models ( 25 ) . indeed , higher degrees of hypoxemia predict both sleepiness and insulin resistance ( 1,26 ) . according to these studies , we can expect that the effect of hypoxemia on glucose metabolism may be more evident in patients with osa and eds . in the current study , we demonstrated that the joint effect of osa and eds on the or for diabetes was highly significant only in nonobese subjects . therefore , the differential impact of eds on glucose metabolism according to obesity status may be responsible for the discrepant effect of osa in subjects with or without obesity in the current study . the association between homa - ir and osa was similar in subjects with and without obesity , as seen in previous research ( 1,3,27 ) . however , few studies have evaluated insulin secretion according to osa status . recently , in healthy young men without diabetes and obesity ( most of them caucasian ) , subjects with osa had increased insulin resistance and compensatory hyperinsulinemia ( 10 ) . in the current study , however , homa- was lower in nonobese subjects with osa than in those without osa but was higher in obese subjects . this finding was more evident in obese subjects after excluding those taking medication for diabetes or dyslipidemia and suggests that inadequate insulin secretion against the increased insulin resistance may be responsible for the abnormal glucose metabolism in nonobese individuals , which is characteristic for diabetes in asians ( 28,29 ) . on the contrary , increased insulin secretion in response to insulin resistance in obese individuals with osa may explain why glucose metabolism is less impaired by osa . however , the hyperinsulinemic - euglycemic clamp technique should be used to further explore the mechanisms of the impairment of glucose metabolism by osa . therefore , the prevalence of metabolically obese normal weight ( monw ) individuals who have not only a normal bmi , but also a cluster of obesity - related risk factors for diabetes and cvd is reportedly higher in asians ( 30,31 ) . higher levels of inflammatory adipokines and atherogenic ldl profiles in monw individuals ( 32,33 ) may mediate the increase in cvd and mortality in this population ( 34 ) . the finding that a worse cardiometabolic profile was more prominent in nonobese subjects with osa in the current study supports the notion that these individuals may have similar characteristics as those with monw . although the relationship between osa and abnormal glucose metabolism has been demonstrated in several previous studies ( 1,2,35 ) , a few have evaluated the association between osa and distinct prediabetic groups ( ifg and/or igt ) , where sleep - disordered breathing was associated with occult diabetes , ifg only , ifg + igt ( 3 ) , or glucose intolerance ( 1,35 ) in population and clinic - based studies . subjects with ifg had a greater impairment of early phase insulin secretion and increased endogenous glucose output , whereas igt was associated with peripheral insulin resistance ( 36 ) . in the current study , however , osa was associated with neither ifg only nor igt only . because more severe metabolic abnormalities are present in individuals with ifg + igt , diabetes develops more rapidly and unfavorable cardiovascular risk factors and mortality are increased compared with individuals with ifg only or igt only ( 3638 ) . from this point of view , the significant association between osa and ifg + igt in the current study suggests that subjects with osa are at a greater risk for developing diabetes and cvd and have a higher risk of mortality . compared with previous studies , which had several methodological limitations in terms of the study population ( 39,40 ) or lacked rigorous control for numerous confounders , especially obesity , the current study used a large community - based sample and differentiated the effect of osa according to the presence of obesity in addition to adjustment for generalized or visceral obesity . another strength of the current study was the evaluation of osa status during sleep at home , which provides a more realistic estimation of osa severity than hospital - based studies because of the maintenance of regular daily habits of sleep , physical activity , and diet in the general population . the major limitation of the current study was its cross - sectional design , which makes it difficult to determine whether there is a causal relationship between osa and impaired glucose metabolism . secondly , because we did not have a sufficient number of subjects with severe osa ( ahi 30 ) , it is unclear whether the present findings would be consistent in those with severe osa . however , the positive association between the ahi tertile and glucose tolerance categories in the nonobese group supports a dose - response relationship . in addition , the small numerical , but significant differences of some metabolic variables between those with and without osa would be more evident if we had more subjects with severe osa . however , the analysis of the association between ahi tertile and glucose tolerance categories showed that subjects with the third ahi tertile had the highest or for ifg + igt and diabetes in the nonobese group , which supports a dose - response relationship ( data not shown ) . finally , because bmi and body fat distribution in this population are different from other ethnicities , the present findings may not be generalizable to non - asian populations . in summary , the current study provides original evidence that the presence of mild to moderate osa in nonobese individuals confers a higher risk for impaired glucose metabolism , even after adjusting for important risk factors . osa can be responsible for the future risk for diabetes and cvd in nonobese individuals , whereas the prognostic implication of osa in obese individuals is unclear . whether this finding is universal across different populations with diverse ethnicity and obesity status should be studied in the future .

objectivethe purpose of this study was to investigate whether the impact of obstructive sleep apnea ( osa ) on glucose metabolism was different according to the presence or absence of obesity.research design and methodsa total of 1,344 subjects > 40 years old from the korean genome and epidemiology study were included . osa was detected by home portable sleep monitoring . plasma glucose , hba1c , and insulin resistance were compared according to osa and obesity status . the associations between osa and impaired fasting glucose ( ifg ) , impaired glucose tolerance ( igt ) , ifg + igt , and diabetes were evaluated in subjects with and without obesity after adjusting for several confounding variables . the effect of visceral obesity on this association was evaluated in 820 subjects who underwent abdominal computed tomography scanning.resultsin subjects without obesity , fasting glucose , 2-h glucose after 75-g glucose loading , and hba1c were higher in those with osa than in those without after controlling for age , sex , and bmi . in addition , the presence of osa in nonobese subjects was associated with a higher prevalence of ifg + igt and diabetes after adjusting for several confounding variables ( odds ratio 3.15 [ 95% ci 1.446.90 ] and 2.24 [ 1.433.50 ] for ifg + igt and diabetes , respectively ) . further adjustment for visceral fat area did not modify this association . in contrast , in those with obesity , none of the abnormal glucose tolerance categories were associated with osa.conclusionsthe presence of osa in nonobese individuals is significantly associated with impaired glucose metabolism , which can be responsible for future risk for diabetes and cardiovascular disease .

cell migration requires the rearrangement of adhesions and cytoskeleton in response to extracellular stimuli to form a leading edge that allows directed locomotion . in most cell types , migration - related adhesive structures are composed of integrin heterodimers and intracellular associated proteins clustered in plaques termed focal complexes and their more mature variant , focal adhesions . in addition to these adhesive structures , dcs also assemble distinctive adhesions named podosomes that are characteristic of cells of the myeloid lineage including macrophages and osteoclasts [ 4 , 5 ] . the molecular composition of podosomes is similar to that of focal complexes and focal adhesions . however , they have a unique organization with actin filaments bundled in discrete foci forming a conical core containing distinctive elements such as gelsolin , cortactin , the actin - nucleating factor arp 2/3 complex , and the scaffolding protein wasp surrounded by a ring of integrins and integrin - associated proteins [ 4 , 5 ] . wasp is an essential component of podosomes , where it activates the arp2/3 complex inducing actin polymerization . wip has also been detected in the core of podosomes in endothelial cells expressing egfp - wip , and although there is evidence indicating that wip plays a major role in regulating the activity of wasp family proteins and the dynamics of actin filaments [ 8 , 9 ] , it remains unknown how wip participates in the assembly of podosomes . to study the role of wip in podosome formation , the distribution of f - actin and vinculin was examined in wild - type and wip dcs by confocal microscopy . as expected , podosomes were grouped in clusters in wild - type dcs ( figures 1a and 1c ) . those few podosomes formed in wip dcs displayed an abnormal structure having less discrete actin cores and disorganized vinculin rings around f - actin ( figure 1d ) . large focal contacts containing vinculin comprised the most striking adhesion structures in wip dcs ( figures 1b and 1e ) , replacing podosomes as the major points of contact with the substratum . the critical involvement of wip in dc podosome formation is also supported by the presence of endogenous wip in the core of podosomes in wild - type cells ( figure 1f ) . our data suggest that the key role of wip in podosome formation is in the organization of f - actin and the clustering of integrins and associated proteins . the absence of wip had a profound effect on the organization of f - actin within dcs . while the bulk of f - actin in wild - type dcs was associated with the core of podosomes ( table 1 , see figure s1a in the supplemental data available online ) , in wip dcs it distributed among various anomalous structures including actin cables ( table 1 , figure s1b ) , large fused actin aggregates , commonly with a circular shape ( table 1 , figure s1c ) , and extensive membrane ruffles ( table 1 , figure s1d ) . clustering of podosomal integrins and the integrin - associated proteins paxillin and talin was also affected . in wild - type dcs , 2-integrins formed rings around the f - actin cores of podosomes ( figure s2a ) as previously described . in contrast , wip dcs failed to cluster 2-integrins into rings , and instead they formed small integrin - containing focal contacts scattered through the basal surface and around the periphery of the cell ( figure s2b ) or large plaques associated with anomalous actin - rich aggregates ( figure s2c ) . facs analysis showed that wild - type and wip dcs expressed equivalent levels of 1- and 2-integrins ( figure s3 ) , indicating that wip does not affect integrin expression . paxillin and talin , which normally array around the podosome core in wild - type dcs ( figures s2d and s2 g ) , in wip dcs localized to focal adhesions ( figures s2e and s2h ) containing 1 integrins ( data not shown ) or formed amorphous clusters colocalizing with the anomalous actin aggregates ( figures s2f and s2i ) . the shift of adhesion composition and structure from podosomes ( short half - life in the range of 30 s to 5 min ) to large focal contacts ( long half life in the range of 30 to 60 min ) in wip dcs had a negative impact on adhesion turnover ( figure 1 g ) . as previously described , interference reflection microscopy ( irm ) analyses showed podosomes in wild - type dcs rapidly assembled and disassembled , giving a high turnover index ( figure 1 g ) . in wip dcs , although multiple transient and unstable lamellar protrusions were developed at the cell periphery , the major adhesion sites in contact with the substratum were highly stable with a significantly lower turnover index compared to wild - type ( figure 1 g ) . interestingly , we found a decrease in podosome turnover in wild - type dcs plated on fibronectin ( fn ) and icam-1 , indicating that integrin ligands promote podosome stability ( figure 1 g ) . this stability of adhesion on integrin ligands correlates with an increase in the percentage of wild - type dcs with well - defined podosomes and small focal contacts ( figure 1a ) and accumulation of podosomal f - actin , integrins , and associated proteins ( data not shown ) . in contrast , no significant differences were detected in turnover index of adhesions between wip dcs plated on different substrata ( figure 1 g ) . the presence of integrin ligands failed to induce major changes in the composition of adhesions of wip dcs ( figure 1b ) except for an increase in the size of the focal adhesions . taken together , our data indicate that wip plays a key role in regulating dc adhesion structure and dynamics on biologically relevant substrata , processes crucial for the migratory capacity of cells . stabilization of the leading edge in the direction of dc movement is provided by podosomes [ 13 , 14 ] . migrating wild - type dcs clearly polarized , forming a patent leading edge , with the rear of the cell retracting at a similar rate , allowing net translocation of the cell body . in contrast , wip dcs failed to develop a major leading front and instead formed multiple simultaneous and unstable lateral lamellae and ruffles ( figures 2a2e ; movies s1 and s2 ) . cortactin is an arp2/3 activator that plays a major role in the formation of podosomes [ 15 , 16 ] , establishment of cell polarization , and also generation of cellular cortical protrusions . it has been proposed that cortactin synergises with the wasp family of proteins to induce actin polymerization , and it is thought that the actin filament meshwork triggered by the n - wasp / cortactin pair may have a different organization from actin meshworks triggered by n - wasp or cortactin alone . importantly , wip has been shown to interact with cortactin and induce cortactin - mediated actin polymerization . our results show that in the absence of wip , cortactin is displaced from podosome cores where it normally colocalizes with f - actin ( figure 2f ) and is instead associated with random lamellar protrusions and ruffles over the cell periphery ( figure 2 g ) . in addition , we found there was a higher proportion of cortactin associated with the cytoskeletal fraction ( triton insoluble ) in adherent wip dcs compared to wild - type dcs ( figure 2h ) . taken together , these results show that in the absence of wip , association of cortactin with the cytoskeleton is deregulated and associated with the formation of random cortical protrusions and loss of dc polarity . this suggests that the wasp / wip complex may couple with cortactin to induce a type of meshwork of actin filaments and associated proteins that allow the clustering of 2-integrins during the assembly of podosomes and dc polarization . in the absence of wip , cortactin can not be recruited to form a complex with wasp , discrete actin foci in podosomes fail to form , and instead , cortactin codistributes with actin filaments at the cell margin , resulting in the random protrusions and reduced motility ( data not shown ) observed in wip dcs . we have previously shown that wasp dcs display equivalent anomalies in 2-integrin clustering and focal contact formation to that observed in wip dcs . also we find here that like wasp dcs , wip dcs fail to polarize and form and stabilize consistent leading edges . recently , it has been proposed that wip contributes to the stabilization of wasp expression and the regulation of wasp - mediated actin rearrangement [ 8 , 22 ] . we find here that wip dcs express low levels of wasp at the protein level ( figure 3a ) , as recently described for wip t - lymphocytes . levels of wip expression were not affected by the lack of wasp in wasp dcs ( figure 3b ) . since mrna levels of wasp in wip dcs were comparable to those of wild - type dcs ( figure 3c ) the levels of expression of the wip binding partners cortactin , nck , and crkl was not affected in wip dcs ( figure s4 ) . it has been predicted that wip may prevent the exposure of wasp family proteins to proteases , because the 25-residue wasp binding motif from wip wraps around the wip binding domain of n - wasp . wasp , but not n - wasp , is a substrate for the protease calpain in platelets , and we have recently found that calpain also cleaves wasp in migrating dcs . our present results show that wip prevents wasp cleavage by calpain in dcs since inhibition of this protease in wip dcs resulted in major recovery of wasp expression ( figures 3d and 3e ) . this suggests that wip may play a crucial role in regulating the accessibility of wasp for calpain cleavage , which takes place during dissolution of podosomes . a role for wip in the maintenance of wasp levels is also suggested by recent findings showing that in was patients , mutations that prevent wasp binding to wip result in diminished expression of wasp [ 24 , 25 ] . we suggest that the low levels of wasp in these was patients may be the result of extensive calpain - mediated cleavage because of failure of wasp - wip interaction . surprisingly , the recovery of wasp levels in wip dcs by treatment with calpain inhibitors was not sufficient to induce podosome formation , and large focal contacts remained the major adhesion structures ( figure 4a ) . in addition , forced expression of egfp - wasp in wip dcs treated with calpain inhibitors alone or in conjunction with proteasome inhibitors failed to restore podosome formation , and egfp - wasp remained diffusely distributed in the cytoplasm or associated with anomalous f - actin aggregates that could contain vinculin forming abnormal plaques colocalizing with f - actin ( figure 4b , top and middle ) . this contrasts with the observed recovery of podosome formation and dynamics in wasp dcs ( where wip levels are normal ) after the expression of egfp - wasp ( figure 4b , bottom ) [ 11 , 13 ] . we excluded the possibility that wip binding is essential for the intrinsic actin polymerizing activity of wasp because specific wasp mutants ( wasp a134 t and wasp r138p ) that interrupt wip binding [ 24 , 27 ] showed an actin polymerizing activity equivalent to wild - type wasp ( figure 4d ) . these results indicate that wip not only protects wasp from calpain cleavage but also facilitates the localization of wasp to sites of actin polymerization and the organization of integrins , vinculin , and other integrin - associated proteins in circular clusters . the mechanisms by which wip regulates wasp activity to allow podosome formation are yet to be fully elucidated . it is possible that wasp / wip interactions may directly facilitate the recruitment of wasp to cell - surface receptors . alternatively , wip may facilitate the bridging of wasp to these receptors by mediating the interaction with other adaptor proteins or may regulate the function of regulatory proteins such as kinases , known to affect wasp activity [ 30 , 31 ] . our data show that wip by itself is not sufficient to promote podosome assembly because wasp dcs , which express normal levels of wip , also lack podosomes and assemble large focal contacts and disorganized actin clumps instead . it is tempting to speculate that the cellular responses ( of dc ) to external soluble ligands such as chemoattractants would recruit wasp / wip complexes coupled to integrin - associated proteins to trigger the assembly of podosomes at appropriate sites in the developing leading edge of the cell . these complexes would provide a cytoskeletal platform that regulates the accurate clustering and activation of 2-integrins , facilitating their function . taken together , our results suggest that wasp and wip work as a functional unit in a cellular context as has been previously proposed [ 8 , 9 ] to regulate podosome formation and dynamics during the establishment of cell polarity as seen in the chemotactic response to extracellular stimuli . in summary , we have shown that in dcs wip is essential for podosome formation and cell polarity by preventing the extensive degradation of wasp by calpain and by facilitating the recruitment of wasp to discrete foci to form the core of podosomes . the tight coupling between wasp and wip is therefore a key contributor to normal podosome dynamics and likely of major importance to the cell trafficking behavior of dcs .

summarythe wiskott - aldrich syndrome protein ( wasp ) is an adaptor protein that is essential for podosome formation in hematopoietic cells [ 1 ] . given that 80% of identified wiskott - aldrich syndrome patients result from mutations in the binding site for wasp - interacting - protein ( wip ) [ 2 ] , we examined the possible role of wip in the regulation of podosome architecture and cell motility in dendritic cells ( dcs ) . our results show that wip is essential both for the formation of actin cores containing wasp and cortactin and for the organization of integrin and integrin - associated proteins in circular arrays , specific characteristics of podosome structure . we also found that wip is essential for the maintenance of the high turnover of adhesions and polarity in dcs . wip exerts these functions by regulating calpain - mediated cleavage of wasp and by facilitating the localization of wasp to sites of actin polymerization at podosomes . taken together , our results indicate that wip is critical for the regulation of both the stability and localization of wasp in migrating dcs and suggest that wasp and wip operate as a functional unit to control dc motility in response to changes in the extracellular environment .

bronchial inflammation represents one of the main pathophysiological changes in bronchial asthma ( ba ) . the intensity of the inflammation influences the frequency and extent of symptoms , as well as the level of disease control . induced sputum provides direct information about inflammation . the non - invasive character and the possibility to perform this test at any disease stage , including exacerbation period , puts it in a special place in the process of evaluation of the child with bronchial asthma . the method is safe , easy to perform and generally well tolerated by children , helping establish the diagnosis and the long - term management . it is also used for research purposes and may be an alternative to bronchoalveolar lavage . our study s main objective was the analysis of the sputum cellularity in patients with ba in correlation with the level of disease control and the controlling therapy with / without inhaled glucocorticoid ( igc ) . the second objective was to establish the correlation between sputum cellularity and other indirect parameters used to evidence bronchial inflammation ( forced expiratory volume in 1 second the study was carried out in a group of children diagnosed with ba , admitted to the 3rd pediatric clinical hospital of cluj - napoca , romania , during july 2008 march 2012 . the study was performed after obtaining informed consent of the parents , according to the regulations of the ethics committee of the emergency clinical hospital for children . the inclusion criteria were : - certified diagnosis of ba;- age between 6 18 years;- absence of airways acute infection signs in the past 6 weeks;- patient s ability to perform respiratory function test correctly;- parents informed consent . - certified diagnosis of ba ; - age between 6 18 years ; - absence of airways acute infection signs in the past 6 weeks ; - patient s ability to perform respiratory function test correctly ; - parents informed consent . we excluded from our study the patients in exacerbation and who could not perform the technique of induced sputum and the spirometric test adequately . ba diagnosis was established based on the clinical manifestations and at least one of the following criteria : - positive bronchial reversibility : increase of fev1 by over 12% after salbutamol administration;- previous admissions for exacerbations;- at least two presentations to the emergency room due to asthma exacerbations . - positive bronchial reversibility : increase of fev1 by over 12% after salbutamol administration ; - previous admissions for exacerbations ; - at least two presentations to the emergency room due to asthma exacerbations . in children with ba included in our study , we performed induced sputum along with spirometry and determination of eno . we started sputum induction with ultrasound nebulization of 3% sodium chloride solution for 5 minutes , after which the patient was asked to expectorate . if sputum was not obtained with this concentration , higher concentrated saline solutions of 4% and 5% were nebulized for another 5 minutes each . after the change of every concentration , , the patients were assessed by spirometry and received 200 g salbutamol at the beginning and at the end of the test . when changing the solutions , the patient was asked to perform the peak expiratory flow ( pef ) using the peak flow meter ( figure 1 ) . the sputum sample was weighed and dithiothreitol ( dtt ) was added in a 4:1 ratio . the sputum - dtt mixture was centrifuged ( 3001500 x g ) for 5 minutes , thus having two phases : the sediment ( represented by cells , which was analyzed under the microscope ) , and the liquid phase . after centrifugation , the cells from the sediment were counted with a hemocytometer , the test being valid for interpretation only if the counting showed at least 400 non - squamous cells on slides . from the centrifugal sediment , a may - grunwald - giemsa stained smear was made . using the optical microscope the number of eosinophils ( eo ) , neutrophils ( n ) , macrophages and bronchial epithelial cells were counted , each value being expressed as percentage from the total of non - squamous cells . the sample obtained was considered unsatisfactory if the percentage of squamous cells was higher than 80% . according to the literature , we considered the upper limit of normal 2.5% for eosinophils and the determination of eno was performed with an appropriate device ( niox - mino sweden ) , the results being expressed in p.p.b . ( parts per billion ) . for children under igc therapy , high levels of eno were considered to be 20 p.p.b . if under 12 years of age , and 25 p.p.b . if over 12 years . in patients not under igc therapy , high eno values were considered 5 p.p.b .. the degree of bronchial obstruction was established by spirometry . the bronchial reversibility test was considered positive in cases in which fev1 increased by more than 12% after salbutamol administration . asthma was labeled as atopic if patients presented one or more of the following : associated allergic rhinitis , positive skin test for one or several allergens , high total immunoglobulin e ( ige ) concentration . we used the prick technique for allergy skin tests and the total ige concentration was determined by elisa . we calculated the statistically significant differences between the means of variables obtained in the study subgroups . when we had 3 subgroups we used the kruskal - wallis test , while for the situations in which we formed 2 subgroups the mann - whitney test was applied . the correlation between the parameters was documented using spearman s correlation coefficient ( direct correlation if r>0.6 and indirect correlation if r<0.6 ) , considered statistically significant if p<0.05 . we calculated the statistically significant differences between the means of variables obtained in the study subgroups . when we had 3 subgroups we used the kruskal - wallis test , while for the situations in which we formed 2 subgroups the mann - whitney test was applied . the correlation between the parameters was documented using spearman s correlation coefficient ( direct correlation if r>0.6 and indirect correlation if r<0.6 ) , considered statistically significant if p<0.05 . the characteristics of children with ba included in the study are shown in table i. sputum for cellularity assessment was obtained from 55 of the 67 patients studied ( 82% ) . during the test only 11 children ( 16.4% ) had mild adverse reactions : rhinorheea ( 7.4% ) , sneezing ( 2.9% ) , cough ( 5.9% ) , fev1 decrease by more than 12% after nebulization ( 4.4% ) . sputum cellularity evidenced the existence of statistically significant differences both for eosinophils ( p=0.049 ) and neutrophils ( p=0.004 ) between patients with controlled ba and those with partial or uncontrolled disease . in patients with controlled ba the eosinophils count was significantly lower than in those with partially controlled or uncontrolled ba . regarding neutrophils , the highest percentage was found in patients with uncontrolled ba , while the lowest count was observed in patents with controlled ba . the analysis of the sputum s cellularity in relation to the controlling therapy did not evidence statistically significant differences between patients on igc therapy and those without this therapy . higher neutrophils levels were found in patients with uncontrolled asthma , both in those under igc therapy and those without this treatment ( table iv ) . analyzing the sputum cellularity in relation to spirometric parameters , we found higher eosinophils counts in patients with fev1 80% as compared with those with normal fev1 ( meansd : 40.330.3% versus 10.611.9% , p=0.003 ) ( table v ) . when analyzing fev1 in patients with high eosinophils values , we observed the existence of a moderate to good indirect correlation ( r=0.56 , p=0.005 ) , but the correlation was not present for patients with normal eosinophils values ( figure 1 ) . we could not establish a correlation between fev1 and the neutrophils count in the sputum , whether the latter was high or normal . of all 55 patients that provided sputum sample , only 24 underwent determination of eno . our study demonstrated the presence of a moderate to good correlation , statistically significant , between the value of eno and the neutrophils percentage in the sputum ( r=0.67 , p=0.0003 ) . our study evidenced higher eosinophils and neutrophils values in the sputum of patients with partially controlled or uncontrolled ba than in those with controlled asthma . we also obtained a statistically significant correlation between the value of eno and the neutrophils percentage in the sputum , which demonstrates the same involvement of neutrophils in the bronchial inflammatory process . the indirect correlation between the high eosinophils percentage and fev1 and the direct correlation between neutrophils and eno point to the value of induced sputum test when it is associated with other indirect parameters used in the assessment of bronchial inflammation . the success rate of induced sputum test in our study was 82% , similar to the reports in literature . during the test we recorded adverse reactions in 16.4% of the patients , similar to other studies . in 4.4% of the patients in which cough was induced , fev1 decreased by more than 12% after saline nebulization , but it was restored after salbutamol administration . the highest percent of eosinophils was observed in patients with partially controlled asthma ( 1825.2 ) , comparing with patients with uncontrolled asthma ( 4.914.4 ) . this finding is not according to most of the studies from literature and the explanation for this result can be the relatively small number of paient included in our study , especially by those with uncontrolled asthma . induced sputum is considered to be one of the most accurate method of assessment of bronchial inflammation . the technique is non - invasive , cheap and may be performed even in patients during a bronchial obstruction episode . the method is successfully used not only in patients with ba , but also in other diseases , such as chronic obstructive pulmonary disease , cystic fibrosis , chronic cough . though most studies have considered the eosinophils count in the sputum to be relevant for bronchial inflammation , more recent data suggest the same relevance regarding neutrophils . various studies evidenced high values of neutrophils in the sputum of patients with ba , especially uncontrolled asthma . a highly increased neutrophils count was evidenced in the sputum of patients during an exacerbation episode , triggered by a viral infection or in which respiratory symptoms were persistent . in the case of these patients , the eosinophils count was generally low , while the response to igc therapy was poor . this explanation is unlikely to be valid for our study , as we excluded patients with signs of acute respiratory infections in the 6 weeks prior the study . various studies analyzing the response to controlling therapy in patients with a high neutrophils level evidenced a lack of response to igc . the lack of studies on the role of neutrophils in the pathogenesis of ba may be explained by the fact that many researchers did not investigate or report all cell types from the sputum , focusing only on eosinophils . another explanation may be related to the method of sputum collection , many authors analyzing only the samples rich in mucus and not the whole amount of sputum obtained . our study supports the theory according to which ba should be also classified according to the inflammatory phenotype of sputum cellularity . the literature describes 3 phenotypes according to sputum cellularity : eosinophilic , neutrophilic and mixed . certain studies mention the neutrophilic type more frequently in severe asthma , refractory to igc therapy . although our study showed that in uncontrolled asthma patients the predominant sputum cells are neutrophils and not eosinophils like we used to know , there are some recent ones that demonstrated the importance of this type of cells . this studies highlights that in uncontrolled asthmatic patients that do not respond to the glucocorticoid therapy , the explanation rises from the presence of a high percent of neutrophils in sputum . the results have clinical implications because in this patients the therapy needs to be changed and this is a motive for more studies to be made . regardless the contradictory results , we all agree in the importance of induced sputum test , especially when it is associated with other indirect parameters used in the assessment of bronchial inflammation .

backgroundinduced sputum in children with bronchial asthma represents a non - invasive method of bronchial inflammation assessment . the main objective of our study was to analyze the cellularity of sputum in patients with bronchial asthma according to the level of disease control and the controlling therapy ( with / without inhaled glucocorticoids ) . the second objective was to establish the correlation between sputum cellularity and other indirect parameters used to evidence bronchial inflammation ( exhaled nitric oxide ) and obstruction ( forced expiratory volume in 1 second).methodsthe study included children with bronchial asthma that were assessed clinically ( physical exam , questionnaire on the control of bronchial asthma in children ) and by medical tests ( induced sputum , exhaled nitric oxide , spirometry).resultsin patients with partially controlled asthma and those with uncontrolled asthma , the eosinophils percentage in the sputum was higher than in patients with controlled asthma ( 19.826.4% respectively 9.220.5% versus 4.514.6% , p<0.001 ) . higher percentage of neutrophils in the sputum was found in the partially controlled and uncontrolled asthma than in the controlled asthma ( 43.920.1% respectively 51.638.3% versus 3519.7% , p=0.009 ) . we also evidenced a direct and statistically significant correlation between the exhaled nitric oxide and the neutrophils percentage in the sputum ( r=0.67 , p=0.0003 ) . also , an indirect , moderate to good correlation ( r=0.56 , p=0.005 ) was evidenced between the values of the forced expiratory volume in 1 second and the high eosinophils percentage in the sputum.conclusionsin partially controlled and uncontrolled bronchial asthma the eosinophils and neutrophils count in the sputum is significantly higher than in patients with controlled asthma . there is an indirect correlation between the high eosinophils count in the sputum and the forced expiratory volume in 1 second , as well as a direct correlation between the neutrophils count and the exhaled nitric oxide , suggesting that induced sputum should be used in combination with other indirect parameters for the evidence of bronchial inflammation .

angular deformities around the knee joint necessitate special consideration to restore normal alignment during total knee arthroplasty ( tka ) . valgus knees are defined as those with > 10 of mechanical tibiofemoral angle on standing hip to ankle anteroposterior ( ap ) radiographs . typically , a valgus deformity may be due to bony deformities such as hypoplastic lateral condyle , tibial bone loss , or due to soft tissue contracture involving iliotibial band ( it band ) , biceps femoris , lateral collateral ligament ( lcl ) or posterolateral capsule,1234 or may be due to both bony and soft tissue involvement . consequently , attenuation of medial soft tissue structures may be a late secondary event.1 an ideal approach for valgus knees must provide adequate exposure with minimal complications due to approach per se . several studies 5678910 had demonstrated the use of lateral capsular approach and modified lateral capsular approach with repositioning of vastus lateralis or tibial tubercle osteotomy ( tto ) . in addition , a lateral subvastus 1112 approach has been described , but it may require snip of vastus lateralis obliquus 11 or tto 12 to shift the patella medially . however , these conventional approaches may result in short and long term deficit in quadriceps muscle function post - tka 131415 or complications related to tto . subsequently , this may reflect lower patient satisfaction due to reduced mobility and significant pain in postoperative period.13 with the recent emphasis on minimally invasive muscle sparing approach in orthopedic surgery including tka , the subvastus approach has been commonly described for varus knees . furthermore , this approach has definite advantages of maintaining extensor mechanism integrity , minimal effect on patellar tracking and preservation of patellar blood supply.1617181920 ultimately , these lead to faster recovery periods , shorter hospital stay , minimal postoperative physiotherapy , improved patellar tracking and less postoperative pain,1317202122 thereby resulting in better functional outcomes . therefore , the present study aimed to evaluate the outcomes of tka and efficacy of minimally invasive subvastus ( mini - subvastus ) approach in valgus knees in terms of : ( 1 ) early functional recovery ( 2 ) postoperative limb alignment and ( 3 ) incidence of complications . we hypothesized that mini - subvastus approach can be well utilized for tka in valgus knees and it results in early functional recovery with minimal complications 112 knees in 108 patients with valgus deformity operated for elective primary tkas by subvastus approach between january 2006 and december 2011 ( out of 3000 consecutive primary tka 's patients ) were included in this retrospective study . exclusion criteria were patients with previous arthrotomy of knee , neurological problems and those requiring revision tka . all surgeries were performed by senior author ( nas ) using mini - subvastus approach without using pneumatic tourniquet . tranexamic acid,23 15 mg / kg , was given 30 minutes prior to surgery and 2 doses 10 mg / kg were given 3 and 6 h postsurgery . a skin incision about 4 inches in length [ figure 1 ] was made slightly medial to the midline of the knee extending from the superior pole of patella to the tibial tubercle in 90 of flexion . intraoperatively , saline adrenaline ( 1:300,000 ) was infiltrated into skin and subcutaneous tissue to reduce bleeding . an l - shaped capsulotomy [ figure 2 ] made with horizontal limb of the l along the inferior margin of vm until the superior pole of patella and vertical limb of l was extended from here until tibial tubercle . the patella was displaced laterally to expose the suprapatellar synovium that was divided medially keeping the suprapatellar pouch intact [ figure 3 ] . peroperative photograph showing mini incision used for subvastus total knee arthroplasty intraoperative photograph showing l - shaped arthrotomy used for subvastus technique intraoperative photograph showing division of suprapatellar synovium at this stage , the femoral trochlea was visible . if present , prominent osteophytes in supratrochlear region were removed . the dissection on medial side was kept to a minimum and no ligament release was performed medially . the distal femoral cut was made first using downsized intramedullary jig with the appropriate valgus angle , generally 3. an extramedullary check was made preoperatively to confirm that this was indeed the correct valgus angle before making the femoral cut . the femoral pulse was felt and a marker ( a stick on electrocardiography lead with metal ) was placed over this . on this , a small vertical marker ( an empty inverted water for injection sterile plastic ampoule ) was strapped on in such a way that it would be easy to feel through the drapes . before cutting the distal femur , the extramedullary femoral guide was used to ascertain that the rod was pointing to this marker applied preoperatively . the tibial cut was made perpendicular to its mechanical axis using an extramedullary jig ( i.e. , neutral varus - valgus ) . the depth of the cut was 810 mm from the intact medial condyle ( in valgus knee ) . the angle that is the slope of the cut was 03. a spacer block was utilized to check extension space . if necessary , lateral release was performed to establish a symmetrical extension space as follows [ figure 4a and b ] : with the knee in extension and distracted with lamina spreader , the posterolateral capsule was released from the tibia . typically , this was done all along the tibial margin from 6 to 9 clock position . if further , posterolateral release was required , it was done with knee flexed at 90. care was taken not to detach the popliteus tendon as it is important to maintain stability in flexion . if still further release was required , it band was released from gerdy 's tubercle . the lcl release although well described in literature , was not required in our cases.82425 the anterior and posterior femoral cuts were made using the ap cutting jig . in all cases , we used the transepicondylar axis as a reference for jig placement [ figure 5 ] . jig was utilized to size and resect the patella if patellar resurfacing was to be carried out . trial components were inserted and careful check was made regarding range of motion ( rom ) , stability and patellar tracking . all knees used were fixed bearing metal backed components ( cruciate retaining [ cr ] or posterioir stabilized [ ps ] ; [ nexgen ; lps ; zimmer inc . , an apical stitch at the angle of the l was first taken to ensure that the capsule was neither advanced nor recessed . the knee was infiltrated with 20 cc of mixture containing 0.25% bupivacaine , cefuroxime and normal saline . at the end of the procedure , a bulky dressing was applied for the first 24 h. intraoperative photograph showing ( a ) asymmetrical extension gap with tight lateral structures . ( b ) released lateral capsule intraoperative photograph showing jig placement parallel to the transepicondylar axis a continuous femoral nerve block technique was used for 12 h postoperatively for pain control and early recovery . static quadriceps exercises and straight leg raising ( slr ) exercise were started from day 0 and rom exercises beginning from day one . chemical prophylaxis for deep vein thrombosis was in form of tablet aspirin 75 mg once a day for 3 weeks . patients were encouraged to get out of bed and walk as tolerated from day 1 . all clinical information was collected from database system maintained from predesigned data sheets by an independent investigator ( one of authors ) . the clinical information included demographic data , preoperative and postoperative clinical status [ figure 6a and b ] including degree of valgus deformity , the correctability , rom , patients categories based on krackow et al.26 classification of valgus knees and postoperative outcomes ( at 2 weeks , 6 weeks , 3 months , 6 months , 12 months and yearly thereafter ) . postoperative pain was assessed using visual analog scale ( vas ) on postoperative day ( pod ) 1 and pod2 . early quadriceps recovery was measured in form of time to active slr and staircase competency . preoperative clinical statuses and postoperative outcomes were evaluated using american knee society ( aks ) knee and functional score.22 postoperatively , a standing whole - limb ap radiograph was taken after surgery and yearly thereafter to measure mechanical tibiofemoral angle [ figure 7a and b ] . knee series including ap view , lateral view [ figure 7c and d ] and an additional merchant view radiograph were also taken and evaluated . clinical photograph showing ( a ) severe valgus deformity before total knee arthroplasty and ( b ) after total knee arthroplasty ( a ) preoperative scanogram of a patient showing valgus deformity . ( b ) postoperative scanogram after total knee arthroplasty showing correction of deformity ( c ) postoperative radiograph with cruciate retaining prosthesis , anteroposterior view . ( d ) postoperative radiograph with cruciate retaining prosthesis , lateral view two of the authors ( npj , nas ) made all radiographic measurements using full length weight bearing ap radiographs . we measured mechanical tibiofemoral angle of knee , which was defined as the angle formed by the intersection between mechanical axes of the femur ( line from the femoral head center to the femoral intercondylar notch center ) and the tibia [ line from the centre of ankle ( talus ) center to the center of knee ( tibial spine tips ) ] . to determine the intra and inter - observer reliabilities of radiographic assessments , two investigators performed all radiographic assessments in 20 randomly selected radiographs twice with an interval of 1-week . the intra- and inter - observer reliabilities of assessments of all radiographic measurements were evaluated using intraclass correlation coefficients ( iccs ) . the iccs of the intra- and inter - observer reliabilities of all measurements were > 0.90 . because the measurements were judged reliable , measurements taken by a single investigator ( npj ) were used in the analyses . statistical analyses were performed using spss for windows ( version 20.0 , ibm , chicago , il , usa ) . the difference was considered significant with p < 0.05 at a probability level of 95% for all comparisons . statistical significance was determined using student 's t - test for continuous variables and the chi - square , or the fisher 's exact test for categorical variables . all surgeries were performed by senior author ( nas ) using mini - subvastus approach without using pneumatic tourniquet . tranexamic acid,23 15 mg / kg , was given 30 minutes prior to surgery and 2 doses 10 mg / kg were given 3 and 6 h postsurgery . a skin incision about 4 inches in length [ figure 1 ] was made slightly medial to the midline of the knee extending from the superior pole of patella to the tibial tubercle in 90 of flexion . intraoperatively , saline adrenaline ( 1:300,000 ) was infiltrated into skin and subcutaneous tissue to reduce bleeding . an l - shaped capsulotomy [ figure 2 ] made with horizontal limb of the l along the inferior margin of vm until the superior pole of patella and vertical limb of l was extended from here until tibial tubercle . the patella was displaced laterally to expose the suprapatellar synovium that was divided medially keeping the suprapatellar pouch intact [ figure 3 ] . peroperative photograph showing mini incision used for subvastus total knee arthroplasty intraoperative photograph showing l - shaped arthrotomy used for subvastus technique intraoperative photograph showing division of suprapatellar synovium at this stage , the femoral trochlea was visible . if present , prominent osteophytes in supratrochlear region were removed . the dissection on medial side was kept to a minimum and no ligament release was performed medially . the distal femoral cut was made first using downsized intramedullary jig with the appropriate valgus angle , generally 3. an extramedullary check was made preoperatively to confirm that this was indeed the correct valgus angle before making the femoral cut . the femoral pulse was felt and a marker ( a stick on electrocardiography lead with metal ) was placed over this . on this , a small vertical marker ( an empty inverted water for injection sterile plastic ampoule ) was strapped on in such a way that it would be easy to feel through the drapes . before cutting the distal femur , the extramedullary femoral guide was used to ascertain that the rod was pointing to this marker applied preoperatively . the tibial cut was made perpendicular to its mechanical axis using an extramedullary jig ( i.e. , neutral varus - valgus ) . the depth of the cut was 810 mm from the intact medial condyle ( in valgus knee ) . the angle that is the slope of the cut was 03. a spacer block was utilized to check extension space . if necessary , lateral release was performed to establish a symmetrical extension space as follows [ figure 4a and b ] : with the knee in extension and distracted with lamina spreader , the posterolateral capsule was released from the tibia . typically , this was done all along the tibial margin from 6 to 9 clock position . if further , posterolateral release was required , it was done with knee flexed at 90. care was taken not to detach the popliteus tendon as it is important to maintain stability in flexion . if still further release was required , it band was released from gerdy 's tubercle . the lcl release although well described in literature , was not required in our cases.82425 the anterior and posterior femoral cuts were made using the ap cutting jig . in all cases , we used the transepicondylar axis as a reference for jig placement [ figure 5 ] . jig was utilized to size and resect the patella if patellar resurfacing was to be carried out . trial components were inserted and careful check was made regarding range of motion ( rom ) , stability and patellar tracking . all knees used were fixed bearing metal backed components ( cruciate retaining [ cr ] or posterioir stabilized [ ps ] ; [ nexgen ; lps ; zimmer inc . , all implants were fixed with cement . an apical stitch at the angle of the l was first taken to ensure that the capsule was neither advanced nor recessed . the knee was infiltrated with 20 cc of mixture containing 0.25% bupivacaine , cefuroxime and normal saline . at the end of the procedure , a bulky dressing was applied for the first 24 h. intraoperative photograph showing ( a ) asymmetrical extension gap with tight lateral structures . ( b ) released lateral capsule intraoperative photograph showing jig placement parallel to the transepicondylar axis a continuous femoral nerve block technique was used for 12 h postoperatively for pain control and early recovery . static quadriceps exercises and straight leg raising ( slr ) exercise were started from day 0 and rom exercises beginning from day one . chemical prophylaxis for deep vein thrombosis was in form of tablet aspirin 75 mg once a day for 3 weeks . patients were encouraged to get out of bed and walk as tolerated from day 1 . all clinical information was collected from database system maintained from predesigned data sheets by an independent investigator ( one of authors ) . the clinical information included demographic data , preoperative and postoperative clinical status [ figure 6a and b ] including degree of valgus deformity , the correctability , rom , patients categories based on krackow et al.26 classification of valgus knees and postoperative outcomes ( at 2 weeks , 6 weeks , 3 months , 6 months , 12 months and yearly thereafter ) . postoperative pain was assessed using visual analog scale ( vas ) on postoperative day ( pod ) 1 and pod2 . early quadriceps recovery was measured in form of time to active slr and staircase competency . preoperative clinical statuses and postoperative outcomes were evaluated using american knee society ( aks ) knee and functional score.22 postoperatively , a standing whole - limb ap radiograph was taken after surgery and yearly thereafter to measure mechanical tibiofemoral angle [ figure 7a and b ] . knee series including ap view , lateral view [ figure 7c and d ] and an additional merchant view radiograph were also taken and evaluated . clinical photograph showing ( a ) severe valgus deformity before total knee arthroplasty and ( b ) after total knee arthroplasty ( a ) preoperative scanogram of a patient showing valgus deformity . ( b ) postoperative scanogram after total knee arthroplasty showing correction of deformity ( c ) postoperative radiograph with cruciate retaining prosthesis , anteroposterior view . ( d ) postoperative radiograph with cruciate retaining prosthesis , lateral view two of the authors ( npj , nas ) made all radiographic measurements using full length weight bearing ap radiographs . we measured mechanical tibiofemoral angle of knee , which was defined as the angle formed by the intersection between mechanical axes of the femur ( line from the femoral head center to the femoral intercondylar notch center ) and the tibia [ line from the centre of ankle ( talus ) center to the center of knee ( tibial spine tips ) ] . to determine the intra and inter - observer reliabilities of radiographic assessments , two investigators performed all radiographic assessments in 20 randomly selected radiographs twice with an interval of 1-week . the intra- and inter - observer reliabilities of assessments of all radiographic measurements were evaluated using intraclass correlation coefficients ( iccs ) . the iccs of the intra- and inter - observer reliabilities of all measurements were > 0.90 . because the measurements were judged reliable , measurements taken by a single investigator ( npj ) were used in the analyses . a continuous femoral nerve block technique was used for 12 h postoperatively for pain control and early recovery . static quadriceps exercises and straight leg raising ( slr ) exercise were started from day 0 and rom exercises beginning from day one . chemical prophylaxis for deep vein thrombosis was in form of tablet aspirin 75 mg once a day for 3 weeks . patients were encouraged to get out of bed and walk as tolerated from day 1 . all clinical information was collected from database system maintained from predesigned data sheets by an independent investigator ( one of authors ) . the clinical information included demographic data , preoperative and postoperative clinical status [ figure 6a and b ] including degree of valgus deformity , the correctability , rom , patients categories based on krackow et al.26 classification of valgus knees and postoperative outcomes ( at 2 weeks , 6 weeks , 3 months , 6 months , 12 months and yearly thereafter ) . postoperative pain was assessed using visual analog scale ( vas ) on postoperative day ( pod ) 1 and pod2 . early quadriceps recovery was measured in form of time to active slr and staircase competency . preoperative clinical statuses and postoperative outcomes were evaluated using american knee society ( aks ) knee and functional score.22 postoperatively , a standing whole - limb ap radiograph was taken after surgery and yearly thereafter to measure mechanical tibiofemoral angle [ figure 7a and b ] . knee series including ap view , lateral view [ figure 7c and d ] and an additional merchant view radiograph were also taken and evaluated . clinical photograph showing ( a ) severe valgus deformity before total knee arthroplasty and ( b ) after total knee arthroplasty ( a ) preoperative scanogram of a patient showing valgus deformity . ( b ) postoperative scanogram after total knee arthroplasty showing correction of deformity ( c ) postoperative radiograph with cruciate retaining prosthesis , anteroposterior view . ( d ) postoperative radiograph with cruciate retaining prosthesis , lateral view two of the authors ( npj , nas ) made all radiographic measurements using full length weight bearing ap radiographs . we measured mechanical tibiofemoral angle of knee , which was defined as the angle formed by the intersection between mechanical axes of the femur ( line from the femoral head center to the femoral intercondylar notch center ) and the tibia [ line from the centre of ankle ( talus ) center to the center of knee ( tibial spine tips ) ] . to determine the intra and inter - observer reliabilities of radiographic assessments , two investigators performed all radiographic assessments in 20 randomly selected radiographs twice with an interval of 1-week . the intra- and inter - observer reliabilities of assessments of all radiographic measurements were evaluated using intraclass correlation coefficients ( iccs ) . the iccs of the intra- and inter - observer reliabilities of all measurements were > 0.90 . because the measurements were judged reliable , measurements taken by a single investigator ( npj ) were used in the analyses . statistical analyses were performed using spss for windows ( version 20.0 , ibm , chicago , il , usa ) . the difference was considered significant with p < 0.05 at a probability level of 95% for all comparisons . statistical significance was determined using student 's t - test for continuous variables and the chi - square , or the fisher 's exact test for categorical variables . the mean duration of followup was 40 months ( range 2484 months ) . in no case , this approach was abandoned and the approach proved effective irrespective of the degree of valgus deformity [ table 1 ] . according to krackow et al.,26 category for valgus knee , 76 knees were type 1 , 32 knees were type 2 and 2 knees belonged to type 3 category . release of the lcl or popliteus was not performed in any case . in all these cases , a cr knees were used in 89 patients and ps knees in 20 patients and constrained condylar knee in one patient with severe combined valgus and recurvatum deformity and patellar resurfacing was done in 36 cases . demographic characteristics of patients undergoing total knee arthroplasty the mean ( standard deviation [ sd ] ) vas on pod1 and pod2 at rest was 2.73 ( 0.67 ) and 2.39 ( 1.11 ) , respectively and after mobilization was 3.28 ( 0.68 ) and 3.08 ( 1.20 ) , respectively ( p < 0.001 ) . the quadriceps recovery was good , and 92 ( 86.7% ) patients were able to do active slr by pod1 with mean ( sd ) time of 21.98 ( 4.09 ) h. the mean ( sd ) time for reciprocal gait and staircase competency was 43.05 ( 6.59 ) h. the mean ( sd ) length of hospital stay was 3.3 ( 0.44 ) days . the aks and function score showed significant improvement from preoperative mean of 39 ( 11.58 ) and 36 ( 11.27 ) to 91 ( 5.04 ) and 79 ( 8.30 ) , respectively at latest followup ( p < 0.001 ) . the mean rom increased from 102 ( 10 ) preoperatively to 119 ( 9 ) at recent followup ( p < 0.001 ) . the mean tibiofemoral valgus angle was corrected from preoperative 16 ( range 1035 ) to 5 ( range 39 ) of valgus ( p < 0.001 ) . there were three outliers with two patient having 9 valgus each and one with 8 valgus . moreover , the component positioning and cementation appeared satisfactory on radiographs with no radiolucency 's and signs of loosening in any patient . furthermore , the outcome of tka using either cr or ps knee showed similar results without any significant difference [ table 2 ] . comparison of outcomes among cr and ps implant groups two patients underwent revision surgery , one for patellar component malposition ; one had liner exchange for hyperextension . also , one patient had foot drop postoperatively probably due to peroneal nerve injury that recovered gradually and completely within 6 weeks . the most important finding of the present study is that medial - subvastus approach provides satisfactory and adequate exposure for tka in valgus knees . tka in valgus knees is considered to be more challenging 122 as it is relatively uncommon in practice . moreover , it has a different pathoanatomy : femur is internally rotated and tibia externally rotated , lateral femoral condyle is deficient in contrast to varus knee where tibia is usually involved , lateral and posterior contractures are present , and ligament balancing is difficult.23427 thus , the present study was undertaken to demonstrate the efficacy of mini - subvastus approach in valgus knees . our findings support the hypothesis that mini subvastus approach can be utilized for tka in valgus knees and that it results in good functional outcome . the present study showed early postoperative recovery in patients post tka with lower mean vas score,28 early mobilization with active slr and staircase competency . the findings of the present study of lower mean vas score is considerably better than the similar vas score assessed by huang et al.22 using median parapatellar approach . moreover , several studies 19202930 in past have evidently endorsed the benefits of muscle sparing subvastus approach in faster recovery and less postoperative pain . also , recently published study 28 on effect of far medial - subvastus technique in fixed valgus type 2 krackow knees showed similar results . the functional outcome at the latest followup assessed by mean aks score showed significant improvement . moreover , the mechanical alignment was good with mean valgus angle of 5. similarly , comparable findings were reported in several studies [ table 3 ] using different approaches for tka in valgus knees . summary of studies reporting outcomes of total knee arthroplasty in valgus knees medial parapatellar is universally accepted due to its familiarity to most surgeons and providing satisfactory exposure in valgus knees , even for lateral soft tissue release.192031 however , several studies 63233 have reported inferior outcomes with medial capsular approach . particularly , it may be due to additional release of lateral soft tissue structures jeopardizing patellar blood supply.34 multiple previous studies 567891011 had described lateral parapatellar and subvastus approaches for valgus knees . moreover , one advantage of lateral approach is that it can directly address the disturbed pathoanatomy of valgus knee . furthermore , lateral approach maintains intact medial soft tissue structures thereby rendering patellar vascularity 34 undisturbed . however , difficulties related to lateral capsulotomy is medial patellar eversion , its unfamiliarity to most surgeons and problems related to tto.2610 in spite of having obvious advantages over other conventional approaches , mini - subvastus approach is seldom used for tka in valgus knees . in fact , it is considered to be relative contraindication in valgus knees.31 moreover , the approach is considered difficult as it is not a familiar approach . theoretically , it can be as good as or even better than median parapatellar approach , as illustrated by several studies.1719202135 nonetheless , it preserves the patellar vascularity even after lateral soft tissue release due to intact quadriceps mechanism . furthermore , major criticism for subvastus technique includes difficulty in subluxation and eversion of patella , poor , and unpredictable exposure , more so of lateral structures and malalignment of components.29 in valgus knees , the patella tends to subluxate laterally due to tight lateral structures and attenuation of medial structures.36 thus , it in fact makes the surgeon 's job easier to dislocate the patella laterally if vm is released adequately from intermuscular septum.35 moreover , synovial division further helps in lateral displacement of patella.35 also , our surgical technique involves eversion of patella after tibial and femoral cuts were taken as quadriceps gets relaxed . although , all the parts of the knee may not be seen simultaneously , symbiotic use of retractors and use of mobile skin window to accomplish the sequential steps with adequate exposure are the key factors in the mini - subvastus approach.3537 in our series , we were able to gain exposure that even suffice for release of the lateral structures including posterolateral capsule and it band . in addition , in this study , posterolateral capsule was released in 86 ( 78.18% ) cases while it band was released in 62 ( 56.36% ) knees using piecrust technique . while none of the cases required extensive lateral release including lcl and popliteus , and medial advancement techniques . likewise , several studies 82425 had revealed the importance of isolated it band release or titrated release of lateral structures by pie crusting method for gap balancing . furthermore , the present study did not observe any component malalignment , which has also been case with similar other studies.1838 however , a detailed analysis of component positioning would form the basis of another study . appropriate exposure of lateral tibial plateau 35 and use of transepicondylar axis for referencing instead of posterior condylar referencing 1 for femur helps us to minimize the component malalignment in valgus knees . in present study , a recent study 39 demonstrated the efficacy of cr implants in valgus knees and that survival of implant is improved when lateral stabilizers like lcl and/or popliteus tendon are preserved . moreover , we assessed the sagittal plane stability intraoperatively by adequacy of posterior cruciate ligament ( pcl ) , and the decision to retain or substitute the pcl was made intraoperatively . sacrifice of pcl was done in cases of laxity and deformity not correctable without the release of pcl . furthermore , the outcome of patients with both cr and ps implant types were similar with no significant differences [ table 2 ] . only one patient needed constrained condylar knee with severe combined valgus and recurvatum deformity . likewise , several studies in past had reported the use of constrained knees using various approaches.5740 patellar resurfacing was done in selected 36 knees , especially those with significant disease of femoral trochlear surface . in our series , one knee had patellar component malpositioning , due to surgical error , which can be avoided by carefully identifying the boundaries of patella after resection . it may be due to peroneal nerve injury,4142 direct traction on the nerve , traction on the surrounding tissues resulting in vascular compromise to the nerve , direct pressure on the nerve from the postoperative dressing , or a combination of these factors . the complication rates were comparable to other series using various conventional approaches [ table 3 ] . the limitations of the study are : first , this is a retrospective study with all its inherent biases and limitations . however , all the patients were managed by single surgeon senior author ( nas ) , using same protocol thereby decreasing the influence of the confounding factors . second , the current study is a midterm followup study with average followup of 40 months . third , this study does not involve any comparison with the other approaches used for valgus knees . but , the senior author exclusively uses subvastus approach for all cases of tka including varus knees and revisions with satisfactory outcomes . fourth , the assessment of vas scores was done on postoperative day 1 and 2 . however , the results may have been affected by the use of femoral nerve block though it has been used for 12 h postoperatively only . a mini - subvastus medial quadriceps approach provides adequate exposure for tka in valgus knees including the lateral structures , without increase in incidence of complications . it provides excellent early recovery phase.13172021 midterm followup showed good outcome in terms of mechanical alignment and the functional scores .

background : an ideal approach for valgus knees must provide adequate exposure with minimal complications due to approach per se . median parapatellar approach is most commonly used approach in tka including valgus knees . a medial subvastus approach is seldom used for valgus knees and has definite advantages of maintaining extensor mechanism integrity and minimal effect on patellar tracking . the present study was conducted to evaluate outcomes of total knee arthroplasty ( tka ) and efficacy of subvastus approach in valgus knees in terms of early functional recovery , limb alignment and complications.materials and methods : we retrospectively reviewed 112 knees with valgus deformity between january 2006 and december 2011 . all patients were assessed postoperatively for pain using visual analog scale ( vas ) and quadriceps recovery in form of time to active straight leg raising ( slr ) and staircase competency and clinical outcomes using american knee society ( aks ) score and radiographic evaluation with average followup of 40 months ( range 2484 months).results : the mean vas on postoperative day ( pod ) 1 and pod2 at rest was 2.73 and 2.39 , respectively and after mobilization was 3.28 and 3.08 , respectively ( p < 0.001 ) . the quadriceps recovery was very early and 92 ( 86.7% ) patients were able to do active slr by pod1 with mean time of 21.98 h while reciprocal gait and staircase competency was possible at 43.05 h. the aks and function score showed significant improvement from preoperative mean score of 39 and 36 to 91 and 79 ( p < 0.001 ) , respectively , and the mean range of motion increased from 102 preoperatively to 119 at recent followup ( p < 0.001 ) . the mean tibiofemoral valgus was corrected from preoperative 16 ( range 1035 ) to 5 ( range 39 ) valgus ( p < 0.001).conclusions : mini - subvastus quadriceps approach provides adequate exposure and excellent early recovery for tka in valgus knees , without increase in incidence of complications .

experimental groups of 20 ( 4- to 6-week old ) c57bl/6 female mice ( charles river , l'arbresle , france ) were injected intracerebrally with 20 l of 10% ( weight / volume ) homogenates per mouse prepared from brain stem samples of 3 cattle tse isolates . two of the isolates were characterized , as previously described ( 7 ) , by a higher molecular mass of unglycosylated prp ( h - type isolates ) and labeling with p4 monoclonal antibody ( table ) . mice were housed and cared for in an appropriate biohazard prevention area ( a3 ) according to european ( directive 86/609/eec ) and french ethical committee ( decree 87848 ) guidelines . mice were checked at least weekly for neurologic clinical signs and were killed when they exhibited signs of distress or confirmed evolution of clinical signs . the whole brain of every second mouse was frozen and stored at 80c before western blot analysis . frozen mouse brain tissues and fixed brain tissues were examined by western blot analysis and immunohistochemical tests as previously described ( 12,13 ) . prp extracted from half of whole brain was detected with monoclonal antibodies sha31 ( 1:10 from tesee sheep / goat western blot , bio - rad , hercules , ca , usa ) ( 14 ) and 12b2 ( 340 ng / ml ) ( 15 ) . these antibodies are directed against the 144-wedryyre-151 and 88-wgqgg-92 murine amino acid prp sequences , respectively . antibody 12b2 , which has an n - terminal specificity similar to that of monoclonal antibody p4 , shows poor binding to bse - derived prp , but unlike p4 , binds with high affinity to prion protein from most mammalian species , including mice and cattle . bound antibodies were detected by using enhanced enzymatic chemiluminescence ( amersham , little chalfont , uk ) or supersignal ( pierce , rockford , il , usa ) and visualized either on film ( biomax , eastman kodak , rochester , ny , usa ) or directly in an image analysis system ( versadoc , bio - rad ) . molecular masses of prp glycoforms were determined as the average of the center positions of the bands from at least 3 repeated electrophoretic procedures , as measured by comparison with a biotinylated marker ( b2787 , sigma , saint louis , mo , usa ) included on each gel . immunologic reactivities of antibodies 12b2 and sha31 were compared in western blots run in parallel with the same samples with both antibodies . after intracerebral injection of cattle brain samples into c57bl/6 mice , disease was observed in mice with the 2 h - type isolates , as well as with the bse sample . survival periods of mice and results of prp detection among mice analyzed by western blot are shown in the table . western blot analysis of prp from h - type infected mouse brains in comparison with bse - infected mice is shown in figure 1 . this pattern showed higher molecular mass prp glycoforms in mice infected with h - type isolates than in mice infected with a typical bse agent ( 1.1- to 1.5-da difference in the unglycosylated prp ( figure 1a ) . studies of prp protease cleavage showed that only the prp of mice infected with h - type isolates was recognized by antibody 12b2 ( figure 1b ) . this finding is in contrast to the result obtained with monoclonal antibody sha31 directed against an epitope in the central region of the protein , which showed that the 12b2 epitope was preserved in h - type infected mice . thus , the molecular features of h - type cattle isolates , which are distinct from those of the bse agent , were maintained after development of disease in mice . western blot analysis of disease - associated prion protein ( prp ) from proteinase k treated brain homogenates of c57bl/6 mice infected with type h ( lanes 2 and 4 ) or bovine spongiform encephalopathy isolates ( lanes 3 and 5 ) . prp of mice infected with an experimental scrapie strain ( c506m3 ) ( 6 ) was used as a control ( lane 1 ) . monoclonal antibodies used for detection of prp were sha31 in panel a and 12b2 in panel b. lane m , molecular mass markers : 39.8 , 29 , 20.1 , and 14.3 kda . histopathologic analysis showed vacuolar lesions in the thalamus ( figure 2a ) that were absent from the hypothalamus , cochlear nucleus , and superior collicules . these 3 neuroanatomic sites were severely affected in c57bl/6 mice brain after primary passage of the bse agent as we and others have reported ( 1 ) . abnormal prp was detected only in amyloid plaques ( figure 2b ) , in contrast to what was reported after bse transmission in c57bl/6 mice ( 1 ) . a ) characteristic vacuolar lesions in the thalamus ( hematoxylin and eosin stained , scale bar = 60 m ) . b ) immunohistochemical analysis of prion protein with monoclonal antibody 12b2 ( diluted 1:200 ) shows the absence of granular deposition , but the presence of plaques in the thalamus . the inset shows that plaques are amyloids since they bind congo red and show birefringence in polarized light ( scale bar = 60 m , scale bar in inset = 16 m ) . our data show that the recently identified bovine h - type isolates involve an infectious agent that can induce development of a disease across a species barrier , while maintaining the specific associated prp molecular signature . this evidence in favor of a new bovine prion strain in cattle suggests that bse is not the only transmissible prion disease in cattle . these cases suggest either the existence of alternative origins of such diseases in cattle or phenotypic changes of prp after infection with the bse agent . however , based on analysis of molecular features of prion diseases in cattle , this situation is similar to that in humans ( 5 ) , in which different subtypes of sporadic creutzfeldt - jakob disease agents are found .

we previously reported that cattle were affected by a prion disorder that differed from bovine spongiform encephalopathy ( bse ) by showing distinct molecular features of disease - associated protease - resistant prion protein ( prpres ) . we show that intracerebral injection of such isolates into c57bl/6 mice produces a disease with preservation of prpres molecular features distinct from bse .

a biloma is a rare abnormal accumulation of intrahepatic or extrahepatic bile caused by traumatic or spontaneous rupture of the biliary tree1 , 2 ) . it is most commonly caused by surgery , percutaneous transhepatic cholangiography ( ptc ) , percutaneous transhepatic biliary drainage ( ptbd ) , and abdominal trauma11 , 12 ) . we report here a patient who complained of chills , fever and right upper quadrant abdominal discomfort . a 78-year - old woman presented to the emergency room with her guardian , with acute fever and chills that developed the day before admission . the patient was diagnosed with diabetes a few years previously , but this was not being treated . on admission to the emergency room , the vital signs were as follows : blood pressure 70/40 mmhg , heart rate 118/min , respiratory rate 30/min and temperature 40.3. the patient appeared to be in acute distress ; she was lethargic , the sclerae were slightly yellow and the conjunctivae were pale . the abdominal examination revealed slight tenderness in the right upper quadrant , but there was no palpable mass . the laboratory studies were as follows : wbc 52,400/mm , hb 12.3 g / dl , platelets 90,000/mm , ast 153 iu / l , alt 135 iu / l , alkaline phosphatase 255 iu / l , total bilirubin 2.07 mg / dl , total protein 5.9 g / dl , albumin 3.0 g / dl , bun / cr 25/1.9 mg / dl , esr 25 mm / hr , and crp 20.4 mg / dl . the serum electrolyte levels were normal except for an elevated potassium 7.0 mmol / l . the abga data were as follows : ph 7.24 , pao271 mmhg , paco2 19 mmhg , hco3 15.1 the hepatitis viral marker tests , including hbsag , anti - hbs ab and anti - hcv , were all negative . standard x - rays on the day of admission revealed lung infiltrates and no specific findings in the abdomen . the abdominal computed tomography ( ct ) revealed a 9.58.5 cm subcapsular cyst with a clear boundary in the left hepatic lobe and enlargement of the intrahepatic bile duct around the cyst . although the left hepatic lobe had some atrophic findings , there were no significant signs of an enlarged bile duct or abnormal gallbladder ( figure 1 ) . on the day of admission after the computed tomography , the abga revealed a ph 7.05 , pco2 64.8 mmhg , po2 73.3 mmhg , hco3 the patient was intubated and put on a respirator ; she was treated with antibiotics and vasopressors . on the second day of admission , percutaneous drainage with an 8.5 fr pigtail catheter , under the guidance of an abdominal ultrasound was performed for diagnosis and treatment . we removed 565 ml of fluid that was dark brown in color . for a diagnosis , biochemical tests were carried out on the bile , and these tests revealed a total bilirubin of 22.3 mg / dl and a direct bilirubin of 18.9 mg / dl . the leukocyte count was 4,560/mm , and gram staining showed a gram negative bacillus . on the third day of admission , her vital signs were as follows : blood pressure 100/65 mmhg , heart rate 110/min and temperature 37.2. there was 200 ml of drained bile , the s - fibrinogen was 894 mg / dl ( 200 - 415 ) , the d - dimer was 9,040 ng / ml ( 0 - 255 ) , and the fdp was elevated up to 97 ug / ml ( normal range : 0 - 5 ) . on the eighth day of admission , the patient 's mental status improved , the vital signs were stable and voluntary respirations recovered . on the thirteenth day of admission , pancreatico - biliary magnetic resonance imaging ( mri ) revealed a biloma that was connected to the intrahepatic bile duct ( figure 2 ) . on the sixteenth day of admission , the biloma that was connected to the intrahepatic duct and common bile duct was noted again via a tubogram performed with a catheter . over time , the amount of bile drainage decreased . on the twentieth day of admission , 64 ml of bile was drained , the leukocyte count was 340/mm and there were no signs of bacteria on culture or gram staining of the bile . the follow - up ct showed that the size of the cyst had decreased to 5.44.6 cm and the density around the cyst had increased . the amount of drainage did not decrease afterward , and the connection between the biloma and the bile duct was still noted . on the 25th day of admission , the drainage tube was blocked and follow - up of the bilirubin in blood and the size of the biloma were assessed . on the 29th day of admission , follow - up ct showed a marked decrease in the size of the cyst ( figure 3 ) , and the serum bilirubin was normal ( 0.22 mg / dl ) . on the 34th day of admission , the drainage tube was removed after evaluating the size and connection of the biloma via a catheter tubogram ( figure 4 ) . they reported a case with extrahepatic bile leakage after trauma to the upper right quadrant of the abdomen ; the bile did not cause peritonitis , but it accumulated in an encapsulated form . this concept was later applied to all cases with biliary tract lacerations caused by bile leaks that formed a capsule inside or outside of the liver1 ) . the mechanism of encapsulation is explained by a large amount of bile leaking at a fast pace that causes biliary peritonitis , or a small amount of bile leaking at a slow pace that causes mild inflammation . this leaked fluid is simultaneously trapped by the greater omentum and mesentery , where it becomes encapsulated2 ) . although the amount of bile leaked may be minor , the occurrence of an inflammatory reaction contributes to the appearance of a growth in size of the biloma3 ) . as diagnostic techniques continue to develop , more cases of biloma are being reported , but cases of spontaneous biloma and biloma accompanied by other diseases have rarely been reported to date . from 1979 to 1997 , 25 cases of spontaneous biloma were analyzed by fujiwara ; the underlying causes included choledocholithiasis ( 16 cases ) , cholangiocarcinoma ( 2 cases ) , acute cholecystitis ( 1 case ) , liver infarction ( 1 case ) , hepatic abscess ( 1 case ) , nephrotic syndrome ( 1 case ) , obstructive jaundice ( 1 case ) , sickle cell anemia ( 1 case ) , and tuberculosis ( 1 case)4 ) . in korea , there have been two cases of choledocholithiasis accompanied by a biloma , and they were treated by nonsurgical methods5 , 6 ) . there is one report of a case of biloma after trauma7 ) , and 1 case of a biloma after endoscopic retrograde cholangiopancreatography ( ercp)8 ) . one of the patients with a choledocholithiasis who suffered a nontraumatic intrahepatic rupture of the bile duct , had a surgical repair9 ) . the causes of biloma include trauma to the liver , which is the most common cause , abdominal surgery , endoscopic surgery and percutaneous catheter drainage10 - 13 ) . currently , the term biloma is generally used to describe intrahepatic or intraperitoneal focal biliary stasis14 ) . the clinical symptoms of a biloma are nonspecific , and they can range from no symptoms to abdominal pain and distention , jaundice and fever ; leukocytosis can also be present15 ) . in the case there were complaints of sudden chills , fever , upper abdominal discomfort and sensations of abdominal distention . physical examinations showed tenderness in the upper right area of the abdomen , slight jaundice , hypotension , leukocytosis and fever over 40. the size and location of the biloma are determined by the cause of the rupture of the bile duct , the location and speed of bile leakage and the rate the bile is reabsorbed by the peritoneum2 ) . fujiwara reported that the locations for bile leakage included the left hepatobiliary duct ( 8 cases ) , the gallbladder ( 5 cases ) , the right hepatobiliary tract ( 2 cases ) , the common bile duct ( 1 case ) , and unknown ( 9 cases ) ; the locations where the bilomas formed were the left lobe ( 11 cases ) , the right lobe ( 10 cases ) , and the upper abdomen ( 4 cases ) . vazquez analyzed 21 cases of biloma and found that 16 cases occurred in the upper abdominal area near the liver and five in the left upper abdominal area4 ) . the diagnosis of a biloma can be assisted by surgery , a history of trauma with abdominal ultrasonography , abdominal ct and mri . a definite diagnosis can be made by a 99mtc - disida scan , percutaneous aspiration or by ercp1 , 2 ) . abdominal ultrasonography shows a mass of low echogenicity with a clear border in the liver , and the follow up tests show changes in size16 ) . the abdominal ct will give a more accurate view of the location of the biloma and show its relationship to the surrounding organs . a standard ct view will show a low - density mass in the liver at a ct number lower than 20 . ultrasonography and abdominal ct are sometimes unable to differentiate bilomas from seromas , lymphoceles and angiomas . mris or hepatobiliary scans can be useful when continuous bile leakage is present , but they are not diagnostic when continuous leakage is not present7 , 17 ) . radiological image - guided aspiration tests , when testing for bilirubin , can be diagnostic18 ) . when the aspirated substance is a clear yellow liquid , then microbiological tests must be done to rule out an infection . ercp can be used to determine the location and severity of an active bile leak . however , the presence of small biliary cysts or bilomas , located in the lower areas of the liver that can be hidden by gastrointestinal shadows , can be difficult to diagnosis19 ) . we were able to identify a cystic lesion by abdominal ct ; a tubogram and magnetic resonance cholangiopancreatography ( mrcp ) were used to confirm a bile leak from the common bile duct . the fluid obtained by the transabdominal ultrasonography assisted percutaneous cyst drainage containing 22.3 mg / dl of total bilirubin and 18.9 mg / dl of direct bilirubin ; these findings confirmed the diagnosis of a biloma . treatment for bilomas that have a diameter of only a few centimeters is not always necessary ; these lesions can be watched . however , most bilomas require treatment . in the past , surgery was the main approach to treatment . currently , there are a much wider variety of options such as percutaneous catheter drainage , endoscopic sphincterotomy ( est ) , endoscopic nasobiliary drainage ( enbd ) and endoscopic drainage4 , 6 , 15 , 19 , 20 ) . surgery is now performed only in cases with a persistent bile leak or for treatment of underlying disease . in this case , a spontaneous biloma with bacterial infection was found after the patient presented in septic shock . after percutaneous drainage was performed , the amount of bile excreted did not decrease to lower than 64 ml per day . as there was no difficulty in flow through the common bile duct , the biochemical tests performed after removal of the drainage catheter showed no increase in total bilirubin or any increase in the size of the biloma , thus demonstrating a rare case of recovery .

a " biloma " is a loculated collection of bile located outside of the biliary tree . it can be caused by traumatic , iatrogenic or spontaneous rupture of the biliary tree . prior reports have documented an association of biloma with abdominal trauma , surgery and other primary causes , but spontaneous bile leakage has rarely been reported . a spontaneous infected biloma , without any underlying disease , is a very rare finding . we recently diagnosed a spontaneous infected biloma by abdominal computed tomography and sonographically guided percutaneous aspiration . the patient was successfully managed with percutaneous drainage and intravenous antibiotics . we report here a case of infected biloma caused by spontaneous rupture of the intrahepatic duct , and review the relevant medical literature .

anteromedial , anterolateral , and superomedial portals were created and a diagnostic arthroscopy was performed . the presence of a concomitant injury to the ligament or cartilage was assessed and meniscal tear was identified . unstable fragments and fibrous tissues were removed with a motorized shaver , rasp , or basket forceps and the surface of the meniscus was exposed . the meniscal surface and adjacent synovium were abraded to stimulate vascularization . for the repair of the medial meniscal tears , an arthroscope was inserted via the anteromedial portal and a zone specific cannula was inserted via the anterolateral portal . vertical mattress sutures using a double arm needle were placed every 4 mm along the length of the tear in an inside out fashion . an additional suture strand was used when the sutures were not considered strong enough . for the lateral meniscal repairs , an arthrosocpe was inserted through the anterolateral portal and sutures were inserted using a zone specific cannula through the anteromedial portal . an accessory incision was made on the medial side for medial tears and lateral side for lateral tears for knot tying and the suture passed through the cartilage was retrieved through it . peripheral blood ( 50 - 60 ml ) was collected from the arm using a sterile syringe and transferred into a 200 ml beaker ( fig . the blood was gently stirred with a glass rod ( 10 ml glass syringe plunger ) for 5 to 10 minutes until 3 - 5 ml fibrin clot precipitated around the rod . then , the clot was placed on damp gauze to remove excess fluid and trimmed to proper size ( fig . a transparent shoulder cannula with its polyethylene diaphragm ( used to prevent reflux of irrigating fluid ) removed to facilitate passage of the fibrin clot was inserted through the anterolateral portal for medial tears and anteromedial portal for lateral tears . a 20 cm - long 18-gauge needle was inserted through the cannula , penetrated the target area where the prepared fibrin clot would be added and the skin . a wire loop attached to a needle was inserted through the 18-gauge needle and was withdrawn from the skin , and the outer 18-gauge needle was removed ( fig . , the suture was passed through the wire loop and pulled out of the skin . irrigation inflow was turned off to proceed the procedure under a " dry scope . " the clot was inserted through the cannula and delivered into the desired intraarticular site by using a plastic pusher and gently pulling the connected suture strand ( fig . after confirming the location of the clot , the sutures that had been pulled through the meniscus out of the joint were ligated . partial weight bearing was performed for 6 postoperative weeks in most of the cases . in cases of radial tears , cast immobilization was applied for 3 postoperative weeks , mobilization exercises were started at 0-30 after the 3rd postoperative week and advanced gradually until the 6th postoperative week , and partial weight bearing was allowed afterwards . between june 2007 and march 2010 , 41 meniscal tears ( 19 radial tears , 12 longitudinal tears in the red - white zone , 7 transverse tears , and 3 oblique tears ) were repaired using a fibrin clot at our institution . second - look arthroscopy or follow - up mri was performed at an average of 8.3 months after surgery . once a meniscus is torn or treated with menisectomy , the load - bearing function of the meniscus changes and the knee joint becomes less stable , which leads to a higher risk of osteoarthritis . an in vitro study showed that 16% to 34% removal of the meniscus led to a 350% increase in the load on the articular surface5 ) . the meniscus should be preserved whenever feasible especially in young or active patients because they become more vulnerable to osteoarthritis after menisectomy6 ) . however , meniscal tear repairs are not always successful and healing can be delayed or fail . accordingly , various methods designed to promote healing have been suggested including meniscal rasping , fibrin clot insertion , vascular access channel creation , and synovial flap transfer . the indications for meniscal repair surgery have been extended with use of those procedures that facilitate healing of lesions in the avascular zones2 ) . webber et al.7 ) reported that delayed healing was attributable to the absence of a hematoma , not a blood supply per se . a hematoma in a torn meniscus acts as a scaffold that contains growth factors and repair cells for fibrocartilage regeneration through chemotactic activity . the frequency of hematoma formation is lower in the white - white zone or white - red zone compare to the red - red zone . arnoczky et al.4 ) reported that an exogenous fibrin clot acted as a hematoma that promotes healing of the meniscus in dogs . in other words , lesions that had been filled with a fibrin clot healed through a proliferation of fibrous connective tissue that modulated into fibrocartilaginous tissue . afterwards , many authors have reported on the efficacy of the use of a fibrin clot in clinical settings . in the study of henning et al.8 ) , the failure rate of meniscal repair using a fibrin clot was 8% . van trommel et al.2 ) reported that all of the 5 lateral meniscus tears involving the popliteal hiatus healed with repair using fibrin clot which were confirmed by second - look arthroscopy . for now , the best clinical results of the fibrin clot use can be expected in isolated meniscal tears . the recent increase in the use of prp has led to a high interest in fibrin clots which has similar healing properties that can be obtained without particular equipment at low costs3 ) . however , due to its fragility , delivering the fibrin clot to the exact desired intraarticular site has been considered challenging . however , studies that document a delivery method that can be standardized are rare . although some studies showed that a fibrin clot could be delivered into the joint using a suture or an injection syringe , they are not described in detail and too difficult to reproduce2,8 ) . in the study of sethi et al.9 ) , the synovium near the meniscus was abraded to induce bleeding and the blood ran down and pooled in the torn site to form an in situ fibrin clot . this method is advantageous in that an exogenous fibrin clot delivery method is not needed . however , there is a possibility that the clot may be irrigated away and it is difficult to create a clot in the exact site . van trommel et al.2 ) reported successful results of meniscal repair using a fibrin clot , but they did not describe the delivery method that they used . arthroscopy forceps can not be helpful in planting fibrin clots in the exact spot10 ) . a fibrin clot tied with a suture can be delivered from the anterior portal to a desired site , but it is highly likely to be destroyed during the procedure . with our method that has high reproducibility , a fibrin clot can be delivered to a desired site with ease without risk of being damaged by soft tissue during insertion . our method can be applied to meniscal tears , especially longitudinal tears in the white - white zone or white - red zone where the likelihood of repair failure is high . in addition , the method is expected to extend the indications for meniscal repair to radial tears where menisectomy is expected to result in functional loss , large - sized transverse tears in young patients , areas between bundles after double - bundle anterior cruciate ligament reconstruction3 ) , and rotator cuff repair . however , we believe a long - term follow - up study should be conducted and biomechanical changes and systemized indications for the use a fibrin clot in meniscal repair should be investigated in future studies .

as meniscal preservation particularly in younger active individuals with a symptomatic meniscal tear remains the preferred treatment option , various methods have been suggested to increase healing and success rates after meniscal repair . the recent increase in clinical use of platelet rich plasma has contributed to the increased use of fibrin clot , which virtually has the same healing property . however , despite the relative ease of acquisition and preparation of fibrin clots , delivering it to the desired target area arthroscopically is challenging . therefore , we report with a pertinent literature review a novel method of planting a fibrin clot to the desired area of meniscal tear arthroscopically using our delivery system to enhance healing .

so far , anthropometric variables and their relation to conventional coronary risk factors in railway employees have been inadequately studied in india . to assess anthropometric variables and their relation to coronary risk factors , this cross - sectional survey was carried out in solapur division of the central railway in the year 2004 . the purpose of this study was to examine the association of obesity with cad risk factors by different anthropometric variables . this particular section of the population was selected , as it comprises all classes of employees , including both sexes , sedentary and nonsedentary job workers of various socioeconomic groups , religions , and from different parts of india . this study was designed to investigate conventional cad risk factors and their relation to anthropometric variables among the solapur division railway employees . this study was conducted among the railway employees of solapur division of the central railway . a proforma was prepared that incorporated information regarding demography ( age and sex ) , anthropometric ( height in meter , weight in kilograms , waist and hip circumferences in centimeters ) variables , occupation ( sedentary or nonsedentary ) , physical examination ( pulse , blood pressure ) , clinical data ( history of diabetes hypertension , smoking , tobacco chewing , exercise ) , and biochemical investigations [ fasting blood sugar level ( bsl ) and complete lipid profile ] . anthropometric variables were calculated by using the above measurements for body mass index ( bmi ) , waist circumference ( wc ) , waist - to - hip ratio ( whr ) , waist - to - height ratio ( whtr ) , and abdominal volume index ( avi ) . all the eligible employees of both sexes underwent physical , anthropometric examination , and biochemical investigations according to the standard guidelines used earlier . a total of 995 railway employees participated in this cross - sectional survey with age 30 and 60 years . railway employees were chosen from railway stations , divisional railway mandal office , railway police force ( rpf ) and railway hospitals . according to the nature of their job , there were 872 men of whom 484 ( 55.5% ) were of age < 45 years and 388 ( 44.5% ) were of age 45 years . a total of 123 were females , 58 ( 48% ) were of age < 45 years and 65 ( 52% ) were of age 45 years . blood pressure was measured using a standard mercury sphygmomanometer under standard conditions as mentioned in cardiovascular survey methods . biochemical investigations were performed on an automated analyzer by using the kit provided by accurex biomedical pvt . ltd . ( iso 13485 : 2003 , iso 9001 : 2008 & ce certified mumbai , india ) . was measured by cholesterol oxidase ( chod) phenol + aminophenazone ( pap ) enzymatic colorimetric technique . triglyceride ( trg ) was measured by glycerol-3-phosphate oxidase ( gpo) peroxidase ( pod ) enzymatic technique . bsl fasting was done by glucose oxidase ( god ) peroxidase ( pod ) enzymatase by autoenzyme technique . each person was examined for height , weight , wc , and hip circumference without shoes and chappals with minimal clothing as per cardiovascular survey methods . bmi was calculated by formula of weight in kg / height m. whr was calculated by wc / hc in centimeters . avi was calculated using volume formulas for cylinder ( v = rh ) and vertical cone v = ( 1/3)rh . the formula developed was avi = [ 2 cm ( waist ) + 0.7 cm ( wc hc)]/1000 , which estimates overall abdominal volume between symphysis of pubis and xiphoid appendix and theoretically includes intra - abdominal fat and adipose tissue volumes . a smoker in india consumes tobacco in the form of bidi / cigarettes and chewing tobacco . a person smoking one or more cigarettes or bidis per day at the time of study was considered as a smoker and one chewing tobacco currently , a tobacco chewer . in the present study , tobacco consumption in the form of smoking and in the form of tobacco chewing were grouped separately.physical activity according to the type of job were classified as , nonsedentary job ( moderate to heavy physical activity)sedentary job ( low physical activity ) physical activity was classified into 2 levels , moderate to high physical activity was referred as nonsedentary ( rpf , drivers , gangmen , and others)low physical activity referred ( sedentary ) to people involved in office work , research work , and so on . a smoker in india consumes tobacco in the form of bidi / cigarettes and chewing tobacco . a person smoking one or more cigarettes or bidis per day at the time of study was considered as a smoker and one chewing tobacco currently , a tobacco chewer . in the present study , tobacco consumption in the form of smoking and in the form of tobacco chewing were grouped separately . physical activity according to the type of job were classified as , nonsedentary job ( moderate to heavy physical activity ) sedentary job ( low physical activity ) physical activity was classified into 2 levels , moderate to high physical activity was referred as nonsedentary ( rpf , drivers , gangmen , and others ) low physical activity referred ( sedentary ) to people involved in office work , research work , and so on . cutoffs for high wc were > 85 cm for females and > 90 cm for males . cutoffs for high avi was calculated by receiver - operator characteristic ( roc ) curve 16.48 liter . diabetes mellitus ( dm ) : if a subject is a known diabetic on treatment with any bsl or if fasting bsl ( f - bsl ) 126 mg / dl . dyslipidemia was defined as if , t - cho 200 mg / dl , ldl , cholesterol 130 mg / dl , hdl , cholesterol 40 mg / dl , trg 150 mg / dl . hypertension was labeled if blood pressure 140 mmhg sbp and 90 mmhg dbp or known to be hypertensive on treatment with any blood pressure . the data were pooled , computerized , and analyzed by evaluation version of epi info . 6 [ ( epi info is public domain statistical software for epidemiology developed by centers for disease control and prevention ( cdc ) in atlanta , georgia ( usa ) ] . correlation of anthropometric variables and coronary risk factors in different age groups were determined using r and multiple linear regression analysis . roc curve is used to find out the cutoff point for particular value of a test as a diagnostic test . cutoff values of whtr and avi were calculated by using this curve as a tool for diagnosing central obesity . correlation ( r ) : r = 0.8 ( high correlation coefficient ) , r= 0.40.7 ( moderate correlation ) , and r= 0.3 and above ( low correlation coefficient ) . each person was examined for height , weight , wc , and hip circumference without shoes and chappals with minimal clothing as per cardiovascular survey methods . bmi was calculated by formula of weight in kg / height m. whr was calculated by wc / hc in centimeters . avi was calculated using volume formulas for cylinder ( v = rh ) and vertical cone v = ( 1/3)rh . the formula developed was avi = [ 2 cm ( waist ) + 0.7 cm ( wc hc)]/1000 , which estimates overall abdominal volume between symphysis of pubis and xiphoid appendix and theoretically includes intra - abdominal fat and adipose tissue volumes . a smoker in india consumes tobacco in the form of bidi / cigarettes and chewing tobacco . a person smoking one or more cigarettes or bidis per day at the time of study was considered as a smoker and one chewing tobacco currently , a tobacco chewer . in the present study , tobacco consumption in the form of smoking and in the form of tobacco chewing were grouped separately.physical activity according to the type of job were classified as , nonsedentary job ( moderate to heavy physical activity)sedentary job ( low physical activity ) physical activity was classified into 2 levels , moderate to high physical activity was referred as nonsedentary ( rpf , drivers , gangmen , and others)low physical activity referred ( sedentary ) to people involved in office work , research work , and so on . a smoker in india consumes tobacco in the form of bidi / cigarettes and chewing tobacco . a person smoking one or more cigarettes or bidis per day at the time of study was considered as a smoker and one chewing tobacco currently , a tobacco chewer . in the present study , tobacco consumption in the form of smoking and in the form of tobacco chewing were grouped separately . physical activity according to the type of job were classified as , nonsedentary job ( moderate to heavy physical activity ) sedentary job ( low physical activity ) physical activity was classified into 2 levels , moderate to high physical activity was referred as nonsedentary ( rpf , drivers , gangmen , and others ) low physical activity referred ( sedentary ) to people involved in office work , research work , and so on . cutoffs for high wc were > 85 cm for females and > 90 cm for males . cutoffs for high avi was calculated by receiver - operator characteristic ( roc ) curve 16.48 liter . diabetes mellitus ( dm ) : if a subject is a known diabetic on treatment with any bsl or if fasting bsl ( f - bsl ) 126 mg / dl . dyslipidemia was defined as if , t - cho 200 mg / dl , ldl , cholesterol 130 mg / dl , hdl , cholesterol 40 mg / dl , trg 150 mg / dl . hypertension was labeled if blood pressure 140 mmhg sbp and 90 mmhg dbp or known to be hypertensive on treatment with any blood pressure . the data were pooled , computerized , and analyzed by evaluation version of epi info . 6 [ ( epi info is public domain statistical software for epidemiology developed by centers for disease control and prevention ( cdc ) in atlanta , georgia ( usa ) ] . correlation of anthropometric variables and coronary risk factors in different age groups were determined using r and multiple linear regression analysis . roc curve is used to find out the cutoff point for particular value of a test as a diagnostic test . cutoff values of whtr and avi were calculated by using this curve as a tool for diagnosing central obesity . correlation ( r ) : r = 0.8 ( high correlation coefficient ) , r= 0.40.7 ( moderate correlation ) , and r= 0.3 and above ( low correlation coefficient ) . a total of 872 ( 87.63% ) were males and 123 ( 12.36% ) were females . all of them underwent physical examination , and anthropometric measurements for ht , wt , hc , and wc , but only 605 males and 95 females underwent biochemical investigations with a response rate of 69.15%in males and 77.23% in females . the subjects were 45 years of age ; 388 ( 53.35% ) males and 65 ( 52.84% ) females . high sbp was present in 290 ( 33.25% ) males and 32 ( 26.03% ) females . high dbp was present in 304 ( 34.86% ) males and 37 ( 30.08% ) females . physical inactivity was present in 767 ( 87.95% ) males and 102 ( 82.92% ) females . tobacco chewing was present in 178 ( 20.41% ) males and 12 ( 9.75% ) females . smoking was present in 151 ( 17.31 % ) males and 2 ( 1.62% ) females . high bmi was present in 172 ( 19.72% ) males and 73 ( 59.34% ) females . high wc was present in 412 ( 47.24% ) males and 98 ( 79.67% ) females . high whr was present in 504 ( 57.79% ) males and 90 ( 73.17% ) females . high whtr was present in 699 ( 80.16% ) males and 103 ( 83.73% ) females . high avi was present in 359 ( 41.16% ) males and 35 ( 28.45% ) females . dm was present in 51 ( 8.42% ) males and 2 ( 2.10% ) females . low hdl was present in 248 ( 40.99% ) males and 15 ( 15.78% ) females . high ldl was present in 117 ( 19.33% ) males and 23 ( 24.21% ) females . high t - cho was present in 151 ( 24.95% ) males and 34 ( 35.78% ) females . high trg was present in 275 ( 45.45% ) males and 19 ( 20% ) females [ table 1 ] . prevalence of coronary artery disease risk factors in both males and females , all the anthropometric variables were high . most prevalent obesity index in male population was high whtr . in female population , the most prevalent obesity index was high whtr . prevalence of overall and central obesity was comparatively more in female population for all the variables as shown in table 2 . mean age , bmi , and low hdl levels were comparatively higher in females , whereas mean sbp , dbp , trg , whr , wc , and whtr were comparatively higher in males . standard deviation values of quantitative data in male and female population are shown in table 3 . prevalence of obesity indices standard deviation values of quantitative data in male and female population total 65 females were with age 45 years and 58 with age < 45 years . in female population , high whr in age 45 years was present in 58 ( 47.15% ) . in females only high bmi was statistically significant in age 45 years with p value < 0.001 . males totalling 388 were of the age 45 years and 484 females were < 45 years of age . in male population , 45 years of age , a high whr was present in 262 ( 30.04% ) . a high bmi in age 45 years was present in 90 ( 10.32% ) . a high wc in age 45 years was present in 207 ( 23.73% ) . high avi in age 45 years was present in 279 ( 31.99% ) . in males , all anthropometric variables , such as whr , whtr , bmi , wc , and avi , were significantly raised , in the age group 45 years with p value < 0.001 . bmi had partial positive correlation ( moderate to low ) with coronary risk factors , such as age weight , sbp , dbp , f - bsl , t - cho , ldl cholesterol , trg , and other anthropometric variables . whr had partial positive correlation ( moderate to low ) with coronary risk factors , such as age , height , weight , sbp , dbp , f - bsl , total , trg , and other anthropometric variables . whtr had partial positive correlation ( moderate to low ) with coronary risk factors , such as age , weight , sbp , dbp , f - bsl , ldl cholesterol , trg , and other anthropometric variables . wc had partial positive correlation ( moderate to low ) with coronary risk factors , such as age , weight , height , sbp , dbp , f - bsl , trg , and other anthropometric variables . avi had partial positive correlation ( moderate to low ) with coronary risk factors , such as age , weight , height , sbp , dbp , f - bsl , trg , and other anthropometric variables [ table 4 ] . in male population , age 45 years had odds ratio of 2.15 with whtr and 1.95 with avi . sbp had an odds ratio of 2.0 with bmi , 1.47 with wc , and 1.48 with avi . correlation of anthropometric indices with coronary artery disease risk factors in female population , age 45 years had an odds ratio of 1.673 with bmi . sedentary job had an odds ratio of 2.0 with whr , 1.86 with wc , and 1.79 with whtr . sbp had an odds ratio of 1.48 with avi and dbp had an odds ratio of 1.34 with wc . we have found that age 45 years was better correlated with high values of bmi , wc , whr , whtr , and avi . high sbp , high dbp , and sedentary job were significantly and positively correlated with high values of bmi , wc , whr , whtr and avi . high values of bmi , wc , whr , whtr , and avi were negatively associated with physical inactivity . tobacco and smoking were negatively correlated with high values of bmi , wc , whr , and whtr . dm was statistically significant and positively associated with high values of bmi , wc , whr , whtr , and avi . in dyslipidemia low hdl was correlated with high values of whr , whtr , and avi , whereas ldl cholesterol , t - cho , and trg were correlated only with high bmi and high whtr in this population [ table 4 ] . in multiple regression analysis a model was utilized that included age , bmi , whr , whtr , wc , avi , sbp , dbp , plasma t - cho , trg , and smoking . this revealed age , whtr , and whr as an independent determinant of most of the cad risk factors in both genders . multiple regression analysis for whtr and whr showed that both of them were significantly associated with most of the cad risk factors , such as age 45 years , high sbp , high dbp , dm , low hdl , high ldl , high t - cho , and high trg [ table 5 ] . relation of anthropometric variables and coronary artery disease risk factors in males and females furthermore , the whr was the single independent variable to all or most of the cad risk factors by multiple regression analysis . multiple logistic regression analysis revealed whtr and whr were significantly associated with age ( p < 0.001 ) , bmi ( p < 0.001 ) dm ( p = 0.005 ) , sbp ( p < 0.001 ) , dbp ( p < 0.001 ) , low hdl in females , and high trg in males ( p < 0.001 ) [ figure 1 ] . bmi had partial positive correlation ( moderate to low ) with coronary risk factors , such as age weight , sbp , dbp , f - bsl , t - cho , ldl cholesterol , trg , and other anthropometric variables . whr had partial positive correlation ( moderate to low ) with coronary risk factors , such as age , height , weight , sbp , dbp , f - bsl , total , trg , and other anthropometric variables . whtr had partial positive correlation ( moderate to low ) with coronary risk factors , such as age , weight , sbp , dbp , f - bsl , ldl cholesterol , trg , and other anthropometric variables . wc had partial positive correlation ( moderate to low ) with coronary risk factors , such as age , weight , height , sbp , dbp , f - bsl , trg , and other anthropometric variables . avi had partial positive correlation ( moderate to low ) with coronary risk factors , such as age , weight , height , sbp , dbp , f - bsl , trg , and other anthropometric variables [ table 4 ] . in male population , age 45 years had odds ratio of 2.15 with whtr and 1.95 with avi . sedentary job had an odds ratio of 1.402 with bmi . sbp had an odds ratio of 2.0 with bmi , 1.47 with wc , and 1.48 with avi . correlation of anthropometric indices with coronary artery disease risk factors in female population , age 45 years had an odds ratio of 1.673 with bmi . sedentary job had an odds ratio of 2.0 with whr , 1.86 with wc , and 1.79 with whtr . sbp had an odds ratio of 1.48 with avi and dbp had an odds ratio of 1.34 with wc . we have found that age 45 years was better correlated with high values of bmi , wc , whr , whtr , and avi . high sbp , high dbp , and sedentary job were significantly and positively correlated with high values of bmi , wc , whr , whtr and avi . high values of bmi , wc , whr , whtr , and avi were negatively associated with physical inactivity . tobacco and smoking were negatively correlated with high values of bmi , wc , whr , and whtr . dm was statistically significant and positively associated with high values of bmi , wc , whr , whtr , and avi . in dyslipidemia low hdl was correlated with high values of whr , whtr , and avi , whereas ldl cholesterol , t - cho , and trg were correlated only with high bmi and high whtr in this population [ table 4 ] . in multiple regression analysis a model was utilized that included age , bmi , whr , whtr , wc , avi , sbp , dbp , plasma t - cho , trg , and smoking . this revealed age , whtr , and whr as an independent determinant of most of the cad risk factors in both genders . multiple regression analysis for whtr and whr showed that both of them were significantly associated with most of the cad risk factors , such as age 45 years , high sbp , high dbp , dm , low hdl , high ldl , high t - cho , and high trg [ table 5 ] . relation of anthropometric variables and coronary artery disease risk factors in males and females furthermore , the whr was the single independent variable to all or most of the cad risk factors by multiple regression analysis . multiple logistic regression analysis revealed whtr and whr were significantly associated with age ( p < 0.001 ) , bmi ( p < 0.001 ) dm ( p = 0.005 ) , sbp ( p < 0.001 ) , dbp ( p < 0.001 ) , low hdl in females , and high trg in males ( p < 0.001 ) [ figure 1 ] . conventional cad risk factors , such as physical inactivity , central obesity , hypertension , low hdl , age 45 years , and hypertriglyceridemia were highly prevalent in males , whereas physical inactivity , central obesity , hypertension , age 45 years , and hypercholesterolemia were highly prevalent in female population . compared to overall obesity , central obesity was better correlated with most of the cad risk factors . whtr was better predictive of cad risk factors than wc and whr in present study . cutoff value for whtr has not been calculated for the indian population , hence further studies are required for calculation in the indian context . in the present study , whtr was a powerful predictor of most of the cad risk factors , whereas whr , wc , and avi were next to it in a decreasing order . we have compared our results with various studies in which lin et al in a general population study reported that , whtr is a better indicator for predicting cvd risk factors than the other 3 indices ( bmi , wc , and whr ) in taiwan , which is comparable with our study . lin et al and hsieh et al also stated that whtr is a better predictor of most of the cad risk factors than whr , wc , and bmi . huang et al in a general population study in taiwan found that the 4 anthropometric indices ( bmi , wc , whr , and whtr ) are closely related to cardiovascular risk factors , which is comparable with our study . the results of misra et al indicated a high prevalence of diabetes , obesity , and dyslipidemia in urban slum population in northern india in middle age , particularly in females , which is comparable with our study . guerrero - romero et al in their study of 746 men and nonpregnant women randomly recruited from durango city in northern mexico , stated that avi is reliable and shown to be strongly related to igt and dm , which is comparable with our study , but they have not correlated the association with other cad risk factors , such as hypertension , dyslipidemia , and physical inactivity . in southern andhra pradesh , india , a study of randomly selected urban and rural population by venkatramana et al suggested that bmi and wc are better indicators of cad risk factors , which is partially comparable with our study as other anthropometric variables have not been used in their study . reeder et al in 5 canadian provinces ( alberta , manitoba , ontario , quebec , and saskatchewan ) general population study , found that wc was the measure of abdominal obesity most highly correlated with cardiovascular disease risk factors , which is partially comparable with our study . in omani population al - riyami and afifi , in their study among male adults aged 20 years and above in a community - based survey as a part of the national health survey , reported that current smokers were of low bmi compared with nonsmokers and exsmokers and currently light smokers were also of low bmi compared with exsmokers , which is comparable with our study . ledoux et al , in a canadian general population study stated that the wc and bmi correlate most closely with blood pressure and plasma lipids , which is comparable with the present study . onat et al , in a random sample of turkish general adult population , observed that the wc and whr were strongly associated with bmi and dbp and plasma trg in turkish women , which is similar to and comparable with our results . hsieh et al , in a study found that , the whtr and whr increased with age in both the genders , which is comparable with our results . rosmond et al , in a selected population study , reported that obesity ( bmi ) and centralization of body fat stores ( whr ) are differently associated with occupational factors as well as leisure time activities . hu et al reported that television watching and other sedentary behaviors are related to risk of obesity and type 2 dm in women . both these studies are comparable with the current study . slattery et al in a study of the us railroad population quoted that occupationally and leisure time physically inactive employees are at a higher risk of developing cad risk factors and cardiovascular death , which is consistent with the current study . several studies showed that smokers have , on an average , lower bmi than nonsmokers and that smoking cessation is often associated with weight gain . in the present study , whtr has been found to be significantly and positively associated with most of the cad risk factors , hence whtr can be used routinely as an anthropometric tool for prediction of cad risk factors in epidemiologic studies.[2529 ] the other parameters of coronary artery risk [ c - reactive protein ( crp ) , homocysteine , and others ] have not been done in the present study because of limitations of study to conventional cad risk factors and resource limitations . other cad risk factors , such as hyperhomocysteinemia , have not shown a robust utility in chd risk stratification , and there are no clinical trial data showing that intervention to lower homocysteine levels reduces chd events . measurement of homocysteine levels should be reserved for individuals with atherosclerosis at a young age or out of proportion to established risk factors . elevations in acute - phase reactants , such as fibrinogen or crp , may reflect overall atherosclerotic burden or extravascular inflammation that could potentiate atherosclerosis or its complications . crp measured by high - sensitivity assay , adds predictive information to that derived from established coronary artery risk factors , such as cholesterol . elevated levels of the acute - phase reactant crp and fibrinogen may merely reflect ongoing inflammation rather than a direct etiologic role for crp in cad . in the present study , the prevalence of metabolic syndrome ( using atp iii criteria modified for indians ) was 25.17% in males and 27.35% in females . among all the components of metabolic syndrome , high wc was most prevalent in females and systolic hypertension in males . in the current study , the most common cad risk factor was truncal obesity in the solapur division railway employees . generalized obesity , overweight , and central obesity problem was more prevalent in subjects with age 45 years , sedentary job , physical inactivity , and female gender . there was a significant lack of physical activity and exercise among sedentary job workers , leading to overweight and obesity . generalized obesity , overweight , and central obesity were significantly correlated with cad risk factors , such as age 45 years , dyslipidemia , hypertension , dm , and physical inactivity . central obesity measured by whtr was strongly correlated with majority of cad risk factors than whr , wc , and avi . these observations serve to underline the central role of overweight / obesity and the additive deleterious effects of abdominal obesity in the pathogenesis of lipid and carbohydrate metabolism , leading to chd and type 2 dm , and hypertension . the present study shows a disturbing burden of coronary risk factors mainly truncal obesity , physical inactivity , hypertension in a sample population of railway employees . there is an urgent need to undertake population - based measures to reverse the trend . out of all the anthropometric variables , whtr was significantly and positively associated with most of the cad risk factors , hence whtr can be used routinely as an anthropometric tool for prediction of cad risk factors in epidemiologic studies . by preventing obesity and overweight , a substantial part of chd mortality may be prevented because a majority of the cad risk factors are strongly correlated with anthropometric indices . we concluded that weight control should be an integral part of the prevention of cardiovascular disease . turn in obesity express highway probably lies in lifestyle modifications , going back to a traditional lifestyle with a judicious use of modern technology .

background and objectives : anthropometric variables and their relation to conventional coronary artery disease ( cad ) risk factors in railway employees have been inadequately studied in india . this cross - sectional survey was carried out in the solapur division of the central railway in the year 2004 , to assess the anthropometric variables in railway employees and their relation to conventional cad risk factors.materials and methods : a total of 995 railway employees , with 872 males and 123 females participated in this cross - sectional study . all subjects underwent anthropometric measurements , fasting lipid profile , and blood sugar level . various anthropometric indices were calculated for body mass index ( bmi ) , waist circumference ( wc ) , waist - to - hip ratio ( whr ) , waist - to - height ratio ( whtr ) , and abdominal volume index ( avi ) . statistical analysis was done by epi info 6 statistical software.results:compared to all other obesity indices , whtr was most prevalent in both genders . high whtr was present in 699 ( 80.16% ) males and 103 ( 83.73% ) females . age 45 years , high systolic bp , high diastolic bp , low hdl , high triglyceride , and diabetes mellitus were positively correlated with high bmi , high wc , high whr , high whtr , and high avi . high bmi , high wc , high whr , high whtr , and high avi were negatively associated with physical inactivity.conclusions:over all , anthropometric variables in both genders were significantly deranged in subjects with coronary risk factors . compared to all other anthropometric variables , whtr was statistically significantly associated with a majority of coronary artery risk factors . hence we recommend inclusion of whtr as a parameter of obesity to predict coronary artery disease risk factor along with wc , whr , and bmi in epidemiologic studies .

aegypti was maintained in the laboratory of chemical ecology of vector insects of the federal university of minas gerais from 2007 . the breeding room was kept at a temperature of 27 1c , 75 - 80% relative humidity ( rh ) and a 12l:12d photoperiod ( eiras & jepson 1991 ) . the larvae were kept in plastic tubs containing water ( approximately 5 cm deep ) and fed reptile food ( reptolife , alcon , brazil ) until they reached the pupa stage . the pupae were collected daily and transferred to adult screened cages ( bugdorm-1 , mega view science education services , taiwan ) ( 30 30 30 cm ) and fed 10% sucrose solution . a selection cage was used to separate the gravid females that were used in the assays . blood meal was prepared using chicken blood ( gallus domesticus ) , which was obtained from a slaughterhouse , including the anticoagulant heparin ( gomes et al . . aegyptifemales ( f6 and f7 ) of five-10 days of chronological age and three days after blood meal ( gomes et al . experimental area - the tests were performed in laboratory and semi - field conditions . in laboratory conditions , four transparent acrylic boxes ( 150 50 41 cm ) were kept in a room with monitored temperature and humidity conditions ( 27 3c , 60 - 90% rh ) and a photoperiod of 12l:12d ( eiras & jepson 1991 ) . the semi - field tests were performed in an experimental area of 14.0 7.0 3.5 m , where eight cages ( 2.5 2.5 2.0 m each ) were installed ( roque & eiras 2008 ) . the temperature , rh and the photoperiod ranged according to the external environment and were monitored with a thermo - hygrometer . the tests were considered valid only when the temperature reached a minimum of 25c ( roque & eiras 2008 ) . bioassays - the tests evaluating the pattern of distribution of eggs by ae . the females were evaluated in laboratory conditions in experiment 1 and in semi - field conditions in experiment 2 . experiment 1 ( laboratory ) was performed in the four acrylic boxes in which four different densities of breeding sites ( 2 , 4 , 8 and 16 ) were simulated . in each box , a single gravid female of ae . aegypti was released and allowed to remain for 96 h in the interior of the box . the breeding sites were ovitraps that were set with 300 ml of tap water and a rectangular wooden paddle ( 12 cm 3 cm ) ( fay & perry 1965 , fay & eliason 1966 ) . for each density level , 12 repetitions of the experiment were performed . 1 ) . sugar solution ( 10% ) absorbed into cotton was provided as food to the insects during the experimental processes . 1:representation of the distribution of the different densities ( 2 , 4 , 8 and 16 breeding sites ) and the positions of the breeding sites in the interior of the acrylic boxes . in the centre , after 96 h , the paddle and the water that were present in each trap were collected , labelled and taken to the laboratory . to verify the distribution of eggs at each breeding site , the number of eggs that were present in the water and in the paddle of each ovitrap was counted using a manual counter and a stereoscopic microscope ( 20 ) . experiment 2 ( semi - field ) was conducted in the interior of the four fabric cages ( 2.5 2.5 2.0 m ) ( roque & eiras 2008 ) . the same densities ( 2 , 4 , 8 and 16 ovitraps ) that were evaluated in the laboratory conditions were evaluated in this environment . for each density the positions of the breeding sites were kept constant and the same methodology as used in experiment 1 was followed . however , to avoid visual effects of the treatment itself , each cage received all 16 of the ovitraps , but only the test traps ( densities 2 , 4 , 8 or 16 ) received water ( fig . 2 ) . fig . 2:diagram of the distribution of the ovitrap with and without water of each treatment / density of breeding sites . statistical analyses - for statistical analyses , the specific locations of egg deposition ( water and paddle ) were called habitats and the local of performance of the experiment ( laboratory or semi - field ) was called environment . to compare the number of eggs that were deposited in each environment / density , data were submitted to a normality test at a significance level of 95% . when the distributions were not in accordance with the criteria of normality , medians were compared using the kruskal - wallis test . the proportions of eggs that were deposited on water by each single female ( n eggs on the water / n total eggs ) at the different densities of breeding sites both in laboratory and semi - field conditions were compared by two - factors anova . before analyses , the data were transformed in arcsin(sqrt ) to normalise the distribution and stabilise the variances . the number of colonised breeding sites in relation to the amount of available ones , both in laboratory and semi - field conditions , was compared by the kruskal - wallis test . by investigating the female groups above and below the regression lines , we obtained two groups : those that colonise various breeding sites and those that colonise few sites . we compared these two groups by the mann - whitney u test to determine whether there was any difference between the groups with higher and lower frequencies of colonised breeding sites and the proportion of eggs that were deposited in the preferential breeding site . the r program ( available from : r-project.org ) environment ( laboratory or semi - field ) of the experiment and egg deposition - the oviposition behaviour of ae . aegypti was maintained in both environments . under laboratory conditions , the mean number [ standard deviation ( sd ) ] of eggs that were laid by one ae . aegyptiper repetition was 73.9 ( 25.64 ) , ranging between 40 - 142 eggs . similarly , under semi - field conditions , the mean number ( sd ) of eggs that were laid by oneae . aegypti per repetition was 72.2 ( 20.57 ) , ranging between 41 - 123 eggs . on average , females in both the experimental environments distributed the same number of eggs and colonised the same number of breeding sites ( anova p > 0.05 ) . therefore , these oviposition behaviours do not seem to be influenced by changes in the tested environments . oviposition on the water - the tested females exhibited a strong tendency to deposit their eggs on the water of the ovitrap . the mean proportion of eggs that were laid on the water surface tended to be higher than that on the paddle when the number of available breeding sites was greater ( fig . habitat received significantly more eggs in semi - field than in laboratory conditions ( anova p < 0.05 ) ( fig . 3:box plot of the proportion of eggs on the water in different density of oviposition breeding sites . 4:box plot of the number of eggs laid on water and paddle in semi - field and laboratory conditions . breeding sites colonised in relation to available ones - the availability of breeding sites directly influenced the dispersion of eggs by females . there was a significant difference ( p < 0.05 ) between the densities 2 * 4 , 2 * 8 , 2 * 16 and 4 * 16 both in laboratory and semi - field conditions ( fig . 5 ) . one female dispersed the eggs among 11 breeding sites , the highest number observed in the study . skip oviposition behaviour , although widely used , may not occur , as observed in eight ( 7.4% ) females . 5:mean and standard deviation of breeding sites colonised on the basis of available ones . different letters mean statistical difference ( kruskal - wallis p < 0.05 ) . one - by counting the number of eggs at each breeding site , we noticed that one of the sites usually received most of the eggs ( 40% or more of the total deposited eggs ) . although all the breeding sites were identical and in semi - field and were not statistically different from each other , it one site generally received more eggs than the others . the amount of eggs that were deposited at the favourite site was similar in both laboratory and semi - field conditions . the average percentage of eggs that were deposited in the favourite site was significantly higher with a density of two breeding sites and did not vary among the other densities ( fig . 6 ) , showing that females tend to aggregate more eggs when there are only two available breeding sites . this number can be reduced by an increase in the dispersion among several breeding sites . there was a negative relationship between the number of colonised breeding sites and the percentages of eggs that were laid at the favourite breeding site ( simple linear regression y = -0.0682x + 0.9256 , f1,106 = 65.9 , r = 0.38 , p < 0.001 ) ( fig . 7 ) . however , even those females that used many breeding sites seemed to deposit at least 40% of their eggs at the favourite breeding site , as shown by the dashed line ( fig . 7 ) . nevertheless , comparing the percentages of eggs that were laid by the ae . aegypti females of the two groups ( above and below the regression lines , fig . 8) , a significant difference was observed in both semi - field ( t = -6.62 , p < 0.001 ) and laboratory ( t = 2.87 , p = 0.001 ) ( fig . 7:percentage of eggs laid on favourite breeding site in relation to the number of colonised breeding sites under laboratory and semi - field conditions . 8:percentage of eggs laid in the favourite breeding site in laboratory by aedes aegypti female s who had higher frequency of breeding site colonisation ( a ) and smaller frequency ( b ) . percentage of eggs laid in the favourite breeding site in semi - field by ae . aegypti female s who had higher frequency of breeding site colonisation ( a ) and smaller frequency ( b ) . in other words , both environments ( laboratory and semi - field ) showed higher percentages of eggs laid at favourite breeding sites by females those that distributed their eggs in small numbers of containers . although all the breeding sites were identical and in semi - field they were not statistically different from each other , one site generally received more eggs than the others . the results that were obtained under laboratory and semi - field conditions were similar , showing that the general aspects of the oviposition behaviour were maintained despite the individual variations that were observed between the tested females ( wong et al . the variation in the number of eggs per female is common in experiments that assess a single gravidae . these variations are probably due to the blood - feeding efficiency ( xue et al . 2008 ) and the size of the adults , which may interfere with the reproductive capacity of males and females ( blackmore & lord 2000 , ponlawat & harrington 2009 ) . the average number of eggs was similar to that found by other authors ( christophers 1960 , madeira et al . as expected , the average number of eggs that were laid did not significantly differ between densities ( 2 , 4 , 8 and 16 breeding sites ) or between environments ( laboratory or semi - field ) . this result is probably due to the duration of the tests , which were performed between the third - seventh day after the consumption of blood meal , during which the females laid almost all of the eggs that were produced ( gomes 2006 , reiter 2007 , chadee 2010 ) . it was demonstrated for the first time that the behavioural response of females depended on the resources ( number of breeding sites ) that were provided to them . in an experiment with eight available containers , chadee ( 2010 ) noted that 87% of females used one to four breeding sites , with a maximum of seven . in our study , the number of containers that were used by the females increased following the increased availability of breeding sites under both laboratory and semi - field conditions . however , the number of breeding sites that were colonised seemed to stabilise at around five , even when there were 16 breeding sites available and reached a maximum of 11 under semi - field conditions . this result has epidemiological importance , as the search for breeding sites seems to be a crucial factor in the dispersal of the females and hence the diseases that they transmit ( edman et al . the determination of the average and maximum number of breeding sites that were used by each female can aid in the development of methodologies for monitoring and controlling the vector . therefore , further studies should be conducted to verify this behaviour in other environments and with different densities of breeding sites , given that few females colonised more than eight ovitraps . the results of the present study support the existence of the skip oviposition aegypti females , as previously observed ( christophers 1960 , fay & perry 1965 , corbet & chadee 1993 , apostol et al . 2003 , reiter 2007 , chadee 2010 ) . the ability of ae . aegypti females to distinguish potential breeding sites that will sustain the survival of their offspring during their development is a crucial factor in the life cycle of mosquitoes ( zahiri & rau 1998 ) . the selective pressure in favour of the females that make choices that may maximise the survival of their offspring ( reiter 2007 , harrington et al . nevertheless , this behaviour does not occur sometimes , as observed in a few females in this study ( 7.4% of females ) and by other authors ( harrington & edman 2001 , chadee 2010 ) . the large proportion of eggs that were laid on water contradicts the findings of most authors , who reported that the number of eggs that were deposited on water is negligible compared to that deposited on the walls , filter paper or paddles of the breeding site ( chadee & corbet 1987,chadee et al . however , a study in brazil demonstrated a large number of eggs that were deposited on water by females of two populations and at different humidities . the obtained figures were 42.9% and 57.3% ( 80% rh ) and 61.4% and 63.2% ( 51% rh ) for populations l and b , respectively ( madeira et al . 2002 ) . these findings were similar to the observations of the present work and almost 10 times higher than those reported by other authors ( chadee & cobert 1987 , chadee et al . aegypti because females of the same population distributed their eggs in different available proportions in oviposition substrates , as observed in our study . considering the behavioural plasticity of ae . aegypti , it is possible that the oviposition in both environments ( water or paddle / side walls of the breeding site ) can be advantageous depending on the circumstance . in anthropic environments , females can lay eggs in a large range of ephemeral containers that are very susceptible to disturbance ( reiter 2007 ) . this behaviour can make the choice of laying most of their eggs out of water relevant in places that can only be achieved by increasing the level of the water and can offer more chances of the larvae hatching and reaching adulthood . furthermore , deposition on the walls of the breeding sites may be an example of germ banking for the future of the ae . aegyptipopulation ( tsunoda et al . 2010 ) . in unfavourable conditions , the maintenance of germ banking until the return of favourable periods may be more advantageous to the offspring than the immediate eclosion of the larvae . on the other hand , the behaviour of laying eggs directly on the water is also relevant to the vector from an epidemiological perspective , as it helps to maintain their populations when there is no rainwater to supply breeding sites , thus allowing the existence of floating populations of mosquitoes during the dry season ( gomes et al . the deposition of eggs on water can also prevent predation by ants and cockroaches and the reduction of viability due to delays in hatching ( madeira et al . this result could perhaps explain why the number of eggs on the water was higher in semi - field conditions , where the eggs would be more exposed to these attacks . the increase in the number of eggs on water as observed in this study could also be explained by the abundance of resources ( breeding sites ) . the quick hatching of the larvae would increase the chances of the offspring finding the same available resources . however , to be considered a viable strategy , the eggs that were laid on the water surface must have hatching rates similar to those that were laid outside of the water . the literature is controversial on this issue because some authors have reported an extremely low hatching rate ( 2% ) ( silva et al . 2003 ) , while others have found higher rates , such as from 47 - 53% ( madeira et al . 2002 ) and from 73 - 80% ( rey & oconnell 2014 ) . therefore , we believe that the deposition of eggs on the water may be , at least in certain cases , a good choice for the survival of ae . aegypti . our results are also relevant in the context of the strategies of monitoring vector populations . in brazil , ovitraps can be used to obtain the indices of infestation ( ovitrap positivity index = number of ovitraps positive 100/number of ovitraps inspected and egg density index = number of eggs / number of positive ovitraps ) by counting the number of eggs on the paddle of the installed ovitraps ( gomes 1998 , ms / svs 2009 ) . in these surveys , the liquids ( water or infusion grass ) that are present in the ovitraps are discarded without performing any type of analysis . however , madeira et al . ( 2002 ) and the present study , both conducted in brazil , found high amounts of eggs in the ovitrap water . additional studies should be performed to determine if this trend is maintained in other populations of the country and under field conditions . the present work demonstrated for the first time the existence of a favourite breeding site and showed that egg distribution was not equal . the term favourite breeding site was used to denote the breeding site that received the highest percentage of eggs . this site was observed in semi - field and in laboratory conditions and with different amounts of available breeding sites . the ae . aegypti females usually choose the most productive containers to deposit most of their eggs . these containers are usually large , dark and unmanaged ( maciel - de - freitas & loureno - de - oliveira 2011 , wong et al . 2012 ) . in the present study , this behaviour was maintained even when all of the breeding sites were identical . either the females were able to recognise clues that were insignificant to us or this was such an intrinsic behaviour that it was maintained even when there was no advantage among the breeding sites . it is not known if the females lay their eggs in a single visit to the breeding site or if the breeding site receives a portion of the eggs and then the female returns to the same place and deposits eggs again . the observations on the proportion of eggs demonstrated the existence of two distinct patterns : ( i ) females lay many eggs at one favourite breeding site ( 40% or more ) and spread the remainder at other breeding sites and ( ii ) females lay few eggs ( less than 40% ) at the favourite breeding site and display a greater potential for spreading the eggs remaining over other breeding sites . ( 2012 ) , who observed an increase in the distribution of eggs in semi - field conditions when the highly productive containers were removed . the first pattern was more frequently observed in our study , although these two strategies can be important , depending on the condition of the breeding site and the environment in which the mosquitoes live . the behaviour of females laying most of their eggs at the favourite breeding site may favour the species in periods during which temporary breeding sites are scarce . this behaviour can also be advantageous when a female finds high - productivity breeding sites containing ideal conditions , such as large volumes and diameters , dark colouring and the presence of co - specific larvae . this behaviour would indicate the capacity to support large quantities of immature forms ( harrington et al . 2008 , maciel - de - freitas & loureno - de - oliveira 2011 , wong et al . females that choose other strategies , spreading the higher proportion of their eggs and depositing some at the favourite breeding site or spreading their eggs without considering the favourite breeding site , can favour their offspring during the rainy season , when there is a wide variety of breeding sites that are constantly supplied by rain water . thus , the plasticity of behaviour that was observed in this study and in previous studies ( madeira et al . the choice of a favourite breeding site and the deposition of large percentage of eggs in water were remarkable behaviours in the assessed population . aegypti ( madeira et al . 2002 , paduan et al . 2006 , hiragi et al . 2009 ) because they inhabit a variety of environmental conditions and present short life cycles . aegyptimosquitoes contain populations of individuals of various origins that have experienced different selective pressures . mosquito populations show great adaptability , primarily because their larval stages develop in different seasons and under different environmental conditions ( becker 1989 ) and because different selective pressures of the environment can lead to populations presenting distinct characteristics and great genetic and behavioural plasticity ( begon et al . 2011 ) . to determine whether the oviposition behaviour is the result of phenotypic plasticity or intraspecific differences between different populations , studies are required . however , regardless of the origin of behavioural plasticity in the oviposition ofae . . therefore , studies that elucidate the vector behaviour are important to the definition of control measures and despite efforts , little is known about the oviposition behaviour . thereby , it is extremely important that further studies investigate these aspects , especially under field and different conditions . finally , it is also important to understand the behaviour of mosquitoes that are used in the most modern and promising control techniques , such as those infected with the bacteria wolbachia and genetically modified mosquitoes .

despite the importance of the mosquito aedes aegypti in the transmission of arboviruses , such as yellow fever , chikungunya fever and dengue fever , some aspects of their behaviour remain unknown . in the present study , the oviposition behaviour of ae . aegypti females that were exposed to different densities of breeding sites ( 2 , 4 , 8 and 16 ) was evaluated in laboratory and semi - field conditions . the number of breeding sites that were used was proportional to the number available , but tended towards stabilisation . females used four - six breeding sites on average , with a maximum of 11 . a high percentage of eggs was observed in the water , along with the presence of a breeding site termed favourite , which received at least 40% of the eggs . the results are discussed in ecological , evolutionary and epidemiological approaches .

molecular diagnostics of cancer predisposition is very important for the medical treatment of the patient and persons belonging to the high risk group . molecular studies enable the detection of mutation carriers and release from unreasonable stress of persons from the group with increased risk of cancer occurrence . the mutation spectrums in the genes predisposing to colorectal cancer in the polish population have been described [ 1 - 4 ] . in the present work we focused on recurrent mutations of the apc gene causing fap . familial adenomatous polyposis ( fap ) is characterized by the appearance of numerous polyps in the large intestine . the correlation between mutations of the apc gene and the occurrence of familial adenomatous polyposis was described in 1991 and since then , mutations of the apc gene have been investigated in research centres leading to identification of various mutation types . mutations of the apc gene , in most cases , are small deletions or insertions with the most frequent mutations , in the greater part of the described populations , being the aaaga deletion at codon 1309 and the acaaa deletion at codon 1061 . clinical diagnoses of fap patients were conducted in collaborating genetic centres or gastroenterology clinics in the place of residence of patients . so far , samples of dna belonging to 280 polish fap families have been collected in the dna bank of polish fap families established in 1997 at the institute of human genetics , polish academy of science in poznan . dna was extracted from peripheral blood cells by the classical phenol purification method and entire coding sequence of apc gene was screened for mutations by pcr - hd and sscp methods in 280 probands . dna fragments showing heteroduplex or additional pattern in sscp analysis were sequenced by direct pcr product sequencing and analyzed using a megabace 500 sequencer according to the manufacturer 's specifications . we identified 72 mutations in 124 of our 280 fap families and observed eight types of recurrent mutations . the mutations and age of onset two of them were localized in exon 11 and the remaining six in the 3 ' part of exon 15 . the most frequent mutation , 39273931delaaaga , occurred in twenty - eight families ( 10% ) ; the second one was 31833187delacaaa , occurring in eight families ( 2.8% ) ; and the third most frequent mutation was 32023205deltcaa , detected in 5 families ( 1.7% ) . in our fap patients y500x occurred in four families ( 1.4% ) while q978x was detected in three families . each of the remaining four types of mutation occurred in two families and the frequencies of these mutations were below one percent . fifty - four recurrent mutations identified in apc gene and age of onset in a group of 124 diagnosed polish fap families nd no data available in the human mutations database at the institute of medical genetics in cardiff , considered the most representative population in the world , seven hundred mutations are listed for the apc gene . the mutation reports describe the frequency of this mutation from 0% in northwest spain , 2.4% in the australian population , 5% in dutch , 7% in israeli to 16% in the group of italian fap patients [ 6 - 9 ] . the second most frequent mutation , 31833187delacaaa , is reported with frequency ranging from 0% in northwest spain , 1.5% in israeli populations to 8.4% in australia [ 6 - 9 ] . a study of over 100 dutch families revealed equal frequency of those two most frequent mutations ( 39273931delaaaga and 31833187delacaaa ) . the latest published report in 2005 involved the analysis of over 1000 patients . in comparison to this study , the representative study of mutation frequency in the neighbouring population indicated two times higher frequency of 39273931delaaaga , whereas a difference in frequency of 31833187del - acaaa was not observed ( germany 3.8% , poland 2.7% ) . the frequency of 32023205deltcaa was equal ( 1.7% ) in both populations . in worldwide comparison differences in the frequency of mutations the polish population of fap patients belongs to the group where 39273931delaaaga occurred with higher frequency , whereas the frequency of mutation 31833187del - acaaa occurred with medium frequency in comparison with other populations . the two recurrent mutations localized on exon 11 were observed only in the polish population . in our two unrelated families with 14901491inst brain tumours were observed . another mutation ( q978x ) did not occur with higher frequency as described for other populations . in our fap patient group q283x , which occurs with frequency of 4.5% in uk fap patients , screening for these mutations permitted us to diagnose 19% of all families in our population but eight types of mutations constitute 43.5% of all our diagnosed families . the mutation study in our population should involve these eight mutations to improve molecular diagnostics of the apc gene . the study was financed by grant 2p05a10728 from the ministry of science and higher education .

the molecular diagnostics of genetically conditioned disorders is based on the identification of the mutations in the predisposing genes . hereditary cancer disorders of the gastrointestinal tracts are caused by mutations of the tumour suppressor genes or the dna repair genes . occurrence of recurrent mutation allows improvement of molecular diagnostics . the mutation spectrum in the genes causing hereditary forms of colorectal cancers in the polish population was previously described . in the present work an estimation of the frequency of the recurrent mutations of the apc gene was performed . eight types of mutations occurred in 19.4% of our fap families and these constitute 43% of all polish diagnosed families .

a total of 2,634 fecal and intestinal samples were collected from pigs exhibiting diarrhea from 569 swine farms in all 9 provinces of south korea , during january 1december 31 , 2008 ; age groups of the pigs are defined in the table . all samples were processed as 10% ( vol / vol ) suspensions with phosphate - buffered saline ( pbs ; 0.1 m , ph 7.2 ) , and viral rna was extracted from them . subsequently , reverse transcription pcr ( rt - pcr ) was performed by using 3 primer pairs as described previously ( 4 ) . to determine complete spike ( s ) gene sequence , we purified , cloned , and sequenced pcr products on an automated dna sequencer by using t7 , sp6 primers , and newly designed s gene specific primers ( primer sequences available on request ) . sequences were analyzed by clustalx version 1.83 program ( http://www.clustal.org ) and megalign software ( dnastar inc . , madison , wi , usa ) , and compared with those of reference strains in genbank . the complete s gene sequence of the pedv variant with a large genomic deletion ( strain mf3809/2008/south korea ) described here has been deposited in genbank under accession no . the pedv variant with a large deletion in the s gene was found in 3 samples ( 2 fecal and 1 intestinal ) of the suckling pigs with diarrhea at 1 farm in chungnam province . other pigs on the same farm were infected with pedv with the full - length s gene ; however , in no instance were both the pedv variant and pedv with the full - length s gene observed in 1 pig . pedv , porcine epidemic diarrhea virus ; s gene , spike gene . of the 2,634 samples , 205 ( 7.8% ; 49/569 [ 8.6% ] farms ) were positive for pedv : 116 ( 16.8% ) of 692 , 12 ( 2.4% ) of 504 , 29 ( 3.6% ) of 808 , 34 ( 24.1% ) of 141 , 1 ( 16.7% ) of 6 , and 13 ( 2.7% ) of 483 samples from the 6 age groups tested ( table ) ; however , when sf2/sr2 primers were subjected to pcr , a strong and single band of unexpected size ( 1,000 bp ) was found in each pcr product from 3 diarrhea samples of the suckling pigs on 1 farm . exact length of the band was 981 nt , and the band was much shorter than that of intact fragment because of 612-nt deletion at positions 2277723388 ( 1,593 vs 981 nt for pedv reference strains and pedv variant , respectively ; technical appendix figure 1 ) . sequence similarity of the 981-nt fragment of mf3809 was found to be in pedv s gene region in genbank by blast analysis ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . the complete s gene ( 3549-nt segment , corresponding to 1182 aa ) of mf3809 had high ( 93.3%98.5% nt , 92.0%98.0% aa ) sequence identity to all known pedv strains for which full - length s gene sequences were available in genbank , except that mf3809 has the large deletion in its s gene . phylogenetic analysis confirmed that mf3809 belonged to a cluster containing a pedv reference strain , not a cluster that included any other coronaviruses , and showed the closest genetic relationship with pedv strains from south korea in 2009 ( figure ) . a ) relationships between the pedv variant ( mf3809/2008/south korea ) and other coronaviruses based on the full - length spike gene . pedv , porcene epidemic diarrhea virus ; tgev , transmissible gastroenteritis virus ; prcv , porcine respiratory coronavirus ; ccov , canine coronavirus ; fcov , feline coronavirus ; hcov , human coronavirus ; mi - batcov , miniopterus bat coronavirus ; sc - batcov , scotophilus bat coronavirus ; hecov , human enteric coronavirus ; bcov , bovine coronavirus ; phev , porcine hemagglutinating encephalomyelitis virus ; eqcov , equine coronavirus ; crcov , canine respiratory coronavirus ; mucov , murine coronavirus ; pi - batcov , pipistrellus bat coronavirus ; ro - batcov , rousettus bat coronavirus ; sarsr - cov , severe acute respiratory syndrome - related coronavirus ; ty - batcov , tylonycteris bat coronavirus ; ibv , infectious bronchitis virus ; tucov , turkey coronavirus ; bwcov , beluga whale coronavirus ; bucov , bulbul coronavirus ; thcov , thrush coronavirus ; muncov , munia coronavirus . b ) phylogenetic tree of the entire spike genes of the pedv variant and all known pedv strains available in genbank . the phylogenetic tree was constructed using the neighbor - joining clustering method in mega version 5.22 with a pairwise distance ( 5 ) . bootstrap values ( based on 1,000 replicates ) for each node are given if > 60% . pedv strains isolated from various countries are marked with colors as follows : europe ( black ) , china ( red ) , japan ( olive green ) , usa ( bright magenta ) and south korea ( blue ) . besides a large ( 204-aa ) deletion at positions 713916 , a 2-aa ( d / ni ) deletion was identified at positions 163164 . we also discovered 2 separate insertions : a 4-aa ( qgvn ) insertion at positions 5962 and a 1-aa ( n ) insertion at position 140 . a total of 15 separate substitutions were identified , and the number(s ) of replaced amino acids ranged from 1 through 5 . these sequence variations were similar to those in highly virulent isolates recently reported in china ( 6 ) and the united states ( 3 ) , as well as in south korea . to further characterize the virus , we conducted rt - pcr with respect to the membrane ( m ) gene using the protocol described previously ( 7 ) . the complete m gene sequence was determined and then submitted , together with the complete s gene sequence , to genbank under accession no . the entire m gene of mf3809 had 96.6%100% nt ( 96.0%100% aa ) sequence identity to all known pedv strains available in genbank , and phylogenetic analysis showed that mf3809 belonged to a cluster containing pedv reference strain and showed the closest genetic relationship with 2007 korean pedv strains ( data not shown ) . cells and supernatants in every passage were collected separately for rna extraction and used to detect the virus and determine the amount of the viral rna in the medium with real - time rt - pcr ( 8) . the cells and supernatants of the 3 passages were positive for the virus , and the control inoculated with pbs was negative ; however , the amount of viral rna in the medium decreased with each passage . whether the positive result was attributed to residual viruses of the initial inoculation or to the decreased propagation of the virus in the cells further studies , such as continuous serial passages and neutralization assays , are needed to determine the final activity of the virus in vero cells . our large - scale study of the incidence of pedv in pigs with diarrhea in south korea found that 7.8% of animals were infected with the virus . moreover , our investigation identified and characterized a new pedv variant with a 612-nt deletion in s gene , corresponding to a 204-aa deletion . the coronavirus s protein plays a pivotal role in regulating interactions with specific host cell receptor glycoproteins to mediate viral entry and stimulate induction of neutralizing antibodies in the natural host ( 1,2,9 ) . mutations or deletions in the coronavirus s gene affect its pathogenicity and tissue tropism ( 1012 ) . porcine respiratory coronavirus ( prcv ) , a naturally occurring deletion mutant of transmissible gastroenteritis virus ( tgev ) , is an example of pathogenic change and tropism switching , apparently associated with s gene change . prcv has a 224-aa deletion at positions 21244 in the n terminal region , which is needed for the enteric tropism of tgev and comprises antigenic sites c and b ( 1315 ) , of s1 compared with tgev . in other words , tgev , a highly enteropathogenic porcine coronavirus , is turned into prcv , a respiratory pathogen with reduced pathogenicity , as a consequence of a large deletion in the s gene . unlike prcv , the pedv variant has a 204-aa deletion at positions 713916 in the c - terminus of s1 and n terminus of s2 , destroying 4 n - linked glycosylation sites at positions 728 , 745 , 783 , and 875 , as well as 2 neutralizing epitopes , ss2 ( 753760 ) and ss6 ( 769776 ) ( technical appendix figure 2 ) . these amino acid mutations might cause the conformational change of s protein and result in antigenicity / immunogenicity alteration of the pedv variant . further studies should be conducted to analyze extensive genomic sequences and determine biological properties , such as pathogenicity , tissue tropism , and transmissibility of the new pedv variant . summary of amino acid mutations in spike ( s ) protein of porcine epidemic diarrhea virus ( pedv ) variant ; schematic diagram of the s genes of classical pedv , recent prevalent pedv and the pedv variant ; and alignment of amino acid sequences of the entire s proteins of the pedv variant and reference strains .

since 1992 , porcine epidemic diarrhea virus ( pedv ) has been one of the most common porcine diarrhea associated viruses in south korea . we conducted a large - scale investigation of the incidence of pedv in pigs with diarrhea in south korea and consequently identified and characterized a novel pedv variant with a large genomic deletion .

there is increasing evidence that higher levels of high sensitivity c - reactive protein ( hs - crp ) is a marker of inflammation and a potential independent predictor of cardiovascular disease , as it may play a role in the development of atherosclerosis , adversely affecting mortality.1)2 ) rate of obesity is increasing globally where studies have shown association of body weight as assessed by body mass index ( bmi ) with clinical outcomes after percutaneous coronary intervention ( pci ) in acute myocardial infarction ( ami ) patients.3 ) previous reports have also indicated that hs - crp level is correlated with bmi and fat distribution.4)5 ) the mechanism for elevation of plasma hs - crp in obese subjects could be through a high production of cytokines , e.g. , interleukin-1 ( il-1 ) , interleukin-6 ( il-6 ) and tumor necrosis factor- ( tnf- ) , by excess adipose tissue which could induce high hs - crp production in liver.4 ) curently there is no data available evaluating the relationship between higher baseline hs - crp levels , body mass index ( bmi ) status , and adverse outcomes in patients with ami undergoing pci . therefore , in the present study we attempted to assess the association between baseline hs - crp level and 12-month clinical outcomes in patients with ami . this retrospective study was carried out at the heart center of chonnam national university hospital , korea . the permission to carry out the study had been sought from the hospital authorities ( irb no.05 - 49 ) and informed consent obtained from the patients . patient medical documents were used to obtain the demographic data , clinical characteristics and relevant laboratory results . patients were enrolled in the registry after admission to participating hospitals with a suspected diagnosis of ami . the korea acute myocardial infarction registry ( kamir ) is a prospective , multicenter , observational registry designed to examine current epidemiology in hospital management and outcome of patients with ami in korea . the registry included 52 community and university hospitals capable of primary pci with one - year clinical follow up . data was collected at each site by a well - trained study coordinator based on a standardized protocol.6 - 8 ) a total of 10974 consecutive patients with ami ( both st - segment elevation mi and non - st - segment elevation mi ) undergoing pci from november 2005 to september 2008 were assessed in this study . patients with unknown bmi ( n=395 ) and missing hs - crp ( n=2405 ) data were excluded from this study ; hence the total study population was 8174 patients . the aim was to evaluate the impact of baseline hs - crp on 12-month clinical outcomes according to bmi status . then , according to bmi categories suggested by the world health organization for asian population , the total study population was divided into three groups that included 1 ) overweight / obese ( bmi 23 kg / m , n=5647 ) , 2 ) normal - weight ( bmi 18.5 to 22.9 kg / m , n=2246 ) and 3 ) underweight ( bmi < 18.5 kg / m , n=281).9 ) each group was then stratified into quartiles based on their hs - crp levels . a final diagnosis of ami was made by the european society of cardiology / american college of cardiology ( esc / acc ) diagnostic criteria for ami.10 ) bmi was calculated as weight ( kg ) divided by height squared ( m ) . diabetes mellitus ( dm ) was defined as requiring the use of oral hypoglycaemic agent or insulin to lower blood glucose levels . hypertension was defined as patient systolic blood pressure > 140 mm hg and/or diastolic blood pressure > 90 mm hg at rest , over a series of repeated measurements , or after treatment with anti - hypertensive medications . for diabetics and patients with chronic renal disease , hypertension was defined as systolic blood pressure 130 mm hg and/or diastolic blood pressure 80 mm hg . hyperlipidemia was defined as patient total cholesterol level > 200 mg / dl or treatment with a lipid - lowering agent . coronary artery disease ( cad ) was defined as patient having a history of myocardial infarction ( mi ) revascularization procedure , or obstructive cad . blood samples were centrifuged and serum was removed and stored at a temperature of -700 until the assay for sugar and proteins was performed . absolute creatine kinase - mb levels were determined by radioimmunoassay ( dade behring , inc . , cardiac specific troponin i levels were measured using a paramagnetic particle , chemiluminescent immunoenzymatic assay ( beckham , coulter , inc . , twelve - hour fasting serum levels of total cholesterol , triglyceride , and low and high density lipoprotein - cholesterol were measured by standard enzymatic methods . blood sample for hs - crp was obtained on admission and was analyzed turbidimetrically with sheep antibodies against human crp ; this has been validated against the dade behring method . overweight / obese quartiles were : first quartile < 0.2 mg / dl , n=1370 , second quartile 0.2 to < 0.82 mg / dl , n=1451 , third quartile 0.82 to < 4.08 mg / dl , n=1413 and fourth quartile 4.08 mg / dl , n=1413 . normal - weight quartiles were : first quartile < 0.2 mg / dl , n=548 , second quartile 0.2 to < 0.92 mg / dl , n=575 , third quartile 0.92 to < 5.23 , n=563 mg / dl and fourth quartile 5.23 mg / dl , n=563 . underweight quartiles were : first quartile < 0.31 mg / dl , n=70 , second quartile 0.31 to < 1.54 mg / dl , n=70 , third quartile 1.54 to < 8.76 mg / dl , n=71 and fourth quartile 8.76 mg / dl , n=70 . two - dimensional echocardiography was performed in all patients and left ventricular ejection fraction ( lvef ) was assessed using a modified simpson 's biplane method . the morphology in coronary angiography was classified by criteria from the american college of cardiology / american heart association ( acc / aha).11 ) the degree of coronary flow was classified by thrombolysis in myocardial infarction ( timi ) score . the presence of left main coronary artery stenosis was defined as a luminal stenosis 50% . multivessel disease was defined as the presence of a lesion with > 50% diameter stenosis in a non - infarct related coronary artery . successful pci was defined as timi flow 3 with residual stenosis 50% in the infarct related artery . in - hospital complications included atrio - ventricular block , bradycardia , ventricular tachycardia / ventricular fibrillation , atrial fibrillation , no reflow , dissection , cardiogenic shock , acute renal failure , metabolic acidosis / lactic acidosis . major advense cardiac event ( mace ) were defined as all - cause death , recurrent mi , or repeat revascularization . the primary endpoint of this study was all - cause mortality at 12-month clinical follow up . all data were recorded on a standardized , electronic , web based registry at http://www.kamir.or.kr . continuous variables were presented as the meansd ; comparisons were conducted by one - way anova test . discrete variables were presented as percentages and frequencies ; comparisons were conducted by chi - square statistics . cox regression analysis was performed to identify a model with independent predictive factors including determination of a hazard ratio and its 95% confidence interval ( ci ) for each variable in the model . cox analysis was performed to identify variables that most affected the hazard ratio of hs - crp , with a cut off at p<0.20 for entry into the model . the korea acute myocardial infarction registry ( kamir ) is a prospective , multicenter , observational registry designed to examine current epidemiology in hospital management and outcome of patients with ami in korea . the registry included 52 community and university hospitals capable of primary pci with one - year clinical follow up . data was collected at each site by a well - trained study coordinator based on a standardized protocol.6 - 8 ) a total of 10974 consecutive patients with ami ( both st - segment elevation mi and non - st - segment elevation mi ) undergoing pci from november 2005 to september 2008 were assessed in this study . patients with unknown bmi ( n=395 ) and missing hs - crp ( n=2405 ) data were excluded from this study ; hence the total study population was 8174 patients . the aim was to evaluate the impact of baseline hs - crp on 12-month clinical outcomes according to bmi status . then , according to bmi categories suggested by the world health organization for asian population , the total study population was divided into three groups that included 1 ) overweight / obese ( bmi 23 kg / m , n=5647 ) , 2 ) normal - weight ( bmi 18.5 to 22.9 kg / m , n=2246 ) and 3 ) underweight ( bmi < 18.5 kg / m , n=281).9 ) each group was then stratified into quartiles based on their hs - crp levels . a final diagnosis of ami was made by the european society of cardiology / american college of cardiology ( esc / acc ) diagnostic criteria for ami.10 ) bmi was calculated as weight ( kg ) divided by height squared ( m ) . diabetes mellitus ( dm ) was defined as requiring the use of oral hypoglycaemic agent or insulin to lower blood glucose levels . hypertension was defined as patient systolic blood pressure > 140 mm hg and/or diastolic blood pressure > 90 mm hg at rest , over a series of repeated measurements , or after treatment with anti - hypertensive medications . for diabetics and patients with chronic renal disease , hypertension was defined as systolic blood pressure 130 mm hg and/or diastolic blood pressure 80 mm hg . hyperlipidemia was defined as patient total cholesterol level > 200 mg / dl or treatment with a lipid - lowering agent . coronary artery disease ( cad ) was defined as patient having a history of myocardial infarction ( mi ) revascularization procedure , or obstructive cad . blood samples were centrifuged and serum was removed and stored at a temperature of -700 until the assay for sugar and proteins was performed . absolute creatine kinase - mb levels were determined by radioimmunoassay ( dade behring , inc . , cardiac specific troponin i levels were measured using a paramagnetic particle , chemiluminescent immunoenzymatic assay ( beckham , coulter , inc . , twelve - hour fasting serum levels of total cholesterol , triglyceride , and low and high density lipoprotein - cholesterol were measured by standard enzymatic methods . blood sample for hs - crp was obtained on admission and was analyzed turbidimetrically with sheep antibodies against human crp ; this has been validated against the dade behring method . overweight / obese quartiles were : first quartile < 0.2 mg / dl , n=1370 , second quartile 0.2 to < 0.82 mg / dl , n=1451 , third quartile 0.82 to < 4.08 mg / dl , n=1413 and fourth quartile 4.08 mg / dl , n=1413 . normal - weight quartiles were : first quartile < 0.2 mg / dl , n=548 , second quartile 0.2 to < 0.92 mg / dl , n=575 , third quartile 0.92 to < 5.23 , n=563 mg / dl and fourth quartile 5.23 mg / dl , n=563 . underweight quartiles were : first quartile < 0.31 mg / dl , n=70 , second quartile 0.31 to < 1.54 mg / dl , n=70 , third quartile 1.54 to < 8.76 mg / dl , n=71 and fourth quartile 8.76 mg / dl , n=70 . two - dimensional echocardiography was performed in all patients and left ventricular ejection fraction ( lvef ) was assessed using a modified simpson 's biplane method . the morphology in coronary angiography was classified by criteria from the american college of cardiology / american heart association ( acc / aha).11 ) the degree of coronary flow was classified by thrombolysis in myocardial infarction ( timi ) score . the presence of left main coronary artery stenosis was defined as a luminal stenosis 50% . multivessel disease was defined as the presence of a lesion with > 50% diameter stenosis in a non - infarct related coronary artery . successful pci was defined as timi flow 3 with residual stenosis 50% in the infarct related artery . in - hospital complications included atrio - ventricular block , bradycardia , ventricular tachycardia / ventricular fibrillation , atrial fibrillation , no reflow , dissection , cardiogenic shock , acute renal failure , metabolic acidosis / lactic acidosis . major advense cardiac event ( mace ) were defined as all - cause death , recurrent mi , or repeat revascularization . the primary endpoint of this study was all - cause mortality at 12-month clinical follow up . all data were recorded on a standardized , electronic , web based registry at http://www.kamir.or.kr . spss 17.0 for windows ( spss , inc . , chicago , il , usa ) was used for all analyses . continuous variables were presented as the meansd ; comparisons were conducted by one - way anova test . discrete variables were presented as percentages and frequencies ; comparisons were conducted by chi - square statistics . cox regression analysis was performed to identify a model with independent predictive factors including determination of a hazard ratio and its 95% confidence interval ( ci ) for each variable in the model . cox analysis was performed to identify variables that most affected the hazard ratio of hs - crp , with a cut off at p<0.20 for entry into the model . a total of 8,174 ami patients , mean age=62.712.4 years and 76% male gender ( n=6217 ) , were stratified into quartiles based on their hs - crp level to study their 12-month clinical outcomes . twelve - month all - cause death and 12-month composite of mace increased as the value of hs - crp increased from 1st to 4th quartile . cox proportional hazard analysis showed 12-month mortality to be highest in patients of 4th quartile { hr=2.80 ( 1.335 - 5.873 ) , p=0.006 } followed by 3rd quartile { hr=2.495 ( 1.186 - 5.248 ) , p=0.016}. it was statistically significant among hs - crp quartiles ( p=0.045 ) . independent factors for 12-month mortality were age greater than 60 years , in - hospital complications , killip class > 1 , use of statin , heart rate > 100 beats / min , n - terminal pro - b - type natriuretic peptide ( nt - probnp ) > 3000 pg / ml , left ventricular ejection fraction ( lvef ) < 55% and creatinine clearance < 60 min / ml ( data not provided ) . as the 12-month mortality was statistically borderline significant among hs - crp quartiles in the total study population , we sought to further evaluate this finding by dividing the total study population into three groups based on their bmi . overweight and obese patients were grouped together as there was no baseline significant difference in their 12-month all - cause mortality ( p=0.076 , data not provided ) . baseline clinical characteristics and laboratory findings of this group are presented in table 1 and 2 respectively . overweight / obese patients with higher hs - crp ( i.e. , 2nd , 3rd and 4th quartiles ) were older , more often female , had higher killip classes , heart rate and nt - probnp ; and had lower blood pressure , lvef and creatinine clearance . they were associated with higher incidence of dm , hypertension , cad , heart failure and cerebrovascular disease . as per past regular medication history , 7.15% of the patients at the time of admission were on statin ( n=404 ) . multivessel involvement , acc / aha lesion type c , and pre - pci timi flow grade 0 were observed to increase with an increase in hs - crp . pci success rate decreased and in - hospital complication rate increased with increasing hs - crp . table 4 shows the clinical outcomes during the 12-month follow - up . in - hospital death , composite mace at 1 month and 12 months , and mortality at 1 month and 12 months were more frequent with increasing hs - crp levels . twelve - month mortality increased as the value of hs - crp increased ; 2.0% ( n=27 ) in 1st , 2.5% ( n=37 ) in 2nd , 4.4% ( n=62 ) in 3rd and highest 6.6% ( n=94 ) in the 4th quartile . cox proportional hazard model was implemented to identify the independent predictors of mortality at the 12-month clinical follow - up . serum hs - crp quartiles showed significant association with 12-month mortality when adjusted for age and gender ( p<0.001 ) . with quartile 1 as a reference , the risk of 12-month mortality was higher in the 3rd quartile { 2.153 ( 1.369 - 3.384 ) , p=0.001 } and 4th quartile { 3.268 ( 2.129 - 5.016 ) , p<0.001}. after adjustment with multiple covariates , 12-month mortality was highest in patients within the 4th quartile compared to other patients { hazard ratio ( hr ) 2.382 ( 1.079 - 5.259 ) , p=0.032 } though statistical significance was not achieved ( p=0.172 ) . independent factors for 12-month mortality were old age ( > 60 years ) , heart rate > 100 beats / min , lvef < 55% , creatinine clearance < 60 min / ml and in - hospital complications . the variables included in cox model were age , male gender , heart rate , systolic blood pressure , smoking history , history of hypertension , diabetes mellitus , coronary artery disease , cerebrovascular disease and heart failure , lvef , killip classes > 1 , glucose , total cholesterol , triglyceride , creatinine clearance , nt - probnp , left main and multivessel involvement , pre - timi flow grade 0 , post - timi flow grade 3 , acc / aha lesion type c , pci success rate , in - hospital complications and use of statin . patients were also stratified into quartiles based on hs - crp level to evaluate their 12-month clinical outcomes . patients with higher hs - crp were found to be older ( p<0.001 ) , with increased heart rate ( p=0.015 ) and higher killip classes ( p=0.002 ) . they had more association with diabetes mellitus ( p=0.019 ) , hypertension ( p=0.049 ) , heart failure ( p=0.001 ) and peripheral vascular disease ( p=0.001 ) . creatinine clearance , lvef and high - density lipoprotein cholesterol decreased with higher hs - crp ( p<0.001 , p<0.001 and p=0.001 respectively ) . total cholesterol , triglyceride and low density lipoproteincholesterol ( ldl - c ) were also significant among quartiles ( p<0.001 , p=0.001 and p=0.001 respectively ) . they were more associated with acc / aha lesion type c , multivessel involvement , pre - timi flow grade 0 and in - hospital complications . twelve - month composite of mace ( p=0.009 ) and all - cause death ( p<0.001 ) increased as the value of hs - crp increased from 1st to 4th quartile . cox proportional hazard model showed 12-month mortality to be statistically insignificant among hs - crp quartiles in this group ( p=0.681 ) . the variables included in the model were age , dm , hypertension , hyperlipidemia , heart failure , peripheral vascular disease , smoking history , clinical presentation st - elevation mi , total cholesterol , triglyceride , creatinine clearance , nt - probnp , systolic blood pressure , heart rate , in - hospital complications , killip class > 1 , lvef < 55% , pre - procedure timi grade 0 flow , acc / aha lesion type c , multivessel involvement , and use of statin . patients from this study were additionally divided into quartiles based on hs - crp level to study their 12-month clinical outcomes . the variables included in the model were age , systolic blood pressure , lvef < 55% , glucose , total cholesterol , ldl - c , creatinine clearance , nt - probnp , smoking history , heart failure , post - procedure timi grade 3 , pci success rate , and in - hospital complications . age , nt - probnp , killip class > 1 and in - hospital complications increased with increasing levels of hs - crp ( p=0.006 , p<0.001 , p=0.009 and p=0.032 respectively ) , while creatinine clearance decreased with rising level of hs - crp ( p=0.001 ) . systolic blood pressure , lvef , total cholesterol and post - timi flow grade iii were statistically significant among the quartiles ( p=0.043 , p=0.023 , p=0.038 and p=0.002 respectively ) . twelve - month composite of mace increased as the value of hs - crp increased from 1st to 4th quartile , but it was statistically insignificant ( p=0.382 ) , and twelve - month all - cause death showed no association among quartiles ( p=0.079 ) . cox proportional hazard model showed 12-month mortality to be statistically insignificant among hs - crp quartiles in this group as well ( p=0.760 ) . 1 shows adjusted survival curves of 12-month mortality in hs - crp quartiles in ( a ) total study population , ( b ) overweight / obese group , ( c ) normal - weight group and ( d ) underweight group . baseline clinical characteristics and laboratory findings of this group are presented in table 1 and 2 respectively . overweight / obese patients with higher hs - crp ( i.e. , 2nd , 3rd and 4th quartiles ) were older , more often female , had higher killip classes , heart rate and nt - probnp ; and had lower blood pressure , lvef and creatinine clearance . they were associated with higher incidence of dm , hypertension , cad , heart failure and cerebrovascular disease . as per past regular medication history , 7.15% of the patients at the time of admission were on statin ( n=404 ) . multivessel involvement , acc / aha lesion type c , and pre - pci timi flow grade 0 were observed to increase with an increase in hs - crp . pci success rate decreased and in - hospital complication rate increased with increasing hs - crp . table 4 shows the clinical outcomes during the 12-month follow - up . in - hospital death , composite mace at 1 month and 12 months , and mortality at 1 month and 12 months were more frequent with increasing hs - crp levels . twelve - month mortality increased as the value of hs - crp increased ; 2.0% ( n=27 ) in 1st , 2.5% ( n=37 ) in 2nd , 4.4% ( n=62 ) in 3rd and highest 6.6% ( n=94 ) in the 4th quartile . cox proportional hazard model was implemented to identify the independent predictors of mortality at the 12-month clinical follow - up . serum hs - crp quartiles showed significant association with 12-month mortality when adjusted for age and gender ( p<0.001 ) . with quartile 1 as a reference , the risk of 12-month mortality was higher in the 3rd quartile { 2.153 ( 1.369 - 3.384 ) , p=0.001 } and 4th quartile { 3.268 ( 2.129 - 5.016 ) , p<0.001}. after adjustment with multiple covariates , 12-month mortality was highest in patients within the 4th quartile compared to other patients { hazard ratio ( hr ) 2.382 ( 1.079 - 5.259 ) , p=0.032 } though statistical significance was not achieved ( p=0.172 ) . independent factors for 12-month mortality were old age ( > 60 years ) , heart rate > 100 beats / min , lvef < 55% , creatinine clearance < 60 min / ml and in - hospital complications . the variables included in cox model were age , male gender , heart rate , systolic blood pressure , smoking history , history of hypertension , diabetes mellitus , coronary artery disease , cerebrovascular disease and heart failure , lvef , killip classes > 1 , glucose , total cholesterol , triglyceride , creatinine clearance , nt - probnp , left main and multivessel involvement , pre - timi flow grade 0 , post - timi flow grade 3 , acc / aha lesion type c , pci success rate , in - hospital complications and use of statin . patients were also stratified into quartiles based on hs - crp level to evaluate their 12-month clinical outcomes . patients with higher hs - crp were found to be older ( p<0.001 ) , with increased heart rate ( p=0.015 ) and higher killip classes ( p=0.002 ) . they had more association with diabetes mellitus ( p=0.019 ) , hypertension ( p=0.049 ) , heart failure ( p=0.001 ) and peripheral vascular disease ( p=0.001 ) . creatinine clearance , lvef and high - density lipoprotein cholesterol decreased with higher hs - crp ( p<0.001 , p<0.001 and p=0.001 respectively ) . total cholesterol , triglyceride and low density lipoproteincholesterol ( ldl - c ) were also significant among quartiles ( p<0.001 , p=0.001 and p=0.001 respectively ) . they were more associated with acc / aha lesion type c , multivessel involvement , pre - timi flow grade 0 and in - hospital complications . twelve - month composite of mace ( p=0.009 ) and all - cause death ( p<0.001 ) increased as the value of hs - crp increased from 1st to 4th quartile . cox proportional hazard model showed 12-month mortality to be statistically insignificant among hs - crp quartiles in this group ( p=0.681 ) . the variables included in the model were age , dm , hypertension , hyperlipidemia , heart failure , peripheral vascular disease , smoking history , clinical presentation st - elevation mi , total cholesterol , triglyceride , creatinine clearance , nt - probnp , systolic blood pressure , heart rate , in - hospital complications , killip class > 1 , lvef < 55% , pre - procedure timi grade 0 flow , acc / aha lesion type c , multivessel involvement , and use of statin . patients from this study were additionally divided into quartiles based on hs - crp level to study their 12-month clinical outcomes . the variables included in the model were age , systolic blood pressure , lvef < 55% , glucose , total cholesterol , ldl - c , creatinine clearance , nt - probnp , smoking history , heart failure , post - procedure timi grade 3 , pci success rate , and in - hospital complications . age , nt - probnp , killip class > 1 and in - hospital complications increased with increasing levels of hs - crp ( p=0.006 , p<0.001 , p=0.009 and p=0.032 respectively ) , while creatinine clearance decreased with rising level of hs - crp ( p=0.001 ) . systolic blood pressure , lvef , total cholesterol and post - timi flow grade iii were statistically significant among the quartiles ( p=0.043 , p=0.023 , p=0.038 and p=0.002 respectively ) . twelve - month composite of mace increased as the value of hs - crp increased from 1st to 4th quartile , but it was statistically insignificant ( p=0.382 ) , and twelve - month all - cause death showed no association among quartiles ( p=0.079 ) . cox proportional hazard model showed 12-month mortality to be statistically insignificant among hs - crp quartiles in this group as well ( p=0.760 ) . 1 shows adjusted survival curves of 12-month mortality in hs - crp quartiles in ( a ) total study population , ( b ) overweight / obese group , ( c ) normal - weight group and ( d ) underweight group . this study demonstrated that higher baseline hs - crp level ( 4.08 mg / dl ) in overweight / obese ami patients showed significant association with 12-month all - cause mortality independent of other prognostic markers . over the past few years , it has become increasingly clear that inflammation plays a pivotal role in cardiovascular disease , and increased circulating inflammation markers may be predictive of cardiovascular events.12)13 ) among the inflammation markers available , hs - crp is one of the independent predictors of cardiovascular events.14)15 ) adiposity is an inflammatory condition and il-6 , tnf- and hs - crp levels are positively correlated with adipocyte size.4)15)16 ) these cytokines induce insulin resistance in adipose cells by inhibiting insulin signaling.17)18 ) crp along with il-6 and other cytokines / adipokines could be deleterious on the arterial wall since it has been found to directly promote endothelial cell inflammation and atherosclerotic processes.19)20 ) thus , the link between adiposity , plaque accumulation / progression / rupture and atherothrombotic events , especially ami , might be explained by higher levels of systemic inflammation and their chronic effect on coronary atherosclerosis over time . marsik et al.2 ) report that patients with crp concentrations > 5 mg / l at the time of hospital admission had a 50% to 330% increase in risk of death from any cause . this increase in risk was present in both short - term and long - term follow - ups , and rose in magnitude as concentrations of crp increased to > another study by koenig et al.1 ) deals with the issue of hs - crp as a predictor of death in the monitoring of trends and determinants in cardiovascular disease . after adjustment for age , smoking , hypertension , hyperlipidemia , diabetes , obesity and socioeconomic markers , those with baseline hs - crp concentrations > 3 mg / l had a 2-fold increase in risk of total mortality . some studies have shown that higher bmi is associated with higher crp concentrations even among young adults 17 to 39 years of age . these findings suggest a state of low - grade systemic inflammation in obese patients.21 ) as previously reported by sutter et al.22 ) it is known that increased levels of hs - crp are greatly associated with extensive tissue destruction or infarct size . in such situations with more severe inflammation and extensive tissue damage , the relationship between adverse outcomes and crp level might be considered independent from bmi status . the present study shows that hs - crp concentrations in the obese rise with increasing age , diabetes mellitus , hypertension , coronary artery disease , heart failure and cerebrovascular disease reaffirming data from earlier studies.23 - 25 ) a prospective study by bruci et al.26 ) on 329 patients with myocardial infarction at the time of admission showed crp to be associated with a strong , positive graded increase in the risk of heart failure and death independently of known risk indicators . ninety - four patients ( 29% ) were in killip class greater than 1 at presentation , and greater killip class was associated with increased crp . interestingly , a study in cairo by fareed et al . noted that patients with hs - crp -17 mg / l were prone to heart failure ( at this value sensitivity=88% , specificity=51.2% ) . our findings are in agreement with a recently published report that showed a trend to decreasing values of lvef in relation to higher crp levels.27 ) the significant relationship between crp and lvef suggests the implication of inflammatory mechanisms in left ventricular dysfunction and in the pathogenesis of congestive heart failure , independently of the underlying atherosclerotic burden . angiographic findings of multivessel involvement , frequency of acc / aha lesion type c and subsequently higher pre - timi grade 0 flow , lower procedure success rate and higher complications support the previous study findings on acute coronary syndrome that demonstrated multiple complex stenoses with high plaque burden suggesting a pan - coronary involvement.28 ) this study showed a negative relation of smoking with hs - crp in the overweight / obese subset . the mechanism whereby smoking is related to crp concentration is unclear , but it may be multi - factorial , involving bronchial injury , endothelial activation and systemic inflammation.29 ) use of a statin is a negative predictor of 12-month mortality in total study population . however , there was no significant association of statin use and hs - crp levels in higher bmi patients though earlier studies have shown that statin reduces hs - crp levels.30)31 ) this could be due to relatively small number of patients on a statin at the time of admission ( 7.15% ) or unavailability of follow - up hs - crp data in our registry . moreover , in anglo - scandinavian cardiac outcomes trial : lipid lowering arm trial neither baseline nor on - treatment crp provided useful information about the efficacy of statin treatment to reduce cardiovascular events beyond ldl - c reduction.32 ) this study should be interpreted in the light of the following limitations . this is not a randomized trial , but a retrospective evaluation on a large - scale multi - center registry . blood samples for hs - crp were obtained at the time of admission , hence higher levels of hs - crp in our study may not be associated with myocardial necrosis.33 ) the time of onset and size of the myocardial infarction may also increase the hs - crp levels . however , we did not determine the serial changes in hs - crp concentration after ami . other inflammatory markers , such as il-6 , tnf- or lipoprotein - associated phospholipase a2 ( lp - pla2 ) that could have added more robust evidence of other inflammatory pathways associated with poor outcome were not a part of this study . extreme variations in hs - crp levels that could have made a difference in standard deviation in each group have not been excluded from this study . higher baseline hs - crp level ( 4.08 mg / dl ) in overweight / obese ami patients showed significant association with 12-month all - cause mortality independent of other prognostic markers . we suggest further long term evaluation with serial changes in hs - crp concentration after ami and other inflammation markers to clearly elucidate its role in more diverse population . this is not a randomized trial , but a retrospective evaluation on a large - scale multi - center registry . blood samples for hs - crp were obtained at the time of admission , hence higher levels of hs - crp in our study may not be associated with myocardial necrosis.33 ) the time of onset and size of the myocardial infarction may also increase the hs - crp levels . however , we did not determine the serial changes in hs - crp concentration after ami . other inflammatory markers , such as il-6 , tnf- or lipoprotein - associated phospholipase a2 ( lp - pla2 ) that could have added more robust evidence of other inflammatory pathways associated with poor outcome were not a part of this study . extreme variations in hs - crp levels that could have made a difference in standard deviation in each group have not been excluded from this study . higher baseline hs - crp level ( 4.08 mg / dl ) in overweight / obese ami patients showed significant association with 12-month all - cause mortality independent of other prognostic markers . we suggest further long term evaluation with serial changes in hs - crp concentration after ami and other inflammation markers to clearly elucidate its role in more diverse population .

background and objectivesserum high sensitivity c - reactive protein ( hs - crp ) is a marker of inflammation and may lead to the development of atherosclerosis , adversely affecting mortality . the aim of this study was to evaluate the relationship between baseline hs - crp level and 12-month clinical outcomes in patients with acute myocardial infarction ( ami ) undergoing percutaneous coronary intervention ( pci ) according to their body mass index ( bmi ) status.subjects and methodsusing data from the korea acute myocardial infarction registry from november 2005 to september 2008 , a total of 8174 consecutive ami patients were studied . cox proportional hazard model revealed that higher baseline levels of hs - crp was associated with 12-month all - cause mortality ( p=0.045 ) . to further understand this association , patients were divided into 3 groups based on their body mass index : 1 ) overweight / obese , 2 ) normal weight , and 3 ) underweight patients . then each group was stratified into quartiles based on their hs-crp.resultsin overweight / obese patients , cox model showed significant association of hs - crp with 12-month mortality when adjusted for age and gender ( p<0.001 ) , however , after adjustment with multiple covariates , mortality was highest in the 4th quartile { hr 2.382 , ( 1.079 - 5.259 ) , p=0.032 } though statistically insignificant ( p=0.172 ) . we observed no significant association of serum hs - crp with 12-month mortality in normal weight ( p=0.681 ) and underweight ( p=0.760 ) patients.conclusionhigher baseline hs - crp level ( 4.08 mg / dl ) in overweight / obese ami patients showed significant association with 12-month all - cause mortality independent of other prognostic markers .

specific learning disabilities ( slds ) are a generic term that refers to a heterogeneous group of neurobehavioral disorders manifested by significant unexpected , specific , and persistent difficulties in the acquisition and use of efficient reading ( dyslexia ) , writing ( dysgraphia ) , or mathematical ( dyscalculia ) abilities despite conventional instruction , intact senses , normal intelligence , proper motivation , and adequate sociocultural opportunity . international classification of diseases-10 describes them as specific developmental disorders of scholastic skills and diagnostic and statistical manual 4 edition - text revision ( dsm - iv ) as learning disorders and dsm-5 as specific learning disorders . dyslexia ( reading disorder ) is the most common among the learning disorders , affecting 80% of all those identified as learning - disabled . the incidence of dyslexia in primary school children in india has been reported to be 218% , dysgraphia 14% , and dyscalculia 5.5% . they are found most commonly associated with other cortical - based disorders such as other learning disorders , language disorders , motor disorders , and organization and executive function disability . the most common comorbidities include attention deficit hyperactivity disorder ( adhd ) , conduct disorder , oppositional defiant disorder , anxiety disorder , and depression . keeping the paucity of literature regarding slds in india , this study aims to document the sociodemographic profile and psychiatric comorbidities in school children . to study the number of children having slds and comorbidities in school settingto study the comorbidities in children with slds who are identified and on remedial educationto study the comorbidities in children who do not have sldsto study the difference in sociodemographic variables as well as a comorbidity in the three groups . to study the number of children having slds and comorbidities in school setting to study the comorbidities in children with slds who are identified and on remedial education to study the comorbidities in children who do not have slds to study the difference in sociodemographic variables as well as a comorbidity in the three groups keeping the paucity of literature regarding slds in india , this study aims to document the sociodemographic profile and psychiatric comorbidities in school children . to study the number of children having slds and comorbidities in school settingto study the comorbidities in children with slds who are identified and on remedial educationto study the comorbidities in children who do not have sldsto study the difference in sociodemographic variables as well as a comorbidity in the three groups . to study the number of children having slds and comorbidities in school setting to study the comorbidities in children with slds who are identified and on remedial education to study the comorbidities in children who do not have slds to study the difference in sociodemographic variables as well as a comorbidity in the three groups the data are collected from a group of sld children in remedial schools ( dyslexia association of andhra pradesh , hyderabad ) . the other in two urban and two semi - urban schools all of them are nongovernment , private organized english medium schools . they were divided into three groups : group 1 consists of thirty children who are already diagnosed as having an sld by a psychiatrist and are on remedial education . after taking written informed consent from the parents , the details as per the intake pro forma are taken and parent is interviewed according to the developmental psychopathology checklist ( dpcl ) . subsequently , the child is administered colored progressive matrices ( cpm)/standard progressive matrices ( spm ) , malin 's intelligence scale for indian children ( misic ) and nimhans sld index . group 2 consists of 32 children diagnosed for the first time at schools during the study . after taking permission from the school management , following the screening , children were administered spm / cpm and those who scored more than the 50 percentile were shortlisted . after taking written informed consent from the parents and using the inclusion and exclusion criteria and information obtained from parents interview and intake pro forma and dpcl , the children were then administered misic and nimhans sld index . group 3 constituted of 34 children who were not included in the list after administering the checklist ; they were administered spm / cpm . the parents of those children who scored more than the 50 percentile were interviewed and information was collected in intake pro forma and dpcl after taking consent . means and standard deviation , chi - square test , and anova were used for analysis . semi - structured intake pro formaintelligence tests : raven 's progressive matrices standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) . progressive matrices are used as a screening instrument to rule out children with low iq misic the misic is an indian adaptation of wechsler intelligence scale for children the nimhans index for slds is a battery of tests used for confirming the diagnosis of sld . it consists of two levels : level 1 : ( 57 years of age ) ; level 2 : ( 812 years)checklist to identify learning disabilitiesdpcl for children . semi - structured intake pro forma raven 's progressive matrices standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) . progressive matrices are used as a screening instrument to rule out children with low iq misic the misic is an indian adaptation of wechsler intelligence scale for children raven 's progressive matrices standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) . progressive matrices are used as a screening instrument to rule out children with low iq standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years ) colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) . progressive matrices are used as a screening instrument to rule out children with low iq the misic is an indian adaptation of wechsler intelligence scale for children the misic is an indian adaptation of wechsler intelligence scale for children the nimhans index for slds is a battery of tests used for confirming the diagnosis of sld . it consists of two levels : level 1 : ( 57 years of age ) ; level 2 : ( 812 years ) checklist to identify learning disabilities dpcl for children . means and standard deviation , chi - square test , and anova were used for analysis . semi - structured intake pro formaintelligence tests : raven 's progressive matrices standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) . progressive matrices are used as a screening instrument to rule out children with low iq misic the misic is an indian adaptation of wechsler intelligence scale for children the nimhans index for slds is a battery of tests used for confirming the diagnosis of sld . it consists of two levels : level 1 : ( 57 years of age ) ; level 2 : ( 812 years)checklist to identify learning disabilitiesdpcl for children . semi - structured intake pro forma raven 's progressive matrices standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) . progressive matrices are used as a screening instrument to rule out children with low iq misic the misic is an indian adaptation of wechsler intelligence scale for children raven 's progressive matrices standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) . progressive matrices are used as a screening instrument to rule out children with low iq standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years ) colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) . progressive matrices are used as a screening instrument to rule out children with low iq the misic is an indian adaptation of wechsler intelligence scale for children the misic is an indian adaptation of wechsler intelligence scale for children the nimhans index for slds is a battery of tests used for confirming the diagnosis of sld . it consists of two levels : level 1 : ( 57 years of age ) ; level 2 : ( 812 years ) checklist to identify learning disabilities dpcl for children . they comprised 40 males ( m ) and 22 females ( f ) with the gender distribution ratio ( m / f ) of 1.8:1 [ table 1 ] . sociodemographic and personal data prematurity ( sld special education group 6 ( 20% ) and sld schools group 1 ( 3.1% ) ( p = 0.006 ) and cesarean section ( sld special education group 63.4% and sld schools group ( 31.3% ) ( p = 0.02 ) are found to be related to sld in children . sld children have delayed milestones particularly problems with speech , which is predominant in sld special education group ( 36.7% ) ( p = 0.02 ) . sld children have significant school problems in the form of school refusal , poor school performance , reading , writing , spelling , and arithmetic when compared to normal children . in sld children , the problems with reading were found to be about 56 ( 90.3% ) , with comprehension 55 ( 88.7% ) , with arithmetic 17 ( 27.4% ) , with spelling 47 ( 75.8% ) , and with writing 28 ( 45.2% ) [ table 2 ] . break up of learning disabilities ( nimhans specific learning disabilities index ) table 3 shows various presentations of learning disabilities , pure reading disability is found in 22 ( 35.48% ) , writing disability in 1 ( 1.61% ) , dyscalculia in 3 ( 4.83% ) whereas combined learning disability in 36 ( 58.06% ) , which is found to be the most common type of learning disability . mathematic disability is found to occur less frequently in sld special education group ( 16.7% ) than in sld schools group ( 34.4% ) . presentation of learning disabilities overall , the prevalence of sld cases in schools is found to be 6.6% . among the subtypes of learning disability , the prevalence of dyslexia is 4.58% , dysgraphia 0.2% , dyscalculia 0.63% , and combined learning disability in 7.5% respectively in school children . children with sld committed significant errors on bender gestalt test ( bgt ) ( p = 0.001 ) as well as number cancellation test ( p = 0.001 ) . no statistical difference was found ( p = 0.37 ) on scores on recall of bgt designs ( memory ) among three groups indicating that there is no statistical difference in memory in the three groups . pre- , peri- , and post - natal problems in mother , epilepsy , head injury or infections in infancy , poor vision , speech and language delay during childhood were found to be significant in children with sld in both groups . developmental psychopathology checklist - developmental history table 5 shows various developmental problems . only speech and articulation problems were found to be of highly statistically significant ( p = 0.001 ) in children with sld . developmental psychopathology checklist - developmental problems adhd [ table 6 ] is found to be the most common comorbidity associated with sld amounting to 26 ( 41.9% ) with trends toward inattentive subtype . attention deficit hyperactivity disorder conduct disorder is found to be about 1 ( 3% ) in sld schools group with trends toward symptoms of disobedience . emotional disorders are found to be about 2 ( 6.3% ) in sld schools group and 4 ( 13.3% ) in sld special education group . none of the children in both sld groups met the criteria for somatic problems , psychosis , or obsessive - compulsive disorder . the family history of alcoholism was found to be significantly higher ( p = 0.001 ) in normal children ( 50% ) [ table 7 ] . the family history of learning problems has been found to be occurring more frequently in both sld groups , which is found to be statistically significant ( p = 0.02 ) . developmental psychopathology checklist - family history stressors such as over expectation ( 26.7% ) ( p = 0.04 ) and over - involvement ( 36.7% ) p = 0.001 ) by parents are found in children with sld special education group . the prevalence of sld in schools is found to be 6.6% with combined learning disability being most common 7.5% . the male : female ratio in children with sld if 1.8:1 , indicating male preponderance . the similar findings were found by karande et al . in his study with a gender distribution ratio of 2.1:1 . a higher representation of middle socioeconomic strata in sld schools group ( 84.4% ) and high socioeconomic strata in sld special education group ( 66.7% ) is observed . the higher representation of middle class is because the study sample is taken from urban and semi - urban areas , with english as the medium of instruction . it was being done due to the lack of standardized assessment tools for sld in vernacular languages . study which states that sld is higher ( 82% ) in middle socioeconomic strata . the higher representation of high socioeconomic strata in sld special education could be because the remedial or special education which was received by the students was from special educators / special education centers which charge a particular amount per month . hence , most of the children who are receiving the special education are the ones whose parents could afford to pay them , apart from regular school fees . the findings are not in continuation of the previous study by murnane who stated that sld are more common in a minority group and those belonging to low socioeconomic status . the parents of children with sld special education group belong to high education group ( mothers 73.3% and fathers 46.7% ) and most of them are professionals ( mothers 23.3% and fathers 46.7% ) while those parents of children with sld schools group are educated up to secondary education ( mothers 46.9% and fathers 25% ) . the previous study conducted by kohli et al . has noticed that parents of sld children had 1215 years of education . mean age of children with sld in schools group is 9.88 years while the mean age of children with sld in special education group is 9.43 years , respectively . the mean age of children with sld as observed by karande et al . was 11.4 years . children with sld in special education group are joined in schools at an early age ( i.e. , 3.02 years ) when compared to that of children with sld schools group ( 3.81 years ) . the difference is statistically significant ( p = 0.001 ) , indicating that children in sld with special education group are being joined in the school at an earlier age than others and due to the high socioeconomic status of these parents of these children who keep them in special education schools for remedial methods . it is to be noted that none of the previous studies compared the age of the child at the time of joining school . prematurity ( sld special education group 20% and sld schools group 3.1% ) and cesarean section ( sld special education group 63.4% and sld schools group 31.3% ) are found to be related to sld in children . the findings go with the result of previous study by wood et al . , where prematurity is found to be a risk factor with sld . furthermore , cesarean section also appears to be a risk factor . however , there are no such reports from the previous studies . sld children have delayed milestones particularly problems with speech , which is predominant in sld special education group ( 36.7% ) indicates that sld special education group has developmental immaturity . significant pre- , peri- , post - natal problems ( 40% ) were found in mothers of children with sld in special education group along with epilepsy , head injury or infections in infancy , poor vision , speech and language delay during childhood are found to be significant in both groups of sld , clearly indicating that sld is a developmental problem . sld children have significant school problems in the form of school refusal , poor school performance , reading , writing , spelling , and arithmetic when compared to normal children . in sld children , the problems with reading were found to be about 90.3% , with comprehension 88.7% , with arithmetic 27.4% , with spelling 75.8% , and with writing 45.2% . pure reading disability is found in 35.48% , writing disability in 1.61% , dyscalculia in 4.83% while combined learning disability in 58.06% , which is found to be the most common type of learning disability . mathematic disability is found to occur less frequently in sld special education group ( 16.7% ) than in sld schools group ( 34.4% ) . these findings are similar to the findings of karande et al . where combined subtype was represented more . however , compared to the study by ramaa where mathematic disability is found to be 5.98% , mathematic disabilities have found to occur less frequently in special education group ; this could be probably due to the reason that most of the children brought to the special educators are having problems with reading and writing . in this study , most common type of learning disability was found to be combined ( reading + writing ) 35.48% which was similar to a finding by karande et al . , 2007 . but as compared to the previous indian studies , mathematic disabilities have found to occur less frequently in special education group , this could be probably due to the reason that most of the children brought to the special educators are having problems with reading , spelling , and writing . children with sld committed statistically significant errors on bgt ( p = 0.001 ) denoting perceptual and visuomotor integration problems in children with sld . this amounts to the high amount of inattentiveness seen in sld and also the influence of other comorbid conditions like adhd . adhd is found to be the most common comorbidity associated with sld amounting to 41.9% with trends toward inattentive subtype . analysis of the characteristics of the 26 ( 41.9% ) children with adhd in sld group , it is found that there are 3 ( 11.5% ) females and 23 ( 88.5% ) males . on subtyping , the number of sld children with adhd inattentive type is found to be 11 ( 42.3% ) , adhd hyperactive type 7 ( 26.9% ) , and combined type of adhd in 8 ( 30.8% ) . the comorbidity of adhd was about 35% . in the present study , trends are toward inattentive type , whereas in other studies on indian and pakistani data found that combined subtype was more common . conduct disorder is found to be about 3% in sld schools group with trends toward symptoms of disobedience . emotional disorders are found to be about 6.3% in sld schools group and 13.3% in sld special education group . none of the children in both sld groups met the criteria for somatic problems , psychosis or obsessive compulsive disorder . on the whole , the comorbidities as assessed by dpcl showed significance on adhd , conduct and emotional disorders which was not found in the study by karande et al . except for adhd . significant family history of learning disabilities is found in children with both groups of sld . learning disorders in family history is found to be significant as also observed by snowling et al . stressors like over expectation and over involvement by parents are found in children with sld special education group . the study was not conducted in government schools as the assessment of sld in vernacular languages was not available . the study was not conducted in government schools as the assessment of sld in vernacular languages was not available . there was a significant association with prematurity , cesarean section , delayed speech and family history of sld . among comorbidities of sld ,

background : specific learning disabilities ( slds ) are an important cause of academic underachievement in children and are also associated with comorbidities like attention deficit hyperactivity disorder ( adhd ) which further have an impact on the child 's education.aims:to estimate the prevalence and psychosocial profile and psychiatric comorbidities in children with sld in two settings , i.e. , on special ( remedial ) education and schools and to compare the findings with normal children.materials and methods : this study was carried out in schools situated in urban and semi - urban areas and special education schools . a total of 96 children were chosen for the study . after taking informed consent from the parents , the details about socioeconomic status , family , developmental history , and school history of the children were collected on a semi - structured pro forma and then the children were screened for sld . they were administered colored / standard progressive matrices and malin 's intelligence scale for assessing their intelligence quotient and nimhans sld index and developmental psychopathology checklist to study psychopathology . chi - square test and anova were done.results:the prevalence of sld in schools is found out to be 6.6% . there was a significant association with prematurity , cesarean section , delayed speech , and family history of sld . among comorbidities of sld , association with adhd alone has been found to be significant.conclusion:the most common type of sld is combined type comorbid with adhd . there is a need for early identification of learning disabilities in schools so that with early recognition and remedial intervention children can be helped with to cope with studies .

the global initiative for chronic obstructive lung disease ( gold ) strategy document recommends that the pharmacological therapy of chronic obstructive pulmonary disease ( copd ) should be predicated according to the individual patient s level of symptoms , airflow limitation , and history of exacerbations.1 the preferred option for maintenance therapy of copd is long - acting bronchodilators,1,2 either alone or in combination with an inhaled corticosteroid ( ics ) . the gold strategy document1 recommends treatment with at least one long - acting bronchodilator for patients with moderate - to - very severe copd . the strategy of combining short - acting bronchodilators with different mechanisms of action , ie , ipratropium bromide and salbutamol , has previously been shown to provide better symptomatic relief and to be cost - saving in patients with copd.3,4 in line with this , more recent studies have shown that also combining long - acting bronchodilators with different mechanisms of action provides additional benefits over the use of a single long - acting bronchodilator , without significantly increasing the risk of adverse effects.57 previous studies have shown that combining a long - acting 2-agonist with a long - acting anti - muscarinic antagonist leads to significant improvements in symptoms and lung function compared with either type of bronchodilator used alone.5,7 acute exacerbations of copd are associated with a poor prognosis in regard to health status , physical activity , decline in lung function , and mortality.813 prevention of exacerbations is therefore of outmost importance in the management of copd,1 and , in keeping with this , a key objective for new drugs for copd . the aim of this review is to provide an overview of the clinical studies evaluating the safety and clinical efficacy of the once - daily fixed - dose dual bronchodilator combination of indacaterol and glycopyrronium bromide for the treatment of copd . in order to perform this review , the general principles of the preferred reporting items for systematic reviews and meta - analyses ( prisma ) guidelines14,15 were adopted . a planned series of systematic searches was carried out , last updated february 2014 , using the databases pubmed , embase , cochrane central register of controlled trials , and clinicaltrials.gov using the following algorithm of mesh terms : glycopyrronium bromide , indacaterol , qva149 , long - acting bronchodilators , qab149 , nva237 , aclidinium bromide , tiotropium bromide , formoterol , salmeterol , and copd . these searches were repeated with these terms in combination with fev1 , dyspnoea , health status , quality of life , day - time symptoms , night - time symptoms , exacerbations , hyperinflation , and exercise capacity , in order to identify published studies . all searches were limited to english - language articles . as the methods and results could not be fully assessed , clinical trials published only in abstract form were excluded from this review . studies were included if they met all of the following criteria : 1 ) published in a peer - reviewed journal ; 2 ) inclusion of adults 40 years of age with stable copd defined according to the gold strategy document1 or the american thoracic society / european respiratory society guideline criteria;2 3 ) comparison of fixed - dose glycopyrronium bromide plus indacaterol with placebo , glycopyrronium bromide , indacaterol , tiotropium bromide , aclidinium bromide , formoterol , or salmeterol ; and 4 ) also reporting at least one of the following outcomes : onset of action ; trough forced expiratory volume in 1 second ( [ fev1 ] 24 hours post - dosing ) at the end of the treatment period ; peak change in fev1 ; health status ( st george s respiratory questionnaire [ sgrq ] ) ; symptom relief transition dyspnea index ( tdi ) ; use of rescue medication ; acute exacerbations ; exercise capacity ; and inspiratory capacity ( ic ) . due primarily to the relatively limited number of published clinical trials fulfilling all inclusion criteria , a meta - analysis was not included in the present review . of the 147 potential relevant citations identified by the series of searches , nine clinical trials fulfilled all inclusion criteria ( 6,166 participants ) . characteristics of the included studies are given in table 1 . all subjects included in the trials were stable but symptomatic at baseline and fulfilled the spirometric criteria for a diagnosis of copd . two studies compared fixed - dose indacaterol / glycopyrronium with indacaterol ( and placebo ) ; one study with indacaterol and glycopyrronium ; two studies with tiotropium ( and placebo ) ; one study with glycopyrronium and tiotropium ; one study with fixed - combination salmeterol / fluticasone ( sfc ) ; one study with indacaterol , glycopyrronium , and tiotropium ( and placebo ) ; and one study with placebo . the cardiovascular safety of fixed - dose indacaterol / glycopyrronium was evaluated by van de maele et al16 in a randomized , double - blind , placebo - controlled , parallel - group study of 257 patients with moderate - to - severe copd . the enrolled patients were randomized to receive fixed - dose indacaterol / glycopyrronium ( 600/100 g , 300/100 g , or 150/100 g ) , indacaterol 300 g , or placebo once daily for 14 days , and the primary endpoint was change from baseline in 24-hour mean heart rate versus placebo on day 14 . no clinical significant difference was observed for the primary endpoint , and once - daily fixed - dose indacaterol / glycopyrronium was , in general , well tolerated among the enrolled copd patients with a cardiovascular safety profile , including qtc interval , and an overall rate of adverse events ( aes ) similar to that of placebo . the safety of fixed - dose indacaterol / glycopyrronium was further investigated in the enlighten study by dahl et al,17 in which 339 patients were randomized to either fixed - dose indacaterol / glycopyrronium or placebo for 52 weeks . no difference between treatment groups was observed for the primary endpoint ; likewise , no clinically relevant differences were observed for vital signs and electrocardiographic parameters . the overall incidence of aes was similar between the two treatment arms ( 57.8% and 56.6% , respectively ) , and the most frequently reported ae was worsening of copd ( 28.5% and 25.7% , respectively , for active and placebo treatment ) . the most common severe ae was exacerbation of copd , and the second most common severe ae was pneumonia , the latter only reported in the indacaterol / glycopyrronium - treated group ( n=8 ) . a post hoc analysis of serious pneumonia aes stratified by copd severity was reported not to provide conclusive evidence that fixed - dose indacaterol / glycopyrronium was associated with a higher incidence of pneumonia than placebo ( rate of events 3.6% , odds ratio 5.11 ; p=0.10 ) . although no statistically significant difference was observed , aes that led to hospitalization or prolonged hospitalization were reported for 15.1% and 8.8% , respectively , of patients treated with fixed - dose indacaterol / glycopyrronium and placebo . in contrast to this , in the illuminate study,18 pneumonia ( confirmed by chest x - ray ) was only reported in patients treated with fixed - combination sfc ( 1.5% ; no cases among patients treated with fixed - dose indacaterol / glycopyrronium ) . the beacon study by dahl et al19 compared fixed - dose indacaterol / glycopyrronium with the concurrent administration of the mono - components indacaterol and glycopyrronium and found that the safety and efficacy profile was similar for the two treatment arms . mahler et al20 investigated the effect of fixed - dose indacaterol / glycopyrronium versus placebo and tiotropium on dyspnea in the blinded , double - dummy , crossover blaze study comprising 247 patients with moderate - to - severe copd ( staging according to the gold 2007 criteria ; mean post - bronchodilator fev1 56% predicted [ pred ] ) . changes in dyspnea were assessed by the self - administered computerized versions of the baseline dyspnea index and the tdi after 6 weeks . the tdi total score was statistically significantly improved with fixed - dose indacaterol / glycopyrronium at 6 weeks compared to both placebo and tiotropium . however , only the improvement with fixed - dose indacaterol / glycopyrronium versus placebo reached the minimal clinically important difference ( mcid ) of 1 point;21 this mcid in total tdi score with fixed - dose indacaterol / glycopyrronium was seen in patients with both moderate ( least squares mean [ lsm ] treatment difference 1.11 ; p<0.001 ) and severe ( lsm treatment difference 1.92 ; p<0.001 ) copd ( defined on spirometric criteria ) compared with placebo . a tdi responder analysis comparing fixed - dose indacaterol / glycopyrronium and tiotropium revealed that the proportion of patients achieving the mcid of at least 1 point was higher with fixed - dose indacaterol / glycopyrronium than with either placebo ( 35.9% and 18.1% , respectively ; p<0.001 ) or tiotropium ( 24.4% ; p=0.012 ) . however , it should be noted that the mcid for comparisons between active treatments has not yet been established . the blaze study20 also showed a significant improvement in percentage of nights with no awakenings , days with no daytime symptoms , and days with usual daily activities compared with placebo , whereas no significant difference in these outcomes was observed between fixed - dose indacaterol / glycopyrronium and tiotropium ; in line with this , no statistically significant differences were observed between the two active treatment arms with regard to mean daily total and individual symptom ( respiratory symptoms , cough , wheeze , and amount of sputum ) scores . however , the patients treated with fixed - dose indacaterol / glycopyrronium used less rescue medication and had a higher percentage of days with no use of rescue medication compared to both placebo ( p<0.001 ) and tiotropium ( p=0.002 and p<0.001 ) . similar findings with regard to symptoms and use of rescue medication were reported in the enlighten study.17 vogelmeier et al18 reported that fixed - dose indacaterol / glycopyrronium significantly increased the tdi focal score after 26 weeks of treatment compared with fixed - combination sfc , with a treatment difference of 0.76 ( p=0.003 ) . although , on average , not reaching the mcid for tdi score , 67.5% of patients treated with fixed - dose indacaterol / glycopyrronium compared with 56.8% of patients on sfc had an increase of at least 1 point in tdi score ( p=0.046 ) . however , no difference in change in total sgrq score was observed between the two treatment groups ; likewise , only modest and , in general , nonsignificant differences were reported with regard to symptoms and use of rescue medication.18 with regard to rescue medication , van noord et al22 found no difference in number of puffs used by patients treated with fixed - dose indacaterol / glycopyrronium and indacaterol , whereas wedzicha et al23 reported a significant decrease in use of rescue medication ( salbutamol ) for patients treated with fixed - dose indacaterol / glycopyrronium compared with glycopyrronium and tiotropium . the shine study24 showed significant improvements in tdi focal score , use of rescue medication , daytime symptoms , nighttime symptoms , and ability to perform usual daily activities for fixed - dose indacaterol / glycopyrronium compared with monotherapy with a long - acting bronchodilator and placebo . furthermore , the shine study also showed a significant improvement in sgrq total score at week 12 and week 26 compared with placebo , whereas no significant improvement was observed for the other active treatments ( indacaterol , glycopyrronium , and tiotropium ) or placebo.24 wedzicha et al23 reported an improvement in sgrq total score from baseline of 89 units with fixed - dose indacaterol / glycopyrronium ; 6 units with glycopyrronium ; and 56 units with tiotropium , and the treatment differences in sgrq total score between fixed - dose indacaterol / glycopyrronium were 1.9 to 2.8 and 1.7 to 3.1 , respectively , compared with glycopyrronium ( p<0.01 ) and tiotropium ( p<0.05 ) . in keeping with this , the percentage of patients achieving the mcid in total sgrq score was significantly higher for fixed - dose indacaterol / glycopyrronium compared with both glycopyrronium and tiotropium up to week 52 . van noord et al,22 in a four - period crossover study ( n=154 ) , compared the efficacy of once - daily fixed - dose indacaterol / glycopyrronium ( 300/50 g ) with indacaterol ( 600 g and 300 g ) and placebo . the lsm trough fev1 on day 7 was significantly higher for fixed - dose indacaterol / glycopyrronium compared with placebo ( treatment difference 0.226 l ; p<0.001 ) and with indacaterol 300 g or 600 g ( treatment difference 0.123 l and 0.117 l , respectively ; p<0.001 ) . furthermore , fixed - dose indacaterol / glycopyrronium had a faster onset of action ( at 5 minutes post - dose on day 1 ) compared with placebo and with indacaterol 300 g or 600 g ( p<0.0001 ) ; the treatment difference between fixed - dose indacaterol / glycopyrronium and placebo was 0.141 l , whereas no exact data were reported for the treatment difference between fixed - dose indacaterol / glycopyrronium and indacaterol . in the shine study , bateman et al24 investigated the efficacy of fixed - dose indacaterol / glycopyrronium versus its mono - components indacaterol and glycopyrronium , tiotropium , and placebo over 26 weeks . patients with moderate - to - severe copd ( defined according to the gold 2007 spirometric criteria ; n=2,144 ) were randomized to once - daily fixed - dose indacaterol / glycopyrronium ( 110/50 g ) , indacaterol ( 150 g ) , glycopyrronium ( 50 g ) , open - label tiotropium ( 18 g ) , or placebo ( table 1 ) . the primary outcome was trough fev1 at week 26 for fixed - dose indacaterol / glycopyrronium versus its mono - components . at week 26 , a significant improvement was seen in trough fev1 for fixed - dose indacaterol / glycopyrronium compared with both indacaterol ( treatment difference 0.07 l ; p<0.001 ) and glycopyrronium ( treatment difference 0.09 l ; p<0.001 ) ; likewise , a significant improvement was observed for fixed - dose indacaterol / glycopyrronium compared with tiotropium and placebo ( treatment difference 0.08 l and 0.20 l , respectively ; p<0.001 ) . the observed statistically significant differences between fixed - dose indacaterol / glycopyrronium versus all active treatments and placebo in trough fev1 were maintained throughout the study period . similar to the findings reported by van noord et al,22 the shine study24 revealed that fixed - dose indacaterol / glycopyrronium provides rapid bronchodilation after administration of the first dose on day 1 , as the fev1 , fev1 4-hour area under the curve , and peak fev1 were higher compared with placebo , glycopyrronium , and tiotropium , whereas data were not given for the comparison with indacaterol . subgroup analysis showed that the improvement in trough fev1 was seen both in patients with moderate and with severe copd ( defined by the spirometric gold criteria ) , although the treatment differences between fixed - dose indacaterol / glycopyrronium and monotherapy with a long - acting bronchodilator ( indacaterol , glycopyrronium , and tiotropium ) was less than 0.10 l. vogelmeier et al,18 in the illuminate study , compared the safety and efficacy of fixed - dose indacaterol / glycopyrronium versus fixed - combination sfc over 26 weeks in patients with moderate - to - severe copd . in this double - blind , double - dummy , parallel - group study , 523 patients ( without exacerbations in the year prior to study entry ) were randomized to either fixed - dose indacaterol / glycopyrronium ( 110/50 g once daily ) or sfc ( 50/500 g twice daily ) . the primary outcome was the standardized area under the fev1 curve from 012 hours post - dose ( fev1auc012 ) at week 26 . thirty - five percent of the enrolled patients were treated with ics at baseline , and the mean post - bronchodilator fev1 was 60.2% pred ( table 1 ) . the fev1auc012 was significantly higher for patients treated with fixed - dose indacaterol / glycopyrronium ( 1.70 l ) than for patients treated with sfc ( 1.56 l ) , with a treatment difference of 0.14 l ( p<0.0001 ) at week 26 ; this treatment difference in fev1auc012 was observed throughout the study period . similar findings were reported for other spirometric parameters , including peak fev1 and forced vital capacity.18 after 6 weeks of treatment in the blaze study,20 a significant and clinically meaningful25 improvement in mean fev1 was seen at every time point from 45 minutes pre - dose to 4 hours post - dose versus both placebo and tiotropium ( p<0.001 ) . the enlighten study17 showed a significant improvement in pre - dose fev1 at week 52 compared with placebo ( treatment difference 0.189 l ; p<0.001 ) , and the treatment difference in pre - dose fev1 from week 3 to week 52 versus placebo was in the range of 0.1520.189 l ( p<0.001 ) . furthermore , the 60-minute post - dose fev1 was significantly higher for patients treated with fixed - dose indacaterol / glycopyrronium compared with placebo ( treatment difference 0.2000.286 l ; p<0.001 ) . trough fev1 in the spark study23 was significantly higher for fixed - dose indacaterol / glycopyrronium at all evaluated time points compared with glycopyrronium ( treatment difference 0.080.09 l ; p<0.0001 ) and tiotropium ( treatment difference 0.060.08 l ; p<0.0001 ) . in the bright study , beeh et al26 investigated the effect of fixed - dose indacaterol / glycopyrronium compared with placebo and tiotropium on exercise tolerance in patients with moderate - to - severe copd ( defined according to the gold 2007 spirometric criteria ; mean fev1 55.9% pred ) ( table 1 ) . a total of 85 patients were randomized to fixed - dose indacaterol / glycopyrronium ( 110/50 g ) , placebo , or tiotropium ( 18 g ) once daily in a blinded , three - period , crossover study for 3 weeks . the primary outcome variable was exercise endurance time at day 21 for fixed - dose indacaterol / glycopyrronium versus placebo . fixed - dose indacaterol / glycopyrronium significantly improved exercise endurance time at day 21 compared with placebo ( lsm treatment difference 60 seconds ) . a similar improvement in exercise endurance time was observed with tiotropium compared with placebo ( lsm treatment difference 66 seconds ) . in line with this , an analysis of data for the subgroup of patients with hyperinflation ( defined as functional residual capacity [ frc ] > 120% pred ) , showed that the mean changes in exercise endurance time from baseline were 85 seconds and 88 seconds , respectively , for indacaterol / glycopyrronium and tiotropium . a significant improvement in ic at peak exercise was observed for fixed - dose indacaterol / glycopyrronium compared with both placebo and tiotropium , and a similar difference was observed for trough ic . in the study by van noord et al,22 changes in ic , although not stated as a secondary outcome variable , were also determined . the authors reported that both lsm ic and trough ic were significantly better for fixed - dose indacaterol / glycopyrronium compared with both indacaterol and placebo ( p=0.02 ) . wedzicha et al , in the spark study,23 investigated the effect of fixed - dose indacaterol / glycopyrronium on exacerbations in patients with severe and very severe copd ( defined according to the gold 2007 spirometric criteria ) . in a parallel - group study , 2,224 patients ( having had at least one exacerbation of copd in the year prior to enrollment ) were randomized to fixed - dose indacaterol / glycopyrronium ( 110/50 g ) , glycopyrronium ( 50 g ) , or open - label tiotropium ( 18 g ) for 64 weeks . the primary outcome variable was to demonstrate superiority of fixed - dose indacaterol / glycopyrronium for the rate of moderate ( defined as worsening of symptoms treated with systemic corticosteroids or antibiotics or both ) and severe ( defined as worsening of symptoms requiring emergency treatment or hospitalization ) exacerbations compared to monotherapy with glycopyrronium . the key secondary outcome variable was to demonstrate superiority of fixed - dose indacaterol / glycopyrronium compared with tiotropium with regard to the rate of moderate and severe exacerbations . twenty - two percent of the patients included in the efficacy analyses had had two or more exacerbations in the previous year , and 88% of the patients had one or more cardiovascular risk factors at baseline , although few patients had a history of cardiovascular disease . furthermore , 75% of the patients in all three treatment arms were treated with icss throughout the study period . the rate of moderate or severe exacerbations was significantly reduced by 12% for the fixed - dose indacaterol / glycopyrronium group compared with the glycopyrronium group ( p=0.038 ) , whereas the 10% reduction in the rate of moderate or severe exacerbations with fixed - dose indacaterol / glycopyrronium compared with tiotropium was nonsignificant . the overall rate of exacerbations ( mild , moderate , and severe ) was significantly reduced with fixed - dose indacaterol / glycopyrronium , by 15% and 14% , respectively , compared with glycopyrronium and tiotropium . the cardiovascular safety of fixed - dose indacaterol / glycopyrronium was evaluated by van de maele et al16 in a randomized , double - blind , placebo - controlled , parallel - group study of 257 patients with moderate - to - severe copd . the enrolled patients were randomized to receive fixed - dose indacaterol / glycopyrronium ( 600/100 g , 300/100 g , or 150/100 g ) , indacaterol 300 g , or placebo once daily for 14 days , and the primary endpoint was change from baseline in 24-hour mean heart rate versus placebo on day 14 . no clinical significant difference was observed for the primary endpoint , and once - daily fixed - dose indacaterol / glycopyrronium was , in general , well tolerated among the enrolled copd patients with a cardiovascular safety profile , including qtc interval , and an overall rate of adverse events ( aes ) similar to that of placebo . the safety of fixed - dose indacaterol / glycopyrronium was further investigated in the enlighten study by dahl et al,17 in which 339 patients were randomized to either fixed - dose indacaterol / glycopyrronium or placebo for 52 weeks . no difference between treatment groups was observed for the primary endpoint ; likewise , no clinically relevant differences were observed for vital signs and electrocardiographic parameters . the overall incidence of aes was similar between the two treatment arms ( 57.8% and 56.6% , respectively ) , and the most frequently reported ae was worsening of copd ( 28.5% and 25.7% , respectively , for active and placebo treatment ) . the most common severe ae was exacerbation of copd , and the second most common severe ae was pneumonia , the latter only reported in the indacaterol / glycopyrronium - treated group ( n=8 ) . a post hoc analysis of serious pneumonia aes stratified by copd severity was reported not to provide conclusive evidence that fixed - dose indacaterol / glycopyrronium was associated with a higher incidence of pneumonia than placebo ( rate of events 3.6% , odds ratio 5.11 ; p=0.10 ) . although no statistically significant difference was observed , aes that led to hospitalization or prolonged hospitalization were reported for 15.1% and 8.8% , respectively , of patients treated with fixed - dose indacaterol / glycopyrronium and placebo . in contrast to this , in the illuminate study,18 pneumonia ( confirmed by chest x - ray ) was only reported in patients treated with fixed - combination sfc ( 1.5% ; no cases among patients treated with fixed - dose indacaterol / glycopyrronium ) . the beacon study by dahl et al19 compared fixed - dose indacaterol / glycopyrronium with the concurrent administration of the mono - components indacaterol and glycopyrronium and found that the safety and efficacy profile was similar for the two treatment arms . mahler et al20 investigated the effect of fixed - dose indacaterol / glycopyrronium versus placebo and tiotropium on dyspnea in the blinded , double - dummy , crossover blaze study comprising 247 patients with moderate - to - severe copd ( staging according to the gold 2007 criteria ; mean post - bronchodilator fev1 56% predicted [ pred ] ) . changes in dyspnea were assessed by the self - administered computerized versions of the baseline dyspnea index and the tdi after 6 weeks . the tdi total score was statistically significantly improved with fixed - dose indacaterol / glycopyrronium at 6 weeks compared to both placebo and tiotropium . however , only the improvement with fixed - dose indacaterol / glycopyrronium versus placebo reached the minimal clinically important difference ( mcid ) of 1 point;21 this mcid in total tdi score with fixed - dose indacaterol / glycopyrronium was seen in patients with both moderate ( least squares mean [ lsm ] treatment difference 1.11 ; p<0.001 ) and severe ( lsm treatment difference 1.92 ; p<0.001 ) copd ( defined on spirometric criteria ) compared with placebo . a tdi responder analysis comparing fixed - dose indacaterol / glycopyrronium and tiotropium revealed that the proportion of patients achieving the mcid of at least 1 point was higher with fixed - dose indacaterol / glycopyrronium than with either placebo ( 35.9% and 18.1% , respectively ; p<0.001 ) or tiotropium ( 24.4% ; p=0.012 ) . however , it should be noted that the mcid for comparisons between active treatments has not yet been established . the blaze study20 also showed a significant improvement in percentage of nights with no awakenings , days with no daytime symptoms , and days with usual daily activities compared with placebo , whereas no significant difference in these outcomes was observed between fixed - dose indacaterol / glycopyrronium and tiotropium ; in line with this , no statistically significant differences were observed between the two active treatment arms with regard to mean daily total and individual symptom ( respiratory symptoms , cough , wheeze , and amount of sputum ) scores . however , the patients treated with fixed - dose indacaterol / glycopyrronium used less rescue medication and had a higher percentage of days with no use of rescue medication compared to both placebo ( p<0.001 ) and tiotropium ( p=0.002 and p<0.001 ) . similar findings with regard to symptoms and use of rescue medication were reported in the enlighten study.17 vogelmeier et al18 reported that fixed - dose indacaterol / glycopyrronium significantly increased the tdi focal score after 26 weeks of treatment compared with fixed - combination sfc , with a treatment difference of 0.76 ( p=0.003 ) . although , on average , not reaching the mcid for tdi score , 67.5% of patients treated with fixed - dose indacaterol / glycopyrronium compared with 56.8% of patients on sfc had an increase of at least 1 point in tdi score ( p=0.046 ) . however , no difference in change in total sgrq score was observed between the two treatment groups ; likewise , only modest and , in general , nonsignificant differences were reported with regard to symptoms and use of rescue medication.18 with regard to rescue medication , van noord et al22 found no difference in number of puffs used by patients treated with fixed - dose indacaterol / glycopyrronium and indacaterol , whereas wedzicha et al23 reported a significant decrease in use of rescue medication ( salbutamol ) for patients treated with fixed - dose indacaterol / glycopyrronium compared with glycopyrronium and tiotropium . the shine study24 showed significant improvements in tdi focal score , use of rescue medication , daytime symptoms , nighttime symptoms , and ability to perform usual daily activities for fixed - dose indacaterol / glycopyrronium compared with monotherapy with a long - acting bronchodilator and placebo . furthermore , the shine study also showed a significant improvement in sgrq total score at week 12 and week 26 compared with placebo , whereas no significant improvement was observed for the other active treatments ( indacaterol , glycopyrronium , and tiotropium ) or placebo.24 wedzicha et al23 reported an improvement in sgrq total score from baseline of 89 units with fixed - dose indacaterol / glycopyrronium ; 6 units with glycopyrronium ; and 56 units with tiotropium , and the treatment differences in sgrq total score between fixed - dose indacaterol / glycopyrronium were 1.9 to 2.8 and 1.7 to 3.1 , respectively , compared with glycopyrronium ( p<0.01 ) and tiotropium ( p<0.05 ) . in keeping with this , the percentage of patients achieving the mcid in total sgrq score was significantly higher for fixed - dose indacaterol / glycopyrronium compared with both glycopyrronium and tiotropium up to week 52 . van noord et al,22 in a four - period crossover study ( n=154 ) , compared the efficacy of once - daily fixed - dose indacaterol / glycopyrronium ( 300/50 g ) with indacaterol ( 600 g and 300 g ) and placebo . the lsm trough fev1 on day 7 was significantly higher for fixed - dose indacaterol / glycopyrronium compared with placebo ( treatment difference 0.226 l ; p<0.001 ) and with indacaterol 300 g or 600 g ( treatment difference 0.123 l and 0.117 l , respectively ; p<0.001 ) . furthermore , fixed - dose indacaterol / glycopyrronium had a faster onset of action ( at 5 minutes post - dose on day 1 ) compared with placebo and with indacaterol 300 g or 600 g ( p<0.0001 ) ; the treatment difference between fixed - dose indacaterol / glycopyrronium and placebo was 0.141 l , whereas no exact data were reported for the treatment difference between fixed - dose indacaterol / glycopyrronium and indacaterol . in the shine study , bateman et al24 investigated the efficacy of fixed - dose indacaterol / glycopyrronium versus its mono - components indacaterol and glycopyrronium , tiotropium , and placebo over 26 weeks . patients with moderate - to - severe copd ( defined according to the gold 2007 spirometric criteria ; n=2,144 ) were randomized to once - daily fixed - dose indacaterol / glycopyrronium ( 110/50 g ) , indacaterol ( 150 g ) , glycopyrronium ( 50 g ) , open - label tiotropium ( 18 g ) , or placebo ( table 1 ) . the primary outcome was trough fev1 at week 26 for fixed - dose indacaterol / glycopyrronium versus its mono - components . at week 26 , a significant improvement was seen in trough fev1 for fixed - dose indacaterol / glycopyrronium compared with both indacaterol ( treatment difference 0.07 l ; p<0.001 ) and glycopyrronium ( treatment difference 0.09 l ; p<0.001 ) ; likewise , a significant improvement was observed for fixed - dose indacaterol / glycopyrronium compared with tiotropium and placebo ( treatment difference 0.08 l and 0.20 l , respectively ; p<0.001 ) . the observed statistically significant differences between fixed - dose indacaterol / glycopyrronium versus all active treatments and placebo in trough fev1 were maintained throughout the study period . similar to the findings reported by van noord et al,22 the shine study24 revealed that fixed - dose indacaterol / glycopyrronium provides rapid bronchodilation after administration of the first dose on day 1 , as the fev1 , fev1 4-hour area under the curve , and peak fev1 were higher compared with placebo , glycopyrronium , and tiotropium , whereas data were not given for the comparison with indacaterol . subgroup analysis showed that the improvement in trough fev1 was seen both in patients with moderate and with severe copd ( defined by the spirometric gold criteria ) , although the treatment differences between fixed - dose indacaterol / glycopyrronium and monotherapy with a long - acting bronchodilator ( indacaterol , glycopyrronium , and tiotropium ) was less than 0.10 l. vogelmeier et al,18 in the illuminate study , compared the safety and efficacy of fixed - dose indacaterol / glycopyrronium versus fixed - combination sfc over 26 weeks in patients with moderate - to - severe copd . in this double - blind , double - dummy , parallel - group study , 523 patients ( without exacerbations in the year prior to study entry ) were randomized to either fixed - dose indacaterol / glycopyrronium ( 110/50 g once daily ) or sfc ( 50/500 g twice daily ) . the primary outcome was the standardized area under the fev1 curve from 012 hours post - dose ( fev1auc012 ) at week 26 . thirty - five percent of the enrolled patients were treated with ics at baseline , and the mean post - bronchodilator fev1 was 60.2% pred ( table 1 ) . the fev1auc012 was significantly higher for patients treated with fixed - dose indacaterol / glycopyrronium ( 1.70 l ) than for patients treated with sfc ( 1.56 l ) , with a treatment difference of 0.14 l ( p<0.0001 ) at week 26 ; this treatment difference in fev1auc012 was observed throughout the study period . similar findings were reported for other spirometric parameters , including peak fev1 and forced vital capacity.18 after 6 weeks of treatment in the blaze study,20 a significant and clinically meaningful25 improvement in mean fev1 was seen at every time point from 45 minutes pre - dose to 4 hours post - dose versus both placebo and tiotropium ( p<0.001 ) . the enlighten study17 showed a significant improvement in pre - dose fev1 at week 52 compared with placebo ( treatment difference 0.189 l ; p<0.001 ) , and the treatment difference in pre - dose fev1 from week 3 to week 52 versus placebo was in the range of 0.1520.189 l ( p<0.001 ) . furthermore , the 60-minute post - dose fev1 was significantly higher for patients treated with fixed - dose indacaterol / glycopyrronium compared with placebo ( treatment difference 0.2000.286 l ; p<0.001 ) . trough fev1 in the spark study23 was significantly higher for fixed - dose indacaterol / glycopyrronium at all evaluated time points compared with glycopyrronium ( treatment difference 0.080.09 l ; p<0.0001 ) and tiotropium ( treatment difference 0.060.08 l ; p<0.0001 ) . in the bright study , beeh et al26 investigated the effect of fixed - dose indacaterol / glycopyrronium compared with placebo and tiotropium on exercise tolerance in patients with moderate - to - severe copd ( defined according to the gold 2007 spirometric criteria ; mean fev1 55.9% pred ) ( table 1 ) . a total of 85 patients were randomized to fixed - dose indacaterol / glycopyrronium ( 110/50 g ) , placebo , or tiotropium ( 18 g ) once daily in a blinded , three - period , crossover study for 3 weeks . the primary outcome variable was exercise endurance time at day 21 for fixed - dose indacaterol / glycopyrronium versus placebo . fixed - dose indacaterol / glycopyrronium significantly improved exercise endurance time at day 21 compared with placebo ( lsm treatment difference 60 seconds ) . a similar improvement in exercise endurance time was observed with tiotropium compared with placebo ( lsm treatment difference 66 seconds ) . in line with this , an analysis of data for the subgroup of patients with hyperinflation ( defined as functional residual capacity [ frc ] > 120% pred ) , showed that the mean changes in exercise endurance time from baseline were 85 seconds and 88 seconds , respectively , for indacaterol / glycopyrronium and tiotropium . a significant improvement in ic at peak exercise was observed for fixed - dose indacaterol / glycopyrronium compared with both placebo and tiotropium , and a similar difference was observed for trough ic . in the study by van noord et al,22 changes in ic , although not stated as a secondary outcome variable , were also determined . the authors reported that both lsm ic and trough ic were significantly better for fixed - dose indacaterol / glycopyrronium compared with both indacaterol and placebo ( p=0.02 ) . wedzicha et al , in the spark study,23 investigated the effect of fixed - dose indacaterol / glycopyrronium on exacerbations in patients with severe and very severe copd ( defined according to the gold 2007 spirometric criteria ) . in a parallel - group study , 2,224 patients ( having had at least one exacerbation of copd in the year prior to enrollment ) were randomized to fixed - dose indacaterol / glycopyrronium ( 110/50 g ) , glycopyrronium ( 50 g ) , or open - label tiotropium ( 18 g ) for 64 weeks . the primary outcome variable was to demonstrate superiority of fixed - dose indacaterol / glycopyrronium for the rate of moderate ( defined as worsening of symptoms treated with systemic corticosteroids or antibiotics or both ) and severe ( defined as worsening of symptoms requiring emergency treatment or hospitalization ) exacerbations compared to monotherapy with glycopyrronium . the key secondary outcome variable was to demonstrate superiority of fixed - dose indacaterol / glycopyrronium compared with tiotropium with regard to the rate of moderate and severe exacerbations . twenty - two percent of the patients included in the efficacy analyses had had two or more exacerbations in the previous year , and 88% of the patients had one or more cardiovascular risk factors at baseline , although few patients had a history of cardiovascular disease . furthermore , 75% of the patients in all three treatment arms were treated with icss throughout the study period . the rate of moderate or severe exacerbations was significantly reduced by 12% for the fixed - dose indacaterol / glycopyrronium group compared with the glycopyrronium group ( p=0.038 ) , whereas the 10% reduction in the rate of moderate or severe exacerbations with fixed - dose indacaterol / glycopyrronium compared with tiotropium was nonsignificant . the overall rate of exacerbations ( mild , moderate , and severe ) was significantly reduced with fixed - dose indacaterol / glycopyrronium , by 15% and 14% , respectively , compared with glycopyrronium and tiotropium . the combination of two long - acting bronchodilators with different mechanisms of action is likely to have the potential to enhance efficacy compared with single long - acting bronchodilators , without a concomitant increase in adverse effects . fixed - dose indacaterol / glycopyrronium is , based on the available evidence , a safe and well - tolerated dual long - acting bronchodilator . in patients with moderate - to - very severe copd ( defined by gold spirometric criteria ) , fixed - dose indacaterol / glycopyrronium has clinically important effects on symptoms , including dyspnea score , health status , level of fev1 , exercise endurance time , and rate of exacerbations . bronchodilation improves airway conductance and airflow and reduces hyperinflation , which subsequently leads to a reduction in dyspnea . sustained bronchodilation is therefore , in accordance with the current gold strategy document,1 recommended as maintenance therapy for patients with symptomatic copd . the published studies show that treatment with once - daily fixed - dose indacaterol / glycopyrronium leads to clinically important improvements , not only in lung function , but also in other important endpoints such as dyspnea index , health status , symptoms , exercise endurance time , and use of rescue medication , although the findings with regard to use of rescue medication were not absolutely consistent . the studies of fixed - dose indacaterol / glycopyrronium are therefore in accordance with several previously published studies that have investigated the efficacy of free combinations of long - acting 2-agonists and long - acting anti - muscarinic agents2730 in patients with copd . furthermore , these studies are also in accordance with a very recently published study by celli et al31 investigating the safety and efficacy of once - daily fixed - dose umeclidinium / vilanterol in patients with copd . sustained bronchodilation is also thought to contribute to the reduction in exacerbations of copd seen with treatment with long - acting bronchodilators , and , although long - acting anti - muscarinic agents are primarily thought to reduce exacerbations by reducing dynamic hyperinflation , both long - acting 2-agonists and long - acting anti - muscarinic agents may have anti - inflammatory effects.3234 the studies published so far investigating the safety and , not least , efficacy of fixed - dose indacaterol / glycopyrronium support the gold 2013 strategy1 alternative - choice recommendation that add - on of a second long - acting bronchodilator in patients with moderate - to - very severe copd ( gold 2013 groups b d ) may improve symptom control , and , in line with this , that patients classified as being ( group b ) , who remain symptomatic on a single long - acting bronchodilator , may significantly benefit from fixed - dose indacaterol / glycopyrronium . furthermore , the reported findings also support the gold 2013 strategy secondary choice recommendation for high - risk patients ( groups c and d based on symptoms and exacerbations ) , as a long - acting 2-agonist plus a long - acting anti - muscarinic agent is recommended as an alternative to a long - acting -agonist plus ics ( group c ) or ics plus long - acting 2-agonist and/or long - acting anti - muscarinic agent ( group d ) . the published studies indicate that once - daily fixed - dose indacaterol / glycopyrronium may become an important therapeutic maintenance option for patients with moderate - to - very severe copd . in years to come , combination therapy with a long - acting antimuscarinic agent and a long - acting 2-agonist , preferably as fixed combination , may become the treatment of choice for maintenance therapy - nave patients with symptomatic copd .

background and aimlong - acting bronchodilators are the preferred option for maintenance therapy of patients with chronic obstructive pulmonary disease ( copd ) . the aim of this review is to provide an overview of the clinical studies evaluating the clinical efficacy of the once - daily fixed - dose dual bronchodilator combination of indacaterol and glycopyrronium bromide in patients suffering from copd.methodsthis study comprised a systematic review of randomized controlled trials identified through systematic searches of different databases of published trials.resultsnine trials ( 6,166 participants ) were included . fixed - dose once - daily indacaterol / glycopyrronium seems to be safe and well tolerated in patients with copd . compared with single therapy with other long - acting bronchodilators ( indacaterol , glycopyrronium , and tiotropium ) and fixed - combination long - acting 2-agonist / inhaled corticosteroid ( salmeterol / fluticasone twice daily ) , once - daily fixed - dose indacaterol / glycopyrronium has clinically important effects on symptoms , including dyspnea score , health status , level of lung function , and rate of moderate or severe exacerbations in patients with moderate - to - very severe copd ( global initiative for chronic obstructive lung disease [ gold ] spirometric criteria ) . furthermore , a very recent study has shown that fixed - dose indacaterol / glycopyrronium improves exercise endurance time compared with placebo , although no significant difference was observed between fixed - dose indacaterol / glycopyrronium and tiotropium.conclusionfixed-dose indacaterol / glycopyrronium has clinically relevant effects on important copd outcome measures and is , in general , superior to therapy with a single long - acting bronchodilator ( with or without inhaled corticosteroid ) indicating long - acting dual bronchodilation as a potential important maintenance therapeutic option for patients with symptomatic copd , possibly also for the treatment of nave patients .

only several cases have been reported in the literature and most of them concerned mucous cysts ( 1 - 3 ) . although many theories have been suggested for the late cyst formation after rhinoplasty , migration or incorporation of mucosal tissue in the subcutaneous space during the surgical procedure has been accepted as an important mechanism ( 4 - 6 ) . careful dissection and meticulous manipulation of tissue and implant are necessary to prevent the mucosa from being grafted with the implant material ( 4 - 6 ) . rarely , a foreign body type cyst resulting from petroleum jelly impregnated with the packing material has been reported . the authors report a case of dorsal nasal cyst that was presumed to have a different pathogenesis . we describe this case with a brief review of the literature with an emphasis on its pathogenesis and treatment . thirty years earlier at a local clinic , she had undergone augmentation rhinoplasty with a material presumed to be silicone . she had had no complication afterwards . however , 5 yr previously , a nasal mass developed that had increased gradually . physical examination showed a 3.52.5 cm round , soft , painless mass on the midline of nasal radix ( fig . the nasal dorsum and the tip were firm on palpation and a small cruciate incision scar was found at the infratip lobule , which was supposed to be the entry of the augmentation material . on computed tomography ( ct ) scan , a heterogeneous cystic mass was observed at the radix and the cyst was continuous with a homogenous density at the nasal dorsum , which was first thought to be a silicone implant ( fig . 2 ) . a presurgical diagnosis of nasal radix cyst associated with previous augmentation rhinoplasty was made , and a direct , open approach was chosen to remove the cyst . after a skin incision on the center of the cyst , it was dissected down to the nasal bone . the cyst was connected to the dorsal mass , which were removed together in en - bloc fashion ( fig . 3a ) . for safe removal of the graft material over the tip area and effective tip - plasty , grossly , the removed cystic mass had a thick , fibrous wall with dirty cheesy material inside . the cystic wall was composed of dense fibrous tissue containing dispersed , microsized amorphous foreign materials ( fig . foreign materials were surrounded by foreign body type multinucleated giant cells and induced granulomatous reactions . a scanning electron microscopy ( sem ) showed a fragment of foreign body ( fig . 4a ) and energy dispersive radiography spectroscopy ( edx ) showed presence of silicone ( si ) in the removed specimen ( fig . the lesion was diagnosed as a foreign - body type cyst associated with silicone material used for the augmentation rhinoplasty . the depressed radix , dorsum , and tip after excision of the mass were reconstructed using auricular and septal cartilages . the redundant and thin skin of the radix was trimmed and reinforced with an underlay of the temporalis fascia . the patient has been followed for 9 months postoperatively with a good aesthetic result and no complication ( fig . exact pathogenesis of the cyst formation in our case is not clear because no information about the material that had been used for dorsal augmentation was available . however , considering the gross and histological findings as well as the results from sem - edx analysis , foreign body inflammatory reaction from silicone material , most possibly a liquid type , which had been injected 30 yr ago , probably caused the cyst . first , sem - edx analysis of the cyst revealed silicone in the cyst , which meant that a type of silicone material was used for dorsal augmentation . second , silicone , whether it is a liquid type or implant type , was and still is the most commonly used material for augmentation rhinoplasty in korea . third , a small , cruciate incision of the infratip lobule was too small to insert any type of hard material including silicone implant . although calcification and inflammatory cell infiltration around a silicone implant have been reported , total degradation is impossible considering the bio - characteristics of the silicone implant ( 7 , 8) . sem - edx used for analyzing the graft material in this case adopted the principles that when the electron generated from the electron microscope collides with the object , a specific radiography characteristic to the object is released from the surface . edx analysis clarifies the object by detecting this radiography , and this technique is widely used in material engineering and archeology . in our case , a small amount of silicone detected in the specimen led us to believe that the foreign body that caused cyst formation was a type of silicone material . silicone implant has been used for rhinoplasty since 1950 and it still remains one of the most widely used implant materials in asian countries , including korea ( 9 - 11 ) . injectable liquid silicone is useful in augmenting the chin , cheek , and glabella area ( 12 ) . it is not recommended for rhinoplasty because the nasal skin is thin and the silicone frequently results in ridging or beading ( 12 ) . serious complications such as severe edema or localized discoloration of the injected area has also been reported ( 13 ) . only one case of nasal dorsal cyst after augmentation rhinoplasty using silicone implant has been reported in the literature but it 's pathologic findings and pathogenesis were not clarified ( 1 ) . the mainstay of treating postrhinoplasty nasal dorsal cyst is complete resection and reconstruction . in this case , a direct open approach using the horizontal incision over the cyst center was used given the location and size of the cyst . to expose the tip , an open approach using separate transcolumellar incision was chosen because a previous cruciate incision was located at the infratip lobule and it was too small to expose the nasal tip . in revision rhinoplasties due to complications of the allograft implant , fortunately , we could augment the depressed dorsum and radix with autogenous septal and auricular cartilages without harvesting the rib cartilage despite the large defect .

development of a cystic mass on the nasal dorsum is a very rare complication of aesthetic rhinoplasty . most reported cases are of mucous cyst and entrapment of the nasal mucosa in the subcutaneous space due to traumatic surgical technique has been suggested as a presumptive pathogenesis . here , we report a case of dorsal nasal cyst that had a different pathogenesis for cyst formation . a 58-yr - old woman developed a large cystic mass on the nasal radix 30 yr after augmentation rhinoplasty with silicone material . the mass was removed via a direct open approach and the pathology findings revealed a foreign body inclusion cyst associated with silicone . successful nasal reconstruction was performed with autologous cartilages . discussion and a brief review of the literature will be focused on the pathophysiology of and treatment options for a postrhinoplasty dorsal cyst .

surgical excision of abnormal parathyroid tissue whether adenoma , carcinoma , or hyperplasia is considered as the definitive treatment of hyperparathyroidism . surgery is the standard of care due to potential long - term skeletal , cardiovascular , and neuropsychiatric effects of even mild hyperparathyroidism . nuclear medicine imaging techniques have played an important role in the detection and localization of parathyroid adenomas , with 99mtc - methoxy - isobutyl - isonitrile ( mibi ) scan currently being the standard test used . the addition of single photon emission computed tomography / computed tomography ( spect / ct ) has had an incremental benefit in exact localization of the adenomas . although f - choline has been used as a photon emission tomography ( pet ) tracer for some years now , its utility in the detection of parathyroid adenomas has only recently been explored . we are currently studying the role of this modality vis - a - vis 99mtc - mibi scan with early spect / ct in patients with primary hyperparathyroidism . a 45-year - old female patient presented with complaints of bone pain and difficulty in walking for last 3 months . a plain radiograph of bilateral hip joints ordered by a local practitioner showed multiple lytic areas in the pelvis and femora . she was referred to a tertiary care institute for a fluorodeoxyglucose ( fdg ) pet / ct , in which multiple poorly fdg avid skeletal lesions were reported with no evidence of primary malignant pathology . the patient was referred to our institution for further management . a review of the fdg pet / ct revealed a rounded lesion measuring 2.2 cm with mild fdg uptake superior to the upper pole of the left lobe of thyroid just inferior to the left submandibular gland lateral to and abutting the left lamina of thyroid cartilage . her renal function was deranged for at least last 1 year , and last serum creatinine was 1.9 mg% . , we ordered a parathyroid hormone ( pth ) assay which showed a value of more than 3000 pg / ml from two different laboratories . her serum calcium was 10.4 mg / dl ( laboratory normal 8.8010.60 ) and serum phosphorus was 3.60 mg / dl ( 2.404.40 ) , and vitamin d levels were consistently in deficient range . we proceeded with a 99mtc - mibi scan and early , delayed and early spect / ct images were taken which revealed high uptake in the above lesion . this was interpreted as an ectopic left superior parathyroid adenoma [ figure 1a and b ] . as part of our ongoing study , she underwent a f - choline pet / ct study post 3 mci intravenous injection of the tracer , which showed intense uptake in the above lesion [ figure 2a and b ] . we found the lesion to thyroid ratio much higher than that seen in the mibi scan , making the images easier to interpret . a diagnosis of ectopic left superior parathyroid adenoma arising in the probable setting of tertiary hyperparathyroidism was made . one month after surgery , the patient reported a significant improvement in her symptoms and pth levels have returned to near normal . ( a ) planar early and delayed images of 99mtc - methoxy - isobutyl - isonitrile scan showing a focus of increased tracer uptake superior to the left lobe of thyroid ( black arrow ) . ( b ) single photon emission computed tomography / computed tomography done after the early image reveals the same lesion ( white arrow ) measuring 2.2 cm 1.5 cm 2.3 cm inferior to the left submandibular gland , closely abutting the left lamina of thyroid cartilage ( a and b ) maximum intensity projection ( posterior view ) and transaxial images of f - choline photon emission tomography / computed tomography image showing the ectopic left superior parathyroid adenoma with intense uptake ( white arrow ) in the era of pet / ct , various tracers have been tried for accurate localization of parathyroid adenomas . however , the efficacy of fdg , fluorodopa , and c - methionine has not been established so far . a search for a pet - based tracer is ongoing that can be as good as mibi scan or better . f - choline has recently been reported as a possible novel pet agent for parathyroid localization preoperatively . the possible mechanism for choline uptake is its conversion into phosphatidylcholine by the enzyme choline kinase and subsequent incorporation into the cell membrane . a recent study by lezaic et al . in 24 patients revealed that the sensitivity and specificity of f - choline pet / ct were 92% and 100% , and sensitivity was much higher than any of the conventional scintigraphic techniques . cazaentre et al . reported incidental uptake of f - choline in parathyroid hyperplasia in a case of prostate cancer . in a study of 12 patients by michaud et al . , the authors found a detection rate of 92% for f - choline pet / ct and it also solved discrepant results between mibi scan and ultrasound . reported a case where f - choline pet / ct enabled a minimally invasive surgery for parathyroid adenoma in a patient with negative ultrasound and mibi scan . no larger studies have been performed in this regard yet . at our institution , we do not have c - choline , and synthesis of f - choline has only recently started . in this particular case , the diagnosis was reliably made on mibi scan , but f - choline pet / ct proved to be as good as mibi scan . furthermore , unlike mibi scan , the choline pet / ct in our case showed a very high lesion to thyroid ratio . thus , it may be easier to report smaller lesions very close to the thyroid with this modality . it appears to provide additional hope specially in cases of low gland mass and multi - gland involvement due to better resolution of pet / ct as compared to spect / ct coupled with good image contrast . we feel that f - choline is , therefore , a promising tracer requiring further investigation .

recently , the role of 18f - choline in the detection of parathyroid adenomas has been reported . at our institution , we are currently studying the role of this tracer in comparison to the standard methoxy - isobutyl - isonitrile.(mibi ) scan with single photon emission tomography / computed tomography . our initial results show that 18f - choline is at least as good as 99mtc - mibi scan . we present here a representative case of a 45-year - old woman with multiple skeletal lytic lesions and a high parathyroid hormone.(pth ) who underwent both these imaging techniques with concordant results , further confirmed by histopathology and postoperative fall in serum pth levels .

stairway falls are a leading cause of injury in patients less than 5 years of age [ 1 , 2 ] . while the majority of these injuries occur when a child steps down the stairs , approximately 3 % of injuries will be associated with a caregiver falling while carrying a child . this specific mechanism has been shown to result in injury patterns that are more likely to necessitate hospitalization and more likely to involve trauma to the head and fractures to the extremities . while it is often necessary for a parent or caregiver to carry children in their arms while walking down stairs , the child may obstruct the caretaker s view on the stairway ( fig . this can lead to a fall by the caretaker and an injury to the child and/or caretaker . to date , several previous studies have been conducted evaluating stairway injuries in children , but none have focused on this specific mechanism ( fall from caregiver s arms ) or the injuries associated with these falls . the goal of this study was to identify injuries that may occur when a caretaker falls while carrying a child on a stairway , to understand the pathomechanics of this injury , and to perform a cost analysis of the injury.fig . d bird s eye view of how the child can obstruct the caretaker s view on the stairway a side view of the caretaker s unobstructed view on the stairway . d bird s eye view of how the child can obstruct the caretaker s view on the stairway emergency department and orthopedic clinic records were reviewed between 2004 and 2012 to identify patients with an orthopedic injury after a fall from stairs . sixteen children were retrospectively found to have a fracture from a fall - in - arms injury sustained while a caregiver was going down stairs , and were included in this study . patient identification occurred at a routine weekly fracture conference , where every emergency department fracture in which the orthopedic service was consulted was presented . if the patient met the inclusion criteria , they were then added to the database . the following demographic and epidemiologic data were recorded for each patient : age , gender , location of injury , and mechanism of fall . additionally , radiographs were reviewed to assess fracture location , type , displacement , and treatment . cost analysis data was obtained from the hospital billing department and included all emergency department care , inpatient care , and subsequent follow - up . this study was granted a waiver of informed consent , including permission and assent , in accordance with 45 cfr 46.116(d ) and 45 cfr 46.408 , and a waiver of hipaa authorization per 45 cfr 164.512(i ) . the study was authorized by the local ethical committee and was performed in accordance with the ethical standards of the 1964 declaration of helsinki as revised in 2000 . sixteen children presented to the emergency room and orthopedic clinic of our hospital after sustaining an injury when their caretaker fell while carrying them down the stairs . our billing records reveal that we , as an institution , treat approximately 9,500 fractures per year , giving an incidence of approximately 1 fracture by this mechanism per 5,000 fractures . interviews with the parents yielded information regarding the specifics of the fall and the possible pathomechanics of the child s injuries . the parent or caregiver noted in all cases that the child was being held in front as they descended the stairs . the child obscured their view and they missed a step and fell ( fig . the age at the time of injury averaged 14 months and ranged from 7 months to 51 months of age . eight ( 50 % ) sustained femur fractures , six ( 38 % ) sustained tibia fractures , and one ( 6 % ) sustained a metatarsal fracture . there was one both bone forearm fracture ( 6 % ) ; this occurred in the eldest child ( 51 months ) . four of these patients , however , required a reduction or manipulation , three of which were performed in the operating room . functionally , all patients did well , with no deficits noted at final follow - up . cost analysis was performed amongst our 16 patients to determine the financial burden accrued in relation to these accidental traumas ( table 1 ) . the average total charge of the treatment of the fractures was $ 6,785 ( standard deviation $ 11,183 ; range $ 948$45,876 ) . five of the 16 children ( 31 % ) had a skeletal survey , as the treating physicians were concerned about possible child abuse ; the average total charge for those receiving a skeletal survey was $ 7,024 . after a social service consultation and a skeletal survey were obtained in these cases , no patient was felt to be the victim of child abuse . for those children not requiring a skeletal survey , three children ( 19 % ) required a closed reduction of their femur fractures in the operating room ; their total care charges averaged $ 23,568 . for the children not treated in the operating room , the average charge was $ 2,912.table 1cost of fall - associated accidental traumasage ( months)sexfractured bone(s)costskeletal survey18mtibia / fibula$2,607no7ftibia$2,024no12ffemur$4,276yes10mfemur$7,861yes12mfemur$9,080no16ftibia$2,801no10mmetatarsal$1,328no17mfemur$19,748yes12ftibia / fibula$2,594no7ffemur$4,231yes51fulna$3,907no15.6 ( avg . age)$5,496 ( avg . injuries incurred on stairs , particularly in patients under the age of five , can occur with relative frequency . three previous studies have been conducted that have evaluated stairway injuries in children [ 3 , 4 , 6 ] . included in these studies were primarily children who fell while walking on the stairs , and none focused on the variable of a caretaker falling while carrying a child . it was noted , however , in these studies that children who sustained injuries while being carried tended to have more severe injuries than those who fell while walking themselves down stairs . in our study , all patients identified had incurred a fracture , and nearly all of these involved the lower extremity . of the 16 children , half ( 50 % ) sustained femur fractures and 38 % sustained tibia fractures . these results show that the pattern of injury differs from that of a child who falls while walking down the stairs . however , a child who is dropped , or fallen upon , while being carried appears more likely to sustain a long bone fracture to the lower extremity . pierce et al . have the only series currently published that examines the incidence of femur fractures and falls down stairs . their series evaluated femur fractures that were due to a reported fall down stairs ( either solo or in a caretaker s arms ) , and evaluated a plausibility model to check whether they could identify cases of nonaccidental trauma . in their subgroup of caretaker falls , this mechanism most commonly caused buckle fractures , followed by transverse / short oblique fractures . they examined the energy absorbed by the patients during falls and noted that the greater number of stairs in the fall correlated to the fracture pattern type , noting that spiral fractures were associated with falls from 1 to 3 steps and buckle fractures with falls from 4 to 15 steps . in our study , we saw an even distribution of spiral and buckle fractures that may be associated with the variability in fall heights that were observed in our patient population . pierce et al . also employed a plausibility model to help identify if certain aspects taken from the history could be used as independent identifiers of child abuse . they found that if a caregiver could not give specific details about the fall dynamics as well as the position of the child before and after the fall , the child may have been a victim of abuse . although this model has not been validated , and was not employed here , it may provide guidance to treating physicians and can reduce costs and avoid exposing patients to unnecessary radiation from a skeletal survey . in our series , all caregivers were able to provide a detailed history regarding the nature of their child s injury , and subsequent non - accidental trauma work - ups suggested no cases of abuse . additionally , no child in our study had a concomitant injury other than the fracture . this is consistent with other studies , where abused children tended to have other injuries such as bruising as well head and trunk injuries . the total charge for the children who received a skeletal survey as part of their work - up was $ 7,024 . it is the duty of treating physicians whether they are primary care physicians , emergency room physicians , or orthopedists to be vigilant in the work - up of suspected nonaccidental trauma , and the skeletal survey is frequently the first test ordered following a history and physical . with that said , in cases where the caregiver can provide a clear history , the skeletal survey can be deferred to minimize costs and radiation exposure to the child . three children in our series had to go to the operating room for a surgical reduction . all of these were for diaphyseal femur fractures that were treated with a spica cast . the costs related to the treatment of these femur fractures dwarf the costs of the other patients in this series . when taken as two separate groups , the average charge for femur fracture treatment in the emergency room was $ 2,912 , compared to $ 23,568 for treatment in the operating room . this study does have limitations and is biased toward orthopedic injuries , as all of the cases reported were obtained through orthopedic emergency room consults and clinic visits . isolated injuries to the head or torso would not involve an orthopedic consult and thus were not included in this group . this most likely underestimates the total number of children seen at our hospital due to a fall down stairs while being carried by a caregiver . additionally , the number of children with relatively minor injuries who did not seek medical treatment further underestimates the true incidence of this mechanism of injury . this paper describes the type of orthopedic injuries sustained during a fall down stairs while in a caregiver s arms . what our study adds to the literature is that nearly one - third of the children underwent a skeletal survey due to concerns regarding child abuse . additionally , the average cost of these injuries was not insignificant and averaged $ 6,785 . thirdly , we believe that the pathomechanics of this injury are as follows : the caregiver descends the stairs carrying a child in front.the size of the child obscures the view of the subsequent stair ( fig . 1a d).the caregiver misses the step and falls.the momentum usually causes the caregiver and child to fall forward down the stairs ( fig . 2).fig . 2they miss the step and fall , often landing on the child as they fall the caregiver descends the stairs carrying a child in front . the size of the child obscures the view of the subsequent stair ( fig . the momentum usually causes the caregiver and child to fall forward down the stairs ( fig . 2they miss the step and fall , often landing on the child as they fall they miss the step and fall , often landing on the child as they fall with a better understanding of this mechanism , the treating er physician can avoid routine skeletal surveys when the parent describes a plausible sequence , as we note above . finally , with awareness of this mechanism , prevention of these injuries may be possible . our recommendations are for the caregiver to hold on to the handrail while traversing the steps . in addition to using the handrail , the caretaker s view of the stairs should be unobstructed ( fig . 3 ) . this can be accomplished by positioning the child on the opposite side of the body to the handrail . we hope that these recommendations serve to remind healthcare providers as to the dangers of carrying children on stairways . at times this may be necessary , and can be performed safely if the proper precautions are taken.fig . in addition to using the handrail , the caretaker s view of the stairs should be unobstructed the caregiver should hold on to the handrail while traversing the steps . in addition to using the handrail , the caretaker s view of the stairs should be unobstructed in conclusion , a fall in a caregiver s arms while going down stairs can result in multiple orthopedic injuries , particularly to the lower extremity . the costs of treating these injuries are not insignificant , averaging nearly $ 7,000 , and the suspicion of child abuse can be both costly and unnecessary in the case of a true accident . while descending the stairs with a child in their arms , the caregiver should hold the child to the side so as not to obscure their vision of the step with one arm , ideally holding the handrail with the other .

backgroundthe purpose of this study was to describe fractures sustained by children and to analyze the associated costs when a caretaker falls down stairs while holding a child.materials and methodsbetween 2004 and 2012 , 16 children who sustained a fracture after a fall down stairs while being carried by a caregiver were identified . parents / caregivers were interviewed to see how the fall occurred , and a cost analysis was performed.resultsthe average age of the patients was 14.5 months ( 751 months ) . the lower extremity was involved in 15 of 16 fractures , with 8 involving the femur . the majority were buckle fractures , but all diaphyseal femur fractures were spiral . three patients required a reduction in the operating room . all fractures healed with cast immobilization . five patients underwent skeletal surveys , as the treating physicians were concerned about potential child abuse . the average cost of treatment was $ 6785 ( range $ 94845,876 ) . detailed histories from the caregivers showed that they missed a step due to the child being carried in front of the caregiver , obscuring their vision.conclusionsa fall in a caregiver s arms while going down stairs can result in multiple orthopedic injuries . the costs of treating these injuries are not insignificant , and the suspicion of child abuse can be both costly and unnecessary in the case of a true accident . while descending the stairs with a child in their arms , the caregiver should hold the child to the side so as not to obscure their vision of the step with one arm , ideally holding the handrail with the other.level of evidenceiv case series .

ubiquitination is a process in which one or multiple ubiquitin moieties are covalently attached to a substrate through an enzymatic cascade involving ubiquitin - activating enzyme ( e1 ) , ubiquitin - carrier protein ( e2 ) , and ubiquitin - protein ligase ( e3 ) . formation of a ubiquitin lys48 chain on the -nh2 group of a substrate 's internal lys residue ( polyubiquitination ) can target the substrate for degradation by the 26s proteasome . ubiquitin can also be attached to the free -nh2 group in a substrate 's n - terminus to promote proteasomal degradation . for example , polyubiquitination at lys63 is involved in inhibitor of nf-b ( ib ) kinase ( ikk ) activation . in addition , a linear polyubiquitin chain can be achieved by conjugating the c - terminal glycine of ubiquitin and the a - nh2 group of the n - terminal methionine of its neighbor ubiquitin . substrates can also undergo monoubiquitination or multi - monoubiquitination adding one ubiquitin to one or multiple lys residues , respectively . recent evidence suggests that ubiquitin can be linked to cys , ser , or thr residues in a substrate through thio- or oxy - ester bonds ( i.e. , esterification ) , though the physiological relevance of these modifications remains to be defined. ubiquitin moieties can be released from a substrate by deubiquitinating enzymes . for an organism to function properly , proteins must be degraded after they undergo specific functions . moreover , proteins that are misfolded or damaged during translation , folding , or translocation must be degraded and eliminated in time . either blocked degradation of oncogenic proteins / growth - enhancing factors or accelerated degradation of growth - suppressing proteins may disrupt the pathways controlling cell cycle progression , cell death , or survival , leading to cancer development , ( table 1 ) . for example , the tumor suppressor cyld is mutated in several cancers , including cylindromatosis . the deubiquitinating activity of cyld for ikk is critical for its cylindromatosis - suppressive function . the ubiquitin ligase itch promotes the polyubiquitination and degradation of large tumor suppressor 1 ( ltsa1 ) , which is closely related to enhanced cell growth and epithelial - to - mesenchymal transition . malt , mucosa - associated lymphoid tissue ; aml , acute myeloid leukemia . due to the critical roles of ubiquitination and the ubiquitin - mediated proteolysis in tumorigenesis and cell growth , it is a dipeptide boronate that specifically and reversibly blocks chymotrypsin - like activity of the proteasome in a variety of cancer cells . although bortezomib inhibits nf-b activation and results in autophagy , this lethal effect of proteasome inhibition is probably due to loss of amino acid homeostasis . notably , bortezomib has been used successfully as an anticancer drug for multiple myeloma and mantle cell lymphoma in the clinic,. because ubiquitination is generally substrate - specific , the components of the ubiquitination pathway might be more specific drug targets for cancer therapy than the proteasome . cullin - ring ubiquitin ligases ( crls ) are involved in cellular processes such as cell cycle progression , cell death signaling , dna damage , and stress responses . nedd8 is a ubiquitin - like protein that modifies cullin and is required for the activity of crls . because nedd8-activating enzyme ( nae ) catalyzes the first step in the nedd8 pathway , targeting crls via inhibition of nae may be a promising anticancer strategy . indeed , mln4924 , a selective inhibitor of nae , has potent tumor - suppressing activity in a wide range of tumors , including acute myeloid leukemia and diffuse large b cell lymphomas , . apoptosis ( i.e. , programmed cell death ) is a cellular suicide process that is important for embryonic development and maintaining the size of cell populations . the extrinsic pathway involves members of the tumor necrosis factor ( tnf ) receptor gene superfamily , which bind extracellular ligands and transduce intracellular signals during cell destruction . the intrinsic pathway does not involve receptor - mediated intracellular signaling , but induces signaling in mitochondria . in mammals , the intrinsic pathway is regulated by the bcl-2 family of proteins , the adaptor protein apoptotic protease - activating factor-1 ( apaf-1 ) , and the caspases . bcl-2 family members include both anti - apoptotic ( bcl-2 , bcl - xl , bcl - w , and mcl-1 ) and pro - apoptotic proteins ( bax , bak , bad , bid , and bim ) . the release of cytochrome c from mitochondria causes the formation of the apoptosome ( apaf-1/caspase-9 complex ) , activates the downstream effector caspases , and finally results in cleavage of crucial substrates . degradation of anti - apoptotic members is necessary for apoptotic progression , whereas degradation of pro - apoptotic members is required for the suppression of apoptosis , . the levels of anti- and pro - apoptotic molecules can be regulated by ubiquitination and proteasomal degradation . for example , trim17-mediated ubiquitination and subsequent degradation of mcl-1 , an anti - apoptotic bcl-2 family member , triggers neuronal apoptosis . the pro - apoptotic bcl-2 member bax can be regulated by ubiquitination indirectly ; the ubiquitin ligase trim39 inhibits apc / c cdh1-mediated ubiquitination and degradation of the bax activator moap-1 , thus enhancing bax activation and apoptosis . moreover , the levels of bcl2l10/bclb , an anti - apoptotic bcl2-like protein , are inversely correlated with survival in patients with several cancer types , including lung adenocarcinomas . the inhibitors of apoptosis proteins ( iaps ) have one to three baculovirus iap repeat ( bir ) domains and can block apoptosis by directly binding and inhibiting caspases , . furthermore , almost all iaps have ubiquitin ligase activity , which is required for the ubiquitination of certain substrates involved in apoptosis . x - linked inhibitor of apoptosis protein ( xiap ) catalyzes the ubiquitination and degradation of caspase-3 , . apoptosis inducing factor ( aif ) is also a substrate of xiap , and ubiquitination at k255 of aif shows a non - degradable role of ubiquitination in caspase - independent cell death . on the other hand , for example , ubiquitin - specific protease 19 ( usp19 ) is responsible for the inhibition of tnf--induced caspase activation and apoptosis in a ciap - dependent manner . the activity of iaps can be suppressed by pro - apoptotic factors , such as second mitochondria - derived activator of caspase ( smac ) . bruce / apollon is a large ( 528 kda ) , membrane - associated , essential iap in mammals . furthermore , bruce / apollon can be degraded in a ubiquitin - dependent manner by the ubiquitin ligase nrdp1 during apoptosis . the tumor suppressor p53 maintains the integrity of the genome and regulates cell cycle , dna repair , and apoptosis . p53 promotes the activation of the pro - apoptotic bcl-2 family proteins and the release of cytochrome c. dysregulation of p53 is reported in numerous types of cancer . several ubiquitin ligases , including mdm2 , have been reported to promote ubiquitination and degradation of p53 , while p53 is deubiquitinated and stabilized by ubiquitin - specific proteases ( usps ) . thus , inhibiting the activity of p53 ubiquitin ligases or activating p53 usps can be a strategy for cancer therapy . hausp regulates the activities of mdm2 and p53 by deubiquitination , while vif1 and vif2 antagonize hausp and promote p53-dependent apoptosis . translationally controlled tumor protein ( tctp ) , which is down - regulated in tumor progression , inhibits mdm2 autoubiquitination and promotes mdm2-mediated ubiquitination and degradation of p53 . in addition , fanconi anemia complementation group f ( fancf ) monoubiquitinates fancd2 , which is involved in the fa / brca dna damage response pathway . silencing fancf elevates p53 activation in mitoxantrone - treated breast cancer cells . as a transcription factor involved in the extrinsic apoptosis pathway , nf-b activates the expression of genes that contribute to cell proliferation , metastasis , and suppression of apoptosis . sharpin , a ubiquitin - binding and ubiquitin - like - domain - containing protein , promotes linear ubiquitination of nemo / ikbkg , an adaptor of ikks , and subsequent activation of nf-b signaling . ib , which inactivates nf-b under normal physiological conditions , can be phosphorylated by activated ikk , ubiquitinated by scf , and finally degraded by the proteasome in response to dna damage , . nrdp1 promotes ubiquitination and degradation of the epidermal growth factor receptor family member erbb3 , which is upstream of nf-b activation , . in a word , ubiquitination plays an important role in the regulation of apoptosis , and the components involved in the ubiquitination of key substrates can be potential targets for cancer therapy ( figure 1 ) . autophagy , once categorized as programmed cell death type ii , is a cellular process by which intracellular proteins , lipids , and organelles are degraded in the lysosomal compartment after delivery from other cellular compartments . there are three types of autophagy : macroautophagy , microautophagy , and chaperone - mediated autophagy . although autophagy is generally thought to be non - selective , certain ubiquitinated proteins ( e.g. , catalase ) , organelles ( e.g. , peroxisomes and mitochondria ) , and invading bacteria have been shown to be selectively targeted for autophagic degradation . , 18 autophagy - related proteins ( atgs ) in yeast , namely atg110 , atg1214 , atg1618 , atg29 , and atg31 , have been found to play a role in autophagosome formation . atg8 , called lc3 in mammals , is a ubiquitin - like protein present on autophagic membranes as a phosphatidylethanolamine ( pe)conjugate . p62/sqstm1 or nbr1 binds both ubiquitin and lc3 , probably providing a selective link between ubiquitinated substrates and autophagy . nuclear dot protein 52 ( ndp52 ) , an autophagy receptor , targets intracellular ubiquitinated bacterial proteins for autophagic degradation . misfolded polypeptides are usually recognized by molecular chaperones and degraded by the proteasome following polyubiquitination by ubiquitin ligases , such as chip and parkin . however , when misfolded proteins can not be sufficiently removed by chaperone - mediated proteasomal degradation , protein aggregation occurs and may in turn inactivate the proteasome , resulting in cytotoxicity . thus , p62/nbr1-mediated autophagic degradation may serve as an important compensatory mechanism for degradation of these ubiquitinated protein aggregates . the crosstalk between autophagy and apoptosis is necessary for controlling the balance between cell survival and death . beclin 1 , a mammalian atg6 ortholog , is a subunit of the class iii pi3-kinase complex . beclin 1 interacts with bcl-2 via the bh3 domain in beclin 1 but can be released in starvation conditions to activate autophagy . this interaction can be terminated through c - jun n - terminal kinase ( jnk)mediated phosphorylation of bcl-2 and tnf receptor - associated factor 6 ( traf6)mediated ubiquitination of beclin 1 , . under extreme conditions that can not be rescued by autophagy , jnk promotes hyperphosphorylation of bcl-2 , resulting in the release of bax to execute apoptosis . caspase - mediated cleavage of beclin 1 inhibits beclin 1induced autophagy , and the cleavage product , the c - terminal region ( ct ) , enhances apoptosis by promoting the release of pro - apoptotic factors from mitochondria . beclin 1 can also indirectly affect the crosstalk between apoptosis and autophagy by controlling the levels of p53 , a tumor suppressor that promotes apoptosis under genotoxic stress , . furthermore , p53 can down - regulate lc3 levels in starved cells , preventing the autophagy burst that may be dangerous for cells . under normal conditions , p53 is kept at low levels by the ubiquitin ligase mdm2 . however , p53 levels can also be controlled by beclin 1 via regulating the deubiquitinating activity of usp10 and usp13 . autophagy can target ubiquitinated misfolded proteins , caspases , and other cargo ( such as damaged mitochondria and invading bacteria ) for degradation , probably through p62 and nbr1 . apoptosis and autophagy are counter - regulated in multiple steps , such as at p53 , the beclin 1/bcl-2 interaction , the cleavage of beclin 1 into the c - terminal region by caspases , and the autophagic degradation of caspases . ubiquitination can promote degradation of both p53 and beclin 1 and thus , controls the mutual regulation of apoptosis and autophagy . ubiquitination can occur on not only the -nh2 group of an internal lys residue , but also the -nh2 group of the n - terminal residue of a substrate . moreover , recent evidence suggests that ubiquitin can be attached to cys , ser , or thr residue on a substrate by esterification . these non - lys ubiquitinations might provide another layer of the regulation of protein functions , and further studies should focus on the identification of relevant substrates and physiological roles of these modifications . future studies should also further explore how the ubiquitination of the critical proteins is involved in tumorigenesis and cancer therapy . recent research efforts on cancer stem cells and personalized cancer genome sequencing are expected to help in this regard . because the cytoprotection of autophagy and the evasion of apoptosis contribute to resistance to cancer therapy , it is important to unravel how these two pathways are mutually regulated . the investigation on this issue has just begun and deserves more attention , especially with regard to how ubiquitination is involved in the counter - regulation of these critical processes .

ubiquitination is crucial for cellular processes , such as protein degradation , apoptosis , autophagy , and cell cycle progression . dysregulation of the ubiquitination network accounts for the development of numerous diseases , including cancer . thus , targeting ubiquitination is a promising strategy in cancer therapy . both apoptosis and autophagy are involved in tumorigenesis and response to cancer therapy . although both are categorized as types of cell death , autophagy is generally considered to have protective functions , including protecting cells from apoptosis under certain cellular stress conditions . this review highlights recent advances in understanding the regulation of apoptosis and autophagy by ubiquitination .

male sprague - dawley rats ( n = 24 ) weighing approximately 250 g were used for the study . permission for the experimental work was obtained from the institutional animal ethics committee ( 767/iaec/13 dated 1 - 1 - 2014 ) . rats were divided into 4 equal groups and injected physiological saline ( twice daily subcutaneously in morning and evening ; group i ) , morphine sulfate ( 10 mg / kg twice daily subcutaneously ; group ii ) , fosaprepitant ( 30 mg / kg once by intraperitoneal route ; group iii ) , and finally , the last group was co - administered both morphine and fosaprepitant ( group iv ) in the same doses as in group ii and iii , respectively , for a week . in the last group , fosaprepitant was injected 30 min before the morphine injection . the dose of fosaprepitant ( 30 mg / kg ) was higher than that used in an earlier study ( 25 mg / kg ) . the rationale for selecting a higher dose was the rapid conversion of fosaprepitant to aprepitant ( half - life ~30 min ) in the rat compared to dogs and humans . hot plate latency or the thermal escape behaviour was determined in the morning , 40 min after saline ( group i ) , fosaprepitant ( group iii ) , or morphine injection ( group ii and iv ) . the hot plate test is commonly employed for screening the putative antinociceptive property of drugs . the advantages of this test are the brief nature of the nociceptive stimulus , which does not produce any tissue damage and that multiple testing can be done in the same animal . the predictability of this test to clinical situations is high for morphine and related opioid substances . testing was done in a quiet room with the ambient temperature between 22c and 25c . on the day of the experiment , the testing platform of the hot plate apparatus ( stoelting , usa ) was set at a constant temperature of 52.5c . the rats were placed on the hot plate , and the behavioral end points were either licking of the hindpaw or jumping . the test was repeated thrice at 57 min intervals and the average of these values was the latency period ( sec ) . a 40 s , cut - off was fixed to prevent damage to the completely analgesic paw after morphine injection . transformation of these values was done by calculating the percent maximum possible effect ( % mpe ) as follows : ( [ drug induced latency basal response time]/[40 s basal response time ] ) 100 on day 8 ( morning ) , rats were anesthetized by pentobarbital injection ( 100 mg / kg intraperitoneal ) . this was followed by intracardiac perfusion with 4% paraformaldehyde solution in 0.1 m phosphate buffer saline . transverse sections of the spinal cord ( 20 m thick ) were obtained in a cryostat and processed for immunohistochemical localization of sp and cgrp using specific antibodies ( anti - sp antibody , abcam , uk ; anti - cgrp antibody , calbiochem , usa ; 1:500 titer ) using the avidin - biotin complex method ( vector labs , usa ) . representative sections ( 3/rat ; systematic random sampling ) were later viewed under a microscope and the images captured . the expression of sp and cgrp in the superficial laminae ( rexed 's laminae i - ii ) of the gray matter was quantitated by image j software ( nih , usa ) . specific expression was obtained after deducting background staining in the white matter ( lateral funiculus ) from the total value obtained from the superficial laminae . some of the cryostat sections were stained with 0.5% cresyl violet for localization of neurons in the dorsal horn . statistical evaluation of data was done by graphpad prism version 5 ( graphpad software , la jolla , san diego , usa ) . values of latency period of the different groups of animals were independently analyzed at each time point by one - way analysis of variance followed by bonferroni multiple comparison test . the various groups of rats had different baseline values , which affected the subsequent comparison of the antinociceptive effect of the drugs [ figure 1a ] . hence , normalization of the data was done by calculating the % mpe [ figure 1b ] . the % mpe values of control and the fosaprepitant - treated groups did not show any statistically significant change during the experiment . morphine injection produced significantly higher antinociceptive effect on day 1 ( p < 0.001 ) and to a lesser extent day 4 ( p < 0.05 ) compared to the control group . the sharp decrease in the antinociceptive effect from day 2 in the morphine treated group indicated the development of tolerance . morphine + fosaprepitant combination delayed the onset of tolerance in comparison to morphine treated group on days 1 ( p < 0.01 ) and 3 ( p < 0.001 ) . compared to control , antinociception was higher between days 1 and 4 ( p < 0.001 ) for this group . ( a ) please note the variability of basal latency period and hence these values were not considered in the experimental results ( b ) % maximum possible effect shows rapid development of tolerance after morphine treatment . however , co - administration of fosaprepitant with morphine increased the antinociceptive effect of morphine ( days 14 ) and also attenuated tolerance ( days 1 and 3 ) . values are represented as mean standard error of mean p < 0.05 , * * p < 0.01 , * * * , ,p < 0.001 cresyl violet staining showed the dorsal horn neurons arranged in various laminae [ figure 2 ] . immunohistochemical study revealed the expression of sp and cgrp over the superficial laminae ( laminae i - ii ) of the dorsal horn [ figure 2 ] . quantitative image analysis revealed increased sp expression in the morphine + fosaprepitant treated group compared to others [ figure 3 ] . statistically significant difference was present between morphine and morphine + fosaprepitant co - treatment groups ( p < 0.001 ) . cresyl violet staining showed the different laminae of the gray matter of the cervical region of the spinal cord ( top panel ) . location of the box showed the area of interest ( laminae i - ii ) . expression of both substance p and calcitonin gene - related peptide was noted over the superficial laminae ( i - ii ) . each group had 6 animals quantitative image analysis of the expression of substance p and calcitonin gene - related peptide ( cgrp ) shows that morphine + fosaprepitant treatment led to higher substance p expression compared to all other groups including the morphine treated group . p the a and c groups of nerve fibers carry pain from the periphery to the spinal cord . the peptidergic subgroup of these nerve fibers contains neuropeptides such as sp and cgrp , which are released into the synaptic cleft following noxious stimulation . these neuropeptides diffuse to the postsynaptic terminal , where they bind to the corresponding receptors . morphine - induced antinociception is linked to decreased release of glutamate , sp , cgrp , etc . , from presynaptic terminals and greater postsynaptic hyperpolarization due to the activation of inwardly rectifying potassium channels . co - administration of fosaprepitant possibly interfered with the binding of sp to the nk1rs expressed by postsynaptic neurons in the spinal cord . the results of this study show that combining morphine with fosaprepitant ( group iv ) delayed the development of morphine tolerance and concurrently increased the antinociceptive effect compared to the control group . as mentioned earlier , this could be due to decreased binding of sp to the nk1r . evidence from electrophysiological experiments indicates that noxious stimuli produce slow and prolonged excitatory potentials in postsynaptic dorsal horn neurons , which are inhibited by nk1r antagonist . since fosaprepitant treatment ( group iii ) alone did not result in an antinociceptive effect , the interaction between morphine and fosaprepitant is likely synergistic in nature . previously , injection of a bifunctional peptide having both -opioid receptor agonist and nk1r antagonist properties relieved pain in neuropathic rats without producing tolerance . in a different study , tumati et al . reported that intrathecal administration of both morphine and a nk1r antagonist reduced subsequent opioid withdrawal - induced hyperalgesia . pharmacokinetic interaction between fosaprepitant / aprepitant and morphine could also contribute to the enhanced antinociceptive effect . hence , morphine concentration in the nervous system could be increased by concurrent administration of these drugs . significant alterations in the expression of sp and cgrp were not observed except in the group treated with morphine + fosaprepitant combination ( group iv ) . this was unexpected because prevailing evidence suggests that nk1r does not regulate the release of sp in the spinal cord . however , contrary to this , nk1r has been recently reported to be crucial for release of sp from dorsal root ganglion neurons . moreover , it has been speculated that there could be sp autoreceptors on presynaptic terminals , which can modulate sp release . but , fosaprepitant treatment alone did not increase sp immunoreactivity in the present work . as noted earlier , morphine inhibits the release of sp under acute conditions but rats chronically treated with morphine in our study did not show a statistically significant increase in sp expression . presumably , both nk1 autoreceptors as well as morphine - induced inhibition of sp release might have contributed to the increased sp expression in group iv [ figure 4 ] . a limitation of this study was that the expression of sp and cgrp was evaluated at the end of the observation period and not between days 1 and 4 , when the antinociceptive effect was maximum . another limitation was the use of a fixed - dose combination of morphine and fosaprepitant . diagrammatic representation of the dorsal horn of the spinal cord ( a ) primary sensory afferents carrying nociceptive sensation ends in the dorsal horn of the spinal cord . ( b ) the presynaptic terminals of the primary sensory afferents contain excess substance p following morphine + fosaprepitant treatment ( red ) . - receptor is present on both the presynaptic and postsynaptic terminals the antinociceptive effect of morphine decreased rapidly following daily administration , indicating development of tolerance . the antinociceptive effect of morphine is likely due to decreased release of pronociceptive neurotransmitters such as glutamate and neuropeptides such as sp from presynaptic nerve terminals which could be lost during tolerance . one of the factors responsible for tolerance could be counter - adapting processes , which maintains the status quo in the spinal cord . similarly , the potentiation of the antinociceptive effect of morphine by fosaprepitant is also lost on continued administration ( day 5 onward ) . identical result was reported with a chimera possessing both opioid agonist and nk1r antagonist properties . moreover , the factors can differ depending upon the specific -opioid receptor agonist used for producing the tolerance . the results indicate that addition of fosaprepitant to morphine can delay morphine tolerance for a limited period of time . to the best of our knowledge , this is the first report on the novel antinociceptive effect of morphine + fosaprepitant combination . further studies are required to further elucidate this novel antinociceptive effect of morphine + fosaprepitant combination .

objectives : opioids such as morphine form the cornerstone in the treatment of moderate to severe pain . however , opioids also produce serious side effects such as tolerance . fosaprepitant is a substance p ( sp ) receptor antagonist , which is used for treating chemotherapy - induced nausea and vomiting . sp is an important neuropeptide mediating transmission of pain at the spinal level . thus , it was hypothesized that combining morphine with fosaprepitant would increase the antinociceptive effect of morphine . the objectives were to evaluate the effect of fosaprepitant on morphine - induced antinociception in rats and to investigate its mechanism of action.methods:sprague-dawley rats were injected with morphine ( 10 mg / kg twice daily ) and/or fosaprepitant ( 30 mg / kg once daily ) for 7 days . pain threshold was assessed by the hot plate test . expression of sp and calcitonin gene - related peptide ( cgrp ) in the spinal cords of these rats was evaluated by immunohistochemistry.results:morphine administration resulted in an antinociceptive effect compared to the control group ( day 1 and to a lesser extent on day 4 ) . the decreased antinociception despite continued morphine treatment indicated development of tolerance . co - administration of fosaprepitant attenuated tolerance to morphine ( days 1 and 3 ) and increased the antinociceptive effect compared to control group ( days 14 ) . expression of sp was increased in the morphine + fosaprepitant group.conclusions:the results show that fosaprepitant attenuates the development of tolerance to morphine and thereby , increases the antinociceptive effect . this is likely linked to decreased release of sp from presynaptic terminals .

collagen is a major connective tissue protein that plays an important role in the extracellular matrix in animals . as such atelocollagen is a type of soluble collagen produced from tropocollagen , the collagen molecule that makes up collagen fibrils , via the elimination of the telopeptide moieties , which are considered to account for most of collagen 's antigenicity [ 1 , 2 ] . thus , atelocollagen is considered to have little immunogenicity , which makes it a safe biomaterial . for example , a minipellet atelocollagen formulation has been demonstrated to sustain the release and maintain stable blood concentrations of protein drugs for more than 1 week . many kinds of protein drugs such as interferon- , interleukin-2 , nerve growth factor , and bone morphogenetic protein , and so forth , have been administered using this drug delivery system , and interferon- and interleukin-2 showed strong antitumor activities in animal models when administered in this manner [ 3 , 4 ] . in the past decade , as well as being used as a solid substrate , dissolved atelocollagen has been used as a drug delivery vehicle for nucleic acid - based medicines for gene conversion , inflammatory disease [ 8 , 9 ] , and tumor therapy . atelocollagen can be used to deliver most kinds of nucleic acid - based medicines including plasmid dna , antisense oligodeoxynucleotides ( odn ) [ 1113 ] , short interference rna ( sirna ) [ 1420 ] , and micro rna ( mirna ) [ 2123 ] . it is also capable of delivering oligonucleotides to subcutaneous xenografts and metastatic tumors after its local and/or systemic administration . many studies , including some involving in vivo tumor models , have evidenced the contribution of atelocollagen to the enhancement of drugs ' antitumor activities , and some of them described the mechanisms . for example , nucleic acids delivered by atelocollagen are protected against degradation by host nucleases [ 8 , 14 , 24 ] , and it has also been shown to improve the delivery efficiency of oligonucleotides to tumors [ 15 , 16 ] . however , the biological functions of atelocollagen and the mechanism by which it enhances delivery efficiency are still not fully understood . it is essential to reveal the biological characteristics of atelocollagen in order to be able to fully exploit its drug delivery potential . while we were studying the basic properties of atelocollagen , we discovered another of its functions : it increases endothelial permeability . here , we describe the results of a study of the effects of atelocollagen on intercellular sealing function . we measured transendothelial electrical residence ( ter ) in order to estimate intercellular barrier function and performed an immunohistochemical examination to see whether any cellular morphological changes were induced . rhodamine red - conjugated atelocollagen was prepared in accordance with the manufacturer 's instructions ( fluoreporter rhodamine red - x protein labeling kit ; life technologies japan , tokyo , japan ) . the oligodeoxynucleotides ( odn ) and double stranded rna ( dsrna ) were synthesized by eurogentec ( seraing , belgium ) . the sequences of the oligonucleotides are listed in table 1 [ 20 , 25 , 26 ] . each atelocollagen - oligonucleotide formulation ( ac formulation ) was prepared by gently mixing aqueous atelocollagen with a solution containing a defined concentration of oligonucleotides . the final oligonucleotide concentration was usually 5 m and that of atelocollagen was 0.1% w / v unless otherwise stated in the text , tables , and/or figures . normal human dermal microvascular endothelial cells ( hmvec ) were purchased from eidia ( tokyo , japan ) . these cells were cultured in egm ( endothelial growth media ; eidia , tokyo , japan ) until they reached confluence on 12 mm transwell filters with a 0.4 m pore size ( corning glass works ; corning japan , tokyo , japan ) coated with rat tail collagen . porcine brain microvascular endothelial cells ( bmvec ) were purified and maintained according to the method described in a previous study . ter was determined using an evom voltohmmeter and an endohm-12 chamber ( world precision instruments , sarasota , fl ) at 37c . cell growth was monitored by measuring ter . once stable intercellular seals had formed ; that is , at confluence , the medium in the inner chamber was exchanged for 400 microliters of culture medium containing 30% v / v of ac formulation . one hour after treatment with the ac formulation , which was performed as described in section 2.2 , 100 l of 0.36% w / v texas red - conjugated dextran ( mw : 40 kda ; life technologies japan , tokyo , japan ) were added to the inner chamber . one hour later , the dextran concentration of the medium in the outer chamber was analyzed by measuring its fluorescence . to investigate the enhancement of paracellular transport by atelocollagen , solute transportation was compared among the ac formulation , bovine serum albumin , and dextran . once stable intercellular seals had formed , the medium in the inner chamber was exchanged for 400 microliters of culture medium containing 30% v / v of the ac formulation , which had been produced using 0.1 or 0.3% w / v rhodamine red - conjugated atelocollagen ( i.e. , approximately 0.03 or 0.1% w / v atelocollagen was added ; molecular weight ( mw ) : 300 kda ) ; 0.1% w / v of fluorescein conjugated dextran ( mw : 70 kda ; life technologies japan , tokyo , japan ) ; 0.1% w / v of alexa fluor 594 conjugated bovine serum albumin ( bsa ; mw : 66 kda ; life technologies japan , tokyo , japan ) . the solute concentrations of the outer chambers were analyzed by measuring their fluorescence at 1 and 2 hours after the medium exchange . after treatment for 1 hr with the ac formulation , oligonucleotide alone , atelocollagen alone , or phosphate - buffered saline ( pbs ) as a control , hmvec cells were fixed with 1% paraformaldehyde for 10 min and then treated with 0.2% triton x-100 for 10 min . after preincubation with 5% skimmed milk , they were incubated for 1 hr at room temperature with rabbit or mouse antibodies against vascular endothelial ( ve)-cadherin ( bd biosciences , san diego , ca ) , zonula occludens-1 ( zo-1 ) ( zymed laboratories , san francisco , ca ) , claudin-5 ( zymed laboratories , san francisco , ca ) , and -tubulin ( amersham , poole , uk ) . then , the samples were incubated for 1 hr with appropriate secondary antibodies labeled with alexa fluor-488 or alexa fluor-596 ( life technologies japan , tokyo , japan ) . actin filaments were labeled with alexa fluor-546 phalloidin ( life technologies japan , tokyo , japan ) . the expression of each protein was examined using a laser scanning confocal microscope ( mrc 1024 ; bio - rad , hercules , ca ) . western blotting was performed according to the method described in a previous report . for western blotting of the total cell lysates , the dishes were washed with pbs , and then 300 l of sample buffer ( 1 mm nahco3 and 2 mm phenylmethylsulfonyl fluoride ) was added to 60 mm culture dishes . the cells were scraped and collected in microcentrifuge tubes and then sonicated for 10 sec . the protein concentrations of the samples were determined using a bca ( bicinchoninic acid ) protein assay reagent kit ( pierce chemical , rockford , il ) . for each sample , aliquots of protein ( 15 g per lane ) were separated by electrophoresis in 4/20% sodium dodecyl sulfate polyacrylamide gels ( cosmo bio , tokyo , japan ) ( sds page ) . after being electrophoretically transferred to nitrocellulose membranes ( immobilon ; millipore , billerica , ma ) , the membranes were saturated with blocking buffer ( trisbuffered saline [ tbs ] supplemented with 0.1% tween 20 and 4% skimmed milk ) for 30 min at room temperature and incubated with antiactin , anti - zo-1 , anti - ve - cadherin ( bd biosciences , san diego , ca ) , anticlaudin-5 ( zymed laboratories , san francisco , ca ) , anti - p38 mitogen - activated protein kinase ( map kinase or mapk ) ( santa cruz biotechnology , santa cruz , ca ) , antiphospho - p38 mapk ( cell signaling , beverly , ma ) , anti - p42/44 mapk ( promega , madison , wi ) , antiphospho - p42/44 mapk ( cell signaling , beverly , ma ) , anti - rho - a , and anti - cdc42 ( santa cruz biotechnology , santa cruz , ca ) antibodies ( 1:1000 ) for 1 h at room temperature . the membranes were then incubated with horseradish peroxidase - conjugated anti - rabbit or mouse igg ( dako a / s , copenhagen , denmark ) at room temperature for 1 h. the immunoreactive bands were detected using an ecl western blotting analysis system ( ge healthcare , little chalfont , uk ) . paracellular flux is dependent on the function of tight junctions [ 30 , 31 ] . we assessed the effects of an ac formulation on the ter of hmvec to evaluate their tight junction function . as shown in figure 1 and table 1(a ) , the odn containing ac formulation caused a time - dependent reduction in ter , while ter was hardly affected by treatment with odn or atelocollagen alone . as for the type of oligonucleotide in the formulation , phosphorothioate odn produced a more significant reduction in ter than phosphodiester odn , which only produced slight alterations . various formulations containing different ratios of odn and atelocollagen were examined in order to understand which parameters have the greatest effect on the change in ter . as a result , we found that the ter change was dependent on the size of the odn and the composition of the formulation , but not the odn sequence , as shown in tables 1(b ) , 1(c ) , and 1(d ) . specifically , odn composed of 15 or more bases were effective and those containing around 30 bases were the most effective , but 10-base - long odn were not effective . the change in tight junction function was also dependent on the concentrations of odn and atelocollagen in the formulation . to verify that the ac formulation increased paracellular flux , the amount of texas red - labeled dextran ( molecular weight : 40 kd ) transported across an endothelial cell layer was examined . as shown in figure 2(a ) , dextran transport was increased approximately twofold in the cell cultures incubated with the ac formulation . next , the paracellular transport of atelocollagen was analyzed and compared with those of bsa and dextran . only very small amounts of bsa and dextran penetrated the cell sheet during the 2-hour study period ; on the other hand , much more atelocollagen passed through , even though the molecular weight of atelocollagen is 4 - 5 times higher than those of bsa and dextran ( figure 2(b ) ) . an examination using bmvec was performed to determine whether the effect of the ac formulation was specific to hmvec . as a result , we found that the ter value of the bmvec was also reduced by the ac formulation ( and only the ac formulation ) , as shown in figure 3 . bmvec forms the blood - brain barrier ( bbb ) , where intercellular sealing function is strictly maintained . these results showed that the ac formulation is able to affect the paracellular flux of endothelial barriers . it is well known that increased endothelial permeability is associated with impaired intercellular contact [ 3235 ] . we carried out an immunohistochemical analysis of the cells treated with the ac formulation to clarify how their intercellular sealing was affected . as shown in figure 4(a ) , treatment with the ac formulation markedly reduced the degree of intercellular contact , as shown by intercellular gap formation , actin stress fiber formation , cellular contraction , and a lack of ve - cadherin . adequate expression of claudin-5 , one of the key components of the endothelial barrier , was noted at the cell periphery . however , zo-1 protein expression was absent from the intercellular gaps . on the contrary , western blotting revealed that treatment with the ac formulation did not affect the expression of these proteins ( figure 4(b ) ) . although the ter value remained low as long as the ac formulation was present in the culture medium , the treatment did not cause toxicity . the cells survived well for at least 24 hrs , and both the ter and morphology of the cells could be recovered by removing the formulation ( data not shown ) . no such morphological changes were induced by treatment with odn or atelocollagen alone ( figure 4(a ) ) . microtubules play an important role in regulating actin formation and hence , endothelial barrier function [ 36 , 37 ] . as shown in figure 5 , -tubulin formed a fine network in the blank control . however , treatment with the ac formulation caused the peripheral fine structure of the -tubulin network to be lost . many studies have shown that increased endothelial permeability and impaired intercellular contact can be induced by signal transduction , mainly that of rho a [ 32 , 38 ] and p38 map kinase [ 36 , 3942 ] . thus , we investigated the effects of the ac formulation on signal transduction . as a result , no differences were found in the expression levels of rho - a , cdc42 , or p42/44 map kinases or their phosphorylated forms . regarding p38 map kinase , although no changes were noted among the control , oligonucleotide alone , or atelocollagen alone groups , the levels of phosphorylated p38 map kinase were markedly increased in the cells treated with the ac formulation ( figure 6 ) , which indicates that the impact of the ac formulation on tissue permeability is associated with the activation of p38 map kinase . collagen plays an important role in the extracellular matrix by supporting cells so that they can form tissues and organs . atelocollagen is produced from type i collagen and is widely used in its solid state as a biomaterial for medical and surgical products because of its biocompatibility and workability . however , the kinetics , dynamics , and biological functions of atelocollagen after its injection into the living body are still poorly understood , and it is essential to elucidate the characteristics of atelocollagen in order to fully exploit its potential . here , we demonstrated a novel biological function of atelocollagen . when endothelial cell sheets were treated with atelocollagen or oligonucleotides alone , the intercellular structure of the sheet was not changed . however , when the atelocollagen and oligonucleotides were administered together , intercellular gaps formed and consequently the paracellular flux of the sheet was elevated . the abovementioned changes were elicited via the activation of p38 map kinase , a signal - transduction - related molecule . in addition , the changes observed in this study are similar to those triggered by thrombin , histamine , tnf- [ 34 , 36 ] , and vegf ( vascular endothelial growth factor ) , and so forth . as shown in section 3.1 , the degree to which endothelial function was affected was dependent on the molecular structure of the oligonucleotides including their size and chemical modifications , suggesting that the three - dimensional structure of the oligonucleotide and atelocollagen complex stimulates a signal transduction pathway that acts as a permeability modulator , although the specific pathway that it stimulates remains unknown . to date , no severe systemic edema or side effects of the ac formulation have been noted , even after the intravenous administration of atelocollagen as an oligonucleotide drug carrier . these findings indicate that atelocollagen could be used as a permeability enhancer at local treatment sites without the adverse systemic effects that cytokines and chemokines sometimes provoke . since tight junction modulators are regarded as practical drug delivery enhancer candidates [ 4446 ] , the function of atelocollagen demonstrated in the present study should be thoroughly investigated . the unique biological functions of atelocollagen have led to the development of unique antitumor therapies and products , such as surgical products ; formulations that sustain the release of antitumor proteins [ 24 ] ; treatments that enhance the antitumor activities of various molecules including antisense odn [ 1113 ] , sirna [ 1420 , 24 ] , and mirna [ 2123 ] . obtaining more information about atelocollagen would allow us to develop the next generation of atelocollagen - mediated drug delivery systems .

atelocollagen is a major animal protein that is used as a highly biocompatible biomaterial . to date , atelocollagen has been used as an effective drug delivery technology to sustain the release of antitumor proteins and to enhance the antitumor activity of oligonucleotides in in vivo models . however , the biological effects of this technology are not fully understood . in the present study , we investigated the effects of atelocollagen on endothelial paracellular barrier function . an atelocollagen formulation containing oligonucleotides specifically increased the permeability of two types of endothelial cells , and the change was dependent on the molecular size , structure of the oligonucleotides used and the concentrations of the oligonucleotide and atelocollagen in the formulation . an immunohistochemical examination revealed that the formulation had effects on the cellular skeleton and intercellular structure although it did not affect the expression of adherens junction or tight junction proteins . these changes were induced through p38 map kinase signaling . it is important to elucidate the biological functions of atelocollagen in order to be able to exploit its drug delivery properties .

cardiac disease and hypertension have been the third and eighth leading causes of death in taiwan since 2000 . according to a recent study , the percentage of the population with a prescription for antihypertensive drugs in taiwan has increased from 2001 to 2006 . the authors of this study report that during this period , the average annual increase in prescriptions for calcium channel blockers ( ccbs ) , angiotensin ii receptor blockers ( arbs ) and angiotensin - converting - enzyme inhibitors ( aceis ) were 10.7 , 22.1 and 4.5 % , respectively . in 2013 , the sale volume of the three leading antihypertensive drugs in taiwan amounted to about us$ 5 million ; in comparison , in the usa the value of prescriptions filled for antihypertensive drugs in 2013 totaled about us$ 678.2 million . the use of antihypertensive agents ( ahs ) has grown globally over the last decade . however , available data on a potential association between the use of ahs and risk of breast cancer are conflicting . recent epidemiological studies suggest that beta - blockers prevent breast cancer progression or reduce recurrence and then improve survival [ 46 ] . in contrast , other studies have reported an increased risk or no association at all between the use of beta - blockers / ccbs and breast cancer risk [ 79 ] . in addition , evidence for any association between the use of aceis / arbs and breast cancer is also inconsistent , with some studies suggesting that aceis / arbs are not associated with cancer risk [ 10 , 11 ] , and others reporting an increased or reduced risk . to address the conflicting evidence from previous studies , the aim of the study reported here was to evaluate the risk of breast cancer associated with long - term use of ahs in hypertensive women . data were retrieved from the national health insurance research database ( nhird ) and registry for catastrophic illness patient dataset ( hv dataset ) between january 1 , 1998 and december 31 , 2011 in taiwan . the nhird contains comprehensive information on demographic characteristics , pharmacy records and medical services from inpatient , outpatient and emergency care under a national health insurance program in which over 99% of the 23 million inhabitants of taiwan are enrolled . the hv dataset comprises specific data subsets of the nhird for research purposes and contains registration files and original claim data on patients registered in the nhird who have / had a catastrophic illness . therefore , this study was exempt from the approval by the ethics review board at our institution . from the hv dataset , we identified 330,699 women with newly diagnosed hypertension [ international classification of disease , ninth revision ( icd-9 cm ) codes 401405 ] who had been treated with any ahs continuously for at least 6 months between january 1 , 1998 and december 31 , 2011 . among these , we further identified women with a first diagnosis of breast cancer ( icd-9 cm codes 174.xx and 175.xx ) ; these women were the cases in our study ( fig aht antihypertensive , h / t hypertension , hv registry for catastrophic illness patient dataset , nhird national health insurance research database study flow diagram . aht antihypertensive , h / t hypertension , hv registry for catastrophic illness patient dataset , nhird national health insurance research database we excluded patients who had a history of breast cancer or any cancer recorded in the hv dataset any time before the initiation of antihypertensive treatment and patients without continuous enrolment in a nhi program . patients were followed from the date of diagnosis of hypertension in 1998 up to december 31 , 2011 ( median duration 13 years ) or death , whichever came first ( fig . we randomly selected hypertensive women registered in the nhird without any diagnosis of breast cancer who were receiving treatment for hypertension in the same period as the cases . these were matched ( 1:4 ) for age ( 5-year categories ) , index date and year of hypertension diagnosis with the cases to establish the control group ( fig . 1 ) . the main exposure of interest was that to beta - blocker , ccb , acei and arb therapy . we collected information on prescribed drug types according to anatomical therapeutic chemical classification system codes ( c07 for beta - blockers ; c02d , c08c , c08d , c08da51 for ccbs ; c02e , c02l , c09a , c09ba for aceis ; c09ca for arbs ) , dosage , date of prescription , supply days and total number of prescriptions from the outpatient and inpatient records . the cumulative defined daily dose ( cddd ) of each ah was calculated as recommended by the world health association . beta - blockers were further classified as nonselective and beta-1 selective beta - blockers , and as selective and nonselective alpha - blockers . several potential covariates , including age and comorbidities at cancer diagnosis , were also measured in the year preceding the index date . we evaluated the sensitivity effects by changing the inclusion criteria of drug prescription for three types of ah beginning at least from 69 months before the index date . logistic regression was used to estimate the crude and adjusted odds ratio ( or ) and 95% confidence interval ( ci ) for breast cancer risk . we calculated a running sum of the duration and ddd of each drug from the date of the initial ah prescription to the index date . we categorized the cumulative use for each patient as follows : 1 , 12 , 23 and 3 years of duration . data were analyzed using the sas statistical package , version 9.3 ( sas institute , cary , nc ) . the significance level was set at p < 0.05 ( two - tailed test ) . data were retrieved from the national health insurance research database ( nhird ) and registry for catastrophic illness patient dataset ( hv dataset ) between january 1 , 1998 and december 31 , 2011 in taiwan . the nhird contains comprehensive information on demographic characteristics , pharmacy records and medical services from inpatient , outpatient and emergency care under a national health insurance program in which over 99% of the 23 million inhabitants of taiwan are enrolled . the hv dataset comprises specific data subsets of the nhird for research purposes and contains registration files and original claim data on patients registered in the nhird who have / had a catastrophic illness . therefore , this study was exempt from the approval by the ethics review board at our institution . from the hv dataset , we identified 330,699 women with newly diagnosed hypertension [ international classification of disease , ninth revision ( icd-9 cm ) codes 401405 ] who had been treated with any ahs continuously for at least 6 months between january 1 , 1998 and december 31 , 2011 . among these , we further identified women with a first diagnosis of breast cancer ( icd-9 cm codes 174.xx and 175.xx ) ; these women were the cases in our study ( fig . 1 ) . aht antihypertensive , h / t hypertension , hv registry for catastrophic illness patient dataset , nhird national health insurance research database study flow diagram . aht antihypertensive , h / t hypertension , hv registry for catastrophic illness patient dataset , nhird national health insurance research database we excluded patients who had a history of breast cancer or any cancer recorded in the hv dataset any time before the initiation of antihypertensive treatment and patients without continuous enrolment in a nhi program . patients were followed from the date of diagnosis of hypertension in 1998 up to december 31 , 2011 ( median duration 13 years ) or death , whichever came first ( fig . 1 ) . we randomly selected hypertensive women registered in the nhird without any diagnosis of breast cancer who were receiving treatment for hypertension in the same period as the cases . these were matched ( 1:4 ) for age ( 5-year categories ) , index date and year of hypertension diagnosis with the cases to establish the control group ( fig . the main exposure of interest was that to beta - blocker , ccb , acei and arb therapy . we collected information on prescribed drug types according to anatomical therapeutic chemical classification system codes ( c07 for beta - blockers ; c02d , c08c , c08d , c08da51 for ccbs ; c02e , c02l , c09a , c09ba for aceis ; c09ca for arbs ) , dosage , date of prescription , supply days and total number of prescriptions from the outpatient and inpatient records . the cumulative defined daily dose ( cddd ) of each ah was calculated as recommended by the world health association . beta - blockers were further classified as nonselective and beta-1 selective beta - blockers , and as selective and nonselective alpha - blockers . several potential covariates , including age and comorbidities at cancer diagnosis , were also measured in the year preceding the index date . we evaluated the sensitivity effects by changing the inclusion criteria of drug prescription for three types of ah beginning at least from 69 months before the index date . logistic regression was used to estimate the crude and adjusted odds ratio ( or ) and 95% confidence interval ( ci ) for breast cancer risk . we calculated a running sum of the duration and ddd of each drug from the date of the initial ah prescription to the index date . we categorized the cumulative use for each patient as follows : 1 , 12 , 23 and 3 years of duration . data were analyzed using the sas statistical package , version 9.3 ( sas institute , cary , nc ) . the significance level was set at p < 0.05 ( two - tailed test ) . we identified 6,463 hypertensive women with breast cancer as cases and 18,987 hypertensive women without breast cancer as controls . among the 6,463 cases , the most commonly prescribed ahs was ccbs ( 52.8% ) , followed by aceis ( 45.5% ) and beta - blockers ( 41.1% ) ( table 1 ) . ever - use of ccbs and beta - blockers for longer than 10 years was significantly associated with breast cancer ( or 1.09 ; 95% ci 1.031.16 ) in an adjusted model . the risk of breast cancer was even higher in patients receiving hormone replacement therapy ( or 1.28 , 95% ci 1.181.39 ) and statins ( or 1.68 , 95 % ci 1.501.83 ) ( table 1).table 1characteristics of hypertensive patients with breast cancer and non - breast cancer during the study period ( 19982011)characteristiccase ( n = 6,463)control ( n = 18,987)odds ratio ( 95 % ci ) n % n % crudeadjustedmean age , years ( sd)61.9(10.7)61.9(10.9 ) 18442724.217854.13 45541,48923.04,40923.2 55642,32035.96,72935.4 65741,61024.94,77825.2 75846459.981,91210.1 85 + 1271.973741.97menopause4,70272.713,79372.6mean cci score ( sd)0.33(0.87)0.34(0.92)0.98 ( 0.951.01) diabetes1,76127.34,80325.31.11 ( 1.041.18)**1.08 ( 1.021.16 ) * hyperlipidemia3,19649.59,20748.51.04 ( 0.981.10)ever users of hrt no5,45084.316,62687.61.00 ( reference)1.00 ( reference ) yes1,01315.72,36112.41.31 ( 1.211.42)***1.28 ( 1.181.39)***ever users of statins no5,72588.617,70093.21.00 ( reference)1.00 ( reference ) yes73811.41,2876.781.77 ( 1.611.95)***1.68 ( 1.521.85)***types of aht acei no3,52054.510,15253.51.00 ( reference ) yes2,94345.58,83546.50.96 ( 0.911.02) arb no4,68272.414,29075.31.00 ( reference)1.00 ( reference ) yes1,78127.64,69724.71.16 ( 1.091.23)***1.04 ( 0.981.12 ) ccbs no3,05247.29,69751.11.00 ( reference)1.00 ( reference ) yes3,41152.89,29048.91.17 ( 1.101.23)***1.09 ( 1.031.16 ) * * beta - blocker no3,80658.911,72161.71.00 ( reference)1.00 ( reference ) yes2,65741.17,26638.31.13 ( 1.061.19)***1.05 ( 0.991.12 ) * p < 0.05 , * * p < 0.01 , * * * p < 0.001 sd standard deviation , cci charlson comorbidity index , hrt hormone replacement therapy , aht antihypertensive therapy , acei angiotensin - converting - enzyme inhibitor , arb angiotensin receptor ii blocker , ccb calcium channel blocker , ci confidence interval characteristics of hypertensive patients with breast cancer and non - breast cancer during the study period ( 19982011 ) * p < 0.05 , * * p < 0.01 , * * * p < 0.001 sd standard deviation , cci charlson comorbidity index , hrt hormone replacement therapy , aht antihypertensive therapy , acei angiotensin - converting - enzyme inhibitor , arb angiotensin receptor ii blocker , ccb calcium channel blocker , ci confidence interval when we stratified the risk of breast cancer associated with different sub - types of beta - blockers , we found a statistically significant risk of breast cancer with most beta-1 selective beta - blockers , such as atenolol ( or 1.14 ; 95% ci 1.051.25 ) acebutolol ( or 1.29 ; 1.001.66 ) and bisoprolol ( or 1.08 ; 1.011.16 ) ( fig . 2 ) . the non - selective beta - blockers , alpha - selective and beta - non selective showed no significant association with breast cancer ( fig . 2).fig . 2forest plot of breast cancer risk associated with use of beta - blockers , 19982011 . or odds ratio , ci confidence interval forest plot of breast cancer risk associated with use of beta - blockers , 19982011 . or odds ratio , ci confidence interval we then stratified beta - blocker , arb and ccb users by exposure duration and the cumulative ddd . the results show that the risk of breast cancer was significantly increased in beta - blocker and ccb users with increasing exposure duration and increasing cddd compared to the controls [ trend test for beta - blocker users : p = 0.003 ( exposure duration ) , p = 0.0003 ( cddd ) ; trend test for ccb users : p = 0.006 ( exposure duration ) , p = 0.002 ( cddd ) ] ( table 2).table 2odds risk and 95% confidence intervals for risk of breast cancer associated with exposure to different types of antihypertensives , duration of exposure and dosagetype of antihypertensive agentno . of study subjectsno . of breast cancer casesmultivariable - adjusted odds ratioodds ratio ( 95 % ci ) p for trendany beta - blocker never use15,5273,8061.00 ( reference ) ever - use exposure duration ( years)0.003 12,0855210.99 ( 0.891.11 ) 122,3005480.91 ( 0.821.01 ) 231,5124021.03 ( 0.921.17 ) > 34,0261,1861.16 ( 1.071.26 ) * * * cumulative ddd 0.0003 cddd < q12,4805970.93 ( 0.841.02 ) q1 cddd < q22,4826210.97 ( 0.881.07 ) q2 cddd < q32,4806841.08 ( 0.981.19 ) cddd q42,4817551.22 ( 1.111.34)***any arb never use18,9724,6821.00 ( reference ) ever - use exposure duration ( years)0.71 11,3553701.05 ( 0.931.19 ) 121,6524391.00 ( 0.891.12 ) 231,0832880.98 ( 0.851.12 ) > 32,3886841.03 ( 0.931.14 ) cumulative ddd 0.53 cddd < q11,6184441.06 ( 0.941.19 ) q1 cddd < q21,6214140.95 ( 0.841.07 ) q2 cddd < q31,6184411.00 ( 0.891.13 ) cddd q41,6214821.07 ( 0.951.21)any ccb never use12,7493,0521.00 ( reference ) ever - use exposure duration ( years)0.006 12,2575721.05 ( 0.941.16 ) 122,6626961.08 ( 0.981.19 ) 231,9585221.09 ( 0.981.22 ) > 35,8521,6211.11 ( 1.031.19 ) * * cumulative ddd 0.002 cddd < q13,1758181.05 ( 0.961.15 ) q1 cddd < q23,1768341.07 ( 0.981.18 ) q2 cddd < q33,1748381.06 ( 0.971.17 ) cddd q41,6214821.16 ( 1.061.28 ) * * * * p < 0.01 , * * * p < 0.001 cddd cumulative defined daily dose adjusted for peripheral vascular disease , diabetes mellitus and medicine use ( included hrt , statin , arb and ccb ) beta - blocker : q1 ( 25% ) = 195.25 ddd , q2 ( 50% ) = 448 ddd , q3 ( 75% ) = 1,012 ddd . ccb : q1 ( 25% ) = 390.1 ddd , q2 ( 50% ) = 851 ddd , q3 ( 75% ) = 1,641.3 ddd . arb : q1 ( 25% ) = 405 ddd , q2 ( 50% ) = 800.5 ddd , q3 ( 75% ) = 1,464 ddd adjusted for peripheral vascular disease , diabetes mellitus and medicine use ( including hrt , statin , beta - blocker and ccb ) adjusted for peripheral vascular disease , diabetes mellitus and medicine use ( including hrt , statin , beta - blocker and arb ) odds risk and 95% confidence intervals for risk of breast cancer associated with exposure to different types of antihypertensives , duration of exposure and dosage * * p < 0.01 , * * * p < 0.001 cddd cumulative defined daily dose adjusted for peripheral vascular disease , diabetes mellitus and medicine use ( included hrt , statin , arb and ccb ) beta - blocker : q1 ( 25% ) = 195.25 ddd , q2 ( 50% ) = 448 ddd , q3 ( 75% ) = 1,012 ddd . ccb : q1 ( 25% ) = 390.1 ddd , q2 ( 50% ) = 851 ddd , q3 ( 75% ) = 1,641.3 ddd . arb : q1 ( 25% ) = 405 ddd , q2 ( 50% ) = 800.5 ddd , q3 ( 75% ) = 1,464 ddd adjusted for peripheral vascular disease , diabetes mellitus and medicine use ( including hrt , statin , beta - blocker and ccb ) adjusted for peripheral vascular disease , diabetes mellitus and medicine use ( including hrt , statin , beta - blocker and arb ) the risk of breast cancer increased with ever - use of atenolol or acebutolol ( table 3 ) . this risk increased with increasing exposure , duration of use ( trend test : p = 0.0003 for atenolol ; p = 0.01 for acebutolol ) and cddd ( trend test : p = 0.002 for atenolol ; p = 0.02 for acebutolol).table 3breast cancer risk associated with exposure duration and dosage of specific beta - blockers during the study period ( 19982011)variableacebutololatenololbisoprolol n / n odds ratio ( 95 % ci ) n / n odds ratio ( 95 % ci ) n / n odds ratio ( 95 % ci)duration of exposure to antihypertensive agent aht non - use6,371/25,1631.00 ( reference)5,661/22,6831.00 ( reference)4,887/19,6971.00 ( reference ) exposure duration ( years ) 130/1041.10 ( 0.711.68)248/9421.01 ( 0.871.17)1,318/4,9131.06 ( 0.991.14 ) 2321/711.10 ( 0.661.84)222/7951.07 ( 0.911.26)121/4021.14 ( 0.921.42 ) 3417/521.37 ( 0.772.46)120/4231.08 ( 0.871.34)57/1791.24 ( 0.901.70 ) > 324/601.85 ( 1.103.12)*212/6071.43 ( 1.201.70)***80/2591.12 ( 0.861.47 ) p for trend0.010.00030.03dosage ( cddd ) < q121/711.10 ( 0.661.84)187/8911.04 ( 0.871.23)384/14571.04 ( 0.921.18 ) q1<q219/720.97 ( 0.571.64)187/6891.03 ( 0.871.23)408/14291.14 ( 1.011.29 ) * q2<q326/721.56 ( 0.962.53)202/6951.13 ( 0.961.34)382/14351.02 ( 0.901.15 ) q326/721.59 ( 0.982.58)226/6921.30 ( 1.101.53)**402/14321.10 ( 0.981.25 ) p for trend0.020.0020.053 * p < 0.05 , * * p < 0.01 , * * * p < 0.001adjusted for diabetes mellitus and medicine use ( including hrt , statins , arbs and ccbs ) n number of breast cancer patients using a specific aht , n total number of study population using a specific aht beta - blocker : q1 ( 25% ) = 195.25 ddd , q2 ( 50% ) = 448 ddd , q3 ( 75% ) = 1,012 ddd . ccb : q1 ( 25% ) = 390.1 ddd , q2 ( 50% ) = 851 ddd , q3 ( 75% ) = 1,641.3 ddd . arb : q1 ( 25% ) = 405 ddd , q2 ( 50% ) = 800.5 ddd , q3 ( 75% ) = 1,464 ddd breast cancer risk associated with exposure duration and dosage of specific beta - blockers during the study period ( 19982011 ) * p < 0.05 , * * p < 0.01 , * * * p < 0.001 adjusted for diabetes mellitus and medicine use ( including hrt , statins , arbs and ccbs ) n number of breast cancer patients using a specific aht , n total number of study population using a specific aht beta - blocker : q1 ( 25% ) = 195.25 ddd , q2 ( 50% ) = 448 ddd , q3 ( 75% ) = 1,012 ddd . ccb : q1 ( 25% ) = 390.1 ddd , q2 ( 50% ) = 851 ddd , q3 ( 75% ) = 1,641.3 ddd . arb : q1 ( 25% ) = 405 ddd , q2 ( 50% ) = 800.5 ddd , q3 ( 75% ) = 1,464 ddd in the sensitivity analysis for exposure duration of ahs , the results were unchanged when the inclusion criteria of ah prescription was changed from < 6 to > 9 months ( table 4).table 4sensitivity analysis for criteria of antihypertensive usevariableany beta - blocker any arb any ccb n / n odds ratio ( 95 % ci ) n / n odds ratio ( 95 % ci ) n / n odds ratio ( 95 % ci)non - user4,107/16,6901.00 ( reference)4,876/19,7001.00 ( reference)3,367/14,0121.00 ( reference)user2,356/8,7601.04 ( 0.981.11)1,587/5,7501.01 ( 0.941.09)3,096/11,4381.09 ( 1.031.16)**drug use ( years ) 2220/9220.94 ( 0.801.10)176/6271.08 ( 0.901.29)257/9941.07 ( 0.921.24 ) 23548/2,3000.91 ( 0.821.01)439/1,6521.00 ( 0.891.12)696/2,6621.07 ( 0.981.18 ) 34402/1,5121.03 ( 0.911.16)288/1,0830.97 ( 0.841.12)522/1,9581.09 ( 0.981.22 ) > 41,186/4,0261.15 ( 1.061.25)***684/2,3881.03 ( 0.931.14)1,621/5,8241.11 ( 1.031.19 ) * * p for trend0.0050.760.005 * p < 0.05 , * * p < 0.01 , * * * p < 0.001 n total number of study population using specific aht , n number of breast cancer patients using specific aht adjusted for peripheral vascular disease , diabetes mellitus and medicine use ( including hrt , statins , arbs and ccbs ) adjusted for peripheral vascular disease , diabetes mellitus and medicine use ( including hrt , statins , beta - blockers and ccbs ) adjusted for peripheral vascular disease , diabetes mellitus and medicine use ( including hrt , statins , beta - blockers and arbs ) sensitivity analysis for criteria of antihypertensive use * p < 0.05 , * * p < 0.01 , * * * p < 0.001 n total number of study population using specific aht , n number of breast cancer patients using specific aht adjusted for peripheral vascular disease , diabetes mellitus and medicine use ( including hrt , statins , arbs and ccbs ) adjusted for peripheral vascular disease , diabetes mellitus and medicine use ( including hrt , statins , beta - blockers and ccbs ) adjusted for peripheral vascular disease , diabetes mellitus and medicine use ( including hrt , statins , beta - blockers and arbs ) the results of this study suggest that the use of acei , arbs , and nonselective beta - adrenergic receptor antagonists ( propranolol or carteolol ) is not associated with breast cancer . these results are consistent with those of most observational studies [ 10 , 11 ] . we also found that ccbs and the beta-1 selective beta - blockers acebutolol , atenolol and bisoprolol may increase the risk of breast cancer . this finding seems to differ from those of previous studies which reported that beta-1 selective beta - blockers and ccbs had marked protective effects [ 15 , 16 ] . however , the authors of a recently published study reported observing a weak inverse association between cardio nonselective beta - blockers and breast cancer risk . however , since the association did not reach statistical significance , the results did not support the hypothesis of beta - blocker usage protecting against breast cancer progression . the results of a recently published network analysis indicated a lack of consistency in the effect of ccbs on breast cancer ; this was attributed to the short duration of the follow - up in the trials included in the network meta - analysis . the results of previous preclinical studies are inconclusive in terms of whether beta - blockers have agonist activity in breast cancer growth . some studies has demonstrated that beta-2 adrenergic signaling plays a role in several pathways involved in breast tumor progression and metastasis [ 17 , 18 ] , but others have found that beta - adrenergic receptor ( ar ) stimulation may both inhibit and promote breast tumor growth [ 1923 ] . a recently published study adds further to the body of evidence on the effect of agonist type , indicating that the beta 2-ar antagonist in particular seems to be the most cytotoxic beta - blocker in non - stimulated cancer cells . however , the majority of clinical observational studies carried out to date have focused on comparing the association between the use of propranolol or atenolol and breast cancer risk or mortality and have not explored the relationship between the subtype of beta - ar expression and breast cancer risk [ 25 , 26 ] . our study is the first from asia to report that treatment with the beta-1 selective blocker but not the nonselective 1/ blocker may increase the risk of breast cancer ( fig . 2 ) . these results appear to be consistent with those of preclinical studies suggesting that the effects of beta - adrenergic signaling on tumor progression and metastasis are inhibited by the 2-receptor antagonists but not by 1 antagonists [ 1824 ] . consequently , better designed observational studies or randomized controlled trials are required before this type of beta - blocker can be considered as a therapeutic option for patients with breast cancer . this finding is consistent with those from a recently published study performed by li et al . . both studies seem to revive an earlier previous hypothesis and focus on the long - term use of ccbs among current or ever - users ( 10 years if the study of li et al . ; 13 years in our study ) . however , other previously published studies found no increased risk of breast cancer associated with ccb use [ 25 , 26 ] . therefore , to date , the results on the effect of ccbs on breast cancer risk are inconsistent . again , larger and more comprehensive studies are needed to confirm the effects of long - term use of ccbs on breast cancer . a major advantage of our study was that we collected information prospectively on healthcare beneficiaries registered in a large population - based database for whom complete data on drug prescriptions and cancer diagnoses were available . first , the health insurance database that we used was developed for administrative purposes and contained de - identified records of each individual registered . second , the database only provided information on the frequency and classes of prescribed medications and did not provide any clinical laboratory data or clinical information ; therefore , we could not estimate patient s responses to drug therapy . finally , the database did not contain information on various lifestyle risk factors for cancer , such as physical activity , alcohol consumption , smoking , body mass index , socioeconomic status and diet ; therefore , these were not included in the analysis . although we adjusted the potential covariates , such as co - morbidities and the use of other medications , the misclassification of these covariates may have some impact on our results . our findings indicate that the long - term use of ccbs or beta-1 selective blockers are likely to be associated with breast cancer risk . further comprehensive and large population - based studies are needed to confirm these findings before any definitive conclusion can be drawn . henry w. c. leung , li - ling hung , agnes l. f. chan , and chih - hsin mou declare no conflict of interest . this study was exempt from the approval by the ethics review board at our institution . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

introductionto evaluate the risk of breast cancer associated with long - term use of antihypertensive agents ( ahs ) in taiwanese women with hypertension.methodsa search of the taiwan national health insurance research database identified 330,699 patients with hypertension who were treated with antihypertensive drugs between january 1 , 1998 and december 31 , 2011 . logistic regression models were used to estimate the odds ratios ( ors ) and 95% confidence intervals ( cis ) between the use of ahs and breast cancer risk , adjusted for other types of antihypertensive drugs , statins and co-morbidities.resultsamong the ahs used to treat the hypertensive women enrolled in our study , our analysis revealed that those treated with one specific particular class of beta - blockers ( beta-1 selective beta - blockers ) had an increased risk for breast cancer . we also found that the ever - use of calcium channel blockers ( ccbs ; i.e. for 13 years ) was associated with breast cancer in an adjusted model ( or 1.09 ; 95% ci 1.031.16 ) . however , the use of nonselective beta - blockers , selective and nonselective alpha - blockers , angiotensin - converting enzyme inhibitors and angiotensin ii antagonists were not associated with breast cancer risk.conclusionbased on the results of our analysis , long - term use of ccbs or beta-1 selective beta - blockers are likely to be associated with the risk of breast cancer . further large comprehensive population - based studies to support our findings are required for confirmation of this conclusion .

the first step in a typical computational pipeline for protein identification through database searching is comparing ms / ms spectra to peptide sequences to identify the best matching peptide for each spectrum , referred to as peptide - to - spectrum matches ( psms ) . these psms are then processed by protein inference algorithms to produce a minimal list of proteins that would need to be present in the sample to explain the identified psms . the proteins in this list are also assigned a confidence score or protein probability calculated on the basis of several factors , including the number of psms supporting the protein and the match score of the psms . to determine the confident identifications from the results of protein inference , false discovery rates ( fdr ) only proteins identified at or above a certain fdr threshold ( typically 1 or 5% ) are chosen as high - confidence identifications for further analyses . although sophisticated algorithms for spectral matching and analysis have been developed , protein identification can still be hampered by issues such as low efficiency of peptide ionization , low - quality or noisy spectra , dynamic range of protein abundances , and the complexity of protein samples . to deal with such issues , there have been continued attempts to incorporate additional information about the ms / ms experiment into analysis pipelines , such as peptide chromatographic retention time , pi , or mass accuracy , some of which are now a routine part of many proteomic analysis pipelines . studies have also investigated using matching ms and ms information , match scores from multiple search engines , and various other information sources to rescore or adjust protein identification probability . another category of methods have investigated utilizing external information ( information from outside the ms / ms experiment ) such as microarray data , protein protein interaction networks , or gene functional networks to improve protein identifications . a recent study by wang et al . described an approach to utilize rna - seq abundance information to limit the size of protein sequence databases and , we describe an alternate method for incorporating external information such as rna - seq abundance and gpmdb identification frequency into proteomic analysis pipelines , through rescoring or adjustment of protein identification probabilities . rna - seq uses short read sequencing technologies to sequence the rna content ( transcriptome profile ) of a sample . on the basis of the central dogma of molecular biology , it is a reasonable assumption that proteins corresponding to high - abundance transcripts are more likely to be found in a sample . the global proteome machine database ( gpmdb ) is a repository storing the results of proteomics experiments . with the large volume of data aggregated in gpmdb , the frequency of identification of a protein in gpmdb can be used as a surrogate measure of a protein s propensity to be observed in a ms / ms experiment . in other words , we can reasonably assume that proteins with a high gpmdb identification frequency ( gpmfreq ) are more likely to be identified in an ms / ms experiment . in this study , we evaluate the utility of incorporating both of the above types of information into proteomics analysis pipelines . data from vcap , a human prostate cancer cell line , and hek293 , a cell line derived from human embryonic kidney cells , were used in this study . the ms / ms and rna - seq data for the vcap cell line were generated in parallel at the same lab . this rna - seq data was also used as the control sample in a paper by sam et al . and is available for download from the ncbi short read archive , sra ( sra accession nos . , ms / ms data was obtained from control samples in a publication by fonslow et al . , whereas the rna - seq data was downloaded from data generated by sultan et al . the gpmdb identification frequencies , which are not cell line specific , were obtained by querying gpmdb for every ensembl protein i d using a perl script . collection of vcap whole cellular protein extract was done in ripa complete buffer supplemented with halt protease and phosphatase inhibitor cocktail ( peirce biotechnology ) . fifty milligram aliquots of total cellullar proteins were first separated by 1d sds - page ( 412% bis - tris novex - invitrogen , carlsbad , ca ) . forty equal - sized gel bands were excised and subjected to in - gel digestion as previously described . extracted peptides were reconstituted with mobile phase a prior to online reverse - phase nanolc peptides were eluted online to the mass spectrometer with a reverse - phase linear gradient from 97% a ( 0.1% formic acid in water ) to 45% b ( 0.1% formic acid in acetonitrille ) . peptides were detected and fragmented in the mass spectrometer in a data - dependent manner , sending the top 12 precursor ions , excluding singly charged ions , for collisional induced dissociation . raw spectra files were converted into mzxml by an in - house version of readw . both of the ms / ms experiments ( vcap and hek293 ) used in this study were seen to have very deep proteome coverage , with about 40006000 protein identifications at 1% fdr . however , most ms / ms experiments do not achieve this level of proteome coverage . to investigate performance under experimental conditions with varying depths of proteome coverage , the ms / ms data was sampled at the level of individual mzxml files to create various subsets of data of varying sizes ( fewer files would be included for a smaller subset , and more files , to get a larger subset ) . the vcap data had a total of 40 mzxml files , whereas the hek293 data consisted of 60 mzxml files . the number of protein identifications in these subsets ranged between about 500 and 5000 protein identifications at 1% fdr . the ms / ms data was searched using the x!tandem ( cyclone ; 2010.12.01.1 ) search engine with a k - score plugin provided by the trans - proteomic pipeline . the search was performed against the ensembl v.66 human proteome with reversed protein sequences appended as decoys . trypsin was specified as the enzyme with no missed cleavages allowed , and cysteine carbamidomethylation and methionine oxidation were set as fixed and variable modifications , respectively . vcap data was searched using a precursor mass error of 1 to + 4 da , whereas the hek293 data ( high mass accuracy data ) was searched with a precursor mass error of 50 ppm . statistical validation of psms was performed using the trans - proteomic pipeline ( tpp v4.6 occupy rev 2 ) software suite . vcap data was processed with + 1 charge state ions set to be ignored and using a semisupervised model for estimating negative distributions . hek293 data was processed using the same settings as above along with additional parameters to use accurate mass binning and the ppm scale for the mass models . the output protxml files from tpp were processed using the abacus software tool to select a representative protein for each protein group , according to heuristic filters built into the tool . rna - seq data was aligned to the ensembl v.66 human genome ( hg19 build 37 ) using the tophat aligner ( tophat v.1.3.2 ) . parameters were set to allow up to one mismatch per alignment , and a gtf file containing ensembl v.66 gene annotations was provided to tophat , using the g transcript abundance , in the form of reads per kilobase per million mapped reads ( rpkm ) ( read count normalized to transcript length and total number of reads in the experiment ) , was calculated for each transcript from the bam file output from tophat . rpkm calculation was performed with a custom r script utilizing functions from the bioconductor packages rsamtools ( v.1.6.3 ) and genomicfeatures ( v.1.6.9 ) . our approach to incorporating rna - seq or gpmdb frequency information ( figure 1 ) is built upon a statistical adjustment of the protein probability . the probability adjustment increases the identification confidence scores of proteins that have significant supporting evidence from external data ( high transcript abundance in rna - seq or high frequency of identification in gpmdb ) , relative to other proteins without such supporting evidence . protein identifications that previously fell just below the fdr threshold based on ms / ms evidence alone , in a gray zone of identification confidence , can be promoted above the threshold when ranked by the adjusted probability . therefore , we are able to obtain more protein identifications at the same fdr . ( a ) external information is added to protein identifications from the analysis pipeline . protein probabilities are adjusted on the basis of transcript abundance ( rpkm ) or gpmdb identification frequency ( gpmfreq ) . ( b ) decoys sequences used to estimate fdr thresholds do not have native rpkm ( or gpmfreq ) values ; they are assigned values by sampling from a set of all forward sequences with similar length ( see methods ) . decoy approach ( reversed decoy sequences are appended to the forward protein sequence database before performing database searching . the number of identifications matched to decoy sequences is used to estimate the rate of random matches in the identifications mapped to forward sequences ) . because decoy sequences do not have inherent rpkm / gpmfreq values , their identification probabilities would be selectively decreased during probability adjustment based on the external information . to be able to estimate fdr after probability adjustment in an unbiased manner , a rational method for assigning rpkm / gpmfreq values to decoy sequences the density distribution of rpkm / gpmfreq values for proteins identified in the ms / ms experiment at 1% fdr ( confident true positive identifications ) is seen to be appreciably different from that of rpkm / gpmfreq for all proteins ( figure 2 ) . on the basis of this observation , sampling from the all proteins distribution allows for the unbiased assignment of rpkm / gpmfreq values to decoy sequences while maintaining discrimination between decoy and forward identifications . weak correlations among the rpkm , gpmfreq values , and protein length were also observed in the data ( data not shown ) . to preserve this structure in the data , our sampling approach was designed to sample rpkm / gpmfreq values together from forward sequences and assign them only to decoys of similar length ( see supporting information methods for a more detailed description of the sampling process ) . in further analysis , it was observed that the improvement from probability could be slightly increased if the sampling for decoy values was weighted to prefer values from proteins not identified in the ms / ms experiment , instead of using a completely random sampling ( supporting information figure 5 ) . however , only results from the more statistically rigorous approach of completely random sampling are reported here . density distributions of rpkm ( a ) and gpmfreq ( b ) values ( log - scaled ) for proteins identified at 1% fdr in the vcap cell line and all proteins in ensembl are plotted . the difference between the two distributions allows us to sample the all proteins distribution to assign values to decoys in an unbiased manner while still maintaining discrimination between true positive and decoy identifications . when performing probability adjustment , pprot , a protein probability score calculated on the basis of maximum peptide probability , was used as the prior probability ( see supporting information methods for details of pprot calculation ) . pprot was used instead of the native protein probability reported by tpp because it has been observed that the maximum peptide probability is a more reliable indicator of true protein identifications than the protein probability value ( supporting information figure 1 ) , especially for large samples . using bayes theorem , the probability adjustment estimates the probability of a protein identification being a true positive given its rpkm / gpmfreq value ( eq 1).1where v can be either an rpkm or gpmfreq value . the prior probability terms p(+ ) and p( ) were substituted with pprot and 1 pprot , respectively . to estimate the conditional probabilities of decoy or forward identifications having value v , p(v| ) and p(v|+ ) , the density distribution of log - scaled rpkm / gpmfreq values was placed into bins of equal width ( supporting information figure 2 ) . conditional probabilities for each bin were calculated as p(vi| ) = ndi / ndt , where vi is any rpkm / gpmfreq value that falls within bin i , ndi is the number of decoys having rpkm / gpmfreq values within bin i , and ndt is the total number of decoys in the sample . values for p(vi|+ ) were also estimated similarly , but instead of number of decoys , the number of forward hits with pprot > 0.5 ( i.e. , forward identifications that are more likely to be true positive than false positive ) were used . because the rpkm / gpmfreq values are assigned through random sampling , the assignment and probability adjustment ( figure 1a ) are repeated multiple times to nullify any sampling artifacts and to obtain stable mean adjusted probability values . in our study , the mean values were typically seen to stabilize after about 200 iterations ( supporting information figure 3 ) , but the process was repeated to 500 iterations for the results reported here . the effect of the probability adjustment was measured by comparing the number of protein identifications at 1% fdr without adjustment to the number of protein identifications at 1% fdr after probability adjustment ( rpkm or gpmfreq based ) . the percent improvement from all of the various subsets was calculated and plotted , as shown in figure 3a , b . percentage improvement due to the probability adjustments ( rpkm and gpmfreq ) for vcap ( a ) and hek293 ( b ) cell lines plotted at various depths of proteome coverage ( no . of proteins ) . the probability adjustment results in improvements of almost 8% in the hek293 cell line and up to 4% in vcap ( figure 3 ) . notably , the amount of improvement observed is similar for both the rpkm and gpmfreq adjustments . furthermore , it appears that using rna - seq data generated in parallel to the ms / ms data ( vcap ) or rna - seq generated at a different time and location from the ms / ms data ( hek293 ) does not significantly affect the results . we believe the probability adjustment works by boosting protein identifications that fall in a gray zone of confidence of identification . to test this hypothesis , the entire analysis described above was repeated using maximum hyperscore instead of maximum peptide probability as the identification confidence score . hyperscore is a spectral matching score calculated and reported by the x!tandem search engine . the maximum hyperscore for a protein can be used as an alternate , albeit less effective than the maximum peptide probability , confidence score for sorting protein identifications and estimating fdr thresholds . because maximum hyperscore is a suboptimal score compared to maximum peptide probability , the resulting protein identifications should have more proteins in the gray zone and therefore the probability adjustment on these identifications should provide increased improvement . as expected , figure 4a , b shows that the percentage improvement is much greater ( 720% ) in the maximum hyperscore - based analysis . these results support the idea that the amount of improvement obtained from probability adjustment is dependent on the number of proteins falling in the gray zone of the confidence of identification . percentage improvement at various depths of proteome coverage when probability adjustment is performed on a maximum hyperscore - based protein identification probability . as expected , the improvement is significantly greater when the suboptimal maximum hyperscore is used instead of maximum peptide probability ( figure 3 ) . in our analysis , a clear trend of the percentage improvement from probability adjustment decreasing as the depth of proteome coverage ( i.e. , number of proteins identified in the data set ) increases can be seen ( figure 3 ) . with deeper coverage of the proteome , such proteins would have low rna - seq abundance and/or low frequency of identification in gpmdb . therefore , these proteins will not benefit from a probability adjustment based on rpkm / gpmfreq evidence and , in fact , may have their confidence scores decreased by it . furthermore , increasing depth of proteome coverage not only increases the number of proteins identified but also increases the amount of ms / ms or spectral evidence collected for each identified protein . this would lead to a decrease in the number of proteins falling in the gray zone . on the basis of this , we believe that the observation of decreased improvement in deeper coverage data sets reflects the fact that in these data sets there are fewer proteins that would benefit from the probability adjustment . a more detailed analysis of the effects of probability adjustment was carried out on one of the sampled data subsets from each cell line , the results of which are shown in table 1 . proteins that were promoted above the 1% fdr threshold as a result of the probability adjustment were selected for manual validation . these selected proteins were compared with the list of proteins identified at 1% fdr in the complete data set ( largest data set without any sub sampling ) of that cell line . a promoted protein being found in the complete sample would suggest that the protein is indeed a true identification . it is possible that there was not sufficient ms / ms evidence in the smaller sampled data set for the protein to be confidently identified , but the probability adjustment using rpkm / gpmfreq information provided the necessary boost to promote it above the fdr threshold . in our analysis , the remaining 2030% of promoted proteins , which were not observed in the complete data set , were seen to have high confidence scores in the same range as that of the validated proteins . in other words , it is possible that these proteins were not observed in the complete data set because , even with the increased amount of ms / ms evidence collected in the complete data set , there still is not sufficient evidence to confidently identify them solely by ms / ms evidence without the aid of external information . in our analysis , a larger proportion of proteins are validated in the vcap sample , which has more ms / ms data collected ( 6000 proteins ) , than in the hek293 sample ( 3000 proteins ) , which appears to support this interpretation . the probability adjustment method described here allows us to utilize external data , such as rna - seq abundance , or gpmdb identification frequency , to improve the sensitivity of protein identification through database searching . although some studies generate rna - seq data in parallel to proteomics data , large amounts of rna - seq data for many common organisms and/or cell lines used in biological research are already freely available from public resources such as the sequence read archive ( sra ) . as we can see from figure 3 , whether rna - seq data is generated in parallel with proteomics data ( vcap ) or independently ( hek293 ) this will allow us effectively leverage the large amounts of publicly available rna - seq data . furthermore , the improvement obtained by adjusting probability based on gpmfreq is similar to , and sometimes better than , improvement from rpkm adjusted probability . this is very convenient , allowing us to make use of readily available gpmdb information in our proteome analysis pipelines . of course , this requires that the gpmdb repository contains enough experiments for the organism of interest for the gpmfreq values to be meaningful . however , for commonly studied organisms of interest such as human or mouse , with numerous experiments in gpmdb , it can be a useful source of external information . computing a combined adjusted probability from both rna - seq and gpmdb information does not result in a marked improvement in protein identification over the individual probability adjustments ( supporting information figure 4 ) , suggesting that rna - seq and gpmdb capture similar types of information about a sample for the purposes of probability adjustment . as mentioned earlier , the improvement obtained from probability adjustment decreases as the depth of proteome coverage of the experiment increases because there are fewer proteins in the gray zone that would benefit from the probability adjustment and there are more rare and low - abundance proteins that could be penalized by it . this is an inherent upper limit to the amount of improvement that is possible by this method and must be taken into consideration when applying this probability adjustment to large samples . however , this method remains useful for ms / ms data of low - to - medium levels of proteome coverage . hence , one potential application of this method may be for data obtained from older instruments or experiments where the amount of instrument time available was low . compared to the customized database approach described by wang et al . , the probability adjustment method was seen to provide better improvement for low - to - medium - coverage samples , whereas the customized database approach performed better for deep - coverage samples ( supporting information figure 9 ) , suggesting nonoverlapping scenarios of usage for the two methods . although the probability adjustment approach has been demonstrated using rna - seq and gpmdb data , it does not include any assumptions that would limit it to only these two kinds of data . therefore , this approach can be utilized to incorporate any external source of data ( with a significant association with protein presence or abundance ) into proteomic analysis pipelines to improve the sensitivity of protein identification .

tandem mass spectrometry ( ms / ms ) followed by database search is the method of choice for protein identification in proteomic studies . database searching methods employ spectral matching algorithms and statistical models to identify and quantify proteins in a sample . in general , these methods do not utilize any information other than spectral data for protein identification . however , considering the wealth of external data available for many biological systems , analysis methods can incorporate such information to improve the sensitivity of protein identification . in this study , we present a method to utilize global proteome machine database identification frequencies and rna - seq transcript abundances to adjust the confidence scores of protein identifications . the method described is particularly useful for samples with low - to - moderate proteome coverage ( i.e. , < 20003000 proteins ) , where we observe up to an 8% improvement in the number of proteins identified at a 1% false discovery rate .

the leading cause of nontraumatic extremity amputation is complications of diabetes , with over 96,000 amputations per year.1 neuropathy associated with diabetes predisposes to pain , numbness , ulceration , infection , gangrene , and amputation . somatic and autonomic diabetic neuropathy contribute to 87% of these amputations.2 it is now appreciated that there are different forms of neuropathy in diabetes.1 damage occurs to both large , myelinated nerve fibers and small , unmyelinated fibers ( c - fibers ) . the small , unmyelinated c - fibers subserve thermal and pain perception , and the small autonomic fibers affect sweating and vascular control.3 damage to small , unmyelinated c - fibers has the greatest impact on survival and quality of life , producing initial symptoms such as pain , numbness , and anhidrotic skin with disordered skin blood flow ( skbf ) predisposing to foot ulcers , infection , gangrene and limb loss.4 diabetic peripheral neuropathy is the primary cause of diabetic ulcers and the most predictive diagnosis for an eventual amputation . current therapies for diabetic peripheral neuropathy are for symptomatic relief of pain and not for the underlying disorder.58 any addition to the therapeutic armamentarium would be welcome . it has been shown that small fiber neuropathies are quantifiable with newer tests of c - fiber integrity that include quantitative measures of skbf and corneal confocal microscopy,911 quantification of intraepidermal nerve fibers ( ienf),1216 and quantitative sensory testing ( qst).17 heat- induced vasodilation ( 44c ) , is reduced in subjects with impaired glucose tolerance , and in type 2 diabetic patients with and without neuropathy.18 we have reported that one of the earliest quantitative sensory abnormalities found in people with diabetic peripheral neuropathy is the impairment of small fiber sensory perception that cosegregates with disordered neurovascular function.19,20 furthermore , there is evidence to suggest that one of the early manifestations of c - fiber dysfunction can be based upon skin biopsies , with measurement of the density of thin , unmyelinated nerve fibers using immunohistochemistry for protein gene product 9.5 ( pgp-9.5 ) as a neuronal marker.12,13,15,16,21,22 since description of the utility of pgp- 9.5 as a marker for ienf in human skin,23 it has been used for identification of small nerve fibers in the dermis and epidermis in a range of small fiber neuropathies , including human immunodeficiency virus , and idiopathic small fiber and postherpetic neuralgia , among others.13,15,2429 in addition , we have now established that there are characteristic changes in ienf that distinguish the metabolic syndrome from diabetes . 30 ienf density was recently given a level a recommendation by the european federation of the neurological societies and the peripheral nerve society as a reliable and efficient technique to confirm the clinical diagnosis of small fiber neuropathy.31 the ienf density has been shown to correlate inversely with cold and heat detection thresholds.32 a oneyear diet and exercise intervention program in patients with small fiber neuropathy and impaired glucose tolerance led to increased ienf density.33 ienf loss is evident early in the disease process , declines with increasing neuropathic pain , and can be used as an outcome measure in clinical trials.31,33 apart from the one - year trial on diet and exercise,33 no drug has been shown to induce c - fiber regeneration . topiramate , a structurally novel antiepileptic drug , is effective in treatment of various types of epilepsy and prevention of migraine headaches . a sulfamate - substituted derivative of d - fructose , topiramate has several pharmacologic properties suggesting it may have potential for overcoming the programmed cell death implicated in the pathogenesis of neuropathy.4 it enhances gamma aminobutyric acid ( gaba ) activity by interacting with a nonbenzodiazepine site on gaba receptors , has negative modulatory effects on voltage - activated na+ channels,34 selectively blocks ampa / kainate glutamate receptors , has negative modulatory effects on l - type high voltage - activated ca channels,35 and inhibits the carbonic anhydrase isozymes , ca - i and ca - ii.34 these multiple mechanisms of action may contribute to its anticonvulsive , antinociceptive , and putative neuroprotective properties . there has been no report on the ability of topiramate to influence the structure and function of ienf , although there are animal studies to suggest that it may exercise these properties.36,37 topiramate thus clearly offers an advantage over agents currently used for neuropathy in as much as it relieves pain and , in animal studies , is neurotrophic and neuroprotective , and may have beneficial effects on weight , blood pressure , and lipids . the american diabetes association and the american society of pain have recently added topiramate and other anticonvulsant medications to the diabetic neuropathy treatment regimen.38 there may be an added benefit with topiramate in treating diabetic patients with neuropathy , because it exerts both neurotrophic and metabolic effects that have not been shown by other anticonvulsants.7,8,39 we have reported on pain reduction with topiramate7 which persisted for at least six months40 and had salutary effects on weight , blood pressure , and serum lipids . the aim of this study was to determine whether topiramate induces regeneration of small cutaneous nerve fibers in concert with improved skbf and sensory measures of c - fiber function . the eastern virginia medical school institutional review board approved the study and all subjects gave written informed consent prior to any study procedures . all subjects participating in this study had type 2 diabetes , were aged 3575 years , and had documented evidence of diabetic peripheral neuropathy . the study was a pure within - subject repeated - measure design comparing skin biopsy data , sensory responses , and skbf before and after treatment with topiramate in subjects with diabetic peripheral neuropathy . all assessments occurred once prior to treatment and again at the end of the 18-week treatment period . neurologic symptom scores and neurologic disability scores were generated by completion of a questionnaire modified from dyck.41 the sum of the three scores ( symptom , motor , and sensory scores ) is the total neuropathy score . neuropathy was established by the criteria suggested by the american diabetes association and american academy of neurology.42 qst included measures of temperature and touch perception thresholds . quantitative autonomic function was assessed by three tests , ie , heart rate variability during deep breathing at six breaths per minute ( e : i ratio ) , the r : r variation in response to the valsalva maneuver , and the r : r variation is response to postural change as suggested by the neurologic disability score position statement on neuropathy.38 qst was performed using the medoc device ( tsa2001/vsa3001 ) following previously published procedures . touch pressure was measured using graded monofilaments ( semmes weinstein).17 measurements were taken 2 cm proximal to the skin biopsy sites on the nondominant limbs . nerve conduction studies were performed on the nondominant peroneal motor , sural sensory , and ulnar motor and sensory nerves , using standard techniques of supramaximal percutaneous nerve stimulation , surface recording , and appropriate filtering and signal amplification . stimulation - to- recording electrode distances , electrode placement , and limb surface temperatures were defined for each nerve conduction study to ensure that all studies were performed under similar conditions . neurologic symptom scores and neurologic disability scores were generated by completion of a questionnaire modified from dyck.41 the sum of the three scores ( symptom , motor , and sensory scores ) is the total neuropathy score . neuropathy was established by the criteria suggested by the american diabetes association and american academy of neurology.42 qst included measures of temperature and touch perception thresholds . quantitative autonomic function was assessed by three tests , ie , heart rate variability during deep breathing at six breaths per minute ( e : i ratio ) , the r : r variation in response to the valsalva maneuver , and the r : r variation is response to postural change as suggested by the neurologic disability score position statement on neuropathy.38 qst was performed using the medoc device ( tsa2001/vsa3001 ) following previously published procedures . touch pressure was measured using graded monofilaments ( semmes weinstein).17 measurements were taken 2 cm proximal to the skin biopsy sites on the nondominant limbs . nerve conduction studies were performed on the nondominant peroneal motor , sural sensory , and ulnar motor and sensory nerves , using standard techniques of supramaximal percutaneous nerve stimulation , surface recording , and appropriate filtering and signal amplification . stimulation - to- recording electrode distances , electrode placement , and limb surface temperatures were defined for each nerve conduction study to ensure that all studies were performed under similar conditions . skbf was measured by continuous laser doppler assessment of the response to varying stimuli , as previously described.20,43 testing was done on the nondominant foot and proximal calf at baseline , after six weeks of maintenance treatment ( ie , a total of 12 weeks treatment ) and at the end of 12 weeks of maintenance treatment . the skbf and temperature probes were placed over the external aspect of the nondominant leg 10 cm below the knee over the area where the skin biopsy sample was taken and on the dorsum of the foot . skbf examinations were done with the patient in a reclined position with the legs elevated and completely extended . after a 10-minute baseline period , the temperature was increased to 32c for 10 minutes , 40c for 10 minutes , and then to 44c for 20 minutes . biopsies were collected from each patient s dorsal forearm , lateral thigh ( 10 cm proximal to patella ) , lateral proximal leg ( 10 cm distal to fibular head ) , and lateral distal leg ( 10 cm proximal to lateral malleolus ) . tissue samples were immediately fixed in 2% buffered paraformaldehyde / lysine / periodate solution for 1224 hours at 4c . they were subsequently cryoprotected in phosphate buffer with 20% glycerol overnight and frozen for later cryosectioning . procedures for identifying neurons in skin biopsies employed immunofluorescence techniques in a modification of the protocol described by mccarthy et al.13 the processing and cutting of the slides were done following procedures previously described.30 the skin biopsy data from this cohort of patients was also compared with healthy control patients taken from the large skin biopsy database created and maintained at our institute . blood samples were obtained at the screening and final visits for determination of glycosylated hemoglobin ( hba1c ) , lipid profile including total serum cholesterol , high - density lipoprotein ( hdl ) cholesterol , low - density lipoprotein ( ldl ) cholesterol , free fatty acids , and triglycerides , and fasting serum glucose and c - peptide . we also measured serum b12 and rapid plasma reagin at screening in order to rule out occult neuropathies . in addition , liver enzymes were measured at screening , and at weeks 4 , 6 , 12 , and 18 . the primary endpoints were skbf , qst , and indices of ienf density and length , all of which are continuous ( parametric ) data . repeated measures analysis of variance was used to examine the treatment effects for skbf , qst , and skin biopsies . significant differences were accepted as p < 0.05 . where significant treatment differences were observed , contrast testing was used to determine the significance at each level , while allowing for multiplicity of comparisons . in addition , nonparametric spearman rank correlations were done to examine the relationship between ienf , skbf , and measures of neurologic function . the patients entered into this study were predominantly caucasian , overweight , male , and hypertensive type 2 diabetics with clinical evidence of distal symmetric polyneuropathy ( table 1 ) . our previous experience prescribing topiramate led us to use a low starting dosage and a slow dosage - escalation protocol . oral topiramate was administered initially at a dose of 25 mg / day and gradually titrated over six weeks to a dose of 100 mg / day or the maximum tolerated dose . fifteen of the 20 subjects were able to tolerate 100 mg / day , two subjects were maintained on 75 mg / day , and three were on 50 mg / day . a 12-week maintenance period followed the titration phase . during this period , the dose of study medication remained constant . one of the 20 patients was discontinued from the study early due to cognitive effects of the medication , including psychomotor slowing , word finding difficulty , and fatigue . we have previously reported16 that mean dendritic length and ienf density progressively decreased from proximal to distal sites in patients with neuropathy . in this cohort of patients , there was a similar decrease in ienf density and mean dendritic length from proximal to distal sites ( figure 1a and 1b ) . nerve fiber density in skin taken from patients before topiramate treatment was significantly lower than in skin taken from comparable sites in healthy control patients in our large skin biopsy database . comparing the results at each site , ienf density in the forearm was significantly lower in patients ( 13.2 3.5 ) compared with controls ( 32.9 3.2 ; p < 0.001 ) . similar deficits in ienf were seen in the thigh ( 8.4 5.7 versus 35.6 7.3 ; p < 0.01 ) , proximal leg ( 3.1 2.2 versus 17.4 2.0 ; p < 0.001 ) , and distal leg ( 4.6 4.6 versus 28.0 3.9 ; p < 0.001 ) . mean dendritic length of nerve fibers in skin from controls was similar , regardless of site , ie , forearm ( 3.28 0.18 m ) , thigh ( 3.69 0.3 m ) , proximal leg ( 3.34 0.23 m ) , and distal leg ( 3.31 0.3 m ) . there was a progressive shortening of fibers in patients before treatment with topiramate from forearm to distal leg . in addition , mean dendritic length in skin from patients before topiramate treatment was significantly lower than skin taken from comparable sites in control subjects , ie , thigh ( 2.28 0.23 ; p < 0.001 ) , proximal leg ( 1.79 0.26 ; p < 0.001 ) , and distal leg ( 1.94 0.35 ; p < 0.005 ) . mean dendritic length was not different from controls in the forearm ( 2.86 0.19 ; p < 0.05 ) . treatment with topiramate significantly increased mean dendritic length in the forearm and proximal leg ( figure 1a ) and ienf density in the proximal leg ( figure 1b ) . this was associated with improvement in total neuropathy scores , including touch , vibration , and prickling pain thresholds , and reduction in weight , body mass index , systolic and diastolic blood pressures , and hba1c ( table 2 ) . a significant increase in skbf was seen at week 12 , but returned to baseline at week 18 . skbf in the leg increased from 87.7 0.1 to 101.3 0.1 ( p < 0.001 ) and in the foot from 102.6 0.2 to 143.7 0.6 ( p < 0.001 ) laser doppler units at 12 weeks . no correlations were found between the changes in measures of neurologic function , ienf , and the changes in metabolic parameters . in this small within - subject pilot study , we have shown that 18 weeks of treatment with topiramate induced improvement in neurologic disability scores , including prickling , vibration , and touch perception . this was associated with improvement in skbf after 12 weeks of treatment , but this returned to baseline at 18 weeks . quantification of ienf density and mean dendritic length in skin biopsies , carried out at entry into the study and repeated at the same sites after 18 weeks of treatment , showed an increase in nerve fiber length at the forearm and proximal leg after treatment with topiramate , and an increase in nerve fiber density at the proximal leg . we have previously shown4,44 that the earliest change found in patients with the metabolic syndrome without hyperglycemia is a reduction in mean dendritic length and that the decrease in ienf density occurs after at least five years of diabetes and is most prominent at the distal site.44 thus , the patients in this study most closely resemble those with diabetes , including those with features of the current definition of the metabolic syndrome ( table 1 ) . there is a distinct clinical entity of neuropathy associated with the metabolic syndrome in the absence of glucose intolerance . however , up to 50% of patients with painful neuropathy may have impaired glucose tolerance.17,45,46 this suggests that treatment of the nonhyperglycemic components of the metabolic syndrome may be important . while we do show an improvement in hba1c , blood pressure , and weight similar to that reported for topiramate in previous neuropathy studies,7,39 we could not find correlations between changes in nerve fibers or cognitive function and changes in the metabolic parameters . in this study we showed that 18 weeks of treatment with topiramate improves total neuropathy scores , touch , prickling pain , and vibration perception , as well as ienf . we have reported16,44 that distal leg ienf density showed significant negative correlations with warm ( p < 0.02 ) and cold ( p < 0.05 ) thermal threshold , heat pain ( p < 0.05 ) , pressure sense ( p < 0.05 ) , sensory score ( p < 0.03 ) , and total neuropathy score ( p < 0.03 ) , and thus objective measures are likely to be related . it is not clear , at this point in time , if these changes can be sustained for greater periods . it has been shown that pain improvement persists for at least six months,40 and anecdotally our patients appeared to continue to improve beyond the defined study period . clearly , longer duration studies are needed to determine if the cross - sectional correlates of nerve fiber densities and mean dendritic length with different sensory modalities can be realized . we have reported that there is a direct correlation between distal leg ienf density and duration of diabetes,47 suggesting that there is a loss of about one nerve fiber per mm per year . a reduction of one nerve fiber per mm translates into clinically significant changes in total neuropathy scores and objective measures of cognitive function . furthermore , an improvement in the proximal leg of three nerve fibers per mm was found to provide clinically meaningful changes in objective parameters of nerve function . therefore , we would propose that a conservative estimate of double the value for change in function , ie , two nerve fibers per mm , could be considered a minimal requirement for success in future studies of agents affecting nerve biology . furthermore , we have also reported that there is a progressive shortening of the mean dendritic length in the distal leg.47 since this change occurs in the metabolic syndrome independent of blood glucose,44 it seems that this could be a valuable endpoint in patients being treated for components of the metabolic syndrome other than hyperglycemia . the pattern of regeneration is unique and dictates a need to use ienf length , rather than density , as an endpoint in clinical studies at sites where viable nerve fibers still exist . because of the demonstrated effects of topiramate on various metabolic aspects , it could be an important addition to therapies aimed at the metabolic syndrome and its sequelae , including neuropathy and diabetes . topiramate has been reported to have positive effects on pain relief in diabetic peripheral neuropathy,7 an effect which has been shown to be durable40 in clinical trials for the management of diabetic peripheral neuropathy pain syndromes . in these studies it was noted that weight loss and changes in metabolic parameters accompanied pain relief , but the role of topiramate in disease modification of neuropathy is limited.48 although the mechanisms underlying the neuroprotective effect of topiramate are not fully understood , there may be added benefits for people with type 2 diabetes . topiramate inhibits weight gain in animals on a high - fat diet , but the mechanism through which change in energy balance is achieved is unclear.49 topiramate causes weight loss50 sustained for one year.51 in our studies , treatment with topiramate not only improved symptoms of neuropathy , but also reduced body weight , body mass index , systolic and diastolic blood pressure , and hba1c . these findings are not unlike those previously reported with topiramate treatment in normal healthy patients , as well as in patients with diabetes.50,51 the caveats , however , are that this was a small open - label trial , with careful attention paid to minimizing the untoward side effects known to occur at higher doses of topiramate . effects of topiramate on cognitive function , including psychomotor slowing , short - term memory loss , fatigue , and anorexia have been reported with topiramate treatment . these common side effects make it difficult to design a true placebo - controlled trial . in our short study , we found if there was a low starting dose ( 15 mg ) , and a slow taper to a low maximum dose of 100 mg , many of these side effects are mitigated . what would be attractive is the discovery of an agent with the beneficial neurologic and metabolic effects of topiramate , but free of cognitive dysfunction and intensive dosing requirements , which would allow a true placebocontrolled trial to be performed .

purposeto assess the effects of topiramate on c - fiber function , nerve fiber morphology , and metabolism ( including insulin sensitivity , obesity , and dyslipidemia ) in type 2 diabetes.patients and methodswe conducted an 18-week , open - label trial treating patients with topiramate . twenty subjects with type 2 diabetes and neuropathy ( 61.5 1.29 years ; 15 male , 5 female ) were enrolled and completed the trial . neuropathy was evaluated by total neuropathy scores , nerve conduction studies , quantitative sensory tests , laser doppler skin blood flow , and intraepidermal nerve fibers in skin biopsies.resultstopiramate treatment improved symptoms compatible with c - fiber dysfunction . weight , blood pressure , and hemoglobin a1c also improved . laser doppler skin blood flow improved significantly after 12 weeks of treatment , but returned to baseline at 18 weeks . after 18 weeks of treatment there was a significant increase in intraepidermal nerve fiber length at the forearm , thigh , and proximal leg . intraepidermal nerve fiber density was significantly increased by topiramate in the proximal leg.conclusionthis study is the first to demonstrate that it is possible to induce skin intraepidermal nerve fiber regeneration accompanied by enhancement of neurovascular function , translating into improved symptoms as well as sensory nerve function . the simultaneous improvement of selective metabolic indices may play a role in this effect , but this remains to be determined .

the cytoskeleton has a well - organized and dynamically regulated subcellular architecture composed of core filaments and various accessory proteins . a variety of cellular signaling events are supported by cytoskeletal / scaffold components , which in some cases constitute structural hubs for signaling and/or in other cases contribute directly or indirectly to signal transduction . such roles of cytoskeletal elements are not limited to purely cytoplasmic events but extend to signals ultimately targeted to the nucleus . nuclear signaling is fundamentally important for cells to properly orchestrate adaptive responses to various environmental changes and functional requirements . the spatio - temporal steps involved in transducing a signal from the plasma membrane to the nucleus occur in different subcellular locations : signal reception / transduction at the plasma membrane , signal amplification and cascade in the cytoplasm , shuttling / signaling through the nuclear pore complex ( npc ) at the nuclear envelope , and modulation of nuclear functions . cytoskeletal involvement in signal transduction is well documented in signal recognition at the plasma membrane and the signal cascade in the cytoplasm . many membrane receptors and transporters interact with the cytoskeleton ( e.g. , ankyrin and spectrin ) and transmit their specific signals to their respective target sites . thus , the cytoskeleton often functions as a platform for signal transduction in the cytoplasm and has been assumed to only indirectly contribute to the subsequent steps of nuclear signaling , that is , signal transmission to the nucleus and regulation of nuclear functions . there is increasing evidence , however , that many types of cytoskeletal proteins are localized to the nucleus , suggestive of their direct involvement in the transmission of nuclear signaling and the regulation of nuclear functions ( table 1 ) . some of these are predominantly localized to the cytoplasmic side and nearly undetectable in the nucleus by using fluorescence techniques under normal conditions ; even so , a small subpopulation of this class of proteins shuttles between the cytoplasm and the nucleoplasm [ 1 , 2 ] . in another case , homologues , variants or fragments of several cytoskeletal proteins are predominantly found in the nucleus [ 3 , 4 ] . such cytoskeletal proteins are strong candidates for components of the nucleoskeleton , the structural basis for nuclear functions . in addition to the linc complex , which links the cytoskeleton and nucleoskeleton by sun - kash protein interactions penetrating through the nuclear membrane , the two compartments may be directly connected via the exchange of structural molecules . to fully understand the biological significance of the cytoskeletal proteins in the nucleus , the molecular mechanisms of their nuclear translocation should be clarified . we will review the studies of cytoskeletal proteins in the nucleus and propose possible mechanisms that regulate their nuclear localization . actin is well known as a component of actin filaments and is one of the most abundant proteins in cells . the first report of actin in the nucleus was published in 1969 , and the association of nuclear actin with rna polymerase ii was found subsequently [ 3335 ] . a report in 1998 revealed the nuclear export signal- ( nes- ) dependent active nuclear export of actin through the npc . thereafter , multiple roles of nuclear actin have been revealed , including transcription [ 3639 ] , chromatin remodeling , and mrna transport [ 41 , 42 ] . furthermore , varieties of other cytoskeletal proteins are localized to the nucleus and are involved in the regulation of nuclear functions ( table 1 ) . the notion that localization of cytoskeletal proteins in the nucleus is indispensable for proper nuclear functions is now gaining widespread acceptance . nuclear actin has been implicated in a variety of nuclear functions , such as transcription [ 3639 ] , chromatin remodeling , and molecular transport of mrna [ 41 , 42 ] and proteins . it associates with an swi / snf - like chromatin remodeling complex , hnrnps [ 39 , 42 , 45 ] , and rna polymerases i , ii , and iii [ 4650 ] . these findings of nuclear actin in various fundamental biological processes substantiate the constitutive requirement for actin in the nucleus . one of the most famous actin - interacting proteins in the cytoplasm , myosin , is also found in the nucleus . nuclear myosin i ( nmi ) is a monomeric and single - headed myosin , which possesses an additional n - terminal 16 amino - acid domain that directs the molecule into the nucleus [ 7 , 8 ] . unlike nuclear actin , nmi is a nuclear - specific isoform , which is almost exclusively localized to the nucleus . nmi functions cooperatively with nuclear actin and especially participates in the association with the chromatin remodeling complex and rna polymerases [ 47 , 51 ] . the precise molecular states of actin in the nucleus , the monomeric or polymeric form , have not yet been fully resolved . a role of nuclear actin in the serum - induced gene activation pathway favors monomeric actin in the nucleus . mal , a coactivator of the serum response transcription factor ( srf ) , is known to interact with monomeric actin . under normal conditions , mal - actin complexes are actively exported from the nucleus , possibly due to the nes of the actin molecule . when serum - induced signaling is triggered , the mal - actin interaction is disrupted , resulting in the accumulation of mal in the nucleus and the activation of srf target genes . on the other hand , polymerized actin is required for the transcription of ribosomal rna by rna polymerase i . nuclear actin and nmi cooperate in the activation of rrna transcription , and actin - dependent motor activity of nmi is required for transcription elongation . coupled with the observation of actin - containing filamentous structures around the npc , it is likely that nuclear actin exists in polymerized states , at least to some extent . considering the cellular concentration of actin , which is estimated to be 100 m , remarkably higher than the critical concentration for polymerization , and also the high abundance of actin in xenopus oocyte nuclei , it is reasonable to assume that both monomeric and polymeric forms of actin exist in the nucleus . mobility measurements of nuclear actin using fluorescence recovery after photobleaching ( frap ) provided important supporting evidence . recovery of the fluorescence of gfp--actin demonstrated that nuclear actin contains several different kinetic populations , suggesting that approximately 16% of the nuclear actin is in a polymeric form . in a recent study demonstrating the role of actin and actin - interacting protein in xenopus oocytes , these findings suggest that dynamic conformational changes of the nuclear actin in association with its interacting partners play significant roles in various cellular events , both for general biological processes and for specific signal responses . several different isoforms of myosins , other than nmi , were also found to localize to the nucleus . myosin ii ( smooth muscle myosin ) is localized to the nucleus in smooth muscle cells and regulates transcription by binding to the promoter regions of target genes and interacting with the rna polymerase ii complex . myosin vi is also detected in the rna polymerase ii complex , and it enhances transcription much like myosin ii . myosin va and vb were recently found in nuclear speckles and nucleoli , and are involved in transcription by rna polymerase i [ 10 , 11 ] . one of the cytoplasmic actin - depolymerizing factors cofilin-1 was also found to function in the nucleus . cofilin-1 associates with a complex containing nuclear actin and phosphorylated rna polymerase ii , and it plays a key role in transcriptional elongation , presumably by regulating polymerization states of nuclear actin along target genes . several different mechanisms were found to target neuronal wiskott - aldrich syndrome protein ( n - wasp ) , a primary cytoplasmic regulator for cortical actin filaments , to the nucleus . n - wasp phosphorylated by focal adhesion kinase at a tyrosine residue y256 is predominantly localized to cytoplasm while wild - type and kinase - insensitive mutant y256f are localized both in cytoplasm and in the nucleus . coprecipitation assays demonstrated that n - wasp interacts with a multifunctional large nuclear protein complex , psf - nono ( polypyrimidine - tract - binding - protein - associated splicing factor - non - pou - domain octamer - binding protein / p54nrb ) , that functions to couple n - wasp with rna polymerase ii to regulate transcription . since n - wasp modulates actin polymerization in nuclear extracts in vitro , it is suggested that nuclear n - wasp promotes polymerization of nuclear actin to regulate transcription . it is likely that the regulation of actin polymerization is a critical factor for nuclear transcription , and there are various factors that cooperatively modulate the process , just as in the cytoplasm . there are at least ten actin family proteins that are conserved among eukaryotes , termed actin - related proteins ( arps ) , which show 3070% similarity with actin and possess a similar atp binding motif . in spite of their well - conserved molecular structures six of them , including arp4 , 5 , 6 , 7 , 8 , and 9 , displayed predominant nuclear localization in s. cerevisiae , and it is also the case for at least four human arps , arp4 , 5 , 6 , and 8 [ 3 , 16 ] . in addition to nuclear actin , these nuclear arps are known to serve as a functional subunit of evolutionally conserved chromatin remodeling complexes , such as ino80 , swr1 , rsc , and adcr [ 6062 ] . taking into account the atp - dependent mechanical sliding or displacement of nucleosomes by a chromatin remodeling complex , nuclear actin and arps may function as a structural element for chromatin dynamics , just as the cytoplasmic actin cytoskeleton serves as a platform for various molecular dynamics . several nuclear arps are known to participate in chromosomal organization and mitotic chromosomal segregation without associating with chromatin remodeling complexes . arp6 binds to several chromatin regions including centromeres and the promoters of ribosomal protein genes . this association is required to anchor a specific chromatin region to the nuclear periphery , suggesting a role of arp6 in organizing proper chromatin regions in the nucleus . when the endogenous arp8 was depleted by sirna transfection , mitotic cells exhibited abnormal chromosome alignment . some of the arps are expressed in a tissue - specific manner . human arpt1 and mouse arpm1 are exclusively expressed in testis [ 66 , 67 ] . arpm1 interacts with profilin iii , a testis - specific nucleotide exchange factor for actin , suggesting that the arpm1-profilin complex functions in spermiogenesis - specific chromosomal organization . it is expected that nuclear arps may participate in specific steps of development or differentiation , while widely expressed nuclear actin is involved in the regulation of general nuclear functions . the spectrin - repeat ( sr ) is a widely conserved domain from bacteria to humans , found in several cytoskeletal crosslinking proteins such as spectrin , actinin , dystrophin , and nesprin . a single sr is a rod - shaped structure consisting of a bundle of three amphiphilic -helices facing each other via hydrophobic interactions , forming a hydrophobic inner surface with a hydrophilic outer surface . sr - containing cytoskeletal crosslinkers possess multiple srs and crosslink their target filaments into variously arranged forms , such as a contractile bundle , parallel array , or meshwork . in the last few years , several sr proteins have been found in the nucleus . -actinin contains four srs at the center of the molecule and forms a homodimer to crosslink actin filaments . one of the nonmuscle -actinin isoforms , actinin-4 , was found to be localized to the nucleus in some cancer cell lines . actinin-4 does not contain an nls and possesses at least one functional nes , which regulates the nucleocytoplasmic shuttling of this molecule in a crm1-dependent manner . nuclear actinin-4 is coprecipitated with the ino80 chromatin remodeling complex , which contains nuclear actin and several arps , and it has also been revealed that a splicing variant of actinin-4 that lacks the amino acids 89478 is predominantly localized to the nucleus and mediates the transcription of the taf55 gene in association with myocite enhancer factor-2 . it is likely that actinin-4 maintains cooperative relationships with actin in the nucleus , just as in the cytoplasm , though the detailed mechanisms of the molecular interaction have not yet been fully revealed . spectrins are large proteins ( over 200 kda ) containing a tandem array of sixteen to twenty srs . as a crosslinker of the actin cytoskeleton in the cytoplasm , spectrins form a tetramer consisting of two each of - and -spectrins . it has been reported that an isoform of nonerythroid -spectrin ( iisp ) is required for recruitment of the dna - repair proteins fanca and xpf to the sites of dna interstrand crosslinks . the iisp isoform from patients of fanconi anemia exhibits reduced stability , which correlates with a decreased level of dna repair activity . this deficiency results in mitotic chromosomal aberrations , suggesting a role of nuclear iisp as a scaffold for dna repair . one of the truncated isoforms of -spectrin , ivsp5 ( 72 kda ) , is localized to the promyelocytic leukemia ( pml ) bodies . this protein is tightly bound to the highly insoluble nuclear scaffold and overexpression of gfp-ivsp5 results in an increase in the number of pml bodies , suggesting that spectrin iv may be involved in the genesis of pml bodies . interestingly , a screen for proteins that co - immunopreciptated with iisp identified several plausible candidate interacting proteins , including ivsp5 , actin , lamin a , emerin , and pml . in addition , the screen identified proteins associated with dna repair ( e.g. , rad51 , rad50 , ku70 , ku80 , fanca , fancg , fancd2 , xpf , xpg , rpa70 ) , chromatin remodeling ( e.g. , brg1 , brm ) , and rna processing ( hnrnp a2/b1 , ribosomal - associated protein p40 ) . many of these interacting proteins associate in their own complexes ; so it is not surprising that immunoprecipitation of iisp pulled down so many candidates . the results are consistent with a role of nuclear spectrins as dynamic scaffolds for coordinating diverse nuclear complexes . several other sr proteins such as nesprins and bpag1 have also been found in the nucleus [ 2022 ] . these proteins are expected to play a variety of roles in the nucleus , such as organization of nuclear lamina and regulation of mapk signaling by tethering erk1/2 at pml bodies . since most of these sr proteins contain actin binding domains , nuclear sr proteins may have cooperative functions with nuclear actin and arps , possibly serving as a structural base or platform for their functions . -catenin , one of the cell adhesion molecules , is also known to be a signal transduction molecule for the canonical wnt signaling pathway . wnt signaling is one of the key signaling pathways in embryonic development and other processes , including bone formation and homeostasis , as well as adult tissue maintenance ; dysregulation of wnt signaling is implicated in some cancer and degenerative diseases [ 23 , 7476 ] . the canonical wnt pathway regulates cell fate by modulating gene expression directed by the nuclear accumulation of -catenin . as the number of reports on wnt signaling is rapidly increasing , we will mainly focus on the molecular interactions related to the nuclear translocation of -catenin . the primary subcellular localization of -catenin is in the adherence junctions . at the junction sites , -catenin is bound to e - cadherin and -catenin and functions to link the actin cytoskeleton to the plasma membrane . the adherence junction complex is dynamically regulated by the association of actin , - and -catenins , cadherin , and also actin - bundling proteins such as actinin and vinculin , which was demonstrated in vitro and on isolated cadherin - containing membrane patches . at the resting stage , these free -catenins are captured by adenomatous polyposis coli ( apc ) and are phosphorylated by glycogen synthase kinase 3 ( gsk3 ) and then ubiquitinated and consigned to proteasomal degradation . when the canonical wnt pathway is activated by the binding of wnt to the membrane receptor , this degradation pathway is inactivated due to the inhibition of -catenin phosphorylation . this results in a high level of the free molecules in the cytoplasm and leads to their nuclear accumulation . in the nucleus , -catenin interacts with the transcription factor lef-1 , which activates transcription of the target genes . thus , the key event in wnt signal transduction is the molecular transport of -catenin from the cytoskeleton to the nucleus . this finding is a remarkable example of cytoskeleton - modulated regulation of nuclear functions , which is mediated by the direct translocation of a cytoskeletal component into the nucleus . zyxin , a 61 kda integrin - associated focal adhesion protein , possesses a functional nes and shuttles between the cytoplasm and the nucleoplasm [ 25 , 80 ] . zyxin promotes nuclear translocation of active akt and participates in antiapoptotic cell survival of cardiomyocytes . zyxin is also translocated into the nucleus in response to mechanical stress and regulates mechanosensitive gene expression . one of the zyxin - family proteins lipoma - preferred partner ( lpp ) , which is predominantly localized to cell adhesion sites and interacts with the tumor suppressor protein scrib , was shown to shuttle into the nucleus in a similar fashion as zyxin . lpp interacts with , and functions as a coactivator of , the transcription factor pea3 , which plays important roles in development and oncogenesis . while many reports describe nuclear localization and functions of actin - related cytoskeletal proteins , several other types of cytoskeletal proteins may also shuttle into the nucleus . in many organisms , including yeast and most fungi , the nuclear membrane does not disassemble during mitosis . in yeast , the -tubulin homologue tub4p is actively imported into the nucleus to form a spindle pole body on the nucleoplasmic side . this nuclear transport is mediated by spc98p , an nls - containing component of the yeast -tubulin complex . a study on fungi has demonstrated that tubulin is excluded from interphase nuclei but is present in mitotic nuclei . live observation of gfp - coupled tubulin revealed that tubulin enters the nucleus seconds before the formation of the mitotic spindle and is removed from the nucleus at the m - to - g1 transition . immunofluorescence analyses of several normal and cancer - derived human cells have revealed ii - tubulin , but not other isotypes , in the nucleoplasm as well as the cytoplasm only in the cancer cell lines . ii - tubulin accumulates in the nucleus after taxol treatment , and it associates with the notch 1 receptor intracellular domain ( n1ic ) , the activated form of notch 1 receptor . it has also been reported that nuclear -tubulin coprecipitated with rad51 and colocalized with rad51 in dna repair foci after treatments with various dna - damaging reagents . though it has not been fully revealed whether nuclear tubulins are a general phenomenon for normal cells , these findings suggest roles for nuclear tubulins in various nuclear events . vimentin , one of the building blocks of type - iii intermediate filaments ( ifs ) , has also been found in the nucleus . vimentin directly interacts with dna through its arg - rich n - terminal head domain . interestingly , coinjection of fitc - labeled vimentin with various single - stranded oligodeoxyribonucleotides or double - stranded circular dna resulted in the nuclear accumulation of fluorescently labeled vimentin . this n - terminal dna binding domain is responsible for the morphological changes of the nucleus induced by the human immunodeficiency virus ( hiv ) type-1 protease , suggestive of the role of nuclear vimentin as a scaffold for chromatin organization . more recently it was reported that vimentin upregulates the expression of p21waf1 , a cyclin - dependent kinase inhibitor in neuroblastoma , and inhibits osteocalcin gene expression in association with activating transcription factor-4 ( atf4 ) in osteoblasts . thus , the direct dna binding property of vimentin suggests that its role in chromatin organization extends to transcriptional regulation . there is also emerging evidence that other cytoplasmic if proteins , such as keratins , localize to the nucleus . considering their conserved molecular structure and polymerization mechanisms , it is conceivable that not only vimentin but also various if components and their accessory proteins can pass through the npc . cytoplasmic ifs are known to cooperatively function to provide mechanical strength to the cells , raising the intriguing possibility that those if factors in the nucleus interact and cooperatively function with nuclear lamins , the nuclear - specific if . the nucleoplasm is separated from the cytoplasm by the nuclear envelope , which harbors the npc as a selective channel / barrier for macromolecules . the npc is an octameric structure composed of ~30 different subunits termed nucleoporins ( nups ) . the nups facing the inner surface of the pore are rich in hydrophobic phenylalanine - glycine repeat motifs ( fg - nups ) that are expected to form a meshwork of hydrophobic barriers inside the pore [ 91 , 92 ] . water , ions , and molecules smaller than ~40 kda seem to pass through the pore by passive diffusion . transportation of larger proteins often requires the help of karyopherins , a family of transport mediators . karyopherins interact with their specific cargos to be transported ; for example , the importin and complex recognizes and imports nls - containing proteins , and crm1 exports nes - containing proteins . these two pathways , passive diffusion and karyopherin - mediated transport , are considered to be the two major mechanisms for molecular transport through the npc . nuclear transport serves as a critical regulatory step for signal transduction from the cytoplasm to the nucleus in some cases . for example , nf-b , a transcriptional factor involved in multiple signaling events related to cell differentiation , apoptosis , and immune responses , is rapidly imported into the nucleus in response to various stimuli . in the tnf-stimulated transport of nf-b p50/p65 heterodimer into the nucleus , importin recognizes the nf-b nls only when nf-b is released from its negative regulatory binding partner , ib . in another case , phosphorylation of the ser385 residue of epstein - barr virus nuclear antigen 1 ( ebna-1 ) upregulates the nls - dependent nuclear import of this molecule , while phosphorylation of ser386 and ser383 downregulates it . these findings illustrate diverse ways with which the nuclear transport of functional molecules is precisely regulated . it may also be reasonable to predict that in cells that reform the nuclear membrane upon completion of mitosis , some cytoskeletal molecules can bind to nuclear material ( e.g. , chromatin ) during mitosis and thereby be passively localized to the interphase nucleus . this may partially contribute to the nuclear localization of some cytoskeletal proteins , especially the proteins with many nuclear interactors such as actin . however , we believe that there should be a transport pathway through the npc to regulate their nuclear populations . considering the case of yeast cells , in which the nuclear membrane does not break down during mitosis , and of terminally differentiated somatic cells that have exited the cell cycle , the nuclear population can not be maintained without transport through the nuclear envelope . though it is not proven , it is posited that nuclear transport is a significant , practical mechanism to regulate nuclear populations of cytoskeletal proteins , even for the constitutively - nuclear proteins . in the following we focus on the molecular mechanisms underlying nucleocytoplasmic shuttling of cytoskeletal proteins through the npc and propose possible regulatory factors determining their nuclear localization ( figure 1 ) . while it is certainly true that karyopherin - dependent active transport contributes to the nuclear localization of many nuclear proteins , the nuclear transport mechanisms of cytoskeletal proteins , in particular , have not yet been fully elucidated . nuclear export , rather than import , is shown to play a central role for regulating the nuclear localization of several cytoskeletal proteins , such as actin , actinin-4 , arp5 , and zyxin [ 1 , 2 , 25 , 98 ] . in some cases karyopherins there is strong evidence that exportin 6 ( exp6 ) critically functions in the nuclear export of actin . interestingly , exp6 recognition of actin is significantly enhanced by the formation of an actin - profilin complex , suggesting a novel role of profilin as a cofactor for the nuclear export of actin . apc itself shuttles between the cytoplasm and the nucleoplasm and enhances active export of -catenin to the cytoplasm in the absence of wnt stimulation . leucine zipper tumor suppressor 2 ( lzts2 ) and ran - binding protein 3 ( ranbp3 ) were also found to function as nuclear exporters of -catenin [ 100 , 101 ] . taken together , nuclear localization of several representative cytoskeletal proteins is largely determined by controlling their nuclear export . there is increasing evidence of karyopherin - independent nuclear transport of several cytoskeletal proteins . whereas it is generally accepted that molecules smaller than the size limit for passive diffusion through the npc ( conventionally around 40 kda ) , such as monomeric actin and some arps , may freely migrate into the nucleus , some of the nuclear - localizing cytoskeletal proteins are substantially larger than the size limitation and are nevertheless able to pass through the npc in a karyopherin - independent manner . for example , in vitro nuclear transport assays using semipermeabilized cells demonstrated that -catenin and actinin-4 can pass through the npc in a karyopherin - independent manner [ 2 , 102 , 103 ] . these proteins possess an amphiphilic structural domain in common : an armadillo repeat for -catenin and sr for actinin-4 , which both play an essential role in their nuclear migration . although the precise mechanism of the molecular transport through the npc is the subject of continuing debate , surface hydrophobicity , promoting interactions with hydrophobic fg - nups , is a critical factor . this was demonstrated with bovine serum albumin ( bsa ) , a protein not normally found in the nucleus . chemical modification of bsa using several hydrophobic moieties on the surface significantly facilitated nuclear transport of the modified protein , demonstrating the role of surface hydrophobicity in the protein 's ability to traverse the npc . by inference , cytoskeletal proteins with amphiphilic domains may adapt to the hydrophilic environment inside the npc and spontaneously migrate into the nucleus . we speculate that this spontaneous migration will be the third mode of molecular transport through the npc , which acts through the amphiphilic property of the molecule . we suggest three possible mechanisms for the nucleocytoplasmic transport of cytoskeletal proteins through the npc : passive diffusion , karyopherin - dependent active transport , and karyopherin - independent spontaneous transport . in vivo , the nucleocytoplasmic distribution of a molecule is determined by the combination of these three factors ( figure 1(a ) ) . it is reasonable to assume that passive diffusion and spontaneous transport permit bidirectional molecular translocation , and karyopherin - dependent directional transport drives the differential distribution of the molecule . for example , in the case of actinin-4 , the amphiphilic sr enables bidirectional spontaneous transport of this molecule through the npc in addition to crm1-dependent nuclear export . this combination of two driving forces not only results in the predominant localization of actinin-4 in the cytoplasm but also allows a small population of the protein in the nucleus . it may be possible to predict similar covert nuclear localizations of proteins by focusing on the size , shape , and hydrophobicity of the molecules . to fully understand the nuclear localization of cytoskeletal proteins , it is also important to consider their accessibility to the npc . in light of the studies we have mentioned in this review , we propose the following regulatory steps of nucleocytoplasmic shuttling of cytoskeletal proteins : ( 1 ) change in the amount of npc - accessible molecules in the cytoplasm , ( 2 ) translocation through the npc , and ( 3 ) retention in the nucleus ( figure 1(b ) ) . the first step includes the changes in the equilibrium of soluble / polymeric molecules , active release from the cytoskeleton , and the inhibition of the degradation system in the case of -catenin under wnt stimulation , which results in the increase of npc - accessible free molecules in the cytoplasm . if the molecule can passively diffuse or spontaneously migrate through the npc , molecules in the cytoplasm and nucleoplasm reach a dynamic equilibrium via bidirectional transport , whereas the association with karyopherins strongly influences the differentially localized steady - state concentrations . retention or release rate of the molecule from a nuclear binding partner may serve as another factor regulating the number of npc - accessible molecules in the nucleoplasm . these three steps can independently modulate the ratio of molecules in the nucleus versus the cytoskeleton . in the case of arp4 in yeast , associations with nuclear factors seem to play a central role in the dominant nuclear localization of this protein . mutation in the putative nls in arp4 did not affect its nuclear targeting , suggesting that arp4 can passively diffuse through the npc , and the binding of arp4 with nuclear proteins prevents its escape from the nucleus . if the concentration of cytoplasmic free molecules is lowered by incorporation into the newly organized cytoskeleton , for example , the result may be the loss of npc - accessible free molecules in the cytoplasm , rebalance of molecular distribution between nucleoplasm and cytoplasm by the molecular transport through the npc , and a subsequent decrease in the population of molecules in the nucleus . we expect that this molecular dynamics , linking cytoplasmic and nuclear functions , may be especially relevant to the regulation of nuclear functions . transmission of environmental signals to the nucleus is fundamentally important for cells in various states to properly organize a response . in contrast to the normal nuclear proteins that are consistently retained in the nucleus , transnuclear signaling molecules should dynamically change their nucleocytoplasmic localization in response to the specific signals . we expect that nuclear shuttling and reorganization of nuclear cytoskeletal proteins will emerge as central to nuclear responses to external signals , just as they are central to transduction of those cues from the plasma membrane . here we propose a model of cytoskeleton - modulated regulation of nuclear functions that are mediated by translocation of cytoskeletal components to the nucleus through the npc ( figure 1 ) . we predict that the dynamic distribution of many of these proteins will depend predominantly on their association with nuclear complexes and on active export by karyopherins . ultrastructural mapping by immunoelectron microscopy revealed the nuclear localization of various cytoskeletal proteins in several foci or territories and also showed somewhat different localization patterns between hela cells and lymphocytes . such structural analyses will broaden our understanding of the role of cytoskeletal proteins in the nucleus . at the same time , a computational approach will help to specify the criteria for their passage through the npc , which , especially for karyopherin - independent transit , remains largely a mystery . silencing of arp4 in arabidopsis results in strong pleiotropic phenotypes such as altered organization of plant organs , early flowering , delayed flower senescence , and high levels of sterility . together with the studies on yeast and mammalian cells , these findings suggest that cytoskeletal proteins are generally involved in various nuclear events in virtually all eukaryotes . considering the roles of the bacterial actin homologue mreb in a wide variety of biological events such as cytoskeletal organization , cell shape maintenance , cell wall biosynthesis , viral dna replication , and gene expression [ 111115 ] , it is no wonder that actin and other associated proteins play roles in both the nuclear and cytoplasmic compartments in eukaryotic cells . furthermore , we suggest that such cytoskeletal proteins may act to functionally link the nucleoplasm and cytoplasm to organize integrated cellular activities that are required for both basal cellular events and for responses to specific environmental changes . while the focus of this review has been on the presence and regulation of cytoskeletal proteins in the nucleus , we would like to extend the discussion to nuclear proteins in general . since all proteins are synthesized in the cytoplasm , all nuclear proteins should pass through the npc , at least once . bioinformatic analysis on whole yeast proteins revealed that only about 57% of steady - state nuclear proteins are predicted to use the classical nuclear import pathway , whereas the remaining 43% may use other mechanisms to enter the nucleus . it also became apparent in that analysis that 40% of cytoplasmic proteins possess nls consensus sequences , suggesting a limited influence of the classical nls in the nuclear localization of molecules in vivo . the idea of export - based regulation of nuclear proteins may extend to other nuclear proteins , and to a general concept for understanding nuclear localization of proteins . this active and selective nuclear export system may also act to remove unneeded / harmful proteins from the nucleus . in contrast to the well - known cytoplasmic ubiquitin - proteasomal system , quality control of proteins in the nucleus is not well understood . recently , a nuclear - specific proteolytic pathway has been identified , and enzymes catalyzing the steps of the ubiquitin - proteosomal degradation in the nucleus are being characterized in both yeast and humans [ 117 , 118 ] . thus , the nuclear border may be leakier than thought , and the nucleus appears to have two options for the clearance of unwanted proteins : export or degradation . currently , nuclear actin and -catenin are two leading examples of constitutively required and specific signal - dependent cytoskeletal molecules , respectively , that shuttle and function in the nucleus . considering the structural and dynamic features of cytoskeletal organizations , it is plausible that various types of cytoskeletal proteins shuttle through the npc and cooperatively function in the nucleus . it is becoming apparent that cytoskeletal proteins in the nucleus play indispensable roles in a variety of nuclear events , even though their nuclear population is quite small . we suggest that there is a need to reconsider the potential functions of apparently cytoplasmic proteins in the nucleus , which may result in the discovery of their hidden talents .

various nuclear functional complexes contain cytoskeletal proteins as regulatory subunits ; for example , nuclear actin participates in transcriptional complexes , and actin - related proteins are integral to chromatin remodeling complexes . nuclear complexes such as these are involved in both basal and adaptive nuclear functions . in addition to nuclear import via classical nuclear transport pathways or passive diffusion , some large cytoskeletal proteins spontaneously migrate into the nucleus in a karyopherin - independent manner . the balance of nucleocytoplasmic distribution of such proteins can be altered by several factors , such as import versus export , or capture and release by complexes . the resulting accumulation or depletion of the nuclear populations thereby enhances or attenuates their nuclear functions . we propose that such molecular dynamics constitute a form of cytoskeleton - modulated regulation of nuclear functions which is mediated by the translocation of cytoskeletal components in and out of the nucleus .

organizations increasingly use teams to do their work which was traditionally given to individuals ( 1 ) . the reason why organizations seek teamwork is that teams can , in most cases , be more successful than individuals who work alone . more effective and better decisions can be made when people work together ( 2 ) . moreover , team works are often more efficient and more effective than individual work ( 3 ) . in addition , using teams can result in increased safety ( 4 ) , improved people s attitudes , and decreased number of absentees ( 5 ) . there is enough evidence on the importance of people s participation in health care teams ( 6 ) . teams in health care organizations are made up of physicians , nurses and other health technicians who officially work to achieve organizational goals ( 7 ) . the basic idea is that patient s health and safety is not only a function of complex treatments and advanced therapeutic technologies but also a function of a degree based on which health care professionals fulfill their duties effectively as a team ( 8) . in addition to promoting mutual support and understanding among members of the health care teams , multidisciplinary teams have the potentials to improve relationships , increase efficiency and coordination and finally improve patient s health ( 9 ) . although there is not much documentation about the evaluation of individual s team skills in health care section ( 6 ) , evaluating teams is an important tool in order to increase efficiency and productivity of teams ( 10 ) . in this case , what is agreed upon is that its members must experience sufficient time and togetherness so as to rate the team s performance in terms of six scales : 1 ) teamwork 2 ) decision - making 3 ) leadership support 4 ) trust and respect 5 ) recognition and reward 6 ) focusing on customers ( 2 ) . different studies show the importance of teams and their effective roles in achieving the goals in health care organizations . brewer and mendelson suggested that multidisciplinary teams were necessary but not enough to be effective . in addition , effective teams need both integrity and diversity ( 11 ) . as well as , nancarrow et al highlighted ten basic effective features of multidisciplinary team working ( 12 ) . in their research on developing and testing self - evaluation tools for cancer improvement multidisciplinary teams , taylor et al stated that self - evaluation of team performance could lead to the development of multidisciplinary teams ( 13 ) . as there is scant evidence regarding the assessment of hospital teams in iran and due to researches on team performance in non - hospital sectors in other countries , we decided to conduct this research because knowing the attitudes of team members in an iranian context can have a significant effect on managing health care teams and the resultant would be better performance . since health care teams present health care in the form of hospital committees in iran hospitals and concerning the effective roles of these teams at hospitals , the aim of this research was to determine the attitudes towards team working among hospital committee members in kerman hospitals . the study population consisted of 171 members of clinical teams in committees of 4 educational hospitals in kerman , iran . the 30 item testing team attitudes questionnaire ( t - taq ) was used to collect data that was jointly proposed in 2008 , as team strategies & tools to enhance performance & patient safety ( team stepps ) , by the american agency for healthcare research and quality and the american department of defense as the american national standard ( 14 ) . this questionnaire has 5 domains in titled : team structure , leadership , situation monitoring , mutual support , communication , in which each domain includes 6 questions . respondents were asked to rate the statements on a five - point scale ranging from 1 to 5 ( 1 strongly agree and 5 strongly disagree ) . the total score of all five domains determined the team work attitude of the hospital committees . external , formal and content validity and reliability of this questionnaire were determined and confirmed in a study by najafi et al in 2012 ( 15 ) . the study population consisted of 220 medical personnel who were members of at least one hospital committee . all questionnaires were completed anonymously during the committee meetings and confidentiality of the data was maintained . to collect data , a written permission was obtained from hospital authorities and kerman university of medical sciences ( kums ) . data were analyzed using descriptive statistics , statistical tests , t - test , anova , and linear regression . of 171 personnel who participated in this study , 111 participants ( 64% ) were women and 144 ( 84% ) participants were married . the average age of participants was 36 years ( sd=7.9 ) , and the average work experience was 10.5 years ( sd = 7.7 ) . 102 participants ( 60% ) worked in the health care field and 69 participants worked ( 40% ) in the administrative field . the total mean score of hospital committees regarding team attitude was 3.9 out of 5 ( sd = 0.31 ) . of all sub - criteria of team attitude , leadership and mutual support had the highest and lowest scores respectively ( 4.9 and 3.7 ) . the mean score of hospital committees in terms of team attitude in all five areas was as follows : leadership 4.19 ( sd = 0.4 ) ; monitoring status 4 ( sd = 0.5 ) ; team structure 3.8 ( sd = 0.4 ) ; communication 3.8 ( sd = 0.5 ) ; and mutual support 3.7 ( sd = 0.5 ) . mean score of teamwork attitude questions the highest team attitude score was related to afzalipour hospital , while the lowest score was given to shahid beheshti hospital and shafa hospital ( table 2 ) . teamwork attitude s mean score in kerman hospitals table 3 shows the relationship between team work attitude sub - scales and variables such as employment , work experience , age , gender , education , marital status , and responsibility . regression analysis indicated that responsibility was the most important factor ( = -0.184 , p = 0.024 ) . among the sub - scales of team work attitude , employment , marital status , and responsibility marital status played a role in leadership ; responsibility had a role in situation monitoring ; and work experience had a role in domains of communication and mutual support . positive attitudes towards behaviors related to effective teamwork and safety among nurses and surgeons were also reported by flin et al ( 16 ) . among the five teamwork attitude sub - scales , this shows that participants had a good attitude towards the role of leadership in health care teams . we can conclude that the team leader had the ability to coordinate activities appropriately , was sure that care programs were completely understood and care duties were fulfilled properly . similarly , edmondson mentioned the proper role of cardiologists in leading health care teams . facilitating communications among team members and developing the art of correct communication among team members , he improved their performance ( 17 ) . in a study carried out by mercer et al , suitable communication i.e. transmitting information between the members of a team or between them and the patients was reported ( 18 ) . by the same token , kilner et al showed suitable communication in a team and positive attitude towards teamwork . in this study , good communication in hospital teams in emergency centers not only improved patients and employees satisfaction , but also reduced errors and improved patient s safety ( 19 ) . in a study by christian et al on patient s safety in the operating rooms , lack of communication and sharing information were two main factors which risked the patient s safety ( 20 ) ; moreover , committee members had a moderate attitude towards communication . campion et al reported that the aim of organizational support is to achieve goals ( 21 ) . according to friedlander , organizational support is having clear objectives and a proper combination of experience , skills and sufficient resources ( 22 ) . monitoring members of a team and their knowledge of conditions of their team members and patients caused people to have more effective roles in their teams ( 23 ) . according to a study by loughry et al , monitoring a team member by his teammates was considered as a benchmark for his effectiveness ( 24 ) . in the present study , however , the participants had a moderate attitude in this regard . participants attitude towards mutual support had the lowest score in the present study . however , mutual support reduced workload and increased patients safety ( 25 ) . in a study by liebman and hyman , health care providers who needed more support were more willing to talk with patients about errors , to answer questions and to express their feelings ( 26 ) . poor attitude of members towards mutual support can be due to cultural aspects of personnel , lack of awareness , and inadequate training . we could also observe that gender and education level had no significant impact on the attitude of team members . this finding is not in line with a study conducted by thomas who showed that team attitude had a significant impact on physicians and nurses ( 27 ) . the results of regression analysis revealed that people who had some responsibilities in health care teams ( compared to those with no responsibility ) had more positive attitude towards their teams . however , curran stated that work experience and age had an impact on the attitude of team members ( 9 ) . the role of responsibility in increasing the team attitude in the present study might be due to in - service training courses held for officials in medical teams . concerning attitude of members of hospital teams towards team structures , responsibility , marital status and area of employment ( health / administrative ) had a significant role . the positive impact of employment on the attitude towards the structure of hospital teams might be due to the attentions of administrative personnel working in teams to establish relationships with other organizations , paying attention to the organization , managing the hospital and their roles in guiding the teams . the role of work experience regarding team attitude towards communication and mutual support could be due to the work experience , having more relationships with others , and having no conflicts . one of the limitations of this study was the small study sample thus ; similar studies with a larger sample size in non - governmental hospitals are recommended . some of the benefits of this research included evaluating the attitude of hospital teams using a team tool to improve performance and patient s safety ( team stepps ) . the efficiency of a health care team and its goals can be achieved if there is a presence of effective communication among health care staff and between members of a team and patients as well as strong team leadership and support within an organization . by training hospital personnel and paying particular attention to main elements of effectiveness in a health care team future studies can be conducted on determining the attitude of health care teams and the relationship between team attitude and productivity , job rotation and patient s safety in iran .

introduction : patients health and safety is not only a function of complex treatments and advanced therapeutic technologies but also a function of a degree based on which health care professionals fulfill their duties effectively as a team . the aim of this study was to determine the attitude of hospital committee members about teamwork in kerman hospitals.methodology:this study was conducted in 2014 on 171 members of clinical teams and committees of four educational hospitals in kerman university of medical sciences . to collect data , the standard team attitude evaluation questionnaire was used . this questionnaire consisted of five domains which evaluated the team attitude in areas related to the team structure , leadership , situation monitoring , mutual support , and communication in the form of a 5-point likert type scale . to analyze data , descriptive statistical tests , t - test , anova , and linear regression were used.results:the average score of team attitude for hospital committee members was 3.9 out of 5 . the findings showed that leadership had the highest score among the subscales of team work attitude , while mutual support had the lowest score . we could also observe that responsibility was an important factor in participants team work attitude ( = -0.184 , p = 0.024 ) . comparing data in different subgroups revealed that employment , marital status , and responsibility were the variables affecting the participants attitudes in the team structure domain . marital status played a role in leadership ; responsibility had a role in situation monitoring ; and work experience played a role in domains of communication and mutual support.conclusions:hospital committee members had a positive attitude towards teamwork . training hospital staff and paying particular attention to key elements of effectiveness in a health care team can have a pivotal role in promoting the team culture .

acute myocardial infarction ( ami ) is a secondary thrombus formation in coronary artery induced by unstable atheromatous plaque rupture and bleeding . the thrombus can further cause coronary artery occlusion , as well as serious and long - term myocardial ischemic and myocardial necrosis [ 15 ] . treatment options for ami include conservative treatment with medicine , thrombolytic therapy in the acute phase , percutaneous coronary intervention , and emergency coronary artery bypass grafting [ 69 ] . the time window for thrombolysis treatment is short and the success rate of infarct - related vessel recanalization remains low . however , pci treatment requires a skilled medical team and is an expensive procedure , thus its availability in primary hospitals is limited . furthermore , 25% of patients have no reflow or slow reflow after infarction - related artery recanalization . emergency coronary artery bypass grafting also requires high technical skill , and very few medical institutions currently are able to implement this technology . therefore , new therapeutic measures are needed to improve the success rate of myocardial infarction rescue . increased neutrophil count has been found in the peripheral blood of ami patients , probably as a result of stress response and local myocardial necrosis . it has been shown that increased leukocytes counts after ami can be used in the prognosis of ami . ami patients with significantly elevated leukocytes count have higher risk of various malignant arrhythmias , acute heart failure , cardiogenic shock , and other acute adverse complications . in ischemic myocardial tissues , leukocytes , especially neutrophils , are activated and aggregated , and the number of infiltrated leukocytes increases significantly with longer duration of myocardial ischemia [ 1517 ] . in addition , the infiltrated leukocytes can induce microthrombosis in coronary arteries by disrupting the functions of endothelial cells [ 1820 ] . insufficient microvascular ischemia reperfusion can further aggravate myocardial ischemia and cause extension of the myocardial infarction area [ 2124 ] . aoki et al . showed that elevated peripheral blood mononuclear cell count serves as an independent predictor for left ventricle remodeling in ami patients . a study of 7651 patients with acute coronary syndromes showed that wbc counts of > 10 000 were associated with increased 30-day and 10-month mortality . further , neutrophil depletion by antiserum reduces ischemic myocardial injury in dogs . taken together , these findings show that leukocyte accumulation in response to myocardial ischemia plays an important role in myocardial injury , and that reduced leukocyte content during this process will help reduce myocardial necrosis . given the relationship between inflammatory response , cytokines production , and ami , we hypothesized that reduced leukocyte count will lead to reduced inflammation , cytokine production , and alleviation of ami lesions . here , we decreased leukocyte counts by hu treatment in a rat ami model to interrupt the inflammatory reaction process . fifty male wistar rats aged 810 weeks and weight 180220 g were purchased from the experimental animal center of shandong university school of medicine , china . the 50 rats were divided into an ami model group with 38 rats and a normal control group with 12 rats . briefly , rats were anesthetized with pentobarbital sodium ( 3040 mg / kg , intraperitoneal injection ) and ventilated mechanically . the heart was then exposed and a ligature using a 3 - 0 prolene was then placed around the proximal portion of the left anterior descending artery ( lad ) . a similar sham operation was performed on rats in the normal control group , except no suture of the lad was performed . two out of the 38 rats used for the construction of ami models died during the process . the remaining 36 ami rats were randomly divided into the hu group and vehicle control ami group ( vehicle ami group ) with 18 rats in each group . for rats in the hu group , this study was approved by the institutional animal use and care committee of the shandong university school of medicine , china . two milliliters ( ml ) of venous blood was harvested from the angulus venosus of the rats and collected in tubes containing 0.109 ml sodium citrate as anticoagulant . after mixing fully , the blood samples were used for the determination of total leukocyte , neutrophil , and leukomonocyte counts using a sysmex xs800i automated hematology analyzer ( sysmex corporation , japan ) . serum sicam-1 level was examined by enzyme - linked immunosorbent assay ( elisa ) ( dia - clone , france ) . serum p - selectin was measured using an elisa kit ( america r&d , usa ) . . electrocardiography ( ecg ) was performed before ami construction and immediately after the successful induction of ami , with a 12-lead ecg instrument ( decg-03a , mindray medical international limited , china ) . four weeks after ami induction , heart function index was measured using a philips sonos 5500 multi - function color ultrasonography device ( philips , usa ) with an 8-mhz transducer . the following equations were used according to the american society of echocardiology ( ase ) : lvm left ventricular mass ; ivsd : interventricular septal end - diastolic dimension ; lvedd left ventricular end - diastolic dimension ; lvpwd left ventricular end - diastolic posterior wall dimension ; sv stroke volume ; lvevd diastolic left ventricular volume ; lvevs systolic left ventricular end volume ; co cardiac output ; hr heart rate ; lvef left ventricular ejection fraction ; lvedv left ventricular end - diastolic volume ; lvesv left ventricular end - systolic volume ; fs fraction shortening ; lvesd left ventricular end - systolic diameter . four weeks after the successful construction of the ami rat model and the cardiac function index measurement , hearts of the rats were harvested and fixed in 4% paraformaldehyde solution . the slices were then stained with hematoxylin and eosin ( he ) and changes of infarction areas were observed under a microscope ( olympus bx51 , japan ) . statistical analysis was conducted using spss 17.0 software , and the results are presented as mean standard deviation ( xsd ) . fifty male wistar rats aged 810 weeks and weight 180220 g were purchased from the experimental animal center of shandong university school of medicine , china . the 50 rats were divided into an ami model group with 38 rats and a normal control group with 12 rats . briefly , rats were anesthetized with pentobarbital sodium ( 3040 mg / kg , intraperitoneal injection ) and ventilated mechanically . the heart was then exposed and a ligature using a 3 - 0 prolene was then placed around the proximal portion of the left anterior descending artery ( lad ) . a similar sham operation was performed on rats in the normal control group , except no suture of the lad was performed . two out of the 38 rats used for the construction of ami models died during the process . the remaining 36 ami rats were randomly divided into the hu group and vehicle control ami group ( vehicle ami group ) with 18 rats in each group . for rats in the hu group , this study was approved by the institutional animal use and care committee of the shandong university school of medicine , china . two milliliters ( ml ) of venous blood was harvested from the angulus venosus of the rats and collected in tubes containing 0.109 ml sodium citrate as anticoagulant . after mixing fully , the blood samples were used for the determination of total leukocyte , neutrophil , and leukomonocyte counts using a sysmex xs800i automated hematology analyzer ( sysmex corporation , japan ) . serum sicam-1 level was examined by enzyme - linked immunosorbent assay ( elisa ) ( dia - clone , france ) . serum p - selectin was measured using an elisa kit ( america r&d , usa ) . activity of paf was also analyzed by elisa kit ( rapid bio , usa ) . electrocardiography ( ecg ) was performed before ami construction and immediately after the successful induction of ami , with a 12-lead ecg instrument ( decg-03a , mindray medical international limited , china ) . four weeks after ami induction , heart function index was measured using a philips sonos 5500 multi - function color ultrasonography device ( philips , usa ) with an 8-mhz transducer . the following equations were used according to the american society of echocardiology ( ase ) : lvm left ventricular mass ; ivsd : interventricular septal end - diastolic dimension ; lvedd left ventricular end - diastolic dimension ; lvpwd left ventricular end - diastolic posterior wall dimension ; sv stroke volume ; lvevd diastolic left ventricular volume ; lvevs systolic left ventricular end volume ; co cardiac output ; hr heart rate ; lvef left ventricular ejection fraction ; lvedv left ventricular end - diastolic volume ; lvesv left ventricular end - systolic volume ; fs fraction shortening ; lvesd left ventricular end - systolic diameter . four weeks after the successful construction of the ami rat model and the cardiac function index measurement , hearts of the rats were harvested and fixed in 4% paraformaldehyde solution . the slices were then stained with hematoxylin and eosin ( he ) and changes of infarction areas were observed under a microscope ( olympus bx51 , japan ) . statistical analysis was conducted using spss 17.0 software , and the results are presented as mean standard deviation ( xsd ) . to verify that the ami model was successfully constructed , electrocardiograms ( ecg ) were measured in rats before and after ami construction . ecg of rats before ami is illustrated in figure 1a , which shows that the st segment was at baseline on lead i and avl . in comparison , the ecg of ami rats ( figure 1b ) showed significant elevation of the st segment on lead i and avl ( 0.5 mv ) . leukocytes , neutrophils , and leukomonocytes were measured 4 weeks after ami construction and hu administration , and the results are presented in table 1 . leukocytes , neutrophils , and leukomonocytes in the vehicle ami group were significantly higher than in the normal control group ( p<0.05 ) , but hu treatment significantly decreased their levels compared to the vehicle ami group ( p<0.05 ) . no significant difference was found between the hu ami group and the normal control group ( p>0.05 ) . the level of sicam ( ng / ml ) , p - selectin ( ng / ml ) and paf ( 10 ) measured by elisa 4 weeks after ami construction and hu administration are shown in table 2 . sicam-1 , p - selectin , and paf level were significantly higher in the vehicle ami group compared with the normal control group ( p<0.05 ) . however , their levels were deceased by hu treatment in the hu ami group compared with the vehicle ami group ( p<0.05 ) . no significant difference was found between the hu ami group and the normal control group ( p>0.05 ) . cardiac function was examined with echocardiography 4 weeks after ami induction ( table 3 ) . lvesd and lvedd levels were significantly higher in the vehicle ami group than in the normal control group ( p<0.01 ) , hu treatment significantly decreased the levels compared with the vehicle ami group ( p<0.05 ) . however , the measurements in the hu ami group were still higher than in the normal control group ( p<0.05 ) ( table 3 ) . lvef and fs percentage was significantly lower in the vehicle ami group than in the normal control group ( p<0.05 ) . hu treatment significantly increased the percentage compared with the vehicle ami group ( p<0.05 ) , but levels in the hu ami group were still lower than in the normal control group ( p<0.05 ) ( table 3 ) . figure 2 shows the representative ultrasonic image of rats from the 3 groups . in the normal control group , cardiac muscle thickness ( figure 2a , red arrow ) and ventricular wall motion were normal , and there was no obvious segmental motion ( figure 2a , green arrow ) . in the vehicle ami group , the cardiac muscle became thinner and the ventricular wall motion in the infarction regional was low ( figure 2b ) . in the hu ami group , cardiac muscle became slightly thicker , and the motion of the infarction regional was slightly lower than in the vehicle group ( figure 2c ) . myocardial tissue was harvested from rats of the 3 groups and observed under a microscope after he staining . as shown in figure 3a , there were no obvious infarcts , and the myocardial nuclei and the myocardial fibers were uniformly arranged and orderly in the normal control group . no infiltration of inflammatory cells in myocardial interstitium was observed ( figure 3a ) . in the vehicle ami group , obvious swelling in necrotic foci of the heart muscle fiber was observed , and the arrangement of nuclei was disordered . there was a considerable amount of infiltration of inflammatory cells in myocardial interstitium ( figure 3b ) . in the hu ami group , nuclear staining and size of focal necrosis of myocardial tissues and residual cardiomyocytes were uniform , and myocardial fiber tissue showed mild swelling . a small amount of fibroblasts and inflammatory cell infiltration was observed ( figure 3c ) . to verify that the ami model was successfully constructed , electrocardiograms ( ecg ) were measured in rats before and after ami construction . ecg of rats before ami is illustrated in figure 1a , which shows that the st segment was at baseline on lead i and avl . in comparison , the ecg of ami rats ( figure 1b ) showed significant elevation of the st segment on lead i and avl ( 0.5 mv ) . leukocytes , neutrophils , and leukomonocytes were measured 4 weeks after ami construction and hu administration , and the results are presented in table 1 . leukocytes , neutrophils , and leukomonocytes in the vehicle ami group were significantly higher than in the normal control group ( p<0.05 ) , but hu treatment significantly decreased their levels compared to the vehicle ami group ( p<0.05 ) . no significant difference was found between the hu ami group and the normal control group ( p>0.05 ) . the level of sicam ( ng / ml ) , p - selectin ( ng / ml ) and paf ( 10 ) measured by elisa 4 weeks after ami construction and hu administration are shown in table 2 . sicam-1 , p - selectin , and paf level were significantly higher in the vehicle ami group compared with the normal control group ( p<0.05 ) . however , their levels were deceased by hu treatment in the hu ami group compared with the vehicle ami group ( p<0.05 ) . no significant difference was found between the hu ami group and the normal control group ( p>0.05 ) . cardiac function was examined with echocardiography 4 weeks after ami induction ( table 3 ) . lvesd and lvedd levels were significantly higher in the vehicle ami group than in the normal control group ( p<0.01 ) , hu treatment significantly decreased the levels compared with the vehicle ami group ( p<0.05 ) . however , the measurements in the hu ami group were still higher than in the normal control group ( p<0.05 ) ( table 3 ) . lvef and fs percentage was significantly lower in the vehicle ami group than in the normal control group ( p<0.05 ) . hu treatment significantly increased the percentage compared with the vehicle ami group ( p<0.05 ) , but levels in the hu ami group were still lower than in the normal control group ( p<0.05 ) ( table 3 ) . figure 2 shows the representative ultrasonic image of rats from the 3 groups . in the normal control group , cardiac muscle thickness ( figure 2a , red arrow ) and ventricular wall motion were normal , and there was no obvious segmental motion ( figure 2a , green arrow ) . in the vehicle ami group , the cardiac muscle became thinner and the ventricular wall motion in the infarction regional was low ( figure 2b ) . in the hu ami group , cardiac muscle became slightly thicker , and the motion of the infarction regional was slightly lower than in the vehicle group ( figure 2c ) . myocardial tissue was harvested from rats of the 3 groups and observed under a microscope after he staining . as shown in figure 3a , there were no obvious infarcts , and the myocardial nuclei and the myocardial fibers were uniformly arranged and orderly in the normal control group . no infiltration of inflammatory cells in myocardial interstitium was observed ( figure 3a ) . in the vehicle ami group , obvious swelling in necrotic foci of the heart muscle fiber was observed , and the arrangement of nuclei was disordered . there was a considerable amount of infiltration of inflammatory cells in myocardial interstitium ( figure 3b ) . in the hu ami group , nuclear staining and size of focal necrosis of myocardial tissues and residual cardiomyocytes were uniform , and myocardial fiber tissue showed mild swelling . a small amount of fibroblasts and inflammatory cell infiltration was observed ( figure 3c ) . increased leukocyte count is part of the inflammation process and participates in the pathogenesis of ami , further affecting the prognosis of ami patients . our results show that the level of inflammatory cytokines , amount of leukocyte infiltration into myocardial interstitium , and cardiac function index were all reduced by hu treatment in a rat ami model , suggesting that leukocyte reduction through hu treatment might be a promising preventive and treatment option for ami . why does leukocyte accumulation exacerbate tissue injury in ami ? the underlying mechanism might be as follows : first , leukocyte accumulation and activation increases the viscosity of peripheral blood ; second , leukocyte accumulation causes capillary plugging and the no - reflow phenomenon , thus leading to coronary artery occlusion ; third , migrating leukocytes release proinflammatory mediators , such as lipoxygenase products , free radicals and hydrolytic enzymes , which causes cardiovascular toxicity and myocardial ischemia . the activated neutrophils in the infarct adhere to vascular endothelial cells , affecting the dilation of the coronary artery , increasing the resistance of blood vessels , and further aggravating myocardial ischemia . a feedback loop exists between ami and leukocytes level : increase in leukocytes promotes the development of the ami , and the further development of ami can in turn induce leukocyte accumulation . leukocytes depletion from the systemic circulation by filtering during cardiopulmonary bypass reduces the expression of neutrophil adhesion molecules . this reduction leads to decreased leukocyte deposition and interaction with the vascular endothelium , and thereby decreases neutrophil - mediated injury . a study showed that 27% of capillaries were plugged after perfusion with whole blood , whereas only 1% capillary had no - reflow with leukocyte - depleted blood . however , leukocyte depletion filters are costly and its benefits need confirmation from large - scale randomized prospective studies . due to the foreseeable difficulty in applying leukocyte filters in rats , we did not consider leukocyte filters as a method of leukocyte reduction in our experiments . busulfan , also called myleran , is a bifunctional alkylating agent that is widely used as an alternative to total body irradiation in conditioning therapy for hematopoietic stem cell transplantation . despite its short half - life ( 23 h ) and quick turnover in vivo , due to the decreased immunity in ami rats , we did not consider busulfan as a leukocyte reduction reagent . hu selectively inhibits cells at the s phase and is used in the treatment of chronic myelogenous leukemia . hu distributes rapidly and widely in the body , and concentrates in leukocytes and erythrocytes . hu has a half - life of 1.55 h , and is probably metabolized and also eliminated through the renal pathway . more than 80% of hu will be secreted 12 hours after administration . based on these advantages , we chose hu for leukocyte reduction purposes in our ami rat . ono - ae-248 , another drug candidate , is a selective prostaglandin e2 ( pge2 ) receptor type 3 ( ep3 ) agonist . however , ono - ae-248 has a short half - life , is still under clinical development , and is costly , thus it was not considered by us . metformin was also found to improve cardiac function in a nondiabetic rate model of post - mi heart failure , but it requires long - term administration ( 12 weeks ) . intercellular adhesion molecule-1 ( icam-1 ) , also called cd54 , is an immunoglobulin ( ig)-like cell adhesion molecule . therefore , monocyte counts and serum icam-1 level can be used as a marker for coronary atherosclerosis . p - selectin ( ps ) is a member of the selectin family of adhesion molecules that is expressed in platelets and is stored on the membrane of alpha granules and on the weibel - palade bodies of endothelia cells . p - selectin mediates the adhesion of leukocytes to stimulate endothelial cells and to activate platelets . platelet - activating factor ( paf ) is a phospholipid mediator and mediates many leukocyte functions , including platelet aggregation . ps and paf function together to promote the adhesion and accumulation of leukocyte to endothelial cells and the subsequent release of inflammatory mediators . our elisa analysis showed that icam-1 , ps and paf were all increased in ami rats , indicating the role of these inflammatory factors in the pathogenesis of ami . further , hu treatment significantly decreased their level , concomitant with an improvement of cardiac function in ami rats . however , the exact molecular mechanism by which these inflammatory factors contribute to ami and how hu down - regulated their expression is unknown . decrease leukocytes count by hu treatment in ami rats can reduce inflammatory reaction significantly and improve heart functions after ami , suggesting that leukocyte reduction through hu treatment might be a promising preventive and treatment option for ami .

backgroundthis study aimed to decrease leukocytes counts by hydroxyurea ( hu ) in an acute myocardial infarction ( ami ) rat model and examine its effect on the inflammatory response of myocardial infarction and cardiac functions.material/methodsami was successfully caused in 36 rats , and 12 control rats received sham operation . rats in the ami group were then randomly divided into hu and vehicle group with 18 rats each . rats in the hu ami group received hu ( 200 mg / kg ) intragastrically while vehicle ami group received saline . leukocytes counts , cardiac functions , myocardial tissue morphology , and levels of soluble intercellular adhesion molecule-1 ( sicam ) , p - selectin and platelet activating factor ( paf ) were measured and compared among the three groups four weeks after ami induction.resultsleukocytes , neutrophils , and leukomonocyte counts in vehicle ami rats were significantly higher than that of the normal control group ( p<0.05 ) . however , hu treatment decreased their counts significantly ( p<0.05 ) . sicam , p - selectin , and paf level in vehicle ami group were significantly higher than those of the normal group , and their level was also decreased by hu treatment ( p<0.05 ) . echocardiography analysis showed that hu treatment increased left ventricular ejection fraction ( lvef ) and left ventricular fractional shortening ( lvfs ) compared to that of vehicle ami group ( p<0.05 ) . histopathological examination showed that hu significantly reduced the swelling of the heart muscle fiber in necrotic foci and the number of inflammatory cells infiltrated into myocardial interstitium compared to vehicle ami group.conclusionsdecrease leukocytes counts by hu significantly reduced inflammatory reaction and improved cardiac functions in ami rats .

road traffic injuries are responsible for the death of 1.23 million people around the world annually ( 1 ) . in terms of disability adjusted life year ( daly ( ( 1 ) . road traffic injuries ranked ninth in 1999 , and are expected to rise to the third place by 2020 ( 2 ) . these events account for 1 - 2% of gross national product ( gdp ) of different countries ( 3 ) . in iran , cost of road traffic injuries is 2.19% of gdp that is significantly higher than the global average ( 4 ) . more than 20% of deaths caused by traffic crashes belong to pedestrians . according to the estimates , about 273000 pedestrians around the world lost their lives due to traffic crashes in 2010 ( 5 ) . in 2013 , 22% of all fatalities as a result of road traffic crashes in the european union(eu ) were fatalities of pedestrians ( 6 ) . studies have shown that most traffic crashes related to pedestrians , particularly in high - income countries , occur in interurban streets and roads ( 7 ) . for example , in the european union countries , 70% of these crashes occur in cities , and this figure is 76% in the u.s . pedestrians are associated with the highest contribution of deaths caused by traffic crashes in the world s densely populated cities . for instance , in mumbai and delhi , pedestrians accounted for 78% and 53% of traffic fatalities , respectively ( 9 ) . according to the statistics of tehran traffic police , more than 6,000 traffic crashes causing injury and more than 100 traffic crashes causing death annually occur in tehran . in fact , more than 40% of deaths from traffic crashes in tehran occur for pedestrians ( 11 ) . given the importance of this issue , in the recent years , many studies have been conducted in different parts of the world , especially in densely populated cities , on the role of environmental and demographic factors in frequency , spatial distribution , and severity of traffic crashes related to pedestrians in urban areas , using different methods of spatial analysis and a variety of statistical models ( 12 - 21 ) . in the recent years , due to the increase in automobile production , traffic load has dramatically increased in urban and suburban streets and roads of iran , while these transport infrastructures have not been developed qualitatively and quantitatively commensurate with car production . since a major proportion of traffic crashes in iran occur due to environmental factors , especially the quality of roads and streets and as the capacity of streets and roads , studying the urban and interurban roads and streets , particularly in densely populated cities with heavy traffic such as tehran , in terms of the environmental factors involved in traffic crashes can be helpful to more accurately identify the environmental factors of such crashes and their contribution . the results of such studies can be used in better planning for reduction of this type of risk factors . hence , this study aimed to determine the high - risk areas and spatially analyze the traffic crashes causing death to pedestrians in tehran in 2013 and 2014 . tehran is the largest city and the capital of iran , with an area of more than 612 square kilometers ( 22 ) . tehran is divided into 22 districts , 370 parishes , and 560 traffic zones , with more than 535 km of highway and 445 arterial streets ( 24 ) . statistical analyses were performed by descriptive and inferential statistics in spss and arc - gis . analytic surveys were conducted in the following steps : first , the exact location of traffic crashes causing death to pedestrians was extracted by reviewing the files of crashes in the database of tehran traffic police . second , according to the universal transverse mercator ( utm ) , geographical coordinates of the traffic crashes locations with the accuracy of one to three meters were recorded and saved in a computer . the utm system divides the earth into 60 zones , each 6 of longitude in width and uses a secant transverse mercator projection in each zone . the point of origin of each utm zone is the intersection of the equator and the zone 's central meridian . third , using arc - gis , different layers of geographic information were put on each other and mapping was extracted . finally , global moran s index with euclidean distance method was used to study and analyze the distribution pattern of traffic crashes related to pedestrians in terms of being either cluster or scattered . moran s i index is the most common index used for measuring the spatial autocorrelation to determine how close are the objects compared to other close objects . a strong autocorrelation occurs when the values of a variable that are spatially close to each other are correlated . in other words , if the spatial objects or the values of variables related to them are randomly distributed in space , there must apparently be no relationship between them . moran s index studies the distribution pattern of these spatial objects by considering the values of the studied variable in terms of being cluster or scattered . if the value of moran 's index is equal to + 1 or near + 1 , it implies a complete cluster pattern or existence of autocorrelation . if this value is zero , it means that the pattern is random or multipolar accumulation . finally , if moran 's index is equal to -1 or near -1 , it shows that the crashes follow a scattered pattern . hot spots analysis : for this analysis , global getis - ord g index with euclidean distance method was calculated for each feature . z - score of this index shows that to what extent the studies feature has been distributed cluster - like , which may be statistically significant . in this study , this index was used to display the areas with high , low or moderate frequency of traffic crashes associated with pedestrians . getis - ord g index values were interpreted based on comparing the observed values and the expected values . if the observed values of getis - ord g index in an area are more than the expected values , that area is considered among the hot spots , and if the observed values are less than the expected values , that area is considered among the cold spots . one hundred ninety - eight incidents were studied ; 92 of which , ( 46.4% ) occurred in april 2013 to march 2014 and other 106 cases ( 63.6% ) occurred in april 2014 to march 2015 . the highest and the lowest frequency of crashes belonged to january ( 26 cases ) and june ( 10 cases ) . figure 1 demonstrates the trend of the traffic crashes causing death to pedestrians in 2013 - 2015 in tehran . distribution of fatal crashes associated with pedestrians in tehran by different months : 2013 - 2015 overall , 158 cases ( 79.8% ) of crashes have occurred in tehran highways . azadegan highway , which stretches from northwest to southeast of tehran , had the highest frequency of crashes ( 25 cases ) . during the study period , fatal road traffic injuries that caused the death of pedestrians occurred most frequently in traffic zones located in the west , east and south of tehran . figure 2 depicts the distribution of studied crashes in tehran and their position to highways , and urban to interurban bus terminals . mapping of fatal crashes associated with pedestrians in tehran and their position relative to highways and urban and interurban passenger terminals : 2013 - 2015 among the 22 districts of tehran , districts located in the south , east , and northwest of tehran , as well as districts located in the center of the city , have shown the highest and the lowest frequency of traffic crashes , respectively . figure 3 displays the distribution of studied crashes in the 22 districts of tehran in 2013 and 2014 . mapping of fatal crashes associated with pedestrians in the 22 districts of tehran in 2013 - 2015 result revealed that the density of events in the areas with dominant transportation , industrial and military land use was higher than the areas with dominant residential land use ( fig . 4 ) . during the study period , fatal road traffic injuries that caused the death of pedestrians occurred most frequently in traffic zones located in the west , east and south of tehran . mapping of fatal crashes associated with pedestrians in tehran and their position relative to land use in 2013 - 2015 figure 5 depicts the distribution pattern of traffic crashes causing death to pedestrians in traffic zones . moran s i index value showed that the studied crashes had a cluster - like distribution ( p<0.001 ) . distribution pattern of fatal crashes associated with pedestrians in tehran by traffic areas in 2013 - 2015 the hot spots in tehran in terms of traffic crashes causing death to pedestrians included the western , southern , northern , partly eastern suburbs , and cold spots were mainly located in the central areas of tehran ( fig . getis - ord general g index showed that the distribution of hot and cold spots of the studied crashes was statistically significant ( p<0.001 ) . low and high - risk traffic areas in terms of frequency of fatal crashes associated with pedestrians in tehran in 2013 - 2015 the results of this study revealed that occurrence of the traffic crashes causing death among pedestrians in tehran during different months of 2013 and 2014 does not follow a specific and fixed trend . for instance , the highest and the lowest frequencies of studied crashes in 2013 have been recorded in january - february and october , respectively , while the highest and the lowest frequencies belonged to may and december , respectively . however , it can be generally concluded that the number of crashes in this period during the summer months ( especially june and august ) is fewer than the winter months ( especially january and march ) . since some studies have shown that the frequency of traffic crashes associated with pedestrians increases with increased pedestrian volume ( 10,25,26 ) , these seasonal differences can be attributed to changes in pedestrian volume . since some studies such as those conducted by cloutier , cottrill , green , miranda - moreno , mcarthur et.al found that the number of schools or students is associated with the frequency of traffic crashes associated with pedestrians in urban areas ( 17,26 - 29 ) , school holidays and reduced traffic of students in june and august can be one of the reasons for the reduced frequency of such crashes during these months . therefore , better organization of students traffic and promotion of the safety of the streets near schools can be considered as one of the strategies for reducing the deaths from traffic crashes related to pedestrians in tehran . this study showed that about 80% of the studied crashes have occurred in highways and freeways . highways located at the main western and southern entrances and exits of tehran such azadegan highway ( from northwest to southeast of tehran ) and karaj special road , which connects karaj to 36 km west of tehran had the highest share of such crashes . these results are consistent with the findings of studies conducted by wier et.al in san francisco and mueler et al . in washington that showed that frequency of traffic crashes associated with pedestrians since traffic load in the western and southern highways of tehran is higher than other areas and there is an intense congestion of different means of transport in some of these areas , one reason for the high frequency of fatal crashes in these areas would be the high traffic load and congestion of different means of transportation . therefore , the reasons of this problem need to be identified by conducting more studies . in addition , the necessary measures should be taken to reduce the rate of traffic crashes through organizing the passage of vehicles and pedestrians and separating the passage of light and heavy means of transportation in the western and southern highways of tehran . distribution of studied crashes in terms of proximity to or remoteness from the west ( azadi ) and south ( khazaneh ) terminals showed that the density of traffic crash was relatively high at the end of the roads leading to azadi passenger terminal such as tehran - karaj freeway , karaj special road , afsarieh bridge , and the eastern part of azadegan highway . thus , one of the reasons for the high frequency of traffic crashes causing death to pedestrians in south and west of tehran is the location of azadi and khazaneh passenger terminals in these areas . on the other hand , distribution of the studied crashes in terms of proximity to or remoteness from urban passenger terminals does not follow a specific pattern and the frequency of traffic crashes associated with pedestrians is very low near these passenger terminals , especially those far from interurban passenger terminals . therefore , it can be concluded that the streets and roads around the urban passenger terminals are of low risk in terms of traffic crashes associated with pedestrians . comparison of the 22 districts of tehran in terms of the frequency of fatal crashes involving pedestrians shows that the highest frequency of this type of crashes has occurred in district 2 , which accounts for 13.6% of the total cases , while this district covers only 8% of the total area of tehran . given that most crashes in district 2 have happened on highways , the relative high length of highways could be mentioned as one of the reasons for the higher frequency of traffic crashes associated with pedestrians in district 2 of tehran . highways in this district , with a length of about 61 km , account for about 11% of all highways of tehran . results of this study revealed that high - risk traffic areas in terms of crashes causing death to pedestrians during the studied years are mostly located in the west and south of tehran . high value of moran s i index indicated the distribution of crashes by the 560 traffic areas of tehran followed a cluster - like pattern . in other words , the location of occurrence of these crashes in tehran did not follow a random distribution . in addition , findings demonstrated that the hot spots of the studied crashes were mostly located in the west and south of tehran , while the cold spots were mainly focused in the central parts of the city . since getis - ord general g index showed that the distribution of hot and cold spots of the studied crashes is statistically significant , it can be stated that some certain factors were involved in non - random distribution traffic crashes causing death to pedestrians in tehran and high frequency of these crashes in high - risk areas . due to the environmental and structural differences between the high - risk and low risk areas , which are mainly located in the center of tehran , environmental factors similar studies in other parts of the world , including the studies conducted by slaughter et.al in new york , anderson in london , taquechel et.al in atlanta , wang and kockelman in texas , siddiqui et.al in florida and kuhlmann et.al in colorado , have also shown that environmental factors are highly involved in non - random distribution of traffic crashes related to pedestrians ( 12,14,16,31 - 33 ) . it seems that one of the factors determining the distribution of the studied crashes in tehran , which is the higher frequency of crashes in the suburban areas than the central part of the city , is the difference between drivers and pedestrians in terms of traffic culture . the drivers and pedestrians in central parts of the city have higher education levels and respect traffic regulations more than those in the suburban areas . higher traffic load on the outskirts of the city compared to the downtown was another reason for this issue . enforcement of regulation of traffic limits and alternative passage of vehicles with even or odd license plates in the central parts of tehran causes the central parts of the city to have a lighter traffic load than the margins of tehran . this was one of the few studies in iran that has used the files drawn by police experts for conducting an applied research to determine the high - risk areas in a metropolis . using these files , we recorded the geographical coordinates of the location of crashes and we prepared a map to determine the distribution pattern of traffic crashes causing death to pedestrians and their position relative to other spatial objects . this study was conducted using only the data of crashes registered by tehran traffic police . comparison of the statistics provided by the police and forensic medicine organization showed that the actual number of fatal crashes in tehran is more than those mentioned in the database of tehran traffic police and the information related to some crashes causing death does not exist in this database . therefore , it is not possible to acquire information on their location of occurrence through the police files . in such studies , it is better that the relevancy of the death of the injured by traffic crashes to these crashes be defined based on the time of death after crash . this time in different countries varies between 6 days to one month , and in iran , this time has been defined to be one month . however , due to the lack of consistency between the data provided by tehran traffic police and forensic medicine organization , relevancy of the death of the injured by traffic crashes to these crashes in this study was determined based on the data extracted from data based on tehran traffic police . various districts of tehran , excluding central districts , are considered high - risk areas . therefore , the majority of traffic crashes causing death to pedestrians have occurred on highways located at the main entrance ; that is , districts located in south , east and northwest of tehran . significant cluster - like distribution of crashes by the 560 traffic zones of tehran and existence of hot spots in terms of the studied crashes in the western and southern parts of tehran indicates the decisive role of environmental factors in occurrence of traffic crashes causing death to pedestrians . moreover , it is necessary to organize the pedestrians traffic in high - risk areas . this study was part of a thesis conducted in shahid beheshti university of medical sciences . we would like to sincerely thank all who helped us in this study , especially gen . hosseini , the commander of tehran traffic police , personnel of crashes department of tehran traffic police , particularly lt . soroush , headquarters of public health faculty and members of the epidemiology department of shahid beheshti university of medical sciences .

background : more than 20% of deaths from traffic crashes are related to pedestrians . this figure in tehran , the capital of iran , reaches to 40% . this study aimed to determine the high - risk areas and spatially analyze the traffic crashes , causing death to pedestrians in tehran . methods : mapping was used to display the distribution of the crashes . determining the distribution pattern of crashes and the hot spots/ low - risk areas were done , using moran s i index and getis - ord g , respectively . results : a total of 198 crashes were studied ; 92 of which , ( 46.4% ) occurred in 2013 to 2014 and other 106 cases ( 63.6% ) occurred in 2014 to 2015 . the highest and the lowest frequency of crashes was related to january ( 26 cases ) and june ( 10 cases ) , respectively . one hundred fifty- eight cases ( 79.8% ) of crashes occurred in tehran highways . moran s index showed that the studied traffic crashes had a cluster distribution ( p<0.001 ) . getis- ord general g index indicated that the distribution of hot and cold spots of the studied crashes was statistically significant ( p<0.001 ) . conclusion : the majority of traffic crashes causing death to pedestrians occurred in highways located in the main entrances and exits of tehran . given the important role of environmental factors in the occurrence of traffic crashes related to pedestrians , identification of these factors requires more studies with casual inferences .

dilated cardiomyopathy ( dcm ) is the most common form of primary cardiomyopathy and is a leading cause for heart transplantations approximately 30%50% of affected individuals have a familial form of dcm . to date , at least 40 disease genes have been reported to be associated with familial dcm , in which ; mutations of scn5a gene which encodes the alpha subunit of the major sodium channel in the heart have been demonstrated to result in myocardial damage and dcm [ 4 , 5 ] . previously , scn5a has been implicated in conduction disturbances and several electrophysiological phenotypes [ 6 , 7 ] , including atrioventricular block ( avb ) , long qt syndrome , sick sinus syndrome , sudden infant death syndrome , and atrial fibrillation [ 8 , 9 ] . meanwhile , more and more scn5a mutations were discovered to be related with dcm , such as a1180v , d1275n , w156x , r225w , and r222q . we first reported finding a1180v , a single nucleotide mutation , c.3539c > t , that occurred in exon 20 of the scn5a gene in a three - generation chinese family with age - related dcm and progressive avb in 2008 . in that study , a1180v was detected in all studied patients and in six younger unaffected members of the pedigree . experimental data revealed that a1180v channels exhibited a negative transform of voltage - dependent inactivation of the cardiac sodium channels , which lead to a rate - dependent sodium current reduction , and a moderate increase in late sodium current . correspondingly , our clinical study revealed qrs widening at high heart rates and qtc prolongation at rest in unaffected carriers . these findings suggest that a1180v is a functional mutation that can contribute to the development of dcm . however , data deposited in the dbsnp database show that a1180v has been found in two small sample groups of the asian population . in the first sample group , a1180v ( dbsnp refsnp number , rs41310765 ) was present in a cohort of 47 han people ( r31_hanla ) who were part of the los angeles panel at a carrier frequency of 6.4% . in another sample group ( her_asian - panel ) of 16 individual asians selected from the coriell cell repository , genetic sequencing yielded a carrier frequency of 6.2% ( http://www.ncbi.nlm.nih.gov/projects/snp/snp_ref.cgi?rs=41310765 ) . to date , the data obtained from the dbsnp database show that a1180v is virtually absent in other ethnic groups . thus , these unpublished data suggest that a1180v might be a common variant in asians , which is inconsistent with our previous observations . in order to further evaluate the pathogenic role of a1180v in familial dcm and make sure the frequency of a1180v among healthy han chinese , we followed up the three - generation chinese family and expanded the control size to screen for a1180v . besides , we analyzed its frequency in other database as well . we followed up all the a1180v carriers and noncarriers of the three - generation chinese family for 6 years . all individuals were interviewed with the symptoms of cardiovascular disease , such as typical chest pain , palpitation , cyanosis , and syncope . 12-lead electrocardiogram ( ecg ) and echocardiography were performed on the family members to make a diagnosis . 460 unrelated healthy han chinese living in shanghai , ranging from 20 to 70 years old , were enrolled to the control group from a national high technology research and development program ( the national 863 program ) of china . individuals with known cardiovascular risk factors other than age , gender , and smoking history were excluded . the enrolled participants did not show any abnormalities under comprehensive clinical evaluation , including review of medical history , physical examination , 12-lead ecg , submaximal exercise ecg , and blood biochemical examinations . each of the participants and individuals whose data are included here signed an informed consent form after a complete and clear explanation of the nature and purpose of the study . the study was approved by the local ethics committee of zhongshan hospital , fudan university . dna was extracted from 2 ml venous blood according to kit procedure ( sigma , usa ) and stored at 80c . a 20 ul mixture was prepared for each reaction and included 1x hotstartaq buffer , 2.0 mm mg , 0.2 mm dntp , 0.2 m of each primer , 1 u hotstartaq polymerase ( qiagen inc . ) and 1 l template dna . the dna was amplified by cycling at 95c for 15 min ; 11 cycles of 94c for 15 s , 62c0.5c per cycle for 40 s , 72c for 1 mins ; 24 cycles of 94c for 15 s , 57c for 30 s , 72c for 1 mins ; 72c for 2 min . the oligonucleotide sequences of the primers were as follows : 5-acccccatcatcgtagctctt-3 and 5-tcctgctctggcctccatac-3. the pcr products were purified by 1 u sap and 6 u exo i , and they were added into 8 l pcr products . the mixture was incubated at 37c for 60 mins , followed by incubation at 70c for 10 mins . reaction mixture included 2 l bigdye3.1 mix , 2 l sequencing primer ( 0.4 m , 5-acccccatcatcgtagctctt3 ) and 1 - 2 l purified pcr product . the cycling program was 96c for 1 min ; 28 cycles of 96c for 10 s , 50c for 5 s , 60c for 4 mins . besides , we have excluded some familial data to make sure that the frequency of a1180v from 1000 genomes project was calculated from unrelated individuals . comparison of the a1180v carrier frequency between our studies and the results of dbsnp database was tested by using the fisher 's exact test . combined with our former study and the 6 years of clinical followup of the affected pedigree , we discovered that a1180v related dcm was preceded by progressive avb which mostly occurred in the third decade of life . during the follow - up period , the condition of an a1180v carrier ( ii-8 in figure 1 ) who had been diagnosed with dcm before aggravated gradually and she now was diagnosed with severe congestive heart failure . besides , iii-1 with a1180v , developed to dcm and complete atrioventricular block ( avb ) and another 3 carriers ( iii-2 , iii-3 , iii-13 ) progressed to different degree of avb . figure 2 displays changes of echocardiograms of iii-1 and ecg recordings of iii-2 in the follow - up period . none of the individuals in the pedigree showed any sign of long - qt or brugada syndrome and other diseases . after screening a1180v in 460 individuals , we found that a1180v was absent in this group ( 0/460 , 0% ) . figure 3(a ) gives an example of the sequencing results , and figure 3(b ) is a sequencing result of a a1180v carrier in 2007 . the combined carrier frequency , 0% ( i.e. , 0 out of 660 cases ) , of a1180v from our studies remarkably suggests that a1180v is not a common variant among healthy chinese , and the data significantly differs from that in the dbsnp database ( p = 5.3 10 ) . data from the 1000 genomes project revealed a carrier frequency of 0.51% ( i.e. , 1 out of 197 cases ) for a1180v among chinese participants . the data was obtained by analyzing the sequencing results of 97 northern and 100 southern chinese adults . since the southern chinese group was familial related , the data from known offspring were excluded from the calculation of the carrier frequency to make sure the value was calculated from unrelated individuals ( http://browser.1000genomes.org/homo_sapiens/variation/population?db=core;r=3:38616415-38617415;v=rs41310765;vdb=variation;vf=12532571 ) . the data also dramatically differs from those deposited in the dbsnp database ( p = 0.013 ) . we have presented the followup and sequencing data identifying that a1180v of scn5a was a potential risk factor for dcm , and it is absent among healthy han chinese . compared with the former results in 2008 , 5 a1180v carriers exhibited age - related progressive avb and dcm , and the a1180v carrier frequency among han chinese in our study is 0% out of 660 control cases . evidence is now being accumulated that scn5a mutations are associated with development of cardiac dilation [ 13 , 1618 ] . mcnair et al . have reported that mutations in scn5a were detected in 1.7% of dcm families with a shared mechanism of disruption of the voltage - sensing mechanism of this channel leading to dcm . moreover , some scn5a mutations which have been found to be a primary factor of leading dcm were characterized by an age - related phenotype of cardiac dilation with different types of arrhythmia [ 11 , 20 ] . and the pathogenic characteristic of a1180v conformed to the pattern as well . since the cardiac sodium channel is principally in charge of the fast depolarization of the myocardium , some suggested that the cardiac dilation may result from long - term arrhythmia [ 22 , 23 ] . however , our experimental evidence indicated that a1180v could alter calcium homeostasis as a consequence of alterations in intracellular sodium concentrations , and the former condition might damage myocardium directly and subsequently lead to cardiac dilation . our data have revealed that a1180v specifically appeared in the affected pedigree and was absent among healthy controls , however , why do such significant discrepancies exist for a1180v among different studies of the same ethnic population ? ( 1 ) different asian ethnic subgroups may have different genetic backgrounds that cause differing prevalence of a genetic variant . for example , r1193q ( another scn5a variant ) is more prevalent in asians living in taiwan ( frequency~12% ) compared to asians living in japan ( ~2% ) . most chinese - americans are descendants of people from southern china , so they may have a genetic background that differs slightly from the majority of the subjects enrolled in our studies . the population of the shanghai area consists mostly of people who have immigrated from other areas of china . ( 2 ) relatively small sample sizes may also account for high carrier frequency of those data deposited in the dbsnp database . ( 3 ) the sequencing depth of different sequencing technologies varies greatly within and between experiments , and it may introduce bias as well . at present , lots of gene mutations were found playing pathogenic roles in both familial and sporadic diseases [ 26 , 27 ] , then another question is that whether a1180v would be a risk factor among sporadic dcm patients as well . in our recent studies of the distributions of candidate mutations in dcm - related genes in sporadic dcm patients conducted using high - throughput sequencing technology , we identified a1180v in 1 out of 68 patients with dcm . the patient , a 41-year - old male , was diagnosed as having dcm with nyha class i , lvedd 78 mm , and lvef 24% , but no other diseases . however , the patient carrying a1180v also carries other mutations in several dcm - related genes , including g1117s , p1119r , y498h of lama4 , w768s of rbm20 , p398s of vcl , and r659w of scn5a . although we found that a1180v might also be a risk factor in sporadic dcm patients , we can not exclude the possibility that a1180v could work together with other gene mutations . in addition to genetic factors , our previous study of the dcm pedigree carrying a1180v demonstrated that the a1180v channel lost current only at high heart rates , which suggested that physical activity and lifestyle that increased average daily heart rate might enhance the phenotype for a1180v carriers . therefore , environmental factors may also play a role in expression of the pathogenic mechanism of a1180v . further studies of genetic and environmental factors and followup of a1180v carriers will provide more information about the relationship between the mutation and the risk of dcm . studies of transgenic mice may also provide a step to test the hypothesis and to explore the underlying mechanism . in conclusion , herein we provide evidence that the a1180v is a potential cause for dcm rather than a common variant in han chinese , although additional genetic or environmental factors may potentiate the risk for dcm in a1180v carriers .

our previous study of a chinese family with dilated cardiomyopathy ( dcm ) suggested that a1180v of the cardiac sodium channel gene ( scn5a ) was associated with the disease within this family . according to data deposited in dbsnp , however , a1180v has been found in some small samples of the asian population . in this study , we followed up the affected pedigree and expanded the investigation of the prevalence of a1180v in 460 unrelated healthy han chinese . besides , we searched and analyzed it in other database as well . during the follow - up period , 1 a1180v carrier 's condition deteriorated alot , and another 4 carriers progressed to dcm or atrioventricular block ( avb ) . we also found that the a1180v was absent among the 460 individuals ( 0% , 0/460 ) , and the carrier frequency of a1180v among chinese was about 0.51% obtained from the 1000 genome project . in conclusion , our finding suggests that a1180v is a potential risk factor for dcm , and it is extremely rare among healthy han chinese .

the risk of stroke and myocardial infarction is considerably increased in subjects with diabetes . already at the time of diagnosis of type 2 diabetes , many patients have manifest cardiovascular disease ( cvd ) . this could be due to a long presymtomatic period with increased glucose level as evident by the increased cvd risk already present with impaired glucose tolerance ( igt ) . thus , interest has focused on factors , not only glucose levels , in the prediabetic state that would increase the atherothrombotic process . a common , possibly genetic , antecedent of both type 2 diabetes and cvd has been proposed as the " common soil " hypotheses . recently , markers of inflammation , such as highly sensitive c - reactive protein , have been found to predict type 2 diabetes in long - time follow up of population samples , although adjusting for measurements of obesity attenuates the relationship [ 8 - 12 ] . such markers are weakly related to variables reflecting endothelial function such as adhesion molecules and von willebrant factor . in a large population - based study , also increased plasma levels of plasminogen activator inhibitor-1 ( pai-1 ) were strongly related to the development of diabetes independent from insulin resistance and obesity . the possibility of elevated pai-1 being a very early risk marker of the insulin resistance syndrome and diabetes was raised . furthermore , haemostatic variables related to endothelial function , such as von willebrant factor and factor viii , also predicted diabetes , especially in women . data are lacking regarding other aspects of the fibrinolytic system and the development of diabetes , such as the activities of pai-1 and tissue plasminogen activator ( tpa ) or the mass concentration of the endothelial - derived tpa ( " tpa antigen " ) , factors related to cvd [ 15 - 19 ] thus , there are two questions related to fibrinolytic variables and the development of diabetes . firstly , do subjects with normal glucose tolerance , who later convert to diabetes , have disturbances in the fibrinolytic system and endothelial function that could explain their increased risk of cvd even before diabetes ensues ? if so , do these changes predict future diabetes , independent of the influence of the insulin resistance syndrome , thereby pointing to other pathways to the diabetic state such as endothelial dysfunction ? we studied fibrinolytic activity , as measured by tpa and pai-1 activity , and a marker of endothelial dysfunction , as measured by tpa antigen , in a population sample with normal glucose tolerance at baseline and analysed data according to conversion to diabetes or not over nine years of follow - up . this study was performed within the framework of the northern sweden monica study . in 1990 a total of 2,000 randomly selected subjects aged 25 to 64 years were invited . in all , 1,583 persons participated ( 79.2 % ) . a 75-gram oral glucose tolerance test ( ogtt ) was performed after an overnight fast in a randomly selected subset of subjects without known diabetes . venous plasma glucose samples were analysed by the hexokinase method ( boehringer mannheim automated analysis for bm / hitachi system 717 ) . glucose tolerance was classified according to who criteria from 1999 : normal glucose tolerance ( ngt ) fasting glucose < 7 mmol / l and post load glucose < 7.8 mmol / l , impaired glucose tolerance ( igt ) fasting glucose < 7 mmol / l and post load glucose 7.811.0 mmol / l , and diabetes fasting glucose 7.0 weight was measured with a balance scale to the nearest 0.2 kg , and height was measured to the nearest centimetre . bmi was calculated as total body weight ( kg ) / height ( m ) . waist circumference was measured midway between the lower rib margin and the iliac crest , which in most occasions was identical with the level of the umbilicus . all measurements were done in a standing position while breathing normally , and participants were asked to wear light underwear and remove shoes . for the measurement of waist circumference , the recording was done after a gentle breath - out . tpa and pai-1 activities were determined by chromogenic assays , spectrolyse / fibrin and spectrolyse / pl kit ( biopool ab , ume , sweden ) , respectively . tpa antigen was measured by an enzyme - linked immunosorbent assay method ( tintelize tpa , biopool ab ) . serum insulin was determined by radio immunoassay with a double - antibody solid - phase technique ( phadaseph insulin ria , pharmacia diagnostics ab , uppsala , sweden ) in 1999 , all participants , alive and still living in the area , were recalled for a follow - up examination . the same questionnaire and anthropometrical measurements as in the baseline survey were used . altogether 1,148 ( 72.5 % ) subjects turned up for re - examination . subjects who previously had performed an ogtt with normal or impaired glucose tolerance , and answered no to the question " participants , who denied having diabetes in the baseline survey and answered affirmative to the question " do you have diabetes mellitus ? " at follow up , were defined as incident cases of known diabetes mellitus according tho the who criteria from 1999 . in november 2002 , a questionnaire exploring details related to the diagnosis of diabetes , and to the type and duration of current hypoglycaemic treatment was sent to them . case records were scrutinised to confirm diabetes diagnosis and time for start of insulin therapy . subjects that started their insulin treatment within two years of diagnosis were classified as type 1 . however , for subjects fulfilling these criteria but with a age of 40 or above at onset , current bmi had to be < 27 to be classified as type 1 . the research ethics committee of ume university and the national computer data inspection board have approved the northern sweden monica study . means or medians are given for those who remained non - diabetic and those who converted to diabetes . because the distributions for serum triglycerides , insulin and the fibrinolytic variables are highly skewed , we used logarithmically transformed values ( i.e. geometric means ) . the risk of incident diabetes was compared across quartiles of the fibrinolytic variables , calculated from the total sample . logistic regression was used for calculation of odds ratios and 95% confidence intervals ( ci ) for the development of diabetes comparing the quartile with highest risk with the other three pooled quartiles . stepwise adjustment for age and sex , waist , serum insulin , triglycerides and diastolic blood pressure was used . five hundred and fifty - one subjects who initially had normal glucose tolerance returned for re - examination . a new ogtt was done in 477 of those with normal glucose tolerance and without clinically diagnosed diabetes . thus , there was no repeated ogtt in 13 % , mostly due to logistic reasons such as not being able to turn up in the morning after an over night fast . among persons with initially normal glucose tolerance , in all 15 subjects ( 2.7 % ) had type 2 diabetes diagnosed , either clinically ( n = 4 ) or by ogtt ( n = 11 ) after a 9-year follow - up or 4,959 person years . fifty - one subjects ( 10,7 % ) worsened from normal glucose tolerance to impaired glucose tolerance . converters from ngt to diabetes were 7 years older and had higher bmi and waist circumference . diastolic blood pressure did not differ but both fasting triglycerides , insulin , fasting and post load plasma glucose were higher in subjects with subsequent diabetes . plasma levels of tpa antigen and pai-1 activity were higher and tpa activity was lower . baseline characteristics of subjects with normal glucose tolerance according to diabetes or not during 9 years of follow - up data are means ( sd ) , median ( interquartile range ) and [ geometric means ] . the risk of developing diabetes increased with increasing quartiles of tpa antigen and pai-1 activity and decreased in a similar way for tpa activity ( figure 1 ) . in the highest quartile of tpa antigen ( above 8.7 g / l ) nearly 9 % had incident diabetes compared to none in the lowest quartile . incidence of type 2 diabetes over nine years in subjects with normal glucose tolerance according to baseline quartiles of fibrinolytic variables . the number of cases in each quartile for tpa activity was 7 , 4 , 3 and 1 . corresponding numbers for tpa antigen was 0 , 1 , 2 and 12 and for pai-1 activity 2 , 1 , 3 and 9 , respectively . p values for x(linear by linear association ) were 0.026 for tpa activity , < 0,001 for tpa antigen and 0,007 for pai-1 activity . logistic regression was performed with an initial model adjusting for age and sex . in a second model , waist circumference was added and in a third model , also diastolic blood pressure , serum insulin and triglycerides were added . quartiles 13 of tpa antigen and pai-1 activity were contrasted with the fourth quartile and , for tpa activity , the first quartiles with quartile 24 ( table 2 ) . logistic regression analysis for fibrinolytic variables at baseline with incident type 2 diabetes as the dependent variable . the lowest quartile of tpa activity was associated with a 3-fold increase in the risk of diabetes , which was attenuated after adjustment for waist circumference . after further adjustment for diastolic blood pressure , fasting insulin and triglycerides , no prediction from low tpa activity on diabetes development was seen . subjects in the highest quartile of tpa antigen experienced more than a ten - fold increase in risk , although the confidence intervals were wide due to few cases . taking waist circumference into account , the odds ratio diminished to 6.7 and diminished further to 5.5 when adjusting for blood pressure , triglycerides and insulin . this lead to somewhat higher age- and gender adjusted odds ratio ( 7.4 ; ci 1.830 , p value 0.005 ) . exchanging diastolic with systolic blood pressure did not change the findings . furter adjustment for leisure time physical activity reduced the odds ratio from 5.5 to 5.1 ( p value 0.03 ) . high pai-1 activity increased the risk of diabetes four - fold , although the result was attenuated to an insignificant or of 2.0 when all factors were adjusted for simultaneously . the risk of developing diabetes or igt during follow up ( n = 66 ) increased stepwise across quartiles of tpa antigen , from 7.8% to 19.8% ( p = 0,003 , test for linear trend ) . when adjusted for age and sex , the odds ratio for the highest vs. the three lower quartiles of tpa antigen was 1.9 ( ci 1.011.6 , p = 0.04 ) but diminished further when also waist circumference was adjusted for , or = 1.3 ( ci 0.636.5 , p = 0.5 ) . subjects with normal glucose tolerance , who subsequently develop type 2 diabetes over the ensuing nine years , are already at baseline characterised by impaired fibrinolysis and endothelial dysfunction . low tpa activity and high pai-1 activity is mainly explained by the presence of abdominal obesity but also by other metabolic disturbances characteristic of the insulin resistance syndrome , such as high serum levels of triglycerides and insulin . a novel finding , in this context , is that a high plasma level of the endothelial - derived tpa is only slightly explained by such factors . thus , the development of type 2 diabetes is preceded by many years of perturbations in fibrinolytic and endothelial function , which increases the risk of atherothrombotic disease long before overt diabetes is present . pa1 - 1 is partly synthesised in fat cells and its activity is strongly related to abdominal obesity as mirrored by high waist circumference or whr . high circulating levels of pai-1 inhibit tpa released from the vessel walls and lead to low levels of free tpa , i.e. low tpa activity and impaired capability of thrombolysis . the only report of an association between fibrinolysis and the development of diabetes is a recent study where pai-1 antigen was an independent predictor of diabetes in american - mexicans that were followed up to 5 years . there is no known pathway whereby impaired fibrinolysis would increase insulin resistance or decrease insulin release and our finding that high pai-1 and low tpa activity precedes diabetes probably mirrors the strong relationship with intraabdominal fat and circulating levels of free fatty acids . a recent experimental study show an almost three fold increase in the level of pai-1 antigen with an infusion of triacylglycerol in healthy subjects without any impact on tpa . this effect was noted with constant levels of insulin and glucose and also increased levels of soluble vascular cell adhesion molecules-1 was found . this support our findings that adjustment for baseline triglyceride levels , which were increased in converters , did not significantly change the relationships noted for tpa antigen but did decrease those for pai-1 . it is thus possible that also tpa concentration is an inflammatory marker although we found low correlations between tpa antigen and fibrinogen and highly sensitive crp ( own unpublished data ) . recently , it was described in type 2 diabetes patients , that increased levels of tpa antigen , among other endothelial markers , predicted the development of urinary albumin excretion , a strong marker for cvd risk . these findings should be related to recent reports where inflammatory markers predict the development of diabetes in longitudinal studies . in most cases , pro - inflammatory substances derived from abdominal fat tissue may be the common background for these findings . we therefore find it plausible that the relation between high tpa and diabetes is a marker of some early disturbance in the vascular wall that operates independent of obesity and inflammation . already borderline hypertension is associated with high concentrations of tpa antigen , even after adjustment for abdominal obesity , insulin and triglycerides . thus , tpa mass may mirror an endothelial dysfunction that is not causally related to the insulin resistance syndrome but acts by a parallel pathogenetic mechanism leading to atherothrombosis [ 30 - 32 ] . insulin resistance , on the other hand , is associated with endothelial dysfunction and impaired vasodilatation due to defects in endothelium - derived nitric oxide . taken together , these findings put focus on the role of the vessel walls in the pathogenesis of type 2 diabetes . the relevance of impaired fibrinolysis , i.e. low activity of tissue plasminogen activator ( tpa ) or high activity of its inhibitor ( pai-1 ) , as independent risk factors for cvd is unclear , as these variables seem to mirror most components of the insulin resistance syndrome . in some studies , decreased fibrinolysis , as measured by high pai-1 activity , has been associated with cardiovascular events . high levels of tpa antigen independently predict cardiovascular events both in a healthy population and in patients with prevalent coronary disease . the major shortcoming of our study is the small number of incident cases of diabetes leading to wide confidence intervals . on the other hand , the odds ratios for tpa antigen are high and consistent and based on a truly representative population sample where basal normal glucose tolerance was well defined , as were the incident cases of diabetes . thus , the risk of bias due to selection or case ascertainment should be low . admittedly , the lack of a renewed ogtt in 13 % of the subjects could contribute to some misclassifiaction and dilute the results somewhat . the early prediabetic state , with normal glucose tolerance , is characterised by both impaired fibrinolysis and endothelial dysfunction years before glucose levels increase . changes in endothelial function are independent from the insulin resistance syndrome and point towards a distinctly different pathway both for cardiovascular disease and for the development of diabetes . to answer a classical question from 1990 yes , the clock for coronary heart disease seems to start ticking before the onset of clinical diabetes ! bmi = body mass index ci = confidence interval crp = c - reactive protein cvd = cardiovascular disease igt = impaired glucose tolerance monica = monitoring of trends and determinants of cardiovascular disease ngt = normal glucose tolerance ogtt = oral glucose tolerance test pai-1 = plasminogen activator inhibitor-1 tpa = tissue plasminogen activator me drafted the research on fibrinolysis within the monica project , performed the statistical analysis and drafted the manuscript . bs and bl participated in the design of northern sweden monica study and participated in its design and coordination . supported by grants from norrbotten and vsterbotten counties , by the joint committee of northern sweden health care region , the swedish public health institute and the swedish medical research council .

backgroundimpaired fibrinolysis is found in impaired glucose tolerance and type 2 diabetes , associated with components of the metabolic syndrome . there are no data concerning fibrinolysis in subjects with normal glucose tolerance that convert to diabetes.methodswe studied the activities of tissue plasminogen activator ( tpa ) and plasminogen activator inhibitor-1 ( pai-1 ) and the levels of tpa antigen ( a marker of endothelial dysfunction ) in 551 subjects with normal glucose tolerance in 1990 in relation to incident diabetes during nine years of follow-up.resultssubjects with diabetes at follow - up ( n = 15 ) had significantly lower baseline tpa activity and higher pai-1 activity and tpa antigen than non - converters . the risk of diabetes increased linearly across quartiles of pai - activity ( p = 0.007 ) and tpa antigen ( p < 0.001 ) and decreased across quartiles of tpa activity ( p = 0.026 ) . the risk of diabetes with low tpa activity or high pai-1 activity persisted after adjustment for age and sex but diminished to a non - significant level after further adjustments . the odds ratio of diabetes for high tpa antigen was 10.4 ( 95% confidence interval 2.740 ) adjusted for age and sex . after further adjustment for diastolic blood pressure , waist circumference , insulin , triglycerides , fasting and post load glucose the odds ratio was 6.5 ( 1.333 , p = 0.024).conclusionsimpaired fibrinolysis and endothelial dysfunction are evident in subjects with normal glucose tolerance who later develop diabetes . high tpa antigen is predictive of future diabetes independent from the metabolic syndrome .

palatal rugae are irregular , asymmetric ridges of mucous membrane extending laterally from the incisive papilla and the anterior part of the median palatal raphe . these structures have been used as internal cast reference points for quantification of tooth migration . a few investigators have shown the medial rugae region to be stable or show predictable changes post orthodontic therapy . the rugae patterns are completely formed by the 12 to 14 week of prenatal life and remain stable thereafter . these are unique to each person and show distinctiveness based on ethnic groups and hence are useful in forensic identification . because they are stable landmarks the palatine rugae play a significant role in clinical dentistry as well . the objective of this study was to analyze and characterize the rugae patterns , compare the rugae dimensions in various age groups and to ascertain any relationship between dimensional analysis and palatal depth . cross - sectional casts of 52 females and 48 males were selected from the archival section of the department of orthodontics , m. m. college of dental sciences and research , mullana ( ambala ) and studied employing following parameters : assessment of age ( according to erupted teeth).gender differentiation ( based upon records).division of medial palatal region into : a : distance between incisive papilla length and anterior limit of the anteriormost rugae.b : distance between incisive papilla and posteriormost rugae limits.lateral rugae dimensions . palatal depth ( measured from healthy gingival margin underneath the mesiolingual cusp to the deepest concavity of the palatal arch using a brass wire between two opposing points and measuring the vertical distance at the center).rugae patterns . division of medial palatal region into : a : distance between incisive papilla length and anterior limit of the anteriormost rugae.b : distance between incisive papilla and posteriormost rugae limits.lateral rugae dimensions . a : distance between incisive papilla length and anterior limit of the anteriormost rugae . palatal depth ( measured from healthy gingival margin underneath the mesiolingual cusp to the deepest concavity of the palatal arch using a brass wire between two opposing points and measuring the vertical distance at the center ) . no patient details were disclosed and ethical guidelines as per the declaration of helsinki were followed . correlations among a , b , lateral rugae patterns and palatal depth were calculated by mean s.d . correlations among a , b , lateral rugae patterns and palatal depth were calculated by mean s.d . followed by evaluation of p values . the rugae patterns identified were : common origin ; separate origin ; lateral branching ; secondary rugae and fragmentary patterns . females were found to have slightly higher predilection towards having the common , fragmentary and lateral branching rugae patterns . there was a stronger female predisposition for rugae with separate origin , whereas , the secondary rugae were equally existent in either gender . significant correlation was found between the a ( p=0.03 ) and b values ( p=0.02 ) on comparing the mean s.d . values of age groups between 12 - 13 years and > 14 years , respectively . hence , there is a corresponding anteroposterior increase in palatal dimensions in these age groups [ table 1 ] . this study showed no changes in the lateral rugae dimension and palatal depth with increasing age . the majority of the study cohort ( 57% ) had a palatal depth in the range of 1.6 - 2.0 cm [ table 2 ] . palatal rugae have been used as reference points for many purposes such as evaluating tooth movement pre- and post - orthodontic treatment , population studies and forensic identification . stability of medial palatal region has been a subject due to differences among various investigators . christou and kiliardis evaluated the vertical changes in the medial aspects of the rugae and concluded that these changes over time are due to the alterations in the vertical positioning of maxillary incisors and increase in lower face height . growth periods ( 12 - 13 years and > 14 years ) studied in this paper , showed a downward and forward movement of the maxilla in relation to the cranial base and also , changes in the size and shape of maxilla by structural remodeling . these phenomena can be explained by the deposition of new bone on the oral surface of the palate and at the alveolar crest . therefore , the changes in rugae dimensions can be the result of this differential growth in the palate and alveolar crest.[24 ] rugae patterns showed a strong female predilection for rugae with separate origins [ figure 1 ] whereas slightly higher incidences were noted for rugae with common origin , fragmentary and lateral branching patterns . there was an equal percentage of gender with secondary rugae pattern in the ethnic segment of north indian population studied [ table 1 ] . photograph depicting rugae patterns with secondary origin and fragmentary nature the purpose of this paper was to evaluate a cross - sectional patient database to analyze the anteroposterior stability of the medial rugae region . analysis of 100 study casts showed a significant difference in the medial rugae region ( p= 0.03 , 0.02 ) in a and b values . no significant changes were noted in the lateral rugae dimensions . the palate growth in this period was found to be non - significant . hence , it can be surmised from the analysis of the results that there is a differential growth spurt in the anterior and posterior palate during adolescence as is marked by the significant differences in the measurement values obtained through this study . palatal rugae can be studied as a strong indicator of ethnicity , gender differentiation and study of growth changes in the anterior maxilla . thus , rugae are important tools in clinical investigations involving forensic anthropology and developmental biology .

introduction : rugae patterns are significant markers for analyzing anteroposterior changes in adolescence , and forensic investigations.aim:the purpose of this study was to ascertain the gender - wise predisposition of rugae patterns and to analyze anteroposterior alterations along with any developmental changes in palatal depth.materials and methods : one hundred pre - treatment study cast models were obtained from the archives of the department of orthodontics , m.m . cdsr , mullana . parameters employed were : division of medial palatal region into a : distance between incisive papilla length and anterior limit of the anteriormost rugae ; b : distance between incisive papilla and most posterior rugae limits ; measurement of lateral rugae dimensions and palatal depth . statistical analysis : mean s.d . values were obtained and p values calculated.results:comparison of a and b showed a significant difference in the p values between the age - groups 12 - 13 years and > 14 years . rugae patterns with separate origins showed a predisposition for female gender.conclusion:a significant change in the anteroposterior medial rugae dimensions was seen in adolescent age groups alongside no variation in palate depth . hence , it can be concluded that differential growth potential is present in the premaxilla and can cause shift in medial palatal dimensions without altering the rugae patterns and palatal depth .

hypophosphatemic rickets ( hr ) is a metabolic bone disorders manifested by musculoskeletal disorders specially in the lower extremities , growth retardation and dental problems[1 , 2 ] . this disorder affects all ages and both sexes and is characterized by a low concentration of serum phosphate , elevated alkaline phosphatase , normal or decreased serum calcium and inappropriate levels of 1,25-dihydroxy - vitamin d [ 1,25 ( oh)2 vit d][1 , 3 ] . musculoskeletal disorders are due to mineralization impairment of the bone matrix and teeth with different etiologies . x - linked hypophosphatemic rickets ( xlhr ) is the most common form of the genetic disorders causing rickets due to hypophosphatemia . this disorder results from mutation in the phex gene and the product of this gene has a role in phosphatonin inactivation . hr is more common in females with positive family history observed in 35.3% patients , nevertheless , it seems this disorder is more frequently and more severe among males in xlh form . common features are a short stature , backache , body deformities , joint pain , and fractures . dental abnormalities include dental taurodontism with increasing mean crown - body to root ratio , ectopic permanent canines , dental abcesses[2 , 5 ] and enamel hypoplasia . treatment of rickets with vitamin d ( vit d ) and phosphate supplements has been shown to prevent and cure the dental anomalies in some but not all patients . from 1970 , dental problems were reported in 23 - 67% xlhr patients[3 , 6 , 7 ] , but these problems have not been described well in previous studies . this study was a cross sectional investigation that was conducted in a referral pediatrics endocrinology clinic in iran during 3 years ( 2008 - 2010 ) . the diagnosis of hypophosphatemic rickets was made on the basis of clinical and radiological findings of rickets in a patient with low level of serum phosphate , normal or low level of serum calcium ( < 8.2 mg / dl ) and normal levels of 25(oh)2-vit d ( < 15 pg / ml ) , and normal or mildly elevated pth with high level of serum alkaline phosphatase ( > 55 pg / ml ) . patients who did not have a clear diagnosis or refused to participate in the study were excluded . after taking a dental history , all subjects had a dental examination ( including dental caries , enamel hypoplasia , taurodontism , dental abscesses and gingivitis ) ; opg was performed if necessary . clinical examination was performed to look for dental deformities by a trained dentist for all patients . information about dental problem including : dental abscess , enamel hypoplasia , dental caries , gingivitis , taurodontism , and delay in the primary or permanent dentition was obtained . for permanent teeth , decay , missing , or filled teeth ( dmft ) index and for primary teeth , decay or filled teeth ( dft ) delay in eruption of teeth was based on times of eruption of the primary and permanent teeth from logan and kronfeld . taurodontism was suggested if there was any change in tooth shape and was confirmed by orthopantomography ( opg ) . in this study we enrolled 19 patients with hypophosphatemic rickets . mean age of these patients was 104.23 years ( range 3 - 17 ) . eleven ( 57.9% ) patients were female and 8 patients ( 42.1% ) male ( female to male ratio : 2/1.3 ) . fifteen ( 79% ) patients had regular follow - up after diagnosis of background disease and 17 ( 89.5% ) patients had at least one dental problem . the most common dental problems were dental caries and delay in eruption of the dentition in 9 ( 47.7% ) patients . enamel hypoplasia was found in 8 ( 42.1% ) patients and 3 ( 15.8% ) patients had taurodontism and all last group were male . both dental abscesses and gingivitis were found in 2 ( 10.5% ) patients . six ( 54.5% ) female and 3 ( 37.5% ) male patients had dental caries . the prevalence of dental caries was significantly more frequent in case group ( p=0.04 ) that was 10.5% in healthy control matched group . this study showed that hr has a variable age of presentation and it was more common in females[2 , 3 ] . dental caries was the most common finding in this study ( 47.7% ) that was similar to chaussain - miller 's study . dental caries is one of the most common factors for pulp infection , so oral hygiene has an important role for caries prevention . dental abscesses were found in 10.5% of the patients compared to 25 - 65% in other studies[4 , 6 , 12 ] . in patients with hr the dentition is highly susceptible to dental caries and bacteria can invade easily from the oral cavity to the dental pulp by means of structural defects in the enamel . difference of prevalence of dental abscess may be due to early diagnostic and treatment of these patients , although shroff et al believe that prophylactic pulpotomy therapy may have been no benefit in preserving the primary dentition in xlhr patients . in our study , enamel hyperplasia has been reported in 16.6% patients with permanent teeth by bender and naidorf , however they said the incidence of this disorder is 36.8% ( 14/38 ) in cases reported in the literature , this is similar to our study . although this phenomena is expectable in hr patients , no significant differences was reported in dental age compared with the respective chronological age in a study . genetic mechanisms have a main role causing defective dentition and dentine mineralization . in this study taurodontism had a significantly increased tendency in male xlhr subjects . similar findings were reported by seow et al . in a controlled , longitudinal study , they showed taurodontism had a significantly higher tendency in male xlhr patients . patients respond well to a combination of oral phosphate and 1,25(oh)2-vitd ( calcitriol)[4 , 6 ] . the beneficial dental effect of phosphate supplements after birth and the possible influence of maternal phosphate homeostasis suggest that most abnormal dental features are linked to hypophosphatemia and deficient renal production of 1,25(oh)2-d . some effects of 1,25(oh)2-d on dentin may be indirect , through an increased intestinal absorption of calcium and phosphorus . 1,25(oh)2-d may also exert some direct effect on dental cells , as odontoblasts and ameloblasts express the vitamin d receptor and respond in vitro to this vitamin . it seems , the best results were achieved when medication started during early childhood[4 , 18 ] . dental care of these patients should consist of periodic examination , topical fluoride application , pit and fissure sealants and maintenance of good oral hygiene . dental caries as common as delay in dentition were the most prevalent problems in xlhr patients that could be reduced by proper dental care and good oral hygiene . so the dentist as well as pediatrician should be made aware of the features of disorder and early intervention .

objectivehypophosphatemic rickets is an uncommon metabolic bone disorder which affects all ages and both sexes . it is characterized by low concentration of serum phosphate levels , impairment of mineralization of bone matrix and teeth with variable etiology . dental problems in this disorder have not been described well in previous studies.methodsall hypophosphatemic rickets patients who came to a referral clinic during 2008 - 2010 enrolled in this study . all patients had low phosphorous and high alp , normal pth and 25-hydroxy - vitamin d and normal or low level of serum calcium . after diagnosis all patients were examined by a dentist for enamel hypoplasia , taurodontism , dental abscess , gingivitis , dental caries , and dentition delay.findingsnineteen patients were enrolled in this study . the average age of the patients was 10 ( 4.23 ) years ( range 3 - 17 ) . seventy - nine percent of patients had regular follow - up after diagnosis of background disease . dental caries and delay in the dentition were most prevalent ( each one 47.7% ) followed by enamel hypoplasia in 42.1% of the patients . other problems were taurodontism in 15.8% patients , dental abscess and gingivitis in 10.9%.conclusionhypophosphatemic rickets is a disease with different clinical features ; one of them is dental problem , dental caries is the most common problem .

melanin is a heterogeneous polymer produced and concentrated within the melanosomes of the melanocytes , and tyrosinase is a key rate - limiting enzyme for melanin biosynthesis . tyrosinase catalyzes three steps in melanin biosynthesis : the hydroxylation of tyrosine to 3 , 4-dihydroxyphenylalanine ( dopa ) , the oxidation of dopa to dopa - quinone , and the oxidation of 5 , 6-dihydroxyindole to indolequinone . because of its central role in melanogenesis , tyrosinase is a key target for the development of new inhibitors [ 13 ] . in the present study , we aimed to directly regulate tyrosinase expression using rna interference ( rnai ) . we screened small interfering rnas ( sirnas ) corresponding to the tyrosinase sequence using the mouse melanoma cell line b16 and then delivered the most effective sequence into c57bl/6 mice to study its effect in vivo . b16 mouse melanoma cells ( shanghai institute of cell biology , chinese academy of sciences ) were cultured in dmem ( gibco , usa ) , supplemented with 10% ( v / v ) fetal bovine serum ( hangzhou sijiqing biological engineering materials co. , ltd . , china ) and 2 mmol / l glutamine ( amresco , usa ) at 37c in a humidified atmosphere with 5% co2 [ 3 , 4 ] . the b16 cells were trypsinized and centrifuged at 1,500 rpm for 5 minutes and then resuspended in dmem culture medium and seeded in a six - well plate ( corning , usa ) at 2 10 per well and 96-well plate ( corning ) at 1 10 per well . cells were cultured for 12 hours and transfected when cells were at 30% to 50% confluence . c57bl/6 mice were purchased from the laboratory animal center at sun yat - sen university in china and were handled in accordance with the research in vision and ophthalmology guidelines for the use of animals in research ( animal quality certificate , 0028619 ; animal experiment license , syxk [ guangdong ] 2005 - 0058 ) . twelve hours after seeding , cells were transfected using lipofectamine 2000 ( invitrogen , usa ) according to the manufacturer 's instructions . sirnas were prepared by serial dilution ( 1060 nmol / l ) in opti - mem i medium ( gibco ) to determine the optimal transfection concentration . the optimal transfection concentration was defined as the minimum concentration of sirnas that can produce the highest fluorescence intensity . the sirna - targeting tyrosinase mrna was designed by the dharmacon online sirna design tool ( http://www.dharmacon.com/homepage.aspx ) and synthesized by ruibo biotechnology co. , ltd . the sirna sequences against tyrosinase were as follows : sinm_011661_001 target sequence , aagcgagtcttgattagaa ; sense ( 5-3 ) , aagcgagucuugauuagaa(dtdt ) ; antisense ( 3-5 ) , ( dtdt)uucgcucagaacuaaucuu ; sinm _ 011661_002 target sequence , acaatgccttacatatctt ; sense , acaaugccuuacauaucuu ( dtdt ) ; antisense , ( dtdt)uguuacggaauguauagaa ; and sinm_011661_003 target sequence , tcataccgctctatagaaa ; sense , ucauaccgcucuauagaaa(dtdt ) ; antisense , ( dtdt ) aguauggcgagauaucuuu . a lipofectamine 2000-containing negative sirna ( green fluorescence protein mrna sequence ) group and a blank group ( lipofectamine solution alone ) were used as controls . after transfection , the cells were harvested at three different time points ( 24 , 48 , and 72 h ) to determine the mrna expression , melanin content , and tyrosinase activity for each treatment . the grouping information is as follows : group1 ( sinm_011661_001 ) , group2 ( sinm_011661_002 ) , group3 ( sinm_011661_003 ) , negative control group ( negative sirna ) , blank group ( lipofectamine ) . tyrosinase mrna in sirna - treated b16 cells was quantitatively analyzed using quantitative real - time pcr ( qrt - pcr ) . cdna was synthesized with random primers using no more than 500 ng of total rna in each 10-l reaction according to the manufacturer 's instruction ( takara , japan ) . sybr green qrt - pcr were done in a real - time detection system ( abi7000 , usa ) according to the manufacturer 's instructions ( takara , japan ) using the comparative cycle threshold method . thermal cycler conditions were 95c for 10 seconds followed by 40 cycles of 5 seconds each at 95c and 31 seconds at 60c . the primer pairs were designed for tyrosinase and -actin using primer 5.0 express software as follows : tyrosinase forward primer , 5-gcccagcatccttcttc-3 ; tyrosinase reverse primer , 5-tagtggtccctcaggtgttc- 3 ; -actin forward primer , 5-cagccttccttcttgggtat- 3 ; and -actin reverse primer , 5-tggcatagaggtctttacgg- 3. tyrosinase activity was assessed by determining the catalysis of l - dopa to dopachrome , which has an absorption peak at 490 nm . in this study , the inhibitory action of sirnas on tyrosinase was assayed as described previously [ 4 , 6 , 7 ] with slight modifications . briefly , b16 cells were seeded at a density of 1 104 cells per well in 96-well plates . after 12 hours , the cells were treated with different sirnas and cultured for 24 to 72 hours at 37c the adherent cells were washed three times with pbs and then lysed in sodium phosphate buffer ( 0.1 mol / l , ph 6.8 ) containing 1% triton x-100 . then , 10 l l - dopa ( 1% ; icn , china ) was added , allowed to react , and incubated for 2 hours at 37c . absorbance was measured at 490 nm using an enzyme - linked immunosorbent assay reader ( powerwave xs , bio - tek , usa ) . the melanin content was determined as described previously [ 4 , 79 ] by alkaline lysis with minor modifications . after transfection , adherent cells ( 1 10 ) were washed with pbs , detached by trypsinization , collected in a test tube , and then washed twice with pbs . the cells were resuspended in 1 mol / l naoh , incubated at 80c for 30 minutes , and then centrifuged at 1,500 rpm for 5 minutes . the absorbance of the supernatant was measured at 475 nm and compared with the standard curve of the serial dilution of standard melanin ( sigma , usa ) . injection of chloral hydrate ( 0.008 ml / kg ) and local anesthesia with 2% pontocaine . with the guidance of an operating microscope ( olympus , japan ) , two groups of mice were given an intravitreous injection of 2.5 l pbs containing either 0.65 or 1.3 nmol sinm_011661_001 , and 2.5 l pbs was injected as control . twenty - four hours after injection , total rna extraction ( qiagen , germany ) was done from the retina of c57bl/6 mice as described previously and analyzed with qrt - pcr . anova was used to compare the differences between the groups using spss 13.0 for windows ( chicago , usa ) . before transfection , most cells appeared fusiform , and some cells were reticular at low density . the cells in the division phase showed a clear outline , an obvious boundary between cells , and a fine refractive index ( figure 1 ) , but after transfection , the cells swelled and the boundary between them became indistinct . twenty - four hours after transfection , the distribution of intracellular fluorescence was sporadic in cells treated with 10 , 20 , 30 , and 60 nmol / l sirna . however , intracellular fluorescence was widespread , and the intensity was strengthened in cells transfected with 40 and 50 nmol / l sirna ( figure 1 ) . in this study , we achieved the best transfection efficacy with the lowest lipofectamine 2000 ( 0.2 l per cell in a 96-well plate ; 4 l per cell in a 6-well plate ) and sirna concentrations ( 40 nmol / l ) . we evaluated the efficiency of each sequence to suppress tyrosinase expression at 24 , 48 , and 72 hours after transfection . the results were 82.81% , 76.19% , and 75.46% for sinm_011661_001 ; 75.75% , 43.49% , and 37.86% for sinm_011661_002 ; 50.54% , 23.66% , and 22.42% for sinm_011661_003 ; 17.22% , 11.34% , and 8.68% for the negative control sequence , respectively ( figure 2 ) . the efficiency of the three fragments decreased gradually from 24 to 72 hours after transfection . the statistical results showed that the sirna sequence sinm_011661_001 was the most effective in suppressing tyrosinase expression ( f = 298.566 and p < .05 at 24 h ; f = 179.217 and p < .05 at 48 h ; f = 188.564 and p < .05 at 72 h ) . we evaluated reductions in tyrosinase activity in the transfected cells at 24 , 48 , and 72 hours after transfection . tyrosinase activity was reduced by 64.73% , 40.42% , and 28.74% for sinm_011661_001 ; 63.37% , 25.06% , and 17.33% for sinm_011661_002 ; 48.16% , 18.33% , and 16.65% for sinm_011661_003 ; 6.44% , 4.90% , and 3.56% for the negative control sequence , respectively ( figure 3 ) . the tyrosinase suppression decreased with time for each sirna sequence , which was similar to the suppression at the mrna level . after 24 hours , there was no significant difference between sinm_011661_001 and sinm_011661_002 ( p > .05 ) , whereas significant differences were observed among the other groups ( f = 58.485 ; p < .05 ) . at 48 hours , significant differences were observed among the groups ( f = 160.208 ; p < .05 ) . at 72 hours , no significant difference was found between sinm_011661_002 and sinm_011661_003 , whereas significant differences were observed between the other groups ( f = 117.372 ; p < .05 ) . the decrease in melanin content in each group was evaluated at days 1 , 2 , and 3 . the percent reduction in melanin was 52.53% , 48.55% , and 32.73% for sinm_011661_001 ; 25.59% , 21.61% , and 6.15% for sinm_011661_002 ; 21.01% , 18.79% , and 1.70% for sinm_011661_003 ; 5.53% , 3.58% , and 1.00% for the negative control sequence , respectively ( figure 4 ) . after 48 hours , the melanin content and interference efficiency of sinm_011661_002 and sinm_011661_003 were not significantly different . however , after 72 hours , cells transfected with sinm_011661_001 contained significantly less melanin than the other groups ( p < .05 ) , whereas comparisons between other groups were not significantly different . because sinm_011661_001 most effectively reduced mrna expression of tyrosinase in b16 cells , we evaluated its effect in c57bl/6 mice . to determine the change in tyrosinase mrna levels , qrt - pcr was carried out using tyrosinase - specific primers and total rna isolated from retinas ( table 1 ) . negative interference sequence and liposome ( blank group ) have nonspecific interference effect during our experiment . all three sirna sequences that we screened suppressed tyrosinase expression , but sinm_011661_001 was most effective , exerting the maximum effect at 24 hours after transfection . it is presumed that tyrosinase mrna possesses a special functional structure that is most similar to sinm_011661_001 . the data presented here suggests a crucial role for sirna structure in rnai [ 8 , 10 ] . in this study , the suppressive effects on melanogenesis in b16 cell line peaked at 24 hours , suggesting that sirna was active for 24 hours after transfection and then was gradually degraded . however , we used modified sirna , which can enhance stability and reduce the potential for off - target effects . sequences of sirna for a target gene do not perform equally , but significant progress has been made in defining sequence features that contribute to sirna potency . low internal stability at the 5 terminus of antisense sirna is an important prerequisite for gene silencing . excessive stability can result in double - strand unwinding , but inferior stability can reduce the affinity of sirna to its mrna target . in the present study , we noted an interesting phenomenon : sinm_011661_001 transfection resulted in the considerable suppression of tyrosinase mrna compared with other control sirnas . furthermore , the negative controls ( sirna sequence and lipofectamine transfection reagent ) were observed to slightly reduce melanin content with time ( figure 4 ) , which was similar to the effect of sinm_011661_002 and sinm_011661_003 72 hours after transfection . this phenomenon also appeared at the level of gene expression and tyrosinase activity , suggesting nonspecific rnai activity . in addition to showing that sinm_011661_001 is the most potent sirna sequence against tyrosinase in b16 cells , we assessed the effect of this sirna sequence on tyrosinase expression in c57bl/6 mice . one of the important factors for rnai effectiveness in vivo may be the delivery and transfection efficiency of sirnas . in mammalian cells , sirna has been administered locally or systemically , and the efficiency of rnai delivery systems is still relatively low . previous studies showed that the systemic delivery of sirnas by tail vein injection does not deliver sirnas efficiently to their target areas because sirna molecules are easily trapped in nonspecific organs . we thought that the optimal efficacy of sirna targeting pigmentary epithelium would be largely deprived via intravenous injection , because the nutrient vessel supporting pigmentary epithelium belongs to distal circulation vessel . what is more , the synthetic sirna pieces are heterogenous to mice ; appearance of these heterogenous pieces in circulation may potentially induce severe immune lesion to mice . in contrast , intravitreous treatment may have its advantage to overcome these problems , because vitreous space has the immediate proximity to retina , the small molecular weight of sirna may be efficiently delivered to retina and pigment epithelium through posterior vitreous membrane and thus have direct effects on target tissues . in addition , the blood - ocular barrier may function to prevent the entry of intravitreously injected sirna into circulation . in summary , intravitreous administration may take many advantages beyond intravenous injection , due to efficient and direct delivery of sirna pieces to target tissues combined with reduced risk of inducing side effects . after considering these factors collectively , we chose intravitreous injection throughout this work . we also believe that this method would provide a new approach to ocular albinism mouse model preparation . in contrast , our results showed that sirna administered locally had significant inhibitory effects on the retina . it strongly suggests that sinm_011661_001 can be delivered effectively in vivo and may also be delivered to other tissues such as skin and hair . in summary , we have shown that sinm_011661_001-targeting tyrosinase is the most potent sequence in vitro and in vivo . rnai is a new tool for gene silencing using antisense nucleic acids and gene targeting . in addition , it gives us a new method to use in the development of pigmentation disease ( e.g. , ocular albinism ) animal models and treatments . furthermore , this technique may be useful in the study of downstream factors of tyrosinase .

tyrosinase is a bifunctional enzyme which oxidizes the initial step of melanin biosynthesis , that is , conversion of tyrosine to dopa and subsequently dopa to dopaquinone . it is a glycosylated protein and a major regulator of melanogenesis . to date , many approaches have been tried to regulate tyrosinase activity and melanin content . to that end , we screened small interfering rna sequences for sequence - inhibited tyrosinase expression in b16 cells and in c57bl/6 mice . we analyzed tyrosinase mrna levels by quantitative real - time pcr and determined tyrosinase activity and melanin content at 24 , 48 , and 72 hours after transfection . results showed that sinm_011661_001 was the most efficient small interfering rna sequence in suppressing tyrosinase mrna expression , and cells transfected with this sequence showed lower tyrosinase activity . moreover , intravitreous injection of sinm_011661_001 in c57bl/6 mice induced an efficient and stable gene - specific inhibition of expression at the posttranscriptional level .

the most recent estimates indicate that the prevalence of autism spectrum disorders ( asd ) in the united states has raised to 1 in 54 boys and 1 in 252 girls . although increased awareness and changes in diagnostic criteria have been proposed as the major contributors to this increased prevalence , as of today , the etiopathology of disorders like asd and schizophrenia ( sz ) remains largely unknown . several studies have suggested that impaired mitochondrial function and altered energy metabolism in individuals with asd may contribute to their social and cognitive deficits [ 35 ] , and recent reports indicate the presence of mitochondrial dysfunction ( md ) in brain , skeletal muscle , and peripheral blood mononuclear cells ( pbmc ) from children with asd . the md in asd is generally characterized by lower complex i activity accompanied , in a subset of cases , by deficits in other complexes [ 68 ] . beside their critical role in a number of pathways , spanning from atp production ( via oxidative phosphorylation ) , one - carbon metabolism regulation , heme biosynthesis , fatty acid catabolism , and branched chain amino acid metabolism for example , human neutrophil mitochondria are involved in several functions such as chemotaxis , respiratory burst activity , maintenance of cell shape , and apoptosis [ 1317 ] . furthermore , neutrophil phagocytosis may involve the incorporation of some mitochondrial proteins into the phagosome . in addition , mitochondria can be involved in the immune response by providing part of the metabolic pathway for gln , in a process named glutaminolysis [ 19 , 20 ] . interestingly , gln is implicated in the expression of nadph oxidase components , cytokine production in lymphocytes , and macrophage , and as a provider for substrates required for nucleic acid synthesis [ 2125 ] . taken together , these lines of evidence unveil a link between mitochondria and immune response [ 1012 ] . indeed , deficits in bioenergetics have been reported in lymphocytes from children with asd enrolled in the case - control population - based childhood autism risk genetics and environment ( charge ) study [ 26 , 27 ] . children with asd in this study display a number of immune dysfunctions including abnormalities in monocytes , t cells and nk cell responses . these observations suggest the presence of a genetic background that results in a distinct immune profile in responses to a variety of triggers , among them psychological stressors , exposure to chemical triggers , and infectious agents [ 29 , 30 ] . considering that ( i ) mitochondria are inherited maternally via oocyte , ( ii ) maternal diet or immune activation during pregnancy has an impact on fetal metabolic and immune programming [ 3133 ] , and ( iii ) offspring born to pregnant mice injected with poly(inosinic : polycytidylic acid ) ( poly(i : c ) ) , a synthetic double - stranded rna that mimics viral infection via activation of toll - like receptor-3 ( tlr3 ) , at embryonic day 12.5 ( e12.5 ) , display core behavioral symptoms of asd [ 34 , 35 ] and sz , it is hypothesized that prenatal exposure of mothers to an immunogenic response , that is , poly(i : c ) elicits changes in mitochondrial function in splenocytes from progeny lasting into adulthood . exposure to tlr ligands can lead to maternal hypertension , vascular dysfunction , and proteinuria in pregnant animals but not in nonpregnant animals [ 3638 ] suggesting the occurrence of a differential immune response / pathway during pregnancy . differences are also evident between pregnant individuals with human placentas and patients with preeclampsia showing greater expression of tlr3 , along with tlr2 , tlr4 , and tlr9 , compared to nonpreeclampsia mothers [ 39 , 40 ] . these data suggest that tlr signaling may be involved in placental deficiencies / abnormalities that may provide a framework for altered fetal programming . of note , trophoblastic inclusions , which are also observed in preeclampsia and other placental defects , were reported to be increased in placenta from mothers of children with asd compared to controls . furthermore , maternal exposure to various pathogens , including viruses , significantly increases the risk for asd and sz [ 4248 ] . considering that maternal exposure to various pathogens is associated with asd and sz , the critical link between prenatal maternal infection and postnatal brain and behavioral pathology seem to be the maternal immune response , including cytokine production [ 47 , 4954 ] , which may contribute to the fetal imprinting of the neuroimmune response and , possibly , mitochondria - mediated metabolic responses . although it is already known that upon poly(i : c ) injection , the induction of maternal cytokines alters the expression of several cytokines in the fetal brain ( with only il-1 remaining elevated at 24 h with only a few changes during adulthood ) , it is unknown if maternal immune activation ( mia ) also causes chronic changes in the bioenergetics of immune cells ( such as splenocytes ) of adult offspring . in this study , we sought to determine whether mia in pregnant dams alters mitochondrial function in splenocytes from affected offspring . to test this , dams were exposed to poly(i : c ) on gestational day 12.5 to induce mia . this stage of gestation correlates with the late first trimester in humans , coincidental with the time that infections are most closely linked to increased incidence of asd and sz [ 47 , 48 ] . the present study reports for the first time that mia activation by poly(i : c ) at early gestation , which can lead to impairments in multiple psychological domains , is associated with mitochondrial changes in immune the cells of adult offspring . male and female c57bl/6j ( jackson laboratory , sacramento , ca , usa ) mice were bred and maintained by the center for laboratory animal research , at university of california , davis , and maintained at ambient room temperature on a 12 h light / dark cycle ( lights on at 06:00 h ) . all procedures were performed with approval by the institutional animal care and use committee , university of california , davis , and in accordance with the guidelines provided by the national institutes of health guide for the care and use of laboratory animals . mice were mated overnight and females were checked daily for the presence of seminal plugs , noted on gestational day 0.5 ( g0.5 ) . on g12.5 , pregnant female mice were weighed and injected with a single dose ( 20 mg / kg ; i.p . ) of poly(i : c ) ( sigma aldrich , st louis , mo , usa ) or saline vehicle ( sham ) as previously described . each dam was returned to its cage and left undisturbed until the birth of its litter . all mice pups remained with the mother until weaning on postnatal day 21 , at which time mice were group - housed 3 - 4 per cage with same - sex littermates . mice born from poly(i : c)-treated dams exhibited autism - like behavioral deficits including reduced social approach , increased ultrasonic vocalizations , and repetitive marble burying behaviors . one week following behavioral testing , 12 wk old mice were sacrificed by cervical dislocation and spleens were collected for tissue processing . while spleen is constituted by a variety of cells relevant to the immune response ( including t- and b - lymphocytes , dendritic cells , and macrophages ) , it has recently been shown that spleens from the offspring of mia mice elicited by poly(i : c ) provide a more homogeneous preparation enriched in granulocytes compared to the preparation obtained from whole blood . briefly , spleens were homogenized into single cell suspensions by gently pushing them through a 100 m nylon mesh filter ( fisher sci ) into pbs at 4c . cells were then pelleted , and rbcs were lysed using ack lysis buffer according to the manufacturer 's instructions ( gibco ) . cell viability was determined by trypan blue staining and found to be about 90% . the oxygen uptake of intact cell suspensions ( 10 cells / ml ) obtained as described above was measured by using a clark - type o2 electrode from hansatech ( king 's lynn , uk ) at 22c . cells were incubated in the presence of 5 mm glucose in calcium and magnesium - supplemented hbss buffer without phenol red at 2022c . nadh , succinate , and cytochrome oxidase activities were evaluated under phosphorylating conditions as described before [ 6 , 30 ] . to this end , cells were permeabilized with a controlled treatment with digitonin by adding 60 g / ml 2x recrystallized digitonin for 2 min . oxygen consumption rates were evaluated in the presence of 1 mm adp plus 1 mm malate-10 mm glutamate followed by the addition of 5 m rotenone ; 10 mm succinate followed by the addition of 3.6 m antimycin a , and 10 mm ascorbate and 0.2 mm n , n , n,n-tetramethyl - p - phenylenediamine followed by the addition of 1 mm kcn . student 's two - tailed t - test was used to evaluate the differences between offspring of poly(i : c)-treated and sham - treated dams . splenocytes from adult mice born to either sham- or poly(i : c)-treated dams were isolated for mitochondrial function testing . given that most of the oxygen uptake by cells is linked to atp production via oxidative phosphorylation , this parameter was evaluated in intact cells in the presence of glucose ( figure 1(a ) ) . the rate of oxygen uptake by intact cells from sham - treated dams was 0.22 0.03 nmol oxygen ( min 10 cells ) . under the same conditions , this rate was decreased by 36% in poly(i : c)-treated dams ( figure 1(a ) ) . addition of oligomycin , an inhibitor of atpase , was used to stop the fraction of oxygen utilized to synthesize atp via mitochondria . in both groups , more than 90% of the total oxygen uptake was inhibited by oligomycin , supporting the previous assumption that most if not all oxygen uptake by these cells was derived from oxidative phosphorylation . the oxygen uptake resistant to oligomycin , considered somewhat equivalent to state 4 ( nonphosphorylating mitochondria ) , was not different between groups . this result indicated that the proton leak across the inner mitochondrial membrane was similar in both groups , suggesting no major mitochondrial membrane damage by either treatment . addition of fccp , an uncoupler of electron transport and atp synthesis , increased significantly the basal oxygen uptake to a similar extent in both groups ( 2.5- to 3-fold ) with no changes between treatments , suggesting that the maximum respiratory capacity was similar in both groups . coupling between oxygen uptake and atp production was evaluated by the respiratory control ratio in intact cells ( rcr ) . mitochondria from either treatment showed a significant coupling with glucose as a substrate ( with malate - glutamate , rcr = 3.5 0.4 and 2.7 0.3 ; with succinate , rcr = 4 1 and 6.2 0.5 , for saline and poly(i : c ) , resp . ) with no statistical differences between treatment groups . this result indicated that mitochondria were highly coupled and provided a means of support for their integrity during the testing process . phosphorylating mitochondria from splenocytes of sham animals in the presence of an nad - linked substrate ( such as malate ) consumed oxygen at a rate of 0.31 0.04 nmol oxygen ( min 10 cells ) . phosphorylating mitochondria from offspring of poly(i : c)-treated dams showed a significant decrease in oxygen consumption ( by 55% ; p < 0.01 ; figure 1(b ) ) . by adding rotenone , an inhibitor of complex i , and succinate , a substrate for complex ii , the segment comprising from complex ii to complex v was evaluated . no differences in terms of oxygen uptake were observed between controls and poly(i : c ) suggesting that the deficit in offspring of poly(i : c)-treated dams was located at the level of complex i. confirming this result , complex iv activity was not different between treatments ( figure 1(b ) ) suggesting that mitochondrial mass was equivalent between groups . however , attempts to directly evaluate complex i activity were unsuccessful due to the limited amount of biological material . the ratios among complexes need to be preserved to provide suitable oxidation of substrates . to this end , the ratios of electron transport chain activities indicated that both treatments allow oxidizing fad - linked substrates ( such as fatty acids ) similarly , whereas a significantly lower oxidation of nad - linked substrates ( such as glucose ) was evident in the poly(i : c)-treated condition compared to controls ( figure 1(c ) ) . this imbalance in the complexes ' ratios suggests that splenocytes from offspring of dams exposed to poly(i : c ) use preferentially fatty acids over glucose as their main substrate for mitochondrial oxidative phosphorylation . these results are consistent with the md observed in lymphocytes from asd children characterized by lower complex i activity and accompanied , in some cases , by deficits in other complexes and/or pyruvate dehydrogenase [ 68 ] . the cytokine production ( il-1 , il-6 , il-10 , il-17 , and tnf- ) from cona - activated splenocytes obtained from adult offspring of poly(i : c)-treated animals was not different from that of sham - treated animals ( see supplementary material available online on http://dx.doi.org/10.1155/2013/609602 ) . this is consistent with the findings of others utilizing a similar mia model in which only a handful of cytokines was still increased in early adulthood ( frontal corteces il-1 , il-6 , il-10 , and il-9 ; cingulate corteces il-10 and ifn- ; none in hippocampus or serum ) . the aim of this study was to evaluate mitochondrial function in splenocytes from offspring gestationally exposed to an acute viral mimetic , that is , poly(i : c ) , to induce mia . our results indicate that the exposure of dams to a single dose of poly(i : c ) at gestational day 12.5 likely triggers a tlr3-mediated response in the mother that is transmitted transplacentally to the offspring . in particular , the proinflammatory cytokine il-6 has been proved to be a key intermediary in the behavioral changes observed in the offspring of dams treated with poly(i : c ) . moreover , blocking il-6 with antibodies prevents behavioral changes in the offspring , and poly(i : c)-induced mia in il-6 ko does not result in behavioral changes in the offspring . these data seem to suggest a role for il-6 in mia - induced behavioral changes , although we can not exclude other inflammatory agents such as type 1 interferons which have also been shown to take part in the response to poly(i : c ) . this mia imprints a fetal programming that can still be detected during adulthood characterized by abnormal behaviors resembling those of asd [ 34 , 35 ] and sz and , biochemically , by a lower oxidative phosphorylation capacity in mitochondria within intact cells and isolated mitochondria . this suggests that prenatal immune changes ensuing the maternal poly(i : c ) administration are likely to imprint the long - lasting changes in the bioenergetics of the adult offspring splenocytes . while , at the normal murine fetomaternal interface , immune cells such as neutrophils , macrophages , and nk cells are assumed to be excluded from the placenta and localized only in the decidua , treatment with poly(i : c ) disrupts this normal distribution and induces a significant increase in the levels of proinflammatory cytokines in the placenta and a large migration of immune cells , primarily nk cells from the decidua towards the placenta , invading the spongiotrophoblast and then the labyrinth . trophoblasts , which express tlr3 , play a role in coordinating the maternal innate immune response to infection at the fetomaternal interface [ 6264 ] and , especially in this case , in response to viral infection . these results beget the question , what is the link between lower complex i activity ( or lower oxidative phosphorylation ) in the offspring and an acute maternal immune response ? a growing body of evidence is placing mitochondria at the center of bioenergetics and immune response / inflammation . immunity to infection is also dependent on mitochondria function by regulating the synthesis of both pro- and anti - inflammatory cytokines [ 6569 ] . more recently , the view that mitochondria act as a platform facilitating innate immune responses adds to our understanding of the molecular complexity of sensor and adaptor interactions that promote effective host defense [ 11 , 70 ] . therefore , an emerging concept is that innate immune signaling is regulated by basic host metabolic functions . for instance , toll - like receptor signaling activates mitochondrial biogenesis during critical illness [ 7173 ] , perhaps in response to increased oxidative damage in host cells [ 71 , 74 , 75 ] . acute inflammation is accompanied by increases in inflammatory cytokines sustained by glycolysis , whereas chronic inflammation is sustained by less inflammatory cytokines , with more reparative features fueled mainly by mitochondria - derived atp . thus , in this study , mia seems to imprint the immune cells of adult offspring with this more glycolytic stage resembling the influence of an acute inflammation , without switching back to the less inflammatory response . without pointing at cause or consequence , it is interesting to note that the changes in bioenergetics in the immune cells ( and not their immune response or the immune response in brain or serum ) segregate with the abnormal behaviors observed in this mia model . however , a number of studies have shown differential mia induction and behavioral responses depending on the gestational exposure stage [ 7779 ] . this would suggest the existence of a window of vulnerability to infection during gestation for the onset of different behavioral defects , which may be reflected also on the mitochondrial function of the offspring . the above effects can be explained by ( i ) the transfer of immune cells and/or cytokines from mother to the fetus at the maternal - fetal interface and ( ii ) a genetic predisposition / susceptibility of the offspring that , in association with maternal viral or bacterial infections , might increase the risk of long - lasting behavioral and immune changes . furthermore , we can not exclude the possibility that the relatively high doses of poly(i : c ) used in this study could have affected the well - being of the mother and therefore that of the fetus . for instance , poly(i : c ) inhibits the development of diabetes in the nod mouse whereas the development of diabetes in diabetes - prone bb rats is poly(i : c)-dose dependent [ 8184 ] . indeed , a recent report indicated that at least some nongenetic risk factors are shared between asd and sz , in particular , diabetes , exposure to drugs , nutritional deficiencies , and infectious agents among others . considering the mechanisms described above , studies are now needed to clearly identify the key players affected in this acute viral response in order to evaluate the increase in risk of either asd or sz that is associated with these ( and other [ 30 , 86 , 87 ] ) modifiable environmental factors to elicit public health interventions .

maternal immune activation ( mia ) is a potential risk factor for autism spectrum disorder ( asd ) and schizophrenia ( sz ) . in rodents , mia results in changes in cytokine profiles and abnormal behaviors in the offspring that model these neuropsychiatric conditions . given the central role that mitochondria have in immunity and other metabolic pathways , we hypothesized that mia will result in a fetal imprinting that leads to postnatal deficits in the bioenergetics of immune cells . to this end , splenocytes from adult offspring exposed gestationally to the viral mimic poly(i : c ) were evaluated for mitochondrial outcomes . a significant decrease in mitochondrial atp production was observed in poly(i : c)-treated mice ( 45% of controls ) mainly attributed to a lower complex i activity . no differences were observed between the two groups in the coupling of electron transport to atp synthesis , or the oxygen uptake under uncoupling conditions . concanavalin a- ( cona- ) stimulated splenocytes from poly(i : c ) animals showed no statistically significant changes in cytokine levels compared to controls . the present study reports for the first time that mia activation by poly(i : c ) at early gestation , which can lead to behavioral impairments in the offspring similar to sz and asd , leads to long - lasting effects in the bioenergetics of splenocytes of adult offspring .

in the era of prostate - specific antigen ( psa ) screening , more and more patients are diagnosed with insignificant prostate cancer . because many of these cancers will not become clinically symptomatic , active surveillance ( as ) with delayed treatment has been introduced and has been reported to have similar therapeutic effects . the criteria for defining patients who are suitable to enter as protocols are mainly based on psa , clinical stage , and tumor grade . however , many studies have shown that the tumor grade obtained by prostate biopsy does not always correlate with the final pathological grade of the surgical specimen resected in a radical prostatectomy . in many cases , gleason score ( gs ) upgrading of up to 57% of cases has been reported in some studies . the incorrect assessment of tumor grade may lead to inappropriate estimation of cancer aggressiveness , resulting in under - treatment of these patients . thus , a critical factor for the success of as is the use of appropriate entry criteria . although a number of prognostic models have been developed to help to identify men who are appropriate candidates for as , it remains controversial whether to adopt psa density as an appropriate entry criterion . accordingly , we conducted a prospective data analysis in patients with low - grade prostate cancer and evaluated the potential of several clinical and pathological variables to predict upgrading of the cancer . after we obtained approval from the ethics committee at institutional review board of daegu catholic university hospital , we conducted a prospective analysis of 60 patients with low - risk prostate cancer who were recruited from 4 university hospitals ( gs6 , psa<10 ng / ml , t1/t2 , 2 positive biopsy cores ) . all patients underwent a retropubic or laparoscopic radical prostatectomy between january 2010 and december 2013 . all clinical , imaging , laboratory , and pathological data were collected and recorded prospectively and were analyzed retrospectively . we excluded patients who had received any preoperative therapy for prostate cancer ( hormone therapy , radiation therapy ) and patients who underwent less than 10-core transrectal ultrasound ( trus)-guided biopsy . biopsy cores were examined by different university - based pathologists and all radical prostatectomy samples were examined according to the standard protocol of the pathology department at each university hospital . we analyzed age , body mass index ( bmi ) , preoperative psa value , psa density , number of cores , and number of positive cores in biopsy material . we analyzed the association of these factors with upgrading of cancer in the radical prostatectomy specimen . any increase in gs between core biopsy and radical prostatectomy specimen preoperative psa was measured before the digital rectal exam , trus , or core biopsy . during trus , prostate volume was calculated according to information on the maximum transverse diameter ( d1 ) , the maximum antero - posterior diameter ( d2 ) , and the maximum longitudinal diameter ( d3 ) and by using the formula based on the prostate ellipse dimension theory : d1d2d3/6 . psa density was calculated by dividing the preoperative psa value and prostate volume . a histological report concerning tumor grade of the surgical specimen and pathological stage we estimated tumor grade by use of the contemporary criteria of the 2005 international society of urological pathology modified gleason system . statistical analysis was performed by using pasw statistics ver . 18.0 ( spss inc . , the normality condition of the numerical variables was studied by means of the kolmogorov - smirnov test . psa was the only variable with a normal distribution ; consequently , student t - test was used to compare means . to analyze other variables , the mann - whitney u test was used , and the chi - square test was used for categorical variables . the optimal cutoff values and sensitivity and specificity for quantitative variables that were found to be significant predictors were estimated by using receiver operating characteristic ( roc ) curve analysis . positive predictive value ( true positive/[true positive+false positive ] ) and negative predictive value ( true negative/[true negative+false negative ] ) were also estimated . in total , 60 patients met our inclusion criteria within the study period and were entered into the analysis . the median patient age was 63 years , the median preoperative psa value was 6.26 ng / ml , and the median psa density was 0.15 ng / ml . as we previously described , every patient underwent trus biopsy with more than 10 cores ; 58 patients ( 96.7% ) underwent 12-core biopsy . on the basis of the pathological results from the radical prostatectomy , 2 patients ( 3.3% ) were diagnosed with gs 5 prostate cancer , 40 patients ( 57.1% ) had gs 6 cancer , 27 patients ( 38.6% ) had gs 7 cancer , and 1 patient ( 1.4% ) had gs 8 . clinical stage on the basis of the digital rectal examination and trus findings was categorized as follows : 61 patients ( 86.7% ) with t1c disease and 9 patients ( 13.3% ) with t2a prostate cancer , respectively . following radical prostatectomy , a tumor upgrade was noticed in 28 patients ( 40.0% ) . extracapsular extensions were noted in 6 patients ( 10.0% ) and positive surgical margins in 8 patients ( 17.3% ) . the demographic , clinical , and pathologic data regarding the presence of pathological tumor upgrading are described in table 1 . a statistically significant correlation between preoperative psa density and postoperative gs upgrading was noted ( p=0.030 ) . no statistical correlation with postoperative gs upgrading was found for age , bmi , preoperative psa level , prostate volume , number of biopsy cores , number of positive biopsy cores , the institute where the biopsy was done , or the operation method . in the analysis of pathological outcomes after radical prostatectomy , tumor upgrading was highly associated with extracapsular cancer extension ( p=0.000 ) . in the multivariate analysis , psa density was the only statistically significant variable to predict gs upgrading ( p=0.030 ) ( table 2 ) . the estimated optimal cutoff value of psa density was 0.13 ng / ml , as obtained by roc analysis ( area under the curve=0.66 ; p=0.020 ; 95% confidence interval , 0.53 - 0.78 ) , as shown in fig 1 . it is well established that tumor grade is the most reliable and valuable parameter for estimating the prognosis of prostate cancer . by being a surrogate for tumor aggressiveness , the tumor grade allows us to stratify patients as either low , intermediate , or high risk . to date , the calculation of gleason grade has been based on pathological evaluation of the cores obtained by prostate biopsy . for patients who enter an as treatment program with close monitoring until disease progression and radical therapy , biopsy results , in combination with psa levels and clinical stage , are the data that guide treatment decisions . nevertheless , upgrading of the gs between the needle biopsy and the radical prostatectomy is not rare . upgrading was found in 30% of patients in one recent meta - analysis , and higher rates have also been published . this observation has important prognostic meaning because a significant percentage of these cases progress outside the prostate capsule . the present data , which showed gs upgrading in 40.0% of samples , also revealed 24.0% extracapsular extension . given that conservative treatment protocols are mainly applied in low - risk patients , these findings are very important . an underestimation of prostate cancer aggressiveness may lead to under - treatment and inappropriate monitoring of biologically aggressive tumors . thus , it is clear that many patients classified as having clinically localized disease and being at low risk actually have highly malignant cancer with a risk of clinical progression . several reports have indicated that the likelihood of gs upgrading will decrease as the number of cores obtained by biopsy increases . king et al . showed that in patients with a biopsy gs of 6 , an extended biopsy strategy reduced gs upgrading from 66.7% to 36.8% . 's study also showed that gs upgrading decreased from 47.9% to 23.5% by taking more than 18 cores in a cohort of patients with low - risk prostate cancer . in coogan . 's study , increasing the number of cores from 6 to10 significantly improved the accuracy of the biopsy gs . the question remains as to what the optimal number of biopsy cores should be . . showed that at least 10-core biopsies may be needed in patients with low - risk prostate cancer . adopting this concept , no statistically significant dif ferences were found in the present study when comparing 12-core biopsies with 10-core biopsies . whereas some groups have shown that an increase in positive cores correlates with an upgrading of gs , other studies could not find any association . in the present study , the association between preoperative psa level and gs upgrading also varies . whereas the studies of mian et al . and king et al . showed that psa level was a statistically significant predictor of gs upgrading . in the present study , we were not able to show an association between psa level and gs upgrading . several reports have shown that an increase in prostate weight reduces the risk of gs upgrading . an explanation for this finding might be that a small prostate size is a surrogate of low in vivo androgenicity , leading to reselection of aggressive cancers in an androgen - depleted hostile environment . however , the results in the literature vary in relation to the weight of the prostate . in the present study however , in the multivariate logistic regression analysis , prostate weight was not an independent predictor of gs upgrading . even though conf licting results regarding the interobserver reproducibility of gleason scoring in prostate biopsies have been reported , we did not observe a significant difference in gs upgrading between university pathologists in a univariate analysis ( table 2 ) . this result could partly be due to the proficiency of the pathologists and also to the number of biopsy cores being more than 10 . corcoran et al . examined the predictive characteristics of psa density in patients with low- and intermediate - risk disease on biopsy and subsequently treated with radical prostatectomy . they found that 58.3% of patients with low - grade disease after prostate biopsy were upgraded to higher scores and that psa density was a significant predictor ( p<0.001 ) of upgrading in patients with gs 6 . similar results were observed by kojima et al . and magheli et al . , with p=0.019 and p=0.037 , respectively . our results add to the above data , revealing a beneficial role of psa density in prediction of upgrading ( p=0.030 ) . a significant association of psa density with the pathological outcomes following radical prostatectomy was observed . we tried to produce a threshold level of psa density , over which the possibility of gleason upgrading increased significantly , triggering either a repeat biopsy or definitive therapy . by using the roc analysis , even though the sensitivity and negative predictive value were low , an increased specificity and positive predictive value were found ( table 3 ) . given the complexity of prostate cancer , it seems to be more reasonable to use psa density combined with other predictive factors . first , the sample size was relatively small and might have decreased the strength of the results . another limitation is that the psa density calculation was based on the estimation of prostate volume during trus made by 4 different operators . even though a standard calculation protocol was followed , the interobserver differences in the volume measurement may have negatively affected the validity of the results . last , even though the data collection was made prospectively , the analysis was done retrospectively . however , the strength of our study was that the evaluation of psa density in low - risk prostate cancer was adjusted solely for patients who underwent biopsies with more than 10 cores . equal distribution of the number of cores obtained by prostate biopsies might have reduced a statistical bias and increased the value of the results for the estimation of tumor grade . with the commonly used criteria for as , gs upgrading in a radical prostatectomy specimen is a challenging problem . psa density was an independent predictor of gs upgrading and may be included as a criterion for patients eligible for as .

purposeto evaluate prospectively the role of prostate - specific antigen ( psa ) density in predicting gleason score upgrading in prostate cancer patients eligible for active surveillance ( t1/t2 , biopsy gleason score6 , psa10 ng / ml , and 2 positive biopsy cores).materials and methodsbetween january 2010 and november 2013 , among patients who underwent greater than 10-core transrectal ultrasound - guided biopsy , 60 patients eligible for active surveillance underwent radical prostatectomy . by use of the modified gleason criteria , the tumor grade of the surgical specimens was examined and compared with the biopsy results.resultstumor upgrading occurred in 24 patients ( 40.0% ) . extracapsular disease and positive surgical margins were found in 6 patients ( 10.0% ) and 8 patients ( 17.30% ) , respectively . a statistically significant correlation between psa density and postoperative upgrading was found ( p=0.030 ) ; this was in contrast with the other studied parameters , which failed to reach significance , including psa , prostate volume , number of biopsy cores , and number of positive cores . tumor upgrading was also highly associated with extracapsular cancer extension ( p=0.000 ) . the estimated optimal cutoff value of psa density was 0.13 ng / ml2 , obtained by receiver operating characteristic analysis ( area under the curve=0.66 ; p=0.020 ; 95% confidence interval , 0.53 - 0.78).conclusionspsa density is a strong predictor of gleason score upgrading after radical prostatectomy in patients eligible for active surveillance . because tumor upgrading increases the potential for postoperative pathological adverse findings and prognosis , psa density should be considered when treating and consulting patients eligible for active surveillance .

traditionally , barium examinations have been the mainstay of radiological investigations of the small and large intestines . however , the standard barium follow - through ( sbft ) examination has high false - negative and false - positive rates for the detection of abnormalities this is because only 2535% of the bowel length can be adequately assessed on a standard examination due to overlapping loops , poor coating , and other factors . several new techniques have therefore evolved that provide better diagnostic capabilities and higher accuracy rates.[24 ] in this article , the newer techniques and modalities being employed for imaging of the bowel are detailed , with an overview of their strengths and weaknesses . innovations in intestinal imaging have been driven by the demand for detailed clinical information and the limitations of standard methods of small - bowel examination . alternative techniques such as the pill camera that allow high - definition color views of the bowel have appeared . at an average frequency of two frames per second , a total of > 50,000 frames are collected in 78 h as the pill camera travels through the bowel . despite the high - resolution images provided by this technique , it has become increasingly apparent that not all mucosal abnormalities seen on wireless capsule endoscopy are clinically relevant . mucosal erosions may be seen in as many as 14% of asymptomatic healthy subjects , with the rate of such abnormalities being more than double this in users of nonsteroidal anti - inflammatory drug ( nsaid ) agents . furthermore , the pill camera does not provide any information about mural and extramural abnormalities . contraindications to the pill camera include bowel obstruction , history of bowel strictures or fistulae . the most accurate conventional radiologic method in the diagnosis of small - bowel obstruction , inflammatory bowel disease ( ibd ) , gastrointestinal bleeding , and neoplasms is double - contrast enteroclysis . however , sbft has remained the most commonly performed method for the investigation of small - bowel diseases because of its ease of performance and the relative technical complexity of enteroclysis as well as the discomfort to patients during intubation . the sbft has a low diagnostic yield and low negative predictive value . in a recent study , 8% of patients with known crohn disease encountered capsule retention despite a previous normal sbft . non - intubation examinations do not fully distend the bowel , and therefore partial strictures , obstructions , and small polyps or masses can be easily missed . the advantage of enteroclysis examinations is that there is optimal bowel distension , which allows detailed evaluation of all segments [ figure 1 ] . methylcellulose bowel examination shows linear filling defects ( webs ) ( arrows ) in the distal ileum secondary to the use of nonsteroidal anti - inflammatory drugs ( nsaids ) enteroclysis examination with barium methylcellulose is the most commonly performed enteroclysis method in many countries . however , the disadvantage with this technique is that methylcellulose has a washout effect on superficial mucosal features . subtle surface abnormalities get effaced as more methylcellulose is infused to achieve a good double - contrast effect and bowel distension . therefore , this method should be reserved for detection of obstruction or mass lesions or it should be used in combination with ct scan or mri exams.[247 ] fluoroscopic evaluation with positive enteral contrast can help distinguish fixed ( stenotic ) segments of small - bowel narrowing from spasm caused by active inflammation in patients who have crohn disease and can also differentiate mild stenosis from normal peristaltic contractions . on the other hand , double - contrast enteroclysis with air / co2 and barium is by far the best method for showing mucosal details of the small bowel.[578 ] a scientific study comparing double - contrast air enteroclysis with histopathology showed good correlation between the two for the visualization of aphthae and small ulcers [ figure 2 ] . magnified view from an air barium double contrast enteroclysis examination shows early linear mucosal breaks and ulcers ( arrow ) in a patient with proven crohn disease therefore , if capsule endoscopy is unavailable or contraindicated , double - contrast air enteroclysis should be the preferred modality for the detection of early ulcerations , irregularities , and erosions of the small bowel . this technique should also be employed if other imaging tests have been negative and a suspicion of small - bowel pathology still persists . this technique can be performed on its own or it can be combined with ct scan or mri . the pill camera remains the most sensitive method for the evaluation of gastrointestinal tract bleeding.[89 ] the most important factor for obtaining good intestinal imaging is optimal distension of the bowel with enteral contrast . enteral contrast can be positive , neutral , or negative , depending on its density ( hu ) . positive enteral contrast agents range from a 415% contrast solution to a dilute 0.36% barium solution . the author prefers an 810% iodine concentration of water - soluble contrast because this density allows diagnostic fluoroscopic observations as well as diagnostic radiographs . neutral contrast agents can be methylcellulose , mannitol , polyethyl glycol , or other bulk fibers and have a density similar to water ( 0 hu ) . negative contrast is mainly used in the imaging of the colon [ ct colonography ( ctc ) ] , where co2 or air is insufflated per rectum . enteral contrast can be administered via the intubation method ( ct enteroclysis / mri enteroclysis ) or the enterographic method [ figure 3 ] . for enterographic examination , the patient ingests a large volume of contrast over a set period of time prior to imaging . the 1300 ml is divided into two aliquots : the first lot is ingested with a prokinetic agent ( metoclopromide ) over 25 min and the second lot is consumed over the next 25 min . just prior to the scan , 200 ml of contrast the main advantage of the enterographic examination is that it is more acceptable to patients than enteroclysis as intubation may be an unpleasant experience for patients . the disadvantage of enterograpic examination is that it may not provide uniform or adequate bowel distension as compared to enteroclysis . axial ct enteroclysis examination demonstrates a segment of kinked bowel ( arrowhead ) and several adhesive bands across other segments ( arrows ) . this patient had undergone several negative ct examinations previously water is also routinely used as a neutral enteral contrast agent at many centers ; however , its use in dedicated small - bowel studies should be discontinued . water has been found to provide inadequate distension ( at various volumes and ingestion times ) of the small bowel in many studies.[1011 ] this is because water undergoes rapid reabsorption in the bowel and fails to adequately distend the distal small - bowel segments . therefore , additives ( such as contrast , mannitol or other agents ) need to be mixed with water to provide an iso - osmolar solution that does not get reabsorbed and remains in the lumen to provide distension . the use of water as a contrast agent may lead to false - negative examinations . furthermore , collapsed loops are known to demonstrate hyperenhancement on post - contrast studies and this may lead to false - positive diagnoses . therefore , the author strongly discourages the use of water as an enteral agent in dedicated studies of the small bowel . the only instance where water may be used as an enteral agent is in the enteroclysis technique where the operator exclusively controls distension of the bowel and rate of infusion . the advantage of current - generation multidetector ct ( mdct ) scanners is increased anatomical coverage with thinner sections , which provide high - quality mutiplanar ( mpr ) images and fewer motion artifacts.[1213 ] positive - contrast ct enteroclysis ( cte ) or enterography ( cteg ) may be used mainly for the diagnostic workup of small - bowel obstruction or in suspected cases of small intestinal tumors or metastases . one of the drawbacks of positive contrast is the production of streak artifacts on mpr reconstruction , particularly on older ct scanners that can not produce isotropic voxels ( slice thickness > 2 mm ) . the advantages of neutral contrast are that it does not obscure the mucosal lining and does not produce streak artifacts . therefore , neutral - contrast cte or cteg is indicated for the workup of patients with inflammatory bowel bleed , gastrointestinal bleeding , and suspected bowel pathologies . several studies have shown that ctc has high sensitivity and specificity ( > 95% ) for the detection of polyps and cancers [ figure 4a ] . datasets of the distended colon are obtained , which can then by manipulated by software to provide virtual colonoscopy images . screenshot ( a ) from a ct colonographic examination shows a polyp in the colon . top two axial images show the location of the polyp within a marker box , whereas the bottom two are virtual 3d images in the supine and prone positions which detail polyp size and distance from the rectum . mucosal views ( b ) show a virtual dissection view of the colon with a small polyp ( arrow ) in the left image . on the right , translucency rendering shows this lesion to be homogenously dense ( red ) , implying this is retained fecal matter mri is an emerging technique for comprehensive and functional bowel imaging . the absence of ionizing radiation and high - contrast resolution are the major advantages of mri over ct scan . the nonionizing aspect of mri makes it particularly suited for use in patients with ibd who may need repeated imaging . the inherent high - contrast resolution can provide high diagnostic confidence and also facilitate the detection of discriminating features of intestinal diseases . t1w and t2w imaging sequences for the imaging of the bowel have been reported in earlier studies.[101718 ] however , in recent years , advances with ultra - fast sequences based on steady - state precession have revolutionized bowel imaging . these sequences can be performed within a single breath - hold , and when combined with bowel paralysis ( using antiperistaltic agents , e.g. , buscopan ) , the technique allows rapid imaging of the small bowel . these sequences are called true fast imaging with steady - state precession ( true - fisp ) , balanced fast field - echo ( ffe ) , or fast imaging employing steady - state acquisition ( fiesta ) . these sequences are relatively insensitive to motion artifacts and provide high contrast between the bowel wall , lumen , and the mesentery . the problem with these sequences is the presence of a black boundary artifact along the bowel wall , which may mask small lesions or abnormalities . the addition of fat suppression may help in reducing the effects of the black boundary artifact [ figure 5 ] . fast sequences also obviate the need for long breath - holds , and thus reduce motion or respiratory artifacts . a patient with crohn disease . mri enterography examination shows good opacification of the small and large bowel with thickening of the inflamed cecal wall ( arrow ) t2w fast sequences based on rapid acquisition and relaxation , such as half - fourier single - shot turbo spin - echo ( haste ) or single - shot fast spin - echo ( ssfse ) sequences , are also used and can provide high contrast between the lumen and the bowel wall , producing images akin to a conventional barium study . fat saturation can be used to increase contrast resolution and also for better assessment of bowel enhancement . it is possible that with this technique , high - resolution three - dimensional datasets comprising isotropic voxels of the entire abdomen could be produced within a single breath - hold . three - dimensional datasets allow high - quality mpr reconstructions , which can increase diagnostic confidence . usg also allows observation of normal or abnormal bowel peristalsis , fixity , and compressibility . some studies have employed bowel distension using oral contrast ( hydrosonography ) although its use remains limited . the current - generation high - frequency probes provide greater spatial resolution than ct scan or mri [ figure 6 ] . contrast - enhanced usg can provide detailed information about vascularity and inflammation of the bowel . high - resolution usg image shows an inflamed bowel segment , with marked enhancement of the mucosal vessels ( arrowheads ) and engorgement of the penetrating blood vessels ( arrow ) the most important factor for obtaining good intestinal imaging is optimal distension of the bowel with enteral contrast . enteral contrast can be positive , neutral , or negative , depending on its density ( hu ) . positive enteral contrast agents range from a 415% contrast solution to a dilute 0.36% barium solution . the author prefers an 810% iodine concentration of water - soluble contrast because this density allows diagnostic fluoroscopic observations as well as diagnostic radiographs . neutral contrast agents can be methylcellulose , mannitol , polyethyl glycol , or other bulk fibers and have a density similar to water ( 0 hu ) . negative contrast is mainly used in the imaging of the colon [ ct colonography ( ctc ) ] , where co2 or air is insufflated per rectum . enteral contrast can be administered via the intubation method ( ct enteroclysis / mri enteroclysis ) or the enterographic method [ figure 3 ] . for enterographic examination , the patient ingests a large volume of contrast over a set period of time prior to imaging . the 1300 ml is divided into two aliquots : the first lot is ingested with a prokinetic agent ( metoclopromide ) over 25 min and the second lot is consumed over the next 25 min . just prior to the scan , 200 ml of contrast the main advantage of the enterographic examination is that it is more acceptable to patients than enteroclysis as intubation may be an unpleasant experience for patients . the disadvantage of enterograpic examination is that it may not provide uniform or adequate bowel distension as compared to enteroclysis . axial ct enteroclysis examination demonstrates a segment of kinked bowel ( arrowhead ) and several adhesive bands across other segments ( arrows ) . this patient had undergone several negative ct examinations previously water is also routinely used as a neutral enteral contrast agent at many centers ; however , its use in dedicated small - bowel studies should be discontinued . water has been found to provide inadequate distension ( at various volumes and ingestion times ) of the small bowel in many studies.[1011 ] this is because water undergoes rapid reabsorption in the bowel and fails to adequately distend the distal small - bowel segments . therefore , additives ( such as contrast , mannitol or other agents ) need to be mixed with water to provide an iso - osmolar solution that does not get reabsorbed and remains in the lumen to provide distension . the use of water as a contrast agent may lead to false - negative examinations . furthermore , collapsed loops are known to demonstrate hyperenhancement on post - contrast studies and this may lead to false - positive diagnoses . therefore , the author strongly discourages the use of water as an enteral agent in dedicated studies of the small bowel . the only instance where water may be used as an enteral agent is in the enteroclysis technique where the operator exclusively controls distension of the bowel and rate of infusion . the advantage of current - generation multidetector ct ( mdct ) scanners is increased anatomical coverage with thinner sections , which provide high - quality mutiplanar ( mpr ) images and fewer motion artifacts.[1213 ] positive - contrast ct enteroclysis ( cte ) or enterography ( cteg ) may be used mainly for the diagnostic workup of small - bowel obstruction or in suspected cases of small intestinal tumors or metastases . one of the drawbacks of positive contrast is the production of streak artifacts on mpr reconstruction , particularly on older ct scanners that can not produce isotropic voxels ( slice thickness > 2 mm ) . the advantages of neutral contrast are that it does not obscure the mucosal lining and does not produce streak artifacts . therefore , neutral - contrast cte or cteg is indicated for the workup of patients with inflammatory bowel bleed , gastrointestinal bleeding , and suspected bowel pathologies . several studies have shown that ctc has high sensitivity and specificity ( > 95% ) for the detection of polyps and cancers [ figure 4a ] . datasets of the distended colon are obtained , which can then by manipulated by software to provide virtual colonoscopy images . screenshot ( a ) from a ct colonographic examination shows a polyp in the colon . top two axial images show the location of the polyp within a marker box , whereas the bottom two are virtual 3d images in the supine and prone positions which detail polyp size and distance from the rectum . mucosal views ( b ) show a virtual dissection view of the colon with a small polyp ( arrow ) in the left image . on the right , translucency rendering shows this lesion to be homogenously dense ( red ) , implying this is retained fecal matter the absence of ionizing radiation and high - contrast resolution are the major advantages of mri over ct scan . the nonionizing aspect of mri makes it particularly suited for use in patients with ibd who may need repeated imaging . the inherent high - contrast resolution can provide high diagnostic confidence and also facilitate the detection of discriminating features of intestinal diseases . t1w and t2w imaging sequences for the imaging of the bowel have been reported in earlier studies.[101718 ] however , in recent years , advances with ultra - fast sequences based on steady - state precession have revolutionized bowel imaging . these sequences can be performed within a single breath - hold , and when combined with bowel paralysis ( using antiperistaltic agents , e.g. , buscopan ) , the technique allows rapid imaging of the small bowel . these sequences are called true fast imaging with steady - state precession ( true - fisp ) , balanced fast field - echo ( ffe ) , or fast imaging employing steady - state acquisition ( fiesta ) . these sequences are relatively insensitive to motion artifacts and provide high contrast between the bowel wall , lumen , and the mesentery . the problem with these sequences is the presence of a black boundary artifact along the bowel wall , which may mask small lesions or abnormalities . the addition of fat suppression may help in reducing the effects of the black boundary artifact [ figure 5 ] . fast sequences also obviate the need for long breath - holds , and thus reduce motion or respiratory artifacts . a patient with crohn disease . mri enterography examination shows good opacification of the small and large bowel with thickening of the inflamed cecal wall ( arrow ) t2w fast sequences based on rapid acquisition and relaxation , such as half - fourier single - shot turbo spin - echo ( haste ) or single - shot fast spin - echo ( ssfse ) sequences , are also used and can provide high contrast between the lumen and the bowel wall , producing images akin to a conventional barium study . fat saturation can be used to increase contrast resolution and also for better assessment of bowel enhancement . it is possible that with this technique , high - resolution three - dimensional datasets comprising isotropic voxels of the entire abdomen could be produced within a single breath - hold . three - dimensional datasets allow high - quality mpr reconstructions , which can increase diagnostic confidence . the main advantages of usg are its nonionizing character and general availability . the dynamic , real - time qualities of usg provide high temporal resolution . usg also allows observation of normal or abnormal bowel peristalsis , fixity , and compressibility . some studies have employed bowel distension using oral contrast ( hydrosonography ) although its use remains limited . the current - generation high - frequency probes provide greater spatial resolution than ct scan or mri [ figure 6 ] . contrast - enhanced usg can provide detailed information about vascularity and inflammation of the bowel . high - resolution usg image shows an inflamed bowel segment , with marked enhancement of the mucosal vessels ( arrowheads ) and engorgement of the penetrating blood vessels ( arrow ) mri fluoroscopy : changes in bowel kinetics can be evaluated on mri fluoroscopy to demonstrate either an obstructive element or abnormalities in peristalsis . mri fluoroscopy can provide functional information regarding bowel motility and may help distinguish between fibrotic strictures and functional bowel spasm . pet / ct : positron emission tomography ( pet)/ct depends on the uptake of tracer by abnormal or highly metabolic tissue . typically , cancers or metastases show up as hot spots due to their higher metabolic rate as compared to the surrounding tissues . the role of pet / ct in intestinal imaging is mainly related to cancer imaging and the detection of distant metastases . pet - ct also has a role is the detection of recurrent cancers and in quantifying malignancy in suspicious nodes [ figure 7].[2324 ] pet / ct image shows a recurrent rectal tumor as a hot spot ( arrow ) just anterior to the presacral fascia diffusion - weighted mri imaging : diffusion - weighted mri signal is derived from the motion of water molecules within cells or extracellular spaces . highly cellular tumors have restricted water diffusion and show up as areas retaining high signal intensity on high b value ( 8001000 s / mm ) images . by performing diffusion imaging using different b values , it is possible to calculate the apparent diffusion coefficient ( adc , measured in m / s ) . areas of restricted diffusion show low adc values and adc values are inversely correlated with tumor cellularity . therefore , reduction in adc has been shown to have good correlation with response to cytotoxic therapy . currently , diffusion imaging is mainly used for detection of abnormal cancerous tissue and involved lymph nodes . studies have also been conducted on the detection of active inflammation in ibd patients using diffusion imaging [ figure 8 ] . diffusion - weighted mri image shows high signal in the cancer tissue ( arrow ) and involved lymph node in the mesorectum ( short arrow ) perfusion imaging : perfusion ct is a technique that integrates anatomic detail with assessment of vascular physiology . analysis of tumor enhancement , tumor blood flow , blood volume , mean transit time , and permeability surface area product are possible . perfusion ct is a reflection of angiogenesis in tumors , and therefore its main use is in the assessment of colorectal tumors and their response to treatment .

in recent years , advances in scanner technology and competition from other specialties have produced rapid changes in the way the intestines are imaged . mri and ct scan along with the traditional enteroclysis examination have emerged at the forefront of intestinal imaging . functional modalities such as diffusion and perfusion imaging are also changing the way tumors and inflammatory bowel diseases are evaluated . ct colonography is now a valid alterative to optical colonoscopy . contrast - enhanced usg is being used for the assessment of inflammation and post - treatment changes . in this review , recent advances in intestinal imaging are described .

the longevity of the world 's population is increasing , and in male patients , so are the complaints of lower urinary tract symptoms ( luts ) . testing to diagnose luts and to differentiate between the various causes should be quick , easy , cheap , specific , not too bothersome for the patient , and noninvasive or minimally so . conventional urodynamic evaluation consists of registering vesical and abdominal pressures during the filling phase and include the flow during the voiding phase , which is invasive , time - consuming , and expensive . nowadays , urodynamic evaluation is the gold standard for diagnosing bladder outlet obstruction ( boo ) , but presents some inconveniences such as embarrassment , pain , and dysuria and with 19% of cases experiencing urinary retention , macroscopic hematuria , or urinary tract infection . a greater number of resources in the diagnostic armamentarium could increase the opportunity for selecting less invasive tests on a patient by patient basis . for instance , in cases in which urodynamic studies would not provide the necessary benefit to overcome the risks of the study , less invasive tests might provide the confirmatory information needed to indicate treatment . the idea is not to replace but to provide alternatives to urodynamics that might better suit the needs of some patients and health care systems . these techniques might feasibly lend themselves to different environments , such as mobile and remote clinics . overall , innovation in health care is how we expand our knowledge , refine practices , and provide better service . a number of groups have risen to this challenge and have formulated and developed ideas and technologies to improve noninvasive methods to diagnosis boo . these techniques start with flowmetry , increased interest in ultrasound , and finally the performance of urodynamic evaluation without a urethral catheter . thus , slow flow can be due to lower detrusor contraction or to increased urethral resistance . by contrast , normal flow can be due to normal detrusor contraction and urethral resistance or to increased contraction of the detrusor and increased urethral resistance . in conclusion , flowmetry is not sufficient for confirming a diagnosis of boo . research has been done to evaluate the contribution of ultrasound to identifying patients with boo . methods such as the measurement of detrusor wall thickness ( dwt ) , intravesical prostatic protrusion ( ipp ) , and ultrasound - estimated bladder weight ( uebw ) are available . to measure the dwt , it is necessary to use a high - frequency transducer ( 7.5 mhz ) and enlarge by approximately 10 the image of the bladder wall ( fig . the measurement of the bladder wall should be at least 250 ml in the bladder . the sonographic measurements of dwt are an accurate alternative for pressure - flow measurements to assess the presence of boo . dwt measurements show a higher diagnostic power than do measurements of maximum flow rate ( qmax ) , average flow rate ( qave ) , postvoid residual urine , or prostate volume . dwt can help to assess boo noninvasively in all men and can be useful for the evaluation of boo at assessment and during treatment of bph patients . the ipp measurement is performed in the sagittal plane with the use of transducer frequencies between 3 and 6 mhz ( fig . 2 ) . the bladder should have at least 100 ml of urine for determining ipp . ipp is defined by the distance from the tip of the prostate 's protrusion into the vesical lumen to the bladder neck measured in millimeters . ipp is divided into three stages : grade i , < 5 mm ; grade ii , 5 to 10 mm ; and grade iii , > 10 mm . ipp grade iii reaches 80% sensitivity and 68% specificity for diagnosing boo when compared with urodynamic evaluation . authors have calculated the uebw from the measurement of bwt and bladder volume and found that a threshold value of 35 g best distinguishes the difference between obstructed and unobstructed bladders . however , other authors have evaluated patients with luts and found that uebw did not present any individual correlation with luts or objective measurements of boo . schafer first described noninvasive urodynamics in 1994 when he used a condom catheter ( fig . mcrae et al . developed a cuff to obstruct the urine flow ( fig . the principle of these tests is to interrupt the flow and measure the bladder pressure . the detrusor contraction is maintained and the urethral sphincters remain open and the column of fluid from the urethra to the bladder is sufficient to measure the bladder pressure ( isometric pressure ) ( fig . 5 ) . in conjunction with the bioengineering department at unicamp ( university of campinas ) , a new device consists of a urethral connector which is placed in the fossa navicularis was developed ( fig . the initial model consisted of only a device to which the transducer was connected and the interruption of the flow was done by the patient himself . the mammograms utilized in conventional urodynamic evaluation are not applied in noninvasive urodynamics . statistical analysis and logistic regression operating characteristic curve was constructed , which showed a sensitivity of 67% and specificity of 79% . a study was performed to compare the time flow , flow max , and volume between noninvasive and conventional urodynamics and the results showed no significant differences . with the objective of facilitating the realization of this study , modifications of the urethral connector were proposed . in a previous study , the system is composed of a pressure transducer , an electrical isolation enlargement board ( national instruments ni usb-6215 ) , and registered software from labview ( national instruments , austin , tx , usa ) . with these , it was possible to test new models ( fig . after various tests , version ii of the urethral connector was developed , in which the transducer is attached to the connector ( fig . 8) . a graph of the vesical pressure registered during a clinical exam with the urethral connector ii is shown in fig . the arrows indicate the approximated movement in which the individual is instructed to close the exit of the device . note that the pressure slowly increases until it reaches the approximated static value , corresponding to the isovolumetric vesical pressure . the flow is stopped several times during voiding , for periods of 2 to 3 seconds , which permits greater accuracy of the vesical pressure . for the comparison of two methods , conventional and connector , the vesical pressure at maximal flow and maximal vesical pressure with the noninvasive method were used . the linear regression resulted in an angular coefficient of 2.000.49 , r=0.8016 , with a 95% confidence interval of 0.6190 to 3.38l . the procedure for occlusion of the flow by use of the urethral connector to avoid hydraulic shock was not adopted without reason . in a previous study , laboratory simulations showed that abrupt occlusion could cause a rapid and significant increase in the pressure . 9 was observed , with a gradual increase of pressure until a static value was reached , which corresponded to the isometric vesical pressure . according to the patients , a closing time of 2 to 3 seconds was not long enough to cause any discomfort . this has already been verified by other methods showing that there is no contraction inhibition of the detrusor during a brief interruption of the flow . the registered vesical pressure values of the urethral connector were compared with the conventional urodynamic method , and the curve can be seen in fig . the conventional method registers the pressure of a free flow , whereas the connector measures the value during an interrupted flow . thus , although the pressures measured for each method reflect the contractile activity of the detrusor , the values are not necessarily identical . however , a linear correlation between the measured pressures is observed , which shows the sensitivity of the connector when registering the vesical pressure . considering that the flows registered by use of both methods were not different , this suggests that the connector does not cause an increase of resistance of the urinary flow , and the necessary interruptions for measuring the vesical pressure do not significantly alter the parameters evaluated . the study of noninvasive urodynamic evaluation has generated much research ; however , it has not been adopted in daily practice . it is difficult to interpret the results and it does not evaluate the phase of vesical filling and has a bit of an engineering characteristic . because it is a noninvasive method , it should be used in the first consultation with the patient , when the symptoms have first begun to appear and not when it is time to schedule surgery . the exam most probably should be placed between the flowmetry and the conventional urodynamic study . the great advantages of using the urethral connector are as follows : minimal discomfort , minimal risk of urinary tract infection , and low cost . this method can be repeated many times , permitting the evaluation of obstruction during clinical treatment . the connector should be used to diagnose boo , for the evaluation for surgery , and in screening for treatment . in the future , noninvasive urodynamics can be used to identify patients with boo , start early medical treatment , and evaluate the results .

the longevity of the world 's population is increasing , and among male patients , complaints of lower urinary tract symptoms ( luts ) are growing . testing to diagnose luts and to differentiate between the various causes should be quick , easy , cheap , specific , not too bothersome for the patient , and noninvasive or minimally so . urodynamic evaluation is the gold standard for diagnosing bladder outlet obstruction ( boo ) but presents some inconveniences such as embarrassment , pain , and dysuria ; furthermore , 19% of cases experience urinary retention , macroscopic hematuria , or urinary tract infection . a greater number of resources in the diagnostic armamentarium could increase the opportunity for selecting less invasive tests . a number of groups have risen to this challenge and have formulated and developed ideas and technologies to improve noninvasive methods to diagnosis boo . these techniques start with flowmetry , an increase in the interest of ultrasound , and finally the performance of urodynamic evaluation without a urethral catheter . flowmetry is not sufficient for confirming a diagnosis of boo . ultrasound of the prostate and the bladder can help to assess boo noninvasively in all men and can be useful for evaluating the value of boo at assessment and during treatment of benign prostatic hyperplasia patients in the future . the great advantages of noninvasive urodynamics are as follows : minimal discomfort , minimal risk of urinary tract infection , and low cost . this method can be repeated many times , permitting the evaluation of obstruction during clinical treatment . a urethral connector should be used to diagnose boo , in evaluation for surgery , and in screening for treatment . in the future , noninvasive urodynamics can be used to identify patients with boo to initiate early medical treatment and evaluate the results . this approach permits the possibility of performing surgery before detrusor damage occurs .

exacerbations of copd are associated with accelerated loss of lung function , poor quality of life , and mortality.1,2 these events can be predicted by numerous clinical factors , including prior exacerbations , airflow obstruction , symptom burden , gastroesophageal reflux , and leukocytosis.3 it is important to detect copd exacerbations early and minimize their severity . patients with copd frequently experience significant decreases in oxygen saturation during exercise , attributed to the imbalance between oxygen delivery and exercise - induced demand.4 exercise - induced oxygen desaturation ( eid ) is reported to be associated with hospitalization and mortality in patients with copd.5 the 6-minute walking test ( 6mwt ) has been suggested as the preferred measure to identify patients with copd and eid.6 eid occurs frequently during the 6mwt in patients with copd.7 eid has been related to forced expiratory volume in 1 second ( fev1 ) , diffusion capacity of lung carbon monoxide ( dlco ) , amount of emphysema , and baseline oxygen saturation.810 pulmonary hypertension ( ph ) is an important factor contributing to acute exacerbation of copd.11 ph appears when airflow limitation is severe , and is associated with chronic hypoxemia . pulmonary vascular remodeling in copd is the main cause of increased pulmonary artery ( pa ) pressure , and is thought to result from the combined effects of hypoxia , inflammation , and capillary loss in severe emphysema.12 the presence of ph has been shown to increase the hospitalization rate and mortality of patients with copd.13,14 computed tomography ( ct)-detected pa enlargement is independently associated with acute exacerbations of copd.15 the pa - to - aorta ( pa : a ) ratio measured by ct scan outperforms echocardiography for diagnosing resting ph in patients with severe copd.16 a pa : a > 1 indicates lower oxygen saturation at rest than a pa : a < 1.15 however , there are no reports on the association between pa : a and eid in patients with copd . we hypothesized that pa : a correlates with the presence of eid and that 6mwt results are useful for predicting the risk of having a pa : a > 1 . the present study aimed to examine the relationship between pa : a and eid and develop a simple screening tool by determining the appropriate cutoff score on the 6mwt to predict a pa : a > 1 in patients with copd . this study analyzed regularly treated outpatients with copd between 2014 and 2015 at the kobe city medical center west hospital . a total of 64 patients with copd were included after applying the exclusion criteria in this study ( figure 1 ) . the criteria for diagnosing copd were a smoking history ( 20 pack - years ) and postbronchodilator fev1/forced vital capacity ( fvc ) < 70% . furthermore , we used the following inclusion criteria to define copd clinically , all of which had to be fulfilled : symptoms , including cough , sputum production , wheezing , dyspnea , smoking history ( 20 pack - years ) , existence of emphysema on chest ct , and a physician diagnosis of copd.1721 study - exclusion criteria were as follows : history of lung surgical procedures , exacerbation - related hospitalization 3 months before 6mwt , and patients on long - term oxygen therapy . this examination included an assessment of body weight , height , and medical history ( eg , pulmonary embolism and sleep apnea syndrome ) , gold ( global initiative for chronic obstructive lung disease ) grade 04 , history of acute exacerbations of copd within the previous year , copd assessment test , level of dyspnea ( using the modified medical research council dyspnea scale ) , postbronchodilator spirometry , dlco , 6mwt ( according to international recommendations ) , emphysema area , and pa enlargement on ct . body mass index ( bmi ) was calculated as weight in kilograms divided by height in meters squared . gold 0 was defined as current and former smokers with a normal postbronchodilator ratio of fev1:fvc exceeding 0.7 and an fev1 of at least 80% , symptoms , including cough , sputum production , wheezing , and dyspnea , smoking history ( 20 pack - years ) , existence of emphysema on chest ct , and a physician diagnosis of copd.1721 the 6mwt was performed according to the 2002 american thoracic society guidelines.22 participants were asked to walk indoors on a flat , round , 25 m walking course supervised by a physician and physical therapist . a pulse oximeter ( wristox 3150 ; nonin medical , plymouth , mn , usa ) with a finger probe measured peripheral oxygen saturation ( spo2 ) during 6mwt , and 6mwt - analysis software ( wristox 2 ; star product , tokyo , japan ) was used . in addition , a modified borg scale was used to quantify the levels of dyspnea perceived by subjects at each minute during the 6mwt . eid was defined as a nadir spo2 < 90% , spo2 88% , and spo2 4%.2325 one reviewer , blinded to hemodynamic information , analyzed ct scans ( optima ct 660 discovery ; ge healthcare , little chalfont , uk ) . measurements of the diameter of the main pa and the diameter of the aorta at the level of the bifurcation were used to calculate the pa : a ratio , as previously reported.1416 in cases where the aorta was not uniform in diameter , two measurements were taken 90 apart and the larger diameter used . pa was measured on the line that joins the origin of the left pa and the center of the adjacent ascending aorta on the axial section at the level of pa bifurcation.26 ct - measured relative pa enlargement was defined as pa : a > 1 ( figure 2).1416 results are expressed as counts or median ( interquartile range ) . cohen s -coefficient measured intraobserver and interobserver agreements for ct measurements of the pa : a ratio . bivariate analyses were performed with the use of pearson s test for categorical data and the mann spearman s rank - correlation coefficient was determined for relationships between the pa : a ratio , lung - function parameters , 6mwt parameters , and ct parameters . receiver operating characteristic ( roc ) curves were used to determine the threshold values with the best sensitivity and specificity to predict pa : a > 1 , with the best being defined as the point on the roc curve with the shortest distance from the upper - left corner . sensitivity , specificity , positive / negative predictive value , and positive / negative likelihood were calculated for lung - function parameters and 6mwt parameters of exacerbation - risk factors on the basis of a previous study.6,27,28 all statistical analyses were performed with ezr ( saitama medical center , jichi medical university , saitama , japan ) , which is a graphical user interface for the r project ( r foundation for statistical computing , vienna , austria).29 more precisely , it is a modified version of r commander designed to add statistical functions frequently used in biostatistics , and p - values < 0.05 were considered statistically significant . this study analyzed regularly treated outpatients with copd between 2014 and 2015 at the kobe city medical center west hospital . a total of 64 patients with copd were included after applying the exclusion criteria in this study ( figure 1 ) . the criteria for diagnosing copd were a smoking history ( 20 pack - years ) and postbronchodilator fev1/forced vital capacity ( fvc ) < 70% . furthermore , we used the following inclusion criteria to define copd clinically , all of which had to be fulfilled : symptoms , including cough , sputum production , wheezing , dyspnea , smoking history ( 20 pack - years ) , existence of emphysema on chest ct , and a physician diagnosis of copd.1721 study - exclusion criteria were as follows : history of lung surgical procedures , exacerbation - related hospitalization 3 months before 6mwt , and patients on long - term oxygen therapy . this examination included an assessment of body weight , height , and medical history ( eg , pulmonary embolism and sleep apnea syndrome ) , gold ( global initiative for chronic obstructive lung disease ) grade 04 , history of acute exacerbations of copd within the previous year , copd assessment test , level of dyspnea ( using the modified medical research council dyspnea scale ) , postbronchodilator spirometry , dlco , 6mwt ( according to international recommendations ) , emphysema area , and pa enlargement on ct . body mass index ( bmi ) was calculated as weight in kilograms divided by height in meters squared . gold 0 was defined as current and former smokers with a normal postbronchodilator ratio of fev1:fvc exceeding 0.7 and an fev1 of at least 80% , symptoms , including cough , sputum production , wheezing , and dyspnea , smoking history ( 20 pack - years ) , existence of emphysema on chest ct , and a physician diagnosis of copd.1721 the 6mwt was performed according to the 2002 american thoracic society guidelines.22 participants were asked to walk indoors on a flat , round , 25 m walking course supervised by a physician and physical therapist . subjects were encouraged using standard methodology every minute of the 6mwt . a pulse oximeter ( wristox 3150 ; nonin medical , plymouth , mn , usa ) with a finger probe measured peripheral oxygen saturation ( spo2 ) during 6mwt , and 6mwt - analysis software ( wristox 2 ; star product , tokyo , japan ) was used . in addition , a modified borg scale was used to quantify the levels of dyspnea perceived by subjects at each minute during the 6mwt . 90% , spo2 88% , and spo2 4%.2325 one reviewer , blinded to hemodynamic information , analyzed ct scans ( optima ct 660 discovery ; ge healthcare , little chalfont , uk ) . measurements of the diameter of the main pa and the diameter of the aorta at the level of the bifurcation were used to calculate the pa : a ratio , as previously reported.1416 in cases where the aorta was not uniform in diameter , two measurements were taken 90 apart and the larger diameter used . pa was measured on the line that joins the origin of the left pa and the center of the adjacent ascending aorta on the axial section at the level of pa bifurcation.26 ct - measured relative pa enlargement was defined as pa : a > 1 ( figure 2).1416 this examination included an assessment of body weight , height , and medical history ( eg , pulmonary embolism and sleep apnea syndrome ) , gold ( global initiative for chronic obstructive lung disease ) grade 04 , history of acute exacerbations of copd within the previous year , copd assessment test , level of dyspnea ( using the modified medical research council dyspnea scale ) , postbronchodilator spirometry , dlco , 6mwt ( according to international recommendations ) , emphysema area , and pa enlargement on ct . body mass index ( bmi ) was calculated as weight in kilograms divided by height in meters squared . gold 0 was defined as current and former smokers with a normal postbronchodilator ratio of fev1:fvc exceeding 0.7 and an fev1 of at least 80% , symptoms , including cough , sputum production , wheezing , and dyspnea , smoking history ( 20 pack - years ) , existence of emphysema on chest ct , and a physician diagnosis of copd.1721 the 6mwt was performed according to the 2002 american thoracic society guidelines.22 participants were asked to walk indoors on a flat , round , 25 m walking course supervised by a physician and physical therapist . a pulse oximeter ( wristox 3150 ; nonin medical , plymouth , mn , usa ) with a finger probe measured peripheral oxygen saturation ( spo2 ) during 6mwt , and 6mwt - analysis software ( wristox 2 ; star product , tokyo , japan ) was used . in addition , a modified borg scale was used to quantify the levels of dyspnea perceived by subjects at each minute during the 6mwt . one reviewer , blinded to hemodynamic information , analyzed ct scans ( optima ct 660 discovery ; ge healthcare , little chalfont , uk ) . measurements of the diameter of the main pa and the diameter of the aorta at the level of the bifurcation were used to calculate the pa : a ratio , as previously reported.1416 in cases where the aorta was not uniform in diameter , two measurements were taken 90 apart and the larger diameter used . pa was measured on the line that joins the origin of the left pa and the center of the adjacent ascending aorta on the axial section at the level of pa bifurcation.26 ct - measured relative pa enlargement was defined as pa : a > 1 ( figure 2).1416 cohen s -coefficient measured intraobserver and interobserver agreements for ct measurements of the pa : a ratio . bivariate analyses were performed with the use of pearson s test for categorical data and the mann spearman s rank - correlation coefficient was determined for relationships between the pa : a ratio , lung - function parameters , 6mwt parameters , and ct parameters . receiver operating characteristic ( roc ) curves were used to determine the threshold values with the best sensitivity and specificity to predict pa : a > 1 , with the best being defined as the point on the roc curve with the shortest distance from the upper - left corner . sensitivity , specificity , positive / negative predictive value , and positive / negative likelihood were calculated for lung - function parameters and 6mwt parameters of exacerbation - risk factors on the basis of a previous study.6,27,28 all statistical analyses were performed with ezr ( saitama medical center , jichi medical university , saitama , japan ) , which is a graphical user interface for the r project ( r foundation for statistical computing , vienna , austria).29 more precisely , it is a modified version of r commander designed to add statistical functions frequently used in biostatistics , and p - values < 0.05 were considered statistically significant . the current analysis comprised 64 patients who were separated into groups on the basis of pa : a > 1 ( n=29 ) and 1 ( n=35 ) . the -values for intraobserver and interobserver agreements for detecting pa : a > 1 were 0.87 ( 95% confidence interval [ ci ] 0.740.99 ) and 0.75 ( 95% ci 0.580.91 ) , respectively . differences in the pa : a ratio between both groups were driven by the diameter of pa ( 2.9 [ 2.73.3 ] cm in pa : a 1 vs 3.7 [ 3.53.9 ] cm in pa : a > 1 , p=0.002 ) , because no differences were detected in the diameter of aortae ( 3.7 [ 3.43.9 ] cm vs 3.5 [ 3.33.7 ] cm , p=0.20 ) . there were no significant differences between the two groups with regard to age , sex , bmi , pack - years , modified medical research council dyspnea scale , gold , copd assessment test , baseline pulse rate , or baseline modified borg scale ( p>0.05 ) . on the other hand , there were significant differences between the two groups with regard to fev1 ( 71.6% [ 60.5%80.8% ] vs 52.6% [ 39.6%72.1% ] , p=0.013 ) , fvc ( 82.3% [ 50.3%93.6% ] vs 75.8% [ 42.7%86% ] , p=0.04 ) , fev1:fvc ratio ( 68% [ 61%73.3% ] vs 53.8% [ 48.8%69.4% ] , p=0.023 ) , dlco ( 72.5% [ 55.5%82.9% ] vs 44.6% [ 37.7%49.6% ] , p=0.005 ) , bode ( bmi , obstruction [ airflow ] , dyspnea , and exercise performance ) index ( 2 [ 13 ] vs 4 [ 25 ] , p<0.001 ) , 6mw distance ( 6mwd ; 450 m [ 400510.5 ] vs 325 m [ 238466 ] , p<0.001 ) , baseline spo2 ( 97% [ 95%97.5% ] vs 95% [ 93%96% ] , p=0.001 ) , lowest spo2 ( 92% [ 91%94% ] vs 86% [ 84%88% ] , p<0.001 ) , highest modified borg scale result ( 2 [ 05 ] vs 5 [ 25 ] , p=0.04 ) , low - attenuation area ( laa ; 6.8% [ 2.8%14.7% ] vs 25.4% [ 11.3%33.4% ] , p<0.001 ) , and history of acute exacerbations of copd within the previous year ( 1 [ 2.9% ] vs 7 [ 24.1% ] , p=0.019 ) ( table 1 ) . the pa : a ratio demonstrated a significant linear correlation with lowest spo2 ( r=0.68 , r=0.46 ; p<0.001 ) , dlco ( r=0.61 , r=0.37 ; p<0.001 ) , 6mwd ( r=0.43 , r=0.18 ; p<0.001 ) , bode index ( r=0.41 , r=0.17 ; p<0.001 ) , baseline spo2 ( r=0.36 , r=0.13 ; p=0.003 ) , laa ( r=0.36 , r=0.13 ; p=0.004 ) , fvc ( r=0.34 , r=0.12 ; p=0.006 ) , fev1 ( r=0.29 , r=0.08 ; p=0.019 ) , and highest pulse rate ( r=0.26 , r=0.07 ; p=0.035 ) ( table 2 ) . using roc curves , the threshold values with the best cutoff point , sensitivity , and specificity to predict pa : a > 1 were determined for spo2 during the 6mwt ( best cutoff point 89% , area under curve [ auc ] 0.94 , 95% ci 0.881 ) , dlco ( best cutoff point 51% , auc 0.87 , 95% ci 0.780.96 ) , 6mwd ( best cutoff point 388 m , auc 0.75 , 95% ci 0.620.87 ) , and bode index ( best cutoff points 4 , auc 0.74 , 95% ci 0.610.87 ) ( table 3 , figure 3 ) . the performance data on the 6mwt and lung function for predicting pa enlargement are depicted in table 4 . spo2 < 90% , spo2 88% , and spo2 4% during 6mwt were 94.3 ( 80.899.3 ) , 97.1 ( 85.199.9 ) , and 45.7 ( 28.863.4 ) , respectively , for specificity , 93.1 ( 77.299.2 ) , 95.8 ( 78.999.9 ) , and 59.6 ( 44.373.6 ) , respectively , for positive predictive value , and 16.2 ( 4.262.8 ) , 27.7 ( 4193.3 ) , and 1.8 ( 1.32.4 ) , respectively , for positive likelihood ratios . first , we found a strong association between the pa : a ratio and lowest spo2 during the 6mwt . for this reason , a consistent finding in patients with copd is the close relationship between severity of hypoxemia and pa pressure or pulmonary vascular resistance , supporting a major role for alveolar hypoxia.30 alveolar hypoxia causes constriction of the resistance pas , and sustained alveolar hypoxia induces pulmonary vascular remodeling.28 pathological studies of lung specimens from patients with copd have shown extensive pulmonary vascular remodeling , with prominent intimal thickening and medial hypertrophy . for this reason , chronic alveolar hypoxia plays an important role in pulmonary vascular remodeling.28 in a previous study , patients with pa : a > 1 showed lower resting spo2 , higher usage rates of supplemental oxygen , and an indirect association with eid.15 in the present study , the lowest spo2 during the 6mwt to predict pa : a > 1 was considered to be a beneficial result based on the roc curves . these results suggest that the lowest spo2 during the 6mwt is a very helpful measure and screening test for the pa : a ratio . for example , it might be possible to easily screen for pulmonary artery expansion by means of the six - minute walking test in a home - care situation , where it would be difficult to perform ct imaging . second , with regard to the relationship between pa : a ratio and lung function , correlations were observed among fev1 , dlco , and laa . one of the factors that may play a role in causing ph to advance in patients with copd is the destruction of lung parenchyma , leading to loss of part of the pulmonary vascular bed,30,31 and the burden of airway remodeling influencing pa - pressure increase.28 a previous study included patients with airflow - obstruction severity greater than moderate , and our study included mild airflow obstruction and smokers with normal spirometry.15 therefore , regardless of the severity of airflow obstruction , pa enlargement may be progressing . undiagnosed copd is a problem worldwide.18 gold 0 has been reported to be an exacerbation risk ; therefore,18 early detection and not just spirometry evaluation is important from multiple perspectives.18,32 from the viewpoint of early detection of pa enlargement , a definition of eid as spo2 < 90% may be a good start . however , these findings may imply that pa : a > 1 is one of the multiple risk factors for acute copd exacerbations . one reason for this is that pa : a is associated with ph16 and ph is also a risk factor for acute copd exacerbations.33 furthermore , a previous study reported an association between the pa : a ratio and acute copd exacerbation.15 these results suggest that screening for the pa : a ratio without ct using the 6mwt may indicate the risk of acute copd exacerbations at an early stage . this study had some limitations , including small size , single - center experience , and retrospective design . in addition , this study also included copd subjects who did not fit the gold criteria . furthermore , because healthy controls do not have respiratory symptoms and there are no control data for the measurement items pertaining to such individuals , healthy controls were not included in the present study . however , it has been reported that the presence of clinical symptoms and low dlco in smokers , even with normal spirometry , increases the risk of progression to airflow obstruction in copd.1720 therefore , the present study s results during the 6mwt could be useful to screen for ph at an early copd stage , even in gold 0 patients . finally , according to a previous study , left ventricular dysfunction causes pa enlargement . however , echocardiography was not performed in all subjects , and this information could not be included because it was unavailable from the medical history , although we observed clinically relevant associations between ct - measured pa : a ratios and 6mwt results . the current study s findings suggest that there is a strong association between pa : a ratio and lowest spo2 during the 6mwt . lowest spo2 during the 6mwt is a very helpful measurement to predict pa : a ratios on ct , and lowest spo2 of < 89% during the 6mwt is excellent to screen for ph in copd .

purposepulmonary hypertension and exercise - induced oxygen desaturation ( eid ) influence acute exacerbation of copd . computed tomography ( ct)-detected pulmonary artery ( pa ) enlargement is independently associated with acute copd exacerbations . associations between pa to aorta ( pa : a ) ratio and eid in patients with copd have not been reported . we hypothesized that the pa : a ratio correlated with eid and that results of the 6-minute walk test ( 6mwt ) would be useful for predicting the risk associated with pa : a > 1.patients and methodswe retrospectively measured lung function , 6mwt , emphysema area , and pa enlargement on ct in 64 patients with copd . the patients were classified into groups with pa : a 1 and > 1 . receiver - operating characteristic curves were used to determine the threshold values with the best cutoff points to predict patients with pa : a > 1.resultsthe pa : a > 1 group had lower forced expiratory volume in 1 second ( fev1 ) , forced vital capacity ( fvc ) , fev1:fvc ratio , diffusion capacity of lung carbon monoxide , 6mw distance , and baseline peripheral oxygen saturation ( spo2 ) , lowest spo2 , highest modified borg scale results , percentage low - attenuation area , and history of acute copd exacerbations 1 year , and worse bode ( body mass index , airflow obstruction , dyspnea , and exercise ) index results ( p<0.05 ) . predicted pa : a > 1 was determined for spo2 during 6mwt ( best cutoff point 89% , area under the curve 0.94 , 95% confidence interval 0.881 ) . spo2 < 90% during 6mwt showed a sensitivity of 93.1 , specificity of 94.3 , positive predictive value of 93.1 , negative predictive value of 94.3 , positive likelihood ratio of 16.2 , and negative likelihood ratio of 0.07.conclusionlowest spo2 during 6mwt may predict ct - measured pa : a , and lowest spo2 < 89% during 6mwt is excellent for detecting pulmonary hypertension in copd .

we thank all the members of the cytoskeleton and cancer group and our collaborators , who collectively participated to the original manuscripts . this work was supported by grants from anr-13-jjc - jsv10005 , la ligue nationale contre le cancer and fondation arc pour la recherche sur le cancer . v.m . is supported by funding from equipe labellise ligue nationale contre le cancer 2011 .

accumulation of type i collagen fibrils in tumors is associated with an increased risk of metastasis . we recently demonstrated that the collagen sensor discoidin domain receptor 1 ( ddr1 ) interacts with type i collagen fibrils to allow proteolysis - based cancer cell invasion through the formation of a new class of invadosomes , termed linear invadosomes .

diosmin ( 3 , 5 , 7-trihydroxy-4-methoxyflavone 7-rutinoside = diosmetin 7-o - rutinoside ) is a naturally occurring flavonoid glycoside found in various plant materials , mainly citrus fruits . due to the high demand for the substance , it is obtained semi - synthetically from hesperidin ( 3 , 5 , 7-trihydroxy-4-methoxyflavanone 7-rutinoside = hesperitin 7-o - rutinoside ) . diosmin ( figure 1a ) is a component of formulations used in the treatment of venous insufficiency , hemorrhoids , lymphedema , diabetes , melanoma , dermatitis , mastalgia , colitis , pre - menstrual syndrome and many other conditions ( 1 , 2 ) . hesperidin ( figure 1b ) is also used with diosmin in various formulations and plant extracts . hesperidin due to their low water solubility , both drugs are barely absorbed from the gastrointestinal tract ; therefore , diosmin is more and more frequently micronised to obtain particles smaller than 2 - 20 m particles . the bioavailability of the formulations is thus much higher , as so their efficiency ( 3 ) . diosmin may be assayed spectrophotometrically with uv - vis detection ( with 4-aminoantypirine using the berthelot reagent , with 3-methyl-2-benzothiazolinone hydrazone ( mbth ) or with 2 , 4 , 6-trimethylaniline ) or densitometrically with uv detection . traces of diosmin in pharmaceuticals are assayed voltamperometrically using a glass carbon electrode ( 4 , 5 , 6 ) . hesperidin , in turn , may be assayed spectrofluorimetrically , by measuring the intensity of the highly fluorescent complex between hesperidin and al ( iii ) ions in a micellar solvent ( sodium dodecylsulfate in water ) ( 7 ) . even though there are several distinct methods for the assay of diosmin and hesperidin in pharmaceutical formulations and body fluids , there is no accurate and rapid method for the co - assay of the substances . diosmin and hesperidin may be assayed simultaneously by reversed - phase hplc with the following eluent : thf / water / acetic acid ( 21:77:2 , v / v / v ) at a wavelength of 280 nm ( 8) or methanol / water ( 60:40 , v / v ) at 345 nm ( 9 ) . the most frequently used column packing for the assay of biological or pharmaceutical samples is silica modified by hydrophobic hydrocarbon chains of various lengths ( usually ods ) or by polar functional groups . when such packing types are used for the separation of certain analytes , in particular basic compounds , long retention times and peak tailing is observed . the effect results from adsorption interactions between free active silanols and the analyte being resolved , which significantly hinders analysis . in order to block active silanols , ionic liquids have been used as mobile phase additives in the separation of ephedrines ( 10 ) , catecholamines ( 11 ) , amines ( 12 ) , basic drugs ( 13 ) , phenoxy acid herbicides and phenols ( 14 ) , vitamins ( 15 ) among other compounds . in this work , didecyldimethylammonium lactate ( figure 2 ) is used as the mobile phase additive for diosmin and hesperidin resolution . chemicals and reagents diosmin and hesperidin were obtained from lkt laboratories , saint paul , usa . the following solvents were used : dimethylsulfoxide ( dmso ) , methanol and chloroform ( high - performance liquid chromatography grade ) were from poch , gliwice , poland . juliusz pernak from the faculty of chemical technology , pozna university of technology , poland . pharmaceutical formulations used in the study standards and samples stock standards ( concentration : 1 mg / ml ) were prepared separately by dissolving 0.0500 g of diosmin and hesperidin in dmso : methanol 1:1 . working solutions were obtained by the successive dilution of the standard solution using the mobile phase used in the chromatographic analysis . samples of the test products were prepared by dissolving 0.0500 g of the powdered product in dmso : methanol 1:1 . a hewlett packard liquid chromatograph ( model hp 1050 , waldbronn , germany ) was used , composed of a quaternary pump , a variable - wavelength uv detector operating at 280 nm and a rheodyne model 7125 injection valve with a 20 l sample loop . the separation was performed on a lichrospher , rp-18 ( 5 m , 250 mm x 4.6 mm i d ) stainless steel column ( merck , darmstadt , germany ) . before use , the mobile phase was vacuum - filtered through a 0.45 m cellulose filter and degassed with helium . the water was distilled and then purified by a milli - q water purification system ( millipore , billerica , usa ) . chromatography conditions the mobile phase consisted of methanol : water = 45:55% , v / v with 0.025% ionic liquid added . analytical method validation parameters calibration curves were obtained based on the results of chromatographic analysis of diosmin and hesperidin standard solutions . the resulting solutions contained 2.5 ; 5 ; 10 ; 25 ; 50 ; 75 and 100 g of each compound per 1 ml of mobile phase . the regression equations were calculated in the form of y = ax + b , where y and x were peak area and sample concentrations , respectively . the repeatability of the chromatographic system was assessed under the chromatographic conditions previously selected by means of 10 replicate injections of a solution with 5 g / ml diosmin and 5 g / ml hesperidin and finding out the peak area by the proposed method . from this peak area % rsd day precision was determined by injecting three different concentrations ( 5 , 50 and 100 g / ml ) for three times in the same day . inter - day precision was determined by injecting three different concentrations ( 5 , 50 and 100 g / ml ) for three days in a week . limit of detection and quantification the limit of detection ( lod ) under the present chromatographic conditions is defined by the concentration of the analyte giving a signal to noise ratio of 3 : 1 . the limit of quantification ( loq ) is the lowest analyte concentration which can be assayed with required precision and accuracy . based on 6 parallel results obtained for each standard solution concentration , relative standard deviations were derived , and loq for rsd = 10% was derived from the relationship between rsd and test glycoside concentrations . the recovery value was determined by adding known quantities of the test compounds to the test formulations ( standard addition method ) ( ich q2a , text of validation of analytical procedures , international conference on harmonization tripartite guidelines , adapted 27 oct . the recovery of the standards added ( diosmin and hesperidin ) was performed for three different concentration levels , corresponding to approx . each sample was tested six times at each concentration level according to the above procedure . optimization of chromatographic analysis reversed - phase chromatography was suggested as a suitable procedure for the co - assay of diosmin and hesperidin in complex matrices , such as pharmaceutical formulations . the optimization of the chromatographic process involved first the determination of the detection wavelength suitable for the co - assay of the test compounds and second , the selection of mobile phase parameters suitable for the total resolution of diosmin and hesperidin as rapidly as possible , with no interference from other ingredients of the test samples . the selection of appropriate conditions involved testing the effects on retention of type and concentration of organic solvent in the mobile phase , and the concentration of the ionic liquid added to the eluent on retention . the analysis of uv spectra for diosmin and hesperidin and initial hplc studies of the test matrices gave appropriate conditions for chromatographic resolution to facilitate the co - assay of both analytes . when an ionic liquid was added , analysis time was significantly reduced and peak symmetry was greatly improved . an example of the chromatograms obtained for a standard mixture of diosmin and hesperidin is shown in figure 3 . chromatogram a was obtained without addition of ionic liquid and chromatogram b with 0.025% ionic liquid in mobile phase . the retention times of diosmin and hesperidin were 8.01 0.03 and 5.73 0.02 min , respectively . chromatograms of diosmin d and hesperidin h for standard without addition of ionic liquid ( a ) and with 0.025% ionic liquid ( b ) in mobile phase we note very good peak symmetry and also the fact that the retention of the test compounds is repeatable with very good precision ( low rsd values ) . the linearity of the method was tested for both flavonoid glycosides assayed in a concentration range of 2.5 to 100 g / ml by injecting standard solutions in the chromatographic conditions as above . the calibration curves for diosmin and hesperidin were derived by the least - squares method . table 2 shows the linearity range tested and parameters of the fitted straight - lines . calibration range and fitted parameters in the simultaneous determination of diosmin and hesperidin , the calibration curves were found to be linear for both compounds in the aforementioned concentrations and the correlations coefficients for the regression lines were 0.9983 and 0.9992 for diosmin and hesperidin , respectively . repeatability expressed as relative standard deviation and determined for both analytes was 0.39 and 0.42% for diosmin and hesperidin , respectively . the precision of the method was demonstrated by inter day and intra day variation studies . in the intra day studies , six repeated injections of sample solutions were made and the mean area of analyte peaks and percentage rsd were calculated and presented in table 3 . in the inter day variation studies , six repeated injections of sample solutions were made for three consecutive days and mean area of analyte peaks and percentage rsd were calculated and presented in table 3 . precision of the method from the data obtained , the developed hplc method was found to be precise . the limits of detection for diosmin and hesperidin were 2.5 and 1.2 g / ml , respectively , and the limits of quantification were 5.5 and 3.5 g / ml , respectively . the method was used for the assay of diosmin and hesperidin in four pharmaceutical products available in poland . product compositions and manufacturers are listed in table 1 . the results of analyses calculated based on calibration curve equations are shown in table 2 . n.a. data not available the results listed in table 4 show that the method can be used for the co - assay of diosmin and hesperidin in various pharmaceutical products . rsd values for six independent determinations in the test pharmaceuticals were within a range of 1.10 to 3.21% and 0.57 to 3.37% of diosmin and hesperidin , respectively . larger differences were noted between analyte quantities assayed and declared by the manufacturers . in order to account for the inconsistencies and to verify the correctness of our method , an experiment was performed to determine analytical accuracy . a solution of the test product was prepared with analyte concentration x within a range of concentrations used for the calibration curve and three series of identically prepared solutions to which diosmin and hesperidin with known concentrations a were added . the value of a for diosmin in pelethrocin and diosminex was 100 , 200 and 300 mg per one tablet , respectively ; for diohespan forte : 120 , 240 and 360 mg per tablet ; for belissa anti red : 15 , 25 and 35 mg per tablet . the value of a for hesperidin in diosminex and diohespan forte was 2.5 , 5.0 and 7.5 mg per tablet ; for diohespan pelethrocin : 5.0 , 7.5 and 10.0 mg per tablet ; for belissa anti red : 10 , 20 and 30 mg per tablet . the determinations were performed for all the test products and both analytes ( six independent analyses for each series of solutions in the same conditions ) . four series of results were obtained , x and x + a , listed in table 5 . the concentration values shown in table 4 correspond to diosmin and hesperidin concentrations with respect to one tablet . method accuracy is determined by recovery values for quantities a of diosmin and hesperidin added to pharmaceutical product solutions . the recovery values are close to 100% , within a range of 96.9 to 103% and 96 to 102.9 for diosmin and hesperidin , respectively . accuracy of diosmin and hesperidin assay in the test products ( n = 6 ) . the results shown in the tables confirm that our method for the co - assay of diosmin and hesperidin in pharmaceuticals has both high precision and accuracy . they confirm the correctness of our method and the procedure used ; the discrepancies between the analytical results and the amounts declared by the manufacturers are consistent with the requirements specified by pharmacopoeia viii ( diosmin should not contain more than 5% hesperidin , considered an impurity ) . the paper puts forward a method for the co - assay of diosmin and hesperidin in pharmaceutical products using hplc in which some ionic liquid is added to the mobile phase . when the ionic liquid was added , analysis time was reduced and resulting peak symmetry was improved . it is the first time an ionic liquid has been used for the resolution and assay of the compounds . the method has high precision from 1.10 to 3.21% and 0.57 to 3.37% for diosmin and hesperidin , respectively ; accuracy from 96.9 to 103% and from 96 to 102.9 for diosmin and hesperidin , respectively and very good repeatability ( 0.39 and 0.42% for diosmin and hesperidin , respectively ) . the precision of the proposed method was carried in terms of the repeatability , inter - day and intra - day time periods . the low % rsd values of repeatability ( 0.39 and 0.42% for diosmin and hesperidin , respectively ) , intra - day ( 0,39 % 1,12% ) and inter - day ( 0.51% - 1.19% ) variations reveal that the proposed method is precise . the limit of detection and quantification is 2.5 and 1.2 g / ml and 5.5 and 3.5 g / ml for diosmin and hesperidin , respectively . the results show that the method may be used for the control of diosmin and hesperidin contents in pharmaceutical products .

diosmin and hesperidin are natural flavonoid glycosides found in various plant materials , mainly in citrus fruits in different concentrations . diosmin for pharmaceutical use is obtained mainly semi - synthetically from hesperidin . hesperidin often accompanies diosmin as a natural impurity in different pharmaceutical formulations ; therefore , a simple , fast and precise method for the simultaneous assay of diosmin and hesperidin in pharmaceutical formulations has been developed to control their contents.chromatographic resolution was performed using a column with c-18 packing and the following mobile phase : methanol / water ( 45:55 , v / v ) with 0.025% added didecyldimethylammonium lactate , which significantly affects retention , shortening analysis time and having a positive impact on the symmetry of resulting chromatographic peaks . the method shows linearity between 2.5 and 100 g / ml , high repeatability ( 0.39 and 0.42% for diosmin and hesperidin , respectively ) and accuracy of 96 to 102% for both the assayed compounds . intraday and interday precision of the new method were less than rsd% 1 , 2 . the limit of detection of the assayed compounds is 2.5 and 1.2 g / ml for diosmin and hesperidin , respectively . the method was tested on several pharmaceutical products available in poland .

carbohydrates play critical roles during pollen growth and development , then in the final phase prior to dispersal carbohydrates change to prepare pollen for dispersal . following dispersal and during pollen storage , the various metabolites comprising the carbohydrate pool change in relative proportion , a behavior that is understood as an adaptation for sustaining pollen viability in time . sucrose in particular may be of particular importance during pollen storage , as variations among species for pollen desiccation tolerance have been linked to sucrose content . sucrose may replace water to preserve native protein structures and spacing between phospholipids in the plasma - membrane during dehydration . the role of starch and sugars in pollen development , dispersal , and maintenance of viability has not been determined for any member of the cycadales , and is therefore a focus of needed research . several authors have conducted robust surveys and noted that starchy pollen occurred disproportionately among anemophilous species . representatives from the cycadales were absent from these surveys and this should be corrected , as cycad pollination studies have only recently illuminated the sophisticated pollination syndromes that characterize the threatened plant group . although arthropods were collected from reproductive structures on various cycad taxa in the past , the scientific community largely ignored their potential role as pollinators due to the established belief that all cycads were anemophilous . the pendulum swung in the 1980s and 1990s when extensive experimental evidence confirmed mutualisms between cycad species and specialist insect pollinators . more recently , ambophily has been confirmed or proposed for several cycas species where wind has been shown to augment insect pollination . furthermore , variations in aggregation tendencies and settling velocity have been reported for pollen from various cycad species . this is an opportune time to add members of the cycadales to studies that have linked the relationship of pollen carbohydrates to insect versus wind pollination strategies . the insect pollinators of cycads gather on male cones , where the adults socialize , mate , and use the post - dispersal male strobilus tissue as larvae food ( fig . the study of idioblasts within cycad sporophyll tissues has illuminated intricacies in how general tissue chemistry may interact with pollinator feeding behaviors . for zamia furfuracea , these idioblasts remained intact in the male sporophyll tissue but appeared to release their contents into female sporophyll tissue prior to ovule receptivity . the authors concluded that toxins remained sequestered within the idioblasts for male tissue only , such that the pollinators could feed on parenchyma tissue around the idioblasts or consume intact idioblasts which protected insect metabolism from the toxins after consumption . many but not all cycad species studied in this context exhibited similar idioblast traits . for example , cycas rumphii and stangeria eriopus strobili did not contain identifiable idioblasts . in contrast , macrozamia lucida and microcycas calocoma exhibited idioblast breakdown prior to pollination stage for both male and female sporophyll tissues . the interactions among carbohydrates , nutrients , and toxins within cycad strobili tissues in relation to these idioblasts may prove to be of crucial importance for developing a full understanding of how pollinators feed on the plant 's reproductive tissues while enacting effectual pollination . conservationists in need of improved knowledge of various pollination syndromes within the cycadales will require more studies within this context . left : microstrobilus tissue is tunneled and consumed by larvae ( arrows ) immediately after pollen dispersal . right : within days the entire microstrobilus is reduced to frass and pupation ( arrow ) heralds in a new generation of pollinators . the role of cycad sugars in mediating left : microstrobilus tissue is tunneled and consumed by larvae ( arrows ) immediately after pollen dispersal . right : within days the entire microstrobilus is reduced to frass and pupation ( arrow ) heralds in a new generation of pollinators . the role of cycad sugars in mediating the majority of cycad pollinators consume strobilus tissue , but direct consumption of cycad pollen may occur for some cycad pollinators . cumulative research on pollen digestion the addition of cycads to studies that determine carbohydrate and nutritional quality of pollen will greatly improve this line of research whether the mutualisms represent ancient associations that pre - dated angiosperms or examples where the contemporary mutualisms were recently derived from an initial antagonistic relationship . the reproductive structures of cycad species exhibit thermogenesis and volatile emissions at the time of pollination , traits that mediate pollinator behavior and maintain pollinator specificity . research is accumulating in areas such as modeling the plant behavior and parsing the influence of specific volatiles on insect behavior . enacting synchronized thermogenesis and volatile biosynthesis is an exceedingly expensive plant behavior , and no studies have determined the tissues of residence , quantities , and stoichiometric relations of the carbohydrate reserves that are mobilized to fund those activities . droplets have been documented at the micropyle location on cycad ovules , and these droplets contain metabolites that may provide a reward for pollinators . studies that marked pollinators then used deposition of the markers to track pollinator behavior have confirmed that the pollen they vector is trapped by the ovule droplets . this remarkable female strobilus behavior represents a system that would be interesting to study in relation to specificity of pollinator attraction , diversity of sugar rewards , and cycad phylogeny . droplets may exhibit taxon - specific carbohydrates that are suited to attracting and nourishing specific specialist pollinators . our recent report of fructose , glucose , and sucrose content in cycad tissues sets the stage for designing continued research on how non - structural carbohydrates are involved in cycad pollination biology . moreover , cycad horticulturists routinely harvest , store , and ship pollen prior to its use for successful pollination . improved understanding of how carbohydrates and other factors influence pollen viability and longevity would improve protocols for artificial pollination . finally , the risks associated with coextinctions are very real during this phase of the anthropocene , and species with complex life history traits , such as cycads , appear to be at greater risk for direct involvement in coextinctions . an increase in knowledge of how cycad carbohydrates influence successful pollination relationships may help reduce the risks of coextinctions in these mutualisms that support contemporary cycad biology .

cycad biology , ecology , and horticulture decisions are not supported by adequate research , and experiments in cycad physiology in particular have been deficient . our recent report on free sugar content in a range of cycad taxa and tissues sets the stage for developing continued carbohydrate research . growth and development of cycad pollen , mediation of the herbivory traits of specialist pollinators , and support of expensive strobilus behavioral traits are areas of cycad pollination biology that would benefit from a greater understanding of the role of carbohydrate relations .

the study is a retrospective , single - center , observational study approved by the institutional review board . there were 1872 patients that underwent ffr assessment between october 2002 and july 2010 at our institution . from 2002 to 2008 , an ffr value 0.75 was used to defer revascularization based on the defer ( ffr to determine appropriateness of angioplasty in moderate coronary stenoses ) study.3 in 2008 , an ffr value > 0.80 was used to defer lesions based on the fame ( fractional flow reserve versus angiography for multivessel evaluation ) trial.4 of these 1872 patients , 742 patients with 906 coronary lesions were deferred revascularization based on ffr . of the 742 patients , 21 patients or 24 lesions were excluded because of loss of follow - up after ffr assessment . furthermore , 47 patients or 68 lesions were excluded because of ffr less than or equal to 0.80 . thus , the final population for this study consisted of 674 patients with 816 lesions that were deferred revascularization based on ffr > 0.80 ( figure 1 ) . ffr was performed according to standard techniques as detailed in a previous observational study using this study population.18 ffr was measured at maximal hyperemia after administering intracoronary ( n=812 lesions ) or intravenous adenosine ( n=4 lesions ) , with the dose determined by the operator . ffr was not performed on lesions with less than thrombolysis in myocardial infarction ( timi ) 3 flow . every patient included in the study was followed from the date of index stenting until march 12 , 2013 by reviewing the medical records and/or telephone interview . all angiograms were reviewed independently by at least 2 authors . for patients with follow - up or hospitalizations outside our institution , the medical records , including angiograms , were obtained for review . the primary outcome was a composite of cardiovascular ( cv ) death , mi , or deferred lesion intervention ( dli ) . secondary outcomes included a composite of cv death or mi and mi or dli , as well as the individual endpoints of cv death , mi , mi - lesion , deferred lesion failure ( dlf ) , and dli . cv death and mi were defined by the academic research consortium guidelines.19 dli was defined as any percutaneous coronary intervention ( pci ) performed within 5-mm proximal or distal to , or any coronary artery bypass graft ( cabg ) placed distal to , a lesion deferred revascularization based on the index ffr assessment.18 mi lesion was defined as any mi that was directly attributable to a lesion deferred revascularization at the index ffr . the study population was divided into acs and non - acs groups based on clinical diagnosis at the time of index ffr assessment . the acs group included unstable angina ( n=257 ) , non - stemi ( nstemi ; n=70 ) , and stemi ( n=7 ) patients . patient characteristics were compared between the acs and non - acs groups using the student 2-sample t test for continuous variables and fisher s exact test for categorical variables . non - normal and ordinal variables were reported as median ( 25th , 75th percentiles ) and compared using the kruskal lesion characteristics were compared using models developed from generalized estimating equation ( gee ) methods to account for patients having multiple lesions ( clustering ) . lesion characteristics that were continuous were evaluated by gee methods using a normal probability distribution . owing to the inherent risk for subsequent adverse cardiac events post - acs that persist despite statistical adjustment , the clinical outcomes between acs and non - acs groups were not compared directly . instead , the association between ffr and clinical outcomes was evaluated within each group using cox proportional hazards modeling . a marginal cox model was used to account for correlated data for patients with multiple lesions.20 tests of interaction were performed to determine whether the ffr association with outcome was different between acs and non - acs groups . furthermore , for the outcomes of a composite of cv death , mi or dli , composite mi or dli , and dlf , a multivariable analysis was performed adjusting for age , diabetes , smoking status , previous cad ( including pci or cabg ) , congestive heart failure , chronic kidney disease , and myocardial jeopardy index.21 for the association between ffr and clinical outcomes as well as tests of interaction , a value of p<0.05 was considered statistically significant.22 all statistical analyses were performed using sas software ( version 9.4 ; sas institute inc . , cary , nc ) . the study is a retrospective , single - center , observational study approved by the institutional review board . there were 1872 patients that underwent ffr assessment between october 2002 and july 2010 at our institution . from 2002 to 2008 , an ffr value 0.75 was used to defer revascularization based on the defer ( ffr to determine appropriateness of angioplasty in moderate coronary stenoses ) study.3 in 2008 , an ffr value > 0.80 was used to defer lesions based on the fame ( fractional flow reserve versus angiography for multivessel evaluation ) trial.4 of these 1872 patients , 742 patients with 906 coronary lesions were deferred revascularization based on ffr . of the 742 patients , 21 patients or 24 lesions were excluded because of loss of follow - up after ffr assessment . furthermore , 47 patients or 68 lesions were excluded because of ffr less than or equal to 0.80 . thus , the final population for this study consisted of 674 patients with 816 lesions that were deferred revascularization based on ffr > 0.80 ( figure 1 ) . ffr was performed according to standard techniques as detailed in a previous observational study using this study population.18 ffr was measured at maximal hyperemia after administering intracoronary ( n=812 lesions ) or intravenous adenosine ( n=4 lesions ) , with the dose determined by the operator . ffr was not performed on lesions with less than thrombolysis in myocardial infarction ( timi ) 3 flow . every patient included in the study was followed from the date of index stenting until march 12 , 2013 by reviewing the medical records and/or telephone interview . all angiograms were reviewed independently by at least 2 authors . for patients with follow - up or hospitalizations outside our institution , the medical records , including angiograms , the primary outcome was a composite of cardiovascular ( cv ) death , mi , or deferred lesion intervention ( dli ) . secondary outcomes included a composite of cv death or mi and mi or dli , as well as the individual endpoints of cv death , mi , mi - lesion , deferred lesion failure ( dlf ) , and dli . cv death and mi were defined by the academic research consortium guidelines.19 dli was defined as any percutaneous coronary intervention ( pci ) performed within 5-mm proximal or distal to , or any coronary artery bypass graft ( cabg ) placed distal to , a lesion deferred revascularization based on the index ffr assessment.18 mi lesion was defined as any mi that was directly attributable to a lesion deferred revascularization at the index ffr . the study population was divided into acs and non - acs groups based on clinical diagnosis at the time of index ffr assessment . the acs group included unstable angina ( n=257 ) , non - stemi ( nstemi ; n=70 ) , and stemi ( n=7 ) patients . patient characteristics were compared between the acs and non - acs groups using the student 2-sample t test for continuous variables and fisher s exact test for categorical variables . non - normal and ordinal variables were reported as median ( 25th , 75th percentiles ) and compared using the kruskal lesion characteristics were compared using models developed from generalized estimating equation ( gee ) methods to account for patients having multiple lesions ( clustering ) . lesion characteristics that were continuous were evaluated by gee methods using a normal probability distribution . owing to the inherent risk for subsequent adverse cardiac events post - acs that persist despite statistical adjustment , the clinical outcomes between acs and non - acs groups were not compared directly . instead , the association between ffr and clinical outcomes was evaluated within each group using cox proportional hazards modeling . a marginal cox model was used to account for correlated data for patients with multiple lesions.20 tests of interaction were performed to determine whether the ffr association with outcome was different between acs and non - acs groups . furthermore , for the outcomes of a composite of cv death , mi or dli , composite mi or dli , and dlf , a multivariable analysis was performed adjusting for age , diabetes , smoking status , previous cad ( including pci or cabg ) , congestive heart failure , chronic kidney disease , and myocardial jeopardy index.21 for the association between ffr and clinical outcomes as well as tests of interaction , a value of p<0.05 was considered statistically significant.22 all statistical analyses were performed using sas software ( version 9.4 ; sas institute inc . , cary , nc ) . baseline patient and lesion characteristics of the study population ( 674 patients with 816 coronary lesions ) are shown in tables1 and 2 . approximately half of the study population was deferred revascularization post - ffr assessment in the setting of non - acs ( n=340 patients ) . the remaining patients ( n=334 ) underwent ffr assessment during an acs , where 77% of these patients presented with unstable angina , 21% with nstemi , and 2% during stemi . the acs group patients had a higher rate of congestive heart failure and were discharged more frequently with angiotensin - converting enzyme inhibitors ( aceis ) or angiotensin receptor blockers ( arbs ) post - ffr assessment , compared to the non - acs group . however , both groups were discharged on a similar median number of optimal medical therapy medications.23 baseline patient - level characteristics values are shown as absolute numbers ( percentages ) , meansd , or median ( 1st , 3rd quartile ) . acei / arb indicates angiotensin - converting enzyme inhibitor / angiotensin receptor blocker ; acs , acute coronary syndrome ; cabg , coronary artery bypass graft ; cad , coronary artery disease ; ffr , fractional flow reserve ; omt , optimal medical therapy ; pci , percutaneous coronary intervention . all remaining comparisons between the acs and non - acs groups were not statistically significant . baseline lesion - level and procedural characteristics values are shown as absolute numbers ( percentages ) , meansd , or median ( 1st , 3rd quartile ) . acs indicates acute coronary syndrome ; cad , coronary artery disease ; ffr , fractional flow reserve ; ic , intracoronary ; lad , left anterior descending artery ; pci , percutaneous coronary intervention ; rca , right coronary artery . all remaining comparisons between the acs and non - acs groups were not statistically significant . multivessel cad was defined as 2 or more significant lesions ( angiographic percent stenosis 50% at the time of ffr assessment . maximum ic dose was the highest dose of adenosine given to induce hyperemia during ffr assessment . the left anterior descending artery ( lad ) was the predominant vessel location ( 42% ) for deferred lesions . the acs group had more ostial lesions ( 25% vs. 18% ) and fewer bifurcation lesions , compared to the non - acs group . the mean ffr value for the study population was 0.880.05 and was not different between the acs and non - acs groups . maximal hyperemia for ffr assessment was induced predominantly using intracoronary adenosine with similar maximum doses for both groups ( 120 g ) . there were no significant differences between patients who were lost to follow - up and those who were included in the analysis except for the history of cad ( 38% vs. 68% ; p=0.008 ) , proximal circumflex lesions ( 27% vs. 13% ; p=0.044 ) , ffr value ( 0.86 vs. 0.88 ; p=0.033 ) , and adenosine dose ( 180 vs. 120 ; p=0.025 ) . patients were followed for a mean of 4.52.1 years . subsequent mi in the lesion that was initially deferred ( ie , mi lesion ) accounted for 30% of the total mis that occurred during follow - up . acs indicates acute coronary syndrome ; dlf , deferred lesion failure ; dli , deferred lesion intervention ; mi , myocardial infarction . table4 stratifies outcomes based on ffr value ranges and demonstrates a numerically higher absolute event rate for each composite outcome within the 0.81 to 0.85 and 0.86 to 0.90 ranges in the acs group , compared to the non - acs group . the associations between ffr values obtained during maximal hyperemia at the time of index ffr assessment and clinical outcome within each group assessed by marginal cox proportional hazard modeling are shown in table5 . in the acs group , lower ffr values were associated with a significantly higher rate of cv death , mi or dli ( hazard ratio [ hr ] , 1.08 per 0.01 decrease ; 95% confidence interval [ ci ] , 1.03 to 1.12 ) , mi or dli ( hr , 1.09 ; 95% ci , 1.04 to 1.14 ) , dlf ( hr , 1.12 ; 95% ci , 1.06 to 1.18 ) , mi ( hr , 1.07 ; 95% ci , 1.00 to 1.14 ) , and dli ( hr , 1.12 ; 95% ci , 1.06 to 1.18 ) . a trend toward a lower ffr value being associated with mi lesion in the acs group was also observed ( hr , 1.12 ; 95% ci , 0.996 to 1.26 ) . the relationship between ffr and each of the clinical outcomes was found to differ significantly between acs and non - acs groups ( p<0.05 for each ) . there was also a trend toward lower ffr being more likely associated with mi in the acs group ( p=0.05 ) . in the non - acs group , lower ffr values were not associated with an increase in the rate of any of the clinical outcomes . using cox proportional hazard modeling adjusting for covariates ( figure 2 ) , lower ffr values remained an independent predictor in the acs group , but not the non - acs group of cv death , mi , or dli ( hr , 1.07 ; 95% ci , 1.02 to 1.11 ; interaction , p=0.08 ) , mi or dli ( hr , 1.08 ; 95% ci , 1.03 to 1.13 ; interaction , p=0.026 ) , and dlf ( hr , 1.11 ; 95% ci , 1.05 to 1.17 ; interaction , p=0.005 ) . a sensitivity analysis was performed including the 21 patients lost to follow - up and did not show any difference in adjusted hrs . acs indicates acute coronary syndrome ; cv , cardiovascular ; dli , deferred lesion intervention ; ffr , fractional flow reserve ; mi , myocardial infarction . cox proportional hr per 0.01 unit decrease in ffr acs indicates acute coronary syndrome ; dlf , deferred lesion failure ; dli , deferred lesion intervention ; ffr , fractional flow reserve ; hr , hazard ratios ; mi , myocardial infarction . for comparing hrs between acs and non - acs groups a marginal cox proportional hazard model was performed to adjust for age , diabetes , smoking status , previous coronary artery disease ( including pci or cabg ) , congestive heart failure , chronic kidney disease , and myocardial jeopardy index . acs indicates acute coronary syndrome ; cabg , coronary artery bypass graft ; dlf , deferred lesion failure ; dli , deferred lesion intervention ; ffr , fractional flow reserve ; hr , hazard ratios ; mi , myocardial infarction ; pci , percutaneous coronary intervention . baseline patient and lesion characteristics of the study population ( 674 patients with 816 coronary lesions ) are shown in tables1 and 2 . approximately half of the study population was deferred revascularization post - ffr assessment in the setting of non - acs ( n=340 patients ) . the remaining patients ( n=334 ) underwent ffr assessment during an acs , where 77% of these patients presented with unstable angina , 21% with nstemi , and 2% during stemi . the acs group patients had a higher rate of congestive heart failure and were discharged more frequently with angiotensin - converting enzyme inhibitors ( aceis ) or angiotensin receptor blockers ( arbs ) post - ffr assessment , compared to the non - acs group . however , both groups were discharged on a similar median number of optimal medical therapy medications.23 baseline patient - level characteristics values are shown as absolute numbers ( percentages ) , meansd , or median ( 1st , 3rd quartile ) . acei / arb indicates angiotensin - converting enzyme inhibitor / angiotensin receptor blocker ; acs , acute coronary syndrome ; cabg , coronary artery bypass graft ; cad , coronary artery disease ; ffr , fractional flow reserve ; omt , optimal medical therapy ; pci , percutaneous coronary intervention . all remaining comparisons between the acs and non - acs groups were not statistically significant . baseline lesion - level and procedural characteristics values are shown as absolute numbers ( percentages ) , meansd , or median ( 1st , 3rd quartile ) . acs indicates acute coronary syndrome ; cad , coronary artery disease ; ffr , fractional flow reserve ; ic , intracoronary ; lad , left anterior descending artery ; pci , percutaneous coronary intervention ; rca , right coronary artery . all remaining comparisons between the acs and non - acs groups were not statistically significant . multivessel cad was defined as 2 or more significant lesions ( angiographic percent stenosis 50% at the time of ffr assessment . maximum ic dose was the highest dose of adenosine given to induce hyperemia during ffr assessment . the left anterior descending artery ( lad ) was the predominant vessel location ( 42% ) for deferred lesions . the acs group had more ostial lesions ( 25% vs. 18% ) and fewer bifurcation lesions , compared to the non - acs group . the mean ffr value for the study population was 0.880.05 and was not different between the acs and non - acs groups . maximal hyperemia for ffr assessment was induced predominantly using intracoronary adenosine with similar maximum doses for both groups ( 120 g ) . there were no significant differences between patients who were lost to follow - up and those who were included in the analysis except for the history of cad ( 38% vs. 68% ; p=0.008 ) , proximal circumflex lesions ( 27% vs. 13% ; p=0.044 ) , ffr value ( 0.86 vs. 0.88 ; p=0.033 ) , and adenosine dose ( 180 vs. 120 ; p=0.025 ) . patients were followed for a mean of 4.52.1 years . clinical outcomes after deferred revascularization based on ffr are shown in table3 . subsequent mi in the lesion that was initially deferred ( ie , mi lesion ) accounted for 30% of the total mis that occurred during follow - up . acs indicates acute coronary syndrome ; dlf , deferred lesion failure ; dli , deferred lesion intervention ; mi , myocardial infarction . table4 stratifies outcomes based on ffr value ranges and demonstrates a numerically higher absolute event rate for each composite outcome within the 0.81 to 0.85 and 0.86 to 0.90 ranges in the acs group , compared to the non - acs group . the associations between ffr values obtained during maximal hyperemia at the time of index ffr assessment and clinical outcome within each group assessed by marginal cox proportional hazard modeling are shown in table5 . in the acs group , lower ffr values were associated with a significantly higher rate of cv death , mi or dli ( hazard ratio [ hr ] , 1.08 per 0.01 decrease ; 95% confidence interval [ ci ] , 1.03 to 1.12 ) , mi or dli ( hr , 1.09 ; 95% ci , 1.04 to 1.14 ) , dlf ( hr , 1.12 ; 95% ci , 1.06 to 1.18 ) , mi ( hr , 1.07 ; 95% ci , 1.00 to 1.14 ) , and dli ( hr , 1.12 ; 95% ci , 1.06 to 1.18 ) . a trend toward a lower ffr value being associated with mi lesion in the acs group was also observed ( hr , 1.12 ; 95% ci , 0.996 to 1.26 ) . the relationship between ffr and each of the clinical outcomes was found to differ significantly between acs and non - acs groups ( p<0.05 for each ) . there was also a trend toward lower ffr being more likely associated with mi in the acs group ( p=0.05 ) . in the non - acs group , lower ffr values were not associated with an increase in the rate of any of the clinical outcomes . using cox proportional hazard modeling adjusting for covariates ( figure 2 ) , lower ffr values remained an independent predictor in the acs group , but not the non - acs group of cv death , mi , or dli ( hr , 1.07 ; 95% ci , 1.02 to 1.11 ; interaction , p=0.08 ) , mi or dli ( hr , 1.08 ; 95% ci , 1.03 to 1.13 ; interaction , p=0.026 ) , and dlf ( hr , 1.11 ; 95% ci , 1.05 to 1.17 ; interaction , p=0.005 ) . a sensitivity analysis was performed including the 21 patients lost to follow - up and did not show any difference in adjusted hrs . acs indicates acute coronary syndrome ; cv , cardiovascular ; dli , deferred lesion intervention ; ffr , fractional flow reserve ; mi , myocardial infarction . cox proportional hr per 0.01 unit decrease in ffr acs indicates acute coronary syndrome ; dlf , deferred lesion failure ; dli , deferred lesion intervention ; ffr , fractional flow reserve ; hr , hazard ratios ; mi , myocardial infarction . for comparing hrs between acs and non - acs groups . a marginal cox proportional hazard model was performed to adjust for age , diabetes , smoking status , previous coronary artery disease ( including pci or cabg ) , congestive heart failure , chronic kidney disease , and myocardial jeopardy index . acs indicates acute coronary syndrome ; cabg , coronary artery bypass graft ; dlf , deferred lesion failure ; dli , deferred lesion intervention ; ffr , fractional flow reserve ; hr , hazard ratios ; mi , myocardial infarction ; pci , percutaneous coronary intervention . this study demonstrates that , for patients with coronary lesions deferred revascularization in the setting of acs based on non - significant ffr values ( ie , ffr > 0.80 ) , lower ffr values were associated with a significantly higher rate of adverse cardiac events . after adjustment , lower ffr values remained an independent predictor of cv death , mi , or dli , cv death or mi , and dlf among acs patients . the association between lower ffr values and adverse clinical outcomes was not observed for any of the composite or individual endpoints among non - acs patients deferred revascularization based on ffr . one third of the subsequent mis that occurred during follow - up were attributable to the lesion initially deferred based on ffr assessment . our study provides new evidence that lesions with lower ffr values deferred in the setting of acs are at higher risk for subsequent adverse cardiac events . in the defer and fame trials , when compared with angiographic guidance , ffr - guided selection of lesions for the deferral or performance of pci improved clinical outcomes , although the majority of patients included in those trials had stable cad.3,4 ffr can improve the diagnostic efficiency over angiography alone in the setting of acs,24 especially in the presence of multivessel cad . however , the reliability of ffr assessment of lesions during acs has been controversial.6 ffr assessment is performed by inducing maximal hyperemia to determine the physiological significance of a coronary stenosis.25 compared with stable cad , acs is a dynamic process with the potential for lesion instability and microvascular dysfunction that has been found in patients with mi to involve both infarct - related and non - infarct - related myocardial segments , and that persists for a poorly defined period , possibly up to 6 months.7 microvascular dysfunction has also been demonstrated in patients with unstable angina , where vasoconstriction and increased distal microvascular resistance reduces coronary blood flow.26 theoretically , when contrasted with intact microvascular function , an inability to induce maximal coronary hyperemia could lead to a smaller gradient and a higher ffr value for a given stenosis.8,27 beyond microvascular dysfunction , lower oxygen consumption in an infarcted territory , abnormal vasomotion of the epicardial and resistance vessels distal to a coronary thrombosis , and/or obstruction of the microvasculature , could limit myocardial blood flow and affect ffr assessment.9 therefore , there are multiple potential mechanisms that could adversely influence the reliability of ffr in the setting of acs . given these considerations and the fact that the decision to revascularize is largely based on an accepted ischemic threshold of 0.80,17 it appears possible that a nonsignificant ffr value that is close to the threshold in a patient with acs could underestimate lesion physiological significance and potential for causing ischemia.4,5 not inconsistent with our data , a recent meta - analysis suggested that ffr has been shown to have a continuous and independent relationship with clinical outcomes , with the suggestion that the ffr threshold for composite major adverse cv events in acs patients may exceed the current threshold of 0.80.28 several studies have previously examined the reliability of ffr for lesion assessment in the setting of acs.911 a study of 57 patients who suffered an mi 6 days before ffr assessment determined that an ffr < 0.75 had an 82% sensitivity and 87% specificity for detecting inducible ischemia determined by stress single - photon emission computed tomography ( spect ) imaging.9 despite concluding that an ffr value of 0.75 was valid 6 days after acute mi , the investigators cautioned that the results should not be extrapolated to ffr measurements obtained during the acute phase of an mi.9 a prospective study performed spect , ffr , and pci of the infarct - related artery 3.71.3 days after an mi ( 73% stemi ; 27% nstemi ) in 48 patients and then repeated spect imaging 11 weeks later to assess for reversibility after revascularization.10 the sensitivity and specificity of an ffr value 0.75 to accurately identify reversible ischemia on spect were 88% and 50% , respectively.10 the findings of these studies provided information regarding ffr post - acs , but they may not be generalizable to our study , given that ffr measurements in the acs group in our study were obtained during the acute phase of a coronary event . to examine the reproducibility of an ffr value measured acutely to one measured after recovery from acs , a prospective study measured ffr in 112 nonculprit lesions in ami patients ( 75% stemi ) at the time of clinical presentation and 354 days later.11 only 2 patients with an ffr value > 0.80 at the time of their mi had an ffr value < 0.75 at follow - up.11 given that microvascular dysfunction may persist for an unknown interval and possibly up to 6 months after an mi,7 the follow - up time between ffr measurements in that study may not have been sufficient for full recovery and restoration of microcirculatory function . observational studies and post - hoc analyses of randomized trials have suggested that ffr - guided revascularization is safe in acs.1216 in fame , 33% of patients ( n=328 ) were studied in the setting of acs ( unstable angina or nstemi ) , where 178 and 150 patients were randomized to angiographic- or ffr - guided pci , respectively.12 the benefit of ffr - guided pci , compared with angiography alone , appeared similar for the acs and stable cad ( n=677 ) groups.12 despite a higher rate of adverse cardiac events in acs patients , compared to stable cad patients , none of the mis during follow - up reportedly occurred in the index lesion deferred pci based on ffr.4,12 nevertheless , it is notable that the number of acs patients in fame undergoing ffr - guided revascularization was relatively small with shorter follow - up , which , as acknowledged by the investigators , may have limited the power of the study to show differences among subgroups . several additional smaller observational studies have assessed the clinical outcomes for lesions that were deferred revascularization in the setting of acs.1316 three of these studies , including a total of less than 250 acs patients , concluded that there were similar outcomes for patients deferred pci based on ffr in the setting of acs or non - acs.14,15 a more recent study of 162 patients deferred pci based on an ffr 0.75 in the setting of acs ( 60% ) and non - acs ( 40% ) demonstrated that patients with an ffr value of 0.75 to 0.85 and microvascular dysfunction ( measured by an abnormal corrected thrombolysis in myocardial infarction frame count [ ctfc ] ) had a significantly higher rate of adverse cardiac events , compared with patients with an ffr value > 0.85 and normal ctfc.16 similar to our study , lower ffr values in their analysis were an independent predictor of later adverse cardiac events ( hr , 1.09 ; 95% ci , 1.01 to 1.19 ; p=0.03).16 previous studies assessing the clinical safety and reliability of ffr for deferring revascularization among patients with acs were limited by small sample sizes1316 and by lesion assessment by ffr beyond the acute phase of acs.12 our study represents one of the largest studies on the clinical outcomes of lesions deferred pci based on ffr , where half of the study population underwent assessment during acs . compared with previous studies,1216 our study has a significantly higher rate of adverse cardiac events , which may be secondary to the nonselected , real - world study population and the longer follow - up ( mean 4.52.1 years ) . our study population also included patients with multivessel disease ( 64% ) and left main lesions ( 7% ) , many of which were excluded from other studies.12,13 the recent prospective randomized fractional flow reserve versus angiography in guiding management to optimize outcomes in nstemi ( famous - nstemi ) trial demonstrated that routine ffr measurement is feasible in nstemi patients , but was not powered to assess for differences in clinical outcomes compared to angiographic - guided care.6 further prospective evidence is needed to fully elucidate the reliability , reproducibility , and safety of ffr measurements in acs . this is a retrospective , observational study conducted at a large , single urban tertiary referral center . the possibility of reporting bias and the absence of an independent clinical events committee are potential limitations . a small number of patients ( n=21 ) were excluded from the study population because of loss to follow - up . the results may not be generalizable to different populations and hospitals of different sizes and/or level of acuity . in the acs group , culprit versus nonculprit lesions were distinguished based on the operator s judgment and this determination was therefore subjective , similar to common clinical practice . however , ffr was not performed on any lesion with less than timi 3 flow . moreover , there may be unmeasured confounders or selection bias that contributed to differences in clinical outcomes between the acs and non - acs groups . although ffr has been well validated as a measure of ischemia , it may have limited relevance for predicting the stability of a plaque . the study can not determine the safety of deferring lesions based on ffr versus angiographic - guidance in the setting of acs . although our study demonstrated an association between lower ffr values and worse clinical outcomes in acs patients , the clinical benefits of ffr - guided pci over angiography alone , as demonstrated in fame,12 may still persist in an acs population . this is a retrospective , observational study conducted at a large , single urban tertiary referral center . the possibility of reporting bias and the absence of an independent clinical events committee are potential limitations . a small number of patients ( n=21 ) the results may not be generalizable to different populations and hospitals of different sizes and/or level of acuity . in the acs group , culprit versus nonculprit lesions were distinguished based on the operator s judgment and this determination was therefore subjective , similar to common clinical practice . however , ffr was not performed on any lesion with less than timi 3 flow . moreover , there may be unmeasured confounders or selection bias that contributed to differences in clinical outcomes between the acs and non - acs groups . although ffr has been well validated as a measure of ischemia , it may have limited relevance for predicting the stability of a plaque . the study can not determine the safety of deferring lesions based on ffr versus angiographic - guidance in the setting of acs . although our study demonstrated an association between lower ffr values and worse clinical outcomes in acs patients , the clinical benefits of ffr - guided pci over angiography alone , as demonstrated in fame,12 may still persist in an acs population . for patients with intermediate severity coronary lesions that are deferred revascularization in the setting of acs based on ffr > 0.80 , lower ffr values are associated with a significantly higher rate of adverse cardiac events . the association between lower ffr values and adverse clinical outcomes was not observed among deferred lesions in non - acs patients . further study is needed to determine the reliability and safety of deferring lesions with relatively lower ffr values in the setting of acs based on the currently accepted ffr threshold validated in predominantly non - acs patients . dr singh is a consultant for abbott vascular , boston scientific , and volcano corp and is on the speakers bureaus of , or receives honoraria from , the medicines company , medtronic vascular , volcano corp , and st . dr bach receives research grants from bristol - myers squibb , eli lilly , glaxosmithkline , merck , and schering - plough and is a consultant ( clinical event committee activity only ) for eli lilly , novo nordisk , and pfizer .

backgroundthe safety of deferring revascularization based on fractional flow reserve ( ffr ) during acute coronary syndrome ( acs ) is unclear . we evaluated the association of ffr and adverse cardiac events among patients with coronary lesions deferred revascularization based on ffr in the setting of acs versus non-acs.methods and resultsthe study population ( 674 patients ; 816 lesions ) was divided into acs ( n=334 ) and non - acs ( n=340 ) groups based on the diagnosis when revascularization was deferred based on ffr values > 0.80 between october 2002 and july 2010 . the association and interaction between ffr and clinical outcomes was evaluated using cox proportional hazards models within each group ( mean follow - up of 4.52.1 years ) . subsequent revascularization of a deferred lesion was classified as a deferred lesion intervention ( dli ) , whereas the composite of dli or myocardial infarction ( mi ) attributed to a deferred lesion was designated as deferred lesion failure ( dlf ) . in the non - acs group , lower ffr values were not associated with any increase in adverse cardiac events . in the acs group , every 0.01 decrease in ffr was associated with a significantly higher rate of cardiovascular death , mi , or dli ( hazard ratio [ hr ] , 1.08 ; 95% confidence interval [ ci ] , 1.03 to 1.12 ) , mi or dli ( hr , 1.09 ; 95% ci : 1.04 to 1.14 ) , dlf ( hr , 1.12 ; 95% ci , 1.06 to 1.18 ) , mi ( hr , 1.07 ; 95% ci , 1.00 to 1.14 ) , and dli ( hr , 1.12 ; 95% ci , 1.06 to 1.18).conclusionlower ffr values among acs patients with coronary lesions deferred revascularization based on ffr are associated with a significantly higher rate of adverse cardiac events . this association was not observed in non - acs patients .

descriptive epidemiologic statistics assist public health planning and provide valuable information about the burden of illness to policy makers , funding agencies , resource planners , healthcare insurers , and manufacturers . information on malignancies that is compatible with their clinical classifications is of particular value to clinicians and public health professionals and increasing efforts are being made to collect data at this detailed level ( e.g. , the haemacare project ) . it is challenging , however , to assemble a database of descriptive epidemiologic statistics from the peer - reviewed literature alone . cancer registries are used worldwide to collect and analyze demographic , diagnostic , and survival data . some registries are fraught with poor quality and infrastructure ; however , there is no standardized system for the collection and reporting of descriptive statistics worldwide [ 2 , 3 ] . comprehensive reviews of descriptive epidemiologic statistics , such as this one , are warranted to better understand the totality of the currently available data and to optimize the utility of such data in the future . this paper uses a particular cancer subtype b - cell malignancies to evaluate a novel approach to assembling a database of worldwide , national - level , descriptive epidemiologic cancer statistics . this approach incorporates information from various sources , including the peer - reviewed literature , online reports , and query systems from cancer registries and health agencies , and direct contact with cancer registries , to provide a current , comprehensive database for a representative group of countries worldwide . the major b - cell malignancies were chosen as the cancer subtype to test this approach largely because their epidemiology has not been well characterized . further , some b - cell malignancy subtypes require detailed diagnostic evaluation , and this paper allowed us to broadly assess the extent to which detailed diagnoses are currently being reported to cancer registries . this is important because an understanding of these subtypes may inform the development of novel treatments . b - cell malignancies emerge in cells of the bone marrow , blood , or other tissues at various stages of b - lymphocyte differentiation and represent a rare ( 3% of all malignancies ) and heterogeneous group of lymphohematopoietic malignancies ( table 1 ) . in 2001 and 2008 , the world health organization ( who ) classified lymphoid hematologic neoplasms into 4 major categories based on the cell linage of the malignancy or the normal cell type that the tumor most resembles : b - cell malignancies , t - cell malignancies , natural killer ( nk ) cell malignancies , and hodgkin 's disease . further divisions were based on cell maturity ( e.g. , precursor versus mature b - cell neoplasms ) , as well as morphologic , genotypic , genetic , immunohistochemical , and clinical criteria . the major b - cell lymphoid malignancies include diffuse large b - cell lymphomas ( dlbcls ) , follicular lymphomas ( fls ) , and plasma cell or multiple myeloma ( mm ) . it is estimated that 85% to 90% of non - hodgkin 's lymphomas ( nhl ) are of b - cell origin , including dlbcl and fl . certain leukemias also arise from the b cells and are categorized based on ( 1 ) whether the disease is acute or chronic and ( 2 ) what type of cell is infected . the leukemias associated with abnormalities of b - cell formation in the blood include b - cell acute lymphoblastic leukemia ( all ) and chronic lymphocytic leukemia ( cll ) or small lymphocytic lymphoma . all represents one of the most common forms of leukemia among children . in this paper , a novel , comprehensive approach to gathering descriptive , epidemiologic statistics was used to develop a current worldwide database of the major b - cell malignancies . the intent of this paper was to evaluate the feasibility of this novel approach for assembling a database of descriptive statistics and to broadly assess the level of detail collected by cancer registries worldwide . we also briefly describe our findings of the descriptive epidemiology of b - cell malignancies . we collected the most recent information available on the incidence , prevalence , and survival of the following b - cell malignancies : dlbcl , fl , cll , mm , adult b - cell all , and pediatric b - cell all . these data were collected for a representative selection of countries worldwide , categorized into the following groups : north and south america ( brazil , canada , and the united states ) , the european union-5 ( france , germany , italy , spain , and the united kingdom ) , asia ( china , india , japan , and south korea ) , and australia . descriptive statistics for the countries constituting the united kingdom ( england , scotland , wales , and ireland [ including northern ireland and the republic of ireland ] ) were also collected . we employed three general strategies : a structured literature review of the published , peer - reviewed literature in pubmed , a review of online documentation from cancer registries and relevant health agencies , and , finally , direct contact via email with personnel at cancer registries and key experts in the field . the first 2 strategies were employed in tandem ; direct contact was used to address any remaining gaps and clarify the reasons behind missing data . various search strings ( see table s1 in supplementary material available online at doi:10.5402/2012/129713 ) were used in the pubmed search to identify general articles describing the epidemiology of these malignancies in the countries of interest . the search was restricted to human studies published from january 1 , 2000 to july 13 , 2011 . approximately 5600 citations were retrieved , and the titles and abstracts of these publications were reviewed . studies that appeared to report descriptive epidemiologic statistics ( i.e. , incidence , prevalence , or survival ) for the b - cell malignancies of interest were included . articles reporting national - level data on the statistics , malignancies , and countries of interest were identified and the relevant information was extracted . in addition , case reports , case series , and letters to the editor were excluded . relevant full - text articles were retrieved and reviewed for inclusion . worldwide , national , and regional cancer registries and online documentation for these registries ( including reports and online query systems ) the websites of other relevant health agencies and government departments were also searched for reports and references to the data of interest . registries and relevant agencies were identified from previous experience , online research into the structure of public health in each country , and involvement in organizations such as the international association of cancer registries , globocan , and the european network of cancer registries . for some non - english language speaking countries ( france , germany , spain , china , and japan ) , persons proficient in the language searched online data and translated some of the published literature ; a translation of non - english language material ( including online documentation and published studies ) was not performed , however . the goal was to identify the most recent estimates of the incidence , prevalence , and survival associated with the relevant b - cell malignancies . if relevant data were reported by more than 1 source , the source with the most recent data and/or the most relevant data ( e.g. , data specific to b - cell all versus all ) was used . once gaps in the availability of the data of interest were identified , cancer registry personnel and key experts in the field were contacted via e - mail to discuss the availability of missing statistics ( these contacts are referred to as personal communication ) . table 2 describes the cancer registries and organizations that were contacted and the outcome of each respective communication . we did not collect data from globocan or the cancer incidence in five continents ( ci5 ) series . these iarc programs provide a valuable resource for modeling estimates on the incidence and mortality of common cancers , including nhl and mm . however , since modeling estimates may not reflect the most recent national - level statistics , we did not rely on these databases for nhl and mm data . there are also standardized efforts in europe to report descriptive epidemiologic data : eurocare and the automated childhood cancer information system . similar to ci5 ; however , these standardized systems are not based on complete ascertainment from registries in the underlying countries , for example , only select regional registries participate in spain , italy , and france . as such , we only used data from these european programs in the absence of national - level data . the following information was extracted from each data source : source of the data , country or countries , malignancy type , including any available information on international classification of diseases ( icd ) codes , and data relevant to incidence , prevalence , and/or survival . we sought to assess the availability of recent statistics ; therefore , only post-2000 diagnoses were assessed for incidence and survival , and only prevalence estimates for a date after 2000 were collected . incidence projections for future years were collected only in the absence of actual incidence estimates . limited - duration prevalence rates and prevalence proportions estimated on a date after 2000 were collected for all available time frames ( e.g. , 5 y , 10 y ) . relative survival rates were collected for all time periods ( e.g. , 5 y survival , 10 y survival ) for diagnoses post-2000 ; it was noted whether survival rates were estimated using period methods . relative survival is defined as the ratio of the proportion of observed survivors in a cohort of cancer patients to the proportion of expected survivors in a comparable set of cancer - free individuals . in the absence of data on relative survival , data were collected by gender and methods for age standardization and any age restrictions were noted . data were reported from multiple citations for a particular malignancy and country if different information sources reported supplementary information . large gaps in the availability of national - level statistics on b - cell malignancies were observed at the outset of our review process and few descriptive statistics were available for the b - cell nhls ( i.e. , dlbcl and fl ) and b - cell all . we , therefore , extended the review to the larger diagnostic categories of nhl and all . data on diffuse nhl , a diagnostic category of nhl that includes dlbcl , were available from numerous countries , and data were collected for this larger diagnostic category in the absence of data specific to dlbcl . as a result , we collected data on the larger diagnostic category lymphoid leukemia . finally , national - level statistics were lacking in several countries ( e.g. , brazil , india , and china ) because of the status of cancer registration in these countries . to compensate for this deficiency , table 3 provides an overview of the availability of statistics by malignancy type and country ; notations are provided where proxy data ( as defined above ) were used . supplementary tables 2 to 7 provide the most recent statistics by country and statistic type for nhl , dlbcl and fl , adult all , pediatric all , mm , and cll , respectively . cancer registries are well recognized as a valuable information source for public health planning and epidemiologic and therapeutic research because they provide essential epidemiologic data on the current burden of disease . nonetheless , we found that the implementation of cancer registries has been slow in some countries , and data were not made readily available or updated in a timely manner in others . in 2006 , almost 80% of the world 's population was not covered by a population - based cancer registry ; most of this unrepresented population was from low- and middle - income countries . the uncoordinated nature of cancer registration worldwide has resulted in wide variation in the geographical areas covered , the methods used to estimate descriptive statistics , and the level of detail collected . this differential evolution of cancer registration worldwide yielded notable between - country variation in the availability of descriptive statistics on b - cell malignancies ( table 3 ) . the use of online resources and direct contact with cancer registries greatly expanded our database . the us surveillance , epidemiology , and end results ( seer ) registry provided the most complete data on b - cell malignancies . the seer registry provides incidence , prevalence , and survival data by icd - o-3 code using the seer*stat software . we were , therefore , able to calculate statistics for the b - cell malignancies dlbcl and b - cell all , which require icd - o-3 coding . nearly all countries in our paper had national - level incidence statistics for nhl and mm , although prevalence and survival data for these malignancies were less complete . data on dlbcl and fl were only available in the united states , the united kingdom , germany , and australia , but supplementary data were available on diffuse nhl from several countries ( brazil , england , ireland , and germany ) . since the course of leukemia varies by its tissue of origin and whether it is acute or chronic , the reporting of data by specific leukemia subtypes is preferable . lymphoid leukemia incidence was often used as a proxy for all and cll , however , and prevalence and survival data were scarce for all categories of lymphoid leukemia . in many developed countries ( e.g. , canada , the united kingdom , and germany ) , only the prevalence of leukemia as a large diagnostic group was reported . france provided no national - level estimates of all incidence , except rates estimated using a statistical method based on unreported mortality data ( belot a and mitchell m , personal communication with universit lyon , january 27 , 2011 ) . in germany , descriptive statistics were only available for leukemia as a large diagnostic group . regional statistics for lymphoid leukemia were found in the bavaria and bremen population - based cancer registries ( i.e. , registries that collected data on every person with incident cancer within a defined geographic region ) . italy , on the other hand , provided pooled data from its 28 regional cancer registries on adult and pediatric all and adult cll . among the asian countries , only japan had national - level data on all , whereas china and south korea had national - level data on lymphoid leukemia . the united states and the republic of ireland were the only countries with incidence data specific to adult b - cell all ( 1.0 and 0.22 ( s. deady and m. wagner , personal communication , with national cancer registry of ireland , september 24 , 2010 ) , respectively , per 100,000 persons ) . in the united states , these data suggest that b - cell all comprises approximately 60% of all adult all . the united states was the only country with statistics on pediatric b - cell all , with an incidence of 1.77 ( 95% ci , 1.701.84 ) per one million children ( representing only 5% of pediatric alls ) . of note , france , germany , and spain reported the incidence of mature b - cell lymphoid leukemias ( international classification of childhood cancer [ iccc ] code ia2 ) among children 0 to 14 years of age ( 1.5 , 1.0 , and 0.6 per million children per year , resp . ) ; these data demonstrate that the vast majority of lymphoid leukemias in children are of the precursor cell type [ 1921 ] . cancer registries in some countries indicated that the provision of descriptive data on specific nhl and leukemia subtypes was not possible because of small population sizes and the associated uncertainty with small numbers . the northern ireland cancer registry , for example , could not provide statistics by nhl or leukemia subtypes because of small numbers ( d. donnelly and m. wagner , personal communication with northern ireland cancer registry , september 27 , 2010 ) . the national cancer registry of the republic of ireland , on the other hand , provided these data after e - mail contact was initiated with the registry ( s. deady and m. wagner , personal communication with national cancer registry of ireland , other cancer registries could not provide the level of detail required for nhl and leukemia subtypes because the data were not coded appropriately to permit such analyses . for example , noted that it is not currently possible to calculate statistics for various hematologic subtypes in france because of issues with reliably classifying cases using a consistent coding scheme . similarly , the office of national statistics ( which releases descriptive data for england ) stated that they were only able to provide data by icd-10 codes because of variability in the coding information provided by regional registries and issues with mapping icd - o-3 ( introduced in 2008 ) to icd - o-2 ( n. jakomis and m. wagner , personal communication with national cancer intelligence center on descriptive cancer statistics , october 11 , 2010 ) . icd-10 codes do not allow for the calculation of statistics on b - cell all or dlbcl . we were able to capture statistics for fl with icd-10 code c82 and for diffuse lymphoma with icd-10 code c83 , however . the icd - o-3 coding system provides the most relevant information for descriptive epidemiologic statistics because malignancies are coded by cell lineage and maturity , as well as morphologic , genotypic , genetic , immunohistochemical , and clinical behavior . icd - o-3 has been adopted by the us and most european countries , but the report of descriptive statistics by icd - o-3 coding is limited by the recent conversion from icd - o-2 in 2000 and the small numbers associated with these rare hematologic malignancies . one author noted that , even considering the whole of europe , some entities were too rare to calculate statistics by icd - o-3 coding . to address this issue , haemacare , a project funded by the european commission to improve the standardization of population - based data on hematologic malignancies , collected incidence data 20002002 from 48 european registries in 20 countries using a grouping system based on who recommendations and the icd - o-3 morphology codes . thus , categories represent cell lineage and prognosis , which are useful for epidemiologic and public health purposes . the haemacare coding system contains detailed information on mature b - cell malignancies , including dlbcl , fl , and cll . in their first publication of incidence data from the haemacare network , sant et al . reported european age - standardized incidence rates ( per 100,000 persons ) for dlbcl , fl , and cll of 3.81 ( 95% ci , 3.733.89 ) , 2.18 ( 95% ci , 2.122.24 ) , and 4.92 ( 95% ci , 4.835.01 ) , respectively . compared to these haemacare european estimates , the incidence of dlbcl is considerably higher in the united states ( 6.83 ; see table s3 ) and the incidence of fl is higher in both the united states ( 3.7 ) and australia ( 4.1 ; see table s3 ) . however , the haemacare european estimate of cll incidence is similar to rates observed in the united states ( 5.0 ) , canada ( 5.0 ) , and australia ( 4.9 ; see table s7 ) . sant et al . noted geographic variability in the incidence of hematologic malignancies across europe , which they attributed to differences in diagnostic and registration criteria . this first publication from the haemacare network suggests that data on dlbcl , fl , and cll are increasingly available in certain registries across europe , but that cancer registries are reluctant to regularly publish and/or provide these statistics . another limitation of the diagnostic classification system used for reporting cancer statistics is the use of the iccc for childhood cancers . in the third version of the iccc , all is categorized along with other lymphatic leukemias in the category iccc-1a , which includes morphology codes 9820 , 9823 , 9826 , 9827 , 98319837 , 9940 , and 9948 . although the vast majority of lymphatic leukemias in childhood are acute in nature , estimates using iccc-1a only provide a proxy for actual all rates . there was wide variation between countries in the methods used for estimating national - level descriptive statistics . some countries used direct estimates from national cancer registries , collecting information on all cancer diagnoses nationwide into 1 registry ( i.e. , wales , northern ireland , republic of ireland , scotland , and south korea ) or a coordinated group of regional registries ( i.e. , england , canada , and australia ) . other countries calculated estimates of the national - level experience using statistical methods to extrapolate data from regional registries to the national level ( i.e. , united states , france , japan , and china ) . another group of european countries estimated the national - level cancer experience by pooling data from regional registries ( i.e. , italy , germany , and spain ) . this variability in the methods used to estimate national - level statistics limits the comparability of statistics between countries . one statistical method used to predict cancer incidence at the national - level is the use of mortality rates ( which are often readily available nationwide ) as a geographic correlate of incidence . observed incidence and mortality data are modeled in this approach using age - cohort techniques to provide estimated incidence rates . this method was recently used to estimate cancer incidence in france and china [ 22 , 25 ] and is frequently used by globocan . while this method is known to be generally reliable , there are some questions about the representativeness of the data used . however , the reliability of this method to estimate the incidence cancers with high survival rates ( e.g. , pediatric all ) is less clear . the vast majority of registries were population - based , but some countries had registries that collected data only on cancer patients seen at a particular hospital ( i.e. , hospital - based registries ) . hospital - based registries are limited compared with population - based cancer registries because the source population is not well - defined . brazil has several hospital - based cancer registries , and south korea has a nationwide , hospital - based cancer registry ( the korea central cancer registry ) that reportedly captures 90% of all cancer cases nationwide . the national registry of childhood tumors is a population - based cancer registries of malignancies and benign brain tumors diagnosed in children living in england , wales , or scotland . the french national registry of childhood hematopoietic malignancies has recorded all cases of hematologic malignancies , including lymphoma , since 1990 ; the french national registry of childhood solid tumors has recorded all cases of solid tumors , except lymphomas , since 2000 . the german childhood cancer registry has collected data on cancer cases nationwide , with an estimated coverage of about 95% . finally , the australian pediatric cancer registry has provided full coverage of all australian states and territories since 1983 . a substantial number of countries do not have a nationwide cancer registration program . rather , cancer registration consists of a group of regional cancer registries , collecting complete data on cancer cases in their respective geographic regions and coordinated by an overarching network . the level of standardization and coordination between these regional registries varies substantially by country . in the united states , seer currently collects and publishes cancer data in a standardized fashion from 17 population - based cancer registries covering approximately 28% of the us population there are 21 regional cancer registries in france coordinated under the association of french cancer registries ( francim ) that cover approximately 16% of the population . in italy , there are 28 registries that cover approximately 26% of the population coordinated through the italian association of cancer registries ( airtum ) . in germany , there are regional registries in 13 states and 1 administrative district , coordinated through the robert koch institute . japan has approximately 30 population - based cancer registries that are coordinated through the japan cancer surveillance research group . some developed countries still lack coordination of their regional registries , which limits what can be done to predict the national - level experience of cancer . spain , for example , currently has 13 regional registries that cover approximately 27% of the population , but no network that coordinates and standardizes these registries ; a network is currently under development , however . in less developed countries , where financial , resource , and logistical challenges dominate , coverage is typically limited to metropolitan areas , and differences between regional registries preclude the development of national estimates . substantial efforts are being made , however , to organize and standardize these regional registries into a coordinated network . in brazil , for example , efforts are being made to collect data from an expanding network of regional registries and to improve data quality to support nationwide cancer prevention and planning . epidemiology surveillance was formally organized and standardized in 1999 and now includes 22 population - based cancer registries , most of which include capital cities and surrounding areas . these population - based cancer registries vary in the source ( hospital , laboratory , etc . ) and quality of their data . because of this variation , pooled data can not be reported and statistics are provided only by individual population - based cancer registries for brazil . the age - adjusted incidence of nhl , for example , varied from 2.0 to14.1 per 100,000 among males in the ten population - based cancer registries with more than 2 years of data . the authors of the latest report cautioned that these results are not generalizable to brazil as a whole because the population - based cancer registries are still of heterogeneous quality , validity , and completeness . underreporting also remains a serious concern in brazil and other less developed countries . despite these limitations , some brazilian population - based cancer registries provided data on detailed diagnostic categories diffuse nhl , follicular lymphoma , and pediatric all . however , although incidence data were readily available from brazilian population - based cancer registries , more complex data ( prevalence and survival ) were not . similarly , india has 26 population - based cancer registries and 6 hospital - based cancer registries coordinated under the national cancer registry programme network and covering 7% of the population ; statistics are only reported by population - based cancer registries . china also has no national - level coordination of its 50 population - based cancer registries , which cover 5.7% of the country . this survey represents the status of data that were available as of mid - july 2011 using comprehensive methods . it is clear from our paper , however , that the breadth and specificity of data reported by cancer registries is continually evolving , and this evolution will ultimately entail the reporting of data at the national level for detailed cancer subtypes . in france , for example , national level data were reported for the first time in 2003 for cancer incidence , in 2006 for cancer survival , and in 2008 for cancer prevalence . lacour et al . described the incidence of childhood cancers nationwide in france for the first time in 2010 . population - based cancer registration in germany improved considerably after the federal cancer registry data act of 2009 , which called on all federal states to report complete data to the german centre for cancer registry data at the robert koch institute , thus expanding the breadth of cancer statistics in germany from data reported only by the saarland cancer registry to pooled analyses of data from cancer registries meeting the minimum requirements for data completeness . current estimates of cancer incidence are based on data from 13 states and 1 administrative district , and the gekid cancer survival working group recently published their first comprehensive monitoring of cancer survival in germany based on a collaboration between 13 population - based cancer registries . some basic patterns in the descriptive epidemiology of b - cell malignancies emerged from reviewing the available data . specifically , the incidence rates of the b - cell neoplasms included in this paper were generally higher in the united states , australia , and the european union-5 , compared with asia and brazil ( possibly reflecting differences in the ascertainment of cases ) . further , the incidence of nhl was generally higher in men , and nhl survival rates were slightly higher in women ( see table s2 ) . the higher incidence of hematologic malignancies in men versus women may be explained by the traditionally higher exposures of men to occupational and environmental carcinogens . many cancer registries reported that the requested statistics were not readily available or were available at a cost ( e.g. , additional data were available at a cost from the office of national statistics , the welsh cancer intelligence service , and the australian institute of health and welfare ) . it is , therefore , possible that statistics beyond those reported in table 3 are available . some attempts to communicate with key experts and cancer registries failed , thus limiting our understanding of the true availability of certain data ( table 2 ) . for example , our attempts to contact the northern and yorkshire cancer registry and information service ( nycris ) and the cote d'or hematological malignancies registry ( the lead registries for hematologic malignancies in england and france , resp . ) yielded no responses . we were also unable to establish contact with cancer registries in italy , japan , and south korea . we limited the data we gathered to diagnoses post-2000 to allow for representation of the availability of recent descriptive statistics . some data were only available , however , for diagnoses that occurred in the late 1990s ( e.g. , survival data in ireland and france and pediatric all incidence in scotland ) . this paper was also limited to select countries , which precludes a full evaluation of the worldwide availability of data and an analysis of geographic differences . a comparison of statistics across countries is also limited by differences in the structure of cancer registries , age standardization methods , methods for estimating the statistics ( e.g. , period estimates for survival ) , and the year(s ) of diagnosis . furthermore , the comparison of hematologic malignancies across time and place was significantly limited by regional differences in disease classification systems . this paper was also limited by reports of cancer data in non - english languages . translations of non - english language material were restricted to languages in which the research team had proficiency and were not carried out in a systematic manner . thus , it is possible that additional data is readily accessible in other languages . updates are published continuously by cancer registries , and investigators are frequently publishing manuscripts with additional , relevant epidemiologic data . we attempted to capture the most recent data available as of approximately mid - july 2011 . at the time of the publication of this study , however , we were aware of numerous updates , namely the release of updated seer data . in addition , the nycris was planning to publish a report on hematologic malignancies for all english cancer networks in 2011 . this paper used a novel approach to gathering descriptive epidemiologic statistics on cancer by combining information from the biomedical literature , internet - based query systems and reports , and direct contact with cancer registry representatives and key experts to provide the most up - to - date , comprehensive picture of a particular malignancy type . this paper highlights the importance of international coordination between cancer registries ( e.g. , haemacare ) to promote standardization in data collection and reporting . we also recommend continued coordination and standardization between regional registries in countries such as brazil , spain , and india to promote the development of nationwide estimates . this novel approach provided a more comprehensive database of the current patterns of b - cell malignancies compared with a simple literature search . in addition , gaps were identified that highlight the need for more detailed reporting to cancer registries and the importance of developing assumptions to estimate the burden of specific b - cell malignancies in the interim . because research indicates that cellular lineage affects prognosis , treatment , and etiology , cancer registries should continue to work to improve reporting of detailed diagnostic information on malignancies .

the compilation of comprehensive , worldwide epidemiologic data can inform hypotheses on cancer etiology and guide future drug development . these statistics are reported by a multitude of sources using varying methods ; thus , compiling a complete database of these statistics is a challenge . to this end , this paper examined the usefulness of a novel , multisource approach extracting data from the peer - reviewed literature , online reports , and query systems from cancer registries and health agencies and directly contacting cancer registry personnel for building a comprehensive , multinational epidemiologic cancer database . the major b - cell malignancies were chosen as the cancer subtype to test this approach largely because their epidemiology has not been well characterized in the peer - reviewed literature . we found that a multisource approach yields a more comprehensive epidemiologic database than what would have been possible with the use of literature searches alone . in addition , our paper revealed that cancer registries vary considerably in their methodology , comprehensiveness , and ability to gather information on specific b - cell malignancy subtypes . collectively , this paper demonstrates the feasibility and value of a multisource approach to gathering epidemiologic data .

the prevalence rate in the general population ranges between 10% and 12% , making this disease one of the most common causes of pain , activity limitation , and restrictions in participation . knee osteoarthritis ( gonarthrosis ) is one of the most frequent forms of degenerative osteoarthritis . in knee osteoarthritis , pain is the primary symptom and probably the most important factor related to activity limitation and disability . although the pain in knee oa is multifactorial , acute exacerbations are usually closely related to synovial inflammation . various studies performed by using arthroscopy have shown synovitis in painful knees of patients with knee oa . on the basis of these reports , many investigators focused on noninvasive imaging techniques , such as magnetic resonance imaging and diagnostic ultrasonography . the term pes anserinus refers to the conjoined insertion of the sartorius , gracilis , and semitendinosus muscles along the proximal medial aspect of the tibia . these three muscles are primarily flexors of the knee ; they also influence internal rotation of the tibia and protect the knee against rotatory and valgus stress . in pes anserine bursitis , the fluid - filled bursa in the conjoined insertion of the three hamstring muscles becomes inflamed and causes pain . patients with this clinical picture may report vague medial knee pain or may present with tenderness and swelling along the proximal medial tibia . a diagnosis of pes anserine bursitis should be considered when there is spontaneous pain inferomedial to the knee joint and a history of trauma or diabetes . conditions associated with pes anserine bursitis include degenerative joint disease of the knee , obesity , valgus knee deformity , pes planus , and sporting activities . pes anserine bursitis affects the quality of life of patients with osteoarthritis , and the treatment methods are different . treatment includes nonsteroidal anti - inflammatory drugs ( nsaids ) , physiotherapy , and injections of corticosteroid , with highly variable responses ; recovery can take 10 days to 36 months . mesotherapy was introduced 50 years ago by michel pistor , a french physician who used this technique as a novel analgesic therapy for a variety of rheumatologic disorders . mesotherapy is a minimally invasive technique that consists of subcutaneous injections of drugs and , occasionally , plant extracts , homeopathic agents , or other bioactive substances ; for this reason , it has been often considered a form of complementary and alternative medicine rather than a conventional medical therapy . the objective of this type of administration is to modulate the pharmacokinetics of the injected substance and to prolong the pharmacologic effects at a local level . one of the main advantages of mesotherapy is that a local pharmacologic effect can be obtained without the need for high systemic concentrations . intradermal injections of small amounts of active substance where the injection site corresponds to the area of the pathologic condition for example , in low back pain may provide clinical benefits when other therapies are not available , are not effective , or can not be used for whatever reason . in addition , intradermal administration of active substances in combination with other systemic therapies can produce synergistic effects , and as a result mesotherapy may have dose - sparing effects . since its introduction , the use of mesotherapy has been expanded , and therapeutic indications have increased . although mesotherapy is primarily used for osteoarticular conditions , this technique has recently become popular in cosmetic medicine for treatment of edematous fibrosclerotic panniculopathy and local adiposity . the aim of the current study was to evaluate the effects of mesotherapy with diclofenac for anserine bursitis in knee osteoarthritis . the multicenter study was conducted at the d'annunzio university in chieti , italy , and at an affiliated rehabilitation center in florence . it was approved by the local ethics committee and was performed in accordance with the 1964 declaration of helsinki . one hundred and seventeen patients ( 59 men and 58 women ) age 1856 years ( average age , 36 years ) were evaluated and treated . all them had anserine bursitis , ascertained clinically and by ultrasonography , in association with grade ii kellgren - lawrence knee osteoarthritis ( fig . inclusion criteria were age between 10 and 60 years , grade ii kellgren - lawrence knee osteoarthritis , re - acutization of pain , and synovial inflammation . exclusion criteria were pregnancy , known hypersensitivity to products , infiltrative therapy with hyaluronic acid , polynucleotides or corticosteroids in progress , drug abuse or alcohol abuse , significant comorbidities ( such as the presence of neurologic abnormalities , concomitant severe rheumatic disease , and systemic abnormalities , such as diabetes ) , a surgical intervention within 3 months before the study , psychiatric conditions , or psychotherapy or physical therapy within 5 weeks before the study . the patients were randomly divided into two groups ( a , mesotherapy ; b , control ) after giving written consent and agreeing to the possible treatment ( local or oral ) . group a ( 60 patients ; 30 men and 30 women ) received nine sessions of mesotherapy with sodium diclofenac ( 25 mg/1 ml ; akis , ibsa , lugano , switzerland ) , 1 ml for each session , three times per week . group b ( 57 patients ; 29 men and 28 women ) received 21 oral administrations of sodium diclofenac ( 50 mg ; voltaren , novartis , parsippany , nj ) , once every day for 3 weeks . all patients reported pain during sleep and after a long period of rest , as well as reduction of pain after about a 200-m walk . all patients were asked to provide standard radiographs ( anterior - posterior and lateral ) of the lumbosacral tract and knee joints . primary outcome measures were pain intensity , quantified by using a 10-cm millimetric visual analogue scale ( vas ) , on which the patient is asked to mark the degree of pain intensity , ranging from 0 ( absence of pain ) to 10 ( the worst pain imaginable ) , along with abilities in activities of daily living ( adls ) , ability to participate in sports , level of pain , symptoms , and quality of life , as assessed by the knee injury and osteoarthritis outcome score ( koos ) . all these assessments , along with ultrasonography , were performed before ( t0 ) and after ( t1 ) the treatment period and at 30 days ' ( t2 ) and 90 days ' ( t3 ) follow - up in all patients . differences between mean values before and after the rehabilitation period were tested for significance by using two - way analysis of variance for repeated measures . graphpad prism ( version 6 ) software ( abacus concepts graphpad software , san diego , ca ) . in group a , the subjective pain levels assessed by vas were 71.05 at t0,20.35 at t1 , 3.80.85 at t2 , and 4.00.85 at t3 ( fig . disability during sports activity at t1 decreased significantly ( p<0.05 ) : t0 score , 7010.7 ; t1 score , 557.9 ; t2 score , 608.5 ; t3 score , 608.7 ( fig . perceived symptoms also were significantly reduced ( p<0.05 ) at t1 and at both follow - up times : t0 score , 71.411.9 ; t1 score , 46.49.4 ; t2 score , 509.7 ; t3 , score 53.69.0 ( fig . was seen in the effect of osteoarthritis on quality of life at t1 and at both follow - up times : t0 score , 68.813.2 ; t1 score , 43.810.9 ; t2 score , 509.7 ; t3 score , 509.8 ( fig . disability with regard to adls also decreased significantly ( p<0.05 ) at t1 and at both follow - up times : t0 score , 7214.2 ; t1 score , 478.7 ; t2 score , 478.7 ; t3 score , 509.2 ( fig . finally , pain perception was significantly reduced ( p<0.01 ) at t1 and at both follow - up times : t0 score , 7510.3 ; t1 score , 30.55.5 ; t2 score , 365.2 ; t3 score , 415.8 ( fig . error bars represent the standard deviation . a , group a ( mesotherapy treatment ) ; b , group b ( oral treatment ) . trend of disability during sports activity ( knee injury and osteoarthritis outcome score [ koos ] ) . trend of disabilities with regard to activities of daily living ( adls ) ( koos ) . b , the subjective pain levels assessed by vas were 7.51.85 at t0 , 31.10 at t1 , and 5 at t2 and t3 ( fig . the koos findings were as follows : disability during sports activity did not significantly decrease : t0 score , 7515.8 ; t1 score , 6515.5 ; t2 score , 6515.7 ; t3 score , 7017.2 ( fig . 3 ) . a significant reduction occurred ( p<0.05 ) in perceived symptoms at t1 : t0 score , 71.413.1 ; t1 score , 60.78.3 ; t2 score , 64.38.7 ; t3 score , 64.39.1 ( fig . the effect of osteoarthritis on quality of life decreased significantly ( p<0.05 ) at t1 : t0 score , 7513.2 ; t1 score , 5011.0 ; t2 score , 68.814.5 ; t3 score , 68.814.7 ( fig . disability with regard to adls was reduced significantly ( p<0.05 ) at t1 : t0 score , 70.612.7 ; t1 score , 61.79.4 ; t2 score , 66.210.9 ; t3 score , 67.612.5 ( fig . finally , a significant reduction ( p<0.05 ) was seen in pain perception at t1 and at both follow - up times : t0 score , 7510.4 ; t1 score , 52.78.3 ; t2 score , 55.68.8 ; t3 score , 58.39.5 ( fig . in particular , group a had pain remission after a 120-m walk at t1 and after a 160-m walk at t2 and t3 . ultrasonography showed a reduction of the hypoechoic area related to anserine bursitis only in group a ( ie , the mesotherapy group ) . the aim of this study was to evaluate the effectiveness of anti - inflammatory drugs administered via mesotherapy in patients with local inflammation in grade ii kellgren - lawrence knee osteoarthritis . present results showed that the administration of nsaids ( diclofenac ) via mesotherapy can provide the same therapeutic benefit as that induced by conventional ( oral ) drug administration in relation to pain after a 3-week treatment period . indeed , both treatments significantly reduced pain intensity and disability in daily life activity ; the effect was maintained up to 3 months ( 30 days ' and 90 days ' follow - up ) only in the group that received mesotherapy . these results are in accordance with previous studies showing that naproxen and diclofenac administered via mesotherapy were more effective than those given orally . the major finding of this study is the similar effectiveness of mesotherapy and conventional systemic therapy , despite the lower amount of drugs administered to patients undergoing mesotherapy ( 25 mg with mesotherapy vs 50 mg with oral administration ) and the lower short - term risk for adverse reactions in the mesotherapy group ( 3 weeks ) . the similar efficacy of mesotherapy and conventional therapy , despite different drug dosages , is difficult to explain . subcutaneous drug administration results in a very slow drug absorption in comparison with other systemic routes , such as oral and intramuscular . thus , it could be hypothesized that anti - inflammatory drugs administered via mesotherapy achieve a higher drug concentration in the subcutaneous tissue and exert local effects close to inflammatory cells , sensory fibers , and vascular mediators that orchestrate inflammation and pain . this hypothesis could be confirmed by the evidence of reduction of the anserine bursitis showed by ultrasonography in group a ( the mesotherapy group ) but not in group b ( the oral administration group ) . although this study did not measure drug plasma levels after the two routes of administration , it is possible to hypothesize that mesotherapy resulted in a lower systemic bioavailability of drugs , with consequent lower incidence of adverse reactions . this could offer a great therapeutic advantage given the high rates of adverse effects associated with nsaid or corticosteroid use in the elderly population . although mesotherapeutic techniques used in dermatologic surgery have been associated with many adverse effects at injection sites , including atypical mycobacterial infections , urticaria , lichenoid drug eruptions , and psoriasis , no evidence of local reactions was found in the present study . in conclusion , results of this study indicate that combined administration of conventional nsaids ( diclofenac ) by mesotherapy is an effective and well - tolerated method for managing anserine bursitis in ii grade knee osteoarthritis in the short term and mid - term compared with oral administration of the same drug with different concentrations . however , future studies could have a larger scale and thus also measure plasma bioavailability of the drug . these observations could be of potential interest for the pharmacologic treatment of anserine bursitis in order to reduce the adverse effects associated with high plasma levels of anti - inflammatory drugs .

abstractbackground : pes anserine bursitis strongly affects quality of life in patients with osteoarthritis . treatment includes nonsteroidal anti - inflammatory drugs ( nsaids ) , physiotherapy , and injections of corticosteroid , with highly variable responses ; recovery can take 10 days to 36 months . mesotherapy is a minimally invasive technique consisting of subcutaneous injections of bioactive substances . the goal is to modulate the pharmacokinetics of the injected substance and prolong the effects at a local level.objective : to evaluate the effects of mesotherapy with diclofenac for anserine bursitis associated with knee osteoarthritis.methods : one hundred and seventeen patients with anserine bursitis associated with grade ii kellgren - lawrence knee osteoarthritis , assessed by clinical , radiographic , and ultrasonographic examination , were evaluated and treated . they were randomly divided into two groups ( a , mesotherapy ; b , control ) . group a completed nine sessions of mesotherapy with sodium diclofenac ( 25 mg/1 ml ; akis , ibsa , lugano , switzerland ) , 1 ml for each session , three times per week . group b received 21 oral administrations of sodium diclofenac ( 50 mg ; voltaren , novartis , parsippany , nj ) , once a day for 3 weeks . primary outcome measures were pain intensity assessed by visual analogue scale ( vas ) , along with ability to perform activities of daily living , ability to participate in sports , level of pain , symptoms , and quality of life , as assessed by the knee injury and osteoarthritis outcome score . these measures were performed before and after the treatment period and at 30 and 90 days ' follow up.results : in both groups pain level decreased significantly after the treatment period . ultrasonography showed a reduction of the hypoechoic area related to anserine bursitis only in group a.conclusion : administration of conventional nsaids ( diclofenac ) by mesotherapy is effective in managing anserine bursitis in knee osteoarthritis in the short term and mid - term . these observations could be of interest for efforts to reduce the adverse effects of oral administration of anti - inflammatory drugs .

antidepressants represent the sixth leading substance category causing intoxication in the usa , with tricyclic antidepressants ( tcas ) being the most important category in that group . tca intoxications led to 69 fatalities in 2012 . with 6305 exposures in 2012 , amitriptyline was the single most important tca involved in intoxication . similar findings are reported all over the world : amitriptyline is the second most responsible drug for death in antidepressant poisoning in the uk , where antidepressant self - poisoning is used in 20% of all poisoning suicides . tcas were the most common suicide poison used in new zealand between 2001 and 2005 . in tehran , iran , amitriptyline is the most used tca in drug intoxication , where tcas are responsible for 16% of all admissions due to intoxication . in adult out - patients , amitriptyline is normally dosed between 75 and 150 mg / day , which provides therapeutic concentrations between 80 and 250 g / l . in cases of overdose , amitriptyline may cause an alteration of consciousness , hypernatraemia , convulsive seizure , arrhythmia and respiratory depression . first - line treatment focuses on general measures as well as correction of electrolyte disorders and arrhythmia . extracorporeal means of toxin removal consist of charcoal haemoperfusion and plasma exchange , which decreased amitriptyline blood concentrations in a few cases . due to its high plasma protein binding and volume of distribution ( vod ) , amitriptyline the extracorporeal treatments in poisoning ( extrip ) workgroup therefore recommends not to use extracorporeal removal of tca , as this approach is not likely to offer a clinical benefit . we report two cases of amitriptyline poisoning in which we measured the amitriptyline elimination using a high cut - off ( hco ) dialyzer . essential information on both patients as recommended by the extrip workgroup is summarized in table 1 . table 1.relevant case report characteristics recommended by the extrip workgroup case 1case 2general information age ( years)5453 weight ( kg)5475 height ( cm)168157 gender concurrent diseasesbreast cancer , chronic pain syndrome , depressionmgus , depression , chronic pain syndrome source providing the history of the poisoningparamedicsparamedics time from ingestion to hospital admission ( h)7.58.5 known co - medicationlorazepampantoprazole , citalopram , mirtazapine other toxinstilidine , ethanolibuprofen , -blockers , alcohol activated charcoal givennoyes icu stay ( days)35 discharge from hospital ( days)320laboratory values albumin ( g / l)34.842 creatinine at baseline ( mol / l)/egfr ckd - epi36/115.3643/6 ( akin iii ) serum amitriptyline peak concentration ( g / l)458412 serum nortipytilin peak concentrations ( g / l)283259 serum tilidine / nortilidin peak concentrations ( g / l)56/849na urine excretion ( ml / days)3100anuria urine amitriptyline concentration ( g / l)623na haematocrit ( % ) 4036.5ectr characteristics modality of ectrintermitted haemodialysisintermitted haemodialysis indication for ectrdetoxificationaki , rhabdomyolysis , detoxification ectr start after admission ( h)2.51.5 dialyser ( material / surface)hco dialyser ( polysulfone , 1.8 m)high - flux dialyser ( polysulfone , 1.3 m)hco dialyser ( polysulfone , 1.8 m ) dialysis time ( min)295 ( first treatment)220 ( second treatment)320 ( first treatment)775 ( second treatment)/350 ( hco ) blood flow ( ml / min)300 ( first treatment)110 ( second treatment)240 ( high - flux)240 ( high - flux ) dialysate flow ( ml / min)300 ( first treatment)55 ( second treatment)240 ( high - flux)240 ( high - flux ) ultrafiltration rate ( ml / h)5050 ( first treatment)/50 ( second treatment)/200 ( hco ) anticoagulationheparinheparin amitriptyline reduction ratio ( % ) 2728 ( hco ) ectr amitriptyline clearance ( ml / min)587 ( high - flux)33 ( hco ) total amount of amitriptyline in the collected dialysate ( g)211920 ( high - flux)na ( hco)aki , acute kidney injury ; akin , acute kidney injury network ; ckd - epi , chronic kidney disease epidemiology collaboration ; egfr , estimated glomerular filtration rate ; hplc , high - performance liquid chromatography ; ms / ms , tandem mass spectrometry.amitriptyline and nortriptyline were quantified ( detection limit 5 g / l ) by hplc followed by electrospray ionization and mass spectrometric detection and quantification of selected ion fragments ( triple quadrupole ms / ms , api2000 , pe sciex ) after simple deproteinization with acetonitrile / methanol . g / l ) by hplc - ms ( waters micromass , quattro micro ) after fluid extraction with cyclohexane.additional membrane characteristics are reported elsewhere .calculations of reduction ratios were executed according to equation ( 1):(1)rr=(cpostcpre)/cpre100 dialyser clearance was measured according to equation ( 2 ) , using the patients hematocrit concentration ( hct ) at the time of clearance sampling:(2)kplasma = qb(1hct/100)((cartcven)/cart ) relevant case report characteristics recommended by the extrip workgroup aki , acute kidney injury ; akin , acute kidney injury network ; ckd - epi , chronic kidney disease epidemiology collaboration ; egfr , estimated glomerular filtration rate ; hplc , high - performance liquid chromatography ; ms / ms , tandem mass spectrometry . amitriptyline and nortriptyline were quantified ( detection limit 5 g / l ) by hplc followed by electrospray ionization and mass spectrometric detection and quantification of selected ion fragments ( triple quadrupole ms / ms , api2000 , pe sciex ) after simple deproteinization with acetonitrile / methanol . g / l ) by hplc - ms ( waters micromass , quattro micro ) after fluid extraction with cyclohexane . calculations of reduction ratios were executed according to equation ( 1):(1)rr=(cpostcpre)/cpre100 dialyser clearance was measured according to equation ( 2 ) , using the patients hematocrit concentration ( hct ) at the time of clearance sampling:(2)kplasma = qb(1hct/100)((cartcven)/cart ) a 54-year - old caucasian woman , who ingested an unknown amount of amitriptyline and tilidine in a suicide attempt , was admitted to our hospital deeply unconscious with a glasgow coma scale score of 5 . electrocardiography showed a broad qrs complex ( 134 ms ) and prolonged qtc ( 517 ms ) , representing increased risk for ventricular arrhythmias . based on the clinical condition , we started a 5 h dialysis using an hco emic2 dialyser ( polysulfone ; 1.8 m ) with a dialysate and blood flow of 300 ml / min to eliminate tilidine and other potentially ingested drugs . the treatment was well tolerated , and immediately afterwards a dialysis with low dialysate and blood flow was started with the aim of performing an extended dialysis . due to clotting of the extracorporeal circuit , this dialysis prematurely ended after 220 min . plasma concentrations of both substances were drawn at different time points ( figure 1a ) . plasma dialyser clearances for amitriptyline and tilidine were 58 and 67 ml / min , respectively , at the beginning and decreased towards the end of the first dialysis session ( supplementary table s1 ) . for comparison , dialyser clearances for creatinine and urea were 115 and 132 ml / min , respectively . dialyser reduction ratios , dialyser clearance and total loss into the collected spent dialysate are listed in supplementary table s1 . open triangles denote myoglobin values at the upper laboratory detection range . * amitriptyline concentration measured in different laboratories . amitriptyline concentration measured in different laboratories . into the sixth hour of treatment , the patient regained consciousness and was transferred to psychiatric care after 3 days in the intensive care unit ( icu ) . a 53-year - old caucasian female who ingested 1750 mg of amitriptyline and 12 g of ibuprofen in a suicide attempt was admitted to our hospital . serum creatinine was 634 mol / l , sodium 137 mmol / l and creatine kinase 27 308 u / l . amitriptyline serum concentration was 412 g / l . the unconscious patient was transferred to our icu , intubated and mechanically ventilated . gastroscopy revealed unabsorbed pills in the stomach , and activated charcoal was administered after they were removed . haemodialysis was started 2 h after admission due to anuric acute kidney injury caused by rhabdomyolysis . dialysis using an fx60s polysulfone dialyser ( fresenius medical care ; 1.3 m ) was performed with a blood and dialysate flow of 240 ml / min . g / l at the start of the second dialysis treatment , amitriptyline concentrations did not show any remarkable decline in the following hours of treatment ( figure 1b ) . to enhance extracorporeal removal , we employed the hco emic2 dialyser ( fresenius medical care , polysulfone ; 1.8 m ) with a dialysate and blood flow of 240 ml / min , decreasing amitriptyline to 163 g / l . the increased removal was also confirmed by a plasma dialyser clearance of 33 ml / min for amitriptyline and 124 ml / min for myoglobin . myoglobin serum concentrations decreased from > 60 000 g / l ( upper detection limit ) to 11 642 five days after admission , the patient was transferred to the nephrology ward , and renal replacement therapy was stopped after 14 days of intermittent haemodialysis . on renal biopsy , twenty days after initial admission , the patient was transferred to a psychiatric hospital . of note , therapy with proton pump inhibitors , a therapy the patient had been prescribed for years , can cause both rhabdomyolysis and interstitial nephritis . a 54-year - old caucasian woman , who ingested an unknown amount of amitriptyline and tilidine in a suicide attempt , was admitted to our hospital deeply unconscious with a glasgow coma scale score of 5 . electrocardiography showed a broad qrs complex ( 134 ms ) and prolonged qtc ( 517 ms ) , representing increased risk for ventricular arrhythmias . based on the clinical condition , we started a 5 h dialysis using an hco emic2 dialyser ( polysulfone ; 1.8 m ) with a dialysate and blood flow of 300 ml / min to eliminate tilidine and other potentially ingested drugs . the treatment was well tolerated , and immediately afterwards a dialysis with low dialysate and blood flow was started with the aim of performing an extended dialysis . due to clotting of the extracorporeal circuit , this dialysis prematurely ended after 220 min . plasma concentrations of both substances were drawn at different time points ( figure 1a ) . plasma dialyser clearances for amitriptyline and tilidine were 58 and 67 ml / min , respectively , at the beginning and decreased towards the end of the first dialysis session ( supplementary table s1 ) . for comparison , dialyser clearances for creatinine and urea were 115 and 132 ml / min , respectively . dialyser reduction ratios , dialyser clearance and total loss into the collected spent dialysate are listed in supplementary table s1 . open triangles denote myoglobin values at the upper laboratory detection range . * amitriptyline concentration measured in different laboratories . * amitriptyline concentration measured in different laboratories . into the sixth hour of treatment , the patient regained consciousness and was transferred to psychiatric care after 3 days in the intensive care unit ( icu ) . a 53-year - old caucasian female who ingested 1750 mg of amitriptyline and 12 g of ibuprofen in a suicide attempt was admitted to our hospital . serum creatinine was 634 mol / l , sodium 137 mmol / l and creatine kinase 27 308 u / l . amitriptyline serum concentration was 412 gastroscopy revealed unabsorbed pills in the stomach , and activated charcoal was administered after they were removed . haemodialysis was started 2 h after admission due to anuric acute kidney injury caused by rhabdomyolysis . dialysis using an fx60s polysulfone dialyser ( fresenius medical care ; 1.3 m ) was performed with a blood and dialysate flow of 240 ml / min . after a quick decrease of amitriptyline to 205 g / l at the start of the second dialysis treatment , amitriptyline concentrations did not show any remarkable decline in the following hours of treatment ( figure 1b ) . to enhance extracorporeal removal , we employed the hco emic2 dialyser ( fresenius medical care , polysulfone ; 1.8 m ) with a dialysate and blood flow of 240 ml / min , decreasing amitriptyline to 163 g / l . the increased removal was also confirmed by a plasma dialyser clearance of 33 ml / min for amitriptyline and 124 ml / min for myoglobin . five days after admission , the patient was transferred to the nephrology ward , and renal replacement therapy was stopped after 14 days of intermittent haemodialysis . on renal biopsy , twenty days after initial admission , the patient was transferred to a psychiatric hospital . of note , therapy with proton pump inhibitors , a therapy the patient had been prescribed for years , can cause both rhabdomyolysis and interstitial nephritis . very recently , the extirp workgroup recommended not performing any extracorporeal treatments ( ectrs ) for intoxication with tcas . this recommendation is based on the fact that conventional haemodialysis is ineffective due to the high protein binding ( > 90% ) and large vod ( 1417 l / kg ) and the lipophilic properties of tca . however , most of the haemodialysis data the workgroup based the recommendation on are from the 1960s and 1970s . hence , data about the performance of modern membranes in tca intoxication are limited . the potential benefit of modern means of renal replacement therapy was suggested in a report on haemodiafiltration after acute amitriptyline intoxication in which the patient showed an improved vigilance during treatment . as no amitriptyline blood concentrations recently , extended dialysis using an hco dialyser has been introduced to remove large quantities of free light chains in patients with multiple myeloma . these dialysers are characterized by a larger pore size , which allows enhanced middle molecule removal without substantial elimination of albumin . myoglobin , accumulated in rhabdomyolysis as in one of our cases , is also dialysed by highly permeable membranes . so far , there are only anecdotal reports on the use of hco dialysers for the removal of toxins . we could show that even with an increase of the amitriptyline dialyser clearance using hco instead of high - flux dialysis , the estimated total eliminated amount of amitriptyline by hco dialysis remains low ( < 0.1% of the ingested dose ) and therefore may not exceed 3% of the ingested dose per hco dialysis session , which is considered to be the lower threshold for dialysability according to extrip criteria . an additional factor influencing amitryptilin concentrations is concomitantly ingested drugs , especially those that are metabolized mainly via cyp2d6 . although these drugs , especially pantoprazole in our cases , might have an effect on metabolization notwithstanding , the inherent limitation of only two reported patients , is an important shortcoming of this article . in summary , even the elevated amitriptyline clearance by hco membranes in the context of extended dialysis is unlikely to confer a clinical benefit , supporting a recent recommendation of extrip to refrain from any ectr in intoxication with tcas . as a finding unrelated to the topic of extracorporeal toxin removal , we could show that hco extended dialysis is very effective in lowering elevated myoglobin in rhabdomyolysis .

antidepressants , especially amitriptyline , are among the most frequent drug classes involved in intoxications . despite its small molecular weight , amitriptyline is not considered to be eliminated by extracorporeal treatment methods due to its high protein binding and large volume of distribution . new high cut - off dialysers have so far not been used for removal of amitriptyline . we report two cases of amitriptyline poisoning in which we measured the amitriptyline elimination using extended high cut - off ( hco ) dialysis . despite dialyser clearances of 33 and 58 ml / min , resulting in the reduction of initial serum concentrations by 30% , only 211 and 920 g of amitryptilin , respectively , ( < 3% of the ingested amount ) could be recovered in the total collected dialysate . hence , due to the high volume of distribution of amitriptyline , even hco dialysis does not contribute substantially to the extracorporeal removal of amitryptilin .

hunter syndrome ( mucopolysaccharidosis type ii or mps ii ) is an x - linked metabolic disorder caused by a deficiency in the lysosomal enzyme iduronate-2-sulfatase ( i2s ) [ 1 , 12 ] . i2s catalyzes a step in the degradation of dermatan sulfate and heparan sulfate , and in affected patients , these enzyme substrates accumulate in cells and tissues and contribute to the multiorgan pathologies associated with hunter syndrome . it has been estimated that the incidence is about 1 in 162,000 live male births . hunter syndrome patients usually appear normal at birth with clinical signs and symptoms emerging between the ages of 2 and 4 years [ 8 , 12 ] . the presenting signs typically include coarse facial features ; respiratory obstruction caused by enlarged tongue , tonsils , and adenoids ; joint stiffness and skeletal abnormalities ; and enlarged liver and spleen [ 1517 ] . the severe phenotype is characterized by progressive central nervous system involvement that results in severe cognitive impairment and developmental regression , with death typically occurring in the second decade of life [ 7 , 12 , 17 ] . patients with the attenuated phenotype do not experience cognitive impairment but may still demonstrate all of the somatic signs and symptoms of the disease [ 8 , 12 , 16 ] . diagnosis of hunter syndrome can be challenging as signs and symptoms of hunter syndrome are not specific to the disease , and not all clinical features will be present in the same order or equally rapidly in each patient . diagnosis may therefore be delayed , especially in patients without a known family history of hunter syndrome . enzyme replacement therapy ( ert ) with recombinant human i2s is available for the treatment of hunter syndrome [ 10 , 11 ] . in addition to ert , there is a hope that hunter syndrome may be responsive to bone marrow transplantation in the future . therefore , although a rare disease , pediatricians and other primary care physicians have an obligation to be aware of the signs and symptoms of hunter syndrome and methods and tools to effectively establish a diagnosis . mnemonics are commonly employed in clinical medicine as screening tools or to guide clinical management [ 2 , 13 ] , and here , we describe the development of a simple mnemonic screening tool that may aid pediatric specialists and primary care physicians in the recognition and diagnosis of hunter syndrome . hunter outcome survey ( hos ) is an international , long - term observational registry designed to increase the knowledge of the natural history of hunter syndrome and to evaluate the safety and effectiveness of long - term ert with idursulfase ( elaprase , shire human genetic therapies , inc . all participating centers receive approval from their local institutional review board or ethics committee before enrolling any patients into hos . hos is controlled by physicians and is overseen by national , regional , and international advisory boards comprising physicians experienced in the management and treatment of hunter syndrome patients . any patient with a biochemically or genetically confirmed diagnosis of hunter syndrome is eligible to enroll in hos independent of whether receiving treatment or not . the hos database consists of data collected from medical examinations conducted during the usual medical care of the patients . the signs , symptoms , and other clinical information ( e.g. , medical history , laboratory values , etc . ) surveyed are described in detail by wraith and colleagues and cover all the major organ and system involvement that occurs in hunter syndrome . shire hgt maintains the database and provides statistical support to hos physicians and advisory boards . for the purposes of this study , the investigators were given permission , by the global executive committee of the hos participating investigators , to query the database regarding the prevalence of specific signs and symptoms present in individual patients . the preliminary assessment was based on the hos database as of january 10 , 2008 , and the final assessment was based on the april 16 , 2009 data extract . the goal of the present study was to develop a sensitive screening tool to aid in the diagnosis of hunter syndrome using medical history data collected in hos . table 1 presents the hunter mnemonic , which was based on the key clinical features observed in patients with hunter syndrome . the january 2008 data extract of the hos database was queried for the prevalence of the signs and symptoms before diagnosis within individual patients as shown in table 2 . a random sample of 25 patients in hos with available information and who did not have a prenatal diagnosis was used to characterize the utility of a uniform weighting ( table 2 , weighting 1 ) of these ten signs and symptoms to specifically identify patients with hunter syndrome . a second weighting scheme was also evaluated ( table 2 , weighting 2 ) using a second random sample of 25 patients in hos with available information who were not included in the first sample and who did not have a prenatal diagnosis or family history of hunter syndrome . with this scheme , signs and symptoms unlikely to be observed in a non - affected child these signs and symptoms included dysmorphic facial features , nasal obstruction , enlarged tongue , enlarged liver , enlarged spleen , and joint stiffness . after this preliminary evaluation of the weighting schemes , weighting 2 was again evaluated using the april 2009 extract of the hos database , which included 237 patients with available data who did not have a prenatal diagnosis or a family history of hunter syndrome and who were not included in the two random samples of 25 patients used to assess the two weighting schemes.table 1the hunter mnemonic screening instrument for identifying patients with hunter syndromemnemonickey clinical featurehherniahearingherniahearing loss or impairment , hearing aids , chronic or acute otitisuunusual facesdysmorphic facial featuresnnasal obstructionnasal obstruction , rhinorrheattongue and tonsilsenlarged tongue , enlarged tonsilseenlarged liver and spleenhepatomegaly , splenomegalyrrespirationrange of motionairway obstruction , sleep apneajoint stiffnesstable 2signs and symptoms used in the development of the hunter score screening toolsigns or symptomsweighting 1weighting 2hernia11hearing loss , use of a hearing aid , acute or chronic otitis11dysmorphic facial features12nasal obstruction or rhinorrhea12enlarged tongue12enlarged tonsils11hepatomegaly12splenomegaly12airway obstruction or sleep apnea11joint stiffness12maximum score for a patient1016 the hunter mnemonic screening instrument for identifying patients with hunter syndrome signs and symptoms used in the development of the hunter score screening tool no formal statistical analysis of the weighting schemes was performed . the investigators relied on their clinical experience to determine if the results appeared to be specific enough to correctly identify patients with hunter syndrome . hunter outcome survey ( hos ) is an international , long - term observational registry designed to increase the knowledge of the natural history of hunter syndrome and to evaluate the safety and effectiveness of long - term ert with idursulfase ( elaprase , shire human genetic therapies , inc . all participating centers receive approval from their local institutional review board or ethics committee before enrolling any patients into hos . hos is controlled by physicians and is overseen by national , regional , and international advisory boards comprising physicians experienced in the management and treatment of hunter syndrome patients . any patient with a biochemically or genetically confirmed diagnosis of hunter syndrome is eligible to enroll in hos independent of whether receiving treatment or not . the hos database consists of data collected from medical examinations conducted during the usual medical care of the patients . the signs , symptoms , and other clinical information ( e.g. , medical history , laboratory values , etc . ) surveyed are described in detail by wraith and colleagues and cover all the major organ and system involvement that occurs in hunter syndrome . shire hgt maintains the database and provides statistical support to hos physicians and advisory boards . for the purposes of this study , the investigators were given permission , by the global executive committee of the hos participating investigators , to query the database regarding the prevalence of specific signs and symptoms present in individual patients . the preliminary assessment was based on the hos database as of january 10 , 2008 , and the final assessment was based on the april 16 , 2009 data extract . the goal of the present study was to develop a sensitive screening tool to aid in the diagnosis of hunter syndrome using medical history data collected in hos . table 1 presents the hunter mnemonic , which was based on the key clinical features observed in patients with hunter syndrome . the january 2008 data extract of the hos database was queried for the prevalence of the signs and symptoms before diagnosis within individual patients as shown in table 2 . a random sample of 25 patients in hos with available information and who did not have a prenatal diagnosis was used to characterize the utility of a uniform weighting ( table 2 , weighting 1 ) of these ten signs and symptoms to specifically identify patients with hunter syndrome . a second weighting scheme was also evaluated ( table 2 , weighting 2 ) using a second random sample of 25 patients in hos with available information who were not included in the first sample and who did not have a prenatal diagnosis or family history of hunter syndrome . with this scheme , signs and symptoms unlikely to be observed in a non - affected child these signs and symptoms included dysmorphic facial features , nasal obstruction , enlarged tongue , enlarged liver , enlarged spleen , and joint stiffness . after this preliminary evaluation of the weighting schemes , weighting 2 was again evaluated using the april 2009 extract of the hos database , which included 237 patients with available data who did not have a prenatal diagnosis or a family history of hunter syndrome and who were not included in the two random samples of 25 patients used to assess the two weighting schemes.table 1the hunter mnemonic screening instrument for identifying patients with hunter syndromemnemonickey clinical featurehherniahearingherniahearing loss or impairment , hearing aids , chronic or acute otitisuunusual facesdysmorphic facial featuresnnasal obstructionnasal obstruction , rhinorrheattongue and tonsilsenlarged tongue , enlarged tonsilseenlarged liver and spleenhepatomegaly , splenomegalyrrespirationrange of motionairway obstruction , sleep apneajoint stiffnesstable 2signs and symptoms used in the development of the hunter score screening toolsigns or symptomsweighting 1weighting 2hernia11hearing loss , use of a hearing aid , acute or chronic otitis11dysmorphic facial features12nasal obstruction or rhinorrhea12enlarged tongue12enlarged tonsils11hepatomegaly12splenomegaly12airway obstruction or sleep apnea11joint stiffness12maximum score for a patient1016 the hunter mnemonic screening instrument for identifying patients with hunter syndrome signs and symptoms used in the development of the hunter score screening tool the investigators relied on their clinical experience to determine if the results appeared to be specific enough to correctly identify patients with hunter syndrome . as of april 16 , 2009 , a total of 541 prospective patients ( i.e. , alive at hos entry ) were enrolled in hos . the mean patient age at their most recent examination as entered into hos was 12.3 years ( median 10.3 years , 10th90th percentile 3.723.4 years ) . the age at onset of signs and symptoms and the age at diagnosis are summarized in table 3.table 3age at onset of signs and symptoms and diagnosis of hunter syndromenumbermean ( years)median ( years)10th to 90th percentileage at onset of symptoms4052.01.50.24.0age at diagnosis4794.03.51.27.1delay between onset of symptoms and diagnosis3991.91.304.4based on prospective patients ( i.e. , alive at hos entry ) from the april 16 , 2009 data extract age at onset of signs and symptoms and diagnosis of hunter syndrome based on prospective patients ( i.e. , alive at hos entry ) from the april 16 , 2009 data extract figure 1 illustrates the results of the preliminary testing of the utility of uniform weighting ( weighting 1 ) of the ten signs and symptoms presented in table 2 as well as the second weighting scheme ( weighting 2 ) using a second random sample . no threshold hunter score was found that could identify a hunter patient without yielding too many false negative results . for example , a score of 1 failed to identify 12 % of the patients with hunter syndrome and a score of 2 failed to identify 20 % of the patients . the failure of uniform weighting to identify hunter syndrome patients was likely due in part to the fact that patients with a family history of hunter syndrome were included in the test sample of 25 randomly selected patients . because a family history of hunter syndrome may result in diagnosis before the emergence of many signs and symptoms , this test sample included many patients with low scores . in addition , many of the signs and symptoms are common in children without hunter syndrome , e.g. , otitis and rhinorrhea.fig . the maximum score for weighting 1 is 10 , and the maximum score for weighting 2 is 16 . n = 25 randomly selected patients from hos . based on two random samples of patients from the january 10 , 2008 data extract . the maximum score for weighting 1 is 10 , and the maximum score for weighting 2 is 16 . based on two random samples of patients from the january 10 , 2008 data extract . hos hunter outcome survey weighting scheme 2 ( table 2 ) was developed to give greater weight to those signs and symptoms that are more likely to be specifically associated with hunter syndrome . preliminary testing with 25 randomly selected patients from hos who did not have a family history of hunter syndrome suggested that a hunter score of 6 or 7 would correctly identify more than 95 % of patients with hunter syndrome . weighting 2 was further evaluated by using the 237 patients in hos who had complete information regarding the prevalence of signs and symptoms and who had no prenatal diagnosis and no family history of hunter syndrome . a score of 6 or greater correctly identified 95 % of this population as having hunter syndrome ( fig . 2 ) . no association between age of diagnosis and hunter score at diagnosis was observed ( fig . the median ( 10th90th percentile ) hunter score for patients < 2 , 2 to 4 , and > 4 years old at diagnosis was 12 ( 716 ) , 13 ( 816 ) , and 13 ( 716 ) , respectively.fig . based on the april 16 , 2009 data extract and excluding patients with missing information , prenatal diagnosis , or family history of hunter . based on the april 16 , 2009 data extract and excluding patients with missing information or with prenatal diagnosis or family history of hunter . . based on the april 16 , 2009 data extract and excluding patients with missing information , prenatal diagnosis , or family history of hunter . based on the april 16 , 2009 data extract and excluding patients with missing information or with prenatal diagnosis or family history of hunter . this simple mnemonic screening tool correctly identified patients as having hunter syndrome with 95 % accuracy at a hunter score of 6 or above . this score appears to be unlikely to be attained by a person without hunter syndrome or other mps disorders . mnemonics are frequently used in clinical practice as screening tools or to guide clinical management , for example , mnemonic or scoring systems have been employed to screen newborns for down syndrome , to identify infants in need of resuscitation , and to describe heart sounds . a newborn screening test is not yet available , and the low awareness of rare , genetic diseases hinders the recognition and diagnosis of this syndrome . the recognition and diagnosis of hunter syndrome is often delayed because its complex and diverse signs and symptoms are not specific to hunter syndrome , making a simple tool to help narrow the differential diagnosis is an asset in pediatric and primary care settings . the goal of the present study was to develop a simple , sensitive , and unified screening tool for identifying hunter syndrome . the first iteration in which the ten signs and symptoms were given equal weight was not sufficiently sensitive and failed to yield a reliable threshold score that would identify hunter syndrome without producing a large number of false negatives ( and presumably false positives ) . the inclusion of patients with a family history of hunter syndrome may have contributed to the failure of uniform weighting to correctly identify patients with hunter syndrome because these patients may have had earlier diagnoses before the emergence of many signs and symptoms of hunter syndrome than patients without a family history . the final iteration in which the hunter syndrome - specific signs and symptoms were weighted 2 points ( table 2 ) appeared to correctly identify 95 % of hunter patients in the study cohort with a hunter score of 6 or higher . additionally , it seems unlikely that patients without hunter syndrome or other mps disorders would be likely to meet or exceed this threshold hunter score . it is important to note that exceeding this threshold is only suggestive of hunter syndrome and biochemical and/or genetic confirmation is still necessary to confirm the presence of this disease and other forms of mps . we acknowledge that this tool requires validation in a clinical setting to assess the tool s sensitivity , specificity , positive and negative predictive values , and patient variability across all types of pediatric patients . this study represents the first step in the development of tool based on hundreds of observations of children with hunter syndrome , which would otherwise be impossible to develop without such a registry , given the rarity of this disorder . the tool at present is likely to generate false positive but may also help in the identification of other children with rare inherited disease , which will still be of great value . we hope this paper will serve as a first step toward a more refined tool with high predictive value . the hunter mnemonic had a sensitivity of over 95 % to detect hunter syndrome at a hunter score of 6 or greater and , given the overlap of clinical features , may also help in the identification of other mpss . validation of this tool in the clinical setting will be required to determine the sensitivity , specificity , as well as positive and negative predictive value of this tool , and to confirm its utility as a screen for hunter syndrome in routine clinical practice . the approach described herein illustrates the utility of disease registries in the development of tools that may assist physicians in the diagnosis and management of rare , genetic disorders . as newborn screening for hunter syndrome is presently unavailable , it is hoped that this simple mnemonic and associated scoring system will prove useful for pediatricians or other primary care clinicians when faced with a patient with hunter syndrome or other mps disease .

the hunter outcome survey ( hos ) , an international , long - term observational registry of patients with hunter syndrome , was used to develop a simple mnemonic screening tool ( hunter ) to aid in the diagnosis of hunter syndrome . data regarding the prediagnosis prevalence of ten specific signs and symptoms present in individual patients enrolled in the hos were used to develop the hunter mnemonic screening tool . a total score of 6 or greater using a weighting scheme in which certain manifestations were assigned a weight of 2 ( facial dysmorphism , nasal obstruction or rhinorrhea , enlarged tongue , enlarged liver , enlarged spleen , joint stiffness ) and others assigned a weight of 1 ( hernia , hearing impairment , enlarged tonsils , airway obstruction or sleep apnea ) correctly identified 95 % of patients who had no family history of hunter syndrome or who were not diagnosed prenatally . no association between age at diagnosis and hunter score was found . conclusion : the hunter mnemonic appears to be a useful screening tool . further validation in the clinical setting will be necessary to confirm its utility .

it is known as the fast enemy that should be treated and destroyed very fast as well . most women do not like to hear the word cancer , and feel worried and stressed over it . it can be the beginning of learning how to fight , getting the facts , and finding hope . this is followed by lack of patient 's personal control over the current treatment method and uncertainty of its outcome . therefore , anxiety is associated with cancer ; it is the most prevalent psychological symptoms perceived by cancer patients as a response to a threat , and so many patients are anxious . in one study done by ashbury et al , 77% of 913 patients within 2 years of treatment recalled experiencing anxiety . however , anxiety after cancer diagnosis is not necessarily abnormal , may not present a problem , or may even be a constructive part of dealing with problems . the most common cancer and the number one cause of cancer death amongst women in malaysia is breast cancer . if not detected and treated promptly , breast cancer can metastasize , spreading to the lymph glands and other parts of the body including the lungs , bones , and liver . usually , cancer is named after the body part in which it originated ; thus , breast cancer refers to the erratic growth and proliferation of cells that originate in the breast tissue . the term breast cancer refers to a malignant tumor that has developed from cells in the breast . the breast is composed of two main types of tissues : glandular tissues and stromal ( supporting ) tissues . glandular tissues house the milk - producing glands ( lobules ) and the ducts ( the milk passages ) , while stromal tissues include fatty and fibrous connective tissues of the breast . the breast is also made up of lymphatic tissue - immune system tissue that removes cellular fluids and waste . breast cancer is characterized by the uncontrolled growth of abnormal cells in the milk - producing glands of the breast or in the passages ( ducts ) that deliver milk to the nipples . the early stage of breast cancer usually refers to the cancer that is confined to the fatty tissue of the breast . it may then spread to underlying tissues of the chest wall and then to other parts of the body . furthermore , worldwide , breast cancer is the leading cause of cancer death in women , and more than one million women are diagnosed each year . in addition to that , more than 500,000 women every year die from the disease worldwide . anxiety , tension , worry , stress , and strain are all common feelings and it is a part of our life today . simple worry or stress will not drive us to look for specialist , but when these feelings become a chronics and interfere with our lives we need to do something and look for ways to manage it in order to function well . anxiety can be defined as an unpleasant subjective experience associated with the perception of real threat ; therefore , it is a common symptom in connection with cancer . furthermore , it can be described as an emotional state characterized by feelings of unpleasant expectation and a sense of imminent danger . according to stark , et al . autonomic hyper - arousal with acceleration of heart rate and respiration , tremor , sweating , muscle tension , and gastrointestinal changes are common physiological experiences . apprehension , feeling powerless , and fearing loss of control are psychological aspects . according to kazdin , anxiety is an emotion that characterized by feelings of tension , worry , and stress as well as physiological changes such as increased blood pressure . furthermore , medical news today defines anxiety as a general term for several disorders that cause nervousness , fear , apprehension , and worrying . these disorders affect how we feel and behave , and they can manifest real physical symptoms . mild anxiety is vague and unsettling , while severe anxiety can be extremely debilitating , having a serious impact on daily life . . we may worry about things that might happen or have a restless night of sleep . but , people with an anxiety problem worry so much that it affects their lives in negative ways . as stated above , anxiety is one of the most dominant psychological challenges associated with cancer . in another word , patients anxiety increases once they discover that they suffer from breast cancer , they may also become more anxious as cancer spreads or treatment becomes more intense . consequently , the level of anxiety experienced by one person with cancer may differ from the anxiety experienced by another . many anxiety cases associated with cancer were treated from this sickness , but others were not . therefore , psychologists need to give support and hope to breast cancer 's patients ; they need to help them cope with their feeling and pain . moreover , cancer 's patients may experience anxiety at different situation as while undergoing a screening test , waiting for the results , receiving a diagnosis , undergoing treatment , or anticipating a recurrence of their cancer . the anxiety associated with cancer may increase feelings of pain , interfere their ability to sleep , causes nausea and vomiting , and interfere with their quality of life . and for most patients , cancer requires facing uncertainty , worries about cancer treatment effects , fear of cancer progression and death , guilt , and spiritual questioning . a study by ashbury et al . , indicated that 77% of patients within 2 years of treatment recalled experiencing anxiety . on the other hand , anxiety after cancer diagnosis is not necessarily to be normal , understanding the nature of the anxiety in cancer patient populations is important because abnormal anxiety is troublesome the psychological wellbeing of the patients sherbourne and sheard . interviewing some breast cancer patients reported that their anxiety is characterized by a number of typical symptoms and signs such as shivering or tremor . the level of anxiety experienced by one person may differ from the level of anxiety experienced by another . anxiety in breast cancer patients is associated with death anxiety , fear of death as a result of their symptoms . according to pollak this type of anxiety is lower for people who have a positive sense of well - being and sense of meaning in life . in addition , evidence indicates that religious beliefs influence their level of anxiety [ figure 1 ] . many researchers have investigated the differences in anxiety level among women receiving different breast cancer treatments . recent study done by lim , indicated that anxiety presents in all treatment types for breast cancer . moreover , the anxiety level in women who underwent chemotherapy was highest before the first chemotherapy infusion , mediated by age and trait anxiety . this result confirms the needs for more research and studies on anxiety among breast cancer patients . in addition , the villadeguadarrama free article reported that the cancer - related anxiety may manifest as physical symptoms , such as rapid heartbeat , tightness in the chest or shortness of breath . the patient may also experience digestive symptoms , such as nausea , vomiting , or diarrhea . thinking about anxiety that can lead to physical symptoms , the overall symptoms of anxiety among cancer patients include : excessive , ongoing worry and tension , an unrealistic view of problems , restlessness or a feeling of being edgy , irritability , muscle tension , headaches , sweating , difficulty concentrating , nausea , the need to go to the bathroom frequently , tiredness , trouble falling or staying asleep , trembling , and being easily startled . symptoms of anxiety and depression have been found to be common in patients with cancer , frequently occurring around the time of diagnosis and during the period of chemotherapy . high - depression burden has been found at the time when patients experience adverse effects of chemotherapy . significant difference in psychological distress has been found depending on age , gender , and living situation , with those living alone experiencing higher levels of distress in a sample of icelandic cancer patients during the treatment period . a study of women at high risk for breast cancer showed significantly higher levels of depressive symptoms and feelings of emotional alienation than did a standardized test group , with 27% of this population defined as having a level of psychological distress justifying psychological counseling . a second study also documented an increase in distress among first - degree relatives of breast cancer patients . an israeli study found that first - degree relatives who have physical symptoms of breast pathology respond with more emotional distress than do women of normal risk in the same situation . patients with cancer face most of the stressors associated with diagnosis , illness , and treatment . cancer diagnosis and treatment brings changes in patients personal paths of life , in their daily activities , work , relationships , and family roles , and it is associated with a high level of patient psychological stress . if you are really caring for someone who is having the symptoms of anxiety , encourage him or her to get help . people are differing in the way they perceive their sickness and they are also differ in the way they cope with their anxiety . some are easily disturbed by their feeling of being anxious and other may take it as a challenge and they try to look for ways to overcome their feeling . there are many treatments for clinical depression including medicines , counseling , or a combination of both . therefore , the diagnosis of anxiety in cancer is usually complicated by the overlap of anxiety and sickness symptoms such as cancer - related fatigue and pain . many common symptoms of major depression were observed in cancer patients who do not endorse full depression symptoms . different studies showed that various kinds of coping strategies are used to overcome the anxiety in different types and stages of cancer . for instance , reuter et al . , stated that patients using ineffective coping strategies have higher levels of anxiety and depression and that benefiting from social support results in a marked reduction in the levels of anxiety and depression . moreover , the importance of social support to good mental health outcomes is well established . as well as the positive effect of good social support , the detrimental effect of negative interactions with significant others in the social environment has also become apparent in psychiatric and other conditions . eventually , people are unique in the way they perceive and cope with the anxiety , also the way one patient uses is different from the way other uses . undoubtedly , it is related to the personality , strength , faith , and hope patients is having toward their sickness . worry , tension , fear , and stress are interrelated to the anxiety and depression among them . patients are differing in the way they perceive their problem as well as the way they cope with the anxiety associate with it . the huge literatures on anxiety , its effects , coping , counseling , and mental health are evidence of the extensive belief that the way people cope is somehow linked to their belief and faith . in conclusion , anxiety has a great effect on the feeling of breast cancer patients and it leads to high level of coping mechanisms .

cancer is a disease wherein abnormal cells divide without control and are able to attack other tissues . most of the patients and their families face some degree of depression , anxiety , and fear when cancer becomes a part of their lives . they feel helpless and eager to find ways on how to get rid of it . the study focuses on anxiety among breast cancer patients . it aims at investigating cancer , its symptoms , and effects the disease has on the anxiety level of patients .

this article briefly reviews the need for emergency care in africa and barriers to its integration into health systems in the region . we describe an example of an emergency care initiative at muhimbili national hospital ( mnh ) in dar es salaam that has integrated a multi - cadre training program with development of a dedicated emergency department in the largest public hospital in tanzania . this article should be of interest to those concerned with prevention and treatment of acute illness in the sub - saharan region , and those with a particular interest in global emergency care and physician training initiatives in resource - limited settings . low - income countries suffer the highest rates of every category of injury from road traffic to drowning ; the highest rates of maternal death from acute complications of pregnancy ; and the highest rates of acute complications of communicable diseases including tuberculosis , malaria , and hiv . the rapidly growing prevalence of cardiovascular and diabetic disease has only increased the burden of acute illness , as patients with chronic disease in low - income countries also have the highest rates of mortality from acute complications . the disease control priorities in developing countries project estimates that 45 per cent of deaths and 36 per cent of disability - adjusted life years ( dalys ) in low- and middle - income countries could be addressed by the implementation of emergency care systems . while definitive specialty care will never be available at all times in all places , several studies suggest that emergency care is an effective and cost - effective means of secondary prevention : prioritizing an integrated approach to early resuscitation and stabilization of acutely ill patients greatly reduces the morbidity and mortality associated with a range of medical , surgical , pediatric , and obstetric conditions . in addition , many acute conditions can be mitigated by primary prevention ; disease surveillance at acute and emergency care facilities has been shown to increase preparedness and disease control capabilities . in general , analysis of acute disease patterns and the distribution of mortality across the acute care continuum is a crucial part of identifying the preventive and training initiatives most likely to improve outcomes . facility - based and pre - facility emergency care are essential components of a continuum ranging from prevention , through primary and chronic care , to inpatient critical care and surgery , and with resolution 60.22 the world health assembly has called for all its member states to consider formal , integrated emergency care systems ' . there are four foundational challenges to integrating emergency care into health systems in sub - saharan africa : the burden of acute disease in sub - saharan africa is severely under - documented . some data exist on the distribution of inpatient diagnoses in the region , but the actual range of acute presentations to health facilities is largely unknown . studies that compare police , hospital , and insurance records with other sources in low - income countries , for example , suggest that as few as 1 in 10 injuries may be documented in official counts . health - care facilities often lack an integrated approach to triage , resuscitation , and stabilization of acutely ill patients . even at a single small district hospital , acutely ill patients may be cared for by several different departments , depending on age , pregnancy status , and specific disease states . this vertical approach means that there is rarely a dedicated acute intake area staffed with nonrotating personnel who can be trained in resuscitation and stabilization.essential components of acute and emergency care have not been determined , and there is no consensus on how to define success . to date , there has been no systematic analysis of the acute care delivery systems most appropriate to these varied contexts . while there are scattered examples of successful interventions , little is known about what makes these programs effective or how others might replicate their success . impact is often quantified by the number of providers trained , rather than by any measure that incorporates quality or performance assessment.there is no current advocacy plan for placing emergency care on the global health agenda . despite the essential role of early resuscitation and stabilization in averting morbidity and mortality , emergency care is conspicuously absent from discussions of global health priority initiatives ( such as the millennium development goals and un consensus statements ) and large - scale global health funding strategies . the burden of acute disease in sub - saharan africa is severely under - documented . some data exist on the distribution of inpatient diagnoses in the region , but the actual range of acute presentations to health facilities is largely unknown . studies that compare police , hospital , and insurance records with other sources in low - income countries , for example , suggest that as few as 1 in 10 injuries may be documented in official counts . health - care facilities often lack an integrated approach to triage , resuscitation , and stabilization of acutely ill patients . even at a single small district hospital , acutely ill patients may be cared for by several different departments , depending on age , pregnancy status , and specific disease states . this vertical approach means that there is rarely a dedicated acute intake area staffed with nonrotating personnel who can be trained in resuscitation and stabilization . essential components of acute and emergency care have not been determined , and there is no consensus on how to define success . to date , there has been no systematic analysis of the acute care delivery systems most appropriate to these varied contexts . while there are scattered examples of successful interventions , little is known about what makes these programs effective or how others might replicate their success . impact is often quantified by the number of providers trained , rather than by any measure that incorporates quality or performance assessment . there is no current advocacy plan for placing emergency care on the global health agenda . despite the essential role of early resuscitation and stabilization in averting morbidity and mortality , emergency care is conspicuously absent from discussions of global health priority initiatives ( such as the millennium development goals and un consensus statements ) and large - scale global health funding strategies . despite these challenges , interest in emergency care training is rapidly expanding in africa , and there are planned or established emergency training programs for specialists in botswana , ethiopia , ghana , kenya , liberia , rwanda , south africa , sudan , tanzania , and uganda ; and for other provider cadres in ghana , tanzania , south africa , and uganda . we review one such initiative at mnh in dar es salaam , with a focus on the development of the emergency medicine residency program . the united republic of tanzania is designated as a low - income country with an estimated population of 46 million , a per capita income of around us$500 , and an average life expectancy of 51 . more than three quarters of the population lives in rural areas and a third lives below the poverty line . the infant mortality rate is around 51 per 1000 population , and there are approximately 3 doctors and 39 nurses and midwives per 100 000 population . in january 2010 , mnh opened tanzania 's first dedicated public emergency department via a public private partnership between tanzania 's ministry of health and social welfare ( mohsw ) , and abbott fund tanzania . the mnh emd is fully staffed with physicians ( registrars have completed medical school and internship , whereas residents have worked as registrars and are now pursuing specialty training ) and nurses 24 hours per day and currently sees 100130 patients daily , or approximately 40 000 patients per year . the department has full resuscitation capacity , and to add to the main hospital - operating suite a single - room operating theater dedicated to emergency cases has recently been opened within the emd . a business plan to ensure the department 's financial sustainability has just been finalized through an agreement between the hospital , the ministry , and abbott fund tanzania . nearly 40 per cent of mnh emd patients are female , 25 per cent are under 18 years of age , and 13 per cent are under 5 years of age ( table 1 ) . approximately , one quarter of all patients and 20 per cent of pediatric patients present with injuries . in adults , intracranial injuries are very common , and road traffic is a very common cause of injury in adults and children . other common diagnoses in adults and children include anemia and infections , particularly pneumonia in children . mental illness is also a top diagnosis in adults , with the majority of these patients presenting with psychosis . while the muhimbili emd project began as an infrastructure - development initiative , the necessity for a coordinated professional development strategy emerged early at the time the emd opened , there were no emergency physicians in tanzania and no dedicated emergency care nurse training programs . abbott fund tanzania initiated and supports an academic collaboration among united states ( us ) , south african , and canadian institutions to provide clinical training for nurses and physicians , with the goal of ensuring long - term sustainability of the muhimbili emd and training leaders to promote , disseminate , and sustain emergency care throughout tanzania . via this program , the emd providers are supported by international emergency nursing and emergency physician faculty year round . visiting faculty serve minimum 1-month stints , and there is an emergency medicine residency program director from the university of california san francisco onsite for the majority of the year ( author tr ) . the collaboration has developed site - specific training materials for a 10-module nursing curriculum , a 1-year registrar program , and a 3-year residency program . ninety per cent of nursing staff have completed the program ; 15 registrars have filed to complete the 1-year emergency care credentialing process ; and the first class of eight residents and second class of six residents will be joined by a third class this fall . the first residency cohort will graduate in july 2013 , and leadership of the program will transition to local faculty by the end of 2014 . the program at muhimbili is the nation 's only emergency medicine residency and is fully integrated into the muhimbili university of health and allied sciences ( muhas ) master of medicine ( mmed ) track . the initial curriculum was developed and implemented in a collaboration between the mnh head of department and the us emergency medicine faculty serving as abbott fund tanzania consultants . the curriculum was subsequently revised as part of a university - wide initiative to introduce a competency - based format . at the time of the curriculum development , the international federation for emergency medicine model curriculum had not been released , but we have since conducted a curricular review to ensure that our curriculum meets its guidelines . residents currently spend approximately half their time in the emd itself , and half on outside rotations in other clinical departments relevant to the practice of emergency medicine , such as surgery , obstetrics gynecology , cardiology , orthopedics , and pediatrics . the competency - based curriculum guides evaluation and allows resident performance to be appraised with reference to context - oriented proficiency , rather than traditional , discipline - based evaluation . this mode of targeted learning is particularly appropriate to the broad , setting specific practice of emergency medicine especially as a newly established field where the transmission of a culture of practice is as essential as the transmission of a body of scientific knowledge . the program is monitored by a combination of resident examinations and other provider performance metrics . each semester residents undergo a multidimensional evaluation including a written multiple - choice exam , a written essay exam , an oral case - based exam , and an observed clinical exam ( with case presentation ) on volunteer hospitalized patients . exams are proctored by local faculty and international outside examiners approved by the university academic senate . performance gaps are recorded and addressed in subsequent review sessions , and any gaps shared by multiple residents trigger review of the relevant component of the didactic and off - service curriculum . in addition , all doctors , nurses , and health attendants undergo regular professional performance audits that evaluate on - time attendance , room - response times , and documentation quality . the emd operations team is currently specifying metrics to evaluate throughput times , triage performance , and resuscitation process . emergency residents have also agreed to participate in a 10-year tracking study , which currently surveys their experiences on off - service rotations , and will continue to track their practice sites and the disease burden they encounter over the next decade . in general , medical trainees in tanzania face an extraordinary burden of disease and take on a degree of individual responsibility that rarely if ever falls on their us counterparts . the ratio of patients to providers is daunting , and the ratio of faculty to residents in most specialties is simply not adequate to the supervision and training needs . whereas this latter condition is mitigated to some extent by the visiting faculty in the muhimbili emergency program , other challenges result from the acuity of the specialty and its nascent status . managing dynamically evolving , unstable patients is new to most trainees as their previous clinical training was often in settings with limited resources for intervening with critically ill patients ( who usually died ) . for now , emergency residents also face the challenge of being taught and supervised by faculty who have not been trained within the same system . although they are guided by faculty in clinical decision making , ultimately the residents themselves are charged with developing the practice of regionally specific emergency care as they build their individual funds of knowledge . they frequently serve as advisors and teachers for each other , and are often in the position of researching a condition as they treat it in a patient . on rotations in other departments , besides mastering the clinical content of that respective specialty , the emergency residents must serve as ambassadors and educate others as to the nature of their own specialty and their learning goals . as the first and only emergency physicians in the country , they are called into institutional and political service to a degree that only a handful of their international colleagues will experience much later in their careers . even in terms of the scientific advancement of the specialty , because the burden of acute disease is so poorly documented in the region , many of these dissertations will be the first , and for a time the definitive literature on the topic . although the practice of emergency medicine has indeed evolved during the past 40 years into a coherent global discipline with a unique set of cognitive , technical , and administrative skills for managing acute illness or injuries , ' there remain notable differences in the scope of practice required of providers in low - income countries . simple translation of curricular content from other regions is unlikely to prepare trainees for what they will face during training and after graduation . acute care facilities serve to fill many gaps in the health systems of low - income countries , and providers at facilities distant from referral centers may be required to perform tasks considered beyond the scope of emergency specialists in other countries , such as basic obstetric , abdominal , and orthopedic surgeries . in this context , off - service rotations serve a different role than they might in areas where consultants are expected to be accessible within minutes to hours . as much as possible , informed by interviews with local providers who have worked in district and sub - district facilities , we have adapted the content of our curriculum to accommodate this reality ( either by emd - based or off - service training ) . in addition , to ensure that our curriculum is appropriate to the local disease burden , we are currently analyzing diagnostic and demographic data from the first 2 years of the mnh emd , and the 2013 curriculum will be adjusted based on these results and other emerging regional data . eventually , we hope that the results of our physician tracking study will also serve to inform future emergency care curricula at muhimbili and elsewhere . in an effort to advocate for the dissemination of high - quality emergency care in tanzania , muhimbili providers formed the emergency medicine association of tanzania ( emat ) , the first emergency medicine professional organization in the region . emat was ratified by the ministry in may of 2011 with the mandate to : promote and improve emergency care through teaching , research , and education.advocate for and provide emergency care across tanzania.collaborate with other emergency care research and educational organizations , regardless of location , to the benefit of emergency care in tanzania . advocate for and provide emergency care across tanzania . collaborate with other emergency care research and educational organizations , regardless of location , to the benefit of emergency care in tanzania . with the approval of the mohsw , a research team including the authors ( tr , hs , jm ) and other emat members has just completed site visits to every district hospital in tanzania to assess emergency care capacity . the results will serve to guide future emergency care planning and development , as well as prevention initiatives . other ongoing emat projects include expanding the registrar training program to accommodate visiting stints by district hospital providers ; onsite training of district hospital providers in regionally appropriate basic and advanced resuscitation and trauma management skills ; the development of a transport medicine skills course ; and a series of needs analysis studies that will facilitate the development of coordinated pre - hospital emergency services . in addition , emat aims to expand collaboration with other emergency provider training efforts in arusha , bugando , moshi , and zanzibar and has begun an exchange of site visits and planned collaborative training projects . to amplify and support its national efforts , emat has affiliated with the african federation for emergency medicine and the international federation for emergency medicine , and plans are underway to establish an east african emergency medicine federation that will allow resource - sharing and coordination of research and training efforts . the first east african regional meeting on emergency care sub - saharan africa faces a disproportionate burden of acute disease and many challenges to meeting the call of world health assembly resolution 60.22 for formal integrated emergency care systems . the abbott fund tanzania - supported program at mnh in dar es salaam represents one effort to integrate a multi - cadre professional development strategy with the establishment of a high - volume emergency care facility . the muhas - based residency will soon enroll its third class and will produce its first graduates in 2013 . their and others ' future efforts will benefit from greater national and regional coordination to allow multi - site research and surveillance and sharing of educational resources . emat has been formed to help expand regional and global advocacy efforts in collaboration with the african and international federations for emergency medicine .

even though sub - saharan africa faces a disproportionate burden of acute injury and illness , few clinical facilities are configured to take an integrated approach to resuscitation and stabilization . emergency care is a high - impact and cost - effective form of secondary prevention ; disease surveillance at facilities delivering acute and emergency care is essential to guide primary prevention . barriers to emergency care implementation in the region include limited documentation of the acute disease burden , a lack of consensus on regionally appropriate metrics to facilitate impact evaluation , and the lack of coordinated advocacy for acute disease prevention and emergency care . despite these challenges , interest in creating dedicated acute care facilities and emergency training programs is rapidly expanding in africa . we describe one such initiative at muhimbili national hospital in dar es salaam , with a focus on the development of the emergency medicine residency program .

sexually transmitted infections ( stis ) , including human immunodeficiency virus ( hiv ) , continue to present major health , social , and economic problem in the developing world , leading to considerable morbidity , mortality , and stigma . unprotected sex with an infected partner is by far the most important risk factor for sti / hiv infection . they show various trends in different parts of the country and constitute a major public health problem for both developing and developed countries . stis increases the risk of transmission of hiv infection causing immense need to understand the patterns of stis prevailing in the regions of a country for proper planning and implementation of sti control strategies . due to the lack of adequate laboratory infrastructure in the country , information regarding the profile of stis relies essentially on syndromic diagnosis . the availability of baseline information on the epidemiology of stis and other associated risk behaviors remain essential for the designing , implementing , and monitoring successful targeted interventions . to study the pattern of common stis and prevalence of hiv infection in patients attending the sti clinic of a tertiary care hospital in northern part of india using a syndromic approach . to study the pattern of common stis and prevalence of hiv infection in patients attending the sti clinic of a tertiary care hospital in northern part of india using a syndromic approach . a retrospective analysis of data collected from the clinical records of 2700 patients over a period of 21 months ( july 2012 to march 2014 ) was carried out . the data were collected from individuals attending the sti clinic at the skin and vd department of sms hospital , jaipur . detailed history , demographical data , and clinical features were recorded from all the patients . all patients were tested for hiv by elisa / rapid tests as recommended by the national aids control organization ( naco ) . stis were categorized in different syndromes as depicted by naco in the syndromic management of stis . the syndromes depicted by naco were urethral discharge , vaginal discharge , genital ulcer disease herpetic and nonherpetic ( gud - h and gud - nh ) , inguinal bubo , lower abdominal pain , scrotal swelling , etc . stis , which were not included in the syndromic management such as molluscum contagiosum , condyloma acuminata , and balanoposthitis were detected clinically . in the study population , 78% ( 2108/2700 ) were males , 22% ( 592/2700 ) were females with male to female ratio being 3.5:1 . the majority of patients attending sti clinic belonged to the age group 25 - 44 years 57.47% ( 1552/2701 ) , followed by the 20 - 24 years 20.7% ( 559/2700 ) , > 44 years 15.84% ( 428/2700 ) , and < 20 years 5.92% ( 161/2700 ) [ figure 1 ] . the overall most common sti was balanoposthitis ( 39.62% ) followed by genital herpes ( 17.5% ) , vaginal / cervical discharge ( 13.4% ) , genital molluscum ( 11.74% ) , genital warts ( 10.77% ) , gud - nh ( 4.59% ) , lower abdominal pain ( 2.66% ) , and urethral discharge ( 2.55% ) in decreasing order . balanoposthitis = bp ; genital ulcer disease herpetic = gud - h ; vaginal/ cervical discharge = vd / cd ; anogenital warts = ca ; molluscum contagiosum = mc ; genital ulcer disease nonherpetic = gud - nh ; lower abdominal pain = lap ; urethral discharge = ud ; scrotal swelling = ss ; inguinal bubo = ib among the male patients , balanoposthitis 50.75% ( 1070/2108 ) was the most common sti , followed by gud - h 19.87% ( 419/2108 ) , condyloma acuminata 12.28% ( 259/2108 ) , and molluscum contagiosum 9% ( 190/2108 ) . in the females , the most common sti was combination of cervical and vaginal discharge 61.04% ( 362/593 ) followed by molluscum contagiosum 20.2% ( 120/593 ) , gud - h 9.1% ( 54/593 ) , and condyloma acuminata 5.3% ( 32/593 ) . of the 4.59% ( 123/2700 ) patients of gud - nh , 62.6% ( 77/123 ) patients were rapid plasma reagin ( rpr ) positive with 56 males and 21 female patients . among the study population , 2.55% ( 69/2700 ) were found to be hiv - positive , in which 84% ( 58/69 ) were males and 16% ( 11/69 ) were females [ figures 3 and 4 ] . pie chart showing prevalence of hiv positivity among the sti patients pie chart showing male and female distribution among hiv positivity patients the pattern of stds differs from country to country and from region to region , especially in large countries such as india . they are responsible for a significant proportion of infertility in both sex , morbidity , economic loss to the family , and increased susceptibility to hiv infection . sti are a major contributor to fetal deaths , abortions , and the delivery of low birth weight babies . early diagnosis and appropriate treatment will definitely curb the transmission of hiv / aids . to achieve this , it is an approach where the health care providers diagnose and treat patients on the basis of signs and symptoms ( syndrome ) rather than specific stis . in the present study , males accounted for 78% cases , females were 22% ( m : f = 3.5:1 ) , which was almost similar to the north eastern study where males constituted 75.45% and females constituted 24.24% ( m : f = 3.09:1 ) . the most common sti was balanoposthitis ( 39.62% ) majority of which were fungal in origin , followed by genital herpes ( 17.5% ) , vaginal / cervical discharge ( 13.4% ) , genital molluscum ( 11.74% ) , genital warts ( 10.77% ) , gud - nh ( 4.59% ) , lower abdominal pain ( 2.66% ) , and urethral discharge ( 2.55% ) in decreasing order . this was in contrast to the north eastern study and study conducted at medical college trivandrum . in the eastern study , gud - h ( 38.1% ) was the most common , followed by vaginal / cervical discharge ( 18.6% ) , urethral discharge ( 13.8% ) , and molluscum contagiosum ( 4.7% ) while in study at trivandrum , the commonest std was syphilis , followed by herpes genitalis and condyloma acuminata . our data compared well with a previous study at a regional std center in new delhi were changing trends of the profile of stis and hiv seropositivity over a 15-year period were analyzed . the sti profile and hiv seropositivity were compared between 1990 and 1993 , 1994 - 1997 , 1998 - 2001 , and 2002 - 2004 . similar to our study , that is , the viral stis such as molluscum contagiosum , gud - h , condyloma acuminata , etc . , were much more prominent than the bacterial stis such as urethral discharge and gud - nh which were more prominent in our institution 10 years back . in the present study , the prevalence of hiv among sti patients was 2.55% ( males - 84% , females - 16% ) with male to female ratio being 3.5:1 which was in concordance with the epidemiological analysis of reported aids cases where disease is common in males than females , ratio being 3:1 . and with the national average ( 2.5% ) as per recent naco estimates . there was a wide variation for seropositivity for hiv among sti patients , 8.21% in zamzachin et al , study , 9.62% in jaiswal et al , study , 17.2% in saikia et al , study , and 10.3% in choudhry et al , study . this difference could be attributed to the high prevalence of hiv infection and intravenous drug abuse in the north - eastern part of india . rpr reactivity was seen in 12.8% of the total ulcerative sti patients , which is contrary to the reports of vora et al , and mewada et al , where the incidence of vdrl reactivity was 19.41% and 53.3% , respectively . hiv and stis are perfect examples of epidemiologic synergy as they are core transmitters of each other . the presence of multiple stis is a risk factor for increased rate of transmission of hiv . this can be controlled by promoting the strategies to reduce high - risk behavior , encouraging condom use , strengthening sti clinics and family health awareness programs , and imparting sex education and awareness regarding sti / hiv among the masses and vulnerable population . our study showed that the most common presenting complaint of the patients was balanoposthitis ( 39.62% ) followed by genital herpes ( 17.5% ) , vaginal / cervical discharge ( 13.4% ) , genital molluscum ( 11.74% ) , genital warts ( 10.77% ) , gud - nh ( 4.59% ) , lower abdominal pain ( 2.66% ) , and urethral discharge ( 2.55% ) in decreasing order . viral stis such as molluscum contagiosum , herpes genitalis , and condylomata acuminata are on the rise among sti / rti clinic attendees due to the occurrence of asymptomatic shedding , partial treatment or modified course of the bacterial stds , thereby leading to apparent reduction in the total number of cases of stds attending std clinics , as well as a decrease in the proportion of bacterial to viral stds .

background : sexually transmitted infections ( stis ) increase the risk of transmission of human immunodeficiency virus ( hiv ) infection causing immense need to understand the patterns of stis prevailing in the regions of a country for proper planning and implementation of sti control strategies . due to the lack of adequate laboratory infrastructure in the country , information regarding the profile of stis relies essentially on syndromic diagnosis.aims and objectives : to study the pattern of common stis and the prevalence of hiv infection in patients attending the sti clinic of a tertiary care hospital in northern part of india using a syndromic approach.materials and methods : a retrospective analysis of data collected from the clinical records of 2700 patients over a period of 21 months ( july 2012 to march 2014 ) was carried out at the skin and vd department of sms hospital , jaipur . detailed history , demographical data , and clinical features were recorded from all the patients . all patients were tested for hiv by elisa and rapid plasma reagin . stis were categorized in different syndromes as depicted by national aids control organization in the syndromic management of stis . the data collected was analyzed statistically . the proportions were calculated for various syndromes and disease prevalence.results:the overall most common sti was balanoposthitis , followed by genital herpes , vaginal / cervical discharge , molluscum contagiosum , genital warts , nonherpetic genital ulcer disease , lower abdominal pain , and urethral discharge in decreasing order . among the study population , 2.55% were found to be hiv-positive.conclusion:viral stis such as molluscum contagiosum , herpes genitalis , and condylomata acuminata are on the rise among sti / rti clinic attendees .

t1d is a chronic autoimmune disease where cd4 + and cd8 + t cells recognizing islet autoantigens are likely the mediators of selective destruction of pancreatic islet beta cells . although direct demonstration of the prominent role of t cells in the disease progression is provided only in animal models , the preclinical period of the disease in humans is marked by the presence of circulating islet - related autoantibodies to beta cell antigens including insulin , glutamic acid decarboxylase ( gad ) , isoforms gad65 and gad67 , the insulinoma - associated antigen ( ia2)/tyrosine phosphatase - like molecule , ia-2 or phogrin , and proinsulin . from the 1990s onwards several laboratories produced an increasing number of reports regarding the detection of t cells directed against these antigens in the peripheral blood . the first attempts employed [ h]thymidine incorporation / proliferation assays setup with pbmc of t1d patients ( at onset or long - standing ) and their high - risk subsequently , elispot assays were implemented for measuring cell - mediated immune ( cmi ) responses in t1d [ 35 ] and immunoblot assays . costimulatory anti - cd28 antibodies were shown to enhance autoreactive t cell responses to gad65 peptides in t1d patients , while , in a previous set of experiments , the expansion of the gad65 ( whole molecule ) reactive t cells was costimulation dependent in healthy controls , as opposed to t1d patients . nevertheless , t cell results were largely inconclusive because autoantigen - specific t cells in an in vitro expansion could indeed be grown both from patients and controls , as evidenced by 4 international workshops of the immunology diabetes society and by multicenter - blinded control trials , organized under the auspices of the immune tolerance network several explanations were put forward for justifying these difficulties including their low precursor frequency , the inhability to identify them from the vast excess of t cells , and their low to moderate affinity to self - antigens . cytotoxicity assays , set in vitro , offered proofs that t lymphocytes are potentially able to kill target cells also in vivo . to this end hla - a*0201 restricted cd8 + cytotoxic t lymphocytes , specific for a gad65 decapeptide ( 114123 ) , were first detected in pbmc of recent onset t1d patients and in high - risk ( ica+ ) individuals by using the classical [ cr ] release cytotoxicity assay . in more recent investigations elispot assays revealed ifn- production when pbmc from t1d patients were challenged with proinsulin peptides ( 3039 ; 3442 ; 4150 ) and the amyloid polypeptide precursor protein ( ppiapp513 ) peptides . the nonapeptides ppiapp917 , igrp152160 , and igrp215223 from the islet - specific glucose-6-phosphatase catalytic subunit related protein and nonapeptides 172180 and 482490 from the islet autoantigen ia-2 that would bind to and stabilize the hla - a2 molecules were also identified . mhc tetramer technology was initially introduced to target antigen - specific cd4 + t cells in patients with viral , bacterial infections , tumors . in reference to human autoimmunity class ii tetramers successfully detected gad65 , proinsulin , ia-2 , and preproinsulin reactive cd4 + t cells in pbmc of t1d patients , low percentages of cd4 + t cells autoreactive to gad65 , the melanocyte differentiation antigen tyrosinase and the testis tumor antigen ny - eso-1 ( epitope 120131 ) in pbmc of healthy individuals , and cd4 + gluten - specific t cells in pbmc of celiac disease patients . the hla class i tetramer technology successfully detected circulating cd8 + t lymphocytes autoreactive to the melan a autoantigen in patients with vitiligo , the pbc - e2 autoantigen in patients with primary biliary cirrhosis ( pbc ) , vimentin in patients who were heart transplanted , and insulin beta chain nonapeptide ( insb10 - 18 ) . in a recent investigation hla - a*0201 gad65 ( 114122 ) pentamers detected an increased percentage of autoreactive t cells in the cd45ro+ subset in t1d patients as compared with controls . in long - standing t1d patients who , after islet transplantation , have a loss of islet allograft and recurrent autoimmunity a high frequency of gad65-specific t cell clones was found within the expanded autoreactive memory t cell compartment . in the light of all the aforegoing , we attempted in this preliminary study to device a more sensitive methodology than those currently available for measuring cmi responses and , in particular , gad65 autoreactive t cells in t1d . preliminary data indicate that it is possible to implement an assay that still will require appropriate standardization before being used in large scale screening programs . in our protocol , after stimulation of the cells with the gad65 114 - 122 epitope , we successfully detected a percentage of cd8 + gad autoreactive t cells in a sample population including 15 t1d patients ( 9 newly diagnosed and 6 long - standing ) by using hla class i tetramers . 9 hla - a*0201 positive pediatric patients ( 4 males and 5 females , age of onset range 9.2 years to 16.4 years , mean 12.8 years ) were recruited from lazio region at the onset of t1d at the unit of pediatric endocrine autoimmune diseases at the children 's hospital bambino ges , rome ( table 1 ) . we also included 6 long - term hla - a*0201 positive pediatric t1d patients ( between 8 months to 4 years and 5 months after diagnosis ) . the control population was recruited at the blood transfusion center of our hospital including 10 hla - a*0201 positive nondiabetic healthy blood donors . they had no history of autoimmunity and no islet - related autoantibodies were detected in their serum . after obtaining informed consent from parents of childrens and normal controls , pbmc were separated by ficoll - hypaque ( histopaque , sigma - aldrich chemical c , st louis , mo , usa ) from 510 ml sodium - heparinized venous blood samples , washed twice in pbs , and then frozen down in liquid nitrogen . hla - a2 typing was initially performed by flow cytometry analysis and subsequently confirmed by standard allele - specific pcr . in the initial screening , 1 10 cells were incubated , for 30 minutes in ice , with an anti - hla - a2 mouse mab ( 1 : 10 dilution , one lambda , inc , canoga park , ca , usa ) . after washing by centrifugation at 1500 rpm for 5 minutes at room temperature ( rt ) in wash buffer [ 0,1% sodium azide , 2% fetal bovine serum ( fbs , hyclone , south logan , ut , usa ) in pbs ] , cells were resuspended in the residual volume ( approximately 50 l ) . 1 l of fluorescent- ( fitc- ) conjugated goat anti - mouse igg ( fc fragment specific antibody ) ( jackson immunoresearch laboratories inc , west grove , pa , usa ) was added . cells were then incubated in ice for 30 minutes in the dark , washed in wash buffer , and acquired for the analysis in a becton & dickinson facscalibur flow cytometer equipped with the cell quest software program . hla - a2 subtyping was carried out using a molecular system ( genovision inc . , west chester , pa , usa ) ; high - resolution dna - based hla typing of polymorphic class i loci hla - a , -b , and -c was also carried out according to a reverse line blot system . at the time of tetramer assay , cells were thawed and resuspended , at a density of 1 10/ml , in rpmi-1640 ( gibco / brl , invitrogen , gaithersburg , ca , usa ) , supplemented with 2 mmol / l l - glutamine , 100 g / ml penicillin / streptomycin , and 10% v / v fbs ; cells were then cultured in the presence of the gad65 peptide aa 114122 ( vmnillqyv ) at a concentration of 30 g / ml , for 4 days in 24-well round - bottomed plates ( falcon , labware bd biosciences , oxnard , ca , usa ) ( 1 10 cells / well ) . the gad65 nonapeptide had been selected for its high affinity binding to hla a*0201 in an hla peptide motif search database ( http://www-bimas.cit.nih.gov/molbio/hla_bind ) . in parallel experiments control cell cultures were set up by incubating pbmc from the same individual with il-2 ( 25 iu / ml , sigma ) for 4 days , in place of the gad65 peptide , in order to ensure that pbmc would live for the entire culture period prior to the flow cytometry analysis ( vide infra ) . at the end of the 4 days , pbmc , either stimulated with the gad65 peptide or incubated with il-2 , were washed in calcium - magnesium free dulbecco 's phosphate - buffered saline 1x ( euroclone , wethersby , west york , uk ) , by centrifugation at 1500 rpm for 5 minutes at rt . this washing was introduced to remove the excess of gad65 peptide and il-2 from the culture , so to maximally reduce the risk of nonspecific binding , when pbmc will subsequently be stained with labeled tetramers . the two sets of pbmc were cultured for additional two days with il-2 ( 25 iu / ml ) in the same medium ( rpmi-1640 , 2 mmol / l l - glutamine , 100 g / ml penicillin / streptomycin , 10% v / v fbs ) . phycoerythrin ( pe ) labeled tetramers were generated , using either the gad65 peptide ( vmnillqyv ) or the flu peptide ( gilgfvftl ) , known to have a high - affinity binding with hla - a*0201 ( purchased from proimmune limited , oxford , uk ) . on day 6 , approximately 5 10 cells , stimulated with the gad65 peptide or cultured with il-2 , were allocated for staining conditions . cells were washed by centrifugation at 1500 rpm for 5 minutes at rt in wash buffer ( 0.1% sodium azide , 2% fbs in pbs ) and resuspended in the residual volume ( approximately 50 l ) . 1 l of phycoerithrin ( pe ) labeled gad65 peptide ( vmnillqyv ) tetramer was added to each cell preparation and incubated in ice for 30 minutes in the dark , then washed in wash buffer . 1 l of mab anti - human cd8 ( fitc labelled ( becton & dickinson , pharmingen , san diego , ca , usa ) ) and 5 l of mab anti - human cd3 [ allophycocyanin ( apc ) labelled ( becton & dickinson ) ] were added for further staining the different cell preparations . in alternative for cd8 staining , 1 l of biotinylated mab anti - human cd8 ( 1 : 10 , becton dickinson ) was used followed by streptavidin cychrome ( cys5 ) conjugate ( 1 : 10 , southern biotechnology , birmingham , al , usa ) . cells were incubated in ice for at least 30 minutes in the dark , washed twice in wash buffer , and then immediately acquired for the analysis in a beckton & dickinson facscalibur flow cytometer and cellquest software program . in order to verify the sensitivity of the hla - a*0201 tetramer assay , the flu nonapeptide ( gilgfvftl ) was used to stimulate pbmc of one hla - a*0201 normal individual . this test was performed in order to verify that we could obtain an increased sensitivity of the flu tetramer staining after flu peptide stimulation . the staining with the flu tetramer was carried out either by direct assay ex vivo or after stimulation of the same pbmc first with the flu peptide ( 3.5 g / ml ) for 4 days followed , after washing the cells , by an incubation with il-2 for 2 days . a positive control of stimulation was introduced in all the experiments in order to prove that in vitro stimulation worked in all subjects , including those from whom no gad specific t cells were detected . the presence of flu tetramer positive cells was assessed in randomly selected samples by staining after flu peptide stimulation . in order to verify the specificity of the reactivity of the gad65 hla a*0201 tetramer in the detection of gad65 nonapeptide reactive t cells and as control of nonspecific binding , pbmc of randomly selected t1d patients and controls were cultured separately with the gad65 nonapeptide , the flu nonapeptide , and il-2 . the flu and gad65 nonapeptides were used at the same concentration ( 1030 g / ml ) . after 4 days , pbmc , cultured in the 3 different conditions , were washed and subsequently incubated with il-2 alone for an additional 2 days . cells were then recovered , washed , and stained with the gad65 nonapeptide hla a*0201 tetramers and analyzed by flow cytometry . differences in the percentages of cd3+/cd8+/gad65 reactive t cells between normal controls and t1d patients in each ( il-2 and gad65 peptide ) stimulated pbmc populations were assessed by mann - whitney - u test . variations in the response of pbmc , either treated with il-2 or stimulated with the gad65 peptide within each group ( i.e. , controls versus t1d patients ) , were evaluated by wilcoxon 's test . it is well known that hla class i molecules bind peptides 810 aminoacids long , but preferentially nonapeptides [ 35 , 36 ] . after database search for nonamers of the gad65 protein sequence ( p48320 , ncbi database ) , we selected peptide gad65 114 - 122 , on the basis of its high score of affinity binding ( 1080.239 ) to hla - a*0201 . direct staining ex vivo with hla a*0201 flu peptide tetramers of pbmc of a normal hla a*0201 positive individual detected 0.03% of cd3+/cd8+/flu reactive t cells . the percentage increased to 0.23% , when pbmc of the same individual were first stimulated for 4 days with the same flu peptide ( data not shown ) . normal controlsin pbmc of normal individuals no significant variations were found in the percentage of cd3/cd8/gad65 reactive t cells between pbmc cultured with either il-2 or the gad65 peptide ( mann - withney test versus pbmc il-2 treated p = .05 , figure 1 ) . in pbmc of normal individuals no significant variations were found in the percentage of cd3/cd8/gad65 reactive t cells between pbmc cultured with either il-2 or the gad65 peptide ( mann - withney test versus pbmc il-2 treated p = .05 , figure 1 ) . td patientsin pbmc of t1d patients the percentage of cd3+/cd8+/gad65 reactive t cells were significantly more pronounced in comparison to those registered with pbmc of controls after stimulation with the same gad65 peptide ( p = .001 t1d versus controls , figures 1 and 2 ) . figure 2 shows pbmc of a representative t1d patient treated with il-2 ( panel ( a ) , ( b ) , ( c ) ) and with gad65 peptide ( panel ( d ) , ( e ) , ( f ) ) . the analysis was conducted only in r1 region ( living cells as evidenced by morphological parameters ) and in the r2 region ( cd3+/cd8 + cells ) . the population of cd3+/cd8+/gad65 reactive cells was shown in panel ( f ) ( 4.04% of total cell population).il-2 treatment affected differentially the detection of cd3+/cd8+/gad65 reactive t cells in t1d patients versus control ( p = .07 t1d patients versus controls ) ( figure 1 ) . in pbmc of t1d patients the percentage of cd3+/cd8+/gad65 reactive t cells were significantly more pronounced in comparison to those registered with pbmc of controls after stimulation with the same gad65 peptide ( p = .001 t1d versus controls , figures 1 and 2 ) . figure 2 shows pbmc of a representative t1d patient treated with il-2 ( panel ( a ) , ( b ) , ( c ) ) and with gad65 peptide ( panel ( d ) , ( e ) , ( f ) ) . the analysis was conducted only in r1 region ( living cells as evidenced by morphological parameters ) and in the r2 region ( cd3+/cd8 + cells ) . the population of cd3+/cd8+/gad65 reactive cells was shown in panel ( f ) ( 4.04% of total cell population ) . il-2 treatment affected differentially the detection of cd3+/cd8+/gad65 reactive t cells in t1d patients versus control ( p = .07 t1d patients versus controls ) ( figure 1 ) . specificity of the gad65 peptide responsethe percentage of cd3+/cd8+/gad65 reactive t cells estimated in pbmc of single patients after gad65 peptide stimulation was higher than after flu peptide stimulation or when cultured with il-2 alone ( data not shown ) . with pbmc of the normal controls , the percentage of cd3/cd8/gad65 reactive t cells was comparable in the 3 stimulation conditions . in principle , we assume that the specific reactivity could be detectable in each single sample only after gad65 peptide stimulation . this test is an alternative to control the nonspecific binding of tetramer to t cells with the use of an irrelevant peptide . the percentage of cd3+/cd8+/gad65 reactive t cells estimated in pbmc of single patients after gad65 peptide stimulation was higher than after flu peptide stimulation or when cultured with il-2 alone ( data not shown ) . with pbmc of the normal controls , the percentage of cd3/cd8/gad65 reactive t cells was comparable in the 3 stimulation conditions . in principle , we assume that the specific reactivity could be detectable in each single sample only after gad65 peptide stimulation . this test is an alternative to control the nonspecific binding of tetramer to t cells with the use of an irrelevant peptide . tetramers containing autoantigenic peptides have clinical utility in autoimmunity for diagnostic and , potentially , therapeutic applications [ 1929 ] . previous studies [ 19 , 20 ] clearly demonstrated that mhc class ii tetramers can efficiently detect gad65 reactive cd4 + t cells in pbmc of t1d patients . gad65-specific hla dr0401-restricted clones could be even derived from a diabetic patient using tetramers as stimulating agent . these encouraging data prompted us to trace gad65 autoreactive t cells in t1d ; in the present investigation hla - a*0201 tetramers were therefore constructed with a natural peptide ( gad65 ( aa114122 ) , half - time disassociation 1080,239 ) . this peptide has been used in previous class i tetramer investigations [ 28 , 29 ] and now employed to establish a more sensitive assay . we could have chosen the gad65 decamer ( aa114123 ) but the peptide ranked low in the affinity binding scale ( half time disassociation , 35.012 ) . in addition , the terminal valine in position 122 seems to be relevant for the antigenic presentation of the motif , as the very low - affinity binding of the nonapeptide 115123 to hla a*0201 ( half time disassociation , 0.316 ) demonstrates . peptides for assembling tetramers employed in previous investigations in order to increase their affinity binding to the tetramer . the first is by direct ex vivo staining with labeled peptide / tetramer complexes ; in the second method pbmc are preincubated and stimulated with the peptide and then stained with the labeled peptide / tetramer or pentamer complexes [ 2529 ] . we have chosen the second approach , because we believe that preincubation with the peptide might induce a limited specific t cell expansion in vitro and , therefore , may increase the chance to awake autoreactive cd8 + t cells . novel steps in the experimental procedure were designed as follows : ( 1 ) a reduced period of antigen - specific cell expansion ( 4 days rather than 21 days , 28 days , 10 days , time course selected in other hla class i tetramer assays ) ; ( 2 ) an intermediate washing step after cells were stimulated with the gad65 nonapeptide ; ( 3 ) a 2-day incubation with il-2 in the absence of the gad65 nonapeptide ; and ( 4 ) a final washing step , before staining the cells with the labeled tetramers . in addition , we relied only on the autologous apcs , present in the pool of the pbmc , not prepulsed with the nonapeptide , as it was done in previous assays , and we set up parallel pbmc cultures either with il-2 alone , an internal cell culture control . the short period of peptide stimulation has the advantage that cultures are terminated before clonal expansion and/or in vitro we need also to underline that the possibility of testing frozen / defrosted pbmc is of paramount importance when planning large retrospective epidemiological investigations . by applying these novel experimental conditions , the recovery of cd3+/cd8+/gad65 reactive t cells in t1d patients ranged from 0.5% to 15.6% after gad65 peptide stimulation of pbmc . to our knowledge , this is the highest percentage of autoreactive cd8 t cells ever captured by hla class i tetramers in pbmc of patients with a human autoimmune disease ( 0,1% to 1% in vitiligo 4,2% in heart transplantation ; up to 0.4% of cd8 + cd45ro+ t cells in newly diagnosed t1d patients ; between 2.6% and 8.3% within the ki-67+cd8 + cell fraction ( 1.65% + /0.25% of the total cd8 + lymphocytes ) in islet transplanted t1d patients ) . this can be achieved by testing pbmc of a large number of t1d patients , normal controls , and especially prediabetic high - risk individuals . a valid statistical evaluation of results will help to establish an appropriate cutoff value of positivity in the assay .

type 1 diabetes ( t1d ) is an autoimmune disease , in which pancreatic cells are destroyed in genetically predisposed individuals . while the direct contribution of autoantibodies to the disease pathogenesis is controversial , it is generally recognised that the mechanism of cell destruction is mediated by autoreactive t cells that had escaped the thymic selection . we aimed to design a method to detect circulating cd8 + t cells autoreactive against an epitope of the glutamic acid decarboxylase autoantigen , isoform 65 ( gad65 ) ex vivo in t1d patients by using hla class i tetramers . low frequencies of gad65 peptide - specific cd8 + cytotoxic t lymphocytes were detected in peripheral blood lymphocytes ( pbmc ) of normal controls after gad65 peptide - specific stimulation . conversely , their frequencies were significantly higher than in controls in pbmc of t1d patients after gad65 peptide stimulation . these preliminary data are encouraging in order to develop a reliable assay to be employed in large - scale screening studies .

small non - coding rnas are transcribed into mrna but remain untranslated in eukaryotic cells . they include sirna ( small interfering rna ) , mirna ( microrna ) , pirna ( piwi - interacting rna ) and snorna ( small nucleolar rna ) . mirnas are a class of multifunctional singled - stranded small rna which are ~20 nt in length and regulate the stability or translational efficiency of targeted messenger rna depending on the base - pairing complementarity between the mirna and its target mrna [ 1 , 2 ] . although over 1,000 mirna sequences have been identified from the tissues or cells of human origin and other species , as many as 1,000 to 10,000 mirnas per genome have been predicted [ 3 , 4 ] . mirnas regulate a broad range of biological processes including timing of development , cell cycle progression , stem cell self - renewal , differentiation , cancer initiation , cancer cell proliferation , metastasis and apoptosis [ 511 ] . cancer is caused by multiple processes including uncontrolled cellular proliferation and inappropriate survival of apoptotic cells . many regulatory factors switch on or off genes that govern cell division and direct cellular proliferation . mirnas regulate gene expression and play important roles in the onset and progression of tumorigenesis . emerging evidence demonstrates the involvement of mirna in mammary gland tumorigenesis , functioning either as tumor suppressors or oncogenes . although the current treatment of radiation therapy , chemotherapy and hormone therapy slow mammary gland tumor growth , prolong survival and improve the quality of patients life , metastatic breast cancer still remains incurable due to our limited understanding of the molecular mechanisms through which tumorigenesis and metastasis occur . as small non - coding rnas regulate gene expression and tumorigenesis , they may represent a novel cancer therapy . unlike mrna , mirnas are transcribed but never translated . some mirnas are transcribed from non - coding regions between genes , deriving from independent transcription unit . other mirnas are transcribed together with coding mrnas from the coding region of the genome , deriving from the introns of gene transcripts [ 13 , 14 ] . mirna gene copy number gain / loss and mirna gene mutation have been observed in breast cancer resulting in the aberrant expression of mirna . the first study about the altered expression of mirnas in human breast cancer patients and human breast cancer cell lines was reported in 2005 by lorio et al . , in which 29 mirnas were identified with aberrant expression based on microarray and northern blot analysis of 76 breast tumor samples and 14 human breast cell lines . zhang and colleagues analyzed 283 human mirna genes on 55 human breast primary tumors and 18 human breast cancer cell lines using array - based comparative genomic hybridization . the results demonstrated a high frequency ( ~72.8% ) of gene copy number abnormality in mirna - containing regions in human breast cancer . wang et al . collected 68 patients with newly diagnosed breast cancer and examined the expression of selected mirnas in tumor and adjacent non - tumor tissues . mir-21 , mir-106a and mir-155 were significantly over - expressed in the tumor specimens compared with normal controls , whereas mir-126 , mir-199a and mir-335 were significantly decreased in expression in the tumor samples . our studies of the mir-17 - 92 cluster demonstrated decreased expression of mir-17/20 in human breast cancer specimens compared with matching normal breast tissue from the same patient . subsequent analysis identified reduced mir-17/20 expression in node - positive compared with node - negative breast cancers and demonstrated that mir-17/20 inhibited breast cancer cell migration and invasion via a heterotypic signaling . although the tendency for a global decrease of mirna expression in human cancers originally suggested a general tumor suppressor function of mirnas , subsequent studies showing the aberrant expression of specific mirnas in breast cancer suggest mirna - specific roles in breast cancer onset and progression . many distinct mirnas have been shown to regulate breast cancer cell proliferation , apoptosis , cancer stem cell expansion , and tumorigenesis . mirna may function as either tumor suppressors or oncogenes depending on the cell type , culture conditions , target genes and pathway . the involvement of mirna in mammary gland tumorigenesis has been reviewed recently [ 21 , 22 ] . le et al . described the expression pattern and regulatory network of key mirnas in breast cancer , including let-7 , mir-34 , mir-125 , mir-200 family , mir-205 , mir-21 , mir-10 and the mir-17 - 92 cluster . adams et al . reviewed the mirna regulation of estrogen signaling pathway and erbb2/her signaling pathway in breast cancer . the understanding of how mirnas are involved in breast cancer through regulating the cell cycle remains rudimentary . herein we summarize the recent literature and research progress on the mechanism by which mirnas regulate the breast cancer cell cycle and cellular proliferation ( fig . 1mirna regulation of mammary gland tumorigenesis in control of the cell cycle . through targeting different genes and different cyclin / cdk complexes , mir-17/20 and let-7 regulate the g1-s transition ; mir-21 and mir-27a regulate the g2-m checkpoint mirna regulation of mammary gland tumorigenesis in control of the cell cycle . through targeting different genes and different cyclin / cdk complexes , mir-17/20 and let-7 regulate the g1-s transition ; mir-21 and mir-27a regulate the g2-m checkpoint cyclin d1 is either overexpressed or amplified in ~50% of breast cancer . the abundance of cyclin d1 is rate - limiting in breast cancer cellular proliferation and g1-s phase transition [ 23 , 24 ] . in addition , cyclin d1 is a critical downstream target of erbb2- , ras- and -catenin- induced breast cancers , and is sufficient for the induction of mammary tumors when targeted to the mammary gland of mice . antisense inhibition of cyclin d1 expression in vivo suppressed the growth of neut - transformed mammary adenocarcinoma cells in nude mice . conserved sequences of the cyclin d1 3utr contain potential binding sites for multiple mirnas including mir-17/20/106 , mir-15/16 , mir-23 and let-7 . mir-17/20 binds the cyclin d1 3utr , inhibiting the expression of cyclin d1 , resulting in cell cycle arrest at the g1 phase and suppression of mcf-7 cell proliferation [ 18 , 26 ] . the regulation of cyclin d1 expression by mir-17 - 92 , as well as mir-15/16 , was confirmed by deshpande et al . . the let-7 family functions as a tumor suppressor in a variety of cancers including lung , colon , ovarian and breast cancer . schultz et al . demonstrated the downregulation of cyclin d1 by mirna let-7 in control of cancer cell growth . the regulation of cyclin d1 by mirna is likely of broad importance as cyclin d1 encodes the regulatory subunit of a kinase that phosphorylates and inactivates the prb family proteins to inhibit dna synthesis , and phosphorylates nuclear respiratory factor 1 ( nrf-1 ) to inhibit mitochondria biogenesis [ 32 , 33 ] . furthermore , cyclin d1 promotes breast epithelial cell angiogenesis and migration , and promotes chromosomal instability which in turn contributes to tumorigenesis . the mir-221/222 cluster regulates the cell cycle , cell growth and epithelial - to - mesenchymal transition ( emt ) in breast cancer . mir-221/222 inhibited p27 and p57 abundance , facilitating g1-s phase transition , thereby promoting cancer cell proliferation [ 36 , 37 ] . moreover , mir-221/222 may contribute to the aggressive clinical behavior of basal - like breast cancers . the breast cancer basal - like subtype - specific mirnas , mir-221 and mir-222 , promote emt in breast cancer by targeting trps1 ( trichorhinophalangeal syndrome type 1 ) which inhibits emt by repressing zeb2 expression . mir-221 and/or mir-222 expression in mcf-7 and t47d breast cancer cells decreased er expression associated with tamoxifen resistance . the onco - mirna mir-21 is overexpressed in a wide variety of cancers including breast cancer [ 40 , 41 ] . mir-21 induced cellular proliferation , migration , invasion , emt , cancer stem cell characteristics and chemotherapy resistance in human breast cancer [ 42 , 43 ] . high mir-21 level is associated with poor prognosis , advanced stage , positive lymph node status and reduced survival time in breast cancer . mir-21 promotes mcf-7 cellular proliferation in part through inhibiting the expression of a tumor suppressor gene programmed cell death 4 ( pdcd4 ) . in colon cancer , mir-21 participates in a dna damage - induced g2-m checkpoint through suppressing the cell cycle regulator cdc25a . a recent report demonstrated the mir-21 regulates the cell cycle through targeting cdc25a in mcf-7 breast cancer cells . with a potential anti - cancer chemical 3,3-diindolylmethane treatment , mir-27a expression is upregulated in human breast cancer cell lines . in mda - mb-231 cells , mir-27a negatively regulated the zinc finger zbtb10 gene and myt-1 , thereby promoting breast cancer cell proliferation . mir-27a suppressed myt-1 , increased cdc2/cyclin b activity and promoted the g2-m checkpoint in mda - mb-231 cells . thus , distinct mirnas affect key genetic targets that govern distinct cell - cycle checkpoints including cyclins , cdks , cdk inhibitors and the g2-m regulation apparatus . in addition to cell - cycle control , breast tumor onset and progression and breast tumor stem cells are also regulated by distinct mirnas . cancer stem cells ( cscs ) are characterized by their self - renewal capacity , an ability to differentiate into non - tumorigenic cell progeny , and their ability to seed tumors when transplanted into animal hosts . cell surface markers such as cd44 , cd24 , cd133 , epithelial - specific antigen and aldehyde dehydrogenase-1 are frequently used to isolate and enrich cscs . the involvement of mirnas in regulating tumor formation by cscs or tumor - initiating cells ( t - ic ) has been widely investigated . let-7 expression is very low to undetectable level in embryonic stem cells ( es cells ) and increases with differentiation . a comparison of mirna expression between breast t - ic and non - t - ic demonstrated reduced let-7 expression in t - ic and increased abundance with differentiation . transduction of breast cscs with let-7 reduced the proportion of undifferentiated cells , inhibited cell proliferation , mammosphere formation , and tumor formation in vivo . clarke and colleagues identified 37 mirnas which were differentially expressed between human breast cscs ( cd44cd24lineage ) and lineage nontumorigenic breast cancer cells . the mir-200 family members were downregulated in human breast cscs and normal mammary stem / progenitor cells . expression of mir-200 inhibited breast cancer stem cell expansion in vitro , and suppressed the tumor formation ability of human breast cancer stem cell in vivo . ectopic mir-34c expression reduced breast t - ics self - renewal , inhibited emt and suppressed tumor cell invasiveness via silencing notch4 . zhu et al . found the reduced mir-128 expression in human breast t - ic was accompanied by bmi-1 and abcc5 overexpression , and associated with chemotherapeutic resistance and poor survival . enforced mir-128 expression increased the sensitivity of breast cancer cells to doxorubicin - induced apoptosis and dna damage . emerging evidence has demonstrated the importance of cscs in cancer initiation , cancer metastasis and drug resistance . cscs are believed to be one of the most promising targets for cure of cancer . the discovery that non - coding rnas regulate cscs widens our understanding of cscs , and may provide potential novel strategies for breast cancer therapy . deletion of chromosome 6q , including region 6q14-q16 , is frequently observed in breast cancer . the small non - coding snorna u50 is a candidate tumor suppressor gene in the 6q14 - 16 region , playing a role in the development and/or progression of breast cancer . genomic deletion and transcriptional downregulation of snorna u50 was detected in breast cancer cell lines . re - expression of snorna u50 inhibited colony formation of the human breast cancer cells hs578 t and mda - mb-231 . pirnas are small non - coding rna that form rna - protein complexes through interactions with piwi proteins . pirna was initially discovered in germ line cells , and considered as germ line - specific small rnas . emerging evidence indicates that pirna expression occurs in somatic cells [ 57 , 58 ] and piwil2 expression has been identified in human breast cancer cells . high - throughput deep sequencing identified a group of small rnas matching pirna sequences in human breast cancer tissues and breast cancer cell lines . the study of these non - coding small rnas in human cancer is just starting . the identification of the expression signature of these non - coding small rnas in breast cancer subtypes , and an understanding of their functional significance to oncogene expression , tumor initiation and tumor cell metastasis may shed important new perspectives on the role of these non - coding small rnas in breast cancer . small non - coding rna - based diagnostic and therapeutic applications for human cancer are expected in the near future . although tumor - targeted delivery and local administration are still major challenges to the practical application of gene therapy for cancer , mirna - based cancer therapeutic approaches are being established and tested in animal models . synthetic mirna mimics or mirna expression vectors have been successfully applied to restore or overexpress mirna in vitro . chemically modified lna anti - sense mirna inhibitor and other approaches have been used to block mirna function in cells . kim et al . recently reported significant anti - tumor effect of virus - mediated delivery of mir-145 combined with 5-fu to treat breast cancer . intranasal delivery of let-7 and intravenous delivery of mir-34a mimics for non - small - cell lung cancer treatment and a virus - mediated delivery of mir-26a for liver cancer treatment in mouse model demonstrate the promise of mirna for treatment of cancer . dysregulated expression of mirnas has implicated components of the non - coding genome as either oncogenes or tumor suppressors of breast cancer . experimental evidence has shown specific mirnas regulating the initiation , progression , metastasis and drug resistance of breast cancer via control of the cell cycle , altering cellular proliferation , altering cellular apoptosis and/or controlling the population of tumor stem cells . dysregulated mirna expression has also been observed in cancer associated fibroblasts ( caf ) and in the systemic circulation [ 65 , 66 ] . the circulating mirnas have the potential to serve as novel diagnostic and prognostic biomarkers for breast cancer . a specific subset of dysregulated mirnas in breast cancer cells may serve as targets for gene therapy either alone or as an adjuvant treatment to current clinical protocols for breast cancer patients .

small non - coding rnas include sirna , mirna , pirna and snorna . the involvement of mirnas in the regulation of mammary gland tumorigenesis has been widely studied while the role for other small non - coding rnas remains unclear . here we summarize the involvement of mirna in breast cancer onset and progression through regulating the cell cycle and cellular proliferation . the regulation of breast cancer stem cells and tumor regeneration by mirna is reviewed . in addition , the emerging evidence demonstrating the involvement of pirna and snorna in breast cancer is briefly described .

turner syndrome ( ts ) is caused by partial or complete monosomy of the x chromosome . only a few cases of ts with ischemic stroke have been reported [ 2 , 3 , 4 , 5 ] . in previously reported cases , various arteriopathies affecting the cerebral arteries such as fibromuscular dysplasia , hypoplasia , moyamoya syndrome , and , we report a case of ts with multiple embolic infarcts caused by a cryptogenic embolism . a 28-year - old woman with ts was referred to our hospital because of abnormal findings on brain magnetic resonance imaging ( mri ) . she was diagnosed with ts [ karyotype : 45 , x/46 , x , + mar ] at the age of 16 years due to amenorrhea . she denied alcohol , tobacco , or drug use and had no family history of neurologic disorders . the patient was afebrile with a blood pressure of 180/130 mm hg and a heart rate of 89 beats / min . she was obese with a body mass index of 27.3 kg / m ( 160 cm height , 70 kg weight ) . she visited hospital due to sudden - onset severe headache with high blood pressure ( 240/140 mm hg ) . she underwent brain computed tomography and mri at the referring hospital for differential diagnosis of thunderclap headache including hypertensive intracranial hemorrhage , subarachnoid hemorrhage , arterial dissection , or reversible cerebral vasoconstriction syndrome . brain computed tomography was unremarkable , but diffusion - weighted imaging showed restricted diffusion in multiple lesions involving multiple vascular territories ( fig 1a ) . cerebral magnetic resonance angiography ( mra ) ( fig 1b ) and carotid sonography demonstrated no extracranial or intracranial arterial stenosis . laboratory tests revealed diabetes mellitus ( fasting plasma glucose 219 mg / dl , hemoglobin a1c 11.0% ) and hyperlipidemia ( low - density lipoprotein 189 mg / dl ) . transthoracic echocardiography revealed normal valvular structure and function with a normal left ventricular ejection fraction . patent foramen ovale , atrial septal aneurysm , or any aortic pathologies including aortic atheroma were not detected in transesophageal echocardiography . the results of the hypercoagulability panel , vasculitis panel , and cerebrospinal fluid examination were all negative . aspirin was administered , and medications for hypertension , hyperlipidemia , and diabetes were also started . within few hours after blood pressure control , the patient was discharged from the hospital without any symptoms of stroke , and recurrence was not observed in the 2 years after discharge . cerebrovascular arteriopathies such as fibromuscular dysplasia , congenital hypoplasia , moyamoya syndrome , and premature atherosclerosis have been shown to be causes of ischemic stroke in previous case reports . in this case furthermore , our patient showed multiple lesions in different vascular territories that were strongly suggestive of embolic stroke . atrial fibrillation ( af ) could also be considered a potential source of embolic stroke in ts , as p - wave dispersion , a potential substrate for af , is increased in ts . the classical risk factors for developing af include hypertension , diabetes mellitus , and valvular disease , and these are more common in ts than in the general population . however , we could not completely exclude the possibility of paroxysmal af , as holter monitoring has a low yield for af detection ( 35% ) . other possible cardio - aortic sources of embolism include intracardiac shunts , thrombi , calcifications / vegetations in the mitral valve , or aortic atheroma plaque . we sought to identify possible cardio - aortic sources on transthoracic echocardiography , transesophageal echocardiography , and transcranial doppler shunt test ; however , all tests were negative . some case reports have described deep venous thrombosis and portal vein thrombosis in ts [ 11 , 12 ] . these factors include fibrinogen , d - dimer , factor viii , von willebrand factor , and proteins c and s . we also considered the possibility of hypercoagulability as a cause of stroke in this case ; however , levels of d - dimer , fibrinogen , protein c and s were normal . the embolic source of stroke in our patient remains unclear ; however , this is the first report of a ts patient with an embolic stroke pattern . our case suggests another possible mechanism of stroke in ts that is different from previous case reports . ischemic stroke in ts could be due to embolism as well as various cerebral arteriopathies . further investigation is needed to more fully understand the various mechanisms of ischemic stroke in ts . ischemic stroke in ts could be due to embolism as well as various cerebral arteriopathies , as documented in previous reports . further studies are needed to determine the extent of the underlying mechanisms of ischemic stroke in ts . this case report has been approved by the institutional review board of the inha university hospital .

only a few cases of turner syndrome ( ts ) with ischemic stroke have been reported . various arteriopathies of the cerebral arteries , including fibromuscular dysplasia , congenital hypoplasia , moyamoya syndrome , and premature atherosclerosis have been assumed to be the cause of ischemic stroke in ts . there has been no case report of a ts patient presenting with an embolic stroke pattern without any cerebral arteriopathy . a 28-year - old woman with ts was referred to our hospital because of abnormal brain magnetic resonance imaging ( mri ) findings . she underwent brain mri at the referring hospital because she experienced sudden - onset diffuse headache . diffusion - weighted imaging revealed multiple acute embolic infarcts in different vascular territories . intracranial and extracranial arterial disease was not detected on cerebral magnetic resonance angiography and carotid sonography . embolic source workups , including transthoracic and transesophageal echocardiography , holter monitoring , and transcranial doppler shunt study , were all negative . hypercoagulability and vasculitis panels were also negative . our patient was diagnosed with cryptogenic embolic stroke . this is the first report of a ts patient with an embolic stroke pattern . our case shows that ischemic stroke in ts could be due to embolism as well as the various cerebral arteriopathies documented in previous reports .

statins or 3-hydroxy - methylglutaryl coenzyme - a ( hmg - coa ) reductase inhibitors are a potent class of cholesterol synthesis inhibitors and an established therapy for primary and secondary prevention of coronary artery diseases . several large studies confirmed reduction in cardiac mortality and morbidity through the treatment with statins . these clinical benefits were reported to be majorly due to lowering of low - density lipoprotein cholesterol ( ldl - c ) . a linear relationship between ldl - c and cardiovascular ( cv ) event rates the study showed landmark reduction in the rate of major coronary event by over 50% after use of statins . this reduction was associated with 2-mmol ( 78 mg / dl ) reduction in ldl - c levels . later several other studies such as the cholesterol treatment trialists meta - analyses , the treating to new target , heart progression study ( hps ) , cholesterol and recurrent events ( care ) trial and long - term intervention with pravastatin in ischaemic disease ( lipid ) studies also confirmed the findings of 4s . accumulating evidence have suggested that statins , in addition to ldl - c lowering , exhibited properties of plaque stabilization and endothelial homeostasis ; anti - inflammatory , antioxidant , anti - proliferative and immunomodulatory effects ; normalization of sympathetic outflow ; and prevention of platelet aggregation . pleion , which means more , and tropy meaning response or stimuli . though pleiotropic effects are defined as a single gene affecting multiple systems or determining more than one phenotype , lately statin pleiotropy is referred as statins exerting multiple pharmacological activities . the pleiotropic effect is the class effect of statins and is exhibited by all statins acting on non - hepatic tissues , including hydrophilic pravastatin , which penetrates poorly through the cell membranes of non - hepatic cells . the rapidity with which statins produce beneficial effects has suggested involvement of mechanisms other than its lipid - lowering activities . over the last more than 10 years , several clinical trials indicated that the benefits of statins extend beyond their effects on cholesterol , supporting the theory of statin pleiotropy [ table 1 ] . clinical studies elucidating statin pleiotropy the program on the surgical control of the hyperlipidemias ( posch ) study reported the benefits of cholesterol lowering after partial ileal bypass surgery after almost 10 years , whereas most statin trials showed benefits at much earlier time points ( e.g. , within 5 years ) . comparing benefits after 5 years for all lipid - lowering trials , non - statin trials no longer were aligned to the same cholesterol : mortality risk reduction slope , whereas all statin trials aligned to this slope . in the posch study , that the 34% reduction in ldl - c within the first 3 months post ileal surgery was although earlier unnoticed could be explained by additional non - lipid effects of statins . in the west of scotland coronary prevention study study , cox regression analyses of data highlighted that pravastatin treatment decreased ldl - c down to 24% and further reducing ldl - c levels did not show additive any benefit , and such phenomenal reduction in coronary heart disease risk was surprising in the pravastatin - treated group as such benefits could not be expected with decrease in ldl - c within this range . this strongly supported non - lipid - lowering theory of statins . in another statin trial with 3000 subjects with unstable angina or non - q - wave acute myocardial infarction , aggressive treatment with statins effectively reduced recurrent ischemic events within 16 weeks after acute coronary events , reduced serum ldl- by 40% and relative risk by 16% . according to the investigators , this period was too short to expect changes in lesion size and plaque stability of such magnitude . therefore , it was assumed that these early changes were due to the direct cellular effects on the vascular wall and improvement of endothelial function with statins . other studies that suggested the involvement of a non - lipid - lowering pleiotropic mechanism of statins were large prospective trials , such as the hps and anglo - scandinavian cardiac outcomes ( ascot ) trials . these studies demonstrated that relative risk reduction with statin treatment was independent of baseline lipid values . the hps trial reported that absolute benefits were related directly to the individual vascular risk of the subjects . patients with low hdl - c and high vascular risk , such as subjects with diabetes , significantly benefited from statin therapy possibly through lipid - independent mechanisms . in 2003 , , reported a randomised trial with hypertensive subjects ( n = 10,305 ) with > 3 cv risk factors . the subjects received either atorvastatin 10 mg / day or placebo and their total cholesterol level was measured to be < 250 mg / dl . the study was halted after a median follow - up of 3.3 years as there was a 36% reduction in the risk of myocardial infarction and fatal coronary heart disease . these very early benefits with large reduction in coronary heart disease events were incredible and highlighted the involvement of mechanisms other than the lipid - lowering effects of stains . this was supported by earlier studies such as the myocardial ischaemia reduction with aggressive cholesterol lowering study ( miracl ) and the pravastatin or atorvastatin evaluation and infection therapy trial ( prove - it ) where early clinical benefits were seen in subjects with coronary heart disease . this rapid time course of event reduction in high - risk subjects with recurrent coronary ischemia suggested non - lipid - lowering effects . in 2006 , vyas et al . , reported over 35% reduced risk of sudden cardiac death or ventricular arrhythmia development due to the plaque - stabilizing effect of statins , increased coronary blood flow and microvascular myocardial perfusion . in 2004 , cannon et al . , reported the prove - it trial , comparing the effect of intensive therapy with atrovastatin ( 80 mg ) daily versus moderate therapy with pravastatin ( 40 mg / day ) in 4162 subjects with acute coronary syndrome . the authors reported that subjects were benefitted from intensive therapy than standard therapy , leading to separation of event curves within 3 weeks of treatment . also , a decrease in hazard ratio was apparent as early as 6 months from treatment . in a sub - study of the prove - it trial , gibson et al . , in the year 2009 reported the benefit of intensive statin therapy in clinical outcomes in subjects undergoing percutaneous coronary intervention for acute coronary syndrome . in 2007 , patti et al . , reported a randomized trial where 12-h pretreatment with 40 mg / day atorvastatin prior to coronary angioplasty showed improvement in clinical outcomes in subjects with acute coronary syndrome no reduction ldl - c in levels are commonly observed , suggested that the anti - inflammatory and pleiotropic properties of statins may be of clinical importance . in 2009 , an observational study showed that statin users hospitalized for acute coronary syndrome were not only at a lower risk of developing in - hospital atrial fibrillation but also had a significantly lower risk of developing ventricular arrhythmias , cardiac arrest and/or death . in these subjects , intensive statin therapy had reduced the risk of major adverse cv events compared with moderate - dose statin therapy . the reduction in the incidence of target vessel revascularization was independent of ldl - c and c - reactive protein ( crp ) lowering , suggesting the statin pleiotropic effect of high - dose therapy . scientists have been intrigued with the magnitude- and timing - reduced cv events in some clinical trials and asserted that these rapid effects mediated by statins could not be explained solely on the basis of the lipid - lowering mechanism of statins . high - sensitivity c - reactive protein ( hscrp ) is a non - specific marker of inflammatory diseases , and has been recognized as an independent risk predictor of cardiovascular diseases . several studies have demonstrated lowering of hscrp upon treatment with statins and this effect was independent of lowering of ldl - c . in patients with high crp values , higher coronary deaths occurred in spite of low ldl - c levels . the air force / texas coronary atherosclerosis prevention study reported lowering of crp values through treatment with lovastatin , and improvement in cardiac outcomes was achieved with lowering of crp levels independent of ldc - c levels . in 2005 , nissen et al . , reported the existence of an independent correlation between low levels of crp and atheroma progression rate from a post - hoc analysis of data of a clinical study . according to the authors , regression of atheroma size was more rapid and significant with greater reduction in crp levels . however , less regression in atheroma size was observed despite greater reduction in ldl - c levels . in the primary prevention also treatment with statin benefitted patients with high hscrp levels and low ldl - c levels and other cv risks . subjects in the early or mild stages of heart failure benefitted from statins and the benefits were reported to be due to anti - inflammatory effects and improvement in endothelial function with statin treatment . the presence of inflammatory components in mediating cv diseases were supported by a retrospective study , the corona ( controlled rosuvastatin multinational trial in heart failure ) trial , and patients benefitting from lowering of hscrp levels suggested a non - lipid effect of rosuvastatin . the finding was further confirmed by the multicenter cosmos ( coronary atherosclerosis study measuring effects of rosuvastatin using intravascular ultrasound in japanese subjects ) trial . these trials with rosuvastatin showed significant reduction in plaque volume after treatment with rosuvastatin independent of ldl - c reduction , suggesting non - lipid - lowering effects . in a double - blind study with 58 subjects with coronary artery disease , comparable reduction in ldl - c occurred through treatment with high- ( 80 mg / day ) than low - dose atorvastatin ( 10 mg / day ) plus ezetimibe ( 10 mg / day ) . statins are known to lower cholesterol by reversibly inhibiting hmg - coa reductase , the well - known and widely established mechanism of action of statins . inhibition of cholesterol synthesis occurs as a result of prevention of mevalonate from producing hmg - coa , since mevalonate is not an immediate precursor of cholesterol synthesis and also acts as precursor for several other key molecules needed for normal functioning of cellular processes . in addition to cholesterol synthesis , mevalonate is required for production of non - steroidal isoprenoid intermediates such as the farnesyl pyrophosphate , geranylgeranyl pyrophosphate , isopentanyl adenosine , dolichols and polyisoprenoid side chains of ubiquinone and heme - a . these isoprenoid intermediates are integral to the post - translation modification and activation of several intracellular / signaling proteins such as the -subunit of heterotrimeric g - proteins ; heme - a ; nuclear lamins ; and small gtp - bound protein ras and ras - like proteins such as rho , rab , rac , ral or rap . post - translational isoprenylation , that is , attachment of isoprenoid chains helps these proteins in their covalent attachment , sub - cellular localization and intracellular trafficking . both ras and rho proteins switch from their gdp - bound inactive state to the gtp - bound active state [ figure 1 ] . these signaling proteins play an indispensable role in multiple cellular processes cell signaling , cell differentiation and proliferation , myelination , cytoskeleton dynamics and endocytotic / exocytotic transport [ figure 2 ] . rho protein cycles between a cytosolic , inactive gdp - bound and an active , membrane , gtp - bound state . inhibition of mevalonate synthesis by statins prevents membrane targeting of rho and its subsequent activation of rock . this cycle is controlled by several cofactors , including guanine nucleotide exchange factors , gtpase - activating proteins , and guanine nucleotide dissociation inhibitors . an important step in the activation of rho gtpases is posttranslational isoprenylation , which allows translocation of rho to the cell membrane and subsequent activation mevalonate pathway for cholesterol biosynthesis showing the effects of inhibition of hmg - coa reductase by statins . statins decrease the isoprenylation of signaling molecules , which leads to modulation ( [increase]/[decrease ] ) of various signaling pathways . enos : endothelial nitric oxide synthetase ; et-1 : endothelin-1 ; etc : electron transport chain ; nadph : nicotinamide adenine dinucleotide phosphate ( reduced ) ; pai-1 : plasminogen activator inhibitor-1 ; t - pa : tissue - type plasminogen activator statins have been reported to prevent isoprenylation of rho- and rho - associated kinases ( rock ) at doses that were used to lower ldl - c levels . rho / rock are involved in controlling important functions such as regulation of contraction , migration and adhesion of vascular smooth muscle cells . statins by inhibiting rho / rock activation increase the bioavailability of nitric oxide ( no ) . the rac protein has been involved in cardiac hypertrophy , actin cytoskeleton remodeling and generation of reactive oxygen species , and statins benefit by inhibiting rac1 . inactivation of rho by statins was reported to upregulate peroxisome proliferator - activated receptor ( ppar ) expression and induce ppar- transcription activity in macrophages , and inhibit lipopolysaccharide . matrix metalloproteinases ( mmps ) are zinc - dependent endopeptidases that promote angiogenesis , intimal proliferation , vessel wall remodeling and extracellular matrix degradation through enhanced migration of vascular smooth muscle cells . the role of rho / rock pathway in the secretion of mmp-2 through stimulation of endothelin-1 and angiotensin - ii has been confirmed with use of small interfering rna and the rock pathway inhibitor , y-27632 , which inhibited mmp-2 release . statins decrease inflammatory cells in atherosclerotic plaques and increase plaque stability through combined reduction of lipids , macrophages and mmps . other cellular molecules inhibited by statins include nuclear factor-b ( nf-b ) , monocyte chemoattractant protein-1 and hscrp . under normal physiological conditions , nf - kb resides in the cytoplasm bound with inhibitor i - kb ( ib ) . in response to inflammatory stimuli , the nf - kb then translocates into the nucleus and induces the expression of inflammatory cytokines , monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 . statins decrease the levels of reactive oxygen species and prevent dislocation of ikb from nf - kb , resulting in inactivation of nf - kb . statins have been reported to reduce the expression of monocyte chemoattractant protein-1 , which lowers the interaction between monocytes and the vascular wall , monocyte chemotaxis , and growth and proliferation of macrophages . antigen - presenting cells express major histocompatibility complex - ii constitutively , and its expression in human endothelial cells and monocytes increases in the presence of interferon-. various statins reduce the expression of major histocompatibility complex - ii on antigen - presenting cells , leading to decreased major histocompatibility complex - ii - mediated activation of t - cells . in addition , statins reduce the production of various inflammatory markers such as tumor necrosis factor- , interleukin-1 and chemotactic cytokines . statins were reported to disrupt the oxidative stress / inflammatory cycles by decreasing lipid peroxidation and release of inflammatory mediators . statins improve endothelial function by inhibiting the isoprenylation of rac and rho , and activation of the rock pathway . inactivation of the rock pathway stabilizes the mrna of endothelium - derived nitric oxide synthase ( enos ) and activates the akt / pi3k cascade , leading to high enos activity , increased no production and bioavailability . protein kinase akt has been associated with improvement of myocardial aerobic metabolism and increase in the levels of angiopoetine ( protein growth factor ) , which accelerate the process of angiogenesis . inactivation of the ras signaling pathway was associated with prevention or reversal of cardiac remodeling in humans . in a preclinical study , atorvastatin reduced the synthesis of collagen , -1-procollagen mrna expression and expression of profibrotic peptide connective tissue growth factor in cell cultures of rat and human cardiac fibroblasts . rosuvastatin administration was reported to decrease the levels of tnf- and mmp-2 and increased the circulating levels of bone marrow - derived stems cells . statins also have inhibitory effects on release of mmp-1 and mmp-3 from macrophages and endothelial cells , and modulate remodeling processes . in endothelial cells , statins activate protein kinase akt leading to stimulation of enos activity , increased no synthesis and neoangiogenesis . in the central nervous system , statins regulate sympathetic and vagal outflow by increasing endothelial no synthesis , and reduce the expression of angiotensin - ii and endothelial receptors . statins by normalizing sympathetic outflow showed beneficial effects in hypertensive patients with a history of mi and cerebral ischemia . statins attenuate the cytokine - mediated proliferation of vascular smooth muscle cells in coronary artery smooth muscle cells , arrest the cell cycle between g1s phase transitions and inhibit smooth muscle cell proliferation through modulation of ras and rho activity . atorvastatin inhibited serotonin - induced mitogenesis and migration by inhibiting gtp - rhoa formation in pulmonary artery vascular smooth muscle cells , which was reversed by geranylgeranyl pyrophosphate but not farnesyl pyrophosphate . the anti - platelet and antithrombotic activities of statins have been attributed to reduction of thromboxane a2 , tissue factor expression and ppars . in heart failure statins activate phosphoinositide-3 kinase and akt , and increase the circulating levels of bone marrow - derived endothelial progenitor cells , which may contribute to improved cardiac regeneration and/or repair . in 2012 , bouitbir et al . , reported statins as a new activating factor of cardiac mitochondrial biogenesis and antioxidant capacities . mitohormesis is defined as a pre - conditioning or adaptive response of mitochondria to low levels of oxidative stress induced by physical exercise , reduced caloric uptake and glucose restriction . this pleiotropic effect of statins was attributed to increased resistance / stress defense against higher levels of oxidative stress and damage to mitochondria and cardiac tissues . use of antioxidants prevented statin - induced mitohormesis since antioxidant vitamins had decreased the clinical benefits of the simvastatin niacin combination in subjects with coronary artery diseases and low levels of hdl - c in the study by brown and co - workers . therefore , some of the cardioprotective mechanisms of statins could be attributed to statin - induced mitohormesis . treatment with simvastatin has been reported to reverse the pulmonary vascular effects of cigarette smoke , including pulmonary hypertension , smoke - induced emphysema , smoke - induced small arterial remodeling and emphysema in guinea pigs exposed to cigarette smoke for 6 months . in cancer , preclinical studies have shown anti - proliferative , pro - apoptotic , anti - invasive and radiosensitizing properties of statins . various lipophilic statins such as lovastatin , simvastatin and fluvastatin exerted direct anticancer activity in vitro by reducing proliferation and survival signals in susceptible breast cancer phenotypes , indicating potential use of statins in breast cancer . in 2008 , bifulco et al . , suggested that statins may have potential to treat various other neurological disorders , including alzheimer 's disease , parkinson 's disease , multiple sclerosis and primary brain tumors as antioxidant , anti - inflammatory , anti - platelet activities and effects on the nitric oxide synthase system have been confirmed previously . in vitro studies , including a few animal studies , have suggested that statins may increase bone mass by enhancing bone morphogenetic protein-2-mediated osteoblast differentiation and activity . in addition , statins also exerted beneficial effects in rheumatoid arthritis by inhibition and downregulation of these cytokines and chemokines . in 2009 , glynn et al . , reported an analysis of the jupiter , which showed that rosuvastatin nearly halved the risk for symptomatic venous thromboembolism vte in apparently healthy patients and the effects were ascribed to the pleiotropic effects of statins . a recent meta - analyses including one randomized and nine observational trials was conducted on nearly 1 million people who were on statin therapy . the results showed a 32% decrease in the risk of vte , 41% decrease in deep vein thrombosis and 30% decrease in pulmonary embolism . in women with polycystic ovary syndrome , statins have been reported to directly or indirectly block oxidative stress - mediated increase in thecal interstitial cell proliferation , steroidogenesis and insulin resistance . in seasonal and avian h5n1 influenza , administration of statins showed modulatory activities on various cytokines and increased the levels of other potential immunomodulatory molecules . the beneficial effect of statins in influenza was partly ascribed to their cardioprotective , anti - inflammatory and immunomodulatory effects . a global consensus on the concept of statin pleiotropy could not be reached as yet and remains a hot topic for debate . the pathophysiological implications of blockade of mevalonate production in the pathway leading to synthesis of cholesterol , that is , the mevalonate pathway suggested multiple mechanisms of statins . over the years , analyses of several clinical studies , including the landmark hps and ascot - lla trial , reported findings with statins that were inexplicable with the lipid - lowering mechanism alone . the most probable mechanism of statin pleiotropy is prevention of isoprenoids synthesis as decrease in levels of circulating isoprenoids have been reported with treatment with statins .

statins or 3-hydroxy - methylglutaryl coenzyme a ( hmg coa ) reductase inhibitors not only prevents the synthesis of cholesterol biosynthesis but also inhibits the synthesis of essential isoprenoid intermediates such as farnesyl pyrophosphate , geranylgeranyl pyrophosphate , isopentanyl adenosine , dolichols and polyisoprenoid side chains of ubiquinone , heme a , and nuclear lamins . these isoprenoid intermediates are required for activation of various intracellular / signaling proteins- small guanosine triphosphate bound protein ras and ras - like proteins like rho , rab , rac , ral , or rap which plays an indispensible role in multiple cellular processes . reduction of circulating isoprenoids intermediates as a result of hmg coa reductase inhibition by statins prevents activation of these signalling proteins . hence , the multiple effects of statins such as antiinflammatory effects , antioxidant effects , antiproliferative and immunomodulatory effects , plaque stability , normalization of sympathetic outflow , and prevention of platelet aggregation are due to reduction of circulating isoprenoids and hence inactivation of signalling proteins . these multiple lipid - independent effects of statins termed as statin pleiotropy would potentially open floodgates for research in multiple treatment domains catching attentions of researchers and clinician across the globe .

chronic hepatitis b virus ( hbv ) infection is a potentially life - threatening liver disease with serious complications such as cirrhosis and hepatocellular carcinoma . the prevalence of hbv infection varies widely , with rates ranging from 0.1% to 20% worldwide . iran is an area of intermediate endemicity and the prevalence of chronic hepatitis b infection is reported to be 1.7% in general population . it has been demonstrated that some hbs - ag negative individuals with positive anti - hbc continue to replicate hbv . thus , the absence of hbs - ag in the blood of apparently healthy individuals may not be sufficient to ensure an hbv - free status . anti - hbc can be detected throughout the course of both acute and chronic hbv infection . it persists longlife after resolution , therefore the routine blood donor screening for anti - hbc has been implemented in some countries , resulting in a decrease in the risk of post - transfusion hbv infection . in iran , the reported rates range from 5.1% in hamadan and 6.5% in shiraz to as high as 34% in sistan - balouchestan . nevertheless , the associated figures for isoalted anti - hbc prevalnce in other studies were as low as 0.56% in the united kingdom to as high as 76% in ghana however , most of previous studies reported isolated anti - hbc rates between 2 - 5% . despite this fact , iranian health policy makers have not introduced anti - hbc screening among blood donors , partly because of the lack of empirical data on the benefit of introducing anti - hbc screening in a donor population with low prevalence of hbv . on the other hand , the indefinite deferral of donors with false - positive anti - hbc is a major disappointing side effect of anti - hbc screening in countries with low endemicity of hbv infection . it is of utmost importance to differentiate whether isolated anti - hbc is due to false positive results or the prior exposure to hbv , because individuals with false - positive anti - hbc can benefit from vaccination and their blood can be safely transfused . to distinguish between the aforementioned conditions , evaluation of response to hb vaccination has been proposed . in the present study , we investigated the anti - hbs seroconversion in 2 groups of blood donors with isolated anti - hbc and their controls after hb vaccination . we also attempted to find the predictors of non - response status among individuals with isolated anti - hbc . ninety individuals with isolated anti - hbc positive test and 100 healthy persons with negative serological markers of hepatitis b were recruited in the study as case and control groups , respectively . the exclusion criteria were : age > 64 years , previous hbv vaccination , organ transplantation , immunodeficiency disorders , hemodialysis , immunosuppressive therapy , contraindication for hbv vaccination ( i.e. prior history of anaphylactic reaction to vaccine ) , positive results for hbs - ag , anti - hbs , anti - hcv , or anti - hiv tests and aspartate aminotransferase ( ast ) or alanine aminotransferase ( alt ) levels above 3 times normal cutoff values . also , none of the participants were positive for signs and symptoms of chronic hepatitis and cirrhosis . prior to intervention , informed consents were obtained from all the participants , in accordance with the guidelines established by the ethics committee of zahedan university of medical sciences . all the participants were tested for hbv - dna by qualitative polymerase chain reaction ( pcr ) ( cinna gen inc . , tehran , iran ) that could detect as low as 100 copy / ml of viral genome . the following reagents were added to each tube on ice : 1x pcr mixture 15 ul and taq - dna polymerase 0.4 ul . then the tubes were shaken and spun thoroughly . the pcr mixture contained primers amplifying 353 bp of the region of the core gene . one drop ( 20 - 25 ul ) of mineral oil was also added to each tube . then , 10 ul dna ( with the use of specified pipette for sampling of dna ) was added to the mixture and spun on microfuge for 3 - 5 seconds . the tubes were transferred to preheated thermocycler with the following program : 1 cycle of 60 seconds at 93c , 20 seconds at 61c and 40 seconds at 72c , followed by 35 cycles of 93c , 61c , and 72c for 20 , 20 , and 40 seconds , respectively . finally , 10 iu / ml of amplified samples were directly analyzed in a 2% agarose gel without adding loading buffer . afterwards , all subjects received three doses of 20 g of hepatitis b recombinant vaccine ( heberbiovac hbr , havana , cuba ) at 0 , 1 , and 6 months , if applicable ( see below ) . the vaccine was injected intramuscularly into the deltoid muscle . in order to quantify anti - hbs antibodies , individuals with high titer anti - hbs response ( anti - hbs > 50 miu / ml ) did not receive additional doses of the vaccine . however , others completed the vaccination course , and another blood sample was collected 30 days after the third dose to measure anti - hbs level . anti - hbs titer 10 miu / ml 30 days after the first vaccination was considered as early primary response . also , late primary response was defined as anti - hbs titer 10 miu / ml 30 days after the third dose of vaccines . non - responders were individuals with < 10 miu / ml anti - hbs titers after receiving three doses of hb vaccine . data were analyzed using spss for windows software , version 11.5 ( spss inc . , odd s ratios ( ors ) were chosen to measure the association between dichotomous variables , and the results were adjusted for potential confounders by multivariate logistic modeling . table 1 presents the demographic and important risk factors of the participants , comparing those with isolated anti - hbc to controls . 1 ) abnormal level was defined as levels > 1.5 times upper than normal limit 2 ) ns : not significant 3 ) linear by linear association ( chi - squared for linear trend ) ; exact method participants of the case group were significantly older , and mostly married . the case group had also higher male / female ratio , and more history of transfusion and tattoo ( p<0.05 ) . however , history of infection in spouse , familial history of hbv infection , and serum ast and alt levels was not significantly different between cases and controls . totally , 19 ( 21.1% ) cases and 3 ( 3% ) controls did not seroconvert ( non - responder ) after three doses of hb vaccine ( p<0.0001 ) . however , primary response was observed in 43 ( 47.8% ) isolated anti - hbc positive cases and 92 ( 92% ) controls ( p<0.0001 ) , of whom 15 cases and two controls seroconverted after the first dose of hb vaccine ( early responder ) . furthermore , anti - hbs titer 50 miu / ml 30 days after the first dose of vaccine was significantly higher among cases ( 31.1% vs. 5% , p<0.0001 ) . two ( 2.2% ) cases with positive isolated anti - hbc had detectable hbv dna and were therefore , excluded from the analysis . both of these two hbv dna positive cases did not seroconvert after receiving 3 doses of vaccination . none of the cases and controls showed adverse effects after receiving recombinant hb vaccine . table 2 compares non - responders ( excluding 2 hbv dna positive cases ) with primary responders ( including early and late responders ) . according to univariate analysis , serum anti - hbc positivity , age , and marital status were significantly different between the two groups ; however , in multivariable analysis , only anti - hbc positivity and age remained independently associated with non - responding status . indeed , anti - hbc positive subjects were 12.2 times ( 95% ci : 3.2 - 46.4 , p<0.0001 ) more likely to fail seroconversion . similarly , age 50 years was 3.6 times more likely to lead in non - responsive state ( 95% ci : 1.0 - 12.3 , p<0.04 ) . also , anti - hbc positive participants were more likely to develop anti - hbs titer 50 miu / ml 30 days after receiving the first dose of vaccine ( table 3 ) . 1 ) abnormal level was defined as levels > 1.5 times upper than normal limit 2 ) ns : not significant 3 ) linear by linear association ( chi - squared for linear trend ) ; exact method . * participants with anti - hbs titer 50 miu / ml 30 days after the first dose of vaccine and those with positive hbv dna were excluded as noted earlier , early response ( anti - hbs titer 10 miu / ml 30 days after the first vaccination ) was found in 17 subjects . we compared early responders to late responders ( the table is not included ) and found a trend toward a higher seroprotective response rate after the first vaccination in the subjects with abnormal baseline alt levels and those aged>50 years ( ors : 6.9 and 8 , p<0.02 and p<0.001 , respectively ) . data were analyzed using spss for windows software , version 11.5 ( spss inc . , odd s ratios ( ors ) were chosen to measure the association between dichotomous variables , and the results were adjusted for potential confounders by multivariate logistic modeling . table 1 presents the demographic and important risk factors of the participants , comparing those with isolated anti - hbc to controls . 1 ) abnormal level was defined as levels > 1.5 times upper than normal limit 2 ) ns : not significant 3 ) linear by linear association ( chi - squared for linear trend ) ; exact method participants of the case group were significantly older , and mostly married . the case group had also higher male / female ratio , and more history of transfusion and tattoo ( p<0.05 ) . however , history of infection in spouse , familial history of hbv infection , and serum ast and alt levels was not significantly different between cases and controls . totally , 19 ( 21.1% ) cases and 3 ( 3% ) controls did not seroconvert ( non - responder ) after three doses of hb vaccine ( p<0.0001 ) . however , primary response was observed in 43 ( 47.8% ) isolated anti - hbc positive cases and 92 ( 92% ) controls ( p<0.0001 ) , of whom 15 cases and two controls seroconverted after the first dose of hb vaccine ( early responder ) . furthermore , anti - hbs titer 50 miu / ml 30 days after the first dose of vaccine was significantly higher among cases ( 31.1% vs. 5% , p<0.0001 ) . two ( 2.2% ) cases with positive isolated anti - hbc had detectable hbv dna and were therefore , excluded from the analysis . both of these two hbv dna positive cases did not seroconvert after receiving 3 doses of vaccination . none of the cases and controls showed adverse effects after receiving recombinant hb vaccine . table 2 compares non - responders ( excluding 2 hbv dna positive cases ) with primary responders ( including early and late responders ) . according to univariate analysis , serum anti - hbc positivity , age , and marital status were significantly different between the two groups ; however , in multivariable analysis , only anti - hbc positivity and age remained independently associated with non - responding status . indeed , anti - hbc positive subjects were 12.2 times ( 95% ci : 3.2 - 46.4 , p<0.0001 ) more likely to fail seroconversion . similarly , age 50 years was 3.6 times more likely to lead in non - responsive state ( 95% ci : 1.0 - 12.3 , p<0.04 ) . also , anti - hbc positive participants were more likely to develop anti - hbs titer 50 miu / ml 30 days after receiving the first dose of vaccine ( table 3 ) . 1 ) abnormal level was defined as levels > 1.5 times upper than normal limit 2 ) ns : not significant 3 ) linear by linear association ( chi - squared for linear trend ) ; exact method . * participants with anti - hbs titer 50 miu / ml 30 days after the first dose of vaccine and those with positive hbv dna were excluded as noted earlier , early response ( anti - hbs titer 10 miu / ml 30 days after the first vaccination ) was found in 17 subjects . we compared early responders to late responders ( the table is not included ) and found a trend toward a higher seroprotective response rate after the first vaccination in the subjects with abnormal baseline alt levels and those aged>50 years ( ors : 6.9 and 8 , p<0.02 and p<0.001 , respectively ) . in our setting , we vaccinated isolated anti - hbc as well as healthy blood donors and monitored their response to hbv vaccination . a total of 19 ( 21.1% ) cases and 3 ( 3% ) controls did not seroconvert after receiving 3 doses of hb vaccine . prior investigators reported a wide range of non - responding state , sunbul 9.1% , coz yatacho 9.7% . therefore , at least 20% of isolated anti - hbc positive cases may suffer from occult hepatitis b infection , for whom a diminished ability to mount an anti - hbs response is proposed . furthermore , in our study , anti - hbs titer 50 miu / ml 30 days after the first dose of vaccine was developed in 28 ( 31.1% ) cases and 5 ( 5% ) controls . scanty studies have addressed anamnestic rates ; however , in a study reported by sunbul and colleagues , out of 33 ( 42.4% ) subjects with isolated anti - hbc showed rapid high seroconversion , while in a report by ural and co - workers , this rate was 41.7% ( 20 out of 48 ) . our lower rate could in part be explained by lower prevalence of hbv in our region . in such patients , further vaccination may not be necessary . finally , 43 ( 47.8% ) subjects with isolated positive anti - hbc and 92 ( 92% ) controls showed primary response after the vaccination protocol . nevertheless , most of previous researchers assigned their subjects in two groups of responders ( either primary or anamnestic ) and non - responders . accordingly , our cumulative responder rate would be 78.9% among subjects with isolated positive anti - hbc , which is in agreement with reports by koh ( 70.6% ) , kabir ( 79.8% ) , and silva ( 80% ) , but lower than what reported by ural ( 89.6% ) , pereira ( 90% ) , yatacho ( 90.3% ) and sunbul ( 90.9% ) . , discrepancies in response rate to hb vaccination among isolated anti - hbc subjects can be explained by serologic kit sensitivity , defining cut off points , and inclusion criteria for borderline results . therefore , in our region , nearly 80% of isolated anti - hbc subjects can be safely included in the donor pool , simply based on a vaccination protocol . notably , the isolated anti - hbc subjects with false positive results ( 47.8% in our setting ) are susceptible to hepatitis b infection and should receive a complete course of hb vaccine . however , they may lose hb vaccination because of misinterpretation of anti - hbc results . on the other hand , our results revealed that individuals with isolated positive anti - hbc were significantly older , mostly married , had higher male / female ratio , history of transfusion , tattoo and abnormal serum ast and alt levels when compared with controls . the higher rate of anti - hbc positivity among older subjects mirrors a cohort effect of hbv infections acquired decades ago when hbv was moderately endemic in iran and hbv national vaccination program had not been commenced . furthermore , only two ( 2.2% ) isolated anti - hbc positive cases had detectable hbv dna . recent studies suggest that 1 - 2% of anti - hbc reactive units contain low levels of hbv dna . however , in studies conducted on 4930 healthy blood donor volunteers in fars and hamadan provinces , 6.5% and 5.1% were isolated anti - hbc positive cases , among whom 16 ( 12.3% ) and 3 ( 1.2% ) were hbv dna positive , respectively . having compared non - responders to primary responders ( table 2 ) , anti - hbc positivity , age , and marital status showed significant differences in univariable analysis . however , in multivariable analysis , only anti - hbc positivity and age ( > 50 years ) remained independently associated with non - responding status . on the other hand , when we compared primary responders with those who developed anti - hbs titer 50 miu / ml 30 days after the first dose of vaccine ( table 3 ) , positivity for anti - hbc and age > 50 years were the most relevant factors in developing higher anti - hbs titers to hbv vaccine . therefore , isolated anti - hbc subjects aged > 50 years would more likely either develop anti - hbs titers 50 miu / ml 30 days after receiving the first dose of vaccine or never respond to hb vaccine . as a result , one month after the first dose of hb vaccine in isolated anti - hbc subjects aged > 50 years , we can make a decision regarding whether to advise a strict follow - up ( occult hbv infection ) or assure the patient that he / she has a resolved hbv infection . interestingly , when we compared the early and late responders ( the table is not included ) , anti - hbc positivity , abnormal alt level , and age > 50 years were associated with a higher probability of early response . therefore , anti - hbc positive subjects aged > 50 years with abnormal alt levels should be offered the first dose of hb vaccine and evaluated one month later in order to decide for their future follow - up . various molecular modalities have been developed to determine if isolated anti - hbc subjects could transmit hbv infection . however , they will further increase the testing costs and require trained personnel and equipment , which may not be possible in many blood banks around the country . thus , studying the cost effectiveness of monitoring the response to hb vaccination could be worth of considering as a diagnostic tool . we also suggest future studies focusing on evaluating the short - course vaccination protocols such as double - dose / double - time vaccination in order to make sooner decision . in conclusion ; using vaccination , we found that half of the subjects with isolated positive anti - hbc were falsely positive for the test and more than 75% of subjects with isolated positive anti - hbc could benefit from vaccination and could be included in donor pool . moreover , one - fifth of subjects were non - responders and may have occult hbv infection . because at least 5% of the donor population in iran is anti - hbc positive , blindly rejecting anti - hbc positive donors would cause the exclusion of a consistent number of donors , most of whom could safely be included in the donor pool . therefore , hb vaccination may be used as a diagnostic tool for clarifying the situation of the subjects with isolated anti - hbc and can help in reducing the blood supply problems . this study was financially supported by zahedan university of medical sciences and iranian blood transfusion organization research center ( zahedan branch ) .

background it is important to differentiate whether isolated anti - hbc is due to false positive results or the prior exposure to hepatitis b virus , because individuals with false - positive anti - hbc can benefit from vaccination and their blood can be safely transfused . to distinguish between these two conditions , we evaluated the serologic response to hepatitis b vaccine . methods ninety subjects with isolated anti - hbc ( cases ) and 100 subjects with totally negative hepatitis b serologic markers ( controls ) were recruited to receive three doses of hepatitis - b ( hb ) vaccine . thirty days after the first dose of the vaccine , anti - hbs titers were checked and individuals with anti - hbs titer > 50 miu / ml did not receive additional doses of the vaccine . however , others completed the vaccination course , and another blood sample was collected 30 days after the third dose to measure anti - hbs level . results nineteen ( 21.1% ) cases and three ( 3% ) controls had no sero - conversion ( anti - hbs titers < 10 miu / ml ) 30 days after the third dose ( p<0.0001 ) . primary response , defined as the development of anti - hbs antibody titers 10 miu / ml 30 days after the third dose , was observed in 43 ( 47.8% ) cases and 92 ( 92% ) controls ( p<0.0001 ) . also , 31.1% of cases developed anti - hbs titers 50 miu / ml 30 days after the first dose of vaccine , but the rate was significantly lower ( 5% ) in the control group ( p<0.0001 ) . furthermore , half of the individuals with positive isolated anti - hbc developed protective levels of anti - hbs after three doses of hb vaccination . conclusion more than 75% of individuals with positive isolated anti - hbc can benefit from vaccination and can be included in donor pool . also , one fifth seemed to have occult hbv infection . so hb vaccination may be used as a diagnostic tool for clarifying the situation of the subjects with isolated anti - hbc .

percutaneous nephrolithotomy ( pcnl ) is a widely accepted treatment for urinary calculi > 2 cm or resistant to other modalities . complications of this procedure are well reported ; however , to our knowledge no mention has occurred in the literature of a retained nephrostomy tube after pcnl . thus , we will present the management of a retained council tip catheter after pcnl . a 76-year - old gentleman underwent a pcnl for a 2.2x1.4-cm lower pole calculus via an uneventful upper pole puncture . incomplete stone fragmentation was obtained using the ultrasonic lithotripter ; therefore , a 24-french council tip nephrostomy tube was placed , the balloon was inflated with 3 ml of sterile water , and the patient was scheduled for a second look nephroscopy when a holmium laser was available . one week later at the second look nephroscopy , attempts to deflate the catheter balloon were unsuccessful . after transection of the balloon port valve , attempted passage of a 0.038 movable core bentson guidewire down the balloon port was unsuccessful . an antegrade nephrostogram outlined the catheter balloon within the renal pelvis ( figure 1 ) . with the c - arm in a vertical orientation , an 18-gauge chiba needle ( cook , bloomington in ) was advanced through a puncture site directly over the balloon under fluoroscopic guidance . the balloon was successfully punctured by the chiba needle with clear efflux through the needle . contrast was then instilled through the chiba needle to evaluate the integrity of the balloon and exclude the possibility of retained balloon fragments from a ruptured balloon ( figure 2b ) . flexible nephroscopy was performed to evaluate for balloon fragments , which were not identified , and to remove the lower pole stone . ( a ) chiba needle ( white arrow ) has punctured and drained the catheter balloon . ( b ) contrast injected via chiba needle has filled the catheter balloon ( dark arrow ) and confirmed its integrity . a retained urethral catheter due to balloon malfunction is a well - recognized urologic complication . many solutions to this problem have been attempted , including overinflation until rupture , puncture of the balloon via a wire through the balloon port , and puncture of the balloon under ultrasound guidance via a suprapubic or transvaginal route . given this infrequent but known complication of balloon - type cystostomy catheters , it is not surprising that a similar complication occurred in our patient . our familiarity with the use of the chiba needle and 3-dimensional fluoroscopy for percutaneous renal access facilitated percutaneous puncture of the catheter balloon . if equipment or experience limits the urologist 's ability to perform this technique , collaboration with an interventional radiologist using conventional c - arm or ultrasound for guidance should be considered . prevention of a retained catheter due to a balloon - port malfunction requires use of sterile water instead of saline and limiting the time of catheter placement . malecot tubes have been utilized for nephrostomy drainage , but these tubes are not without difficulties . tissue bridge formation over flanges of the tube has occurred , requiring endoscopic removal . tubeless pcnl would avoid these issues entirely ; however , the tubeless technique is contraindicated in the face of significant postoperative bleeding , urinary extravasation , ureteral obstruction , or anticipated need for a second - look nephroscopy . pigtail catheters following pcnl may represent a good alternative for drainage and access , while minimizing the risk of catheter malfunction or retention .

percutaneous nephrolithotomy is a widely accepted treatment for urinary calculi , but it is not without complications . we present the case of a 76-year - old male with a retained council tip catheter after percutaneous nephrolithotomy . fluoroscopic guidance was used to perform percutaneous puncture of the catheter balloon , and the catheter was removed without complication . advantages of various nephrostomy tube designs and additional measures to prevent this type of complication are discussed .

high - throughput biochemical screens , used to identify pharmacologically active small - molecule compounds , are a staple of modern drug discovery . in these screens , hundreds of thousands to even millions of compounds are tested in highly automated assays . although robotics and miniaturization have led to increased efficiency , traditional high - throughput screens are nevertheless expensive and labor intensive . with a few notable exceptions , the financial and labor requirements of such screens place them beyond the reach of most academic institutions . consequently , academic researchers , as well as some in industry , are increasingly turning to virtual screens . virtual screens rely on computer docking programs to position models of potential ligands within target active sites and predict the binding affinity . precise physics - based computational techniques for binding - energy prediction , such as thermodynamic integration , single - step perturbation,(3 ) and free energy perturbation,(4 ) are too time- and calculation - intensive for use in virtual screens . instead , researchers have developed simpler scoring functions that sacrifice some accuracy in favor of greater speed . because of these accepted inaccuracies , scoring functions are unable to explicitly identify ligands in silico ; rather , they serve only to enrich the pool of candidate ligands with potential hits . the compounds with current scoring functions fall into three general classes.(8 ) the first class , based on molecular force fields , predicts binding energy by estimating electrostatic and van der waals forces explicitly . docking programs using force - field - based scoring functions include autodock,(1 ) dock,(9 ) glide , icm,(12 ) and gold ( goldscore).(13 ) a second class of empirical scoring functions include those used by glide and ehits ( sfe empirical scoring function ) , as well as the chemscore(16 ) and piecewise linear potential ( plp)(17 ) functions . these functions estimate binding energy by calculating the weighted sum of all hydrogen - bond and hydrophobic contacts . a third class of scoring function , called knowledge based , pairs of atom types that are frequently found in close proximity are judged to be energetically favorable . examples include the astex statistical potential ( asp)(18 ) and the sfs statistical scoring function used by ehits . these approaches to binding - affinity prediction have proven very useful ; virtual - screening efforts routinely identify predicted ligands that are subsequently validated experimentally ( see , for example , refs ( 19 ) and ( 20 ) ) . however , modern scoring functions produce many false - positive and false - negative results . surprisingly , a human being with the proper training can often analyze a docked structure visually and correctly assess inhibition with greater accuracy.(8 ) remarkably , the human mind can characterize ligand binding without employing physical or chemical equations and without requiring the explicit calculation of affinity constants . although any computational model pales in comparison to the complexity of the brain , the mind s ability to categorize proteinligand complexes nevertheless suggests that a neural network , a computer model designed to mimic the microscopic organization of the brain , might be at least as suited to the prediction of proteinligand binding affinities as equation- and statistics - based scoring functions . herein , we describe a fast and accurate neural - network - based scoring function that can be used to rescore the docked poses of candidate ligands . the function is in some ways knowledge based , as it draws upon protein - structure databases to however , the use of neural networks in this context is largely unprecedented.(21 ) although useful in its own right , the neural - network approach to affinity prediction is also orthogonal to existing physics - based and statistics - based scoring functions and , so , might prove useful in consensus - scoring projects as well . neural networks are computer models designed to mimic , albeit inadequately , the microscopic architecture and organization of the brain . in brief , various neurodes , analogous to biological neurons , are joined by the behavior of the network is determined not only by the organization and number of the neurodes , but also by the weights ( i.e. , strengths ) of the connections . the first , called the input layer , receives information about the system the network is to analyze . additionally , optional hidden layers receive input from the input layer and transmit it to the output layer , allowing for even more complex behavior ( figure 1 ) . all neural networks have an input layer , through which information about the system to be analyzed is passed , and an output layer , which encodes the results of the analysis . optional hidden layers receive input from the input layer and transmit it to the output layer , allowing for even more complex behavior . in designing a neural network to analyze a complex data set , the specific formulas that describe the relationships between data - set characteristics need not be explicitly delineated ; rather , the designer need only provide the network with an adequate description of the system so that the network can infer those relationships on its own . in the current context , creating neural networks to characterize the binding affinity of proteinligand complexes does not require that we implement or even understand the specific formulaic relationships that describe van der waals , electrostatic , and hydrogen - bond interactions , though the energies calculated by these formulas can in theory be included in the network input.(21 ) rather , we must determine what characteristics of a proteinligand complex the network needs to see in order to correctly analyze and characterize the complex on its own . specific atomatom interactions , including electrostatic , hydrogen - bond , van der waals , , and cation interactions , contribute to the enthalpic component of the binding energy . in contrast , the entropic contribution is determined in part by the number of ligand rotatable bonds and the challenges of disordering and rearranging the ordered hydration shells surrounding the unbound ligand and the apo active site . the entropic penalty related to hydration is difficult to calculate explicitly and is likely a function of many factors , including the hydrophobicity and volume of the ligand , the number of buried but unsatisfied hydrogen - bond donors and acceptors upon binding , and the proteinligand contact surface area . in the current work , we therefore sought to identify the characteristics of proteinligand complexes that might affect these enthalpic and entropic factors . first , the proximity of ligand and protein atoms likely contributes to the binding affinity by affecting enthalpic factors ( i.e. , electrostatic , van der waals , hydrogen - bond , , and cation interactions ) , as well as entropic factors ( i.e. , buried but unsatisfied hydrogen bonds and the size of the proteinligand contact area ) . in the current context , this proximity information is stored in a proximity list . the distances between the atoms of the ligand and the protein are considered ; atoms that are close to each other are subsequently grouped by their corresponding autodock atom types , and the number of each atom - type pair is tallied . second , electrostatic interactions contribute to the enthalpic component of the binding energy through salt bridges and hydrogen bonds . the electrostatic energy is certainly dependent on proximity , but it is also dependent on partial atomic charges . consequently , for each of the atom - type pairs in the proximity list described above , a summed electrostatic energy is also calculated from the assigned gasteiger charges . third , certain ligand characteristics related to the quantity and identity of ligand atom types , such as ligand hydrophobicity and volume , could affect the entropy of binding as well . consequently , all of the atoms of the ligand are categorized by their corresponding atom types . finally , the number of rotatable bonds in the ligand is explicitly counted , as ligand flexibility can also have an important impact on entropy . when all of these atom - type pairs , ligand atom types , and other metrics are considered separately , a given proteinligand complex can be characterized across 194 dimensions ; thus , we created neural networks with input layers containing 194 neurodes . as output , only two neurodes are needed to distinguish between good and poor binders : ( 1 , 0 ) indicates that a given proteinligand complex has a dissociation constant kd < 25 m , and ( 0 , 1 ) indicates that a given complex has kd > 25 m . although somewhat arbitrary , our experience with virtual screening has led us to believe that 25 m is a reasonable cutoff for distinguishing between inhibitors that warrant further study and optimization and poor inhibitors that are best not pursued further . to train candidate neural networks , 4141 proteinligand complexes were downloaded from the protein data bank(22 ) and characterized across the 194 dimensions described above . these 4141 complexes included 2695 unique , diverse protein structures mapping to over 600 uniprot primary accession numbers . of these 4141 complexes , 2710 had kd values that had been experimentally measured ; 2022 of these were good binders ( kd < 25 m ) , and 688 were poor binders ( kd 25 m ) . recognizing that the poor binders were underrepresented , additional complexes of poorly binding ligands were obtained by docking compounds of the nci diversity set ii into the same protein receptors as used previously . one thousand four hundred thirty - one of these dockings into 571 unique pdb structures had highest - ranked ligand poses with predicted binding energies between 0 and 4 kcal / mol . these were also included in the database of proteinligand complexes as examples of weak binders . multiple neural networks were trained to study the influence of network architecture and training - set size on accuracy and to judge the robustness of the network output . ultimately , a network architecture consisting of a single hidden layer of five neurodes was selected . training sets of 1 , 10 , 25 , 50 , 100 , 250 , 500 , 1000 , 2000 , 3000 , and 4000 proteinligand complexes were generated by randomly selecting complexes from among the 4141 complexes previously characterized . random training sets were generated for each network to ensure that network accuracy was independent of the complexes chosen for inclusion . in all cases , the remaining complexes were used as a validation set to verify that the networks had not been overtrained and to judge training effectiveness . each individual network has its own unique strengths and weaknesses ; to obtain consistent results across multiple proteinligand complexes , it is better to take the average prediction of multiple networks rather than to trust the prediction of any single network . for each training - set size described above , we therefore trained 10 independent neural networks and averaged the corresponding outputs ( figure 2 ) . the x axis shows the size of the training set , and the y axis shows the percent accuracy . each data point represents the average accuracy of 10 independent neural networks with one hidden layer of five neurodes . are shown the accuracies with which the various networks were able to characterize the binding constants of the proteinligand complexes in their respective training sets . in green are shown the accuracies with which the various networks were able to characterize the binding constants of the complexes in their respective validation sets . in purple is shown the likelihood that a given proteinligand complex has a kd value less than 25 m given that the network predicts high - affinity binding ( i.e. , the true - positive rate when the respective validation sets were analyzed ) . in red is shown the likelihood that a given proteinligand complex has a binding affinity greater than 25 m given that the network predicts poor binding ( i.e. , the true - negative rate when the respective validation sets were analyzed ) . figure 2 depicts the accuracy of these neural networks , that is , the frequency with which they accurately characterized the proteinligand complexes of their respective training and validation sets as either having high affinity ( kd < 25 m ) or low affinity ( kd > 25 m ) . although trained to give a binary response [ ( 1 , 0 ) for strong binding , ( 0 , 1 ) for weak binding ] , network output was in fact continuous [ ( a , b ) , where a + b = 1.0 because network outputs were normalized ] . to evaluate each proteinligand complex if n > 0 , the network output was interpreted to predict kd < 25 m ; otherwise , it predicted kd > 25 m . the accuracy with which the networks were able to characterize the binding constants of the proteinligand complexes in their respective training sets ( i.e. , those complexes to which the network had already been exposed ) is shown in figure 2 , in blue . interestingly , training - set accuracy was good regardless of training - set size . the networks ability to correctly characterize the proteinligand complexes of their respective validation sets , sets comprising complexes that had never been seen before , was far more indicative of true predictive ability . the accuracies of these predictions ( figure 2 , in green ) clearly demonstrate that overtraining had not occurred , as accuracy consistently improved with exposure to larger training sets . after having been exposed to only 1000 examples , the networks were already quite good at characterizing proteinligand complexes ; however , additional examples did result in moderate improvements in accuracy . we note also that , for training - set sizes greater than 1000 , the standard deviation of the outputs of the 10 networks associated with each training - set size was relatively small , suggesting that network output was largely independent of the training set selected . the single best network of all those tested correctly characterized the proteinligand complexes of its training and validation sets with 94.8% and 87.9% accuracy , respectively . the true - positive rate ( figure 2 , in purple ) indicates the likelihood that a given proteinligand complex has a kd value less than 25 m given that the network predicts high - affinity binding . the true - negative rate ( figure 2 , in red ) indicates the likelihood that a given proteinligand complex has a binding affinity greater than 25 m given that the network predicts poor binding . the true - positive and true - negative rates of these networks are roughly equal regardless of training - set size ; these networks are just as good at identifying true inhibitors as they are at identifying poor ones . having confirmed that the networks were not overtrained and that network output was robust regardless of the composition of the training sets , we next sought to train additional networks to determine whether accuracy could be improved . ten networks with training sets of 4000 randomly selected complexes were generated in the preliminary studies described above . to determine whether even more accurate networks could be trained , we generated an additional 1000 independent neural networks with similar training sets of 4000 randomly selected proteinligand complexes . in each case , the remaining 141 complexes were again used as a validation set . three of these 1000 networks emerged as the most accurate ( 89.4% accuracy on the validation set ) ; 24 had validation - set accuracies greater than 87.5% . recalling that each network is unique and that consistent results are best obtained when the average prediction of multiple networks is considered , we defined a single score , called an nnscore ( n ) , obtained by averaging the outputs of these 24 networks . to assess how the nnscore ( n ) varied according to the experimentally measured kd values , we considered the scores of the 2710 characterized proteinligand complexes with known kd values described above . to facilitate visualization , moving averages of both the log10(kd ) values and the associated n values were calculated over 100 points and are plotted in figure 3 . it is interesting to note that the data - averaged function crosses the x axis at roughly 25 m [ log10(25 10 ) = 4.60 ] , as expected . so remarkable is this result that one again wonders whether the networks were overtrained ; however , as figure 2 demonstrates , these networks were consistently able to predict the binding of ligands to which they had never been exposed , suggesting the development of a genuine inductive bias . average score ( n ) over 24 networks as a function of the experimentally measured kd value . to facilitate visualization , the data were ordered by log10(kd ) value . moving averages of both the log10(kd ) values and the associated n values this data - averaged function ( shown in black ) crosses the x axis at 25 m [ log10(25 10 ) = 4.60 , shown as a dotted line ] . despite the fact that the networks were trained to answer what is essentially a yes - or - no question , figure 3 demonstrates that they can nevertheless perceive certain shades of gray , that is , they can distinguish not only between good and poor binders , but , to a certain extent , even between good and better binders . given that n decreases somewhat monotonically as log10(kd ) increases , it might therefore be possible to use n as a scoring function . a good scoring function should be able to distinguish between ligands that are well docked and ligands that are poorly docked . to determine whether or not the nnscore could make this distinction , we generated a database of poorly docked ligands separate from the training / testing database described above . selected ligands from the 4141 previously characterized proteinligand complexes were redocked back into their corresponding receptors using autodock vina.(23 ) in 287 cases , the predicted binding energy of the worst - docked pose was greater than 4 kcal / mol . these 287 worst - docked poses , together with their associated protein receptors , were included in the poorly docked database . the top three neural networks of the 1000 generated were each used to characterize these 287 poorly docked binding poses . these three networks correctly identified the ligands as poor binders 94.1% , 88.9% , and 95.5% of the time . thus , despite the fact that these three networks characterized the proteinligand complexes of their respective validation sets with the same accuracy , they were somewhat less consistent when presented with new data . under most circumstances , it is not possible to know a priori which network is best suited for a given database of proteinligand complexes ; a more consistent result can be obtained by considering the average score over multiple networks ( n ) . indeed , when the average score over the top 24 networks was used to characterize these proteinligand complexes , an accuracy of 94.1% was achieved . although the networks were successful at identifying poorly docked ligands , the ability to distinguish between true high - affinity binders and poor binders when both are well docked is far more challenging . to test the networks abilities , we docked 103 small - molecule compounds into the active site of influenza n1 neuraminidase ( n1 , pdb i d : 3b7e)(24 ) using autodock vina.(23 ) three of these compounds were known neuraminidase inhibitors : oseltamivir , peramivir , and zanamivir . the remaining 100 ligands were decoys selected at random from the nci diversity set ii . we note that , of the 4141 proteinligand complexes used in the training and validation sets , one consisted of zanamivir bound to n1 , and two consisted of oseltamivir bound to n1 . however , no examples of peramivir bound to n1 were present in the protein data bank;(22 ) the networks had never been exposed to this proteinligand complex . the results of this small virtual screen are shown in table 1 . when the autodock vina scoring function(23 ) was used to rank the compounds , the known inhibitors ranked 5th , 10th , and 55th . when the docked poses were rescored using each of the top three individual neural networks , the known inhibitors fared substantially better , with the predicted poorest binder ranking 24th , 31st , and 9th , respectively . one of the individual neural networks performed particularly well , ranking the known inhibitors second , eighth , and ninth . however , this network could not have been identified a priori . when the compounds were ranked by the average score over the top 24 networks ( n ) , the known inhibitors performed equally well . had the top 10 compounds from this virtual screen been subsequently tested experimentally , only the network - based scoring functions would have permitted the identification of all three inhibitors . five scoring functions compared : autodock vina score , predictions of the top three individual neural networks ( nn1 , nn2 , and nn3 , respectively ) , and average prediction of the top 24 networks ( n ) . rank of the known inhibitor peramivir . to further validate the predictive potential of these neural networks , we repeated the above virtual - screening protocol using a crystal structure of t. brucei rna editing ligase 1 ( tbrel1 ) , a protein that was not included in the 4141 proteinligand complexes used in the training and validation sets . as three positive controls ( i.e. , known inhibitors ) , we chose atp , the natural substrate , and compounds v1 and s5 , tbrel1 inhibitors recently identified by amaro et al.(19 ) when the compounds were ranked by the average nnscore score over the top 24 networks ( n ) , two of the three known inhibitors ranked in the top ten , giving an enrichment factor of 6.87 . this enrichment was equal to that obtained when the compounds were ranked by the vina scoring function ( table 2 ) . five scoring functions compared : autodock vina score , predictions of the top three individual neural networks ( nn1 , nn2 , and nn3 , respectively ) , and average prediction of the top 24 networks ( n ) . first , as mentioned above , each of the individual networks has its own unique strengths and weaknesses . for example , the network labeled nn1 was far better at identifying compound v1 than was vina , nn2 was better at identifying atp , and nn3 was better at identifying both atp and v1 ( table 2 ) . as we could not have known a priori which network is best suited for this system , it is wise not to trust the results of any single network . when the compounds were ranked by the average output of the top 24 networks , atp and v1 still ranked in the top 10 compounds , but the result was not dependent on a single network output . a second point of interest illustrated by this screen is that , for at least some systems , the networks unique approach to binding - affinity prediction might be orthogonal to more traditional approaches . for example , in this second screen , vina identified s5 as a true binder , but the networks did not ; in contrast , the networks tended to be better at identifying v1 and , with the notable exception of nn1 , atp . by using the networks in conjunction with vina , perhaps a useful consensus score could be developed . to test this hypothesis , a consensus score for each compound was calculated by averaging the ranks obtained when the vina score and the nnscore score were used . when the compounds were reranked by this consensus score , atp , v1 , and s5 ranked first , second , and eighth , respectively . assuming that the top 10 predicted inhibitors were subsequently tested experimentally , ranking by this consensus score would yield an enrichment factor of 10.3 , superior to the enrichment obtained when the compounds were ranked by the vina scoring function or the network outputs alone . we recommend using positive controls ( i.e. , known inhibitors ) to determine whether a single scoring function or a consensus score is best suited to a given virtual - screening project . the research presented here demonstrates that neural networks can be used to successfully characterize the binding affinities of proteinligand complexes . not only were these networks able to distinguish between well - docked and poorly docked ligands , they were also able to distinguish between true ligands and decoy compounds when both were well docked . a user - friendly scoring function based on these networks has been implemented in python and can be downloaded from http://www.nbcr.net/software/nnscore . although the networks success with the neuraminidase and tbrel1 systems was promising , predictive accuracy might be system dependent . regardless , one strength of the neural - network scoring function is that it is largely orthogonal to other kinds of functions based on force fields , linear regression , and statistical analyses . thus , in many cases , it could be useful to rank by consensus scores that combine neural - network scoring functions with other more traditional functions . to build a database of proteinligand complexes of known binding affinity , we identified x - ray crystal and nmr structures from the protein data bank ( pdb)(22 ) that had kd values listed in the moad(25 ) and pdbbind - cn databases . where multiple similar kd values were present in these databases , kd values were averaged to give one value per proteinligand complex . where multiple differing kd values were present , the corresponding complex was discarded . additionally , complexes with peptide or dna ligands , ligands with rare atom types ( e.g. , gold , copper , iron , zinc ) , receptors with rare ligand - binding atom types ( e.g. , copper , nickel , cobalt ) , and complexes with kd values greater than 0.5 m were likewise discarded . ultimately , 2710 complexes remained . hydrogen atoms were added to the ligands of these complexes using schrdinger maestro ( schrdinger ) . all protonation states were verified by visual inspection . for those complexes with ligand - binding metal cations , the partial charge of each metal atom the geometries of the hydrogen bonds between the ligand and the receptor were optimized using an in - house script . autodocktools 1.5.1(28 ) was used to add hydrogen atoms to the receptors , to merge nonpolar hydrogen atoms with their parent atoms , and to assign atom types and gasteiger charges . most of the proteinligand interactions listed in the moad and pdbbind - cn databases were high - affinity ; of those used in the current study , for example , only about 25% had kd values greater than 25 m . to include adequate examples of weak - binding ligands , 20 randomly selected ligands from the nci diversity set ii , a set of freely available , diverse compounds provided by the developmental therapeutics program ( nci / nih ) , were docked into each of the receptors described above using autodock vina.(23 ) in all , 1431 ligands with best predicted binding energies between 0 and 4 kcal / mol were identified and included in the database as examples of weak - binding ligands . in addition to the database of crystallographic and well - docked poses described above , we also generated a separate database of poorly docked proteinligand complexes . some of the ligands of the training / testing database described above were docked back into their corresponding receptors using autodock vina.(23 ) rather than identifying the ligand pose with the best predicted binding energy , the pose with the worst predicted binding energy was considered . in 287 cases , this worst predicted binding energy was greater than 4 kcal / mol ; these 287 proteinligand complexes were included in the poorly docked database . to create a database of compounds docked into influenza neuraminidase , three known neuraminidase inhibitors ( oseltamivir , peramivir , and zanamivir ) were docked into a neuraminidase crystal structure obtained from the pdb ( pdb i d : 3b7e).(24 ) additionally , 100 compounds were randomly selected from the nci diversity set ii to serve as decoys . all 103 compounds were docked into the neuraminidase active site using autodock vina.(23 ) a database of compounds docked into tbrel1 was similarly generated . three known ligands ( atp , v1 , and s5(19 ) ) were docked into a tbrel1 crystal structure ( pdb i d : 1xdn).(29 ) as before , 100 compounds were randomly selected from the nci diversity set ii to serve as decoys . all proteinligand complexes were characterized in four ways . pairs of ligand and protein atoms within 2 of each other were first identified . these proteinligand atom pairs were then characterized according to the autodock atom types of their two constituents , and the number of each of these close - contact atom - type pairs was tallied in a list . fourteen proteinligand atom - type pairs were permitted : ( a , hd ) , ( c , hd ) , ( c , oa ) , ( c , sa ) , ( fe , hd ) , ( hd , hd ) , ( hd , mg ) , ( hd , n ) , ( hd , na ) , ( hd , oa ) , ( hd , zn ) , ( mg , oa ) , ( na , zn ) , and ( oa , zn ) . a similar list of close - contact atom - type pairs was tallied for all proteinligand atom pairs within 4 of each other . eighty - three atom - type pairs were permitted : ( a , a ) , ( a , br ) , ( a , c ) , ( a , cl ) , ( a , f ) , ( a , fe ) , ( a , hd ) , ( a , i ) , ( a , n ) , ( a , na ) , ( a , oa ) , ( a , p ) , ( a , s ) , ( a , sa ) , ( a , zn ) , ( br , c ) , ( br , hd ) , ( br , n ) , ( br , oa ) , ( c , c ) , ( c , cl ) , ( c , f ) , ( c , fe ) , ( c , hd ) , ( c , i ) , ( cl , hd ) , ( cl , n ) , ( cl , oa ) , ( cl , sa ) , ( c , mg ) , ( c , mn ) , ( c , n ) , ( c , na ) , ( c , oa ) , ( c , p ) , ( c , s ) , ( c , sa ) , ( c , zn ) , ( fe , hd ) , ( fe , n ) , ( fe , oa ) , ( f , hd ) , ( f , n ) , ( f , oa ) , ( f , sa ) , ( hd , hd ) , ( hd , i ) , ( hd , mg ) , ( hd , mn ) , ( hd , n ) , ( hd , na ) , ( hd , oa ) , ( hd , p ) , ( hd , s ) , ( hd , sa ) , ( hd , zn ) , ( i , n ) , ( i , oa ) , ( mg , na ) , ( mg , oa ) , ( mg , p ) , ( mn , n ) , ( mn , oa ) , ( mn , p ) , ( na , oa ) , ( na , sa ) , ( na , zn ) , ( n , n ) , ( n , na ) , ( n , oa ) , ( n , p ) , ( n , s ) , ( n , sa ) , ( n , zn ) , ( oa , oa ) , ( oa , p ) , ( oa , s ) , ( oa , sa ) , ( oa , zn ) , ( p , zn ) , ( sa , sa ) , ( sa , zn ) , and ( s , zn ) . second , the electrostatic - interaction energy between protein and ligand atoms within 4 of each other was calculated and summed for each of the atom - type pairs described above : where qr is the partial charge of receptor atom r , ql is the partial charge of ligand atom l , and d is the distance between the two . the same atom - type pairs permitted for close - contact proteinligand atoms within 4 of each other were again used . third , a list of ligand atom types was likewise tallied , and the number of atoms of each type was counted . thirteen ligand atom types were permitted : a , br , c , cl , f , hd , i , n , na , oa , p , s , and sa . finally , the number of ligand rotatable bonds was likewise counted . in all , each proteinligand complex was thus characterized across 194 ( 14 + 83 + 83 + 13 + 1 ) dimensions . all neural networks were feed - forward networks created using ffnet(30 ) with 194 inputs and 2 outputs . all nodes in the hidden and output layers had log - sigmoid activation functions of the form where t is the input sum of the respective node . network inputs and outputs were normalized with a linear mapping to the range ( 0.15 , 0.85 ) so that each variable was given equal initial importance independent of its scale . there were no direct connections between the input and output layers ; all nodes of the hidden layer were connected to all nodes of the input layer , and all nodes of the output layer were connected to all nodes of the hidden layer . the networks were trained to return ( 1 , 0 ) for proteinligand complexes with experimentally measured kd values less than 25 m , and ( 0 , 1 ) for complexes with kd values greater than 25 m . to train each network , the weights of the connections between neurodes were first randomly assigned ; these weights were subsequently optimized by applying 10000 steps of a constrained truncated newton algorithm(31 ) as implemented in scipy.(32 ) training sets of varying sizes were employed . in all cases , training sets consisted of proteinligand complexes picked at random from the 4141 complexes of the training / testing database described above . the remaining complexes constituted the validation set , used to judge the network s predictive accuracy . abbreviations : ef , enrichment factor ; nci , national cancer institute ; nih , national institutes of health ; pdb , protein data bank .

as high - throughput biochemical screens are both expensive and labor intensive , researchers in academia and industry are turning increasingly to virtual - screening methodologies . virtual screening relies on scoring functions to quickly assess ligand potency . although useful for in silico ligand identification , these scoring functions generally give many false positives and negatives ; indeed , a properly trained human being can often assess ligand potency by visual inspection with greater accuracy . given the success of the human mind at proteinligand complex characterization , we present here a scoring function based on a neural network , a computational model that attempts to simulate , albeit inadequately , the microscopic organization of the brain . computer - aided drug design depends on fast and accurate scoring functions to aid in the identification of small - molecule ligands . the scoring function presented here , used either on its own or in conjunction with other more traditional functions , could prove useful in future drug - discovery efforts .

t cells and b cells derived from the lymphoid lineage belong to the adaptive immune system . function via production of effector cytokines after differentiation and activation . in comparison to the cytotoxic feature of both adaptive cd8 t cells and innate cnk cells , the helper feature of cd4 th cells was considered to be a unique characteristic of the adaptive system acquired during evolution . however , over the past few years several groundbreaking works on a novel member of the innate immune system , the innate lymphoid cell ( ilc ) , have dramatically changed our knowledge about the composition of the innate lymphoid lineage and led us to reconsider the relationship between innate and adaptive lymphoid lineages in the context of evolution . like other lymphocytes , ilcs also develop from the common lymphoid progenitors ( clps ) found in fetal liver and adult bone marrow . they were not discovered and classified as a new lymphocyte family until recently , partly due to their distinct enrichment in nonlymphoid tissues such as mucosal tissues , skin , and adipose tissues , with scarce distribution in lymphoid tissues . their lack of any known lineage surface markers may also contribute to their belated discovery . in actuality , scientists noticed certain subsets of ilcs such as lymphoid tissue inducers ( ltis ) as early as the 1990s [ 3 , 4 ] , but it was not until three independent reports on type 2 cytokine producing innate lymphoid cells ( ilc2s ) in 2010 [ 57 ] that people began to recognize the possible existence of an innate population with a helper feature mirroring adaptive th cells . the nomenclature of innate lymphoid cells ( ilcs ) was then formed based on the existence of helper lymphocyte in the innate arm of the immune system . similar to the classification of th cells , mature helper - like ilcs can be categorized into three groups based on their master regulator expression and signature effector cytokine production . ilc2s , the innate counterpart of th2 cells , express high levels of gata-3 and are capable of producing type 2 cytokines such as il-5 and il-13 [ 57 ] . ilc3s express rort and are capable of producing il-22 and il-17 , similar to th17/th22 cells [ 810 ] . the ccr6 lineage includes lymphoid tissue inducer ( lti ) cells , while ccr6ilc3 can give rise to a special population of rort ilc3s that express the natural cytotoxicity receptor ( ncr ) nkp46 ( encoded by ncr1 ) in mice and nkp44 ( encoded by ncr2 ) in humans . ccr6ilc3s can also express t - bet which drives further development of this lineage into the ncr stage [ 1113 ] . finally , ilc1s are the innate counterpart of th1 cells . they are t - bet positive and better ifn- producers than cnk cells [ 14 , 15 ] . absence of rort and eomes expression in ilc1s distinguishes them from rort - expressing ncr ilc3 and eomes - expressing cnk cells . initially , the nomenclature of ilcs included cnk cells , a notion many scientists still hold . but in this review , we limit ilcs to helper - like ilcs to distinguish these special innate lymphocytes from cnk cells . a defining function of ilcs is their production of a similar set of effector cytokines as those produced by cd4 th cells in the adaptive immune system . this feature of ilcs enables them to mount robust immune responses at the innate stage via acting on other immune or structure cells . through production of il-5 and il-13 , ilc2s may induce the first wave of eosinophil recruitment and stimulate epithelial and smooth muscle cells , during type 2 responses to helminth infection or allergen inoculation [ 17 , 18 ] . steady state production of il-22 by ilc3s is crucial for the homeostasis between host and commensals within the mucosal tissues . upon infection , ilc3s are also the major innate source of il-22 after receiving il-23 stimulation [ 8 , 10 ] . ilc1 cells are relatively scarce in the gut but enriched in the liver in steady state [ 14 , 20 ] . during early type 1 responses , ilc1s are better ifn- producers than cnk cells and they provide the initial protection in mice infected with t. gondii . ilc2s and th2 cells may collaborate to mount robust type 2 immune responses during the effector phase . some ilc2s express mhc class ii and thus are able to stimulate th2 cells to produce il-2 , which in turn promotes ilc2 proliferation and cytokine production [ 21 , 22 ] . however , possibly due to the lack or low level of costimulatory molecules cd80/cd86 on ilc3s , this type of antigen presentation functions through a suppressive mechanism to maintain the homeostasis of commensal specific th cell in the colon . ilcs have additional functions , which may or may not be shared by th cells . for example , ilc2 can produce amphiregulin , which facilitates the repair and reorganization of damaged tissues after viral infection . for example , ilc2s are enriched in adipose tissues and contribute to the beiging of white adipose tissue through production of cytokines and/or methionine - enkephalin [ 26 , 27 ] . through il-22 and lymphotoxin production , ilc3s can induce intestinal epithelial cell expression of fucosyltransferase 2 ( fut2 ) and thus regulate the epithelial fucosylation , which provides the metabolic substrates for commensals [ 28 , 29 ] . the development , maturation , and maintenance of distinct ilc subsets are regulated by a set of specific transcriptional regulators , including t - bet , gata-3 , and rort , similar to the regulation of effector th cell differentiation . a master regulator that determines th cell differentiation towards a particular subset also seems to direct the development of the related ilc subset . in addition , other factors such as ror , bcl11b , and ahr are involved in regulating the development and functions of ilc subsets . mirroring their critical functions during th1 , th2 , and th17 differentiation , the master regulators t - bet , gata-3 , and rort are also implicated in fate determination of ilc subsets . furthermore , gata-3 is required for the maintenance and function of fully developed ilc2s [ 30 , 31 ] . deletion of gata3 in mature ilc2s results in dramatic diminution of il-5 and il-13 production followed by the rapid disappearance of these cells . gata-3 is also expressed in ilc1 and ilc3 cells as well as in ilc progenitors , in which gata-3 has a critical function ; we will discuss gata-3 function during early ilc development in detail in the progenitor section below . t - bet induction in ilc3s may be driven by notch signals , il-23 stimulation , and the microbiota . the gradient increment of t - bet levels directs further development of ccr6ilc3s into ncr ilc3s . some cells may even turn off rort expression to become t - betncr ex - ilc3s . in addition , t - bet regulates ifn- production by the ncr ilc3s . besides ccr6ilc3s accordingly , t - bet deficient mice lack ilc1s and ncr ilc3s but have normal ilc2s and ccr6 ilc3s . ilc1s were previously confused with cnk cells as both express t - bet and are capable of producing ifn-. however , cnk cells express eomes while ilc1s do not . in addition , ilc1s may also express cell surface markers such as cd49a , cd160 , and/or cd127 , which are usually absent on cnk cells . ror is highly expressed by ilc2s and is necessary for their development [ 32 , 33 ] . ror deficiency results in dramatic reduction of ilc2s but does not affect the development of other ilc subsets . thus , mice reconstituted with ror deficient bone marrow are useful tools for studying ilc2 functions during immune responses . bcl11b is a critical factor during early stages of t cell development [ 34 , 35 ] . but during ilc development , bcl11b deficiency only blocks the development of ilc2s as shown by two independent studies published this year [ 3638 ] . bcl11b is expressed as early as the common ilc progenitor stage and it may suppress the development of ilc3s . the mechanism of how bcl11b specifies ilc2 fate is yet to be determined . unlike gata-3 function in mature ilc2s , bcl11b deletion does not affect the maintenance or function of mature ilc2s despite bcl11b being highly expressed in mature ilc2s . the aryl hydrocarbon receptor ( ahr ) is well known to be involved in th17 cell differentiation and function . ahr is also expressed by both ccr6 and ccr6 ilc3s [ 40 , 41 ] . in adult ahr deficient mice , ilc3 cell number in the gut is dramatically reduced , probably due to the defective accumulation and/or enhanced apoptosis of ilc3s . ahr is also critical for the function of ilc3s by regulating il-22 production and the ahr effects on ilcs seem to be ilc3 specific . ilcs , cnk cells , and t cells all develop from clps found in fetal liver and adult bone marrow . at late stages of t cell development , nave cd4 and cd8 t cells develop from the cd4cd8 double positive cells in the thymus whereas cd4 t effector th cells are differentiated from nave cd4 t cells in the periphery . considering the functional similarity between mature ilcs and cd4 th cells thus , a hypothesis concerning ilc development is that , like t cells , there may be a common progenitor for all innate lymphocytes , including ilcs and cnk cells , after the clp stage . in a subsequent stage , a common ilc progenitor would be capable of giving rise to all ilcs , in parallel with the potential of nave cd4 t cells to become different th effector cells . this hypothesis is supported by the fact that certain gene deletions affect the development of all innate lymphocytes and/or all ilcs . indeed , during the last year , we have witnessed a few breakthrough studies in identifying the common progenitors for ilcs [ 14 , 42 ] . id2 is required for the development of all ilc populations since its deficiency results in the loss of all ilcs . using an id2 reporter mouse strain , a lineagecd127flt-3integrin47 population has been reported in a stage after clp and has lost the potential to become t , b , and cnk cells . id2 expression within this population has been shown by expression of an id2 fluorescent reporter . previously reported immature ilc2s in bone marrow are also id2 ; however , they can be excluded by cd25 staining . the multipotential capacity of these id2cd25 progenitors for all ilc subsets was also confirmed by in vitro single cell development assay . these lineagecd127flt-3integrin47id2cd25 progenitors are thus termed as common helper - like innate lymphoid progenitors ( chilps ) . in addition to the critical role in maintenance and function of ilc2s , gata-3 is also crucial in the general development of all ilcs . it has been recently reported that gata3 deficiency prior to the clp stage affects the development of all ilcs in a cell intrinsic manner , indicating that gata-3 is a critical regulator for ilc development . in mice carrying a cre construct driven by the vav1 promoter to conditionally delete gata3 at the hematopoietic stem cell stage , dramatic defects in all il-7r-expressing ilcs in the periphery are noted . moreover , these gata3 conditional - deficient mice do not generate lymph nodes or peyer 's patches , consistent with the finding that the development of functional lti cells is defective at the fetal stage . they also succumb to citrobacter rodentium infection , consistent with another report showing that gata-3 mediates the development of ilc3s by using chimera mice with hematopoietic precursor cells from e12.513.5 gata3 embryos . the essential role of gata-3 during ilc development is consistent with its indispensable function during cd4 t cell development after the cd4cd8 stage in the thymus . consistent with its critical role during ilc development , gata-3 expression levels in the common ilc progenitors , such as chilps , are comparable to that in ilc2s . thus , gata-3 is likely a master regulator for the development of common ilc progenitors . given that both id2 and gata-3 are highly expressed in ilc progenitors , it is reasonable to speculate that while id2 may direct the acquisition of the innate feature of ilcs similar to cnk cells gata-3 may play an indispensable role in directing the helper feature of ilcs similar to cd4 t cells . because of this , gata-3 may distinguish the helper lineage from cytotoxic cnk lineage during innate cell development . altogether , id2 and gata-3 coexpression during the common ilc progenitor stage may establish a special regulatory network that determines the innate and helper features of the ilcs . by analyzing a plzf fate mapping mouse strain , researchers found that the majority of mature ilcs have expressed plzf with the exception of the ccr6 ilc3 lineage . plzf is hardly detectable in mature ilcs but is transiently expressed by a subset of the chilp population in fetal liver and adult bone marrow . these plzf progenitors are multipotential cells and are able to give rise to all ilcs except ccr6 lti cells . furthermore , these plzf progenitors do not develop into t cells , b cells , or cnk cells . plzf progenitors may develop after the chilp stage and have lost the potential to develop into lti lineage . the function of plzf during ilc development remains elusive particularly because plzf is only transiently expressed during the early development stage . furthermore , although zbtb16 mice with plzf deficiency have altered ilc development especially for ilc2s , all the ilcs can still be detected in these mice . an obvious issue for these defined common ilc progenitors is that they are actually heterogeneous populations . within the chilp cells , there should be some ccr6 ilc3 progenitors within the plzf population , which will never turn on plzf expression . even within the plzf population , only a minority of the cells have the potential to become multiple ilcs during in vitro single cell development assay . it is likely that both id2 chilps and plzf common ilc progenitors contain a large fraction of partially committed ilc immediate progenitors . common progenitor multipotent for all ilcs is still required . meanwhile , in addition to fetal liver and adult bone marrow , the common ilc progenitors may also exist in other tissues , such as fetal intestine , as was shown in a recent study using arginase-1 reporter mice . this report suggests that we should not limit ourselves to study fetal liver and adult bone marrow cells to gain further knowledge on ilc progenitors . as mentioned above , many ilc progenitors found in fetal liver and adult bone marrow may have already committed partially to a specific ilc lineage . indeed , bcl11b - expressing progenitors appear within the chilp population ; yet these cells have already committed to the ilc2 fate . there are also abundant immature ilc2s , presumably the most immediate ilc2 precursors , present in the bone marrow ; these cells express mature ilc2 markers sca-1 and cd25 . plzf fate mapping analysis indicates that most immature ilc1s have expressed plzf and thus are derived from the plzf ilc progenitors rather than the cnk progenitors . a couple of other transcription factors , nfil3 [ 4851 ] and tox , have also been found to be involved in the development of both ilcs and cnk cells . since their expression is detected earlier than id2 expression , which occurs at the chilp stage , the expression of these transcription factors may mark even earlier common progenitors for both ilcs and cnk cells , similar to cd4cd8 thymocytes during t cell development . however , the phenotypes of ilc development in mice deficient in either of these regulators are not as dramatic as those resulting from id2 or gata3 deficiency . the mechanisms through which nfil3 and tox function during ilc and cnk cell development require further investigation . given the functional similarities between the innate ilcs and cnk cells and the adaptive cd4 and cd8 t cells , it is reasonable to propose that ilcs and cnk cells follow a similar developmental pathway to that of t cells . a clp may give rise to a common progenitor for all innate lymphocytes including ilcs and cnk cells , which subsequently gives rise to chilps . distinct ilc subsets are further developed from the plzf chilp stage as the differentiation of th effectors from nave cd4 t cells . thus , the development of innate lymphocyte subsets , including ilcs and cnk cells , to a certain extent , mirrors that of t cells ( figure 1 ) . as discussed above , more and more regulators are being found to be involved in the development of ilcs . interestingly , most of these regulators are also involved with t cell development . however , distinct from t cell development , the regulatory functions of the various transcription factors during ilc development are not related to tcr - mediated thymic selection , which , to some extent , may explain why the deficiency of certain regulators has distinct effects on ilc and t cell development . although deletion of either one of the newly identified genes , including nfil3 , tox , or tcf7 [ 53 , 54 ] , results in defective ilc development to a various extent , none of these defects are as severe as that resulting from id2 or gata3 deficiency . thus , it is possible that in the early progenitors of the ilc lineage , these regulators may function through a network designed for fine - tuning ilc development . id2 and gata-3 might form the core complex , while other regulators are involved in tuning the optimal function of the core components . id2 or gata3 deficiency dramatically affects ilc development , whereas deletion of other regulatory components results in various incomplete defects of ilc development . nuclear factor interleukin-3 ( nfil3 ; also known as e4-binding protein 4 , e4bp4 ) , a basic region leucine zipper transcription factor , is critical for cnk cell development . based on this , it is possible that nfil3 is expressed and functions at a progenitor stage common to both ilcs and cnk cells . nfil3 deficient mice showed dramatic defects during the transition from clp to chilp stage [ 48 , 51 ] . however , ilc development is not completely abolished in nfil3 deficient mice ; in the periphery , there are still substantial numbers of ilcs that have escaped the developmental block , although the population is too small to control infectious challenges . nfil3 expression is induced by il-7 signaling , which is also critical for the development of ilcs . after development , nfil3 maintains a very high level of expression in mature ilcs , a level higher than that in cnk cells . nfil3 is dispensable for ilc3 maintenance ; however , its function in other mature ilcs remains unclear . thymocyte selection - associated high - mobility group box protein ( tox ) is a member of the high - mobility group box superfamily . it contains a dna - binding domain and is required for the development of t [ 56 , 57 ] , nk , nkt , and lti cells . a new study this year showed that tox also has broad effects in ilc development . in tox deficient mice , the chilps as well as mature ilcs were severely reduced compared with these cells in wild type mice . there is an intrinsic defect in the notch signaling pathway in tox deficient chilps , which may explain the defect in ilc development in tox deficient mice . t cell factor 1 ( tcf-1 ) is a critical transcription factor for t cell fate specification at the early development stage . tcf7 ( gene encode tcf-1 ) deficiency affects both ilcs [ 53 , 54 ] and cnk cells . similar as during t cell development , tcf-1 is regulated by notch signaling and may be involved in the induction of il-7r and gata-3 expression during ilc development [ 53 , 54 ] . based on the sequential expression of nfil3 , tox , tcf-1 , id2 , and gata-3 and the knowledge we have concerning their relationships in different systems , it is likely that nfil3 expression initially increases after the clp stage , which in turn regulates the expression of tox and id2 . after id2 is turned on , the core assembly involved in directing the innate features of ilc development begins to function . although gata-3 is expressed at low levels at the clp stage , its function during ilc development requires high expression levels . tox is involved in the regulation of notch signaling pathway , which in turn regulates tcf-1 and gata-3 . tcf-1 is then required for optimal expression of gata-3 . upon increased expression of gata-3 , the helper transcription factor assembly nfil3 , tox , and tcf-1 , in connection with the notch pathways , form a network to prepare for the upregulation of id2 and gata-3 , which together form the executive regulatory network to direct lineage fate determination of ilcs , possibly with continued assistance from the initiation transcription factors such as tcf7 , tox , and nfil3 ( figure 2 ) . additional experiments are required to confirm this proposed regulatory network model and it is likely that additional regulators for ilc development may soon be discovered . studies on the newly identified ilcs in recent years have enriched our knowledge on the innate lymphoid lineage development and have provided us with more evidence supporting the close relationship between the innate and adaptive immune systems during evolution . the classification of the ilc population was initially based on their capacity to produce a similar set of effector cytokines compared to th cells . subsequent studies have revealed that the development of ilcs is also regulated by a similar set of key transcription factors required for t cell development . after the clp stage , innate lymphoid lineages and t cells lineages start to develop separately but the regulatory mechanisms may still be shared . nfil3 , tox , and tcf-1 expression at an early stage immediately after clp in the innate lymphoid lineage may mark the common progenitor for both ilcs and cnk cells . ilc and cnk lineages are further separated by the induced expression of id2 and gata-3 in the common ilc progenitors . at later stages , additional regulators are upregulated to drive the distinct fates of different ilc subsets . in conclusion , the ilc cell fate is precisely regulated during development by a network of multiple serially expressed regulators , which may form a regulatory network during development and render the developed ilcs with both innate and helper features .

recent studies on innate lymphoid cells ( ilcs ) have expanded our knowledge about the innate arm of the immune system . helper - like ilcs share both the innate feature of conventional natural killer ( cnk ) cells and the helper feature of cd4 + t helper ( th ) cells . with this combination , helper - like ilcs are capable of initiating early immune responses similar to cnk cells , but via secretion of a set of effector cytokines similar to those produced by th cells . although many studies have revealed the functional similarity between helper - like ilcs and th cells , some aspects of ilcs including the development of this lineage remain elusive . it is intriguing that the majority of transcription factors involved in multiple stages of t cell development , differentiation , and function also play critical roles during ilc development . regulators such as id2 , gata-3 , nfil3 , tox , and tcf-1 are expressed and function at various stages of ilc development . in this review , we will summarize the expression and functions of these transcription factors shared by ilcs and th cells . we will also propose a complex transcriptional regulatory network for the lineage commitment of ilcs .

sarcoidosis is a multisystemic granulomatous disease of unknown etiology characterized by the presence of non - caseating granuloma consisting of epithelioid cells . heart , spleen , bone marrow and less often eye , skin and salivary glands are common extrapulmonary sites of disease manifestation . the liver has sarcoid nodules on occasions , however these nodules are generally multiple and diffuse . granulomatous lesions in hepatic sarcoidosis are most often located at the catchment area of glisson 's capsule . in these patients , portal hypertension is the predominant symptom . the diagnosis of hepatic sarcoidosis might be easy because patients with hepatic sarcoidosis have generally pulmonary lesions or hilar lymph node swelling at the same time . we present a case of hepatic sarcoidosis with a solitary giant nodule in the liver , but with radiological findings typical of hilar cholangiocarcinoma . a 51-year - old female was referred to our institution with abnormal laboratory data of liver function and the biliary system . the laboratory data on admission showed slight elevation of alkaline phosphatase ( alp ) and gamma - glutamyl transpeptidase ( -gtp ) , but a normal level of tumor marker ( table 1 ) . her physical condition upon admission was generally good ; there was no anemia or jaundice , and the liver was not palpable . abdominal ultrasonography showed a low echoic lesion of 37 mm diameter with an unclear margin at segment 4 . the intrahepatic bile ducts ( ihbds ) of the left lobe were dilated to 6 mm with obstruction by this tumor . a swelling round lymph node of 11 mm diameter was revealed in the hilar region of the liver . abdominal dynamic computed tomography ( ct ) revealed a 4.0 cm tumor of low density at segment 4 , which partially invaded segments 1 , 5 , and 8 . , the tumor was detected as a low - density lesion with an irregular shape ( fig . the contrast enhancement of the tumor was slightly intense at the margin of the tumor during the equilibrium phase ( fig . 1c ) and became more intense during the delayed phase , but the margin became unclear ( fig . magnetic resonance cholangiopancreatography showed remarkable dilatations of the ihbds of segment 2 and 3 with an extreme stenosis of the left main hepatic duct . endoscopic retrograde cholangiopancreatography also revealed conspicuous stenosis at the junction of the left hepatic duct and the common hepatic duct as well as stenosis and rigidity of the anterior and posterior branches of the right hepatic duct . moreover , the junction of the hepatic duct draining the right caudate lobe had stenosis ( fig . when deep cannulation was performed beyond the stenotic hepatic duct , dilated ihbds of segment 2 , 3 and 4 were detected . 2b ) . extended left lobectomy with caudate lobectomy and bile duct resection was performed with a diagnosis of hilar cholangiocarcinoma located predominantly at the left main hepatic duct . some daughter nodules were found in the left lobe , which were considered to be intrahepatic metastasis . pathological findings confirmed the presence of non - caseating granuloma with multinucleated giant cells . there were multiple nodules with severe fibrosis in the resected specimen . a lot of small non - caseating granulomas consisting of epithelioid cells were found in the dissected lymph nodes . the case reported here is important because of its morphologically distinct characteristics from radiological findings for hepatic sarcoidosis . sarcoidosis is a systemic disease that primarily affects the lungs and lymphoid tissues of the body . in japan , sarcoidosis occurs mainly in the age group of 50- to 60-year - olds with a predilection for females . hepatic involvement of sarcoidosis was described in 11.5% of 736 patients in the access study . hepatic granulomas due to sarcoidosis were recognized most commonly as multiple and small nodules with less than 1.0 cm diameter . liver involvement ranges from asymptomatic incidental granulomas to portal hypertension from granulomas in the portal triad , usually with relative preserved liver function . angiotensin - converting enzyme ( ace ) level may be elevated in 70% of patients with sarcoidosis and is elevated in almost all patients with hepatic sarcoidosis . in general , in 2040% of patients with sarcoidosis , alp and -gtp levels are elevated , but in those with hepatic sarcoidosis the levels are usually much higher . ultrasonography findings include parenchymal echogenicity , coarsening of the liver parenchyma with or without discrete nodules , and focal calcifications as well as contour irregularity . on ct , hepatic granulomas are visualized as multiple , discrete , low - attenuating , non - contrast - enhancing nodules of variable size . as they increase in size , they tend to become confluent and have to be differentiated from various infectious and neoplasmatic conditions . magnetic resonance imaging also shows multiple diffuse , densely packed , uniform nodular foci with normal signal intensity with t1- and hypodense with t2-weighted sequence . on dynamic ct imaging , our case was compatible with hepatic sarcoidosis due to a poor enhancement effect on early arterial phase and a slight enhancement from the margin on delayed phase , even though this intrahepatic nodule was huge and solitary . in most patients with sarcoidosis , the course of hepatic involvement is asymptomatic . in a few patients , chronic intrahepatic cholestasis or portal hypertension portal hypertension could be due to obstruction of portal flow because of granulomas in the portal area , with fibrosis and hyalinization of the portal triad causing presinusoidal block . another possible mechanism was advocated in which there might be arteriovenous shunts that increase portal blood flow . asymptomatic patients with mild elevation in liver function enzymes should not be treated but should be followed with serial blood work . it has been reported that the majority of untreated patients who are clinically asymptomatic have spontaneous improvement in their liver function . indeed , perry and vuitch reported that the mortality rate of patients with sarcoidosis was 1 of 28 at autopsy and 1 of 18 with portal hypertension . for patients with refractory liver dysfunction , chronic progressive cholestatic disease and portal hypertension that develop more severe liver disease , corticosteroids should be administered . however , corticosteroids can not prevent disease progression , including development of portal hypertension in patients with early or established cirrhosis . for such cases , when medications are no longer efficacious , liver transplantation is the only option . there are other causes of hepatic granulomas , including bacterial infections such as tuberculosis , brucellosis , viral infections and parasite infections , primary liver disease such as primary biliary cirrhosis , autoimmune cholangiopathy and many other systemic illnesses . as mentioned above , there is a wide differential for granulomas in the liver . in western countries , a large portion is attributed to primary biliary cirrhosis ( up to 55% ) and sarcoidosis ( 1530% ) , but infective cases , like tuberculosis and brucellosis , should always be kept in mind during initial investigation [ 12 , 13 ] . evaluating hepatic granulomas the majority of cases in which granulomas appear only in the liver are caused by liver disease rather than multisystemic disease . in that case , the differential diagnosis may be frequently difficult compared to other tumor - forming diseases , such as neoplasm in the liver . the diagnostic approach to hepatic sarcoidosis must be made on the histological evidence of non - caseating granulomas in a liver biopsy . in this report , we describe a rare experience with hepatic sarcoidosis , which we presented with clinical and radiological findings characteristic of hilar cholangiocarcinoma . our experience suggests that the diagnosis of patients with tumors in the liver with atypical radiological findings , who have a case history of sarcoidosis , need to be established by comprehensive and cautious consideration . there should be no hesitation to perform liver biopsy if there is difficulty in making a definitive diagnosis . despite utilizing several detailed and extensive diagnostic modalities , we may occasionally encounter a patient with a solitary tumor in the liver for whom it is extremely difficult to make a differential diagnosis from the malignant neoplasm , much like our experience . in this case , a surgical treatment including liver resection should be seriously considered , because a patient with hepatic sarcoidosis occasionally had to undergo liver transplantation in the end after a short period of observation without any treatment due to the difficulty of diagnosis like our case .

sarcoidosis is a multisystemic granulomatous disease of unknown etiology . hepatic involvement was reported in about 11% of patients with sarcoidosis . however , cases of sarcoidosis in which the granuloma is solitary and limited in the liver are very rare . a 51-year - old woman with tumors in the liver underwent extended left lobectomy with caudate lobectomy and bile duct resection . the tumor was located between segment 4 and the hilar region . some daughter nodules were found in the left lobe , which were regarded as intrahepatic metastasis . our case displayed clinical and radiologically distinct findings , which are very similar to those of hilar cholangiocarcinoma restricted to the liver . this report demonstrates that sarcoidosis can show solitary hepatic involvement in the absence of thoracic lymphadenopathy . in such a case , it is difficult to distinguish the diagnosis from other malignant neoplasms . in conclusion , the diagnosis of hepatic sarcoidosis has to be made through prudent and comprehensive investigations that include a full clinical history of sarcoidosis in other organs . despite utilizing several detailed diagnostic modalities , the definitive diagnosis of cases of solitary sarcoidosis may remain difficult . in these cases , surgical treatment including liver resection should be considered in order to avoid missing a suitable opportunity for treatment .

our survey covered all small molecule new molecular entity ( nme ) drugs that are intended for systemic use and were approved by the fda between january 2013 and august 2016 . the new drug application ( nda ) review documents ( drugs@fda , http://www.fda.gov/drugsatfda ) and product labels were examined for pbpkrelated information for the 85 products that met the above criteria . there were a total of 18 products for which pbpk models were considered in the nda review documents ( tables 14 ) . in the majority of cases , pbpk models were used for the prediction of the effect of metabolic enzyme mediated drugdrug interactions ( ddis ; figure 1a ; tables 14 ) . importantly , the frequency of model use is consistent with the perceived level of reliability.1 , 3 , 5 overview of physiologically based pharmacokinetic ( pbpk ) information in product labels or us food and drug administration ( fda ) review documents for drugs approved by the fda between january 2013 and august 2016 . ( a ) number of new molecular entities ( nmes ) with information of pbpk for respective areas of applications . ( b ) proportion of product labels / reviews containing pbpk information for drugs in all nmes , anticancer agents , and nmes with breakthrough therapy designation and/or accelerated approval status at the time of approval . seven of eight nmes with pbpk and breakthrough / accelerated approval status were anticancer agents . a key caveat of our survey was that it was limited to publicly available information . the utilization of pbpk models for sponsor 's internal decisionmaking and earlier stage and postapproval regulatory interactions , such as postmarketing requirement ( pmr ) or supplementary nda , were not captured . pbpk models have great potential to influence decisionmaking at different stages of drug development , such as initial dosing recommendations for pediatric clinical trials , design of ddi studies , etc.5 the current evaluation should be interpreted in the context of pbpk use during the final phase in the development of a new drug , because pbpk modeling strategies and the level of model validation can vary at different stages of drug development . this survey also did not capture the potential value of cost and speed savings by using pbpk vs. conducting clinical studies . interestingly , we observed wider acceptance of pbpk models in the field of oncology compared with other therapeutic areas ( figure 1b ) . one possible explanation is the difficulty of conducting clinical ddi studies in oncology , due to ( 1 ) shortage of appropriate patient populations or ( 2 ) ethical and safety concerns over exposing healthy volunteers to oncology medications . the first point is supported by the observation that three of eight nmes with pbpk in the nononcology field were for rare diseases ( such as eliglustat , macitentan , and obeticholic acid ) . another possible explanation is that the higher levels of toxicity and narrower therapeutic windows for oncology drugs compared with drugs in other therapeutic areas warrant precise optimization of drug exposure . it is also noteworthy that 7 of 10 anticancer agents were given regulatory incentives to accelerate drug development , either with breakthrough therapy designation or accelerated approval , which may have contributed to a greater reliance on pbpk simulations . pbpk models have been most extensively used for the prediction of the effect of ddi or pharmacogenetic effect on the pharmacokinetics of nme as a victim ( figure 1a ) . in particular , the extrapolation of the effect of strong inhibitors or inducers to less potent perpetrators constituted the majority of the applications ( 11 among 13 nmes ; table 1 , ids 111 ) , and all applications resulted in labeling recommendations ( table 1 ) . in these cases , existing clinical data with strong perpetrator(s ) were used to anchor the pbpk model performance , namely by accurately providing fraction metabolized by a particular enzyme , which provides a higher level of confidence in ddi prediction.1 , 5 for example , a fourfold dose reduction of ibrutinib was recommended for patients taking moderate cytochrome p450 ( cyp)3a inhibitors based on the pbpk model validated with clinical ddi data using strong perpetrators . pbpk models in product labels or fda review documents : new drug as a victim of ddis or genetic variations cyp , cytochrome ; ddi , drugdrug interaction ; i d , identification ; nme , new molecular entity ; pbpk , physiologically based pharmacokinetic ; pmc , postmarketing commitment ; pmr , postmarketing requirement ; ugt1a1 , udpglucuronosyltransferase 1a1 . the numbers in the reference column represent pubmed i d ( if physiologically based pharmacokinetic models were published in scientific journals ) . the elimination pathways for all the 11 nmes involve cyp3amediated metabolism , whereas two are metabolized by cyp2d6 in addition to cyp3a . this observation is not surprising , considering that cyp3a and cyp2d6 are involved in metabolism of a large proportion of marketed drugs , and wellestablished probe perpetrators are available for these two enzymes . for two cyp3a and cyp2d6 dual substrates , aripiprazole lauroxil and eliglustat , complex interactions involving the inhibitors of these enzymes and cyp2d6 genotype anchoring of model prediction with both a strong inhibitor and an inducer has been conducted in most cases , but there were two cases in which model validation with inhibitors was used for the prediction of inducer effect ( cobimetinib , panobinostat ) , both of which resulted in a conservative labeling language of avoiding concomitant use with the strong inducers . this may suggest that the required level of pbpk model qualification depends on the context of pbpk application . because modification of recommended dose in the product label application of pbpk,2 the use of a certain amount of clinical pharmacokinetic data as an external validation dataset has generally been required for including dosing recommendations in the product label . the case of belinostat is intriguingalthough there were no external data , the simulation performed by the fda during the nda review resulted in label language recommending a 25% reduction in the starting dose for homozygous carriers of a genetic polymorphism of its major metabolic enzyme , udpglucuronosyltransferase 1a1 . this is likely , in part , because the recommended starting dose is equal to the maximum tolerated dose . pmr to definitively examine the effect of pharmacogenetic alteration on belinostat pharmacokinetics and safety was included in the nda approval . therefore , even when the pbpk approach could not obviate the conduct of a dedicated clinical study , the model can inform the optimal use of medications . the second most frequent use of pbpk modeling is to predict the ddi potency of an nme as a perpetrator ( inhibitor / inducer ; table 2 ) . four nmes ( ids 1417 ) have successfully utilized a pbpk modeling approach to demonstrate the lack of a clinically significant effect on the metabolic pathways of interest for the particular drug . one method was to use a negative clinical ddi result with other cyp enzymes as an external validation ( alectinib , panobinostat ) , whereas another was to use a sensitivity analysis on inhibition parameters ( alectinib , canagliflozin ) . in the case of lenvatinib , an external model validation was seemingly not performed , presumably supported by general perception that pbpkbased prediction of mechanismbased inhibition leads to overestimation of ddis.5 these observations based on limited cases suggest that required levels of model validation could be flexible , and that we may expect wider application of pbpk in this category . pbpk models in product labels or fda review documents : new drug as a perpetrator of drugdrug interactions cyp , cytochrome ; ddi , drugdrug interaction ; i d , identification ; mate , multidrug and toxin extrusion transporter ; nme , new molecular entity ; oct2 , organic cation transporter 2 ; pbpk , physiologically based pharmacokinetic ; pmc , postmarketing commitment ; pmr , postmarketing requirement . the numbers in the reference column represent pubmed i d ( if physiologically based pharmacokinetic models were published in scientific journals ) . there were two nmes with pbpk models for which additional dedicated clinical ddi studies with cyp3a substrates were requested as pmr . for ceritinib , in addition , neither of these two nmes had supporting clinical data to verify model performance . further examples will be needed to evaluate the ability of the pbpk approach to quantitatively predict positive ddi effects.1 most of the pbpk applications on absorptionrelated pharmacokinetic changes were exploratory and did not impact the labeling recommendation ( table 3 ) . altered panobinostat absorption was not observed with drugs that elevate the gastric ph in pbpk simulation . pbpk models in product labels or fda review documents : drug absorption ara , acid reducing agents ; auc , area under the curve ; cmax , peak plasma concentration ; fda , us food and drug administration ; i d , identification ; nme , new molecular entity ; pbpk , physiologically based pharmacokinetic ; pgp , pglycoprotein . the numbers in the reference column represent pubmed i d ( if pbpk models were published in scientific journals ) . other pbpk applications observed in the nda review documents include prediction of the effect of hepatic impairment ( hi ; table 4 ) , but pmrs were issued for three of four nmes to conduct or complete dedicated clinical studies , presumably because of the low level of reliability.1 in the case of obeticholic acid , the effect of hi on hepatic exposure was explored with pbpk simulations , and this helped to inform the dosing recommendation in patients with hi . pbpk models in product labels or fda review documents : other areas of pbpk applications cyp , cytochrome ; ddi , drugdrug interaction ; hi , hepatic impairment ; i d , identification ; nme , new molecular entity ; oatp , organic aniontransporting polypeptide ; pbpk , physiologically based pharmacokinetic ; pmr , postmarketing requirement . the numbers in the reference column represent pubmed i d ( if physiologically based pharmacokinetic [ pbpk ] models were published in scientific journals ) . the regulatory impacts of pbpkbased predictions have been limited in areas other than metabolismbased ddis and pharmacogenetics , such as in transportermediated ddis or effect of acid reducing agents . accumulated experience of pbpk application in these areas will help to establish the level of confidence necessary to inform regulatory decisions . also , some degree of model validation with clinical data using the nme has generally been required for highimpact regulatory decisions.2 currently , validation of system components , such as hepatic metabolic activity in patients with renal impairment , based on other molecules that share same elimination pathways has not been considered sufficient . such limitations have impeded the application of pbpk models to replace clinical studies for evaluating socalled intrinsic factors , such as in pediatric populations , to evaluate ethnic differences in pharmacokinetics , or in patient populations with organ impairment . the authors believe that future verification efforts of pbpkbased prediction performance with cross learning from other molecules , including both xenobiotics and endogenous biomarkers , will greatly expand the use of pbpk beyond ddi and pharmacogenetic applications and will contribute to acceleration of new drug development .

physiologically based pharmacokinetic ( pbpk ) modeling can be used to predict drug pharmacokinetics in virtual populations using models that integrate understanding of physiological systems . pbpk models have been widely utilized for predicting pharmacokinetics in clinically untested scenarios during drug applications and regulatory reviews in recent years . here , we provide a comprehensive review of the application of pbpk in new drug application ( nda ) review documents from the us food and drug administration ( fda ) in the past 4 years .